Guanine nucleotides protect adenylate cyclase against inhibition by Pb2+

The inhibition of rat liver adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) by Pb²⁺ could be separated into an irreversible and a reversible component.Evidence was obtained that both types of inhibition were due to free Pb²⁺, rather than Pb/ATP, and that Pb²⁺ did not act via the site wherein Mg²⁺ and Mn²⁺ activate the cyclase.Guanine nucleotides strongly counteracted the reversible inhibition of cyclase by Pb²⁺, providing onother example of guanine nucleotide effects on adenylate cyclase function.It is suggested that the Pb²⁺-inhibited cyclase may be of value in the study of guanine nucleotide-cyclase interactions.     

Regulation of calmodulin-sensitive adenylate cyclase by the stimulatory G-protein, Gs

Studies in bovine and rat brain membranes have suggested that calmodulin can potentiate neurotransmitter- and GTP-stimulated adenylate cyclase activities. To examine whether calmodulin and the stimulatory G-protein, Gs, are potentiative at a calmodulin-sensitive adenylate cyclase, Gs was purified from rabbit liver and reconstituted with a partially purified calmodulin-sensitive adenylate cyclase from bovine brain. Activated Gs (G*s) stimulated basal adenylate cyclase activity and enhanced the stimulation by calmodulin. The potentiation of the calmodulin-stimulated adenylate cyclase activity was dose-dependent with respect to G*s concentration. At the highest concentration of G*s tested (3 nM), a 2-fold enhancement of the calmodulin-stimulated adenylate cyclase activity was observed at all concentrations of calmodulin. The synergistic activation of adenylate cyclase by calmodulin and Gs was dependent on the presence of Ca2+ and occurred at physiologically relevant Ca2+ concentrations. The potentiation was not observed when either a nonactivated Gs or a mixture of activated Gi/Go was used. G*s was not able to stimulate or potentiate a calmodulin-stimulated adenylate cyclase purified from membranes pretreated with the nonhydrolyzable GTP analog, guanyl-5'-yl beta,gamma-imidodiphosphate. Photochemical cross-linking of 125I-calmodulin-diazopyruvamide to proteins having an Mr corresponding to the known Mr of adenylate cyclase was not enhanced by G*s. The results demonstrate that the guanyl nucleotide-dependent enhancement of calmodulin-stimulated adenylate cyclase activity is mediated by G*s and suggest that G*s modulates the enzymatic turnover of the calmodulin-stimulated activity.     

Gossypol inhibition of adenylate cyclase

Gossypol, a polyphenolic binaphthalene -dialdehyde reputed to exert contraceptive action in males, reversibly inhibits adenylate cyclase [ATP pyrophosphate lyase (cyclizing), EC 4.6.1.1] in a concentration-dependent manner. In membranes prepared from a variety of organs, the half-maximal inhibitory concentration (IC50) ranges from 75 microM (rat Leydig tumor cells) to 250 microM (rat liver membranes). Kinetic studies using partially purified catalytic subunit isolated from bovine testis show that gossypol is competitive with ATP with an apparent Ki of 110 microM. These data suggest that gossypol inhibition of adenylate cyclase is due to direct interaction at the nucleotide-binding domain of the catalytic subunit of the enzyme.     

Inhibition of adenylate cyclase from luteinized rat ovary by monovalent cations: roles of the stimulatory guanine nucleotide-binding regulatory component and stimulatory hormone receptor

Sodium and other monovalent cations (added as chloride salts) inhibited adenylate cyclase of luteinized rat ovary. Sodium chloride (150 mM) inhibited basal enzyme activity by 20%. Sodium chloride inhibition was enhanced to 34-54% under conditions of enzyme stimulation by guanine nucleotides (GTP and its nonhydrolyzable analog 5'-guanylyl imidodiphosphate), fluoride anion, and agonists (ovine luteinizing hormone (oLH) and the beta-adrenergic catecholamine isoproterenol) acting at stimulatory receptors linked to adenylate cyclase. Sodium chloride inhibition was dependent on salt concentration over a wide range (25-800 mM) as well as the concentrations of GTP and oLH. Inhibition by NaCl was of rapid onset and appeared to be reversible. The order of inhibitory potency of monovalent cations was Li+ greater than Na+ greater than K+. The role of individual components of adenylate cyclase in the inhibitory action of monovalent cations was examined. Exotoxins of Vibrio cholerae and Bordetella pertussis were used to determine respectively the involvement of the stimulatory and inhibitory guanine nucleotide-binding regulatory components (Ns and Ni) in NaCl inhibition. Sodium chloride inhibited cholera toxin-activated adenylate cyclase activity by 29%. Ni did not appear to mediate cation inhibition of adenylate cyclase because pertussis toxin did not attenuate inhibition by NaCl. Enzyme stimulation by agents (forskolin and Mn2+) thought to activate the catalytic component directly was not inhibited by NaCl but was instead significantly enhanced. Sodium chloride (150 mM) increased both the Kd for high-affinity binding of oLH to 125I-human chorionic gonadotropin binding sites and the Kact for oLH stimulation of adenylate cyclase by sevenfold. In contrast, NaCl had no appreciable effect on either isoproterenol binding to (-)-[125I]iodopindolol binding sites or the Kact for isoproterenol stimulation of adenylate cyclase. The results suggest that in luteinized rat ovary monovalent cations uncouple, or dissociate, Ns from the catalytic component and, in a distinct action, reduce gonadotropin receptor affinity for hormone. Dissociation of the inhibitory influence of Ni from direct catalytic activation could account for NaCl enhancement of forskolin- and Mn2+-associated activities. On the basis of these results, the spectrum of divergent stimulatory and inhibitory effects of monovalent cations on adenylate cyclase activities in a variety of tissues may be interpreted in terms of differential enzyme susceptibilities to cation-induced uncoupling of N and catalytic component functions.     

Ni-mediated inhibition of human platelet adenylate cyclase by thrombin

Cefoxitin, a poor substrate of the RTEM beta-lactamase (penicillin amido-beta-lactam hydrolase, EC 3.5.2.6), induces a reversible change in the conformation of the enzyme. The change is manifested in gradual loss of catalytic activity and increased susceptibility to proteolytic inactivation. It is prevented by antibodies, which stabilize the native conformation. By contrast, divalent cations, which have no effect on the native enzyme, delay recovery from the cefoxitin-induced state, presumably by reacting with sites made accessible in the partly unfolded enzyme. Prolonged exposure to excess of cefoxitin causes a similar delay. The kinetic evidence, namely, the initial burst of consumption of cefoxitin and the subsequent gradual recovery of activity with better substrates, appears to be consistent with acylation of the active site by cefoxitin followed by a slower deacylation step [Fisher et al. (1980) Biochemistry 19, 2895-2901]. However, additional evidence leads us to conclude that the kinetics observed reflect deformation of the active site, rather than its blockage, by cefoxitin. Of most significance is the transient change in specificity, i. e. a preferential interaction of the recovering enzyme with substrates which are closest in structure to cefoxitin.     

Guanine nucleotide activation of adenylate cyclase in saponin permeabilized glioma cells

We have compared the regulation of adenylate cyclase activity in membrane fractions from C6 glioma cells and in monolayer cultures of C6 cells that had been permeabilized with saponin. Guanine nucleotides (GTP and GTP gamma S) and isoproterenol increase adenylate cyclase activity in C6 membranes and in permeabilized C6 cells. In C6 membranes, guanine nucleotides activate adenylate cyclase in the presence or absence of isoproterenol; in permeabilized cells, however, guanine nucleotides increase adenylate cyclase activity only in the presence of isoproterenol. We suggest that the properties of the permeabilized cells more closely resemble those of intact cells, and that some component which is present in permeabilized cells but is lost following cell disruption may be important for the normal regulation of adenylate cyclase activity.     

Alpha-adrenergic inhibition of renal cortical adenylate cyclase

Adenylate cyclase in homogenates of rat renal cortex was inhibited by alpha-adrenergic agonists. Inhibition required sodium ion and GTP. A maximum inhibition of 17.8 +/- 1.4% (S.E.M.) was produced by l-epinephrine in the presence of 0.2 M NaCl, 10 microM GTP and 10 microM propranolol. Similar inhibition was produced by l-norepinephrine and alpha-methylnorepinephrine. The EC50 values for l-epinephrine, l-norepinephrine and alpha-methylnorepinephrine were respectively 1.9 +/- 0.7 microM, 2.3 +/- 1.6 microM and 5.1 +/- 1.8 microM. Clonidine was a partial agonist causing 50% as much inhibition as epinephrine. Phenylephrine and methoxamine did not inhibit at concentrations up to 100 microM. Micromolar concentrations of phentolamine and yohimbine prevented the inhibition of adenylate cyclase by epinephrine. However, prazosin was ineffective. Thus the adenylate cyclase coupled alpha-receptors have alpha-2 specificity. Inhibition of adenylate cyclase by alpha-adrenergic agonists was not observed in homogenates of renal medulla.     

Guanine nucleotide regulation of adenylate cyclase in permeabilized cells of Saccharomyces cerevisiae

Adenylate cyclase in permeabilized cells of Saccharomyces cerevisiae was examined. Among various permeabilization procedures, including organic solvents, detergents and other reagents, dimethylsulfoxide (DMSO) and digitonin treatments resulted in the highest recovery of adenylate cyclase activity. Incubation of cells at 30 degrees C with digitonin at 0.01% to 0.1%, or DMSO at 20% to 40% for 15 to 30 min gave optimal adenylate cyclase activity. The enzyme activity in digitonin-permeabilized cells could be supported only by Mn2+, whereas Mg2+ with or without guanine nucleotides did not support cyclase activity. DMSO-permeabilized cells exhibit efficient Mn2+- and Mg2+/Gpp[NH]p-dependent stimulation. Furthermore, digitonin added to yeast membranes at a 1:50 detergent to protein ratio (w/w) abolishes guanyl nucleotide regulation without significantly affecting the Mn2+-supported cyclase activity. The superiority of DMSO is further supported by the fact that recovery of adenylate cyclase activity is better in the DMSO-treated cells than in the digitonin-treated cells. DMSO most probably causes less disturbance of the fabric of the native cell. We conclude that digitonin, but not DMSO, uncouples the catalytic unit of adenylate cyclase from the regulatory GTP binding (ras) proteins.     

Induction of catecholamine-responsive adenylate cyclase in HeLa cells by sodium butyrate. Evidence for a more efficient stimulatory regulatory component

HeLa cells, when exposed to 5 mM sodium butyrate, increased their responsiveness to isoproterenol and their number of beta-receptors. As untreated HeLa cells have a substantial number of receptors but respond poorly to isoproterenol, the effect of butyrate could be due to quantitative or qualitative changes in beta-receptors or other components of the adenylate cyclase system. Receptors were analyzed by membrane/membrane and membrane/cell fusion techniques. HeLa donor membranes, treated to inactivate regulatory and catalytic components of adenylate cyclase, were fused with Fc cells, which lack beta-receptors. Isoproterenol-stimulated adenylate cyclase activity in the fusates was proportional to the number of receptors present. There appeared to be only quantitative but not qualitative differences in beta-receptors from control and butyrate-treated HeLa. Prostaglandin E1 receptors from neuroblastoma cell membranes were similarly coupled to HeLa adenylate cyclase. The hybrid prostaglandin E1-stimulated activity was lower when acceptor membranes were from control HeLa than when they were from butyrate-treated HeLa cells. These results suggested that butyrate was altering the ability of the regulatory component to interact with receptors. HeLa membranes were extracted with sodium cholate and the extracts used to reconstitute effector-stimulated adenylate cyclase activity in S49 cyc- membranes, which lack a functional regulatory component. Whereas extracts from control and butyrate-treated HeLa were equally effective in restoring NaF-stimulated activity in cyc- membranes, extracts from control HeLa were less efficient in reconstituting isoproterenol- and prostaglandin E1-stimulated activities. We conclude that the poor response of control HeLa to beta-agonists is due to a limited activity of the regulatory component but not the receptor. Butyrate induces quantitative changes in the receptor and qualitative changes in the regulatory component that facilitate its ability to couple to receptors but do not alter its ability to interact with the catalytic component of adenylate cyclase.     

Gi protein mediates adenylate cyclase inhibition by neurohypophyseal hormones in fish gill.

Adenylate cyclase activity was measured on membrane fractions from the gill epithelium of rainbow trout Salmo gairdneri. Basal and glucagon-stimulated activities responded negatively to homologous neurohypophyseal peptides (arginine-vasotocin and isotocin). This inhibitory effect was totally abolished in the presence of pertussis toxin (IAP). The guanine nucleotide dependence of the enzyme was further explored by using GTP, GDP, and their stable analogs Gpp(NH)p, GTP gamma S, and GDP beta S. The results suggest that neurohypophyseal peptides at low concentrations inhibit the adenylate cyclase system directly by way of a Gi-protein, thus implying the intervention of a new type of membrane receptor for these hormones in fish gills.     

Uncoupling of alpha-adrenoceptor-mediated inhibition of human platelet adenylate cyclase by N-ethylmaleimide

Human platelet adenylate cyclase is stimulated by prostaglandin E1 (PGE1) and is inhibited by epinephrine via alpha-adrenoceptors. Both agonists, epinephrine more than PGE1, increase the activity of a low Km GTPase in platelet membranes. Pretreatment of intact platelets or platelet membranes with the sulfhydryl reagent, N-ethylmaleimide (NEM), abolished the inhibition of the adenylate cyclase and the concomitant stimulation of the GTPase by epinephrine. In contrast, stimulation of the adenylate cyclase by PGE1 was not affected or even increased by NEM pretreatment; only at high NEM concentrations were both basal and PGE1-stimulated activities decreased. Similarly, the PGE1-induced activation of the low Km GTPase was not or was only partially reduced by NEM. Adenylate cyclase activation by stable GTP analogs, NaF, and cholera toxin was also not decreased by NEM pretreatment. Exposure of intact platelets to NEM did not reduce alpha-adrenoceptor number and affinities for agonists and antagonists, as determined by [3H]yohimbine binding in platelet particles. The data indicate that NEM uncouples alpha-adrenoceptor-mediated inhibition of platelet adenylate cyclase, leaving the receptor recognition site and the adenylate cyclase itself relatively intact. Although the effect of NEM may be based on a reaction with the alpha-adrenoceptor site interacting with a coupling component, the selective loss of the adenylate cyclase inhibition together with an even increased stimulation of the enzyme by PGE1 suggests that there are two at least partially distinct regulatory sites involved in opposing hormonal regulations of adenylate cyclase activity, with that involved in hormonal inhibition being highly susceptible to inactivation by NEM.     

Receptor-mediated elevation of adenylate cyclase by luteinizing hormone in Candida albicans

Human luteinizing hormone (hLH) and the GTP analogue guanosine 5'-O-(3-thio)triphosphate stimulated morphogenesis in the dimorphic fungal pathogen Candida albicans. hLH bound specifically to subcellular fractions from C. albicans and stimulated adenylate cyclase activity in C. albicans microsomes. We provide the first demonstration of guanine-nucleotide-binding proteins (G-proteins) in C. albicans, and propose that the stimulation of C. albicans morphogenesis by hLH is mediated by a receptor-coupled adenylate cyclase system involving G-proteins.     

Activation of adenylate cyclase by molybdate.

The effect of molybdate on adenylate cyclase (EC 4.6.1.1) in rat liver plasma membranes has been examined. The apparent K alpha for molybdate activation of the enzyme is 4.5 mM, and maximal, 7-fold stimulation is achieved at 50 mM. The observed increase in cAMP formation in the adenylate cyclase assay is not due to: (a) an inhibition of ATP hydrolysis; (b) a molybdate-catalyzed conversion of ATP to cAMP; (c) an inhibition of cAMP hydrolysis; or (d) an artifact in the isolation of cAMP formed in the reaction. Molybdate activation of adenylate cyclase is a general phenomenon exhibited by the enzyme in brain, cardiac, and renal tissue homogenates and in erythrocyte ghosts. However, like fluoride and guanyl-5'-yl imidodiphosphate (Gpp(NH)p), molybdate does not activate the soluble rat testicular adenylate cyclase. Molybdate is a reversible activator of adenylate cyclase. Activation is not due to an increase in ionic strength and is independent of the salt used to introduce molybdate. Molybdate does not activate adenylate cyclase previously stimulated with Gpp(NH)p or fluoride. At concentration greater than 20 mM, molybdate inhibits fluoride-stimulated adenylate cyclase, and at concentrations greater than 100 mM, molybdate stimulation of basal adenylate cyclase activity is diminished.     

Guanine nucleotides regulate the effect of substance P on striatal adenylate cyclase of the rat.

Substance P was incubated in an adenylate cyclase assay of a particulate fraction of caudate-putamen tissue of the rat in order to examine the effect of the peptide on D-1 receptor coupled adenylate cyclase in vitro. Substance P did not influence basal adenylate cyclase activity or the stimulation of the enzyme by dopamine. No influence of substance P was seen on the effects of calcium and magnesium chloride as a cofactor of adenylate cyclase. Also the inhibition of adenylate cyclase activity by the dopamine antagonist fluphenazine was not influenced by substance P. However, substance P was able to enhance cyclic AMP formation in the presence of guanosine-imidodiphosphate (Gpp(NH)p), whereas the stimulatory effect of guanosine-triphosphate (GTP) was inhibited by substance P. In our study we suggest that substance P interacts with the guanine nucleotide regulatory subunit without directly affecting D-1 dopamine receptors in the caudate-putamen of the rat.     

A dose-response study of forskolin, stimulatory hormone, and guanosine triphosphate analog on adenylate cyclase from several sources

We have described relationships involving forskolin stimulation of adenylate cyclase (AC) from a variety of sources and the potentiation of forskolin effects by stimulatory hormones (glucagon, ACTH, and epinephrine) and beta, gamma-imidoguanosine 5'-triphosphate (Gpp(NH)p). The effects on AC were examined using membrane preparations of rabbit adipocytes, rat adipocytes, rat erythrocytes, and rat liver. Also examined was the AC of liver membranes of rat pretreated with pertussis toxin as well as that solubilized from rat liver membranes. Maximal forskolin stimulation of AC in all preparations studied revealed a consistent 10-fold increase in AC activity. The EC50 for forskolin was 10 microM for rat liver, 15 microM for rabbit and rat adipocytes and 17 microM for rat erythrocyte AC stimulation. In all cases the AC activity attained by forskolin stimulation was further enhanced by stimulatory hormones in a dose-dependent manner. Furthermore, a combination of all three activators (forskolin, stimulatory hormone, and Gpp(NH)p) resulted in an even greater overall stimulation to levels ranging from 25- to 30-fold over unstimulated activity levels. In the presence of saturating levels of each stimulatory hormone and Gpp(NH)p, the EC50 for forskolin diminished markedly to the range of 0.5 to 4.0 microM. In the absence of any apparent tissue specificity for forskolin stimulation, the general pattern of these results further implicates the catalytic site of the AC complex as the site of forskolin activation. Furthermore, activation of additional components of the complex by Gpp(NH)p and tissue specific hormones may further influence the AC activity and thereby potentiate the stimulation by forskolin.