Ectopic beta-adrenergic receptor binding sites. possible molecular basis of aberrant catecholamine responsiveness of an adrenocortical tumor adenylate cyclase.

The molecular basis for the aberrant catecholamine responsiveness of the adenylate cyclase of adrenocortical carcinoma 494 was explored. The adenylate cyclase of this corticosteroid-producing, transplanted, adrenal cancer of the rat was stimulated not only by adrenocorticotropic hormone and fluoride, but also by the beta-adrenergic agonist, isoproterenol. The adenylate cyclase of normal adrenal tissue was unresponsive to isoproterenol. Direct binding studies with the specific high affinity B-adrenergic ligand, (-)[3H]dihydroalprenolol, demonstrated the pressure of 0.094 pmol of specific binding sites per milligram of tumor membrane protein. By contrast, normal adrenal membranes contained too few binding sites to accurately measure and study using these techniques. The tumor binding sites had high affinity for (-)[3H] dihydroalprenolol with an equilibrium dissociation constant of 2.1 nM. Adrenergic agonists competed for the binding sites in an order of potency, [(-) isoproterenol greater than (-) epinephrine (-) norepinephrine], paralleling their order of potency as beta-adrenergic agonists. The beta-adrenergic antagonist, (-) propranolol, competed for binding, causing half-mzximal inhibition of specific binding at a concentration of 6 nM. The alpha-adrenergic antagonist, phentolamine, and several catecholamine metabolites and precursors did not effectively compete for the binding sites at high concentrations. Binding was stereospecific, the (+) stereoisomers of beta-adrenergic agonists and antagonists requiring 40- to 300-fold higher concentrations than the corresponding (-) stereoisomers to half maximally inhibit (-) [3H] dihydroalprenolol binding. These results indicate that adrenocortical carcinoma 494 membranes contain beta-adrenergic receptor-binding sites which are not normally present in membranes of adrenal tissue. These ectopic beta-adrenergic receptors presumably confer on the neoplastic tissue the catecholamine sensitivity of its adenylate cyclase.     

Identification of beta-adrenergic receptors in human lymphocytes by (-) (3H) alprenolol binding.

Human lymphocytes are known to posessess a catecholamine-responsive adenylate cyclase which has typical beta-adrenergic specificity. To identify directly and to quantitate these beta-adenergic receptors in human lymphocytes, (-) [3H] alprenolol, a potent beta-adrenergic antagonist, was used to label binding sites in homogenates of human mononuclear leukocytes. Binding of (-) [3H] alprenolol to these sites demonstrated the kinetics, affinity, and stereospecificity expected of binding to adenylate cyclase-coupled beta-adrenergic receptors. Binding was rapid (t1/2 less than 30 s) and rapidly reversible (t1/2 less than 3 min) at 37 degrees C. Binding was a saturable process with 75 +/- 12 fmol (-) [3H] alprenolol bound/mg protein (mean +/- SEM) at saturation, corresponding to about 2,000 sites/cell. Half-maximal saturation occurred at 10 nM (-) [3H] alprenolol, which provides an estimate of the dissociation constant of (-) [3H] alprenolol for the beta-adrenergic receptor. The beta-adrenergic antagonist, (-) propranolol, potently competed for the binding sites, causing half-maximal inhibition of binding at 9 nM. beta-Adrenergic agonists also competed for the binding sites. The order of potency was (-) isoproterenol greater than (-) epinephrine greater than (-)-norepinephrine which agreed with the order of potency of these agents in stimulating leukocyte adenylate cyclase. Dissociation constants computed from binding experiments were virtually identical to those obtained from adenylate cyclase activation studies. Marked stereospecificity was observed for both binding and activation of adenylate cyclase. (-)Stereoisomers of beta-adrenergic agonists and antagonists were 9- to 300-fold more potent than their corresponding (+) stereoisomers. Structurally related compounds devoid of beta-adrenergic activity such as dopamine, dihydroxymandelic acid, normetanephrine, pyrocatechol, and phentolamine did not effectively compete for the binding sites. (-) [3H] alprenolol binding to human mononuclear leukocyte preparations was almost entirely accounted for by binding to small lymphocytes, the predominant cell type in the preparations. No binding was detectable to human erythrocytes. These results demonstrate the feasibility of using direct binding methods to study beta-adrenergic receptors in a human tissue. They also provide an experimental approach to the study of states of altered sensitivity to catecholamines at the receptor level in man.     

The effects of chronic caffeine administration on peripheral adenosine receptors

Rat platelets and adipocytes were used as models to investigate alterations of the A2- and of the A1-adenosine receptor-adenylate cyclase system of peripheral cells caused by chronic caffeine administration. The maximum effects of 5'-N-ethylcarboxamidoadenosine (NECA) to stimulate adenylate cyclase activity in suspensions of platelet membranes and to inhibit aggregation were significantly greater with platelets from caffeine-treated rats than from control rats. The effects of 1 to 100 nM prostaglandin E1 to inhibit platelet aggregation and to stimulate adenylate cyclase activity in platelet membranes were also significantly greater with caffeine-treated than with control rats. These data suggest that the increased ability of NECA to inhibit platelet aggregation after chronic caffeine ingestion was a result of increased cyclic AMP accumulation induced by this agonist. The increased stimulatory effect of NECA on adenylate cyclase in platelet membranes could be due to an increased A2-adenosine receptor number and/or an increased functional coupling between A2-adenosine receptor and stimulatory guanine nucleotide binding proteins. In contrast, although A1-receptor number was 37% higher in fat cell membranes from caffeine-treated rats than in those from control rats, increased A1-adenosine receptor-mediated inhibition of lipolysis and of adenylate cyclase was not detected. Thus, chronic caffeine consumption causes alterations in the response of some but not all peripheral cell types to agonists of adenosine receptors.     

Pineal beta adrenergic receptor: correlation of binding of 3H-l-alprenolol with stimulation of adenylate cyclase.

3H-l-Alprenolol, a potent competitive beta adrenergic antagonist, binds to sites in rat pineal gland membranes. The properties of these binding sites were compared to those of the receptors which mediate the beta adrenergic activation of pineal adenylate cyclase. Both sites are highly stereospecific. The l-stereoisomers of alprenolol and propranolol were at least two orders of magnitude more potent than the d-stereoisomers in inhibiting isoproterenol-stimulated adenylate cyclase or 3H-l-alprenolol binding. The dissociation constants (Kd) of the l-stereoisomers of both alprenolol and propranolol were 10 to 22 nM as determined by competition for binding sites or by inhibition of isoproternol-stimulated adenylate cyclase. Beta adrenergic agonists which stimulated adenylate cyclase also competitively inhibited the binding of 3H-l-alprenolol. They showed the same order of potency (isoproterenol greater than norepinephrine greater than or equal to epinephrine) and the same individual affinities in the two systems. Alpha adrenergic blockers were ineffective in inhibiting either adenylate cyclase stimulation or 3H-l-alprenolol binding. Isoproternol stimulation of adenylate cyclase acrivity, and 3H-l-alprenolol binding, were rapid and rapidly reversible. The 3H-l-alprenolol binding sites were saturable and bound 0.6 pmol of ligand per mg of added protein. The data suggest that the binding of 3H-l-alprenolol occurs at sites indistinguishable from the pineal beta adrenergic receptor.     

Effects of ethanol in vitro on the beta adrenergic receptor-coupled adenylate cyclase system

The effects of ethanol on the beta adrenergic receptor-coupled adenylate cyclase system were examined in vitro using membranes prepared from S49 lymphoma cells. Ethanol caused a dose-dependent increase in adenylate cyclase activity in membranes prepared from wild-type cells when the activity was measured in the presence of GTP. Activity measured in the presence of isoproterenol was also increased by ethanol, but the fold-stimulation by isoproterenol was lower in the presence of ethanol. Ethanol also shifted the dose-response curve for stimulation of the enzyme by isoproterenol to the right. This shift was due to a decrease in the affinity of the beta adrenergic receptor for isoproterenol. A decrease in the affinity of the receptor for the antagonists [125I]iodopindolol and propranolol was also observed, but the magnitude of this effect was less than that seen with the agonist isoproterenol. The density of binding sites for [125I]iodopindolol was not affected by ethanol. Dose-response curves for NaF and guanosine-5'-O-(3-thiotriphosphate), both of which stimulate adenylate cyclase activity through an effect on the stimulatory guanine nucleotide-binding protein (Gs), were shifted to the left by the addition of ethanol. In membranes prepared from the CYC- variant of S49 cells, which lacks the alpha subunit of Gs, guanosine-5'-O-(3-thiotriphosphate) inhibited forskolin-stimulated adenylate cyclase activity. The inhibition by guanosine-5'-O-(3-thiotriphosphate) was not affected by ethanol. In membranes prepared from both wild-type and CYC- S49 cells, ethanol inhibited forskolin-stimulated adenylate cyclase activity. Whereas the inhibition of this activity by GTP was greatly attenuated in membranes prepared from CYC- S49 cells which had been pretreated with pertussis toxin, the inhibition by ethanol was not affected by pretreatment with pertussis toxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Norepinephrine and BRL 37344 stimulate adenylate cyclase by different receptors in rat brown adipose tissue

The beta adrenergic activation of adenylate cyclase was examined in membrane homogenates of rat interscapular brown adipose tissue (IBAT). In control membranes, isoproterenol and norepinephrine (NE) stimulated adenylate cyclase with activation constants of about 20 and 300 nM, respectively. Exposure of rats to 4 degrees C for 3 days increased the maximal stimulation of adenylate cyclase to these agonists but did not alter the respective activation constants. The beta 1-selective antagonist 1-(2-cyanophenoxy)-3-beta-(3-phenylureido)ethylamino-2-pr opa nol blocked isoproterenol stimulation of adenylate cyclase in control and cold-exposed membranes at a concentration 100 times lower than did the beta 2-selective antagonist erythro-dl-1-(7-methylindan-4-yloxy)-3-isopropylaminobuta n-2-ol. These data indicate that typical adrenergic agonists stimulate IBAT adenylate cyclase via beta 1 receptors. (R*,R*)-4-[2-[2 [9 3-chlorophenyl)-2-hydroxyethyl]amino)propyl) phenyl]phenoxyacetic acid (BRL 37344), an atypical agonist with activity at the beta 3 receptor, stimulated adenylate cyclase in control membranes with an activation constant of approximately 700 nM. Membranes of cold-exposed rats exhibited a high affinity response to BRL 37344 similar to that seen in control membranes and, in addition, a low affinity response. BRL 37344 stimulation of adenylate cyclase was unaffected by 1-(2-cyanophenoxy)-3-beta-(3-phenylureido)ethyl-amino-2-prop anol, whereas stimulation by NE or epinephrine was potently blocked.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological and biochemical characterization of the D-1 dopamine receptor mediating acetylcholine release in rabbit retina

Superfusion with dopamine (0.1 microM-10 mM) evokes calcium-dependent [3H]acetylcholine release from rabbit retina labeled in vitro with [3H]choline. This effect is antagonized by the D-1 dopamine receptor antagonist SCH 23390. Activation or blockade of D-2 dopamine, alpha-2 or beta receptors did not stimulate or attenuate the release of [3H]acetylcholine from rabbit retina. Dopamine receptor agonists evoke the release of [3H]acetylcholine with the following order of potency: apomorphine greater than or equal to SKF(R)82526 greater than SKF 85174 greater than SKF(R)38393 greater than or equal to pergolide greater than or equal to dopamine (EC50 = 4.5 microM) greater than SKF(S)82526 greater than or equal to SKF(S)38393. Dopamine receptor antagonists inhibited the dopamine-evoked release of [3H]acetylcholine: SCH 23390 (IC50 = 1 nM) greater than (+)-butaclamol greater than or equal to cis-flupenthixol greater than fluphenazine greater than perphenazine greater than trans-flupenthixol greater than R-sulpiride. The potencies of dopamine receptor agonists and antagonists at the dopamine receptor mediating [3H]acetylcholine release is characteristic of the D-1 dopamine receptor. These potencies were correlated with the potencies of dopamine receptor agonists and antagonists at the D-1 dopamine receptor in rabbit retina as labeled by [3H]SCH 23390, or as determined by adenylate cyclase activity. [3H]SCH 23390 binding in rabbit retinal membranes was stable, saturable and reversible. Scatchard analysis of [3H]SCH 23390 saturation data revealed a single high affinity binding site (Kd = 0.175 +/- 0.002 nM) with a maximum binding of 482 +/- 12 fmol/mg of protein. The potencies of dopamine receptor agonists to stimulate [3H]acetylcholine release were correlated with their potencies to stimulate adenylate cyclase (r = 0.784, P less than .05, n = 7) and with their affinities at [3H]SCH 23390 binding sites (r = 0.755, P greater than .05, n = 8). The potencies of antagonists to inhibit dopamine-evoked [3H]acetylcholine release were correlated with their potencies to inhibit the dopamine-stimulated adenylate cyclase (r = 0.759, P less than .05, n = 5) and with their affinities at [3H]SCH 23390 binding sites (r = 0.998, P less than .01, n = 7). We conclude that in rabbit retina dopamine evokes calcium-dependent [3H]acetylcholine release through activation of a site with the pharmacological characteristics of a D-1 dopamine receptor.     

Decreased stimulatory guanosine triphosphate binding protein in dogs with pressure-overload left ventricular failure.

Alterations in the level and function of the stimulatory guanyl nucleotide binding protein (Gs) from the cardiac sarcolemma were examined in a canine model of heart failure. The present study is based on our previous investigations that demonstrated both a loss of beta-adrenergic agonist high-affinity binding sites and a decreased adenylate cyclase activity in sarcolemma from failing hearts. Using cholera toxin and [32P]NAD, we labeled the alpha subunit of Gs (Gs alpha) and found a 59% reduction in the level of this protein. Further, a 50% reduction in Gs activity was noted in a reconstitution assay utilizing membranes from the mouse S49 lymphoma cell line cyc-, which is deficient in Gs. These data suggest that, in this model of pressure-overload left ventricular failure, the acquired defect in the beta-adrenergic receptor/adenylate cyclase system involves a deficiency in the coupling protein Gs. Such an abnormality may explain the decreased adrenergic responsiveness of the failing left ventricle.     

Cellular mechanisms of impaired adrenergic responsiveness in neonatal dogs.

The myocardial responsiveness of conscious, instrumental dogs to exogenously administered isoproterenol and norepinephrine was investigated in neonatal, 6-wk-old, and adult animals. Comparable base-line values for peak left ventricular derivative of pressure with respect to time were observed in all age categories. However, when compared with adult responses, the sympathomimetic amine-induced increases in neonatal left ventricular dP/dt were significantly blunted at each concentration of adrenergic agonist examined, whereas the 6-wk-old puppies displayed an intermediate inotropic response. To investigate the cellular mechanisms of this blunted neonatal response, we correlated physiologic and biochemical measurements of the myocardial responses to catecholamines in each age category. When compared with adult myocardial membrane preparations, neonatal cardiac membranes were characterized in vitro by an increased density of beta-adrenergic binding sites, comparable affinity for adrenergic agonists and antagonists, and an enhanced coupling of adenylate cyclase activation to receptor occupancy. Simultaneous changes in either the serum catecholamine concentration or the membrane content of other intrinsic proteins failed to account for the observed neonatal increase in beta-adrenergic receptor density. These findings are most consistent with a compensatory mechanism of the cardiac cell membrane, whereby an inherent depression in the adrenergic responsiveness of the immature myocardium appears to induce the increase in receptor density and activation of adenylate cyclase.     

Biochemical and functional identification of a novel dopamine receptor subtype in rat brown adipose tissue. Its role in modulating sympathetic stimulation-induced thermogenesis.

Various dopamine (DA) agonists including propylnorapomorphine, lisuride, bromocriptine, apomorphine and quinpirole were found to reduce adenylate cyclase activity in rat brown adipose tissue homogenates. These inhibitory effects were antagonized, with a very low stereoselectivity, by DA receptor antagonists with the following rank order of potency: haloperidol > (+)-butaclamol > or = (-)-butaclamol > clozapine > or = (-)-sulpiride > or = (+)-sulpiride > or = eticlopride, but not by the alpha-2 adrenoceptor antagonists, phenoxybenzamine and yohimbine or the serotonin receptor antagonists, ketanserin and metergoline. The selective D1 agonist, fenoldopam, was completely inactive in modifying the basal enzyme activity. DA as well as various DA agonists (lisuride > propylnorapomorphine > bromocriptine > apomorphine > quinpirole) dose-dependently reduced the stimulation of adenylate cyclase activity induced either by forskolin or by the beta adrenoceptor agonist, (-)-isoproterenol. Similar results were obtained also in dispersed brown adipocytes. We also found that DA and various DA receptor agonists induced a significant decrease of beta adrenoceptor-stimulated glycerol and nonesterified fatty acids release from brown adipocytes. This effect was selectively antagonized by haloperidol and butaclamol. Thus, the receptors present on the BAT membranes appear to be dopaminergic in nature although they differ from the classical D2 receptor for the following: 1) the low affinity for the most selective D2, D3 and D4 receptor agonist and antagonist (quinpirole, sulpiride and clozapine); 2) the absence of stereoselectivity for various DA antagonists (butaclamol and sulpiride); and 3) the lack of detectable mRNA encoding D2 or D3 DA receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

In rat hippocampus, somatostatin 14 and muscarinic receptor ligands modulate an adenylate cyclase belonging to a common domain of the receptor

In hippocampal slices, somatostatin 14 and its stable analog L363 [cyclo(Phe-Pro-Phe-D-Trp-Lys-Thr)] fail to modify muscarinic signal transduction mediated by stimulation of phosphoinositide breakdown, whereas somatostatin 14 mimics oxotremorine in inhibiting adenylate cyclase activity of hippocampal membranes. The simultaneous addition of somatostatin 14 and oxotremorine elicits a nonadditive convergent inhibition of adenylate cyclase activity. Both L363 and oxotremorine nonadditively stimulate a high-affinity guanosine 5'-triphosphatase activity of hippocampal membranes. This stimulation could be operative in mediating the convergent inhibition of adenylate cyclase activity elicited by the binding of specific ligands to somatostatin and muscarinic recognition sites present in hippocampal membranes. Because L363 competitively displaces muscarinic agonists fand antagonists from their specific recognition sites, one might infer that the two recognition sites interact functionally; that is, somatostatin reduces the efficacy of oxotremorine and/or vice versa.     

Thermodynamic properties of agonist interactions with the beta adrenergic receptor-coupled adenylate cyclase system. I. High- and low-affinity states of agonist binding to membrane-bound beta adrenergic receptors

The properties associated with ligand interactions with membrane-bound beta adrenergic receptors prepared from L6 myoblasts were examined at five temperatures between 10 degrees and 30 degrees C. The interactions of antagonists with membrane-bound receptors were insensitive to temperature, whereas the interactions of agonists were temperature-dependent. The affinity constants for the low-affinity binding states of agonists (KL) decreased slightly with decreasing assay temperature. The small temperature-dependent changes in KL values were similar to the changes in Kd values observed in studies of the binding of agonists in the presence of GTP. The high-affinity dissociation constants (KH) for binding of full agonists to membrane-bound receptors in the absence of GTP were approximately 50-fold lower at 10 degrees than at 30 degrees C. The KH values for partial agonists also decreased with decreasing temperature, but the changes were smaller in magnitude. Thermodynamically, the binding of antagonists was primarily entropy-driven, whereas the binding of agonists was enthalpy-driven. The energetics of the low-affinity component of agonist binding to membrane-bound receptors were similar to the energetics for binding of agonists to membrane-bound receptors in the presence of GTP. Under these conditions, standard enthalpy change (delta H degree) and standard entropy change (delta S degree) values for binding of agonists were more negative than the corresponding values for binding of antagonists, possibly reflecting a conformational change in the receptor or an increased ordering of the lipids surrounding the receptor. The interaction of the receptor with the guanine nucleotide-binding protein to form the high-affinity component of agonist binding was thermodynamically described by larger negative changes in enthalpy and entropy than the values for formation of the low-affinity component of agonist binding. There was a correlation between the efficacies of ligands in activating adenylate cyclase and the delta H degree and delta S degree values for high-affinity binding of agonists. Thus, the extent or nature of the interaction between the guanine nucleotide-binding protein and the receptor may determine the efficacies of ligands.     

Pharmacological control of the human gastric histamine H2 receptor by famotidine: comparison with H1, H2 and H3 receptor agonists and antagonists

Histamine 0.1 microM-0.1 mM increased adenylate cyclase activity five- to ten-fold in human fundic membranes, with a potency Ka = 3 microM. The histamine dose-response curve was mimicked by the H3 receptor agonist (R) alpha-MeHA, but at 100 times lower potency, Ka = 0.3 mM. Histamine-induced adenylate cyclase activation was abolished by H2, H1 and H3 receptor antagonists, according to the following order of potency IC50: famotidine (0.3 microM) greater than triprolidine (0.1 mM) thioperamide (2 mM), respectively. Famotidine has no action on membrane components activating the adenylate cyclase system, including the Gs subunit of the enzyme stimulated by forskolin and cell surface receptors sensitive to isoproterenol (beta 2-type), PGE2 and VIP. The Schild plot was linear for famotidine (P less than 0.01) with a regression coefficient r = 0.678. The slope of the regression line was 0.64 and differs from unity. Accordingly, famotidine showed a slow onset of inhibition and dissociation from the H2 receptor in human cancerous HGT-1 cells. The results demonstrate that famotidine is a potent and selective H2 receptor antagonist with uncompetitive actions in human gastric mucosa. Consequently, famotidine might be a suitable drug with long-lasting actions in the treatment of Zollinger-Ellison syndrome. The results also confirm and extend the previous observations that (R) alpha-MeHA and thioperamide are two selective ligands at histamine H3 receptor sites. In the human gastric mucosa, these drugs are respectively 330 and 6700 times less potent than histamine and famotidine on the adenylate cyclase system. The possible involvement of histamine H3 receptors in the regulation of gastric secretion is proposed.     

Characterization of serotonin-1B receptors negatively coupled to adenylate cyclase in OK cells, a renal epithelial cell line from the opossum

We have previously reported the presence of a 5-HT1 (serotonin)-like receptor coupled in an inhibitory manner to adenylate cyclase in the opossum kidney cell line, which is derived from the kidney of a North American opossum. Pharmacological data from binding and cyclic AMP production studies indicate that this receptor does not have characteristics of a 5-HT1A, 5-HT1C or 5-HT1D receptor, but is similar to 5-HT1B receptors found in rodent tissues. Many serotonergic drugs, including 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyrindinyl)1H-indol, 5-HT and methysergide, but not (+/-)-8-hydroxy-2-(di-N-propylamino)tetralin hydrobromide or buspirone, were full agonists at this receptor as defined by the inhibition of bovine parathyroid hormone peptide fragment 1-34-stimulated cyclic AMP production in an intact cell assay. Several classical beta adrenergic antagonists including propranolol and cyanopindolol were also full agonists at this receptor. Radioligand binding studies using [125I](-)-iodocyanopindolol gave a Bmax of 88 fmol/mg of protein and a KD of 47 pM for saturation experiments carried out in the presence of GTP. In the absence of GTP, the binding data were significantly better fit by a two-site model with KD values of 10 and 345 pM. Inhibition binding experiments were consistent with the results of the cyclic AMP experiments. The identification of 5-HT1B receptors in a tissue derived from the opossum kidney suggests that these receptors may be distributed more widely than previously thought, inasmuch as other studies have found them only in neuronal tissues of rodents.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the adenosine receptor in cultured embryonic chick atrial myocytes: coupling to modulation of contractility and adenylate cyclase activity and identification by direct radioligand binding

Adenosine receptors in a spontaneously contracting atrial myocyte culture from 14-day chick embryos were characterized by radioligand binding studies and by examining the involvement of G-protein in coupling these receptors to a high-affinity state and to the adenylate cyclase and the myocyte contractility. Binding of the antagonist radioligand [3H]-8-cyclopentyl-1,3-diproylxanthine ([3H]CPX) was rapid, reversible and saturable and was to a homogeneous population of sites with a Kd value of 2.1 +/- 0.2 nM and an apparent maximum binding of 26.2 +/- 3 fmol/mg of protein (n = 10, +/- S.E.). Guanyl-5-yl-(beta, gamma-imido)diphosphate had no effect on either the Kd or the maximum binding and CPX reversed the N6-R-phenyl-2-propyladenosine-induced inhibition of adenylate cyclase activity and contractility, indicating that [3H] CPX is an antagonist radioligand. Competition curves for [3H] CPX binding by a series of reference adenosine agonists were consistent with labeling of an A1 adenosine receptor and were better fit by a two-site model than by a one-site model. ADP-ribosylation of the G-protein by the endogenous NAD+ in the presence of pertussis toxin shifted the competition curves from bi to monophasic with Ki values similar to those of the KL observed in the absence of prior pertussis intoxication. The adenosine agonists were capable of inhibiting both the adenylate cyclase activity and myocyte contractility in either the absence or the presence of isoproterenol. The A1 adenosine receptor-selective antagonist CPX reversed these agonist effects. The order of ability of the reference adenosine receptor agonists in causing these inhibitory effects was similar to the order of potency of the same agonists in inhibiting the specific [3H]CPX binding (N6-R-phenyl-2-propyladenosine greater than N6-S-phenyl-2-propyladenosine or N-ethyladenosine-5'-uronic acid). These data indicate that the adenosine receptor coupled to inhibition of adenylate cyclase activity and to the negative inotropic effect is the A1 subtype. Pertussis treatment uncoupled the adenosine receptor from both inhibition of adenylate cyclase activity and negative inotropic effect. Taken together, the present study indicates that adenosine receptors of the A1 subtype are present on the spontaneously contracting atrial myocytes and are negatively coupled to adenylate cyclase and to the contractile state. The cultured embryonic chick atrial myocyte preparation represents a useful model system for characterizing the cardiac A1 adenosine receptor.     

Coupling of subtypes of the muscarinic receptor to adenylate cyclase in the corpus striatum and heart

The binding properties of a series of muscarinic antagonists were compared with their ability to antagonize muscarinic receptor mediated inhibition of adenylate cyclase activity in homogenates of the corpus striatum and heart of rats. When measured by the competitive inhibition of the binding of the muscarinic antagonist N-[3H]methylscopolamine, the binding properties of selective muscarinic antagonists in the corpus stratum and cerebral cortex were consistent with a model incorporating a minimum of three populations of muscarinic receptors, a high affinity site for pirenzepine (M1), a high affinity site for AF-DX 116 [11] [2-[ (diethylamino)methyl]-1-piperidinyl] acetyl] -5, 11-dihydro-6H-pyrido [2,3-b] 1,4] benzodiazepine-6-one (M2) and a third population (non-Ml, non-M2 sites) displaying low affinity for the latter antagonists. The results of similar experiments on the heart showed that this tissue contained a uniform population of M2 muscarinic receptors. The binding properties of the M2 receptor in cerebral cortex and corpus stratum were also investigated directly in antagonist [3H] AF-DX 116 competition experiments and, although the high affinity AF-DX 116 site in brain (M2) exhibited selectivity for the cardioselective antagonists AF-DX 116 and gallamine, some differences were noted between M2 sites in brain and heart. The muscarinic adenylate cyclase response in the corpus striatum was relatively insensitive to the M2 selective antagonists AF-DX 116 and gallamine as well as the M1 selective antagonist pirenzepine, suggesting that non-M1, non-M2 sites inhibit adenylate cyclase activity in the corpus striatum. In contrast, the effects of muscarinic antagonists on the muscarinic adenylate cyclase response in the heart were consistent with the postulate that M2 receptors inhibit adenylate cyclase activity in this tissue.     

Effects of acute ischemia in the dog on myocardial blood flow, beta receptors, and adenylate cyclase activity with and without chronic beta blockade.

We ligated the left anterior descending coronary artery for 1 or 2 h in 31 purebred beagles. We did not detect any changes in beta-adrenergic receptor density or affinity when normal and ischemic zones were compared, either in the subendocardium or in the subepicardium. In the ischemic zones, there was a significant decline in all measures of adenylate cyclase activity, including activity mediated by the beta-adrenergic receptor. By contrast, after chronic beta-adrenergic blockade (1.5 mg/kg propranolol i.v. twice daily for 7 d), there was an increase in adenylate cyclase activity stimulated by (-)-isoproterenol relative to adenylate cyclase activity stimulated by guanyl-5'imidodiphosphate (GppNHp) in both normal and ischemic tissue, suggesting that one effect of chronic beta blockade may be to enhance coupling between the stimulatory guanine nucleotide regulatory protein (Gs) and the beta-adrenergic receptor, despite a reduction in the number or function of Gs units. Chronic beta blockade also led to up regulation of beta-adrenergic receptor density in subepicardial regions. After 20 min of reperfusion following 2 h of ischemia, adenylate cyclase activity tended to return to control levels, particularly in the subepicardium, where (-)-isoproterenol-stimulated adenylate cyclase activity was not different from normal myocardium. We conclude that chronic beta-adrenergic blockade may have beneficial effects during prolonged episodes of myocardial ischemia by preserving signal transduction mediated by the beta-adrenergic receptor.     

Chronic caffeine ingestion sensitizes the A1 adenosine receptor-adenylate cyclase system in rat cerebral cortex.

Caffeine consumption causes significant physiologic effects due to its antagonism of adenosine receptors. The A1 adenosine receptor is coupled in an inhibitory manner to adenylate cyclase. To study the effects of chronic caffeine ingestion, rats were provided with 0.1% caffeine drinking solution for 28 d. The A1 adenosine receptor agonist radioligand [3H]phenylisopropyladenosine identifies two affinity states in control rat cerebral cortex membranes with a high affinity dissociation constant (KH) of 0.40 +/- 0.08 nM and low affinity dissociation constant (KL) of 13.7 +/- 3.9 nM, with 33% of the receptors in the high affinity state. In membranes from caffeine-treated animals, all of the A1 receptors are shifted to the high affinity state with a dissociation constant (KD) of 0.59 +/- 0.06 nM. Guanylyl-imidodiphosphate (10(-4) M) decreases binding by 43% in control membrane, with no change in KH or KL, while membrane binding in caffeine-treated animals decreases by 45% with a threefold shift in KD to 1.5 +/- 0.3 nM. Concomitant with the enhanced high affinity A1 receptor state and increased sensitivity to guanine nucleotides, membranes from treated animals show a 35% enhancement in (-)-N6-(R-phenylisopropyl)adenosine-mediated inhibition of adenylate cyclase compared with controls (P less than 0.03). Photoaffinity crosslinking the receptors with [125I]N6-2-(3-iodo-4-aminophenyl)ethyladenosine reveals that A1 receptors from both groups migrate as Mr 38,000 proteins. beta-adrenergic receptor binding with [125I]iodocyanopindolol shows a decrease in the number of beta-receptors from 233 +/- 7 fmol/mg protein in control membranes to 190 +/- 10 fmol/mg protein in treated membranes (P = 0.01). These data indicate that the adenosine receptor antagonist, caffeine, induces a compensatory sensitization of the A1 receptor-adenylate cyclase system and downregulation of beta-adrenergic receptors, and provides a molecular mechanism for the caffeine withdrawal syndrome.     

Adenosine and muscarinic cholinergic receptors attenuate cyclic AMP accumulation by different mechanisms in 1321N1 astrocytoma cells

An adenosine receptor has been characterized to unambiguously demonstrate that the inhibitory guanine nucleotide regulatory protein, Gi, of 1321N1 human astrocytoma cells is fully capable of functionally coupling to adenylate cyclase. Adenosine receptor agonists attenuated cyclic AMP accumulation by 35 to 75% with the order of potency of N6(R-phenylisopropyl)-adenosine greater than adenosine = 2-chloroadenosine greater than N6-methyladenosine = N6-benzyladenosine. 3-Isobutyl-1-methylxanthine competitively antagonized the effect of adenosine receptor agonists. Adenylate cyclase activity measured in cell-free preparations from 1321N1 cells was inhibited by N6(R-phenylisopropyl)-adenosine. Pretreatment of 1321N1 cells with pertussis toxin blocked both adenosine receptor-mediated inhibition of adenylate cyclase activity and attenuation of cyclic AMP accumulation. In contrast to the effects on responses to adenosine receptor agonists, 3-isobutyl-1-methylxanthine noncompetitively antagonized muscarinic receptor-mediated attenuation of cyclic AMP accumulation and pertussis toxin had no effect. These data are consistent with the ideas that Gi is fully functional in 1321N1 cells and links inhibitory adenosine receptors to adenylate cyclase, and that the muscarinic receptor of these cells couples to the phosphoinositide response system, but is incapable of functionally coupling through Gi to inhibit adenylate cyclase.