Binding of [3H]forskolin to solubilized preparations of adenylate cyclase

The binding of [3H]forskolin to proteins solubilized from bovine brain membranes was studied by precipitating proteins with polyethylene glycol and separating [3H]forskolin bound to protein from free [3H]forskolin by rapid filtration. The Kd for [3H]forskolin binding to solubilized proteins was 14 nM which was similar to that for [3H]forskolin binding sites in membranes from rat brain and human platelets. Forskolin analogs competed for [3H]forskolin binding sites with the same rank potency in both brain membranes and in proteins solubilized from brain membranes. [3H]forskolin bound to proteins solubilized from membranes with a Bmax of 38 fmol/mg protein which increased to 94 fmol/mg protein when GppNHp was included in the binding assay. In contrast, GppNHp had no effect on [3H]forskolin binding to proteins solubilized from membranes preactivated with GppNHp. Solubilized adenylate cyclase from non-preactivated membranes had a basal activity of 130 pmol/mg/min which was increased 7-fold by GppNHp. In contrast, adenylate cyclase from preactivated membranes had a basal activity of 850 pmol/mg/min which was not stimulated by GppNHp or forskolin. Thus, the number of high affinity binding sites for [3H]forskolin in solubilized preparations correlated with the activation of adenylate cyclase by GppNHp via the guanine nucleotide binding protein (GS).     

Cholesterol modulation of β-adrenergic receptor characteristics

Cholesterol, a major structural component of plasma membranes, has a profound influence on cell surface receptor characteristics and on adenylate cyclase activity. β-Adrenergic receptor number, adenylate cyclase activity, and receptor-cyclase coupling were assessed in rat lung membranes following preincubation with cholesteryl hemisuccinate. β-Adrenergic receptor number increased by 50% without a change in antagonist affinity. However, β-adrenergic receptor affinity for isoproterenol increased 2-fold as a result of an increase in the affinity of the isoproterenol high-affinity binding site. This increase in agonist affinity did not potentiate hormone-stimulated adenylate cyclase activity, which decreased 3-fold following cholesterol incorporation. However, the ratio of isoproterenol to GTP-stimulated activity was unchanged with cholesterol. Stimulation distal to the receptor by GTP, NaF, GppNHp, Mn²⁺ and forskolin also demonstrated 50–80% reduced enzyme activity following cholesterol incorporation. These data suggest that membrane cholesterol incorporation decreases catalytic unit activity without affecting transduction of the hormone signal.     

Cholesterol modulation of beta-adrenergic receptor characteristics

Cholesterol, a major structural component of plasma membranes, has a profound influence on cell surface receptor characteristics and on adenylate cyclase activity. beta-Adrenergic receptor number, adenylate cyclase activity, and receptor-cyclase coupling were assessed in rat lung membranes following preincubation with cholesteryl hemisuccinate. beta-Adrenergic receptor number increased by 50% without a change in antagonist affinity. However, beta-adrenergic receptor affinity for isoproterenol increased 2-fold as a result of an increase in the affinity of the isoproterenol high-affinity binding site. The increase in agonist affinity did not potentiate hormone-stimulated adenylate cyclase activity, which decreased 3-fold following cholesterol incorporation. However, the ratio of isoproterenol to GTP-stimulated activity was unchanged with cholesterol. Stimulation distal to the receptor by GTP, NaF, GppNHp, Mn2+ and forskolin also demonstrated 50-80% reduced enzyme activity following cholesterol incorporation. These data suggest that membrane cholesterol incorporation decreases catalytic unit activity without affecting transduction of the hormone signal.     

Influence of pertussis toxin on the effects of guanine nucleotide on adenylate cyclase in rat striatal membranes

The influence of pertussis toxin on the effects of guanine nucleotide on adenylate cyclase activity were investigated in rat striatal membranes. GTP promoted and inhibited the activity at 1 and 100 microM, respectively. The inhibitory effects of GTP were abolished by pretreatment of the membranes with pertussis toxin. GppNHp (guanyl-5'-y1-beta,gamma-imidodiphosphate) exerted only stimulatory effects and pertussis toxin did not affect the effects of GppNHp. GDP at 10 and 100 microM caused significant inhibition which was completely suppressed by pertussis toxin. It is suggested that guanine nucleotide regulates the affinity of as in stimulatory GTP-binding regulatory protein to either beta gamma or catalytic units of adenylate cyclase in a flip-flop manner. Inhibitory GTP-binding regulatory protein seems to play a regulatory role in inhibiting alpha s activity supplying the beta gamma heterodimer.     

Prostaglandin E-induced heterologous desensitization of hepatic adenylate cyclase. Consequences on the guanyl nucleotide regulatory complex

Prostaglandin E (PGE) receptor density in hepatic plasma membranes can be down-regulated by in vivo exposure to the 16,16-dimethyl analog of PGE2, and this is associated with desensitization of PGE-sensitive adenylate cyclase. These studies examined adenylate cyclase response to other agonists in membranes whose PGE receptor density was 51% decreased and whose maximal PGE-stimulated adenylate cyclase activity was 31% decreased. Down-regulated membranes had a 37% decrease in their maximal response to glucagon, indicating that treatment with the PGE analog had induced both homologous and heterologous desensitization. To determine whether adenylate cyclase had been affected, stimulation with NaF, guanyl 5'-yl imidodiphosphate (GppNHp), and forskolin was examined in both intact and solubilized membranes. Intact membranes had decreased adenylate cyclase responses to all three stimulators (NaF, -41%; GppNHp, -25%; forskolin, -41%) as did solubilized membranes (NaF, -51%; GppNHp, -50%; forskolin, -50%), suggesting alterations in adenylate cyclase rather than indirect membrane effects. Cholera toxin activation and labeling were examined to more directly assess whether the guanine nucleotide (G/F) regulatory component of adenylate cyclase had been affected. Cholera toxin activation was 42% less in down-regulated membranes, and these membranes incorporated less label when the incubation was performed in the presence of [32]NAD. Solubilized G/F subunit activity from down-regulated membranes was less effective in reconstitution of adenylate cyclase activity from cyc- cell membranes than G/F activity from control membranes. These data indicate that in vivo exposure to the PGE analog causes both homologous and heterologous desensitization of adenylate cyclase as well as an apparent quantitative decrease in G/F.     

Adenylate cyclase in membrane fractions of RIN-A2-cells: studies with forskolin, NaF, GppNHp and NEM

In crude membrane fractions of rat pancreatic islets and of RIN-A2-cells, forskolin and NaF stimulated adenylate cyclase activity. Basal and stimulated enzyme activity was approximately 3 to 6 fold higher in membranes of RIN-A2-cells than in membranes of islet cells. In RIN-A2-cells GppNHp and NEM inhibited forskolin-stimulated enzyme activity. The inhibitory effect of GppNHp could be reduced by NEM. It is suggested that the adenylate cyclase system of RIN-A2-cells contains inhibitory and stimulatory N-proteins and that there are critical thiols related to Ni, Ns and/or the catalytic unit. Thus, membrane fractions of RIN-A2-cells may be an appropriate model for studies on the adenylate cyclase system of insulin-producing cells.     

Effect of proteases and protease inhibitors on adenylate cyclase activity in rat cerebral cortical membranes

Mild proteolysis of membrane preparations from rat cerebral cortex with low concentrations of endopeptidases such as trypsin or chymotrypsin caused a 50–400% increase in the basal adenylate cyclase activity. Maximal activation of adenylate cyclase was obtained by including the protease in the adenylate cyclase assay, although an activated preparation could be obtained by pretreatment of the membranes with proteolytic enzymes. The proteolytically activated enzyme showed an increased V, with very little change in the K_m for the substrate, ATP. The proteolytically activated enzyme retained responsiveness to activation by sodium fluoride and 5′-guanylylimidodiphosphate (GppNHp), but was no longer activated by gangliosides or calcium-dependent activator protein. Activation by alcohols and detergent was lost or reduced in magnitude. The activity of adenylate cyclase after protease treatment showed a very marked temperature dependence, with maximal activity expressed in the 30–40 °C range and no activation due to the prior protease treatment expressed at either 10 or 50 °C. Basal adenylate cyclase activity was usually slightly inhibited in the presence of various protease inhibitors. Activation by fluoride, gangliosides, or GppNHp was little affected by protease inhibitors although one inhibitor, N-α-tosyl-l-lysine chloromethyl ketone, caused an inhibition of the ganglioside and GppNHp responses, slightly inhibited the fluoride response, and blocked the norepinephrine response normally seen in the presence of gangliosides or GppNHp. This inhibitor caused a loss of β-adrenergic binding sites for dihydroalprenolol in rat cortical membranes which paralleled the loss of the responsiveness of adenylate cyclase to a GppNHp-norepinephrine combination.     

Modulation byn-alkanols of rat cardiac adenylate cyclase activity

Summary n-Alkanols (from methanol to decanol) have a biphasic effect on rat cardiac adenylate cyclase either basal or stimulated by GTP, GppNHp, NaF or hormones (isoproterenol, glucagon, secretin) in the presence of GTP. At high concentration, all the enzyme activities are inhibited. At low concentration, adenylate cyclase activity is either unchanged or potentiated depending on both the stimulus and the alkanols involved. Potentiation is due to an increase of maximum velocity with no change in the activation constant of the enzyme. Basal activity is unchanged as well as the isoproterenol-and glucagon-stimulated enzyme. The secretin-stimulated enzyme is potentiated. It is the guanyl nucleotide regulatory protein-mediated stimulation of adenylate cyclase which is mainly affected. An attempt was made to relate these effects on adenylate cyclase with physical parameters of the alkanols (partition coefficient). From the data obtained as a function of the alkanol chain-length and of temperature on the adenylate cyclase stimulated by GTP, GppNHp, NaF and permanently activated, it is concluded that the increase in efficacy observed in the presence of alkanol is due to an interaction with the protein moeity particularly with the guanyl nucleotide regulatory protein.     

Immunological distinction between calmodulin-sensitive and calmodulin-insensitive adenylate cyclases

Previous studies using calmodulin-Sepharose affinity chromatography have suggested that bovine brain may contain a mixture of calmodulin-sensitive and -insensitive adenylate cyclase activities (Wescott, K. R., La Porte, D. C., and Storm, D. R. (1979) Proc. Natl. Acad. Sci. U.S.A. 82, 3086-3090). In this study, mice were immunized with a purified preparation of the calmodulin-sensitive adenylate cyclase from bovine brain, and a polyclonal antiserum was obtained which was specific to the calmodulin-sensitive form of the enzyme. The antiserum was not inhibitory and precipitated enzyme activity from a homogeneous preparation of the calmodulin-sensitive adenylate cyclase catalytic subunit. Furthermore, the antiserum did not interact with calmodulin-insensitive adenylate cyclase which was resolved from the calmodulin-sensitive form of the enzyme by calmodulin-Sepharose affinity chromatography. Since the only polypeptide specifically precipitated by the antiserum had an Mr of 135,000, which was identical to the Mr of the catalytic subunit of the enzyme, it is concluded that the antiserum interacted directly and specifically with the catalytic subunit of the calmodulin-sensitive isozyme of adenylate cyclase. Detergent-solubilized membranes from several rat tissues were examined for the presence of calmodulin-sensitive adenylate cyclase using anti-calmodulin-sensitive adenylate cyclase antiserum. Approximately 40-60% of the total adenylate cyclase activity of rat brain and kidney were immunoprecipitated by the antiserum, whereas liver and testes contained no detectable calmodulin-sensitive adenylate cyclase. Approximately 15% of the total adenylate cyclase activity in rat heart and lung was the calmodulin-sensitive form. These data indicate that the calmodulin-sensitive and insensitive adenylate cyclases from bovine brain are immunologically distinct and support the proposal that there may be two or more distinct adenylate cyclase isozymes in brain.     

Calmodulin stimulation of adenylate cyclase in rat brain membranes does not require GTP

Calcium stimulates adenylate cyclase activity in rat cerebral cortical membranes with either ATP or AppNHp as substrate. In contrast, isoproterenol stimulates the cerebral cortical enzyme with ATP as substrate but not with AppNHp as substrate unless exogenous GTP is added. In rat striatal membranes, calcium or dopamine stimulate adenylate cyclase activity with ATP as substrate, but not with AppNHp as substrate. GTP restores the dopamine but not the calcium response. The inhibitory guanine nucleotide GDP-βS antagonizes dopamine and GppNHp stimulation of the brain adenylate cyclases, but not stimulation by calcium of either rat cerebral cortical or striatal enzymes. Results indicate that GTP is not requisite to calcium-calmodulin activation of adenylate cyclases in brain membranes. In addition, calcium-calmodulin cannot activate striatal adenylate cyclases with a nonphosphorylating nucleotide, AppNHp, as substrate.     

Role of calmodulin in the effect of guanyl nucleotides on rat hippocampal adenylate cyclase: involvement of adenosine and opiates

Abstract: The adenylate cyclase activity of rat hippocampal plasma membranes can be stimulated by vaso-active intestinal polypeptide (VIP). Low concentrations (10⁻⁹ to 10⁻⁷M) of 5'-guanylyl-imido diphosphate (GppNHp) evoke a transient inhibition of the enzyme, which is followed by stimulation with increasing GppNHp concentrations (10⁻⁶ to 10⁻⁴M). Inclusion of ethyleneglycol - bis - (β - aminoethylether) - N,N' - tetraacetic acid (EGTA) during incubation abolishes the GppNHp inhibition while preserving GppNHp activation. The stimulation induced by GppNHp is amplified by VIP, but the inhibition is unaffected. Adenosine analogs and opiates are inhibitory ligands in the presence of GTP, and their effects can be reversed by the appropiate receptor antagonists, 3-isobutyl-1-methylxanthine and naloxone. Treatment of membranes with trypsin abolishes the GppNHp-induced inhibition without affecting the GppNHp stimulation. The inhibition induced by GppNHp is also abolished by EGTA treatment followed by washing, which coincides wtih a reduction in the adenosine- and opiate-mediated, GTP-dependent inhibition. The GppNHp inhibition can be restored in EGTA-treated but not in trypsin-treated membranes by addition of calcium-calmodulin but not by Ca²⁺ or Mg²⁺. Calcium-calmodulindepleted membranes lack calcium stimulation as well as GppNHp-induced inhibition, whereas untreated membranes and calcium-calmodulin-depleted membranes plus exogenous calcium-calmodulin showed calcium stimulation and GppNHp inhibition. These results suggest that calmodulin is involved in both Ca²⁺ stimulation and guanine nucleotide-mediated inhibition of rat hippocampal adenylate cyclase.     

Pertussis toxin reverses Gpp(NH)p inhibition of basal and forskolin activated adipocyte adenylate cyclase

Inhibition of basal adenylate cyclase by GTP or guanyl-5'-yl imidodiphosphate was abolished in membranes isolated from rat adipocytes previously incubated with pertussis toxin. Forskolin (0.1 microM) stimulated adenylate cyclase about 4-fold and inhibition of cyclase by GTP or guanyl-5'-yl imidodiphosphate was also abolished by pertussis toxin treatment of rat adipocytes. Forskolin (1 microM) increased adenylate cyclase activity at least ten-fold and the inhibitory effect of GppNHp was reduced but not abolished by pertussis toxin. In rabbit adipocytes, pertussis toxin reversed the inhibition of adenylate cyclase activity by GppNHp to the same extent as that by GTP in the presence of 1 microM forskolin. The present results indicate that pertussis toxin can reverse the inhibition of adipocyte adenylate cyclase by nonhydrolyzable GTP analogs as well as that by GTP.     

Phosphorylated adenosine derivatives as low-affinity adenosine-receptor agonists. Methodological implications for the adenylate cyclase assay.

In cellular systems provided with activatory (Ra-site) receptors for adenosine, such as rat cerebral microvessels and rat liver plasma membranes, the adenosine-receptor antagonist 8-phenyltheophylline (10 microM) significantly decreased adenylate cyclase activity if ATP was the substrate and only if GTP was present. With dATP as substrate, adenylate cyclase activities in both preparations remained unaffected by 8-phenyltheophylline. In rat cerebral-cortical membranes, with inhibitory (Ri-site) receptors for adenosine, 8-phenyltheophylline significantly enhanced adenylate cyclase activity only in the presence of GTP and if ATP was the substrate. In rat cardiac ventricular membranes, which are devoid of any adenylate cyclase-coupled adenosine receptor, the methylxanthine had no GTP-dependent effect, irrespective of the substrate used. All assay systems contained sufficiently high amounts of adenosine deaminase (2.5 units/ml), since no endogenous adenosine, formed from ATP, was found chromatographically. In order to demonstrate a direct influence of phosphorylated adenosine derivatives on adenylate cyclase activity, we investigated AMP in a dATP assay system. AMP was verified chromatographically to remain reasonably stable under the adenylate cyclase assay conditions. In the microvessels, AMP increased enzyme activity in the range 0.03-1.0 mM, an effect competitively antagonized by 8-phenyltheophylline. In the cortical membranes, 0.1 mM-AMP inhibited adenylate cyclase, which was partially reversed by the methylxanthine. The presence of GTP was again necessary for all observations. In the ventricular membranes, AMP had no effect. Since the efficacy of adenosine-receptor agonists and, probably, that of other hormones on adenylate cyclase activity can be more efficiently measured with dATP as the enzyme substrate, this nucleotide seems preferable for adenylate cyclase measurements in systems susceptible to modulation by adenosine.     

Calmodulin-Sensitive and Calmodulin-Insensitive Components of Adenylate Cyclase Activity in Rat Striatum Have Differential Responsiveness to Guanyl Nucleotides

The interaction between the Ca2+-binding protein, calmodulin, and guanyl nucleotides was investigated in a rat striatal particulate fraction. We found that the ability of calmodulin to stimulate adenylate cyclase in the presence of guanyl nucleotides depends upon the type and concentration of the guanyl nucleotide. Adenylate cyclase activity measured in the presence of calmodulin and GTP reflected additivity at every concentration of these reactants. On the contrary, when the activating guanyl nucleotide was the nonhydrolyzable analog of GTP, guanosine-5'-(beta,gamma-imido)triphosphate (GppNHp), calmodulin could further activate adenylate cyclase only at concentrations less than 0.2 microM GppNHp. Kinetic analysis of adenylate cyclase by GppNHp was compatible with a model of two components of adenylate cyclase activity, with over a 100-fold difference in sensitivity for GppNHp. The component with the higher affinity for GppNHp was competitively stimulated by calmodulin. The additivity between calmodulin and GTP in the striatal particulate fraction suggests that they stimulate different components of cyclase activity. The calmodulin-stimulatable component constituted 60% of the total activity. Our two-component model does not delineate, at this point, whether there are two separate catalytic subunits or one catalytic subunit with two GTP-binding proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Domoic acid inhibits adenylate cyclase activity in rat brain membranes

Adenylate cyclase activity measured by the formation of cyclic AMP in rat brain membranes was inhibited by a shellfish toxin, domoic acid (DOM). The inhibition of enzyme was dependent on DOM concentration, but about 50% of enzyme activity was resistant to DOM-induced inhibition. Rat brain supernatant resulting from 105,000×g centrifugation for 60 min, stimulated adenylate cyclase activity in membranes. Domoic acid abolished the supernatant-stimulated adenylate cyclase activity. The brain supernatant contains factors which modulate adenylate cyclase activity in membranes. The stimulatory factors include calcium, calmodulin, and GTP. In view of these findings, we examined the role of calcium and calmodulin in DOM-induced inhibition of adenylate cyclase in brain membranes. Calcium stimulated adenylate cyclase activity in membranes, and further addition of calmodulin potentiated calcium-stimulated enzyme activity in a concentration dependent manner. Calmodulin also stimulated adenylate cyclase activity, but further addition of calcium did not potentiate calmodulin-stimulated enzyme activity. These results show that the rat brain membranes contain endogenous calcium and calmodulin which stimulate adenylate cyclase activity. However, calmodulin appears to be present in membranes in sub-optimal concentration for adenylate cyclase activation, whereas calcium is present at saturating concentration. Adenylate cyclase activity diminished as DOM concentration was increased, reaching a nadir at about 1 mM. Addition of calcium restored DOM-inhibited adenylate cyclase activity to the control level. Similarly, EGTA also inhibited adenylate cyclase activity in brain membranes in a concentration dependent manner, and addition of calcium restored EGTA-inhibited enzyme activity to above control level. The fact that EGTA is a specific chelator of calcium, and that DOM mimicked adenylate cyclase inhibition by EGTA, indicate that calcium mediates DOM-induced inhibition of adenylate cyclase activity in brain membranes. While DOM completely abolished the supernatant-, and Gpp (NH)p-stimulated adenylate cyclase activity, it partly blocked calmodulin-, and forskolin-stimulated adenylate cyclase activity in brain membranes. These results indicate that DOM may interact with guanine nucleotide-binding (G) protein and/or the catalytic subunit of adenylate cyclase to produce inhibition of enzyme in rat brain membranes.     

Calcitonin-sensitive adenylate cyclase in rat renal tubular membranes.

1. Renal tubular membranes from rat kidneys were prepared, and adenylate cyclase activity was measured under basal conditions, after stimulation by NaF or salmon calcitonin. Apparent Km value of the enzyme for hormone-linked receptor was close to 1 x 10(-8) M. 2. The system was sensitive to temperature and pH. pH was found to act both on affinity for salmon calcitonin-linked receptor and maximum stimulation, suggesting an effect of pH on hormone-receptor binding and on a subsequent step. 3. KCl was without effect areas whereas CoCl and CaCl2 above 100 muM and MnCl2 above 1 muM inhibited F- -and salmon calcitonin-sensitive adenylate cyclase activities. The Ca2+ inhibition of the response reflected a fall in maximum stimulation and not a loss of affinity of salmon calcitonin-linked receptor for the enzyme. 4. The measurement of salmon calcitonin-sensitive adenylate cyclase activity as a function of ATP concentration showed that the hormone increases the maximum velocity of the adenylate cyclase. GTP, ITP and XTP at 200 muM did not modify basal, salmon calcitonin- and parathyroid hormone-sensitive adenylate cyclase activities. 5. Basal, salmon calcitonin- and F- -sensitive adenylate cyclase activities decreased at Mg2+ concentrations below 10 mM. High concentrations of Mg2+ (100 mM) led to an inhibition of the F- -stimulated enzyme. 6. Salmon calcitonin-linked receptor had a greater affinity for adenylate cyclase than human or porcine calcitonin-linked receptors. There was no additive effect of these three calcitonin peptides whereas parathyroid hormone added to salmon calcitonin increased adenylate cyclase activity, thus showing that both hormones bound to different membrane receptors. Human calcitonin fragments had no effect on adenylate cyclase activity. 7. Salmon calcitonin-stimulated adenylate cyclase activity decreased with the preincubation time. This was due to progressive degradation of the hormone and not to the rate of binding to membrane receptors.     

Relationship between the inhibition of adenylate cyclase by pentobarbital and the functional coupling of Ns and the catalytic unit

The effect of barbiturate on adenylate cyclase system was examined in rat brain. Pentobarbital inhibited the enzyme activities in both synaptic membrane and solubilized catalytic unit of the system in dose and time-dependent manners. The inhibitory effect of pentobarbital was more potent on the activation of the system by NaF-AlCl3 than on the basal activity. The inhibitory effect, however, was less in the synaptic membrane in which the catalytic unit was prestimulated through coupling with Ns by the treatment with NaF-AlCl3. Similar results were obtained with the solubilized preparation which was pretreated with guanylyl-5'-imidodiphosphate before solubilization. On the other hand, the effect of pentobarbital was not modified by the treatment of the synaptic membrane with pertussis toxin. These findings indicate that barbiturates suppress primarily the activation of the catalytic unit through the coupling with guanine nucleotide-binding stimulatory protein (Ns) without affecting the inhibitory protein (Ni).     

Adenosine receptor permanently coupled to turkey erythrocyte adenylate cyclase.

The mode of coupling of the adenosine receptor to adenylate cyclase in turkey erythrocyte membranes was probed by two independent approaches. The progressive inactivation of the adenosine receptor by an adenosine receptor affinity label resulted in the proportional reduction in the adenosine plus GppNHp dependent specific activity. In contrast, the intrinsic rate constant (k3), characterizing the process of adenylate cyclase activation by the adenosine-adenosine receptor complex, is independent of the extent of receptor inactivation. This behavior favors the precoupled mechanism, A + R.E: formula: (see text), where the receptor R and the enzyme E are permanently coupled to each other and the adenosine A binds to the receptor and induces the first-order process of cyclase activation to its active form ARE'. The finding that adenosine receptor is permanently coupled to the cyclase catalytic unit is corroborated by the observation that the progressive increase in membrane fluidity has no effect on the rate constant (k3) of adenylate cyclase activation by the adenosine-adenosine receptor complex and that the dose-response curve for adenosine is noncooperative.     

Pertussis Toxin Attenuates D2 Inhibition and Enhances D1 Stimulation of Adenylate Cyclase by Dopamine in Rat Striatum

The response of adenylate cyclase to GTP and to dopamine (DA) was investigated in synaptic plasma membranes isolated from rat striatum injected with pertussis toxin, which inactivates the inhibitory guanine nucleotide-binding regulatory protein (Ni) of adenylate cyclase. Pertussis toxin treatment reverted the inhibitory effects on the enzyme activity elicited by micromolar concentrations of GTP and reduced by 50% the DA inhibition of cyclase activity via D2 receptors. The toxin treatment enhanced the net stimulation of enzyme activity by DA in the presence of micromolar concentrations of GTP. However, the stimulatory effect of the selective D1 receptor agonist SKF 38393 was not significantly affected. The data indicate that Ni mediates D2 inhibition of striatal adenylate cyclase and participates in the modulation of D1 stimulation of the enzyme activity by DA.     

Muscarinic cholinergic receptor modulation of beta-adrenergic receptor affinity for catecholamines.

The effects of the muscarinic cholinergic agonist methacholine on affinity of beta-adrenergic receptors for isoproterenol and on isoproterenol-induced stimulation of adenylate cyclase activity were assessed in canine myocardium. GTP and guanyl-5'-yl imidoiphosphate both decreased the affinity of beta-adrenergic receptors for isoproterenol without altering the affinity of these receptors for propranolol. Methacholine (10 nM to 10 micronM) antagonized the guanine nucleotide-induced reduction in beta-adrenergic receptor affinity for isoproterenol. This effect of methacholine was reversed by atropine. The choline ester had no effect on the affinity of beta-adrenergic receptors for isoproterenol in the absence of guanine nucleotides. Likewise, methacholine had no effect on the affinity of beta-adrenergic receptors for propranolol, either in the presence or absence of guanine nucleotides. Methacholine also attenuated GTP-induced activation of adenylate cyclase or isoproterenol-induced activation of the enzyme in the presence of GTP. The effects of methacholine on myocardial adenylate cyclase activity were apparent only in the presence of GTP. These effects were also reversed by atropine. The choline ester had no effect on adenylate cyclase activity in the presence of guanyl-5'-yl imidodiphosphate or NaF. The results of the present study suggest that muscarinic cholinergic agonists can regulate both beta-adrenergic receptors and adenylate cyclase by modulating the effects of GTP.