Association of a human G-protein beta3 subunit variant with hypertension.

Hypertension is a common disorder of multifactorial origin that constitutes a major risk factor for cardiovascular events such as stroke and myocardial infarction. Previous studies demonstrated an enhanced signal transduction via pertussis toxin-sensitive G proteins in lymphoblasts and fibroblasts from selected patients with essential hypertension. We have detected a novel polymorphism (C825T) in exon 10 of the gene encoding the beta3 subunit of heterotrimeric G proteins (GNB3). The T allele is associated with the occurrence of a splice variant, GNB3-s (encoding G beta3-s), in which the nucleotides 498-620 of exon 9 are deleted. This in-frame deletion causes the loss of 41 amino acids and one WD repeat domain of the G beta subunit. By western-blot analysis, G beta3-s appears to be predominantly expressed in cells from individuals carrying the T allele. Significant enhancement of stimulated GTPgammaS binding to Sf9 insect cells expressing G beta3-s together with G alpha(i)2 and G gamma5 indicates that this splice variant is biologically active. Genotype analysis of 427 normotensive and 426 hypertensive subjects suggests a significant association of the T allele with essential hypertension.

A new phospholipase-C-calcium signalling pathway mediated by cyclic AMP and a Rap GTPase.

Stimulation of phosphoinositide-hydrolysing phospholipase C (PLC) generating inositol-1,4,5-trisphosphate is a major calcium signalling pathway used by a wide variety of membrane receptors, activating distinct PLC-beta or PLC-gamma isoforms. Here we report a new PLC and calcium signalling pathway that is triggered by cyclic AMP (cAMP) and mediated by a small GTPase of the Rap family. Activation of the adenylyl cyclase-coupled beta2-adrenoceptor expressed in HEK-293 cells or the endogenous receptor for prostaglandin E1 in N1E-115 neuroblastoma cells induced calcium mobilization and PLC stimulation, seemingly caused by cAMP formation, but was independent of protein kinase A (PKA). We provide evidence that these receptor responses are mediated by a Rap GTPase, specifically Rap2B, activated by a guanine-nucleotide-exchange factor (Epac) regulated by cAMP, and involve the recently identified PLC-epsilon isoform.

Human fat cell lipolysis is primarily regulated by inhibitory modulators acting through distinct mechanisms.

The effects of adenosine deaminase and of pertussis toxin on hormonal regulation of lipolysis were investigated in isolated human fat cells. Adenosine deaminase (1.6 micrograms/ml) caused a two-to threefold increase in cyclic AMP, which was associated with an increase in glycerol release averaging 150-200% above basal levels. Clonidine, N6-phenylisopropyladenosine, prostaglandin E2, and insulin caused a dose-dependent inhibition of glycerol release in the presence of adenosine deaminase. Pretreatment of adipocytes with pertussis toxin (5 micrograms/ml) for 180 min resulted in a five- to sevenfold increase in cyclic AMP. Glycerol release was almost maximal and isoproterenol caused either no further increase or only a marginal additional increase of lipolysis after pretreatment with pertussis toxin, whereas cyclic AMP levels were 500 times higher than in controls. The effects of antilipolytic agents known to affect lipolysis by inhibition of adenylate cyclase activity, i.e., clonidine, N6-phenylisopropyladenosine, and prostaglandin E2, were impaired. In contrast, the antilipolytic action of insulin was preserved in adipocytes pretreated with pertussis toxin. As in controls, the peptide hormone had no detectable effect on cyclic AMP after pertussis toxin treatment. The findings support the view that the antilipolytic effect of insulin does not require adenylate cyclase or phosphodiesterase action. In addition, the results demonstrate that, upon relief of endogenous inhibition, human fat cell lipolysis proceeds at considerable (adenosine deaminase) or almost maximal (pertussis toxin) rates. A certain degree of inhibition, therefore, appears to be necessary for human fat cell lipolysis to be susceptible for hormonal activation.

Enhanced G protein activation in immortalized lymphoblasts from patients with essential hypertension.

Epstein-Barr virus-immortalized B lymphoblasts obtained from hypertensive patients with enhanced Na+/H+ exchanger activity (HT cells) proliferate distinctly faster upon serum stimulation than those from normotensive controls with low exchanger activity (NT cells) (Rosskopf, D., E. Frömter, and W. Siffert. 1993. J. Clin. Invest. 92:2553-2559). Stimulation with platelet-activating factor (PAF) as well caused an enhanced proliferation of HT cells. In analyzing possible differences in signal transduction between the immortalized NT and HT lymphoblasts, we observed that cell stimulation with PAF and somatostatin caused a twofold higher increase in [Ca2+]i in HT than in NT cell lines. This difference was completely abrogated by pertussis toxin (PTX) treatment. Furthermore, PAF-stimulated formation of inositol 1,4,5-trisphosphate (IP3) was twofold enhanced in HT cell lines. On the other hand, PAF receptor density and affinity, total cellular phospholipase C activity, expression of PTX-sensitive G proteins, and control binding of the stable GTP analogue, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S), to membrane G proteins were not different in NT and HT cell lines. However, PAF- and mastoparan-stimulated binding of GTP gamma S to G proteins, which was fully PTX-sensitive, was 2.5-fold higher in HT than NT cell lines. These data suggest an enhanced receptor-mediated activation of PTX-sensitive G proteins despite unchanged receptor and G protein expression. Thus, this study not only suggests that enhanced signal transduction and cell proliferation are abnormalities in a certain group of patients with essential hypertension but also explains these findings as a result of an enhanced G protein activation in this common disorder.

Renal mesangial cells: moving on sphingosine kinase-1.

Sphingosine kinase-1 (SphK1) catalyses the phosphorylation of sphingosine to sphingosine-1-phosphate (S1P), which acts on at least five specific G-protein-coupled receptors and also intracellularly. SphK1 has been implicated in cell proliferation, cancer growth, chemoresistance, immune cell functions and cell migration. In this issue of the British Journal of Pharmacology, Klawitter et al. demonstrate that extracellular nucleotides stimulate the migration of renal mesangial cells. The nucleotides furthermore upregulated SphK1 expression and activity, and this enzyme was required for nucleotide-induced migration. Together with previous findings, these data raise exciting questions: by which mechanism does SphK1 regulate migration in mesangial cells, how is the interplay of purinoceptors and S1P receptors organized in these cells, and how would SphK1-deficient mice respond to kidney damage?

Stimulation and inhibition of rat basophilic leukemia cell adenylate cyclase by forskolin.

The influence of the diterpene, forskolin, was studied on adenylate cyclase activity in membranes of rat basophilic leukemia cells. Forskolin increased basal adenylate cyclase activity maximally 2-fold at 100 microM. However, adenylate cyclase activity stimulated via the stimulatory guanine nucleotide-binding protein, Ns, by fluoride and the stable GTP analog, guanosine 5'-O-(3-thiotriphosphate), was inhibited by forskolin. Half-maximal and maximal inhibition occurred at about 1 and 10 microM forskolin, respectively. The inhibition occurred without an apparent lag phase, whereas the enzyme stimulation by forskolin was preceded by a considerable lag period. The inhibition was not affected by treating intact cells or membranes with pertussis toxin and proteolytic enzymes, respectively, which have been shown in other cell types to prevent adenylate cyclase inhibition mediated by the guanine nucleotide-binding regulatory component, Ni. The forskolin inhibition of the stable GTP analog-activated adenylate cyclase was impaired by increasing the Mg2+ concentration and was reversed into a stimulation by Mn2+. Under optimal inhibitory conditions, forskolin even decreased basal adenylate cyclase activity. Finally, forskolin largely reduced the apparent affinity of the rat basophilic leukemia cell adenylate cyclase for its substrate, MgATP, which reduction resulted in an apparent inhibition at low MgATP concentrations and a loss of the inhibition at higher MgATP concentrations. The data indicate that forskolin can cause both stimulation and inhibition of adenylate cyclase and, furthermore, they suggest that the inhibition may not be mediated by the Ni protein, but may be caused by a direct action of forskolin at the adenylate cyclase catalytic moiety.

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.

Regulation of platelet adenylate cyclase by adenosine.

The stimulatory and inhibitory effects of adenosine on the adenylate cyclases of human and pig platelets were studied. Stimulation occurred at lower concentrations than did inhibition, and the stimulatory effect was prevented by methylxanthines. Stimulation by adenosine was immediate in onset and was reversible, under conditions when cyclic AMP formation was linear with respect to time and protein concentration. The stimulatory and inhibitory effects could be distinguished further by the use of various analogues of adenosine and could be prevented by adenosine deaminase. The data suggest that both stimulation and inhibition were due to adenosine itself and not one of its degradation products and that in the platelet preparation, neither formation nor degradation of adenosine during the adenylate cyclase incubation appreciably influenced measured activity. Stimulation by adenosine was additive with the effects of GMP-P(NH)P, and alpha- or beta-adrenergic stimulation, but was abolished by prostaglandin E1 or by NaF. Prostaglandin E1 and NaF increased the sensitivity of adenylate cyclase to inhibition by adenosine. The data suggest that guanyl-5'-yl-(beta-gamma-imino)diphosphate and/or adrenergic stimulation and adenosine exert their effects on adenylate cyclase by distinct mechanisms, but that prostaglandin E1 or F- and adenosine increase enzyme activity by mechanisms which may involve common intermediates in the coupling to adenylate cyclase.

Activation of human platelet adenylate cyclase by a bovine sperm component.

The interaction of bovine sperm particles and human platelet membranes was studied with regard to adenylate cyclase activity. When sperm and platelet membrane preparations were combined, greater than additive adenylate cyclase activity was measured. Data obtained after inactivation of the sperm enzyme indicated that the observed increase in adenylate cyclase activity was not due to an activation of the sperm cyclase but that a sperm component, which could be extracted from the sperm particles, activated the platelet adenylate cyclase. Platelet cyclase activation by the sperm particles was a saturable and time-dependent process. The extent of activation was particularly high (up to 10-fold) in the presence of a stable GTP analog. Under this condition, the apparent affinity of the platelet enzyme for Mg2+ was increased by about one order of magnitude, whereas with Mn2+ only a minor effect of the sperm particles was observed. Platelet adenylate cyclase stimulation by prostaglandin E1 (up to 20-fold) was more than doubled by the sperm particles, whereas the inhibition of the platelet enzyme by epinephrine was abolished. The sperm factor was trypsin-sensitive and was inactivated by boiling. The data indicate that bovine sperm particles contain an extractable protein, which activates platelet adenylate cyclase probably by inactivating an inhibitory site.

Cholera toxin does not impair hormonal inhibition of adenylate cyclase and concomitant stimulation of a GTPase in adipocyte membranes.

The influence of cholera toxin on hormonal inhibition of adenylate cyclase and concomitant stimulation of low Km GTPase was studied in adipocyte membrane preparations. In hamster adipocyte ghosts, cholera toxin caused an about 8-fold activation of the adenylate cyclase. The antilipolytic hormonal factors, prostaglandin E1 (1 micro M), N6-phenylisopropyladenosine (1 micro M) and nicotinic acid (30 micro M) reduced both basal and cholera toxin-stimulated enzyme activities. Similar data with regard to inhibition of cholera toxin-stimulated adenylate cyclase were obtained in mouse and rat adipocyte ghosts. As studied in hamster adipocyte ghosts, prostaglandin E1 (1 micro M) increased GTP hydrolysis by a low Km GTPase by about 3-4 fold. Pretreatment of the membrane preparation with cholera toxin did not impair prostaglandin E1-induced GTPase stimulation. The data suggest that cholera toxin does not directly affect the GTPase enzyme stimulated by adenylate cyclase inhibitory hormones.

Stable GDP analog-induced inactivation of G(i) proteins promotes cardiac adenylyl cyclase inhibition by guanosine 5'-(beta gamma-imino)triphosphate and muscarinic acetylcholine receptor.

Low concentrations of GDP and its stable analog guanosine 5'-O-(2-thio)diphosphate (GDP beta S) have been shown to stimulate adenylyl cyclase activity in canine cardiac sarcolemmal membranes independent from a phosphate transfer reaction. The mechanism of this stimulation was further examined. The stable GTP analog guanosine 5'-(beta gamma-imino)triphosphate (Gpp(NH)p) increased basal adenylyl cyclase activity and inhibited forskolin-stimulated activity with EC50 (half-maximal effective concentration) values of 0.7 mumol/l and 10 nmol/l, respectively. In the presence of GDP beta S (5 mumol/l), which increased basal activity by about 150%, addition of Gpp(NH)p inhibited adenylyl cyclase activity by up to 50% with an EC50 value of 40 nmol/l. Activation of cardiac muscarinic acetylcholine receptors by carbachol amplified this Gpp(NH)p-induced inhibition of GDP beta S-stimulated adenylyl cyclase activity. The stimulatory effect of GDP beta S and the inhibitory effect of Gpp(NH)p on GDP beta S-stimulated adenylyl cyclase activity were both attenuated by increasing the Mg2+ concentration or substituting Mn2+ for Mg2+ in the assay. Furthermore, both effects were strongly reduced or abolished upon pretreatment of the sarcolemmal membranes with a low concentration of the SH reagent N-ethylmaleimide (10 mumol/l). These results suggest that the stimulatory effect of GDP beta S on basal adenylyl cyclase activity in canine cardiac sarcolemmal membranes is caused by inactivation of G(i) proteins, which are then rendered susceptible to activation by Gpp(NH)p and inhibitory receptors.

Activation of solubilized G-proteins by muscarinic acetylcholine receptors.

Binding of GTP and its analogue, guanosine 5'-O-[gamma-thio]triphosphate (GTP[S]) to G-proteins, and release of GTP[S] from G-proteins are stimulated by muscarinic acetylcholine (mACh) receptors in intact cardiac membranes. Upon solubilization of receptors and G-proteins by membrane extraction with the detergent, 3-[(cholamidopropyl)dimethylammonio]-1-propanesulphonate, followed by sucrose density gradient centrifugation, agonist-liganded mACh receptors stimulated binding of GTP[S] and hydrolysis of GTP by G-proteins with similar requirements as in intact membranes. One soluble agonist-activated mACh receptor induced binding of GTP[S] to several (about seven) soluble G-proteins. In contrast to intact membranes, however, agonist activation of mACh receptors did not induce release of GTP[S] from solubilized G-proteins. The data presented indicate that mACh receptors can interact with and efficiently activate G-proteins even in solution, whereas the possible interaction of receptors with GTP[S]-liganded G-proteins observed in intact membranes is lost upon solubilization of these components.

Receptor-stimulated dissociation of GTP[S] from Gi-proteins in membranes of HL-60 cells.

Formyl peptides stimulate binding of the stable GTP analogue, guanosine 5'-O-[gamma-thio]triphosphate (GTP[S]), to G-proteins in membranes of myeloid differentiated human leukaemia (HL-60) cells. On the other hand, agonist-activated formyl peptide receptors can also cause rapid and substantial release of GTP[S] bound to HL-60 membrane G-proteins. For fMet-Leu-Phe-stimulated dissociation of labelled GTP[S], an additional guanine nucleotide, in the potency order, unlabelled GTP[S] >> GTP >> guanosine 5'-[beta,gamma-imino]triphosphate > or = guanosine 5'-O-[beta-thio]diphosphate > or = GDP > GMP = ATP (no effects at 1 mM), was absolutely necessary. While with unlabelled GTP[S] and GTP similar concentrations were required for control and fMet-Leu-Phe-stimulated release, about 50-100-fold higher concentrations of the other nucleotides were necessary for agonist-stimulated than for basal release of bound GTP[S]. The receptor action appeared to be catalytic, required Mg2+ and was pertussis toxin sensitive. The data indicate that binding of GTP[S] to HL-60 membrane G-proteins is reversible and that agonist-activated formyl peptide receptors can interact, either directly or indirectly, with GTP[S]-liganded Gi-proteins, resulting in release of bound GTP[S].

Altered guanine nucleoside triphosphate binding to transducin by cholera toxin-catalysed ADP-ribosylation.

The influence of cholera toxin (CTX)-catalysed ADP-ribosylation on binding of guanine nucleoside triphosphates to transducin was studied by measuring the binding of the GTP analogue, guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), to illuminated bovine rod outer segment (ROS) membranes treated with or without CTX. Besides the well-documented inhibition of the transducin GTPase activity, CTX treatment inhibited binding of GTP[gamma S] to illuminated ROS membranes. This inhibition was due to an approximately two-fold lower apparent affinity for the nucleotide, while the density of binding sites was not altered. CTX decreased the association rate of GTP[gamma S] by a factor of about two. Competition experiments with GTP, guanosine 5'-[beta, gamma]iminotriphosphate or GDP showed that the apparent affinities for both guanine nucleoside triphosphates, but not for GDP, were lowered by about two-fold upon CTX treatment. In contrast to CTX, pertussis toxin treatment of ROS membranes reduced the density of binding sites available to GTP[gamma S], while the apparent affinity of the remaining sites was unchanged. It is concluded that ADP-ribosylation of transducin by CTX not only inhibits its GTPase activity but also decreases the affinity for guanine nucleoside triphosphates, data which suggest that the arginine moiety modified by CTX is involved in both binding and hydrolysis of GTP.

Nitroimidazole derivatives inhibit anterior pituitary cell function apparently by a direct effect on the catalytic subunit of the adenylate cyclase holoenzyme.

Nitroimidazole derivatives dose-dependently decreased basal and CRF-stimulated ACTH release, basal and GRF-stimulated rat GH release, and basal rat PRL release in primary cultures of rat anterior pituitary cells. In addition, basal and CRF-stimulated mRNA coding for the ACTH precursor were reduced after preincubation with the nitroimidazole derivatives. Miconazole, econazole, isoconazole, clotrimazole, and bifonazole had similar or more pronounced effects on anterior pituitary function compared to ketoconazole, whereas metronidazole and etomidate were less effective. The positive correlation between the number of phenylated side-chains or phenolic rings of the imidazole molecule and the efficacy to inhibit activity on pituitary hormone secretion suggests a structure-activity relationship of these compounds. The effects of the nitroimidazole derivatives on anterior pituitary hormone release and biosynthesis were mediated by cAMP. Thus, basal and CRF-, cholera toxin-, and forskolin-stimulated adenylate cyclase activities in rat anterior pituitary cell membranes determined by cAMP formation were suppressed by the nitroimidazole derivatives. Pertussis toxin did not diminish the nitroimidazole derivative effect on cAMP formation. The adenylate cyclase inhibitory effect of these substances was independent of the presence of GTP in the assay system, underlining a direct effect on the catalytic subunit. In addition, basal and forskolin-stimulated cAMP generation in membranes of S49 lymphoma cyc-variants, which lack a functional Gs protein, was efficiently suppressed (by up to 90%) by the nitroimidazole derivatives. In conclusion, ketoconazole and other nitroimidazole derivatives inhibit anterior pituitary hormone synthesis and secretion apparently by a direct effect on the catalytic subunit of the adenylate cyclase system.

Phorbol ester treatment impairs hormone- but not stable GTP analog-induced inhibition of adenylate cyclase.

Treatment of intact human platelets and S49 lymphoma cyc- cells with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, impairs GTP-dependent and hormone-induced inhibition of adenylate cyclase, an action mediated by the inhibitory coupling protein Ni. In contrast, receptor-independent activation of Ni with subsequent adenylate cyclase inhibition induced by the stable GTP analog, guanosine 5'-[gamma-thio]triphosphate, was affected in neither the potency nor onset of Ni activation by the stable GTP analog, in both membrane systems studied. The data indicate that modification of Ni following phorbol ester treatment does not impair its activation by stable GTP analogs.

Receptor-mediated ADP-ribosylation of a phospholipase C-stimulating G protein.

In membranes of myeloid differentiated HL 60 cells, the chemotactic peptide FMLP stimulates phospholipase C via a pertussis toxin-sensitive G protein. FMLP markedly stimulates the cholera toxin-dependent ADP-ribosylation of a 40 kDa protein in these membranes. This effect of FMLP is inhibited by GTP and GTP[S], and is almost completely abolished in membranes of pertussis toxin-pretreated HL 60 cells. Treatment of HL 60 membranes with cholera toxin and NAD markedly inhibits FMLP-stimulated high affinity GTPase. These results suggest that a 40 kDa G protein sensitive to both pertussis and cholera toxin functionally interacts with the formyl peptide receptor of HL 60 cells and, thus, very likely is the G protein that stimulates phospholipase C in this system.

Stimulation and inhibition of human platelet membrane high-affinity GTPase by neomycin.

The effect of the inositol phospholipid-binding antibiotic neomycin was studied on high-affinity GTPase in human platelet membranes. At low concentrations (up to 1 mM), neomycin by itself stimulated a high-affinity GTPase. This GTPase stimulation was additive with that caused by the hormonal factors, prostaglandin E1 and epinephrine, but not with thrombin. At concentrations higher than 1 mM, neomycin reduced control GTPase activity and eliminated the stimulation caused by thrombin. The data suggest that neomycin by a presently unknown mechanism can regulate activity states of signal transducing GTP-binding proteins.

Receptor-regulated formation of GTP[gamma S] with subsequent persistent Gs-protein activation in membranes of human platelets.

Preincubation of human platelet membranes with the ATP analog ATP[gamma S] led to persistent adenylate cyclase activation. This stimulation was increased by copreincubation with PGE1 and obliterated by removing endogenous GDP by the NTP-regenerating system, creatine phosphate plus creatine kinase. PGE1 partially reversed the action of the regenerating system. Control formation of GTP[gamma S] from ATP[gamma S] and GDP in platelet membranes was apparently not stimulated by PGE1. In contrast, in the presence of creatine phosphate plus creatine kinase, which prevented formation of GTP[gamma S], PGE1 stimulated formation of this GTP analog, by partially reversing the action of the NTP-regenerating system. The data indicate that GTP[gamma S] can be formed by a membrane-associated nucleoside diphosphokinase from ATP[gamma S] and GDP, resulting in persistent Gs-protein activation, and that this process can be stimulated by an agonist-activated receptor.

Adenylate cyclase inhibition and GTPase stimulation by somatostatin in S49 lymphoma cyc- variants are prevented by islet-activating protein.

cyc--Variants of S49 lymphoma cells are defective in the stimulatory guanine nucleotide site of the adenylate cyclase but contain an inhibitory site. Treatment of cyc- cells with islet-activating protein (IAP), which causes ADP-ribosylation of an Mr 40 000 polypeptide in cyc- membranes, abolishes adenylate cyclase inhibition by GTP and the peptide hormone, somatostatin, but not that induced by GTP gamma S. Furthermore, somatostatin-induced stimulation of GTP hydrolysis is lost. Thus, the data indicate that IAP interferes with the adenylate cyclase system by an action at the inhibitory guanine nucleotide site.