Adenosine 3', 5'-monophosphorothioate (Rp-isomer) induces down-regulation of surface cyclic AMP receptors without receptor activation in Dictyostelium discoideum

cAMP induces the activation and subsequent desensitization of adenylate cyclase in Dictyostelium discoideum. cAMP also induces down-regulation of surface cAMP receptors. Desensitization of adenylate cyclase is composed of a rapidly reversible component (adaptation) and a slowly reversible component related to down-regulation of surface cAMP receptors (Van Haastert, P.J.M. (1987) J. Biol. Chem. 262, 7700-7704). The agonistic and antagonistic activities of the cAMP derivative adenosine 3',5'-monophosphorothioate ((Rp)-cAMPS) for these responses were investigated. (Rp)-cAMPS competes with cAMP for binding to different receptor forms with an apparent Ki = 5 microM. (Rp)-cAMPS does not activate adenylate cyclase and antagonizes the cAMP-induced activation with an apparent Ki = 5 microM. (Rp)-cAMPS induces down-regulation of surface cAMP receptors with EC50 = 5 microM. (Rp)-cAMPS induces desensitization of adenylate cyclase, which is not rapidly reversible. These results indicate that desensitization of adenylate cyclase by (Rp)-cAMPS is due to down-regulation of surface cAMP receptors and not to adaptation. We conclude that down-regulation of surface cAMP receptors does not require their activation or modification involved in adaptation.     

Down-regulation of gonadotropin and beta-adrenergic receptors by hormones and cyclic AMP

Loss of gonadotropin receptors in murine Leydig tumor cells and of beta-adrenergic receptors in rat glioma C6 cells occurred following exposure of the cells to human chorionic gonadotropin and isoproterenol, respectively. Down-regulation of receptors was mimicked in part by other agents that elevated cyclic AMP levels in the cells such as cholera toxin and dibutyryl cyclic AMP. Whereas agonist-mediated receptor loss was rapid and almost total, down-regulation by cyclic AMP was slower and less extensive. Down-regulation of receptors did not appear to be accompanied by loss of the regulatory and catalytic components of adenylate cyclase. Hormone-mediated down-regulation was preceded by desensitization of hormone-stimulated adenylate cyclase. In contrast, there was no evidence that cyclic AMP caused desensitization. Finally, loss of receptors induced either by agonists or cyclic AMP required protein synthesis as cycloheximide inhibited down-regulation. We conclude that down-regulation of receptors in these cells is a complex process involving both cyclic AMP-independent and -dependent events.     

Reducing reagent-induced activation of adenylate cyclase in the cellular slime mold, Dictyostelium discoideum.

Binding of an intrinsic agonist (cAMP) to specific receptors on the cell surface induces transmembrane signals for activation and desensitization (adaptation and down regulation) of adenylate cyclase in the cellular slime mold, Dictyostelium discoideum. It is generally believed that dithiothreitol (DTT) induces the activation through interaction between the receptor and gradually accumulated cAMP, since DTT is known to inhibit cAMP-phosphodiesterase which degrades cAMP. In the present paper, we investigated the mechanism of activation of adenylate cyclase by the thiol-reducing agents, DTT and 2,3-dimercapto-1-propanol (BAL). We found that BAL activated adenylate cyclase transiently even under conditions where the intrinsic agonist supersaturated the cAMP-receptors and competitively inhibited phosphodiesterase. This result is inconsistent with the generally accepted notion. We conclude that BAL has an independent effect from those of the intrinsic agonist (cAMP) and phosphodiesterase in activation of adenylate cyclase. Since BAL could induce activation just after the activation induced by a supersaturating concentration of the intrinsic agonist had ceased, the independent effect of BAL is not a simple enhancement of the cAMP-induced activation. Our result also suggests that the cAMP-induced adaptation (but not down regulation) suppresses the BAL-induced activation while BAL itself does not induce adaptation to cAMP or BAL. We propose that the thiol-reducing reagent induces or modifies the transmembrane activation signal for adenylate cyclase.     

Regulation of cyclic AMP accumulation in lymphoid cells

We have examined several features of the regulation of cyclic AMP accumulation in lymphoid cells isolated from peripheral blood of human subjects and in the murine T-lymphoma cell line, S49, S49 cells are unique because of the availability of variant clones with lesions in the pathway of cyclic AMP generation and response. We found that human lymphoid cells prepared at 4 degrees C showed substantially greater cyclic AMP accumulation in response to histamine and the beta-adrenergic agonist isoproterenol than did cells prepared at ambient temperature. The muscarinic cholinergic agonist carbamylcholine and peptide hormone somatostatin failed to inhibit cyclic AMP accumulation in human lymphoid cells and treatment with pertussis toxin (which blocks function of Gi, the guanine nucleotide binding protein that mediates inhibition of adenylate cyclase) only minimally increased cyclic AMP levels in these cells. Thus the Gi component of adenylate cyclase appears to play only a small role in modulating cyclic AMP levels in this mixed population of lymphoid cells. Incubation of whole blood with isoproterenol desensitized human lymphocytes to subsequent stimulation with beta agonist. This desensitization was associated with a redistribution of beta-adrenergic receptors such that a substantial portion of the receptors in intact cells could no longer bind a hydrophilic antagonist. Wild-type S49 lymphoma cells showed a similar redistribution of beta-adrenergic receptors after a few minutes' incubation with agonist. Based on studies in S49 variants, this redistribution is independent of components distal to receptors in the adenylate cyclase/cyclic AMP pathway. By contrast, a more slowly developing, agonist-mediated down-regulation of beta-adrenergic receptors was blunted in variants with defective interaction between receptors and Gs, the guanine nucleotide binding protein that mediates stimulation of adenylate cyclase. Unlike results in human lymphoid cells, S49 cells show a prominent inhibition of cyclic AMP accumulation mediated by Gi; this inhibition is promoted by somatostatin and blocked by pertussis toxin. Inhibition by Gi is unable to account for the marked decrease in ability of the diterpene forskolin to maximally stimulate adenylate cyclase in S49 variants having defective Gs. These results emphasize that both Gs and Gi component are important in modulating cyclic AMP accumulation and receptors linked to adenylate cyclase in S49 lymphoma cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Histidine regulation of cyclic AMP metabolism in cultured renal epithelial LLC-PK1 cells.

L-Histidine and imidazole (the histidine side chain) significantly increase cAMP accumulation in intact LLC-PK1 cells. This effect is completely inhibited by isobutylmethylxanthine (IBMX). Histidine and imidazole stimulate cAMP phosphodiesterase activity in soluble and membrane fractions of LLC-PK1 cells suggesting that the IBMX-sensitive effect of these agents to stimulate cAMP formation is not due to inhibition of cAMP phosphodiesterase. Histidine and imidazole but not alanine (the histidine core structure) increase basal, GTP-, forskolin-, and AVP-stimulated adenylate cyclase activity in LLC-PK1 membranes. Two other amino acids with charged side chains (aspartic and glutamic acids) increase AVP-stimulated but neither basal- nor forskolin-stimulated adenylate cyclase activity. This suggests that multiple amino acids with charged side chains can regulate selected aspects of adenylate cyclase activity. To better define the mechanism of histidine regulation of adenylate cyclase, membranes were detergent-solubilized which prevents histidine and imidazole potentiation of forskolin-stimulated adenylate cyclase activity and suggests that an intact plasma membrane environment is required for potentiation. Neither pertussis toxin nor indomethacin pretreatment alter imidazole potentiation of adenylate cyclase. IBMX pretreatment of LLC-PK1 membranes also prevents imidazole to potentiate adenylate cyclase activity. Since IBMX inhibits adenylate cyclase coupled adenosine receptors, LLC-PK1 cells were incubated in vitro with 5'-N-ethylcarboxyamideadenosine (NECA) which produced a homologous pattern of desensitization of NECA to stimulate adenylate cyclase activity. Despite homologous desensitization, histidine and imidazole potentiation of adenylate cyclase was unaltered. These data suggest that histidine, acting via an imidazole ring, potentiates adenylate cyclase activity and thereby increases cAMP formation in cultured LLC-PK1 epithelial cells. This potentiation requires an intact plasma membrane environment, occurs independent of a pertussis toxin-sensitive substrate and of products of cyclooxygenase, and is inhibited by IBMX. This IBMX-sensitive pathway does not involve either inhibition of cAMP phosphodiesterase activity or a stimulatory adenosine receptor coupled to adenylate cyclase.     

Differential effects of temperature on cAMP-induced excitation, adaptation, and deadaptation of adenylate and guanylate cyclase in Dictyostelium discoideum

Extracellular cAMP induces excitation of adenylate and guanylate cyclase in Dictyostelium discoideum. Continuous stimulation with cAMP leads to adaptation, while cells deadapt upon removal of the cAMP stimulus. Excitation of guanylate cyclase by cAMP has a lag time of approximately 1 s; excitation of adenylate cyclase is much slower with a lag time of 30 s. Excitation of both enzyme activities is less than twofold slower at 0 degrees C than at 20 degrees C. Adaptation of guanylate cyclase is very fast (t1/2 = 2.4 s at 20 degrees C), and virtually absent at 0 degrees C. Adaptation of adenylate cyclase is much slower (t1/2 = 110 s at 20 degrees C) but not very temperature sensitive (t1/2 = 290 s at 0 degrees C). At 20 degrees C, deadaptation of adenylate cyclase is about twofold slower than deadaptation of guanylate cyclase (t1/2 = 190 and 95 s, respectively). Deadaptation of adenylate cyclase is absent at 0 degrees C, while that of guanylate cyclase proceeds slowly (t1/2 = 975 s). The results show that excitation, adaptation, and deadaptation of guanylate cyclase have different kinetics and temperature sensitivities than those of adenylate cyclase, and therefore are probably independent processes.     

Dictyostelium discoideum lipids modulate cell-cell cohesion and cyclic AMP signaling.

During Dictyostelium discoideum development, cell-cell communication is mediated through cyclic AMP (cAMP)-induced cAMP synthesis and secretion (cAMP signaling) and cell-cell contact. Cell-cell contact elicits cAMP secretion and modulates the magnitude of a subsequent cAMP signaling response (D. R. Fontana and P. L. Price, Differentiation 41:184-192, 1989), demonstrating that cell-cell contact and cAMP signaling are not independent events. To identify components involved in the contact-mediated modulation of cAMP signaling, amoebal membranes were added to aggregation-competent amoebae in suspension. The membranes from aggregation-competent amoebae inhibited cAMP signaling at all concentrations tested, while the membranes from vegetative amoebae exhibited a concentration-dependent enhancement or inhibition of cAMP signaling. Membrane lipids inhibited cAMP signaling at all concentrations tested. The lipids abolished cAMP signaling by blocking cAMP-induced adenylyl cyclase activation. The membrane lipids also inhibited amoeba-amoeba cohesion at concentrations comparable to those which inhibited cAMP signaling. The phospholipids and neutral lipids decreased cohesion and inhibited the cAMP signaling response. The glycolipid/sulfolipid fraction enhanced cohesion and cAMP signaling. Caffeine, a known inhibitor of cAMP-induced adenylyl cyclase activation, inhibited amoeba-amoeba cohesion. These studies demonstrate that endogenous lipids are capable of modulating amoeba-amoeba cohesion and cAMP-induced activation of the adenylyl cyclase. These results suggest that cohesion may modulate cAMP-induced adenylyl cyclase activation. Because the complete elimination of cohesion is accompanied by the complete elimination of cAMP signaling, these results further suggest that cohesion may be necessary for cAMP-induced adenylyl cyclase activation in D. discoideum.     

Beta-adrenergic receptor desensitization of wild-type but not cyc lymphoma cells unmasked by submillimolar Mg2+

Treatment with low physiological concentrations of epinephrine (5-50 nM) rapidly desensitizes beta-adrenergic stimulation of cAMP formation in S49 wild-type (WT) lymphoma cells. Previous attempts to detect this early phase of desensitization in cell-free assays of adenylate cyclase (EC 4.6.1.1) after intact cell treatment were unsuccessful. We have now found that reducing the Mg2+ concentrations in the adenylate cyclase assays to less than 1.0 mM unmasked this rapid phase of desensitization of the WT cells, and that high Mg2+ concentrations (5-10 mM) largely obscured the desensitization. Submillimolar Mg2+ conditions also revealed a two- to threefold decrease in the affinity of epinephrine binding to the beta-adrenergic receptor after desensitization with 20 nM epinephrine. Detection of 4 beta-phorbol 12-myristate 13-acetate (PMA) desensitization of the WT beta-adrenergic receptor was also dependent on low Mg2+ as measured either by the decrease in epinephrine stimulation of adenylate cyclase or by the reduction in the affinity of epinephrine binding. Unexpectedly, when cyc- cells were pretreated with 50 nM epinephrine, the beta-adrenergic stimulation of reconstituted adenylate cyclase was not desensitized. The characteristics of the Mg2+ effect on epinephrine- and PMA-induced desensitizations suggest a similar mechanism of action with the most likely events being phosphorylations of the beta-adrenergic receptors. Our data indicate that cAMP-dependent protein kinase (EC 2.7.1.37) may play a role in the desensitization caused by low epinephrine concentrations inasmuch as this phase of desensitization did not occur in the cyc-. For the PMA-induced desensitization, the phosphorylation may be mediated by protein kinase C (EC 2.7.1.37).     

Homologous Regulation of the MSH Receptor in Melanoma Cells

Abstract

We have examined the mechanism of homologous regulation of MSH receptor binding and receptor-mediated adenylate cyclase activation in three human and two mouse melanoma cell lines. Pretreatment with α-MSH resulted in a time- and dose-dependent up-regulation of MSH receptors in human D10 and 205 melanoma cells whereas in human HBL and in mouse B16–F1 and Cloudman S91 cells α-MSH induced receptor down-regulation. Up-regulation of receptors was maximal after a 24–h incubation period and an α-MSH concentration of 100 nM (EC₅₀ = 2.4 nM). The increase in α-MSH binding was independent of adenylate cyclase activation and protein synthesis and appeared to be caused by recruitment of spare receptors. The structural requirements of the peptide for triggering this process differed from those found in receptor-binding analyses. Receptor down-regulation was maximal after 12 h and hence more rapid than up-regulation. In B16–F1 cells, 10 nM α-MSH caused the disappearance of 85–90% of the MSH receptors, the EC₅₀ of 0.23 nM lying exactly between that for α-MSH-induced melanogenesis (0.027 nM) and the dissociation constant of receptor binding (1.31 nM). Down-regulation in B16–F1 cells appears to be the consequence of receptor internalization following MSH binding and seems to be initiated during an early step in MSH signalling, preceding the activation of adenylate cyclase and the cAMP signal. Receptor up- and down- regulation were not accompanied by an alteration in affinity to a-MSH, as demonstrated by Scatchard analysis of the binding curves.     

Pertussis toxin inhibits CAMP-induced desensitization of adenylate cyclase in Dictyostelium discoideum

cAMP binds to surface receptors of Dictyostelium discoideum cells, transducing the signal to adenylate cyclase, guanylate cyclase and to chemotaxis. The activation of adenylate cyclase is maximal after 1 min and then declines to basal levels due to desensitization, which is composed of two components: a rapidly reversible adaptation process, and a slowly reversible down-regulation of cAMP receptors. Adaptation is correlated with receptor phosphorylation.The chemotactic response and the cAMP-induced cGMP response were not significantly altered in D. discoideum cells pretreated with pertussis toxin. The initial increase of cAMP levels was identical in control and toxin treated cells, suggesting that activation of adenylate cyclase was also not affected. However, cAMP synthesis continued in toxin treated cells, due to a strongly diminished desensitization. Pertussis toxin inhibited the adaptation of adenylate cyclase stimulation, but not the down-regulation or phosphorylation of the cAMP receptors. Adenylate cyclase in D. discoideum membranes can be stimulated or inhibited by GTP, depending on the conditions used. Pertussis toxin did not affect the stimulation of adenylate cyclase but nullified the inhibition. In membranes from desensitized control cells, stimulation of adenylate cyclase by GTP was lost, whereas inhibition was retained. Stimulation of adenylate cyclase in membranes from desensitized pertussis toxin treated cells was diminished but not absent. These results indicate that receptor phosphorylation is not sufficient for adaptation of adenylate cyclase, and that a pertussis toxin substrate, possibly Gi, is also involved in this process.Abbreviations used ATPS Adenosine 5-0-(3-Thiotriphosphate) - GTPS Guanosine 5-0-(3-thiotri-phosphate) - (Sp)-cAMPS Adenosine 3,5-monophosphorothioate-Sp-isomer - dcAMP 2-deoxyadenosine 3,5-monophosphate - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - DTT Dithiothreitol - buffer A 10 mM KH₂PO₄/Na2HPO₄, pH 6.5 - buffer B 40 mM Hepes/NaOH, 0.5 mM EDTA, 250 mM sucrose, pH 7.7     

cAMP activates adenylate and guanylate cyclase of Dictyostelium discoideum cells by binding to different classes of cell-surface receptors. A study with extracellular Ca2+

cAMP induces a transient increase of cAMP and cGMP levels in Dictyostelium discoideum cells. Fast binding experiments reveal three types of cAMP-binding site (S, H and L), which have different off-rates (t0.5, 0.7-15 s) and different affinities (Kd, 15-450 nM). A time- and cAMP-concentration-dependent transition of H- to L-sites occurs during the binding reaction (Van Haastert, P.J.M. and De Wit, R.J.W. (1984) J. Biol. Chem. 13321-13328). Extracellular Ca2+ had multiple effects on cAMP-binding sites. (i) The number of H + L-sites increased 2.5-fold, while the number of S-sites was not strongly affected. (ii) The Kd of the S-sites was reduced from 16 nM to 5 nM (iii) The conversion of H-sites to L-sites was inhibited up to 80%. The kinetics of the cAMP-induced cAMP accumulation was not strongly altered by Ca2+, but the amount of cAMP produced was inhibited up to 80%. The kinetics of the cAMP-induced cGMP accumulation was strongly altered; maximal levels were obtained sooner, and the Ka was reduced from 15 to 3.5 nM cAMP. Ca2+, Mg2+ and Mn2+ increased the number of binding sites, all with EC50 = 0.5 mM. The S-sites and the cGMP response were modified by equal Ca2+ concentrations and by higher concentrations of Mg2+ and Mn2+ (EC50 are respectively 0.4 mM, 2.5 mM and about 25 mM). The conversion of H- to L-sites and the cAMP response were specifically inhibited by Ca2+ with EC50 = 20 microM. It is concluded that cAMP activates guanylate cyclase through the S-sites; adenylate cyclase is activated by the H + L-sites, in which the appearance of the L-sites during the binding reaction represents the coupling of occupied surface cAMP receptors to adenylate cyclase.     

Desensitization of adenylate cyclase and cyclic AMP flux during the early stages of liver regeneration

Liver regeneration is controlled by a complex network of interactions between hormones, growth factors, and a variety of hepatotrophic factors. Transient increases in cAMP in the early stages of liver regeneration that are necessary for DNA synthesis and subsequent mitosis have been reported; however, studies on the mechanisms that control cellular cAMP levels during liver regeneration, namely adenylate cyclase activity, cAMP-dependent phosphodiesterase activity, and cAMP efflux from the cell, have been generally incomplete. In this study we have shown that although there are three peaks in intracellular cAMP levels in the first 24 hours after partial hepatectomy, the adenylate cyclase activity stimulated by glucagon, prostaglandin E2, adrenaline, and fluoride in vitro decreases with time. KD and BMAX of hepatocyte glucagon and beta receptors were similar to the sham controls. Our results are consistent with a mixed homologous/heterologous desensitization of the adenylate cyclase system. There was also a loss of cAMP-dependent phosphodiesterase activity after partial hepatectomy. We speculate that even though the hormone-stimulated adenylate cyclase system has been desensitized, the system retains the ability to respond to the transient pulses of the variety of hormones secreted after partial hepatectomy and thus raise the intracellular concentration of cAMP. The decrease in cAMP-dependent phosphodiesterase may be necessary to prevent rapid breakdown of cAMP.     

Kinetics and nucleotide specificity of a surface cAMP binding site in Dictyostelium discoideum, which is not down-regulated by cAMP

Dictyostelium cells exhibit four types of kinetically distinct surface cAMP binding sites, the AH, AL, BS, and BSS sites, which are down-regulated during persistent stimulation with cAMP. Although most cAMP-induced responses are subject to desensitization during constant stimulation, some responses, notably the induction of post-aggregative gene expression, require persistent cAMP stimulation. The kinetics and specificity of residual cAMP-binding activity in cells treated for 4 h with micromolar cAMP were investigated. It was found that around 4000 rapidly dissociating binding sites per cell with an affinity of about 300 nM are retained after down-regulation. The nucleotide specificity of the remaining sites was very similar, but not completely identical to the AH, AL and B sites, suggesting that these sites belong to the same class of cell surface cAMP receptors and may be utilized to mediate responses requiring continuous cAMP stimulation.     

In vivo regulation of cyclic AMP phosphodiesterase in Xenopus oocytes. Stimulation by insulin and insulin-like growth factor 1

Cyclic AMP phosphodiesterase activity was measured in vivo after microinjection of [3H]cAMP into intact Xenopus oocytes. This activity was inhibited by extracellular application of methylxanthines, and the dose-dependent inhibition of phosphodiesterase activity correlated with the abilities of isobutylmethylxanthine and theophylline to inhibit oocyte maturation induced by progesterone, with IC50 values of approximately 0.3 and 1.5 mM, respectively. Insulin stimulated in vivo phosphodiesterase activity measured after microinjection of 200 microM [3H]cAMP in a time- and dose-dependent fashion without affecting phosphodiesterase activity measured after microinjection of 2 microM [3H]cAMP. Although progesterone alone had no effect on in vivo phosphodiesterase activity, low concentrations of progesterone (0.01 microM) accelerated the time course of insulin stimulation of both phosphodiesterase activity and oocyte maturation. The EC50 for stimulation of in vivo phosphodiesterase activity by insulin correlated with the IC50 for inhibition of oocyte membrane adenylate cyclase activity measured in vitro (2 and 4 nM, respectively). Twenty-fold higher concentrations of insulin were required to stimulate oocyte maturation. In contrast, insulin-like growth factor 1 stimulated in vivo phosphodiesterase, inhibited in vitro adenylate cyclase, and induced oocyte maturation at concentrations of 0.3-1.0 nM. These results demonstrate a dual regulation of oocyte phosphodiesterase and adenylate cyclase by insulin and insulin-like growth factor 1.     

Inhibitory action of forskolin on adenylate cyclase activity and cyclic AMP generation

In testicular Leydig cells, forskolin causes the expected stimulation of cAMP and testosterone production and potentiates gonadotropin-induced responses, when present in concentrations of 1-10 microM. In addition, when added at lower doses that did not affect cAMP generation and testosterone responses (100 nM), forskolin caused an increase in sensitivity to hormonal stimulation for all cAMP pools (extracellular, intracellular, and receptor-bound) and a 70% reduction in the ED50 for human chorionic gonadotropin (hCG) stimulation of testosterone production. Forskolin-induced increases in receptor-bound cAMP were less effective than those elicited by hCG in stimulating steroidogenesis. In contrast to the well-known stimulatory actions of forskolin, low doses of the diterpene (in the picomolar to nanomolar range) markedly inhibited the production of cAMP and testosterone. Such inhibitory actions of low-dose forskolin were prevented by preincubation of Leydig cells with pertussis toxin before addition of forskolin and/or hCG. Low concentrations of forskolin also inhibited adenylate cyclase activation by GTP and luteinizing hormone, and this effect was prevented by pretreatment of cell membranes with pertussis toxin. These studies have defined the stimulatory effects of forskolin on Leydig-cell cAMP pools, including potentiation of the hormonal increase in receptor-bound cyclic AMP by forskolin, and have provided additional evidence for the functional importance of cAMP compartmentalization during hormonal stimulation of steroidogenesis. We have also demonstrated a novel, high-affinity inhibitory action of forskolin upon adenylate cyclase activity and cyclic AMP generation, an effect that appears to be mediated by the Ni guanine nucleotide regulatory subunit of adenylate cyclase.     

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.     

Phorbol 12-myristate 13-acetate and vasopressin potentiate the effect of corticotropin-releasing factor on cyclic AMP production in rat anterior pituitary cells. Mechanisms of action

The potentiation of corticotropin-releasing factor (CRF)-stimulated cAMP production by vasopressin (VP) in the pituitary cell was investigated by studies on the interaction of CRF, VP, and the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA) on cAMP, adenylate cyclase and phosphodiesterase. Addition of VP or PMA (0.01-100 nM) alone did not alter cellular cAMP content, but markedly increased the effect of 10 nM CRF with ED50 of about 1 nM. Treatment of the cells with 200 ng/ml pertussis toxin for 4 h increased CRF-stimulated cAMP accumulation by 3.2-fold, an effect that was not additive to those of VP and PMA. Incubation of pituitary cells with 2 mM 1-methyl-3-isobutylxanthine increased CRF-stimulated cAMP accumulation and decreased the relative effect of VP and PMA, suggesting that the actions of VP and PMA are partially due to inhibition of phosphodiesterase. This was confirmed by the demonstration of a 30% inhibition of the low-affinity phosphodiesterase activity in cytosol and membranes prepared from cells preincubated with VP or PMA. In intact cells, following [3H]adenine prelabeling of endogenous ATP pools, measurement of adenylate cyclase in the presence of 1-methyl-3-isobutylxanthine showed no effect of VP and PMA alone, but did show a 2-fold potentiation of the effect of CRF. Measurement of adenylate cyclase in pituitary homogenates by conversion of [alpha-32P]ATP to [32P]cAMP showed a paradoxical GTP-dependent inhibition by VP of basal and CRF-stimulated adenylate cyclase activity, suggesting that the VP receptor is coupled to an inhibitory guanyl nucleotide-binding protein. Pertussis toxin pretreatment of the cells prevented the VP inhibition of adenylate cyclase activity observed in pituitary cell homogenates. These findings indicate that besides inhibition of phosphodiesterase, VP has a dual interaction with the pituitary adenylate cyclase system; a direct inhibitory effect, manifested only in broken cells, that is mediated by a receptor-coupled guanyl nucleotide-binding protein, and a physiologically predominant indirect stimulatory effect in the intact cell, mediated by protein kinase C phosphorylation of one of the components of the CRF-activated adenylate cyclase system.     

Increased cyclic AMP content directly correlated with morphological transformation of cells infected with a temperature-sensitive mutant of mouse sarcoma virus

Normal rat kidney cells infected with a cold-sensitive mutant of mouse sarcoma virus [NRK(MSV-lb)] morphologically transform when exposed to adenosine 3':5' cyclic monophosphate (cAMP) at the restrictive temperature. The cAMP-induced morphological changes occur rapidly and are reversible. Agents capable of elevating endogenous levels of cAMP [prostaglandin E1 (PGE1) and cholera toxin (CT)] induced morphological transformation of NRK(MSV-lb) cells at the restrictive temperature that was concentration dependent, potentiated by cAMP phosphodiesterase inhibitors, and not prevented by inhibitors of DNA, RNA, and protein synthesis. Prostaglandin E1 stimulated a transient increase in the intracellular level of cAMP with a concomitant morphological transformation and reversion of cells as cAMP levels decline. The maximum increase is reached by 10 min, followed by a decline to near basal level by 80 min. In contrast, incubation of cells with CT resulted in irreversible morphological transformation and increased levels of cAMP first detectable by 1 hr with maximum levels reached by 24 hr. Heated CT (100 degrees C, 20 min) was without effect. Addition of CT to reverted PGE1-treated cells resulted in morphological transformation suggesting the existence of discrete receptors in NRK(MSV-lb) cells.     

Involvement of cyclic AMP cell surface receptors and G-proteins in signal transduction during slug migration of Dictyostelium discoideum.

The presence of G-proteins, interacting with cAMP surface receptors, was investigated in vegetative cells, aggregation-competent cells, and migrating slugs of Dictyostelium discoideum. Our results indicate that G-proteins are present in all stages. In vegetative cells there is a limited number of cAMP receptors but no effect of GTP tau S on cAMP binding could be detected; in addition, no effect of cAMP on GTP tau S binding or GTPase activity was observed. In both aggregation-competent cells and slugs GTP tau S inhibits cAMP binding, while cAMP stimulates GTP tau S binding and high-affinity GTPase. Since the presence of G-proteins coupled to cAMP receptors could be demonstrated in slugs, the involvement of the effector enzymes adenylate cyclase and phospholipase C was investigated. The results show that adenylate cyclase activity is stimulated by GTP tau S in both stages and that in cells from migrating slugs the Ins(1,4,5)P3 production is increased upon stimulation with cAMP. The possible involvement of G-proteins in signal transduction during the slug stage of D. discoideum is discussed.     

Aminergic and peptidergic amplification of intracellular cyclic AMP levels in a molluscan neural network

1. Serotonin (5-hydroxytryptamine; 5-HT), dopamine (DA), and small cardioactive peptide B (SCPB) can activate adenylate cyclase and increase the intracellular cyclic AMP (cAMP) levels in the Limax procerebrum (PC), with differing time courses and to differing extents. 5-HT and SCPB are potent stimulators of adenylate cyclase, and when both were applied simultaneously, an additive effect was observed. 2. In contrast, DA shows a great variability in the time course of cAMP synthesis and is a weak stimulator. Ergonovine, a DA antagonist, failed to inhibit cyclase activation, indicating that ergonovine-sensitive receptors are absent or ergonovine-sensitive DA receptors are not coupled to adenylate cyclase. 3. 5-HT and SCPB cause a rapid synthesis of cAMP, reaching the maximum 20- to 30-fold increase within a minute. DA's effect is slow in onset and very prolonged, reaching a maximum of only a two- to three-fold increase in the cAMP level. Reasons for variability in DA action are discussed.