Involvement of a high-affinity GTPase in the inhibitory coupling of striatal muscarinic receptors to adenylate cyclase

The stimulation of GTP hydrolysis has been proposed as a mechanism by which hormones inhibit receptor-coupled adenylate cyclase activity. The present study attempts to verify whether this mechanism is also operative in transmitter-mediated receptor-coupled attenuation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] located in synaptic plasma membrane preparations. As a model, we used the inhibition of adenylate cyclase activity by muscarinic receptor activation in rat striatum. This striatal preparation contains high-affinity GTPase (EC 3.6.1-) activity which is stimulated when the recognition site for muscarinic agonists is occupied. Acetylcholine (ACh), but not nicotine, increases the Vmax of the high-affinity GTPase, and the stimulatory effect is antagonized by atropine but not by d-tubocurarine. The rank order of potency of various cholinergic agonists to stimulate GTPase correlates with their ability to inhibit adenylate cyclase activity of striatal membranes. Pre-exposure of striatal membranes to guanosine-5'-O-(3-thiotriphosphate) causes a parallel decrease in the basal and ACh-stimulated GTPase activities and in the ACh-induced inhibition of adenylate cyclase. Treatment of the membranes with cholera toxin does not affect the ACh-stimulated GTPase activity but amplifies the extent of adenylate cyclase inhibition elicited by the cholinergic agonist. These results indicate that the stimulation of a high-affinity GTPase parallels the inhibitory coupling of central muscarinic receptors to adenylate cyclase.     

GTP-dependent inhibition of cardiac adenylate cyclase by muscarinic cholinergic agonists

Summary In membrane-containing particles of rabbit myocardium, inhibition of adenylate cyclase (EC 4.6.1.1) by cholinergic agonists was studied. The cholinergic agonists, in the potency order oxotremorine > acetylcholine > carbachol, reduced basal adenylate cyclase activity with half-maximal effects at about 0.3, 1 and 3 M, respectively; maximal inhibition observed was about 40%. The enzyme inhibition occurred without apparent lag phase and was reversed by the muscarinic cholinergic antagonist, atropine, which finding indicates that muscarinic cholinergic receptors are involved in this process.As for stimulation of cardiac adenylate cyclase by the -adrenergic agonist, isoproterenol, the addition of GTP was necessary for maximal enzyme inhibition by the cholinergic agonists. The effects of GTP were half-maximal at about 0.2 and 1 M and maximal at about 3 and 30 M GTP for stimulation and inhibition, respectively. NaCl, which increased cardiac adenylate cyclase activity, facilitated the GTP-dependent cholinergic inhibition; the NaCl effect was maximal between 50 and 100 mM.In the presence of GTP, the cholinergic agonist, carbachol, not only reduced basal adenylate cyclase activity, but also inhibited adenylate cyclase stimulated by isoproterenol (100 M) or NaF (10 mM). In the presence of cholera toxin (40 g/ml), the GTP-induced activation of the enzyme was counteracted by carbachol. However, the stable GTP-analogues, guanylyl-5-imidodiphosphate and guanosine-5-O-(3-thiotriphosphate), which caused a persistent adenylate cyclase activation, reversed or prevented the carbachol-induced inhibition.The data indicate that cholinergic agonists inhibit cardiac adenylate cyclase by a process that requires (the hydrolyzable) GTP and involves sodium ions.Some of the data were presented in abstract form (Aktories and Jakobs, 1979)     

Positive coupling of cholinergic muscarinic receptors to adenylate cyclase activity in membranes of rat olfactory bulb

Summary In membranes of rat olfactory bulb acetylcholine stimulated adenylate cyclase activity in a concentration-dependent manner. The maximal stimulation corresponded to 53% increase of basal enzyme activity and was obtained with 100 M acetylcholine. The concentration of the cholinergic agonist eliciting a half-maximal effect was 0.4 M. The stimulatory effect of acetylcholine was antagonized by 0.1 M atropine but not by 10 M (+)-tubocurarine. Moreover, the addition of micromolar concentrations of GTP was absolutely required for the enzyme stimulation by acetylcholine. The results demonstrate the presence in rat olfactory bulb of muscarinic receptors coupled to stimulation of adenylate cyclase probably via a GTP regulatory protein and provide evidence for a novel signal transduction mechanism of central muscarinic receptors. Send offprint requests to P. Onali at the above address     

Differential coupling of subtypes of the muscarinic receptor to adenylate cyclase and phosphoinositide hydrolysis in the longitudinal muscle of the rat ileum

The binding affinities of selective muscarinic antagonists were compared with their ability to block receptor-mediated inhibition of adenylate cyclase and stimulation of phosphoinositide hydrolysis in the longitudinal muscle of the rat ileum. When measured by competitive inhibition of the binding of N-[3H]methylscopolamine, the binding properties of selective muscarinic antagonists were consistent with a two-site model. Approximately 84% of the binding sites (major sites) had high affinity for the M2-selective antagonists methoctramine and AF-DX 116 (11[[2-[(diethylamino)methyl]-1- piperidinyl]acetyl]-5,11-dihydro-6H-pyrido [2,3-b][1,4]benzodiazepine-6-one), whereas the remainder of the sites (minor sites) had high affinity for hexahydrosiladifenidol and its para-fluoro derivative. There was good agreement between the estimates of the dissociation constants of muscarinic antagonists for the major binding site and those measured by antagonism of the adenylate cyclase response. There was also good agreement between the dissociation constants of muscarinic antagonists for the minor binding site and those measured by antagonism of the phosphoinositide response and the contractile response. Our data indicate that there are at least two types of muscarinic receptors in the longitudinal muscle of the ileum, the more abundant being an M2 receptor, which mediates an inhibition of adenylate cyclase activity, and the less abundant being an M3 receptor, which triggers contraction and phosphoinositide hydrolysis.     

The relationship between muscarinic receptor occupancy and adenylate cyclase inhibition in the rabbit myocardium

The muscarinic receptor-binding properties of a series of muscarinic drugs were compared with their effects on adenylate cyclase in membranes of the rabbit myocardium. When measured by competitive inhibition of [3H]-N-methylscopolamine binding, the competition curves of the various agonists were adequately described by the ternary complex model. This model assumes that the receptor can bind reversibly with a guanine nucleotide binding protein in the membrane and that the affinity of the agonist for the receptor-guanine nucleotide-binding protein complex is higher than that for the free receptor. A satisfactory fit of the ternary complex model to the data could only be achieved assuming that very little receptor is precoupled with the guanine nucleotide-binding protein in the absence of agonist. There was good agreement between the efficacy of each agonist as measured by inhibition of adenylate cyclase and the estimate of the positive cooperativity between the binding of the agonist receptor complex and the guanine nucleotide-binding protein. Guanosine 5'-triphosphate (0.1 mM) had no significant effect on the binding of [3H]N-methylscopolamine but caused an increase in the concentration of the various agonists required for half-maximal receptor occupancy. There was good correlation between efficacy as measured by inhibition of adenylate cyclase and the influence of guanosine 5'-triphosphate on binding properties.     

Beta-adrenergic receptor coupled adenylate cyclase in rat kidney. Differential coupling in glomeruli and tubules

[125I]Iodocyanopindolol ([125I]ICYP) was used to identify and characterize the beta-adrenoceptors in isolated rat kidney glomeruli and cortical tubules. In both the tissues, specific binding of [125I]ICYP was saturable with time and ligand concentration and showed appropriate stereospecificity and agonist rank order potency characteristic of binding to beta-adrenoceptors. Scatchard analysis revealed that the beta-adrenoceptor concentration in the glomeruli (111.1 +/- 8.9 fmol/mg protein) was about three times that in the tubules (40.1 +/- 1.8 fmol/mg protein). The dissociation constants (KD) were similar in the two tissues. Both beta 1- and beta 2-adrenoceptor subtypes were present in the glomeruli and tubules, but the beta 1-subtype was predominant, constituting greater than 80% of the total beta-adrenoceptors in the two tissues. Isoproterenol was twice as potent in competing for [125I]ICYP binding in the tubules as in the glomeruli (P less than 0.05). The slope factor (pseudo-Hill coefficient) for the isoproterenol competition curve was 0.74 +/- 0.04 in the glomeruli and 0.54 +/- 0.02 in the tubules (P less than 0.05). The nonmetabolized guanyl nucleotide analogue Gpp(NH)p caused a steepening and a 3-fold shift of the isoproterenol competition curve in both tissues. Isoproterenol-stimulated cAMP accumulation in the glomeruli was only 31% of the value in the tubules. The concentration of isoproterenol producing half-maximal stimulation (EC50) was 114 +/- 13 nM in the glomeruli and 19 +/- 3 nM in the tubules (P less than 0.05). Gpp(NH)p and forskolin caused a similar increase in cAMP accumulation over basal value in the glomeruli as in the tubules. Overall, our results indicate a decreased efficiency in the interaction between the beta-adrenergic agonist hormone, the receptor and the guanine nucleotide regulatory protein in the glomeruli as compared to the tubules.     

Adenosine regulation of canine cardiac adenylate cyclase

Adenosine has a biphasic, [Mg²⁺]-dependent effect on the catalytic activity of dog heart adenylate cyclase. In the presence of 0.5 mM Mg²⁺, adenosine stimulated cyclic AMP formation, but when the cyclase was activated with 4 mM Mg²⁺ plus 0.5 mM Mn²⁺, adenosine inhibited catalytic activity in a dose-dependent fashion. Adenine, 3'-deoxyadenosine and selected purine-modified adenosine analogs stimulated the enzyme, whereas 2'-deoxyadenosine, 5'-deoxyadenosine and adenine-α-l-lyxofuranoside mimicked the inhibitory effect of adenosine on the Mg²⁺ plus Mn²⁺ stimulated enzyme. These results are consistent with the ‘two receptor’ model of Londos and Wolff [C. Londos and J. Wolff, Proc. natn. Acad. Sci. U.S.A.74, 5482 (1978)], but they raise the possibility of subtle organ and species differences in the chemical determinants of adenosine binding. Adenosine in both intracellular and extracellular compartments may modulate adenylate cyclase activity in the beating heart, in addition to its putative role in the regulation of coronary vascular resistance.     

Evidence for reduced beta-adrenoceptor coupling to adenylate cyclase in femoral arteries from spontaneously hypertensive rats.

1. Arterial relaxant responses via beta-adrenoceptors have been demonstrated to be decreased in spontaneously hypertensive rats (SHR) when compared with normotensive Wistar-Kyoto rats (WKY). To determine which process of the beta-adrenoceptor.adenylate cyclase (AC) system is involved in the decreased responsiveness to beta-adrenoceptor stimulation, relaxant responses to forskolin and dibutyryl cyclic AMP (db cyclic AMP) were compared strips of femoral and mesenteric arteries isolated from 13 week-old SHR and age-matched WKY. 2. The relaxant response to either forskolin, an activator of AC, or db cyclic AMP was not significantly different between the SHR and WKY, when the strips of both arteries from both strains were contracted with K+ to an equivalent magnitude (85% of the maximum). 3. Under the same conditions, however, the relaxant response to noradrenaline (NA) via beta-adrenoceptors was significantly decreased in the SHR arteries. 4. When the strips of femoral arteries were contracted with the same concentration of K+, there was a precontraction of greater magnitude in response to the K+ and a decreased relaxation in response to forskolin, db cyclic AMP or NA in the SHR. On the other hand, when the strips of mesenteric arteries were contracted with the same concentration of K+, the precontraction was smaller in magnitude and there was an increased relaxation in the SHR. 5. The relationship between the relaxant responses and the K+-induced precontractions clearly showed that the ability of forskolin and NA to relax the K+-contracted strips depends on the magnitude of precontraction. Therefore, a difference in magnitude of precontraction between the two groups may produce a meaningless difference. 6. The relaxant responses to forskolin and NA were significantly potentiated by the addition of isobutyl methylxanthine (IBMX), an inhibitor of cyclic AMP phosphodiesterase. Even in the presence of IBMX, relaxant responses to forskolin were the same for the two strains. The difference in the pD2 value for NA-induced relaxation between the two strains was the same in the presence and absence of IBMX. 7. The relaxant effect of either nitroprusside or nifedipine, agents which are independent of this system, was not significantly different between the strips from SHR and WKY. These relaxations were not potentiated by IBMX. 8. From these results, it is concluded that the reduced beta-adrenoceptor coupling to AC is mainly involved in the decreased responsiveness to beta-adrenoceptor stimulation. Furthermore, for an accurate comparison to be made, it is necessary to minimize the influence of variations in the magnitude of precontraction on the relaxant responses.     

Cooperative enhancement of insulinotropic action of GLP-1 by acetylcholine uncovers paradoxical inhibitory effect of beta cell muscarinic receptor activation on adenylate cyclase activity

The cooperative effect of glucagon-like peptide 1 (GLP-1) and acetylcholine (ACh) was evaluated in a beta cell line model (BRIN BD11). GLP-1 (20 nM) and ACh (100 microM) increased insulin secretion by 24-47%, whereas in combination there was a further 89% enhancement of insulin release. Overnight culture with 100 ng/mL pertussis toxin (PTX) or 10nM PMA significantly reduced the combined insulinotropic action (P<0.05 and P<0.001, respectively) and the sole stimulatory effects of GLP-1 (PTX treatment; P<0.01) or ACh (PMA treatment; P<0.05). Under control conditions, ACh (50nM-1mM) concentration-dependently inhibited by up to 40% (P<0.001) the 10-fold (P<0.001) elevation of cyclic 3',5'-adenosine monophosphate (cAMP) induced by 20 nM GLP-1. The paradoxical inhibitory action of ACh was abolished by PTX pre-treatment, suggesting involvement of G(i) and/or G(o) G protein alpha subunit. Effects of selective muscarinic receptor antagonists on the concentration-dependent insulinotropic actions of ACh (50 nM-1 mM) on 20 nM GLP-1 induced insulin secretion revealed inhibition by rho-FHHSiD (M3 antagonist, P<0.05), stimulation with pirenzepine (M1 antagonist, P<0.001) and no significant effects of either methoctramine (M2 antagonist) or MT-3 (M4 antagonist). Antagonism of M2, M3 and M4 muscarinic receptor effects with methoctramine (3-100 nM), rho-FHHSiD (3-30 nM) or MT-3 (10-300 nM) did not significantly affect the inhibitory action of ACh on GLP-1 stimulated cAMP production. In contrast, M1 receptor antagonism with pirenzepine (3-30 0nM) resulted in a concentration-dependent decrease in the inhibitory action of ACh on GLP-1 stimulated cAMP production (P<0.001). These data indicate an important functional cooperation between the cholinergic neurotransmitter ACh and the incretin hormone GLP-1 on insulin secretion mediated through the M3 muscarinic receptor subtype. However, the insulinotropic action of ACh was associated with a paradoxical inhibitory effect on GLP-1 stimulated cAMP production, achieved through a novel PTX- and pirenzepine-sensitive M1 muscarinic receptor activated pathway. An imbalance between these pathways may contribute to dysfunctional insulin secretion.     

Desensitization of epinephrine-initiated platelet aggregation does not alter binding to the alpha 2-adrenergic receptor or receptor coupling to adenylate cyclase

Several investigators have shown that incubating unstirred platelets with epinephrine blunts subsequent aggregation when the platelets are stirred. Using aspirin-treated platelets, we further characterized this desensitization of alpha 2-adrenergic receptor-initiated aggregation. Desensitization occurred with a t1/2 of 3-6 min and was maximal at 20-30 min, at which time the initial rate of aggregation and its maximal extent were about half that of control platelets. When we preincubated platelets with epinephrine, and then added phentolamine to block the alpha 2-receptors, ADP-initiated aggregation occurred normally. Thus, the desensitization of epinephrine-initiated aggregation was not associated with a generalized impairment of aggregation. At concentrations too low to initiate aggregation, epinephrine is known to potentiate aggregation initiated by other agents. Clonidine also acts at alpha 2-receptors to potentiate aggregation initiated by other agents, but it does not initiate aggregation by itself. Preincubating clonidine with platelets for 30 min abolished its potentiating effect on ADP-initiated aggregation. Thus, the ability of alpha 2-receptors to both potentiate and initiate aggregation desensitizes after a brief preincubation with agonist. We performed several types of experiments to investigate the mechanism of this desensitization. Platelet alpha 2-receptors are coupled to an inhibition of adenylate cyclase. We found, however, that alpha 2-mediated inhibition of prostaglandin E1-stimulated cAMP accumulation occurred normally in desensitized platelets. Similarly, epinephrine inhibited basal adenylate cyclase activity normally in membranes prepared from desensitized platelets. In membranes prepared from desensitized platelets, epinephrine competed normally for [3H]rauwolscine binding, and this competition was modulated normally by guanine nucleotides. Thus, the properties of the alpha 2-receptors, as measured in radioligand binding experiments, were unchanged by densensitization. In conclusion, desensitization of alpha 2-adrenergic receptor-mediated aggregation occurs without change in the alpha 2-adrenergic receptors or in their coupling to an inhibition of adenylate cyclase.     

Effects of delta 9-tetrahydrocannabinol on glucagon receptor coupling to adenylate cyclase in rat liver plasma membranes

Delta-Tetrahydrocannabinol (delta 9-THC), the principal psychoactive constituent of Cannabis sativa, was found to increase glucagon activation of liver plasma membrane adenylate cyclase. In the presence of 30 microM delta 9-THC, the EC50 for glucagon was decreased by 60% from 7.6nM to 3.1 nM. 11-OH-delta 9-THC, a psychoactive metabolite of delta 9-THC, also increased glucagon activation of adenylate cyclase while two cannabinoids without marihuana-like psychoactive potency, cannabinol and cannabidiol, did not. At 30 microM, delta 9-THC either slightly decreased or had no effect on the activation of adenylate cyclase by GTP, Gpp(NH)p, fluoride ion, forskolin or ATP alone. Delta 9-THC had no effect on the binding of [125I] glucagon to liver plasma membranes. Arrhenius plots demonstrated that delta 9-THC and 11-OH-delta 9-THC, but not CBD, decreased the activation energy above the break temperature. Therefore, delta 9-THC increased the coupling of the glucagon receptor to adenylate cyclase apparently by removing a constraint on receptor-Ns coupling.     

Phosphatidate and monooleylphosphatidate inhibition of fibroblast adenylate cyclase is mediated by the inhibitory coupling protein, Ni

It has previously been shown that monooleylphosphatidate (MOPA) and phosphatidate inhibit cAMP accumulation in VA13 and WI-38 fibroblasts. In this study we investigated whether this inhibition might be due to a decrease in adenylate cyclase activity. Our results showed that both MOPA and phosphatidate inhibit prostaglandin E1-stimulated adenylate cyclase in WI-38 membranes in a concentration-dependent manner with half-maximal inhibitions at 0.1 and 0.5 microM, respectively, and maximal inhibitions of 35-55%. A 5 microM concentration of structurally similar lipids caused no significant inhibition. The inhibitory effects of MOPA and phosphatidate on adenylate cyclase were GTP dependent, greater at low concentrations of Mg2+, eliminated following treatment of cells with islet-activating protein, nonadditive with carbachol, and noncompetitive with prostaglandin E1. Collectively these data suggested that MOPA and phosphatidate inhibitions of cAMP accumulation were due at least in part to an Ni-mediated inhibition of adenylate cyclase. Furthermore, the inhibitions showed the same characteristics normally associated with hormonal inhibition of this enzyme.     

Prostaglandin receptors in NIH 3T3 cells: coupling of one receptor to adenylate cyclase and of a second receptor to phospholipase C.

In intact NIH 3T3 murine fibroblasts, prostaglandins (PGs) F2 alpha and E2 induce dose-dependent stimulation of inositol monophosphate generation. PGF2 alpha is greater than 50-fold more potent than PGE2 in eliciting this response. In streptolysin O-permeabilized NIH 3T3 cells, PGF2 alpha and PGE2 induced dose-dependent accumulations of inositol bis- and trisphosphates, which were dependent on the presence of the guanine nucleotide guanosine-5'-O-(3-thio)triphosphate (GTP gamma S) (10 microM). Pretreatment of cells for 16 hr with 100 nM PGF2 alpha resulted in a significant reduction of not only subsequent PGF2 alpha- and PGE2-induced but also GTP gamma S-induced stimulation of inositol phosphate formation in permeabilized cells. PGF2 alpha-induced accumulation of inositol phosphates was partially inhibited by pretreatment with pertussis toxin (1 microgram/ml, 4 hr). The inhibition by pertussis toxin was small but was not related to cyclic AMP formation, because forskolin, which activates adenylate cyclase, did not mimic pertussis toxin-induced inhibition. In the same cell line, PGF2 alpha and PGE2 induced a dose-dependent accumulation of cAMP and a dose-dependent potentiation of 0.5 microM forskolin-stimulated cAMP formation. PGF2 alpha and PGE2 were almost equipotent in eliciting both responses. However, PGF2 alpha was less efficacious than PGE2 and, in the presence of forskolin, PGF2 alpha at 10 microM induced an inhibitory effect on cAMP accumulation. Such inhibition may be related to PGF2 alpha-mediated phospholipase C activation and subsequent stimulation of protein kinase C, because the phorbol ester phorbol 12-myristate-13-acetate, which directly activates protein kinase C, also inhibited forskolin- and PGE2-induced cAMP accumulation. Pretreatment with PGF2 alpha for 16 hr did not reduce subsequent stimulation of cAMP accumulation by PGF2 alpha or PGE2. The results indicate that in NIH 3T3 cells two receptors for PGs are present, one that couples to adenylate cyclase, probably through Gs, and does not exhibit selectivity between PGF2 alpha and PGE2 and a second receptor that couples to phospholipase C through a guanine nucleotide-binding protein that is not sensitive to pertussis toxin pretreatment. The latter shows at least 40-fold selectivity towards PGF2 alpha over PGE2. Because long treatment with PGF2 alpha resulted in desensitization of the GTP gamma S-induced response, it is possible that long exposure to PGF2 alpha may down-regulate the guanine nucleotide-binding involved in phospholipase C signal transduction.     

Differences in muscarinic receptor reserve for inhibition of adenylate cyclase and stimulation of phosphoinositide hydrolysis in chick heart cells

Carbachol is 100 times more potent for inhibiting cyclic AMP formation than for stimulating phosphoinositide (PI) hydrolysis in chick heart cells. To determine whether this reflects differences in agonist affinity of the receptor(s) coupled to the two responses, we measured these functional responses following removal of receptor reserve with propylbenzilycholine mustard (PrBCM). Conditions of PrBCM treatment that led to progressive loss of up to 95% of the [3H]-N-methylscopolamine-binding sites decreased the potency but not the maximal capacity of carbachol to inhibit cyclic AMP formation. In contrast, there was a marked decrease in the maximal PI response to carbachol. The KA for carbachol, calculated by measuring functional responses following receptor inactivation, was similar whether the cyclic AMP or the PI response was examined. These KA values (approximately 40 microM) were similar to the KD calculated by examining carbachol competition for [3H]-N-methylscopolamine-binding sites on the intact cell. PrBCM treatment also decreased the maximal effect of oxotremorine on cyclic AMP formation under conditions in which carbachol remained a full agonist for this response. We interpret our data as indicating that: there is much greater receptor reserve in the coupling of muscarinic receptors to adenylate cyclase than to PI hydrolysis; this, rather than differences in receptor affinity underlies the disparate dose-response relationships for the two responses; and differences in the effects of weak agonist on the two responses may also reflect differences in receptor reserve. We suggest that muscarinic receptors with the same affinity for carbachol interact with different efficiency with the transducers (Gi and Gx) that regulate adenylate cyclase and phospholipase C.     

A comparison between muscarinic receptor occupancy,adenylate cyclase inhibition,and inotropic response in human heart

Summary Binding to muscarinic receptors was compared with adenylate cyclase inhibition in membranes derived from human heart auricles, and with inhibition of the contraction of auricular muscle fibers.In the absence of GTP, agonists recognized two classes of receptors both of which bound antagonists with the same affinity. In the presence of GTP, both classes of receptors for agonists were converted into a single low affinity state.Carbachol and oxotremorine inhibited adenylate cyclase activity by 43%, pilocarpine being less efficient (–28%). The 3 agonists exerted similar inhibitory effects on the inotropic response, in 7 out of 9 preparations of electrically- and norepinephrine-stimulated fibers. Dose-effect curves suggested that spareness (or an amplification mechanism) was implicated in the occupancy of low affinity binding sites by carbachol and oxotremorine (but not by the partial agonist pilocarpine) and the resulting inhibition of both adenylate cyclase activity and contractile force.Abbreviations [³H] NMS, [N-methyl-³H] scopolamine methyl chloride - Gpp(NH)p guanosine 5-0-(2-3 imido) triphosphate - EGTA ethylene glycol bis (2-aminoethyl ether)-N,N,N,N-tetraacetic acid     

Halothane attenuation of muscarinic inhibition of adenylate cyclase in rat heart

Halothane stimulated basal adenylate cyclase activity in rat cardiac membranes. Maximal stimulation (54%) was obtained after equilibrating the membranes with 2% halothane. Halothane did not affect the fractional stimulation of adenylate cyclase activity produced by either forskolin or isoproterenol. However, halothane decreased carbamylcholine inhibition of adenylate cyclase activity stimulated by both forskolin and isoproterenol. Maximal depression of carbamylcholine inhibition of stimulated cyclase activity was obtained after equilibration with 1% halothane. These results are consistent with evidence from ligand binding studies and indicate that halothane disrupts muscarinic receptor-G-protein interactions.     

Effects of prostaglandins on rat cardiac adenylate cyclase

A significant stimulatory action of PGE₁ on adenylate cyclase of the particulate membrane preparation of rat heart was observed at 1 × 10^?4 M. PGE₁ caused a positive inotropic effect on isolated spontaneously beating rat atria at 1 × 10^?5 M, and an increase in atrial cyclic AMP level at 1 × 10^?6 M. PGE_2α had no effect on these enzyme preparations. Our results suggest that: (i) The inotropic response of isolated spontaneously beating rat atria to PGE₁ may be connected with an increase in cyclic AMP level; (ii) the effect of prostaglandins on heart in vivo must be indirect, because the concentrations needed to stimulate isolated atria and particulate adenylate cyclase were high, compared with the doses effective in vivo.     

Alpha 2-adrenoceptor coupling to adenylate cyclase in adrenaline insensitive human platelets

Coupling of the alpha 2-adrenoceptor to adenylate cyclase was assessed in platelets from 3 groups of subjects: normal controls and patients with myeloproliferative disorders whose platelets were either sensitive or specifically insensitive to the aggregatory effects of adrenaline. The ability of adrenaline to induce the formation of a complex between the alpha 2-adrenoceptor and the inhibitory guanine nucleotide binding protein was examined in all three groups by assessment of the effect of mono and divalent cations and Gpp(NH)p on the displacement of [3H]yohimbine binding to platelet membranes by adrenaline. Coupling to adenylate cyclase was also assessed in separate studies of the inhibition by adrenaline of PGE1 stimulated cyclic AMP accumulation in whole platelets. Both the formation of the ternary complex and the inhibition by adrenaline of cyclic AMP accumulation was not significantly different in platelets sensitive or insensitive to adrenaline. These results suggest that inhibition of cyclic AMP alone does not explain the mechanisms of adrenaline induced platelet aggregation.