Influence of pertussis toxin on the effects of guanine nucleotide on adenylate cyclase in rat striatal membranes

The influence of pertussis toxin on the effects of guanine nucleotide on adenylate cyclase activity were investigated in rat striatal membranes. GTP promoted and inhibited the activity at 1 and 100 microM, respectively. The inhibitory effects of GTP were abolished by pretreatment of the membranes with pertussis toxin. GppNHp (guanyl-5'-y1-beta,gamma-imidodiphosphate) exerted only stimulatory effects and pertussis toxin did not affect the effects of GppNHp. GDP at 10 and 100 microM caused significant inhibition which was completely suppressed by pertussis toxin. It is suggested that guanine nucleotide regulates the affinity of as in stimulatory GTP-binding regulatory protein to either beta gamma or catalytic units of adenylate cyclase in a flip-flop manner. Inhibitory GTP-binding regulatory protein seems to play a regulatory role in inhibiting alpha s activity supplying the beta gamma heterodimer.     

Calmodulin-Sensitive and Calmodulin-Insensitive Components of Adenylate Cyclase Activity in Rat Striatum Have Differential Responsiveness to Guanyl Nucleotides

The interaction between the Ca2+-binding protein, calmodulin, and guanyl nucleotides was investigated in a rat striatal particulate fraction. We found that the ability of calmodulin to stimulate adenylate cyclase in the presence of guanyl nucleotides depends upon the type and concentration of the guanyl nucleotide. Adenylate cyclase activity measured in the presence of calmodulin and GTP reflected additivity at every concentration of these reactants. On the contrary, when the activating guanyl nucleotide was the nonhydrolyzable analog of GTP, guanosine-5'-(beta,gamma-imido)triphosphate (GppNHp), calmodulin could further activate adenylate cyclase only at concentrations less than 0.2 microM GppNHp. Kinetic analysis of adenylate cyclase by GppNHp was compatible with a model of two components of adenylate cyclase activity, with over a 100-fold difference in sensitivity for GppNHp. The component with the higher affinity for GppNHp was competitively stimulated by calmodulin. The additivity between calmodulin and GTP in the striatal particulate fraction suggests that they stimulate different components of cyclase activity. The calmodulin-stimulatable component constituted 60% of the total activity. Our two-component model does not delineate, at this point, whether there are two separate catalytic subunits or one catalytic subunit with two GTP-binding proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correction by dexamethasone treatment of the altered lipolytic cascade induced by adrenalectomy in rat adipocytes

The ability of glucocorticoid-treatment to reverse the metabolic alterations caused by adrenalectomy in rat adipocytes was studied. Correction of the enhanced adenosine antilipolytic effect and of the defect in lipolysis, cyclic AMP and adenylate cyclase responsiveness to guanine nucleotides were all achieved after a 24 h dexamethasone treatment, whereas correction of the defect in beta-adrenoceptor-density required a 48 h treatment. The latter treatment, however, failed to reverse the defect in both the adenylate cyclase catalytic activity and protein content per fat cell. These different kinetics of restoration indicate that correction by dexamethasone of the defective cyclic AMP and lipolytic responses on one hand and of the guanine nucleotide control of adenylate cyclase on the other one are two related phenomenoms.     

Mechanism of guanine nucleotide regulatory protein-mediated inhibition of adenylate cyclase. Studies with isolated subunits of transducin in a reconstituted system

The retinal nucleotide regulatory protein, transducin, can substitute for the inhibitory guanine nucleotide-binding regulatory protein (Ni) in inhibiting adenylate cyclase activity in phospholipid vesicle systems. In the present work we have assessed the roles of the alpha (alpha T) and beta gamma (beta gamma T) subunit components in mediating this inhibition. The inclusion of either a preactivated alpha T . GTP gamma S (where GTP gamma S is guanosine 5'-O-(thiotriphosphate)) complex, or the beta gamma complex, in phospholipid vesicles containing the pure human erythrocyte stimulatory guanine nucleotide-binding regulatory protein (Ns) and the resolved catalytic moiety of bovine caudate adenylate cyclase (C) resulted in inhibition of the GppNHp-stimulated (where GppNHp is guanyl-5'-yl imidodiphosphate) activity (by approximately 30-60 and 90%, respectively, at 2 mM MgCl2). The inhibitions by both of these subunit species are specific for the Ns-stimulated activity with neither alpha T . GTP gamma S nor beta gamma T having any direct effect on the intrinsic activity of the catalytic moiety. Increasing the MgCl2 concentration in the assay incubations significantly decreases the inhibitions by both alpha T . GTP gamma S and beta gamma T. Similarly, when the pure hamster lung beta-adrenergic receptor is included in the lipid vesicles with Ns and C, the levels of inhibition of the GppNHp-stimulated activity by both alpha T . GTP gamma S and beta gamma T are reduced compared to those obtained in vesicles containing just Ns and C (but not stimulatory receptor). These inhibitions are reduced still further under conditions where the agonist stimulation of adenylate cyclase activity is maximal, i.e. when stimulating with isoproterenol plus GTP. In these cases the alpha T . GTP gamma S inhibitory effects are completely eliminated and the inhibitions observed with holotransducin can be fully accounted for by the beta gamma T complex. The ability of the beta-adrenergic receptor to relieve these inhibitions suggests that the receptor may remain coupled to Ns (or alpha s) during the activation of the regulatory protein and the stimulation of adenylate cyclase. These results also suggest that under physiological conditions the beta gamma subunit complex is primarily responsible for mediating the inhibition of adenylate cyclase activity.     

Roles of GTP and GDP in the regulation of the thyroid adenylate cyclase system

Effects of guanine nucleotides on the adenylate cyclase activity of thyroid plasma membranes were investigated by monitoring metabolism of the radiolabeled nucleotides by thin-layer chromatography (TLC). When ATP was used as substrate with a nucleotide-regeneratign system, TSH stimulated the adenylate cyclase activity in the absence of exogenous guanine nucleotide. Addition of GTP and GDP equally enhanced the TSH stimulation. Effects of GTP and GDP were indistinguishable in regard to their inhibitory effects on NaF-stimulated activities. The results from TLC suggested that GDP could be converted to GTP by a nucleotide-regenerating system. Even in the absence of nucleotide-regenerating system, addition of GDP to the adenylate cyclase assay mixture int he parallel decrease in ATP levels and formation of GTP indicating that thyroid plasma membrane preparatiosn possessed a transphosphorylating activity. When an ATP analog, App[NH]p, was used as substrate without a nucleotide-regenerating system, no conversion of GDP to GTP was observed. Under such conditions, TSH did not stimulate the adenylate cyclase activity unless exogenous GTP or Gpp[NH]p was added. GDP no longer supported TSH stimulation and caused a slight decrease in the activity. GDP was less inhibitory than Gpp(NH)p to the NaF-stimulated adenylate cyclase activity. These results suggest: (1) TSH stimulation of thyroid adenylate cyclase is absolutely dependent on the regulatory nucleotides. (2) In contrst to GTP, GDP cannot support the coupling of the receptor-TSH complex to the catalytic componenet of adenylate cyclase. (3) The nucleotide regulatory site is more inhibitory to the stimulation of the enzyme by NaF when occupied by Gpp[NH]p than GDP.     

Prostaglandin E-induced heterologous desensitization of hepatic adenylate cyclase. Consequences on the guanyl nucleotide regulatory complex

Prostaglandin E (PGE) receptor density in hepatic plasma membranes can be down-regulated by in vivo exposure to the 16,16-dimethyl analog of PGE2, and this is associated with desensitization of PGE-sensitive adenylate cyclase. These studies examined adenylate cyclase response to other agonists in membranes whose PGE receptor density was 51% decreased and whose maximal PGE-stimulated adenylate cyclase activity was 31% decreased. Down-regulated membranes had a 37% decrease in their maximal response to glucagon, indicating that treatment with the PGE analog had induced both homologous and heterologous desensitization. To determine whether adenylate cyclase had been affected, stimulation with NaF, guanyl 5'-yl imidodiphosphate (GppNHp), and forskolin was examined in both intact and solubilized membranes. Intact membranes had decreased adenylate cyclase responses to all three stimulators (NaF, -41%; GppNHp, -25%; forskolin, -41%) as did solubilized membranes (NaF, -51%; GppNHp, -50%; forskolin, -50%), suggesting alterations in adenylate cyclase rather than indirect membrane effects. Cholera toxin activation and labeling were examined to more directly assess whether the guanine nucleotide (G/F) regulatory component of adenylate cyclase had been affected. Cholera toxin activation was 42% less in down-regulated membranes, and these membranes incorporated less label when the incubation was performed in the presence of [32]NAD. Solubilized G/F subunit activity from down-regulated membranes was less effective in reconstitution of adenylate cyclase activity from cyc- cell membranes than G/F activity from control membranes. These data indicate that in vivo exposure to the PGE analog causes both homologous and heterologous desensitization of adenylate cyclase as well as an apparent quantitative decrease in G/F.     

Antibodies directed against transducin beta subunits interfere with the regulation of adenylate cyclase activity in brain membranes

In an attempt to study the mechanisms of action of membrane-bound adenylate cyclase, we have applied to rat brain synaptosomal membranes antibodies raised against purified bovine transducin (T) beta gamma subunits. The antibodies recognized one 36-kDa protein in Western blots of the membranes. Adenylate cyclase activation by GTP non-hydrolyzable analogues was greatly decreased in immune, as compared to preimmune, antibody-treated membranes, whereas the enzyme basal activity was unaffected by both types of antibodies. The inhibition of forskolin-stimulated adenylate cyclase by guanine 5'-(beta, gamma-imino)triphosphate (Gpp-(NH)p) was decreased in membranes preincubated with immune, but not preimmune, antibodies. Anti-T beta antibodies moderately decreased the extent of subsequent adenylate cyclase activation by forskolin, while not affecting activation by Al3+/F-. The enzyme activation by Gpp(NH)p in untreated membranes remained the same upon further incubation in the presence of either type of antibodies. Such results were consistent with the decreased exchange of guanine nucleotides which occurred in membrane treated with immune, but not preimmune antibodies, upon addition of GTP. The blockade of the regulation of adenylate cyclase by Gpp(NH)p observed in membranes pretreated by anti-T beta antibodies thus appears to be caused by the impairment of the guanine nucleotide exchange occurring on Gs alpha subunits. The G beta subunits in the adenylate cyclase complex seem to be instrumental in the guanine nucleotide exchange on G alpha subunits, just as T beta subunits are in the transducin complex.     

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.     

The subunits of the stimulatory regulatory component of adenylate cyclase. Resolution of the activated 45,000-dalton (alpha) subunit

Activation of the stimulatory guanine nucleotide-binding regulatory component (G/F) of adenylate cyclase by guanine nucleotides or by Al3+, Mg2+, and F-stabilizes the protein to thermal denaturation or to inactivation by LiBr, guanidine HCl, or urea. Such activation allows the resolution of the active 45,000-Da alpha subunit from the 35,000-Da beta subunit by a high performance gel filtration procedure. Separation of the active alpha subunit has allowed definitive evaluation of the subunit dissociation model for the activation of G/F. The resolved alpha subunit is sufficient to reconstitute the adenylate cyclase activity of the cyc-S49 cell mutant. The alpha subunit alone is also sufficient to activate a preparation of the catalyst of adenylate cyclase that had been resolved from all other identified components of the enzyme system. The resolved alpha subunit displays hydrodynamic properties characteristic of activated G/F. The alpha subunit contains a high affinity guanine nucleotide-binding site. Activation of G/F by guanine nucleotides or by Al3+ + Mg2+ + F- allows resolution of the activated alpha subunit. Reversal of the activated state of the resolved alpha subunit occurs only slowly. Addition of beta subunit enhances the rate of deactivation. Deactivation of the activated alpha subunit by the beta subunit changes the S20,w for G/F activity from 2.0 to 4.0 (in Lubrol), consistent with a formation of the alpha X beta heterodimer. These data, taken in aggregate, constitute proof for the proposed mechanism of activation of G/F by non-hydrolyzable analogs of GTP and by Al3+, Mg2+, and F-. They are analogous to data obtained for transducin, the GTP-binding regulatory protein from vertebrate rod outer segment discs, and for the putative inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase (the substrate for islet-activating protein). The model provides several powerful tests for study of mechanisms of hormonal regulation of adenylate cyclase in membranes.     

Metabolism of arachidonic acid by isolated rat hepatocytes, renal cells and by some rabbit tissues. Detection of vicinal diols by mass fragmentography

Effects of guanine nucleotides on the adenylate cyclase activity of thyroid plasma membranes were investigated by monitoring metabolism of the radiolabeled nucleotides by thin-layer chromatography (TLC). When ATP was used as substrate with a nucleotide-regenerating system, TSH stimulated the adenylate cyclase activity in the absence of exogenous guanine nucleotide. Addition of GTP or GDP equally enhanced the TSH stimulation. Effects of GTP and GDP were indistinguishable in regard to their inhibitory effects on NaF-stimulated activities. The results from TLC suggested that GDP could be converted to GTP by a nucleotide-regenerating system. Even in the absence of a nucleotide-regeneration system, addition of GDP to the adenylate cyclase assay mixture resulted in the parallel decrease in ATP levels and formation of GTP indicating that thyroid plasma membrane preparations possessed a transphosphorylating activity. When an ATP analog, App[NH]p, was used as substrate without a nucleotide-regenerating system, no conversion of GDP to GTP was observed. Under such conditions, TSH did not stimulate the adenylate cyclase activity unless exogenous GTP or Gpp[NH]p was added. GDP no longer supported TSH stimulation and caused a slight decrease in the activity. GDP was less inhibitory than Gpp(NH)p to the NaF-stimulated adenylate cyclase activity. These results suggest: (1) TSH stimulation of thyroid adenylate cyclase is absolutely dependent on the regulatory nucleotides. (2) In contrast to GTP, GDP cannot support the coupling of the receptor-TSH complex to the catalytic component of adenylate cyclase. (3) The nucleotide regulatory site is more inhibitory to the stimulation of the enzyme by NaF when occupied by Gpp[NH]p than GDP.     

Differential effects of GTP on the coupling of beta-adrenergic receptors to adenylate cyclase from frog and turkey erythrocytes. Application of new methods for the analysis of receptor-effector coupling.

A detailed comparison of the interaction of beta-adrenergic receptors with adenylate cyclase stimulation and modification of this interaction by guanine nucleotides has been made in two model systems, the frog and turkey erythrocyte. Objective analysis of the data was facilitated by the development of new graphical methods which involve the use of logit-logit transformations of percent receptor occupancy versus percent enzyme stimulation plots (coupling curves). Receptor-cyclase coupling in turkey erythrocyte membranes demonstrates a proportional relationship between receptor occupancy and adenylate cyclase activation and is unaffected by exogenous guanine nucleotides. By comparison, the proportional relationship of receptor occupancy and adenylate cyclase activation observed in frog erythrocyte membranes in the absence of guanine nucleotides is modified by the addition of exogenous guanine nucleotides such that a greater fractional enzyme stimulation is elicited by low receptor occupancy. Methodological criteria crucial for valid comparison of receptor occupancy and adenylate cyclase activity are delineated. In addition, the possible molecular mechanisms of receptor-cyclase coupling which might give rise to the coupling curves observed are discussed.     

Differential effects of GTP on the coupling of β-adrenergic receptors to adenylate cyclase from frog and turkey erythrocytes application of new methods for the analysis of receptor-effector coupling

A detailed comparison of the interaction of β-adrenergic receptors with adenylate cyclase stimulation and modification of this interaction by guanine nucleotides has been made in two model systems, the frog and turkey erythrocyte. Objective analysis of the data was facilitated by the development of new graphical methods which involve the use of logit-logit transformations of percent receptor occupancy versus percent enzyme stimulation plots (coupling curves). Receptor-cyclase coupling in turkey erythrocyte membranes demonstrates a proportional relationship between receptor occupancy and adenylate cyclase activation and is unaffected by exogenous guanine nucleotides. By comparison, the proportional relationship of receptor occupancy and adenylate cyclase activation observed in frog erythrocyte membranes in the absence of guanine nucleotides is modified by the addition of exogenous guanine nucleotides such that a greater fractional enzyme stimulation is elicited by low receptor occupancy. Methodological criteria crucial for valid comparison of receptor occupancy and adenylate cyclase activity are delineated. In addition, the possible molecular mechanisms of receptor-cyclase coupling which might give rise to the coupling curves observed are discussed.     

Adenylate Cyclase of Torpedo Synaptosomes Is Inhibited by Calcium and Not Affected by Muscarinic Ligands

Abstract: Cholinergic synaptosomes isolated from the electric organ of Torpedo contain membrane-bound adenylate cyclase activity (∼6 pmol/mg proteidmin), which is dependent on the presence of guanine nucleotides. The activity is strongly dependent on temperature and only slightly affected by NaCl. The Torpedo adenylate cyclase is completely inhibited by low levels of free Ca²⁺ (K₀∼ 0.5 μ M ). This effect is not altered by either trifluoperazine or addition of exogenous calmodulin. Ca³⁺ has no effect on the activation step of the adenylate cyclase by guanyl-5'-yl imidodiphosphate (GppNHp), and Mn²⁺ abolishes the Ca²⁺-dependent inhibition of cyclic AMP synthesis. These findings suggest that Ca²⁺ exerts its effect by direct interaction with a site located on the catalytic subunit. Torpedo synaptosomes contain presynaptic inhibitory muscarinic receptors. The binding of muscarinic agonists to the receptors is modulated (to lower affinity) by GTP. However, muscarinic ligands, examined under a variety of assay conditions, have no effect on adenylate cyclase activity. These results suggest that although both the muscarinic receptor and the adenylate cyclase are coupled to G proteins, they either interact with different G proteins or are situated in different regions of the presynaptic membrane.     

Transient complexes. A structural model for the activation of adenylate cyclase by hormone receptors (guanine nucleotides/irradiation inactivation)

1. The irradiation-inactivation procedure was used to study changes in the state of association of the protein components of adenylate cyclase in intact rat liver plasma membranes by measurement of alterations in the target size determined from the catalytic activity of the enzyme. 2. A decrease in target size at 30 degrees C in response to p[NH]ppG (guanosine 5'-[betagamma-imido]triphosphate) or GTP was demonstrated, which we take to reflect the dissociation of a regulatory subunit. The effect of GTP is potentiated by glucagon. This effect is not observed at 0 degrees C. 3. An increase in target size was observed in response to glucagon in the absence of guanine nucleotides, which we take to reflect the association of glucagon receptor with adenylate cyclase. 4. We propose a model for the activation of adenylate cyclase by glucagon in which the binding of the hormone to its receptor causes an initial association of the receptor with the catalytic unit of the enzyme and a regulatory subunit to form a ternary complex. The subsequent activation of the adenylate cyclase results from the dissociation of the ternary complex to leave a free catalytic unit in the activated state. This dissociation requires the binding of a guanine nucleotide to the regulatory subunit. 5. The effects of variation of temperature on the activation of adenylate cyclase by glucagon and guanine nucleotides were examined and are discussed in relation to the irradiation-activation data. 6. The effectiveness of hormones, guanine nucleotides and combinations of hormone and guanine nucleotides as activators of adenylate cyclase in both rat liver and rat fat-cell plasma membranes was studied and the results are discussed in relation to the model proposed, which is also considered in relation to the observations published by other workers.     

Fasting increases fat cell adenylate cyclase sensitivity to stimulatory agonists through enhanced ability of the stimulatory regulatory component Ns to dissociate

In rat fat cell membranes, a 72-hour fasting fails to alter the adenylate cyclase stimulatory responses to Mn2+, forskolin and cholera toxin and the cholera toxin catalyzed [alpha-32P] ADP ribose incorporation into the Mr = 42,000 and 46,000/48,000 alpha s peptides of Ns. In contrast, dose-response curves for GTP-stimulation of basal and isoproterenol-stimulated adenylate cyclase display higher maximal responses in fasted rats under conditions restraining (2 mM Mg2+) but not promoting (10 mM Mg2+) the dissociation of Ns. Moreover, at 10 mM Mg2+, the sensitivity of isoproterenol-stimulated adenylate cyclase to GTP is clearly increased in fasted rats. Finally, fasting reduces by 40% the lag-phase of adenylate cyclase activation by Gpp(NH)p. Taken together, these results are consistent with the hypothesis that the permissive effect of fasting on the fat cell adenylate cyclase response to stimulatory agonists is related to increased ability of Ns and the ternary H.R.Ns. complex to dissociate which is likely due to enhanced Ns affinity for guanine nucleotides.     

Interaction of guanine nucleotides with adenylate cyclase in normal and spontaneously transformed RL-RP-C cloned rat hepatocytes

Spontaneous transformation of RL-PR-C hepatocytes leads to alterations in the adenylate cyclase complex which include a lower than normal basal level of activity, a loss of sensitivity to exogenous GTP, and a decreased sensitivity to isoproterenol. Both normal and transformed membranes possess substantial GTPase activity. Treatment of transformed hepatocyte membranes with either isoproterenol plus GMP or with cholera toxin, under conditions that displace tightly bound GDP, restored the GTP effect on adenylate cyclase, and eliminated the lag in the activation by guanyl-5'-yl-imidodiphosphate. Such pretreatment also enhanced guanine nucleotide effects on the adenylate cyclase of normal hepatocytes. These results are explainable on the basis that transformation increases adenylate cyclase-associated GTPase activity, and increases occupancy of nucleotide regulatory sites by inactive or inhibitory guanine nucleotides, e.g., GDP. Seemingly, both catecholamines and cholera toxin promote an exchange reaction at the regulatory sites, resulting in clearance of these sites of inhibitory nucleotides.     

The inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. Subunit dissociation and the inhibition of adenylate cyclase in S49 lymphoma cyc- and wild type membranes

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 subunits are functionally indistinguishable. GTP-dependent hormonal inhibition of adenylate cyclase and that caused by guanine nucleotide analogs seem to result from dissociation of the subunits of Gi. Such inhibition can be explained by reduction of the concentration of the free alpha subunit of Gs as a result of its interaction with the beta subunit of Gi in normal Gs-containing membranes. However, inhibition in S49 lymphoma cyc- cell membranes presumably cannot be explained by the Gi-Gs interaction, since the activity of the alpha subunit of Gs is not detectable in this variant. Several characteristics of Gi-mediated inhibition of adenylate cyclase have been studied in both S49 cyc- and wild type membranes. There are several similarities between inhibition of forskolin-stimulated adenylate cyclase by guanine nucleotides and somatostatin in cyc- and wild type membranes. 1) Somatostatin-induced inhibition of the enzyme is dependent on GTP; nonhydrolyzable GTP analogs are also effective inhibitors. 2) The effect of guanosine-5'-(3-O-thio)triphosphate (GTP gamma S) is essentially irreversible, and somatostatin accelerates GTP gamma S-induced inhibition. 3) Inhibition of adenylate cyclase by somatostatin or Gpp(NH)p is attenuated by treatment of cells with islet-activating protein (IAP). 4) Both cyc- and wild type membranes contain the substrate for IAP-catalyzed ADP-ribosylation (the alpha subunit of Gi). 5) beta Subunit activity in detergent extracts of membranes is liberated by exposure of the membranes to GTP gamma S. The alpha subunit of Gi in such extracts has a reduced ability to be ADP-ribosylated by IAP, which implies that this subunit is in the GTP gamma S-bound form. The resolved subunits of Gi have been tested as regulators of cyc- and wild type adenylate cyclase under a variety of conditions. The alpha subunit of Gi inhibits forskolin-stimulated adenylate cyclase activity in cyc-, while the beta subunit stimulates; these actions are opposite to those seen with wild type membranes. The inhibitory effects of GTP plus somatostatin (or GTP gamma S) and the alpha subunit of Gi are not additive in cyc- membranes. In wild type, the inhibitory effects of the hormone and GTP gamma S are not additive with those of the beta subunit.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction of guanine nucleotides with adenylate cyclase in normal and spontaneously transformed RL-PR-C cloned rat hepatocytes

Spontaneous transformation of RL-PR-C hepatocytes leads to alterations in the adenylate cyclase complex which include a lower than normal basal level of activity, a loss of sensitivity to exogenous GTP, and a decreased sensitivity to isoproterenol. Both normal and transformed membranes posses substantial TGPase activity. Treatment of transformed hepatocyte membranes with either isoproterenol plus GMP or with cholera toxin, under conditions that displace tightly bound GDP, restored the GTP effect on adenylate cyclase, and eliminated the lag in the activation by guanyl-5′-yl-imidodiphosphate. Such pretreatment also enhanced guanine nucleotide effects on the adenylate cyclase of normal hepatocytes. These results are explainable on the basis that transformation increases adenylate cyclase-associated GTPase activity, and increase occupancy of nuceotide regulatory sites by inactive or inhibitory guanine nucleotides, e.g., GDP. Seemingly, both catecholamines and cholera toxin promote an exchange reaction at the regulatory sites, resulting in clearance of these sites of inhibitory nucleotides.     

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.     

Role of calmodulin in the effect of guanyl nucleotides on rat hippocampal adenylate cyclase: involvement of adenosine and opiates

Abstract: The adenylate cyclase activity of rat hippocampal plasma membranes can be stimulated by vaso-active intestinal polypeptide (VIP). Low concentrations (10⁻⁹ to 10⁻⁷M) of 5'-guanylyl-imido diphosphate (GppNHp) evoke a transient inhibition of the enzyme, which is followed by stimulation with increasing GppNHp concentrations (10⁻⁶ to 10⁻⁴M). Inclusion of ethyleneglycol - bis - (β - aminoethylether) - N,N' - tetraacetic acid (EGTA) during incubation abolishes the GppNHp inhibition while preserving GppNHp activation. The stimulation induced by GppNHp is amplified by VIP, but the inhibition is unaffected. Adenosine analogs and opiates are inhibitory ligands in the presence of GTP, and their effects can be reversed by the appropiate receptor antagonists, 3-isobutyl-1-methylxanthine and naloxone. Treatment of membranes with trypsin abolishes the GppNHp-induced inhibition without affecting the GppNHp stimulation. The inhibition induced by GppNHp is also abolished by EGTA treatment followed by washing, which coincides wtih a reduction in the adenosine- and opiate-mediated, GTP-dependent inhibition. The GppNHp inhibition can be restored in EGTA-treated but not in trypsin-treated membranes by addition of calcium-calmodulin but not by Ca²⁺ or Mg²⁺. Calcium-calmodulindepleted membranes lack calcium stimulation as well as GppNHp-induced inhibition, whereas untreated membranes and calcium-calmodulin-depleted membranes plus exogenous calcium-calmodulin showed calcium stimulation and GppNHp inhibition. These results suggest that calmodulin is involved in both Ca²⁺ stimulation and guanine nucleotide-mediated inhibition of rat hippocampal adenylate cyclase.