Guanyl nucleotide and divalent cation regulation of cortical S2 serotonin receptors

Computer-assisted quantitative analysis of radioligand binding to rat cortical S2 serotonin receptors indicates the existence of two affinity states of the same receptor population. Monophasic antagonist competition curves for [3H]ketanserin-labelled sites suggest a uniform population of receptors with one affinity state for antagonists. Biphasic competition curves of agonists suggest that agonists discriminate high- and low-agonist-affinity forms of the S2 receptors. The affinities of agonists for the high- and low-affinity states, and the apparent percentages of high agonist-affinity forms varies with different agonists. The guanine nucleotides GTP and guanyl-5'-imido-diphosphate [Gpp(NH)p], as well as divalent cations, modulate the proportion of the sites with high affinity for agonists as evidenced by their ability to shift the agonist competition curves for [3H]ketanserin-labelled S2 receptors. GTP and Gpp(NH)p effects appear to be agonist-specific, as they do not affect antagonist competition for [3H]ketanserin-labelled S2 receptors, or [3H]ketanserin binding to S2 receptors. ATP and ADP have little or no effect on the binding properties of S2 serotonin receptors, whereas GDP is less potent than GTP. The presence of these specific nucleotide effects are the first evidence suggesting involvement of a guanine nucleotide-binding protein in the mechanism of agonist interaction with the S2 serotonin receptor. In general, the binding properties of [3H]ketanserin-labelled S2 serotonin receptors strongly resemble those of adenylate-cyclase coupled receptors such as the beta-adrenergic, the alpha 2-receptor, and the D-2 dopamine receptor. This may indicate the S2 serotonin receptor is coupled to adenylate cyclase activity, through a GTP binding 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.     

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.     

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.     

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.     

Evidence for specific binding sites for guanine nucleotides in adipocyte and hepatocyte plasma membranes. A difference in fate of GTP and guanosine 5'-(beta, gamma-imino) triphosphate.

Binding and degradation of GTP and guanosine 5'-(beta, gamma-imino)triphosphate (Gpp(NH)p by plasma membranes from rat liver and fat cells were investigated. Gpp(NH)p is hydrolyzed predominantly by nucleotide pyrophosphohydrolases in the membranes, whereas GTP is hydrolyzed primarily by nucleotide phosphohydrolases. These enzymes are not specific for the guanine nucleotides since co-addition of the analogous adenine nucleotides spares their hydrolysis. Both Gpp(NH)p and GTP are taken up by the membranes at sites which, to the extent that high concentrations of the corresponding adenine nucleotides fail to inhibit uptake, appear to be specific for guanine nucleotides. Gpp(NH)p taken up at these sites remains essentially intact irrespective of the degree of hydrolysis of unbound Gpp(NH)p by nucleotide pyrophosphohydrolases, indicating that the binding siteis incapable of degrading Gpp(NH)p. GTP and GDP inhibit competitively the binding of Gpp(NH)p; the binding constants for the three nucleotides are similar (0.1 to 0.4 muM) and are in the same range required for their effects on adenylate cyclase activity. Binding of the nucleotides is inhibited by sulfhydryl agents, suggesting that a sulfhydryl group is involved in the binding process. In contrast to binding of Gpp(NH)p, uptake of GTP is accompanied by substantial hydrolysis, primarily to GDP, under incubation conditions (high [ATP] plus ATP regenerating system) in which [GTP] in the medium remains essentially constant. GDP bound to the membranes is progressively hydrolyzed to 5'-GMP. Thus, GTP and Gpp(NH)p, although binding to the same specific sites, are differentially susceptible to hydrolysis at their terminal phosphates when bound to these sites. These findings are discussed in terms of the markedly different potencies of GTP and Gpp(NH)p as activators of adenylate cyclase systems.     

Adenylate cyclase from Hirudo medicinalis segmental ganglia: Modulation by physiological and non-physiological agents

1. In Hirudo medicinalis segmental ganglia GTP is essential for the full expression of the stimulatory action of serotonin on the adenylate cyclase activity. The amine, in turn, increases the overall affinity of the enzymatic system for GTP.2. GTPγS and Gpp(NH)p, non-hydrolysable analogues of GTP, dose-dependently enhance the basal enzyme activity, but impair the stimulatory effect of serotonin.3. Fluoride ions biphasically modulate the leech adenylate cyclase both in the absence and in the presence of GTP. The ion effect is also influenced by non-physiological guanine nucleotides     

Guanosine 5', alpha-beta-methylene, triphosphate, a novel GTP analog, causes persistent activation of adenylate cyclase: evidence against pyrophosphorylation mechanism.

To test the hypothesis that guanine nucleotides activate adenylate cyclase by a covalent mechanism involving pyrophosphorylation of the enzyme, we studied the effect of a novel GTP analog, guanosine 5′, α-β-methylene triphosphate (Gp(CH₂)pp), with a methylene bond in the α-β-position that is stable to enzymatic hydrolysis. Gp(CH₂)pp was as effective as GTP in stimulating rat reticulocyte adenylate cyclase in the presence of isoproterenol. Previously only guanine nucleotides with modified terminal phosphates such as guanylyl 5′-imidodiphosphate (Gpp(NH)p) were thought capable of causing persistent activation of adenylate cyclase. Gp(CH₂)pp, however, caused persistent activation of rat reticulocyte and turkey erythrocyte adenylate cyclase. We conclude that guanine nucleotides do not activate adenylate cyclase by a pyrophosphorylation mechanism and that a modified γ-phosphate is not essential in guanine nucleotides for generation of the irreversibly-activated enzyme state.     

Differential effects of guanine nucleotides on the first step of VIP and glucagon action in membranes from liver cells

Despite the presence of a similar number of glucagon and VIP receptors in liver membranes, VIP induces a negligeable stimulation of adenylate cyclase when compared with glucagon effect. In order to elucidate these discrepancies, the effects of guanine nucleotides on the VIP and glucagon-responsive adenylate cyclase of liver were compared using pure ATP as substrate. 10^?8 M VIP accounted for a 1.5-fold increase of basal activity. In the presence of GTP or Gpp(NH)p (10^?9 to 10^?5 M), the level of cAMP production induced by VIP was no more than additive. In contrast, Gpp(NH)p potentiated the effect of glucagon on liver adenylate cyclase. These discrepancies are not explained by a difference in the peptide binding process. These data suggest that, in liver membranes, a GTP-binding protein N₂ is associated with the glucagon-sensitive adenylate cyclase, but is not detected for VIP. It is suggested that N₂ appears to be specific for the peptidic receptor.     

Guanosine diphosphate binding,metabolism and regulation of follitropin-sensitive adenylate cyclase activity in Sertoli cell membranes

Nucleotides such as GTP and GDP appear to be involved in signal transduction via G protein modulation of adenylate cyclase activity. Studies on direct binding of [³H]GDP to membranes prepared from cultured immature rat Sertoli cells indicated that this process was reversible, approached steady state within 10 min, had a K_a of 4.5 ·10⁶M⁻¹ and was specific for guanine nucleotides. The non-hydrolyzable analog, guanosine 5′-O-[3-thio]triphosphate (GPPP[S]), was most effective as an inhibitor of [³H]GDP binding (ED₅₀ = 4.8·10⁻⁸M), whereas guanosine 5′-O-[2-thio]diphosphate (Gpp[S]) was less potent (ED₅₀ = 3.4·10⁻⁷M). Release of bound GDP was enhanced by follitropin (FSH) in the presence of Gppp[S], although not by FSH alone. Sertoli cell membranes possess guanine nucleotide hydrolase activity, where 95% of added nucleotide was rapidly degraded to guanosine. Binding kinetics were significantly influenced by nucleotide metabolism, which was prevented by controlling the Mg²⁺ concentration with EDTA and including App[NH]p to reduce nonspecific hydrolysis. Kinetic studies indicated that Gpp[S] inhibited (P < 0.05) Gppp[S]-stimulated adenylate cyclase activity (K_i = 1.8·10⁻⁷M), whereas basal activity remained unaffected. Addition of Gpp[S] to pre-activated enzyme (FSH plus GTP) resulted in a time-dependent decay of adenylate cyclase activity with a K_off value of 6 ± 1·min⁻¹. Using a two-stage pre-inculbation technique, adenylate cyclase activity was demonstrated to be sensitive to the nucleotide bound. When FSH was included, catalytic activity was not altered by the order of pre-incubation with the nucleotides. This suggested that the exchange of bound Gpp[S] for Gppp[S] was enhance by FSH. Activation and attenuation of FSH-sensitive adenylate cyclase activity is dependent on a nucleotide exchange mechanism which is driven by (1) the higher affinity of G for GTP than GDP, (2) enhanced release of GD when FSH is present and (3) GTP hydrolysis coupled to rapid metabolism of guanine nucleotides.     

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.     

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.     

Multiple inhibitory and activating effects of nucleotides and magnesium on adrenal adenylate cyclase.

Adenylate cyclase in particulate fractions from rat adrenal glands is subject to regulation by purine nucleotides, particularly guanine nucleotides. While GTP activates the enzyme, this effect is not evident in all particulate fractions. Following dialysis of the refractory fractions activation by GTP is observed, an indication that endogenous nucleotides may obscure the effects of added GTP. The analog, guanyl-5'-yl imidodiphosphate (Gpp(NH)p gives considerable more activity than does GTP. GDP, on the other hand, is inhibitory, an effect revealed only in the absence of a nucleotide-regenerating solution. GDP blocks the action of both GTP and Gpp(NH)p. These results show that the gamma-phosphate of the nucleotide is required for but need not be metabolized in the activation process. At low substrate concentration (0.1 mM ATP or adenyl-5'-yl imidodiphosphate) stimulation of the enzyme by ACTH occurs only in the presence of added guanine nucleotide (GTP or Gpp(NH)p); the hormone and nucleotide act synergistically. While both GTP and Gpp(NH)p inhibit fluoride-stimulated activity, the level of fluoride required to demonstrate such inhibition appears not to be related to the level of fluoride required for activation of the enzyme. In the presence of GTP, or GTP plus ACTH, the enzyme exhibits normal Michaelis-Menten kinetics with respect to substrate utilization (K-m equal to 0.16 mM). In the activated state, produced with ACTH plus GTP, the enzyme is less susceptible to inhibition by a species of ATP uncomplexed with Mg2+, but is more susceptible to inhibition by Mg2+. These results demonstrate that fundamental differences exist between different states of the adenylate cyclase. The difficulties in describing kinetically the regulation of adenylate cyclase systems in view of the multiple actions of nucleotides and magnesium are discussed.     

Essential role of GTP in the expression of adenylate cyclase activity after cholera toxin treatment.

Expression of activation of rat liver adenylate cyclase by the A1 peptide of cholera toxin and NAD is dependent on GTP. The nucleotide is effective either when added to the assay medium or during toxin (and NAD) treatment. Toxin treatment increases the Vmax for activation by GTP and the effect of GTP persists in toxin-treated membranes, a property seen in control membranes only with non-hydrolyzable analogs of GTP such as Gpp(NH)p. These observations could be explained by a recent report that cholera toxin acts to inhibit a GTPase associated with denylate cyclase. However, we have observed that one of the major effects of the toxin is to decrease the affinity of guanine nucleotides for the processes involved in the activation of adenylate cyclase and in the regulation of the binding of glucagon to its receptor. Moreover, the absence of lag time in the activation of adenylate cyclase by GTP, in contrast to by Gpp(NH)p, and the markedly reduced fluoride action after toxin treatment suggest that GTPase inhibition may not be the only action of cholera toxin on the adenylate cyclase system. We believe that the multiple effects of toxin action is a reflection of the recently revealed complexity of the regulation of adenylate cyclase by guanine nucleotides.     

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.     

ADP- and epinephrine-elicited release of [3H]guanylylimododiphosphate from platelet membranes: Implications for receptor-Ni stoichiometry

Epinephrine-promoted release of [³H]guanylylimidodiphosphate ([³H]Gpp(NH)p) from human platelet membranes has been used to probe the interactions between alpha₂-adrenergic recpetors and N_i, the guanine nucleotide binding protein that couples those receptors to an inhibition of adenylate cyclase activity. We show here that ADP, which also acts through specific platelet receptors to inhibit adenylate cyclase activity, also promotes the release of [³H]Gpp(NH). The amount of [³H]Gpp(NH)-release elicited by epinephrine and by ADP together is equal to the sum of the amounts released by the two agents acting individually. Furthermore the maximal amounts of [³H]Gpp(NH)-release elicited by each of the two agents approximates the numbers of receptors for ADP and epinephrine present in the platelet membranes. These results suggest that the two receptor types interact with distinct portions of the pool of N_i molecules and that each receptor initiates guanine-nucleotide exchange on a single molecule of N_i.     

Calcium-dependent adenylate cyclase from rat cerebral cortex: Activation by guanine nucleotides

The adenylate cyclase activity of a participate preparation of rat cerebral cortex is composed of at least two contributing components, one of which requires a Ca²⁺-dependent regulator protein (CDR) for activity (Brostrom, C. O., Brostrom, M. A., and Wolff, D. J. (1977) J. Biol. Chem.252, 5677–5685). Each of these components of the activity was activated by GTP and its synthetic analog, 5-guanylylimidodiphosphate (Gpp(NH)p). The component of the adenylate cyclase activity which did not respond to CDR (CDR-independent activity) was stimulated approximately 60% by 100 μm GTP and 3.5-fold by 100 μm Gpp(NH)p. Concentrations of GTP required for maximal activation of the CDR-dependent adenylate cyclase component decreased as CDR concentrations in the assay were increased. Similarly, GTP pr Gpp(NH)p lowered the concentration of CDR required to produce half-maximal activation of this enzyme form. At saturating CDR concentrations, however, increases in activity were not observed with the addition of these nucleotides. The CDR-dependent component responded biphasically (activation followed by inhibition) to increasing free Ca²⁺ concentrations; both phases of this response occurred at lower free Ca²⁺ concentrations with GTP present in the assay. The concentration of chlorpromazine which inhibited activation of adenylate cyclase by CDR was elevated when GTP was present. The CDR-dependent form of activity, which is stabilized by CDR to thermal inactivation, was also stabilized by Gpp(NH)p. The increase in stability produced by Gpp(NH)p did not require the presence of CDR, and stabilization with both Gpp(NH)p and CDR was greater than that obtained with either Gpp(NH)p or CDR alone.     

Effect of Guanine Nucleotides on Dopaminergic Agonist and Antagonist Affinity for [3H]Sulpiride Binding Sites in Rat Striatal Membrane Preparations

Abstract: [³H]Sulpiride bound to rat striatal membrane preparations with a saturable, high affinity component. This binding was displaced potently by dopamine antagonists (both classic neuroleptics and the benzamide, sulpiride) and less potently by dopamine agonists. GTP and its stable analogue Gpp(NH)p did not affect [³H]sulpiride binding to the membranes but altered the affinity for dopaminergic agonists. This effect was specific in that antagonist binding was not affected and only GTP, GDP, and Gpp(NH)p produced the effect. Similar alterations in ligand binding affinity caused by guanine nucleotides have been observed for binding sites linked to an adenylate cyclase. Such an interpretation for the case of [³H]sulpiride is contrary to suggestions that sulpiride labels only those dopamine receptors that are not cyclase linked.     

Attenuation of chick heart adenylate cyclase by muscarinic receptors after pertussis toxin treatment

Pertussis toxin selectively modifies the function of Ni, the inhibitory guanine nucleotide binding protein of the adenylate cyclase complex. In chick heart membranes, guanine nucleotide activation of Ni resulted in a decrease in the apparent affinity of the muscarinic receptor for the agonist oxotremorine, inhibition of basal adenylate cyclase activity, and the attenuation of adenylate cyclase by oxotremorine. Treatment of chicks with pertussis toxin caused the covalent modification of 80-85% of cardiac Ni. After this treatment Gpp(NH)p had no effect on muscarinic receptor affinity and GTP stimulated basal adenylate cyclase activity. In contrast, the GTP-dependent attenuation of adenylate cyclase caused by muscarinic receptors was unaffected.     

The adenylate cyclase system of planaria Polycelis tenuis: activation by serotonin and guanine nucleotides

The particulate fraction prepared after homogenization of planaria Polycelis tenuis in a buffer containing 3 mM EDTA and 15 mM 2-mercaptoethanol possesses an adenylate cyclase activity which was enhanced two-fold by serotonin and 20-fold by the nucleotide analog guanosine 5'-(beta-gamma-imino)triphosphate, Gpp(NH)p; when present together, the two activators exhibited a marked synergistic effect. The effect of serotonin was dose dependent, with a KA of 2 micrometer and a Hill coefficient of 0.4. In the presence of 10 micrometer Gpp(NH)p, these values became 45 nM and 1.5, respectively. The effect of serotonin was due to an increase in the maximal velocity of the enzyme and was specifically inhibited by methiotepin. The effect of methiotepin was half-maximal at 0.2 micrometer in the absence of Gpp(NH)p and at 5.0 micrometer in its presence. Planaria thus appear to be the lowest organisms in which guanine nucleotides are active upon adenylate cyclase. As serotonin is normally present in planaria, it is postulated that a serotonin-dependent regulation of adenylate cyclase activity plays a physiological role in this species.