Stimulative effect of guanylylimidodiphosphate on the vasopressin-induced increment of osmotic water flow of the toad bladder.

The effect of guanylylimidodiphosphate [Gpp(NH)p] on vasopressin-induced osmotic water flow across the bladder of the toad, $\text{Bufo}\text{bufo}\text{japonicus}$ was examined. Gpp(NH)p significantly enhanced vasopressin-induced osmotic water flow of the bladder at a concentration of 1 × 10^?5M, while it showed no effect on the water flow without vasopressin. Gpp(NH)p alone could not enhance cyclic AMP-induced osmotic water flow of the toad bladder. Adenylylimidodiphosphate [App(NH)p] could not enhance vasopressin-induced osmotic water flow of the bladder at a concentration of 1 × 10^?5M. The results suggest that Gpp(NH)p can enhance the physiological effect of vasopressin by stimulating vasopressin activation of adenylate cyclase during substrate and hormone depletion of the toad bladder.     

Multiphasic activation of smooth muscle adenylate cyclase by pretreatment with guanyl-5′-yl imidodiphosphate (Gpp(NH)p) suggests multiple enzyme populations

The activation of uterine smooth muscle adenylate cyclase was studied by pretreating the particulate form of the enzyme with the GTP analog guanyl-5′-yl imidodiphosphate (Gpp(NH)p). Pretreatment with Gpp(NH)p left the enzyme in an irreversibly activated state which survived subsequent washing in guanyl nucleotide-free buffer. Activation under these conditions was multiphasic with rapid and slow components. At 23 °C slow activation proceeded at about $\text{1}\text{10}\text{th}$ the rate of rapid activation. The onset of the slow phase took longer at lower temperatures. Routine adenylate cyclase assay conditions (conversion of [³²P]ATP to cyclic [³²P]AMP) carried out without pretreatment probably characterized the rapidly activated component. The simplest kinetic model suggests not only the generally accepted two-step association reaction, but also implies the existence of more than one enzyme form, each of which is characterized by a separate activation rate. The complex kinetics of activation might be explained by a heterogeneous mixture of unassociated and preassociated nucleotide binding and catalytic subunits.     

Association of nucleosidediphosphate kinase with microtubules.

A nucleosidediphosphate kinase activity (EC 2.7.4.6) which phosphorylates GDP to GTP is present in bovine brain microtubule protein prepared by cycles of assembly-disassembly. This activity persists through 5 cycles of assembly-disassembly and sediments with microtubules in sucrose density gradients, but is not associated with the tubulin dimer. It is proposed that the kinase is an integral part of the microtubule and is therefore a microtubule associated protein (MAP). Several isozymes of nucleosidediphosphate kinase exist in our preparations with a pI 7.6 form predominant. It may be speculated that this enzyme affects tubulin assembly $\text{in vivo}$ by modulating the $\text{GTP}\text{GDP}$ ratio in the microtubule environment.     

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.     

The influence of magnesium on calcium-activated, vascular smooth muscle actomyosin ATPase activity

Histamine activation of adenylyl cyclase activity in sonicated enriched rat gastric parietal cells showed a time, temperature, and concentration dependence upon guanine diphosphoimide (Gpp(NH)p). Enzyme activation was first order with Gpp(NH)p alone or Gpp(NH)p plus histamine. The K_a for Gpp(NH)p was ~2 μm and was not influenced by histamine. GTP and GDP were inactive alone or with histamine and were competitive with Gpp(NH)p, showing apparent K_i's of near 0.4 and 0.3 μm, respectively. In the presence of Gpp(NH)p, parietal cell adenylyl cyclase was activated by histamine with an EC₅₀ of 24 μm, the most potent in a series of histamine analogs, further substantiating an H₂-receptor classification for this response. H₂-Receptor antagonists were competitive inhibitors with submicromolar K_i's. Preincubation of parietal cells with histamine and Gpp(NH)p resulted in adenylyl cyclase activity up to 15 times the basal level. The activated state was retained after washing the cells free of histamine and Gpp(NH)p and was not reversed by the subsequent addition of either histamine, cimetidine, or GTP. The other gastric acid secretagogues, pentagastrin and carbamylcholine, were without effect upon histamine activation or the activated state of adenylyl cyclase. These results describe a level of control of histamine-sensitive adenylyl cyclase that requires consideration in the activation of the parietal cell H₂-receptor system by histamine to modulate acid secretion.     

Exchange of Guanine Nucleotides Between Tubulin and GTP-Binding Proteins That Regulate Adenylate Cyclase: Cytoskeletal Modification of Neuronal Signal Transduction

Tubulin, the primary constituent of microtubules, is a GTP-binding proteins with structural similarities to other GTP-binding proteins. Whereas microtubules have been implicated as modulators of the adenylate cyclase system, the mechanism of this regulation has been elusive. Tubulin, polymerized with the hydrolysis-resistant GTP analog, 5'-guanylylimidodiphosphate [Gpp(NH)p], can promote inhibition of synaptic membrane adenylate cyclase which persists subsequent to washing. Tubulin with Gpp(NH)p bound was slightly less potent than free Gpp(NH)p in the inhibition of adenylate cyclase, but tubulin without nucleotide bound had no effect on the enzyme. A GTP-binding protein from the rod outer segment (transducin), with Gpp(NH)p bound, was also without effect on adenylate cyclase. Tubulin (regardless of the nucleotide bound to it) did not alter the activity of the adenylate cyclase catalytic unit directly. When tubulin was polymerized with the hydrolysis-resistant photoaffinity GTP analog, [32P]P3(4-azidoanilido)-P1-5'-GTP ([32P]AAGTP), and this protein was added to synaptic membranes, AAGTP was transferred from tubulin to the inhibitory GTP-binding protein, Gi. This transfer was blocked by prior incubation of the membranes with Gpp(NH)p or covalent binding of AAGTP to tubulin prior to exposure of that tubulin to membranes. Incubation of membranes with Gpp(NH)p subsequent to incubation with tubulin-AAGTP results in a decrease in AAGTP bound to Gi and a compensatory increase in AAGTP bound to the stimulatory GTP-binding protein, Gs. Likewise, persistent inhibition of adenylate cyclase by tubulin-Gpp(NH)p could be overridden by the inclusion of 100 microM Gpp(NH)p in the assay inhibition. Whereas Gpp(NH)p promotes persistent inhibition of synaptic membrane adenylate cyclase without incubation at elevated temperatures, tubulin [with AAGTP or Gpp(NH)p bound] requires 30 s incubation at 23 degrees C to effect adenylate cyclase inhibition. Photoaffinity experiments yield parallel results. These data are consistent with synaptic membrane tubulin regulating neuronal adenylate cyclase by transferring GTP to Gi and, subsequently, to Gs.     

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.     

Characterization of beta-adrenergic receptor and adenylate cyclase in canine cerebellum.

Binding of (?)-[${}^3\text{H}$]dihydroalprenolol to the synaptic membrane fractions of canine cerebellum was rapid and reversible with rate constants of 1.62 × 10⁸m^?1 min^?1 and 0.189 min^?1 for the forward and reverse reactions, respectively. The binding was of high affinity and saturable with an equilibrium dissociation constant (K_D) of 5 to 7 nm. Bound (?)-[${}^3\text{H}$]-dihydroalprenolol was displaceable with β-adrenergic agonists and antagonists, but not with a variety of other neuroactive substances such as acetylcholine, histamine, serotonin, dopamine, tyramine, (?)-phenylephrine, γ-aminobutyric acid, glycine, and glutamic acid. Adenylate cyclase of the membranes was stimulated at most three times by β-adrenergic agonists, but not significantly by the other neuroactive substances. Guanine nucleotides such as GTP and guanyl-5′-yl imidodiphosphate (Gpp(NH)p) were strictly required for β-adrenergic stimulation of adenylate cyclase with their optimum concentrations of 50 μm, although the nucleotides alone elevated virtually no basal activity. The affinities of β-adrenergic ligands including some stereoisomers for (?)-[${}^3\text{H}$]dihydroalprenolol binding sites were very similar to those for adenylate cyclase in the presence of GTP. Binding of β-adrenergic agonists to the membranes exhibited an apparent negative cooperativity as determined by displacement of (?)-[${}^3\text{H}$]dihydroalprenolol in the absence of purine nucleotides. This negative cooperativity was entirely abolished by addition of either GTP or Gpp(NH)p at 50 μm. Both (?)-isoproterenol-stimulated adenylate cyclase activity and binding of (?)-[${}^3\text{H}$]dihydroalprenolol were not affected by β₁-selective antagonists, (±)-atenolol, and (±)-practolol, at concentrations which completely inhibit peripheral β₁-responses in vitro, whereas β₂-selective agonists such as YM-08316 (BD-40A) and (±)-salbutamol not only stimulated adenylate cyclase but also competitively inhibited binding of (?)-[${}^3\text{H}$]dihydroalprenolol. These results indicate that canine cerebellar adenylate cyclase may be coupled specifically with β₂-adrenergic receptor.     

Adriamycin inactivates cytochrome c oxidase by exclusion of the enzyme from its cardiolipin essential environment

The regulation of ligand binding to the muscarinic acetylcholine receptor in developing chick heart has been studied using the radiolabeled antagonist [³H]quinuclidinyl benzilate (QNB). In assays containing only buffer and a source of receptor protein, the antagonist radioligand bound to a single, high affinity state of the receptor. If Mg²⁺ and EDTA were added, [³H]QNB bound to a single, low affinity state. The guanine nucleotide analog, guanylylimidodiphosphate [Gpp(NH)p], reversed the effect of $\text{Mg}{}^{\mathrm{2+}}\text{EDTA}$ so that [³H]QNB again bound only to a single, high affinity state. Sodium could also reverse the effect of $\text{Mg}{}^{\mathrm{2+}}\text{EDTA}$ on antagonist binding but the effects of sodium and Gpp(NH)p on [³H]QNB binding were not additive.     

On the mechanism of activation of fat cell adenylate cyclase by guanine nucleotides. An explanation for the biphasic inhibitory and stimulatory effects of the nucleotides and the role of hormones.

Adenylate cyclase activity in purified plasma membranes from rat fat cells displays transient kinetic characteristics in the absence and presence of guanyl=5'=yl imidodiphosphate (Gpp(NH)p). Gpp(NH)p causes immediate inhibition of enzyme activity; the inhibitory phase is followed by a slow increase in activity which, depending on incubation temperature, exceeds activity stimulated in the presence of hormones (glucagon, secretin, epinephrine, or adrenocorticotropin). Basal activity displays an initial high rate of activity which decays to a low state of activity within 2 min of incubation. Hormones do not alter the initial rate but prevent the decay in enzyme activity. The inhibitory phase of Gpp(NH)p action and the previously reported (Harwood, J.P., Low, H., and Rodbell, M. (1973) J. Biol. Chem. 248, 6239-6245) inhibitory effects of GTP are abolished by increasing (Mg2+) and pH to 50 mM and 8.5, respectively. Under these conditions, Gpp(NH)p and GTP cause marked stimulation of activity, the stimulatory effect of Gpp(NH)p being greater than that of GTP both in the absence and presence of hormones...     

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.     

Guanine nucleotides modulate cell surface cAMP-binding sites in membranes from Dictyostelium discoideum

D. discoideum contains kinetically distinguishable cell surface cAMP binding sites. One class, S, is slowly dissociating and has high affinity for cAMP (K_d = 15 nM, $\text{t}\text{1}\text{2}\text{= 15 s}$). A second class is fast dissociating ($\text{t}\text{1}\text{2}$ about 1 s) and is composed of high affinity binding sites H (K_d ≈ 60 nM), and low affinity binding sites L (K_d = ≈ 450 nM) which interconvert during the binding reaction. Guanine nucleotides affect these three binding types in membranes prepared by shearing D.discoideum cells through Nucleopore filters. The affinity of S for cAMP is reduced by guanine nucleotides from 13 nM to 25 nM, and the number of S-sites is reduced about 50%. The number of fast dissociating sites is not altered by guanine nucleotides, but these sites are mainly in the low affinity state. Half-maximal effects are obtained at about 1 μM GTP, 2 μM GDP and 10 μM Gpp(NH)p(guanyl-5′-yl-imidodiphosphate); ATP and ADP are without effect up to 1 mM. These results indicate that D.discoideum cells have a functionally active guanine nucleotide binding protein involved in the transduction of extracellular cAMP signals via cell surface cAMP receptors.     

Assembly and disassembly properties of microtubules formed in the presence of GTP, 5'-guanylyl imidodiphosphate, and 5'-guanylyl methylenediphosphate

We have examined the properties of microtubules formed in the presence of GTP, 5'-guanylyl imidodiphosphate (GMPP(NH)P), and 5'-guanylyl methylenediphosphate (GMPP(CH2)P) to identify features of the assembly or disassembly reactions uniquely related to hydrolysis. The assembly of microtubules with GTP or GMPP(NH)P was similar in terms of rates and extents of assembly, the length distributions, and podophyllotoxin-induced depolymerization. The greater rapidity of GMPP(CH2)P-supported assembly, however, resulted in shorter, more numerous microtubules and the rate of podophyllotoxin-induced depolymerization was consistent with an increased number of concentration of microtubules. Experiments with GTP or analogue incorporation and release indicated that GTP-tubule turnover corresponded to a rate of about 8% of the microtubule protein taken up or released per h. With GMPP(NH)P- and GMPP(CH2)P-tubules, the rates of label uptake by unlabeled microtubules were considerably lower than observed with guanosine triphosphate. We suggest that exchange experiments can reflect contributions from head-to-tail polymerization and polymer length redistribution, but it is not as yet possible to evaluate the relative contributions of each process.     

Uroporphyria development in cultured chick embryo fibroblasts long-term treated with chloramphenicol and ethidium bromide

In brain cortex, low concentrations of GTP or Gpp(NH)p activated the membrane-bound low Km cyclic AMP phosphodiesterase while higher concentrations of GTP, but not of Gpp(NH)p, reversed this activation. The adenosine analog N6-phenylisopropyladenosine (N6-PIA) elicited biphasic effect on this enzyme (activation up to 10(-8) M, complete reversion at 10(-5) M), provided that GTP was present. N6-PIA activation was reduced in the presence of Gpp(NH)p and blocked by sodium (80 mM). In contrast, the soluble low Km cyclic AMP phosphodiesterase was insensitive to GTP or N6-PIA. This study suggests that guanine nucleotides and N6-PIA exert their effects on the membrane-bound enzyme through guanine nucleotide regulatory proteins.     

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.     

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.     

Differential Sensitivity of Basal and Opioid-Stimulated Low Km GTPase to Guanine Nucleotide Analogs

In membranes derived from NG108-15 cells, the opioid peptide [D-Ala2,D-Leu5]enkephalin (DADLE) stimulates a low Km GTPase. The nucleotide analogs guanosine 5'-O-(2-thio)diphosphate (GDP beta S), guanosine 5'-(beta,gamma-imido)triphosphate [Gpp(NH)p] and guanosine 5'-O-(3-thio)-triphosphate (GTP gamma S) inhibit the basal enzymatic activity with the order of potency GTP gamma S greater than Gpp (NH)p greater than GDP beta S. In the presence of DADLE, the inhibition isotherms of GDP beta S and Gpp(NH)p are shifted to the right five- and fourfold, respectively, compared to the inhibition observed in the absence of DADLE. In contrast, the IC50 of GTP gamma S for inhibiting the enzyme is reduced by 55% in the presence of the opioid. Both Gpp(NH)p and GTP gamma S produce a concentration-dependent increase in the Km(app) of GTPase, without affecting its Vmax, indicating a competitive inhibition. However, the replots of Km(app) versus inhibitor concentration are hyperbolic, suggesting a partial type of inhibition. Both Gpp(NH)p and GTP gamma S, but not GTP, induce an increase in the EC50 of DADLE for stimulating GTPase. These findings indicate that the basal and the opioid-stimulated low Km GTPase differ in their respective sensitivities to inhibition by guanine nucleotide analogs.     

Effects of drugs on pigeon erythrocyte membrane and asymmetric control of adenylate cyclase by the lipid bilayer

In pigeon erythrocyte membrane, the β-adrenergic receptor and the enzyme adenylate cyclase can be uncoupled in two different ways depending on the type of drug used.Cationic drugs: chlorpromazine, methochlorpromazine, tetracaine, $\text{n-}\text{octylamine}$ and a neutral alcohol, octanol, abolished alprenolol receptor binding ability and in the same range of concentration of the drug, sensitized adenylate cyclase to fluoride or Gpp(NH)p stimulation. Anionic drugs: di- and trinitrophenols, indomethacin and octanoic acid did not affect the total number of β-adrenergic receptor sites and, with the exception of trinitrophenol, did not change the association constant for alprenolol but they abolished the stimulation of adenylate cyclase by isoproterenol, fluoride or Gpp(NH)p. These modifications of the adenylate cyclase system occurred in a range of drug concentration where cell shape and protection against hemolysis were also affected.As chemical composition varies widely from one drug to another, it is suggested that these effects are largely nonspecific and mediated by the lipid bilayer. They are probably related to a preferential sidedness of action of the drugs in the lipid bilayer, displaying the role of an asymmetric control of the adenylate cyclase system in the membrane by the two halves of this bilayer.     

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.     

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.