Effect of thioacetamide on cytochrome P-450 synthesis in rat liver.

Both calcitonin and prostaglandin E2 (PGE2) stimulate adenylate cyclase activity in the human breast cancer cell line (T 47D). The maximum cyclic AMP response to calcitonin exceeds that of PGE2. When maximal concentrations of the two hormones were added simultaneously to the cells, the amount of cyclic AMP generated was less than that seen with calcitonin alone. When cells were treated with the protein toxin of Bordetella pertussis (islet-activating protein; IAP) which inactivates the inhibitory regulatory component (Ni) of adenylate cyclase, there was no change in basal or calcitonin-responsive adenylate cyclase in intact cells. However, the PGE2 response was augmented at all dose levels, and this effect was dependent on the concentration of IAP. Moreover, in cells pretreated with IAP, simultaneous addition of PGE2 and calcitonin resulted in additivity rather than in inhibition of cyclic AMP production. The additivity of the response to calcitonin and PGE2 after IAP treatment implies activation of separate pools of adenylate cyclase catalytic subunit by the two hormones. These data are consistent with a model in which calcitonin acts on adenylate cyclase in T 47D cells through stimulatory regulatory components alone, whereas PGE2 acts on the same cells through both stimulatory and inhibitory components. The Ni input can limit the maximum effect of PGE2 and is capable of limiting calcitonin effects when the two agonists are used simultaneously.     

Loss of the inhibitory function of the guanine nucleotide regulatory component of adenylate cyclase due to its ADP ribosylation by islet-activating protein, pertussis toxin, in adipocyte membranes

Adenylate cyclase of rat adipocyte membranes exhibited dual responses in a strictly GTP-dependent manner; an activation took place in the presence of certain receptor agonists such as isoproterenol or secretin, whereas an inhibitory phase was observed with other agonists such as prostaglandin E1 or purine-modified adenosine as well as with the stimulatory agonists at higher GTP concentrations. Treatment of membrane donor cells with islet-activating protein (IAP), pertussis toxin, abolished the inhibitory phase while preserving the activatory phase. This unique action of IAP was associated with ADP-ribosylation of a membrane Mr = 41,000 protein. In contrast, the inhibitory phase was preserved in membranes from cholera toxin-treated cells. Monophasic and persistent activation of the cyclase was provoked by guanyl-5'-yl beta,gamma-imidodiphosphate. The time lag normally observed for the guanyl-5'-yl beta,gamma-imidodiphosphate activation was decreased by isoproterenol or cholera toxin but was not altered by IAP treatment. Our conclusion is that the sole site of IAP action is the guanine nucleotide regulatory protein (Ni) that is required for transmission of inhibitory signals from receptors to the catalytic unit of adenylate cyclase; the function of Ni is lost upon IAP-catalyzed ADP ribosylation of the Mr = 41,000 protein which appears to be an active subunit of Ni. A possibility is discussed that rather diverse effects of IAP so far reported with various cell types are accounted for in terms of such interference with the function of Ni.     

Modulation of adenylate cyclase of human platelets by phorbol ester. Impairment of the hormone-sensitive inhibitory pathway

The influence of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), a direct activator of the Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C), was studied on regulation of human platelet adenylate cyclase. Intact platelets were pretreated with the phorbol ester and, thereafter, membranes were prepared and the regulation of the hormone-sensitive adenylate cyclase in these membranes was studied. The following data were obtained: The TPA treatment applied had apparently no effect on the activity of the catalytic moiety of the platelet adenylate cyclase nor on the stimulatory NS protein nor on stimulatory hormone receptors (prostaglandin E1) and the mutual interactions of these components of the stimulatory hormone-sensitive pathway. However, the TPA treatment of intact platelets largely impaired the GTP-dependent, hormone-sensitive inhibitory pathway to the adenylate cyclase, involving the inhibitory Ni protein. The pretreatment led to a large reduction or loss of adenylate cyclase inhibition by GTP itself and by the inhibitory agonists, epinephrine and thrombin, inhibiting the untreated enzyme via separate receptors by an Ni-mediated process. In contrast, platelet adenylate cyclase inhibition not involving the Ni protein was not affected by the TPA treatment. The observed effects of TPA were very rapid in onset and were not shared by a derivative of TPA which did not activate protein kinase C. The data obtained suggest than protein kinase C activated by the phorbol ester interferes with the platelet adenylate cyclase system, leading to a specific alteration of the Ni-protein-mediated signal transduction to the adenylate cyclase.     

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

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

Adenylate cyclase inhibition and GTPase stimulation by somatostatin in S49 lymphoma cyc- variants are prevented by islet-activating protein

cyc--Variants of S49 lymphoma cells are defective in the stimulatory guanine nucleotide site of the adenylate cyclase but contain an inhibitory site. Treatment of cyc- cells with islet-activating protein (IAP), which causes ADP-ribosylation of an Mr 40 000 polypeptide in cyc- membranes, abolishes adenylate cyclase inhibition by GTP and the peptide hormone, somatostatin, but not that induced by GTP gamma S. Furthermore, somatostatin-induced stimulation of GTP hydrolysis is lost. Thus, the data indicate that IAP interferes with the adenylate cyclase system by an action at the inhibitory guanine nucleotide site.     

The Escherichia coli adenylate cyclase complex. Stimulation by potassium and phosphate

In Escherichia coli, adenylate cyclase activity in toluene-treated cells can be inhibited by glucose while the activity in a broken cell preparation cannot. Adenylate cyclase activity in the permeabilized but not in broken cells is stimulated somewhat specifically and additively by potassium and phosphate. Kinetic studies show sigmoid substrate-velocity curves for the toluene-treated cells but hyperbolic curves for the broken cells. The stimulatory effects of potassium and phosphate on adenylate cyclase activity in tolulene-treated cells are associated with increases in the Vmax and Km for ATP. While the enzyme activity in toluene-treated cells shows a preference for magnesium over manganese, the reverse is observed in broken cells. Stimulation of adenylate cyclase activity in toluene-treated cells requires the presence of the proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The PTS proteins can be phosphorylated in a P-enolpyruvate-dependent reaction. The stimulatory effects of ions will not occur if the PTS proteins are not phosphorylated. Since potassium phosphate stimulates both adenylate cyclase and PTS activities in toluene-treated cells, it is proposed that the effect of potassium phosphate on adenylate cyclase activity is mediated through an effect on the PTS. A model for dual regulation by glucose of adenylate cyclase activity is proposed. This model involves regulation of both the condition of the PTS proteins as well as the cellular concentration of phosphate.     

G-protein-mediated regulation of the insulin-responsive glucose transporter in isolated cardiac myocytes.

Isolated muscle cells from adult rat heart were used to study the involvement of G-proteins in the regulation of the glucose transporter by insulin and isoprenaline. Efficient modification of G-protein functions was established by measuring isoprenaline-stimulated cyclic AMP production, viability and ATP content after treating the cells with cholera toxin and pertussis toxin for 2 h. Under these conditions cholera toxin decreased the stimulatory action of insulin on 3-O-methylglucose transport by 56%, but pertussis toxin had no effect. Basal transport was not affected by toxin treatment. Isoprenaline increased 3-O-methylglucose transport by 63%. This effect was not mimicked by dibutyryl cyclic AMP, but was completely blocked by cholera toxin. Streptozotocin-diabetes abolished isoprenaline action and decreased stimulation of transport by 64%. Concomitantly, cholera-toxin sensitivity of glucose transport was lost in cells from diabetic animals. This was paralleled by a large decrease (87 +/- 4%) in mRNA expression of the insulin-regulatable glucose transporter, as shown by Northern-blot analysis of RNA isolated from cardiomyocytes of diabetic rats. These data suggest a functional association between the insulin-responsive glucose transporter and a cholera-toxin-sensitive G-protein mediating stimulation by insulin and isoprenaline.     

Glycogen synthesis stimulation by adenylate cyclase inhibition in rat epididymal adipocytes

The effects of adenylate cyclase inhibition on the transport of glucose and fructose and their incorporation into glycogen were investigated in order to assess the extent to which lowered cAMP levels can take part in the various components of glycogen synthesis regulation in isolated rat epididymal adipocytes. The dose-response characteristics of (R)-N-(2-phenylisopropyl)adenosine (PIA), a potent and specific adenylate cyclase inhibitor, on glycogen synthesis were compared with those effectively inhibiting lipolysis, a measure of functional cAMP levels. PIA had no effect on basal glucose or fructose transport but stimulated glucose and fructose incorporation into glycogen. Their respective incorporation was 10 and 69% of that achieved in the presence of insulin. These effects of PIA were shown to be in part the result of increased glycogen synthase I activity. PIA was 20% as effective as insulin in this action. Thus, were insulin to lower cAMP levels and/or inhibit cAMP-dependent protein kinase, this action would be irrelevant to glucose transport but would contribute to the stimulation of glycogen metabolism. However, an additional mechanism(s) involving neither increased glucose transport nor lowered cAMP levels is required to account for the full action of insulin. Fat cells in the absence of medium glucose and in the presence of 10(-7) M PIA and adenosine deaminase constitute a system functionally depleted of cAMP where this mechanism can be studied in isolation.     

Guanine nucleotide activation and inhibition of adenylate cyclase as modified by islet-activating protein, pertussis toxin, in mouse 3T3 fibroblasts

Guanine nucleotide regulation of membrane adenylate cyclase activity was uniquely modified after exposure of 3T3 mouse fibroblasts to low concentrations of islet-activating protein (IAP), pertussis toxin. The action of IAP, which occurred after a lag time, was durable and irreversible, and was associated with ADP-ribosylation of a membrane Mr = 41,000 protein. GTP, but not Gpp(NH)p, was more efficient and persistent in activating adenylate cyclase in membranes from IAP-treated cells than membranes from control cells. GTP and Gpp(NH)p caused marked inhibition of adenylate cyclase when the enzyme system was converted to its highly activated state by cholera toxin treatment or fluoride addition, presumably as a result of their interaction with the specific binding protein which is responsible for inhibition of adenylate cyclase. This inhibition was totally abolished by IAP treatment of cells, making it very likely that IAP preferentially modulates GTP inhibitory responses, thereby increasing GTP-dependent activation and negating GTP-mediated inhibition of adenylate cyclase.     

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.     

Cholera toxin ADP-ribosylates the islet-activating protein substrate in adipocyte membranes and alters its function

In adipocyte membranes, cholera toxin may ADP-ribosylate the islet-activating protein (IAP) substrate, under certain conditions. Covalent modification is maximal in the absence of a guanosine triphosphate; in the presence of 5'-guanylylimidodiphosphate, incorporation of [32P]ADP-ribose is markedly reduced. ADP-ribosylation by cholera toxin has similar functional consequences as does IAP-mediated modification, i.e. the biphasic response of isoproterenol-stimulated adenylate cyclase to GTP and the inhibition by N6-phenylisopropyladenosine is abolished, and only the stimulatory phase remains. In contrast, membranes treated with cholera toxin in the presence of GTP display both the stimulatory and inhibitory responses to GTP. The binding of the adenosine analog [3H]N6-phenylisopropyladenosine is increased in the presence of GTP. Treatment of the membranes with IAP, but not with cholera toxin in the absence of GTP, reverses this GTP effect on [3H]N6-phenylisopropyladenosine binding. However, [3H]N6-phenylisopropyladenosine binding is still sensitive to GTP in membranes treated with cholera toxin in the presence of GTP. In adipocyte and cerebral cortical membranes, the IAP substrate appears as a 39,000/41,000-Da doublet which does not appear to reflect protease activity. On two-dimensional polyacrylamide gels, these two proteins migrate with approximate pI values 6.0 and 5.6, respectively. Although both behave similarly under all conditions explored in this study, it is unknown whether both, or only one, are involved in inhibition of adenylate cyclase activity. These results extend the already striking homology between the adenylate cyclase complex and the visual system. Ni, as well as transducin, may be ADP-ribosylated by cholera toxin and by IAP, and, in all cases, there are functional consequences.     

Inhibition of glucose transport by cyclic GMP in cardiomyocytes

Recent evidence points to a potential role of cyclic GMP (cGMP) in the control of cardiac glucose utilization. The present work examines whether the glucose transport system of cardiac myocyte is a site of this cGMP-dependent regulation. Treatment of isolated rat cardiomyocytes (for 10 min) with the membrane-permeant cGMP analogue 8-(4-chlorophenylthio)-cGMP (8-p-CPT-cGMP, 200 microM) caused a decrease in glucose transport in non-stimulated (basal) myocytes, as well as in cells stimulated with insulin or with the mitochondrial inhibitor oligomycin B by up to 40%. An inhibitory effect was also observed with another cGMP analogue (8-bromo-cGMP), and in cells stimulated by hydrogen peroxide or anoxia. In contrast, 8-p-CPT-cAMP (200 microM), or the beta-adrenergic agonist isoprenaline (which increases cAMP levels) did not depress glucose transport, and even potentiated the effect of insulin. Blockade of endogenous cGMP formation with the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM) significantly increased basal and insulin-dependent glucose transport (by 25%), whereas addition of the guanylate cyclase activator 3-(5'-hydroxymethyl-2'furyl)-1-benzylindazol (YC-1, 30 microM) produced a depression of glucose transport (by 20%). Confocal laser scanning microscopic studies revealed that cGMP partially prevents the insulin-induced redistribution of the glucose transporter GLUT4 from intracellular stores to the cell surface. These observations suggest that the glucose transport system of cardiomyocytes represents a metabolic target of inhibition by cGMP, and that this regulation occurs at the level of the trafficking of glucose transporters.     

The inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. Subunit dissociation and guanine nucleotide-dependent hormonal inhibition

The inhibitory and stimulatory guanine nucleotide-binding regulatory components (Gi and Gs) of adenylate cyclase both have an alpha X beta subunit structure, and the beta (35,000 Da) subunits are functionally indistinguishable. Gi and Gs both dissociate in the presence of guanine nucleotide analogs or Al3+, Mg2+, and F- in detergent-containing solutions. Several characteristics of Gi- and Gs-mediated regulation of adenylate cyclase activity have been studied in human platelet membranes. The nonhydrolyzable analog of GTP, guanosine-5'-(3-O-thio)triphosphate (GTP gamma S) mimics GTP-dependent hormonal inhibition or stimulation of adenylate cyclase under appropriate conditions. This inhibition or stimulation follows a lag period. The combined addition of epinephrine or prostaglandin E1 with GTP gamma S results in the immediate onset of steady state inhibition or activation. The effects of the GTP analog are essentially irreversible. Fluoride is also an effective inhibitor of prostaglandin E1-stimulated adenylate cyclase, while it markedly stimulates the basal activity of the enzyme. The addition of the resolved 35,000-Da subunit of Gi to membranes results in inhibition of adenylate cyclase, and the resolved 41,000-Da subunit has a stimulatory effect on enzymatic activity. The inhibitory action of the 35,000-Da subunit is almost completely abolished in membranes that have been irreversibly inhibited by GTP gamma S plus epinephrine; this irreversible inhibition is almost completely relieved by the 41,000-Da subunit. Detergent extracts of membranes that have been treated with GTP gamma S plus epinephrine contain free 35,000-Da subunit. The 41,000-Da subunit of Gi contained in such extracts has a reduced ability to be ADP-ribosylated by islet-activating protein (IAP), which implies that this subunit is in the GTP gamma S-bound form. The irreversible inhibition of adenylate cyclase caused by GTP gamma S (plus epinephrine) in membranes is highly correlated with the liberation of free 35,000-Da subunit activity and is inversely related to the 41,000-Da IAP substrate activity in detergent extracts prepared therefrom. The increase in free 35,000-Da subunit activity in extracts and the inhibition of adenylate cyclase activity in GTP gamma S (plus epinephrine)-treated membranes are both markedly inhibited by treatment with IAP.(ABSTRACT TRUNCATED AT 400 WORDS)     

The mode of action of chloride on rabbit heart adenylate cyclase

The mechanism by which chloride stimulates adenylate cyclase was investigated. Depletion of GDP increased basal adenylate cyclase activity and reduced the stimulation by isoprenaline. Restoration of bound GDP partially reversed these effects. Chloride stimulated cyclase activity by the same proportion in control, GDP-depleted and GDP-restored preparations, as did Gpp(NH)p. Fluoride increased adenylate cyclase activity to the same final level in both GDP-depleted and GDP-restored membranes; addition of Gpp(NH)p as well as fluoride had no further effect. Solubilisation of adenylate cyclase reduced the stimulatory effect of Gpp(NH)p only slightly, but greatly attenuated the activation by chloride. We conclude that chloride does not stimulate cyclase activity by an action on GDP exchange. Activation by chloride may be due to a disrupting or chaotropic effect on membrane/protein interactions.     

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.     

Dual pathways of receptor-mediated cyclic GMP generation in NG108-15 cells as differentiated by susceptibility to islet-activating protein, pertussis toxin

The cellular cGMP content increased in response to a variety of receptor agonists, which activate [e.g., prostaglandin (PG) E1, E2, and F2 alpha] or inhibit (e.g., alpha-adrenergic, muscarinic, and opiate agonists) adenylate cyclase in neuroblastoma X glioma hybrid NG108-15 cells. The responses were additive when PGF2 alpha and enkephalin were mixed. The inhibitory guanine nucleotide regulatory protein (Ni) is involved in adenylate cyclase inhibition; this function of Ni is lost when it is ADP-ribosylated by islet-activating protein (IAP), pertussis toxin [H. Kurose, T. Katada, T. Amano, and M. Ui (1983) J. Biol. Chem. 258, 4870-4875]. The cGMP rise induced by stimulation of the receptors linked to adenylate cyclase inhibition was also diminished by IAP; the time course and dose response for the IAP-induced diminution were the same between adenylate cyclase inhibition and cGMP generation. Ni thus appears to mediate guanylate cyclase activation as well as adenylate cyclase inhibition initiated via the same receptors. Melittin also increased cGMP. No additivity was shown when enkephalin and melittin were combined, suggesting that phospholipase A2 might play a role in Ni-mediated guanylate cyclase activation. On the other hand, the PGF2 alpha-induced cGMP rise was associated with increased incorporation of 32Pi into phosphatidylinositol; was not affected by cholera toxin, IAP or forskolin; and showed no additivity when combined with A23187, which increased cGMP by itself. PGs would occupy receptors linked to phosphatidylinositol breakdown, thereby increasing the availability of intracellular Ca2+, which is responsible for guanylate cyclase activation. Thus, dual pathways are proposed for a receptor-mediated cGMP rise in NG108-15 cells.     

Altered effect of insulin and catecholamines in brown adipose tissue of cold-acclimated rats.

Data are presented indicating that in brown adipose tissue (BAT) of cold-acclimated (CA), but not cold-exposed (CE) rats, there was an alteration in the relative response to catecholamines and insulin as evidenced by increased binding of alprenolol and decreased binding of insulin to plasma membrane enriched fractions. In addition, the stimulatory effect of insulin on glucose incorporation into glycogen and its inhibitory action on adenylate cyclase activity were both blunted in the CA tissues. It is proposed that shifts in the capacity of BAT to respond to catecholamines and insulin may be involved in the mechanism of cold acclimation.     

Does the guanine nucleotide regulatory protein Ni mediate progesterone inhibition of Xenopus oocyte adenylate cyclase?

In Xenopus laevis oocytes progesterone is able to inhibit directly the plasma membrane adenylate cyclase activity and induce reinitiation of meiotic maturation. To determine whether progesterone inhibition is mediated by the inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase, Ni, the effect of the Bordetella pertussis toxin (IAP) and limited proteolysis on progesterone action in oocytes was investigated. Treatment of oocyte membranes with islet activating protein (IAP) in the presence of [32P]NAD led to incorporation of radiolabel into a 41 000-dalton membrane protein. However, exposure of isolated oocytes to 100 ng/ml IAP for up to 24 h, or oocyte membranes with concentrations of toxin as high as 100 micrograms/ml, had no effect on either progesterone inhibition of adenylate cyclase or induction of maturation. Similarly, limited alpha-chymotrypsin proteolysis of oocyte membranes failed to modify progesterone-induced inhibition of adenylate cyclase. In contrast, inhibition of human platelet adenylate cyclase by epinephrine, acting via a GTP-dependent, alpha 2-adrenergic receptor-mediated pathway, is almost completely abolished by both IAP treatment and limited proteolysis of platelet membranes. These data indicate that unlike attenuation of platelet enzyme activity, the inhibition of adenylate cyclase in oocyte membranes by progesterone does not occur via a classical Ni-mediated pathway.     

Receptor-mediated inhibition of adenylate cyclase and stimulation of arachidonic acid release in 3T3 fibroblasts. Selective susceptibility to islet-activating protein, pertussis toxin

Thrombin exhibited diverse effects on mouse 3T3 fibroblasts. It (a) decreased cAMP in the cell suspension, (b) inhibited adenylate cyclase in the Lubrol-permeabilized cell suspension in a GTP-dependent manner, increased releases of (c) arachidonic acid and (d) inositol from the cell monolayer prelabeled with these labeled compounds, (e) increased 45Ca2+ uptake into the cell monolayer, and (f) increased 86Rb+ uptake into the cell monolayer in a ouabain-sensitive manner. Most of the effects were reproduced by bradykinin, platelet-activating factor, and angiotensin II. The receptors for these agonists are thus likely to be linked to three separate effector systems: the adenylate cyclase inhibition, the phosphoinositide breakdown leading to Ca2+ mobilization and phospholipase A2 activation, and the Na,K-ATPase activation. Among the effects of these agonists, (a), (b), (c), and (e) were abolished, but (d) and (f) were not, by prior treatment of the cells with islet-activating protein (IAP), pertussis toxin, which ADP-ribosylates the Mr = 41,000 protein, the alpha-subunit of the inhibitory guanine nucleotide regulatory protein (Ni), thereby abolishing receptor-mediated inhibition of adenylate cyclase. The effects (a), (c), (d), and (e) of thrombin, but not (b), were mimicked by A23187, a calcium ionophore. The effects of A23187, in contrast to those of receptor agonists, were not affected by the treatment of cells with IAP. Thus, the IAP substrate, the alpha-subunit of Ni, or the protein alike, may play an additional role in signal transduction arising from the Ca2+-mobilizing receptors, probably mediating process(es) distal to phosphoinositide breakdown and proximal to Ca2+ gating.     

The inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. Properties and function of the purified protein

Treatment of membranes with islet activating protein (IAP), a toxin from Bordetella pertussis, results in abolition of GTP-dependent, receptor-mediated inhibition of adenylate cyclase. This appears to result from IAP-catalyzed ADP-ribosylation of a 41,000-Da membrane-bound protein. A protein with 41,000- and 35,000-Da subunits has been purified from rabbit liver membranes as the predominant substrate for IAP. This protein has now been shown to be capable of regulating membrane-bound adenylate cyclase activity of human platelets under various conditions. The characteristics of the actions of the IAP substrate are as follows. 1) Purified 41,000/35,000-Da dimer is capable of restoring the inhibitory effects of guanine nucleotides and the alpha 2-adrenergic agonist, epinephrine, on the adenylate cyclase activity of IAP-treated membranes. 2) The subunits of the dimer dissociate in the presence of guanine nucleotide analogs or A1(3+), Mg2+, and F-. The 41,000-Da subunit has a high affinity binding site for guanine nucleotides. 3) The resolved 35,000-Da subunit of the dimer mimics guanine nucleotide- and epinephrine-induced inhibition of adenylate cyclase. 4) The resolved (unliganded) 41,000-Da subunit stimulates adenylate cyclase activity and relieves guanine nucleotide- +/- epinephrine-induced inhibition of the enzyme. In contrast, the GTP gamma S-bound form of the 41,000-Da subunit inhibits adenylate cyclase activity, although with lower apparent affinity than does the 35,000-Da subunit. 5) The 35,000-Da subunit increases the rate of deactivation of Gs, the stimulatory regulatory protein of adenylate cyclase. In contrast, the 41,000-Da subunit can interact with Gs and inhibit its deactivation. These data strongly suggest that the IAP substrate is another dimeric, guanine nucleotide-binding regulatory protein and that it is responsible for inhibitory modulation of adenylate cyclase activity.