Acidic phospholipid species inhibit adenylate cyclase activity in rat liver plasma membranes.

Incubation of rat liver plasma membranes with liposomes of dioleoyl phosphatidic acid (dioleoyl-PA) led to an inhibition of adenylate cyclase activity which was more pronounced when fluoride-stimulated activity was followed than when glucagon-stimulated activity was followed. If Mn2+ (5 mM) replaced low (5 mM) [Mg2+] in adenylate cyclase assays, or if high (20 mM) [Mg2+] were employed, then the perceived inhibitory effect of phosphatidic acid was markedly reduced when the fluoride-stimulated activity was followed but was enhanced for the glucagon-stimulated activity. The inhibition of adenylate cyclase activity observed correlated with the association of dioleoyl-PA with the plasma membranes. Adenylate cyclase activity in dioleoyl-PA-treated membranes, however, responded differently to changes in [Mg2+] than did the enzyme in native liver plasma membranes. Benzyl alcohol, which increases membrane fluidity, had similar stimulatory effects on the fluoride- and glucagon-stimulated adenylate cyclase activities in both native and dioleoyl-PA-treated membranes. Incubation of the plasma membranes with phosphatidylserine also led to similar inhibitory effects on adenylate cyclase and responses to Mg2+. Arrhenius plots of both glucagon- and fluoride-stimulated adenylate cyclase activity were different in dioleoyl-PA-treated plasma membranes, compared with native membranes, with a new 'break' occurring at around 16 degrees C, indicating that dioleoyl-PA had become incorporated into the bilayer. E.s.r. analysis of dioleoyl-PA-treated plasma membranes with a nitroxide-labelled fatty acid spin probe identified a new lipid phase separation occurring at around 16 degrees C with also a lipid phase separation occurring at around 28 degrees C as in native liver plasma membranes. It is suggested that acidic phospholipids inhibit adenylate cyclase by virtue of a direct headgroup specific interaction and that this perturbation may be centred at the level of regulation of this enzyme by the stimulatory guanine nucleotide regulatory protein NS.     

Elevated membrane cholesterol concentrations inhibit glucagon-stimulated adenylate cyclase.

A method was devised which increases the cholesterol concentration of rat liver plasma membranes by exchange from cholesterol-rich liposomes at low temperature (4 degrees C). When the cholesterol concentration of liver plasma membranes is increased, there is an increase in lipid order as detected by a decrease in mobility of an incorporated fatty acid spin probe. This is accompanied by an inhibition of adenylate cyclase activity. The various ligand-stimulated adenylate cyclase activities exhibit different sensitivities to inhibition by cholesterol, with inhibition of glucagon-stimulated greater than fluoride-stimulated greater than basal activity. The bilayer-fluidizing agent benzyl alcohol is able to reverse the inhibitory effect of cholesterol on adenylate cyclase activity in full. The thermostability of fluoride-stimulated cyclase is increased in the cholesterol-rich membranes. Elevated cholesterol concentrations abolish the lipid-phase separation occurring at 28 degrees C in native membranes as detected by an incorporated fatty acid spin probe. This causes Arrhenius plots of glucagon-stimulated adenylate cyclase activity to become linear, rather than exhibiting a break at 28 degrees C. It is suggested that the cholesterol contents of both halves of the bilayer are increased by the method used and that inhibition of adenylate cyclase ensues, owing to the increase in lipid order and promotion of protein-protein and specific cholesterol-phospholipid interactions.     

The activity of glucagon-stimulated adenylate cyclase from rat liver plasma membranes is modulated by the fluidity of its lipid environment.

1. The local anaesthetic benzyl alcohol progressively activated glucagon-stimulated adenylate cyclase activity up to a maximum at 50 mM-benzyl alcohol. Further increases in benzyl alcohol concentration inhibited the activity. The fluoride-stimulated adenylate cyclase activity was similarly affected except for an inhibition of activity occurring at low benzyl alcohol concentrations (approx. 10 mM. 2. The fluoride-stimulated adenylate cyclase activity of a solubilized enzyme preparation was unaffected by any of the benzyl alcohol concentrations tested. 3. Increases in 3-phenylpropan-1-ol and 5-phenylpentan-1-ol concentrations progressively activated both the fluoride- and glucagon-stimulated adenylate cyclase activities up to a maximum, above which further increases in alcohol concentration inhibited the activities. 4. The 'break' points in Arrhenius plots of glucagon-stimulated adenylate cyclase activity in native plasma membranes, and in plasma membranes fused with synthetic dimyristoyl phosphatidylcholine so as to constitute 60% of the total lipid pool, were decreased by approx. 6 degrees C by addition of 40 mM-benzyl alcohol. This was accompanied by a fall in the associated activation energies. 6. Arrhenius plots of fluoride-stimulated adenylate cyclase activity in the presence and absence of 40 mM-benzyl alcohol were linear, although addition of benzyl alcohol caused a dramatic decrease in the associated activation energy of the reaction. 7. 5'-Nucleotidase activity was stimulated by benzyl alcohol, and the 'break' point in the Arrhenius plot of its activity was decreased by about 6 degrees C by addition of 40 mM-benzyl alcohol to the assay. 8. It is suggested that benzyl alcohol effects a fluidization of the bilayer, which is clearly demonstrated by its ability to lower the temperature of a lipid phase separation occurring at 28 degrees C in the outer half of the bilayer to around 22 degrees C. The increase in bilayer fluidity relieves a physical constraint on the membrane-bound adenylate cyclase, activating the enzyme. 9. The various inhibition phenomena are discussed in detail, together with the suggestion that the interaction between the uncoupled catalytic unit of adenylate cyclase and the lipids of the bilayer is altered on its physical coupling to the glucagon receptor.     

The selective effects of charged local anaesthetics on the glucagon- and fluoride-stimulated adenylate cyclase activity of rat-liver plasma membranes

The cationic local anaesthetics carbocaine and unpercaine were found to increase the fluoride-stimulated adenylate cyclase up to a maximum level; above this maximum level further increases in drug concentration inhibited the enzyme. At concentrations where this activity was stimulated, a fatty acid spin label detected an increase in bilayer fluidity, which, it is suggested, is responsible for the activation of the enzyme. A solubilized enzyme was unaffected by the drugs, a finding consistent with this proposal. These cationic drugs began to inhibit the glucagon-stimulated activity at concentrations where they activated the fluoride-stimulated activity. It is suggested that this is due to their effect on the coupling interaction between the receptor and catalytic unit. The anionic drugs, phenobarbital, pentobarbital, and salicylic acid, all inhibited the fluoride-stimulated enzyme. This may be due in part to a direct effect on the protein and in part to the interaction of the drugs with the bilayer. The drugs had small inhibitory effects on the lubrol-solubilized enzyme. The glucagon-stimulated enzyme was initially inhibited by the anionic drugs at low concentrations, then activated, and finally inhibited with increasing drug concentration. The reasons for such changes are complex, but there was no evidence from electron spin resonance studies to suggest that the elevations in activity were due to increases in bilayer fluidity.     

Glucagon-stimulated adenylate cyclase detects a selective perturbation of the inner half of the liver plasma-membrane bilayer achieved by the local anaesthetic prilocaine.

Prilocaine can increase the fluidity of rat liver plasma membranes, as indicated by a fatty acid spin-probe. This led to the activation of the membrane-bound fluoride-stimulated adenylate cyclase activity, but not the Lubrol-solubilized activity, suggesting that increased lipid fluidity can activate the enzyme. With increasing prilocaine concentrations above 10 mM, the membrane-bound fluoride-stimulated activity was progressively inhibited, even though bilayer fluidity continued to increase and the activity of the solubilized enzyme remained unaffected. Glucagon-stimulated adenylate cyclase was progressively inhibited by increasing prilocaine concentrations. Prilocaine (10 mM) had no effect on the lipid phase separation occurring at 28 degrees C and attributed to those lipids in the external half of the bilayer, as indicated by Arrhenius plots of both glucagon-stimulated adenylate cyclase activity and the order parameter of a fatty acid spin-probe. However, 10 mM-prilocaine induced a lipid phase separation at around 11 degrees C that was attributed to the lipids of the internal (cytosol-facing) half of the bilayer. It is suggested that prilocaine (10 mM) can selectively perturb the inner half of the bilayer of rat liver plasma membranes owing to its preferential interaction with the acidic phospholipids residing there.     

Quinidine and melittin both decrease the fluidity of liver plasma membranes and both inhibit hormone-stimulated adenylate cyclase activity

Increasing concentrations of either quinidine or melittin gave a dose-dependent inhibition of both the glucagon- and fluoride-stimulated activities of adenylate cyclase in the liver plasma membranes. At similar concentrations these agents increased the order of liver plasma membranes as detected by a fatty acid ESR probe, doxyl stearic acid. This increase in bilayer order (decrease in 'fluidity') is suggested to explain the inhibitory action of quinidine on adenylate cyclase activity but only in part contributes to the inhibitory action of melittin on adenylate cyclase. Arrhenius plots of fluoride-stimulated activity became non-linear in the presence of either quinidine or melittin, with a single well-defined break occurring at around 12 degrees C in each instance. Arrhenius plots of the glucagon-stimulated activity also exhibited such a novel break at around 12 degrees C when either quinidine or melittin were present as well as exhibiting a break at around 28 degrees C, as was seen in the absence of these ligands. The fatty acid spin probe inserted into liver plasma membranes detected a novel lipid phase separation occurring at around 12 degrees C when either quinidine or melittin was present and showed that the lipid phase separation occurring at around 28 degrees C in native membranes was apparently unaffected by these ligands.     

Activation by GTP of liver adenylate cyclase in the presence of high concentrations of ATP

Effect of GTP on adenylate cyclase of liver plasma membrane was examined using ATP which was extensively purified by DEAE-cellulose column chromatography. In the incubation containing 2mM purified ATP as substrate, GTP enhanced basal and glucagon- or fluoride-stimulated activities. When the unpurified ATP at 2mM was used, all the activities were high and the stimulatory effect of GTP was not detected. The substance(s) which was recovered from a small but significant peak on DEAE-cellulose column was equivalent to 10–100μM GTP in stimulating adenylate cyclase. These results indicate that, if highly purified ATP is used as substrate, GTP can enhance adenylate cyclase activity in the presence of millimolar concentration of ATP and that GTP enhances not only the glucagon-stimulated adenylate cyclase but also the basal as well as fluoride-stimulated adenylate cyclase activities.     

Evidence that the mitochondrial activator of phosphorylated branched-chain 2-oxoacid dehydrogenase complex is the dissociated E1 component of the complex

The ability of glucagon (10 nM) to increase hepatocyte intracellular cyclic AMP concentrations was reduced markedly by the tumour-promoting phorbol ester TPA (12-O-tetradecanoyl phorbol-13-acetate). The half-maximal inhibitory effect occurred at 0.14 ng/ml TPA. This action occurred in the presence of the cyclic AMP phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) indicating that TPA inhibited glucagon-stimulated adenylate cyclase activity. TPA did not affect either the binding of glucagon to its receptor or ATP concentrations within the cell. TPA did inhibit the increase in intracellular cyclic AMP initiated by the action of cholera toxin (1 microgram/ml) under conditions where phosphodiesterase activity was blocked. TPA did not inhibit glucagon-stimulated adenylate cyclase activity in a broken plasma membrane preparation unless Ca2+, phosphatidylserine and ATP were also present. It is suggested that TPA exerts its inhibitory effect on adenylate cyclase through the action of protein kinase C. This action is presumed to be exerted at the point of regulation of adenylate cyclase by guanine nucleotides.     

Studies of binding of parathyroid hormone to a detergent-dispersed preparation from bovine kidney cortex plasma membranes.

Bovine kidney plasma membranes containing parathyroid hormone-sensitive adenylate cyclase activity were dispersed with 1% Triton X-100 and centrifuged at 150,000 X g for 2 h. Approximately 40% of the total membrane protein was extracted by this procedure. The extraction greatly reduces the fluoride-stimulated and the parathyroid hormone-sensitive adenylate cyclase activity of the membranes and yields a supernatnat which binds biologically active, tritiated parathyroid hormone. Hormone binding is stable for up to 15 h and has a linear dependence on protein concentration in the extract. Binding of the labeled hormone at concentrations of 5 to 10 nM is inhibited by preincubation with unlabeled min, and displays a dependence on temperature, time, and pH. Binding specificity is maximal at physiological pH, being inhibited by only the native hormone or its synthetic 1-34 NH2-terminal, biologically active fragment. Binding increases dramatically at pH 6.0, but is nonspecific in character. Half-maximal inhibition of the binding was achieved at 3.2 X 10(-7) M concentrations of the native hormone and 5.0 X 10(-7) M concentrations of the synthetic 1-34 NH2-terminal fragment. Calcium does not inhibit either total or specific binding. Inhibition, kinetic, and pH dependence data suggest that the extracted component(s) represent the parathyroid hormone binding protein(s) formerly identified in particulate membrane preparations.     

The rapid desensitization of glucagon-stimulated adenylate cyclase is a cyclic AMP-independent process that can be mimicked by hormones which stimulate inositol phospholipid metabolism.

Treatment of intact hepatocytes with glucagon, TH-glucagon [( 1-N-alpha-trinitrophenylhistidine, 12-homoarginine]glucagon), angiotensin or vasopressin led to a rapid time- and dose-dependent loss of the glucagon-stimulated response of the adenylate cyclase activity seen in membrane fractions isolated from these cells. Intracellular cyclic AMP concentrations were only elevated with glucagon. All ligands were capable of causing both desensitization/loss of glucagon-stimulated adenylate cyclase activity and stimulation of inositol phospholipid metabolism in the intact hepatocytes. Maximally effective doses of angiotensin precluded any further inhibition/desensitizing action when either glucagon or TH-glucagon was subsequently added to these intact cells, as has been shown previously for the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) [Heyworth, Wilson, Gawler & Houslay (1985) FEBS Lett. 187, 196-200]. Treatment of intact hepatocytes with these various ligands caused a selective loss of the glucagon-stimulated adenylate cyclase activity in a washed membrane fraction and did not alter the basal, GTP-, NaF- and forskolin-stimulated responses. Angiotensin failed to inhibit glucagon-stimulated adenylate cyclase activity when added directly to a washed membrane fraction from control cells. Glucagon GR2 receptor-stimulated adenylate cyclase is suggested to undergo desensitization/uncoupling through a cyclic AMP-independent process, which involves the stimulation of inositol phospholipid metabolism by glucagon acting through GR1 receptors. This action can be mimicked by other hormones which act on the liver to stimulate inositol phospholipid metabolism. As the phorbol ester TPA also mimics this process, it is proposed that protein kinase C activation plays a pivotal role in the molecular mechanism of desensitization of glucagon-stimulated adenylate cyclase. The site of the lesion in desensitization is shown to be at the level of coupling between the glucagon receptor and the stimulatory guanine nucleotide regulatory protein Gs, and it is suggested that one or both of these components may provide a target for phosphorylation by protein kinase C.     

Protease inhibitors block hormonal activation of adenylate cyclase

To investigate the mechanism of serine protease stimulation of rat ovarian adenylate cyclase, a variety of synthetic protease inhibitors were used. These inhibitors blocked trypsin, chymotrypsin and hCG stimulation of adenylate cyclase in nearly the same manner. The inhibition of hormone stimulated adnylate cyclase could not be explained by a loss of [¹²⁵I]hCG binding. Cholera toxin and epinephrine stimulation of adenylate cyclase were similarly inhibited, whereas basal and fluoride-stimulated activities were only affected by higher doses of the inhibitors. The results suggest that adenylate cyclase in the ovary may be regulated by membrane protease activity.     

Role of cellular redox state and glutathione in adenylate cyclase activity in rat adipocytes.

Adenylate cyclase in rat adipocyte membranes was inactivated as a result of treatment with sulfhydryl oxidants or with p-chloromercuribenzoate as well as by S-alkylating agents. The inhibition of the basal and isoproterenol- or glucagon-stimulated enzyme activity by the oxidants or the mercurial could be reversed by adding thiols to the isolated membranes. The activity of the enzyme paralleled the cellular glutathione (GSH) content. Lowering of intracellular glutathione by incubating the cells with specific reactants resulted in the inhibition of both basal and hormone-stimulated adenylate cyclase activity in the isolated membranes. Activity could be partly restored by supplying glucose to the incubation medium of intact cells. The fluoride-stimulated adenylate cyclase was also inhibited by the oxidants or the sulfhydryl inhibitors. The results suggest that adenylate cyclase may be partly regulated by oxidation-reduction. Thus, a direct relationship between both basal and hormone-stimulated adenylate cyclase activity and the cellular redox potential, determined by the cellular level of reduced glutathione, may be ascribed to the protection of the catalytic -SH groups of the enzyme from oxidative or peroxidative reactions and maintenance of the redox optimum for the reaction.     

Effects of 4-hydroxynonenal on adenylate cyclase and 5'-nucleotidase activities in rat liver plasma membranes

Adenylate cyclase and 5'-nucleotidase activities in rat liver plasma membranes were assayed in vitro in the presence of 4-hydroxy-2,3-trans-nonenal (HNE), a major end-product of microsomal lipid peroxidation. Both basal and glucagon-stimulated adenylate cyclase were inhibited in a dose-dependent manner, even at micromolar HNE concentrations, whereas fluoride-stimulated activity increased. A biphasic, dose- and time-dependent effect was noted when the basal activity was monitored at increasing doses. 5'-Nucleotidase activity was also decreased by HNE, but only at millimolar concentrations. These findings are related to the view that aldehydes, especially HNE, may act as diffusible cytotoxic compounds when lipid peroxidative derangement of membrane lipids is provoked by toxic conditions.     

Treatment of streptozotocin-diabetic rats with metformin restores the ability of insulin to inhibit adenylate cyclase activity and demonstrates that insulin does not exert this action through the inhibitory guanine nucleotide regulatory protein Gi.

Insulin caused the inhibition of glucagon-stimulated adenylate cyclase activity in liver plasma membranes, but failed to inhibit this activity in liver membranes from rats made diabetic by treatment with either alloxan or streptozotocin. Treatment of streptozotocin-diabetic rats with insulin, to normalize their blood glucose concentrations, restored this action of insulin. Rats treated with the biguanide drug metformin exhibited a decreased content of the inhibitory guanine nucleotide regulatory protein Gi in liver plasma membranes assessed both structurally, by using a specific polyclonal antibody (AS7), and functionally. Treatment of normal rats with metformin did not alter insulin's ability to inhibit adenylate cyclase in liver plasma membranes; however, metformin treatment of streptozotocin-diabetic rats completely restored this inhibitory action of insulin. Liver plasma membranes from streptozotocin-diabetic animals which either had or had not been treated with metformin had contents of Gi which were less than 10% of those seen in control animals. We conclude that: (i) insulin does not inhibit adenylate cyclase activity through the inhibitory guanine nucleotide regulatory protein Gi; (ii) streptozotocin- and alloxan-induced diabetes elicit a selective insulin-resistant state; and (iii) metformin can exert a post-receptor effect, at the level of the liver plasma membrane, which restores the ability of insulin to inhibit adenylate cyclase.     

Inhibition of a parathyroid hormone-stimulated renal adenylate cyclase by cyclic AMP.

Plasma membranes were isolated from bovine renal cortex. This particulate, adenylate cyclase-containing fraction was stimulated to produce cyclic AMP by parathyroid hormone and fluoride. When the time-course of adenylate cyclase activity was investigated, it was found that while PTH-stimulated cyclic AMP production comes to a halt in about 15 minutes after the initiation of the reaction, fluoride-stimulated activity continues unabated for at least an hour. Experiments to determine the cause of this showed that the cyclase enzyme is not degraded under our experimental conditions, but is inhibited by a soluble, unbound product of the reaction which requires ATP for its synthesis. In our experiments degradation of parathyroid hormone was relatively slow and could not account for the rapid inhibition of PTH-stimulated cyclase activity. Of the various agents tested, cyclic AMP was found capable of inhibiting PTH-stimulated cyclic AMP production by our purified membrane preparation. Half-maximal inhibition was observed at around 10(-6) M concentrations of the nucleotide. Pyrophosphate, adenosine, 5'-AMP and ADP had no effects. The significance of these results in relation to the regulation of adenylate cyclase activity is discussed.     

Perturbations of liver plasma membranes induced by Ca2+ are detected using a fatty acid spin label and adenylate cyclase as membrane probes

Ca2+ decreased the lipid fluidity of rat liver plasma membranes labeled with 5-nitroxide stearate, I(12,3), as indicated by the order parameter (S). These effects form a reversible, saturable process with an association constant of 1 x 10(3) M-1. Arrhenius-type plots of S indicated that the lipid phase separation, present in the external leaflet of native membranes between 28 and 19 degrees C, is perturbed by mM Ca2+ such that the high temperature onset is elevated to 32-34 degrees C. Fluoride-stimulated adenylate cyclase was similarly inhibited by Ca2+ (ID50 = 1 mM) for the enzyme in membrane-bound or solubilized states. The glucagon-stimulated activity was more sensitive to Ca2+ inhibition with an ID50 of 0.2 mM. These inhibitory effects are due neither to perturbations of glucagon binding to its receptor nor to fluidity changes, but are instead attributed to direct Ca2+-enzyme interactions. Such binding desensitizes the enzyme to fluidity alterations induced by temperature elevation or benzyl alcohol addition. With Ca2+, Arrhenius plots of glucagon-stimulated activity indicated breaks at 32 and 16 degrees C, whereas those of fluoride-stimulated activity showed one break at 17 degrees C. Without Ca2+, Arrhenius plots exhibited one break at 28 degrees C for glucagon-stimulated activity, whereas fluoride-stimulated plots were linear. We propose that Ca2+ achieves these effects through asymmetric perturbations of the membrane lipid structure.     

The hepatic angiotensin II receptor. II. Effect of guanine nucleotides and interaction with cyclic AMP production

Guanine nucleotides were observed to modify the binding of 125I-angiotensin II to rat hepatic plasma membrane receptors. GTP and its nonhydrolyzable analogues greatly increased the dissociation rate of bound 125I-angiotensin II and altered hormone binding to the receptor under equilibrium conditions. In the absence of GTP, 125I-angiotensin II labeled both high affinity sites (Kd1 = 0.46 nM, N1 = 650 fmol/mg) and low affinity sites (Kd2 = 4.1 nM, N2 = 1740 fmol/mg). In the presence of guanine nucleotides, the affinities of the two sites were unchanged, but the number of high affinity sites decreased markedly to 52 fmol/mg. In analogous experiments using the angiotensin II antagonist, 125I-sarcosine1,Ala8-angiotensin II (125I-saralasin), guanine nucleotides minimally affected the interaction of 125I-saralasin with its receptor, increasing the dissociation rate 1.9-fold and the Kd 1.4-fold. The guanine nucleotide inhibition of agonist binding required a cation such as Na+ or Mg2+, with a maximal effect occurring at about 1 mM Mg2+. In liver plasma membranes prepared in EDTA, angiotensin II inhibited basal and glucagon-stimulated adenylate cyclase activities by 30% and 10%, respectively. Angiotensin II also caused a 40% inhibition of glucagon-stimulated cyclic AMP accumulation in intact hepatocytes, with a half-maximal effect occurring at 1 nM. The inhibition by angiotensin II of adenylate cyclase in membranes and of cAMP levels in intact cells could be reversed by the antagonist sarcosine1,Ile8-angiotensin II. Vasopressin caused a smaller 26% inhibition of glucagon-stimulated cyclic AMP accumulation. The ability of angiotensin II to inhibit cyclic AMP synthesis may provide an explanation for the observed effects of guanine nucleotides on 125I-angiotensin II binding to plasma membranes.     

Inhibition of gonadotropin-stimulated adenylate cyclase by dansyl-arginyl-(4'-ethyl)piperidine amide (DAPA)

Protease inhibitors are known to suppress basal, fluoride-, and hormone-stimulated adenylate cyclase activities. The thrombin inhibitor, dansyl-arginyl-(4'-ethyl)piperidine amide (DAPA), also specifically inhibits the binding of gonadotropins to their receptors. Our studies were undertaken to find a concentration of DAPA that would specifically inhibit gonadotropin-stimulated adenylate cyclase without significantly altering basal, fluoride-, isoproterenol-, or prostaglandin E1-stimulated cyclase. Basal adenylate cyclase activity was not inhibited by DAPA in either human chorionic gonadotropin (hCG)- or follicle-stimulating hormone (FSH)-responsive rat ovarian plasma membranes. Human chorionic gonadotropin-stimulated cyclase was completely inhibited by DAPA at a concentration of 2.96 mM; the ID50 was 1.32 mM. Follicle-stimulating hormone-stimulated cyclase was completely inhibited by a DAPA concentration of 4.44 mM, and the ID50 was 1.75 mM. Dansyl-arginyl-(4'-ethyl)piperidine amide (2.96 mM) inhibited isoproterenol-, prostaglandin E1-, and fluoride-stimulated cyclase in hCG-responsive membranes by 11%, 28%, and 35%, respectively. Dansyl-arginyl-(4'-ethyl)piperidine amide (4.44 mM) inhibited fluoride- and prostaglandin-stimulated cyclase in FSH-responsive membranes by 10% and 11%, respectively. The data show that appropriate concentrations of DAPA can antagonize gonadotropin-stimulated adenylate cyclase while only minimally affecting fluoride- and other receptor-activated cyclase activities.     

Evidence that muscarinic receptors in islet cells are not coupled functionally to adenylate cyclase through the inhibitory guanine nucleotide binding protein (Ni)

The effect of muscarinic agonist on adenylate cyclase was investigated in neonatal islet cells and in a clonal pituitary cell line (GH4C1) following labelling of the intracellular ATP pool with [2,8 3H]adenine. In islet cells carbamylcholine was without effect on basal or glucagon-stimulated adenylate cyclase activity, measured as 3H cyclic AMP production, but inhibited 3H cyclic AMP production in the clonal pituitary cells. The involvement of the inhibitory guanine nucleotide binding protein of adenylate cyclase (Ni) was investigated by the use of the Bordetella pertussis exotoxin, islet activating protein (IAP). Pre-treatment of islet cells with IAP was without effect on adenylate cyclase following carbamylcholine but in the clonal pituitary line abolished the inhibition of 3H cyclic AMP production. It is concluded that in the islet cell, in contrast to the clonal pituitary cell, muscarinic receptors are not effectively coupled through Ni to inhibit adenylate cyclase.     

Involvement of calcium in the inhibition by insulin of the glucagon-stimulated adenylate-cyclase activity.

1. The inhibitory effect of adenosine on the glucagon-stimulated adenylate cyclase activity of liver plasma membranes, prepared from PVG/c rats, was potentiated by insulin. In the presence of EGTA, such potentiating effect of insulin was lost. 2. Calcium (10 microM) potentiated the inhibitory effects of both adenosine and insulin on the glucagon-stimulated cyclase activity. The synergestic effect of calcium + insulin required the presence of adenosine as judged from the use of adenosine deaminase. 3. Insulin had no significant inhibitory effect on the glucagon-stimulated cyclase activity of liver plasma membranes, prepared from young Wistar rats, unless both adenosine (50 microM) and calcium (10 microM) were added externally. 4. Results demonstrate an interaction of calcium and insulin at membrane level that, in the presence of adenosine, results in the inhibition of the glucagon-stimulated adenylate cyclase activity.