Characterization of some carbostyril congeners having novel beta-adrenoreceptor agonist properties

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Characterization of some carbostyril congeners having novel beta-adrenoreceptor agonist properties
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vi, 118 leaves : ill. ; 29 cm.
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Standifer, Kelly Marie, 1962-
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Thesis:
Thesis (Ph.D.)--University of Florida, 1988.
Bibliography:
Bibliography: leaves 108-116.
Statement of Responsibility:
by Kelly Marie Standifer.
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Typescript.
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Vita.

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CHARACTERIZATION OF SOME
CARBOSTYRIL CONGENERS HAVING NOVEL BETA-ADRENORECEPTOR
AGONIST PROPERTIES












By

KELLY MARIE STANDIFER


A DISSERTATION SUBMITTED 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


1988















This dissertation is dedicated to my parents and
grandparents whom I love very much. They have taught me that
even the most outrageous goals can be more than just dreams.















ACKNOWLEDGMENTS


While I have enjoyed my brief stay in Gainesville, there

have been times when I would never have finished without the

support of good friends. For this reason, I will always be

indebted to James S. Meyer, Karen Reynolds, John Carl, Paula

Papanek, Dave Sherry, and Lynne Fleming. I have unfailing

gratitude and respect for my mentor and friend, Steve Baker,

who believed in me when no one else would. I send a big

"Thank You!" to Dr. Joseph Pitha for supplying us with the

wonder drug and for consenting to allow a mere graduate

student to carry out its initial characterization. Many

thanks are also expressed to my helpful and friendly

committee: Steve Childers, Allen Neims, Matt Knight and Phil

Posner. I never knew committee meetings could be so much

fun. Thanks also to my animal care mentor Thomas Muther --

Let's hear it for cell culture! My best wishes to all my

fellow (and former fellow) graduate students; thanks for the

friendship and moral support. I would also like to thank

other faculty members who worked so hard to improve the

program, and the secretarial and administrative staff who

keep the department running. Special thanks are given also

to Judy Adams for patiently answering my endless questions;

she is priceless.


iii
















TABLE OF CONTENTS




ACKNOWLEDGMENTS................................. iii

ABSTRACT ...................................... v

CHAPTERS

1 INTRODUCTION ........ .. .................. 1

Function............................... 1
Regulation........ ................... 3
Structure........ ..................... 6

2 MEMBRANE STUDIES.......................... 11

Introduction.......................... 11
Experimental Procedures............... 13
Results................................ 19
Discussion............................ 38

3 INTACT CELL STUDIES...................... 46

Introduction.......................... 46
Experimental Procedures................ 47
Results ............................... 54
Discussion............................ 71

4 WHOLE ANIMAL STUDIES..................... 77

Introduction .................. ... ... 77
Experimental Procedures................ 78
Results................ ................ 84
Discussion............................ 99

5 SUMMARY AND CONCLUSIONS.................. 105

LIST OF REFERENCES........................... 108

BIOGRAPHICAL SKETCH........................... 117















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 SOME
CARBOSTYRIL CONGENERS HAVING NOVEL BETA-ADRENORECEPTOR
AGONIST PROPERTIES

By

KELLY MARIE STANDIFER

August 1988


Chairman: Stephen P. Baker, Ph.D.

Major Department: Pharmacology and Therapeutics

The interaction of two new derivatives of 8-hydroxy-

carbostyril with the beta-adrenoreceptor system was partially

characterized using isolated membranes, cultured cells, and

animals. The carbostyril congeners contained an amino

(Carbo-Am) or bromoacetamido (Carbo-Br) moiety in the para

position of the phenyl ring.

In rat reticulocyte membranes, binding studies under

different conditions with [125I]iodocyanopindolol revealed

that Carbo-Br and Carbo-Am were 17 to 107-fold more potent

than (-)isoproterenol. Carbo-Am and Carbo-Br were 13.5 and

30-fold, respectively, more potent than (-)isoproterenol at

stimulating adenylate cyclase activity, but exhibited the

same intrinsic activity. Adenylate cyclase activation by

Carbo-Br, Carbo-Am or (-)isoproterenol was blocked with

concurrent addition of propranolol. If, however,










propranolol was added 7 min into the assay, further cAMP

production by Carbo-Br and Carbo-Am was unaffected while

further cAMP production by (-)isoproterenol was immediately

blocked. Both Carbo-Br and Carbo-Am induced a receptor loss

that was blocked by nadolol. The tightly bound Carbo-Am

could be partially dissociated by heat treatment whereas

Carbo-Br could not.

Using DDT1-MF-2 cells, Carbo-Br produced a desensitiz-

ation much like (-)isoproterenol, yet appeared to be binding

irreversibly to the beta-adrenoreceptor.

In vivo, Carbo-Br stimulated rat heart and lung orn-

ithine decarboxylase activity. This stimulation was blocked

by propranolol. Three hours after an injection of Carbo-Br

(0.5-10.0 mg/kg), there was a dose-dependent loss of up to

75% of beta-adrenoreceptors in lung and spleen. Maximal loss

of binding in heart and submaxillary gland was only 25%. In

the four tissues tested, the beta2-receptor population showed

the greatest loss of specific binding.

These results indicated that the two carbostyril con-

geners were potent, full, beta-adrenoreceptor agonists that

produced sustaineded activation effects in membranes and

bound receptors tightly and/or irreversibly. Carbo-Br

produced desensitization in intact cells and appeared to show

irreversible selectivity for the beta2 subtype. Both

congeners could be useful tools with which to further probe

beta-adrenoreceptor turnover, processing, and regulation.















CHAPTER 1
INTRODUCTION


Function

The catecholamines, epinephrine (Epi) and norepinephrine

(NE), have been shown to be responsible for a wide spectrum

of biochemical, physiological, and pharmacological effects.

Ahlquist (1948) clarified the previous confusion concerning

the actions of norepinephrine, epinephrine, and isoproterenol

causing excitation or inhibition of smooth muscle contrac-

tion, depending on the site, dose and catecholamine given.

He found norepinephrine to be the most potent excitatory

catecholamine, with very low activity as an inhibitor. Iso-

proterenol (Iso) exhibited the reverse actions. Epinephrine

was found to be relatively potent in both actions. On the

basis of these observations, Ahlquist proposed the terms

alpha- and beta-receptors for the sites on smooth muscle

where catecholamines produced these responses. He distin-

guished the two receptor sites by the following potency

series: alpha = Epi > NE >> Iso, and beta = Iso > Epi > NE.

These distinctions were later supported by the finding that

there are antagonistic drugs which are specific for each

receptor as well (alpha: phentolamine, phenoxybenzamine;

beta: propranolol, dichloroisoproterenol). In 1967, Lands et

al.(a,b) proposed dividing beta-adrenoreceptors into two








2

subtypes, betal and beta2, based on the relative selectivity

of both excitatory agents and antagonists. Betal-adreno-

receptors, found primarily in the heart and adipose tissue,

showed the potency series Iso > Epi = NE. Beta2-adreno-

receptors were found in the bronchi and vascular smooth

muscle and showed the series Iso > Epi > NE. Though selec-

tivity has been seen only at low doses, further support for

this classification has come from observations that antag-

onists practolol, metaprolol and atenolol were more effective

at blocking responses in heart, whereas certain agonists

(salbutamol and terbutaline) were more selective for beta2-

receptors. Both betal- and beta2-adrenoreceptors were shown

by Sutherland and coworkers (1965) to be coupled to the

membrane bound enzyme adenylate cyclase. Occupancy of the

receptor by an agonist stimulated the enzyme which increased

the intracellular concentration of adenosine 3'5'-cyclic

monophosphate (cAMP). The beta-adrenoreceptor regulated

cyclase system was composed of three separate components:

receptor (R), stimulatory guanine nucleotide regulatory

protein (Ns), and the adenylate cyclase catalytic moiety (De

Lean et al., 1980; Limbird et al., 1980a; Levitzki, 1978;

Rodbell, 1980). Interaction of an agonist (H) with the

receptor has been shown to result in the initial formation of

a low affinity, freely reversible complex, H-R (Heidenreich

et al., 1980; Kent et al., 1980; Williams & Lefkowitz, 1977;

Lefkowitz et al., 1978). A conformational change was then

induced in the receptor such that the receptor became coupled










to the Ns-protein (Citri & Schramm, 1980; Limbird et al.,

1980a). The ternary complex, or H-R-Ns, has been shown to

include high affinity agonist binding which was slowly

dissociable (Heidenreich et al., 1980; Kent et al., 1980;

Williams & Lefkowitz, 1977; Wessels et al., 1978). Formation

of this ternary complex has been shown to be a prerequisite

for activation of cyclase (De Lean et al., 1980; Limbird et

al., 1980a). In forming this complex there was a loss of

tightly bound guanosine 5'-diphosphate (GDP) from Ns, and a

subsequent facilitation of binding of guanosine 5'-

triphosphate (GTP) to the Ns-site (Cassel & Selinger, 1978).

Interaction with GTP destabilized the complex, freeing H-R

and releasing free Ns-GTP. Free NS-GTP then interacted with

the inactive enzyme and activated it (Pfeuffer, 1979).

Cleavage of GTP to GDP by GTPase located on Ns returned the

cycle to its basal state (Cassel & Selinger, 1976; 1977).

Regulation

The term desensitization has been defined as a "phenom-

enon in which the response of a tissue or cell to a biolog-

ically active agent becomes attenuated with time in the

presence of a continuing stimulus of constant intensity"

(Sibley & Lefkowitz, 1985). Desensitization has been com-

monly observed in the beta-adrenoreceptor-coupled adenylate

cyclase system.

Two types of desentization have been described (Stiles

et al., 1984; Harden, 1983). The first, homologous (or

agonist-specific) desensitization, occurred when an










attenuated responsiveness was observed only to the desensi-

tizing agonist, with no loss of responsiveness to any other

agonist. Conversely, a heterologous desensitization was

observed when exposure of one agonist to a cell decreased the

response to multiple agonists operating through distinct

receptors (such as prostaglandin El, PGE1, and glucagon).

In both cases, the beta-adrenoreceptor has been shown to

be phosporylated during the desensitization process. Recent

evidence linked this covalent modification to the decreased

functionality of the receptor (Benovic et al., 1985; Sibley

et al., 1984; Strulovici et al., 1984; Stadel et al., 1983).

These studies suggested that phosphorylation of the receptor

was involved in regulating the hormonal responsiveness of the

adenylate cyclase system. Several patterns of phosphor-

ylation of these receptors by various kinases have been

characterized with respect to their dependence on agonist

occupancy.

Phosphorylation of the beta2-adrenoreceptor by protein

kinase C has been shown to be an agonist-independent process

(Bouvier et al., 1987; Fishman et al., 1987), whereas the

rate of phosphorylation of beta2-adrenoreceptors by cAMP-

dependent protein kinase was increased by agonist occupancy

of the receptors (Bouvier et al., 1987; Yamashita et al.,

1987; Benovic et al., 1985). In the latter case, however,

the cAMP-dependent protein kinase was responsible for only

one-half of the desensitzation, the rest attributed to other

kinases. Since many different drugs or hormones have been










shown to activate cAMP-dependent protein kinase and protein

kinase C in vivo, these types of phosphorylation reactions

were good candidates for the development of heterologous

desensitization.

Identification of a new cAMP-independent protein kinase,

beta-adrenergic receptor kinase (BARK) (Benovic et al.,

1986), which preferentially phosphorylated the agonist-

occupied form of the receptor, suggested a mechanism for

homologous desensitization. The enzyme phosphorylated the

receptor on several serine and threonine residues near the

carboxyl terminus of the receptor (Benovic et al., 1986).

Purification of BARK (Benovic et al., 1987a) yielded a

cytosolic protein with molecular weight (Mr) of 80,000.

Agonist occupancy of the beta-adrenoreceptor caused a trans-

location of BARK from the cytosol to the plasma membrane, and

this translocation was concurrent with the time courses of

receptor phosphorylation, desensitization, and sequestration

(Strasser et al., 1986). Agonist binding to another stimu-

latory adenylate-cyclase coupled receptor, PGE1-receptor, and

to an inhibitory adenylate cyclase-coupled receptor, alpha2-

adrenoreceptor (Benovic et al., 1987b), also promoted trans-

location of BARK, suggesting that BARK may have served to

phosphorylate and desensitize other adenylate cyclase-coupled

receptors as well. However, only agonist occupied receptors

were substrates for the kinase, consistent with homologous

desensitization.










The mechanisms by which receptor phosphorylation led to

desensitization have remained unclear. The possibility that

the phosphorylated receptor has diminished ability to

interact with the guanine nucleotide regulatory protein was a

mechanism that was consistent with the actions of cAMP-

dependent protein kinase in heterologous desensitization.

Studies on human astrocytoma cells (Su et al., 1980; Harden

et al., 1979) demonstrated that desensitization preceded

receptor sequestration, an event characterized by a loss of

binding of cell surface receptors. It has been shown that

receptor phosphorylation triggers the sequestration event

(Benovic et al., 1986). Only agonist-occupied receptor

phosphorylation, and not cAMP-induced receptor phosphory-

lation, has resulted in receptor sequestration or internal-

ization (Sibley et al., 1987 and Toews et al., 1987).. An

alternative mechanism (Clark et al., 1988) suggested that

phosphorylation was only a signal that resulted in desens-

itization; i.e., desensitization did not occur until the

physical isolation of the receptor from the NS-protein was

complete.

Structure

The beta-adrenergic receptors have been shown to be

integral membrane glycoproteins of Mr = 64,000 (Kobilka et

al., 1987a). The complete primary amino acid sequences of

the mammalian betal- (Frielle et al., 1987) and beta2-adreno-

receptors (Dixon et al., 1987a; Kobilka et al., 1987a) were

recently deduced. Though the amino acid sequence was highly










conserved, distinct genes code for each subtype (Emorine et

al., 1987). Each protein has been shown to consist of 418

amino acids which form seven membrane spanning regions. The

striking sequence homology between the beta-adrenergic,

alpha-adrenergic, muscarinic cholinergic, and opsin families

of receptors have led many to theorize that a large "Super-

gene" or multi-gene family of receptors exists (Gocayne et

al., 1987; Lefkowitz & Caron, 1988). On the basis of the

homology between the amino acid sequence of the beta-adreno-

receptors and the opsin proteins, Dixon et al. (1987b)

proposed that the ligand binding domain must lie within the

seven hydrophobic domains. By producing genes for hamster

beta2-adrenoreceptor with sequential deletions, the same

group showed that the transmembrane regions were required for

the structural integrity of the receptor, and that two

cysteine residues in the receptor were important for agonist

binding affinity. A deletion of eight amino acids in the

fourth membrane spanning region resulted in the ability of

the agonist to bind with high affinity but not to stimulate

adenylate cyclase activity (Dixon et al., 1987a). Substi-

tution on amino acid 79 in the second membrane spanning

region resulted in a receptor that displayed normal binding

for antagonists, but reduced affinity for agonists (Strader

et al., 1987b; Chung et al., 1988).

Site-directed mutagenesis was also used to study the

importance of specific receptor regions in desensitization

(Strader et al., 1987a; Kobilka et al., 1987b; Fraser et al.,










1987). Mutations were introduced that resulted in deletions

of the carboxyl terminus, putative cAMP-dependent protein

kinase substrate sites, and the NS-protein coupling site.

Only the mutation which perturbed coupling of the receptor

and NS-protein caused an altered sequestration response.

This suggested a correlation between coupling of the receptor

to cyclase and the ability of the receptor to undergo

agonist-mediated sequestration.

As briefly reviewed above, much has been established

about the subtypes of the beta-adrenoreceptor, the mechanism

of receptor mediated cyclase activation, receptor regulation,

and desensitization. In addition, the recent cloning of the

receptor has led to a major focus on structural studies of

the receptor. Even with these advances, a great deal has

remained in question: the precise interaction of the

receptor with the stimulatory guanine nucleotide binding

protein, a detailed examination of the relationship between

receptor number and the ability of the receptor to produce a

response, a description of the basal turnover of the

receptor, the cellular components involved in receptor

processing, and the processing of the receptor after agonist-

induced internalization. These questions could only be

answered by studying the receptors in their homeostatic

environment. In the past, a number of irreversible antag-

onists have been synthesized to attempt to study some of

these questions (Pitha et al., 1980; Dickinson et al., 1985;

Kusiak & Pitha, 1987). However important differences between










agonist and antagonist binding have been shown to exist.

Agonist binding showed several affinity states (Stiles et

al., 1984), and was modulated by guanine nucleotides

(Lefkowitz et al., 1976; Maguire et al., 1976), divalent

cations (Bird & Maguire, 1978; Williams et al., 1978),

temperature (Scarpace et al., 1986; Baker & Potter, 1981;

Briggs & Lefkowitz, 1980; Weiland et al., 1979; Insel &

Sanda, 1979), and N-ethylmaleimide (Vauquelin et al., 1980;

Vauquelin & Maguire, 1980). In contrast, antagonist binding

usually showed only a single affinity state and was not

affected by those modulators (Heidenreich et al., 1980;

Lefkowitz et al., 1978). In light of the differences between

agonist and antagonist binding, it would be useful to charac-

terize the effects of an agonist that binds irreversibly to

the beta-adrenoreceptor. Another important question has been

whether an irreversibly bound agonist could produce a sus-

tained beta-adrenergic response in a multicomponent receptor

system. Towards this end, a program was initiated to design

and synthesize some potent and stable beta-adrenergic ligands

based on a carbostyril nucleus (Milecki et al., 1987), the

structure of two of which can be seen in Figure 1-1. The

goal of this work has been to examine beta-adreno-receptor

mediated properties of these ligands with the implicit hypo-

thesis that the bromoacetylated compound was an irreversible

agonist.














CH3
I
CHOH-CH,-NH-C-CH R
I
CH3


I
NN 0
OH H


= NH2
= NHCOCH2Br


Figure 1-1. Structures of Carbostyril compounds Carbo-Am
(R1) and Carbo-Br (R2). The 8-hydroxycarbostyril moiety is
the double-ringed structure on the lower left.















CHAPTER 2
MEMBRANE STUDIES

Introduction


Beta-adrenergic activation of adenylate cyclase activity

has been shown to involve a complex set of interactions of

several components in the plasma membrane. These components

included at least the beta-adrenoreceptor, a stimulatory

guanine nucleotide binding protein (Ns) and the catalytic

unit of adenylate cyclase (Levitzki, 1978; Rodbell, 1980).

One useful approach in the study of the beta-adrenoreceptor

has been the development of several irreversible antagonists.

Irreversible receptor antagonists have been used to study the

relationship between receptor number and biological responses

(Homburger et al., 1985; Posner et al., 1984; Venter, 1979;

Tolkovsky & Levitzki, 1978), receptor recovery after irre-

versible blockade in cultured cells (Hughes & Insel, 1986;

George et al., 1986; Homburger et al., 1984; Fraser & Venter,

1980) or whole animals (Baker et al., 1986; Nelson et al.,

1986; Baker & Pitha, 1982), and the antagonist binding sub-

unit of the receptor (Minneman & Mowry, 1986; Stiles et al.,

1984; Atlas & Levitzki, 1978). To date, most agonist-recep-

tor interaction studies have been performed indirectly by

competing unlabelled agonists with labelled antagonists.










Although some studies have used labelled agonists, these

compounds were generally unstable, had low specific activ-

ities, and showed high nonspecific binding (Giudicelli et

al., 1982; Heidenreich et al., 1980; Lefkowitz & Williams,

1977). An irreversible agonist would have been helpful in

characterizing the differences between agonist and antagonist

interaction with the beta-adrenoreceptor and the complex

interactions of the components in that system. Towards that

end, an alkylating derivative of norepinephrine was recently

reported to be a partial beta-adrenergic agonist which could

irreversibly bind to the beta-adrenoreceptor (Baker et al.,

1985). However, though this compound initially stimulated

adenylate cyclase activity, it appeared to act as an

antagonist after receptor alkylation and was, like most

catecholamines, unstable. Recently we reported on the

synthesis of several stable carbostyril derivatives which

preliminary evidence indicated were potent beta-agonists

(Milecki et al., 1987). The synthesis of this type of

compound was based on an earlier report that carbostyril-

based compounds were stable beta-agonists (Yoshizaki et al.,

1976). Figure 1-1 showed two of the carbostyril compounds

used in the present study. A more detailed examination was

made on the interaction of these two derivatives with the

beta-adrenoreceptor system of rat reticulocytes. Evidence

has been provided to indicate that, as hypothesized in the

first chapter, they produce an extremely tight and/or










irreversible activation of adenylate cyclase activity in

vitro.


Experimental Procedures


Source of Materials

The radioligands (-)-[1251]Iodocyanopindolol ([125I]CYP;

2000-2200 Ci/mmol) and [2,8-3H]adenosine 3'5'-cyclic mono-

phosphate ([3H]cAMP; 31.2 Ci/mmol) were obtained from

Amersham Corp. (Arlington Heights, IL, USA) and New England

Nuclear (Boston, MA, USA), respectively. ATP, guanyl-5'-yl-

imididodiphosphate (Gpp(NH)p), GTP, protein kinase, ()al-

prenolol, hydroxyapatite, Iso, theophylline, creatine

phosphokinase, phosphocreatine, penicillin G, streptomycin

sulfate, and amphotericin B were purchased from Sigma

Chemical Co. (St. Louis, MO, USA). Nadolol was a gift from

Dr. A. L. Bassett, University of Miami (Miami, FL, USA), and

propranolol was a gift from Ayerst Laboratories Inc. (New

York, NY, USA). The GH3 cell line was obtained from American

Type Culture Collection (Rockville, MD, USA). Media, fetal

bovine serum and horse serum were purchased from Gibco (Grand

Island, NY, USA). Liquiscint was purchased from National

Diagnostics (Somerville, NJ, USA).

Methods

Drug Preparations. The synthesis and chemical

characterization of 5-[2-[[1-(4-aminophenyl)-2-methylprop-2-

yl]amino]-l-hydroxyethyl]-8-hydroxycarbostyril (Carbo-Am) and










5-[2-[[3-[4-(bromoacetamido)phenyl]-2-methylprop-2-yl]amino]-

l-hydroxyethyl]-8-hydroxycarbostyril (Carbo-Br) has been

previously described in detail (Milecki et al. 1987), and was

received from this group. Stock solutions (1 mM) were made

with ethanol and diluted with either water or buffer to give

the desired concentrations.

In some experiments, the Carbo-Br compound (0.5 mM) was

incubated without and with 2 mM cysteine in 50 mM sodium

phosphate buffer pH 7.4 for 18 hr at 250C. At the end of the

incubation, samples were run on silica-gel TLC plates with

chloroform/methanol (1:1). The Rf for Carbo-Br alone was

0.34 whereas for Rf for the reaction product of Carbo-Br and

cysteine was 0.05. Virtually complete conversion of the

Carbo-Br occurred. The reaction product, when used, was

referred to as carbo-cysteine.

Buffer. Unless otherwise noted, buffer was 50 mM Tris-

HC1 pH 7.4 containing 5 mM MgC12.

Rat Reticulocyte Preparation. Reticulocyte production

was induced by a slight modification of a previously

described technique (Baker et al., 1985). Briefly, male

Sprague-Dawley rats (175-225 g) were injected (subcutan-

eously) on days one, three, and four with 25, 30 and 35

mg/kg, respectively, of phenylhydrazine hydrochloride to

induce hemolysis. The drug was prepared by dissolving in

water and adjusting the pH to 7.0 with solid sodium bicarb-

onate. On day seven, blood was withdrawn by cardiac puncture

and the red cells washed twice at 40C in 20 mM Tris-HC1 at pH










7.4 containing 150 mM NaCI and 20 mM sodium citrate. The

cells were then washed (by centrifugation at 800xg for 10 min

and gentle resuspension) twice more in 20 mM Tris-HCl at pH

7.4 containing 150 mM NaC1. The cells were then lysed by a

20-fold dilution in ice-cold 10 mM Tris-HC1 pH 7.4 containing

2 mM MgCl2 followed by homogenization in a Waring blender at

top speed for 2 min. The suspension was centrifuged at

48,000xg for 10 min and the top layer of the bilayered pellet

was swirled free. The top layer of membranes was then washed

(by centrifugation at 48,000xg for 10 min and resuspension)

twice in buffer and stored in buffer under liquid nitrogen

until used. The protein content was determined by the method

of Lowry et al. (1951) using bovine serum albumin as

standard. Maximal specific [125I]CYP binding in these

membranes ranged from 0.5 to 0.8 pmol/mg protein.

Rat erythrocyte membranes were prepared by withdrawing

blood by cardiac puncture, washing and lysing the red blood

cells as described above for reticulocytes. After lysis, the

suspension was centrifuged at 48,000xg for 10 min and the

pellet resuspended in ice-cold buffer. The suspension was

centrifuged again at 48,000xg for 10 min and the pellet was

washed (by resuspension and centrifugation) once more with

buffer. The final pellet was resuspended in 1 vol of buffer

for assays and stored under liquid nitrogen.
Cell Culture. Rat pituitary tumor (GH3) cells were

grown in 57 cm2 culture dishes with Dulbecco's Modified

Eagle's Medium containing 5% horse serum, 5% fetal calf










serum, 100 U/ml penicillin G, 0.1 mg/ml streptomycin sulfate,

and 0.25 mg/ml amphotericin B. The cells were grown in a 5%

C02/95% air humidified atmosphere at 370C. Plates were

seeded at 1 x 105 cells/ml and the cells harvested at 80-90%

confluence. The cells were washed twice on the plate with 5

mM sodium phosphate buffer at pH 7.4 containing 150 mM NaCl

(PBS) and scraped free in 1 ml of PBS with a rubber

policeman. The cells were then centrifuged at 1000xg for 5

min and the cell pellet resuspended in 20 vol of ice-cold

buffer. The suspension was then homogenized (Tekmar SDT-100

EN, setting 3, 5 sec) and centrifuged at 48,000xg for 10 min.

The pellet was then washed twice more (by resuspension and

centrifugation) and the final pellet resuspended in 1 vol of

buffer for assays.

Antagonist Binding Assays. Beta-adrenoreceptor content

was determined by incubating membrane protein (5-10 gg) in a

total volume of 0.25 ml with 50 mM Tris-HC1 pH 7.4, 5 mM

MgCl2, 3-100 pM [125I]CYP in the presence and absence of 1 lM

()alprenolol for 60 min at 360C. At the end of the

incubation, each suspension was diluted with 3 ml of 50 mM

Tris-HCl at pH 7.4 (36C) and poured onto a Whatman GF/B

glass fiber filter under reduced pressure. The filter was

washed with a further 6 ml of buffer, placed in a vial and

radioactivity determined. Specific [125I]CYP binding to the

beta-adrenoreceptor was calculated as the difference between

the total binding in the absence of ()alprenolol and the

nonspecific binding determined in the presence of 1 gM










()alprenolol. Nonspecific binding was the same if the

()alprenolol was replaced with 100 pM Iso and specific

binding was 90-95% of the total bound.

In some experiments, the ability of the carbostyril

congeners or Iso to inhibit specific [125I]CYP binding was

determined. Assays were the same as above except the

[125I]CYP concentration was 30 pM and 0.1% sodium ascorbate

was included when Iso was used. The competitive binding

assays were also performed in the presence and absence of 100

JIM Gpp(NH)p, a non-hydrolyzable analog of GTP. All binding

assays were performed in triplicate, the results varying by

less than 5%.

Adenylate Cyclase assays. Enzyme activity was deter-

mined by a slight modification of a competitive protein

binding assay described previously (Baker et al., 1985).

Membrane protein (20-40 gg) was incubated in a total volume

of 0.15 ml with 1.6 mM ATP, 5 mM MgCl2, 1.0 mM ethylene

glycol bis(beta-aminoethylether-N,N,N'N'-tetraacetic acid

(EGTA), 10 mM theophylline, 0.1% bovine serum albumin, 50 mM

Tris-HC1 at pH 7.4, creatine phosphokinase (67 units/ml) and

phosphocreatine (2.5 mM) for 10 min at 320C. These were the

basal conditions. When stimulation of enzyme activity was

measured, the binding assay also contained 500 gM GTP, in the

presence or absence of Iso or the carbostyril congeners. At

the end of the incubation, 0.3 ml of 10 mM Tris-HCl buffer pH

7.0 containing 5 mM ethylenediamine tetraacetic acid (EDTA)

was added to each tube and the tubes placed in a boiling










water bath for 5 min. After cooling to room temperature, the

tubes were centrifuged for 5 min at 1,200xg.

The cAMP content of the supernatant was determined by

incubation in a total volume of 0.2 ml containing 25 mM Tris-

HC1 buffer pH 7.0, 10 mM theophylline, 0.8 pmol of [3H]cAMP,

an aliquot of the supernatant and 8 gg of bovine heart cAMP

dependent protein kinase for 60 min at 40C. At the end of

the incubation, 70 pl of a 50% (w/v) hydroxyapatite suspen-

sion was added to each tube followed by 4 ml of ice-cold 10

mM Tris-HCl buffer at pH 7.0. The suspension was then poured

onto a Whatman GF/C glass fiber filter under reduced

pressure. The filter was washed with a further 8 ml of ice-

cold 10 mM Tris-HCl pH 7.0, and placed in a scintillation

vial with 1 ml of 0.3 N HC1. After the hydroxyapatite had

dissolved (about 10-15 min), 9 ml of Liquiscint was added and

the radioactivity determined. The amount of cAMP present was

calculated from a standard curve determined with unlabelled

cAMP. The production of cAMP was linear with time and

protein through 14 min and 0.1 mg, respectively.

Membrane Pretreatments. Membrane protein (2-3 mg/ml) was

suspended in 50 mM Tris-HCl buffer pH 7.4 containing 5 mM

MgC12 and other additions as indicated in the text. The

suspensions were then incubated for varying times at 300C.

At the end of the incubation, the suspensions were diluted

with 20 ml of ice-cold incubation buffer containing 5 mM

MgCl2 and centrifuged at 48,000xg for 10 min. The pellet was

resuspended in 20 ml of ice-cold incubation buffer and










centrifuged again at 48,000xg for 10 min. The pellet was

washed twice more by centrifugation and resuspension and the

final pellets were resuspended in 1-2 ml of buffer for

assays.

In heat treatment experiments, membrane protein (2-3

mg/ml) was incubated in 50 mM sodium phosphate buffer pH 7.4

containing 5 mM MgCl2 and other additions as indicated in the

text at 450C for 30 min. At the end of the incubation, the

suspensions were cooled in an ice-water bath and centrifuged

at 48,000xg for 10 min. The pellets were then washed (by

centrifugation and resuspension) three more times with buffer

and the final pellets were resuspended in 2 ml of buffer for

assays.

Data Analysis. The receptor concentrations and KD

values were determined from regression analysis of Scatchard

(1949) plots. The concentrations of compounds which

inhibited ligand binding by 50% (IC50) or the effective

concentrations of drugs which gave 50% of a maximal response

(EC50) were determined from a dose-effect analysis using an

Apple IIe microcomputer as described by Chou and Talalay

(1983). Statistical analysis of the data was performed using

the Student's t-test.

Results

Effects of carbostyril derivatives on reticulocyte beta-

adrenoreceptors. The structures of Carbo-Am and Carbo-Br

were shown in Figure 1-1. Both compounds were racemic

mixtures. Figure 2-1 showed the ability of Iso and the two










carbostyril derivatives to inhibit specific [125I]CYP binding

to rat reticulocyte membranes. In the absence of Gpp(NH)p,

the concentration of Iso that inhibited [125I]CYP binding by

50% (IC50) was 49 3 nM and the Hill slope was 0.51 0.03.

In the presence of 100 pM Gpp(NH)p, the IC50 value increased

16.5-fold to 813 66 nM and the Hill slope increased to 0.94

0.02 (Figure 2-1A). Figure 2-1A also showed the competi-

tion curves for Carbo-Am. In the absence of Gpp(NH)p, the

IC50 value was 5.9 0.2 nM whereas in the presence of

Gpp(NH)p the IC50 value was 21 0.6 nM. The ability of

Carbo-Br to inhibit [125I]CYP binding was shown in Figure 2-
1B. In the absence of Gpp(NH)p, the IC50 value was 3.3 0.3

nM and the addition of Gpp(NH)p only shifted the IC50 value

2.2-fold to 7.6 0.3 nM.

Figure 2-2 showed a representative Scatchard plot of

[125I]CYP binding after pretreatment of reticulocyte mem-

branes with Carbo-Br or Carbo-Am. As Iso is easily washed

from membranes, no control for Iso pretreatment was neces-

sary. Membranes incubated with 25 nM Carbo-Br for 30 min at

320C followed by four washes had an 82% reduction of specific
[125I]CYP binding sites with no change in the KD value of

[125I]CYP for the remaining receptors (control, 14 pM; Carbo-

Br-treated, 12 pM). When 100 gM Gpp(NH)p was added during

the pretreatment period, the loss of specific binding sites
was reduced to 65% with no alteration of the KD value (13 pM)

as compared to the control for the remaining receptors

(Figure 2-2A). Figure 2-2B showed a Scatchard plot after









Figure 2-1. Inhibition of specific [125I]CYP binding in rat
reticulocyte membranes by Iso, Carbo-Am (A) and Carbo-Br (B).
Membranes were incubated with buffer at pH 7.4, 30 pM
[125I]CYP, the indicated concentrations of (A) Iso (squares),
Carbo-Am (circles) or (B) Carbo-Br (circles) and without
(open symbols) or with (closed symbols) 100 JiM Gpp(NH)p for
45 min at 360C. In the Iso competition assays, 0.1%
ascorbate was also present. At the end of the incubation,
the specific binding was determined as described in the
"Methods" section. Each point on the graph is the mean of
three determinations assayed in triplicate. Control
[125I]CYP binding ranged from 524 to 583 fmol/mg protein.













0-%


n


0
-C








LO
cm





C
0







n
a-
U
"-
H
If
TJ


Ligand (-Log M)










Figure 2-2. Scatchard plot of specific [125I]CYP binding to
rat reticulocyte membranes after treatment with (A) Carbo-Br
or (B) Carbo-Am. In A, membranes were incubated in buffer
without (open circles) and with (closed circles) 25 nM Carbo-
Br or with 25 nM Carbo-Br plus 100 pM Gpp(NH)p (squares) for
30 min at 320C. In B, membranes were incubated without (open
circles) and with (closed circles) 0.1 pM Carbo-Am or with
0.1 JM Carbo-Am plus 100 IM Gpp(NH)p (squares) for 30 min at
32 oC. At the end of the incubation, the membranes were
washed four times with buffer and assayed with 3 to 100 pM
[125I]CYP as described in the "Methods" section. The data
were plotted as the ratio of the amount of specifically bound
ligand (pmol/mg protein) to free ligand (pmol/l) versus the
amount of specifically bound ligand/mg protein. Data points
were the mean of triplicate determinations and are
representative of three experiments.















0
T-
x
320-






O X
10
40
50-


40









100 300 500
125CYP Bound(f mg protein)
c20


i 10-

.0
100 300 500
EC125JCYP Bound (fmdl/mg protein)










pretreatment of reticulocyte membranes with 0.1 g1M Carbo-Am

for 30 min at 320C followed by four wash cycles. The Carbo-

Am-treated membranes showed a 73% reduction in specific
[125I]CYP binding with no change in the KD value of [125I]CYP

for the remaining receptors (control, 8 pM; Carbo-Am-

treated,10.4 pM). Inclusion of 100 JM Gpp(NH)p in the

preincubation reduced the Carbo-Am-induced receptor loss to

51% with no change in the KD value for [125I]CYP (10.8 pM).

In one other series of experiments, the ability of various

doses of the two derivatives to induce a receptor loss was

determined. Incubation of reticulocyte membranes at 320C for

30 min with 10, 1, 0.1 and 0.01 JM of Carbo-Br followed by

four membrane wash cycles reduced specific [125I]CYP binding

by 97, 93, 86 and 80%, respectively. In contrast, using the

same concentrations of Carbo-Am, the receptor content was

reduced by 73, 73, 72 and 62%, respectively.

Table 2-1 showed the effects of several treatments on

the ability of the carbostyril derivatives to bind to the

beta-adrenoreceptor in reticulocyte membranes. Preincubation

of membranes with nadolol (10 gM) followed by washing did not

reduce specific [125I]CYP binding indicating that this beta-

antagonist is easily washed off the receptor and out of the

membranes (unlike propranolol). Furthermore, membrane

heating at 450C for 30 min had no significant effect on the

receptor. Preincubation with Carbo-Am (1 JM) followed by

membrane washing, reduced the binding by 73%, which could be









Table 2-1. Effects of nadolol and heat treatment on the
Carbo-Am and Carbo-Br-induced receptor loss in reticulocyte
membranes.


[125I]CYP Bound
Pretreatment A Pretreatment B (%Control)


----- ---- 100

Nadolol (10 gM) ------- 110 6

----- 450C 97 4

Carbo-Am (1 LM) ----- 27 3

Carbo-Am (1 gM) plus
Nadolol (10 rM) ----- 87 5

Carbo-Am (1 iM) Nadolol (10 gM) 29 6

Carbo-Am (1 IgM) 450C plus Nadolol (10 pM) 82 4

Carbo-Br (1 RM) ----- 12 4

Carbo-Br (1 gM) plus
Nadolol (10 gM) ----- 96 2

Carbo-Br (1 gM) 450C plus Nadolol (10 IM) 16 2

Carbo-cysteine (1 iM) ------ 25 4

Carbo-cysteine (1 IM) 450C plus Nadolol (10 pM) 86 6


In pretreatment A, membrane protein was incubated with
buffer and the additions indicated for 30 min at 320C. At
the end of the incubation, the membranes were washed four
times and assayed with [125I]CYP (100 pM). Alternatively, at
the end of pretreatment A, the membranes were washed once,
resuspended in 50 mM sodium phosphate buffer plus the
additions under pretreatment B and incubated for 30 min at
45C or at 320C for 20 min in the presence of 10 iM nadolol.
At the end of the incubation, the membranes were washed four
times with ice-cold buffer and assayed with [125I]CYP at 100
pM. Carbo-cysteine is Carbo-Br (0.5 mM) reacted for 18 hr at
25C with cysteine (2 mM) and diluted to the final concentra-
tion indicated. Values are the mean of triplicate determin-
ations S.D, n=3-4.










largely prevented (87% of control) by concurrent incubation

with 10 gM nadolol. However, no receptor recovery occurred

if the membranes were incubated with 10 .LM nadolol after the

Carbo-Am pretreatment. The initial Carbo-Am-induced loss of

receptors could be substantially recovered (82% of control)

by subsequent membrane heating at 450C in the presence of

nadolol. Table 2-1 also showed that membranes pretreated

with Carbo-Br (1 pM) followed by washing had an 88% decrease

in specific [125I]CYP binding which was largely attenuated by

concurrent pretreatment with 10 pM nadolol. When membranes

were pretreated with Carbo-Br, washed and then incubated with

nadolol at 450C for 30 min followed by washing, there was

little reversal (<10%) of the lost specific binding sites.

In contrast, when a solution of Carbo-Br was incubated with

cysteine (250C, 18 hr), diluted to 1 gM and incubated with

membranes followed by washing, there was a 75% loss of

binding sites. These lost sites were largely recovered (86%

of control) if the pretreated membranes were further

incubated with nadolol at 450C for 30 min followed by

washing. A diluted sample of cysteine alone had no effect on

[125I]CYP binding.

Current evidence indicated that in the absence of a

guanine nucleotide, beta-agonists promoted the formation of a

ternary complex composed of the agonist, the beta-adreno-

receptor and Ns. Agonist binding in the complex was of high

affinity. In the presence of a guanine nucleotide, the

complex destablized and agonist affinity decreased (De Lean










et al., 1980; Kent et al., 1980). To explore if the small

Gpp(NH)p-induced potency shift for the Carbo-Am was due to a
tightly bound receptor-Ns complex resistant to guanine

nucleotide destabilization, competition experiments were

performed under conditions where guanine nucleotide modu-

lation of receptor affinity (receptor-Ns interactions) was

greatly reduced. Two preparations were used: 1) rat

erythrocyte membranes which have a greatly reduced content of
Ns (Limbird et al., 1980a), and 2) reticulo-cyte membranes

heated at 500C to destroy the ability of guanine nucleotides

to modulate receptor affinity (Baker et al., 1985). Figure

2-3A showed the ability of Iso and Carbo-Am to inhibit

specific [125I]CYP binding in rat erythrocyte membranes. In

the absence of Gpp(NH)p, the IC50 value for Iso was 941 67

nM. In the presence of 100 LM Gpp(NH)p, there was only a

slight increase in the IC50 value to 1263 60 nM. The IC50

values for the Carbo-Am were not different when the assays

were performed in the presence or absence of Gpp(NH)p (IC50

values: minus Gpp(NH)p, 27 1.2 nM; plus Gpp(NH)p, 29 2.5

nM). Figure 2-3B showed the competition curves in reticulo-

cyte membranes. In control membranes, the IC50 value for Iso

was shifted 20.4-fold from 58 7.5 nM in the absence of

Gpp(NH)p to 1182 20 nM in the presence of 100 gM Gpp(NH)p.
After treatment of membranes for 45 min at 500C, the IC50

value for Iso in the absence of Gpp(NH)p was 750 57 nM.

The IC50 value for Carbo-Am in control membranes and in the

absence of Gpp(NH)p was 7.3 0.3 nM which was increased









Figure 2-3. Inhibition of specific [125I]CYP binding in rat
erythrocyte (A) and reticulocyte (B) membranes by Iso and
Carbo-Am. In A, erythrocyte membranes were incubated with
buffer, 30 pM [125I]CYP and the indicated concentrations of
Iso (squares), Iso plus 100 JM Gpp(NH)p (triangles), Carbo-Am
(open circles) or Carbo-Am plus 100 p!M Gpp(NH)p (closed
circles) for 45 min at 360C. In B, control reticulocyte
membranes were incubated with buffer, 30 pM [125I]CYP and the
indicated concentration of Iso (open squares) and Iso plus
100 JM Gpp(NH)p (closed squares), Carbo-Am (open circles) or
Carbo-Am plus 100 IM Gpp(NH)p (closed circles), for 45 min at
360C. In addition, reticulocyte membranes in 50 mM sodium
phosphate buffer at pH 7.4 were incubated at 500C for 45 min,
washed 1 time in buffer and the competition assay carried out
with the indicated concentration of Iso (closed triangles)
and Carbo-Am (open triangles). In all assays utilizing Iso,
0.1% ascorbate was also present. At the end of the incuba-
tions, the specific binding was determined as described in
the "Methods" section. Each point on the graph was the mean
of three determinations assayed in triplicate. The control
[125I]CYP binding values were 39 7, 622 13 and 644 12
fmol/mg protein for the erythrocyte, control reticulocyte and
heat-treated reticulocyte membranes, respectively.













0
.Co
c-













10(
45
5 8'







6(



o
4(
0..
U
-l
H 2(
if)
C\1
Ti_


Ligand (-Log M)










to 26 1.1 nM in the presence of Gpp(NH)p. After heat

treatment, the IC50 value in the absence of Gpp(NH)p was 21

1.5 nM. The IC50 values of the Carbo-Am in the presence of

Gpp(NH)p in control reticulocytes (26 nM) was virtually

identical to the heat-treated membranes (21 nM) in the

absence of Gpp(NH)p and erythrocyte membranes both in the

presence or absence of Gpp(NH)p (27-29 nM).

Effects of the carbostyril derivatives on reticulocyte

adenylate cyclase activity. Figure 2-4 showed the ability of

Iso and the two carbostyril derivatives to stimulate cAMP

formation. The concentration that produced half-maximal

formation was 8.2 2.1, 17.8 3.1 and 241 17 nM for

Carbo-Br, Carbo-Am and Iso, respectively. In addition, the

maximal formation of cAMP was the same for all 3 compounds.

Unlike Iso, both of the carbostyril derivatives appear to be

quite stable agonists: even after several weeks in solution

they retained full potency and efficacy to stimulate

adenylate cyclase. Table 2-2 showed the partial specificity

of Carbo-Br in the adenylate cyclase system. In reticulocyte

membranes, Forskolin (1 gM) stimulated cAMP production by

17.9-fold. When Iso (10 gM) or Carbo-Br (10 IM) was added in

addition to Forskolin, the fold stimulation for both was 23.

Using GH3 cell membranes, forskolin (1 IM) stimulated cAMP

formation 15.9-fold. However, Carbo-Br (10 gM) did not

stimulate cAMP formation above the basal level. Membranes

from GH3cells did not appear to contain beta-adrenoreceptors







32








200
C


01160-
E
o,


E



1 80 -

4O
40-



10 9 8 7 6 5 4
Ligand (-LogM)





Figure 2-4. Stimulation of reticulocyte adenylate cyclase
activity by Iso, Carbo-Am and Carbo-Br. Cyclase activity was
determined by incubating membrane protein (35 gg) in buffer
pH 7.4 containing 500 lM GTP, the indicated concentration of
Iso (squares), Carbo-Am (closed circles) and Carbo-Br (open
circles) for 10 min at 320C. Other standard cyclase assay
components and the determination of the cAMP content were
performed as described in the "Methods" section. Each data
point is the mean S.D., n = 3. Basal activity in the
presence of GTP was 17 4 pmol/min/mg protein and was
subtracted from the stimulated values.









Table 2-2. Effect of Carbo-Br on adenylate cyclase
activity in rat reticulocyte and GH3 cell membranes.


cAMP Formed
(pmol/min/mg protein)


Additions


A Reticulocytes


Basal


Forskolin (1 JiM)


Isoproterenol (10 gM)
plus Forskolin (1 IM)
Carbo-Br (10 pM)
plus Forskolin (1 IM)


B. GH3 membranes

Basal plus GTP

Forskolin (1 IM)


Carbo-Br (10 pLM) plus GTP (500 WLM)


14.7 + 1.4

264 8.0

347 11.0a

339 9.0a


10.0 3.3

159.0 11.0


9.6 1.4


Membrane protein (reticulocytes, 35 gg; GH3, 50 gg) was
incubated with the standard cyclase assay components and the
additions indicated for 10 min at 320C. At the end of the
incubation, the cAMP formed was determined as described under
"Experimental Procedures". Values are the means of tripli-
cate determinations S.E., n=3-4.

aSignificantly different from the Forskolin group (p <0.01).










as no specific [125I]CYP (3-100 pM) binding was detectable

(data not shown).

The time course of cAMP formation in reticulocyte

membranes induced by Iso (10 1M), Carbo-Br (1 !LM) and Carbo-

Am (1 1M) was shown in Figures 2-5A, B and C, respectively.

Cyclic AMP formation in the presence of all three compounds

was linear for at least 14 min of incubation. When propran-

olol (20 1M) was added after 7 min of incubation, the Iso-

stimulated formation of cAMP was blocked (Figure 2-5A).

However, when propranolol (20 J!M) was added after 7 min of

incubation with Carbo-Br (Figure 2-5B) or Carbo-Am (Figure 2-

5C), no effect on the rate of cAMP formation was observed.

In contrast, if propranolol was added at time zero with the

three compounds, then cAMP formation by all three was largely

blocked. Figure 2-6 showed the time course of Carbo-Am and

Carbo-Br-induced receptor loss in reticulocyte membranes.

Incubations were carried out at 320C with 1 gM of both com-

pounds plus all of the additions used in the assay of adeny-

late cyclase activity. At various times during the

incubation, membrane samples were washed four times and

assayed for beta-adrenoreceptors. After 2 min of incubation,

specific [125I]CYP binding was decreased by 51 and 61% in

membranes incubated with Carbo-Am and Carbo-Br, respectively.

This loss continued slowly until, by the end of 12 min,

specific binding sites had decreased 61% in the Carbo-Am

treated membranes and 75% in those treated with









Figure 2-5. Time course of reticulocyte adenylate cyclase
activation by Iso (A), Carbo-Am (B) and Carbo-Br (C).
Cyclase activity was determined by incubating membrane
protein (35 gg) in buffer containing 500 pM GTP, 10 JIM Iso
(A), 1 gLM Carbo-Br (B) or 1 LIM Carbo-Am (C) at 320C.
Activity was determined with the agonists alone (open
circles), agonists plus 20 pIM propranolol added at time 0
(squares) or after 7 min of incubation with the agonists
alone, propranolol (20 AM) was added (closed circles). The
arrow indicated the time when propranolol was added. Other
standard cyclase components and the determination of the cAMP
content were as described in the "Methods" section. Each
data point was the mean of triplicate determinations and was
representative of four experiments. Basal cAMP formation in
the presence of GTP alone was subtracted from the stimulated
values.




























































Time (min)

















2 80
C
0
U
60-


0




H
S20
(,1
c"u
LTJ

I I I I I I
0 2 4 6 8 10 12
Time (min)



Figure 2-6. Time course of specific [125I]CYP binding loss
by Carbo-Am and Carbo-Br in reticulocyte membranes. Membrane
protein (0.26 mg/ml) was incubated in buffer containing 1.6
mM ATP, 500 IM GTP, 1.0 mM EGTA, 10 mM theophylline, 0.1%
BSA, creatine phosphokinase (67 units/ml), 2.5 mM phospho-
creatine and without (squares) and with 1 pM Carbo-Am (closed
circles) or 1 gM Carbo-Br (open circles) at 320C. At the
times indicated, samples were removed, the membranes washed
four times with ice-cold buffer and assayed with 100 pM
[125I]CYP as described in the "Methods" section. Data points
are the mean of three determinations. The control [125I]CYP
binding values were 566 17 fmol/mg of protein in the Carbo-
Am experiments and 575 25 fmol/mg protein in the Carbo-Br
experiments.










Carbo-Br. Less than 5% of the binding sites were lost over

the 12 min incubation period in control membranes.


Discussion


The data from this study showed that both of the

carbostyril derivatives were highly potent beta-adrenergic

agonists. The Carbo-Am and Carbo-Br compounds were 14- and

29-fold, respectively, more potent than Iso in stimulating

adenylate cyclase activity. That enzyme activation by the

carbostyril derivatives was mediated through the beta-adreno-

receptor rather than a nonspecific effect was indicated by

two lines of evidence. First, concurrent addition of

propranolol, a beta-antagonist, blocked the enzyme activation

by both compounds. Second, the enzyme in GH3 cell membranes

was vigorously stimulated by forskolin but not by Carbo-Br.

These membranes did not contain any detectable beta-adreno-

receptors (Henneberry et al., 1986). The observation that

both of the carbostyril congeners produced the same maximal

stimulation of adenylate cyclase activity as Iso indicated

that both compounds were full beta-agonists.

Current evidence has suggested that a beta-agonist, the

beta-adrenoreceptor and Ns interact to form a ternary complex

(Citri and Schramm, 1980; De Lean et al., 1980; Kent et al.,

1980; Limbird et al., 1980b). When a guanine nucleotide has

bound to Ns, it destabilized the ternary complex, causing Ns

to dissociate from the receptor and reduced the receptor










affinity for the agonist which can then dissociated from the

receptor. Thus, in the absence of a guanine nucleotide, a

substantial fraction of the beta-adrenoreceptors showed high

affinity for the receptor ternaryy complex formation) whereas

in the presence of a guanine nucleotide all of the receptors

showed an agonist low affinity binding state (Lefkowitz et

al., 1976; Maguire et al., 1976; De Lean et al., 1980). The

initial ternary complex formation appeared to be a necessary

prerequisite for a beta-agonist to stimulate adenylate

cyclase activity (De Lean et al., 1980; Kent et al., 1980).

From competition studies in the absence of guanine nucle-

otide, the Carbo-Am and Carbo-Br compounds were 8- and 14-

fold more potent than Iso. In the presence of Gpp(NH)p,

however, the Carbo-Am and Carbo-Br were 39- and 106-fold

(respectively) more potent than Iso. This substantial

increase in the difference in potency between the carbostyril

derivatives and Iso in the presence of guanine nucleotides

was apparently due to the large Gpp(NH)p-induced decrease in

the affinity of Iso for the receptor (17-fold) whereas

Gpp(NH)p reduced the potency of the carbostyril compounds

only slightly (2- to 3.5-fold).

It has been reported that there is a direct relationship

between the ability of a beta-agonist to stimulate maximally

adenylate cyclase activity (intrinsic activity) and the

Gpp(NH)p-induced shift in agonist affinity (Lefkowitz et al.,

1976; Kent et al., 1980). Full agonists have large affinity

shifts whereas partial agonists have small affinity shifts.










Since both of the carbostyril congeners act as full agonists

with relatively small Gpp(NH)p-induced potency shifts, they

would appear to be exceptions to the above relationship.

However, there were several possible reasons for the small

Gpp(NH)p-induced shift in potency for these compounds. These

agonists could promote an extremely tight receptor-Ns complex

such that even in the presence of a guanine nucleotide little

complex destabilization (and hence a reduction in agonist

potency) is observed. If this is correct, then the loss of

Ns should reduce the potency beyond that observed in the

presence of Gpp(NH)p. However, this possibility was unlikely

since the IC50 values for Carbo-Am were very similar using

control reticulocyte membranes in the presence of Gpp(NH)p,

using reticulocyte membranes treated at 500C (to reduce

functional Ns) [Baker et al., 1985], and using erythrocyte

membranes (which lack Ns) [Limbird et al., 1980a]. An

alternative explanation for the small Gpp(NH)p-induced shift

could be an extremely tight (quasi-irreversible) or irre-

versible covalentt) binding of the carbostyril derivatives to

the receptor. Previous studies have shown that the major

effect of guanine nucleotides is to markedly increase the

dissociation rate of the agonist from the receptor (Williams

& Lefkowitz, 1977; Heidenreich et al., 1980). Thus an

extremely tight binding or covalent attachment of the agonist

to the receptor might greatly reduce or prevent the Gpp(NH)p-

induced agonist dissociation resulting in a small potency










shift. As discussed below, this was likely for the

carbostyril derivatives.

Several lines of evidence suggested that the carbostyril

derivatives bound in an extremely tight and/or irreversible

manner to the beta-adrenoreceptor. Scatchard analysis of

[125I]CYP binding after incubation with Carbo-Br or Carbo-Am

and membrane washing indicated a quasi-irreversible inter-

action as the receptor capacity decreased, whereas the KD for

[125I]CYP binding to the remaining receptors did not change.

In keeping with the small Gpp(NH)p-induced shifts to lower

potency for both compounds, the inclusion of Gpp(NH)p during

the membrane preincubation reduced the receptor loss to a

relatively small degree. Furthermore, the receptor loss

induced by the carbostyril derivatives was largely attenuated

by concurrent incubation with nadolol. These results

suggested that the initial interaction of both carbostyril

derivatives with the receptor was competitive followed by a

longer term quasi-irreversible binding. The Carbo-Am

compound contained no highly reactive moiety, suggesting that

its tight binding to the receptor was noncovalent in nature.

This was supported by the finding that a substantial reversal

of Carbo-Am binding occurred (>50%) by incubation of

membranes at 450C in the presence of nadolol. This effect

may have been due to a high temperature-induced dissociation

of the ligand from an intact receptor structure or a

dissociation induced by a reversible denaturation of the

receptor at 450C. A quasi-irreversible binding of two










beta-adrenoreceptor antagonists has been previously reported

(Terasaki et al., 1979; Lucas et al., 1979). In contrast to

the Carbo-Am, the Carbo-Br compound contained a reactive

bromoacetyl moiety. Thus its binding to the receptor may

have involved a loss of the bromo group resulting in a

reactive electrophilic ligand that could undergo an

irreversible alkylation of a nucleophile in the receptor.

This was supported by the observation that only a small

reversal of Carbo-Br binding (<10%) occurred by membrane

heating at 450C. Furthermore, after reacting Carbo-Br with

cysteine to inactivate the bromoacetyl moiety, a relatively

large reversal (58%) of its (carbo-cysteine) binding to the

receptor was found after membrane heating at 450C. Although

these data were consistent with a covalent attachment of

Carbo-Br to the receptor, another alternative was that Carbo-

Br bound reversibly with even higher affinity than Carbo-Am

such that little dissociation occurred, even at 450C.

Definitive proof of a covalent interaction would require more

experiments, perhaps using a radiolabelled compound in

conjunction with purified receptors as previously described

for an alkylating antagonist (Dickinson et al., 1985).

Interestingly, the maximal receptor loss induced by a wide

range of Carbo-Am concentrations was about 75%, suggesting

that a small fraction of receptors was resistant to the tight

binding of this compound. In contrast, the maximal receptor

loss induced by Carbo-Br approached 100%.










The data from the time course of reticulocyte adenylate

cyclase activation suggested that the two carbostyril

compounds acted as quasi-irreversible agonists. This was

indicated by the observation that addition of a 20-fold

excess of propranolol after seven min of incubation with the

two derivatives alone did not affect the rate of cAMP

accumulation. During the first seven min of incubation alone

with Carbo-Am or Carbo-Br, there was a 59 and 71% reduction

in specific [125I]CYP binding sites, respectively. In

contrast, when propranolol was added after seven min of

incubation with Iso alone, there was a complete blockade of

further cAMP production, consistent with Iso being a fully

reversible agonist. In addition, when propranolol was added

concurrently with both of the carbostyril compounds and Iso,

stimulation of the enzyme was virtually prevented. Thus the

cyclase activation data in conjunction with the previously

discussed binding data indicated that the initial interaction

of the carbostyril derivatives with the beta-adrenoreceptor

was competitive followed by a longer term quasi-irreversible

interaction. The activation of receptors by agonists has

been explained by two basic theories. The occupation theory

involved receptor activation as long as the receptor was

occupied whereas the rate theory predicted that activation

was proportional to the rate of combination between the

receptor and agonist (see Bowman & Rand, 1980; Yamamura et

al., 1985 for reviews). The data showing an apparent irre-

versible activation of adenylate cyclase by the carbostyril










congeners supported the occupancy theory for the beta-

adrenergic system.

In comparison to the apparent irreversible agonist

effects of the carbostyril derivatives, we recently reported

that bromoacetylaminomenthylnorepinephrine (BAAN) could,

under defined conditions, bind to the beta-adrenoreceptor in

an irreversible manner (Baker et al., 1985). Although BAAN

initially stimulated adenylate cyclase activity, after

irreversible binding it acted as an antagonist. The reasons

for the lack of an irreversible agonist effect by BAAN

whereas the carbostyril derivatives produced an apparent

irreversible agonist action were not obvious. These

differences might be related to the structures of the

compounds whereby different ligand-induced conformational

changes in the receptor were produced. Alternatively, the

differences might be related to the observation that BAAN was

a weak partial agonist whereas the carbostyril derivatives

were potent full agonists. Further studies would be neces-

sary to delineate the apparent irreversible effects of these

compounds. Although, to our knowledge, this was the first

report describing an apparent irreversible agonist for the

beta-adrenergic system, ligands showing irreversible or

sustained agonist effects have been reported for the insulin

(Brandenburg et al., 1980), adrenocorticotropin (Ramachandran

et al., 1981), adenosine (Lohse et al., 1986), and opiate

(Schoenecker et al., 1987) receptors.








45

In summary, the experimental results indicated that the

two carbostyril derivatives were stable, potent full beta-

adrenergic agonists that produced sustained activation

effects in vitro. The tight binding of the Carbo-Am to the

beta-adrenoreceptor appeared to be noncovalent whereas the

quasi-irreversible binding of the Carbo-Br might have

involved receptor alkylation.
















CHAPTER 3
INTACT CELL STUDIES


Introduction


All of the experiments described thus far were performed

on rat reticulocyte membranes. This preparation offered a

simplified, cell-free system within which various components

could be easily manipulated. Some interesting results per-

taining to the interactions of the carbostyril congeners with

beta2-adrenoreceptors were obtained through examination of

this simplified system. Nonetheless, beta-adrenoreceptor

agonists and antagonists have been shown to interact in vivo

with living cells, not membrane fragments (Abramson &

Molinoff, 1984). Beta-adrenoreceptors on intact cells

possessed properties not found in simple membrane

preparations. One example of this was the agonist-induced

process of desensitization. The desensitization sequence

began with a rapid uncoupling of cell surface beta-adreno-

receptors from stimulation of adenylate cyclase (Su et al.,

1980; Harden et al., 1979). This was followed by an apparent

internalization or sequestration of the beta-adrenoreceptor

from the cell surface (Toews et al., 1984; Toews & Perkins,

1984; Harden et al., 1980), and eventual down-regulation

(loss of binding function) of total receptor number (Su et










al., 1980). The ability of internalized receptors to recycle

back to the cell surface depended on the length of exposure

to the agonist. In most cases (i.e., acute agonist

pretreatment), when the agonist was removed, receptors would

reappear on the cell surface without further protein

synthesis (Hertel & Staehelin, 1983; Stadel et al., 1983;

Doss et al., 1981). In contrast, after chronic agonist

treatment, receptors were internalized and degraded.

Therefore, because of the differences between an intact cell

system and isolated membranes, we have characterized the

agonist-induced changes of Carbo-Br in the DDT1-MF-2 smooth

muscle cell line. The ability of Carbo-Br to bind to alphal-

adrenoreceptors present in DDT membranes was examined also.

Evidence was provided to indicate that while Carbo-Br bound

either extremely tightly or irreversibly, it still caused a

desensitization effect in the same manner as Iso.

Experimental Procedures

Source of Materials

The radioligands (-)-[3H]CGP-12177 ([3H]CGP; 38.8-53.1

Ci/mmol) and [3H]Prazosin (82 Ci/mmol) were obtained from New

England Nuclear, Boston, MA, USA. The antagonist CGP-20712A

was a generous gift from Dr. L. Maitre at Ciba-Geigy, Ltd.,

Basle, Switzerland. Three-isobutyl-one-methylxanthine

(IBMX), and snake venom (crotalus atrox, western diamondback

rattlesnake) were obtained from Sigma Chemical Co., St.

Louis, MO, USA. Dowex 1-X8 anion exchange resin (200-400

mesh, chloride form) was purchased from Bio-Rad Laboratories,










Richmond, VA, USA and 2-mercaptoethanol was a gift from Dr.

S.R. Childers, University of Florida. Cultured cell lines

DDT1-MF-2 and C6 were obtained from American Type Culture

Collection, Rockville, MD, USA. The C62B cell line was a

gift from Dr. Mark Rasenick, University of Illinois College

of Medicine.

Methods

Buffer. Studies with intact cells were carried out

using Hank's Balanced Salt Solution (HBSS) unless otherwise

noted. The solution contains 5 mM KC1, 0.4 mM KH2P04, 137 mM

NaC1, 4 mM NaHCO3, 0.6 mM Na2HPO4, 6 mM D-glucose, 0.5 mM

MgCl2, 0.4 mM MgSO4, and 1 mM CaC12, pH 7.4.

Cell culture. The DDTI-MF-2 cell line (a smooth muscle

cell line derived from a leiomyosarcoma of the ductus def-

erens of Syrian hamster) and both lines of C6 cells (from rat

glial tumors) were grown in Dulbecco's modified Eagle's

medium supplemented with 5% fetal calf serum, 2.75 gg/ml

amphotericin B, 100 U/ml penicillin G, and 0.1 mg/ml strep-

tomycin sulfate in a humidified atmosphere of 95% air/5% C02.

Cells were routinely subcultured every seven days with

trypsin (0.01%) from an initial inoculum of 4-6 x 105 c/ml.

Cell harvesting and treatment. Cells were harvested at

approximately 50% confluence. Cell monolayers were washed

twice with PBS to remove culture medium. Cells used in

intact cell studies were incubated for 5 min at 360C in 3-10

ml of PBS containing 1 mM EGTA. The cells were lifted with

repeated pipetting, centrifuged at 1000xg for 5 min, and










resuspended in HBSS. In pretreatment studies, cells were

incubated for various times in the presence or absence of 10

gM Iso or 1 pM Carbo-Br (final concentrations) at 360C.

Cells were then washed five times with cold HBSS by centrif-

ugation at 1000xg followed by resuspension. Whole cell

homogenates were obtained by resuspending the final pellet in

50 mM Tris-HCl pH 7.4 containing 5 mM MgC12 and homogenizing

for 15 sec at setting 2.5. Binding assays were performed as

described above with [125I]CYP using 0.5 mg/ml protein.

Antagonist Binding Assays. Beta-adrenoreceptor content

of DDT and C6 membranes was determined by incubating membrane

protein (25 gg) in a total volume of 0.25 ml with 50 mM Tris-
HCl at pH 7.4, 5 mM MgCl2, 3-100 pM [125I]CYP and with and

without 1 JM ()alprenolol for 60 min at 360C. At the end of

the incubation, each suspension was diluted with 3 ml of 50

mM Tris-HCl pH 7.4 (360C) and poured onto a Whatman GF/B

glass fiber filter under reduced pressure. The filter was

washed with a further 6 ml of buffer, placed in a vial and

the radioactivity determined. Specific [125I]CYP binding to

the beta-adrenoreceptor was calculated as the difference

between the total binding in the absence of ()alprenolol and

the nonspecific binding determined in the presence of 1 gM

()alprenolol. Nonspecific binding was the same if

()alprenolol was replaced with 100 gM Iso. Specific binding

was 90-95% of the total bound.

In some experiments, the ability of the carbostyril

congeners, Iso, or the betal-selective antagonist CGP-20712A










to inhibit specific [125I]CYP binding was determined. Assays

were the same as above except the [125I]CYP concentration was

30 pM and 0.1% sodium ascorbate was included when Iso was

used. The competitive binding assays were also performed in

the presence and absence of 100 gM Gpp(NH)p, a non-hydro-

lyzable analog of GTP. All binding assays were performed in

triplicate, the results varying by less than 5%.

The recently introduced beta-antagonist CGP-20712A has

been shown to have an extremely high selectivity (about

10,000-fold) for the betal-adrenoreceptor subtype (Dooley &

Bittinger, 1984; Lemoine et al., 1985). In competition

assays with a nonselective labelled ligand, low concentra-

tions of CGP-20712A bound to the betal-subtype. As the

concentration of CGP-20712A was increased, a plateau region

appeared where no further displacement of labelled ligand

occurred until a high enough concentration of CGP-20712A was

present to displace binding of the labelled ligand to the

beta2-subtype. The ratio of betai to beta2 receptors could be

estimated from the plateau region of the inhibition curve or

calculated using various curve-fitting programs for the

analysis of multiple site binding data.

Beta-adrenoreceptor content on the surface of intact DDT

cells were measured by incubating cells (0.2 mg/tube) in a

total volume of 1.0 ml containing HBSS, 0.2 to 6.3 nM

[3H]CGP, and in the presence and absence of 1 JLM

()alprenolol for 2 hr at 40C. At the end of the incubation,

4 ml of 50 mM Tris-HCl pH 7.4 containing 5 mM MgC12 at 40C










was added to each tube, and the suspension was poured onto a

GF/C glass fiber filter under reduced pressure. Filters were

washed quickly with an additional 8 ml of the same buffer,

placed in scintillation vials with 7 ml of Liquiscint and the

radioactivity determined. Specific ligand binding was

calculated as previously described.

In some experiments, intact cells, in suspension (2

mg/ml) or plated on 143 cm2 plates, were pretreated for

various times with 10 gM Iso or 1 1M Carbo-Br at 360C. The

cells (or plates) were promptly removed to ice, washed five

times with HBSS at 40C, and resuspended in HBSS (cells) or

reinoculated in fresh media (plates). The suspended cells

were then assayed with a single concentration of [3H]CGP

(0.75 nM) as previously described.

Alphal-adrenoreceptors were measured by incubating

membrane protein (0.4 mg/tube) in a total volume of 2.0 ml of

50 mM Tris-HCl pH 7.4, containing 5 mM MgCl2, 0.25 nM

[3H]Prazosin, and in the presence or absence of 10 (M phent-

olamine for 30 min at 300C. At the end of the incubation, 4

ml of 50 mM Tris-HCl pH 7.4, containing 5 mM MgCl2 at 40C was

added to each tube, and the suspensions poured onto GF/C

glass fiber filters under reduced pressure. Filters were

washed quickly with an additional 8 ml of the same buffer,

placed in scintillation vials with 7 ml Liquiscint and the

radioactivity determined. Specific ligand binding was

determined as described earlier.










Measurement of cAMP production in intact cells. Intact

cell cAMP accumulation was measured by incubating detached

cells with various concentrations of Carbo-Br or Iso for 5

min at 360C in HBSS that contained 0.5 mM of the phospho-

diesterase inhibitor IBMX. At the end of the incubation,

cells were quickly (20 sec) sedimented by centrifugation at

1000xg to remove the drug as well as any extracellular cAMP.

The supernatant was aspirated, the pellet resuspended in 25

mM Tris-HCl buffer (pH 7.0, 40C), and samples placed in a

boiling water bath for 5 min. After boiling, samples were

centrifuged at 1000xg and the supernatant saved for cAMP

assay as described in Chapter two. For the time course of

cAMP accumulation or phosphodiesterase activity assay, cells

were incubated with either 1 iM Carbo-Br or 10 pM Iso for

various times in the absence of IBMX. The phosphodiesterase

inhibitor was added to some cell suspensions after the

incubation and before the first spin to prevent further cAMP

degradation.

DDT Membrane Adenylate Cyclase Assay. Activity was

determined as described earlier in Chapter two "Methods"

except that the membrane protein assayed was 65 ig/tube.

"Basal" conditions included membrane protein and the regen-

erating system which included ATP, creatine phosphokinase,

phosphocreatine, EGTA, BSA, and theophylline. "Carbo-Br"

conditions included basal additions plus GTP and Carbo-Br.

"Propranolol" conditions included Carbo-Br additions plus










propranolol. The cAMP content of the supernatant was

determined as described in Chapter two, "Methods".

Cyclic AMP Phosphodiesterase Assay. The activity of

cAMP phosphodiesterase was examined by utilizing the assay

procedure of Thompson & Appleman (1971) with modifications by

Meeker & Harden (1982). With all additions made on ice,

whole DDT cell homogenates (300 gg/200 .1) were added to

tubes containing 20 mM MgCl2, 4 mM 2-mercaptoethanol, 60 ig

bovine serum albumin, 0.5-400 gM cAMP, and 100,000 cpm/assay

of [3H]cAMP. All dilutions from stock were made with 40 mM

Tris-HCl (pH 7.4 at 300C). The total assay volume was 0.4

ml.

After incubation at 300C for 20 min, the reaction was

terminated by transferring the tubes to a boiling water bath

for 2 min. The tubes were then placed in a 40C bath where

100 p. of snake venom toxin (1 mg/ml) was added. The tubes

were incubated for another 10 min at 300C, and transferred

back to the 40C bath were the incubation was stopped by the

addition of 1.0 ml of a 1:2 slurry of Dowex 1-X8. The tubes

were mixed by vortexing and the Dowex sedimented by centrifu-

gation for 5 min at 1000xg. An aliquot of 0.5 ml was taken

from each tube, added to 9.5 ml Liquiscint and counted for 5

min. Blanks were determined in the presence of boiled

protein.

Data analysis. The competition data using CGP-20712A

were analyzed both as a one- and two-site model with a "By

Hand" curve-fitting program for the analysis of multiple site










radioligand binding data (Richardson & Humrich, 1984). This

analysis provided estimates of the competitor's affinity for

and the concentration of the two sites. The best fit of the

experimental data was obtained as defined by a minimum

deviation. The analysis assumed the ligand binding to both

sites followed mass-action kinetics. Arguments supporting

this assumption have been presented by Minneman et al.

(1979). Statistical analysis of all other data was performed

using the Student's t-test and was presented as mean S.E.

Results


Inhibition of specific f125I1CYP binding in DDT membranes

by Carbo-Br and Iso. Figure 3-1 showed the ability of Iso

and Carbo-Br to inhibit specific binding of [125I]CYP to DDT

cell membranes in the presence of 0.1 mM Gpp(NH)p. The IC50

values for Carbo-Br and Iso were 9 2 (n=3) and 400 23 nM

(n=3), respectively.

Effects of Carbo-Br on DDT cell cAMP production and

phosphodiesterase activity. Figure 3-2 showed the ability of

Iso and Carbo-Br to stimulate cAMP accumulation in intact DDT

cells. Carbo-Br was 9-fold more potent then Iso at stimu-

lating cAMP accumulation with an EC50 value of 9 1.6 nM

(n=3) compared to 80 12 nM (n=3) for Iso. A time course of

cAMP accumulation in DDT cells induced by 1 LM Carbo-Br or 10

AM Iso was shown in Figure 3-3. The time of cAMP

accumulation by both agonists was the same over the 60 min

time period. After 3 min of incubation there was an





















o 100 a Iso
5 a Carbo-Br
80


60


o 40


u 20 -


-w 0 L -
-10 -9 -8 -7 -6 -5
Ligand (Log M)




Figure 3-1. Inhibition of specific [125I]CYP binding in DDT
cell membranes by Iso and Carbo-Br. Membranes (25 gg/tube)
were incubated with buffer at pH 7.4, 30 pM [125I]CYP, 100 IM
Gpp(NH)p, and the indicated concentrations of Iso and Carbo-
Br for 45 min at 360C. In the Iso competition assays, 0.1%
ascorbate was also present. At the end of the incubation,
the specific binding was determined as described under
"Methods". Each point on the graph was the mean of 3
determinations assayed in triplicate. Specific [125I]CYP
binding ranged from 63 to 69 fmol/mg protein.














.$120-
C L

90-



S60-


30-



o i
10 9 8 7 6 5
Agonist (-Log M)



Figure 3-2. Stimulation of cAMP production in intact DDT
cells by Iso and Carbo-Br. Cyclic AMP production was
determined by incubating intact cells (0.36 mg) in HBSS
buffer containing the indicated concentrations of Iso (closed
circles) or Carbo-Br (open circles) for 5 min at 360C. The
determination of cAMP content was performed as described
under "Methods". Each data point was the mean + S.E., n=3.
Basal activity was 10 pmol/min/mg protein and was subtracted
from the stimulated values.





















-~. 60

2 50-
0)
E 40

E
- 30-

E 20-
O T
S1 0 l1 *


0 10 20 30 40 50 60
Time (min)



Figure 3-3. Time course of cAMP accumulation in intact DDT
cells by Iso and Carbo-Br. Cyclic AMP accumulation was
determined by incubating intact cells (0.36 mg) in HBSS
buffer containing 10 gM Iso (closed squares) or 1 JiM Carbo-Br
(open squares) at 360C for the indicated times. Determin-
ation of cAMP was performed as described under "Methods".
Each data point was the mean of 3-4 determinations S.E.
Basal cAMP formation (10 pmol/min/mg protein) has been
subtracted from the stimulated values.




















80-

0
- 60
0)
E

E 40



S 20-

a.

o 0-
0 10 20 30 40 50 60
Time (min)


Figure 3-4. Time course of cAMP accumulation in intact DDT
cells by Iso and Carbo-Br after addition of propranolol.
Cyclic AMP accumulation was determined by incubating intact
cells (0.36 mg) in HBSS buffer containing 10 gM Iso (closed
squares) or 1 JM Carbo-Br (open squares) plus 20 pM propran-
olol added to all tubes after 3 min of incubation at 360C.
Determination of cAMP was performed as described under
"Methods". Each data point was the mean of 3-4 determin-
ations S.E. Basal cAMP formation has been subtracted from
stimulated values.










increase (6-fold above basal) in cellular cAMP content

followed by a relatively slow reduction over the next 57 min.

When 20 AM propranolol was added to the system at the 3 min

time point (Figure 3-4), there was an immediate drop in Iso-

induced cAMP accumulation to basal levels. This was not the

case, however, in the Carbo-Br-induced system. When

propranolol was added after 3 min of incubation with Carbo-

Br, the loss of cAMP accumulation over the next 57 min was

similar to that observed in the absence of propranolol. This

suggested that Carbo-Br had already bound to the receptors in

a tight and/or irreversible manner. Phosphodiesterase

activity was also measured after a time course of Carbo-Br

and Iso pretreatment (Table 3-1). There was no difference in

total phosphodiesterase activity over time with either

pretreatment group as compared to control values.

Effects of Carbo-Br pretreatment on membrane adenylate

cvclase activity. Table 3-2 showed the ability of Carbo-Br

to stimulate cAMP production in DDT cell membranes under

"Basal" cyclase conditions after intact cell pretreatment

with Carbo-Br for 3, 30, and 60 min. The membranes from

those cells which were pretreated with Carbo-Br (1 iM) and

washed exhibited a 2.5-fold higher basal activity than the

controls. In fact, the basal activity in membranes from 3

min Carbo-Br pretreated cells was as great as the Carbo-Br

stimulated activity in membranes from control cells. There

did appear to be a desensitization of this effect as the









Table 3-1. Effect of Carbo-Br and Iso pretreatment on
phosphodiesterase activity in DDT cells


[3H]Adenosine formed (pmol/min/mg protein)



Pretreatment time (min) Carbo-Br Iso


10 699.8 80.5 326.8 40.6

20 633.2 97.0 558.2 129.0

30 654.3 67.8 510.8 140.0

40 607.8 43.0 492.2 162.0

50 614.4 26.9 533.5 83.2

60 638.3 10.6 542.7 133.0

control 658.9 26.0 517.2 82.7



Intact cells (2.4 mg) were incubated for various times
in HBSS buffer pH 7.4 containing 1 JLM Carbo-Br or 10 LM Iso
at 360C. At the end of the incubations, cells were
sedimented by centrifugation at 1000xg and resuspension in
0.9 ml of40 mM Tris-HCl. Cells were then homogenized, and
the whole cell homogenate (0.3 mg/tube) assayed for
phosphodiesterase activity as described in "Methods".


Each value is the mean SE, n=3.









Table 3-2. Stimulation of adenylate cyclase activity in DDT cell
membranes after pretreatment of intact cells with Carbo-Br


Membrane cAMP production (pmol/10


min/mg protein)


Cellular Carbo-Br plus
Preincubation Basal Carbo-Br Propranolol
Conditions (1 iM) (201M)


Control 37.3 4.4 115.3 24.0 43.8 6.3
(3 min)

Carbo-Br 92.6 9.6a 104 11.0 90.0 10.2c
(3 min)

Control 40.0 3 ---- ---
(30 min)

Carbo-Br 74.4 4.1a --- -
(30 min)

Control 38.1 2.8 ---- ----
(60 min)

Carbo-Br 53.2 7.1b
(60 min)


Intact cells (1.5 mg) were incubated for various times in
HBSS buffer at pH 7.4 in the presence or absence of 1 JM Carbo-Br.
At the end of the incubations, the cells were washed four times by
centrifugation at 1,000xg and resuspension. After the final wash,
cells were resuspended in 50 mM Tris-HC1 pH 7.4, homogenized, and
the membranes sedimented by centrifugation at 35,000xg for 10 min.
DDT cell membranes (65 gg/tube) were assayed for adenylate cyclase
activity as described in "Methods", and the results were listed
under the appropriate assay condition heading.

a Significantly different from the respective control, p 0.005;
b Significantly different from the respective control, p 0.05;
c Significantly different from the respective control, p < 0.01,
as determined by unpaired student's t-test.
All values were means S.E., n=3-4.










basal activity declined in membranes from Carbo-Br treated

cells over an hr, from 2.5-fold at 3 min to 1.4-fold

stimulation at 60 min.

It was important to notice also that propranolol blocked

Carbo-Br stimulated cAMP production in control membranes when

the two were added together, but had no effect on cAMP

production in membranes from cells that were pretreated with

Carbo-Br (Table 3-2). This was consistent with our findings

in reticulocytes and whole cells that Carbo-Br had bound

tightly and/or irreversibly by at least 3 min, yet still

stimulated cAMP production.

Effects of Carbo-Br and Iso pretreatment on DDT cell

beta-adrenoreceptors. Receptor content was assayed in two

ways: whole cell homogenates were assayed using [125I]CYP,

and intact cells were assayed using the hydrophilic beta-

antagonist [3H]CGP which measured only those receptors found

on the cell surface (Hertel et al., 1983; Wilkinson &

Wilkinson, 1985). Figure 3-5 was a representative Scatchard

plot of [125I]CYP binding in whole cell homogenates after a 3

min pretreatment of intact cells with 1 LM Carbo-Br, 10 LM

Iso or buffer alone followed by cell washing. The slopes of

the lines were parallel, indicating that all of the unbound

drugs have washed out (KD values: control, 22 4; Iso-

treated, 16 3; Carbo-Br-treated, 29 8 pM, n=4-7). There

was a 50% loss of binding sites after only a 3 min

pretreatment with Carbo-Br (Bmax values: control, 58 3,

Iso-treated, 57 6; Carbo-Br-treated, 34 5 fmol/mg


















5.0

o
o 4.0 -
o

3.0


2.0
C !
0 A
00 1.0


0.0
0 10 20 30 40 50 60 70

[1251]CYP Bound (fmol/mg protein)

Figure 3-5. Scatchard plot of specific [125I]CYP binding to
whole DDT cell homogenates after intact cell treatment with
Iso and Carbo-Br. Intact cells (3 mg) were incubated in HBSS
buffer containing 10 pM Iso (triangles), 1 pM Carbo-Br
(closed squares) or buffer alone (open squares) at 360C for 3
min. Samples were removed, intact cells washed five times
with ice-cold HBSS buffer, resuspended in 50 mM Tris
containing 5 mM MgCl2, and homogenized. The homogenates were
assayed with 3 to 100 pM [125I]CYP as described under
"Methods". The data were plotted as the ratio of the amount
of specifically bound ligand (pmol/mg protein) to free ligand
(pmol/l) versus the amount of specifically bound ligand/mg
protein. Data points were the mean of triplicate determin-
ations and were representative of three experiments.



















= 100


80
o 80


60


o 40 -


20


0
0 10 20 30 40 50 60
Time (min)




Figure 3-6. Time course of specific [125I]CYP binding loss
in whole DDT cell homogenates after intact cell treatment by
Iso and Carbo-Br. Intact cells (3 mg) were incubated in HBSS
buffer containing 10 LM Iso (closed squares), 1 JM Carbo-Br
(open squares), or buffer alone (circles) at 360C. At the
times indicated, samples were removed, intact cells washed
five times with ice-cold HBSS buffer, resuspended in 50 mM
Tris-HCl containing 5 mM MgC12 and homogenized. The homog-
enates were assayed with 75 pM [125I]CYP as described under
"Methods". Data points were the mean of four determinations.
The control [125I]CYP values were 48 4 fmol/mg protein.










protein, n=4-7). Similar results were obtained with [3H]CGP

(results not shown). There was no loss of binding sites

after a 3 min pretreatment with Iso. Figure 3-6 presented a

time course of the loss of specific binding sites with Carbo-

Br, as measured with [125I]CYP. There was a 42% loss of

sites by 3 min with Carbo-Br that continued gradually, so

that by 60 min there was a 70% loss of binding sites.

Comparison with Iso-treated cells indicated that while the

response may have been desensitized, there was no loss of

binding sites as measured by [125I]CYP binding in whole cell

homogenates. Figure 3-7 demonstrated the loss of binding

sites from the intact cell surface with both Iso and Carbo-Br

pretreatment as measured by [3H]CGP. After pretreatment with

10 pM Iso there was a 21% decrease in binding sites at 3 min

that reached a 77% loss by 60 min. Carbo-Br showed a drop of

42, 76, and 82% over 3, 20, and 60 min, respectively. Figure

3-8 showed the recovery of specific [3H]CGP binding sites

after 1 hr of Carbo-Br or Iso pretreatment followed by a 24

hr incubation after the washout of unbound agonists. After

the 1 hr pretreatment there was an 80 and 87% loss of binding

sites with Iso and Carbo-Br, respectively. After 24 hr, Iso

treated cells showed only a 27% decrease from controls, while

Carbo-Br treated cells showed an 80% drop in binding sites.

Effects of Carbo-Br pretreatment on C6 cell membrane

beta-adrenoreceptors. Since reticulocytes and DDT cells

contained only receptors of the beta2-subtype, it was of


















80

*Control
c Isoproterenol



40


















Figure 3-7. Time course of specific [3H]CGP binding loss by
Iso and Carbo-Br in intact DDT cells. Intact cells (6 mg)
were incubated in HBSS buffer containing 10 M Iso or 1
3 20 60
Time (min)






Figure 3-7. Time course of specific [3H]CGP binding loss by
Iso and Carbo-Br in intact DDT cells. Intact cells (6 mg)
were incubated in HBSS buffer containing 10 pM Iso or 1 gM
Carbo-Br at 360C. At the times indicated, samples were
removed to ice, intact cells washed five times with ice-cold
HBSS buffer, resuspended in 40C HBSS and assayed with 0.75 nM
[3H]CGP as described under "Methods". Data points were the
mean of three determinations (significantly different from
control, *p 0.005; **p 5 0.05). The control [3H]CGP binding
values ranged from 60.2 to 69.6 fmol/mg protein.














90-
Control
U Isoproterenol


c 60
-I


E
6 30




0

1 24
Time (hours)




Figure 3-8. Loss of specific [3H]CGP binding in intact DDT
cells immediately or 24 hr after a 60 min treatment with Iso
or Carbo-Br. DDT cells plated on 143 cm2 plates were
incubated in HBSS buffer containing 10 lM Iso or 1 LM Carbo-
Br for 60 min at 360C. At that time, the plates were removed
to 4C and washed five times with ice-cold HBSS buffer. One-
half the cells from each treatment group were given DMEM plus
5% FCS and removed to the cell incubator. The remaining
cells were lifted with ice-cold HBSS containing 1 mM EGTA,
resuspended in 40C HBSS and assayed with 0.75 nM [3H]CGP as
described under "Methods". Twenty-four hr later the rest of
the cells were lifted and assayed as the former. Data points
were the mean of three determinations (significantly dif-
ferent from control; *p < 0.0005, **p 5 0.025). The control
[3H]CGP binding values were 74.3 1.63 fmol/mg protein for 1
hr and 76.7 0.76 for 24 hr (mean + S.E.).










Table 3-3. Loss of binding sites in two C6 clones after
Carbo-Br pretreatment


Bmax (fmol/mg protein)


Cell line Control Carbo-Br


C6 33.3 0.1 25.2 1.5a

C62B 16.3 1.4 13.6 1.6



Cell membranes (4.0 mg) were preincubated in the
presence or absence of 1 1M Carbo-Br for 15 min at 360C in 50
mM Tris-HC1 buffer pH 7.4 containing 5 mM MgCl2 and 100 M
Gpp(NH)p. At the end of that time, membranes were washed 5
times by centrifugation at 35,000xg and resuspension.
Membranes (25 gg/tube) were assayed with 3-100 pM [125I]CYP
as described in "Methods".

a Significantly different from control, p 5 0.025, as
determined by unpaired student's t-test.


All values given as means S.E., n=3.








69













S100


80


60
60


o 40

Control
S20 Carbo-Br

P "
0 i I I I
-11 -10 -9 -8 -7 -6 -5 -4

Log [CGP-20712AR




Figure 3-9. Inhibition of specific [125I]CYP binding in C6
cell membranes by CGP-20712A after Carbo-Br treatment.
Membranes (4.0 mg)were incubated with buffer at pH 7.4 in the
presence or absence of 1 JLM Carbo-Br for 15 min at 360C. The
membranes were washed five times with ice-cold buffer and
assayed with 30 pM [125I]CYP, 100 gM Gpp(NH)p, and the
indicated concentrations of CGP-20712A for 45 min at 360C.
The curves were representative of three experiments.
Specific [125I]CYP binding totals were 18.6 and 13.8 fmol/mg
protein for control and Carbo-Br treated membranes,
respectively.










interest to determine the effects of Carbo-Br binding on

betal-adrenoreceptors. Table 3-3 compared the loss of

binding sites in two other cell lines, C6 and its subclone

C62B, after Carbo-Br pretreatment. The C62B cell membranes,

which appeared to contain only betal-adrenoreceptors, showed

no loss of binding sites after Carbo-Br pretreatment.

Membranes from C6 cells, on the other hand, exhibited a 25%

loss in binding sites after Carbo-Br pretreatment. The IC50

values for Carbo-Br inhibition of specific [125I]CYP binding

in C6 and C62B membranes in the presence of 100 I.M Gpp(NH)p

were the same, 40 nM 5 (n=2-5). Figure 3-9 showed the

ability of CGP-20712A to inhibit specific [125I]CYP binding in

membranes from C6 cells. In control membranes the inhibition

curve was biphasic. Using a multisite analysis, the data

best fit a 2-site model with 82% of the sites showing high

affinity for CGP-20712A (betal subtype) and 18% of the sites

with low affinity (beta2 subtype). After pretreatment of the

C6 membranes with Carbo-Br (1 IM), the CGP-20712A competition

data best fit a single binding site model with only high

affinity for the antagonist. There may have been a small

number of low affinity sites present, but they were within

the error of the assay. The data indicated that Carbo-Br

mainly bound in a tight and/or irreversible manner to the low

affinity (beta2 subtype) CGP-20712A binding sites.

Effects of Carbo-Br on alpha-adrenoreceptor binding.

The fact that the DDT cells contained alphal- (Cornett &

Norris, 1982) as well as beta2-adrenoreceptors allowed for










the examination of Carbo-Br binding to alphal-adreno-

receptors. Carbo-Br bound to the beta2-adrenoreceptors of

DDT cell membranes with an IC50 value of 9 nM. In contrast,

Carbo-Br displaced [3H]Prazosin binding with an IC50 value of

16,000 nM for the alphal-adrenoreceptor. Therefore, there

was a large difference in potencies for Carbo-Br for the

alphal- and beta2-adrenoreceptors.

Discussion


We have shown that Carbo-Br produced sustained acti-

vation of adenylate cyclase in reticulocyte membranes after

binding tightly and/or irreversibly to the receptor. We have

further investigated the effects of Carbo-Br using an intact

cell system. Similar to the reticulocyte studies, Carbo-Br

was more potent than Iso at inhibiting specific [125I]Cyp

binding and stimulating cAMP production in DDT cell membranes

and intact cells. It also exhibited the same intrinsic

activity as Iso, indicating that Carbo-Br was a full agonist

in this system as well as in the reticulocyte membrane

system.

That Carbo-Br produced desensitization of beta-adreno-

receptor-coupled adenylate cyclase activity was shown in two

ways. First, cAMP levels in intact cells dropped to near

basal levels within 57 min of being stimulated 6-fold by 1 AM

Carbo-Br (almost identical to the pattern elicited by 10 gM

Iso). The decline in cAMP levels cannot be attributed to

differential effects of Carbo-Br on phosphodiesterase










activity, as neither Iso nor Carbo-Br affected the activity

of that enzyme. Second, studies of cAMP production in

membranes produced from intact cells which had been

pretreated with Carbo-Br also appeared to undergo a

desensitization. Membranes from cells preincubated for 30

and 60 min with 1 gM Carbo-Br only produced 80 and 57%,

respectively, as much cAMP as the 3 min treated ones.

Interestingly, the desensitization observed in the membrane

assays appeared to be slower than that observed in the intact

cells. This discrepancy may be a reflection of the intrinsic

differences in assay procedure, the presence (membrane

assays) or absence (intact cell assays) of a phosphodi-

esterase inhibitor, or an active cellular extrusion mechanism

to remove cAMP from intact cells. Nonetheless, both experi-

mental approaches indicated that Carbo-Br was producing a

desensitization. A desensitization phenomenon in DDT cells

has been reported by others (Strasser et al., 1986; Cowlen &

Toews, 1987; Toews et al., 1987).

That Carbo-Br was binding either quasi-irreversibly or

covalently to the beta-adrenoreceptors of DDT cells was

supported by both binding and cAMP stimulation studies. The

inability of propranolol to reverse Carbo-Br-induced cAMP

accumulation in intact cells or cell membranes after 3 min

implied that Carbo-Br had bound tightly and/or irreversibly

by that time. A 40-50% loss of binding sites was detected

after only a 3 min pretreatment of intact cells with Carbo-Br

with no change in the KD of [125I]CYP or [3H]CGP for the










remaining receptors. By measuring beta-adrenoreceptor

binding in two ways, we showed the pattern of Iso-induced

receptor redistribution and compared it to Carbo-Br-induced

receptor loss. Using whole cell homogenates allowed any

internalized receptors to be accessible to [125I]CYP, and

measured all structurally intact beta-adrenoreceptors

(Motulsky et al., 1986). Because [3H]CGP is hydrophilic, it

has be used to measure only those beta-adrenoreceptors found

on the cell surface (Hertel et al., 1983; Wilkinson &

Wilkinson, 1985). It was shown that acute Iso treatment

reduced [3H]CGP binding to intact cells with no change in

[125I]CYP binding to cell homogenates indicating an agonist-

induced receptor redistribution, not receptor loss. In

contrast, there was a receptor loss during Carbo-Br pretreat-

ment that was similar when measured with either [125I]CYP or

[3H]CGP. At this point it was unclear whether Carbo-Br-bound

receptors were internalized or not. What was clear, however,

was that after 24 hr, almost all of the Iso-internalized

receptors had reappeared on the cell surface. While the

cellular location of Carbo-Br-bound receptors was not clear,

it appeared that Carbo-Br had not dissoci-ated from them,

even after 24 hr. The other possibility was that the Carbo-

Br-bound receptors were signalled for degradation. Current

evidence has indicated that acute agonist treatment uncoupled

the receptor from Ns. Agonist affinity for the receptor in

the uncoupled state was low and suggested that freely rever-

sible agonists would dissociate during or before receptor










internalization (Hertel & Perkins, 1984). Since Carbo-Br was

tightly or irreversibly bound to the receptor, it seems

probable that if internalization occurred, Carbo-Br would be

still attached to the receptor and might have increased the

rate of receptor degradation as has been observed with

chronic agonist treatment (Hertel & Perkins, 1984). Once

Carbo-Br is radiolabelled, it might be possible to learn what

was happening to the receptor. If indeed it was being inter-

nalized, a radiolabelled irreversible agonist might give us

the most accurate picture yet of agonist-induced receptor

processing. There has been little doubt that agonist-induced

internalization and down-regulation set in motion a different

chain of events than normal basal receptor turnover. For

example, agonist-induced internalization appeared to increase

the rate of receptor clearance from the cell surface and

degradation (Mahan et al., 1987). It seemed logical,

therefore, that an irreversible radiolabelled agonist might

provide different information than a radiolabelled irrevers-

ible antagonist. Together, they might be able to provide a

more detailed picture of basal and agonist-induced receptor

turnover.

Since all of our studies to this point had included only

systems composed of pure beta2-adrenoreceptor populations, we

were curious as to whether Carbo-Br would show the same

affinity and irreversible properties for betal-adreno-

receptors. The C6 glioma line contained 80% betal- and 20%

beta2-receptors (Homburger et al., 1981; Harden & McCarthy,










1982), C62B membranes consisted primarily of betal-adreno-

receptors. Pretreatment of membranes from both cell types

with Carbo-Br at a concentration that occupied greater than

90% of the receptors resulted in a 25% loss of binding sites

from C6 membranes with no loss of sites in C62B membranes.

It was of interest to determine if the 25% loss of binding

sites in C6 membranes corresponded to the 20% beta2-receptor

population there. Using the betal-selective antagonist CGP-

20712A (Dooley et al., 1986; Brodde, 1987), we demonstrated a

change in the betal to beta2 ratio after Carbo-Br pretreatment

of C6 membranes. It appeared that 1 ILM Carbo-Br bound quasi-

irreversibly to the beta2-adrenoreceptors on those membranes,

with only one receptor population remaining (betal). If

Carbo-Br was only binding tightly to the beta2-adreno-

receptor, it argues strongly for important structural dif-

ferences in the ligand binding sites between the two sub-

types. If Carbo-Br could be radiolabelled, it could be used

to distinguish differences between the ligand binding sites

of the two subtypes by using the method recently described by

Dohlman et al. (1988).

Finally, it appeared that Carbo-Br was able to compete

with [3H]Prazosin for alphal-adrenoreceptor binding sites.

While the drug was much more selective for beta- than alphal-

receptors, the IC50 of Carbo-Br for inhibiting [3H]Prazosin

binding was comparable to both the alpha-antagonist yohimbine

(4.6 pM) and the agonist epinephrine (27.8 gM) as measured in

DDT cells (Toews, 1987). Whether or not Carbo-Br was an








76

alpha agonist or antagonist, and if it bound in an irrever-

sible manner to the alpha-adrenoreceptor would require

further study.
















CHAPTER 4
WHOLE ANIMAL STUDIES


Introduction


As shown in the previous chapters, Carbo-Br was a

potent, stable, beta-adrenoreceptor agonist that seemed to

bind irreversibly to membranes and intact cells. Though

Carbo-Br produced a sustained activation of adenylate cyclase

in reticulocyte membranes, it appeared to trigger a desen-

sitization response in intact cells, similar to other freely

reversible beta-agonists. Moreover, the extremely tight

and/or irreversible binding of Carbo-Br was specific for the

beta2-receptor subtype. Because of the complex interaction

of Carbo-Br with the beta-adrenoreceptor, it was important to

characterize its effects when administered to animals. Most

tissues in vivo contained beta-adrenoreceptors with the

subtype ratio varying considerably. Furthermore, beta-

adrenoreceptors mediated a variety of important physiological

responses, and both beta-agonists and antagonists have

salutary effects in the treatment of a number of disorders.

In the long-term, the development of beta-adrenoreceptor

compounds which act differently than those of the

catecholamines may provide further insight on the regulation

and role of the beta-adrenoreceptor in health and disease.











Therefore, the interaction of Carbo-Br with beta-

adrenoreceptors from several tissues of the rat were

partially characterized.


Experimental Procedures


Source of Materials

The radiolabelled compounds L-[14C]ornithine (52-59

Ci/mmol) and (-)-[3H]dihydroalprenolol ([3H]DHA; 48.6-60.0

Ci/mmol), Econofluor and Protosol were purchased from New

England Nuclear, Boston, MA, USA. Dithiothreitol and

pyridoxyl phosphate were purchased from Sigma Chemical Co.,

St. Louis, MO, USA.

Methods

Drug treatments. Stock solutions of Carbo-Br (1 mM)

were prepared fresh monthly by dissolving the compound in

ethanol. Dilutions from the stock solution were made with

distilled water only. Male Sprague-Dawley rats (200 g) were

injected i.p. or s.c. with 0.5, 2.0, 5.0, or 10.0 mg/kg of

the drug. Control rats were injected i.p. or s.c. with the

ethanol-water vehicle only.

Membrane preparation. Rats were decapitated and the

hearts, lung lobes, spleens, submaxillary glands, and brain

cortices removed to ice-cold saline. Atria, valves and fat

were dissected from the heart ventricles. The tissues were

rinsed, blotted, weighed, and all tissues but the atria,










ventricles and brain were frozen in liquid nitrogen for

assays performed no longer than one week later.

Tissues were cut into small pieces and homogenized in 15

volumes (w/v) of ice-cold 50 mM Tris-HCl buffer pH 7.4

containing 5 mM MgCl2 and 0.32 M sucrose with a Tekmar SDT-

182EN homogenizer at setting 5 for 15 sec. For atria, the

homogenate was placed on ice for 1 min and homogenized again.

The suspensions were diluted with 20 ml of homogenization

buffer and centrifuged at 48,000xg for 10 min in a Sorvall

RC-5B centrifuge. The supernatant was discarded and the

pellet resuspended (setting 3, 10 sec) in 20 ml of ice-cold

50 mM Tris-HCl buffer pH 7.4 containing 5 mM MgCl2, passed

through a tea sieve (30 mesh nylon sieve for heart) to remove

large pieces of connective tissue, and centrifuged at

48,000xg for 10 min. The resulting pellets were washed twice

more and resuspended in 1 volume of 50 mM Tris-HCl buffer pH

7.4 containing 5 mM MgC12 for assays.

Membrane pretreatments. In the isolated membrane

studies, membrane protein (3.5 mg/ml, heart; 2.0 mg/ml, lung;

3.0 mg/ml, spleen; and 3.0 mg/ml, submaxillary gland) was

incubated with 50 mM Tris-HCl buffer pH 7.4 containing 5 mM

MgCl2, Carbo-Br and with 100 gLM Gpp(NH)p at 300C for the

times indicated. At the end of the incubation, suspensions

were diluted with 30 ml cold buffer pH 7.4 and centrifuged at

48,000xg for 10 min. Pellets were resuspended and centri-

fuged three more times as above, and final pellets suspended

in 3 ml buffer for assays.










Antagonist binding assays. Beta-adrenoreceptor

concentration was determined by incubating membrane protein

(0.07-0.08 mg, heart; 0.05 mg, cerebral cortex, spleen and

submaxillary gland) in a total volume of 0.25 ml with 50 mM

Tris-HCl buffer pH 7.4 containing 5 mM MgCl2, 6.25-100 pM

[125I]CYP, and in the presence and absence of 1 iLM

()alprenolol for 60 min at 360C. At the end of the

incubation, each suspension was diluted with 3 ml of 50 mM

Tris-HCl buffer at pH 7.4 (360C) containing 5 mM MgCl2,

poured onto a Whatman GF/C glass fiber filter under reduced

pressure. Filters were quickly washed with an additional 6

ml of buffer, placed in vials, and radioactivity determined.

Specific binding was determined as previously described.

In some experiments, the ability of various concen-

trations of Iso and Carbo-Br to inhibit specific [125I]CYP

binding was performed. Assays were the same as above except

the [125I]CYP concentration was 30 pM and 0.1% sodium ascor-

bate was included to retard the oxidation of Iso. These

competitive binding assays were performed in the presence or

absence of 100 pM Gpp(NH)p. All binding assays were

performed in triplicate.

Beta-adrenoreceptor content in lung membranes was deter-

mined by incubating membrane protein (0.25 mg/tube) in 50 mM

Tris-HCl pH 7.4 containing 5 mM MgCl2, 0.15-5.0 nM [3H]DHA,

and in the presence and absence of 1 pM ()alprenolol for 30

min at 36C. At the end of that time, 4 ml of 50 mM Tris-HC1

containing 5 mM MgCl2 at 40C was added to each tube, and the










suspensions were poured onto GF/C glass fiber filters under

reduced pressure. Filters were quickly washed with an

additional 8 ml of buffer, placed in scintillation vials with

7 ml Liquiscint and radioactivity determined. Specific

binding was determined as previously described. The use of

[3H]DHA instead of [125I]CYP was necessitated by the inclusion

of incompletely homogenized tissue in a given assay tube.

The large disparity in the data observed with the use of

[125I]CYP (with its high specific activity) was reduced with

[3H]DHA.

Beating atrial preparations. Rats were killed by

decapitation and the hearts quickly removed. The atria

containing the sinus node (S-A) were immersed in modified

Tyrode's solution (NaC1, 136.9 mM; KC1, 5.36 mM; NaH2PO4,

0.33 mM; MgCl2, 2.3 mM; CaCl2, 2.0 mM; HEPES, 5.0 mM; D-

glucose, 5.0 mM; sodium ascorbate, 50 pM; bubbled with 100%

02, pH 7.4). Tissue containing the S-A pacemaker cells,

adjacent segments of the crista terminals and atrial

appendage, were dissected free and mounted (endocardial

surface up) in a constant temperature perfusion bath (360C).

The preparation was superfused with modified Tyrode's

solution and allowed to beat spontaneously. Changes in

beating rate were measured by using standard microelectrode

techniques to record transmembrane action potentials from the

S-A-node-atrial preparation as described previously (Baker &

Posner, 1986; Carpentier et al., 1984).










The spontaneous beating rate was allowed to stabilize at

least 1 hr, at which time the action potential configuration

was observed and, if not normal, the preparation not used.

At the end of the equilibration period, cumulative dose

response data were obtained by superfusing the drug for 5

min, at which time the beating rate was recorded. For time

course studies, the preparations were superfused with 0.1 pM

Iso or Carbo-Br for 20 min. After that time, the tissues

were immediately superfused with 50 nM nadolol until the

beating rates returned to basal levels and stabilized.

Ornithine decarboxylase (ODC) activity. Rats were

decapitated 3 and 24 hr after a 0.5 ml s.c. injection con-

taining 0.1 or 5.0 mg/kg of either Iso or Carbo-Br. Basal

ODC activity was determined 3 or 24 hr after a s.c. injection

of saline as described by Nelson et al. (1987). Briefly, the

hearts and lungs were rapidly removed after decapitation and

rinsed in ice-cold saline. The atria were dissected free of

the heart, and the ventricles and lungs were each homogenized

in 10 volumes of a 25 mM Tris-HCl buffer pH 7.2 containing

0.5 mM dithiothreitol and 50 gM pyridoxyl phosphate for 20

sec at setting 5 with Tekmar SDT-100EN. The homogenates were

centrifuged at 40,000xg for 15 min, and the supernatant

fraction saved for the assay. Supernatant (200 .l) was added

to 50 .1 of homogenization buffer containing 60 .M

[14C]ornithine in an air-tight 10-ml side arm flask (Kontes,

Vineland, NJ, USA) and incubated for 60 min at 360C. The

assay was stopped by adding 0.5 ml of 1 M citric acid to the










incubation mixture. Protosol (0.2 ml) was added to the

center well of the flask by syringe to absorb the 14C02. The

flasks were incubated for an additional 30 min at 360 C,

after which the center wells were removed, placed in 10 ml

Econofluor, and the radioactivity determined. Blanks were

determined in the absence of protein.

Synaptosomal preparation. Male Sprague-Dawley rats were

decapitated and the cortical tissues rapidly dissected. A

crude synaptosomal (P2) fraction was prepared essentially as

described by Gray & Whittaker (1962). Briefly, the tissue

was homogenized in 20 volumes of ice-cold 0.32 M sucrose

using a Teflon homogenizer (10 strokes, 900 rpm). The

homogenate was centrifuged at 1,000xg for 20 min, and the

supernatant discarded. The pellet (P2) was gently

resuspended in oxygenated incubation buffer pH 7.4 containing

10 mM glucose, 20 mM HEPES, 145 mM NaC1, 4.5 mM KC1, 1.2 mM

MgCl2, and 1.5 mM CaCl2 for assays.

13H1 Norepinephrine uptake assay. Synaptosomal protein

(0.2-0.3 mg) was suspended in a total volume of 1.0 ml

containing oxygenated incubation buffer, 10 JM pargyline (to

inhibit monoamine oxidase), 200 ILM sodium ascorbate, and

other drugs as indicated in the text. Synaptosomal protein

(5 mg/ml) was incubated with oxygenated incubation buffer in

the presence and absence of desired concentrations of Carbo-

Br (10-9 M to 10-4 M) at 370C for 30 min. At that time, 50 nM

unlabelled L-NE plus 0.45 JCi of [3H]NE was added to the

assay tubes. Incubations were carried out at 370C for 6 min,










after which time the suspensions were diluted with 4 ml of

ice-cold incubation buffer. The suspensions were poured onto

0.45 pm Millipore filters under reduced pressure. The

filters were washed with a further 8 ml of ice-cold buffer,

placed in a scintillation vial with 8 ml of Liquiscint, and

the radioactivity determined. Net NE uptake was calculated

by subtacting the blank values obtained by the substitution

of LiC1 for NaCl. All assays were performed in triplicate.

Data analysis. Data was analyzed using a Student's t-

test or analysis of variance as described in text.


Results


Binding of Carbo-Br to beta-adrenoreceptors in vivo.

Male Sprague-Dawley rats were injected i.p with various doses

of Carbo-Br (0.5, 2.0, 5.0, or 10.0 mg/kg). Figure 4-1

showed the receptor loss in 5 tissues, 3 hours following the

injection. The observation that no loss of receptors was

seen in the cerebral cortex (and cerebellum, data not shown)

suggested that this compound was not crossing the blood-

brain-barrier. There was a dose-dependent reduction in

binding sites in lung and spleen to a maximal loss of about

70-75% at 5.0 and 10.0 mg/kg. In contrast, the heart and

submaxillary glands showed only a 15-25% loss of binding

sites, even at high doses of Carbo-Br. Similar losses of

receptor binding at the 5 mg/kg dose were observed when the

drug was given s.c. (data not shown). No changes in the KD

values for [125I]CYP or [3H]DHA were observed using any


















100


80 -*
0o cortex
o heart
S60 submax
S* 8 lung
S40 f spleen
CO
~, 20


0
.5 2 5 10
Dose Carbo-Br (mg/kg)




Figure 4-1. Effect of in vivo Carbo-Br treatment on beta-
adrenoreceptors in 5 tissues of the rat. Male rats were
injected i.p. with 0.5, 2, 5, or 10 mg/kg of Carbo-Br. After
3 hr, cerebral cortices, submaxillary glands, hearts, lungs,
and spleens were removed. Membranes were prepared and
assayed for beta-adrenoreceptor content with 3-100 pM
[125I]CYP or 0.15-5 nM [3H]DHA as described under "Methods".
Specific binding for each tissue was as follows: submaxil-
lary gland, 98 5.8; heart, 22.3 1.0; lung, 658 29;
spleen, 53.2 1.8; cerebral cortex, 119 10 fmol/mg
protein. The values for each group were the mean of 7-12
animals S.D (significantly different from control: *p 5
0.0005; **p < 0.05).










of the tissues or at any dose of Carbo-Br as compared to

control values (data not shown).

To test for a selective loss of beta-adrenoceptor sub-

type, the ability of the highly betal-selective antagonist,

CGP-20712A, to inhibit subtype non-selective [125I]CYP

binding after animal treatment with Carbo-Br was examined.

The results were shown in Figure 4-2. In control membranes,

the ratio of betal to beta2 estimated from the plateau region

was: lung, 22/78; heart, 75/25; spleen, 17/83; submaxillary

gland, 82/18. Three hours after a 5 mg/kg injection of

Carbo-Br, the ratio of betal to beta2 was increased to:

63/37, lung; 43/57, spleen; 95/5, submaxillary gland; and

90/10, heart. This seemed to indicate that the majority of

receptor loss was at the beta2-receptor, with little or no

irreversible binding of Carbo-Br to the betal-receptor.

Binding of Carbo-Br to beta-adrenoceptors in vitro,

Table 4-1 showed the ability of Iso and Carbo-Br to inhibit

specific radioligand binding to membranes from four rat

tissues. Carbo-Br was found to be 4.6-fold more potent in

the heart and submaxillary gland and 22 to 26-fold more

potent in the lung and spleen than Iso at inhibiting specific

ligand binding.

Figure 4-3 showed a representative Scatchard plot of

[3H]DHA binding to lung membranes after a preincubation with

0.5 jM Carbo-Br. Membranes were preincubated with the

compound for 30 min at 300C in the presence and absence of

100 jM Gpp(NH)p and washed four times. Compared to the









Figure 4-2. Inhibition of specific [125I]CYP and [3H]DHA
binding in rat tissue membranes by CGP-20712A following in
vivo Carbo-Br treatment. A) Heart (squares), spleen
(circles); B) sumaxillary gland (squares), lung (circles).
Male rats were injected i.p. with 5 mg/kg Carbo-Br (closed
symbols) or vehicle (open symbols). After 3 hr, submaxillary
glands, hearts, lungs, and spleens were removed. Membranes
were prepared and assayed with 30 pM [125I]CYP or 5 nM
[3H]DHA, and the indicated concentrations of CGP-20712A for
45 min at 360C. At the end of the incubations, specific
binding was determined as described under "Methods". Each
point on the graph was the mean of four determinations
assayed in triplicate. The control [125I]CYP binding values
were 48 2, 14.5 0.5, and 50 2.6 fmol/mg protein for the
submaxillary gland, heart, and spleen, respectively. The
control [3H]DHA binding value for the lung was 343 28
fmol/mg protein. Specific [125I]CYP binding values for
Carbo-Br treated tissues were submaxillary gland, 37.5 1.8;
heart, 8.8 0.25; and spleen, 6.2 0.3 fmol/mg protein.
Specific [3H]DHA binding for the Carbo-Br treated lung tissue
was 76.6 6.3 fmol/mg protein. Each value was the mean of
six to eight determinations.


















C

E 20-

H
c- O
0

c 100-
a B
o

c
S80
CD
' 60


40-


20-

I I
10 9 8 7 6
CGP-20712A (-Log M)









Table 4-1. Concentration of Iso and Carbo-Br that inhibit
specific ligand binding to the beta-adrenoreceptors from
several rat tissues by 50% (IC50)


IC50 nM a

Tissue Iso Carbo-Br


Heart 443 55 95 7b

Submaxillary gland 449 41 96 llb

Spleen 613 23 23 6b

Lung 543 22 24 5b


Membranes from each tissue were incubated with 50 mM
Tris-HCl buffer at pH 7.4 containing 5 mM MgCl2, various
concentrations of Iso or Carbo-Br, and 100 IM Gpp(NH)p for 45
min at 360C. The incubations also included 30 pM [125I]CYP
for heart, submaxillary gland, and spleen assays, and 5 nM
[3H]DHA for lung assays. The total specific binding values
were similar to those given as control values in Figure 4-2.

a Each value was the mean S.E., n= 4-8.
b Significantly different from Iso values, p < 0.0005 as
determined with unpaired student's t-test.











control, there was a 71% decrease in specific [3H]DHA binding

with no effect of Gpp(NH)p on receptor loss. In addition,

there was no change in the KD value for [3H]DHA binding to the

remaining receptors after Carbo-Br pretreatment as compared

to the control (control, 0.85 nM; Carbo-Br pretreated, 0.74

nM; Carbo-Br plus Gpp(NH)p pretreated, 0.87 nM). Figure 4-4

showed the same experimental protocol using cardiac ventric-

ular membranes. After pretreatment with Carbo-Br or Carbo-Br

plus Gpp(NH)p there was a 30 and 15% loss of specific

[125I]CYP binding sites, respectively. However, there was an

increase in the KD value for [125I]CYP binding (control, 20

pM; Carbo-Br pretreated, 38 pM; Carbo-Br plus Gpp(NH)p

pretreated, 38 pM).

Physiological effects of Carbo-Br. Figures 4-5 and 4-6

showed the ability of Iso and Carbo-Br to stimulate lung and

heart ODC activity, respectively. Three hours after a 0.1

mg/kg injection of Iso there was an 8.0- and 8.6-fold

increase in lung and heart ODC activity, respectively.

Increasing the dose of Iso to 5 mg/kg increased the fold

stimulation to 21- for both tissues. At a dose of 0.1 mg/kg

of Carbo-Br in both tissues, there was an 8-fold stimulation

of enzyme activity that was increased to 12-fold 3 hr after a

5 mg/kg dose. When propranolol (20 mg/kg) was given 30 min

before the 5 mg/kg dose of either agonist, enzyme stimulation

was completely blocked in both tissues. Finally, Figures 4-5


















0.8



S 0.6- 03



g 0.4- [





II !
1 0.2




0 100 200 300 400 500 600

[3HIDHR Bound (fmol/mg protein)





Figure 4-3. Scatchard plot of specific (3H]DHA binding to
rat lung membranes after treatment with Carbo-Br in vitro.
Membranes were incubated without (open squares), with 0.5 LM
Carbo-Br (closed squares), and with 0.5 gM Carbo-Br plus 100
JM Gpp(NH)p (triangles) for 30 min at 300C. At the end of
the incubation, the membranes were washed four times with
buffer and assayed with 0.31-10 nM [3H]DHA as described under
"Methods". The data were plotted as the ratio of the amount
of specifically bound ligand (pmol/mg protein) to free ligand
(pmol/l) versus the amount of specifically bound ligand/mg
protein. Data points were the mean of triplicate determin-
ations and were representative of three experiments.


















0.6


o 0.5

0.4 -


S0.3
u_

i 0.2


0.1



0 2 4 6 8 10 12 14 16
[1251]CYP Bound (fmol/mg protein)




Figure 4-4. Scatchard plot of specific [125I]CYP binding to
rat heart membranes after treatment with Carbo-Br in vitro.
Membranes were incubated without (open squares), with 0.5 gLM
Carbo-Br (closed squares), and with 0.5 pM Carbo-Br plus 100
pM Gpp(NH)p (triangles) for 30 min at 300C. At the end of
the incubation, membranes were washed four times with buffer
and assayed with 3 to 100 pM [125I]CYP as described under
"Methods". The data were plotted as the ratio of the amount
of specifically bound ligand (fmol/mg protein) to free ligand
(fmol/l) versus the amount of specifically bound ligand/mg
protein. Data points were the mean of triplicate determin-
ations and were representative of three experiments.






















40 -
40- basal
,c Lung isoproterenol

S30 Carbo-Br

E

20
-a


0 10 *
0



basal 0.1 mg 5 mg 5+P 5/24

Figure 4-5. Stimulation of ornithine decarboxylase activity
in rat lung 3 and 24 hr after in vivo Iso and Carbo-Br
treatment. Male rats were injected i.p. with 0.1 or 5 mg/kg
Iso or Carbo-Br, 20 mg/kg propranolol followed by 5 mg/kg Iso
or Carbo-Br, or with vehicle alone. After 3 or 24 hr, lungs
were removed and assayed for ODC activity as described in
"Methods". Each response was the mean of three determin-
ations assayed in triplicate (significantly different from
basal levels, *p 5 0.0005).




















30 -
30 Heart M Basal
-E Isoproterenol
E L Carbo-Br
0o
E 20




om 10
o *


O-

Basal 0.1 mg 5 mg 5+P 5/24


Figure 4-6. Stimulation of ornithine decarboxylase activity
in rat heart 3 and 24 hr after in vivo Iso and Carbo-Br
treatment. Male rats were injected i.p. with 0.1 or 5 mg/kg
Iso or Carbo-Br, 20 mg/kg propranolol followed by 5 mg/kg Iso
or Carbo-Br, or with vehicle alone. After 3 or 24 hr, hearts
were removed and assayed for ODC activity as described in
"Methods". Each response was the mean of three determin-
ations assayed in triplicate (significantly different from
basal levels, *p < 0.0005).




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