Influence of cholera toxin on the regulation of adenylate cyclase by GTP.

In the presence of NAD⁺, cholera toxin activates adenylate cyclase in membranes of S49 mouse lymphoma cells. The following evidence supports the hypothesis that the toxin acts by inhibiting a specific GTPase associated with a guanyl nucleotide regulatory component of hormone-responsive cyclase: 1. GTP alone markedly stimulates cyclase activity in toxin-treated, but not in untreated membranes; 2. The poorly hydrolyzable GTP analog, guanosine 5′-(β,γ-imino) triphosphate (Gpp(NH)p), stimulates cyclase equally well in toxin-treated and untreated membranes; 3. Cyclase activation by isoproterenol plus GTP persists in toxin-treated membranes, but not in controls, after addition of propranolol; 4. GTP is a more potent competitive inhibitor of the irreversible activation of cyclase by Gpp(NH)p in toxin-treated than in untreated membranes.     

Regulation of human platelet adenylate cyclase by epinephrine, prostaglandin E1, and guanine nucleotides. Evidence for separate guanine nucleotide sites mediating stimulation and inhibition.

A method for preparing human platelet membranes with high adenylate cyclase activity is described. Using these membranes, epinephrine and GTP individually are noted to inhibit adenylate cyclase slightly. When present together, epinephrine and GTP act synergistically to cause a 50% inhibition of basal activity. The epinephrine effect is an alpha-adrenergic process as it is reversed by phentolamine but not propranolol. The quasi-irreversible activation of adenylate cyclase by Gpp(NH)p is time, concentration, and Mg2+-dependent but is not altered by the presence of epinephrine. Adenylate cyclase activated by Gpp(NH)p, and extensively washed to remove unbound Gpp(NH)p, is inhibited by the subsequent addition of Gpp(NH)p, GTP, and epinephrine. This effect of epinephrine is also an alpha-adrenergic phenomenon. In contrast to epinephrine which inhibits the cyclase, PGE1 addition results in enzyme stimulation. PGE1 stimulation does not require GTP addition. PGE1 accelerates the rate of Gpp(NH)p-induced activation. Low GTP concentrations (less than 1 x 10(-6) M) enhance PGE1 stimulation while higher GTP concentrations cause inhibition. These observations suggest that human platelet adenylate cyclase possesses at least two guanine nucleotide sites, one which interacts with the alpha-receptor to result in enzyme inhibition and a second guanine nucleotide site which interacts with the PGE1 receptor and causes enzyme stimulation.     

Calcium and adenosine affect human sperm adenylate cyclase activity

The adenylate cyclase activity of human ejaculated spermatozoa in broken-cell preparations was investigated. In the presence of 5 mM metal cations and 0.1 mM ATP, the relative enzyme activity with Mn²⁺, Ca²⁺, Mg²⁺, Ba²⁺ was 1.00, 0.28, 0.22, and 0.03, respectively. Added Ca²⁺ appeared to activate the enzyme in the presence of Mn²⁺ or Mg²⁺. The human sperm adenylate cyclase was stimulated by ～ 2-fold by free Ca²⁺ (lmM) in the presence of Mg²⁺ (5 mM). If the GTP analogue, 5′-guanylyl imidophosphate (Gpp(NH)p) was added to the sperm homogenate in the presence of 200 μM ethylene-glycol-bis (β-aminoethylether) N,N′-tetraacetic acid (EGTA), the adenylate cyclase activity was increased by approximately 25%, but with the addition of 280 μM Ca²⁺ there was a decrease in enzyme activity. A similar response to low concentrations of Ca²⁺ was obtained after complementation of the sperm enzyme with the guanine nucleotide regulatory component from human erythrocytes, where the addition of 40 μM Gpp(NH)p, 200 μM EGTA, and Ca²⁺ (≤ 160 μM) stimulated the sperm enzyme ～ 3–4-fold, but the further addition of Ca²⁺ (280 μM, final) neutralized the stimulatory effect. The addition of adenosine, and the nucleotides 5′-AMP and 5′-ADP inhibited the enzyme, whereas guanine and 5′-GMP had no appreciable effect. Human follicular fluid and serum also had little direct effect on the sperm adenylate cyclase. These resuls suggest that Ca²⁺ might be an important physiological modulator of the human sperm adenylate cyclase.     

Forskolin-activated adenylate cyclase. Inhibition by guanyl-5'-yl imidodiphosphate

Forskolin activated adenylate cyclase of purified rat adipocyte membranes in the absence of exogenous guanine nucleotides. Guanyl-5'-yl imidodiphosphate (Gpp(NH)p) inhibited the forskolin-activated cyclase immediately upon addition of the nucleotide at concentrations too low to activate adenylate cyclase (10(-9) to 10(-7) M). Inhibition seen with a very high concentration of Gpp(NH)p (10(-4) M) lasted for 3-4 min and was followed by an increase in the synthetic rate which remained constant for at least 15 min. The length of the transient inhibition did not vary with forskolin concentrations above 0.05 microM but low Gpp(NH)p (10(-8) M) exhibited a lengthened (6-7 min) inhibitory phase. The transient inhibitory effects of Gpp(NH)p were eliminated by 10(-7) M isoproterenol, high (40 mM) Mg2+, or preincubation with Gpp(NH)p in the absence of forskolin. While forskolin stimulated fat cell cyclase in the presence of Mn2+, this ion blocked the inhibitory effects of Gpp(NH)p. The well documented inhibitory effects of GTP on the fat cell adenylate cyclase system were also observed in the presence of forskolin. However, the inhibition by GTP is not transitory. These findings indicate that Gpp(NH)p regulation of forskolin-stimulated cyclase has at least two components: 1) an inhibitory component which acts through an undetermined mechanism and which acts immediately to decrease cyclase activity; and 2) an activating component which modulates the inhibited cyclase activity through the guanine nucleotide regulatory protein.     

Effect of Dopamine on Activation of Rat Striatal Adenylate Cyclase by Free Mg2+ and Guanyl Nucleotides1

Abstract: Stimulation of rat striatal adenylate cyclase by guanyl nucleotides was examined utilizing either MgATP or magnesium 5′-adenylylimidodiphos-phate (MgApp(NH) p) as substrate. GTP and 5′- guanylylimidodiphosphate (Gpp(NH) p) stimulate adenylate cyclase under conditions where the guanyl nucleotide is not degraded. The apparent stimulation of adenylate cyclase by GDP is due to an ATP-dependent transphosphorylase present in the tissue which converts GDP to GTP. We conclude that GTP is the physiological guanyl nucleotide responsible for stimulation of striatal adenylate cyclase. Dopamine lowers the K_a for Gpp(NH) p stimulation twofold, from 2.4 μM to 1.2 μM and increases maximal velocity 60%. The kinetics of Gpp(NH) p stimulation indicate no homotropic interactions between Gpp(NH) p sites and are consistent with one nonessential Gpp(NH) p activator site per catalytic site. Double reciprocal plots of the activation by free Mg²⁺ were concave downward, indicating either two sets of sites with different affinities or negative cooperativity (Hill coefficient = 0.3, K_0.5= 23 mM). The data conform well to a model for two sets of independent sites and dopamine lowers the K_a for free Mg²⁺ at the high-affinity site threefold, from 0.21 mM to 0.07 mM. The antipsy-chotic drug fluphenazine blocks this shift in K_a due to dopamine. Dopamine does not appreciably affect the affinity of adenylate cyclase for the substrate, MgApp(NH) p. Therefore, dopamine stimulates striatal adenylate cyclase by increasing the affinity for free Mg²⁺ and guanyl nucleotide and by increasing maximal velocity.     

Characterization of an ATP-Mg2+-dependent guanine nucleotide-stimulated adenylate cyclase from Neurospora crassa

A novel adenylate cyclase activity was found in crude homogenates of Neurospora crassa. The adenylate cyclase had substantial activity with ATP-Mg2+ as substrate differing significantly from the strictly ATP-Mn2+-dependent enzyme characterized previously. Additionally, the ATP-Mg2+-dependent activity was stimulated two- to fourfold by GTP or guanyl-5'-yl-imido-diphosphate (Gpp(NH)p). We propose that the ATP-Mg2+-dependent, guanine nucleotide-stimulated activity is due to a labile regulatory component (G component) of the adenylate cyclase which was present in carefully prepared extracts. The adenylate cyclase had a pH optimum of 5.8 and both the catalytic and G component were particulate. The Km for ATP-Mg2+ was 2.2 mM in the presence of 4.5 mM excess Mg2+. Low Mn2+ concentrations had no effect on adenylate cyclase activity whereas high concentrations of Mn2+ or Mg2+ stimulated the enzyme. Maximal Gpp(NH)p stimulation required preincubation of the enzyme in the presence of the guanine nucleotide and the K1/2 for Gpp(NH)p stimulation was 110 nM. Neither fluoride nor any of a variety of glycolytic intermediates or hormones, including glucagon, epinephrine, and dopamine, had an effect on ATP-Mg2+-dependent adenylate cyclase activity. However, the enzymatic activity was stimulated not only by GTP but also by 5'-AMP and was inhibited by NADH.     

Calmodulin regulation of adenylate cyclase activity in human platelet membranes

The mechanism of calmodulin dependent regulation of adenylate cyclase has been studied in human platelet membranes. Calmodulin activated adenylate cyclase exhibited a biphasic response to both Mg2+ and Ca2+. A stimulatory effect of Mg2 on adenylate cyclase was observed at all Mg2+ concentrations employed, although the degree of activation by calmodulin was progressively decreased with increasing concentrations of Mg2+. These results demonstrate that the Vmax of calmodulin dependent platelet adenylate cyclase can be manipulated by varying the relative concentrations of Mg2+ and Ca2+. The activity of calmodulin stimulated adenylate cyclase was always increased 2-fold above respective levels of activity induced by GTP, Gpp(NH)p and/or PGE. The stimulatory influence of calmodulin was not additive but synergistic to the effects of PGE1, GTP and Gpp(NH)p. GDP beta S inhibited GTP-and Gpp(NH)p stimulation of adenylate cyclase but was without effect on calmodulin stimulation. Since the inhibitory effects of GDP beta S have been ascribed to apparent reduction of active N-protein-catalytic unit (C) complex formation, these results suggest that the magnitude of calmodulin dependent adenylate cyclase activity is proportional to the number of N-protein-C complexes, and that calmodulin interacts with preformed N-protein-C complex to increase its catalytic turnover. Our data do not support existence of two isoenzymes of adenylate cyclase (calmodulin sensitive and calmodulin insensitive) in human platelets.     

Potentiation by guanine nucleotides of the VIP-induced adenylate cyclase stimulation in intestinal epithelial cell membranes

The GTP analog 5′-quanylyl-imidodiphosphate Gpp(NH) p potentiated the action of VIP on adenylate cyclase from intestinal epithelial cell membranes. The other nucleotides tested were also active on adenylate cyclase with the following order of potency GTP>GDP>GMP>ITP>UTP=CTP. Guanine nucleotides act by increasing the Vmax of the enzyme activity and by decreasing the Km of enzyme activation by VIP. Activation of the peptide-induced adenylate cyclase activity by Gpp (NH) p was inhibited by GTP and the other nucleotides with the same order and range of potency than those observed for their intrinsic stimulatory effect on adenylate cyclase. These data demonstrate the potent and specific action of quanine nucleotides on the VIP-sensitive adenylate cyclase.     

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.     

Guanine Nucleotides Modulate Cortical S2 Serotonin Receptors

Abstract

Many radiolabelled receptors coupled to intracellular adenylate cyclase activity have been found to be modulated by physiological modulators such as GTP (guanosine triphosphate) and Gpp(NH)p (guanosine-imido-diphosphate). In particular, the apparent affinity of agonists competing for the binding of ³H-antagonist-labelled receptors is reduced in the presence of GTP and Gpp(NH)p. We report herein the agonist-specific effects of GTP and Gpp(NH)p on rat brain cortical S² serotonin receptors. The agonists serotonin, 5-methoxytryptamine, bufotenine, and tryptamine display threefold lower affinities for S₂ serotonin receptors in the presence of 10^-4M GTP or Gpp(NH)p than in the absence of the nucleotides. The antagonists spiperone, cinanserin, cyproheptadine and methysergide are unaffected by the guanine nucleotides. The Hill coefficients of the agonists increase from between 0.70–0.80 to 0.90–1.00 due to guanine nucleotides. ATP, ADP, and GDP have little or no effect. This pattern of guanine nucleotide effects has been found with receptors which are modulated by a guanine nucleotide regulatory protein and may indicate that the S₂ serotonin receptor may be coupled to intracellular adenylate cyclase activity.     

Adenosine and gpp(NH)p modulate mouse sperm adenylate cyclase

The possible roles of adenosine and the GTP analogue Gpp(NH)p in regulating mouse sperm adenylate cyclase activity were investigated during incubation in vitro under conditions in which after 30 min the spermatozoa are essentially uncapacitated and poorly fertile, whereas after 120 min they are capacitated and highly fertile. Adenylate cyclase activity, assayed in the presence of 1 mM ATP and 2 mM Mn²⁺, was determined by monitoring cAMP production. When adenosine deaminase (1 U/ml) was included in the assay to deplete endogenous adenosine, enzyme activity was decreased in the 30-min suspensions but increased in the 120-min samples (P < 0.02). This suggests that endogenous adenosine has a stimulatory effect on adenylate cyclase in uncapacitated spermatozoa but is inhibitory in capacitated cells. Since the expression of adenosine effects at low nucleoside concentrations usually requires guanine nucleotides, the effect of adding adenosine in the presence of 5 x 10^–5 M Gpp(NH)p was examined. While either endogenous adenosine or adenosine deaminase may have masked low concentration (10^?9?10^?7 M) effects of exogenous adenosine, a marked inhibition (P < 0.001) of adenylate cyclase activity in both uncapacitated and capacitated suspensions was observed with higher concentrations (>10^?5 M) of adenosine. Similar inhibition was also observed in the absence of Gpp(NH)p, suggesting the presence of an inhibitory P site on the enzyme. In further experiments, the effects of Gpp(NH)p in the presence and absence of adenosine deaminase were examined. Activity in 30-min suspensions was stimulated by the guanine nucleotide and in the presence of adenosine deaminase this stimulation was marked, reversing the inhibition seen with adenosine deaminase alone. In capacitated suspensions the opposite profile was observed, with Gpp(NH)p plus adenosine deaminase being inhibitory; again, this was a reversal of the effects obtained in the presence of adenosine deaminase alone, which had stimulated enzyme activity. These results suggest the existence of a stimulatory adenosine receptor site (R_a) on mouse sperm adenylate cyclase that is expressed in uncapacitated spermatozoa and an inhibitory receptor site (R_i) that is expressed in capacitated cells, with guanine nucleotides modifying the final response to adenosine. It is concluded that adenosine and guanine nucleotides may regulate mouse sperm adenylate cyclase activity during capacitation.     

Mechanism of adenylate cyclase activation by the rat lung cytoplasmic factors

Epinephrine, histamine and prostaglandin E₁ stimulated adenylate cyclase activity in lung membranes and their stimulation of the enzyme activity was completely blocked by propranolol, metiamide and indomethacin, respectively. A partially-purified activator from the adult rat lung also enhanced adenylate cyclase activity in membranes. However, stimulation of adenylate cyclase by the rat lung activator was not abolished by the above receptor antagonists. Further, epinephrine, NaF and Gpp(NH)p stimulated adenylate cyclase activity rather readily, whereas stimulation of the enzyme activity by the lung activator was evident after an initial lag phase of 10 min. Also, the lung activator produced additive activation of adenylate cyclase with epinephrine, NaF and Gpp(NH)p. These results indicate that the lung activator potentiates adenylate cyclase activity in membranes by a mechanism independent from those known for epinephrine, NaF and Gpp(NH)p. Incubation of lung membranes for 30 min at 40°C resulted in a loss of adenylate cyclase activation by NaF and Gpp(NH)p. Addition of the released proteins to the heat-treated membranes did not restore the enzyme response to these agonists. However, heat treatment of lung membranes in the presence of 2-mercaptoethanol or dithiothreitol prevented the loss of adenylate cyclase response to NaF and Gpp (NH)p. N-ethylmaleimide abolished adenylate cyclase activation by epinephrine, NaF, Gpp(NH)p and the lung activator. These results indicate that the sulfhydryl groups are important for adenylate cyclase function in rat lung membranes.Abbreviations Gpp(NH)p 5-Guanylimidodiphosphate     

A reconstitution assay for the guanine nucleotide regulatory protein of the adenylate cyclase system using turkey erythrocyte membranes as the acceptor preparation

The turkey erythrocyte beta-adrenergic receptor-adenylate cyclase system has the unusual property that neither GTP nor Gpp(NH)p are effective in activating adenylate cyclase unless a beta-agonist is present simultaneously. This property results in essentially no basal activity and the inability of GTP or Gpp(NH)p alone to activate the catalytic moiety. In this study, we have exploited these characteristics to utilize turkey erythrocyte membranes as the acceptor preparation in a reconstitution assay. Rat reticulocyte or turkey erythrocyte membranes that have been activated with isoproterenol and Gpp(NH)p followed by solubilization with sodium cholate serve as the donor source of the guanine nucleotide regulatory protein (N). By reconstituting this Gpp(NH)p-activated N protein, it has been found that: (1) exogenous Gpp(NH)p-associated N could activate the catalytic unit of adenylate cyclase in turkey erythrocyte membranes; (2) this system can be used to assay N protein activity; (3) the endogenous pathway for activation of turkey erythrocyte membrane adenylate cyclase by hormones and fluoride remains qualitatively functional; and (4) the effects of combined activation via the endogenous and exogenous pathways are additive and saturable.     

Solubilization and separation of the glucagon receptor and adenylate cyclase in guanine nucleotide-sensitive states.

Adenylate cyclase in liver membranes was solubilized with Lubrol PX and partially purified by gel filtration. The partially purified enzyme was susceptible to activation by guanyl-5'-yl imidodiphosphate (Gpp(NH)p). Studies on the binding of [3H]Gpp(NH)p to various fractions eluted from the gels revealed that an upper limit of 1% of the Gpp(NH)p binding sites is associated with adenylate cyclase activity stimulated by the nucleotide. The glucagon receptor, pretagged with 125I-glucagon in the membranes, solubilized with Lubrol PX, and fractionated on the same gel columns, eluted in a peak fraction that overlaps with, but is separate from, adenylate cyclase in its Gpp(NH)p-stimulated form. Addition of GTP to the solubilized glucagon-receptor complex caused complete dissociation of the complex, as has been shown with the membrane-bound form of the complex. Since the GTP-sensitive form of the glucagon receptor complex separates from the Gpp(NH)p-sensitive form of adenylate cyclase, it is concluded that the receptor and the enzyme are separate molecules, each associated with a distinct nucleotide regulatory site or component. These findings are discussed in terms of the possible structure of the hormone-sensitive state of adenylate cyclase.     

Evidence that forskolin activates turkey erythrocyte adenylate cyclase through a noncatalytic site

The diterpene forskolin has been reported to activate adenylate cyclase in a manner consistent with an interaction at the catalytic unit. However, some of its actions are more consistent with an interaction at the coupling unit that links the hormone receptor to the adenylate cyclase activity. This report adds support to the latter possibility. Under conditions that lead to stimulation of adenylate cyclase in turkey erythrocyte membranes by GTP, forskolin also becomes more active. Additional evidence to support an influence of forskolin upon adenylate cyclase via the GTP-coupling protein N includes the following: (i) forskolin, at submaximal concentrations, leads to enhanced sensitivity and responsiveness of isoproterenol-dependent adenylate cyclase activity in turkey erythrocyte membranes; (ii) under specified conditions, the nucleotide GDP, an inhibitor of the stimulating nucleotide GTP and its analog, guanyl imidodiphosphate (Gpp(NH)p), also markedly inhibits the action of forskolin; (iii) both Gpp(NH)p and forskolin are associated with a decrease in agonist affinity for the beta-adrenergic receptor. However, actions of forskolin in the turkey erythrocyte are not identical to those of GTP: (i) forskolin is never as potent as Gpp(NH)p in activating adenylate cyclase; (ii) the magnitude of synergism between isoproterenol and forskolin is not equal to that observed with isoproterenol and Gpp(NH)p; (iii) at high concentrations, forskolin inhibits antagonist binding to the beta-receptor. Forskolin appears to have several sites of action in the turkey erythrocyte membrane, including an influence upon the adenylate cyclase regulatory protein N.     

Interaction of the Inhibitory GTP Regulatory Component with Soluble Cerebral Cortical Adenylate Cyclase

Functional interaction of the inhibitory GTP regulatory component (Ni) with the adenylate cyclase catalytic subunit has not previously been demonstrated after detergent solubilization. The present report describes a sodium cholate-solubilized preparation of rat cerebral cortical membrane adenylate cyclase that retains guanine nucleotide-mediated inhibition of activity. Methods of membrane preparation, cholate extraction, and assay conditions were manipulated such that guanosine-5'-(beta-gamma-imido)triphosphate [Gpp(NH)p] inhibited basal activity 40-60%. The rank order of potency among various GTP analogs was similar in cholate extracts and in membranes: guanosine-5'-0-(3-thiotriphosphate) greater than Gpp(NH)p greater than GTP. Inclusion of 0.1 mM EGTA reduced basal activity 70-90% and abolished Gpp(NH)p inhibition of basal activity in both membranes and cholate extracts. Forskolin-stimulated activity was also inhibited by Gpp(NH)p. Treatment of either membranes or cholate extracts with N-ethylmaleimide abolished Gpp(NH)p inhibition. Gel filtration of the cholate extract over a Sepharose 6B column in 0.1% Lubrol PX partially resolved the adenylate cyclase components. However, Gpp(NH)p inhibition of basal activity (60% of the control) was maintained in select column fractions. Sucrose gradient centrifugation totally resolved the catalytic subunit from both functional Ni and stimulatory GTP regulatory component (Ns) activities. The sedimentation of functional Ni activity was detected by assaying the ability of sucrose gradient fractions to confer Gpp(NH)p inhibition of the resolved catalytic activity. Labeling of gradient or column fractions with pertussis toxin and [32P]NAD revealed that both the 39,000- and 41,000-dalton substrates comigrated with the functional Ni activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of β-adrenergic receptors in untreated and butyrate-threated HeLa cells

HeLa cells contain receptors on their surface which are β-adrenergic in nature. The binding of (?)-[³H]dihydroalprenolol is rapid, reversible, stereo-specific and of relatively high affinity. The HeLa cells also contain an adenylate cyclase which is activated by (?)-isoproterenol > (?)-epinephrine > (?)-norepinephrine. The adenylate cyclase of HeLa is also activated by guanyl-5′-yl-imidodophosphate (Gpp(NH)p), a nonhydrolyzable analogue of GTP. Inclusion of both (?)-isoproterenol and Gpp(NH)p leads to approximately additive rathen than synergistic activation of adenylate cyclase. After treatment of HeLa cells with 5 mM sodium butyrate there is an increase in the number of β-adrenergic receptors, but not in their affinity, which is reflected in an increased ability of (?)-isoproterenol to activate adenylate cyclase. Other properties of the β-adrenergic receptor including association and dissociation rates, temperature optimum of adenylate cyclase and response to Gpp(NH)p are relatively unaffected by butyrate pretreatment of the cells.     

Studies on the mechanism of inhibition of amphibian oocyte adenylate cyclase by progesterone

Progesterone treatment induces the meiotic maturation of Xenopus laevis oocytes. Previous evidence indicates that this hormonal effect may be due to inhibition of oocyte adenylate cyclase. The present work studies several aspects of the mechanism of adenylate cyclase inhibition by this hormone. Forskolin greatly stimulates oocyte adenylate cyclase in the absence of guanine nucleotides and this activity is not sensitive to progesterone inhibition. In addition the forskolin-activated enzyme is not inhibited by a wide range of guanine nucleotide, in the presence or absence of hormone. The time course of cAMP synthesis catalyzed by oocyte adenylate cyclase in the presence of guanyl-5′_l-imidodiphosphate (Gpp(NH)p) shows an initial lag period that does not depend on the concentration of Gpp(NH)p. Progesterone causes a very significant increase in the hysteresis of the reaction, at least doubling the half-time of enzyme activation. The hormonal effect on the lag cannot be reversed by saturating concentrations of Gpp(NH)p. Progesterone also decreases the steady-state rates of the reaction. This effect, however, depends on the concentration of Gpp(NH)p. High concentrations of Gpp(NH)p almost completely reverse the inhibition of the steady-state rates. Progesterone does not inhibit if it is added to the reaction after the initial lag period. Guanosine-5′-O-(2-thiodiphosphate) (GDP-β-S) is an efficient competitive inhibitor of Gpp(NH)p activation of adenylate cyclase. Progesterone inhibition is observed at all concentrations of GDP-β-S and is potentiated at high ratios of GDP-β-S to Gpp(NH)p. These data indicate that progesterone inhibits by interfering with the activation of the N_s subunit of the enzyme by guanine nucleotides, rather than through a mechanism involving a separate N_i subunit.     

Synergistic activation of bovine cerebellum adenylate cyclase by calmodulin and β-adrenergic agonists

The relationship between calmodulin-dependent and β-adrenergic-sensitive adenylate cyclase activities was examined in membrane preparations from bovine cerebellum. Although stimulation by β-adrenergic agonists or calmodulin can occur independently, it is shown that their simultaneous presence has a strong synergistic effect on enzyme activity. Calmodulin did not influence the regulatory components of the neurotransmitter-dependent pathway as shown by the lack of effect on (1) receptor affinity, (2) GTP requirement for receptor-mediated activation, (3) rate of activation by guanyl 5′-yl imidodiphosphate [Gpp(NH)p]. Conversely, isoproterenol and guanine nucleotides did not modify to a significant extent the characteristics of enzyme stimulation by Ca²⁺ and calmodulin. Furthermore, calmodulin and Gpp(NH)p-dependent activities displayed different sensitivities to thermal inactivation.Our results indicate that β-adrenergic agonists and calmodulin interact with the same catalytic activity in cerebellar membranes, but presumably via two independent pathways.     

Antagonism of the prostaglandin endoperoxide imhibition of hormone-stimulated adenylate cyclase by guanosine triphosphate and 5'-guanylyl-imidodiphosphate.

The prostaglandin endoperoxide prostaglandin H2 (15-hydroxy-9alpha, 11alpha-peroxidoprosta-5,13-dienoic acid) inhibits basal and hormone-stimulated adenylate cyclase in fat cell ghosts. This inhibition by prostaglandin H2 has been found to be antagonized by GTP and Gpp(NH)p. Dose response studies have shown GTP and Gpp(nh)p to be maximally effective at 3.3 muM, the lowest concentration tested. Although the system is exceedingly sensitive to modulation by GTP or Gpp(NH)p UTP, CTP, GMP, and cyclic GMP did not antagonize the antihormone activity of prostaglandin H2. Kinetic studies indicate that the GTP or Gpp(NH)p antagonism of prostaglandin H2 is observable on initial rates of cyclic AMP synthesis, and persists throughout the adenylate cyclase measurements. Preincubation of fat cell ghosts with GTP followed by washing and resuspension results in a prostaglandin H2-sensitive adenylate cyclase system. However, the same preincubation experiment with Gpp(NH)p produces an irreversible antagonism of the prostaglandin H2 inhibition of hormone-stimulated adenylate cyclase. It is suggested that prostaglandin H2 stabilizes the fat cell adenylate cyclase system in a state that is resistant to hormone stimulation, and GTP or Gpp(NH)p overcome this stabilization.