Characterization of kinin action and kinin receptors in central nervous system tissue

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Characterization of kinin action and kinin receptors in central nervous system tissue
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Thesis (Ph.D.)--University of Florida.
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Bibliography: leaves 124-137.
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by Robert Edgar Lewis.
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Photocopy of typescript.
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Vita.

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CHARACTERIZATION OF KININ ACTION AND KININ RECEPTORS
IN CENTRAL NERVOUS SYSTEM TISSUE







BY

ROBERT EDGAR LEWIS


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


1984





























To Mom and Dad












ACKNOWLEDGEMENTS


I wish to express my deepest appreciation to the chairman of my

supervisory committee, Dr. M. Ian Phillips, for tremendous support

through this long and sometimes arduous project.

I wish to express my appreciation and gratitude to Dr. Stephen R.

Childers for the invaluable knowledge and advice which he willingly

gave me, and without which this project could not have been

completed.

My sincere thanks are extended to Drs. Mohan K. Raizada and Colin

Sumners for helpful advice and the generous use of their facilities.

I also wish to express my gratitude to Laurie Tudeen and Birgitta

Stenstrom for invaluable technical assistance, and to Sheri Jones for

typing this manuscript.

To Kenneth and Mary Jo Lewis, who have supported and encouraged

me through every endeavor, mere thanks cannot express the debt of

gratitude and pride I feel for you.

This project or any other endeavor I might undertake would be

irrelevant without the love, patience and support Sally willingly

gave.














TABLE OF CONTENTS


PAGE


ACKNOWLEDGEMENTS.............................................. ... iii

KEY TO ABBREVIATIONS............... ...............................vi

ABSTRACT.........................................................viii

CHAPTER

I INTRODUCTION............................................ 1

A Brief History of Kinins and Kinin Research.............1
The Kallikrein-Kinin System ..............................3
Kinin Action in the Central Nervous System..............12

II LOCALIZATION OF THE CENTRAL PRESSOR ACTION OF
BRADYKININ TO THE THIRD VENTRICLE.......................19

Rationale.............................. .................19
Methods.................................................20
Results.................................................24
Discussion..................................... ..... 29

III INVESTIGATING THE CENTRAL ACTION OF BRADYKININ:
POSSIBLE INTERACTIONS WITH ANGIOTENSIN II...............38

Rationale ............... ......................... ...38
Methods.................................................39
Results.................................................40
Discussion..............................................53

IV DEGRADATION STUDIES WITH A RADIOACTIVE ANALOGUE
OF BRADYKININ................................... .......58

Rationale ......................................... .....58
Methods................................................ 58
Results................................................ 60
Discussion.............................................. 72








V SPECIFIC BINDING OF 1251-TYR-BRADYKININ
IN CULTURED RAT BRAIN CELLS.............................76

Rationale...............................................76
Methods.................................................76
Results.................................... ............. 79
Discussion.............................................104

VI SUMMARY................................................120

Specific Kinin Recognition Sites.......................120
Exogenous Administration of Kinins.....................122
Peptide Inactivation .................................. 123
Conclusion.............................................123

REFERENCES.......................... .................... .........124
BIOGRAPHICAL SKETCH............................................... ..138














KEY TO ABBREVIATIONS


ACTH

ADH

Bmax

cm

cpm

CSF

DMEM

FBS

fmoles

g
HPLC

HS

IC50


adrenocorticotropic hormone

antidiuretic hormone

maximum number of sites bound

centimeter

counts per minute

cerebrospinal fluid

Dulbecco's modified Eagle medium

fetal bovine serum

femtomoles

gram or acceleration due to gravity

high pressure liquid chromatography

horse serum

concentration which inhibits 50%
of maximum value

intracerebroventricular

equilibrium dissociation constant

Michaelis-Menten constant

kallikrein inhibitory unit

lysine

millicurie

methionine

milliliter


ivt

KD

Km

KIU

Lys

mCi

Met

ml








mmHg millimeters of mercury

pCi microcurie

pg microgram

1l microliter

yM micromolar

pmole micromole

ng nanogram

nM nanomolar

nmoles nanomoles

pg picogram

pM picomolar

pmoles picomoles

Rf distance of substance migration
divided by distance of solvent front
migration

w/v weight to volume ratio













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

CHARACTERIZATION OF KININ ACTION AND KININ RECEPTORS
IN CENTRAL NERVOUS SYSTEM TISSUE

by

Robert Edgar Lewis

April, 1984


Chairman: M. Ian Phillips
Major Department: Physiology


Kinins are nine to ten amino acid peptides cleaved from kininogen by

kallikrein. The ability of kinins injected into the brain to induce

changes in blood pressure and the ability of kinins to bind to specific,

high affinity recognition sites in rat brain cell culture were examined.

The central pressor response in rats to kinins was studied by

intracerebroventricular (ivt) pretreatment with different

pharmacological agents. Saralasin and Captopril potentiated, and

indomethacin (ivt) attenuated, the central kinin pressor response.

These results suggest that the centrally mediated pressor response to

ivt kinins is modified by an inhibitory action of the brain

renin-angiotensin system, and that prostaglandins may be formed as

intermediaries is this kinin-induced response.

The central kinin pressor response was localized to the ventral

third ventricle by cream plugs which block drug access to discrete


viii








regions of the ventricular surface. Ventral third ventricle plugs

blocked the pressor response to ivt bradykinin, while fourth ventricle

plugs did not.

The possibility that kinins bind to specific, high affinity

recognition sites in brain tissue was examined. Rat brain cell culture

provided a system whereby nonspecific binding and degradation of the

ligand could be minimized. Using the iodinated ligand 1251-Tyr-

bradykinin, binding was observed to be time and pH dependent. Scatchard

analysis of saturation experiments yielded two components with

dissociation constant and maximum binding site concentration averaging

1 nM and 100 fmoles/mg protein, and 16 nM and 1000 fmoles/mg protein,

respectively. The binding sites were specific for kinins and kinin

analogues, and the order of potency in competing for 1251-Tyr-bradykinin

binding was Lys-bradykinin > bradykinin > Tyr-bradykinin > Tyr8-

bradykinin >>> Des-Arg9-bradykinin. Monovalent and divalent cations

inhibited kinin binding. Comparison of competition curves performed in

glial-enriched versus neuron-enriched cultures suggested that the kinin

binding sites resided primarily on neurons. These data enhance the

existing evidence suggesting kinins as neurotransmitters or

neuromodul ators.













CHAPTER I
INTRODUCTION

A Brief History of Kinins and Kinin Research

The roots of kinin research reach back to 1909 with the observation

by two French surgeons that intravenous injections of fractions

extracted from urine resulted in a transient fall in blood pressure (1).

This hypotensive effect was attributed by Frey and Kraut (2) to a

thermolabile, nondialysable substance they termed "Kreislaufhormon" or

essentially "circulating hormone." Search for the tissue of origin led

to the discovery of a hypotensive factor in pancreas which was given the

name kallikrein (3) derived from the Greek word for pancreas, kalli-

kreas. Werle and colleagues (4) observed the inactivity of kallikrein

on isolated guinea pig intestine but discovered that a marked increase

in activity occurred following preincubation of the substance with

blood. Kallikrein was postulated to cause the formation of an intesti-

nal contracting substance which was eventually termed kalladin (5). The

following year, 1949, Rocha e Silva and co-workers (6) reported their

observation of a factor formed in the blood upon incubation with trypsin

which contracts the isolated guinea pig ileum. Because the contractile

response was slow relative to that induced by histamine, the factor was

named bradykinin, derived from the Greek bradys kinein meaning "slow to

move." Eventually bradykinin was shown to be a peptide (7) and purified

to homogeneity (8). In 1960 Boisonnas et al. (9) reported the correct

amino acid sequence and successfully synthesized the peptide.








Subsequently, the biologically active kinins kalladin, Lys-bradykinin,

and Met-Lys-bradykinin were isolated and purified (10, 11). With the

availability of the synthetic peptides, work on the biological actions

of kinins extracted from various sources could be confirmed and

consolidated.
The discovery by Ferreira and Vane (12) in 1967 that kinins were

rapidly degraded to inactive peptides upon passage through the lung

placed in doubt any idea that bradykinin might be a circulating hormone.

Based on this observation Rocha e Silva (13) suggested that kinins might

be better thought of as "tissue" hormones autocoidss). Classically a

hormone is defined as a substance formed in one part of the body and

carried via the blood to another organ or region where the substance

acts. A "tissue" or local hormone varies from this classical definition

in that it is formed at the site of action.

Numerous investigations have led to the uncovering of a variety of

biological actions elicited by kinins on peripheral tissues. Four

biological activities characterize kinins: contraction of nonvascular

smooth muscle (except for rat duodenum), vasodilation, increased

vascular permeability, and pain production (for a review see 14).

Kinins have been suggested as regulators in local circulatory control

(15), fibrinolysis and clotting (16, 17), inflammation (18), control of

electrolyte and water excretion by the kidney (19), metabolic and

mitotic activity of different cell types (20-22) and modulators within

the nervous system (23), as well as other physiological and pathological

states. Nevertheless, the kallikrein-kinin system, though implicated,

has yet to be conclusively demonstrated as an active participant in any








physiological process. Several technological insufficiencies may

explain, at least in part, this deficiency. Until recently (24) one

problem has been lack of a sensitive radioimmunoassay for plasma kinins.

It is difficult to raise an antibody to bradykinin with little or no

crossreactivity to kininogen. One must also be able to insure against

the de novo formation of kinins during sample preparation.

Consequently, accurate measurement of kinin levels has been difficult.

Another technological shortcoming which is currently being corrected

(25, 26) is the development of an adequate assay for probing

kinin-receptor interactions. Both RIA and radioligand binding are

necessary to complement bioassays which may be subsensitive to peptide

concentrations in the picomolar range. Finally, the study of kinin

action lacks an adequate antagonist, a fundamental component to the

study of any putative physiological system. It is through these modes

of attack that investigators are approaching current problems in kinin

research.

The Kallikrein-Kinin System

Perhaps the most useful means of understanding the kallikrein-kinin

system is by dissecting it into its component parts. An outline of this

system is in Figure 1.

Kallikreins

Kallikreins can be roughly divided into two categories, plasma and

glandular. Kininogens are their only known natural substances. Plasma

kallikreins, by definition, are found in blood, whereas glandular

kallikreins have been isolated from tissues. The two enzymes are

immunologically (27,28) and enzymatically distinct. Plasma kallikreins







4














4 4J







4J M






























11
EC .
CA 4)
0 00





















4-C .- ---
o- -







4 O .0
CC C
) .-J E Ci-









t O 9-



0 10


I 0 t I (








cleave high molecular weight kininogens to form bradykinin, while

glandular kallikreins enzymatically release kalladin (Lys-bradykinin)

(12, 29). Moreover, soybean trypsin inhibitor inhibits plasma
kallikreins but not glandular kallikreins (30). Plasma prekalllkreins,

generated in the liver (31), can be activated by Hageman factor (32) or

trypsin (33). Activation yields the functional serine protease (34).

Human plasma kallikrein is 108,000 daltons as measured by sodium dodecyl

sulfate disc gel electrophoresis (32, 33), and has demonstrated several

peaks between pH 7.7 and 9.4 during isoelectric focusing. Plasma

kallikreins from other animals have similar molecular weights of around

100,000 (35).
Glandular kallikreins, which occur mainly in the pancreas, salivary

glands and kidney, as well as in their exocrine secretions, are

glycoproteins of 25,000 to 40,000 molecular weight (36, 37) and

isoelectric points near 4.0 (37). There is also heterogeneity

associated with distinct glandular kallikreins. Habermann separated pig

pancreatic kallikrein into two forms, A and B, by electrophoresis (38).
Glandular kallikreins are immunologically identical. Antibody to

human urinary kallikrein forms lines of identity with human salivary and

pancreatic kallikrein as well as with kallikrein exuded in human sweat

(39). Urinary kallikrein is generally believed to be identical to renal

kallikrein. The biochemical properties of the two enzymes are

essentially indistinguishable (40).
Kallikreins can be assayed by several different methods. Bioassay,

the assessment of kallikreins' ability to release kinins from plasma

substrate, is one method of assay. The amount of kinin liberated is








measured by its ability to contract smooth muscle preparations (e.g.,

guinea pig ileum or rat uterus), or to lower blood pressure by

decreasing vascular resistance. Difficulties arise, however, unless

care is taken to assure that samples are free from contaminating

proteases with kininogenase activity and that all kininase activity is

blocked to prevent diminution of the oxytocic or vasodilatory effects.

Alternatively, assays have been developed to take advantage of

plasma and glandular kallikreins' ability to hydrolyze esters of a-N-

acylated arginine. Examples of synthetic substances are a-N-tosyl-L-

arginine methyl ester (TAMe), benzoyl-L-arginine ethyl ester (BAEe) and

benzylcarboxyl-L-arginine ethyl ester (CBZ-AEe). Hydrolysis of these

substrates can be followed spectrophotometrically, and titrimetrically

or radiolabelled substrates can be used and the hydrolysis products

separated and counted (41). These assays can reveal unusual

characteristics such as time dependence of enzymatic activity (42).

However, esterase activity does not parallel activity in bioassay or

radioimmunoassay.

Radioimmunoassays (RIA) using specific antibodies to plasma or

glandular kallikreins have been developed (24, 27). A specific RIA

eliminates the need to purify kininogens or inhibit kininases, and

assures greater specificity than a bioassay.

Kallikreins can be inhibited by several substances. Natural
inhibitors are C1 esterase inhibitors (43), soybean trypsin inhibitor

(plasma kallikrein only) (30) and bovine pancreatic trypsin inhibitor

(Trasylol, Aprotinin) (44) which has been identified in several bovine

organs. The major synthetic inhibitor is di-isopropyl-fluorophosphate









(DFP) which has been used to demonstrate the serine protease activity of

kallikreins (45).

Plasma kallikrein is localized in the blood. It may be completed

endogenously to a2-macroglobulin which allows the enzyme some protease

activity while preventing binding of circulating inhibitors (e.g., Cl

esterase inactivator). Glandular kallikrein has localized to various

cell types depending on the tissue studied. In rat submandibular gland

kallikrein immunoreactivity has been observed in the granular tubules,

striated ducts and some main duct cells, but not acinar cells (46, 47).

Subcellularly, the salivary gland has been suggested to be localized to

zymogen granules (48, 49). The cellular localization of pancreatic

kallikrein (acinar cells or beta cells) has continued to be debated (50,

51). While the discrepancy is still unresolved, efforts have been made

to explain the conflicting results (52).

Much work has been done on the cellular localization of renal

kallikrein (53). A recent report utilizing radioimmunoassay

demonstrated the highest kallikrein content in the outer cortex with

progressively decreasing immunoreactivity toward the papillary tip (54).

Microdissected cortical nephrons had their highest kallikrein content in

the connecting tubule followed by the initial collecting tubule and the

distal convoluted tubule. Little or no kallikrein was identified in

glomeruli, proximal convoluted tubule, proximal straight tubule or

cortical thick ascending limbs (54).

Functionally, plasma kallikrein can cause the formation of

bradykinin potentially leading to changes in local vascular tone, and

can potentiate Hageman factor activation (55) leading to the clotting









cascade. Glandular kallikreins may have physiological roles in local

circulatory regulation (15) and control of electrolyte and water balance

by the kidney (19).

Kininogens

Kininogens, formed in the liver (56), are glycoproteins that can be

categorized by their molecular weight and susceptibility to enzymatic

cleavage. Low molecular weight (LMW) kininogen has been reported to be

between 50,000 (57) and 78,000 (58) daltons, and its isoelectric point

is 4.7 (57). LMW kininogen has been resolved into two types: Type I

(32% of LMW kininogen) loses its kinin generating ability upon extensive

treatment with carboxypeptidase B; Type II (68% of LMW kininogen) is

insensitive to carboxypeptidase B. These observations were interpreted

as indicating the kinin moiety resides on the C-terminal of LMW

kininogen I, but is buried within the Type II molecule (59).

Human high molecular weight (HMW) kininogen is between 108,000 and

120,000 daltons (58, 60). HMW and LMW kininogen are immunologically

similar (61); however, plasma kallikrein prefers HMW kininogen as a

substrate whereas glandular kallikrein can form kallikrein from either

substrate (62, 63).

Kinins

The primary mammalian kinins, bradykinin and kalladin

(Lys-bradykinin), are nona- and decapeptides, respectively (Figure 2).

The arginine residues and additional lysine in kalladin make these

peptides very basic. Kinins are typically found in pg/ml quantities in

the blood (64).








Delineation of such low levels, however, requires a sensitive

radioimmunoassay, and there are problems inherent to radioimmunoassay of

kinins (65, 66). Among these problems are the poor immunogenicity of

kinins and the frequent observation that kinin antibodies cross-react

with kininogens. Another difficulty which must be carefully controlled

for is prevention of kinin formation or degradation during the

collection of biological fluid. Bioassays have been used for

determination of kinin levels but the problems of sensitivity and

specificity are the same for kinins as they are for other substances.

The biological actions of kinins have been listed above. A direct

physiological role for kinins has not been determined. However, they

have been suggested as playing roles in clotting and fibrinolysis (16);

sodium retention by the kidney (67), vasodilation in exocrine glands

(15) and the heart (68); uterine contraction (69); sperm-motility (21);

catechol release by the adrenal (70); ovulation (71); and cardiovascular

regulation by the brain (72). Pathologically, kinins have been

suggested for roles in allergy (73), arthritis (74), carcinoid and

"dumping" syndromes (75, 76), headache (77), hypertension (78) and

inflammation (79).

Kininases

Some proteolytic enzymes which can degrade bradykinin are shown in

Figure 2 along with their sites of cleavage. An aminopeptidase has been

isolated from blood (29, 80) of approximately 95,000 daltons which can

cleave lysine from the amino terminal of kalladin, but which will not
cleave the Arg1-Pro2 bond of bradykinin. Trypsin will also cleave

lysine from kalladin, but under normal conditions it is not active in























try sin -brain endopeptidase
aminopeptidase kininase II

NH2-LYS-ARG PRO-PRO-GLY-PHE SER-PRO-PHE ARG-COOH

prolidase kininase I
I LI
imidopeptidase chymotrypsin


Figure 2. Primary structure of kalladin (lys-
bradykinin) and bradykinin. Site of action of
known proteolytic enzymes.








blood (81). Except for indirect evidence provided in one report (82)

prolidase and imidopeptidase are believed to be intracellular

constituents (80, 83). Consequently highly charged molecules like

kinins would not be expected to be degraded by these enzymes unless

specific transport systems or receptor-mediated endocytosis shifted the

peptide to an intracellular compartment. In general, degradation of

kinins at the amino terminal is not believed to play a major role in the

circulation.

Enzymes with specificity toward the carboxy terminal of kinins are

considered the most important in degrading these peptides. Primary

among these peptides are kininase I and kininase II. Kininase I,

synonymous with carboxypeptidase N, is responsible for 90% of the

degradation of kinins in plasma (84, 85). It is a circulating

carboxypeptidase which can cleave the C-terminal arginine from

bradykinin and kalladin (86). Kininase II has been shown to be

identical to angiotensin converting enzyme (87, 88). Kininase II is a

membrane bound enzyme with a wide distribution. It has been identified

in various vascular beds (89, 90), kidney (91), testicular tissue (92),

brain (93, 94) and choroid plexus (95). The enzyme has specificity for

the Pro7-Phe8 and Phe5-Ser6 bonds of bradykinin (88).

Bradykinin is an excellent substrate for kininase II. The Km for

degradation of bradykinin is lower than the Km for angiotensin I (96).

Kalladin is not as susceptible to kininase II as bradykinin (97).

Kininase II has been identified as glycoprotein with a wide molecular

weight range (129,000 to 480,000) (88, 89) due primarily to the

variability of its carbohydrate moiety (88).








Both kininase I and kininase II are metalloproteins as indicated by

their inhibition with 1,10-o-phenanthroline or other metal chelators

(98). Other natural and synthetic inhibitors of kinin degradation have

been discovered. The venom of Bothrops jararaca has been shown to

contain peptide inhibitors whose isolation leads to the subsequent

synthesis of similar peptides with enhanced abilities to inhibit kinin

degradation (99). Other synthetic chelators that inhibit kininase II,

such as SQ 14,225 (Captopril), have also been developed (100).

Chymotrypsin and an endopeptidase described in rabbit brain can

also degrade kinins (101, 102). Chymotrypsin can split the C-terminal

residue or the Phe5-Ser6 bond (102). The Phe5-Ser6 bond is also

a site of cleavage by the brain endopeptidase (101).

Circulating kinins are kept at a low level by both low rates of

production and rapid degradation. Kininase I and II are the major

enzymes in the blood. If kinins are formed discretely in various organs

(11), the mode of degradation may be quite different than that observed

in blood.

Kinin Action in the Central Nervous System

Study of the biochemistry and physiology of kinins has centered

around their properties and actions in peripheral tissue. However,

kinins may also directly influence the actions of the central nervous

system (CNS) either by entering from the periphery (103), or by being

synthesized within CNS structures by an endogenous kallikrein-kinin

system (104, 105). One approach to studying the central biological

properties of a substance is by injecting the substance into the brain

(106). If the dose injected into the brain is lower than the dose which









is effective peripherally, or if the response under observation is

opposite to that seen peripherally, it can be ascertained that the

substance is acting on central structures. Additionally, electro-

physiology can be useful to show that a particular substance can modify

the electrical activity of brain cells. However, evidence demonstrating

that a substance is part of an endogenous system in the brain requires

both physiological and biochemical applications.

Brain Kininogenases

Kallikrein-like activity has been isolated in the microsomal

fraction of rabbit brain (107, 108). Furthermore acetone treatment

(which activates prekallikrein) increases kallikrein-like activity in

these homogenates. Shikimi et al. (109) determined that kallikrein-

like activity in brain was greatest in the cerebral cortex, but lowest

in brain stem.

Kinin-Like Activity in Brain

Correa et al. (104) are the only investigators to immunocytochem-

ically document the presence of kinin-like antigens in brain tissue.

All positively staining cells were localized to the hypothalamus,

whereas positively staining fibers were more widespread. Fibers were

observed in periaqueductal gray matter, hypothalamus, lateral septal

region, perirhinal and singulate cortex and ventral portions of the

caudate-putamen. Low levels (5 pmoles/g brain) of kinin-like material

have also been measured in brain by RIA (105). Hori (107) reported the

presence of a partially purified kinin-like peptide from rabbit brain

which had similarities to bradykinin in bioassay and electrophoretic

mobility. Similarly, Pela and colleagues (110) also extracted

kinin-like activity from rabbit brain.








Brain Kininases

Kininase activity in brain has been described in rat (109), mouse

(111), rabbit (107, 112), dog (113), cat (113) and guinea pig (114).

The majority of kinin degrading activity appears in the soluble fraction

during subcellular fractionation (107, 112).

While kininase II is found abundantly in brain (94) it is certainly

not the sole inactivator of kinins in the brain. Kininases distinct

from kininase II have been described by Carmargo et al. (101), Marks and

Pirotta (115) and Oliveira et al. (116) in brain tissue. Nevertheless,

the high concentration of kininase II observed in choroid plexus (94) is

probably the primary cause for the rapid degradation of kinins injected

into the brain ventricles (105).

Biological Effects of Central Bradykinin

The biological actions of bradykinin which are mediated through the

CNS have been studied primarily by injecting the peptide into the brain

or into blood vessels which perfuse the brain. Of the various biological

effects that bradykinin elicits through the CNS, the most striking is a

sustained rise in blood pressure (72, 117, 118). The central pressor

response is in direct contrast to the short-lived but potent
vasodilation observed when the peptide was injected into the peripheral

circulation.

The characteristics of the central pressor response varied in dif-

ferent laboratories. Generally, anesthetized animals receiving brady-

kinin via intracarotid or intracerebroventricular (ivt) injections have

demonstrated biphasic pressor responses--a short depressor effect

followed by a sustained pressor response (103, 119, 120). This biphasic








response did not appear to be the result of a direct action of

bradykinin on the vasculature as it persisted in animals whose cerebral

blood flow was separated from the peripheral circulation in a

cross-perfused preparation (103). The initial fall in blood pressure

has been suggested to be mediated through activation of a-adrenergic

vasodepressor mechanisms in supramedullary centers (103, 120).

Conscious and free-moving animals typically elicit only a pressor

response when bradykinin is injected centrally (117, 118, 121), although

an exception has been observed (119). This pressor response to ivt

bradykinin is believed to be mediated through prostaglandins (103, 121,

122) as well as noradrenergic systems (72, 103, 117). Pain production

resulting in an elevation of blood pressure has been discounted by the

observation that intravenous analgesics do not block the central pressor

response (123, 124). Interestingly, centrally administered bradykinin

has been reported to have an antinocioceptive effect of its own (124).

Consistent with the suggestion that the central action of kinins on

blood pressure is mediated through noradrenergic systems (72, 103),

Capek et al. (125) observed a 40% reduction in norepinephrine one hour

after ivt injection of 1-5 ug of bradykinin. However, work with

bradykinin effects on dopamine (123, 126, 127) and serotonin (123, 127)

are equivocal and no clear conclusion can be drawn about ivt kinin

effects on these amines. Bradykinin has also been suggested to release

vasopressin and ACTH from the pituitary. Median eminence lesions

abolished the urine-concentrating effects of ivt bradykinin (118).

Similarly, intracarotid bradykinin elevated coricosterone levels (used

as an index of ACTH release) and this effect was abolished by









hypophysectomy (128). However, an effect of bradykinin causing

secondary, vasopressin-induced, release of ACTH cannot be ruled out by

the above.

A hyperthermic response to kinins has been observed in rabbits

(129). Bradykinin, kalladin, and Met-Lys-bradykinin were equivalent in

elevating rectal temperature, whereas Des-Arg9-bradykinin had no

effect. The hyperthermia was antagonized by acetominophen and

indomethacin suggesting prostaglandin synthesis as a mediating event in

the response.

Electrophoretic studies with kinins on brain tissue have been

carried out. Phillis and Limacher (130) iontophoretically applied

bradykinin to spontaneously active cortical neurons including Betz

cells. Ninety-one percent of all Betz cells and 76% of all other

unidentified neurons responded to bradykinin. Guyenet and Aghajanian

(131) found no response to bradykinin while recording from cells in the

locus coeruleus. Cultured glioma and neuroblastoma X glioma hybrids

were found to be hyperpolarized by bradykinin (132). By altering the

content of specific ions in the culture dish and using specific ion

channel blockers, the hyperpolarization to bradykinin was found to be

due to increased potassium influx. In iontophoretic experiments, as

well as with the injection of drugs directly into the brains of experi-

mental animals, it is not possible to know the effective concentration

of a drug at its site of action. Thus, these biological experiments are

inadequate for demonstrating that brain tissue has a reasonable

specificity and sensitivity to kinins. This shortcoming could be

reduced by reversal of biological responsiveness by an appropriate









antagonist. Unfortunately, no suitable antagonist exists, and as a

consequence the specificity of kinin action in brain cannot be assured.

Significance

The kallikrein-kinin system is a diverse and complex system whose

function is not completely understood. Though several physiological

mechanisms have been suggested to include kinins (15-23), none are

considered to explicitly require the presence of these peptides. This

is particularly true concerning kinin actions on CNS tissue. While an

action of kinins in brain distinct from peripheral effectors has clearly

been demonstrated (72, 118, 121), questions concerning the location,

pathways, and mechanisms of action by kinins remain. For kinins to be

identified as neurotransmitters or neuromodulators they should meet

eight criteria (modified from 133):

1. They should be localized to the presumed releasing cell(s).

2. They should be synthesized or accumulated in the releasing

cell(s) by a specific mechanism.

3. They should be released from the releasing cell(s) when the

system is physiologically activated.

4. They should have access to specific receiving cells.

5. Specific recognition sites should be present on the receiving

cell(s).

6. Exogenous administration of kinins to receiving cells should

mimic the response produced by physiological activation of the

system.

7. A system for inactivation of kinins should exist in the

vicinity of the receptor.








8. Kinin antagonists should block the action of exogenous kinins

and antagonize the physiologically activated system.

Within this dissertation will be presented evidence directed at helping

to fulfill the fifth, sixth and seventh criteria in the list above.

Data demonstrating specific recognition sites in brain tissue will be

presented. The injection of kinins into the brain ventricles of

conscious rats will be used to demonstrate the biological activity of

the peptide, as well as help localize the biological activity to a

specific region of the ventricular surface. Additionally, data showing

the presence of kinin degrading activity in brain tissue and the

inhibition of degradation by known protease inhibitors will be

presented.














CHAPTER II
LOCALIZATION OF THE CENTRAL PRESSOR ACTION OF
BRADYKININ TO THE THIRD VENTRICLE

Rationale

Bradykinin and kalladin (Lys-bradykinin), which are potent

vasodilators in the peripheral circulation (134), have been found to

elicit a pressor response when injected into the brains of rats (72,

118, 119). The mechanism of action of the pressor response may involve

antidiuretic hormone (ADH). Baertschi and colleagues (135, 136)

suggested the kinin-induced ADH release is the result of a central

action of the peptide on the hindbrain. Hoffman and Schmid (118) showed

that the antidiuretic response to central bradykinin appears to be at

least partially responsible for the rise in systemic blood pressure.

An attempt has been made to localize this effect. Using ivt injections

of bradykinin into the lateral ventricle prior to and following

selective electrolytic lesions, Correa and Graeff (137) concluded that

the ventrolateral septal region was the locus for the kinin induced

central pressor response. In the present report ventricular plugging

(138-140) is used to determine the periventricular site or sites of

action. Bradykinin injections combined with intraventricular plugs to

restrict the flow of CSF were used to localize the pressor response.

The data suggest that access to the ventral third ventricle is essential

for the central kinin pressor response.









Methods

Under chloral hydrate anesthesia (400 mg/kg), female Sprague-Dawley

retired breeders (Holtzman, 340-420g) were stereotaxically fitted with

two intraventricular cannulae made from 23 gauge stainless steel tubing

14 mm in length. Each animal received a lateral ventricle cannula

1.0 mm posterior to bregma, 1.0 mm lateral from the midline and 5.0 mm

deep from the dura. Each animal was also simultaneously implanted with

either a third ventricle or fourth ventricle cannula of the same

material (see Figure 3). Third ventricle cannulae were inserted 1.0 mm

posterior to bregma, 1.0 mm lateral of the midline and 7.0 mm deep from

the dura at an angle of 100 from the vertical. Coordinates of cannulae

implanted into the fourth ventricle were 3.0 mm posterior from lambda,

0.0 mm lateral from the midline and 7.0 mm deep from the dura. Cannulae

were anchored to the skull by jewelers screws embedded in dental acrylic

cement. All rats were allowed three days recuperation before insertion

of an indwelling catheter for measurement of blood pressure. Each

silastic catheter was filled with heparinized sterile saline and

inserted into the right femoral artery. After being secured by suture,

the catheter was slipped underneath the skin and exteriorized on the

back of the animal. A closed-end stylus plugged the free end of the

tubing to maintain patency. This scheme allowed recording of blood

pressure while the animal was conscious and unrestrained. Testing was

performed on the day following catheterization.

Cream plugs injected into specific ventricular locations through

properly oriented cannulae deny drug access to those locations (139,

140). The plug consisted of cold cream (Nivea) and was injected by





























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filling a short section of PE 50 tubing calibrated to a known volume of

10 pl (third ventricle plugs) or 6 pl (fourth ventricle plugs). The

filled polyethylene tube was then fitted to a saline-filled 1 ml syringe

at one end and to the appropriate cannula via a short section of 23

gauge tubing at the other end.

For plug injections animals were lightly anesthetized with ether.

The saline-filled syringe was slowly compressed so that injection of the

cream could be observed as it moved through the short length of

calibrated tubing. Each injection took approximately two minutes. Two

hours of recovery were allowed before testing. Plug placement had no

outward effect on the animals' behavior. Each animal was alert and

responsive. Mean basal blood pressure was unchanged following plug

injection (Third ventricle plugs: 106 3 mm Hg before plug vs

104 3 mm Hg following plug; Fourth ventricle plugs: 96 4 mm Hg

before plug vs 96 6 mm Hg following plug).

One hundred nanograms angiotensin II (Ciba-Geigy) or 5 ug

bradykinin triacetate (Sigma) were injected in 1 pl volumes through 30

gauge injectors attached via polyethylene tubing to a Hamilton syringe.

The injectors were constructed so that when inserted their tips ended

just beyond the tip of the lateral ventricle cannula.

At the end of each experiment the rat was injected with 1 3 p1 of

black ink and after several minutes was decapitated. The brain was

excised and immersed in 40% formaldehyde. On subsequent days brains

were sectioned parasagittally on a freezing microtome. The location and

patency of each plug were recorded.








All data presented are paired values given as mean + standard

error. Results were evaluated by paired Student t-tests.

The study was performed in two parts. First, pressor responses to

ivt angiotensin II and bradykinin were recorded. In the case of

angiotensin II drinking was also measured. Following these initial

tests a ventral third ventricle plug was inserted as described above.

Following a two-hour recovery period each animal was tested a second

time with each drug. One hour was permitted between drug injections and

the order of both pre- and post-plug injections was randomized.

The second experiment consisted of intravenous injections of

bradykinin before and after cream plug insertion into the fourth

ventricle. The same procedure was followed as described above.
Results

Third Ventricle Plugs

Previously published results using cream plugs (140) suggested that

the site of angiotensin II action was the ventral anterior third

ventricle. In an attempt to confirm this finding and to pharmacologi-

cally verify the location of the cream plugs, five rats were challenged

with 100 ng angiotensin II injected into the lateral ventricle before

and after plugging. Figure 4 and Figure 5b show the pressor and

drinking responses recorded. Ventricular plugs significantly depressed

the pressor response (22 + 3 mm Hg pre-plug vs 4 + 2 post-plug, p < .005,

N = 5) and drinking response (3.7 + 0.6 ml pre-plug vs. 0.9 + 0.6 ml

post-plug, p < .005, N = 5).

Pressor responses to 5 ug bradykinin injected into the left

ventricle following third ventricle plugging were significantly lower































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than before insertion of the plugs (Figure 5a and Figure 6, left panel,

27 + 5 mm Hg pre-plug vs 5 + 5 mm Hg post-plug, p < .025, N = 7).

Histology of rat brains with third ventricle plugs confirmed the

correct position of all plugs. In every animal cream plugs obstructed

access of CSF to the anterior ventral third ventricle. In five of seven

cases plugs prevented access of ink injected into the lateral ventricle

beyond the foramen of Munro. In the two instances when ink passed the

foramen and traversed the third ventricle, the plugs securely blocked

access to all sites on the anterior ventral and posterior ventral third

ventricle. An example of the third ventricle plug is shown in Figure 7.

Fourth Ventricle Plugs

Pressor responses to 5 ug bradykinin before and after fourth

ventricle plugging are illustrated in Figure 5c and Figure 6 (right

panel). Lateral ventricular injections elevated mean arterial blood

pressure 27 + 8 mm Hg before plug insertion and 35 + 9 mm Hg after plug

insertion (p > .05, N = 5). Histological analysis following testing

confirmed plug localization within the fourth ventricle. Figure 8

demonstrates a typical fourth ventricle plug.
Discussion

The major finding of this study is that the central pressor

response elicited by injected bradykinin into the lateral ventricle is

localized in or requires access to the ventral portion of the third

ventricle. CSF flows from the lateral ventricle to the third and fourth

ventricles and out the arachnoid villi (141). Cream plugs blocking drug

access from the lateral to the third ventricle blocked the pressor

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fourth ventricle, but not to the cerebral aqueduct or third ventricle,

did not block the bradykinin pressor response. In two instances where

cream plugs denied drug access to the ventral third ventricle, but

allowed passage to the dorsal third ventricle, cerebral aqueduct and

fourth ventricle structures, the bradykinin pressor response was still

absent. The patency of third ventricle plugs was confirmed by ink

injection and histological section and also by cream plug obliteration

of the pressor and drinking responses to angiotensin II injected into

the lateral ventricles.

The results of the cream plug on intravenous angiotensin II action

support previous findings using this technique (140). That report

concluded that the site of action of angiotensin II for the pressor and

drinking responses was the anterior ventral third ventricle.

Correa and Graeff (137) suggested from lesion experiments that the

site of action of the central kinin pressor response is the ventral

lateral septal region. However, their study does not rule out two

alternative explanations of their results: (1) the possibility that they

lesioned a portion of the pathway mediating the central kinin pressor

response and not the receptor site for bradykinin; and (2) the site of

the lesion was so close to the foramen of Munro that post-operative

swelling effectively blocked passage of the drug to distal structures.

Baertschi and colleagues (135, 136) concluded from responses to

bradykinin systemically injected so as to pass through the vertebral

arteries that the peptide had a rhombencephalic locus of action on the

neurohypophysis. ADH release has been reported as a consequence of









bradykinin action on the brain when injected into the blood (142) or

intracerebroventricularly (118). Hoffman and Schmid (118) report that

the release of ADH is partially responsible for the central kinin

pressor response. Presumably for bradykinin to penetrate to the brain

it would require access to a circumventricular organ where there is no

blood brain barrier. In the hindbrain only the area postrema is known

to lack a blood brain barrier. Results from fourth ventricle plugs can

be used to argue against this site of action. The possibility that

systemically injected bradykinin acts on the blood side of the neural

tissue cannot be excluded from the results of Baertschi et al. (135,

136), nor, however, can the possibility that bradykinin in their study

traversed the vertebral and basilar arteries to reach the posterior

cerebral artery. The posterior cerebral artery supplies the caudal

diencephalon among other regions (143). Results reported here are

consistent with this possibility.

Correa et al. (104) recently reported kinin-like immunoreactivity

in rat brain. Immunofluorescent cell bodies were observed only in the

medial basal hypothalamus--a ventral third ventricle structure.

Evidence exists for an endogenous brain-kinin system (107, 112);

however, specific kinin receptors have yet to be identified in brain

tissue.

That the site of action of bradykinin and angiotensin II can be

localized to the ventral third ventricle is interesting in light of

previous comparisons between the central actions of angiotensin II and

bradykinin (118, 125).













CHAPTER III
INVESTIGATING THE CENTRAL ACTION OF BRADYKININ:
POSSIBLE INTERACTIONS WITH ANGIOTENSIN II

Rationale

Recent advances in research on central actions of peptides suggest

that central mechanisms of cardiovascular control may be mediated or

modulated by brain peptides. In this study a peptide-peptide

interaction on blood pressure was investigated by focusing on the

cardiovascular responses of angiotensin II and bradykinin. When these

peptides are injected into the brain ventricles they both cause an

increase in mean arterial blood pressure (72, 117, 118, 144). The

peripheral action of these peptides, however, is quite different.

Intravenous angiotensin II increases blood pressure but intravenous

bradykinin produces vasodilation. Bradykinin is degraded by the enzyme

kininase II, the same enzyme that has the role of converting angiotensin

I to angiotensin II (145). Thus, the two peptides would seem to have an

interaction which could be tested by the use of converting enzyme

inhibitors and angiotensin II antagonists. In addition, angiotensin II

has more prolonged effects in the brain when prostaglandin synthesis is

inhibited (146), whereas the central pressor effect of bradykinin is

attenuated by the prostaglandin synthesis inhibitor indomethacin (121).

It has been reported that prostaglandin El and E2 will inhibit

angiotensin II-induced drinking in the rat (147).








To test if there is a central interaction between two neuropeptides

with cardiovascular effects we have studied selected doses of bradykinin

that are known to be effective and reliable in inducing pressor

responses when injected into the brain. We have found an interaction

between angiotensin II and bradykinin on pressor responses to ivt

injections.

Methods
Surgery

All experiments were performed on conscious unrestrained male

Sprague-Dawley rats. Each rat was surgically prepared under chloral

hydrate anesthesia (400 mg/kg) with 23 gauge, 14 mm long stainless steel

cannula at least 3 days before testing. Each cannula was placed in the

lateral ventricle: 0.5 or 1.0 mm posterior, 1.0 mm lateral, 5.0 mm deep

(from dura) with respect to bregma (flat skull). For blood pressure

recording a chronic indwelling catheter was inserted into the femoral

artery and vein under ether anesthesia. Each catheter was slipped

underneath the skin and exteriorized on the back of the animal.

Equipment and Solutions

Blood pressure was continuously measured with a Statham P23Gb

pressure transducer and heart rate was measured by a cardiotachometer.

Changes in blood pressure and heart rate were recorded on a Beckman R411

Dynograph Recorder. When a central drug pretreatment was called for,

the infusion was given through the same intracranial cannula used for

kinin injections. Drug infusions were administered in 1-5 pl volumes.

Bolus intracranial injections were given by hand with a 10 pl or 25 pl

Hamilton syringe.








Solutions containing angiotensin II (Ciba), bradykinin, Lys-brady-

kinin, Des-Arg9-bradykinin, Tyr-bradykinin (Penninsula and Bachem),

(Sigma), saralasin acetate (Calbiochem-Behring), or SQ 14,225

(Captopril, D-3-mercapto-2-methylpropanoyl-L-proline, Squibb) were

prepared in 0.9% sterile saline at neutral pH. Indomethacin (Upjohn)

was dissolved in 1.0 M NaOH and titrated to pH 7.4 with HC1 or dissolved

by stirring at 400 C in 0.9% saline buffered to pH 7.4 with 23 mM

NaHCO3. The doses of bradykinin, indomethacin and angiotensin II were

chosen from dose response curves published previously (118, 144, 148).

Procedures

Separate tests were carried out in different groups of rats. Blood

pressure responses to kinins and angiotensin II were conducted in

separate tests. In all tests the order of testing was randomized. In

tests conducted where a change in responsiveness followed indomethacin

treatment, at least 2 hours were allowed between tests. All other tests

were separated by 45 minutes or more. All values are reported as mean

standard error. Comparisons were made with a paired or unpaired

Student t-test, or analysis of variance and Newman-Keuls test, where

appropriate.

Results

Figure 9 demonstrates the difference in blood pressure changes to

intravenous and ivt injections of bradykinin. The intravenous routes

produced a brief depressor and the ivt route of injection produced a

long-lasting pressor effect.

Pressor responses were of short latency for ivt injections of

200 ng angiotensin II (20 3 mm Hg, 25 4 sec) and 5 ug bradykinin





























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(18 + 5 mm Hg, 37 + 10 sec). Duration of the pressor response, i.e.,

the time from beginning of response until return to baseline, was

15 + 4 min for angiotensin II and 18 + 6 min for bradykinin. Treatment

with 10 pg saralasin ivt 3-5 minutes prior to injection of 200 ng

angiotensin II significantly reduced the pressor response (Figure 10,

P < .005 measured by a paired t-test) and associated bradycardia (Figure

11, P < .05 measured by a paired t-test). The same dose of saralasin

ivt, however, potentiated the pressor response to 5 ug bradykinin ivt

(Figure 10, P < .01 measured by a paired t-test) with no significant

effect on the associated tachycardia (Figure 11). Saralasin alone at

the dose used had no effect on basal heart rate or blood pressure.

Pretreatment with 2 ug of the angiotensin converting enzyme inhibi-

tor Captopril potentiated 1 Ug ivt bradykinin but when given 5 minutes

prior to 5 yg bradykinin ivt a drop in pressure resulted (Figure 12).

Captopril alone did not affect blood pressure. The Captopril solution

was acidic (pH 3.5) and therefore the vehicle (1 pl) with pH 3.5 was

tested alone. No effect on blood pressure or drinking was observed.

The pressor and heart rate responses to 5 ug ivt bradykinin were

also attenuated by 10 ug ivt indomethacin administered 3-5 minutes

before injection of the peptide (Figure 13). Ivt injection of indo-

methacin alone did not alter blood pressure or heart rate.

The central pressor response to bradykinin was compared with Lys-

bradykinin and other kinin analogs (Figure 14). Bradykinin, Lys-brady-

kinin and Tyr-bradykinin were all capable of elevating blood pressure above

saline control injections. Des-Arg9-bradykinin, however, did not

































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FIGURE 12: Change in blood pressure to different doses of
bradykinin ivt with (open circles) and without
(closed circles) Captopril ivt. Captopril alone
demonstrated no effect on blood pressure.
BK = bradykinin; data are given as mean S.E.;
P < .001.


















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elicit pressor responses at the doses tested which were significantly

different from control injections.

Discussion

Angiotensin II has an action in the brain which elevates blood

pressure (144, 149). Increases in arterial blood pressure following

central administration of the nonapeptide bradykinin have also been

reported (72, 118, 119). Additionally, Yang et al. (150) recognized

that angiotensin converting enzyme and kininase II were one and the

same. Given these facts we hypothesized that angiotensin II and brady-

kinin shared some relationship with respect to their pressor actions in

the brain. We tested whether the peptides were in series, i.e., brady-

kinin causing the release of angiotensin II which, in turn, elicits the

pressor response. If this were true it would be predicted that the

pressor effect of central bradykinin could be abolished by the

angiotensin II competitive antagonist saralasin. The two doses were

selected to produce nearly equivalent rises in blood pressure on their

own (20 3 mm Hg for angiotensin II vs 18 5 mm Hg for bradykinin).

However, saralasin potentiated the pressor responses to bradykinin while

attenuating the response to angiotensin II (Figure 10). The experiments

suggest that the two peptides do not act in series and that angiotensin

II inhibits the pressor action of bradykinin.

The possibility of saralasin inhibition of converting enzyme to

produce greater amounts of bradykinin was considered. Chiu et al. (151)

found a reduction in the conversion of angiotensin I to angiotensin II

by porcine lung converting enzyme in the presence of an equimolar








concentration of saralasin. However, Fitz and co-workers (152) found

little if any inhibition of the enzyme extracted from human lung or

porcine plasma by saralasin. Further, converting enzyme/kininase II has

a 10- to 100-fold greater affinity for bradykinin than for angiotensin I

(88). Therefore, since the molarities of bradykinin and saralasin

administered in this study were similar, it is unlikely that saralasin

would have been a better competitor for kininase II than bradykinin.

If angiotensin II is inhibiting the bradykinin pressor response as

the saralasin data indicate, then inhibition of angiotensin II formation

should also enhance the bradykinin response. Figure 12 demonstrates

potentiation of the pressor response to bradykinin during converting

enzyme inhibition by Captopril. A similar result has recently been

reported by Unger et al. (145). Their interpretation was that more

bradykinin was available. However, the potentiation also supports the

notion of a decrease in the inhibitory action of angiotensin II on the

central bradykinin pressor response. At the highest dose, 5 ug of

bradykinin in the presence of 2 ug Captopril resulted in a depressor

rather than a pressor response. The decrease in blood pressure can be

explained by leakage of the higher dose into the circulation.

Peripherally, bradykinin is a vasodilator and peripheral vasodilation

may override the central pressor response. Thus, Captopril may not only

inhibit degradation of central bradykinin, but may also prevent its

central inhibition by angiotensin II.

Injection of 50 ng angiotensin II with 5 ug bradykinin into the

brain ventricles of conscious rats does not result in a pressor response

significantly different from the response observed when either of the








two peptides is injected alone (153). It might be anticipated that the

addition of two separate central pressor substances would have an

additive effect upon blood pressure. However, the lack of additivity

with coinjection of angiotensin II and bradykinin could be interpreted

as the result of angiotensin II inhibition of bradykinin pressor

activity. Five micrograms bradykinin ivt elicits a maximal pressor

response, and additivity of an effect would not be expected when the

system is already maximally stimulated.

Bradykinin's centrally induced tachycardia (Figure 11, Figure 13)

is difficult to interpret in light of other studies. Hoffman and Schmid

(118) report a mild tachycardia in conscious rats with 1.0 Ug doses of

ivt bradykinin, but they observed a bradycardia with 5.0 Ug ivt

bradykinin. Conflicting with the results of Hoffman and Schmid (118)

are other reports of tachycardia induced by ivt bradykinin injections in

conscious rats (154) and rabbits (123). A tachycardia to ivt bradykinin

is consistent with the tachycardia observed with ivt prostaglandin E1

or E2 (155, 148), potential mediators of the central kinin response

(Figure 13).

A relationship between bradykinin and prostaglandins in the

peripheral circulation is well demonstrated. Bradykinin induces the

formation of prostaglandins in kidney (156) and isolated blood vessels

(157). Prostaglandins also raise blood pressure when injected into the

brain ventricles (149). Consequently, we looked for a relationship

between the central action of bradykinin and prostaglandin synthesis.

In fact, indomethacin did reverse the heart rate and attenuate the

pressor effects of centrally administered bradykinin (Figure 13). This









confirms the results of Kondo et al. (121) and suggests that

prostaglandin formation plays a role in bradykinin's central pressor

effect.

These experiments provide evidence for an interaction between two

peptides in the brain, angiotensin and bradykinin. Evidence exists for

the presence of both angiotensin and bradykinin in the brain (107, 144).

Both peptides have been demonstrated immunocytochemically in different

brain regions (104, 158). Specific angiotensin receptors have been

demonstrated in brain membranes (159) and neuronal cell culture (160)

although bradykinin receptors in the brain have not yet been

established.

Several analogues of bradykinin showed similar abilities to induce

a pressor response when injected into the brain ventricles. While Lys-

bradykinin demonstrated the most potent pressor response, it was not

statistically different from either bradykinin or Tyr-bradykinin. That

Des-Arg9-bradykinin could not elevate blood pressure significantly

more than control saline injections suggests that the central kinin

pressor response is not mediated through receptors with affinity for

this peptide. Such receptors, designated B1 by Regoli, have only been

identified in selected peripheral vasculature (14).














CHAPTER IV
DEGRADATION STUDIES WITH A RADIOACTIVE
ANALOGUE OF BRADYKININ

Rationale

The biological activity of kinins in CNS tissue (72, 117-132)

suggests the presence of specific kinin receptors in brain as mediators

of these functions. Investigating the existence of kinin receptors in

CNS tissue would require demonstrating the specific, saturable binding

of a radioactive kinin molecule to a suspension of brain membranes, or

using the ligand in an in vitro preparation of brain cells. However,

technical considerations preclude direct application of standard

radioligand binding techniques. Two criteria must be fulfilled prior to

a search for specific, high affinity kinin receptors in CNS tissue:

1) choice of an appropriate radioactive ligand, and 2) inhibition of

degradation of the ligand by brain proteases. The following describes

the procedures used for meeting these criteria.

Methods

Iodination

lodination of Tyr-bradykinin (Penninsula, Bachem) was accomplished

by a modification of the dilute chloramine T method of DeMeyts (161).

Twenty-five microliters of sodium phosphate buffer (pH 6.7) and 0.5 ug

Tyr-bradykinin in 10 pl phosphate buffer were added to 0.5 mCi

Na125I (Amersham) and 10 ul chloramine T (40 pg/ml) and incubated









for five minutes at 24* C to react all of the oxidizing reagent. This

precludes the addition of reducing reagents such as sodium metabisulfite

to quench the reaction. The reaction mixture was applied to a 5 ml

Dowex 1 x 4, 200 mesh column (Sigma). lodinated Tyr-bradykinin was

eluted from the column with water. Twenty-drop fractions were

collected.

Specific activity was determined by counting 10 pl aliquots of each

fraction collected. Recovery was 95%. Thus (using a counting

efficiency of 74%) the calculated number of vCi collected from the Dowex

column was divided by 0.5 pg Tyr-bradykinin to yield the specific

activity. Peak fractions from the Dowex column were pooled, diluted

50:50 with 100% ethanol and stored at -200 C.

Purity of the iodinated peptide was determined by HPLC. An aliquot

of the pooled fractions from iodination was eluted on a methanol-

ammonium acetate gradient. One milliliter fractions were collected at a

flow rate of 1 ml/minute. The purity of the peptide was assessed by

dividing the cpm in the kinin peak by the total cpm added. The location

of the kinin peak was previously determined by addition of fresh, cold

Tyr-bradykinin and observing an absorbance peak at 230 nM.

Degradation

Degradation experiments were carried out by incubation of

1251-Tyr-bradykinin with either homogenates of whole brain or with

cultured brain cells in the presence or absence of various inhibitors.

Whole brain homogenate was prepared by homogenizing the brains of female

Sprague-Dawley rats in 10 volumes of 25 mM potassium phosphate buffer

(pH 7.2) with a polytron at maximum speed for 30 seconds followed by









centrifugation at 30,000 x g for 20 minutes. The pellet was resuspended

in phosphate buffer, and membranes were incubated at 10 mg/ml (wet

weight) with inhibitors and 1251-Tyr-bradykinin. The final

reaction volume was 400 pl. After incubation of the reaction mixture

was centrifuged and an aliquot of the supernatant was applied to a

silica gel plate for chromatographic analysis. Chromatography was

performed with a mixture of ethyl acetate:pyridine:water:acetic acid

(65:18:9:4). In this system the intact peptide remained at the origin

while major hydrolysis product migrated with an Rf value of 0.47.

For degradation experiments in cultured brain cells, 1251-Tyr-bradykinin

was incubated with each culture in 5 ntM potassium phosphate (pH 7.2)

containing 320 mM sucrose. Integrity of the incubated radioactive

ligand was determined by chromatography on cellulose plates and a

solvent system of butanol:acetic acid:water (25:4:10, Rf = 0.5). The

second system (butanol:acetic acid:water) was used to identify any

hydrolysis products which might remain at the origin in the first

system.

Results
Iodination

The dilute chloramine T method consistently yielded 1251-Tyr-

bradykinin of high specific activity (500-800 Ci/mmole) and purity

(greater than 90% as determined by HPLC, Figure 15). The degree of

iodination appeared to be most dependent on the "age" of the Na1251.

Fresh deliveries of Na125I from the manufacturer gave higher

specific activities than did older lots which had been used previously.
































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Degradation studies in brain homogenate

A search for adequate inhibitors of brain kininases was designed

along two strategies. First, conditions were used which might inhibit

kininase activity, but which would probably not inhibit a receptor

binding (Figure 16). Second, another set of conditions was designed

which would certainly destroy enzymatic activity but would probably also

inhibit receptor binding (Figure 17). This second set of conditions

would assure that enzymatic degradation was present, and that it could

be inhibited. Figure 16 demonstrates that Captopril, EDTA,

dithiotheitol, bacitracin, trasylol, ovomucoid trypsin inhibitor and pH

8.9 were ineffective in inhibiting degradation, while of 1 mM 1,10-

o-phenanthroline inhibited 40% of degradation. Higher concentrations of

phenathroline were compared to severe conditions such as 0.1 M HC1 and

pretreatment of the tissue by boiling (Figure 17). Fifty and 100 mM

1,10-o-phenanthroline completely inhibited degradation, as did boiling

and acid, but Triton X-100, DTNB and trypsin did not. A dose-response

experiment (Figure 18) shows that a concentration as low as 5 mM

phenanthroline was effective in blocking kinin degradation at 40 C for

five minutes. Since the radioactive ligand would be required to be in

the presence of the tissue for more than five minutes in a receptor

assay the ability of 1,10-o-phenanthroline to inhibit degradation for

one to two hours was assessed. Figure 19 shows that 5 mM phenanthroline

completely inhibited degradation of 1251-Tyr-bradykinin for one-

and two-hour incubations.

Degradation studies in brain cell culture

Characterization of kinin receptors required the use of cultured

brain cells to decrease non-specific absorption (see Chapter V). Thus,


























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it was prudent to investigate the integrity of the iodinated ligand

following incubation with these intact cells. Because whole cells in an

isotonic medium will retain their intracellular enzymes and consequently

decrease the potential for proteolytic degradation, the dose of 1,10-o-

phenanthroline was decreased by half from the previous experiments to

2.5 mM. However, 2 yM SQ 20,881 was added to the incubation medium

because subsequent experiments for characterization of kinin binding

sites required assurance that the radioactive ligand was not bound to

kininase II (Chapter V). Table 1 shows that two hours of incubation at

4 C resulted in only a 3% loss in intact 1251-Tyr-bradykinin.

Incubation of the ligand for two hours at room temperature caused

slightly more degradation and a 24-hour incubation resulted in a 14%

loss of 1251-Tyr-bradykinin.

Discussion

The ultimate application of the radioactive kinin analogue is

characterization of specific, high affinity kinin binding sites in CNS

tissue. Proper choice of ligand is necessary to maximize receptor

affinity. A variety of radioactive analogues of bradykinin have been

prepared for use in binding studies and radioimmunoassay (25, 26, 162,

163). In these studies 1251-Tyr-bradykinin (1251-Tyr1-kalladin) was

selected as the radioactive probe for two reasons: biological activity

and cost of preparation. Previous investigations (25) have shown

Tyr-bradykinin and its iodinated form to retain almost all of their

biological activity in smooth muscle preparations, whereas other

iodinated kinin analogues with tyrosine substitutions at the 8 and 5





















TABLE 1
Degradation of 1251-Tyr-bradykinin in Cultured Brain Cells


Incubation conditions


Control


2 hrs., 40 C


2 hrs., 240 C


24 hrs., 40 C


Percent intact


95.5 + 0.7


93.0 + 0


89.0 + 4.0


82.0 + 1.0








positions have little or no biological activity in these same

preparations (14, 25). Tyr-bradykinin is also active when injected into

the brain (Figure 14) and thus is a suitable probe for investigating

kinin receptors in the CNS.

Tritiated analogues of bradykinin have been used (26, 163) but

these ligands must be purchased by commercial suppliers and are

expensive. lodinated kinins are preferable because the molecule can be

radioactivity tagged and purified in the laboratory by simple procedures

for relatively little cost. Additionally, the specific activity of the

iodinated kinin is much greater than tritated kinins available from

commercial sources (500-800 Ci/mmole vs 50 Ci/mmole, respectively).

The presence of kininases has been extensively reported in brain

(93-95, 101, 107, 111-116) as well as other tissues which elicit

biological activity in the presence of kinins (92, 164). Inhibition of

degradation is a prerequisite to a successful receptor binding assay,

without which receptor-ligand equilibrium cannot be achieved. A variety

of enzyme inhibitors were unable to stop degradation of the radioactive

ligand even at 40 C. The inability of Captopril to stop degradation

suggests that the radioactive kinin is rapidly destroyed by enzymes

other than kininase II (angiotensin-converting enzyme), an enzyme known

to be present in various brain regions (93, 94). The metal chelator

1,10-o-phenanthroline is capable of binding metal ions often found as

co-factors in enzymes. Since 5 mM phenanthroline was effective in

blocking 1251-Tyr-bradykinin degradation in brain homogenate for up

to two hours at 40 C, the major proportion of kininase activity in cold

brain tissue is through enzymes with metal co-factors. Phenanthroline





75


was equally effective in blocking degradation of kinins in cultured

brain cells. The anatomical integrity of cultured cells keeps the

majority of proteases sequestered within intracellular compartments and

probably decreases the degradation potential of this preparation.

Selection of an appropriate ligand, and inhibition of degradation

of that ligand in the proper tissue preparation should allow an accurate

search for kinin binding sites in brain tissue.














CHAPT 05V
SPECIFIC BINDING OF 5I-TYR-BRADYKININ
IN CULTURED RAT BRAIN CELLS

Rationale

Given the biological actions of exogenous kinins in vivo it is

reasonable to postulate that the biological response occurs by kinins

binding to these receptors. Binding sites have been identified in brain

tissue for a variety of peptides (159, 160, 165, 166), but kinin binding

sites have not previously been identified in CNS tissue and have only

recently been described in peripheral tissue (25, 26, 163, 164).

Binding studies in CNS tissue tend to amplify the technical problems

normally found when other tissues are utilized. The high lipid content

of brain may contribute to the high levels of nonspecific binding, and

the high levels of protease activity can cause rapid degradation of the

radioactive ligand. The methods described in Chapter IV will be

combined with brain cell culture to overcome these technical problems.
Methods

Brain cell culture

Neonatal rats were obtained from Sprague-Dawley rats mated within

the laboratory. Pregnant Wistar-Kyoto and spontaneously hypertensive

rats were obtained from University of Florida animal resources. Cells

from neonatal rat brain were cultured according to the methods of

Razaida et al. (165) with minor modifications. All procedures were









performed aseptically. Neonates of 0-1 day in age were placed in an

isotonic salt solution (136 mM NaCi, 5.4 mM KC1, 0.16 mM Na2HPO4,

0.22 mM KH2PO4, 5.5 mM glucose and 59.0 mM sucrose) containing 100

units penicillin G, 100 ug streptomycin and 0.25 ug Fungizone (Gibco)

per ml, pH 7.2. The cranium was opened, and the whole brain was exised

at the level of the medulla and placed in the isotonic salt solution.

Under magnification each brain was cleaned free of pia mater and minced

with iris scissors. The minced tissue was then incubated twice for 15

minutes at 37 C with a total volume of 0.25% trypsin (Worthington) in

isotonic salt solution and 25 ml of trypsin added prior to each

incubation. The mixture was constantly agitated. At the second

incubation 160 ug DNAase I (Sigma) in isotonic salt solution was also

added. Next, the dissociated cells were centrifuged at 800 x g for 5

minutes and the supernatant was aspirated. The pellet was triturated

and suspended in 40 ml Dulbecco's modified Eagle's medium (DMEM, Gibco)

containing 10% fetal bovine serum (FBS, Gibco) and filtered through

sterile gauze into a sterile bottle. The cell suspension was diluted to

the appropriate concentration and cells were pipetted onto sterile

Falcon culture dishes. Mixed cultures containing both glia and neuronal

cell types were plated on 35 mm dishes at a density of 2.8 x 106

cells/plate. Neuronally enriched cultures were plated at a density of 5

x 106 cells/plate on 60 mm culture dishes precoated with .001% poly-L-

lysine. Cells were fed on day three after plating with DMEM containing

5% FBS and 10% horse serum (HS, Gibco). Successive feedings occurred at

four-day intervals and consisted of DMEM containing 10% HS. Neuronally

enriched cultures were treated with cytosine arabinoside (10 PM) on days

2 and 3 to minimize glial growth and proliferation in these cultures.









Measurement of 1251-Tyr-bradykinin binding

The specific binding of 1251-Tyr-bradykinin to membrane

receptors was measured on intact brain cells attached to sterile plastic

culture dishes. Prior to each assay cells were washed twice with small

volumes of 5 mM potassium phosphate buffer (pH 7.2) containing 0.32 M

sucrose. Cultures were typically incubated in triplicate for two hours

at 40 C in 5 mM potassium phosphate/0.32 M sucrose containing 2.5 mM 1,

10-o-phenanthroline (Sigma), 2 yM SQ 20,881 (Penninsula, Bachem), and

0.3 nM 1251-Tyr-bradykinin. For determination of nonspecific

binding some plates also contained 0.1 0.2 yM unlabelled bradykinin or

kinin analogues where indicated. Following incubation the plates were

rapidly rinsed (<1 minute) three times with the ice cold potassium

phosphate sucrose buffer. The monolayer in each dish was then dissolved

with 0.5 ml of 2.0 M NaOH. The dissolved tissue was transferred to 12 x

75 cm test tubes, the plates were rinsed once with 0.5 ml water and this

was pooled with the original sample. Samples were counted in a Beckman

gamma 5500 counter (74% efficiency). Specific binding was determined by

subtracting cpm bound in the presence of excess unlabelled kinin from

cpm bound in cultures without unlabelled peptide added. Protein was

determined by the method of Lowry et al. (167) from samples used in each

experiment or from identically grown cells. Biphasic competition curves

were fitted according to IC50 values and the percentage of the high

affinity sites calculated by an iterative program based on a one- or

two-site receptor model(168).









Results

Morphology

Cultures contained cells having morphology consistent with that of

glia and neurons. In developed cultures the larger glial cells formed a

flat monolayer on the bottom of each plate upon which neurons attached

and developed. Neurons were distinguished by their extensive neurite

development. A photograph of a typical culture is shown in Figure 20.

Characteristics and specificity of binding

Figure 21 shows that binding to these cultures is linear in

proportion to cell number within the range examined. Typical

experiments were carried out at cell densities of 2 x 104 cells/cm2

in 35 mm dishes, and corresponded to approximately 300 ug of protein per

dish. Optimum pH was within the neutral range (pH 7.2-7.5, Figure 22),

so subsequent experiments were carried out at pH 7.2.

Incubation of 1251-Tyr-bradykinin with whole brain cells resulted

in a time dependent increase in specific binding at 4* C (Figure 23),

reaching a maximum in two hours. In subsequent experiments no increase

in specific binding was observed at ten hours association as compared to

two hours. Nonspecific binding was stable and did not change

significantly beyond five minutes. For all experiments specific binding

(i.e., total amount bound minus the amount bound in the presence of

excess unlabelled ligand) ranged between 35 and 40 percent of total

binding. Five percent of the total radioactivity added (typically

200,000 cpm) was bound to cultures in the absence of unlabelled ligand.

Saturation experiments plotted by Scatchard analysis suggested two

distinct high affinity components to 1251-Tyr-bradykinin binding

































FIGURE 20:


Fetal rat brain cell culture. Note two
distinct cell morphologies. Larger, flat
cells are identified as glia cells. Cells
with extensive neuritic processes on top
of the glial monolayer are identified as
neurons.


































FIGURE 21:


Specific 1251-Tyr-bradykinin (1251-Tyr-BK)
binding is linear with increasing tissue
concentration in culture. Varying densities
of cells were grown in 60 mm plates. Cell
density in subsequent assays was performed
near 2 x 10 cells/cm2, which corresponds
to 300 pg of protein. Data are presented as
mean S.E. of triplicate determination.
















































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FIGURE 22:


Specific 1251-Tyr-bradykinin(125-Tyr-BK)
binding in brain cell culture demonstrates a
pH optimum in the neutral range (pH 7.2-7.5).
Subsequent experiments were performed at pH
7.2. Data are presented as mean S.E. of
triplicate determinations.





















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in cultured cells. As shown in Figure 24 these components were resolved

by linear regression into two lines with different slopes. For the

higher affinity site the calculated KD was 0.5 nM with a Bmax

of 72 fmoles/mg protein. The lower affinity site had KD and

Bmax values of 21 nM and 1193 fmoles/mg protein, respectively.

From this experiment the higher affinity sites represent only 6% of the

total number of kinin binding sites.

When saturation experiments were done by competition for
1251-Tyr-bradykinin binding with unlabelled kinin, displacement

curves were biphasic when the competing kinin was added in

concentrations greater than 0.2 pM. Figure 25 illustrates a biphasic

competition curve with high affinity sites (0.9 nM KD, 31% of total

sites), and micromolar affinity sites (11 yM KD, 69% of total sites)

as determined by computer analysis. The micromolar affinity site

appeared to be due to nonspecific, electrostatic attraction because

binding in plates devoid of cells was displaceable when 10 yM, but not

0.2 pM, unlabelled bradykinin was added to the dishes.

Competition for radioligand binding was assessed with various kinin

analogues, other unrelated peptides and prostaglandin El (Figure 26

and Table 2). Most kinin analogues were potent in displacing binding

with the order of potency Lys-bradykinin > bradykinin > Tyr-bradykinin >

Tyr8-bradykinin. Des-Arg9-bradykinin did not compete for
125I-Tyr-bradykinin at 1 uM or 10 pM. Other peptides (Table 2)

displaced less than 10% of specific binding for high affinity binding at

concentrations up to 1 pM.




























FIGURE 24:


Scatchard analysis of 1251-Tyr-bradykinin
binding. The experiment was performed by addition
of increasing amounts of 1251-Tyr-bradykinin
alone or in the presence of 0.2 yM unlabelled
Tyr-bradykinin in identical cultures. The plot
was resolved into two components by linear
regression. The higher affinity component had a
KD of 0.5 nM and Bmax equal to 72
fmoles/mg protein. The lower affinity component
revealed KD and Bmax values of 21 nM and
1193 fmoles/mg protein, respectively. Data are
the means of triplicate determinations.












































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