Metabolic antagonism between insulin action and activators of adenylate cyclase in rat fat cells

1. Short-term effects of lipolytic agents in the absence or in the presence of insulin on fatty acid biosynthesis have been examined, in terms of the control rate of [1-14C]acetate incorporation into labeled fatty acids in the presence of glucose, as stimulator of lipogenesis by generating NADPH for the process. 2. The relationship between lipogenesis and lipolysis in the absence or in the presence of insulin was compared with a variety of adenylate cyclase activators. 3. The data obtained reveal that a reciprocal relationship exists between lipogenesis and lipolysis. 4. The changes in the activity of hexose monophosphate shunt produced by activation or inhibition of lipogenic process has been studied. 5. The regulation of the hexose monophosphate shunt activity mainly by the intracellular fatty acyl-CoA concentration and NADPH/NADP ratio is discussed.     

Pertussis toxin effects on adenylate cyclase activity, cyclic AMP accumulation and lipolysis in adipocytes from hypothyroid, euthyroid and hyperthyroid rats

Adipocytes from hypothyroid rats have a decreased responsiveness to agents that activate adenylate cyclase, whereas cells from hyperthyroid rats have an increased responsiveness as compared to the controls. This is reflected in cyclic AMP accumulation as well as lipolysis. Administration of pertussis toxin to rats or its in vitro addition to adipocytes increased basal lipolysis and cyclic AMP accumulation as well as the response to norepinephrine or forskolin. The effects of thyroid status was not abolished by toxin treatment. Pertussis toxin-catalyzed ADP ribosylation of Ni was increased in adipocyte membranes from hypothyroid rats as compared to those from euthyroid rats. However, no change in sensitivity to N6-(phenylisopropyl)adenosine was observed. The data suggest that the amount of Ni might not be rate-limiting for the inhibitory action of adenosine. A consistent decrease in maximal lipolysis was observed in freshly isolated adipocytes from hypothyroid animals as compared to those from the controls. Such defective maximal lipolysis was not corrected by adenosine deaminase or in vivo administration of pertussis toxin. The relationship between cyclic AMP levels and lipolysis suggests that in fat cells from hypothyroid rats either the cyclic AMP-dependent protein kinase or the lipase activity itself may limit maximal lipolysis. There appears to be multiple effects of thyroid status on lipolysis involving factors other than those affecting adenylate cyclase activation.     

Characteristics of high-affinity [3H]adenosine binding to rat brain synaptosomes and turkey erythrocyte membranes

High affinity binding sites for [³H]adenosine in rat brain and in turkey erythrocytes can be identified by binding experiments. Displacement experiments using a number of adenosine analogs indicate that these high affinity sites do not represent the R-type adenosine receptors which mediate activation of adenylate cyclase, although the binding is theophylline sensitive. Similarly, the binding of [³H]adenosine is not to the P-site, which mediates inhibition of adenylate cyclase, since the high affinity binding persists in the presence of 2′,5′-dideoxyadenosine. Furthermore, these results remain qualitatively similar also in the presence of dipyridamole which blocks adenosine transport sites. We conclude that theophylline sensitivity does not indicate that [³H]adenosine binding sites correspond to adenosine receptors coupled to adenylate cyclase.     

Adenosine receptor-mediated changes in cyclic AMP production and DNA synthesis in cultured arterial smooth muscle cells

The effects of adenosine and two analogs, L-phenylisopropyladenosine (L-PIA) and 5'-N-ethylcarboxamidoadenosine (NECA), on cAMP production and on platelet-derived growth factor (PDGF)-stimulated initiation of DNA synthesis in growth-arrested cultures of rat arterial smooth muscle cells (SMC) were studied. The intracellular cAMP concentration was dose-dependently enhanced by micromolar concentrations of adenosine and its analogs, with the potency order NECA greater than adenosine greater than L-PIA. The effect was antagonized, in a competitive manner, by the adenosine receptor antagonist 8-phenyltheophylline (8-PT). The stimulatory effect of adenosine was enhanced by 3 microM dipyridamole an adenosine-uptake blocker. DNA synthesis was inhibited in a parallel manner, showing the same potency order. The inhibition was antagonized by 8-PT. Forskolin, a diterpene with the ability to stimulate the catalytic unit of adenylate cyclase and thereby cAMP formation, potentiated the effects of micromolar concentrations of NECA and L-PIA. Forskolin, by itself, stimulated cAMP production and inhibited DNA synthesis. The forskolin-stimulated increase in cAMP was inhibited by L-PIA at nanomolar concentrations. L-PIA in the nanomolar concentration range also stimulated DNA synthesis when initiation was stimulated with suboptimal concentrations of PDGF. These findings suggest the presence of adenosine receptors of both the A1- and A2-subtype on SM-mediating bidirectional changes of cAMP and DNA synthesis.     

The antilipolytic effect of insulin does not require adenylate cyclase or phosphodiesterase action

Insulin antagonized the lipolytic actions of epinephrine in rat epididymal adipocytes when the phosphodiesterase inhibitor, Ro 20-1724, was present. Adipocytes were depleted of functional cAMP by inhibiting adenylate cyclase with N6-phenylisopropyladenosine in the presence of adenosine deaminase such that Ro 20-1724 no longer stimulated lipolysis. The cAMP analogs 8-thioisopropyl-cAMP or 8-thiomethyl-cAMP, which are resistant to phosphodiesterase hydrolysis, were subsequently added to bypass adenylate cyclase and phosphodiesterase action. Under these conditions, insulin antagonized the lipolytic effects of these analogs, even in the presence of Ro 20-1724.     

Evaluation of the effects of adenosine on hepatic and adipocyte adenylate cyclase under conditions where adenosine is not generated endogenously.

Direct effects of adenosine on adipocyte and hepatic adenylate cyclase have been demonstrated in an assay system where adenosine is not generated. The substrate used, 2'-deoxy ATP may, on metabolism, only give rise to 2'-deoxyadenosine, which does not act at adenosine receptors. With a slight modification of existing assay techniques this assay system has been used to detect a hitherto undiscovered adenosine receptor on liver plasma membranes, which is antagonised by methylxanthines and which stimulates adenylate cyclase activity in a GTP-dependent manner. The potent inhibitory effects of purine-modified adenosine analogs on fat cell adenylate cyclase are reproduced by adenosine in this assay system. An application of this approach to the study of adenylate cyclase not only simplifies detection of the role of adenosine, but also yields insights into the interaction between guanine nucleotides and hormones.     

Free fatty acids as feedback regulators of adenylate cyclase and cyclic 3':5'-AMP accumulation in rat fat cells.

Rat fat cells incubated with lipolytic agents released substances to the medium which acted as feedback regulators of cyclic adenosine 3':5'-monophosphate (cyclic AMP) accumulation. The feedback regulators were not removed by adenosine deaminase. Dialyzed medium that had previously been incubated with fat cells in the presence of norepinephrine markedly inhibited cyclic AMP accumulation by fresh cells, whereas dialyzed medium from control cells did not inhibit cyclic AMP accumulation. The effects of lipolytic agents could be mimicked by adding dialyzed medium previously incubated with fat cells in the presence of oleic acid. This suggested that free fatty acids were the nondialyzable and adenosine deaminase-insensitive inhibitors of cyclic AMP accumulation released to the medium by fat cells incubated with lipolytic agents. The regulatory function of free fatty acids was related to the molar ratio of fatty acid to albumin. Profound inhibition of both lipolysis and cyclic AMP accumulation was seen as the free fatty acid/albumin ratio exceeded 3. The inhibition of cyclic AMP accumulation by oleate was seen as soon as there was a detectable increase in cyclic AMP due to lipolytic agents. Protein kinase activity (in the presence of cyclic AMP) of the infranatant obtained after centrifugation of fat cell homogenates at 48,000 x g was inhibited by medium from cells incubated with lipolytic agents or added oleate. Adenylate cyclase activity of rat fat cell ghosts was also inhibited by dialyzed or nondialyzed medium that previously had been incubated with lipolytic agents or added fatty acids. The direct addition of oleate markedly inhibited adenylate cyclase activity as the free fatty acid/albumin ratio exceeded 2. These data suggest that the prolonged drop in cyclic AMP accumulation seen during the incubation of rat fat cells with lipolytic agents is due to the inhibition of adenylate cyclase. This occurs when the free fatty acid/albumin ratio exceeds 3.     

Activation of adenylate cyclase from rat striatum and tuberculum olfactorium by adenosine

Adenosine caused a dose-dependent stimulation of adenylate cyclase in homogenates from rat striatum and tuberculum olfactorium (200 and 300% stimulation by 100 muM adenosine). The effect of adenosine was not antagonized by haloperidol. Subcellular fractionation suggested that adenosine stimulates a different adenylate cyclase than dopamine. Basal adenylate cyclase activity in freshly prepared homogenates was reduced by dialysis and by the addition of adenosine deaminase. Basal adenylate cyclase activity was enchanced by papaverine and dipyridamole, but reduced by theophylline and isobutylmethylxanthine. The results are compatible with the opinion that endogenous adenosine is capable of activating adenylate cyclase in these areas of the rat brain.     

Adenosine receptor-adenylate cyclase system in the trout testis: involvement in the regulation of germ cell proliferation

To ascertain the presence of adenosine receptors in the trout testis, cells isolated from testes at different spermatogenetic stages were cultured in the presence or absence of adenosine, adenosine receptor agonists, or antagonists and of cAMP analogs, for up to 20 min, or 20 hr, or 4.5 days. Cyclic AMP production was then assayed or 3H-thymidine incorporation was measured. Cellular content of cAMP was enhanced by adenosine, by the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), and by 2-p(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680), an adenosine A2A receptor-selective agonist. The increase in cAMP induced by the adenylate cyclase activator L-858051 was inhibited by the adenosine A1)receptor-selective agonists R-N6-(2-phenylisopropyl)adenosine (R-PIA) and N6-cyclopentyladenosine (CPA). These effects were antagonized by the two adenosine A2)receptor antagonists 3,7-dimethyl-1-propargylxanthine (DMPX) and 8-(3-chlorostyryl)caffeine (CSC), and by the adenosine A1)receptor-selective antagonist 8-cyclopentyl-1,3dipropylxanthine (CPX), respectively. Increase in the cAMP content induced by adenosine was inhibited by the cell permeable adenylate cyclase inhibitor 2',5'-dideoxyadenosine. These data suggest that A(1) and A(2) adenosine receptors which respectively inhibit and stimulate adenylate cyclase activity are present on trout testicular cells (unidentified), while the presence of A3 adenosine receptor subtype was not apparent. 3H-thymidine incorporation decreased in the presence of the adenylate cyclase activator L-858051 and of the cAMP analogs 8-CPT cAMP and Sp-5,6-DCI-cBiMPS, regardless of the presence or absence of the phosphodiesterase inhibitor RO 20-1724. This suggests that an increase in testicular cAMP may act as a negative growth regulator for the mitotic germ cells. In agreement with these data, the activation of A2 stimulatory receptors inhibited short-term (20 hr) DNA synthesis. However, the activation of A1 inhibitory receptors had the same effect. This suggests that events, cAMP-dependent or independent, induced by the activation of testicular adenosine receptors, may participate in the regulation of trout male germ cell proliferation.     

Adenosine potentiates lutropin-stimulated cyclic AMP production and inhibits lutropin-induced desensitization of adenylate cyclase in rat Leydig tumour cells.

The action of adenosine on lutropin (LH)-stimulated cyclic AMP production and LH-induced desensitization of adenylate cyclase in rat Leydig tumour cells was investigated. Adenosine and N6-(phenylisopropyl)adenosine caused a dose-dependent potentiation of LH-stimulated cyclic AMP production at concentrations (0.01-10 microM) which alone did not produce an increase in cyclic AMP production. However, 2-deoxyadenosine had no effect either alone or in combination with LH on cyclic AMP production. The potentiation produced by adenosine was unaffected by concentrations of the specific nucleoside-transport inhibitor dipyridamole, which inhibited [3H]adenosine uptake by up to 90%. The phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine, but not RO-10-1724, inhibited the adenosine-induced potentiation. In the presence of adenosine, the kinetics of LH-stimulated cyclic AMP production were linear with time up to 2h, compared with those with LH alone, which showed a characteristic decrease in rate of cyclic AMP production after the first 15-20 min. Consistent with the altered kinetics, adenosine also inhibited the LH-induced desensitization of adenylate cyclase. These results suggest that adenosine has effects on rat tumour Leydig cells through receptors on the external surface of the plasma membrane. This receptor has characteristics similar to those of the R-type receptors, which have been shown either to stimulate or to inhibit adenylate cyclase. However, the effects of adenosine in the present studies does not involve a direct inhibition or activation of adenylate cyclase, but may involve an as yet undefined receptor-mediated modulation of adenylate cyclase.     

Multiple mechanisms of growth inhibition by cyclic AMP derivatives in rat GH1 pituitary cells: isolation of an adenylate cyclase-deficient variant

GH pituitary cells have been widely utilized for studies of hormone response mechanisms. Studies reported here were motivated by the desirability of isolating characterized GH clones defective in cyclic AMP synthesis or action. Spontaneously occurring GH1 cell variants resistant to the growth-inhibitory effects of cyclic AMP analogs were isolated. Characterization of four variants showed that these were deficient in adenosine kinase and had acquired resistance to the cytotoxic effects of purine nucleoside derivatives formed in the culture medium. A second-stage selection was undertaken with mutagenized adenosine kinase-deficient cells. One 8 Br cAMP-resistant variant was found to have normal cyclic AMP-dependent protein kinase activity but exhibited altered adenylate cyclase activity. Activation of cyclase activity by fluoride, guanyl nucleotides, cholera toxin, and hormone (VIP) was subnormal in the variant. Mn-dependent cyclase activity was also subnormal, suggesting that the 8 Br cAMP-resistant variant may have a deficiency in the catalytic moiety of adenylate cyclase. Surprisingly, adenosine 3':5'-monophosphate and 5'-monophosphate derivatives were found to be equally potent in growth-inhibiting adenosine kinase-deficient cells. Cross-resistance to 8 Br AMP was observed in the 8 Br cAMP-resistant variant. We conclude that cyclic AMP derivatives inhibit growth of GH cells by an unanticipated mechanism that is, nonetheless, related to endogenous cyclic AMP synthesis.     

cAMP-dependent protein kinase and lipolysis in rat adipocytes. III. Multiple modes of insulin regulation of lipolysis and regulation of insulin responses by adenylate cyclase regulators

The relationship between cAMP-dependent protein kinase (A-kinase) activity ratios and lipolysis in the presence of insulin was compared to the standard relationship between these two parameters established with a variety of adenylate cyclase modulators (Honnor, R. C., Dhillon, G., and Londos, C. (1985) J. Biol. Chem. 260, 15130-15138). Three phases of insulin action were observed. First, when tested in control cells exhibiting A-kinase activity ratios up to approximately 0.25, insulin inhibition of lipolysis could be accounted for by the decrease in A-kinase activity. Second, in cells exhibiting A-kinase activity ratios greater than 0.3, the decrease in kinase activity by insulin did not account for the decrease in lipolysis. Finally, as the A-kinase activity ratio approached 0.6 the insulin effect on lipolysis was lost. The data suggest that protein phosphatase activation accounts for the cAMP-independent insulin action. Moreover, the insulin effect not accounted for by a decrease in A-kinase activity appears to be elicited only upon elevation of A-kinase activity. The method by which cells were stimulated determined the IC50 for insulin inhibition of: 1) A-kinase activity ratios, 2) lipolysis explained by the decrease in A-kinase activity ratios, and 3) lipolysis not explained by a decrease in A-kinase activity ratios. For all three parameters, cells stimulated by lipolytic hormones were approximately 5 times more sensitive to insulin than cells stimulated by incubation in a ligand-free environment achieved with adenosine deaminase; insulin IC50 values were approximately 120 and 600 pM, respectively. Such data establish a link between insulin actions in modifying cAMP concentrations and in modifying events apparently independent of changes in cAMP. It is proposed that the receptors and regulatory components associated with adipocyte adenylate cyclase are associated also with components of the insulin response system separate from cyclase.     

Inhibition of rat and human adipocyte adenylate cyclase in the antilipolytic action of insulin, clofibrate, and nicotinic acid.

Clofibrate (Atromid-S), nicotinic acid, and insulin are known to be potent hypolipidemic and antilipolytic agents. The present study was undertaken to define the mechanism of action of this latter effect on isolated rat and human fat cells. Sodium clofibrate (0.42 mM), nicotinic acid (0.42 mM), and insulin (100 microU/mL) were shown to inhibit norepinephrine-stimulated lipolysis in rat and human adipose cells and this inhibition was associated with a reduction in intracellular 3',5'-cyclic AMP levels. A similar cyclic AMP lowering effect was demonstrated with insulin in the presence of procaine-HCL, which uncouples the adenylate cyclase system from lipolysis. This insulin effect was attributed to inhibition of adenylate cyclase. A direct and significant inhibition of adenylate cyclase in membrane fractions obtained from isolated human adipocytes was demonstrated for all three antilipolytic agents. The common membrane site of action of these agents whereby adenylate cyclase activity is depressed, thus decreasing cyclic AMP production and free fatty acid (FFA) mobilization from adipose stores, implies a central role for the adenylate cyclase system. These findings are consistent with the view that the hypotriglyceridemic effects of clofibrate, nicotinic acid, and insulin may be partly explained by deprivation of FFA substrate for hepatic very low density lipoprotein synthesis.     

Regulation of platelet adenylate cyclase by adenosine.

The stimulatory and inhibitory effects of adenosine on the adenylate cyclases of human and pig platelets were studied. Stimulation occurred at lower concentrations than did inhibition, and the stimulatory effect was prevented by methylxanthines. Stimulation by adenosine was immediate in onset and was reversible, under conditions when cyclic AMP formation was linear with respect to time and protein concentration. The stimulatory and inhibitory effects could be distinguished further by the use of various analogues of adenosine and could be prevented by adenosine deaminase. The data suggest that both stimulation and inhibition were due to adenosine itself and not one of its degradation products and that in the platelet preparation, neither formation nor degradation of adenosine during the adenylate cyclase incubation appreciably influenced measured activity. Stimulation by adenosine was additive with the effects of GMP-P(NH)P, and alpha- or beta-adrenergic stimulation, but was abolished by prostaglandin E1 or by NaF. Prostaglandin E1 and NaF increased the sensitivity of adenylate cyclase to inhibition by adenosine. The data suggest that guanyl-5'-yl-(beta-gamma-imino)diphosphate and/or adrenergic stimulation and adenosine exert their effects on adenylate cyclase by distinct mechanisms, but that prostaglandin E1 or F- and adenosine increase enzyme activity by mechanisms which may involve common intermediates in the coupling to adenylate cyclase.     

Regulation of Dictyostelium discoideum adenylate cyclase by manganese and adenosine analogs

Adenylate cyclase is the critical enzyme in the chemotactic signal relay mechanism of the slime mold amoeba, Dictyostelium discoideum. However, few studies examining the regulation of this enzyme have been performed in vitro due to the instability of enzyme activity in crude lysates. For studies presented in this communication, a membrane preparation has been isolated that exhibits a high specific activity adenylate cyclase that is stable during storage at -70 degrees C and under assay conditions at 27 degrees C. The enzyme was activated by micromolar concentrations of MnCl2. GTP and its non-hydrolyzable analog, guanosine 5'-(beta, gamma-imino)triphosphate, inhibited the enzyme non-competitively in the presence of either Mg2+ or Mn2+. However, this inhibition was more pronounced in the presence of Mn2+. Since guanylate cyclase activity in the D. discoideum membranes was less than 10% of the adenylate cyclase activity, there could not be a significant contribution by guanylate cyclase toward the production of cyclic AMP. Experiments indicate that D. discoideum adenylate cyclase was also regulated by adenosine analogs. The enzyme was inhibited by 2',5'-dideoxyadenosine and 2'-deoxyadenosine and inhibition was augmented by the presence of Mn2+. However, the inhibition was not entirely consistent with that which would be expected for the P-site of eukaryotic systems because some purine-modified adenosine analogs also inhibited the enzyme. Guanine nucleotides had no effect on the inhibition by either purine-modified or ribose-modified adenosine analogs. The binding of cyclic AMP to its receptor on the D. discoideum membranes was not affected by either MnCl2 or adenosine analogs.     

Adenosine regulates the release of adrenocorticotropic hormone (ACTH) from cultured anterior pituitary cells

We have recently shown the presence of adenosine receptors coupled to adenylate cyclase in anterior pituitary and in the present studies we have investigated the effects of adenosine on ACTH release. The R-site specific analogs of adenosine such as N-Ethylcarboxamide adenosine (NECA), L-N⁶-phenylisopropyl adenosine (PIA), 2-chloro-adenosine (2-Cl-Ado) all stimulated ACTH release in a dose-dependent manner. NECA was the most potent analog and stimulated ACTH release by about 170% with an apparent Ka of 0.1 µM, whereas PIA and 2-Cl-Ado were less potent and stimulated the release by about 110% and 125% with an apparent Ka of 0.2 and 0.4 µ-M respectively. The stimulation of ACTH release by NECA was inhibited by 3-isobutyl-1-methylxanthine (IBMX). On the other hand, adenosine deaminase (ADA) treatment of the cells also stimulated ACTH release as well as adenylate cyclase activity by about 2-fold, suggesting that endogenous adenosine plays an inhibitory role in the release of ACTH. Other agents, such as corticotropin-releasing factor (CRF), vasoactive intestinal peptide (VIP) and forskolin (FSK) also stimulated ACTH release from these cells. In addition, the stimulation by an optimal concentration of NECA was almost additive with maximal stimulation caused by VIP and FSK. These data suggest that adenosine modulates ACTH release from anterior pituitary through its interaction with adenosine receptors coupled to adenylate cyclase.Abbreviations NECA N-Ethylcarboxamideadenosine - PIA L-N⁶-Phenylisopropyladenosine - 2-Cl-Ado 2-chloroadenosine - FSK Forskolin - VIP Vasoactive Intestinal Peptide - CRF Corticotropin Releasing Factor - ADA Adenosine Deaminase - IBMX 3-Isobutyl-1-methylxanthine     

Adenosine Inhibition of Calmodulin-Sensitive Adenylate Cyclase from Bovine Cerebral Cortex

Calmodulin (CaM)-sensitive adenylate cyclase has recently been purified extensively from bovine brain. In this study, the sensitivity of the CaM-sensitive adenylate cyclase to adenosine and adenosine analogs was examined. The highly purified enzyme preparation retained sensitivity to inhibition by adenosine and adenosine analogs with ribose ring modifications, but not to those with purine ring modifications. Adenosine inhibition of this enzyme was not dependent on GTP and was noncompetitive with respect to ATP. Enzyme that had been dissociated from functional guanine nucleotide binding protein interactions by gel filtration in the presence of the zwitterionic detergent 3-[3-(cholamidopropyl)-dimethylammonio]-propanesulfonate and Mn2+ retained sensitivity to adenosine inhibition. The Ki for adenosine inhibition of the CaM-sensitive adenylate cyclase was approximately 2.6 X 10(-4) M. 5'-Guanylylimidodiphosphate and CaM did not affect the Ki of 3'-deoxyadenosine for the enzyme, but the presence of Ca2+ in the millimolar range raised the Ki by a factor of 5. These results show that the CaM-sensitive form of adenylate cyclase from bovine brain is subject to adenosine inhibition, and strongly suggest that this inhibition is due to interaction of ligands with a purine-specific ("P") site located on the catalytic subunit of the enzyme.     

Definition of subclasses of adenosine receptors associated with adenylate cyclase: interaction of adenosine analogs with inhibitory A1 receptors and stimulatory A2 receptors

The structure-activity relationships of 63 adenosine analogs as agonists for the A1 adenosine receptors that mediate inhibition of adenylate cyclase activity in rat fat cells and for the A2 adenosine receptors that mediate stimulation of adenylate cyclase in rat pheochromocytoma PC12 cells and human platelets were determined. The lack of correspondence between the structure-activity relationships of these analogs at the A1 and A2 receptors appear definitive in terms of establishing the existence of A1 and A2 subclasses of adenosine receptors. However, significant differences in the agonist profiles at A2 receptors of platelet and PC12 indicate a certain degree of structural heterogeneity within the members of the A2 adenosine receptor subclass. Whether such differences are due to different species or different cell types is not known. A set of adenosine analogs, such as N6-cyclohexyl-, N6-R-, and S-1-phenyl-2- propyladenosines, 5'-N-ethylcarboxamidoadenosine and its N6-cyclohexyl derivative, 2-chloroadenosine, and 2-phenylaminoadenosine, appear to represent a series of analogs useful for pharmacological characterization of A1 and A2 classes of adenosine receptors.     

CD3-dependent increase in cyclic AMP in human T-cells following stimulation of the CD2 receptor.

We have previously shown that stimulation of the Ti/CD3 receptor complex on human T-cells potentiates adenylate cyclase activation by adenosine or forskolin. Anti-CD2 receptor antibodies shared with anti-CD3 antibodies the ability to potentiate dose dependently the adenosine- and forskolin-stimulated cyclic adenosine monophosphate (cAMP) accumulation, whereas stimulation of the CD45 receptor had no effect on cyclase activity. Modulation of the CD3 complex with anti-CD3 antibodies was found to decrease the CD2 receptor effect on adenylate cyclase activity greatly. The possible involvement of CD3-stimulated phospholipase C (PLC) activation on the cAMP potentiation was examined using HPB-ALL cells that express a CD3 complex with a defect coupling to PLC. Stimulation of the CD3 complex on HPB-ALL cells had only slight effects on adenosine-stimulated cAMP formation, whereas the effect on forskolin-stimulated cAMP was virtually unchanged. The CD3 effect was further analyzed in Jurkat cell membranes. In contrast to the results obtained after stimulation of intact cells, it was found that OKT3 stimulation of membranes did not potentiate the forskolin response. Finally, we tested whether inhibition of endogenous adenylate cyclase agonist production affected the CD3 effect. Inhibition of adenosine production or adenosine breakdown with 8-p-sulphophenyl theophylline (8-PST) or adenosine deaminase (ADA), respectively, did not alter the CD3 effects. Indometacin, which inhibits prostaglandin production, also had no effect. Together, these data show that stimulation of the CD2 receptor potentiates adenylate cyclase responses by a mechanism that is dependent on CD3 expression. Furthermore, the CD3 effect on cAMP appears to be mediated by two different mechanisms, one which is, and one which is not dependent on PLC. Finally, this effect is not due to an endogenous production of adenylate cyclase agonists.