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Mechanisms of neuronal angiotensin II receptor regulation

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Mechanisms of neuronal angiotensin II receptor regulation
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Kalberg, Christopher John, 1961-
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vi, 162 leaves : ill. ; 29 cm.

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Angiotensin II -- physiology ( mesh )
Receptors, Angiotensin ( mesh )
Physiology thesis Ph.D ( mesh )
Dissertations, Academic -- Physiology -- UF ( mesh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis (Ph.D.)--University of Florida, 1989.
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Bibliography: leaves 143-161.
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Typescript.
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Vita.
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by Christopher John Kalberg.

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Full Text
MECHANISMS OF NEURONAL
ANGIOTENSIN II RECEPTOR REGULATION
By
CHRISTOPHER JOHN KALBERG
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA

1989




ACN4OWE GEMENIS
I wish to extent my sincerest gratitude to the chairman of my ccmnittee, Dr. Colin Sumners, for his unending support, understanirng and breadth of knowledge. I am indebted to Colin for the respect and independence he gave me which allowed me to grow both as a student and an individual. My thanks go to the other members of my cuittee, Drs. Melvin Fregly, Mohan Raizada and Ed Meyer, for helping me view my research from their enlightened perspectives. Additionally, I would like to thank the chairman of the physiology department, Dr. Ian Phillips, for his support and development of the graduate program. For their expert technical assistance, I thank Jacqueline Perez and Tanmy Gault. For their invaluable assistance with administrative and technical affairs, I sincerely thank Kevin Fortin and Victoria LaPlaca. I am indebted to my colleagues Drs. Laura Mudd and Brain Masters for helping me weather some moribund times and classes, and I thank the distinguished Jenny Chou for making it fun to come to the lab. My sincerest thanks go to Drs. Elaine Sumners and Linda Myers for all their help in the lab.
I am most indebted to my mother, father, Tutu, and grandmother for their support and understarding throughout the years. Finally, I wish to thank Jacqueline Perez, again, for being my best friend and compatriot over the last four years. I look forward to many happy years together.




TABLE OF CONTENTS
ACKNOWLEDGEMENTS ......... ..................ii
ABSTRACT ........... ...................... v
CHAPTERS
I. INTRODUCTION ........... .........1
The Renin-Angiotensin System in the Brain 1
Angiotensin II Receptors in the Brain ... 5
Regulation of the Renin-Angiotensin System
in the Brain......... . . 11
Regulation of Angiotensin II Receptors . 12
Neuronal Cultures: A Model to Study
Angiotensin II Receptor Regulation ... 18
Intracellular Mechanisms Involved in
Angiotensin II Receptor Regulation ... 23
General Hypothesis .... ............. 28
II. CHARACTERIZATION OF PROTEIN KINASE C
IN NEURONAL CULTURES .... ............. 32
Introduction ...... ................ 32
Methods ........ ................... 34
Results ........ ................... 39
Discussion ...... ................. 45
III. IDENTIFICATION AND CHARACTERIZATION OF
PROTEIN KINASE C INVOLVEMENT IN ANGIOTENSIN II
RECEPTOR EXPRESSION .... ............. 57
Introduction ...... ................ 57
Methods ........ ................... 58
Results ........ ................... 63
Discussion ...... ................. 77

iii




IV. ADRENERGIC REGULATION OF PROTEIN KINASE C
SUBCELLULAR DISTRIBUTION ... ........... 87
Introduction ...... ................ 87
Methods ........ ................... 89
Results ........ ................... 92
Discussion ....... ................. 102
V. REGULATION OF ANGIOTENSIN II RECEPTORS UNDER
DEPOLARIZING CONDITIONS ... ........... 108
Introduction ...... ................ 108
Methods ........ ................... 109
Results ........ ................... 112
Discussion ....... ................. 119
VI. SUMMARY ........ .................... 128
REFERENCES ......... ..................... 143
BIOGRAPHICAL SKETCH ....... ................. 162




Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy MECHANISMS OF REGULATION
OF NEURONAL ANGIOTENSIN II RECEPTORS By
Christopher John Kalberg
December, 1989
Chairman: Dr. Colin Sumners
Major Department: Physiology
The activity of calcium, phospholipid-dependent protein
kinase, protein kinase C, was characterized in primary neuronal cultures prepared from the brains of neonatal rats. Several protocols were used to determine that protein kinase C is integrally involved in the stimulation of angiotensin II receptor expression in culture. These included the use of non-phorbol ester activators of protein kinase C, the use of an antagonist of protein kinase C and the downregulation of protein kinase C activity.
The involvement of protein kinase C in the upregulation of angiotensin II receptors produced by the activation of a,adrenergic receptors was investigated. Incubations of the neuronal cultures with epinephrine for 1-5 min resulted in a




translocation of protein kinase C from the cytosol to the
particulate fraction. This effect was transient and was blocked by prazosin indicating the involvement of eq-adrenergic receptors. Thus, protein kinase C may mediate a1-adrenergic receptorstimulated increases in angiotensin II receptor expression.
Conversely, the influx of calcium has been implicated in the downregulation of angiotensin II receptors. In this study, under depolarizing conditions, which were shown to significantly
increase the uptake of 4Ca2+, a decrease in the specific binding of [(26 I]angiotensin II occurred in the neuronal cultures after incubations of 2-4 hrs. Both the uptake of 4Ca2+ and the decrease in the specific binding of [nsI]Ang II observed under depolarizing conditions were blocked by the calcium channel antagonist, nifedipine. These data indicate that the influx of
Ca+ through voltage sensitive calcium channels is a mechanism involved in the downregulation of angiotensin II receptors.
This study suggests that two mechanisms are involved in the regulation of angiotensin II receptors in the neuronal brain cells cultures. An overview of the possible scenarios in which these regulatory mechanisms may act is presented.




CHAPTER I
INTRODUCTION
The Renin-Angiotensin System in the Brain
The discovery by Goldblatt et al. in 1934 that renal artery constriction produced a prolonged elevation of systemic arterial pressure initiated the search for endogenously produced pressor substances. In 1956, this search was, in part, culminated by the isolation and identification of the vasoactive octapeptide angiotensin II (Ang II) (Skeggs et al., 1956a; Lentz et al.. 1956; Bumpus et al., 1957). Originally termed hypertensin, Ang II (Braun-Menendez and Page, 1958) is formed by a series of
proteolytical events. The initial event in Ang II production involves the cleavage of the decapeptide angiotensin I (Ang I) from the c-globulin angiotensinogen by the enzyme renin. Ang I is then cleaved of two amino acids by angiotensin converting enzyme (ACE) to produce Ang II. Using pithed cats, initial investigators presumed the pressor actions of Ang II were limited to the periphery (Bumpus et al., 1957). However, in 1961, Bickerton and Buckley identified an action of Ang II in the brain using a cross-perfusion preparation. In this study, the blood supply to the head of a recipient dog was detached from its peripheral circulation by ligation of the carotid arteries. The




blood supply to the head of a donor dog was rerouted to the head of the recipient dog. The peripheral circulation of the recipient
dog was left intact. The injection of Ang II (0.2 /g/kg-0.4 Ag/kg) into the carotid artery of the donor dog produced a pressor response in the recipient dog. This pressor response in the recipient dog occurred even though its central blood flow was detached from its peripheral circulation. Thus, a central pressor effect of Ang II which appeared to be neurogenic in origin was
identified.
Presently, the actions of Ang II in the brain are known to extend well beyond the pressor response identified by Bickerton
and Buckley. These actions are manifested by both physiological and behavioral responses which interact in a complex manner to maintain body-fluid homeostasis. The intraventricular (i.v.t) administration of Ang II (eg in the picogram range) produces the following effects: the stimulation of drinking (Booth, 1968; Epstein et al., 1969), the facilitation of adrenocorticotropin (ACTH) release from the anterior pituitary (Maran and Yates, 1977; Spinedi and Negro-Vilar, 1983; Keller-Wood et al., 1986), and an increased salt appetite (Fluharty and Epstein, 1983). Severs et al. (1967, 1970) and Severs and Daniels-Severs (1973) further characterized the pressor effect of centrally
administered Ang II. They determined that the increase in blood pressure elicited by Ang II was due to an activation of the sympathetic nervous system and the release of vasopressin (AVP)




from the posterior pituitary. Thus, several actions of Ang II on the brain are well established. However, some controversy exists as to the site of production of the Ang II which acts centrally. This is because the peripheral administration of Ang II (eg in the nanogram range) can mimic many of the effects of centrally administered Ang II. This raises the possibility that Ang II produced peripherally could affect the brain. Ang II cannot cross the blood-brain barrier under conditions where the blood-brain barrier is intact (Phillips, 1980). However, certain small areas of the brain, the circumventricular organs, lie outside of the blood-brain barrier. It is likely that peripherally administered Ang II acts at the circumventricular organs to affect the
function of the brain.
Peripherally, the existence of a functional reninangiotensin system is firmly established. The main loci of Ang II production in the periphery are the kidneys, liver and lungs (Peach, 1977). Ang II has several sites of action in the periphery including the vascular smooth muscle, the sympathetic
nervous system and the adrenal glands (Peach, 1977). Similar to the central nervous system, the peripheral actions of Ang II act to maintain body fluid homeostasis. The establishment of a functional renin-angiotensin system in the brain has proven to be more difficult. This is primarily due to the inherent complexity of the brain. However, over the last 18 years much research has
confirmed the presence of a renin-angiotensin system in the




4
brain. The existence of Ang II in the brain was first inferred by the identification of Ang II-like material in cerebral spinal fluid. (Finkielman et al., 1972; Hutchinson et al., 1975). More recently, Ang II in the brain has been characterized using high
performance liquid chromatography (Ganten et al., 1983; Hermann et al., 1984; Phillips and Stenstrom, 1985) and localized to specific brain regions using immunohistochemical and immunofluorescence techniques (Lind et al., 1985; Weyhenmeyer and Phillips, 1982). These studies indicate Ang II is primarily located in the hypothalamus and brainstem which are areas of the brain closely associated with the central actions of Ang II (eg pressor and dipsogenic effects).
Ang II is not synthesized de novo, but is produced from a
series of proteolytic steps. Therefore, for Ang II to be produced in the brain the intermediaries involved in Ang II production must be present in the brain. The initial precursor of Ang II, angiotensinogen, has been found in specific brain areas (Hawkins
and Printz, 1983; Healy and Printz, 1985; Lewicki et al., 1978). These include the hypothalamus, the organum vasculosum lamina
terminalis (OVLT), and the area postrema (AP) of the brainstem. Further, angiotensinogen mRNA is expressed in the brain (Dzau et
al., 1986; Lynch et al., 1986; Ohkumbo et al., 1986). Renin-like activity is present in the brain as identified by bioassay (Ganten et al.. 1971; Fischer-Ferraro et al., 1971) and immunocytochemical techniques (Fuxe et al., 1980). The presence




of renin mRNA in the brain is still being debated although one group has reported the presence of low levels of renin mRNA in the brains of rats (Dzau et al., 1986). Additionally, ACE has been localized in the brain (Yang and Neff, 1972).
Angiotensin II Receptors in the Brain
The biological actions of Ang II are elicited at the cellular level. Here, Ang II binds to specific cell surface receptors to affect cellular functions. The integration of the actions of Ang II at the cellular level results in a given biological response. Specific, high affinity binding sites for Ang II were first identified in peripheral tissues using
monoiodinated ([Q'I]) Ang II ([I]Ang II) as the receptor ligand. High levels of [ I]Ang II binding were found in the adrenal cortex (Glossman et al., 1974) and in aortic tissue (Devynk et al., 1974). Bennett and Snyder (1976) first characterized Ang II binding sites in the brain using the brains of calves and rats.
[MI]Ile5-Ang II was used as the receptor ligand. Here, classical radioligand membrane binding techniques were used to satisfy the basic criteria necessary for the identification of receptors.
That is, [n2I]Ang II binding was shown to be saturable, reversible, specific, and kinetically consistent. Saturation of the Ang II binding sites was achieved by incubating calf cerebellar cortex membranes with increasing concentrations of [UI]Ang II. The binding saturated at approximately 0.5 nM
[MI]Ang II and the specific binding of [125I]Ang II accounted for




80-90% of the total binding. Transformation of the saturation data by Scatchard analysis (Scatchard, 1949) revealed a linear plot. This indicates a single, homogeneous class of binding
sites. The dissociation constant (kd) and number of binding sites as determined by Scatchard analysis were 0.2 nM and 1600 fmol/g tissue, respectively. The binding specificity was determined by
competing Ang II and Ang II-related compounds with [sI]Ang II for the binding sites. The relative order of potency for the displacement of [uI]Ang II binding was Sar'leu!-Ang II > (des Asp')Iles-Ang II > Iles-Ang II >> Ile5-Ang I >> 3-8 Ang II hexapeptide > 4-8 Ang II pentapeptide. Thus, Ile6-Ang II displaced [lsI]Ang II binding with high specificity. As expected, the antagonist Sar leu8-Ang II was slightly more potent than Iles-Ang II, but interestingly (des Asp) Iles-Ang II which is the heptapeptide angiotensin III (Ang III) displaced [s5I]Ang II
binding to a greater extent than Iles-Ang II. Antagonists generally have a higher receptor affinity than the natural ligand. However, the reason why Ang III was more potent at displacement than Ang II is not clear. Further kinetic analysis was obtained by incubating ['I]Ang II with brain membranes over time. At 37-C, the binding was linear for 10 min and reached
equilibrium after 30 min. An association constant (k,) of 3.0 X 106 M1 S-i was estimated using the linear portion of the binding time course. After the binding reached equilibrium, the dissociation constant (k2) of 2.6 X 10- S-' was determined. The




ratio of k/k gives a value of 0.87 nM and can be used as an estimate of the Kd. Additionally, the binding of ['lI]Ang II was linear with respect to the membrane tissue concentration and showed pH and ion dependency. The distribution of ["I]Ang II binding sites varied in the brains from calves and rats. In the calf, the binding was highest in the cerebellum with much lower levels in the forebrain, midbrain, cortex and brainstem. In contrast, [MI]Ang II binding sites in the brains of rats were highest in the thalamus, hypothalamus, midbrain and brainstem. The cortex and cerebellum contained only about 25% of the binding levels found in the thalamus and hypothalamus. This initial study confirmed the presence of Ang II receptors in the brain. However, the demonstration of a correlation between the binding kinetics of [MI]Ang II and the time-course of a biological response induced by Ang II was not shown. This is often cited as a criterion for receptor identification, but is often difficult to show especially using membrane binding techniques. This deficiency was partially remedied by localizing high levels of Ang II binding to the hypothalamus and brainstem of the rat. These areas are primary sites of action for centrally administered Ang II.
Sirett et al. (1977) further characterized and localized [125I]Ang II binding sites in the brains of rats. A block of tissue containing the hypothalamus, thalamus, septum and midbrain
(H-T-S-M) was used to study the binding of [nI]Ang II. The




specific binding of ["I]Ang II accounted for approximately 70% of the total binding. The relative order of potency for the displacement of [MI]Ang II binding was not well established, but it appeared to be Ang II = Ang III > saralasin >> Ang I >> ACTH >> vasopressin. Interestingly, Ang III was equipotent to Ang II in competing for binding sites. This is similar to the results obtained by Bennett and Snyder (1976). Further, the binding of I I]Ang II was reversible and saturable. The linear transformation of the saturation analysis gave a Kd of 0.9 nM and a Bm= of 11 fmoles/mg protein. This kd is similar to the d obtained by Bennett and Snyder (1976) using the cerebellar cortex of calves. The regional distribution of [nI]Ang II binding in the brains of rats had a relative order of septum > thalamusmidbrain > hypothalamus > medulla >> cerebrum > cortex > hippocampus. Thus, the binding of ['15I]Ang II was concentrated in the H-T-S-M and medullary regions of the brain.
Unfortunately, the use of membrane binding techniques to
study Ang II receptor localization is limited. This is due to the
difficulty in locating and obtaining adequate amounts of tissue from discrete brain areas. However, with the advent of in vitro autoradiographic techniques in combination with computerized
densitometry the identification of Ang II receptor populations in discrete brain nuclei became possible. Mendelsohn et al. (1984) first used in vitro autoradiography to localize Ang II receptor
populations in the brain. High affinity receptor sites were found




in the subfornical organ (hypothalamus), the OVLT (hypothalamus), the median eminence (hypothalamus) and the AP (brainstem). These sites are all circumventricular organs which lie outside the blood-brain barrier. High concentrations of receptor sites for
Ang II located within the blood-brain barrier were found in the paraventricular and periventricular nuclei (hypothalamus), the nucleus tractus solitarius (brainstem), the suprachiasmatic nucleus (hypothalamus), the locus coereleus (brainstem), the subthalamic nucleus (hypothalamus), and the inferior olive nucleus (brainstem). In general, the distribution of Ang II binding sites was shown to be concentrated in specific brain areas, namely the hypothalamus and brainstem, which are associated with the dipsogenic and pressor effects of Ang II. This study did not quantify the binding of Ang II. However, using autoradiography, other studies have provided detailed quantitative analyses of discrete Ang II receptor populations in the brain (Gehlert et al., 1986; Israel et al., 1985; Saavedra et al., 1986). The Ang II receptor affinities and capacities obtained by
autoradiography generally agree with those obtained using membrane binding techniques. However, using autoradiography the binding affinity was shown to vary depending of the receptor population studied. For example, Saavedra et al. (1986) reported K values of 1.5 X 10 M-1, 0.56 X 10' Ki and 0.36 X 109 1 for the subfornical organ, area postrema and nucleus tractus solitarious, respectively. Additionally, the Bm,, values obtained




by autoradiography were in some cases 20-200 times greater than Bm= values determined using membrane binding techniques (eg Israel, 1985; Saavedra, 1986). Two possible explanations for this discrepancy are 1) the receptor populations analyzed by autoradiography are more concentrated and are not diluted by
tissue which does not contain Ang II receptors or 2) the methodological and analytical differences between membrane binding techniques and in vitro autoradiographic techniques result in different relative values for saturation analyses.
The localization of Ang II receptors to specific brain areas that are sites of action for Ang II indirectly proves the existence of functional Ang II receptors in the brain. More direct evidence has come from studies using Ang II receptor antagonists and from electrophysiological studies. According to the occupancy theory, an antagonist binds to a receptor but has zero efficacy. Thus, the antagonist does not elicit a biological
response. [SarValAla]Ang II (saralasin) is a competitive antagonist commonly used to antagonize the effects of Ang II. Saralasin has been used to block the dipsogenic response of water
deprived rats (Malvin et al., 1977) and in rats administered Ang II i.v.t. (Epstein et al., 1973; Hoffman and Phillips, 1976). Similarly, the pressor effect of Ang II given i.v.t. is antagonized by saralasin (Hoffman and Phillips, 1976). The central pressor effect of Ang II is produced by both sympathetic
nervous system activation and AVP release. The AVP component of




this response was studied using organ explants of the hypothalamus and the posterior pituitary (Sladek and Joynt, 1979). Ang II administration resulted in the release of AVP into the explant growth medium. This effect of Ang II was antagonized by saralasin which indicates that Ang II acts at specific receptors to stimulate the release of AVP.
The use of electrophysiological iontophoretic techniques has identified several regions of the brain which contain Ang II
sensitive neurons. Areas which exhibit excitatory responses to Ang II that are blocked by Ang II receptor antagonists include the subfornical organ (Phillips and Felix, 1976), the preoptic region (Akaishi et al., 1981; Gronan and York, 1978), the paraventricular and supraoptic nuclei (Akaishi et al., 1980) and the lateral septum (Huwyler and Felix, 1980; Simonnet et al., 1980). These studies correspond favorably to the autoradiographic receptor binding studies. For example, Gehlert et al. (1986) in the most definitive study of Ang II binding sites to date, identified high concentrations of Ang II receptors in the paraventricular nucleus, the lateral septum, the subfornical organ and the supraoptic nucleus.
Regulation of the Renin-Ancriotensin System in the Brain
Past studies have primarily sought to investigate the
function and the existence of the brain renin-angiotensin system. Only recently have investigators focused on the regulation of the




12
brain renin-angiotensin system. Contrary to the peripheral reninangiotensin system, the mechanisms involved in the control of the brain renin-angiotensin system remain obscure. Presently, two loci of regulation have been identified. They are 1) the regulation of the production/expression of angiotensinogen and 2) the regulation of Ang II receptors.
Angiotensinogen mRNA is distributed in a variety of tissues including the brain (Dzau et al., 1986; Lynch et al., 1986; Ohkumbo et al., 1986). In the brain, angiotensinogen mRNA
expression is increased by treatment with the synthetic glucocorticoid, dexamethasone (Kalinyak and Perlman, 1987), or by the multiple administration of ethynylestradiol, dexamethasone and triiodothyronine (Campbell and Habener, 1986).
Reulation of Ancriotensin II Receptors Receptor Reqculation: Concepts
It is well established that various receptor agonists can alter the responsiveness of their effector systems. This results in either a supersensitivity or a subsensitivity of the effector system to the agonist. A major mechanism involved in agonistinduced changes in sensitivity is the alteration of receptor number. For example, supersensitivity of skeletal muscle following denervation results, in part, from an increase in the number of postsynaptic nicotinic acetylcholine receptors (Miledi and Potter, 1971). Conversely, the prolonged exposure to Preceptor agonists in a variety of systems leads to a decrease in




13
p-receptor number and a decreased ability of P-receptor agonists to stimulate adenylate cyclase (Kebabian et al., 1975; Mikey et al., 1976; Mukherjee et al., 1975). This type of receptor regulation in which a receptor agonist regulates the receptor system with which it is associated is termed homologous regulation. Mechanisms involved in homologous regulation not only
include a change in receptor number (i.e., downregulation), but a change in receptor affinity and/or an alteration of the receptorsecond messenger coupling. In addition to homologous regulation, receptors can be regulated in a heterologous manner. In this case, agonists act at their specific receptor to alter the function of other receptor systems. The mechanisms involved in heterologous regulation are believed to be similar to those described for homologous regulation. Peripheral Requlation of Angiotensin II Receptors
Ang II receptors in the periphery are regulated in both a
homologous and heterologous manner depending on the tissue studied. In urinary bladder smooth muscle, the mesenteric artery and the uterus, Ang II infusion decreases the Ang II receptor number (Bm) with no change in affinity (Aguilera and Catt, 1981; Devynk et al., 1976; Schiffrin et al., 1984). However, in the glomerulosa zone of the adrenal gland the intravenous (i.v.) infusion of Ang II increases the Bmm while either decreasing or not changing the receptor affinity (Hauger et al., 1978; Mendelsohn et al., 1983). Heterologous regulation of Ang II




14
receptors has been shown using steroid hormones. The infusion of
mineralocorticoids, either deoxycorticosterone acetate (DOCA) or aldosterone, into rats increases the number of Ang II binding
sites in the mesenteric artery and the uterus with no change in affinity (Douglas and Brown, 1982; Schiffrin et al., 1983, Schiffrin et al., 1984). However, Douglas and Brown (1982) and Douglas (1987) reported a decrease in Ang II receptor number in the adrenal glomerulosa and in glomerular mesangial cells following aldosterone infusion. Alternatively, estrogen treatment has been shown to decrease Ang II receptor number in the uterus and the anterior pituitary of rats (Chen and Printz, 1983; Schirar et al., 1980).
Regulation of Angiotensin II Receptors in the Brain
In vivo studies of the homologous regulation of Ang II
receptors in the brain have produced mixed results. Singh et al.
(1984) found the i.v.t. infusion of Ang II (500 ng/~l/hr) for six days significantly increased the water intake of rats, but had no effect on the binding of [5I]Ang II in the H-T-S-M or medullary regions of the brain. This experiment was repeated using the Ang II receptor antagonist [SarIle ]Ang II. Similarly, this treatment did not alter the binding of [1sI]Ang II in the H-T-S-M region of the brain of rats (Singh et al., 1986). Alternatively, Thomas and Sernia (1985) noted that the i.v. infusion of Ang II (25 ng/kg/hr) decreased the number of Ang II receptors in a block of tissue containing the hypothalamus, thalamus and septum (H-T-S),




but increased the number of receptors in the medulla. This differential effect was reported for the receptor affinities in the H-T-S and medulla which were increased and decreased, respectively, by Ang II infusion. Thus, the route of administration of Ang II and the brain area of study may be
important variables when investigating the homologous regulation of Ang II receptors. Certainly more studies are necessary, particularly using in vitro autoradiography, to determine whether Ang II receptors in the brain are homologously regulated.
More discernable evidence exists for the heterologous
regulation of Ang II receptors by steroid hormones. Estradiol
benzoate (EB) was shown by both Fregly et al. (1985) and Jonklaas and Buggy (1985) to alter the binding of [nsI]Ang II in the brain. Jonklaas and Buggy reported that after two days of treatment, rats given EB (500 Ag in 0.3 ml of vehicle) subcutaneously (s.c.) had decreased Ang II receptor binding in
membrane homogenates prepared from tissue blocks which included the preoptic area, the septum and the thalamus. This effect was associated with a decrease in Ang II-stimulated drinking. Fregly et al. (1985) chronically administered EB peripherally (30-46
ag/kg/day for 8 weeks) and found a similar decrease in Ang II binding in a diencephalic block of tissue with a concomitant
reduction in the dipsogenic response to Ang II.
In contrast to estradiol benzoate, it appears
mineralocorticoids act to increase the number of Ang II receptors




16
in the brain. Wilson et al. (1986) treated rats for 8 weeks with DOCA (240 g/kg/day, s.c. implants) and found a significant increase in the number of Ang II binding sites in the H-T-S region of the brain. The receptor affinity was unaffected. This increase in receptors likely involved functional Ang II receptors as either the peripheral or central administration of Ang II elicited greater drinking and pressor responses in DOCA-treated rats. King et al. (1988) further showed that DOCA (500 Ag/day, s.c. for four days) significantly increased the specific binding of [nsI]Ang II in the area postrema, superior colliculus, midbrain, olfactory bulb, septum/subfornical organ and the anterioventral third ventricle (AV3V) regions of the brain of rats. In this case, increased Ang II binding was linked to an increased sodium appetite in DOCA-treated rats elicited by Ang II. Using autoradiography, Gutkind et al. (1988) have localized
the increase in Ang II receptors in DOCA and DOCA-salt treated rats to specific brain nuclei. In both DOCA and DOCA-salt treated animals, Ang II binding was increased in the median preoptic nucleus, the subfornical organ and the paraventricular nucleus. However, only DOCA-salt animals had additional elevated binding levels in the nucleus tractus solitarious and AP areas of the brainstem. In another autoradiographic study, Wilson et al.
(1989) found increased levels of [nsI]Ang II binding in the subfornical organ and the medial preoptic area of rats made hypertensive by DOCA-salt treatment. This is in contrast to both




rats made hypertensive by the clipping of their renal artery (two-kidney, one-clip, and one-kidney, one clip) and salt loaded rats which did not have elevated levels of [I]Ang II binding in the brain. Thus, from these studies, either DOCA alone or DOCA in combination with certain forms of hypertension acts to increase the number of Ang II receptors in discrete brain areas.
Other investigators have studied the role of sodium balance in the regulation of Ang II receptors in the brain. These studies have produced mixed results, undoubtably due to the variety of protocols used and the nonspecific nature of the treatments. For
example, Mann et al. (1980) found a 30% reduction in the binding of [n5I]Ang II in the H-T-S-M region of the brains of rats fed a low sodium diet for 7 days. This effect was associated with decreased drinking and blood pressure responses to icv-injected Ang II. However, Cole et al. (1980) and Speth et al. (1984) found no effect of low sodium diets on the binding of [mI]Ang II in the H-T-S-M region of the brain of rats. Conversely, Ashida et
al. (1982) reported that Ang II receptors in the H-T-S-M region of normotensive (Wistar Kyoto) rats, but not spontaneously hypertensive (SHR) rats, were reduced by a high sodium diet. Thomas and Sernia (1985) found a differential effect on the binding of Ang II in the brain after the administration of a low sodium diet to rats for 21-30 days. The density of Ang II binding sites was decreased in the midbrain, olfactory bulb, and cerebellum and increased in the H-T-S region and the medulla.




Additionally, the Ang II receptor affinity was increased in the cerebellum, midbrain and medulla.
In summary, in vivo studies have demonstrated both the
homologous and heterologous regulation of Ang II receptors in the brain, although the homologous regulation of Ang II receptors needs more clarification. The heterologous regulation of Ang II receptors in the brain by steroid hormones is more conclusive. In general, estrogen treatment leads to a decrease in the number of Ang II receptors, while the administration of the mineralocorticoid DOCA results in an increase in Ang II
receptors. In both cases, changes in Ang II receptor number in discrete brain regions has been associated with a decreased biological responsiveness to Ang II, indicating that functional Ang II receptors are involved.
Neuronal Cultures: A Model to Study Angiotensin II Receptor Regulation
In vivo techniques have contributed greatly to the study of Ang II receptor regulation. However, the efficacy of these techniques is limited when studying the brain. This is mainly due to the inherent complexity of the brain. Ang II receptors are located in small, inaccessible regions of the brain which are undoubtably subject to a variety of neuroendocrine influences.
This makes the controlled study of centrally located Ang II receptors in an isolated in vivo system very difficult. This is particularly true for studying the effects of non-steroidal




agents such as peptides and amino acid derivatives on Ang II receptors. These compounds are not 1 ipophilic and have a limited access to discrete brain regions.
Recently, the study of Ang II receptor regulation in the brain has been greatly facilitated by the use of cell cultures. In these cultures, neural tissue, dissected from various regions of the brains of neonatal rats, is dissociated into individual cells and plated on tissue cultures dishes. The cells are then grown in a relatively defined medium which can be experimentally
manipulated in a quantifiable manner. The growth medium is not completely defined due to the presence of horse serum which
contains variable amounts of hormones, growth factors, vitamins, trace elements and other unknown elements. Although the addition of serum to the growth medium adds some variability, it is essential for the long-term viability of the neuronal cultures.
Thus, by using neuronal cultures, Ang II receptor regulation can be studied in a partially controlled, easily accessible environment which is unaffected by nonspecific peripheral
influences such as stress and locomotion. Further, the intracellular mechanisms involved in Ang II receptor regulation are amenable to study. However, the study of cellular function in an artificial environment is itself a caveat. This means, if possible, in vitr studies should be correlated with In vivo tudies.




Characterization of the Renin-Angiotensin System in the Brain Usin Neuronal Cultures
As in the brain, all the components of the renin-angiotensin system have been identified in neuronal cultures prepared from the brains of neonatal rats. Neuronal cultures prepared from the hypothalamus and brainstem of neonatal rats contain and synthesize angiotensinogen and angiotensin II (Hermann et al., 1988a; Hermann et al., 1988b) and contain angiotensinogen mRNA (Kumar et al., 1988), while neuronal cultures prepared from the whole brains of neonatal rats have been used to identify the
presence of renin (Hermann et al., 1987) and specific Ang II receptors (Raizada et al., 1984). These Ang II binding sites were shown to satisfy the requirements for receptor identification using radioligand binding techniques as described previously. The
B= and affinity constant obtained by Scatchard analysis were 2.6 X 104 binding sites/cell and 1.0 nM, respectively. The K obtained by taking the ratio of k2 to k, was 0.15 nM. Functionally, in neuronal cultures, Ang II has been shown to modulate [H]norepinephrine uptake (Sumners et al., 1985; Sumners and Raizada, 1986a), cause an increase in the norepinephrine (NE)
content in both neuronal cultures and the growth media (Sumners et al., 1983), and stimulate monoamine oxidase (MAO) activity (Sumners et al., 1987a). All of the effects listed above were blocked by specific Ang II receptor antagonists, indicating the presence of functional Ang II receptors in the neuronal cultures.




Regulation of Angiotensin II Receptors in Neuronal Cultures
Both catecholamines and steroid hormones regulate Ang II receptors in neuronal cultures. However, the homologous regulation of Ang II receptors does not appear to occur (Sumners and Raizada, unpublished results). Wilson et al. (1986) extended and confirmed their in vivo work by demonstrating that DOCA (1.4 nM, 15-20 hrs) and aldosterone (1.35 nM, 15-20 hrs) could each significantly increase the specific binding of [MI]Ang II in neuronal cultures prepared from whole brains. This effect was due to an increase in the Bm of the receptor. The Bm of DOCAtreated cells was 439 fmol/mg protein and represents a 52% increase over the Bm of 288 fmol/mg protein in control cells.
Sumners and Fregly (1989) studied the specificity of
mineralocorticoid regulation of Ang II receptors in cultures prepared from the brainstem and hypothalamus of neonatal rats. Neither testosterone, 9-estradiol or the glucocorticoid
dexamethasone mimicked the effect of DOCA and aldosterone to increase [mII]Ang II specific binding. Additionally, an increase in [MI]Ang II binding was not detected in neuronal cultures cotreated with DOCA and mineralocorticoid type I receptor antagonists (either mespirinone or ZK 97894). This indicates that
DOCA acts via a type I mineralocorticoid receptor to stimulate the binding of ['I]Ang II binding.
The heterologous regulation of Ang II receptors in culture in not limited to steroid hormones. Initial studies by Sumners




and Raizada (1984) identified an inverse relationship between catecholamines (CA) and Ang II receptors. Treatment of neuronal cultures prepared from whole brains with the tyrosine hydroxylase inhibitor, o-methyl-tyrosine (c-MT), induced a decrease in neuronal NE and dopamine (DA) contents and an increase in the specific binding of [(I]Ang II. Conversely, pargyline, a MAO inhibitor, increased NE and DA contents in the neuronal cultures and decreased the specific binding of [nsI]Ang II. The respective increase and decrease in [sI]Ang II binding with a-MT and pargyline treatments was attributed to a increased receptor affinity in a-MT treated cells and a decreased receptor affinity in pargyline treated cells. The Bm was unchanged in both cases as determined by Scatchard analysis.
Using more direct means to study the effects of CA on Ang
II receptor regulation, Sumners et al. (1986b) incubated neuronal
cultures prepared from the hypothalamus and brainstem with either NE or DA. These compounds acted to dose-dependently decrease the specific binding of [125I]Ang II. The decrease in [121I]Ang II binding elicited by NE (1 AM, 4 hrs) was blocked by the receptor antagonists, prazosin (10 AM) and phentolamine (10 AM), but not by the oc-receptor antagonist, yohimbine (10 AM), or by the Preceptor antagonist, propranolol (10 pM). Saturation analysis
determined a 60% decrease in the B, of NE-treated cells as compared to control cells. Conversely, short-term incubations with higher concentrations of NE (5-50 MM, 15-60 min) resulted in




an increase in the specific binding of [m6I]Ang II (Myers and Sumners, in press). This effect was blocked by prazosin (1 MM), indicating the involvement of specific o,-adrenergic receptors. Additionally, the increase in [mI]Ang II binding elicited by NE was due to an increase in the receptor B. with little change in the receptor affinity. Thus, the activation of oc-adrenergic receptors, which are present in these neuronal cultures (Feldstein et al., 1986), appears to have a biphasic regulatory effect on Ang II receptors depending on the concentration of agonist used and the duration of agonist incubation.
Intracellular Mechanisms Involved in Angiotensin II Receptor Requlation
Presently, the mechanisms involved in the regulation of Ang II receptors in the brain remain unclear. Mineralocorticoid mediated upregulation of Ang II receptors appears to involve protein synthesis. Sumners and Fregly (1989) demonstrated that cycloheximide (3.5 AM and 35 pM) blocks the increase in the specific binding of [1sI]Ang II produced by aldosterone. Presumably mineralocorticoids act by increasing the de novo synthesis of Ang II receptors.
The c1-adrenergic regulation of Ang II receptors has been linked to both the activation of protein kinase C and calcium mobilization. Protein kinase C is a calcium, phospholipiddependent enzyme which affects cellular function by phosphorylating key membrane proteins (Nishizuka, 1986). The




initial step in the activation of protein kinase C involves the
receptor mediated activation (in this case the ac-adrenergic receptor) of phospholipase C. Phospholipase C then hydrolyzes the specific membrane phospholipid phosphatidylinositol-4,5bisphosphate. This results in the generation of diacylglycerol
(DAG) and inositol triphosphate (IP3). DAG is the endogenous activator of protein kinase C (Takai et al., 1979b). The involvement of protein kinase C in Ang II receptor regulation was inferred by Sumners et al. (1987b) using phorbol esters. Phorbol esters are compounds which activate protein kinase C by
substituting for DAG (Castagna et al., 1982). The phorbol ester, phorbol 12-myristate-13-acetate (TPA), was found to increase the specific binding of [M I]Ang II in neuronal cultures. This effect was rapid, occurring by 15 min and reaching a maximal level between 1 and 2 hr. Scatchard analysis revealed the increase in the specific binding of [(s5I]Ang II was due to an increase in the Bm= with little change in the receptor affinity. The involvement of protein kinase C is further implied from the studies by Myers
and Sumners (in press). Here, o-adrenergic receptor stimulation was found to increase significantly the hydrolysis of phosphatidylinositol. This effect slightly preceded, and was associated with, an increase in the specific binding of [12I]Ang II elicited by the short-term (15-60 min) activation of eqadrenergic receptors. These studies suggest that the stimulation
of oc-adrenergic receptors leads to the activation of protein




25
kinase C which acts acutely to increase the expression of Ang II receptors.
An alternative mechanism of Ang II receptor regulation in
neuronal cultures prepared from neonatal rats appears to involve calcium. Sumners et al. (1988) reported that the calcium ionophore A23187 enhanced the effects of TPA to upregulate Ang II receptors in culture. The ability of A23187 to potentiate the effects of TPA were not observed in calcium-free medium. Further, A23187 alone, at concentrations which significantly increased
4Ca+ influx, caused a decrease in Ang II receptor number. This effect was negated in calcium-free medium. Thus, Ang II receptor regulation (ie downregulation) may involve calcium flux. However, the question remains whether a more physiologically relevant stimulation of calcium flux such as by depolarization or direct receptor activation can downregulate Ang II receptors. It is tempting to speculate that the long-term effect of NE (2-6 hrs) incubations to downregulate Ang II receptors in culture is the delayed result of cc-adrenergic stimulation of calcium influx. Preliminary evidence suggests this is the case as COC12, which blocks certain calcium channels, attenuated the effect of longterm NE incubations to decrease the specific binding of [inI]Ang
II in neuronal cultures (Sumners, personal correspondence). This effect could be mediated by the putative cxl-adrenergic receptor subtype which is linked to calcium mobilization (Han et al., 1987).




A model for protein kinase C activation
Woodgett et al. (1988) have proposed a general model for the activation of protein kinase C. Here, protein kinase C exists in
a cytosolic state that is closely associated with the plasma membrane. Receptor-mediated activation of phospholipase C results in phosphatidylinositol-4,5-bisphosphate hydrolysis and the
generation of DAG and IPs. DAG binds to protein kinase C and acts to lower the affinity of protein kinase C for Ca+. This promotes the binding of protein kinase C to the plasma membrane. At the membrane, protein kinase C forms a ternary complex with Ca+, DAG and specific membrane phospholipids (eg phosphatidylserine) that results in the full activation of protein kinase C. In this scenario the process is enhanced by IP3 which liberates Ca+ from intracellular stores. The dissociation of protein kinase C from
the membrane and its subsequent inactivation follows the rapid metabolism of DAG by specific lipases and kinases. Phorbol esters as a tool to study protein kinase C
Phorbol esters are potent activators of protein kinase C (Castagna et al., 1982). Initially isolated from croton oil, which is derived from the plant croteus tiqclium, these compounds were first identified as tumor promoters involved in the twostage model of carcinogenesis (Blumberg, 1980). These highly lipophilic compounds were later found to have saturable, high affinity cellular binding sites (Delcos et al., 1980; Drieger and Blumberg, 1980). At the present, it appears that protein kinase C




27
is the major intracellular binding site for phorbol esters. This was determined by showing the co-purification of the phorbol ester receptor with protein kinase C using various separation
techniques (Ashdell et al., 1983; Kikkawa et al., 1983; Leach et al., 1983). Mechanistically, phorbol esters activate protein kinase C by substituting for DAG, the endogenous activator of protein kinase C (Castagna et al., 1982).
Recently, phorbol esters have been used to implicate protein kinase C in an astonishing array of cellular processes. As
reviewed by Woodgett et al. (1988), phorbol esters have a wide variety a cellular effects which include carcinogenic effects, effects on cellular differentiation, the alteration of gene expression, the modulation of ion flux and substrate transport,
modulatory secretagogue effects, the modulation of enzyme activities, interactions with other second messenger systems, receptor regulation and cytoskeletal alterations. Thus, by using phorbol ester, a plethora of possible actions of protein kinase C has been discerned. However, the implication of protein kinase C involvement in cellular processes using phorbol esters must be viewed with some skepticism. This statement is based on made for two reasons. First, phorbol esters have been shown to alter the fluidity of the cell membrane (Tran et al., 1983). This may be due to their lipophilic nature which allows them to bind to the cell membrane and could potentially alter various membrane proteins such as receptors, transporters, ion channels and




cytoskeletal components independently of protein kinase C activation. Second, in some instances, the effects of phorbol esters are not mimicked by DAGs or are not blocked by protein kinase C antagonists. For example, the ability of TPA to induce the differentiation of HL-60 leukemia cells was not mimicked by the synthetic DAG, l-oleoyl-2-acetylglycerol (OAG) (Yamamoto et al., 1985; Kreutter et al., 1985). This occurred in spite of the continued application of OAG to the cells to account for the higher rate of metabolism of this compound (Kreutter et al., 1985). Similarly, OAG does not increase the binding of [MI]Ang II in neuronal cultures (Kalberg, unpublished results). Further, polymyxin B, an inhibitor of protein kinase C (Mazzei et al., 1982) does not inhibit TPA-stimulated protein phosphorylation and differentiation of HL 60 cells (Kiss et al., 1987).
General Hypothesis
The data reviewed to this point were provided to show that the brain contains a functional renin-angiotensin system. Further, it was argued that the use of neuronal cultures prepared from the brains of neonatal rats provides an adequate in vitro
model with which the renin-angiotensin system can be studied at the cellular level. Presently, both the homologous and the heterologous regulation of Ang II receptors have been studied in culture. The heterologous regulation of Ang II receptors is known to involve both steroid hormones and a-adrenergic receptor agonists. The activation of cq-adrenergic receptors by eq-




adrenergic receptor agonists acts both to upregulate and
downregulate Ang II receptors depending on the concentration and the duration of incubation of the agonist. Two mechanisms are proposed for this biphasic effect. The first mechanism identifies a means by which the activation of oc-adrenergic receptors leads to an increase in the expression of Ang II receptors. Here, the stimulation of oc-adrenergic receptors results in the activation of protein kinase C which, through a phosphorylation-mediated event, leads to the increased expression of Ang II receptors. Previous studies using neuronal cultures have associated the
stimulation of cc-adrenergic receptors with increases in both phosphatidylinositol hydrolysis and Ang II receptor number. In these studies it is assumed that phosphatidylinositol hydrolysis leads to the activation of protein kinase C. However, the c1adrenergic receptor-mediated activation of protein kinase C has not been demonstrated in these cultures. Additionally, the involvement of protein kinase C in the regulation of Ang II receptors has been implicated using phorbol esters. These compounds are known to activate protein kinase C. However, they have a variety of nonspecific effects which may secondarily act to increase Ang II receptors. Clearly additional investigation is needed to confirm the involvement of protein kinase C in the regulation of Ang II receptors.
A second mechanism is proposed to explain the downregulation
of Ang II receptors by oc-adrenergic receptors. In this case, the




activation of cq-adrenergic receptors causes an increase in Ca"2+ influx. This increase in Ca+ mobilization acts to decrease the number of Ang II receptors possibly by the activation of a calcium, calmodulin-dependent enzyme. To speculate further, this effect could be mediated by the putative c, receptor subtype which has been linked to the activation of voltage sensitive calcium channels (VSCC). Thus, the activation of the oac receptor could mediate the downregulation of Ang II receptors. Past studies used the calcium ionophore A23187 to demonstrate the possible involvement of calcium in the regulation of Ang II receptors. Presently it is not known whether calcium influx through VSCC is involved in the regulation of Ang II receptors in neuronal cultures. Finally, interactions between the two proposed mechanisms may exist. Saitoh and Dobkins (1986) reported that the calcium binding protein, calmodulin, inhibits the phosphorylation by protein kinase C of certain substrates in the brain. This raises the possibility that calcium-calmodulin dependent enzymes/kinases and protein kinase C may interact. Thus, a further degree of control of Ang II receptor regulation may exist if the regulatory pathways involved in Ang II receptor regulation
are interconnected.
Specific Aims
The following specific aims are designed to define further the involvement of protein kinase C and calcium in the regulation




of Ang II receptors in neuronal cultures prepared from neonatal rats.
1) Confirm the involvement of protein kinase C in the regulation of Ang II receptors. These experiments were performed due to the nonspecific nature of phorbol esters. They include the use of the following: a protein kinase C antagonist, non-phorbol ester activators of protein kinase C, and protein kinase C deficient cells.
2) Characterize of protein kinase C activity in neuronal
cultures.
3) Qualitatively analyze protein kinase C involvement in the
regulation of Ang II receptors.
4) Determine whether protein kinase C is activated by OC1adrenergic receptors in neuronal cultures.
5) Determine the role of calcium flux through VSCC in the
regulation of Ang II receptors.




CHAPTER II
CHARACTERIZATION OF PROTEIN KINASE C ACTIVITY IN NEURONAL CULTURES
Introduction
The isolation and characterization of calcium, phospholipiddependent protein kinase (protein kinase C) has occurred at an incredibly fast rate through the use of the latest scientific
technology. Protein kinase C was first isolated by Takai et al. (1977) in the cerebellum of cows. They identified protein kinase C as a cyclic nucleotide-independent enzyme which phosphorylated
serine residues and was activated by Ca+-dependent proteolysis (Inoue et al., 1977; Takai et al., 1977). Later, the same group determined that the activation of this enzyme occurred in the
presence of Ca+ and phosphatidylserine and did not involve proteolysis (Takai et al., 1979a). The further discovery that
diacylglycerol (DAG) was required for the complete activation of protein kinase C irrevocably linked protein kinase C with the phosphoinositide signalling pathway (Kishimoto et al., 1980; Takai et al., 1979b). Protein kinase C was initially described as a single enzyme which is distributed in a wide variety of tissues and animal phyla and is especially enriched in the brain and lymphoid tissue (Inoue et al., 1977; Kuo et al., 1980). However, hydroxylapatite column chromatography has been used to isolate




33
three isozymes of protein kinase C in the brains of rats, rabbits and monkeys (Huang et al., 1986; Jaken and Kiley, 1987; Yoshida et al., 1987). These isozyme are distinguishable by their sites of autophosphorylation, immunoreactivity, activation by lipids, tissue distribution and susceptibility to proteolysis (Huang et al., 1987a, Huang et al., 1987b, Huang et al., 1988; Huang et al., 1989; Sekiguchi et al., 1987; Shearman et al., 1989; Yoshida et al., 1988). Three additional isozymes have been identified using complementary DNA probes for protein kinase C (Ono et al., 1988).
Further characterization of protein kinase C has analyzed the unique subcellular distribution of this enzyme and what factors affect its distribution. As discussed in the Chapter I,
protein kinase C is thought to exist in a loose association with the plasma membrane (Kraft and Anderson, 1983a; Woodgett et al., 1988). Upon stimulation, this enzyme becomes tightly bound to the membrane and in combination with specific membrane phospholipids
it becomes fully activated. Together, C2+ and DAG regulate the binding of protein kinase C to the plasma membrane (Wolf et al., 1985a). However, calcium alone has profound effects on the attachment of protein kinase C to cellular membranes (Farrago et
al., 1988; Halsey et al., 1987; Kikkawa et al., 1982; Phillips et al., 1989; Wolf et al., 1985a; Wolf et al., 1985b, Yoshida et al., 1988). This ability of calcium to affect the binding of
protein kinase C to cell membranes is a unique characteristic of




the enzyme and has both physiological and experimental importance.
The following study deals with the characterization of protein kinase C in primary neuronal cultures prepared from neonatal rats. Previously, the binding of [3H]phorbol esters (Raizada et al., 1988) and immunological techniques (Mudd, 1989) have been used to identify phorbol ester binding sites and
immunoreactive protein kinase C activity in these cultures. However, protein kinase C has yet to be characterized in these cultures in terms of its activity. In this study, the activity of protein kinase C in neuronal whole brain cultures has been characterized, in part, relative to the prior discussion. The activity of protein kinase C in the neuronal cultures exhibited many of the standard characteristics identified for this enzyme. Further, the subcellular distribution of protein kinase C was found to be dependent on calcium.
Methods
Preparation of neuronal cultures
The neuronal cultures were prepared from the whole brains of one-day-old Sprague-Dawley (SD) rats. The brains were dissected from one-day-old SD rats and placed in an isotonic salt solution containing 100 U penicillin G, 100 gg of streptomycin and 0.25 g amphotericin B (fungizone) per milliliter, pH 7.4. The blood vessels and pia mater were then removed from the brain after which the brains were chopped into -2.0 mm chunks. The brain




pieces were suspended in 25 ml of 0.25% trypsin (wt/vol) in isotonic salt solution (pH 7.4), pipetted into a flask, and placed in a shaking water bath for 6 min at 37*C to dissociate the cells. After this time, 160 Ag of deoxyribonuclease I (DNase
I) was added to the cells and the flask was replaced in the shaker bath for an additional 6 min at 37*C. The dissociated cells were diluted with 100 ml of Dulbeco's modified Eagle's medium (DMEM) containing 10% plasma derived horse serum (PDHS) and were centrifuged at 1,000 X g for 10 min. The resulting pellet was resuspended in 10-15 ml of DMEM containing 10% PDHS and pipetted through sterile gauze into a 500 ml bottle to remove debris. The cells were diluted to 100 ml, counted and further diluted to the desired volume in the same media. The cell recoveries were normally 50-55 X 106 cells per brain. The cells suspended in DMEM containing 10% PDHS were plated at a concentration of 18 X 106 cells per 9 ml per dish on 100 mm Falcon tissue-culture dishes that were precoated with poly-L-lysine. The cells were incubated for 3 days at 37*C in a
humidified incubator with 10% C02-90% air. On day three, all the cells were treated with 10 jM cytosine arabinoside (ARC) in 9.0 ml of DMEM-10% PDHS. This treatment inhibits the division of
nonneuronal cells and provides cultures that are enriched with neuronal cells. After 2 days, this medium was removed and replaced with 9 ml of fresh DMEM-10% PDHS. The cultures were placed back into a humidified incubator (10% CO2-90% air) and




36
were used for experiments after a total of between 12 and 21 days
in culture. This length of time is consistent with the occurrence of high levels of protein kinase C. Burgess et al. (1986) reported that a 20-fold induction of protein kinase C occurs in primary neuronal cultures prepared from the brains of embryonic
rats during the first week of culture. In the neuronal cultures described here, the cells started to produce projection within 24 hrs of being plated on the tissue cultures dishes. Imunofluorescent examination of the cultures using fluorescent antibodies against the monoclonal neurofilament antibody NE-14
revealed that the cultures contained > 85% neuronal cells (Richards et al., 1989). The remaining cells were nonneuronal as evidenced by inmunofluorescent staining against glial fibrillary acid protein antibody (GFAP).
Preparation of cellular fractions
To characterize the protein kinase C in the neuronal
cultures, the cellular fractions were isolated by one of two different protocols. For the first protocol, 1-3 large 100 mm dishes containing neuronal cultures were washed three times with 2 ml of homogenization buffer A (20 mM Tris HC1, pH 7.5, 2.0 mM EGTA, 0.5 mM EGTA, 0.25 M sucrose, 0.2 mM PMSF and 2.0 Ag/ml leupeptin), scraped with 1-2 ml of homogenization buffer A and homogenized in a dounce homogenizer (15-20 strokes) at 4*C. When more than 1 100 mm dish was used, the cells were pelleted in a Sorvall RT 6000 centrifuge at setting 5 at 4*C before




homogenization in 1 ml of homogenization buffer A. In either case, the homogenate was centrifuged at 3000 rpm for 8 min at 4C to remove debris and the supernatant was centrifuged at 20,000 rpm (-43,000 X g) for 45 min at 4C. The soluble fraction was used as the cytosol fraction and the pellet was resuspended in 1 ml of homogenization buffer A contain 0.1 % triton X-100 for 30 min at 4C. The suspension was centrifuged at -43,000 X g for 45 min at 40C and the supernatant was used as the particulate fraction. The second protocol was identical to the first except the low-speed centrifugation (3000 rpm, 8 min) was omitted and only the high-speed centrifugation (-43,000 X g) was used. DEAE column chromatoQraphy
In some experiments, DEAE column chromatography was used to characterize and partially purify protein kinase C. The preparation and elution of the DEAE columns was performed at 40C. Graduated 0.8 X 4 cm disposable polypropylene columns with a 12 ml volume were packed with 0.6 ml of diethylaminoethyl cellulose (DE 52) which was equilibrated with homogenization buffer B (20 mM Tris HC1, pH 7.5, 2.0 mM EDTA, 0.5 mM EGTA, 0.2 mM PMSF and
1.0 Ag/ml leupeptin). The packed columns were washed with 2 ml of homogenization buffer B, the respective cytosolic and particulate samples were applied, and the columns were washed with 2 ml of homogenization buffer B. Protein kinase C was eluted from the columns by either a linear NaCl gradient (0-0.3 M NaCl in buffer




38
B) or by a single-step elution with 5 ml of homogenization buffer B containing 0.15 M NaCl.
Assay of protein kinase C activity
Protein kinase C activity was determined by measuring the
incorporation of yuP from [Cy"P]ATP into lysine rich histone (type III-S) using a slight modification of the method described by Hirota et al. (1985). The final assay mixture (250 Al) contained 20 mM Tris HCl, pH 7.5, 5.0 mM MgSO4, 0.5 mM CaCl2, 50 Ag histone, 6 gg phosphatidylserine (PS), 0.4 Ag 1,2-diolein, 10 /M [' 32P]ATP (500 cpm/pmol) and 1-5 Ag protein. The lipids were prepared immediately before their use by adding 120 Al of PS stock solution (5.0 mg/ml in chloroform stored at -20*C) and 40 Al of diolein stock solution (1 mg/ml in chloroform stored at 20*C) to a glass scintillation vial kept on ice (4C). The lipids were evaporated under nitrogen and were resuspended in 5 ml of 20 mM Tris HC1, pH 7.5, using two 10 second bursts of a ultrasonic homogenizer (Cole Parmer 4710 series) fitted with a I/" microtip at an output setting of 4. The lipids were added to the reaction mixture just prior the start of the assay which was initiated by the addition of 50 l of enzyme preparation. The samples were assayed in triplicate. After a 3 min incubation at 30C, the reaction was stopped by the addition of 1 ml of 25% trichloroacetic acid (4C). The precipitates were collected by vacuum filtration onto 0.45 AM cellulose filters and washed 4 times with 2 ml of 5% trichloroacetic acid (40C). Additionally,




the non-Ca2+, phospholipid-dependent protein kinase activity of each sample was determined in duplicate under the same conditions but in the absence of lipids and with 1.0 mM EGTA substituted for CaCl2. These samples were used as blank values. The filters were placed in plastic scintillation vials and 10 ml of liquiscint was added. Samples were counted on a open window of 0-1000 in a Beckman LS 1801 counter with a counting efficiency of 88% for
3 P. In most cases, protein kinase C activity was expressed as
nmol "P/min/mg protein.
Protein determination
The protein content of the cytosol and particulate fractions was determined by the method of Lowry et al. (1951).
Results
Identification of protein kinase C activity
The presence of protein kinase C activity in the cytosol fraction from neuronal cultures was determined by assaying a 50 Al (5 jg protein) aliquot of the cytosol fraction in the presence or absence of calcium, phosphatidylserine (PS) and the synthetic DAG diolein (Table 2-1). The incorporation of 7y32 P into lysinerich histone in the presence of either 0.5 mM Ca2+ or 6 Mg PS was negligible. When 0.5 mM Ca2+ and 6 Ag PS were added to the reaction mixture the activity increased 987% over the levels found with Ca2+ alone. The further addition of 0.4 Mg diolein to the reaction mixture resulted in an additional 79% increase in activity.




Table 2-1 Calcium, phospholipid-dependence and diolein
stimulation of protein kinase C
Assay conditions Protein kinase activity
(pmol/min/mg protein)
Ca+ (0.5 mM) 129 + 87
PS (6 Ag) 54 + 33
Ca2+ + PS 1402 + 62
Ca+ + PS + diolein (0.4 Mg) 2511 + 124
Protein kinase C from the cytosol fraction of neuronal cultures was assayed as detailed in the Methods. Calcium (Ca2+), phosphatidylserine (PS) and diolein were added to 5 Mg of protein in the concentrations indicated. Data are the means + SEM of triplicate determinations from a representative experiment which was repeated twice with similar percent changes in protein kinase C activity.
Time and protein dependence of protein kinase C activity
The activity of protein kinase C under the assay conditions described in the Methods increased in a linear manner between 30 sec and 3 min of incubation (Fig 2-1). The activity plateaued after 10 min of incubation.
The dependence of the protein kinase C activity on protein concentration is shown in Fig 2-2. The activity from the cytosol fraction was approximately linear between 1 Mg and 5 Mg of protein and began to plateau between 5 Mg and 20 Mg of protein. Conversely, the activity of protein kinase C from the particulate fraction was linear between 1 Mg and 10 jg of protein with a flattening of the protein curve occurring between 10 Ag and 20 Mg of protein. Both the cytosol and particulate fractions used to




E
a.
E
C

0 5 10 15
time (min)

Figure 2-1.

Time-dependence of protein kinase C activity. 5 Ag of protein from the cytosol fraction isolated from the homogenate of neuronal whole brain cultures was assayed for protein kinase C activity over time. Data are means + SEM of triplicate determinations from a representative
experiment which was repeated twice with similar percent changes in protein kinase C activity.




.
E &.10
5--0
0
0
Figure 2-2.

5 10 15 20
/4g PROTEIN

Effect of protein concentration on protein kinase C activity. Various protein concentrations from
the cytosol (0) or particulate (0) fractions were assayed for 3 min as described' in the Methods. Data are means + SEM of triplicate determinations from a representative experiment
which was repeated two times with similar percent changes in protein kinase C activity.




43
generate the protein curves were prepared from crude homogenates and without the use of DEAE chromatography. Fig 2-2 shows a representative experiment which was repeated twice with similar results.
In vitro inhibition of protein kinase C
The activity of protein kinase C was further characterized by determining the efficacy of kinase inhibitors to inhibit the enzyme in vitro. Two inhibitors were used, H-7 and HA 1004. H-7 is a potent inhibitor of protein kinase C while HA 1004 is less potent and was used as a control for H-7. H-7 dose-dependently inhibited protein kinase C and had a Kj similar to the reported Y<, value (Table 2-2). HA 1004 was approximately 4 times less potent at inhibiting protein kinase C (Table 2-2). Table 2-2 Effects of H-7 and HA 1004 on protein kinase C in
vitro
H-7 8 M 6 UM
HA 1004 30 pM 40 1M
Data are from experimentally obtained resultsa or from reported valuesb (Inagaki et al., 1984; Hidaka et al., 1984). The experimental values are means obtained from the compilation of 3
separate experiments for each H-7 and HA 1004 in which the cytosol fraction from neuronal whole brain cultures was used.
Effect of Ca2+ on the distribution of protein kinase C
Neuronal cultures were homogenized either in a buffer
containing Ca2+ chelators (homogenization buffer B) or a buffer containing 0.1 AM Ca2+ (20 mM Tris HCl, pH 7.5, 0.1 pM CaCI2, 0.2




44
mM PM4SF and 2.0 Ag/ml leupeptin). In cells homogenized with Ca2+ chelators, approximately 70% of the protein kinase C was isolated in the cytosol fraction (Fig 2-3). However, the homogenization of cells with 0. 1AM CaC12 resulted in a 96% reduction in the activity in the cytosol fraction and a 244% increase in the protein kinase C in the particulate fraction (Fig 2-3). The total activities (ie cytosol + particulate) of cells homogenized with Ca2+ chelators or with Ca were not different and had respective values of 1.29 + .03 and 1.32 + .10 nmol 32P/min/mg protein. Anion-exchange chromatoqraphic analysis and purification of protein kinase C
The elution profiles of the cytosol and particulate fractions of cell homogenates isolated by high-speed centrifugation (-43,000 X g) following a low-speed (3000 rpm, 8 min) spin are shown in Fig 2-4A. The first major peak of protein kinase C activity from the cytosol fraction eluted between .04 M0.12 M NaCl (Fig 2-4A). A plateau or minor peak of activity eluted at approximately 0.15 M NaCl. The activity in the cytosol fraction accounted for 68% of the total protein kinase C activity. The major peak of activity from the particulate fraction eluted at 0.045 M NaCl. After approximately 0.14 M NaCl, the elution profile of the particulate fraction exhibited a flattening of the slope although a second peak of activity is not apparent.




The elution profiles of the cytosol and particulate fractions of cell homogenates isolated by high-speed centrifugation (-43,000 X g), but without a prior low-speed spin are shown in Fig 2-4B. The major peak of protein kinase C activity in the cytosol fraction eluted at a low ionic strength (0.04 M NaCl). Additionally, a minor peak of activity appears to have eluted at 0.1 M NaCl. The first major peak of protein kinase
C activity in the particulate fraction eluted at 0.4 M NaCl (Fig 2-4B). However, a second major peak of activity eluted at 0.14 M NaCl. In this case, when a low-speed spin was not included in the preparation of the cellular fractions, the particulate fraction contained the majority of the total protein kinase C activity
(53%). Fig 2-4B is part of an experiment described elsewhere (Chapter IV). It is used here for comparative purposes only.
Protein kinase C from crude extracts of homogenates was
partially purified using DEAE cellulose chromatography (Table 23). Using a single-step elution procedure, the detergentsolubilized particulate fractions were purified to an extent approximately three times greater than the cytosol fraction.
Discussion
Primary neuronal cultures prepared from the whole brains of neonatal rats were shown to contain high levels of calcium, phospholipid-dependent protein kinase (protein kinase C). Under in vitro assay conditions, the activity of this enzyme was dependent on the concentration of protein used in the assay and




C
E
1.0
.C
0.5
E
C
0
Figure 2-3.

A B A B
cytosol particulate

Effect of Ca2+ on the subcellular distribution of protein kinase C. Neuronal cultures were
homogenized in the presence of Ca2+-chelators (A) or with 0.1 pM CaC12 (B). The homogenates were spun at 3000 rpm for 8 min at 4*C. The supernatant fraction was centrifuged at ~43,000 X g for 45 min at 40C to separate the cytosol and particulate fractions. Data are means + SEM of two separate experiments.




-YT2SCL

!
N'
K 'N

p ,T-,.-,A T.3

10/
5 /
/
/
Io /r /
/
.L~

5 10
FRACTION

--*+PL and Ci2+
*.PL od Ca2+
0. 3
CYT, C 0OL

1> .K

PARTICULATE

30
0.1 20
10

15 20

/
S0.2

FRACTION

Figure 2-4.

Anion-exchange chromatographic analysis of protein kinase C. (A), a homogenate of neuronal cultures was centrifuged at 3,000 rpm for 8 min at 4 *C. The supernatant was centrifuged at ~43,000 X g for 45 min at 40C to isolate the
cytosol and particulate fractions. The fractions were eluted with a linear (0-0.3 M) NaCl gradient
and assayed in the presence (0) or absence (0) of Ca2+, phosphatidylserine and diolein. (B), the cytosol and particulate fractions from neuronal
cultures were isolated by high-speed centrifugation (-43,000 X g) without performing a prior low-speed centrifugation (3,000 rpm). The fractions were eluted and assayed as in (A).

-0.2
4J

/

C.2
,-0.1




Table 2-3

Partial purification of protein kinase
exchange chromatography

C by anion-

step volume protein specific activity purification
(ml) (mg) (units/mg protein) (fold)
crude
cytosol 1.0 1.39 0.73 ----extract
DEAE
cytosol 5.0 0.84 3.44 4.7
fraction
crude
particulate 1.0 1.08 0.28 ----fraction
DEAE
particulate 5.0 0.66 4.24 15.3
fraction
The activity of protein kinase C from the cytosol and detergentsolubilized particulate fractions isolated by high-speed centrifugation (43,000 X g) was assayed before and after elution from DEAE columns with a single 5.0 ml aliquot of homogenization buffer B containing 0.15 M NaCl. Data are means from 2 separate experiments.

C bv anion..

Table 2-3




was stimulated by the synthetic diacylglycerol diolein. However, the activity of protein kinase C in the cytosol fraction was greater per mg protein than the activity in the particulate fraction. The protein curves were generated using crude homogenates upon which DEAE chromatography was not used. Thus, the lower activity in the particulate fraction may be due to the presence of triton X-100 or a greater abundance of endogenous inhibitors of protein kinase C. This appears to be true as the partial purification of the crude extracts by DEAE chromatography resulted in a purification of the particulate fraction which was approximately three times greater than that of the cytosol fraction. Schwantke and Le Peuch (1984) isolated a proteinaceous inhibitory factor(s) of protein kinase C from the supernatant fraction of homogenates of rat brain. This inhibitory factor was resolved by DEAE cellulose chromatography as two peaks which eluted a higher ionic strength than protein kinase C. The two fractions of inhibitor activity were completely inactivated by trypsin proteolysis. Two additional inhibitors of protein kinase C were isolated in the high-speed supernatants of bovine brain. (McDonald and Walsh, 1986). These inhibitors were identified as M4 17,000 and Mr 12,000 Ca2-binding proteins. In both of the previous studies, a quantification of the cytosol and particulate distribution of these inhibitors was not performed. Additional non-protein inhibitors of protein kinase C have been identified. These are primarily metabolic derivatives of membrane




sphingolipids such as sphingosine (Hunan et al., 1986) and various lysosphingolipids (Hunan and Bell, 1987). These compounds have Kj values of 25-180 AM, act in a reversible manner and have implicated the inhibition of protein kinase C as a mechanisms in the pathogenesis of sphingolipidoses (e.g., Krabbe's and TaySachs disease). Thus, these membrane-associated inhibitors may be responsible for the lower activity (on a per mg protein basis) and higher purification found for the crude particulate fraction.
Isoquinalinesulfonamides are synthetic competitive
inhibitors of cyclic nucleotide-dependent protein kinases and protein kinase C (Hidaka et al., 1984). H-7 is a isoquinalinesulfonamide compound used to inhibit protein kinase C (Hidaka et al., 1984; Hidaka and Hagiwara, 1987). However, because it inhibits c-AMP and c-GMP-dependent protein kinases the actions of H-7 in vivo or in situ cannot be directly attributed to the inhibition of protein kinase C. An alternative isoquinalinesulfonamide derivative is HA 1004 which strongly inhibits c-AMP and c-GMP-dependent protein kinases but has a higher Kj for protein kinase C than H-7. Due to their different inhibitory abilities, HA 1004 can be used as a control for H-7. In this study, these compounds were used for characterization purposes by demonstrating the differential inhibition of protein kinase C by H-7 and HA 1004. These compounds inhibited protein
kinase C isolated from the cytosol fraction of neuronal cultures at K~ values similar to their reported values.




Protein kinase C in the cytosol and particulate fractions eluted from the DE 52-cellulose columns at a low NaCl
concentration (-.04 M) which is characteristic for the enzyme (Kikkawa et al., 1982). Two peaks of activity appeared to elute from the columns of both the cytosol and the particulate fractions which were prepared without a low-speed spin (Fig 24B). In the cytosol, the second peak was much smaller than the first peak of activity. However, in the particulate fraction the second peak was equal in height to the first peak of activity. Anion-exchange chromatography is not used to separate the isozymes of protein kinase C. Hydroxylapatite columns are used
for this purpose (Huang et al., 1986). However, it is possible that a partial separation of the isozymes was achieved in this study due to charge differences of the isozymes. In other studies using DEAE chromatography, separate minor peaks of protein kinase C activity were shown in the brain (Kikkawa et al., 1982), embryonic carcinoma cells (Kraft and Anderson, 1983b) and NIH 3T3 cells (Uratsuyi et al., 1985). Conversely, separate non-calcium, phospholipid-dependent kinase activity has been shown to elute from DEAE-cellulose columns at higher ionic concentrations than protein kinase C (Hirota et al., 1985; Lang and Vallotton, 1986). This activity was not found in this study.
When the cytosol and particulate fractions were isolated
after performing a low-speed spin much less protein kinase C was found in the particulate fraction. From this study it appears




that approximately 20-25% of the total protein kinase C was in the discarded pellet obtained by low-speed centrifugation. This pellet would roughly be the P1 or nuclear pellet which was shown
by Kikkawa et al. (1982) to contain 20.5% of the total protein kinase C activity isolated from homogenates of the brains of rats.
The inclusion of Ca2+ during the homogenization of the
neuronal cultures dramatically increased the amount of protein kinase C in the particulate fraction by 244%. A corresponding 96% decrease in the amount of enzyme occurred in the cytosol fraction. The percent changes are not equal as a smaller amount of protein kinase C was found in the particulate fraction under control conditions due to the inclusion of a low-speed spin in the preparation of the cellular fractions. The total amount of protein kinase C (ie cytosol + particulate) in the cells was unchanged regardless of the homogenization conditions. This
indicates that the protein kinase C was not degraded by Ca2+dependent proteases such as calpain (Kishimoto et al., 1989). This may be due to the inclusion of protease inhibitors in the homogenization buffers. However, in neutrophils, a decrease in the total protein kinase C activity was reported in cells homogenized in the presence of calcium even though protease inhibitors were included in the homogenization buffer (Phillips et al., 1989).




53
The ability of Ca+ to affect the subcellular distribution of protein kinase C has both physiological and experimental
implications. Physiologically, agents which act to mobilize intracellular calcium may cause the translocation and activation of protein kinase C independent of the production of DAG. Wolf et al. (1985b) found Ca2+ at concentrations between 100 nM and 10 p1M increased the binding of protein kinase C to erythrocyte membranes. These levels of Ca2+ are near steady-state levels and are reached during the stimulation of Ca2+ mobilization. However, DAG activates protein kinase C by lowering the affinity of the
enzyme for calcium and phosphatidylserine (Takai et al., 1979b). Thus, the mobilization of calcium and the production of DAG probably work in a synergistic manner to activate protein kinase C. This would be the case during the hydrolysis of phosphoinositides where the mobilization of Ca2+ by IP3 would act to enhance the actions of DAG.
Experimentally, the amount of Ca2+ present during
homogenization can have an important bearing on the reported subcellular distribution of protein kinase C. For example,
Farrago et al. (1988) found 35% of the total protein kinase C in the particulate fraction when adrenal glomerulosa cells were
homogenized in the presence of Ca2+-chelators. This percentage increased to 80% when the cells were homogenized with 0.1 mM Ca2+. The preparation of cellular membranes for the quantification of the subcellular distribution of protein kinase C is usually




performed in the presence of Ca2+-chelators. This may act to artificially dissociate protein kinase C from the cell membrane as intracellular Ca2+ concentrations under steady-state conditions may be high enough to bind some protein kinase C to the cell membrane. However, a certain amount of protein kinase C is tightly bound to the membrane and requires detergent solubilization to remove it (Kikkawa et al., 1982; Kraft and Anderson, 1983a). The membrane distribution of this protein
kinase C is unaffected by Ca2+-chelators. Thus, the protein kinase C recovered in the particulate fraction in the presence of Ca"+chelators represents chelator-stable protein kinase C and may not reflect the true subcellular distribution of this enzyme. Perhaps a more precise means of determining the subcellular distribution of protein kinase C is through immunological methods in which intact cells are studied (Kiss et al., 1988). In any case, when
the neuronal cultures were homogenized with Ca2+-chelators and without a low-speed spin, the amount of the total cellular protein kinase C isolated in the cytosol and particulate fractions was 47% and 53%, respectively. Weiss et al. (1989) used a similar protocol to isolate 35% of the total protein kinase C in the particulate fraction of neuronal cultures prepared from the striatum. Kikkawa et al. (1982) recovered approximately 67% of the total protein kinase C activity in the particulate fraction from homogenates of rat brain. This is in contrast to many peripheral cell lines in which negligible amounts of protein




kinase C are detected in the particulate fraction using similar protocols to prepare the cellular fractions. For example, 80-95% of the total protein kinase C is recovered in the cytosol fraction prepared from pinealocytes (Sugden et al., 1985), MadinDarby canine kidney cells (Slivka et al., 1988), adrenal glomerulosa cells (Lang and Vallotton, 1986), neutrophils (Phillips et al., 1989), PC 12 cells (Messing et al., 1989), GH3 pituitary cells (Drust and Martin, 1985) and basophilic leukemia cells (Farrar and Anderson, 1985a). Additionally, Neary et al. (1988) recovered 91% of the total protein kinase C in the cytosol fraction of primary astrocyte cultures. The high amount of membrane-associated protein kinase C in neural tissue from the brain may be due to high levels of the type I isozyme. This isozyme of protein kinase C has not been found in peripheral tissues and 80-90% of it is recovered in the particulate fraction of homogenates prepared from rat brain in the presence of Ca+chelators (Yoshida et al., 1988). This is in contrast to the type II and type III isozymes which are located peripherally and are more readily recovered in the cytosol fraction (Yoshida et al., 1988).
In summary, neuronal cultures prepared from the whole brains of neonatal rats contain high levels of protein kinase C activity which exhibited properties characteristic for this enzyme. The subcellular distribution of protein kinase C was affected by Ca2+ and when the neuronal cultures were homogenized in the presence




56
of Ca2+-chelators the majority of this enzyme was located in the particulate fraction.




CHAPTER III
IDENTIFICATION AND CHARACTERIZATION OF PROTEIN KINASE C
INVOLVEMENT IN ANGIOTENSIN II RECEPTOR EXPRESSION
Introduction
Prior studies in our lab have investigated the mechanisms of oc-adrenergic receptor regulation of Ang II receptors. Two lines of evidence from these experiments suggest protein kinase C is involved. First, oc-adrenergic agonists stimulate the hydrolysis of phosphatidylinositol in neuronal cultures (Gonzales et al., 1987; Myers and Sumners, in press). Phosphatidylinositol hydrolysis results in the generation of diacylglycerol which is an endogenous activator of protein kinase C (Nishizuka, 1986). Second, phorbol esters which are known activators of protein kinase C (Castagna et al., 1982) significantly increase the specific binding of [5I]Ang II) in neuronal cultures (Sumners et al., 1987b). Taken together these studies suggest a role for protein kinase C in the regulation of Ang II receptor expression. However, phorbol esters have a wide variety of biological effects in addition to the activation of protein kinase C which may secondarily act to increase the binding of [ MsI]Ang II. These include tumor promotion, cytoskeletal alterations, cellular differentiation and modulatory secretagogue capabilities (see Chapter I). Presently, the full extent of protein kinase C




involvement in producing these effects is not known. Additionally, the synthetic diacylglycerol, l-oleoyl-2acetylglycerol (OAG), does not mimic the effect of phorbol esters
to increase the binding of [nsI]Ang II in neuronal cultures (Kalberg, unpublished results). Thus, the first objective of this study was to confirm the involvement of protein kinase C in the regulation of specific Ang II receptors. This was accomplished by determining the effect on the specific binding of [CnsI]Ang II of protein kinase C activators which are chemically unrelated to phorbol esters, using a protein kinase C antagonist, and depleting the neuronal cultures of protein kinase C activity. Our results suggest that protein kinase C is directly involved in the stimulation of Ang II receptor expression. We next investigated the nature of protein kinase C involvement in the regulation of Ang II receptors by determining if changes in protein kinase C distribution and activity are associated with changes in [MI]Ang II specific binding.
Methods
Preparation of neuronal cultures
Neuronal cultures used for the [sI]Ang II binding studies were prepared from the hypothalamus and brain stem (co-cultures) of one-day-old Sprague-Dawley (SD) rats essentially as described in Chapter II. The rationale for choosing these brain regions is that they contain the major concentrations of [nI]Ang II binding sites (Simonnet et al., 1982). For cultures used in [12I]Ang II




59
binding experiments, cells were suspended in Dulbecco's modified Eagle's medium (I)4M) containing 10% plasma derived horse serum (PDHS) and plated on 35 mm diameter Falcon tissue culture dishes
at a density of 3.0 x 106 cells per dish. For the experiments in which the activity of protein kinase C was measured, both cocultures and neuronal cultures prepared from whole brains of oneday-old rats were used. For each culture type, the cells were plated on 100 mm diameter dishes at a density of 18 x 10 cells per dish. All cultures were grown for 10-14 days in a humidified incubator with 10% 002/90% air, and after this time were used in experiments. Neuronal cultures prepared in this way contained
-90% neuronal cells with the remaining cells being predominantly astrocyte glia (Richards et al., 1989). [15I]Ang II binding assay
The specific binding of [mI]Ang II was determined using intact neuronal cultures attached to 35 mm diameter dishes.
Growth media was removed and the cells were washed twice with phosphate buffered saline (PBS), pH 7.2. For each experimental datum point, total binding was determined by incubating triplicate cultures for 60 min at 24C with 500 Al of PBS containing 0.2 nM ['2I]Ang II (150,000 cpm) and 0.16% heat inactivated bovine serum albumin (BSA). Nonspecific binding was
determined by incubating triplicate cultures under the same conditions except the reaction mixtures contained a 10,000 fold excess of unlabeled Ang II. Following the incubation, the cells




were rinsed twice with ice-cold PBS containing 0.8% BSA. Cells were dissolved with 1.0 ml of 2.0 N NaOH and transferred to plastic tubes. Each plate was rinsed with 500 Ml of deionized water, which was combined with the original sample. Specific binding was calculated as the mean of triplicate samples obtained by subtracting nonspecific radioactivity bound from the total radioactivity bound and was expressed as fmol of [ I]Ang II/mg protein.
Preparation of cellular fractions for protein kinase C analysis
Neuronal cultures were washed 3 times with ice-cold PBS,
scraped with 1.0 ml of PBS into 50 ml centrifuge tubes and centrifuged at 2500 rpm for 3 min at 40C. The pellets were washed, resuspended in 2.0 ml of homogenization buffer (20 mM Tris HCl, pH 7.5, 20 mM P-mercaptoethanol, 2.0 mM EDTA, 0.5 mM EGTA, 0.2 mM PMSF, and 1.0 Ag/ml leupeptin), homogenized in a Broeck tissue grinder (15-20 strokes) and centrifuged at 3000 X g for 8 min to remove nuclei and debris. The supernatant was centrifuged 100,000 X g for 60 min. The resulting supernatant was
used as the cytosol fraction and the pellet was resuspended in homogenization buffer containing 0.1% triton X-100 for 30 min at 4C. This suspension was centrifuged 100,000 X g for 60 min and
the supernatant was used as the particulate fraction. Protein kinase C assay
The activity of protein kinase C was assayed by measuring the incorporation of 32 P from [y32P]ATP into lysine rich histone




61
(type III-S) exactly as described in Chapter II. All samples were assayed in triplicate and the activity of protein kinase C was
expressed as nmol 3P/min/mg protein. Desensitization of protein kinase C
The desensitization of protein kinase C in neuronal cultures was achieved using long-term treatments with TPA. The method is as follows and describes 24 hr chronic TPA and control vehicle treatments. A group of neuronal cultures was divided into two equal groups. At time zero, one group received control vehicle and the other group received 0.16 jUM TPA. Cells were treated under sterile conditions, including the use of sterile vehicle and TPA solutions. After treatment, the cells were kept at 37C in a humidified incubator. Following 24 hrs, the growth media was aspirated off all the cells, which were next washed with DMEM containing 10% PDHS and then 2.0 mls of the same media was placed on each dish. At this time, half of the control-treated cells (24 hrs) received 0.16 gM TPA and the other half received control vehicle. Likewise, half of the TPA treated cells (24 hrs) received more TPA (0.16 AM) and the other half received control vehicle. All cultures were placed back into the incubator for one hour after which [inI]Ang II specific binding was determined for all the cells. The same procedure was followed for cells treated for 48, 72 and 96 hrs with TPA or control vehicle.




Protein determination
The protein content in both the [nIl]Ang II binding assay and the assays of protein kinase C was determined by the method of Lowry et al. (1951). In the [inI]Ang II binding studies, the cells were rinsed twice with PBS and then dissolved with 500 gl of 2.0 N NaOH. The protein was transferred to plastic tubes and each plate was washed with 500 ul of deioninzed water which was combined with the original sample and assayed for protein content.
DruQ incubations
The effects of the agonists of protein kinase C teleocidin A, mezerein, phorbol 12,13-dibutyrate (PDB) and phorbol-12myristate-13-acetate (TPA) on ['I]Ang II binding and the activity of protein kinase C were determined by incubating cultures with the drugs at various times and concentrations prior to performing the assays. The antagonist of protein kinase C, H-7, was added to the cultures 30 min prior to the addition of TPA. Control incubations were performed using drug vehicles. All incubations were carried out at 37*C in a humidified incubator (10% C02/90% air).
Preparation of drug solutions
Teleocidin A, mezerein, PDB and TPA were diluted in
dimethylsulfoxide (DMSO) at stock concentrations of 1.0 mM, 1.5
mM, 2.0 mM and 1.0 mM respectively and stored at -20C. Each was diluted in PBS to the desired concentration prior to use. H-7 was




63
dissolved directly into deionized water at a concentration of 5.0 mM. Further dilutions were made into PBS. Final DMSO concentrations did not exceed 0.1%. Statistical analyses
All results are expressed as means + SEM. Comparisons of
multiple means were made by one-way analysis of variance (ANOVA1) followed by a Newman-Keuls test to assess statistical differences between individual means when applicable. A difference at the 1% level was accepted as statistically significant. Statistical analyses of the data were performed with an Apple IIe computer using a ED-SCI statistical (Interactive microware) program.
Results
Effects of teleocidin A and mezerein on [rsI]Anq II specific binding in neuronal cultures
The first approach to determine whether the specific
activation of protein kinase C is responsible for an increase in the specific binding of [5I]Ang II involved testing two agonists of protein kinase C, teleocidin A and mezerein, which
are chemically unrelated to phorbol esters. Fig 3-1 is a compilation of separate representative time course experiments for each agonist of protein kinase C with PDB included as a positive control. Mezerein (0.5 gM), teleocidin A (0.76 pM), and PDB (0.99 AM) at concentrations which stimulate maximal increases
in the specific binding of [nsI]Ang II all rapidly increased the




specific binding of [1I]Ang II. At the earliest time points tested, 15 min for teleocidin A and mezerein and 30 min for PDB, all of the agonists significantly elevated the specific binding of C I]Ang II. In all cases, the stimulation of ['MI]Ang II binding reached a maximal level after 1 hr of incubation. In neuronal cultures treated with PDB and mezerein, the levels of [I]Ang II specific binding returned to control levels after 24 hrs. However, in cells receiving teleocidin A, ['sI]Ang II specific binding was still significantly higher than control levels after 24 hrs (data not shown). Time course experiments
were repeated four to five times for each compound tested and showed similar percent changes in ['MI]Ang II specific binding.
Representative dose response curves are shown in Fig 3-2 and each of these was repeated at least five times with similar percent changes in binding. Teleocidin A was found to be the most effective at increasing the specific binding of ['MI]Ang II with
an ED of 32 nM, while PDB and mezerein had ED values of 63 and 79 nM respectively. In prior studies, TPA was found to have an ED of 5 nM with respect to its ability to increase [ I]Ang II specific binding (Sumners et al., 1987). None of the compounds, tested at the highest concentrations used, were cytotoxic as
determined by trypan blue exclusion or significantly altered the protein content of the cultures as compared to controls (Table 31).




1 I I I I I I I p

60
Time (min)

Figure 3-1.

Effects of protein kinase C agonists on [125I]Ang II binding to neuronal cultures as a function of time. Neuronal cultures were incubated with teleocidin A (0.5 PgM;rD, mezerein (0.76 AM; 0), or PDB (0.99 pM; 0), in DMEM containing 10% PDHS at 37*C. The data presented here are a compilation of representative experiments performed for each agonist of protein kinase C and numbers are means + SEM of triplicate determinations. Individual time course
experiments for each agonist were repeated four to five times with similar results. ANOVA 1, p <
0.01 for each treatment. In the treated cells, the specific [25I]Ang II binding at each time point was significantly different from controls. See text for description.

40+

30
2010
0

30

90

1 0
120




9 8 7 6
[PKC agonist] -log M

Figure 3-2.

Binding of [12I]Ang II to neuronal cultures as a function of protein kinase C agonist concentration. Neuronal cultures were incubated with the indicated concentrations of teleocidin A
(0), mezerein (0), or PDB (0) in DMEM containing 10% PDHS for 1 hr at 370C in a humidified incubator (10% C02/90% air). The data are from
representative experiments and the numbers are means + SEM of triplicate determinations. Control specific binding values are plotted on the yaxis. All experiments were repeated a minimum of five times for each compound with similar changes
in [12I]Ang II specifically bound at each agonist dose. ANOVA 1, p < 0.01 for each dose-response curve. The comparisons of control-teleocidin A, control-mezerein and control-PDB were
significantly different at each dose at the 1% level, except for 1 nM teleocidin A, 10 nM teleocidin A and 1.56 nM mezerein. See text for description.




Effects of H-7 on TPA stimulated increases in [nsIAnq II binding.
In the next series of experiments, the effects of the inhibitor of protein kinase C H-7 (Hidaka, 1984; Hidaka and Hagiwara, 1987) on TPA-stimulated increases in the specific binding of [MI]Ang II were analyzed. Preincubation of neuronal cultures for 30 min with H-7, followed by TPA treatment (0.8 gM,
1 hr) resulted in a dose-dependent inhibition of TPA-stimulated increases in [M I]Ang II specific binding. A representative experiment is shown in Fig 3-3. TPA (0.8 pM, 1 hr) alone increased binding by 154%. Co-incubation of the cultures with TPA
and H-7 (100-300 AM) resulted in levels of [nsI]Ang II binding which were not significantly different from those seen in controls. At 50 and 75 pM, H-7 significantly reduced the TPA stimulated increases in [nI]Ang II specific binding, though levels were still elevated as compared to control values. H-7 (300 AM, 30 min) alone did not significantly alter the specific binding of [sI]Ang II as compared to control values and was not cytotoxic as determined by protein content (Table 3-1) and trypan blue exclusion.
Effects of phorbol esters on [5IlAnq II specific binding in neuronal cultures depleted of protein kinase C activity.
In the neuronal cultures used in this study and in other
cell cultures systems, prolonged phorbol ester treatment (24 hrs) results in a depletion of total cellular protein kinase C
activity (Matthies et al., 1987; Neary et al., 1988). This




strategy was used here to determine if TPA can stimulate the specific binding of ['2I]Ang II in neurons depleted of protein kinase C. Fig 3-4A shows 24 hr and 48 hr pretreatment groups. The addition of TPA (0.16 AM, 1 hr) to neuronal cultures that had received a 24 hr TPA pretreatment (D), as detailed in the Methods, failed to significantly increase the specific binding of [I]Ang II as compared to control cells (A) or cells that only received a TPA pretreatment (C). TPA (0.16 AM) significantly
increased [n6I]Ang II binding in neuronal cultures that were not pretreated with TPA (B). Similar results occurred in the 48 hr groups.
TABLE 3-1 Effects of protein kinase C activators and H-7 on
neuronal culture protein contents.
Treatment Protein content (mcr)
Control (vehicle) .46 + .02
TPA (0.8 jIM) .45 + .02
PDB (1.9 AM) .46 + .01
Mezerein (2.3 jAM) .44 + .01
Teleocidin A (1.5 AM) .46 + .02
H-7 (300 AM) .45 + .01
Neuronal cultures were incubated with the activators of protein kinase C for 1 hr or with H-7 for 30 min at 37C in a humidified incubator. Cultures were washed with PBS and their protein contents were determined as described in the Methods. Protein contents of treated cells were not significantly different from controls. Data are means + SEM from six 35 mm culture dishes.




Desensitization of the neuronal cultures to TPA-stimulated increases in the specific binding of ['sI]Ang II was not irreversible. The addition of TPA to neuronal cultures pretreated
for 72 and 96 hrs with TPA (0.16 AM) significantly increased specific [I]Ang II binding levels as compared to control levels (Fig 3-4B). At 72 hrs, TPA (1 hr) increased ['I]Ang II binding levels in TPA pretreated cells by 43% above control binding levels. After 96 hrs of TPA pretreatment, ['MI]Ang II specific binding was increased 44% over control levels by TPA. Both Fig 34A and 3-4B show representative experiments, and each was repeated 4 to 5 times with similar results.
Phorbol esters induce cell growth, cell differentiation and
mitogenesis in a variety of cell types (Woodgett et al., 1988; Blumberg, 1980). Thus, it is possible that the stimulation of
cell growth in the neuronal cultures by TPA may affect the binding levels of [nI]Ang II on a per mg protein basis. This was tested by determining whether chronic incubations of TPA alter the protein content of the neuronal cultures. As shown in table 3-2, the incubation of neuronal co-cultures with 0.16 pM TPA for 24, 48, 72, of 96 hrs did not significantly effect the protein content of the cultures as compared to control vehicle-treated cells.




A B C D E F

Figure 3-3.

Effects of H-7 on TPA stimulated increases in [15I]Ang II specific binding. Neuronal cultures
were incubated with (A) vehicle (0.1% DMSO in PBS) or (B) TPA (0.8 MM) for 60 min at 37C. Other groups received a 60 min TPA treatment following a 30 min preincubation with (C) 10 AM H-7, (D) 50 MM H-7, (E) 75 MM H-7, (F) 100 MM H7, (G) 150 juM H-7 or (H) 300 AM H-7. The final group (I) received 300 MM H-7 only. Following these incubations, [15 I]Ang II binding was measured as detailed in the Methods. The data presented here are from a representative experiment and numbers are means + SEM of triplicate determinations. This experiment was repeated five times with the same dose-response relationships observed each time. ANOVA 1, p <
0.01. Significantly different from TPA treatment.




251
201

fl.L

-% 50
e
o.40
C.
E
Z
E30
o 20.
10.
'*9

Figure 3-4.

Ii.

A B C D
24 hre

A B C D
72 hrs

~I1

B C 48 hre

A 8 C D 96 hrz

(Panel A). The effects of 24 hr and 48 hr
phorbol ester pretreatments on TPA stimulated increases in the specific binding of [12I]Ang II. Neuronal cultures received a pretreatment with vehicle (0.02% IMSO in PBS) or TPA (0.16 pM) for 24 or 48 hrs. Following this, vehicle treated groups received an additional 1 hr treatment of
(A) vehicle or (B) 0.16 /M TPA and groups pretreated with TPA received a 1 hr treatment of (C_ vehicle or (D) 0.16 pM TPA after which [ 1I]Ang II specific binding was determined in all groups. These data are from a representative experiment and numbers are means + SEM of triplicate determinations. ANOVA 1, p < 0.01. Significantly different from controls (A). (Panel B). Effects of 72 and 96 hr phorbol ester pretreatments on TPA stimulated increases in [25I]Ang II specific binding. Cells were grouped as described in panel A. ANOVA p < 0.01. Significantly different from controls (A). ** Significantly different from cells receiving TPA pretreatment followed by a 60 min vehicle
treatment (C). Experiments in figs 4A and 4B were repeated four times each with similar results
obtained each time.




Effect of protein kinase C activators on the distribution and activity of protein kinase C in neuronal cultures.
Neuronal cultures prepared from whole brains were incubated with either mezerein (0.76 1M), teleocidin A (0.5 JM) or TPA (0.8 j1M) for 15 min, 60 min, and 24 hrs. All three compounds caused the translocation of protein kinase C from the cytosol to the particulate fraction after 15 min of incubation. Thereafter, the activity of protein kinase C was gradually decreased below control levels (Fig 3-5, Fig 3-6, and Fig 3-7). In the three experiments shown (Figs 3-5, 3-6, 3-7), the activity of protein
kinase C in the cytosol and particulate fractions of control cells accounted for an average of 75% and 25%, respectively, of the total activity of protein kinase C. Following 15 min of incubation with mezerein, teleocidin A or TPA, the activity of protein kinase C in the cytosol fraction accounted for only 15%, 33%, and 15%, respectively, of the total protein kinase C activity, while the particulate fractions showed a corresponding increase in total protein kinase C activity. The total activity of protein kinase C (i.e., cytosol activity plus particulate
activity) in cells treated with mezerein, teleocidin A or TPA for 24 hrs was reduced to 8%, 25%, and 3%, respectively, of the total activity of protein kinase C found in drug-vehicle treated control cells.
In the next series of experiments, the effect of long-term TPA treatments on the activity of protein kinase C in neuronal




0 .25 1 24
time (hrs)

Figure 3-5.

The effect of mezerein (0.76 jiM) on the distribution of protein kinase C activity in
neuronal cultures. The cells were incubated with mezerein for 15 min, 60 min or 24 hrs at 37C. Cytosol (0) and particulate (0) fractions were prepared from the cultures and the protein kinase C activity was assayed as described in the Methods. The data presented here are means + SEM of triplicate determinations from a representative experiment which was replicated 3 times with similar percent changes in the activity of protein kinase C.




0o
0
Figure 3-6.

.25 1 24
time (hrs)

The effect of teleocidin A (0.5 AM) on the distribution of protein kinase C activity in neuronal cultures. The cells were incubated with teleocidin A for 15 min, 60 min or 24 hrs at
37C. Cytosol (0) and particulate (0) fractions were prepared from the cultures and the protein kinase C activity was assayed as described in the Methods. The data presented here are means + SEM of triplicate determinations from a representative experiment which was replicated 3
times with similar percent changes in the activity of protein kinase C.




0 .25 1
time (hrs)

Figure 3-7.

The effect of TPA (0.8 11M) on the distribution of protein kinase C activity in neuronal cultures. The cells were incubated with TPA for 15 min, 60 min or 24 hrs at 37-C. Cytosol (0) and particulate (0) fractions were prepared from the cultures and the protein kinase C activity was assayed as described in the Methods. The data
presented here are means + SEM of triplicate determinations from a representative experiment which was repeated 4 times with similar percent changes in the activity of protein kinase C.




cultures was examined. After 24 hrs of TPA (0.16 p1M) treatment, the total activity of protein kinase C (cytosol + particulate fractions) was significantly reduced as compared to controls. This is clearly seen in Fig 3-8 which illustrates the total activity of protein kinase C in TPA-treated cells as a percentage of the activity in control cells. However, following 72 and 96 hrs of TPA treatment, the total activity of protein kinase C had partially returned to 31 + 11% and 53 + 3% of the total control activity (Fig 3-8). The amount of protein kinase C in the cytosol and particulate fractions used to calculate the total activity (i.e., Fig 3-8) is shown in Fig 3-9. After 24, 72, and 96 hrs, the protein kinase C activities in the cytosol fractions of TPAtreated cells were 8%, 26%, and 48%, respectively, of the activity found in the control cytosol fractions. In the particulate fractions of cells treated with TPA for 24, 48, and 72 hrs, the amount of protein kinase C was 20%, 42%, and 57%, respectively, of the enzyme levels found in the control
particulate fractions.
The treatment of neuronal co-cultures prepared from the hypothalamus and brainstem with TPA (0.8pM) caused similar percent changes in the activity of protein kinase C. In control cells, the activity of protein kinase C in the cytosol accounted for 85% of the total protein kinase C activity. Incubation with TPA for 15 min reduced the activity of protein kinase C in the cytosol fraction to 15% of the total protein kinase C activity




77
with a corresponding increase in the activity of protein kinase C in the particulate fraction. In co-cultures treated with TPA for 24 hrs, the total activity of protein kinase C was reduced to 6% of the total protein kinase C activity found in vehicle treated cells (data not shown).
Table 3-2 Effect of chronic TPA incubations on the protein
content of neuronal co-cultures
Treatment Protein Content (mg)
24 hrs 48 hrs 72 hrs 96 hrs
Control .41 +.01(7) .49 +.01(6) .44 +.02(6) .48+.02(8) TPA .38 +.02(7) .50 +.01(6) .46 +.03(6) .49+.02(8)
Neuronal cultures were incubated with TPA (0.16 UM) for the indicated times. The cultures were washed twice with PBS and their protein contents were assayed as described in the Methods. Data are the means + SEM from values obtained from individual dishes. The number of dishes assayed is indicated in parenthesis.
Discussion
This study has clearly demonstrated the specific and
integral involvement of protein kinase C in the regulation of Ang II receptor expression in neuronal cultures. Two activators of protein kinase C, the diterpine ester mezerein and the indole alkaloid teleocidin A which are chemically unrelated to phorbol esters, both increased the specific binding of [n5I]Ang II in a dose and time dependent manner. These effects were similar to those obtained with the phorbol ester PDB. All the compounds tested increased [m2I]Ang II specific binding within 30 min and




100
80

n20
0
Figure 3-8.

24 72 96
(hrs)

The effect of long-term treatment with TPA on protein kinase C activity in neuronal cultures. Neuronal cultures were treated with TPA (0.16 pM) for 24 hrs, 72 hrs or 96 hrs, and the activity of protein kinase C was assayed in the cytosol and particulate fractions. The results represent total protein kinase C activity (obtained by adding cytosol and particulate activities) in TPA treated cells, expressed as a percentage of the total protein kinase C activity found in control cells. Data are means + SEM from two separate experiments.




Q cytosol E particulate
-Tfl

Figure 3-9.

The effect of long-term TPA treatment on the protein kinase activity in the cytosol and particulate fractions. (A) control vehicletreated, (B) 24 hr TPA (0.16 A.M treatment, (C) 72 hr TPA (0.16 jtM) treatment, (D), 96 hr TPA (0.16 1M) treatment. Data are means + SEM from two separate experiments.

3,-

.
N.
C
E
C




80
exerted a maximal effect by 1 hr. With mezerein and PDB, [ I]Ang II specific binding returned to control levels by 24 hrs. However, with teleocidin A, binding was still elevated after 24 hrs. This suggests teleocidin A is a longer acting substance or utilizes other mechanisms in the stimulation of Ang II receptor
binding. This was supported by the fact that teleocidin A did not downregulate total activity of protein kinase C to the same extent as mezerein and TPA (Fig 3-6). The EDm values for stimulation of [nI]Ang II specific binding by mezerein, teleocidin A and PDB generally agreed with their reported ED values for activation of protein kinase C (Couturier et al., 1984; Kraft et al., 1982). For example, our EDm of 32 nM for
teleocidin A is similar to an ED of 18 nM obtained using purified protein kinase C (Arcoleo and Weinstein, 1985). Although mezerein and teleocidin A are chemically and structurally unrelated to phorbol esters, the hydrophilic and hydrophobic moieties of these compounds are arranged in a similar spatial manner (Jeffery and Liskamp, 1986). This apparently accounts for their similar functionalities even though they are chemically unrelated.
The isoquinalinesulfonamide inhibitor of protein kinase C H7 has been useful in elucidating the role of protein kinase C in various biological functions (Hidaka and Hagiwara, 1987). H-7 is a direct inhibitor of protein kinase C which acts in a competitive manner with respect to ATP (Hidaka et al., 1984). In




this study, we showed that 50 14M H-7 significantly lowered (p <
0.01) TPA-stimulated increases in [2MI]Ang II specific binding in neuronal cultures. At 100 AM, 150 pM and 300 jAM, H-7 completely inhibited TPA-stimulated increases in [ I]Ang II specific binding. The concentrations of H-7 used were several fold higher than concentrations used to inhibit purified protein kinase C in vitro (K = 6 AM) (Hidaka et al., 1984) and in our own assay of protein kinase C in which H-7 had a I of 8 AM (see Chapter II). However, the concentrations used are comparable to those used in other intact cell preparations (Inagaki et al.,1984; Pandol and Schoefield, 1986; Turner et al., 1987). Higher concentrations of H-7 are necessary in intact cell studies probably due to the lipid insolubility of this compound (Hidaka et al., 1984), so restricting its entry into cells. Additionally, the intracellular concentration of ATP may alter the effectiveness of H-7 due to the competitive interaction between H-7 and ATP. For example, if the intracellular concentration of ATP in the neuronal cultures is in the millimolar range as it is in muscle cells (McGilvery, 1983) a relatively high concentration of H-7 would be needed to displace the binding of ATP to the ATP-binding site on the catalytic domain of protein kinase C.
The specific and direct involvement of protein kinase C in the increased expression of Ang II receptors was further suggested by depleting our neuronal cultures of protein kinase C activity. This resulted in a complete abolition of TPA-stimulated




increases in the specific binding of [sI]Ang II. Prolonged treatment of the cultures for 24 and 48 hrs with the phorbol ester TPA (0.16 AM) completely blocked TPA-stimulated increases in the specific binding of [MI]Ang II. Using PC-12 cells, Matthies et al. (1987) reported a depletion of protein kinase C activity in both the cytosol and particulate fractions after 24, 48 and 67 hr of incubation with 1 M TPA. This treatment did not effect cyclic AMP-dependent and Ca2+/calmodulin dependent protein kinase activities. Our [s25I]Ang II binding results indicate that after 24 and 48 hrs of TPA treatment the activity of protein kinase C was depleted. However, 72 and 96 hr TPA pretreatments did not abolish TPA-stimulated increases in [MsI]Ang II specific binding. In addition to activating protein kinase C, phorbol esters are potent tumor promotors. Concomitant with their tumor promoting ability are the actions of phorbol esters to induce cellular differentiation and mitosis and to promote cell growth (Woodgett et al., 1988; Blumberg, 1980). However, chronic incubation of the neuronal cultures for 24, 48, 72 and 96 hrs did not alter the morphology of the cells and was not cytotoxic. Additionally, chronic incubations with TPA did not affect the protein content of the cultures. Thus, the levels of [M I]Ang II binding expressed on a per mg protein basis were not altered due
to changes in the protein content of the cultures.
The stimulation of the translocation of protein kinase C
from the cytosol to the cell membrane by phorbol esters is




followed by the gradual degradation of protein kinase C at the cell membrane and this degradation is enhanced by phorbol esters
(Ballester and Rosen, 1985; Young et al., 1987). Thus, it is likely that the return of the stimulatory effects of TPA on ['5I]Ang II binding by 72 hours represents synthesis of new protein kinase C. This was confirmed by the our studies which showed a partial return of the total protein kinase C activity after 72 and 96 hrs of TPA treatment of approximately 30% and 50%, respectively. The partial return of the total protein kinase
C activity was due to an increase in activity in both the cytosol and particulate fractions (Fig 3-9), although the activity in neither of the fractions increased to control levels. Presumably, the activity found in the cytosol represents "translocatable" protein kinase C and is more important in the regulation of Ang receptors. However, one cannot rule out the possibility that a membrane bound form of protein kinase C may be involved in the regulation of Ang II receptors by an unknown mechanism (for a discussion of protein kinase C isozymes and their subcellular distribution see (Chapter II). Further, it is apparent that a complete return of protein kinase C activity is not required for TPA to stimulate increases in the specific binding of [mI]Ang II. Thus, in the unstimulated-control state, spare protein kinase C exists with respect to the regulation of Ang II receptors. Additionally, only one isozyme of protein kinase C may be involved in the regulation of Ang II receptors in cultures and




the return of protein kinase C activity after 72 and 96 hrs represents that single isozyme. Since the protein kinase C assay used in this study cannot distinguish the isozymes of protein kinase C, further studies are necessary to identify the isozymes present in the neuronal cultures and what roles they may have in the regulation of Ang II receptors. Recently, Mochly-Rosen et al. (1987) raised several monoclonal antibodies against protein kinase C, one of which inhibited protein kinase C. Thus the possibility exists that antibodies with inhibitory actions against specific protein kinase C isozymes can be isolated. These would be powerful tools for deciphering the individual functions of the protein kinase C isozymes.
Finally, the complete downregulation of protein kinase C by treatment of the neuronal cultures with TPA for 24 hrs did not alter steady-state levels of the specific binding of [='I]Ang II. This suggesting that protein kinase C is not involved in the steady-state expression of Ang II-specific receptors.
The data presented here further indicate that translocation of protein kinase C from the cytosol to the cell membrane is a prerequisite for an increase in Ang II receptor expression. The translocation of protein kinase C produced by mezerein, teleocidin A, and TPA appeared to be complete by 15 min while the increases in [6I]Ang II binding reached maximal levels by 1 hr. Presumably, the translocation of protein kinase C from the cytosol to cellular membranes allows for complete activation of




protein kinase C and is required for an increased expression of Ang II receptors.
Phorbol esters act to increase the specific binding of
[Il]Ang II in neuronal cultures by primarily increasing the Bmax (Sumners et al., 1987b). The above studies suggest that protein kinase C is directly involved in this action. There are several mechanisms by which the activation of protein kinase C may lead to an increase in the specific binding of [nI]Ang II. These include phosphorylation of a membrane protein which leads to the
unmasking of membranous Ang II receptors, the direct phosphorylation of refractory Ang II receptors which may induce a conformational change that permits ligand binding or phosphorylation by protein kinase C of an cellular component involved in the translocation of Ang II receptors to the membrane surface. The latter mechanisms would be analogous to an increase in the expression of transferrin receptors in Chinese hamster ovary (CHO) fibroblasts by TPA which is due to an increased exocytosis of transferrin receptors without a corresponding change in their endocytotic rate (McGraw et al., 1988).
Protein kinase C is an intracellular second-messenger and
presumably mediates the regulatory actions of another hormone(s) or neurotransmitter(s) on Ang II receptors. Possibilities include acetylcholine, NE and bradykinin, all of which stimulate the hydrolysis of phosphatidylinositol in the brain (Batty et al., 1985; Gonzales et al., 1987; Myers and Sumners in press; Yano et




al., 1987) which putatively leads to the generation of diacylglycerol, an endogenous activator of protein kinase C (Nishizuka, 1986). Recently we have shown that acute treatment of
neuronal cultures with NE (30-60 mins) at concentrations which cause large increases in phosphatidylinositol hydrolysis increases
the specific binding of [sI]Ang II via o-adrenergic receptors (Myers and Sumners in press). This is in contrast to past
experiments in which NE at longer incubations (2-6 hrs) and at concentrations which caused small or no increases in phosphatidylinositol hydrolysis was shown to decrease the
specific binding of [25I]Ang II (Sumners et al., 1986b). Therefore, at least acutely, protein kinase C may mediate NEstimulated increases in the specific binding of [MI]Ang II.
This study has shown that protein kinase C plays a specific and integral role in the regulation of Ang II receptor expression in neuronal cultures prepared from the brains of one-day-old rats and suggests that receptor systems which activate protein kinase C, presumably by translocation from the cytosol to the cellular membranes, may act to regulate Ang II receptor expression.




CHAPTER IV
ADRENERGIC REGULATION OF PROTEIN KINASE C SUBCELLUIAR DISTRIBUTION
Introduction
The activation of oc-adrenergic receptors mediates the
hydrolysis of phosphoinositides in a variety of tissues. In the
central nervous system, the stimulation of oc-adrenergic receptors elicits the hydrolysis of phosphoinositides in brain slices
(Kendall et al., 1985; Gonzales and Crews, 1985; Minneman and Johnson, 1984) and primary neuronal cultures (Gonzales et al., 1985; Gonzales et al., 1987; Myers and Sumners, in press; Weiss
et al., 1988). As reviewed in Chapter I the hydrolysis of phosphoinositides, specifically phosphatidyl inositol 4,5 bisphosphate, results in the generation of two products
(Berridge, 1984). Inositol 1,4,5 triphosphate (IP3), which mobilizes intracellular calcium and diacylglycerol (DAG), which is the endogenous activator of protein kinase C (Takai et al., 1979b). Following the stimulation of phosphoinositide hydrolysis, calcium and DAG promote the association of protein kinase C with the plasma membrane where, in combination with calcium, phosphatidylserine and DAG, it becomes fully activated (Nishizuka, 1986). Experimentally, the binding of protein kinase C to the plasma membrane is identified by an increased level of




protein kinase C in the particulate fraction isolated from stimulated cells. A corresponding decrease in protein kinase C is observed in the cytosol fraction. This subcellular redistribution is termed translocation and is used as an index of the activation of protein kinase C.
Previous studies from our lab have implied that protein
kinase C is activated by a1-adrenergic receptors. In these studies, the short-term stimulation of o-adrenergic receptors was associated with an increase in both the hydrolysis of phosphoinositides and the number of Ang II receptors in primary neuronal cultures (Myers and Sumners, in press). Additionally, protein kinase C was shown to be integrally involved in phorbol ester-stimulated increases in the expression of Ang II receptors (Chapter III; Sumners et al., 1987b). Together, these studies
indicate that the upregulation of Ang II receptors due to the stimulation of c-adrenergic receptors involves the translocation and activation of protein kinase C. However, the c-adrenergic receptor-mediated translocation of protein kinase C has not been demonstrated in primary neuronal cultures prepared from the brains of rats. Alternatively, the translocation of protein
kinase C by a1-adrenergic receptor agonists and other receptor agonists has been demonstrated in a variety of peripheral cell types. These include angiotensin II (Ang II) in adrenal glomerulosa cells (Lang and Vallotton, 1986), interleukin 2 in murine CT6 cells and interleukin 3 in FDC-Pl cells (Farrar and




Anderson, 1985; Farrar et al., 1985), thyrotropin-releasing hormone in GB cells (Drust and Martin, 1985), gonadotropinreleasing hormone in pituitary gonadotrophs (Hirota et al.,
1985), leukotriene D4 in basophilic leukemia cells (Vegesna et al., 1988), and nicotinic and muscarinic receptor agonists in PC 12 cells (Messing et al., 1989). The translocation of protein
kinase C by e1-adrenergic receptor agonists has been demonstrated in pinealocytes using phenylephrine (Sugden et al., 1985) and in Madin-Darby canine kidney cells using epinephrine (Slivka et al., 1988).
In this study, epinephrine was used to study the effects of c-adrenergic receptor activation on the subcellular distribution of protein kinase C in primary neuronal cultures. This study was carried out to further investigate the mechanisms involved in the regulation of Ang II receptors by oc-adrenergic receptors.
Results from this investigation suggest that the activation
of oc-adrenergic receptors by epinephrine causes a transient translocation of protein kinase C in neuronal cultures prepared from the whole brains of neonatal rats.
Methods
Preparation of neuronal cultures
The neuronal cultures used in this study were prepared from the whole brains of one-day-old Sprague-Dawley (SD) rats exactly as described in Chapter II.




Preparation of cellular fractions and treatment of neuronal
cultures
The isolation of supernatant and particulate fractions and the DEAE chromatography protocols used here are similar to those described in Chapter II. In brief, the neuronal cultures were treated with the desired compounds (ie phorbol esters or epinephrine) for various times by adding the compounds directly into the growth media. Untreated neuronal cultures (ie controls) were treated with vehicle solutions. The vehicle solution for epinephrine-treated cells was PBS containing 1 PM ascorbic acid while the vehicle solution for TPA-treated cells was 0.02% DMSO in PBS. After treatment, the growth media was aspirated off and the cells were washed twice with 3 ml of ice-cold (4C) homogenization buffer A (20 mM Tris HCI, pH 7.5, 2.0 mM EDTA, 0.5
mM EGTA, 0.25 M sucrose, 0.2 mM phenylmethylsulfonylfloride (PMSF), and 2.0 1g/ml leupeptin). The cells were rapidly scraped from the dish with 1 ml of homogenization buffer A (4C), homogenized in a dounce homogenizer (15-20 strokes), and centrifuged at 20,000 RPM (-43,000 X g) for 45 min at 40C. The supernatant was used as the cytosol fraction and the pellet was resuspended in 1 ml of homogenization buffer A (4C) containing
0.1 % Triton X-100 for 30 min at 4C. The suspension was centrifuged at 20,000 RPM (-43,000 X g) for 45 min at 40C and the supernatant was used as the particulate fraction.




DEAE column chromatographv
The preparation and elution of the DEAE columns was performed at 4C as described in Chapter II. In brief, the protein kinase C was eluted from the columns by either a linear
NaCI gradient (0-0.3 M NaCl in buffer B) or by a single-step elution with 5.0 ml of homogenization buffer B containing 0.15 M NaCl.
Assay of protein kinase C activity
The protein kinase C activity was determined by measuring the incorporation of -y2P from [-Y3P]ATP into lysine rich histone (type III-S) exactly as described in Chapter II. The activity was expressed as nmol3P/min/mg protein. Protein determination
The protein content of the cytosol and particulate fractions collected by DE 52 cellulose chromatography was determined by the method of Lowry et al. (1951). Statistical analyses
All results are expressed as means + SEM. Comparisons of
multiple means were made by one-way analysis of variance followed by a Newman-Keuls test to assess statistical differences between individual means when applicable. A difference at the 5% level was accepted as statistically significant. Statistical analyses of the data were performed using an analysis of variance program
designed by Human Systems Dynamics (Northridge, Ca.).




Results
Anion-exchange chromatographic analysis of protein kinase C distribution in neuronal cultures treated with epinephrine
The elution profile of protein kinase activity in the
cytosol fraction of control and epinephrine-treated neuronal cultures is shown in Fig 4-1. In control cells, the first major peak of calcium, phospholipid-dependent protein kinase (protein
kinase C) activity was eluted between 0.04-0.1 M NaCl. A second minor peak of activity eluted at 0.1 M NaCl. Treatment of the
neuronal cultures for 5 min with 50 pM epinephrine resulted in a 29% decrease in the protein kinase C collected in the cytosol fractions from 0.04-0.1 M NaCl. Additionally, a second peak of protein kinase C activity in the cytosol fraction was not detected in cells treated with epinephrine. A concentration of 50
jiM epinephrine was used as this is a concentration which maximally stimulates the hydrolysis of phosphoinositides (Crews et al., 1988; Myers and Sumners, in press). Typically, between 10 pM and 100 1M epinephrine or norepinephrine is used in either neuronal cultures or brain slices to elicit a maximal hydrolysis of phosphoinositides.
The first major peak of protein kinase C activity from the particulate fraction of control cells and cells treated with epinephrine eluted at 0.04 M NaCl (Fig 4-1). Additionally, what
appeared to be a second major peak eluted at 0.14 M NaCl. The treatment of the cultures with 50 1M epinephrine did not result




in a corresponding increase the protein kinase C content of the particulate fraction which eluted from 0.04-0.2 M NaCl, although the elution profile of the cultures treated with epinephrine appears broader than that of control cells. However, using a single step elution procedure, an increase in the protein kinase
C content in the particulate fraction of epinephrine-treated cells was detected (see below).
The results from Fig 4-1 raise the possibility that
treatment with epinephrine reduces the total protein kinase C activity of the neuronal cultures. However, the total activity of protein kinase C was unchanged in neuronal cultures treated with 50 AM epinephrine for 5 min (Fig 4-2). Thus, the decrease in the protein kinase C in the cytosol fraction was not due to a
reduction or inhibition of the enzyme activity. This is a representative experiment which was repeated twice with no difference between the protein kinase C activity of control and epinephrine-treated cells observed in each case. The analysis of protein kinase C distribution usinq a single-step elution
The effect of epinephrine on the distribution of protein kinase C was further analyzed using a single-step elution procedure. In these experiments protein kinase C was eluted from columns packed with DE 52 cellulose with a single aliquot of 0.15 M NaCl. Using this method, the translocation of protein kinase C
from the cytosol to the particulate fraction is readily apparent




CONTROL CYTO SOL

50f~

0
x ,
a.
C
U
W
I) z
2
I
Figure 4-1.

CO-NTROL

4 (NJ
I iv
I '
/
/ /
k
,L ~f'
'I
KU

EPI
/
/
/

/,P
/

CYT0SOL 94
/ i0.2 / 1
RTICULATE
/ r 0.2 / 0.
'9-

Sz1 15 10 15
FRACTION
A single 100 mm dish (18 X 106 cells) of neuronal cultures prepared from the whole brains of neonatal rats was treated with either phosphate buffered saline-vehicle (PBS) or 50 gM epinephrine for 5 min. The cytosol or the detergent-solubilized particulate fraction
(membranes) were prepared as described in the Methods and applied to a column (0.8 X 4 cm) packed with 0.6 ml of DE 52 cellulose equilibrated with homogenization buffer B. The columns were eluted with a linear (0-0.3 M) NaCl gradient as 1.0 ml fractions. Protein kinase activity was determined in 50 Al aliquots of the
indicated fractions in the presence (0) or absence (0) of 0.5 mM CaCl2, 6 Mg phosphatidylserine, and 0.4 jg diolein. Protein kinase activity is expressed as cpm of 32P incorporated into histone (type III-S)/3 min/50 Al aliquot. NaCl concentrations (dashed lines) were determined by conductance.




Full Text

PAGE 1

MECHANISMS OF NEURONAL ANGIOTENSIN II RECEPI'OR REGUIATION By CHRISTOPHER JOHN KALBERG A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FIDRIDA IN PARTIAL FULFILIMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCI'OR OF PHIIDSOPHY UNIVERSITY OF FIDRIDA 1989

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ACKNCMI.E[X;EMEN I wish to extent my sincerest gratitude to the chairman of my canmittee, Dr. COlin SUmners, for his unen::lin;J support, urrlerst:an:ti.rg an:l breadth of knowledge. I am i.rrlebted to Colin for the respect an:l irrleperrlence he gave ne which allowed ne to grow both as a student an:l an irrlividual. My thanks go to the other members of my canmittee, Ors. Melvin Fregly, Mchan Raizada an:l F.d Meyer, for helpi.n] ne vie-w my research from their enlightened perspectives. Additionally, I would like to thank the chairman of the physiology department, Dr. Ian Fhillips, for his support an:l developnent of the graduate program. For their expert technical assistance, I thank Jacqueline Perez an:l Tammy Gault. For their invaluable assistance with administrative an:l technical affairs, I sincerely thank Kevin Fortin an:l Victoria I.aPlaca. I am i.rrlebted to my ex>lleagues Ors. laura Mudd an:l Brain Masters for helpi.n] ne -weather sane iooriburrl tiloos an:l classes, an:l I thank the distir)3uished Jenny Olou for makin3 it fun to cx::me to the lab. My sincerest thanks go to Ors. Elaine SUmners an:l Lirrla Myers for all their help in the lab. I am IOOSt irrlebted to my ioother, father, TU.tu, an:l granjroc>ther for their support an:l urrlerstan:lin;J throughout the years. Finally, I wish to thank Jacqueline Perez, again, for bei.n] my best frien:i an:l canpatriot over the last four years. I look forward to many happy years tog-ether. ii

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TABLE OF CONTENTS ACKNOWLEDGEMENTS ABSTRACT CHAPTERS ii V I. INTRODUCTION . . . . 1 II. The Renin-Angiotensin System in the Brain 1 Angiotensin II Receptors in the Brain. 5 Regulation of the Renin-Angiotensin System in the Brain . . . 11 Regulation of Angiotensin II Receptors ... 12 Neuronal Cultures: A Model to Study Angiotensin II Receptor Regulation .... 18 Intracellular Mechanisms Involved in Angiotensin II Receptor Regulation 23 General Hypothesis ............. 28 CHARACTERIZATION OF PROTEIN KINASE C IN NEURONAL CULTURES. .... 32 Introduction ........... 32 Methods . . . . 34 Results . . . . 39 Discussion . . . 45 III. IDENTIFICATION AND CHARACTERIZATION OF PROTEIN KINASE C INVOLVEMENT IN ANGIOTENSIN II RECEPTOR EXPRESSION . . 57 Introduction .... Methods Results ...... Discussion iii 57 58 63 77

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IV. v. VI. ADRENERGIC REGULATION OF PROTEIN KINASE C SUBCELLULAR DISTRIBUTION Introduction Methods .. Results .. Discussion REGULATION OF ANGIOTENSIN II RECEPTORS UNDER DEPOLARIZING CONDITIONS ........ Introduction Methods Results Discussion SUMMARY REFERENCES BIOGRAPHICAL SKETCH. iv 87 87 89 92 102 108 108 109 112 119 128 143 162

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MECHANISMS OF REGUIATION OF NEURONAL ANGIOTENSIN II RECEPl'ORS By Christopher John Kalberg December, 1989 Chairman: Dr. Colin Sumners Major Department: Physiology The activity of calcium, phospholipid-dependent protein kinase, protein kinase c, was characterized in primary neuronal cultures prepared from the brains of neonatal rats. Several protocols were used to determine that protein kinase C is integrally involved in the stimulation of angiotensin II receptor expression in culture. These included the use of non-phorbol ester activators of protein kinase c, the use of an antagonist of protein kinase C and the downregulation of protein kinase c activity. The involvement of protein kinase C in the upregulation of angiotensin II receptors produced by the activation of ~ 1 adrenergic receptors was investigated. Incubations of the neuronal cultures with epinephrine for 1-5 min resulted in a V

PAGE 6

translocation of protein kinase C from the cytosol to the particulate fraction. This effect was transient and was blocked by prazosin indicating the involvement of ~ 1 -adrenergic receptors. Thus, protein kinase C may mediate ~ 1 -adrenergic receptor stimulated increases in angiotensin II receptor expression. Conversely, the influx of calcium has been implicated in the downregulation of angiotensin II receptors. In this study, under depolarizing conditions, which were shown to significantly increase the uptake of 45 ca 2 +, a decrease in the specific binding of [ 125 I]angiotensin II occurred in the neuronal cultures after incubations of 2-4 hrs. Both the uptake of 45 ca 2 + and the decrease in the specific binding of [u 5 I]Ang II observed under depolarizing conditions were blocked by the calcium channel antagonist, nifedipine. These data indicate that the influx of Ca 2 + through voltage sensitive calcium channels is a mechanism involved in the downregulation of angiotensin II receptors. This study suggests that two mechanisms are involved in the regulation of angiotensin II receptors in the neuronal brain cells cultures. An overview of the possible scenarios in which these regulatory mechanisms may act is presented. vi

PAGE 7

CHAPl'ER I INTRODUCTION The Renin-Angiotensin System in the Brain The discovery by Goldblatt et al. in 1934 that renal artery constriction produced a prolonged elevation of systemic arterial pressure initiated the search for endogenously produced pressor substances. In 1956, this search was, in part, culminated by the isolation and identification of the vasoactive octapeptide angiotensin II (Ang II) (Skeggs et al., 1956a; I.entz et al 1956; Bumpus et al., 1957). Originally tenned hypertensin, Ang II (Braun-Menendez and Page, 1958) is fonned by a series of proteolytical events. The initial event in Ang II production involves the cleavage of the decapeptide angiotensin I (Ang I) from the ~-globulin angiotensinogen by the enzyme renin. Ang I is then cleaved of two amino acids by angiotensin converting enzyme (ACE) to produce Ang II. Using pithed cats, initial investigators presumed the pressor actions of Ang II were limited to the periphery (Bumpus et al., 1957). However, in 1961, Bickerton and Buckley identified an action of Ang II in the brain using a cross-perfusion preparation. In this study, the blood supply to the head of a recipient dog was detached from its peripheral circulation by ligation of the carotid arteries. The 1

PAGE 8

2 blood supply to the head of a donor dog was rerouted to the head of the recipient dog. The peripheral circulation of the recipient dog was left intact. The injection of Ang II (0.2 g/kg-0.4 g/kg) into the carotid artery of the donor dog produced a pressor response in the recipient dog. This pressor response in the recipient dog occurred even though its central blood flow was detached from its peripheral circulation. Thus, a central pressor effect of Ang II which appeared to be neurogenic in origin was identified. Presently, the actions of Ang II in the brain are known to extend well beyond the pressor response identified by Bickerton and Buckley. These actions are manifested by both physiological and behavioral responses which interact in a complex manner to maintain body-fluid homeostasis. The intraventricular (i.v.t) administration of Ang II (eg in the picogram range) produces the following effects: the stimulation of drinking (Booth, 1968; Epstein et al., 1969), the facilitation of adrenocorticotropin (ACTH) release from the anterior pituitary (Maran and Yates, 1977; Spinedi and Negro-Vilar, 1983; Keller-Wood et al., 1986), and an increased salt appetite (Fluharty and Epstein, 1983). Severs et al. (1967, 1970) and Severs and Daniels-Severs (1973) further characterized the pressor effect of centrally administered Ang II. They determined that the increase in blood pressure elicited by Ang II was due to an activation of the sympathetic nervous system and the release of vasopressin (AVP)

PAGE 9

3 from the posterior pituitary. Thus, several actions of Ang II on the brain are well established. However, some controversy exists as to the site of production of the Ang II which acts centrally. This is because the peripheral administration of Ang II (eg in the nanogram range) can mimic many of the effects of centrally administered Ang II. This raises the possibility that Ang II produced peripherally could affect the brain. Ang II cannot cross the blood-brain barrier under conditions where the blood-brain barrier is intact (Phillips, 1980). However, certain small areas of the brain, the circumventricular organs, lie outside of the blood-brain barrier. It is likely that peripherally administered Ang II acts at the circumventricular organs to affect the function of the brain. Peripherally, the existence of a functional renin angiotensin system is firmly established. The main loci of Ang II production in the periphery are the kidneys, liver and lungs (Peach, 1977). Ang II has several sites of action in the periphery including the vascular smooth muscle, the sympathetic nervous system and the adrenal glands (Peach, 1977). Similar to the central nervous system, the peripheral actions of Ang II act to maintain body fluid homeostasis. The establishment of a functional renin-angiotensin system in the brain has proven to be more difficult. This is primarily due to the inherent complexity of the brain. However, over the last 18 years much research has confirmed the presence of a renin-angiotensin system in the

PAGE 10

4 brain. The existence of Ang II in the brain was first inferred by the identification of Ang II-like material in cerebral spinal fluid. (Finkielman et al., 1972; Hutchinson et al., 1975). More recently, Ang II in the brain has been characterized using high performance liquid chromatography (Ganten et al., 1983; Hermann et al., 1984; Phillips and Stenstrom, 1985) and localized to specific brain regions using immunohistochemical and immunofluorescence techniques (Lind et al., 1985; Weyhenmeyer and Phillips, 1982). These studies indicate Ang II is primarily located in the hypothalamus and brainstem which are areas of the brain closely associated with the central actions of Ang II (eg pressor and dipsogenic effects). Ang II is not synthesized de novo, but is produced from a series of proteolytic steps. Therefore, for Ang II to be produced in the brain the intermediaries involved in Ang II production must be present in the brain. The initial precursor of Ang II, angiotensinogen, has been found in specific brain areas (Hawkins and Printz, 1983; Healy and Printz, 1985; Lewicki et al., 1978). These include the hypothalamus, the organum vasculosum lamina terminalis (OVLT), and the area postrema (AP) of the brainstem. Further, angiotensinogen mRNA is expressed in the brain (Dzau et al., 1986; Lynch et al., 1986; Ohkumbo et al., 1986). Renin-like activity is present in the brain as identified by bioassay (Ganten et al 1971; Fischer-Ferraro et al., 1971) and immunocytochemical techniques (Fuxe et al., 1980). The presence

PAGE 11

5 of renin mRNA in the brain is still being debated although one group has reported the presence of low levels of renin mRNA in the brains of rats (Dzau et al., 1986). Additionally, ACE has been localized in the brain (Yang and Neff, 1972). Angiotensin II Receptors in the Brain The biological actions of Ang II are elicited at the cellular level. Here, Ang II binds to specific cell surface receptors to affect cellular functions. The integration of the actions of Ang II at the cellular level results in a given biological response. Specific, high affinity binding sites for Ang II were first identified in peripheral tissues using monoiodinated ( [ 125 !]) Ang II ( [ 125 I]Ang II) as the receptor ligand. High levels of [ 125 I]Ang II binding were found in the adrenal cortex (Glossman et al., 1974) and in aortic tissue (Devynk et al., 1974). Bennett and Snyder (1976) first characterized Ang II binding sites in the brain using the brains of calves and rats. [ 125 I]Ile 5 -Ang II was used as the receptor ligand. Here, classical radioligand membrane binding techniques were used to satisfy the basic criteria necessary for the identification of receptors. That is, [ 125 I]Ang II binding was shown to be saturable, reversible, specific, and kinetically consistent. Saturation of the Ang II binding sites was achieved by incubating calf cerebellar cortex membranes with increasing concentrations of [ 125 I]Ang II. The binding saturated at approximately 0.5 nM [ 126 I]Ang II and the specific binding of [ 125 I]Ang II accounted for

PAGE 12

6 80-90% of the total binding. Transfonnation of the saturation data by Scatchard analysis (Scatchard, 1949) revealed a linear plot. This indicates a single, homogeneous class of binding sites. The dissociation constant (~) and nl.llnber of binding sites as determined by Scatchard analysis were 0.2 nM and 1600 fmol/g tissue, respectively. The binding specificity was determined by competing Ang II and Ang II-related compounds with [ 126 I]Ang II for the binding sites. The relative order of potency for the displacement of [ 126 I]Ang II binding was sar1leu 8 -Ang II > (des Asp 1 ) Ile 6 -Ang II > Ile 6 -Ang II >> Ile 6 -Ang I >> 3-8 Ang II hexapeptide > 4-8 Ang II pentapeptide. Thus, Ile 6 -Ang II displaced [ 126 I]Ang II binding with high specificity. As expected, the antagonist sar1 leu 8 -Ang II was slightly more potent than Ile 6 -Ang II, but interestingly (des Asp 1 )Ile 6 -Ang II which is the heptapeptide angiotensin III (Ang III) displaced [u 6 I]Ang II binding to a greater extent than Ile 6 -Ang II. Antagonists generally have a higher receptor affinity than the natural ligand. However, the reason why Ang III was more potent at displacement than Ang II is not clear. Further kinetic analysis was obtained by incubating [u 6 I]Ang II with brain membranes over time. At 37C, the binding was linear for 10 min and reached equilibrium after 30 min. An association constant (k 1 ) of 3.0 X 10 6 g1 s1 was estimated using the linear portion of the binding time course. After the binding reached equilibrium, the dissociation constant (~) of 2. 6 X 10 4 s1 was determined. The

PAGE 13

7 ratio of kvfk 1 gives a value of 0.87 nM and can be used as an estimate of the Kn Additionally, the binding of [u 6 I]Ang II was linear with respect to the membrane tissue concentration and showed pH and ion dependency. The distribution of [mI]Ang II binding sites varied in the brains from calves and rats. In the calf, the binding was highest in the cerebellum with much lower levels in the forebrain, midbrain, cortex and brainstem. In contrast, [ 126 I]Ang II binding sites in the brains of rats were highest in the thalamus, hypothalamus, midbrain and brainstem. The cortex and cerebellum contained only about 25% of the binding levels found in the thalamus and hypothalamus. This initial study confirmed the presence of Ang II receptors in the brain. However, the demonstration of a correlation between the binding kinetics of [mI]Ang II and the time-course of a biological response induced by Ang II was not shown. This is often cited as a criterion for receptor identification, but is often difficult to show especially using membrane binding techniques. This deficiency was partially remedied by localizing high levels of Ang II binding to the hypothalamus and brainstem of the rat. These areas are primary sites of action for centrally administered Ang II. Sirett et al. (1977) further characterized and localized [u 6 I]Ang II binding sites in the brains of rats. A block of tissue containing the hypothalamus, thalamus, septum and midbrain (H-T-S-M) was used to study the binding of [ 126 I]Ang II. The

PAGE 14

8 specific binding of [ 126 I]Ang II accounted for approximately 70% of the total binding. The relative order of potency for the displacement of [ 126 I]Ang II binding was not well established, but it appeared to be Ang II= Ang III~ saralasin >> Ang I>> ACTH >> vasopressin. Interestingly, Ang III was equipotent to Ang II in competing for binding sites. This is similar to the results obtained by Bennett and Snyder (1976). Further, the binding of [u 6 I]Ang II was reversible and saturable. The linear transformation of the saturation analysis gave a~ of 0.9 nM and a of 11 fmoles/mg protein. This is similar to the obtained by Bennett and Snyder (1976) using the cerebellar cortex of calves. The regional distribution of [u 6 I]Ang II binding in the brains of rats had a relative order of septum> thalamus midbrain >hypothalamus> medulla>> cerebrum> cortex> hippocampus. Thus, the binding of [u 6 I]Ang II was concentrated in the H-T-S-M and medullary regions of the brain. Unfortunately, the use of membrane binding techniques to study Ang II receptor localization is limited. This is due to the difficulty in locating and obtaining adequate amounts of tissue from discrete brain areas. However, with the advent of in vitro autoradiographic techniques in combination with computerized densitometry the identification of Ang II receptor populations in discrete brain nuclei became possible. Mendelsohn et al. (1984) first used in vitro autoradiography to localize Ang II receptor populations in the brain. High affinity receptor sites were found

PAGE 15

9 in the subfornical organ (hypothalamus), the OVLT (hypothalamus), the median eminence (hypothalamus) and the AP (brainstem). These sites are all circumventricular organs which lie outside the blood-brain barrier. High concentrations of receptor sites for Ang II located within the blood-brain barrier were found in the paraventricular and periventricular nuclei (hypothalamus), the nucleus tractus solitarius (brainstem), the suprachiasmatic nucleus (hypothalamus), the locus coereleus (brainstem), the subthalamic nucleus (hypothalamus), and the inferior olive nucleus (brainstem). In general, the distribution of Ang II binding sites was shown to be concentrated in specific brain areas, namely the hypothalamus and brainstem, which are associated with the dipsogenic and pressor effects of Ang II. This study did not quantify the binding of Ang II. However, using autoradiography, other studies have provided detailed quantitative analyses of discrete Ang II receptor populations in the brain (Gehlert et al., 1986; Israel et al., 1985; Saavedra et al., 1986). The Ang II receptor affinities and capacities obtained by autoradiography generally agree with those obtained using membrane binding techniques. However, using autoradiography the binding affinity was shown to vary depending of the receptor population studied. For example, Saavedra et al. (1986) reported Ka values of 1. 5 X 10 9 M1 0. 56 X 10 9 M" 1 and O. 36 X 10 9 M" 1 for the subfornical organ, area postrema and nucleus tractus solitarious, respectively. Additionally, the~ values obtained

PAGE 16

10 by autoradiography were in some cases 20-200 times greater than values determined using membrane binding techniques (eg Israel, 1985; Saavedra, 1986). Two possible explanations for this discrepancy are 1) the receptor populations analyzed by autoradiography are more concentrated and are not diluted by tissue which does not contain Ang II receptors or 2) the methodological and analytical differences between membrane binding techniques and in vitro autoradiographic techniques result in different relative values for saturation analyses. The localization of Ang II receptors to specific brain areas that are sites of action for Ang II indirectly proves the existence of functional Ang II receptors in the brain. More direct evidence has come from studies using Ang II receptor antagonists and from electrophysiological studies. According to the occupancy theory, an antagonist binds to a receptor but has zero efficacy. Thus, the antagonist does not elicit a biological response. [Sar1Val 5 Ala 8 JAng II (saralasin) is a competitive antagonist commonly used to antagonize the effects of Ang II. Saralasin has been used to block the dipsogenic response of water deprived rats (Malvin et al., 1977) and in rats administered Ang II i.v.t. (Epstein et al., 1973; Hoffman and Phillips, 1976). Similarly, the presser effect of Ang II given i.v.t. is antagonized by saralasin (Hoffman and Phillips, 1976). The central presser effect of Ang II is produced by both sympathetic nervous system activation and AVP release. The AVP component of

PAGE 17

11 this response was studied using organ explants of the hypothalamus and the posterior pituitary (Sladek and Joynt, 1979). Ang II administration resulted in the release of AVP into the explant growth medium. This effect of Ang II was antagonized by saralasin which indicates that Ang II acts at specific receptors to stimulate the release of AVP. The use of electrophysiological iontophoretic techniques has identified several regions of the brain which contain Ang II sensitive neurons. Areas which exhibit excitatory responses to Ang II that are blocked by Ang II receptor antagonists include the subfornical organ (Phillips and Felix, 1976), the preoptic region (Akaishi et al., 1981; Gronan and York, 1978), the paraventricular and supraoptic nuclei (Akaishi et al., 1980) and the lateral septum (Huwyler and Felix, 1980; Simonnet et al., 1980). These studies correspond favorably to the autoradiographic receptor binding studies. For example, Gehlert et al. (1986) in the most definitive study of Ang II binding sites to date, identified high concentrations of Ang II receptors in the paraventricular nucleus, the lateral septum, the subfornical organ and the supraoptic nucleus. Regulation of the Renin-Angiotensin System in the Brain Past studies have primarily sought to investigate the function and the existence of the brain renin-angiotensin system. Only recently have investigators focused on the regulation of the

PAGE 18

12 brain renin-angiotensin system. Contrary to the peripheral renin angiotensin system, the mechanisms involved in the control of the brain renin-angiotensin system remain obscure. Presently, two loci of regulation have been identified. They are 1) the regulation of the production/expression of angiotensinogen and 2) the regulation of Ang II receptors. Angiotensinogen mRNA is distributed in a variety of tissues including the brain (Dzau et al., 1986; Lynch et al., 1986; Ohkumbo et al., 1986). In the brain, angiotensinogen mRNA expression is increased by treatment with the synthetic glucocorticoid, dexamethasone (Kalinyak and Perlman, 1987), or by the multiple administration of ethynylestradiol, dexamethasone and triiodothyronine (Campbell and Habener, 1986). Regulation of Angiotensin II Receptors Receptor Regulation: Concepts It is well established that various receptor agonists can alter the responsiveness of their effector systems. This results in either a supersensitivity or a subsensitivity of the effector system to the agonist. A major mechanism involved in agonist induced changes in sensitivity is the alteration of receptor number. For example, supersensitivity of skeletal muscle following denervation results, in part, from an increase in the number of postsynaptic nicotinic acetylcholine receptors (Miledi and Potter, 1971). Conversely, the prolonged exposure to fi receptor agonists in a variety of systems leads to a decrease in

PAGE 19

13 ~-receptor number and a decreased ability of ~-receptor agonists to stimulate adenylate cyclase (Kebabian et al., 1975; Mikey et al., 1976; Mukherjee et al., 1975). This type of receptor regulation in which a receptor agonist regulates the receptor system with which it is associated is termed homologous regulation. Mechanisms involved in homologous regulation not only include a change in receptor number (i.e., downregulation), but a change in receptor affinity and/or an alteration of the receptor second messenger coupling. In addition to homologous regulation, receptors can be regulated in a heterologous manner. In this case, agonists act at their specific receptor to alter the function of other receptor systems. The mechanisms involved in heterologous regulation are believed to be similar to those described for homologous regulation. Peripheral Regulation of Angiotensin II Receptors Ang II receptors in the periphery are regulated in both a homologous and heterologous manner depending on the tissue studied. In urinary bladder smooth muscle, the mesenteric artery and the uterus, Ang II infusion decreases the Ang II receptor number (Bmax) with no change in affinity (Aguilera and catt, 1981; Devynk et al., 1976; Schiffrin et al., 1984). However, in the glomerulosa zone of the adrenal gland the intravenous (i.v.) infusion of Ang II increases the~ while either decreasing or not changing the receptor affinity (Hauger et al., 1978; Mendelsohn et al., 1983). Heterologous regulation of Ang II

PAGE 20

14 receptors has been shown using steroid hormones. The infusion of mineralocorticoids, either deoxycorticosterone acetate (DOCA) or aldosterone, into rats increases the number of Ang II binding sites in the mesenteric artery and the uterus with no change in affinity (Douglas and Brown, 1982; Schiffrin et al., 1983, Schiffrin et al., 1984). However, Douglas and Brown (1982) and Douglas (1987) reported a decrease in Ang II receptor number in the adrenal glomerulosa and in glomerular mesangial cells following aldosterone infusion. Alternatively, estrogen treatment has been shown to decrease Ang II receptor number in the uterus and the anterior pituitary of rats (Chen and Printz, 1983; Schirar et al., 1980). Regulation of Angiotensin II Receptors in the Brain In vivo studies of the homologous regulation of Ang II receptors in the brain have produced mixed results. Singh et al. (1984) found the i.v.t. infusion of Ang II (500 ng/l/hr) for six days significantly increased the water intake of rats, but had no effect on the binding of [u 6 I]Ang II in the H-T-S-M or medullary regions of the brain. This experiment was repeated using the Ang II receptor antagonist [Sar 1 Ile 8 ]Ang II. Similarly, this treatment did not alter the binding of [ 126 I)Ang II in the H-T-S-M region of the brain of rats (Singh et al., 1986). Alternatively, Thomas and Sernia (1985) noted that the i.v. infusion of Ang II (25 ng/kg/hr) decreased the number of Ang II receptors in a block of tissue containing the hypothalamus, thalamus and septum (H-T-S),

PAGE 21

15 but increased the number of receptors in the medulla. This differential effect was reported for the receptor affinities in the H-T-S and medulla which were increased and decreased, respectively, by Ang II infusion. Thus, the route of administration of Ang II and the brain area of study may be important variables when investigating the homologous regulation of Ang II receptors. Certainly more studies are necessary, particularly using in vitro autoradiography, to determine whether Ang II receptors in the brain are homologously regulated. More discernable evidence exists for the heterologous regulation of Ang II receptors by steroid hormones. Estradiol benzoate (EB) was shown by both Fregly et al. (1985) and Jonklaas and Buggy (1985) to alter the binding of [u 6 I]Ang II in the brain. Jonklaas and Buggy reported that after two days of treatment, rats given EB (500 gin 0.3 ml of vehicle) subcutaneously (s.c.) had decreased Ang II receptor binding in membrane homogenates prepared from tissue blocks which included the preoptic area, the septum and the thalamus. This effect was associated with a decrease in Ang II-stimulated drinking. Fregly et al. (1985) chronically administered EB peripherally (30-46 g/kg/day for 8 weeks) and found a similar decrease in Ang II binding in a diencephalic block of tissue with a concomitant reduction in the dipsogenic response to Ang II. In contrast to estradiol benzoate, it appears mineralocorticoids act to increase the number of Ang II receptors

PAGE 22

16 in the brain. Wilson et al. (1986) treated rats for 8 weeks with IX>CA (240 g/kg/day, s.c. implants) and found a significant increase in the nmnber of Ang II binding sites in the H-T-S region of the brain. The receptor affinity was unaffected. This increase in receptors likely involved functional Ang II receptors as either the peripheral or central administration of Ang II elicited greater drinking and pressor responses in IX>CA-treated rats. King et al. (1988) further showed that IX>CA (500 g/day, s.c. for four days) significantly increased the specific binding of [u 5 I]Ang II in the area postrerna, superior colliculus, midbrain, olfactory bulb, septumjsubfornical organ and the anterioventral third ventricle (AV3V) regions of the brain of rats. In this case, increased Ang II binding was linked to an increased sodium appetite in IX>CA-treated rats elicited by Ang II. Using autoradiography, Gutkind et al. (1988) have localized the increase in Ang II receptors in IX>CA and IX>CA-salt treated rats to specific brain nuclei. In both IX>CA and OOCA-salt treated animals, Ang II binding was increased in the median preoptic nucleus, the subfornical organ and the paraventricular nucleus. However, only IX>CA-salt animals had additional elevated binding levels in the nucleus tractus solitarious and AP areas of the brainstem. In another autoradiographic study, Wilson et al. (1989) found increased levels of [u 5 I]Ang II binding in the subfornical organ and the medial preoptic area of rats made hypertensive by IX>CA-salt treatment. This is in contrast to both

PAGE 23

17 rats made hypertensive by the clipping of their renal artery (two-kidney, one-clip, and one-kidney, one clip) and salt loaded rats which did not have elevated levels of [ 125 I]Ang II binding in the brain. Thus, from these studies, either DOCA alone or DOCA in combination with certain forms of hypertension acts to increase the number of Ang II receptors in discrete brain areas. Other investigators have studied the role of sodium balance in the regulation of Ang II receptors in the brain. These studies have produced mixed results, undoubtably due to the variety of protocols used and the nonspecific nature of the treatments. For example, Mann et al. (1980) found a 30% reduction in the binding of [u 6 I]Ang II in the H-T-S-M region of the brains of rats fed a low sodium diet for 7 days. This effect was associated with decreased drinking and blood pressure responses to icv-injected Ang II. However, Cole et al. (1980) and Speth et al. (1984) found no effect of low sodium diets on the binding of [u 6 I]Ang II in the H-T-S-M region of the brain of rats. Conversely, Ashida et al. (1982) reported that Ang II receptors in the H-T-S-M region of normotensive (Wistar Kyoto) rats, but not spontaneously hypertensive (SHR) rats, were reduced by a high sodium diet. Thomas and Sernia (1985) found a differential effect on the binding of Ang II in the brain after the administration of a low sodium diet to rats for 21-30 days. The density of Ang II binding sites was decreased in the midbrain, olfactory bulb, and cerebellum and increased in the H-T-S region and the medulla.

PAGE 24

18 Additionally, the Ang II receptor affinity was increased in the cerebellum, midbrain and medulla. In summary, in vivo studies have demonstrated both the homologous and heterologous regulation of Ang II receptors in the brain, although the homologous regulation of Ang II receptors needs more clarification. The heterologous regulation of Ang II receptors in the brain by steroid hormones is more conclusive. In general, estrogen treatment leads to a decrease in the number of Ang II receptors, while the administration of the mineralocorticoid OOCA results in an increase in Ang II receptors. In both cases, changes in Ang II receptor number in discrete brain regions has been associated with a decreased biological responsiveness to Ang II, indicating that functional Ang II receptors are involved. Neuronal cultures: A Model to Study Angiotensin II Receptor Regulation In vivo techniques have contributed greatly to the study of Ang II receptor regulation. However, the efficacy of these techniques is limited when studying the brain. This is mainly due to the inherent complexity of the brain. Ang II receptors are located in small, inaccessible regions of the brain which are undoubtably subject to a variety of neuroendocrine influences. This makes the controlled study of centrally located Ang II receptors in an isolated in vivo system very difficult. This is particularly true for studying the effects of non-steroidal

PAGE 25

19 agents such as peptides and amino acid derivatives on Ang II receptors. These compounds are not lipophilic and have a limited access to discrete brain regions. Recently, the study of Ang II receptor regulation in the brain has been greatly facilitated by the use of cell cultures. In these cultures, neural tissue, dissected from various regions of the brains of neonatal rats, is dissociated into individual cells and plated on tissue cultures dishes. The cells are then grown in a relatively defined medium which can be experimentally manipulated in a quantifiable manner. The growth medium is not completely defined due to the presence of horse serum which contains variable amounts of hormones, growth factors, vitamins, trace elements and other unknown elements. Although the addition of serum to the growth medium adds some variability, it is essential for the long-term viability of the neuronal cultures. Thus, by using neuronal cultures, Ang II receptor regulation can be studied in a partially controlled, easily accessible environment which is unaffected by nonspecific peripheral influences such as stress and locomotion. Further, the intracellular mechanisms involved in Ang II receptor regulation are amenable to study. However, the study of cellular function in an artificial environment is itself a caveat. This means, if possible, in vitro studies should be correlated with in vivo tudies.

PAGE 26

Characterization of the Renin-Angiotensin System in the Brain Using Neuronal cultures 20 As in the brain, all the components of the renin-angiotensin system have been identified in neuronal cultures prepared from the brains of neonatal rats. Neuronal cultures prepared from the hypothalamus and brainstem of neonatal rats contain and synthesize angiotensinogen and angiotensin II (Hennann et al., 1988a; Hennann et al., 1988b) and contain angiotensinogen mRNA (Kumar et al., 1988), while neuronal cultures prepared from the whole brains of neonatal rats have been used to identify the presence of renin (Hermann et al., 1987) and specific Ang II receptors (Raizada et al., 1984). These Ang II binding sites were shown to satisfy the requirements for receptor identification using radioligand binding techniques as described previously. The Bmax and affinity constant obtained by Scatchard analysis were 2.6 X 10 4 binding sites/cell and 1.0 nM, respectively. The~ obtained by taking the ratio of k:i to k 1 was 0.15 nM. Functionally, in neuronal cultures, Ang II has been shown to modulate [3tt]norepinephrine uptake (Sumners et al., 1985; Sumners and Raizada, 1986a), cause an increase in the norepinephrine (NE) content in both neuronal cultures and the growth media (Sumners et al., 1983), and stimulate monoamine oxidase (MAO) activity (Sumners et al., 1987a). All of the effects listed above were blocked by specific Ang II receptor antagonists, indicating the presence of functional Ang II receptors in the neuronal cultures.

PAGE 27

21 Regulation of Angiotensin II Receptors in Neuronal cultures Both catecholamines and steroid honnones regulate Ang II receptors in neuronal cultures. However, the homologous regulation of Ang II receptors does not appear to occur (Sumners and Raizada, unpublished results). Wilson et al. (1986) extended and confinned their in vivo work by demonstrating that DOCA (1.4 nM, 15-20 hrs) and aldosterone (1.35 nM, 15-20 hrs) could each significantly increase the specific binding of [ 125 I]Ang II in neuronal cultures prepared from whole brains. This effect was due to an increase in the~ of the receptor. The~ of DOCA treated cells was 439 fmol/mg protein and represents a 52% increase over the~ of 288 fmol/mg protein in control cells. Sumners and Fregly (1989) studied the specificity of mineralocorticoid regulation of Ang II receptors in cultures prepared from the brainstem and hypothalamus of neonatal rats. Neither testosterone, p-estradiol or the glucocorticoid dexamethasone mimicked the effect of DOCA and aldosterone to increase [ 125 I]Ang II specific binding. Additionally, an increase in [u 5 I]Ang II binding was not detected in neuronal cultures co treated with DOCA and mineralocorticoid type I receptor antagonists (either mespirinone or ZK 97894). This indicates that DOCA acts via a type I mineralocorticoid receptor to stimulate the binding of [ 125 I]Ang II binding. The heterologous regulation of Ang II receptors in culture in not limited to steroid honnones. Initial studies by Sumners

PAGE 28

22 and Raizada (1984) identified an inverse relationship between catecholamines (CA) and Ang II receptors. Treatment of neuronal cultures prepared from whole brains with the tyrosine hydroxylase inhibitor, ~-methyl-tyrosine (~-MT), induced a decrease in neuronal NE and dopamine (DA) contents and an increase in the specific binding of [ 125 I]Ang II. Conversely, pargyline, a MAO inhibitor, increased NE and DA contents in the neuronal cultures and decreased the specific binding of [u 6 I)Ang II. The respective increase and decrease in [u 6 I]Ang II binding with ~-MT and pargyline treatments was attributed to a increased receptor affinity in ~-MT treated cells and a decreased receptor affinity in pargyline treated cells. The~ was unchanged in both cases as determined by Scatchard analysis. Using more direct means to study the effects of CA on Ang II receptor regulation, Sumners et al. (1986b) incubated neuronal cultures prepared from the hypothalamus and brainstem with either NE or DA. These compounds acted to dose-dependently decrease the specific binding of [ 126 I]Ang II. The decrease in [ 126 I]Ang II binding elicited by NE (1 .M, 4 hrs) was blocked by the receptor antagonists, prazosin (10 .M) and phentolamine (10 .M), but not by the '2-receptor antagonist, yohirnbine (10 .M), or by the receptor antagonist, propranolol (10 .M). Saturation analysis determined a 60% decrease in the~ of NE-treated cells as compared to control cells. Conversely, short-term incubations with higher concentrations of NE (5-50 .M, 15-60 min) resulted in

PAGE 29

23 an increase in the specific binding of (u 6 I]Ang II (Myers and Sumners, in press). This effect was blocked by prazosin (1 M), indicating the involvement of specific ~ 1 -adrenergic receptors. Additionally, the increase in (mI]Ang II binding elicited by NE was due to an increase in the receptor 8nwc with little change in the receptor affinity. Thus, the activation of ~-adrenergic receptors, which are present in these neuronal cultures (Feldstein et al., 1986), appears to have a biphasic regulatory effect on Ang II receptors depending on the concentration of agonist used and the duration of agonist incubation. Intracellular Mechanisms Involved in Angiotensin II Receptor Regulation Presently, the mechanisms involved in the regulation of Ang II receptors in the brain remain unclear. Mineralocorticoid mediated upregulation of Ang II receptors appears to involve protein synthesis. Sumners and Fregly (1989) demonstrated that cycloheximide (3.5 Mand 35 M) blocks the increase in the specific binding of [ 126 I]Ang II produced by aldosterone. Presumably mineralocorticoids act by increasing the de novo synthesis of Ang II receptors. The ~ 1 -adrenergic regulation of Ang II receptors has been linked to both the activation of protein kinase C and calcium mobilization. Protein kinase C is a calcium, phospholipid dependent enzyme which affects cellular function by phosphorylating key membrane proteins (Nishizuka, 1986). The

PAGE 30

24 initial step in the activation of protein kinase C involves the receptor mediated activation (in this case the ~ 1 -adrenergic receptor) of phospholipase c. Phospholipase C then hydrolyzes the specific membrane phospholipid phosphatidylinositol-4,5bisphosphate. This results in the generation of diacylglycerol (DAG) and inositol triphosphate (IP 3 ). DAG is the endogenous activator of protein kinase C (Takai et al., 1979b). The involvement of protein kinase c in Ang II receptor regulation was inferred by Sumners et al. (1987b) using phorbol esters. Phorbol esters are compounds which activate protein kinase c by substituting for DAG (castagna et al., 1982). The phorbol ester, phorbol 12-myristate-13-acetate (TPA), was found to increase the specific binding of [u 5 I]Ang II in neuronal cultures. This effect was rapid, occurring by 15 min and reaching a maximal level between 1 and 2 hr. Scatchard analysis revealed the increase in the specific binding of [ 125 I]Ang II was due to an increase in the 8= with little change in the receptor affinity. The involvement of protein kinase C is further implied from the studies by Myers and Sumners (in press). Here, ~ 1 -adrenergic receptor stimulation was found to increase significantly the hydrolysis of phosphatidylinositol. This effect slightly preceded, and was associated with, an increase in the specific binding of [u 5 I]Ang II elicited by the short-term (15-60 min) activation of ~ 1 adrenergic receptors. These studies suggest that the stimulation of ~ 1 -adrenergic receptors leads to the activation of protein

PAGE 31

25 kinase C which acts acutely to increase the expression of Ang II receptors. An alternative mechanism of Ang II receptor regulation in neuronal cultures prepared from neonatal rats appears to involve calcium. Sumners et al. (1988) reported that the calcium ionophore A23187 enhanced the effects of TPA to upregulate Ang II receptors in culture. The ability of A23187 to potentiate the effects of TPA were not observed in calcium-free medium. Further, A23187 alone, at concentrations which significantly increased ~ca 2 + influx, caused a decrease in Ang II receptor number. This effect was negated in calcium-free medium. Thus, Ang II receptor regulation (ie downregulation) may involve calcium flux. However, the question remains whether a more physiologically relevant stimulation of calcium flux such as by depolarization or direct receptor activation can downregulate Ang II receptors. It is tempting to speculate that the long-term effect of NE (2-6 hrs) incubations to downregulate Ang II receptors in culture is the delayed result of ~ 1 -adrenergic stimulation of calcium influx. Preliminary evidence suggests this is the case as CoC1 2 which blocks certain calcium channels, attenuated the effect of long term NE incubations to decrease the specific binding of [ 1 ~I]Ang II in neuronal cultures (Sumners, personal correspondence). This effect could be mediated by the putative ~ 1 a-adrenergic receptor subtype which is linked to calcium mobilization (Han et al., 1987).

PAGE 32

26 A model for protein kinase C activation Woodgett et al. (1988) have proposed a general model for the activation of protein kinase c. Here, protein kinase C exists in a cytosolic state that is closely associated with the plasma membrane. Receptor-mediated activation of phospholipase C results in phosphatidylinositol-4,5-bisphosphate hydrolysis and the generation of DAG and IP 3 DAG binds to protein kinase C and acts to lower the affinity of protein kinase C for ca 2 +. This promotes the binding of protein kinase c to the plasma membrane. At the membrane, protein kinase C forms a ternary complex with Ca 2 +, DAG and specific membrane phospholipids (eg phosphatidylserine) that results in the full activation of protein kinase C. In this scenario the process is enhanced by IP 3 which liberates ca 2 + from intracellular stores. The dissociation of protein kinase C from the membrane and its subsequent inactivation follows the rapid metabolism of DAG by specific lipases and kinases. Phorbol esters as a tool to study protein kinase C Phorbol esters are potent activators of protein kinase C (castagna et al., 1982). Initially isolated from croton oil, which is derived from the plant croteus tiglium, these compounds were first identified as tumor promoters involved in the two stage model of carcinogenesis (Blumberg, 1980). These highly lipophilic compounds were later found to have saturable, high a fi finity cellular binding sites (I)elcos et al., 1980; Drieger and B ] umberg, 1980). At the present, it appears that protein kinase C

PAGE 33

27 is the major intracellular binding site for phorbol esters. This was determined by showing the co-purification of the phorbol ester receptor with protein kinase C using various separation techniques (Ashdell et al., 1983; Kikkawa et al., 1983; leach et al., 1983). Mechanistically, phorbol esters activate protein kinase C by substituting for DAG, the endogenous activator of protein kinase C (castagna et al., 1982). Recently, phorbol esters have been used to implicate protein kinase C in an astonishing array of cellular processes. As reviewed by Woodgett et al. (1988), phorbol esters have a wide variety a cellular effects which include carcinogenic effects, effects on cellular differentiation, the alteration of gene expression, the modulation of ion flux and substrate transport, modulatory secretagogue effects, the modulation of enzyme activities, interactions with other second messenger systems, receptor regulation and cytoskeletal alterations. Thus, by using phorbol ester, a plethora of possible actions of protein kinase C has been discerned. However, the implication of protein kinase C involvement in cellular processes using phorbol esters must be viewed with some skepticism. This statement is based on made for two reasons. First, phorbol esters have been shown to alter the fluidity of the cell membrane (Tran et al., 1983). This may be due to their lipophilic nature which allows them to bind to the cell membrane and could potentially alter various membrane proteins such as receptors, transporters, ion channels and

PAGE 34

28 cytoskeletal components independently of protein kinase C activation. Second, in some instances, the effects of phorbol esters are not mimicked by DAGs or are not blocked by protein kinase C antagonists. For example, the ability of TPA to induce the differentiation of HL-60 leukemia cells was not mimicked by the synthetic DAG, 1-oleoyl-2-acetylglycerol (OAG) (Yamamoto et al., 1985; Kreutter et al., 1985). This occurred in spite of the continued application of OAG to the cells to account for the higher rate of metabolism of this compound (Kreutter et al., 1985). Similarly, OAG does not increase the binding of [ll 5 I]Ang II in neuronal cultures (Kalberg, unpublished results). Further, polymyxin B, an inhibitor of protein kinase C (Mazzei et al., 1982) does not inhibit TPA-stimulated protein phosphorylation and differentiation of HL 60 cells (Kiss et al., 1987). General Hypothesis The data reviewed to this point were provided to show that the brain contains a functional renin-angiotensin system. Further, it was argued that the use of neuronal cultures prepared from the brains of neonatal rats provides an adequate in vitro model with which the renin-angiotensin system can be studied at the cellular level. Presently, both the homologous and the heterologous regulation of Ang II receptors have been studied in culture. The heterologous regulation of Ang II receptors is known to involve both steroid hormones and ~ 1 -adrenergic receptor agonists. The activation of ~ 1 -adrenergic receptors by ~ 1

PAGE 35

29 adrenergic receptor agonists acts both to upregulate and downregulate Ang II receptors depending on the concentration and the duration of incubation of the agonist. TWo mechanisms are proposed for this biphasic effect. The first mechanism identifies a means by which the activation of ~ 1 -adrenergic receptors leads to an increase in the expression of Ang II receptors. Here, the stimulation of ~ 1 -adrenergic receptors results in the activation of protein kinase C which, through a phosphorylation-mediated event, leads to the increased expression of Ang II receptors. Previous studies using neuronal cultures have associated the stimulation of ~ 1 -adrenergic receptors with increases in both phosphatidylinositol hydrolysis and Ang II receptor number. In these studies it is assumed that phosphatidylinositol hydrolysis leads to the activation of protein kinase c. However, the ~ 1 adrenergic receptor-mediated activation of protein kinase Chas not been demonstrated in these cultures. Additionally, the involvement of protein kinase C in the regulation of Ang II receptors has been implicated using phorbol esters. These compounds are known to activate protein kinase c. However, they have a variety of nonspecific effects which may secondarily act to increase Ang II receptors. Clearly additional investigation is needed to confirm the involvement of protein kinase c in the regulation of Ang II receptors. A second mechanism is proposed to explain the downregulation of Ang II receptors by ~ 1 -adrenergic receptors. In this case, the

PAGE 36

30 activation of cx 1 -adrenergic receptors causes an increase in ca 2 + influx. This increase in ca 2 + mobilization acts to decrease the number of Ang II receptors possibly by the activation of a calcium, calmodulin-dependent enzyme. To speculate further, this effect could be mediated by the putative cx 1 a receptor subtype which has been linked to the activation of voltage sensitive calcium channels (VSCC) Thus, the activation of the cx 1 a receptor could mediate the downregulation of Ang II receptors. Past studies used the calcium ionophore A23187 to demonstrate the possible involvement of calcium in the regulation of Ang II receptors. Presently it is not Jmown whether calcium influx through VSCC is involved in the regulation of Ang II receptors in neuronal cultures. Finally, interactions between the two proposed mechanisms may exist. Saitoh and Dobkins (1986) reported that the calcium binding protein, calmodulin, inhibits the phosphorylation by protein kinase C of certain substrates in the brain. This raises the possibility that calcium-calmodulin dependent enzymes/kinases and protein kinase C may interact. Thus, a further degree of control of Ang II receptor regulation may exist if the regulatory pathways involved in Ang II receptor regulation are interconnected. Specific Aims The following specific aims are designed to define further the involvement of protein kinase c and calcium in the regulation

PAGE 37

31 of Ang II receptors in neuronal cultures prepared from neonatal rats. 1) Confirm the involvement of protein kinase C in the regulation of Ang II receptors. These experiments were performed due to the nonspecific nature of phorbol esters. They include the use of the following: a protein kinase c antagonist, non-phorbol ester activators of protein kinase c, and protein kinase C deficient cells. 2) Characterize of protein kinase c activity in neuronal cultures. 3) Qualitatively analyze protein kinase C involvement in the regulation of Ang II receptors. 4) Determine whether protein kinase C is activated by ~ 1 adrenergic receptors in neuronal cultures. 5) Determine the role of calcium flux through VSCC in the regulation of Ang II receptors.

PAGE 38

CHAPl'ER II CHARACTERIZATION OF PROTEIN KINASE C ACTIVITY IN NEURONAL CULTURES Introduction The isolation and characterization of calcium, phospholipid dependent protein kinase (protein kinase C) has occurred at an incredibly fast rate through the use of the latest scientific technology. Protein kinase C was first isolated by Takai et al. (1977) in the cerebellum of cows. They identified protein kinase Casa cyclic nucleotide-independent enzyme which phosphorylated serine residues and was activated by ca 2 +-dependent proteolysis (Inoue et al., 1977; Takai et al., 1977). later, the same group determined that the activation of this enzyme occurred in the presence of ca 2 + and phosphatidylserine and did not involve proteolysis (Takai et al., 1979a). The further discovery that diacylglycerol (DAG) was required for the complete activation of protein kinase C irrevocably linked protein kinase C with the phosphoinositide signalling pathway (Kishimoto et al., 1980; Takai et al., 1979b). Protein kinase C was initially described as a single enzyme which is distributed in a wide variety of tissues and animal phyla and is especially enriched in the brain and lymphoid tissue (Inoue et al., 1977; Kuo et al., 1980). However, hydroxylapatite column chromatography has been used to isolate 32

PAGE 39

33 three isozymes of protein kinase C in the brains of rats, rabbits and monkeys (Huang et al., 1986; Jaken and Kiley, 1987; Yoshida et al., 1987). These isozyme are distinguishable by their sites of autophosphorylation, inununoreactivity, activation by lipids, tissue distribution and susceptibility to proteolysis (Huang et al., 1987a, Huang et al., 1987b, Huang et al., 1988; Huang et al., 1989; Sekiguchi et al., 1987; Sheannan et al., 1989; Yoshida et al., 1988). Three additional isozymes have been identified using complementary DNA probes for protein kinase C (Ono et al., 1988). Further characterization of protein kinase Chas analyzed the unique subcellular distribution of this enzyme and what factors affect its distribution. As discussed in the Chapter I, protein kinase C is thought to exist in a loose association with the plasma membrane (Kraft and Anderson, 1983a; Woodgett et al., 1988). Upon stimulation, this enzyme becomes tightly bound to the membrane and in combination with specific membrane phospholipids it becomes fully activated. Together, ca 2 + and DAG regulate the binding of protein kinase C to the plasma membrane (Wolf et al., 1985a). However, calcium alone has profound effects on the attachment of protein kinase C to cellular membranes (Farrago et al., 1988; Halsey et al., 1987; Kikkawa et al., 1982; Phillips et al., 1989; Wolf et al., 1985a; Wolf et al., 1985b, Yoshida et al., 1988). This ability of calcium to affect the binding of protein kinase C to cell membranes is a unique characteristic of

PAGE 40

34 the enzyme and has both physiological and experimental importance. The following study deals with the characterization of protein kinase C in primary neuronal cultures prepared from neonatal rats. Previously, the binding of [3H]phorbol esters (Raizada et al., 1988) and innnunological techniques (Mudd, 1989) have been used to identify phorbol ester binding sites and innnunoreactive protein kinase c activity in these cultures. However, protein kinase Chas yet to be characterized in these cultures in terms of its activity. In this study, the activity of protein kinase C in neuronal whole brain cultures has been characterized, in part, relative to the prior discussion. The activity of protein kinase C in the neuronal cultures exhibited many of the standard characteristics identified for this enzyme. Further, the subcellular distribution of protein kinase C was found to be dependent on calcium. Methods Preparation of neuronal cultures The neuronal cultures were prepared from the whole brains of one-day-old Sprague-Dawley (SD) rats. The brains were dissected from one-day-old SD rats and placed in an isotonic salt solution containing 100 U penicillin G, 100 g of streptomycin and 0.25 g amphotericin B (fungizone) per milliliter, pH 7.4. The blood vessels and pia mater were then removed from the brain after which the brains were chopped into -2.0 nun chunks. The brain

PAGE 41

35 pieces were suspended in 25 ml of 0.25% trypsin (wt/vol) in isotonic salt solution (pH 7.4), pipetted into a flask, and placed in a shaking water bath for 6 min at 37C to dissociate the cells. After this time, 160 g of deoxyribonuclease I (DNase I) was added to the cells and the flask was replaced in the shaker bath for an additional 6 min at 37C. The dissociated cells were diluted with 100 ml of Dulbeco's modified Eagle's medium (DMEM) containing 10% plasma derived horse serum (PDHS) and were centrifuged at 1,000 X g for 10 min. The resulting pellet was resuspended in 10-15 ml of DMEM containing 10% PDHS and pipetted through sterile gauze into a 500 ml bottle to remove debris. The cells were diluted to 100 ml, counted and further diluted to the desired volume in the same media. The cell recoveries were normally 50-55 X 10 6 cells per brain. The cells suspended in DMEM containing 10% PDHS were plated at a concentration of 18 X 10 6 cells per 9 ml per dish on 100 nun Falcon tissue-culture dishes that were precoated with poly-L-lysine. The cells were incubated for 3 days at 37C in a humidified incubator with 10% C0 2 -90% air. On day three, all the cells were treated with 10 .M cytosine arabinoside (ARC) in 9.0 ml of DMEM-10% PDHS. This treatment inhibits the division of nonneuronal cells and provides cultures that are enriched with neuronal cells. After 2 days, this medium was removed and replaced with 9 ml of fresh DMEM-10% PDHS. The cultures were placed back into a humidified incubator (10% co 2 -90% air) and

PAGE 42

36 were used for experiments after a total of between 12 and 21 days in culture. This length of time is consistent with the occurrence of high levels of protein kinase C. Burgess et al. (1986) reported that a 20-fold induction of protein kinase C occurs in primary neuronal cultures prepared from the brains of embryonic rats during the first week of culture. In the neuronal cultures described here, the cells started to produce projection within 24 hrs of being plated on the tissue cultures dishes. Inununofluorescent examination of the cultures using fluorescent antibodies against the monoclonal neurofilament antibody NE-14 revealed that the cultures contained> 85% neuronal cells (Richards et al., 1989). The remaining cells were nonneuronal as evidenced by innnunofluorescent staining against glial fibrillary acid protein antibody (GFAP). Preparation of cellular fractions To characterize the protein kinase C in the neuronal cultures, the cellular fractions were isolated by one of two different protocols. For the first protocol, 1-3 large 100 nun dishes containing neuronal cultures were washed three times with 2 ml of homogenization buffer A (20 mM Tris HCl, pH 7.5, 2.0 mM EGTA, 0.5 mM EGTA, 0.25 M sucrose, 0.2 mM PMSF and 2.0 g/ml leupeptin), scraped with 1-2 ml of homogenization buffer A and homogenized in a dounce homogenizer (15-20 strokes) at 4C. When more than 1 100 nun dish was used, the cells were pelleted in a Sorvall RT 6000 centrifuge at setting 5 at 4C before

PAGE 43

37 homogenization in 1 ml of homogenization buffer A. In either case, the homogenate was centrifuged at 3000 rpm for 8 min at 4C to remove debris and the supernatant was centrifuged at 20,000 rpm (-43,000 X g) for 45 min at 4C. The soluble fraction was used as the cytosol fraction and the pellet was resuspended in 1 ml of homogenization buffer A contain 0.1 % triton X-100 for 30 min at 4C. The suspension was centrifuged at -43,000 X g for 45 min at 4C and the supernatant was used as the particulate fraction. The second protocol was identical to the first except the low-speed centrifugation (3000 rpm, 8 min) was omitted and only the high-speed centrifugation (-43,000 X g) was used. DEAE column chromatography In some experiments, DEAE column chromatography was used to characterize and partially purify protein kinase c. The preparation and elution of the DEAE columns was performed at 4C. Graduated 0.8 X 4 cm disposable polypropylene columns with a 12 ml volume were packed with 0.6 ml of diethylaminoethyl cellulose (DE 52) which was equilibrated with homogenization buffer B (20 mM Tris HCl, pH 7.5, 2.0 mM Effi'A, 0.5 mM EGTA, 0.2 mM PMSF and 1.0 g/ml leupeptin). The packed columns were washed with 2 ml of homogenization buffer B, the respective cytosolic and particulate samples were applied, and the columns were washed with 2 ml of homogenization buffer B. Protein kinase C was eluted from the columns by either a linear NaCl gradient (0-0.3 M NaCl in buffer

PAGE 44

38 B) or by a single-step elution with 5 ml of homogenization buffer B containing 0.15 M NaCl. Assay of protein kinase C activity Protein kinase C activity was determined by measuring the incorporation of ,~P from [1~P]ATP into lysine rich histone (type III-S) using a slight modification of the method described by Hirota et al. (1985). The final assay mixture (250 l) contained 20 mM Tris HCl, pH 7.5, 5.0 mM MgSO4, 0.5 mM cac12, 50 g histone, 6 g phosphatidylserine (PS), 0.4 g 1,2-diolein, 10 .M [1~P]ATP (500 cpm/pmol) and 1-5 g protein. The lipids were prepared immediately before their use by adding 120 l of PS stock solution (5.0 mg/ml in chloroform stored at -20"C) and 40 l of diolein stock solution (1 mg/ml in chloroform stored at 20"C) to a glass scintillation vial kept on ice (4"C). The lipids were evaporated under nitrogen and were resuspended in 5 ml of 20 mM Tris HCl, pH 7.5, using two 10 second bursts of a ultrasonic homogenizer (Cole Parmer 4710 series) fitted with a 1 / 8 microtip at an output setting of 4. The lipids were added to the reaction mixture just prior the start of the assay which was initiated by the addition of 50 l of enzyme preparation. The samples were assayed in triplicate. After a 3 min incubation at 30C, the reaction was stopped by the addition of 1 ml of 25% trichloroacetic acid (4"C). The precipitates were collected by vacuum filtration onto 0.45 .M cellulose filters and washed 4 times with 2 ml of 5% trichloroacetic acid (4"C). Additionally,

PAGE 45

39 the non-ca 2 +, phospholipid-dependent protein kinase activity of each sample was detennined in duplicate under the same conditions but in the absence of lipids and with 1.0 mM EGTA substituted for CaC1 2 These samples were used as blank values. The filters were placed in plastic scintillation vials and 10 ml of liquiscint was added. Samples were counted on a open window of 0-1000 in a Beckman lS 1801 counter with a counting efficiency of 88% for 1~P. In most cases, protein kinase C activity was expressed as nmol 32 P /min/mg protein. Protein detennination The protein content of the cytosol and particulate fractions was detennined by the method of IDwry et al. (1951). Results Identification of protein kinase C activity The presence of protein kinase C activity in the cytosol fraction from neuronal cultures was detennined by assaying a 50 l (5 g protein) aliquot of the cytosol fraction in the presence or absence of calcium, phosphatidylserine (PS) and the synthetic DAG diolein (Table 2-1). The incorporation of 1~P into lysine rich histone in the presence of either 0.5 mM ca 2 + or 6 g PS was negligible. When 0.5 mM ca 2 + and 6 g PS were added to the reaction mixture the activity increased 987% over the levels found with ca 2 + alone. The further addition of 0.4 g diolein to the reaction mixture resulted in an additional 79% increase in activity.

PAGE 46

Table 2-1 calcium. phospholipid-dependence and diolein stimulation of protein kinase C Assay conditions ca 2 + (o. 5 mM) PS (6 g) ca 2 + + PS ca 2 + + PS + diolein ( o. 4 g) Protein kinase activity (pmol/min/mg protein) 129 + 87 54 + 33 1402 + 62 2511 124 40 Protein kinase C from the cytosol fraction of neuronal cultures was assayed as detailed in the Methods. calcium (ca 2 +), phosphatidylserine (PS) and diolein were added to 5 g of protein in the concentrations indicated. Data are the means SEM of triplicate determinations from a representative experiment which was repeated twice with similar percent changes in protein kinase C activity. Time and protein dependence of protein kinase c activity The activity of protein kinase C under the assay conditions described in the Methods increased in a linear manner between 30 sec and 3 min of incubation (Fig 2-1). The activity plateaued after 10 min of incubation. The dependence of the protein kinase C activity on protein concentration is shown in Fig 2-2. The activity from the cytosol fraction was approximately linear between 1 g and 5 g of protein and began to plateau between 5 g and 20 g of protein. Conversely, the activity of protein kinase C from the particulate fraction was linear between 1 g and 10 g of protein with a flattening of the protein curve occurring between 10 g and 20 g of protein. Both the cytosol and particulate fractions used to

PAGE 47

41 12 10 0 C ., .... 8 e 9/ 0. E 6 / Q. N t") 0 4 E j C 2 0 0 5 10 15 time (min) Figure 2-1. Time-dependence of protein kinase C activity. 5 g of protein from the cytosol fraction isolated from the homogenate of neuronal whole brain cultures was assayed for protein kinase C activity over time. Data are means SEM of triplicate determinations from a representative experiment which was repeated twice with similar percent changes in protein kinase C activity.

PAGE 48

30 25 C 20 e 15 N I') 0 E 0. 10 5 0 0 Figure 2-2. 42 1 5 10 15 20 g PROTEIN Effect of protein concentration on protein kinase C activity. Various protein concentrations from the cytosol (0) or particulate <> fractions were assayed for 3 min as described in the Methods. Data are means SEM of triplicate determinations from a representative experiment which was repeated two times with similar percent changes in protein kinase C activity.

PAGE 49

43 generate the protein curves were prepared from crude homogenates and without the use of DEAE chromatography. Fig 2-2 shows a representative experiment which was repeated twice with similar results. In vitro inhibition of protein kinase C The activity of protein kinase C was further characterized by determining the efficacy of kinase inhibitors to inhibit the enzyme in vitro. Two inhibitors were used, H-7 and HA 1004. H-7 is a potent inhibitor of protein kinase C while HA 1004 is less potent and was used as a control for H-7. H-7 dose-dependently inhibited protein kinase C and had a~ similar to the reported~ value (Table 2-2). HA 1004 was approximately 4 times less potent at inhibiting protein kinase C (Table 2-2). Table 2-2 Effects of H-7 and HA 1004 on protein kinase C in vitro H-7 HA 1004 8 .M 30 .M 6 .M 40 .M Data are from experimentally obtained resultsa or from reported valuesb (Inagaki et al., 1984; Hidaka et al., 1984). The experimental values are means obtained from the compilation of 3 separate experiments for each H-7 and HA 1004 in which the cytosol fraction from neuronal whole brain cultures was used. Effect of Ca 2 + on the distribution of protein kinase C Neuronal cultures were homogenized either in a buffer containing ca 2 + chelators (homogenization buffer B) or a buffer containing 0.1 .M ca 2 + (20 mM Tris HCl, pH 7.5, 0.1 .M cac1 2 0.2

PAGE 50

44 mM PMSF and 2.0 g/ml leupeptin). In cells homogenized with ca 2 + chelators, approximately 70% of the protein kinase C was isolated in the cytosol fraction (Fig 2-3). However, the homogenization of cells with 0.1 .M cacl 2 resulted in a 96% reduction in the activity in the cytosol fraction and a 244% increase in the protein kinase C in the particulate fraction (Fig 2-3). The total activities (ie cytosol + particulate) of cells homogenized with ca 2 + chelators or with ca 2 + were not different and had respective values of 1.29 + .03 and 1.32 .10 nmol ~P/min/mg protein. Anion-exchange chromatographic analysis and purification of protein kinase C The elution profiles of the cytosol and particulate fractions of cell homogenates isolated by high-speed centrifugation (-43,000 X g) following a low-speed (3000 rpm, 8 min) spin are shown in Fig 2-4A. The first major peak of protein kinase C activity from the cytosol fraction eluted between .04 M0.12 M NaCl (Fig 2-4A). A plateau or minor peak of activity eluted at approximately 0.15 M NaCl. The activity in the cytosol fraction accounted for 68% of the total protein kinase c activity. The major peak of activity from the particulate fraction eluted at 0.045 M NaCl. After approximately 0.14 M NaCl, the elution profile of the particulate fraction exhibited a flattening of the slope although a second peak of activity is not apparent.

PAGE 51

45 The elution profiles of the cytosol and particulate fractions of cell homogenates isolated by high-speed centrifugation (-43,000 X g), but without a prior low-speed spin are shown in Fig 2-4B. The major peak of protein kinase C activity in the cytosol fraction eluted at a low ionic strength (0.04 M NaCl). Additionally, a minor peak of activity appears to have eluted at 0.1 M NaCl. The first major peak of protein kinase C activity in the particulate fraction eluted at 0.4 M NaCl (Fig 2-4B). However, a second major peak of activity eluted at 0.14 M NaCl. In this case, when a low-speed spin was not included in the preparation of the cellular fractions, the particulate fraction contained the majority of the total protein kinase C activity (53%). Fig 2-4B is part of an experiment described elsewhere (Chapter IV). It is used here for comparative purposes only. Protein kinase C from crude extracts of homogenates was partially purified using DEAE cellulose chromatography (Table 23). Using a single-step elution procedure, the detergent solubilized particulate fractions were purified to an extent approximately three times greater than the cytosol fraction. Discussion Primary neuronal cultures prepared from the whole brains of neonatal rats were shown to contain high levels of calcium, phospholipid-dependent protein kinase (protein kinase C). Under in vitro assay conditions, the activity of this enzyme was dependent on the concentration of protein used in the assay and

PAGE 52

C u +# e 0. 1.0 0' E C e Q. N 0.5 t") 0 E C Figure 2-3. 46 Effect of Ca 2 + on the subcellular distribution of protein kinase c. Neuronal cultures were homogenized in the presence of Ca 2 +-chelators (A) or with 0.1 .M CaC1 2 (B). The homogenates were spun at 3000 rpm for 8 min at 4C. The supernatant fraction was centrifuged at -43,000 X g for 45 min at 4C to separate the cytosol and particulate fractions. Data are means+ SEM of two separate experiments.

PAGE 53

Ill "' 0 C 20 I A 1 : r i -~ 15 '= V 0 ... Cl. \ 'I I I I \ I \ SYTOSOL / I / I I / PARTIC~L.AE I I I 0 3 0 .3 0. 2 70 B 6J .SQ 4 0 i \ I \ l I : \ + PL and Ca 2 + -PL ond ca 2 + 0.3 CYTCSOL / / / / / C 2 2: I I \ / /~\ 0 1 I I ,o~ Y I I : / ,: \_ I 0' ---..:::...~ PARTIC !J ~TE / / I / 0.2 / 47 6 u 0 z : ~ t ~ I d-0. 1 50 4 0 30 20 1 0 r 0. 1 0 Figure 2-4. 5 10 15 20 0 ~~:::;::::=::::::::: =:: ==::::===:t O 0 5 10 15 F R A C T ION FRA CT IO N Anion-exchange chromatographic analysis of protein kinase C. (A), a homogenate of neuronal cultures was centrifuged at 3,000 rpm for 8 min at 4C. The supernatant was centrifuged at -43,000 X g for 45 min at 4C to isolate the cytosol and particulate fractions. The fractions were eluted with a linear (0-0.3 M) NaCl gradient and assayed in the presence (e) or absence (0) of ca 2 +, phosphatidylserine and diolein. (B), the cytosol and particulate fractions from neuronal cultures were isolated by high-speed centrifugation (-43,000 X g) without performing a prior low-speed centrifugation (3,000 rpm). The fractions were eluted and assayed as in (A).

PAGE 54

48 Table 2-3 Partial purification of protein kinase c by anion exchange chromatography step crude cytosol extract DEAE cytosol fraction volume (ml) 1.0 5.0 crude particulate 1.0 fraction DEAE particulate 5.0 fraction protein (mg) 1.39 0.84 1.08 0.66 specific activity (units/mg protein) 0.73 3.44 0.28 4.24 purification (fold) 4.7 15.3 The activity of protein kinase C from the cytosol and detergent solubilized particulate fractions isolated by high-speed centrifugation (43,000 X g) was assayed before and after elution from DEAE columns with a single 5.0 ml aliquot of homogenization buffer B containing 0.15 M NaCl. Data are means from 2 separate experiments.

PAGE 55

49 was stimulated by the synthetic diacylglycerol diolein. However, the activity of protein kinase C in the cytosol fraction was greater per mg protein than the activity in the particulate fraction. The protein curves were generated using crude homogenates upon which DEAE chromatography was not used. Thus, the lower activity in the particulate fraction may be due to the presence of triton X-100 or a greater abundance of endogenous inhibitors of protein kinase C. This appears to be true as the partial purification of the crude extracts by DEAE chromatography resulted in a purification of the particulate fraction which was approximately three times greater than that of the cytosol fraction. Schwantke and le Peuch (1984) isolated a proteinaceous inhibitory factor(s) of protein kinase C from the supernatant fraction of homogenates of rat brain. This inhibitory factor was resolved by DEAE cellulose chromatography as two peaks which eluted a higher ionic strength than protein kinase C. The two fractions of inhibitor activity were completely inactivated by trypsin proteolysis. Two additional inhibitors of protein kinase c were isolated in the high-speed supernatants of bovine brain. (McDonald and Walsh, 1986). These inhibitors were identified as~ 17, ooo and 12, ooo Ca 2 + -binding proteins. In both of the previous studies, a quantification of the cytosol and particulate distribution of these inhibitors was not performed. Additional non-protein inhibitors of protein kinase C have been identified. These are primarily metabolic derivatives of membrane

PAGE 56

50 sphingolipids such as sphingosine (Hunan et al., 1986) and various lysosphingolipids (Hunan and Bell, 1987). These compounds have~ values of 25-180 .M, act in a reversible manner and have implicated the inhibition of protein kinase Casa mechanisms in the pathogenesis of sphingolipidoses (e.g., Krabbe's and Tay Sachs disease). Thus, these membrane-associated inhibitors may be responsible for the lower activity (on a per mg protein basis) and higher purification found for the crude particulate fraction. Isoquinalinesulfonamides are synthetic competitive inhibitors of cyclic nucleotide-dependent protein kinases and protein kinase c (Hidaka et al., 1984). H-7 is a isoquinalinesulfonamide compound used to inhibit protein kinase C (Hidaka et al., 1984; Hidaka and Hagiwara, 1987). However, because it inhibits c-AMP and c-GMP-dependent protein kinases the actions of H-7 in vivo or in situ cannot be directly attributed to the inhibition of protein kinase c. An alternative isoquinalinesulfonamide derivative is HA 1004 which strongly inhibits c-AMP and c-GMP-dependent protein kinases but has a higher~ for protein kinase C than H-7. Due to their different inhibitory abilities, HA 1004 can be used as a control for H-7. In this study, these compounds were used for characterization purposes by demonstrating the differential inhibition of protein kinase C by H-7 and HA 1004. These compounds inhibited protein kinase C isolated from the cytosol fraction of neuronal cultures at~ values similar to their reported values.

PAGE 57

51 Protein kinase C in the cytosol and particulate fractions eluted from the DE 52-cellulose columns at a low NaCl concentration (-.04 M) which is characteristic for the enzyme (Kikkawa et al., 1982). Two peaks of activity appeared to elute from the columns of both the cytosol and the particulate fractions which were prepared without a low-speed spin (Fig 24B). In the cytosol, the second peak was much smaller than the first peak of activity. However, in the particulate fraction the second peak was equal in height to the first peak of activity. Anion-exchange chromatography is not used to separate the isozymes of protein kinase c. Hydroxylapatite columns are used for this purpose (Huang et al., 1986). However, it is possible that a partial separation of the isozymes was achieved in this study due to charge differences of the isozymes. In other studies using DEAE chromatography, separate minor peaks of protein kinase C activity were shown in the brain (Kikkawa et al., 1982), embryonic carcinoma cells (Kraft and Anderson, 1983b) and NIH 3T3 cells (Uratsuyi et al., 1985). Conversely, separate non-calcium, phospholipid-dependent kinase activity has been shown to elute from DEAE-cellulose columns at higher ionic concentrations than protein kinase C (Hirota et al., 1985; I.ang and Vallotton, 1986). This activity was not found in this study. When the cytosol and particulate fractions were isolated after performing a low-speed spin much less protein kinase c was found in the particulate fraction. From this study it appears

PAGE 58

52 that approximately 20-25% of the total protein kinase C was in the discarded pellet obtained by low-speed centrifugation. This pellet would roughly be the Pl or nuclear pellet which was shown by Kikkawa et al. (1982) to contain 20.5% of the total protein kinase C activity isolated from homogenates of the brains of rats. The inclusion of ca 2 + during the homogenization of the neuronal cultures dramatically increased the amount of protein kinase C in the particulate fraction by 244%. A corresponding 96% decrease in the amount of enzyme occurred in the cytosol fraction. The percent changes are not equal as a smaller amount of protein kinase C was found in the particulate fraction under control conditions due to the inclusion of a low-speed spin in the preparation of the cellular fractions. The total amount of protein kinase C (ie cytosol + particulate) in the cells was unchanged regardless of the homogenization conditions. This indicates that the protein kinase C was not degraded by Ca 2 dependent proteases such as calpain (Kishimoto et al., 1989). This may be due to the inclusion of protease inhibitors in the homogenization buffers. However, in neutrophils, a decrease in the total protein kinase C activity was reported in cells homogenized in the presence of calcium even though protease inhibitors were included in the homogenization buffer (Phillips et al., 1989).

PAGE 59

53 The ability of ca 2 + to affect the subcellular distribution of protein kinase Chas both physiological and experimental implications. Physiologically, agents which act to mobilize intracellular calcium may cause the translocation and activation of protein kinase c independent of the production of DAG. Wolf et al. (1985b) found ca 2 + at concentrations between 100 nM and 10 .M increased the binding of protein kinase C to erythrocyte membranes. These levels of ca 2 + are near steady-state levels and are reached during the stimulation of ca 2 + mobilization. However, DAG activates protein kinase c by lowering the affinity of the enzyme for calcium and phosphatidylserine (Takai et al., 1979b). Thus, the mobilization of calcium and the production of DAG probably work in a synergistic manner to activate protein kinase C. This would be the case during the hydrolysis of phosphoinositides where the mobilization of ca 2 + by IP 3 would act to enhance the actions of DAG. Experimentally, the amount of ca 2 + present during homogenization can have an important bearing on the reported subcellular distribution of protein kinase C. For example, Farrago et al. (1988) found 35% of the total protein kinase C in the particulate fraction when adrenal glomerulosa cells were homogenized in the presence of Ca 2 +-chelators. This percentage increased to 80% when the cells were homogenized with 0.1 mM Ca 2 +. The preparation of cellular membranes for the quantification of the subcellular distribution of protein kinase C is usually

PAGE 60

54 performed in the presence of ca 2 +-chelators. This may act to artificially dissociate protein kinase C from the cell membrane as intracellular ca 2 + concentrations under steady-state conditions may be high enough to bind some protein kinase C to the cell membrane. However, a certain amount of protein kinase C is tightly bound to the membrane and requires detergent solubilization to remove it (Kikkawa et al., 1982; Kraft and Anderson, 1983a). The membrane distribution of this protein kinase c is unaffected by ca 2 +-chelators. Thus, the protein kinase c recovered in the particulate fraction in the presence of ca 2 chelators represents chelator-stable protein kinase C and may not reflect the true subcellular distribution of this enzyme. Perhaps a more precise means of determining the subcellular distribution of protein kinase C is through immunological methods in which intact cells are studied (Kiss et al., 1988). In any case, when the neuronal cultures were homogenized with ca 2 +-chelators and without a low-speed spin, the amount of the total cellular protein kinase C isolated in the cytosol and particulate fractions was 47% and 53%, respectively. Weiss et al. (1989) used a similar protocol to isolate 35% of the total protein kinase C in the particulate fraction of neuronal cultures prepared from the striatum. Kikkawa et al. (1982) recovered approximately 67% of the total protein kinase C activity in the particulate fraction from homogenates of rat brain. This is in contrast to many peripheral cell lines in which negligible amounts of protein

PAGE 61

55 kinase Care detected in the particulate fraction using similar protocols to prepare the cellular fractions. For example, 80-95% of the total protein kinase C is recovered in the cytosol fraction prepared from pinealocytes (Sugden et al., 1985), Madin Darby canine kidney cells (Slivka et al., 1988), adrenal glomerulosa cells (Lang and Vallotton, 1986), neutrophils (Phillips et al., 1989), PC 12 cells (Messing et al., 1989), GH:i pituitary cells (Drust and Martin, 1985) and basophilic leukemia cells (Farrar and Anderson, 1985a). Additionally, Neary et al. (1988) recovered 91% of the total protein kinase C in the cytosol fraction of primary astrocyte cultures. The high amount of membrane-associated protein kinase C in neural tissue from the brain may be due to high levels of the type I isozyme. This isozyme of protein kinase Chas not been found in peripheral tissues and 80-90% of it is recovered in the particulate fraction of homogenates prepared from rat brain in the presence of ca 2 chelators (Yoshida et al., 1988). This is in contrast to the type II and type III isozymes which are located peripherally and are more readily recovered in the cytosol fraction (Yoshida et al., 1988). In sununary, neuronal cultures prepared from the whole brains of neonatal rats contain high levels of protein kinase c activity which exhibited properties characteristic for this enzyme. The subcellular distribution of protein kinase C was affected by Ca 2 + and when the neuronal cultures were homogenized in the presence

PAGE 62

56 of ca 2 + -chelators the majority of this enzyme was located in the particulate fraction.

PAGE 63

CHAPl'ER III IDENTIFICATION AND CHARACTERIZATION OF PROTEIN KINASE C INVOLVEMENT IN ANGIOTENSIN II RECEPIDR EXPRESSION Introduction Prior studies in our lab have investigated the mechanisms of ~ 1 -adrenergic receptor regulation of Ang II receptors. Two lines of evidence from these experiments suggest protein kinase C is involved. First, ~ 1 -adrenergic agonists stimulate the hydrolysis of phosphatidylinositol in neuronal cultures (Gonzales et al., 1987; Myers and Sumners, in press). Phosphatidylinositol hydrolysis results in the generation of diacylglycerol which is an endogenous activator of protein kinase C (Nishizuka, 1986). Second, phorbol esters which are known activators of protein kinase C (castagna et al., 1982) significantly increase the specific binding of [u 6 I]Ang II) in neuronal cultures (Sumners et al., 1987b). Taken together these studies suggest a role for protein kinase C in the regulation of Ang II receptor expression. However, phorbol esters have a wide variety of biological effects in addition to the activation of protein kinase C which may secondarily act to increase the binding of [ 126 I]Ang II. These include tumor promotion, cytoskeletal alterations, cellular differentiation and modulatory secretagogue capabilities (see Chapter I). Presently, the full extent of protein kinase C 57

PAGE 64

58 involvement in producing these effects is not known. Additionally, the synthetic diacylglycerol, 1-oleoyl-2acetylglycerol (OAG), does not mimic the effect of phorbol esters to increase the binding of [u 6 I]Ang II in neuronal cultures (Kalberg, unpublished results). Thus, the first objective of this study was to confirm the involvement of protein kinase C in the regulation of specific Ang II receptors. This was accomplished by determining the effect on the specific binding of [u 6 I]Ang II of protein kinase C activators which are chemically unrelated to phorbol esters, using a protein kinase C antagonist, and depleting the neuronal cultures of protein kinase c activity. Our results suggest that protein kinase c is directly involved in the stimulation of Ang II receptor expression. We next investigated the nature of protein kinase C involvement in the regulation of Ang II receptors by determining if changes in protein kinase C distribution and activity are associated with changes in [u 6 I]Ang II specific binding. Methods Preparation of neuronal cultures Neuronal cultures used for the [u 6 I]Ang II binding studies were prepared from the hypothalamus and brain stem (co-cultures) of one-day-old Sprague-Dawley (SD) rats essentially as described in Chapter II. The rationale for choosing these brain regions is that they contain the major concentrations of [ 1 ~I)Ang II binding sites (Simonnet et al., 1982). For cultures used in [ 126 I]Ang II

PAGE 65

59 binding experiments, cells were suspended in Dulbecco's modified Eagle's medium (EM) containing 10% plasma derived horse serum (PDHS) and plated on 35 nun diameter Falcon tissue culture dishes at a density of 3.0 x 10 6 cells per dish. For the experiments in which the activity of protein kinase C was measured, both co cultures and neuronal cultures prepared from whole brains of one day-old rats were used. For each culture type, the cells were plated on 100 nun diameter dishes at a density of 18 x 10 6 cells per dish. All cultures were grown for 10-14 days in a humidified incubator with 10% COi/90% air, and after this time were used in experiments. Neuronal cultures prepared in this way contained -90% neuronal cells with the remaining cells being predominantly astrocyte glia (Richards et al., 1989). [u 6 I]Ang II binding assay The specific binding of [mI]Ang II was determined using intact neuronal cultures attached to 35 nun diameter dishes. Growth media was removed and the cells were washed twice with phosphate buffered saline (PBS), pH 7.2. For each experimental datum point, total binding was determined by incubating triplicate cultures for 60 min at 24C with 500 l of PBS containing 0.2 nM [u 6 I]Ang II (150,000 cpm) and 0.16% heat inactivated bovine serum albumin (BSA). Nonspecific binding was determined by incubating triplicate cultures under the same conditions except the reaction mixtures contained a 10,000 fold excess of unlabeled Ang II. Following the incubation, the cells

PAGE 66

60 were rinsed twice with ice-cold PBS containing 0.8% BSA. Cells were dissolved with 1.0 ml of 2.0 N NaOH and transferred to plastic tubes. Each plate was rinsed with 500 l of deionized water, which was combined with the original sample. Specific binding was calculated as the mean of triplicate samples obtained by subtracting nonspecific radioactivity bound from the total radioactivity bound and was expressed as fmol of [u 5 I]Ang II/mg protein. Preparation of cellular fractions for protein kinase C analysis Neuronal cultures were washed 3 times with ice-cold PBS, scraped with 1.0 ml of PBS into 50 ml centrifuge tubes and centrifuged at 2500 rpm for 3 min at 4C. The pellets were washed, resuspended in 2.0 ml of homogenization buffer (20 mM Tris HCl, pH 7.5, 20 mM p-mercaptoethanol, 2.0 mM EDrA, 0.5 mM EGTA, O. 2 mM PMSF, and 1. O g/ml leupeptin) homogenized in a Broeck tissue grinder (15-20 strokes) and centrifuged at 3000 X g for 8 min to remove nuclei and debris. The supernatant was centrifuged 100,000 X g for 60 min. The resulting supernatant was used as the cytosol fraction and the pellet was resuspended in homogenization buffer containing 0.1% triton X-100 for 30 min at 4C. This suspension was centrifuged 100,000 X g for 60 min and the supernatant was used as the particulate fraction. Protein kinase C assay The activity of protein kinase c was assayed by measuring the incorporation of ..,S 2 P from [..,S 2 PJATP into lysine rich histone

PAGE 67

61 (type III-S) exactly as described in Chapter II. All samples were assayed in triplicate and the activity of protein kinase C was expressed as nmol ~P/min/mg protein. Desensitization of protein kinase C The desensitization of protein kinase C in neuronal cultures was achieved using long-term treatments with TPA. The method is as follows and describes 24 hr chronic TPA and control vehicle treatments. A group of neuronal cultures was divided into two equal groups. At time zero, one group received control vehicle and the other group received 0.16 .M TPA. Cells were treated under sterile conditions, including the use of sterile vehicle and TPA solutions. After treatment, the cells were kept at 37C in a humidified incubator. Following 24 hrs, the growth media was aspirated off all the cells, which were next washed with EM containing 10% PDHS and then 2.0 mls of the same media was placed on each dish. At this time, half of the control-treated cells (24 hrs) received 0.16 .M TPA and the other half received control vehicle. Likewise, half of the TPA treated cells (24 hrs) received more TPA (0.16 .M) and the other half received control vehicle. All cultures were placed back into the incubator for one hour after which [ 125 I]Ang II specific binding was determined for all the cells. The same procedure was followed for cells treated for 48, 72 and 96 hrs with TPA or control vehicle.

PAGE 68

62 Protein determination The protein content in both the [ 126 I]Ang II binding assay and the assays of protein kinase C was determined by the method of I.Dwry et al. (1951). In the [ 126 I]Ang II binding studies, the cells were rinsed twice with PBS and then dissolved with 500 l of 2.0 N NaOH. The protein was transferred to plastic tubes and each plate was washed with 500 l of deioninzed water which was combined with the original sample and assayed for protein content. Drug incubations The effects of the agonists of protein kinase C teleocidin A, mezerein, phorbol 12,13-dibutyrate (PDB) and phorbol-12myristate-13-acetate (TPA) on [u 6 I)Ang II binding and the activity of protein kinase C were determined by incubating cultures with the drugs at various times and concentrations prior to performing the assays. The antagonist of protein kinase C, H-7, was added to the cultures 30 min prior to the addition of TPA. Control incubations were performed using drug vehicles. All incubations were carried out at 37C in a humidified incubator (10% C0:/90% air). Preparation of drug solutions Teleocidin A, mezerein, PDB and TPA were diluted in dimethylsulfoxide (DMSO) at stock concentrations of 1.0 mM, 1.5 mM, 2.0 mM and 1.0 mM respectively and stored at -20c. Each was diluted in PBS to the desired concentration prior to use. H-7 was

PAGE 69

63 dissolved directly into deionized water at a concentration of 5.0 mM. Further dilutions were made into PBS. Final mso concentrations did not exceed 0.1%. statistical analyses All results are expressed as means SEM. Comparisons of multiple means were made by one-way analysis of variance (ANOVA1) followed by a Newman-Keuls test to assess statistical differences between individual means when applicable. A difference at the 1% level was accepted as statistically significant. Statistical analyses of the data were performed with an Apple IIe computer using a ED-SCI statistical (Interactive microware) program. Results Effects of teleocidin A and mezerein on [ 126 I]Ang II specific binding in neuronal cultures The first approach to determine whether the specific activation of protein kinase C is responsible for an increase in the specific binding of [u 6 I]Ang II involved testing two agonists of protein kinase c, teleocidin A and mezerein, which are chemically unrelated to phorbol esters. Fig 3-1 is a compilation of separate representative time course experiments for each agonist of protein kinase C with PDB included as a positive control. Mezerein (0.5 M), teleocidin A (0.76 M), and PDB (0.99 M) at concentrations which stimulate maximal increases in the specific binding of [ 126 I]Ang II all rapidly increased the

PAGE 70

64 specific binding of [ 126 I]Ang II. At the earliest time points tested, 15 min for teleocidin A and mezerein and 30 min for PDB, all of the agonists significantly elevated the specific binding of [ 126 I]Ang II. In all cases, the stimulation of [ 126 I]Ang II binding reached a maximal level after 1 hr of incubation. In neuronal cultures treated with PDB and mezerein, the levels of [u 6 I]Ang II specific binding returned to control levels after 24 hrs. However, in cells receiving teleocidin A, [ 126 I]Ang II specific binding was still significantly higher than control levels after 24 hrs (data not shown). Time course experiments were repeated four to five times for each compound tested and showed similar percent changes in [u 6 I]Ang II specific binding. Representative dose response curves are shown in Fig 3-2 and each of these was repeated at least five times with similar percent changes in binding. Teleocidin A was found to be the most effective at increasing the specific binding of [ 126 I]Ang II with an EDso of 32 nM, while PDB and mezerein had EDso values of 63 and 79 nM respectively. In prior studies, TPA was found to have an EDso of 5 nM with respect to its ability to increase [ 126 I]Ang II specific binding (Sumners et al., 1987). None of the compounds, tested at the highest concentrations used, were cytotoxic as determined by trypan blue exclusion or significantly altered the protein content of the cultures as compared to controls (Table 31)

PAGE 71

"" C .; 40 e Q. c,, E 30 0 E It....., "'0 C 20 :::, 0 .0 c,, C 10 <( I ,--, U") N L-J 0 Figure 3-1. 0 65 30 60 90 1 20 Time (min) Effects of protein kinase C agonists on [ 125 I]Ang II binding to neuronal cultures as a function of time. Neuronal cultures were incubated with teleocidin A (0. 5 .M; CJ)., mezerein (0. 76 .M; >, or PDB (0.99 .M; 0), in DMEM containing 10% PDHS at 37C. The data presented here are a compilation of representative experiments performed for each agonist of protein kinase C and numbers are means SEM of triplicate determinations. Individual time course experiments for each agonist were repeated four to five times with similar results. ANOVA 1, p < 0.01 for each treatment. In the treated cells, the specific [ 125 I]Ang II binding at each time point was significantly different from controls. See text for description.

PAGE 72

Figure 3-2. "O C: :::, 0 .c CJ'\ C: <: 35 30 25 20 15 I 10 .--, l[) N 66 9 8 7 6 5 [PKC agonist] -log M Binding of [ 125 I]Ang II to neuronal cultures as a function of protein kinase C agonist concentration. Neuronal cultures were incubated with the indicated concentrations of teleocidin A (D), mezerein (e), or PDB (0) in DMEM containing 10% PDHS for 1 hr at 37c in a humidified incubator ( 10% C0:/90% air) The data are from representative experiments and the numbers are means SEM of triplicate determinations. Control specific binding values are plotted on they axis. All experiments were repeated a minimum of five times for each compound with similar changes in [ 125 I]Ang II specifically bound at each agonist dose. ANOVA 1, p < 0.01 for each dose-response curve. The comparisons of control-teleocidin A, control-mezerein and control-PDE were significantly different at each dose at the 1% level, except for 1 nM teleocidin A, 10 nM teleocidin A and 1.56 nM mezerein. See text for description.

PAGE 73

Effects of H-7 on TPA stimulated increases in [ 126 I]Ang II binding. 67 In the next series of experiments, the effects of the inhibitor of protein kinase C H-7 (Hidaka, 1984; Hidaka and Hagiwara, 1987) on TPA-stimulated increases in the specific binding of [ 126 I]Ang II were analyzed. Preincubation of neuronal cultures for 30 min with H-7, followed by TPA treatment (0.8 .M, 1 hr) resulted in a dose-dependent inhibition of TPA-stimulated increases in [ 126 I]Ang II specific binding. A representative experiment is shown in Fig 3-3. TPA (0.8 .M, 1 hr) alone increased binding by 154%. Co-incubation of the cultures with TPA and H-7 (100-300 .M) resulted in levels of [u 5 I]Ang II binding which were not significantly different from those seen in controls. At 50 and 75 .M, H-7 significantly reduced the TPA stimulated increases in [u 5 I]Ang II specific binding, though levels were still elevated as compared to control values. H-7 (300 .M, 30 min) alone did not significantly alter the specific binding of [u 5 I]Ang II as compared to control values and was not cytotoxic as determined by protein content (Table 3-1) and trypan blue exclusion. Effects of phorbol esters on [u 5 I]Ang II specific binding in neuronal cultures depleted of protein kinase C activity. In the neuronal cultures used in this study and in other cell cultures systems, prolonged phorbol ester treatment (24 hrs) results in a depletion of total cellular protein kinase C activity (Matthies et al., 1987; Neary et al., 1988). This

PAGE 74

68 strategy was used here to determine if TPA can stimulate the specific binding of [u 6 I]Ang II in neurons depleted of protein kinase c. Fig 3-4A shows 24 hr and 48 hr pretreatment groups. The addition of TPA (0.16 .M, 1 hr) to neuronal cultures that had received a 24 hr TPA pretreatment (D), as detailed in the Methods, failed to significantly increase the specific binding of [mI]Ang II as compared to control cells (A) or cells that only received a TPA pretreatment (C). TPA (0.16 .M) significantly increased [u 6 I]Ang II binding in neuronal cultures that were not pretreated with TPA (B). Similar results occurred in the 48 hr groups. TABLE 3-1 Effects of protein kinase c activators and H-7 on neuronal culture protein contents. Treatment Protein content (mg) Control (vehicle) .46 + .02 TPA (0.8 .M) .45 + .02 PDB (1.9 .M) .46 + .01 Mezerein (2.3 .M) .44 + .01 Teleocidin A (1.5 .M) .46 + .02 H-7 (300 .M) .45 + .01 Neuronal cultures were incubated with the activators of protein kinase c for 1 hr or with H-7 for 30 min at 37C in a humidified incubator. cultures were washed with PBS and their protein contents were determined as described in the Methods. Protein contents of treated cells were not significantly different from controls. Data are means SEM from six 35 nnn culture dishes.

PAGE 75

69 Desensitization of the neuronal cultures to TPA-stimulated increases in the specific binding of [ 125 I]Ang II was not irreversible. The addition of TPA to neuronal cultures pretreated for 72 and 96 hrs with TPA (0.16 M) significantly increased specific [ 125 IJAng II binding levels as compared to control levels (Fig 3-4B). At 72 hrs, TPA (1 hr) increased [ 125 I]Ang II binding levels in TPA pretreated cells by 43% above control binding levels. After 96 hrs of TPA pretreatment, [ 125 I]Ang II specific binding was increased 44% over control levels by TPA. Both Fig 34A and 3-4B show representative experiments, and each was repeated 4 to 5 times with similar results. Phorbol esters induce cell growth, cell differentiation and mitogenesis in a variety of cell types (Woodgett et al., 1988; Blumberg, 1980). Thus, it is possible that the stimulation of cell growth in the neuronal cultures by TPA may affect the binding levels of [ 126 I]Ang II on a per mg protein basis. This was tested by determining whether chronic incubations of TPA alter the protein content of the neuronal cultures. As shown in table 3-2, the incubation of neuronal co-cultures with 0.16 M TPA for 24, 48, 72, of 96 hrs did not significantly effect the protein content of the cultures as compared to control vehicle-treated cells.

PAGE 76

...... 25 C I) e a. 0\ 20 E ,, ., 0 15 E ... .,, C :::, 10 0 ..0 0\ cl 5 I ,.., in N 0 '--' Figure 3-3. 70 A B C D E F G H Effects of H-7 on TPA stimulated increases in [u 5 I]Ang II specific binding. Neuronal cultures were incubated with (A) vehicle (0.1% DMSO in PBS) or (B) TPA (0.8 M) for 60 min at 37C. Other groups received a 60 min TPA treatment following a 30 min preincubation with (C) 10 M H-7, (D) 50 M H-7, (E) 75 M H-7, (F) 100 M H7, (G) 150 .M H-7 or (H) 300 .M H-7. The final group (I) received 300 M H-7 only. Following these incubations, [ 125 I]Ang II binding was measured as detailed in the Methcxls. The data presented here are from a representative experiment and numbers are means SEM of triplicate determinations. This experiment was repeated five times with the same dose-response relationships observed each time. ANOVA 1, p < 0.01. Significantly different from TPA treatment.

PAGE 77

Figure 3-4. 'c 25 ii e o.. 20 a, 0 E 15 ... ...... "O C g 10 .J = 'c 50 e Q. 40 a, 0 E 30 ... "O C g 20 .J = O'I 10 I ,...., ,if N '-' 0 A B ..L A B C D 24 hrt ,.&. A 8 C D 72 hrs ., ... A B C D 48 hra A B C D 96 hrs 71 (Panel A). The effects of 24 hr and 48 hr phorbol ester pretreatments on TPA stimulated increases in the specific binding of [ 125 I]Ang II. Neuronal cultures received a pretreatment with vehicle (0.02% mso in PBS) or TPA (0.16 M) for 24 or 48 hrs. Following this, vehicle treated groups received an additional 1 hr treatment of (A) vehicle or (B) 0.16 M TPA and groups pretreated with TPA received a 1 hr treatment of (Cl vehicle or (D) 0.16 M TPA after which [ 12 I]Ang II specific binding was determined in all groups. These data are from a representative experiment and numbers are means SEM of triplicate determinations. ANOVA 1, p < 0.01. Significantly different from controls (A). (Panel B). Effects of 72 and 96 hr phorbol ester pretreatments on TPA stimulated increases in [u 5 I]Ang II specific binding. Cells were grouped as described in panel A. ANOVA p < 0.01. Significantly different from controls (A). ** Significantly different from cells receiving TPA pretreatment followed by a 60 min vehicle treatment (C). Experiments in figs 4A and 4B were repeated four times each with similar results obtained each time.

PAGE 78

Effect of protein kinase C activators on the distribution and activity of protein kinase C in neuronal cultures. 72 Neuronal cultures prepared from whole brains were incubated with either mezerein (0.76 M), teleocidin A (0.5 M) or TPA (0.8 M) for 15 min, 60 min, and 24 hrs. All three compounds caused the translocation of protein kinase C from the cytosol to the particulate fraction after 15 min of incubation. Thereafter, the activity of protein kinase c was gradually decreased below control levels (Fig 3-5, Fig 3-6, and Fig 3-7). In the three experiments shown (Figs 3-5, 3-6, 3-7), the activity of protein kinase c in the cytosol and particulate fractions of control cells accounted for an average of 75% and 25%, respectively, of the total activity of protein kinase C. Following 15 min of incubation with mezerein, teleocidin A or TPA, the activity of protein kinase c in the cytosol fraction accounted for only 15%, 33%, and 15%, respectively, of the total protein kinase C activity, while the particulate fractions showed a corresponding increase in total protein kinase C activity. The total activity of protein kinase C (i.e., cytosol activity plus particulate activity) in cells treated with mezerein, teleocidin A or TPA for 24 hrs was reduced to 8%, 25%, and 3%, respectively, of the total activity of protein kinase C found in drug-vehicle treated control cells. In the next series of experiments, the effect of long-term TPA treatments on the activity of protein kinase C in neuronal

PAGE 79

6 C 5 u e ci c,, 4 E .E 3 E Q. N I"') 2 0 E C: 1 0 0 Figure 3-5. .25 73 1 24 time (hrs) The effect of mezerein (0.76 .M) on the distribution of protein kinase C activity in neuronal cultures. The cells were incubated with mezerein for 15 min, 60 min or 24 hrs at 37C. cytosol (0) and particulate (e) fractions were prepared from the cultures and the protein kinase c activity was assayed as described in the Methods. The data presented here are means SEM of triplicate determinations from a representative experiment which was replicated 3 times with similar percent changes in the activity of protein kinase c.

PAGE 80

4 C .i ..., 3 e 0. "' E C 2 E c.. N n 0 1 E al C: 0 Figure 3-6. I .25 74 I 1 24 time (h r s) The effect of teleocidin A (0.5 M) on the distribution of protein kinase C activity in neuronal cultures. The cells were incubated with teleocidin A for 15 min, 60 min or 24 hrs at 37C. cytosol (0) and particulate <> fractions were prepared from the cultures and the protein kinase C activity was assayed as described in the Methods. The data presented here are means SEM of triplicate determinations from a representative experiment which was replicated 3 times with similar percent changes in the activity of protein kinase C.

PAGE 81

4 C 'ii +' .3 e a. c:,I E C 2 E a.. N n 0 1 E C 0 0 Figure 3-7. .25 75 1 24 time (hrs) The effect of TPA (0.8 M) on the distribution of protein kinase C activity in neuronal cultures. The cells were incubated with TPA for 15 min, 60 min or 24 hrs at 37C. cytosol (0) and particulate <> fractions were prepared from the cultures and the protein kinase C activity was assayed as described in the Methods. The data presented here are means SEM of triplicate determinations from a representative experiment which was repeated 4 times with similar percent changes in the activity of protein kinase C.

PAGE 82

76 cultures was examined. After 24 hrs of TPA (0.16 .M) treatment, the total activity of protein kinase C (cytosol + particulate fractions) was significantly reduced as compared to controls. This is clearly seen in Fig 3-8 which illustrates the total activity of protein kinase c in TPA-treated cells as a percentage of the activity in control cells. However, following 72 and 96 hrs of TPA treatment, the total activity of protein kinase Chad partially returned to 31 11% and 53 3% of the total control activity (Fig 3-8). The amount of protein kinase C in the cytosol and particulate fractions used to calculate the total activity (i.e., Fig 3-8) is shown in Fig 3-9. After 24, 72, and 96 hrs, the protein kinase c activities in the cytosol fractions of TPA treated cells were 8%, 26%, and 48%, respectively, of the activity found in the control cytosol fractions. In the particulate fractions of cells treated with TPA for 24, 48, and 72 hrs, the amount of protein kinase C was 20%, 42%, and 57%, respectively, of the enzyme levels found in the control particulate fractions. The treatment of neuronal co-cultures prepared from the hypothalamus and brainstem with TPA (0.8.M) caused similar percent changes in the activity of protein kinase C. In control cells, the activity of protein kinase c in the cytosol accounted for 85% of the total protein kinase c activity. Incubation with TPA for 15 min reduced the activity of protein kinase C in the cytosol fraction to 15% of the total protein kinase C activity

PAGE 83

77 with a corresponding increase in the activity of protein kinase C in the particulate fraction. In co-cultures treated with TPA for 24 hrs, the total activity of protein kinase C was reduced to 6% of the total protein kinase C activity found in vehicle treated cells (data not shown). Table 3-2 Effect of chronic TPA incubations on the protein content of neuronal co-cultures Treatment Protein Content (mg) Control TPA 24 hrs 48 hrs 72 hrs 96 hrs .41 -01(7) .49 -01(6) .44 -02(6) .48.02(8) .38 -02(7) .50 -01(6) .46 -03(6) .49.02(8) Neuronal cultures were incubated with TPA (0.16 M) for the indicated times. The cultures were washed twice with PBS and their protein contents were assayed as described in the Methods. Data are the means SEM from values obtained from individual dishes. The number of dishes assayed is indicated in parenthesis. Discussion This study has clearly demonstrated the specific and integral involvement of protein kinase C in the regulation of Ang II receptor expression in neuronal cultures. Two activators of protein kinase C, the diterpine ester mezerein and the indole alkaloid teleocidin A which are chemically unrelated to phorbol esters, both increased the specific binding of [ 126 I]Ang II in a dose and time dependent manner. These effects were similar to those obtained with the phorbol ester PDB. All the compounds tested increased [u 6 I]Ang II specific binding within 30 min and

PAGE 84

100 80 e +' C 0 u 60 ....... s: 40 +' u 0 u 20 0 Figure 3-8. T 24 72 (hrs) 78 T l 96 The effect of long-term treatment with TPA on protein kinase C activity in neuronal cultures. Neuronal cultures were treated with TPA (0.16 M) for 24 hrs, 72 hrs or 96 hrs, and the activity of protein kinase C was assayed in the cytosol and particulate fractions. The results represent total protein kinase C activity (obtained by adding cytosol and particulate activities) in TPA treated cells, expressed as a percentage of the total protein kinase C activity found in control cells. Data are means SEM from two separate experiments.

PAGE 85

79 3 Dcytosol particulate C: I) +' e 0. 2 C1\ E C: E 0.. N 1 n 0 E C 0 ..,__ ___._~......,..__ ............ .a..ll~--._ ........ ~..._-....L..---J.~~A Figure 3-9. 8 C D The effect of long-term TPA treatment on the protein kinase activity in the cytosol and particulate fractions. (A) control vehicle treated, (B) 24 hr TPA (0.16 M treatment, (C) 72 hr TPA (0.16 .M) treatment, (D), 96 hr TPA (0.16 .M) treatment. Data are means SEM from two separate experiments.

PAGE 86

80 exerted a maximal effect by 1 hr. With mezerein and PDB, [ 126 I]Ang II specific binding returned to control levels by 24 hrs. However, with teleocidin A, binding was still elevated after 24 hrs. This suggests teleocidin A is a longer acting substance or utilizes other mechanisms in the stimulation of Ang II receptor binding. This was supported by the fact that teleocidin A did not downregulate total activity of protein kinase C to the same extent as mezerein and TPA (Fig 3-6). The EDso values for stimulation of [u 6 I]Ang II specific binding by mezerein, teleocidin A and PDB generally agreed with their reported EDso values for activation of protein kinase C (Couturier et al., 1984; Kraft et al., 1982). For example, our EDso of 32 nM for teleocidin A is similar to an EDso of 18 nM obtained using purified protein kinase C (Arcoleo and Weinstein, 1985). Although mezerein and teleocidin A are chemically and structurally unrelated to phorbol esters, the hydrophilic and hydrophobic moieties of these compounds are arranged in a similar spatial manner (Jeffery and Liskamp, 1986). This apparently accounts for their similar functionalities even though they are chemically unrelated. The isoquinalinesulfonamide inhibitor of protein kinase CH7 has been useful in elucidating the role of protein kinase C in various biological functions (Hidaka and Hagiwara, 1987). H-7 is a direct inhibitor of protein kinase C which acts in a competitive manner with respect to ATP (Hidaka et al., 1984). In

PAGE 87

81 this study, we showed that 50 .M H-7 significantly lowered (p < 0.01) TPA-stimulated increases in [u 6 I]Ang II specific binding in neuronal cultures. At 100 .M, 150 .Mand 300 .M, H-7 completely inhibited TPA-stimulated increases in [mI]Ang II specific binding. The concentrations of H-7 used were several fold higher than concentrations used to inhibit purified protein kinase C in vitro(~= 6 .M) (Hidaka et al., 1984) and in our own assay of protein kinase C in which H-7 had a~ of 8 .M (see Chapter II). However, the concentrations used are comparable to those used in other intact cell preparations (Inagaki et al.,1984; Pandol and Schoefield, 1986; Turner et al., 1987). Higher concentrations of H-7 are necessary in intact cell studies probably due to the lipid insolubility of this compound (Hidaka et al., 1984), so restricting its entry into cells. Additionally, the intracellular concentration of ATP may alter the effectiveness of H-7 due to the competitive interaction between H-7 and ATP. For example, if the intracellular concentration of ATP in the neuronal cultures is in the millimolar range as it is in muscle cells (McGilvery, 1983) a relatively high concentration of H-7 would be needed to displace the binding of ATP to the ATP-binding site on the catalytic domain of protein kinase c. The specific and direct involvement of protein kinase C in the increased expression of Ang II receptors was further suggested by depleting our neuronal cultures of protein kinase c activity. This resulted in a complete abolition of TPA-stimulated

PAGE 88

82 increases in the specific binding of [ 125 I]Ang II. Prolonged treatment of the cultures for 24 and 48 hrs with the phorbol ester TPA (0.16 .M) completely blocked TPA-stimulated increases in the specific binding of [ 125 I]Ang II. Using PC-12 cells, Matthies et al. (1987) reported a depletion of protein kinase C activity in both the cytosol and particulate fractions after 24, 48 and 67 hr of incubation with 1 .M TPA. This treatment did not effect cyclic AMP-dependent and ca 2 +/calmodulin dependent protein kinase activities. OUr [u 5 I]Ang II binding results indicate that after 24 and 48 hrs of TPA treatment the activity of protein kinase C was depleted. However, 72 and 96 hr TPA pretreatments did not abolish TPA-stimulated increases in [u 5 I]Ang II specific binding. In addition to activating protein kinase C, phorbol esters are potent tumor promoters. Concomitant with their tumor promoting ability are the actions of phorbol esters to induce cellular differentiation and mitosis and to promote cell growth (Woodgett et al., 1988; Blumberg, 1980). However, chronic incubation of the neuronal cultures for 24, 48, 72 and 96 hrs did not alter the morphology of the cells and was not cytotoxic. Additionally, chronic incubations with TPA did not affect the protein content of the cultures. Thus, the levels of [u 5 I]Ang II binding expressed on a per mg protein basis were not altered due to changes in the protein content of the cultures. The stimulation of the translocation of protein kinase C from the cytosol to the cell membrane by phorbol esters is

PAGE 89

83 followed by the gradual degradation of protein kinase Cat the cell membrane and this degradation is enhanced by phorbol esters (Ballester and Rosen, 1985; Young et al., 1987). Thus, it is likely that the return of the stimulatory effects of TPA on [ 126 I]Ang II binding by 72 hours represents synthesis of new protein kinase c. This was confirmed by the our studies which showed a partial return of the total protein kinase C activity after 72 and 96 hrs of TPA treatment of approximately 30% and 50%, respectively. The partial return of the total protein kinase C activity was due to an increase in activity in both the cytosol and particulate fractions (Fig 3-9), although the activity in neither of the fractions increased to control levels. Presumably, the activity found in the cytosol represents "translocatable" protein kinase Candis more important in the regulation of Ang receptors. However, one cannot rule out the possibility that a membrane bound form of protein kinase C may be involved in the regulation of Ang II receptors by an unknown mechanism (for a discussion of protein kinase C isozymes and their subcellular distribution see (Chapter II). Further, it is apparent that a complete return of protein kinase C activity is not required for TPA to stimulate increases in the specific binding of [u 6 I]Ang II. Thus, in the unstimulated-control state, spare protein kinase C exists with respect to the regulation of Ang II receptors. Additionally, only one isozyme of protein kinase C may be involved in the regulation of Ang II receptors in cultures and

PAGE 90

84 the return of protein kinase C activity after 72 and 96 hrs represents that single isozyme. Since the protein kinase C assay used in this study cannot distinguish the isozymes of protein kinase c, further studies are necessary to identify the isozymes present in the neuronal cultures and what roles they may have in the regulation of Ang II receptors. Recently, Mochly-Rosen et al. (1987) raised several monoclonal antibodies against protein kinase c, one of which inhibited protein kinase c. Thus the possibility exists that antibodies with inhibitory actions against specific protein kinase C isozymes can be isolated. These would be powerful tools for deciphering the individual functions of the protein kinase C isozymes. Finally, the complete downregulation of protein kinase C by treatment of the neuronal cultures with TPA for 24 hrs did not alter steady-state levels of the specific binding of [u 5 I]Ang II. This suggesting that protein kinase C is not involved in the steady-state expression of Ang II-specific receptors. The data presented here further indicate that translocation of protein kinase C from the cytosol to the cell membrane is a prerequisite for an increase in Ang II receptor expression. The translocation of protein kinase C produced by mezerein, teleocidin A, and TPA appeared to be complete by 15 min while the increases in [u 5 I]Ang II binding reached maximal levels by 1 hr. Presumably, the translocation of protein kinase C from the cytosol to cellular membranes allows for complete activation of

PAGE 91

85 protein kinase c and is required for an increased expression of Ang II receptors. Phorbol esters act to increase the specific binding of [ 1 ~I]Ang II in neuronal cultures by primarily increasing the Brnax (Sumners et al., 1987b). The above studies suggest that protein kinase C is directly involved in this action. There are several mechanisms by which the activation of protein kinase C may lead to an increase in the specific binding of [mI]Ang II. These include phosphorylation of a membrane protein which leads to the unmasking of membranous Ang II receptors, the direct phosphorylation of refractory Ang II receptors which may induce a conformational change that permits ligand binding or phosphorylation by protein kinase C of an cellular component involved in the translocation of Ang II receptors to the membrane surface. The latter mechanisms would be analogous to an increase in the expression of transferrin receptors in Chinese hamster ovary (CHO) fibroblasts by TPA which is due to an increased exocytosis of transferrin receptors without a corresponding change in their endocytotic rate (McGraw et al., 1988). Protein kinase C is an intracellular second-messenger and presumably mediates the regulatory actions of another hormone(s) or neurotransmitter(s) on Ang II receptors. Possibilities include acetylcholine, NE and bradykinin, all of which stimulate the hydrolysis of phosphatidylinositol in the brain (Batty et al., 1985; Gonzales et al., 1987; Myers and Sumners in press; Yano et

PAGE 92

86 al., 1987) which putatively leads to the generation of diacylglycerol, an endogenous activator of protein kinase C (Nishizuka, 1986). Recently we have shown that acute treatment of neuronal cultures with NE (30-60 mins) at concentrations which cause large increases in phosphatidylinositol hydrolysis increases the specific binding of [u 6 I]Ang II via ~ 1 -adrenergic receptors (Myers and Sumners in press). This is in contrast to past experiments in which NE at longer incubations (2-6 hrs) and at concentrations which caused small or no increases in phosphatidylinositol hydrolysis was shown to decrease the specific binding of [ 126 I]Ang II (Sumners et al., 1986b). Therefore, at least acutely, protein kinase C may mediate NE stimulated increases in the specific binding of [u 6 I]Ang II. This study has shown that protein kinase C plays a specific and integral role in the regulation of Ang II receptor expression in neuronal cultures prepared from the brains of one-day-old rats and suggests that receptor systems which activate protein kinase C, presumably by translocation from the cytosol to the cellular membranes, may act to regulate Ang II receptor expression.

PAGE 93

CHAPl'ER IV ADRENERGIC REGUIATION OF PROTEIN KINASE C SUBCELI.lJIAR DISTRIBUTION Introduction The activation of ~ 1 -adrenergic receptors mediates the hydrolysis of phosphoinositides in a variety of tissues. In the central ne:rvous system, the stimulation of ~ 1 -adrenergic receptors elicits the hydrolysis of phosphoinositides in brain slices (Kendall et al., 1985; Gonzales and Crews, 1985; Minneman and Johnson, 1984) and primary neuronal cultures (Gonzales et al., 1985; Gonzales et al., 1987; Myers and Sumners, in press; Weiss et al., 1988). As reviewed in Chapter I the hydrolysis of phosphoinositides, specifically phosphatidyl inositol 4,5 bisphosphate, results in the generation of two products (Berridge, 1984). Inositol 1,4,5 triphosphate (IP 3 ), which mobilizes intracellular calcium and diacylglycerol (DAG), which is the endogenous activator of protein kinase C (Takai et al., 1979b). Following the stimulation of phosphoinositide hydrolysis, calcium and DAG promote the association of protein kinase C with the plasma membrane where, in combination with calcium, phosphatidylserine and DAG, it becomes fully activated (Nishizuka, 1986). Experimentally, the binding of protein kinase C to the plasma membrane is identified by an increased level of 87

PAGE 94

88 protein kinase C in the particulate fraction isolated from stimulated cells. A corresponding decrease in protein kinase C is observed in the cytosol fraction. This subcellular redistribution is termed translocation and is used as an index of the activation of protein kinase c. Previous studies from our lab have implied that protein kinase C is activated by ~ 1 -adrenergic receptors. In these studies, the short-term stimulation of ~ 1 -adrenergic receptors was associated with an increase in both the hydrolysis of phosphoinositides and the number of Ang II receptors in primary neuronal cultures (Myers and Sumners, in press). Additionally, protein kinase C was shown to be integrally involved in phorbol ester-stimulated increases in the expression of Ang II receptors (Chapter III; Sumners et al., 1987b). Together, these studies indicate that the upregulation of Ang II receptors due to the stimulation of ~ 1 -adrenergic receptors involves the translocation and activation of protein kinase C. However, the ~ 1 -adrenergic receptor-mediated translocation of protein kinase Chas not been demonstrated in primary neuronal cultures prepared from the brains of rats. Alternatively, the translocation of protein kinase C by ~ 1 -adrenergic receptor agonists and other receptor agonists has been demonstrated in a variety of peripheral cell types. These include angiotensin II (Ang II) in adrenal glomerulosa cells (Iang and Vallotton, 1986), interleukin 2 in murine CT6 cells and interleukin 3 in FOC-Pl cells (Farrar and

PAGE 95

89 Anderson, 1985; Farrar et al., 1985), thyrotropin-releasing hormone in Gffs cells (Drust and Martin, 1985), gonadotropin releasing hormone in pituitary gonadotrophs (Hirota et al., 1985), leukotriene D 4 in basophilic leukemia cells (Vegesna et al., 1988), and nicotinic and muscarinic receptor agonists in PC 12 cells (Messing et al., 1989). The translocation of protein kinase C by ~ 1 -adrenergic receptor agonists has been demonstrated in pinealocytes using phenylephrine (Sugden et al., 1985) and in Madin-Darby canine kidney cells using epinephrine (Slivka et al., 1988). In this study, epinephrine was used to study the effects of ~ 1 -adrenergic receptor activation on the subcellular distribution of protein kinase C in primary neuronal cultures. This study was carried out to further investigate the mechanisms involved in the regulation of Ang II receptors by ~ 1 -adrenergic receptors. Results from this investigation suggest that the activation of ~ 1 -adrenergic receptors by epinephrine causes a transient translocation of protein kinase c in neuronal cultures prepared from the whole brains of neonatal rats. Methods Preparation of neuronal cultures The neuronal cultures used in this study were prepared from the whole brains of one-day-old Sprague-Dawley (SD) rats exactly as described in Chapter II.

PAGE 96

90 Preparation of cellular fractions and treatment of neuronal cultures The isolation of supernatant and particulate fractions and the DEAE chromatography protocols used here are similar to those described in Chapter II. In brief, the neuronal cultures were treated with the desired compounds (ie phorbol esters or epinephrine) for various times by adding the compounds directly into the growth media. Untreated neuronal cultures (ie controls) were treated with vehicle solutions. The vehicle solution for epinephrine-treated cells was PBS containing 1 .M ascorbic acid while the vehicle solution for TPA-treated cells was 0.02% DMSO in PBS. After treatment, the growth media was aspirated off and the cells were washed twice with 3 ml of ice-cold (4C) homogenization buffer A (20 mM Tris HCl, pH 7.5, 2.0 mM EIYI'A, 0.5 mM EGTA, 0.25 M sucrose, 0.2 mM phenylmethylsulfonylfloride (PMSF), and 2.0 g/ml leupeptin). The cells were rapidly scraped from the dish with 1 ml of homogenization buffer A (4C), homogenized in a dounce homogenizer (15-20 strokes), and centrifuged at 20,000 RPM (-43,000 X g) for 45 min at 4C. The supernatant was used as the cytosol fraction and the pellet was resuspended in 1 ml of homogenization buffer A (4C) containing 0.1 % Triton X-100 for 30 min at 4C. The suspension was centrifuged at 20,000 RPM (-43,000 X g) for 45 min at 4C and the supernatant was used as the particulate fraction.

PAGE 97

91 DEAE column chromatography The preparation and elution of the DEAE columns was performed at 4C as described in Chapter II. In brief, the protein kinase c was eluted from the columns by either a linear NaCl gradient (0-0.3 M NaCl in buffer B) or by a single-step elution with 5.0 ml of homogenization buffer B containing 0.15 M NaCl. Assay of protein kinase C activity The protein kinase C activity was determined by measuring the incorporation of 1~P from [1~P]ATP into lysine rich histone (type III-S) exactly as described in Chapter II. The activity was expressed as nmol~P/min/mg protein. Protein determination The protein content of the cytosol and particulate fractions collected by DE 52 cellulose chromatography was determined by the method of I.J:Jwry et al. (1951). statistical analyses All results are expressed as means SEM. Comparisons of multiple means were made by one-way analysis of variance followed by a Newman-Keuls test to assess statistical differences between individual means when applicable. A difference at the 5% level was accepted as statistically significant. Statistical analyses of the data were performed using an analysis of variance program designed by Human Systems Dynamics (Northridge, ca.).

PAGE 98

Results Anion-exchange chromatographic analysis of protein kinase C distribution in neuronal cultures treated with epinephrine 92 The elution profile of protein kinase activity in the cytosol fraction of control and epinephrine-treated neuronal cultures is shown in Fig 4-1. In control cells, the first major peak of calcium, phospholipid-dependent protein kinase (protein kinase C) activity was eluted between 0.04-0.1 M NaCl. A second minor peak of activity eluted at 0.1 M NaCl. Treatment of the neuronal cultures for 5 min with 50 .M epinephrine resulted in a 29% decrease in the protein kinase C collected in the cytosol fractions from 0.04-0.1 M NaCl. Additionally, a second peak of protein kinase C activity in the cytosol fraction was not detected in cells treated with epinephrine. A concentration of 50 .M epinephrine was used as this is a concentration which maximally stimulates the hydrolysis of phosphoinositides (Crews et al., 1988; Myers and Sumners, in press). Typically, between 10 .Mand 100 .M epinephrine or norepinephrine is used in either neuronal cultures or brain slices to elicit a maximal hydrolysis of phosphoinositides. The first major peak of protein kinase C activity from the particulate fraction of control cells and cells treated with epinephrine eluted at 0.04 M NaCl (Fig 4-1). Additionally, what appeared to be a second major peak eluted at 0.14 M NaCl. The treatment of the cultures with 50 .M epinephrine did not result

PAGE 99

93 in a corresponding increase the protein kinase C content of the particulate fraction which eluted from 0.04-0.2 M NaCl, although the elution profile of the cultures treated with epinephrine appears broader than that of control cells. However, using a single step elution procedure, an increase in the protein kinase c content in the particulate fraction of epinephrine-treated cells was detected (see below). The results from Fig 4-1 raise the possibility that treatment with epinephrine reduces the total protein kinase C activity of the neuronal cultures. However, the total activity of protein kinase C was unchanged in neuronal cultures treated with 50 .M epinephrine for 5 min (Fig 4-2). Thus, the decrease in the protein kinase C in the cytosol fraction was not due to a reduction or inhibition of the enzyme activity. This is a representative experiment which was repeated twice with no difference between the protein kinase C activity of control and epinephrine-treated cells observed in each case. The analysis of protein kinase C distribution using a single-step elution The effect of epinephrine on the distribution of protein kinase C was further analyzed using a single-step elution procedure. In these experiments protein kinase c was eluted from columns packed with DE 52 cellulose with a single aliquot of 0.15 M NaCl. Using this method, the translocation of protein kinase C from the cytosol to the particulate fraction is readily apparent

PAGE 100

::f 50 r 4 0 1 n 0 X 30 a.. (.) 2 0 '' > 1 0 i== u 4:: u 0 w (/ ) 4: z 6't 5: z w f0 (l_ 4 0 .3C 2n ., I 1C u Figure 4-1. CO NTROL PARTICULATE / / F K A C:: "'."lmJ r o. 3 EPI C Y. OSOL ; / 1 / / 0 2 I / I / I I i I 0 E PI PARTICULATE / / / i r I I 0 2 94 u 0 z .._, A single 100 mm dish (18 X 10 6 cells) of neuronal cultures prepared from the whole brains of neonatal rats was treated with either phosphate buffered saline-vehicle (PBS) or 50 .M epinephrine for 5 min. The cytosol or the detergent-solubilized particulate fraction (membranes) were prepared as described in the Methods and applied to a column (0.8 X 4 cm) packed with 0.6 ml of DE 52 cellulose equilibrated with homogenization buffer B. The columns were eluted with a linear (0-0.3 M) NaCl gradient as 1.0 ml fractions. Protein kinase activity was determined in 50 aliquots of the indicated fractions in the presence (0) or absence (0) of 0.5 mM CaC1 2 6 g phosphatidylserine, and 0.4 g diolein. Protein kinase activity is expressed as cpm of 32 P incorporated into histone (type III-S)/3 min/50 l aliquot. NaCl concentrations (dashed lines) were determined by conductance.

PAGE 101

4 C 3 u +I e E 2 C E N n 1 0 E C 0 Figure 4-2. 95 T l A 8 Effect of epinephrine on the total protein kinase C activity in neuronal cultures. Neuronal cultures were treated with either PBS (control) (A) or 50 .M epinephrine (B) for 5.0 min. The cells were scraped and homogenized in homogenization buffer A containing 0.1 % triton X-100. The homogenate was placed on a shaker for 30 min at 4C to solubilize the membranes and centrifuged at 43,000 X g for 45 min at 4C. The supernatant fraction was placed on a column packed with 0.6 ml of DE 52 cellulose and eluted with a single aliquot of homogenization buffer B containing 0.15 M NaCl and used as a measure of the total cellular protein kinase C activity. Protein kinase C activity was determined as described in the methods and is expressed as nmol 32 P transferred to histone/min/mg protein. The data are means SEM from triplicate assays of a representative experiment which was repeated twice with similar results.

PAGE 102

96 in cells treated with the phorbol ester, phorbol 12-myristate, 13-acetate (TPA) (Fig 4-3). After 15 min of incubation with TPA (0.8 M), the activity of protein kinase C in the cytosol fraction significantly decreased by 65%. This was associated with a significant 41% increase in the protein kinase C activity in the particulate fraction. The translocation of protein kinase C was followed by the downregulation of the protein kinase C activity after 1 hr of treatment with TPA. This downregulation of protein kinase C activity is characteristic of prolonged treatment with phorbol esters (Chida et al., 1986). The effect of epinephrine on the distribution of protein kinase C was detennined next. In unstimulated control cells, the activity of protein kinase C in the cytosol and particulate fractions represented 44% and 56%, respectively, of the total protein kinase C activity (Fig 4-4). Treatment of the neuronal cultures for 1 and 5 min with 50 M epinephrine significantly decreased the activity of protein kinase C in the cytosol fractions by 23% and 31%, respectively (Fig 4-4). This was associated with corresponding significant increases of 16% and 15% in the amount of protein kinase C activity in the particulate fraction of cells treated with epinephrine for 1 and 5 min, respectively. The percent changes in the amount of protein kinase C in the cytosol and particulate fractions are not equal due to the greater amount of protein kinase C in the particulate fraction under control conditions. This experiment is a

PAGE 103

Figure 4-3. C: .; ..., 0 ... a. C7I E C: e N ,.., 0 E C: C: C) ..., 0 ... a. C7I E C: e a. N n 0 E C 3 97 2 0 ~o 0 +-,~-+--+-+-+--+--+-~i--..-... .. 5 2 0 ~-------+-+--I--+--+-.+ 0 1 O 20 30 40 50 60 time (min) Effect of TPA on the distribution of protein kinase C in neuronal cultures using a single-step elution. Neuronal cultures (18 X 10 6 cells) were treated with 0.8 .M phorbol 12-myristate, 13acetate (TPA) for 15 min or 60 min. The cytosol (0) and particulate (e) fractions were prepared as described in the Methods. The fractions were placed on separate columns packed with 0.6 ml of DE 52 and eluted with a single aliquot of homogenization buffer B containing 0.15 M NaCl. A 50 l aliquot of the eluate from the fractions was assayed for protein kinase C activity as described in the Methods. Unstimulated control values are shown at time zero. The data are the means of triplicate assays and the results are representative of one additional experiment. ANOVA 1, p<0.05 for both cytosol and particulate fractions. significantly different from controls. See text for description.

PAGE 104

Figure 4-4. 2.75 C: 98 'ii 2 5 0 0 ... a. \. 01 -( 2.25 C: e 0 ........ 2.0 Q. N I") 0 E 1.75 C: 0 1.5 4.0 C: l "ii 3.75 e a. 01 E 3.5 l ........ C: e Cl. 3.25 N I") 0 E 3.0 C: 1 2.75 0 2 3 4 5 time (min) Effect of epinephrine on the distribution of protein kinase C using a single-step elution. A single large plate of (18 X 10 6 cells) of neuronal cultures was treated with 50 .M epinephrine for either 1 or 5 min. Unstimulated control values are shown at time zero. The cytosol (0) and particulate (e) fractions were prepared as described in the Methods and placed on individual columns packed with 0.6 ml of DE 52 cellulose. The fractions were eluted with a single 5.0 ml aliquot of homogenization buffer containing 0.15 M NaCl and 50 l of the eluate was assayed for protein kinase C activity. Data are the means SEM of triplicate assays and are representative of three similar experiments which showed similar percent changes in the protein kinase C activity. ANOVA 1, p<0.05 for both cytosol and particulate fractions. significantly different from controls. See text for description.

PAGE 105

99 representative experiment which was repeated three times with similar percent changes in protein kinase C activity occurring each time. A translocation of protein kinase C was not observed in neuronal cultures treated with epinephrine for less than 1 min (data not shown). An extended time course of epinephrine treatment is shown in Fig 4-5. A translocation of protein kinase C in cells treated with epinephrine was detected after 5 min of incubation. This effect was transient as the levels of protein kinase C in the cytosol and particulate fractions returned to control levels after 10 and 15 min of incubation with epinephrine. This is representative experiment which was repeated twice with similar results. The specific ~ 1 -adrenergic receptor antagonist prazosin blocked the translocation of protein kinase C in cultures treated with epinephrine (Fig 4-6). The activity of protein kinase C in the cytosol fraction of unstimulated control cells was 4.160 0.12 nmol 32 P/min/mg protein (n=3 experiments). Treatment with epinephrine (50 .M, 5 min) decreased the protein kinase C activity in the cytosol fraction to 3.00 0.17 nmol/min/mg protein (n=3 experiments). This represents significant decrease of 28% in the activity of protein kinase c. The levels of protein kinase C in cells treated with prazosin (1.M) and epinephrine (50 .M, 5 min) were not significantly different from control levels ( 3 84 7 0. 12 nmol 32 P /min/mg protein; n=3 experiments) Prazosin

PAGE 106

Figure 4-5. 100 4.5 C ) -4.25 Cit -4.0 C I N J.75 .., 0 E 3.5 C 3.25 7.25 C 'i 7.0 .. e Q. c,t ~6.75 C l 0.. 6.5 N I') 0 E C 6,25 6.0 0 5 10 15 time(min) Extended time course of the distribution of protein kinase C in neuronal cultures treated with epinephrine. Neuronal cultures (18 X 10 6 cells) were treated with 50 .M epinephrine for the indicated times. The cytosol (0) and particulate (e) fractions were isolated as described in the Methods and eluted from columns packed with 0.6 ml of DE 52 with 5.0 ml of homogenization buffer B containing 0.15 M NaCl. The data are means SEM and are representative of two similar experiments.

PAGE 107

C: .; +I e 4.0 3.0 E C: 2.0 a. N n 0 E 1.0 C 0 ... ... ... ... ... .. ... Figure 4-6. 101 T l T l T i A 8 C D Effect of prazosin on protein kinase C redistribution in neuronal cultures treated with epinephrine. Neuronal cultures (18 X 10 6 cells) were treated for 5 min with (A) PBS, (B) 50 .M epinephrine, (C) 50 .M epinephrine+ 1 .M prazosin, (D) 1 .M prazosin. The cytosol and particulate fractions were isolated as described in the Methods and eluted from columns packed with o. 6 ml of DE 52 with 5. o ml of homogenization buffer B containing 0.15 M NaCl. The activity of protein kinase C found in the cytosol fractions was used as index of the translocation of protein kinase C. The data are means SEM of three separate experiments. ANOVA 1, p<0.05. significantly different from controls. See text for description.1

PAGE 108

102 (lM) alone had no effect (3.723 0.09 nmol ~P/min/mg protein; n=3 experiments). A similar inhibition of an increase in the membrane-associated protein kinase C in epinephrine treated cells was found in cells treated with prazosin (data not shown). Discussion The present study demonstrates that the incubation of neuronal cultures with epinephrine results in the redistribution of protein kinase C in neuronal cultures prepared from the brains of neonatal rats. A decrease in the protein kinase C levels of approximately 20-30% was detected in the cytosol fraction of cells treated for 5 min with epinephrine as compared to unstimulated control cells. This effect was associated with an increase of approximately 10-15% in the protein kinase C levels in the particulate fraction of neuronal cultures treated with epinephrine. The translocation of protein kinase C was transient and was not detected after 10 or 15 min of incubation with epinephrine. The percent changes in the protein kinase c activity in the cytosol and particulate fraction were not equal which was due, in part, to the greater amount of protein kinase C in the particulate fraction of unstimulated control cells. Thus, the amount of protein kinase C (in te:nns of nmol~P/min/mg protein) which translocated is more representative of the redistribution of activity which occurred in epinephrine-treated cells. However, in general, the decrease in the activity of protein kinase C in the cytosol was greater than the increase in activity found in

PAGE 109

103 the particulate fraction. The reasons for this are not clear, although several possibilities exist. The first may involve a partial degradation of protein kinase C upon translocation to the plasma membrane in epinephrine-treated cells. The plasma membrane is the site of metabolism for protein kinase C (Young et al., 1987). However, this is unlikely as the total activity in the homogenate of cells treated with epinephrine was unchanged as compared to control values (Fig 4-2). Additionally, if the protein kinase c activity in the cytosol and particulate fractions are added together as a measure of total activity, cells treated with epinephrine for 1 and 5 min contained 98% and 92%, respectively, of the total protein kinase C activity found in unstimulated control cells (n=3 experiments). Secondly, using crude homogenates, the activity of protein kinase C in the particulate fraction is less on a per mg protein basis than the activity in the cytosol fraction (see Chapter II). This could be due to an overabundance of inhibitors of protein kinase C in the particulate fraction (see Chapter II). It is possible that some of the inhibitory activity in the particulate fraction was not completely removed by DEAE chromatography and acted to partially obscure the changes in activity in epinephrine-treated cells. Finally, Thomas et al. (1987) reported that in NIH 3T3 cells the protein kinase C from the particulate fraction of TPA-treated cells eluted from DEAE-cellulose columns at higher NaCl concentrations as compared to control cells. They postulated this

PAGE 110

104 was due to an alteration in the physical properties of the enzyme by TPA and phospholipids which are still bound to protein kinase c even after elution from DEAE-cellulose columns (Uratsuyi et al., 1985). Thus in epinephrine-treated cells, the protein kinase C in the particulate fraction may have been in a complex with DAG and Phospholipids and only partially eluted from the columns at the NaCl concentrations used (0.15 M) for the single-step elution procedure. In fact, the elution profile of the particulate fraction of the epinephrine-treated cells does appear to be shifted slightly to the right (Fig 4-1). The elution profile of protein kinase C activity in cells treated with epinephrine did not show an increase of protein kinase C in the particulate fraction, although a 29% decrease in protein kinase C was detected in the cytosol fraction. The reason for this discrepancy is not clear. It was not due to an inhibition of protein kinase C as the treatment of cells with epinephrine did not reduce the total activity protein kinase c. It is possible that the membranes were not completely solubilized in this experiment by treatment with triton x-100. This would cause a portion of the membrane-associated protein kinase C to remain on the anion exchange column. Prazosin was shown to inhibit the translocation of protein kinase C in neuronal cultures treated with epinephrine. This indicates that the activation of ~ 1 -adrenergic receptors stimulates the translocation of protein kinase c in cultures

PAGE 111

105 treated with epinephrine. A similar ~ 1 -adrenergic receptor mediated translocation of protein kinase C by epinephrine was reported in MDCK-Dl cells (Slivka et al., 1988). However, in MDCK-Dl cells the hydrolysis of phosphatidylcholine was associated with increased DAG production and the translocation of protein kinase C. The hydrolysis of phosphatidylcholine by ~ 1 adrenergic receptors has not been characterized in primary neuronal cultures. However, the activation of ~ 1 -adrenergic receptors produces a large stimulation of phosphoinositide hydrolysis in primary neuronal cultures (Gonzales et al., 1987; Weiss et al., 1988). Obviously, norepinephrine is the primary neurotransmitter in the brain which utilizes ~ 1 -adrenergic receptors., In this study, epinephrine was used to stimulate ~ 1 adrenergic receptors and it is not assumed that epinephrine is the actual endogenous neurotransmitter which acts at ~ 1 -adrenergic receptors to regulate Ang II receptors in the brain. It is likely that norepinephrine fulfills this role and in a preliminary experiment norepinephrine was found to stimulate the translocation of protein kinase C in neuronal cultures (data not shown). However, the involvement of epinephrine cannot be ruled out as some epinephrine cell groups are found in the medulla which send projections anterioventrally to the hypothalamus (Palkovits and Brownstein, 1989; Routledge and Marsden, 1987). In fact, epinephrine has a greater efficacy for phosphoinositide hydrolysis and produces a greater fractional release of inositol

PAGE 112

106 phosphates in the brain than either norepinephrine or phenylephrine (Crews et al., 1988; Minneman and Johnson, 1984). Compared with epinephrine, the phorbol ester TPA induced a more pronounced translocation of protein kinase c. Phorbol esters are not readily metabolized as opposed to DAG which is rapidly produced and inactivated (Welsh and cabot, 1987). This may contribute to both the smaller degree and the transient nature of protein kinase C translocation in epinephrine-treated cells. Further, TPA may stimulate the translocation of all of the isozymes of protein kinase c which are located in the cytosol as opposed to epinephrine which may affect only a single isozyme or a subpopulation of isozyme(s). In previous experiments, the incubation of neuronal cultures with epinephrine or norepinephrine for 15-60 min at concentrations which maximally stimulate the hydrolysis of phosphoinositides (ie 5-50 .M) has been shown to increase the number of Ang II receptors by a mechanism specific for ~ 1 adrenergic receptors (Myers and Sumners, in press). Further, protein kinase C is known to be integrally involved in the increased expression of Ang II receptors in neuronal cultures (Chapter III). These findings have led to the hypothesis that the short-term activation of ~ 1 -adrenergic receptors leads to the hydrolysis of phosphoinositides and the activation of protein kinase C which acts to increase the expression of Ang II receptors in primary neuronal cultures prepared from the brains

PAGE 113

107 of neonatal rats. The present study furthers this hypothesis by showing that ~ 1 -adrenergic receptors mediate the translocation of protein kinase C in neuronal cultures. A translocation is used as an index of the activation of protein kinase C (Nishizuka, 1986). In this study, the translocation of protein kinase C occurred by 5 min of incubation with epinephrine. This precedes the time required to increase the number of Ang II receptors by the activation of ~ 1 -adrenergic receptors. Possibly, the translocation and activation of protein kinase C initiates a phosphorylation event which leads to the increase expression of Ang II receptors. In sunnnary, the incubation of primary neuronal cultures prepared from neonatal rats with epinephrine results in the time dependent redistribution of protein kinase C. This effect was blocked by prazosin which indicates that the translocation of protein kinase C was mediated by specific ~ 1 -adrenergic receptors. These findings, together with previous results, suggest protein kinase C mediates the increase in the expression of Ang II receptors produced by the short-term activation of ~ 1 -adrenergic receptors in neuronal cultures.

PAGE 114

CHAPI'ER V REGUIATION OF ANGIOTENSIN II RECEPI'ORS UNDER DEPOIARIZING CONDITIONS Introduction The regulation of Ang II receptors by oc 1 -adrenergic receptors occurs in a biphasic manner in primary neuronal cultures prepared from the hypothalamus and brainstem of neonatal rats. In the short term, the activation of oc 1 -adrenergic receptors acts to upregulate the number of Ang II receptors (Myers and Sumners, in press). The mechanisms which may be involved in this short-term effect were discussed in Chapters III and IV, by Sumners et al. (1987b) and by Myers and Sumners (in press). These studies strongly suggest that the hydrolysis of phoshoinosites and the activation of protein kinase Care integrally involved in the short-term regulation of Ang II receptors by oc 1 -adrenergic receptors. Conversely, the long-term (2-6 hr) activation of oc 1 adrenergic receptors acts to downregulate the number of Ang II receptors in primary neuronal cultures (Sumners et al., 1986b). The mechanisms involved in the long-term regulation of Ang II receptors by OCi-adrenergic receptors are not well understood. However, previous studies have indicated that ca 2 + influx may be involved. In these studies, the incubation of neuronal co cultures with the ca 2 + ionophore A23187 at concentrations which 108

PAGE 115

109 significantly increased the uptake of ~ca 2 + resulted in a decrease in the number of Ang II receptors (Sumners et al., 1988). An influx of ca 2 + is a plausible mechanism by which cx: 1 -adrenergic receptors act to downregulate Ang II receptors as certain cx: 1 adrenergic receptors have been linked to the activation of voltage sensitive calcium channels (VSCC) (Han et al., 1987). The following study had two objectives. The first was to identify VSCCs in the primary neuronal cultures. If vscc were present in these cultures, the second objective was to detennine the effect of ca 2 + flux through vscc on the specific binding of [u 5 I]Ang II. The results obtained in this study indicate that vsccs are present in neuronal co-cultures and that ca 2 + influx through specific VSCCs is involved in the downregulation of Ang II receptors. Methods Preparation of neuronal cultures The neuronal cultures used for the ( 125 1] Ang II binding studies and the ~ca 2 + uptake studies were prepared from the hypothalamus and brain stem of (co-cultures) of one-day-old Sprague-Dawley (SD) rats as described in Chapters II and III. Measurement of ~ca 2 + flux into neuronal cultures The flux or uptake of ca 2 + into neuronal cultures was detennined using ~ca 2 +. Intact neuronal co-cultures grown on 35 mm diameter dishes were used. The growth media was removed and the cells were washed twice with a control buffer (130 mM NaCl, 5 mM

PAGE 116

110 KCl, 10 mM glucose, 1.2 mM cac1 2 1 mM MgC1 2 and 25 mM Hepes, pH 7.4, 37C) and 2 ml of the same buffer was placed on the cells. The cultures were placed in a water bath (37C) for a 10 min equilibrium period. After this time, the buffer was aspirated off and 1 ml of control buffer or high~ buffer containing 2 Ci/ml of ~ca 2 + was added to the cells. The cells were placed back into the water bath and incubated for various times. The high~ buffers were the same as the control buffer except NaCl was isosmotically replaced by KCl. The incubation was terminated by aspirating off the reaction mixture and washing the cells three times with ice-cold wash buffer (135 mM NaCl, 5 mM cacl 2 5 mM KCl, 1 mM MgC1 2 and 25 mM Hepes, pH 7.4). The cells were dissolved in 0.5 ml of 0.1 N NaOH and transferred to plastic tubes. Each dish was washed with 0.5 ml of deionized ~O which was combined with the original sample. A 0.5 ml aliquot of each sample was added to a scintillation vial with 10 ml of liquiscint and counted on a Beckman LS 1801 counter with a counting efficiency of 93% for 46 ca 2 +. The uptake of ~Ca 2 + was expressed as pmol ~ca 2 + /mg protein. ru 6 I]Ang II binding assay The specific binding of [u 6 I]Ang II was determined exactly as described in Chapter III. Treatment of neuronal cultures for [u 6 I]Ang II binding experiments Depolarizing conditions for cultures used in the [u 6 I]Ang II binding studies were obtained by adding a concentrated KCl

PAGE 117

111 solution directly into the growth media to give a final concentration of 35 mM r. Sucrose was used as an osmotic control for r-treated cells. With sucrose-treated cells, a concentrated sucrose solution was added directly to the growth media to give a final concentration of 60 mM or 70 mM. In some experiments, neuronal cultures were incubated under isosmotic conditions with the same buffers used in the ~ca 2 + uptake experiments in which KCl was isosmotically substituted for NaCl. Protein determination The protein contents of cells used for the [u 6 I]Ang II binding assay or the measurement of 45 ca 2 + uptake were determined by the method of IJ:Jwry (1951). The preparation of the protein samples from the [u 6 I]Ang II binding assay was described in Chapter III. For the ~ca 2 + flux studies, the cells were dissolved in 0.5 ml of 0.1 N NaOH. The protein was transferred to a plastic tube and the plates were washed with 0.5 ml of deionized water which was combined with the original sample. The protein content was then assayed. Statistical analysis All results are expressed as means SEM. Comparisons of multiple means were made by one-way analysis of variance followed by a Newman-Keuls test, when applicable, to asses statistical differences between individual means. A difference at the 5% level was accepted as statistically significant. Statistical

PAGE 118

112 analyses of the data were performed using an analysis of variance program designed by Human Systems Dynamics (Northridge, ca). Preparation of drug solutions and treatment of neuronal cultures Nifedipine was diluted in ethanol and deionized ~o (1:1) at a stock concentration of 10 mM. Further dilutions were made into PBS. The maximal final ethanol concentration used was 0.1% which did not alter the binding of [ 126 I]Ang II or the uptake of 45 Ca 2 +. Control vehicle solutions contained an equivalent amount of ethanol as the test solutions. Results Effects of r on 45 Ca 2 + uptake A time course for the uptake of 45 ca 2 + in neuronal co cultures under control and depolarizing conditions is shown in Fig 5-1. The accumulation of 45 ca 2 + during control (5 mM r) conditions occurred rapidly and reached equilibrium by 1 min. The incubation of the cells with 35 mM r or 60 mM r resulted in an increase in the initial rate of 45 ca 2 + uptake and an increase in the maximal uptake of 45 ca 2 + by 41% and 124%, respectively, as compared to control values. The uptake of 45 ca 2 + under depolarizing conditions (35 mM or 60 mM r) plateaued between 1 and 5 min of incubation. A shorter time course for the uptake of 45 ca 2 + is shown in Fig 5-2. At the earliest time point tested (5 sec), the amount of uptake under depolarizing conditions (60 mM r) was 1.5 fold greater than control levels (5 mM r). After 30 sec, control

PAGE 119

C V _,_. 0 L 0. CJ) E + N 0 u t.n tj0 E r, ..... 113 oo control 50 35 mM K+ A-A6 0 mM K+ 40 I I I T T A 6. l .l 3 0 ~i-0 2 0 T 0 --~ l 1 0 0 ----t------r-----+--------1----------+0 5 Figure 5-1. 10 15 ti~ 20 2 5 30 Effect of 35 and 60 mM K+ on the uptake of 45 ca 2 + over time. Values are means SEM of quadruplicate determ i nations from a representative experiment which wa s reEeated twice with similar percent changes in 5 ca 2 + uptake.

PAGE 120

C Q) -+-' 0 L a. 01 E + N 0 u tn s;t 0 E Q_ 30 20 0 Figure 5-2. l 1 5 10 15 20 time (sec ) oo control 60 mM K+ 2 5 20 114 Effect of 60 mM K+ on 45 Ca 2 + uptake at time points less than 1 min. Values are means SEM of quadruplicate determinations from a representative experiment which was reEeated twice with similar percent changes in 5 ca 2 + uptake.

PAGE 121

115 uptake appeared to equilibrate while the uptake under depolarizing conditions continued to increase. The stimulation of 45 ca 2 + flux was dependent of the concentration of KCl (Fig 5-3). The incubation of the neuronal cultures with 30 mM, 40 mM and 60 mM KCl for 5 min significantly increased the uptake of 45 ca 2 + above control levels by 57%, 80% and 204%, respectively. The uptake of 45 ca 2 + was not significantly altered by 10 mM and 2 0 mM KCl. Effect of nifedipine on 45 Ca 2 + uptake under depolarizing conditions In the next series of experiments, the dihydropyridine (DHP) antagonist of vscc, nifedipine, was used to determine if 45 ca 2 + flux under depolarizing conditions occurred through VSCC. Nifedipine was found to dose-dependently inhibit the uptake of 45 ca 2 + in the presence of 35 mM (Fig 5-4). At concentration of 10 .M and 50 .M, nifedipine inhibited 45 ca 2 + uptake by 60% and 90%, respectively. Concentrations of nifedipine less than 10 .M were generally ineffective at inhibiting ~-stimulated increases in 45 ca 2 + uptake, although an 11% inhibition was observed with 1 .M nifedipine. Effect of depolarizing conditions on [ 125 I]Ang II binding Incubation of neuronal co-cultures with 35 mM resulted in a time-dependent decrease in the specific binding of [u 5 I]Ang II (Fig 5-5). A significant decrease in binding was detected between 2-6 hrs, with a maximal decrease of 49% occurring at 2 hrs. After 24 hrs of treatment with 35 mM ~, the specific binding of

PAGE 122

35 C 30 ., e 25 0. Cl E 20 + N C 0 15 U') 0 10 E 0. 5 0 Figure 5-3. 116 A 8 C D E F Concentration-dependence of K+-stimulated increases in the uptake of 45 Ca 2 +. Neuronal cultures were incubated with A, 5 mm K+; B, 10 mM K+; C, 20 mM K+; D, 30 mM K+; E, 40 mM K+; E, 50 mM K+; or F, 60 mM K+ for 5 min in the presence of 2 Ci/ml of 45 Ca 2 +. Values are means SEM of quadruplicate determinations from a representative experiment which was repeated twice with similar results. significantly different from controls. See text for description.

PAGE 123

120 C 100 QJ -+J 0 L a. 80 0' 0 E !i ,c + 8 60 N 't-C 0 Ji'cll. 40 v'--' 0 E a. 20 0 Figure 5-4. 117 ...-.----, 9 8 7 6 5 4 -log (mole) Concentration-dependent inhibition of 45 ca 2 + uptake by nifedipine. Neuronal cultures were incubated with various concentrations of nifedipine 10 min prior to the addition of a isosmotic reaction mixture containing 35 mM K+ and 2 Ci/ml of 45 Ca 2 + for 5 min. Results are the means from two separate experiments and are expressed as% of control.

PAGE 124

118 [u 6 I]Ang II was no longer significantly different from control binding levels. Additionally, the specific binding of [u 6 I]Ang II was assayed after incubating the cells with 60 mM r for various time under isosmotic conditions (Fig 5-6). Similarly, a significant decrease in the specific binding of [ 126 I]Ang II was observed after 4 hrs of incubation. The incubation of the neuronal cultures with 60 mM or 70 mM sucrose, which were used as osmotic controls for cells treated with 35 mM r under hypertonic conditions, did not effect the specific binding of [ 126 I]Ang II (Table 5-1). Table 5-1. Effects of 60 mM and 70 mM sucrose on r 125 I] Ang II binding Treatment control 60 mM sucrose 70 mM sucrose n 4 4 4 [u 6 I]Ang II bound (fmoles/rng protein 10. 36 o. 58 9.23 0.49 9.98 1.55 Values are means SEM; n, no. of separate experiments. Neuronal cultures were treated for 2 hrs and the values were not significantly different as determined by lillOVA. The next set of experiments were performed to determine if ca 2 + influx through DHP-sensitive vscc may be involved in the decrease in the specific binding of [u 6 I]Ang II observed under depolarizing conditions. Treatment of the neuronal cultures with 50 .M nifedipine under depolarizing conditions (35 mM r) resulted in specific binding levels of [u 6 I]Ang II which were not

PAGE 125

119 significantly different from control binding levels (Fig 5-7). This is in contrast to a 50% decrease in the specific binding levels of [ 125 I]Ang II of cells treated with 35 mM ~. The treatment of the cells with 50 .M nifedipine alone did not significantly alter the binding levels of [ 125 I]Ang II. Discussion This study was conducted to further investigate the mechanisms involved in the downregulation of Ang II receptors in neuronal cultures. Previous studies using the ca 2 + ionophore A23187 implicated the influx of ca 2 + from extracellular stores in the downregulation of Ang II receptors (Sumners et al., 1988). In the present study, depolarization was used as a more physiologically relevant stimulus of ca 2 + to study the downregulation of Ang II receptors. The incubation of neuronal co-cultures with 30-60 mM significantly increased the uptake of ~ca 2 + over time indicating the presence of vsccs. Further, it appears dihydropyridine (DHP) sensitive-vsccs are present as the uptake of ~ca 2 + under depolarizing conditions was attenuated by the DHP antagonist, nifedipine, at concentrations of 10 .Mand 50 .M. These concentrations are several fold higher than those used by Thayer et al. (1986) to inhibit ca 2 + influx under depolarization conditions in isolated neurons cultured from specific brain regions. This group used a concentration of 1 .M of the DHP antagonist, nitrendipine, to block the influx of ca 2 + in

PAGE 126

120 20 C t u "' e 1 s Q. \ Cl E V c:,110 ,..., 1 in' N .... '--' 5 0 E .... 0 0 1 2 3 4 5 6 24 time {hrs) Figure 5-5. Effect of depolarizini conditions on the specific binding of [ 5 I]Ang II over time. Neuronal cultures were incubated with 35 mM K+ for various times after which the binding of [" 5 I]Ang II was assayed as described in the Methods. Values are means SEM of triplicate determinations from of representative experiment which was repeated twice with similar percent changes in the binding levels. significantly different from controls. See text for description.

PAGE 127

6 C I) 5 e 0.: 01 4 E 01 3 ,...., an N 2 '--' f) Q) 0 1 E -... 0 Figure 5-6. 121 ~ 0 /9 t 1 2 3 4 5 6 time {hrs) Effect of depolarizing conditions on the specific binding of [ 5 I]Ang II over time using an isosmotic buffer. Neuronal cultures were incubated with the same buffer used to measure 45 ca 2 + uptake in which 60 mM K+ was isosmotically substituted for Na+. Values are means SEM of quadruplicate determinations. significantly different from controls. See text for details.

PAGE 128

15 -~ C .; .. e E 10 -~ ,....., 5 N -~ '--' 0 E .... 0 Figure 5-7. 122 T l a A 8 C D Effect of nifedipine on the specific binding of [" 5 I]Ang II binding under depolarizing conditions. Neuronal cultures were incubated with A, control-vehicle (PBS); B, 35 mM KCl; c, 35 mM KCl + 50 M nifedipine; or D, 50 M nifedipine for 2 hrs. Values are means SEM of quadruplicate determinations from a representative experiment which was repeated twice with similar percent differences in the binding levels. significantly different from controls. See text for details.

PAGE 129

123 hippocampal and striatal neurons by 79% and 31% respectively. A concentration of 1 .M nifedipine only reduced the uptake of ~ca 2 + by 11% in the present study. The results of Thayer et al. (1986) indicate that neurons from different brain regions are heterogeneous in their responsiveness to DHP antagonists. This may explain the higher concentrations of nifedipine required to inhibit ~ca 2 + uptake in the neuronal co-cultures which represent of heterogenous collection of neurons. Additionally, the experiments conducted by Thayer et al. (1986) were performed on single neurons in a Na+-free buffer using microspectrofluorimetry and the fluorescent ca 2 +-chelator, fura-2. Using a Na+-free buffer eliminates alterations in intracellular Ca 2 + due to the Na+;ca 2 + exchange mechanism while the microspectrofluorimetric technique is much more sensitive to changes in intracellular ca 2 + than the measurement of ~ca 2 + uptake. Both of these factors may have necessitated the use of higher concentrations of nifedipine to detect the inhibition of ~Ca 2 + uptake in the neuronal co-cultures. Under depolarizing conditions (35 mM r), which were found to stimulate the uptake of ~ca 2 +, the specific binding of [u 6 I]Ang II was found to decrease over time. This effect was maximal between 1-6 hrs and was not detected after 24 hrs of incubation. These findings suggest that ca 2 + influx through vsccs is involved in the regulation of Ang II receptors. It is unlikely that the decrease in the specific binding of [u 6 I]Ang II observed under depolarizing conditions was due to direct effects of r.

PAGE 130

124 Feldstein et al. (1986) found a increase in the specific binding of [u 6 I]Ang II when the binding assay was performed in the presence of excess r which was not the case in this study where the binding of [u 6 I]Ang II was performed in isotonic PBS. Further, osmotic effects were not responsible for the decreased in the specific binding of [ 126 !] Ang II as the incubation of the neuronal cultures with 60 mM and 70 mM sucrose, which were used as osmotic controls, did not alter the binding levels. To better associate the binding data with the ~ca 2 + studies, the binding of [ 126 I]Ang II was assayed after the incubation of the neuronal cultures with 60 mM r under isosmotic conditions. In this situation, a significant decrease in the specific binding of [1 26 I]Ang II was observed after 4 hrs of incubation. The antagonism by nifedipine of the decrease in the specific binding of [u 6 I]Ang II observed under depolarizing conditions indicates that ca 2 + influx through specific DHP-sensitive ca 2 + channels is involved in the regulation of Ang II receptors. ca 2 + influx under depolarizing conditions occurs through multiple voltage sensitive ca 2 + channels (Miller, 1986; Nowycky et al., 1985). Using dorsal root ganglion cells, these VSCCs have been differentiated into three subtypes based on several properties including their activation potentials, duration of activation and susceptibility to blockage by DHP antagonists (Nowychy et al., 1985). Only one channel, the L channel, is highly sensitive to DHP antagonists. Additionally, the widespread occurrence of

PAGE 131

125 putative vsccs have been identified in the brain using radiolabelled DHPs. The highest binding levels were detected in the hippocampus, cerebral cortex, certain thalamic nuclei, and the olfactory bulb, while intennediate to low levels were found in the midbrain, hypothalamus and brain stem (Gould et al., 1985; Quirion, 1983). These results are somewhat incongruous with the results obtained in this study which showed 10 .Mand 50 .M nifedipine inhibited the uptake of ~ca 2 + under depolarizing conditions by 60% and 90%, respectively, in neuronal co-cultures prepared from the hypothalamus and brainstem. These results indicate a majority of the ca 2 + channels in the co-cultures were DHP-sensitive. Further, DHP-sensitive VSCCs appear to be integral in the regulation of Ang II receptors as nifedipine blocked the decrease in the specific binding of [u 6 I]Ang II observed under depolarizing conditions. Certainly further experiments are needed to clarify the role of ca 2 + influx in the regulation of Ang II receptors in culture. Specific experiments which remain to be perfonned include 1) the analysis of [u 6 I]Ang II binding after incubations under depolarizing conditions in the absence of external ca 2 +, although these experiments may prove difficult if long-tenn incubations are studied due to cell adhesion problems in the absence of Ca 2 +; 2) the analysis of [u 6 I]Ang II binding after treating the cultures with specific ~ 1 -adrenerg i c receptor antagonists under depolarizing conditions to eliminate the possibility that

PAGE 132

126 depolarization-stimulated. release of NE is not responsible for the downregulation of Ang II receptors (preliminary experiments using prazosin indicate this is not the case); 3) the determination of the effects on ~ca 2 + uptake and the binding of [ 125 I]Ang II of DHP-agonists such as BAY K8644 which activates DHP-sensitive VSCC; and 4) saturation analysis of the effects of decreased. specific binding of [ 125 I]Ang II observed. under depolarizing conditions to determine if this effect is actually due to a downregulation of Ang II receptors. The incubation of neuronal cultures with NE for 2-6 hrs results in the downregulation of Ang II receptors in neuronal co cultures (Sumners et al., 1986b). The time course for this effect parallels that of the depolarization-induced. decreases in the specific binding of [u 5 I]Ang II observed. in this study suggesting a conunon mechanism of action. Recent studies have linked ~ 1 adrenergic receptors to the activation of vscc. In these studies, depending on the tissue examined., selective ~ 1 -adrenergic receptor antagonists differentially displaced. the binding of the ~ 1 adrenergic receptor antagonist [u 5 I]BE 2254 and differentially inhibited. functional responses to NE (Han et al., 1987) Further, the differential ability of the antagonists to block NE stimulated responses in different tissues was associated. with different means of ca 2 + mobilization by NE. For example, in the vas deferens, NE-stimulated. contraction was associated. with the influx of ca 2 + from extracellular stores through DHP-sensitive

PAGE 133

127 VSCC. Conversely, the NE-stimulated contractions of the spleen were associated with inositol phospholipid hydrolysis and the mobilization of intracellular ca 2 +. Using nomenclature established by others (Morrow and Creese, 1986) the ~-receptor linked to VSCC the was termed the ~ 1 a-receptor while the receptor linked to the hydrolysis of phosphoinositides was termed the ~ 1 b-receptor. Further, based on competitive binding studies and the measurement of phosphoinositide hydrolysis, the ~la and ~ 1 b-receptor subtypes were identified in brain slices from the hippocampus. Thus, the possibility exists that the NE-stimulated downregulation of Ang II-specific receptors is mediated by the influx of ca 2 + through vscc possibly due to the activation of a ~ 1 -adrenergic receptors which are linked to VSCCs. In summary, this study associated the uptake of 46 ca 2 + through DHP-sensitive vsccs with decreases in the specific binding of [ 126 I]Ang II obtained under depolarizing conditions. It was postulated that the influx of ca 2 + through vsccs is a mechanism involved in the downregulation of Ang II receptors produced by the activation of ~ 1 -adrenergic receptors.

PAGE 134

CHAPl'ER VI SUMMARY The work presented in the previous chapters was perfonned using primary neuronal cultures prepared from the brains of neonatal rats. These cultures were used as a model to study the mechanisms involved in the regulation of Ang II receptors in the brain. Previous investigators have shown that these cultures are a suitable model to study the brain renin-angiotensin system as they contain all the components of the renin-angiotensin system and have functional Ang II receptors (see Chapter I). Primary brain cell cultures are extremely useful for studying the regulation of receptors for the following reasons: 1) specific cell types (ie neurons or glia) from specific brain regions may be analyzed, 2) the cells are readily accessible, 3) the environment of the cells may be manipulated in a quantifiable manner, and 4) the cells may be studied irrespective of nonspecific peripheral influences such as stress and locomotion. Alternatively, there are drawbacks to using neuronal cultures. These include the use of serum to grow the cells, which adds variability to the growth media, and the inability to reproduce exactly in vivo conditions. In spite of these limitations, primary neuronal cultures are a powerful analytical in the tool 128

PAGE 135

129 in the study of cell biology and their use is gaining wide acceptance by the scientific community. Using primary neuronal cultures, it was demonstrated that these cultures contain high levels of calcium, phospholipid dependent protein kinase (protein kinase C). The cultures used were previously shown to contain -80-90% neuronal cells with the remainder being astrocyte glia cells (Richards et al., 1989). In addition to being dependent on ca 2 + and phospholipids, the activity of protein kinase C was stimulated by a synthetic diacylglycerol (DAG), diolein Previously, both the binding of [3tt] -phorbol esters (Raizada et al. 1988) and immunological methods (Mudd, 1989) were used to identify protein kinase c in these cultures. However, there are drawbacks to using these methods. The binding of [3tt]-phorbol esters may identify, in part, enzyme fragments which represent non-calcium, phospholipid dependent enzyme. The same is true for immunological methods which, additionally, may only identify certain isozymes of protein kinase c. Further, phorbol esters act to downregulate protein kinase C (Chida et al., 1986; Ballester and Rosen, 1985; Matthies et al., 1987; Neary et al., 1988) which may hamper studies that use [3ttJ-phorbol ester-binding to identify this enzyme. Using a single high speed centrifugation step, approximately 50-60% of the total cellular protein kinase C activity was found in the particulate fraction. This is in accord with studies using

PAGE 136

130 rat brain homogenate (Kikkawa et al., 1982) and is slightly higher than the levels found in primary neuronal cultures prepared from the striatum (Weiss et al., 1989). However, when a low speed spin was performed prior to the isolation of the particulate fraction by high speed centrifugation only approximately one-third of the protein kinase c activity was found in the particulate fraction. The studies presented here did not quantify the amount of protein kinase C in the specific constituents of the particulate fraction. However, preliminary experiments indicate that approximately 20-25% of the protein kinase c in the particulate fraction was associated with the nuclear fraction. Additionally, the subcellular distribution of protein kinase C was found to be dependent on calcium. The implications of this are two-fold. First, the calcium concentration obtained during the homogenization of cell or tissue preparations has an important bearing on the final distribution of protein kinase C obtained in isolated cytosol and particulate fractions. Second, it is possible that the intracellular mobilization of ca 2 + may cause the translocation of protein kinase C independent of the production of DAG. This may provide an alternative means of activation of protein kinase C by receptor systems linked to calcium channels. It would be interesting to determine if r depolarization or ca 2 + ionophores act to alter the subcellular distribution of protein kinase C in the neuronal cultures.

PAGE 137

131 The primary reason for identifying and characterizing protein kinase C activity in neuronal cultures was to further investigate the role of this enzyme in the regulation of Ang II receptors. Past studies in which phorbol esters were used (Sumners et al., 1987) or where the hydrolysis of phosphoinositides was analyzed (Myers and Sumners, in press) implicated the involvement of protein kinase C in the short-tenn regulation of Ang II receptors. Phorbol esters, which activate protein kinase C by substituting for diacylglycerol (DAG), increase the number of Ang II receptors in culture. However, phorbol esters have many nonspecific biological effects which may occur independent of protein kinase C activation (see Chapter I). Additionally, phorbol esters have cellular actions which are not mimicked by DAG or synthetic DAGs (see Chapter I). For example, the increased expression of Ang II receptors elicited by phorbol esters does not occur using the synthetic DAG, 1-oleoyl-2-acetal glycerol (OAG) (Kalberg, unpublished results). Thus, further investigation was necessary to confinn the involvement of protein kinase C in the regulation of Ang II receptors. Several strategies were used to demonstrate the integral involvement of protein kinase c in the regulation of the expression Ang II receptors in neuronal cultures. These included the use of non phorbol ester activators of protein kinase c, the use of an antagonist of protein kinase c, and the depletion of protein kinase C activity in the neuronal cultures. Additionally, a

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132 qualitative understanding of the role of protein kinase C in the regulation of Ang II receptors was obtained. From the results obtained, it appears that the translocation of protein kinase C is required for an increased expression of Ang II receptors, that protein kinase c is not involved in the steady-state expression of Ang II receptors, and that not all the protein kinase C present under steady-state conditions is required for the stimulation of Ang II receptor expression. The determination of the integral involvement of protein kinase C in the regulation of Ang II receptors aids in the identification of the ho:rmone(s) or neurotransmitter(s) which may act to regulate Ang II receptors in the brain. As discussed in previous chapters, the hydrolysis of phosphoinositides and possibly phosphatidylcholine are linked to the activation of protein kinase C. Receptor agonists which stimulate the hydrolysis of these membrane phospholipids may act to regulate Ang II receptors. Several compounds act on brain slices and primary neuronal cultures to potently increase the hydrolysis of phosphoinositides. These include norepinephrine, epinephrine, carbachol, glutamate, and neurotensin (Gonzales et al., 1985; Myers and Sumners, in press; Weiss et al., 1988). However, only norepinephrine and epinephrine are known to alter the number of Ang II receptors in neuronal cultures. Both glutamate and carbachol have no effect on the binding of [u 6 I]Ang II in culture

PAGE 139

133 (Myers and Sumners, in press), while the effects of neurotensin remain to be tested. In the short term, the incubation of neuronal cultures with either norepinephrine or epinephrine for 15-60 min results in an increase in the number of Ang II receptors (Myers and Sumners, in press). This effect of norepinephrine and epinephrine is blocked by prazosin indicating the involvement of specific ~ 1 -adrenergic receptors (Myers and Sumners, in press). Additionally, the concentrations of norepinephrine and epinephrine (10-500 M) required to increase the number of Ang II receptors are similar to those required to significantly stimulate the hydrolysis of phosphoinositides (Myers and Sumners, in press). Thus, these studies and previous studies using phorbol esters (i.e., Sumners et al., 1987 and those presented in Chapter III) strongly suggest that the ~ 1 -adrenergic receptor is functionally linked to the activation of protein kinase c in primary neuronal cultures. This led to the investigation of the interaction between ~ 1 -adrenergic receptors and protein kinase C in culture. In these studies, it was determined that the treatment of neuronal cultures with epinephrine leads to the rapid and reversible translocation of protein kinase C from the cytosol to the particulate fraction. This effect resulted in a maximal decrease of 30% in the protein kinase C content of the cytosol fraction with an associated increase of approximately 15% in the particulate fraction. Additionally, the ~ 1 -adrenergic receptor antagonist, prazosin,

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134 blocked the stimulation of protein kinase C translocation by epinephrine indicating the involvement of specific ~ 1 -adrenergic receptors. Considering that less than 50% of the protein kinase C found in the unstimulated control condition is required for an increased expression of Ang II receptors (Chapter III), it is not surprising that a greater percent of the cellular protein kinase C was not involved in the epinephrine-stimulated translocation process. Additionally, it is unlikely that all of the "translocatable" protein kinase c in the neuronal cultures is functionally linked to only ~ 1 -adrenergic receptors. A time scale which summarizes the studies that implicate the activation of ~ 1 -adrenergic receptors and protein kinase C in the regulation of Ang II receptor expression is shown in Fig 6-1. Temporally, the sequence of events initiated by the activation of ~ 1 -adrenergic receptors occurs as expected. That is, during the maximal period of phosphoinositide hydrolysis the translocation of protein kinase C occurs which is followed by an increase in the number of Ang II receptors. Critical to the completion of the time course presented in Fig 6-1 is the measurement of the production of DAG production due to the activation of ~ 1 adrenergic receptors. Presumably, the production of DAG would precede the translocation of protein kinase C. It is interesting that the epinephrine-stimulated translocation of protein kinase c was no longer detected after 10 and 15 min despite the continued hydrolysis of inositol phospholipids. Thus, it is unlikely that

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135 0D PKC translocation by phorbol esters Oo ------------- t Ang II binding by phorbol esters 0D ------------ t Ang II binding by a 1 receptors PKC translocotion by a1 receptors ---- max. rote of Pl hydrolysis by a 1 receptors I I I I 0 5 10 15 30 time (min) 60 time points < 15 min not tested Figure 6-1. Time scale of events implicated in the upregulation of angiotensin II receptors.

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136 the transient nature of protein kinase C translocation was due to the inactivation of ~ 1 -adrenergic receptors. Slivka et al. (1988) reported a transient translocation of protein kinase C by epinephrine in spite of the continued hydrolysis of phosphatidylinositol and the production of DAG. They postulated this may be due to the negative feedback inhibition of protein kinase C by sphingolipid metabolites which are endogenous inhibitors of this enzyme. Alternatively, the autophosphorylation of protein kinase C, which occurs in the presence of ca 2 +, phospholipids, and diolein (Huang et al., 1986), may be a self regulatory mechanism involved in the inactivation of protein kinase C. However, it is presently not known what effect autophosphorylation has on the activity of this enzyme. Further inspection of Fig 6-1 reveals a slight time lag occurs between the translocation of protein kinase C and the increased expression of Ang II receptors produced by the stimulation of ~ 1 -adrenergic receptors. This may not be true as the effect of ~ 1 -adrenergic receptor-stimulation on Ang II receptor expression at time points less than 15 min has not been tested. Neverless, it is of interest to consider the mechanisms by which protein kinase C may act to increase in the number of Ang II receptors. As briefly discussed in Chapter III, a direct phosphorylation by protein kinase C of refractory Ang II receptors which permits ligand binding or the phosphorylation of a protein which "masks" Ang II receptors may be involved.

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137 Additionally, by several means, protein kinase C may alter the cycle of events which regulates the cell-surface expression of Ang II receptors. These include 1) a decrease in the internalization rate of Ang II receptors, 2) an increase in the externalization rate of Ang II receptors while the rate of internalization is unchanged or decreased, and 3) an increase in the externalizable pool of Ang II receptors. The direct phosphorylation of the Ang II receptor or a masking protein would likely occur very rapidly and not result in a time lag between the translocation of protein kinase C and the increased expression of Ang II receptors. However, an alteration in the kinetics of Ang II receptor cycling may involve a time lag depending on the rate of Ang II receptor cycling. If this does occur, the mechanisms may involve the phosphorylation of key transport proteins such as microtubules or the activation of enzyme(s) utilized in the processing of Ang II receptors. Certainly, futures studies are necessary to identify the mechanisms by which protein kinase C regulates the expression of Ang II receptors in neuronal cultures. In contrast to the upregulation of Ang II receptors by short-term (15-60 min) incubations with ~ 1 -adrenergic agonists, long-term (2-6 hrs) incubations with ~ 1 -adrenergic receptor agonists leads to the downregulation of Ang II receptors (Sumners et al., 1986b). These long-term effects may be explained two ways. First, the decreased number of Ang II receptors may simply

PAGE 144

138 reflect the overshoot of a rectification of the Ang II receptor levels. This compensatory mechanism would act to decrease Ang II receptors following the short-term stimulation of Ang II receptor expression. Second, the downregulation of Ang II receptors may be a separate regulatory mechanism. Investigations into the possible mechanisms involved in the downregulation of Ang II receptors showed that the calcium channel antagonist, nifedipine, at concentrations which inhibited ~ca 2 + uptake by 90%, blocked the decrease in the specific binding of [u 6 I]Ang II observed under depolarizing conditions. This suggests that ca 2 + influx through vscc initiates the downregulation of Ang II receptors. Thus, it was postulated that ~ 1 receptor agonists may act to downregulate Ang II receptors by acting at a receptor subtype <~1a> which is linked to the activation of voltage sensitive calcium channels (VSCC). The stimulation of ~ca 2 + uptake under depolarizing conditions occurred rapidly and reached equilibrium between 1 and 5 min. Conversely, a significantly decrease in the specific binding of [u 6 I]Ang II under depolarizing conditions was observed after 2-4 hrs. Thus, a considerable time lag exists between the influx of ca 2 + and decrease in the specific binding of [ 126 I]Ang II. A temporal comparison can be made between the initiation of intracellular events associated with the activation of muscarinic acetylcholine (mAch) receptors, which leads to their subsequent downregulation, and the rapid influx of ~Ca 2 + through VSCC which

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139 is associated with a decrease in [ 126 I]Ang II binding. The downregulation of muscarinic acetylcholine (mAch) receptors occurs after 9-18 hrs of incubation with carbachol in NG 108-15 cells and is associated with an internalization and degradation of the receptor (Ray et al., 1989). Additionally, the downregulation of mAch receptors occurs in spite of the rapid inactivation of the receptors within 15 min of agonist treatment. Thus, it is possible that the rapid influx of ca 2 + initiates the clustering, endocytosis and degradation of Ang II receptors and that this process takes several hours. The bidirectional regulation of Ang II receptors by ~ 1 adrenergic receptor agonists implies that the ~ 1 -adrenergic receptors which mediate the actions are heterogeneously distributed. Thus, ~ 1 -adrenergic receptors which act to upregulate Ang II receptors would be located on separate neurons than those ~ 1 -adrenergic receptors which have the opposite effects. However, this does not exclude the possibility that ~ 1 -adrenergic receptors with differential regulatory properties could not exist in separate regions of the same neuron (eg pre and post synaptically). Alternatively, if the downregulation of Ang II receptors is a rectification mechanism, this could result from the dual activation of VSCC and protein kinase C by the same ~ 1 -adrenergic receptor. This could occur by the following series of hypothetical events: the influx of Ca 2 + through vscc could

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140 initially act to enhance the actions of protein kinase C resulting in the increased expression of Ang II receptors and, in fact, ca 2 + was shown in Chapter II to increase the binding of protein kinase c to the plasma membrane. After this initial activation of protein kinase c, ca 2 + could act to inactivate protein kinase c through the calcium binding protein calmodulin which has been shown to inhibit protein kinase c-mediated phosphorylation of proteins (Albert et al., 1984; Saitoh and Dobkins, 1986). Finally, ca 2 + or ca 2 +-calmodulin activated kinases or phosphatases could initiate events leading to the internalization of surface Ang II receptors. If the regulation of Ang II receptors by ~ 1 -adrenergic receptors is analyzed on a larger scale, it appears that catecholamines may act as neuromodulators to alter the actions of Ang II in the brain. A couple of possible scenarios could exist if this is true. First, a noradrenergic neuron (or adrenergic) may act at a synapse between a presynaptic Ang II neuron and a postsynaptic neuron of any type to alter the number of Ang II receptors on the postsynaptic neuron via ~ 1 -adrenergic receptor activation. In this case, the effects of Ang II released presynaptically would be modulated on the postsynaptic neuron. For this to occur, the ~ 1 -adrenergic and Ang II receptors would have to be co-localized on the postsynaptic neuron and reside in close proximity. However, at the present, the co-localization of Ang II receptors and ~ 1 -adrenergic receptors on the same neuron

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141 has not been identified due to technical limitations. Further, other compounds (ie carbachol and glutamate) which stimulate the hydrolysis of phosphoinositides by acting at receptors other than ~ 1 -adrenergic receptors, but do not affect the binding of [ 125 I]Ang II, presumably are not co-localized with Ang II receptors. This is assuming that these compounds activate protein kinase C. An alternative scenario is that catecholamines act presynaptically to alter the number of presynaptic Ang II receptors. Ang II has been shown to facilitate the release of norepinephrine from the hypothalamus of rabbits and the A2 brainstem region of rats (Garcia-Sevilla et al., 1979; Meldrum et al., 1984) and to stimulate the uptake and metabolism of catecholamines in neuronal cultures (Sumners et al., 1986a; Sumners et al., 1987a). Any of these actions of Ang II may be due to the activation of presynaptic Ang II receptors and could be modulated by increasing or decreasing the number these receptors. This study has presented the hypothesis that changes in the number of Ang II receptors, specifically by the activation of ~ 1 adrenergic receptors, is a means by which the actions of Ang II are modulated in situ. However, critical to the advancement of this hypothesis is the determination that the Ang II receptors which are affected by phorbol esters or catecholamines are functional. This is currently being studied in our lab in spite of limitations due to the identification of a functional aspect

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142 of Ang II receptors which is not influenced by phorbol esters or catecholamines. In conclusion, this study has provided important insights into the mechanisms involved in the regulation of Ang II receptors in primary neuronal cultures. The calcium, phospholipid-dependent protein kinase, protein kinase c, was found in high levels in these cultures and was shown to be integrally involved in the stimulation of Ang II receptor expression. Additionally, epinephrine was found to transiently activate protein kinase C and this effect was blocked by the ~ 1 adrenergic receptor antagonist, prazosin. These results suggest that the short-tenn stimulation of Ang II receptor expression by ~ 1 -adrenergic receptor agonists is mediated by protein kinase c. Conversely, the influx of ca 2 + through voltage sensitive calcium channels was associated with a decrease in the specific binding of [u 6 I]Ang II. This suggests that receptor systems which are linked to the activation of voltage sensitive calcium channels may act to downregulate Ang II receptors.

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REFERENCES Aguilera, G. and catt, K. Regulation of angiotensin II receptors in the rat during altered sodium intake. Circ. Res. 49: 751758, 1981. Akaishi, T. Negoro, H. and Kabayasi, s. Responses of paraventricular and supraoptic units to angiotensin II, sar1Ile 6 angiotensin II and hypertonic NaCl administered to the cerebral ventricle. Brain Res. 198: 499-511, 1980. Akaishi, T., Negro, H. and Kobayasi, s. Electrophysiological evidence for multiple sites of action of angiotensin II for stimulating paraventricular neurosecretory cells in the rat. Brain Res. 220: 386-390, 1981. Albert, K.A., Wu, w. c-s, Nairn, A.C. and Greengard, P. Inhibition by calmodulin of calciumjphospholipid-dependent protein phosphorylation. P.N.A.S. 81: 3622-3625, 1984. Arcoleo, J.P., and Weinstein, I.B. Activation of protein kinase c by tumor promoting phorbol esters, teleocidin and aplysiatoxin in the absence of added calcium. carcinogenesis 6: 213-217, 1985. Ashdell, C.L., Staller, J.M. and Boutwell, R.K. Identification of a calcium-and phospholipid-dependent phorbol ester binding activity in the soluble fraction of mouse tissues. Biochem. Biophys. Res. Connn. 111: 340-345, 1983. Ashida, T., Ohuchi, Y., Saito, T. and Yazaki, Y. Effects of dietary sodium on brain angiotensin II receptors in spontaneously hypertensive rats. Jap. Circ. J. 46: 1328-1336, 1982. Ballester, R., and Rosen, O.M. Fate of innnunoprecipitable protein kinase C in Gffs cells treated with phorbol-12myristate-13-acetate. J. Biol. Chem. 260: 15194-15199, 1985. Batty, I.R., Nahorski, S.R. and Irvine, R.F. Rapid formation of inositol (1,3,4,5) tetrakisphosphate following muscarinic stimulation of rat cerebral cortical slices. Biochem. J. 232: 211-215, 1985. 143

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144 Bennett, J.P. and Snyder, S.H. Angiotensin II binding to mammalian brain membranes. J. Biol. Chem. 251: 7423-7430, 1976. Berridge, M.J. Inositol triphosphate and diacylglycerol as second messengers. Biochem. J. 220: 345-360, 1984. Berg, O.K., Kelly, R.B., Sargent, P.B., Williamson, P. and Hall, z.w. Binding of ~-bungarotoxin to acetylcholine receptors in mannnalian muscle. Proc. Natl. Acad. Sci. 69: 147-151, 1972. Bickerton, R.K. and Buckley, J.P. Evidence for a central mechanism in angiotensin induced hypertension. Proc. Soc. Exp. Biol. Med. 106: 834-836, 1961. Blumberg, P.M. In vitro studies on the mode of action of the phorbol esters, potent tumor promotors. CRC Rev. Toxicol. 8: 153-234, 1980. Booth, D. Mechanism of action of norepinephrine in eliciting an eating response on injection into the rat hypothalamus. J. Pharmacol. Exp. Therap. 160: 336-348, 1968. Braun-Menendez, E. and Page, I.H. Science 127: 242, 1958. Bumpus, F. M. Schwarz, H. and Page, I. H. Synthesis and pharmacology of the octapeptide angiotonin. Science 125: 886887, 1957. Burgess, S.K., Sahyoun, N., Blanchard, S.G., I.eVine III, H., Chang, K-J., and cuatrecasas, P. Phorbol ester receptors and protein kinase C in primary neuronal cultures: Developement and stimulation of endogenous phosphorylation. J. Cell Biol. 103: 312-319, 1986. Campbell, O.J. and Habener, J.F. Angiotensinogen gene is expressed and differentially regulated in multiple tissues of the rat. J. Clin. Invest. 78: 31-39, 1986. castagna, M. Takai, Y. Kaibuchi, K. Sano, K. Kikkawa, u. and Nishizuka, Y. Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor promoting phorbol esters. J. Biol. Chem. 257: 7847-7851, 1982. Chen, F.C.M. and Printz, M.P. Chronic estrogen treatment reduces angiotensinogen II receptors in the anterior pituitary. Endocrinology 113: 1503-1510, 1983. Chida, K., Kato, N. and Kuroki, T. Down regulation of phorbol diester receptors by proteolytic degradation of protein kinase Cina cultured cell line of fetal rat skin keratinocytes. J. Biol. Chem. 261: 13013-13018, 1986.

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145 Cole, F.E., Blakesley, H.L., Graci, K.A., Frohlich, E.D. and MacPhee, A.A. Brain angiotensin II receptor affinity and capacity in SHR and WKY rats: effects of acute dietary changes in NaCl. Brain Res. 190: 272-277, 1980. Couturier, A. Bazgar, S. and castagna, M. Further characterization of tumor-promoter-mediated activation of protein kinase c. Biochem. Biophys. Res. Conun. 121: 448-455, 1984. Crews, F.T., Raulli, R., Gonzales, R., Sumners, c. and Raizada, M.K. ~ 1 -adrenergic receptor stimulated responses. J. cardiovasc. Pharmacol. 11: S99-S106, 1988. Delcos, B.K., Nagle, D.S. and Blumberg, P.M. Specific binding of phorbol ester tumor promoters to mouse skin. Cell 19: 10251032, 1980. Devynk, M.A., Pernollet, M.G., Meyer, P., Fermandjian, s. and Fromageot, P. Solubilization of angiotensin II receptors in rabbit aortae membranes. Nature (Lend.) 249: 67-69, 1974. Devynk, M.A., Rauzaire-Dubois, B., Chevillotte, E. and Meyer, P. Variation in the number of uterine angiotensin receptors following changes in plasma angiotensin levels. Eur. J. Pharm. 40: 27-37, 1976. Douglas, J.G. Corticosteroids decrease glomerular angiotensin receptors. Am. J. Physiol. 252: F453-F457, 1987. Douglas, J.G. and Brown, G.P. Effects of prolonged low dose infusion of angiotensin II and aldosterone on rat smooth muscle and adrenal angiotensin II receptors. Endocrinology 111: 988-992, 1982. Drieger, P.E. and Blumberg, P.M. Specific binding of phorbol ester tumor promoters. Proc. Natl. Acad. Sci. 77: 567-571, 1980. Drust, D.S. and Martin, T.F.J. Protein kinase C translocates from cytosol to membrane upon hormone activation: Effects of thyrotropin-releasing hormone in Gffs cells. Biochem. Biophys. Res. Conun. 128: 531-537, 1985. Dusterdiek, G. and McElwee, G. Iodination of angiotensin II and purification of the labelled hormone. In: Radioinnnunoassay Methods., edited by K.E. Kirkham and W.M. Hunter. London: Churchill-Livingston. 1971, p. 24-30.

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Dzau, V.J., Ingelfinger, J., Pratt, R.E. and Ellison, K.E. Identification of renin and angiotensinogen messenger RNA sequences in mouse and rat brains. Hypertension 8: 544-548, 1986. 146 Epstein, A.N., Fitzsimons, J.T. and Johnson, A.K. Peptide antagonists of the renin-angiotensin system and the elucidation of the receptors for angiotensin-induced drinking. J. Physiol. 238: 34-35, 1973. Epstein, A.N., Fitzsimons, J.T. and Rolls, B.J. Drinking caused by the intracranial injection of angiotensin into the rat. J. Physiol. (London). 200: 98-100, 1969. Farrago, A., Seprodi, J. and Spat, A. Subcellular distribution of protein kinase C in rat adrenal glomerulosa cells. Biochem. Biophys. Res. Comm. 156: 628-633, 1988. Farrar, W.L. and Anderson, W.B. Interleukin-2 stimulates association of protein kinase C with plasma membrane. Nature 315: 233-235, 1985. Farrar, W.L., Thomas, T.P. and Anderson, W.B. Altered cytosol/membrane enzyme redistribution on interleukin-3 activation of protein kinase C. Nature 315: 235-237, 1985. Feldstein, J.B., Pacitti, A.J., Sumners, C. and Raizada, M.K. Alpha 1-adrenergic receptors in neuronal cultures from rat brain: Increased expression in the spontaneously hypertensive rat. J. Neurochem. 47: 1190-1198, 1986a. Feldstein, J.B., Sumners, C. and Raizada, M.K. Sodium increases the angiotensin II receptors in neuronal cultures from brains of normotensive and hypertensive rats. Brain Res. 370, 265272, 1986b. Finkielman, s., Fisher-Ferraro, L., Diaz, A., Goldstein, P.J. and Nahmod, V.E. A presser substance in the cerebral spinal fluid of normotensive and hypertensive patients. Proc. Natl. Acad. Sci. 69: 3341-3344, 1972. Fischer-Ferraro, c., Nahmod, V.E., Goldstein, D.J. and Finkielman, s. Angiotensin and renin in rat and dog brain. J. Exp. Med. 133: 353-361, 1971. Fluharty, S.J. and Epstein, A.N. Sodium appetite elicited by angiotensin II in the rat: synergistic interaction with systemic mineralocorticoids. Behav. Neurosci. 97: 746-758, 1983.

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147 Fregly, M.J., Rowland, N.E., Sumners, C., and Gordon, D.B. Reduced dipsogenic responsiveness to intracerebroventricularly administered angiotensin II in estrogen-treated rats. Brain Res. 338: 115-121, 1985. Fuxe, K., Ganten, D. and Hokfelt, T. Renin-like immunocytochemical activity in the rat and mouse brain. Neurosci. Lett. 18: 245-250, 1980. Ganten, D., Hennann, K., Bayer, c., Unger, T. and Lang, R.E. Angiotensin synthesis in the brain and increased turnover in hypertensive rats. Science 221: 869-871, 1983. Ganten, D. Marquez, J. A. Granger, P. Hayduk, K. Karsunisky, K.D. Boucher, R. and Genest, J. Renin in dog brain. Am. J. Physiol. 221: 1733-1737, 1971. Garcia-Sevilla, J.A., Dubocovich, M.L. and Langer, s.z. Angiotensin II facilitates the potassium evoked release of 3H noradrenaline from the rabbit hypothalamus. Eur. J. Pharmacol. 56: 171-176, 1979. Gehlert, D.R., Speth, R.C. and Wamsley, J.K. Distribution of [ 125 1] angiotensin II binding sites in the rat brain: A quantitative autoradiographic study. Neurosci. 18: 837-856, 1986. Glossman, H., Baukal, A.J. and catt, K.J. Properties of angiotensin II receptors in the bovine and rat adrenal cortex J. Biol. Chem. 249: 825-836, 1974. Goldblatt, H., Lynch, J., Hanzel, R.F. and Summerville, W.W. Studies on experimental hypertension. I. The production of persistent elevation of systolic blood pressure by means of renal ischemia. J. Exp. Med. 59: 347-380, 1934. Gonzales, R.A. and Crews, F.T. Cholinergicand adrenergic stimulated inositide hydrolysis in brain: Interaction, regional distribution, and coupling mechanisms. J. Neurochem. 45: 10761084, 1985a. Gonzales, R.A., Crews, F.T., Sumners, C. and Raizada, M.K. Norepinephrine regulation of alpha-1 receptors and alpha-1 receptor stimulated phosphoinositol hydrolysis in primary neuronal cultures. J. Pharmacol. Exp. Therap. 242: 764-771, 1987. Gonzales, R.A., Feldstein, J.B., Crews, F.T. and Raizada, M.K. Receptor-mediated inositol hydrolysis is a neuronal response: Comparison of primary neuronal and glial cultures. Brain Res. 345: 350-355, 1985b.

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148 Gould, R.J., Murphy, K.M. and Snyder, S.H. Autoradiographic localization of calcium channel antagonist receptors in rat brain with [8H]nitrendipine. Brain Res. 330: 217-223, 1985. Gronan, R.J. and York, D.H. Effects of angiotensin II and acetylcholine on neurons in the preoptic area. Brain Res. 154: 172-177, 1978. Gutkind, J .s., Kurihara, M. and Saavedra, J.M. Increased angiotensin II receptors in brain nuclei of DOCA-salt hypertensive rats. Am. J. Physiol. 255: H646-H650, 1988. Halsey, D.L., Girard, P.R., Kuo, J.F. and Blackshear, P.J. Protein kinase c in fibroblasts. J. Biol. Chem. 262: 22342243, 1987. Han, C. Abel, P. W. and Minneman, K. P. o: 1 -adrenoceptor subtypes linked to different mechanisms for increasing intracellular ca 2 + in smooth muscle. Nature 329: 333-335, 1987. Hauger, R.C., Aguilera, G., catt. K.J. Angiotensin II regulates its receptor sites in the adrenal glomerulosa zone. Nature 271: 176-178, 1978. Hawkins, R.L. and Printz, M.P. Distribution of angiotensinogen in Battleboro rat brain. Brain Res. Bull. 10: 163-166, 1983. Healy, D.P. and Printz, M.P. Angiotensinogen levels in the brain and cerebral spinal fluid of genetically hypertensive rats. Hypertension 7: 752-759, 1985. Hermann, K., McDonald, w., Unger, T., Lang, R.E. and Ganten, D. Angiotensin biosynthesis and concentrations in brain of normotensive and hypertensive rats. J. Physiol. (Paris) 79: 471-480, 1984. Hermann, K., Phillips, M.I., Hilgenfeldt, U. and Raizada, M.K. Biosynthesis of angiotensinogen and angiotensins by brain cells in primary cultures. J. Neurochem. 51: 398-405, 1988b. Hermann, K., Raizada, M.K., Sumners, C. and Phillips, M.I. Presence of renin in primary neuronal and glia cells from rat brain. Brain Res. 437: 205-213, 1987. Hermann, K., Raizada, M.K., Sumners, c. and Phillips, M.I. Immunocytochemical and biochemical characterization of angiotensin I and II in cultured neuronal and glial cells from rat brain. Neuroendocrinology 47: 125-132, 1988a. Hidaka, H. and Hagiwara, M. Pharmacology of the isoquinaline sulfonamide protein kinase c inhibitors. Trends Pharmacol. Sci. 8: 162-164, 1987.

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149 Hidaka, H., Inagaki, s., Kawamoto, s. and Sasaki, Y. Isoquinalinesulfonamides, novel and potent inhibitors of protein kinase and protein kinase c. Biochem. 23: 5036-5041, 1984. Hirota, K., Hirota, T., Aguilera, G. and catt, K.J. Honnone induced redistribution of calcium-activated phospholipid dependent protein kinase in pituitary gonadotrophs. J. Biol. Chem. 260: 3243-3246, 1985. Hoffman, W.E. and Phillips, M.I. Regional study of cerebral ventricular sensitive sites to angiotensin II. Brain Res. 110: 313-330, 1976. Huang, F.L., Yoshida, Y., CUnha-Melo, J.R., Beaven, M.A. and Huang, K-P. Differential down-regulation of protein kinase C isozymes. J. Biol. Chem. 264: 4238-4243, 1989. Huang, F.L., Yoshida, Y., Nakabayashi, H. and Huang, F-P. Differential distribution of protein kinase C isozymes in various regions of brain. J. Biol. Chem. 262: 15714-15720, 1987a. Huang, F.L., Yoshida, Y., Nakabayashi, H., Knop, J.L., Young, s.w., III. and Huang, K-P. Inununochemical identification of protein kinase C isozymes as products of discrete genes. Biochem. Biophys. Res. Conun. 149: 946-952, 1987b. Huang, K-P., Huang, F.L., Nakabayashi, H. and Yoshida, Y. Biochemical characterization of rat brain protein kinase C isozymes. J. Biol. Chem. 263: 14839-14845, 1988. Huang, K-P., Nakabayashi, H. and Huang, F.L. Isozymic forms of rat brain ca 2 +-activated and phospolipid-dependent protein kinase. Proc. Natl. Acad. Sci. 83: 8535-8539, 1986. Hunan, Y.A. and Bell, R.M. Lysosphingolipids inhibit protein kinase C: Implications for sphingolipidoses. Science 235: 670674, 1987. Hunan, Y.A., Loomis, C.R., Merrill ,A.H., Jr. and Bell. R.M. Sphingosine inhibition of protein kinase c activity and of phorbol dibutyrate binding in vitro and in human platelets. J. Biol. Chem. 261: 12604-12609, 1986. Hutchinson, J.S., Schelling, P. and Ganten, D. Effect of centrally administered angiotensin II and Pll3 on blood pressure in conscious rats. Pflugers Arch. Eur. J. Physiol. 355 (suppl.): R28, 1975. Huwyler, T. and Felix, D. Angiotensin II-sensitive neurons in septal area of rats. Brain Res. 195: 187-195, 1980.

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150 Inagaki, M., Kawamoto, s. and Hidaka, H. Serotonin secretion from human platelets may be modified by ca 2 +-activated, phospholipid-dependent myosin phosphorylation. J. Biol. Chem. 259: 14321-14323, 1984. Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. studies on a cyclic nucleotide-independent protein kinase and its proenzyme im maimnalian tissues. J. Biol. Chem. 252: 7610-7616, 1977. Israel, A., Plunkett, L.M. and Saavedra, J.M. Quantitative autoradiographic characterization of receptors for angiotensin II and other neuropeptides in individual brain nuclei and peripheral tissues from single rats. Cell. Mol. Neurobiol. 5: 211-222, 1985. Jaken, s. and Kiley, S.C. Purification and characterization of three types of protein kinase C from rabbit brain cytosol. Proc. Natl. Acad. Sci. 84: 4418-4422, 1987. Jeffery, A.M. and Liskamp, R.M.J. Computer-assisted molecular modelling of tumor promoters rational for the activity of phorbol esters, teleocidin Band aplysiatoxin. Proc. Nat. Acad. Sci. 83: 241-245, 1986. Jonklaas, J. and Buggy, J. Angiotensin-estrogen central interaction: localization and mechanism. Brain Res. 326: 239249, 1985. Kalinyak, J.E. and Perlman, A.J. Tissue specific regulation of angiotensinogen mRNA accumulation by dexamethasone. J. Biol. Chem. 262: 460-464, 1987. Kebabian, J.W., Zatz, M., Romero, J.A. and Axelrod, J. Rapid changes in rat pineal ~-adrenergic receptor: Alterations in (1I)alprenolol binding and adenylate cyclase. Proc. Natl. Acad. Sci. 72: 3735-3739, 1975. Keller-Wood, M., Stenstrom, B., Shinsako, J. and Phillips, M.I. Interaction between CRF and Ang II in control of ACTH and adrenal steroids. Am. J. Physiol. 250: R396-R402, 1986. Kendall, D.A., Brown, E. and Nahorski, S.R. ~ 1 -adrenoceptor stimulated-mediated inositol phospholipid hydrolysis in rat cerebral cortex: Relationship between receptor occupancy and response and effects of denervation. Eur. J. Pharmacol. 114: 41-52, 1985. Kikkawa, U., Takai, Y., Minakuchi, R., Inohara, s. and Nishizuka, Y. calcium-activated, phospholipid-dependent protein kinase from rat brain. J. Biol. Chem. 257: 13341-13348, 1982.

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151 Kikkawa, U., Takai, Y., Tanaka, Y., Miyake, R. and Nishizuka, Y. Protein kinase Casa possible receptor protein of tmnor promoting phorbol esters. J. Biol. Chem. 258: 11442-11445, 1983. King, S.J., Harding, J.W. and Moe, K.E. Elevated salt appetite and brain binding of angiotensin II in mineralocorticoid treated rats. Brain Res. 448: 140-149, 1988. Kishimoto, A., Mikawa, K., Hashimoto, K., Yashudo, I., Tanaka, s., Tominaga, M., Kuroda, T. and Nishizuka, Y. Limited proteolysis of protein kinase c subspecies by calcimn dependent neutral protease (calpain). J. Biol. Chem. 264: 4088-4092, 1989. Kishimoto, A., Takai, Y., Mori, T., Kikkawa, u. and Nishizuka, Y. Activation of calcimn and phospholipid-dependent protein kinase by diacylglycerol. Its possible relation to phosphatidylinositol turnover. J. Biol. Chem. 255: 2273-2276, 1980. Kiss, z., Deli, E., Girard, P.R., Pettit, G.R. and Kuo, J.F. Comparative effects of polymyxin B, phorbol ester and bryostatin on protein kinase c translocation, phospholipid metabolism and differentiation of HL60 cells. Biochem. Biophys. Res. Conun. 146: 208-215, 1987. Kiss, Z. Deli, E. and Kuo. J. F. Temporal changes in intracellular distribution of protein kinase C during differentiation of hmnan leukemia HL 60 cells induced by phorbol ester. Febs. Iett. 231: 41-46, 1988. Kraft, A.S. and Anderson, W.B. Phorbol esters increase the amount of ca 2 +, phospholipid-dependent protein kinase associated with plasma membrane. Nature 301: 621-623, 1983a. Kraft, A.S. and Anderson, W.B. Characterization of cytosolic calcimn-activated phospholipid-dependent protein kinase activity in embryonic carcinoma cells. J. Biol. Chem. 258: 9178-9183, 1983b. Kraft, A.S., Anderson, W.B., Cooper, H.L. and Sando, J.J. Decrease in cytosolic calciumjphospholipid-dependent protein kinase activity following phorbol ester treatment of EL4 thymoma cells. J. Biol. Chem. 257: 13193-13196, 1982. Kreutter, D., caldwell, A.B. and Morin, M.J. Dissociation of protein kinase C activation from phorbol ester-induced maturation of HL-60 leukemia cells. J. Biol. Chem. 260: 59795984, 1985.

PAGE 158

152 Kumar, A., Rassoli, A. and Raizada, M.K. Angiotensinogen gene expression in neuronal and glial cells in primary cultures of rat brain. J. Neurosci. Res. 19: 287-290, 1988. Kuo, J.F., Andersson, R.G.G., Wise, B.C., Mackerlova, L., Salomonsson, I., Brackett, N.L., Katoh, N., Shoji, M. and Wrenn, R.R. calcium-dependent protein kinase: Widespred occurrence in various tissues and phyla of the animal kingdom and comparison of effects of phospholipid, calmodulin, and trifluoroperazine. Proc. Natl. Acad. Sci. 77: 7039-7043, 1980. Lang, u. and Vallotton, M.B. Angiotensin II but not potassium induces subcellular redistribution of protein kinase C in bovine adrenal glomerlulosa cells. J. Biol. Chem. 262: 80478050, 1986. Leach, K.L., James, M.C. and Blumberg, P.M. Characterization of a specific phorbol ester aporeceptor in mouse brain cytosol. Proc. Natl. Acad. Sci. 80: 4208-4212, 1983. Lentz, K.E., Skeggs, L.T., Woods, K.R., Kahn, J.R. and Shumway, N.P. The amino acid composition of hypertensin II and its biochemical relationship to hypertensin I. J. Exp. Med. 104: 183-191, 1956. Lewicki, J.A., Fallon, J.H. and Printz, M.P. Regional distribution of angiotensinogen in rat brain. Brain Res. 158: 359-371, 1978. Lind, R.W., Swanson, L.W. and Ganten, D. Organization of angiotensin II innnunoreactive cells and fibers in the rat central nervous system. Neuroendocrinolology 40: 2-24, 1985. Lowry, O. H. Rosenbourough, N. J. Farr, A. L. and Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 165-175, 1951. Lynch, K.R., Simnad, V.I., Ben-Ari, E.T. and Garrison, J.C. Localization of angiotensinogen mRNA sequences in rat brain. Hypertension 8: 540-543, 1986. Malvin, R.C., Mouw, D. and Vander, A.J. Angiotensin: physiological role in water-deprivation-induced thirst in rats. Science 197: 171-173, 1977. Mann, J.F.E., Schiffrin, E.L., Schiller, P.W., Rascher, w., Boucher, R. and Genest, J. Central actions and brain receptor binding of angiotensin II: influence of sodium intake. Hypertension 2: 437-443, 1980.

PAGE 159

153 Maran, J.W. and Yates, F.E. Cortisol secretion during intrapituitary infusion of angiotensin II in conscious dogs. Am. J. Physiol. 233: E273-E285, 1977. Matthies, H.J.G., Palfrey, H.C., Hirning, L.D. and Miller, R.J. Downregulation of protein kinase C in neuronal cells: Effects on neurotransmitter release. J. Neuroscience 7: 1198-1206, 1987. Mazzei, G.J., Katoh, N. and Kuo, J.F. Polymyxin Bis a more selective inhibitor of phospholipid-sensitive ca 2 +-dependent protein kinase than for calmodulin-sensitive ca 2 +-dependent protein kinase. Biochem. Biophys. Res. Comm. 109: 1129-1133, 1982. McDonald, J.R. and Walsh, M.P. Regulation of protein kinase C activity by natural inhibitors. Biochem. Soc. Trans. 14: 585586, 1986. McGilvery, R.W. In: Biochemistry. a functional approach Ed. McGilvery, R.W. W.B. Saunders, Philadelphia, PA, p. 377, 1983. McGraw, T.E., Dunn, K.W. and Maxfield, F. Phorbol ester treatment increases the exocytotic rate of the transferrin receptor recycling pathway independent of serine-24 phosphorylation. J. Cell Biol. 106: 1061-1066, 1988. Meldrum, M.J., Xue, c-s, Badino, L. and Westfall, T.C. Angiotensin facilitation of noradrenergic neurotransmission in central tissues of the rat: effects of sodium restriction. J. cardiovasc. Pharmacol. 6: 989-995, 1984. Mendelsohn, F.A.O., Aguilera, G., Saavedra, J.M., Quirrion, R. and catt, K.J. Characteristics and regulation of angiotensin II receptors in pituitary, circurnventricular organs and kidney. Clin. Exp. Hypertension (A] A5: 1081-1087, 1983. Mendelsohn, F.A.O., Quirion, R., Saavedra, J.M., Aguilera, G. and catt, K.J. Autoradiographic localization of angiotensin II receptors in rat brain. Proc. Natl. Acad. Sci. 81: 1575-1579, 1984. Messing, R.O., Stevens, A.M., Kiyasu, E. and Sneade, A.B. Nicotinic and muscarinic agonists stimulate rapid protein kinase C translocation in PC-12 cells. J. Neurosci. 9: 507512, 1989. Mickey, J., Tate, R., Mullikin, P. and Lefkowitz, R.J. Regulation of adenylate cyclase-coupled-beta adrenergic receptor binding sites by beta adrenergic catecholamines in vitro. Mol. Pharmacol. 12: 409-419, 1976.

PAGE 160

154 Miledi, R. and Potter, L.T. Acetylcholine receptors in muscle fibers. Nature 233: 599-603, 1971. Miller, R.J. Multiple calcium channels and neuronal function. Science 235: 46-52, 1986. Minneman, K.P. and Johnson, R.P. Characterization of alpha-1 adrenergic receptors linked to [3:HJinositol metabolism in rat cerebral cortex. J. Phannacol. Exp. Ther. 230: 317-323, 1984. Mochly-Rosen, Basbaum, A.I. and Koshland, D.E., Jr. Distinct cellular and regional localization of inununoreactive protein kinase C in rat brain. Proc. Natl. Acad. Sci. 84: 4660-4664, 1987. Morrow, A. L. and Creese, I. Characterization of o: 1 -adrenergic receptor subtypes in the brain: A reevaluation of [3:HJ4104 and [3:H]prazosin binding. Molecular Phannac. 29: 321-330, 1986. Mudd, L.M. Regulation of insulin effector systems in the brain. Doctoratal dissertation. Univ. of Florida, 1989. Mukherjee, c., caron, M.G. and Lefkowitz, R.J. catecholamine induced subsensitivity of adenylate cyclase associated with loss of p-adrenergic receptor binding sites. Proc. Natl. Acad. Sci. 72: 1945-1949, 1975. Myers, L. M. and Sumners, c. Regulation of angiotensin II binding sites in neuronal cultures by catecholamines. Am. J. Physiol. 257: (Cell Physiol. 26), in press. Neary, J.T., Norenberg, L.O.B. and Norenberg, M.D. Protein kinase C in primary astrocyte cultures: Cytoplasmic localization and translocation by a phorbol ester. J. Neurochem. 50: 1179-1184, 1988. Nishizuka, Y. Studies and perspectives of protein kinase C. Science 233: 305-312, 1986. Nowycky, M.C., Fox, A.P. and Tsien, R.W. Three types of neuronal calcium channel with different calcium agonist sensitivity. Nature 316: 440-443, 1985. Ohkumbo, H., Nakayama, K., Tanaka, T. and Nakanishi, S. Tissue distribution of rat angiotensinogen mRNA and structural analysis of its heterogeneity. J. Biol. Chem. 261: 319-323, 1986. Ono, Y., Fujii, T., Ogita, K., Kikkawa, u., Igarashi, K. and Nishizuka, Y. The structure, expression, and properties of additional members of the protein kinase c family. J. Biol. Chem. 263: 6927-6933, 1988.

PAGE 161

155 Palkovits, M. and Brownstein, M.J. catecholamines in the central nervous system. In: Handbook of experimental pharmacology: catecholamines II. Eds. Trendelenburg, u. and Weiner, N. Springer-Verlag, New York, p. 6-11, 1989. Pandol, S.J. and Schoefield, M.S. 1,2-diacylglycerol, protein kinase c and pancreatic enzyme secretion. J. Biol. Chem. 261: 4438-4444, 1986. Peach, M.J. Renin-angiotensin system: biochemistry and mechanisms of action. Physiol Rev. 57: 313-370, 1977. Phillips, M.I. Biological effects of angiotensin in the brain. In: Enzymatic release of vasoactive peptides. Eds. Gross, F. and Vogel, G. Raven Press, New York, p. 339, 1980. Phillips, M.I. Functions of angiotensin in the central nervous system. Ann. Rev. Physiol. 49: 413-435, 1987. Phillips, M.I. and Felix, D. Specific angiotensin II receptive neurons in the cat subfornical organ. Brain Res. 109, 531-540, 1976. Phillips, M.I. and Stenstrom, B. Angiotensin II co-migrates with authentic angiotensin II in high pressure liquid chromatography. Circ. Res. 56: 212-219, 1985. Phillips, W.A., Fujiki, T., Rossi, M.W., Korchak, H.M. and Johnson, R.B., Jr. Influence of calcium on the subcellular distribution of protein kinase C in human neutrophils. J. Biol. Chem. 264: 8361-8365, 1989. Quirion, R. Autoradiographic localization of calcium channel antagonist, [3HJnitrendipine, binding sites in rat brain. Neurosci. Lett. 36: 267-271, 1983. Raizada, M.K., Morse, C.A., Gonzales, R.A., Crews, F.T. and Sumners, c. Receptors for phorbol esters are primarily localized in neurons: Comparison of neuronal and glial cultures. Neurochem. Res. 14: 51-56, 1988. Raizada, M.K., Muther, T.F. and Sumners, c. Increased angiotensin II receptors in neuronal cultures from hypertensive rat brain. Am. J. Physiol. 247: C364-C372, 1984. Ray, P., Middleton, w. and Berman, J.D. Mechanism of agonist induced down-regulation and subsequent recovery of muscarinic acetylcholine receptors in a clonal neuroblastoma X glioma hybrid cell line. J. Neurochem. 52: 402-409, 1989.

PAGE 162

156 Richards, E.M., Sumners, c., Chou, Y.C., Raizada, M.K. and Phillips, M.I. ~-2 adrenergic receptors in neuronal and glial cultures: Characterization and comparison. J. Neurochem. 53: 287-296, 1989. Routledge, c. and Marsden, C.A. Adrenaline in the CNS: In vivo evidence for a functional pathway innervating the hypothalamus. Neurophann. 26: 823-830, 1987. Saavedra, J.M., Israel, A., Plunkett, L.M., Kurihara, M., Shigematsu, K. and Correa, F.M.A. Quantitative distribution of angiotensin II binding sites in rat brain by autoradiography. Peptides 7: 679-687, 1986. Saitoh, T. and Dobkins, K.R. Protein kinase C in human brain and its inhibition by calmodulin. Brain Res. 379: 196-199, 1986. Scatchard, G. The attraction of protein for small molecules and ions. Ann. N.Y. Acad. Sci. 51: 660-672, 1949. Schiffrin, E.C., Franks, D.J. and Genest, J. Aldosterone up regulates vascular angiotensin II receptors in vivo and in cultured smooth muscle cells in vitro. Clin. Res. 31: 688A, 1983. Schiffrin, E.C., Gutkowska, J. and Genest, J. Effect of angiotensin II and deoxycorticosterone infusion on vascular angiotensin II receptors in rats. Am. J. Physiol. 246: H608H614, 1984. Schirar, A., capponi, A. and catt, K.J. Regulation of uterine angiotensin II receptors by estrogen and progesterone. Endocrinology 106: 5-12, 1980. Schwantke, N. and Le Peuch, c. J. A protein kinase C inhibitory activity is present in rat brain homogenate. Febs. Lett. 177: 36-40, 1984. Sekiguchi, K., Tsukuda, M., Ogita, K., Kikkawa, U. and Nishizuka, Y. Three distinct forms of rat brain protein kinase C: Differential response to unsaturated fatty acids. Biochem. Biophys. Res. Cornn. 145: 797-802, 1987. Severs, W.B. and Daniels-Severs, A.E. Effects of angiotensin on the central nervous system. Phannacol. Rev. 25: 415-449, 1973. Severs, W.B., Daniels, A.E. and Buckley, J.P. On the central hypertensive effect of angiotensin II. Int. J. Phannacol. 6: 199-205, 1967.

PAGE 163

Severs, W.B., Sunnny-long, J., Taylor, J.S. and Connor, J.D. A central effect of angiotensin: Release of pituitary pressor material. J. Pharmacol. Exp. Therap. 174: 27-34, 1970. Shearman, M.S., Naor, z., Sekiguchi, K., Kishimoto, A. and Nishizuka, Y. Selective activation of the 1-subspecies of protein kinase C from bovine cerebellum by arachidonic acid and its lipoxygenase metabolites. Febs. I.ett. 243: 177-182, 1989. Simonnet, G., Bioulac, B., Rodriguez, F. and Vincent, J.D. 157 Evidence for the direct action of angiotensin II on neurons in the septum and in the medial preoptic area. Pharmacol. Biochem. Behav. 13: 359-363, 1980. Simonnet, G. and Vincent, J.D. Characterization of angiotensin II binding sites in the neostriatum of the rat brain. Neurochem. Int. 4: 124-155, 1982. Singh, R., Harding, J.W. and Speth, R.C. Effect of intraventricular infusion of an angiotensin II antagonist on u 6 I-angiotensin II binding in rats. Eur. J. Pharmacol. 120: 319-327, 1986. Singh, R., Husain, A., Ferraro, C. and Speth, R.C. Rat brain angiotensin II receptors: Effects of intracerebroventricular angiotensin II infusion. Brain Res. 303: 133-139, 1984. Sirett, N.E., Mclean, A.S., Bray, J.J. and Hubbard, J.I. Distribution of angiotensin II receptors in rat brain. Brain Res. 122: 299-312, 1977. Skeggs, L.T., Kahn, J.R. and Shumway, N.P. The purification of hypertensin II. J. Exp. Med. 103: 295-307, 1956a. Skeggs, L.T., Lentz, K.E., Kahn, J.R., Shumway, N.P. and Woods, K.R. The amino acid sequence of hypertensin II. J. Exp. Med. 104: 193-197, 1956b. Skeggs, L.T., Marsh, W.H., Kahn, J.R. and Shumway, N.P. The existence of two forms of hypertensin. J. Exp. Med. 99: 275282, 1954a. Skeggs, L.T., Marsh, W.H., Kahn, J.R. and Shumway, N.P. The purification of hypertensin I. J. Exp. Med. 100: 363-370, 1954b. Sladek, C.D. and Joynt, R.J. Angiotensin stimulation of vasopressin release from the rat hypothalamo-neurohypophyseal system in organ culture. Endocrinology. 104: 148-153, 1979.

PAGE 164

158 Slivka, S.R., Meier, K.E. and Insel, P.A. ~ 1 -adrenergic receptors promote phosphatidylcholine hydrolysis in MDCK-D1 cells. J. Biol. Chem. 263: 12242-12246, 1988. Speth, R.C., Singh, R., Smeby, R.R., Ferrario, C.M., and Husain, A. Restricted dietary sodium intake alters peripheral but not central angiotensin II receptors. Neuroendocrinology 38: 387392, 1984. Spinedi, E. and Negro-Vilar, A. Angiotensin II and ACI'H release: Site of action and potency relative to corticotropin-releasing factor and vasopressin. Neuroendocrinology 37: 446-453, 1983. Sudgen, D., Vanecek, J., Klein, D.C., Thomas, T.P. and Anderson, W.B. Activation of protein kinase C potentiates isoprenaline induced cyclic AMP accumulation in rat pinealocytes. Nature 314: 359-361, 1985. Sumners, C. and Fregly, M.J. Modulation of angiotensin II binding sites in neuronal cultures by mineralocorticoids. Am. J. Physiol. 256: Cl21-Cl29, 1989. Sumners, c., Muther, T.F. and Raizada, M.K. Altered norepinephrine uptake in neuronal cultures from spontaneously hypertensive rat brain. Am. J. Physiol. 248: C488-C497, 1985. Sumners, c., Phillips, M.I. and Raizada, M.K. Angiotensin II stimulates changes in the norepinephrine content of primary cultures of rat brain. Neurosci. I..ett. 36: 305-309, 1983. Sumners, C. and Raizada, M.K. catecholamine-angiotensin II receptor interaction in primary cultures of rat brain. Am. J. Physiol. 246: C502-C509, 1984. Sumners, c. and Raizada, M.K. Angiotensin II stimulates norepinephrine uptake in hypothalamus-brainstem neuronal cultures. Am. J. Physiol. 250: C236-C244, 1986a. Sumners, c., Reuth, S.M., Crews, F.T. and Raizada, M.K. Protein kinase C agonists increase the expression of angiotensin II receptors in neuronal cultures. J. Neurochem. 48: 1954-1961, 1987b. Sumners, C., Reuth, S.M., Myers, L.M., Kalberg, C.J., Crews, F.T. and Raizada, M.K. Phorbol ester-induced upregulation of angiotensin II receptors in neuronal cultures is potentiated by a calcium ionophore. J. Neurochem. 51: 153-162, 1988. Sumners, c., Shalit, S.L., Kalberg, C.J. and Raizada, M.K. Norepinephrine metabolism in neuronal cultures is increased by angiotensin II. Am. J. Physiol. 252: C650-C656, 1987a.

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159 Sumners, C., Watkins, L.L. and Raizada, M.K.
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160 Weiss, s., Ellis, J., Hendley, D.D. and Ienox, R.H. Translocation and activation of protein kinase C in striatal neurons in primary culture: Relationship to phorbol dibutyrate actions on the inositol phosphate generating system and neurotransmitter release. J. Neurochem. 52: 530-536, 1989. Weiss, S., Schmidt, B.H., Sebben, M., Kemp, D.E., Bockaert, J. and Sladeczek, F. Neurotransmitter-induced inositol phosphate formation in neurons in primary culture. J. Neurochem. 50: 1425-1433, 1988. Welsh, C.J. and cabot, M.C. sn-1,2-diacylglycerols and phorbol diesters: Uptake, metabolism, and subsequent assimilation of the diacylglycerol metabolites into complex lipids of cultured cells. J. Cell. Biochem. 35: 231-245, 1987. Weyhenmeyer, J .A. and Phillips, M. I. Immunocytochemical localization of angiotensin in the CNS of Wistar Kyoto and spontaneously hypertensive rats. Hypertension 4: 514-523, 1982. Wilson, K., Sumners, c., Hathaway, s. and Fregly, M.J. Mineralocorticoids modulate central angiotensin receptors in rats. Brain Res. 382: 87-96, 1986. Wilson, S.K., Lynch, D.R., and Ladenson, P.W. Angiotensin II and atrial natriuretic factor binding sites in various tissues in hypertension: Comparative receptor localization and changes in different hypertension models in the rat. Endocrinology 124: 2799-2808, 1989. Wolf, M., cuatrecasas, P. and Sahyoun, N. Interaction of protein kinase c with membranes is regulated by ca 2 +, phorbol esters, and ATP. J. Biol. Chem. 260: 15718-15722, 1985a. Wolf, M., I.eVine, H., III, May, w.s., Jr., CUatrecasas, P. and Sahyoun, N. A model for intracellular translocation of protein kinase C ivolving synergism between ca 2 + and phorbol esters. Nature 317: 546-549, 1985b. Woodgett, J .R., Hunter, T. and Gould, K.L. Protein kinase C and its role in cell growth. In: Cell membranes methods and reviews. Eds. Elson, E. and Frazier, W. Plenum Press, New York, pp. 275-287, 1988. Yamamoto, s., Gotoh, H., Aizu, E. and Kato, R. Failure of 1oleoyl-2-acetylglycerol to mimic the cell-differentiation action of 12-0-tetradecanoyl 13-actetate in HL-60 cells. J. Biol. Chem. 260: 14230-14234, 1985. Yang, H.Y. and Neff, N.H. Distribution and properties of angiotensin converting enzyme of rat brain. J. Neurochem. 19: 2443-2450, 1972.

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161 Yano, K., Higashida, H., Hattori, H. and Zozawa, Y. Bradykinin induced transient accumulation of inositol triphosphate in neuron-like cell line NG 108-15 cells. Febs. Iett. 181: 403406, 1985. Yoshida, Y., Huang, F.L, Nakabayashi, H. and Huang, K-P. Tissue distribution and developemental expression of protein kinase C isozymes. J. Biol. Chem. 263: 9868-9873, 1988. Young, s., Parker, P.J., Ullrich, A. and Stabel, s. Down regulation of protein kinase C is due to an increased rate of degradation. Biochem. J. 244: 775-779, 1987.

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BIOGRAPHICAL SKETCH Christopher John Kalberg was born October 21, 1961, in Placerville, california. He has one brother, Michael, who works as a mechanical engineer in Sacramento, california. In 1979, he graduated from Cordova Senior High School in Rancho Cordova, California. His undergraduate education culminated upon graduation from the infamous California State University, Chico in 1984 with a B.A. degree in biology (major) and chemistry (minor). After a year of work and travel, he entered graduate school at the University of Florida under the tutelage of Dr. Colin Sumners. As of November, 1989, Mr. Kalberg will relinquish his bachelorhood to Ms. Jacqueline Perez and begin working as a post doctoral research associate with Dr. John Kessler in the Department of Neuroscience at the Albert Einstein College of Medicine in New York City. 162

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Dr. Colin Sumners, Ph.D. Associate Professor of Physiology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentat i on and is fully adequate, in scope and quality, as a dissertat i on for the degree of Doctor of Philosophy. Melvin Ph.D. Graduate Researc Professor of Physiology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentat i on and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Edwin M. Meyer, Ph.D. Associate Professor of Pharmacology and Therapeutics

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This dissertation was submitted to the Graduate Faculty of the College of Medicine and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. December, 1989 Dean, College of Medicine '->0.-t..,