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.     

Resensitization of hepatocyte glucagon-stimulated adenylate cyclase can be inhibited when cyclic AMP phosphodiesterase inhibitors are used to elevate intracellular cyclic AMP concentrations to supraphysiological values.

Treatment of intact hepatocytes with glucagon led to the rapid desensitization of adenylate cyclase, which reached a maximum around 5 min after application of glucagon, after which resensitization ensued. Complete resensitization occurred some 20 min after the addition of glucagon. In hepatocytes which had been preincubated with the cyclic AMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), glucagon elicited a stable desensitized state where resensitization failed to occur even 20 min after exposure of hepatocytes to glucagon. Treatment with IBMX alone did not elicit desensitization. The action of IBMX in stabilizing the glucagon-mediated desensitized state was mimicked by the non-methylxanthine cyclic AMP phosphodiesterase inhibitor Ro-20-1724 [4-(3-butoxy-4-methoxylbenzyl)-2-imidazolidinone]. IBMX inhibited the resensitization process in a dose-dependent fashion with an EC50 (concn. giving 50% of maximal effect) of 26 +/- 5 microM, which was similar to the EC50 value of 22 +/- 6 microM observed for the ability of IBMX to augment the glucagon-stimulated rise in intracellular cyclic AMP concentrations. Pre-treatment of hepatocytes with IBMX did not alter the ability of either angiotensin or the glucagon analogue TH-glucagon, ligands which did not increase intracellular cyclic AMP concentrations, to cause the rapid desensitization and subsequent resensitization of adenylate cyclase. It is suggested that, although desensitization of glucagon-stimulated adenylate cyclase is elicited by a cyclic AMP-independent process, the resensitization of adenylate cyclase can be inhibited by a process which is dependent on elevated cyclic AMP concentrations. This action can be detected by attenuating the degradation of cyclic AMP by using inhibitors of cyclic AMP phosphodiesterase.     

Glucagon desensitization of adenylate cyclase and stimulation of inositol phospholipid metabolism does not involve the inhibitory guanine nucleotide regulatory protein Gi, which is inactivated upon challenge of hepatocytes with glucagon.

Brief exposure of hepatocytes to glucagon, angiotensin or the protein kinase C activator TPA (12-O-tetradecanoylphorbol 13-acetate) caused the inactivation of the inhibitory guanine nucleotide regulatory protein Gi. Glucagon-mediated desensitization of glucagon-stimulated adenylate cyclase activity was seen in hepatocytes from both normal rats and those made diabetic with streptozotocin, where Gi is not functionally expressed. Normal glucagon desensitization was seen in hepatocytes from young animals, 6 weeks of age, which had amounts of Gi in their hepatocyte membranes which were some 45% of that seen in mature animals (3.4 pmol/mg of plasma-membrane protein). Streptozotocin-induced diabetes in young animals abolished the appearance of functional Gi in hepatocyte plasma membranes. Pertussis-toxin treatment of hepatocytes from both normal mature animals and those made diabetic, with streptozotocin, blocked the ability of glucagon or angiotensin or TPA to elicit desensitization of adenylate cyclase. The isolated B (binding)-subunit of pertussis toxin was ineffective in blocking desensitization. Neither induction of diabetes nor treatment of hepatocytes with pertussis toxin inhibited the ability of glucagon and angiotensin to stimulate the production of inositol phosphates in intact hepatocytes. Thus (i) Gi does not appear to play a role in the molecular mechanism of glucagon desensitization in hepatocytes, (ii) the G-protein concerned with receptor-stimulated inositol phospholipid metabolism in hepatocytes appears not to be a substrate for the action of pertussis toxin, (iii) in intact hepatocytes, treatment with glucagon and/or angiotensin can elicit the inactivation of the inhibitory G-protein Gi, and (iv) pertussis toxin blocks desensitization by a process which does not involve Gi.     

Desensitization of Sertoli cell-enriched cultures by FSH, L-isoproterenol and glucagon. Influence on subsequent stimulation of 17 beta-estradiol production

Sertoli cell-enriched cultures derived from 19-day old rats were exposed to FSH, L-isoproterenol, glucagon and dbcAMP for 24 up to 96 h. The influence of primary stimulation with these agonists on the response of the cells to subsequent stimulation with the homologous or heterologous agonists was investigated. Particular attention was paid to the response of the aromatase system defined as the ability of the cells to convert testosterone into 17 beta-estradiol. The responsiveness of this system was compared with the responsiveness of two other systems: adenylate cyclase and phosphodiesterase. It could be demonstrated that preincubation with the mentioned agonists results in a decreased responsiveness (maximal response) and a decreased sensitivity (ED50) upon re-stimulation with the homologous agonists. Preincubation with FSH also provokes partial desensitization for glucagon and L-isoproterenol. This heterologous desensitization can be mimicked by dbcAMP. The mentioned desensitization reactions are accompanied by a marked decrease in the accumulation of cAMP in the medium. Whereas desensitization of the adenylate cyclase occurs rapidly, desensitization of the aromatase response requires protracted stimulation for 3-4 days. In contrast with the adenylate cyclase and the aromatase system, the responsiveness of phosphodiesterase to FSH, L-isoproterenol, glucagon and dbcAMP is not affected by repeated stimulation for 96 h. It is concluded that hormonal desensitization affects both early (cAMP) and late (aromatase) responses of the Sertoli cell. However, some responses, such as phosphodiesterase activity seem to escape the desensitization process.     

Homologous and heterologous beta-adrenergic desensitization in hepatocytes. Additivity and effect of pertussis toxin

In hepatocytes obtained from hypothyroid rats, phorbol myristate acetate (PMA) and vasopressin diminished the accumulation of cyclic AMP and the stimulation of ureagenesis induced by isoprenaline or glucagon without altering significantly the accumulation of cyclic AMP induced by forskolin. Pretreatment with PMA markedly reduced the stimulation of ureagenesis and the accumulation of cyclic AMP induced by isoprenaline or glucagon. In membranes from cells pretreated with PMA, the stimulation of adenylate cyclase induced by isoprenaline + GTP, glucagon + GTP or by Gpp[NH]p were clearly diminished as compared to the control, whereas forskolin-stimulated activity was not affected. The data indicate heterologous desensitization of adenylate cyclase. It was also observed that the homologous (García-Sáinz J.A. and Michel, B. (1987) Biochem. J. 246, 331-336) and this heterologous beta-adrenergic desensitizations were additive. Pertussis toxin treatment markedly reduced the heterologous desensitization of adenylate cyclase but not the homologous beta-adrenergic desensitization. It is concluded that the homologous and heterologous desensitizations involve different mechanisms. The homologous desensitization seems to occur at the receptor level, whereas the heterologous probably involves the guanine nucleotide-binding regulatory protein, Ns.     

Homologous and heterologous β-adrenergic desensitization in hepatocytes. Additivity and effect of pertussis toxin

In hepatocytes obtained from hypothyroid rats, phorbol myristate acetate (PMA) and vasopressin diminished the accumulation of cyclic AMP and the stimulation of ureagenesis induced by isoprenaline or glucagon without altering significantly the accumulation of cyclic AMP induced by forskolin. Pretreatment with PMA markedly reduced the stimulation of ureagenesis and the accumulation of cyclic AMP induced by isoprenaline or glucagon. In membranes from cells pretreated with PMA, the stimulation of adenylate cyclase induced by isoprenaline + GTP, glucagon + GTP or by Gpp[NH]p were clearly diminished as compared to the control, whereas forskolin-stimulated activity was not affected. The data indicate heterologous desensitization of adenylate cyclase. It was also observed that the homologous (García-Sáinz J.A. and Michel, B. (1987) Biochem. J. 246, 331–336) and this heterologous β-adrenergic desensitizations were additive. Pertussis toxin treatment markedly reduced the heterologous desensitization of adenylate cyclase but not the homologous β-adrenergic desensitization. It is concluded that the homologous and heterologous desensitizations involve different mechanisms. The homologous desensitization seems to occur at the receptor level, whereas the heterologous probably involves the guanine nucleotide-binding regulatory protein, Ns.     

Mechanism of molybdate activation of adenylate cyclase

Molybdate activation of rat liver plasma membrane adenylate cyclase has been examined and compared with the effect of glucagon, Gpp(NG)p and fluoride. Glucagon does not stimulate the detergent solubilized enzyme, though molybdate, fluoride, and Gpp(NH)p are effective in this regard. The stimulatory effects of either fluoride or molybdate are additive with those of GTP and do not require guanyl nucleotide to evoke their activation. Neither fluoride nor molybdate can substitute for GTP when glucagon is the activator of rat liver adenylate cyclase. The stimulatory effects of either ion on adenylate cyclase are additive with that produced by glucagon. Activation of adenylate cyclase by either molybdate or fluoride occurs by a mechanism distinct from that of glucagon or guanyl nucleotide. The data presented here suggest that fluoride and molybdate may act via a similar mechanism of action. Neither ion displays a lag in activation of adenylate cyclase. The pH profiles of fluoride and molybdate-stimulated adenylate cyclase activity are similar, and distinct from guanyl nucleotide-stimulated activity. Cholera toxin treatment of adenylate cyclase blocks fluoride and molybdate stimulation of the enzyme to the same extent, while enhancing the activation obtained with GTP and hormones.     

Challenge of hepatocytes by glucagon triggers a rapid modulation of adenylate cyclase activity in isolated membranes.

Membrane fractions obtained from hepatocytes treated with glucagon exhibited a decreased glucagon (with or without GTP)-stimulated adenylate cyclase activity. A maximum effect was seen in around 5 min. No change in the rate of cyclic AMP production was observed for the basal, NaF-, p[NH]ppG (guanosine 5'-[beta, gamma-imido]-triphosphate)- and GTP-stimulated states of the enzyme. The lag observed in the p[NH]ppG-stimulated adenylate cyclase activity of native membranes was abolished when membranes from glucagon-pretreated cells were used. When Mn2+ replaced Mg2+ in the assays, the magnitude of the apparent desensitization was decreased. Mn2+ abolished the lag of onset of p[NH]ppG-stimulated activity in native membranes. The desensitization process was dose-dependent on glucagon, which exhibited a Ka of 4 X 10(-10) M. Depletion of intracellular ATP did not affect this process. It is suggested that this desensitization occurs at the level of the guanine nucleotide-regulatory protein.     

Uncoupling of the Glucagon Receptor-Adenylate Cyclase System by Glucagon in Cloned Differentiated Rat Hepatocytes

Abstract

The ability of glucagon to induce a state of desens it ization to glucagon responsiveness has been examined in a cloned line of normal, differentiated, diploid rat hepatocytes (RL-PR-C). These cells, which respond to glucagon with increased production of cyclic AMP, become refractory to further stimulation of cyclic AMP synthesis following a 4 hour exposure period of the cells to the hormone. Refractoriness to glucagon was demonstrated over a wide range of hormone concentrations (10^?12 to 10^?6 M). In desensitized cells that were subsequently washed free of the hormone, recovery from refractoriness was complete by 20 hours. The mechanism underlying this desensitization does not appear to involve decreased receptor numbers, increased efflux of cyclic AMP from the cells, increased degradation of cyclic AMP by phosphodtesterase, or an alteration in the catalytic unit of the adenylate cyclase enzyme system. By elimination, the diminished cellular cyclic AMP responsiveness to glucagon in normal RL-PR-C hepatocytes may involve a reversible uncoupling of glucagon receptors from adenylate cyclase. In addition, late passage, spontaneously transformed RL-PR-C hepatocytes were found to exist in a state in which glucagon receptors are permanently uncoupled from adenylate cyclase.     

The relationship between beta-adrenoceptor regulation and beta-adrenergic responsiveness in hepatocytes. Studies on acquisition, desensitization and resensitization of isoproterenol-sensitive adenylate cyclase in primary culture

The role of beta-adrenoceptor regulation in the mechanisms controlling beta-adrenergic responsiveness in hepatocytes was explored, using primary monolayer cultures. When plated in vitro, these cells gradually acquire a strong catecholamine-sensitive adenylate cyclase activity and an enhanced ability to bind the beta-adrenoceptor ligand [125I]iodocyanopindolol (125ICYP). Examination of the time course showed that the increase in the number of 125ICYP binding sites was detectable within 1-2 h of culturing and slightly preceded the elevation of isoproterenol-responsive activity. Then the responsiveness rose steeply and between about 5-24 h it closely followed the increase in beta-receptor binding. Addition of isoproterenol (10 microM) to cells after 20 h of culturing caused a rapid homologous desensitization of the adenylate cyclase (50% after about 5 min). This was paralleled by a down-regulation of beta-adrenoceptors measured both in membrane particles and in total cell lysates. Removal of isoproterenol led to a resensitization of the adenylate cyclase, which was rapid and protein-synthesis-independent after a brief (10-min) desensitization, or slow and cycloheximide-sensitive after prolonged (4-h) exposure to the agonist. In both cases an up-regulation of the 125ICYP binding paralleled the recovery from refractoriness. In contrast, no concurring changes in 125ICYP binding were measured when the beta-adrenoceptor-linked adenylate cyclase activity was enhanced by pretreatment with pertussin toxin (islet-activating protein, IAP) or was desensitized by exposure of the cells to glucagon or 8-bromo-cAMP; however, these modulations of the adenylate cyclase were nonselective, since the pretreatments with IAP, glucagon or 8-bromo-cAMP affected both isoproterenol-sensitive and glucagon-sensitive activities. The results suggest that, in hepatocytes, regulation at the beta-adrenoceptor level is a major determinant for both short-term and long-term selective changes of the beta-adrenergic responsiveness.     

Specific Muscarinic-Cholinergic Desensitization in the Neuroblastoma-Glioma Hybrid NG108-15

Abstract: The cholinergic agonist carbachol, epinephrine, and the opiate morphine all inhibit prostaglandin E₁ (PGE₁)-stimulated adenylate cyclase in homogenates from the neuroblastoma-glioma hybrid NG108-15. Pretreatment of the hybrid with 100 μ M carbachol resulted in the rapid loss (desensitization) of the carbachol inhibition of adenylate cyclase (tM_1/2< 3 min). The desensitization of the carbachol inhibition was blocked by 0.1 μ M atropine. Pretreatment with carbachol (1–24 h) did not significantly affect the inhibition of adenylate cyclase by either epinephrine or morphine, nor did it alter the PGE₁-stimulated activity, that is, no supersensitization was observed. Cholate extracts of the particulate fraction from either carbachol-desensitized or of control NGlOS-15 were able to reconstitute adenylate cyclase activities of the coupling proteins (G/F)-deficient cyclymphoma cell membranes with equal efficacy. These results suggested that the coupling proteins of the adenylate cyclase were not altered by the carbachol pretreatment and that desensitization occurs at the receptor or at a receptor-associated level. However, the possibility remained that specific domains of the G/F, which interact only with muscarinic receptors, were altered.     

Adenosine and the regulation of insulin secretion by isolated rat islets of Langerhans.

The effect of adenosine in insulin secretion and adenylate cyclase activity of rat islets of Langerhans was investigated. Adenosine inhibited insulin secretion stimulated by glucose, glucagon, prostaglandin E2, tolbutamine and theophylline. Adenosine decreased basal adenylate cyclase activity of the islets as well as that stimulated by glucagon prostaglandin E2 and GTP, although fluoride-stimulated activity was not affected. Neither insulin secretion nor adenylate cyclase activity of the islets was affected by adenine, AMP or ADP. The inhibitory effect of adenosine on adenylate cyclase activity was not altered by either phenoxybenzamine (alpha-adrenergic blocker) or propranolol (beta-adrenergic blocker), suggesting that the effect is not mediated through the adrenergic receptors of the islet cells. These results suggest that the intracellular concentration of adenosine in the beta-cell may play a role in regulating insulin secretion and that this effect may be mediated via alterations in the activity of adenylate cyclase in the beta-cell.     

Adenylate cyclase responsiveness of human insulinomas

Adenylate cyclase activity was assayed in a crude particulate fraction of one benign and one malignant human insulinoma. Adenylate cyclase of both tumours responded to 5'-guanylyl-imidodiphosphate, sodium fluoride, glucagon and prostaglandin E2, and in addition the adenylate cyclase of the benign tumour responded to isoprenaline. Glucose and prostaglandin I2 (prostacyclin) did not stimulate the adenylate cyclase in either tumour, although prostaglandin I2 stimulated insulin secretion in cultures of the benign tumour. The in vitro responsiveness of the adenylate cyclase to glucagon did not correlate closely with the effect of glucagon on insulin secretion in vivo.     

Phorbol esters and beta-adrenergic agonists mediate desensitization of adenylate cyclase in rat glioma C6 cells by distinct mechanisms

Exposure of rat glioma C6 cells to either isoproterenol or 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in desensitization of isoproterenol-stimulated adenylate cyclase activity. After either treatment, the affinity of beta-receptors for isoproterenol was reduced. Thus, desensitization by TPA or isoproterenol appeared to involve an "uncoupling" of the beta-receptor from the stimulatory regulatory component (Ns) of adenylate cyclase. The activity of Ns, assayed by reconstitution of S49 cyc- adenylate cyclase activity, was found to be unchanged after desensitization. The activity of beta-receptors was measured by inactivating Ns and the catalytic component of adenylate cyclase in C6 membranes and fusing them with membranes lacking beta-receptors. Receptors from isoproterenol-treated C6 cells were less active in "coupling" to the foreign adenylate cyclase than receptors from untreated cells, whereas receptors from TPA-treated cells were fully active. This unexpected latter result was explored further. Lysates from C6 cells were centrifuged on linear sucrose density gradients and the gradient fractions assayed for beta-receptor binding activity. Most of the receptors were recovered in a "heavy" plasma membrane peak but some receptors also appeared in a "light" membrane peak. After treatment of the cells with isoproterenol or TPA, the proportion of receptors in the light peak increased. Prior treatment of the cells with concanavalin A prevented the increase in light receptors caused by isoproterenol or TPA. In addition, the concanavalin A treatment prevented the desensitization of adenylate cyclase caused by TPA but not that caused by isoproterenol. Finally, desensitization of adenylate cyclase was reversed by polyethylene glycol-induced fusion of membranes from cells treated with TPA but not isoproterenol. We conclude that beta-agonists and phorbol esters desensitize adenylate cyclase by distinct mechanisms. Agonists cause a reduction in the functional activity of the beta-receptors followed by a segregation of the receptors into a light membrane fraction devoid of Ns. Phorbol esters do not alter the activity of the receptors but do cause their segregation.     

Islet-activating protein blocks glucagon desensitization in intact hepatocytes.

Treatment of intact hepatocytes with islet-activating protein, from Bordatella pertussis, led to a pronounced increase in the ability of glucagon to raise intracellular cyclic AMP concentrations. Islet-activating protein, however, caused no apparent increase in the intracellular concentration of cyclic AMP under basal conditions. These effects were attributed to an enhanced ability of adenylate cyclase, in membranes from hepatocytes treated with islet-activating protein, to be stimulated by glucagon. When forskolin was used to amplify the basal adenylate cyclase activity, elevated GTP concentrations were shown to inhibit adenylate cyclase activity in membranes from control hepatocytes. This inhibitory effect of GTP was abolished if the hepatocytes had been pre-treated with islet activating protein. In isolated liver plasma membranes, islet-activating protein caused the NAD-dependent ribosylation of a Mr-40000 protein, the putative inhibitory guanine nucleotide regulatory protein, Ni. This effect was inhibited if guanosine 5'-[beta-thio]diphosphate rather than GTP was present in the ribosylation incubations. The ability of glucagon to uncouple or desensitize the activity of adenylate cyclase in intact hepatocytes was also blocked by pre-treating hepatocytes with islet-activating protein. Islet-activating protein thus heightens the response of hepatocytes to the stimulatory hormone glucagon. It achieves this by both inhibiting the expression of desensitization and also removing a residual inhibitory input expressed in the presence of glucagon.     

Cholera toxin and adenylate cyclase: properties of the activated enzyme in liver plasma membranes.

Adenylate cyclase (EC 4.6.1.1) activity in mouse liver plasma membranes is increased fivefold when animals are pretreated with cholera toxin. The increase in activity is detectable within 20 min of an intravenous injection of the toxin. The response of the control and cholera-toxin-activated adenylate cyclase to hormones, GTP, and NaF is complex. GTP causes the same fold stimulation of control and toxin-activated cyclase, but glucagon and NaF remain the most potent activators of liver adenylate cyclase irrespective of whether the enzyme is activated by cholera toxin. Determination of kinetic parameters of adenylate cyclase indicates that cholera toxin, hormones, and NaF do not change the affinity of the enzyme for ATP-Mg nor do they alter the Ka for free Mg2+. High concentrations of Mg2+ inhibit adenylate cyclase that is stimulated by either cholera toxin, glucagon, or NaF. These same Mg2+ concentrations have no effect on the basal activity of the enzyme or its activity in the presence of GTP.     

Biological activities of catfish glucagon and glucagon-like peptide

The ability of catfish glucagon and glucagon-like peptide to bind and activate mammalian glucagon receptors was investigated. Neither catfish peptide binds to glucagon receptors of rat liver, hypothalamus or pituitary. Neither stimulates adenylate cyclase activity in liver membranes. Catfish glucagon fails to activate adenylate cyclase in hypothalamic or pituitary membranes in contrast to mammalian glucagon. However, catfish glucagon-like peptide does stimulate hypothalamic and pituitary adenylate cyclase (EC50 approximately 1 pM) possibly through mammalian glucagon-like peptide receptors.     

The glucagon receptor of rat liver plasma membrane can couple to adenylate cyclase without activating it.

1. Activation of adenylate cyclase in rat liver plasma membranes by fluoride or GMP-P (NH)P yielded linear Arrheniun plots. Activation by glucagon alone, or in combination with either fluoride or GMP-P(NH)P resulted in biphasic Arrhenius plots with a well-defined break at 28.5 +/- 1 degrees C. 2. The competitive glucagon antagonist, des-His-glucagon did not activate the adenylate cyclase but produced biphasic Arrhenius plots in combination with fluoride or GMP-P(NH)P. The break temperatures and activation energies were very similar to those observed with glucagon alone, or in combination with either fluoride or GMP-P(NH)P. 3. It is concluded that although des-His-glucagon is a potent antagonist of glucagon, it nevertheless causes a structural coupling between the receptor and the catalytic unit.     

N6-(Phenylisopropyl)adenosine prevents glucagon both blocking insulin''s activation of the plasma-membrane cyclic AMP phosphodiesterase and uncoupling hormonal stimulation of adenylate cyclase activity in hepatocytes.

Glucagon (10nM) prevented insulin (10nM) from activating the plasma-membrane cyclic AMP phosphodiesterase. This effect of glucagon was abolished by either PIA [N6-(phenylisopropyl)adenosine] (100nM) or adenosine (10 microM). Neither PIA nor adenosine exerted any effect on the plasma-membrane cyclic AMP phosphodiesterase activity either alone or in combination with glucagon. Furthermore, PIA and adenosine did not potentiate the action of insulin in activating this enzyme. 2-Deoxy-adenosine (10 microM) was ineffective in mimicking the action of adenosine. The effect of PIA in preventing the blockade by glucagon of insulin's action was inhibited by low concentrations of theophylline. Half-maximal effects of PIA were elicited at around 6nM-PIA. It is suggested that adenosine is exerting its effects on this system through an R-type receptor. This receptor does not appear to be directly coupled to adenylate cyclase, however, as PIA did not affect either the activity of adenylate cyclase or intracellular cyclic AMP concentrations. Insulin's activation of the plasma-membrane cyclic AMP phosphodiesterase, in the presence of both glucagon and PIA, was augmented by increasing intracellular cyclic AMP concentrations with either dibutyryl cyclic AMP or the cyclic AMP phosphodiesterase inhibitor Ro-20-1724. PIA also inhibited the ability of glucagon to uncouple (desensitize) adenylate cyclase activity in intact hepatocytes. This occurred at a half-maximal concentration of around 3 microM-PIA. However, if insulin (10 nM) was also present in the incubation medium, PIA exerted its action at a much lower concentration, with a half-maximal effect occurring at around 4 nM.     

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.