Interactions of nucleotide analogues with rod outer segment guanylate cyclase.

Light activation of cyclic GMP hydrolysis in rod outer segments is mediated by a G-protein which is active in the GTP-bound form. Substitution of GTP with a nonhydrolyzable GTP analogue is thought to leave the G-protein in a persistently activated state, thereby prolonging the hydrolysis of cyclic GMP. Restoration of cyclic GMP concentration in the cell also depends upon GTP since it is the substrate for guanylate cyclase, but little is known about the effects of GTP analogues on this enzyme. We report here the effects of the analogues of GTP and ATP as inhibitors and substrates of rod disk membrane guanylate cyclase. The rate of cyclic GMP synthesis from GTP in rod disk membranes was about 50 pmol min-1 (nmol of rhodopsin)-1. Analogues of GTP and adenine nucleotides competitively inhibited the cyclase activity. The order of inhibition, with magnesium as metal cofactor, was ATP greater than GMP-PNP greater than AMP-PNP approximately GTP-gamma-S; with manganese, AMP-PNP was more inhibitory than GTP-gamma-S. The inhibition constants, with magnesium as cofactor, were 0.65-2.0 mM for GTP-gamma-S, 0.4-0.8 mM for GMP-PNP, 1.5-2.3 mM for AMP-PNP, and 0.07-0.2 mM for ATP. The fraction of cyclase activity inhibited by analogues was similar at 1 and 0.03 microM calcium. Besides inhibition of cyclase, the analogues also served as its substrates. GTP-gamma-S substituted GTP with about 85% efficiency while GMP-PNP and ATP were about 5 and 7% as efficient, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of pertussis-toxin-sensitive G protein in muscarinic-receptor-mediated inhibition of K(+)-activated 4-nitrophenylphosphatase activity of cardiac sarcolemma

The effects of the cholinergic agonist carbachol on ouabain-sensitive K(+)-activated 4-nitrophenylphosphatase (K(+)-O2NPhPase) activity of rabbit and pig ventricular sarcolemma were examined. Carbachol (0.01-1000 microM) alone had no effect on K(+)-O2NPase. However, in the presence of GTP (100 microM) or its analog guanosine 5'-[gamma-thio]triphosphate (GTP[S], 1 microM) the agonist reduced this enzymatic activity (IC50 = 0.3 microM) by about 45% in a concentration-dependent manner. The GTP[S]-dependent effect of carbachol was blocked by 10 microM atropine, an antagonist of muscarinic acetylcholine receptor (mAcChoR). In the presence of micromolar concentrations of ATP or the GDP analog guanosine 5'-[beta-thio]diphosphate, carbachol did not change sarcolemmal K(+)-O2NPhPase activity. GTP[S] alone reduced this activity (IC50 = 2 microM) by about 40% in a concentration-dependent manner with a lag period of about 3 min. This lag disappeared in the presence of carbachol. Treatment of sarcolemmal membranes with 20 micrograms/ml pertussis toxin, which catalyzed ADP-ribosylation of the 40-41-kDa alpha-subunits of inhibitory GTP-binding protein (Gi), abolished the GTP[S]-promoted inhibitory effect of carbachol. Immunochemically, these alpha-subunits were identified as alpha 12- and alpha i3-subunits. It is suggested that the carbachol-induced inhibition of ouabain-sensitive K(+)-O2NPhPase activity of mammalian myocardial sarcolemma is a result of a negative coupling between mAcChoR and Na+/K(+)-ATPase via Gi protein.     

Characterization of an octopamine-sensitive adenylate cyclase from insect brain (Mamestra configurata Wlk.).

An adenylate cyclase present in the brain of the moth Mamestra configurata Wlk. that is stimulated selectively by low (micromolar) concentrations of octopamine has been characterized with respect to several properties. The optimum pH, optimum ATP:Mg2+ ratio, the concentration of ATP required for half-maximal and maximal reaction velocity, metal ion specificity, effect of NaF, and effects of GTP and 5'-guanylylimidodiphosphate were in general similar to those of catecholamine-sensitive adenylate cyclases from various regions of mammalian brain. However, ethylene glycol bis-(beta-aminoethyl ether)-N,N-tetraacetic acid (EGTA), a calcium chelator, stimulated both basal and octopamine-sensitive enzyme activity in the insect brain, whereas in mammalian brain EGTA is usually observed to inhibit basal activity but not catecholamine-stimulated activity. Adenylate cyclase activity of the 47,000 g particulate fraction of the insect brain was almost undetectable in the absence of added GTP. Addition of saturating concentrations (100 micrometer) of GTP to the particles restored about 30% of the basal and octopamine-sensitive enzyme activity present in the homogenate. Addition of 100,000 g supernatant to the particles doubled both basal and octopamine-sensitive enzyme activity in the presence of saturating concentrations of GTP, indicating that in addition to GTP, a cytosolic factor(s) is necessary for enhanced adenylate cyclase activity.     

Evidence of GTP-binding protein regulation of phospholipase A2 activity in isolated human platelet membranes

G protein regulation of human platelet membrane phospholipase A2 activity was investigated at pH 8.0 and 9.0 by studying the effects of the nonhydrolyzable GTP analogue, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), and of F-/Al3+ ions on arachidonic acid (AA) release. The membrane acted as the source of the enzyme, the substrate, and the G protein. At pH 8.0, 10 and 100 microM GTP gamma S stimulated AA mobilization at least 6-fold. Optimum AA release conditions required 1 mM Ca2+ and 5 mM Mg2+. Nonspecific nucleotide effect was excluded since similar stimulatory effects on AA release were not observed by ATP, GTP, ADP, and NADP. Although at pH 9.0 the GTP gamma S-stimulated AA release was greater than at pH 8.0, it constituted only 26% of the total. At both pH values the effect of F- (10 mM) in the presence of Al3+ (2 microM) was similar to that of GTP gamma S. The G protein inhibitor, guanosine 5'-O-(2-thiodiphosphate), inhibited the GTP gamma S-stimulated AA release by about 80% at pH 8.0 and by 100% at pH 9.0. To determine a possible contribution to AA mobilization by the phospholipase C and diacylglycerol lipase pathway, the effects of neomycin, a phospholipase C inhibitor, were investigated. 100 microM neomycin did not inhibit the GTP gamma S-stimulated AA release at pH 8.0 and only slightly so (17%) at pH 9.0. At pH 8.0 in the presence of Ca2+ the released fatty acids consisted mainly of arachidonic and docosahexaenoic acids (80 and 8%, respectively). GTP gamma S had no effect on the fatty acid profile but only on their quantity. These results provide evidence of G protein regulation of phospholipase A2 activity in isolated platelet membranes.     

Nuclear protein phosphorylation in isolated nuclei from HeLa cells. Evidence that 32P incorporation from [gamma-32P]GTP is catalyzed by nuclear kinase II

A nuclear system for studying nuclear protein phosphorylation is characterized, using as phosphate donor either low levels of [gamma-32P]GTP, low levels of [gamma-32P]ATP, or low levels of labeled ATP plus excess unlabeled GTP. Since nuclear casein kinase II is the only described nuclear protein kinase to use GTP with high affinity, low levels of GTP should specifically assay this enzyme. ATP should measure all kinases, and ATP plus unlabeled GTP should measure all kinases except nuclear casein kinase II (ATP-specific kinases). The results are consistent with these predictions. In contrast with the ATP-specific activity, endogenous phosphorylation with GTP was enhanced by 100 mM NaCl, inhibited by heparin and quercetin, stimulated by polyamines, and did not use exogenous histone as substrate. The GTP- and ATP-specific kinases phosphorylated different subsets of about 20 endogenous polypeptides each. Addition of purified casein kinase II enhanced the GTP-supported phosphorylation of the identical proteins that were phosphorylated by endogenous kinase. These results support the hypothesis that activity measured with GTP is catalyzed by nuclear casein kinase II, though other minor kinases which can use GTP are not ruled out. Preliminary observations with this system suggest that the major nuclear kinases exist in an inhibited state in nuclei, and that the effects of polyamines on nuclear casein kinase II activity are substrate specific. This nuclear system is used to determine if the C-proteins of hnRNP particles, previously shown to be substrates for nuclear casein kinase II in isolated particles, is phosphorylated by GTP in intact nuclei. The results demonstrate that the C-proteins are effectively phosphorylated by GTP, but in addition they are phosphorylated by ATP-specific kinase activity.     

GTP hydrolysis by pure Ni, the inhibitory regulatory component of adenylyl cyclases

The stimulatory and inhibitory regulatory components of adenylyl cyclase (Ns and Ni), purified to apparent homogeneity without the use of regulatory ligands such as Mg, NaF, and guanyl-5'-yl imidodiphosphate, were tested for GTPase activity by incubating them with [gamma-32P]GTP and measuring 32Pi liberation using a charcoal adsorption assay to separate hydrolyzed from nonhydrolyzed radioactivity. We found that Ni is capable of hydrolyzing GTP. The activity was shown to be due to Ni itself and not to presence of one of its minor contaminants by correlating activity with abundance of the 40,000 Da alpha i subunit throughout the last stages of purification and by showing co-migration on a sucrose density gradient of the GTP-hydrolyzing activity with the alpha i, beta, and gamma subunits of Ni and not with any one of three minor contaminants present in the preparation tested. Preparations of Ns, free of detectable Ni, exhibited less than 10% the capacity to hydrolyze GTP, as compared to Ni on an equal protein basis. The basic properties of the GTP-hydrolyzing activity of Ni were determined. The activity is dependent on Mg ion (apparent Km = 5 to 15 nM), and is rapidly lost upon incubation with Mg2+ in the absence of GTP. MgGTP and free GTP serve equally well as substrate (apparent Km about 40 nM). Isotopic dilution studies indicate that the GTP binding site has a relative affinity for guanine nucleotides in the order GTP = GTP gamma S greater than GDP = GMP-P(NH)P greater than GDP beta S with the highest difference (GTP versus GDP beta S) being about 10-fold. NaF inhibited GTP hydrolysis by Ni at concentrations at which it activates Ni in intact membranes.     

Effects of phospholipase A2 and filipin on the activation of adenylate cyclase.

Rat liver plasma membranes were incubated with phospholipase A2 (purified from snake venom) or with filipin, a polyene antibiotic, followed by analysis of the binding of glucagon to receptors, effects of GTP on the glucagon-receptor complex, and the activity and responses of adenylate cyclase to glucagon + GTP, GTP, Gpp(NH)p, and F-. Phospholipase A2 treatment resulted in concomitant lossess of glucagon binding and of activation of cyclase by glucagon + GTP. Greater than 85% of maximal hydrolysis of membrane phospholipids was required before significant effects of phospholipase A2 on receptor binding and activity response to glucagon were observed. The stimulatory effects of Gpp(NH)p or F- remained essentially unaffected even at maximal hydrolysis of phospholipids, whereas the stimulatory effect of GTP was reduced. Detailed analysis of receptor binding indicates that phospholipase A2 treatment affected the affinity but not the number of glucagon receptors. The receptors remain sensitive to the effects of GTP on hormone binding. Filipin also caused marked reduction in activation by glucagon + GTP. However, in contrast to phospholipase A2 treatment, the binding of glucagon to receptors was unaffected. The effect of GTP on the binding process was also not affected. The most sensitive parameter of activity altered by filipin was stimulation by GTP or Gpp(NH)p; basal and fluoride-stimulated activities were least affected. It is concluded from these findings that phospholipase A2 and filipin, as was previously shown with phospholipase C, are valuable tools for differentially affecting the components involved in hormone, guanyl nucleotide, and fluoride action on hepatic adenylate cyclase.     

Inhibition of tubulin polymerization with ribose-modified analogs of GDP and GTP reduced inhibition with microtubule-associated proteins and magnesium

Inhibitory effects of ribose-modified GDP and GTP analogs on tubulin polymerization were examined to explore nucleotide structural requirements at the exchangeable GTP binding site. With microtubule-associated proteins and Mg²⁺, GTP-supported polymerization was only modestly inhibited by GDP, and still weaker inhibitory activity was found with two analogs, dGDP and 9-β-D-arabinofuranosylguanine-5′-diphosphate (araGDP). Omission of Mg²⁺ significantly enhanced the inhibitory effects of GDP, dGDP and araGDP and resulted in weak inhibition of the reaction by several other GDP analogs. The relative inhibitory activity of the GDP analogs had no discernable relationship to the relative activity of cognate GTP analogs in supporting microtubule-associated protein-dependent polymerization. One GTP analog, 2′,3′-dideoxyguanosine 5′-triphosphate (ddGTP), supports polymerization both with and without microtubule-associated proteins. The inhibitory activity of GDP and GDP analogs in ddGTP-supported polymerization was much greater in the absence of microtubule-associated proteins than in their presence; and both reactions were more readily inhibited than was microtubule-associated protein-dependent, GTP-supported polymerization. Microtubule-associated protein-independent, ddGTP-supported polymerization was also potently inhibited by GTP and a number of GTP analogs. GTP was in fact twice as inhibitory as GDP. The relative inhibitory activity of the GTP analogs was comparable to the relative inhibitory activity of the cognate GDP analogs and very different from their relative activity in supporting polymerization.     

Synergistic inhibition of human platelet adenylate cyclase by stable GTP analogs and epinephrine

Adenylate cyclase inhibition by stable GTP analogs and their interaction with epinephrine were studied in human platelet membranes. Whereas basal enzyme activity was increased by these nucleotides, the stable GTP analogs decreased the adenylate cyclase activity stimulated by fluoride or forskolin by maximally 60 to 70%, with the potency order, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) greater than guanyl-5'-ylimidodiphosphate greater than guanyl-5'-ylmethylenediphosphate. The inhibition of the forskolin-stimulated enzyme by GTP gamma S was half-maximal at about 4 nM, occurred after a time lag period, which was inversely related to the GTP gamma S concentration, and was resistant to washing of the membranes. Prostaglandin E1-stimulated activity exhibited a biphasic response towards GTP gamma S, with activation occurring at low (1 nM) and inhibition at higher GTP gamma S concentrations. The inhibitory effect of GTP gamma S was competitively antagonized by GTP. This antagonism was prevented by epinephrine, which inhibited the stimulated platelet adenylate cyclase in the presence of GTP to the same degree as observed with GTP gamma S alone. In the absence of GTP, epinephrine largely diminished the time lag required for the inhibitory action of GTP gamma S. Furthermore, the decrease in final activity induced by GTP gamma S was amplified by epinephrine. Whereas the acceleration of the inhibitory action of GTP gamma S was observed at low and high GTP gamma S concentrations, the amplification by epinephrine was observed only at submaximally effective concentrations of GTP gamma S.     

Nuclear protein phosphorylation in isolated nuclei from HeLa cells. Evidence that 32P incorporation from [γ-32P]GTP is catalyzed by nuclear kinase II

A nuclear system for studying nuclear protein phosphorylation is characterized, using as phosphate donor either low levels of [γ-³²P]GTP, low levels of [γ-³²P]ATP, or low levels of labeled ATP plus excess unlabeled GTP. Since nuclear casein kinase II is the only described nuclear protein kinase to use GTP with high affinity, low levels of GTP should specifically assay this enzyme. ATP should measure all kinases, and ATP plus unlabeled GTP should measure all kinases except nuclear casein kinase II (ATP-specific kinases). The results are consistent with these predictions. In contrast with the ATP-specific activity, endogenous phosphorylation with GTP was enhanced by 100 mM NaCl, inhibited by heparin and quercetin, stimulated by polyamines, and did not use exogenous histone as substrate. The GTP- and ATP-specific kinases phosphorylated different subsets of about 20 endogenous polypeptides each. Addition of purified casein kinase II enhanced the GTP-supported phosphorylation of the identical proteins that were phosphorylated by endogenous kinase. These results support the hypothesis that activity measured with GTP is catalyzed by nuclear casein kinase II, though other minor kinases which can use GTP are not ruled out. Preliminary observations with this system suggest that the major nuclear kinases exist in an inhibited state in nuclei, and that the effects of polyamines on nuclear casein kinase II activity are substrate specific. This nuclear system is used to determine if the C-proteins of hnRNP particles, previously shown to be substrates for nuclear casein kinase II in isolated particles, is phosphorylated by GTP in intact nuclei. The results demonstrate that the C-proteins are effectively phosphorylated by GTP, but in addition they are phosphorylated by ATP-specific kinase activity.     

A Genetic Analysis of the Pteridine Biosynthetic Enzyme, Guanosine Triphosphate Cyclohydrolase, in DROSOPHILA MELANOGASTER

Strains with mutant eye color were surveyed for levels of GTP cyclohydrolase (GTP CH), the first enzyme acting in the biosynthesis of pteridines, the pigments causing red eye color in Drosophila. Six strains were found to have reduced GTP CH activity. In five of the six strains, the reduction of activity is apparent only in the adult head of homozygous mutants. We show that mutations in Punch (2-97, Pu) have severe effects on GTP CH activity. In most cases, the reduction of activity is apparent in all tissues and stages that express the enzyme. The activity of GTP CH is shown to be closely correlated with the number of Pu+ genes in the genome. One ethyl methanesulfonate (EMS)-induced Pu mutant has a GTP CH enzyme that is unstable when compared with the wild-type enzyme. Mutations in Pu fall into three general classes. The largest class has a recessive lethal and eye color phenotype, 50% or higher GTP CH activity in heterozygotes, and equivalent defects in all tissues. A second class is dominant in eye color phenotype and recessive lethal, with less than 50% GTP CH activity in heterozygotes. The third class is homozygous viable and has severe reduction of activity in the adult head, but no or less severe loss in other tissues.     

Immunological cross-reactivity of gamma-glutamyltranspeptidases from human and rat kidney, liver, and bile

Antisera to purified gamma-glutamyltranspeptidase (gamma GTP) from human and rat kidney were prepared, and their reactivities toward purified gamma GTP from kidney, liver, and bile were tested. The following results were obtained: 1. On double immunodiffusion, Triton-solubilized gamma GTP, and papain-solubilized gamma GTP from rat kidney gave single precipitin lines which fused completely against antiserum to the purified enzyme from rat kidney. 2. An antigen-antibody complex of human kidney gamma GTP retained about 50% of the catalytic activity of the antigen. 3. Double immunodiffusion showed that the enzymes from human liver, kidney, and bile were immunologically identical. 4. Antiserum to rat kidney gamma GTP partially cross reacted with human gamma GTP, but antiserum to human gamma GTP reacted only very weakly with rat gamma GTP. It is concluded that gamma GTP of human liver, kidney, and bile are immunologically identical and that rat gamma GTP and human gamma GTP have certain antigenic determinants in common.     

Effect of guanosine triphosphate on the release and uptake of Ca2+ in saponin-permeabilized macrophages and the skeletal-muscle sarcoplasmic reticulum.

The effects of GTP, with or without polyethylene glycol (PEG), on the release and uptake of Ca2+ were examined by using saponin-treated macrophages and sarcoplasmic reticulum isolated from skeletal muscles. The application of GTP in concentrations in the range 0.1-10 microM induced a gradual, small but sustained release of Ca2+ from the saponin-treated macrophages. The addition of PEG to GTP markedly enhanced the GTP-mediated Ca2+ release. GTP at the same concentration ranges used for Ca2+ release decreased the amount of Ca2+ uptake, at a steady state, but stimulated the rate of Ca2+ accumulation in the presence of oxalate, the Ca2+-precipitating anion. The addition of PEG abolished the GTP-evoked stimulation of Ca2+ accumulation in the presence of oxalate. The stimulating effect on the rate of Ca2+ accumulation by GTP and its elimination by PEG were not due to changes in the permeability of oxalate by either GTP or PEG, or both. The Ca2+-releasing effect of GTP without PEG was enhanced by eliminating the uptake activity by decreasing the content of ATP. These results indicate that GTP has an inherent activity to release Ca2+ from non-mitochondrial intracellular stores of saponin-treated macrophages, and PEG enhances the GTP-mediated Ca2+ release, partly owing to its eliminating effect on GTP-stimulated Ca2+ uptake activity. These effects of GTP observed with saponin-permeabilized macrophages were not apparent in the isolated skeletal-muscle sarcoplasmic reticulum.     

Dopamine stimulated increase of GTP levels in the rat retina

Dopamine stimulates a 7-10-fold increase of GTP concentration in whole rat retina maintained in vitro. Half-maximal stimulation of GTP levels were obtained with 10(-6) M dopamine, and significant increases in GTP levels were seen with 10(-7) M dopamine. Intracellular GTP levels were significantly increased within 4 min after exposure to dopamine and maximal effects were reached within 30 min. Dopamine agonists, apomorphine and bromocriptine, also stimulate a 7-10-fold increase in GTP concentration, whereas other catecholamines (norepinephrine, epinephrine, and isoproterenol) were less potent. Several other neurotransmitters present in rat retina (gamma-aminobutyric acid, glycine, glutamine, and taurine) had no effect on GTP levels. Although dopamine also stimulates increases in cyclic AMP levels in the retina, dibutyryl cyclic AMP and 8-bromo-cyclic AMP had no effect on GTP levels, indicating that the dopamine-stimulated increase of GTP is independent of the catalytic production of cyclic AMP by adenylate cyclase. Since dopamine-stimulated adenylate cyclase activity requires GTP, the dopamine-stimulated increase in GTP concentration described in this report may serve to facilitate dopamine stimulation of adenylate cyclase activity.     

Dual effect of fluoride on phosphoinositide metabolism in rat brain cortex. Stimulation of phospholipase C and inhibition of polyphosphoinositide synthesis.

We have studied the effects of fluoride, guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and carbachol on phospholipase C and polyphosphoinositide synthesis. The experimental system consisted of membranes from rat brain cortex, with exogenous [3H]phosphatidylinositol ([3H]PtdIns) as substrate. In such systems, we have not found evidence to support carbachol and/or GTP[S] stimulation of PtdIns phosphorylation. Fluoride inhibited synthesis of PtdIns4P and PtdIns(4,5)P2 from PtdIns. Consequently, under conditions where breakdown of polyphosphoinositides by phospholipase C was dependent on PtdIns kinase activity, fluoride inhibited activation by GTP[S] plus carbachol of phospholipase C. When conditions allowed direct breakdown of PtdIns and precluded PtdIns kinase activity, the stimulatory effects of fluoride and GTP[S] plus carbachol on phospholipase C activity were additive.     

Differential sensitivity to guanine nucleotides of basal and insulin-stimulated glucose transporter activity reconstituted from adipocyte membrane fractions

The effects of GTP gamma S on glucose transport activity reconstituted from adipocyte membrane fractions were studied in order to test the hypothesis that intrinsic activity changes of the insulin-sensitive glucose transporter may be mediated by guanine nucleotide-dependent mechanisms. GTP gamma S and GTP inhibited reconstituted glucose transport activity by 50% in membrane fractions from insulin-treated cells in a concentration-dependent manner; no inhibitory effect was observed in membrane fractions obtained from basal cells. GDP, GMP and guanosine were less effective than GTP, whereas the adenine nucleotides ATP gamma S and AMP failed to reduce the reconstituted transport activity. The data indicate that guanine nucleotides may modulate the activity of the adipocyte glucose transporter. Since the effect is dependent on treatment of cells with insulin, the hormone appears to induce a specific functional alteration of the glucose transporter.     

Polyethylene glycol-stimulated microsomal GTP hydrolysis. Relationship to GTP-mediated Ca2+ release

It has recently been observed that GTP mediates Ca2+ release from internal Ca2+ stores. In contrast to effects on permeabilized cells, GTP-dependent Ca2+ release in isolated microsomes requires the presence of polyethylene glycol (PEG). We have investigated the effects of PEG on microsomal GTPase activity and report that PEG stimulates a high-affinity (Km = 0.9 microM) GTPase. The effects of PEG reflect an increase in the Vmax of this activity; no effects on Km were observed. The concentration dependence for PEG-dependent stimulation of the high-affinity GTPase exactly mimicked that for GTP-dependent Ca2+ release. The stimulation of GTP hydrolysis by PEG was specific for the microsome fraction; only small effects were obtained with plasma membrane or cytosol fractions. As observed for GTP-dependent Ca2+ release, the microsomal PEG-stimulated GTPase was competitively inhibited by the GTP analog GTP gamma S (Ki = 60 nM). It is proposed that the PEG-stimulated GTPase may represent an intrinsic activity of the guanine nucleotide binding protein involved in the regulation of reticular Ca2+ fluxes.     

Muscarinic cholinergic receptor modulation of beta-adrenergic receptor affinity for catecholamines.

The effects of the muscarinic cholinergic agonist methacholine on affinity of beta-adrenergic receptors for isoproterenol and on isoproterenol-induced stimulation of adenylate cyclase activity were assessed in canine myocardium. GTP and guanyl-5'-yl imidoiphosphate both decreased the affinity of beta-adrenergic receptors for isoproterenol without altering the affinity of these receptors for propranolol. Methacholine (10 nM to 10 micronM) antagonized the guanine nucleotide-induced reduction in beta-adrenergic receptor affinity for isoproterenol. This effect of methacholine was reversed by atropine. The choline ester had no effect on the affinity of beta-adrenergic receptors for isoproterenol in the absence of guanine nucleotides. Likewise, methacholine had no effect on the affinity of beta-adrenergic receptors for propranolol, either in the presence or absence of guanine nucleotides. Methacholine also attenuated GTP-induced activation of adenylate cyclase or isoproterenol-induced activation of the enzyme in the presence of GTP. The effects of methacholine on myocardial adenylate cyclase activity were apparent only in the presence of GTP. These effects were also reversed by atropine. The choline ester had no effect on adenylate cyclase activity in the presence of guanyl-5'-yl imidodiphosphate or NaF. The results of the present study suggest that muscarinic cholinergic agonists can regulate both beta-adrenergic receptors and adenylate cyclase by modulating the effects of GTP.     

IMP dehydrogenase inhibitors reduce intracellular tetrahydrobiopterin levels through reduction of intracellular GTP levels. Indications of the regulation of GTP cyclohydrolase I activity by restriction of GTP availability in the cells.

GTP cyclohydrolase I exhibits a positive homotropic cooperative binding to GTP, which raises the possibility of a role for GTP in regulating the enzyme reaction (Hatakeyama, K., Harada, T., Suzuki, S., Watanabe, Y., and Kagamiyama, H. (1989) J. Biol. Chem. 264, 21660-21664). We examined whether or not the intracellular GTP level is within the range of affecting GTP cyclohydrolase I activity, using PC-12 rat pheochromocytoma and IMR-32 human neuroblastoma cells. Since GTP cyclohydrolase I was the rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin in these cell lines, the intracellular activities of this enzyme were reflected in the tetrahydrobiopterin contents. We found that the addition of guanine or guanosine increased GTP but not tetrahydrobiopterin in these cells. On the other hand, three IMP dehydrogenase inhibitors, tiazofurin, 2-amino-1,3,4-thiadiazole, and mycophenolic acid, decreased both GTP and tetrahydrobiopterin in a parallel and dose-dependent manner, and these effects were reversed by the simultaneous addition of guanine or guanosine. There was no evidence suggesting that these inhibitors inhibited other enzymes involved in the biosynthesis and regeneration of tetrahydrobiopterin. Comparing intracellular activities of GTP cyclohydrolase I in the inhibitor-treated cells with its substrate-velocity curve, we estimated that the intracellular concentration of free GTP is 150 microM at which point the activity of GTP cyclohydrolase I is elicited at its maximum velocity. Below this GTP concentration, GTP cyclohydrolase I activity is rapidly decreased. Therefore GTP can be a regulator for tetrahydrobiopterin biosynthesis.     

Nerve growth factor increases the cyclic GMP level and activates the cyclic GMP phosphodiesterase in PC12 cells

Nerve growth factor (NGF) rapidly increases the cyclic GMP (cGMP) level about 2-3-fold and enhances the cGMP phosphodiesterase (PDE) activity about 2-fold in rat pheochromocytoma PC12 cells. No changes in the level of cyclic AMP (cAMP) and in the activity of cAMP PDE were found. GTP and a nonhydrolysable analog of GTP, GMP-PCP, at 100 microM, were able to mimic the effect of NGF on the cGMP PDE activity. These results suggest that the cGMP system may be one of the second messengers of NGF action in PC12 cells.