Multiple effects of guanine nucleotides on human platelet adenylate cyclase

We report that the adenylate cyclase system in human platelets is subject to multiple regulation by guanine nucleotides. Previously it has been reported that GTP is either required for or has little effect on the response of the enzyme to prostaglandin E₁. We have found that when platelet lysates were prepared in the presence of 5 mM EDTA, GTP lowered the basal and prostaglandin E₁-stimulated adenylate cyclase activity when the enzyme was assayed in the presence of Mg²⁺. The basal and prostaglandin E₁-stimulated adenylate cyclase activities were also increased by washing, which presumably removes endogenous GTP. The analog, guanyl-5′-yl-imidodiphosphate mimics the inhibitory effect of GTP on prostaglandin E₁-stimulated adenylate cyclase activity but it stimulates basal enzyme activity. The onset of the inhibitory effect of GTP on the adenylate cyclase system is rapid (1 min) and is maintained at a constant rate during incubation for 10 min. GTP and guanyl-5′-yl-imidodiphosphate were noncompetitive inhibitors of prostaglandin E₁. An increase in the concentration of Mg²⁺ gradually reduces the effect of GTP while having little influence on the effect of guanyl-5′-yl-imidodiphosphate. Neither the substrate concentration nor the pH (7.2–8.5) is related to the inhibitory effect of guanine nucleotides. The inhibition by nucleotides was found to show a specificity for purine nucleotides with the order of potency being guanyl-5′-yl-imidodiphosphate > dGTP > GTP > ITP > XTP > CTP > TTP. The inhibitory effect of GTP is reversible while the effect of guanyl-5′-yl-imidodiphosphate is irreversible. The GTP inhibitory effect was abolished by preparing the lysates in the presence of Ca²⁺. However, the inhibitory effect of guanyl-5′-yl-imidodiphosphate persisted. Substitution of Mn²⁺ for Mg²⁺ in the assay medium resulted in a diminution of the inhibitory effect of GTP on basal activity and converted the inhibitory effect of GTP on prostaglandin E₁-stimulated activity to a stimulatory effect. At a lower concentration of Mn²⁺ (less than 2 mM) guanyl-5′-yl-imidodiphosphate inhibited prostaglandin E₁-stimulated adenylate cyclase activity, but at a higher concentration of Mn²⁺, it caused an increase in enzyme activity exceeding that occuring in the presence of prostaglandin E₁. In the presence of Mn²⁺, dGTP mimics the effect of GTP and is 50% as effective as GTP. Our data suggest that the inhibitory effect of GTP on prostaglandin E₁-stimulated adenylate cyclase is mainly due to its direct effect on the enzyme itself, whereas the stimulatory effect of GTP on prostaglandin E₁-stimulated adenylate cyclase is due to enhancement of the coupling between the prostaglandin E₁ receptor and adenylate cyclase. These studies also indicate that the method of preparation of platelet lysates can profoundly alter the nature of guanine nucleotide regulation of adenylate cyclase.     

Regulation of catecholamine-responsive adenylate cyclase activity in rat reticulocyte membranes by endogenous factors General characteristics and resolution into protein and nucleotide components

A 100 000 × g soluble, supernatant fraction obtained from the hemolysate of rat reticulocytes was studied for its effect upon catecholamine-sensitive adenylate cyclase activity in reticulocyte membranes. The supernatant material, devoid of adenylate cyclase activity itself, amplified isoproterenol-dependent activity in responsive membranes and was an essential requirement for the expression of hormone sensitivity in membranes rendered unresponsive to isoproterenol alone. The increment in catecholamine-associated activity conferred upon reticulocyte membranes by the supernatant material was β-adrenergic because it did not affect basal or fluoride-related activity and was completely inhibited by propranolol. Guanine nucleotides were present in the supernatant but could account for only a fraction of the total activity because the supernatant was able to cause greater stimulation than maximal concentrations of GTP and when specified concentrations of exogenous GTP were compared with equivalent nucleotide concentrations in the supernatant, the supernatant always led to greater activity. The supernatant was resolved into protein- and nucleotide-containing components by ion-exchange chromatography. Each component was approximately one-half as active in amplifying catecholamine-dependent adenylate cyclase as the unresolved, crude supernatant material. The activity eluted in the first peak of the DEAE chromatogram was resistant to alkaline phosphatase, sensitive to trypsin, not dialyzable and contained no detectable concentrations of GTP or GDP. In contrast, the activity eluted in the second peak of the DEAE chromatogram was sensitive to alkaline phosphatase, resistant to trypsin, completely dialyzable and contained both GTP (30 μM) and GDP (10 μM) in significant concentrations. Neither the crude supernatant not its two active components affected the binding of [¹²⁵I]-iodohydroxybenzylpindolol to reticulocyte membranes. These observations establish in rat reticulocytes the presence of protein and guanine nucleotide constituents which have independent influences upon the catecholamine-responsive adenylate cyclase of reticulocyte membranes.     

[3H]Vasopressin binding to rat hippocampal synaptic plasma membrane: Kinetic and pharmacological characterization

Characterization of specific vasopressin binding sites to rat hippocampal membranes has been assayed using tritiated lysine-vasopressin labelled on the tyrosyl residue. At 30°C specific [³H]vasopressin binding was saturable. The estimated equilibrium dissociation constant was 7.1 nM, the mean maximal binding capacity was 78 fmol/mg protein. Arginine-vasopressin has a high affinity (K_d = 2.8 nM) and dDAVP has a low affinity (K_d = 249 nM) for hippocampal synaptic membranes. (OH)AVP and Phe²Orn⁸VT are at least as active as AVP in inhibiting [³H]vasopressin binding. Adenylate cyclase was activated by VIP and inhibited by PIA, but not affected by lysine-vasopressin.     

Adenosine Inhibition of Calmodulin-Sensitive Adenylate Cyclase from Bovine Cerebral Cortex

Calmodulin (CaM)-sensitive adenylate cyclase has recently been purified extensively from bovine brain. In this study, the sensitivity of the CaM-sensitive adenylate cyclase to adenosine and adenosine analogs was examined. The highly purified enzyme preparation retained sensitivity to inhibition by adenosine and adenosine analogs with ribose ring modifications, but not to those with purine ring modifications. Adenosine inhibition of this enzyme was not dependent on GTP and was noncompetitive with respect to ATP. Enzyme that had been dissociated from functional guanine nucleotide binding protein interactions by gel filtration in the presence of the zwitterionic detergent 3-[3-(cholamidopropyl)-dimethylammonio]-propanesulfonate and Mn2+ retained sensitivity to adenosine inhibition. The Ki for adenosine inhibition of the CaM-sensitive adenylate cyclase was approximately 2.6 X 10(-4) M. 5'-Guanylylimidodiphosphate and CaM did not affect the Ki of 3'-deoxyadenosine for the enzyme, but the presence of Ca2+ in the millimolar range raised the Ki by a factor of 5. These results show that the CaM-sensitive form of adenylate cyclase from bovine brain is subject to adenosine inhibition, and strongly suggest that this inhibition is due to interaction of ligands with a purine-specific ("P") site located on the catalytic subunit of the enzyme.     

Cyclic AMP, calcium and control of cell growth

The role of cyclic AMP and calcium in the control of normal and tumour cell growth is considered in relation to the question whether cyclic AMP is a true mitogen or co-mitogen. It is proposed that cyclic AMP normally controls the cell cycle at a point in G1 phase only by virtue of its ability to exclude calcium required by cells to progress past this point into S phase. Therefore increased influx of calcium by other routes induced by various factors can bypass the inhibitory effect of cyclic AMP and stimulate growth. In these circumstances cyclic AMP or calcium may or may not facilitate further progress into S phase according to the metabolic requirements of individual cells. The relevance to cancer cells is considered.     

小牛睾丸中G蛋白对FSH受体亲和性和腺苷酸环化酶活性的调节

本文用NEM(N-ethylmaleimide)为探针研究了G蛋白(鸟嘌呤核苷酸调节蛋白,Gp)对小牛睾丸中FSH受体的亲和性及腺苷酸环化酶活性的调节作用。证据表明,①在小牛睾丸细胞膜G蛋白上存在两种类型鸟嘌呤核苷酸结合位点(下简称GTP结合位点),高亲和性低容量结合位点及低亲和性高容量结合位点;②高亲和性结合位点(对NEM敏感)调节腺苷酸环化酶活性,而对NEM相对不敏感的低亲和性位点则不直接参与该酶活性的调节;③G蛋白对受体亲和性的调节则不仅要高亲和性位点的参与,而且主要受低亲和性位点的调节。     

Equilibrium binding of [3H]tubocurarine and [3H]acetylcholine by Torpedo postsynaptic membranes: stoichiometry and ligand interactions

Studies are presented of the equilibrium binding of [3H]-d-tubocurarine (dTC) and [3H]acetylcholine (AcCh) to Torpedo postsynaptic membranes. The saturable binding of [3H]dTC is characterized by two affinities: Kd1 = 33 +/- 6 nM and Kd2 = 7.7 +/- 4.6 microM, with equal numbers of binding sites. Both components are completely inhibited by pretreatment with excess alpha-bungarotoxin or 100 microM nonradioactive dTC and competitively inhibited by carbamylcholine with a KI = 100 nM, but not affected by the local anesthetics dimethisoquin, proadifen, and meproadifen. The biphasic nature of [3H]dTC binding was unaltered in solutions of low ionic strength and by preparation of Torpedo membranes in the presence of N-ethylmaleimide, a treatment which yields dimeric AcCJ receptors. dTC competitively inhibits the binding of [3H]AcCH and decreases the fluorescence of 1-(5-dimethylaminonaphthalene-1-sulfonamido)ethane-2-trimethylammonium (Dns-Chol) in a manner quantitatively consistent with its directly measured binding properties. It decreases the initial rate of 3H-labeled Naja nigricollis alpha-toxin binding by 50% at 60 nM with an apparent Hill coefficient of 0.58. The stoichiometry of total dTC, AcCh, and alpha-neurotoxin binding sites in Torpedo membranes was determined by radiochemical techniques and by a novel fluorescence assay utilizing Dns-Chol as an indicator, yielding ratios of 0.9 +/- 0.1:0.9 +/- 0.2:1, respectively. The biphasic equilibrium binding function is not unique to dTC since other ligands inhibited [3h]acCh binding in a biphasic manner with apparent inhibition constants as follows: gallamine triethiodide (K11 = 2 microM, K12 = 1 mM); Me2dTC (K11 = 500 nM, K12 = 10 microM); decamethonium (K11 = 100 nM, K12 = 1.6 microM). Carbamylcholine, however, inhibited [3H]AcCh binding with a single KI = 100 nM. The observed competition between those ligands and [3H] AcCh cannot be completely accounted for by competitive interaction with two different affinities, and the deviations are discussed in terms of the positive cooperativity of the [3H] AcCh binding function itself. It is concluded that dTC binds only to the AcCh sites in Torpedo membranes and that those sites display two affinities for dTC but only one for AcCh.     

Different specific binding sites of [3H]glycine and [3H]strychnine in synaptosomal membranes isolated from frog retina

Synaptosomal fractions were isolated from frog retina: a fraction enriched in photoreceptor terminals (P1) and a second one (P2) containing interneurons terminals. We compared the binding of [³H]glycine and [³H]strychine to membranes of these synaptosomes. The binding of both radioactive ligands was saturable and Na⁺-independent. [³H]Glycine bound to a single site in P1 and P2 synaptosomal fractions, with K_D=12 and 82 nM and B_Max=3.1 and 3.06 pmol/mg protein respectively. [³H]Strychnine bound to two sites in each one of the synaptosomal fractions. For P1 K_D values were 3.9 and 18.7 nM, and B_Max values were 1.1 and 7.1 pmol/mg protein, respecitively. Membranes from the P2 synaptosomal fraction showed K_D's of 0.6 and 48 nM and B_Max's of 0.4 and 4.5 pmol/mg. Specific [³H]glycine binding was displaced by -alanine, l-serine, d-serine and HA966, but not by strychnine 7-chlorokynurenic or 5,7-dichloro-kynurenic acids. Specific [³H]strychnine, binding was partially displaced by glycine and related aminoacids and totally displaced only by 2-NH₂-strychnine. Our results indicate the presence of high affinity binding sites for glycine and strychnine in frog retinal synaptosomal membranes. The pharmacological binding pattern indicates the presence of the strychnine sensitive glycine receptor as well as other sites. These might not include the NMDA receptor-associated glycine site.     

Calmodulin activates adenylate cyclase from rabbit heart plasma membranes

It was shown that calmodulin (CM) activates the adenylate cyclase (AC) of rabbit heart light sarcolemma in the presence of micromolar free Ca2+ concentrations and this effect is blocked by trifluoroperazine and troponin I. GTP (in the presence of isoproterenol) and Gpp(NH)p are able to increase the CM-dependent activity of enzyme. It was concluded that there is no special CM-dependent "form' of AC in the heart and the common catalytic component of AC can be regulated both by CM and guanine nucleotide-binding regulatory component (N-protein). In the presence of Ca2+ and guanine nucleotide heart AC exists as a complex: CM-catalytic component-N-protein.     

A Kinetic Analysis of k-Opioid Agonist Binding Using the Selective Radioligand [3H]U69593

The interaction of the nonselective opioid ligand [3H]bremazocine and of the kappa-opioid [3H]U69593 with the kappa-receptor was investigated in guinea-pig cortical membranes. Each radioligand bound to a single population of high-affinity sites, although [3H]U69593 apparently recognised only 70% of those sites labelled by [3H]bremazocine. Naloxone and the kappa-selective ligands U69593 and PD117302 exhibited full inhibition of the binding of both radioligands. Kinetic analysis demonstrated biphasic rates of association and dissociation for both [3H]bremazocine and [3H]U69593. Detailed analysis of the binding of [3H]U69593 revealed that the fast rate of association was dependent on radioligand concentration, in contrast to the slow rate, which was independent of ligand concentration. Guanylyl-5'-imidodiphosphate (GppNHp) inhibited binding of [3H]U69593; saturation analysis demonstrated that the inhibitory effects of GppNHp resulted in a decrease in affinity without any significant change in binding capacity. GppNHp attenuated the formation of the slow component of [3H]U69593 binding, while accelerating the fast component. The data are consistent with the formation of a high-affinity complex between the kappa-receptor and a guanine nucleotide binding protein. Guanine nucleotides promote the dissociation of this ternary complex and the stabilisation of a lower-affinity state of the receptor.     

Hippocampal and Cerebellar β-Adrenergic Receptors and Adenylate Cyclase Are Differentially Altered by Chronic Ethanol Ingestion

Chronic ethanol ingestion by mice resulted in the loss of high-affinity beta-adrenergic agonist binding sites and a significant decrease in activation of adenylate cyclase by guanine nucleotides and beta-adrenergic agonists in the hippocampus, although no significant change was noted in the total number of beta-adrenergic receptors, as defined by the binding of the antagonist [125]iodocyanopindolol. In cerebellum, chronic ethanol ingestion resulted in a 16% decrease in the total concentration of beta-adrenergic receptors and in a decrease in the affinity for agonist of the high-affinity beta-adrenergic agonist binding sites. However, neither the amount of the high-affinity agonist binding sites nor the activation of adenylate cyclase by agonist was affected. The different responses to ethanol in hippocampus and cerebellum may result from quantitative differences in distribution of beta 1- and beta 2-adrenergic receptors in the tested brain areas and/or differential effects of ethanol on stimulatory guanine nucleotide binding protein in these brain areas.     

Characterization of [3H]Guanine Nucleotide Binding Sites in Brain Membranes

[3H]GTP [guanosine triphosphate] and [3H]GMP-PNP [guanosine 5'-(beta, 8-imino)triphosphate, a nonmetabolized analog of GTP] have been utilized as ligands to characterize binding sites of guanine nucleotides to rat brain membranes. Binding of both [3H]GTP and [3H]GMP-PNP is saturable, with respective KD values of 0.76 and 0.42 microM. The number of binding sites for GMP-PNP (4 nmol/g) is three times greater than for GTP (1.5 nmol/g). This discrepancy is caused by rapid degradation of GTP to guanosine by brain membranes, which can be partially prevented by addition of 100 microM-ATP. The binding of [3H]guanine nucleotides is selective, with approximately equipotent inhibition by GTP, GDP, and GMP-PNP (at 0.2--1.0 microM), but no inhibition by other nucleotides at 100 microM concentrations. The bindings sites for guanine nucleotides in brain membranes appear not to be associated with microtubules, since treatments that reduce [3H]colchicine binding by 65% have no effect on [3H]GTP binding. [3H]Guanine nucleotide binding is widely distributed in various organs, with highest levels in liver and brain and lowest levels in skeletal muscle. The characteristics of these binding sites in brain show specificity properties of sites that regulate neurotransmitter receptors and adenylate cyclase.     

Effects of proteolysis on the actions of monovalent cations and 1-O-octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine on platelet adenylate cyclase

NaCl stimulated the adenylate cyclase activities of human and rabbit platelet particulate fractions prepared in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetate, but inhibited the activities of particulate fractions proteolysed by endogenous Ca2+-activated protease or treatment with alpha-chymotrypsin. Studies with other monovalent cations showed that LiCl had weak effects similar to those of NaCl, whereas KCl inhibited the enzyme in both proteolysed and non-proteolysed preparations. The results suggest that NaCl exerts stimulatory and inhibitory effects through different sites. NaCl potentiated and proteolysis greatly reduced the inhibition of platelet adenylate cyclase by 1-O-octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (platelet-activating factor).     

[3H]Tyramine binding: A comparison with neuronal [3H]-dopamine uptake and [3H]mazindol binding processes

[³H]Spiperone ([³H]SPI) binding sites in rat or bovine striata have been solubilized using CHAPS or digitonin detergents. Solubilized sites retained the binding characteristics of those in native membrane preparations. The same solubilized material, however, did not bind [³H]tyramine ([³H]PTA), thus indicating that [³H]PTA binding sites and DA receptors are different chemico-physical entities. In membrane preparations or crude synaptosomes obtained from the c.striatum of neonatally-rendered hypothyroid rats, when central DA-pathways are impaired, both [³H]PTA binding and [³H]DA uptake processes were markedly decreased, with no effect on [³H]mazindol ([³H]MAZ) binding, compared to euthyroids. Reserpine, a well-known inhibitor of DA-uptake into a variety of secretory vesicles, and a potent in vivo andin vitro inhibitor of [³H]PTA binding, did not affect the [³H]MAZ binding process. This further supported the suggestion that while [³H]PTA binding sites are almost totally associated with the vesicular transporter for DA, [³H]MAZ does label a site involved in the DA-translocation across the neuronal membrane. The latter process seems to be rather insensitive to thyroid hypofunction, when however the intracellular storage of DA might be consistently impaired. In conclusion, PTA might be well exploited as a marker of the DA vesicular transporter through its molecular characterization, whenever possible.Special issue dedicated to Dr. Paola S. Timiras     

Distinct effects of the C-terminal octapeptide of cholecystokinin and of a cholera toxin pretreatment on the kinetics of rat pancreatic adenylate cyclase activity

(1) The kinetic parameters of rat pancreatic adenylate cyclase were evaluated, using GTP, p[NH]ppG or GTPγS as nucleotide activator, cholecystokinin as peptide hormone, and GDPβS and dibutyryl cyclic GMP as inhibitors of guanosine triphosphate and CCK-8, respectively. The time courses of activation and the degree of activation at steady state (E_A/E_TOT) were compatible with a simple two-state model of activation-deactivation based on a pseudo-monomolecular activation process (rate constant k₊₂, and a deactivation process (rate constant k_off) that included, depending on the activating nucleotide, the hydrolysis of GTP (rate constant k₂) and/or the dissociation of the intact nucleotide (rate constant k_?1), so that E_A/E_TOT = k₊₁/(k₊₁ + k₂ + k_?a). (2) The hormone CCK-8 increased the value of k₊₁ with GTP dose-dependently, from 0.2 to 10.9 min^?1. The value of k_?1 increased 0.01 to 0.3 min^?1 but the value of k₂ was unaltered at 7 min^?1, so that E_A/E_TOT increased 15-fold, from 4% to 61%. (3) A cholera toxin pretreatment at 30 μg/ml allowed also a large increase in E_A/E_TOT with GTP (up to 51%) but the underlying mechanism was different. It consisted of a 14-fold decrease in the k_off value of the GTP-activated enzyme (from 7 min^? to 0.5 min^?1) that corresponded to a reduction in GTPase activity. When testing the system with p[NH]ppG, two added effects of the cholera toxin pretreatment were observed: a 4-fold increase in the value of k₊₁ (from 0.2 to 0.8 min^?1) and the occurrence of a significant 0.3 min^?1 value for k_?1.     

Inhibition of adenylate cyclase activity of human thyroid membranes by gangliosides

Gangliosides inhibit basal, thyrotropin-induced and fluoride-induced adenylate cyclase activity of human thyroid membranes in physiological conditions. In contrast neutral glycolipids, phospholipids and neuraminic acid containing oligosaccharides show no effect. The efficacy of inhibition is more dependent upon the position of the sialic acid residues than upon their absolute number. In general gangliosides with disialyl groups are more inhibitory than those with single sialyl moieties. The inhibitory effects of the individual gangliosides on the two modes of stimulation are parallel. This parallelism suggests that the inhibitory effect is located at the postreceptor level and that the gangliosides interact directly with the adenylate cyclase system. A possible role of thyroid membrane gangliosides as suppressive cofactors of adenylate cyclase is discussed in relation to recent findings of stimulating anti-ganglioside antibodies in Graves' disease.     

The release of [3H]GABA formed from [3H]glutamate in rat hippocampal slices

to compare the storage and release of endogenous GABA, of [3H]GABA formed endogenously from glutamate, and of exogenous [14C]GABA, hippocampal slices were incubated with 5 microCi/ml [3,4-3H]1-glutamate and 0.5 microCi/ml [U-14C]GABA and then were superfused in the presence or absence of Ca+ with either 50 mM K+ or 50 microM veratridine. Endogenous GABA was determined by high performance liquid chromatography which separated labeled GABA from its precursors and metabolites. Exogenous [14C]GABA content of the slices declined spontaneously while endogenous GABA and endogenously formed [3H]GABA stayed constant over a 48 min period. In the presence of Ca+ 50 mM K+ and in the presence or absence of Ca2+ veratridine released exogenous [14C]GABA more rapidly than endogenous or endogenously formed [3H]GABA, the release of the latter two occurring always in parallel. The initial specific activity of released exogenous [14C]GABA was three times, while that of endogenously formed [3H]GABA was only 50% higher than that in the slices. There was an excess of endogenous GABA content following superfusion with 50 mM K+ and Ca2+, which did not occur in the absence of Ca2+ or after veratridine. The observation that endogenous GABA and [3H]GABA formed endogenously from glutamate are stored and released in parallel but differently from exogenous labelled GABA, suggests that exogenous [3H] glutamate can enter a glutamate pool that normally serves as precursor of GABA.