An intrinsic guanine nucleotide exchange inhibitor in Gi2 alpha. Significance of G-protein self-suppression which antagonizes receptor signal

The alpha subunit of Gi2 (Gi2 alpha) is a member of the heterotrimeric G protein family, which transduces receptor signals as a proto-oncogene product. We have found a novel self-suppressive region in Gi2 alpha near its C terminus. A polypeptide consisting of residues 338-352 of Gi2 alpha (Gi2 alpha-339-352) antagonizes receptor- and receptor peptide-stimulated Gi2 alpha activation, without affecting basal activity. Antagonism by Gi2 alpha-338-352 is attributable to an interaction with activated Gi2 alpha, which is not competitive with receptor polypeptides. Combined with the reports suggesting the presence of self-suppressive domains in a juxta-C-terminal portion of Gi2 alpha and G(o) alpha, this study supports the hypothesis that Gi2 alpha-338-352 constitutes an intrinsic guanine nucleotide exchange inhibitor, which in turn antagonizes receptor stimulation, suggesting that G proteins are activated by receptors through relaxation of a self-suppressive conformation.     

Signal transduction through trimeric G proteins in megakaryoblastic cell lines

The biogenesis of trimeric G proteins was investigated by measurement of the expression of alpha-subunits in the megakaryoblastic cell lines MEG-01, DAMI, and CHRF-288-11, representing stages of increasing maturation, and compared with platelets. Megakaryoblasts and platelets contained approximately equal amounts of Gi alpha-1/2, Gi alpha-3, Gq alpha, and G12 alpha protein. Maturation was accompanied by (1) downregulation of mRNA for Gs alpha and disappearance of iloprost-induced Ca2+ mobilization, (2) upregulation of the long form of Gs alpha protein (Gs alpha-L) and an increase in iloprost-induced cAMP formation, and (3) upregulation of G16 alpha mRNA and G16 alpha protein and appearance of thromboxane A2-induced signaling (Ca2+ mobilization and stimulation of prostaglandin I2-induced cAMP formation). Gz alpha protein was absent in the megakaryoblasts despite weak expression of Gz alpha mRNA in DAMI and relatively high levels of Gz alpha mRNA and Gz alpha protein in platelets. These findings reveal major changes in G protein-mediated signal transduction during megakaryocytopoiesis and indicate that G16 alpha couples the thromboxane receptor to phospholipase C beta.     

Activation of various subtypes of G-protein alpha subunits by partial agonists of the adenosine A1 receptor

The activation of different G protein subtypes by the rat adenosine A1 receptor initiated by stimulation with the full agonist 2-chloro-N6-cyclopentyladenosine (CCPA) and by six structurally distinct partial agonists of this receptor was investigated. Endogenous G protein alpha subunits in rat cortical membranes were inactivated by N-ethylmaleimide (NEM). Activation of rat recombinant myristoylated alpha(o), alpha(i1), alpha(i2) and alpha(i3) by partial agonists in comparison to the full agonist was assessed by guanosine-5'-(gamma-[35S]thio)triphosphate ([35S]GTPgammaS) binding after reconstitution of G protein alpha subunits with the adenosine A1 receptor in N-ethylmaleimide-treated membranes. 2-Chloro-N6-cyclopentyladenosine and 3' -deoxy-N6-cyclopentyladenosine (3'-d-CPA), the partial agonist with the highest intrinsic activity, were significantly more potent in activation of alpha(i) subtypes than alpha(o). In contrast, 5'-methylthioadenosine (MeSA), 2'-deoxy-2-chloroadenosine (cladribine), 2'-deoxy-N6-cyclopentyladenosine (2'-d-CPA), 2-phenylaminoadenosine (CV 1808) and C8-aminopropyl-N6-cyclopentyladenosine (C8-aminopropyl-CPA) did not exhibit higher potency for Go or any Gi subtype. All partial agonists, although carrying structurally different modifications, showed higher relative intrinsic activities in activation of Gi than of Go, indicating that Gi-coupled pathways may be activated selectively via the A1 receptor by partial agonists, but not Go-mediated responses.     

Myocardial Gi alpha-protein levels in patients with hypertensive cardiac hypertrophy, ischemic heart disease and cardiogenic shock

OBJECTIVE: Increased inhibitory G-protein alpha-subunits (Gi alpha) have been reported to be related to adenylyl cyclase desensitization in the failing human heart. In order to investigate whether this cellular alteration occurs already at the stage of hypertensive cardiac hypertrophy or in catecholamine-refractory cardiogenic shock, Gi alpha levels were studied in myocardial samples from patients with hypertensive cardiac hypertrophy, coronary heart disease without heart failure and from patients with cardiogenic shock on high-dose catecholamine therapy as well as from patients without evidence of heart disease. METHODS: Gi alpha was quantified with pertussis-toxin-catalyzed 32P-ADP-ribosylation and with a radioimmunoassay in myocardial samples from patients within 16 h of death. The radioimmunoassay was constructed with recombinant G-protein alpha-subunits (rGi alpha 1) from transformed E. coli harbouring the full-length cDNA of Gi alpha 1, iodinated peptide 125I-KENLKDCGLF and immunoprecipitating antiserum (MB 1) raised against the synthetic peptide (KENLKDCGLF) in rabbits. RESULTS: Pertussis toxin substrates and immunochemical Gi alpha remained stable up to 80 h following storage at room temperature in myocardium obtained during cardiac transplantation. Gs alpha, adenylyl cyclase, beta-adrenoceptors and inhibitory receptors were not stable and could not be determined. Increases in myocardial Gi alpha of 65-82% of both pertussis toxin substrates and immunologically quantified Gi alpha were observed in hypertensive cardiac hypertrophy. Catecholamine therapy in patients who died of catecholamine-refractory shock increased myocardial Gi alpha by 225% compared to myocardium from patients with coronary heart disease without heart failure and without catecholamine therapy or compared to control myocardium. CONCLUSION: These findings provide evidence than an increase in myocardial Gi alpha-proteins could be of relevance in pathological conditions other than chronic heart failure. Since an increase in Gi alpha levels already occurs in hypertensive cardiac hypertrophy, it could play a role in contributing to the development of contractile dysfunction and heart failure in later stages of the syndrome. Finally, an increase in Gi alpha could be one mechanism contributing to catecholamine refractoriness in shock. This could provide a target for pharmacological treatment in this condition.     

Selective reconstitution of gastrin-releasing peptide receptor with G alpha q

Identification of the molecular mechanisms that determine specificity of coupling interactions between gastrin-releasing peptide receptors (GRPrs) and their cognate heterotrimeric GTP-binding proteins is a fundamental step in understanding the signal transduction cascade initiated by receptor-ligand interaction. To explore these mechanisms in greater detail, we have developed an in situ reconstitution assay in chaotrope-extracted membranes from mouse fibroblasts expressing the GRPr, and we have used it to measure GRPr-catalyzed binding of GTP gamma S to purified G protein alpha subunits. Binding studies with 125I-labeled [D-Tyr6]bombesin(6-13) methyl ester (125I-Tyr-ME), a GRPr specific antagonist, show a single binding site with a Kd = 1.4 nM +/- 0.4 (mean +/- SD, n = 3) and capacity of 15-22 pmol of receptor per mg of protein in the extracted membrane preparations, representing a 2- to 3-fold enrichment of binding sites compared with the membranes before extraction. Quantitative ligand displacement analysis using various unlabeled GRPr agonists shows a rank order of potency characteristic of the GRPr: bombesin > or = GRP > > neuromedin B. Reconstitution of urea extracted membranes with a purified G alpha q showed that receptor-catalyzed binding of GTP gamma S was dependent on agonist (GRP) and G beta gamma subunits. The EC50 for GRP was 3.5 nM, which correlates well with the reported Kd of 3.1 nM for GRP binding to GRPr expressed in mouse fibroblasts [Benya, R. V., et al. (1994) Mol. Pharmacol. 46, 235-245]. The apparent Kd for bovine brain G beta gamma in this assay was 60 nM, and the Km for squid retinal G alpha q was 90 nM. The GRPr-catalyzed binding of GTP gamma S is selective for G alpha q, since we did not detect receptor-catalyzed exchange using either G alpha i/o or G alpha t. These data demonstrate that GRPr can functionally couple to G alpha q but not to the pertussis toxin-sensitive G alpha i/o or retinal specific G alpha t. This in situ receptor reconstitution method will allow molecular characterization of G protein coupling to other heptahelical receptors.     

Gi3 mediates somatostatin-induced activation of an inwardly rectifying K+ current in human growth hormone-secreting adenoma cells

SRIF activates an inwardly rectifying K+ current in human GH-secreting adenoma cells. Activation of this K+ current induces hyperpolarization of the membrane and abolishment of action potential firing. This mechanism is an essential mechanism for SRIF-induced decrease in intracellular Ca2+ concentration and inhibition of GH secretion. The activation of the inwardly rectifying K+ current is mediated by a pertussis toxin-sensitive G protein. In this article, the expression of the pertussis toxin-sensitive G protein alpha-subunits in the human GH-secreting adenoma cells were analyzed by RT-PCR, and the G protein transducing the SRIF-induced activation of this inwardly rectifying K+ current was investigated. RT-PCR of the messenger RNA from two human GH-secreting adenomas revealed that all G alpha(i1), G alpha(i2), G alpha(i3), and G alpha(o) were expressed in these adenomas. Primary cultured cells from these two adenoma cells were investigated under the voltage clamp of the whole-cell mode. Specific antibodies against the carboxyl terminus of G protein alpha-subunits were microinjected into the cells. Microinjection of antibody against the carboxyl terminal sequence of G alpha(i3) attenuated the SRIF-induced activation of the inwardly rectifying K+ current, whereas antibody against the common carboxyl terminal sequence of G alpha(i1) and G alpha(i2) did not. These data indicate that the G protein transducing the SRIF-induced activation of the inwardly rectifying K+ current is Gi3.     

G proteins of the Gq family couple the H2 histamine receptor to phospholipase C

In several cell systems histamine has been shown to stimulate both adenylyl cyclase and phospholipase C through activation of a G protein-coupled H2 receptor. To analyze the bifurcating signal emanating from the activated H2 receptor and to identify the G proteins involved, H1 and H2 histamine receptors were functionally expressed in baculovirus-infected insect cells. Histamine challenge lead to concentration-dependent cAMP formation and Ca2+ mobilization in Sf9 cells infected with a virus encoding the H2 receptor, whereas H1 receptor stimulation only resulted in pronounced phospholipase C activation. To analyze the G protein coupling pattern of histamine receptors, activated G proteins were labeled with [alpha-32P]GTP azidoanilide and identified by selective immunoprecipitation. In insect cell membranes expressing H1 histamine receptors, histamine led to incorporation of the label into alpha q-like proteins, whereas activation of the H2 receptor resulted in labeling of alpha q- and alpha s-like G protein alpha-subunits. In COS cells transfected with H2 receptor complementary DNA, histamine caused concentration-dependent accumulation of cAMP and inositol phosphates; the latter effect was insensitive to pertussis toxin treatment. Histamine stimulation led to a pronounced increase in inositol phosphate production when complementary DNAs coding for alpha q, alpha 11, alpha 14, or alpha 15 G protein alpha-subunits were cotransfected. This increase was specific for Gq family members, as overexpression of alpha 12 or alpha s did not enhance histamine-stimulated phospholipase C activation. In membranes of guinea pig heart, addition of [alpha-32P]GTP azidoanilide resulted in labeling of alpha q and alpha 11 via the activated H1 and also via H2 receptors. These data demonstrate that dual signaling of the activated H2 histamine receptor is mediated by coupling of the receptor to Gs and Gq family members.     

Glucocorticoid regulation of G-protein subunits in neonatal liver

The effect of dexamethasone administration in vivo on the steady-state levels of G-protein subunits in liver of neonatal rabbits was investigated using specific antibodies to each subunit as well as bacterial toxin-mediated ADP-ribosylation assays. Parallel measurements were also made of the activity of adenylyl cyclase, as influenced by a variety of activators. Dexamethasone administration modulated the levels of G-protein subunits in liver in an age-dependent and subunit-specific manner but not in 24-h-old newborns. The inductive effect of dexamethasone was observed in animals older than 24 h, the greatest effect being on 2- to 3-day-old neonates. In 48-h-old animals the alpha-subunits Gs alpha-1, Gs alpha-2, Gi alpha and the beta-subunit G beta increased 2.0-, 2.1-, 4.3- and 2.8-fold, respectively, compared to the control. The increases were much less for older animals. Dexamethasone treatment also modulated effector-mediated stimulation of adenylyl cyclase activity in vitro and mimicked its effects on G-protein levels; the greatest increase (approximately 2-fold) in the activation of adenylyl cyclase occurred in membranes isolated from 2- to 3-day-old animals. In older animals there was either no effect of dexamethasone or a decrease in activity. The degree of change in enzyme activity paralleled the change in the amount of Gs alpha rather than of Gi alpha or G beta. These results suggest development-dependent regulation of hepatic G-proteins by glucocorticoids.     

Growth hormone-binding protein in the goat: characterization, evolution under exogenous growth hormone treatment, and correlation with liver growth hormone receptor levels

This report describes the identification and characterization of a specific, high-affinity growth hormone-binding protein (GHBP) in lactating goat serum. Serum samples were incubated with [125I]human GH as ligand and in the absence or in the presence of bovine GH as competitor. GH-GHBP complex formation was performed by high-performance liquid chromatography, and the radioactivity was recorded on-line with a Berthold LB detector connected to a computer. The results showed that a serum protein was able to bind specifically to human GH and bovine GH but not to ovine prolactin. Scatchard plots indicated an affinity constant of 4.5 x 10(8) M-1 and a maximum binding capacity of 4.8 x 10(-10) mol/l. In addition, we conducted a 4-wk study to determine the effects of recombinant bovine GH administration on milk production in lactating goats. The effects of recombinant bovine GH treatment on milk production and on the regulation of GHBP and hepatic GH receptor levels were studied. As expected, recombinant bovine GH injected daily increased yields of milk, fat, protein (40, 61, and 40%, respectively), and circulating insulin-like growth factor 1 concentrations compared with controls. During the pretreatment and treatment periods, the control goats exhibited a constant amount of GHBP in serum. No consistent effect of GH treatment on GHBP level was observed. The binding of [125I]bovine GH to hepatic microsomal membranes of GH-treated goats was significantly decreased compared with that of control goats. After MgCl2 desaturation of membranes, the results demonstrated that the down-regulation of GH hepatic receptors, observed for the treated goat group, was induced by receptor occupancy without modification of binding affinity. The GH receptor gene expression, analyzed by slot blot and hybridization with an [alpha-32P]GH receptor cDNA probe, was not modified by the GH treatment. In lactating goats, the galactopoietic effect of exogenous GH involved a hepatic receptor occupancy. The individual concentration of GHBP in serum cannot explain the individual variations of responses to GH treatment in goats.     

Rescue of functional interactions between the alpha2A-adrenoreceptor and acylation-resistant forms of Gi1alpha by expressing the proteins from chimeric open reading frames

Co-expression of the alpha2A-adrenoreceptor with a pertussis toxin-resistant (C351G), but not with an also palmitoylation-resistant (C3S/C351G), form of the alpha subunit of Gi1 resulted in agonist-induced, pertussis toxin-independent, GTP hydrolysis. Construction and expression of a chimeric fusion protein between the receptor and C351G Gi1alpha generated a membrane protein in which the G protein element was activated by receptor agonist. An equivalent fusion protein containing C3S/C351G Gi1alpha rescued the ability of receptor agonist to activate this mutant. Fusion proteins of a palmitoylation-resistant (C442A) alpha2A-adrenoreceptor and either C351G or C3S/C351G Gi1alpha also responded effectively to agonist. Myristoylation resistant (G2A/C351G) and combined acylation-resistant (G2A/C3S/C351G) mutants of Gi1alpha are cytosolic proteins. Expression of these as chimeric alpha2A-adrenoreceptor-G protein fusions restored membrane localization and activation of the G protein by receptor agonist. These studies demonstrate the general utility of generating chimeric fusion proteins to examine receptor regulation of G protein function and that the lack of functional activation of acylation-negative G proteins by a co-expressed receptor is related to deficiencies in cellular targeting and location rather than an inherent incapacity to produce appropriate protein-protein interactions and signal transmission.     

Agonist-mediated downregulation of G alpha i via the alpha 2-adrenergic receptor is targeted by receptor-Gi interaction and is independent of receptor signaling and regulation

One mechanism of long-term agonist-promoted desensitization of alpha2AR function is downregulation of the cellular levels of the alpha subunit of the inhibitory G protein, Gi. In transfected CHO cells expressing the human alpha2AAR, a 40.1 +/- 3.3% downregulation of Galphai2 protein occurred after 24 h of exposure of the cells to epinephrine, which was not accompanied by a decrease in Galphai2 mRNA. The essential step that targets Gi for degradation by agonist occupancy of the receptor was explored using mutated alpha2AAR lacking specific structural or functional elements. These consisted of 5HT1A receptor and beta2AR sequences substituted at residues 113-149 of the second intracellular loop and 218-235 and 355-371 of the N- and C-terminal regions of the third intracellular loop (altered Gi and Gs coupling), deletion of Ser296-299 (absent GRK phosphorylation), and substitution of Cys442 (absent palmitoylation and receptor downregulation). Of these mutants, only those with diminished Gi coupling displayed a loss of agonist-promoted Gi downregulation, thus excluding Gs coupling and receptor downregulation, palmitoylation, and phosphorylation as necessary events. Furthermore, coupling-impaired receptors consisting of mutations in the second or third loops ablated Gi downregulation, suggesting that a discreet structural motif of the receptor is unlikely to represent a key element in the process. While pertussis toxin ablated Gi downregulation, blocking downstream intracellular consequences of alpha2AAR activation or mimicking these pathways by heterologous means failed to implicate cAMP/adenylyl cyclase, phospholipase C, phospholipase D, or MAP kinase pathways in alpha2AAR-mediated Gi downregulation. Taken together, agonist-promoted Gi downregulation requires physical alpha2AAR-Gi interaction which targets Gi for degradation in a manner that is independent of alpha2AAR trafficking, regulation, or second messengers.     

Identification of a distinct molecular mass G alpha(h) (transglutaminase II) coupled to alpha1-adrenoceptor in mouse heart

Our previous studies on alpha1-adrenoceptor signaling suggested that G alpha(h) family is a signal mediator in different species. To elucidate the species-specificity of G alpha(h) family in molecular mass, we used the solubilized membranes from mouse heart and the ternary complex preparations containing alpha1-agonist/receptor/G-protein. Binding of [35S]GTPgammaS and the intensity of the [alpha-32P]GTP photoaffinity labeled protein resulting from activation of the alpha1-adrenoceptor were significantly attenuated by the antagonist, phentolamine. The molecular mass of the specific GTP-binding protein was approximately 72-kDa; homologous with G alpha(h) (transglutaminase II) family. Furthermore, immunological cross-reactivity of ternary complex from mouse heart and purified G alpha(h) from rat, guinea pig, and bovine using anti-G alpha(h7) antibody showed that their molecular masses were distinctly different and approximately 72-kDa G alpha(h) from mouse heart was the lowest molecular mass. Consistent with these observations, in co-immunoprecipitation and co-immunoadsorption of the alpha1-adrenoceptor in the ternary complex preparation by anti-G alpha(h7) antibody, the G alpha(h) family protein tightly coupled to alpha1-adrenoceptor. These results demonstrate the species-specificity of G alpha(h) family in molecular mass, especially the lowest molecular mass in mouse.     

Alpha adrenergic receptor subtypes involved in prostaglandin synthesis are coupled to Ca++ channels through a pertussis toxin-sensitive guanine nucleotide-binding protein

Previously, we have shown that alpha-2C and alpha-1A adrenergic receptors (AR) stimulate prostacyclin (PGI2) synthesis through a pertussis toxin-sensitive guanine nucleotide-binding protein (G protein) in vascular smooth muscle cells (VSMC). The purpose of this study was to assess the role of Ca++ in PGI2 production elicited by alpha-AR activation and to investigate the modulation of the Ca++ channel by G proteins coupled to these alpha-AR in VSMC. PGI2 was measured as immunoreactive 6-keto-PGF1 alpha by radioimmunoassay and cytosolic calcium ([Ca++]i) by spectrofluorometry using fura-2. Norepinephrine, methoxamine and UK-14304 enhanced 6-keto-PGF1 alpha production and [Ca++]i, which was inhibited by depletion of extracellular Ca++ and by Ca++ channel antagonists (verapamil, nifedipine and PN 200-110). Moreover, the Ca++ channel activator Bay K 8644 increased 6-keto-PGF1 alpha production in a nifedipine-sensitive manner, indicating the involvement of dihydropyridine-sensitive Ca++ channels in VSMC. Pertussis toxin inhibited AR agonist-induced 6-keto-PGF1 alpha production and the increase in [Ca++]i. Alpha AR agonists increase Ca++ influx in the presence of guanosine 5'-0-(2- thiodiphosphate) (GTP-gamma-S), and this effect was blocked in the presence of guanine 5'-O-(2-thiodiphosphate) (GDP-beta-S) and antiserum against Gi alpha 1-2 protein in reversibly permeabilized cells with beta-escin. VSMC of rabbit aortae contain a G protein(s) that was recognized by Gi alpha 1-2 but not Gi alpha 3 or G0 antibodies at 1:200 dilution. The calmodulin inhibitor W-7 blocked AR agonist and Bay K 8644-stimulated 6-keto-PGF1 alpha production. The phospholipase A2 inhibitors 7,7-dimethyleicosadienoic acid and oleoyloxyethyl phosphocholine but not phospholipase C inhibitor U-73122 reduced 6-keto-PGF1 alpha production in VSMC. These data suggest that a pertussis toxin-sensitive G protein, probably Gi alpha 1-2, coupled to alpha AR regulates Ca++ influx, which, in turn, by interacting with calmodulin, increases phospholipase A2 activity to release arachidonic acid for PGI2 synthesis in VSMC of rabbit aortae.     

Reconstitution of recombinant N-formyl chemotactic peptide receptor with G protein

A recombinant human neutrophil N-formyl peptide receptor (rFPR) expressed in transfected mouse fibroblasts (TX2 cells) was analyzed for its ability to couple physically with the heterotrimeric G protein, Gi. Immunoprecipitation of photoaffinity-labeled rFPR and endogenous neutrophil formyl peptide receptor (nFPR) with an anti-FPR peptide antibody demonstrated that the receptors were identical in both size and extent of glycosylation. Coupling of rFPR with endogenous TX2 Gi was demonstrated by coimmunoprecipitation of the two proteins with an anti-Gi antibody. Moreover, rFPR was able to form a physical complex with purified Gi in a soluble reconstitution system. We observed similar affinities of rFPR and nFPR for Gi. This report provides the first direct evidence that rFPR associates physically with Gi and provides a foundation for analysis of the G protein coupling capacity of mutant rFPRs.     

Direct or C5a-induced activation of heterotrimeric Gi2 proteins in human neutrophils is associated with interaction between formyl peptide receptors and the cytoskeleton

The binding of ligands to N-formyl peptide chemoattractant receptors in human neutrophils results in a rapid association of these receptors with a cytoskeletal fraction and a specific activation and release of Gi2 alpha-subunits from this fraction. In the present study we could show that pretreating neutrophils with GDPbetaS prevented the fMet-Leu-Phe-induced association of its receptor with a cytoskeletal fraction and also blocked the release of Gi2 alpha-subunits from the same cytoskeletal fraction. In contrast, direct activation of Gi2 proteins by addition of GTPgammaS or AlF4- not only caused a release of Gi2 alpha-subunits from the cytoskeleton but also an association of formyl peptide receptors with the cytoskeleton. The receptor for complement fragment 5a, which transduces its signaling through the same Gi2 protein, triggers both a release of Gi2 alpha-subunits from the cytoskeleton fraction and, of even greater interest, an association between formyl peptide receptors and the cytoskeleton. The close relationship between the activation and release of Gi2 alpha-subunits from the cytoskeleton and the association of formyl peptide receptors with the cytoskeleton might, however, not be a matter of protein-protein exchange, since the increased binding of formyl peptide receptors to the cytoskeleton occurs more rapidly than the release of Gi2 alpha-subunits from the cytoskeleton. The present findings suggest a possible mechanism for the initiation of formyl peptide receptor desensitization during neutrophil locomotion.     

Muscarinic regulation of intracellular signaling and neurosecretion in gonadotropin-releasing hormone neurons

Agonist activation of cholinergic receptors expressed in perifused hypothalamic and immortalized GnRH-producing (GT1-7) cells induced prominent peaks in GnRH release, each followed by a rapid decrease, a transient plateau, and a decline to below basal levels. The complex profile of GnRH release suggested that acetylcholine (ACh) acts through different cholinergic receptor subtypes to exert stimulatory and inhibitory effects on GnRH release. Whereas activation of nicotinic receptors caused a transient increase in GnRH release, activation of muscarinic receptors inhibited basal GnRH release. Nanomolar concentrations of ACh caused dose-dependent inhibition of cAMP production that was prevented by pertussis toxin (PTX), consistent with the activation of a plasma-membrane Gi protein. Micromolar concentrations of ACh also caused an increase in phosphoinositide hydrolysis that was inhibited by the M1 receptor antagonist, pirenzepine. In ACh-treated cells, immunoblot analysis revealed that membrane-associated G(alpha q/11) immunoreactivity was decreased after 5 min but was restored at later times. In contrast, immunoreactive G(alpha i3) was decreased for up to 120 min after ACh treatment. The agonist-induced changes in G protein alpha-subunits liberated during activation of muscarinic receptors were correlated with regulation of their respective transduction pathways. These results indicate that ACh modulates GnRH release from hypothalamic neurons through both M1 and M2 muscarinic receptors. These receptor subtypes are coupled to Gq and Gi proteins that respectively influence the activities of PLC and adenylyl cyclase/ion channels, with consequent effects on neurosecretion.     

Localization of a G protein Gi2 in the cilia of rat ependyma, oviduct and trachea

In previous studies, the localization of a pertussis toxin-sensitive G protein was demonstrated in ependymal cilia, but the identification of the subtype of G protein was inconsistent. To clarify this issue, we studied the localization of Goalpha, Gi1alpha, Gi3alpha and Gi2alpha in the ciliated ependymal cells and in the cilia of some other tissues of rats using specific antibodies. The cilia of the ependymal cells that line the ventricular cavity of the brain were intensely immunoreactive for Gi2alpha, but not for Goalpha, Gi1alpha or Gi3alpha. Immunoblot analysis demonstrated higher levels of Gi2alpha in the ependymal cilia-rich pellet than in the motor area of the parietal cortex. At the ultrastructural level, the immunoreactivity specific for Gi2alpha was found predominantly in the cilia, but rarely in the microvilli or the basal bodies of ependymal cells. In cross-sections, the immunoreactivity specific for Gi2alpha was observed only in cell membranes, in particular, in the inner electron-dense leaflet of the trilaminar structure. In addition to that in the ependymal cilia, such specific localization of Gi2alpha was observed in the motile cilia in other tissues, including the oviduct and trachea. By contrast, the stereocilia in the ductus deferens were not immunopositive for Gi2alpha. These findings suggest that Gi2 might play an important role in the signal transduction in ciliary membrane-associated function(s) of the ependymal cells, oviduct and trachea.     

Expression of a functional endothelin (ETA) receptor in human meningiomas

Endothelin (ET) receptor subtypes (ETA and ETB) in human meningiomas were characterized using quantitative receptor autoradiography. A single class of high-affinity 125I-ET-1 binding sites was localized in all meningioma tissue studied (dissociation constant: 2.4 +/- 0.3 nM, maximum binding capacity: 319 +/- 66 fmol/mg (mean +/- standard error of the mean for 13 tumors)). Unlabeled ET-1 showed a strong affinity for 125I-ET-1 binding to tissue sections of the tumors with a 50% inhibiting concentration (IC50) of 2.9 +/- 0.7 x 10(-9) M, whereas ET-3 showed a much lower affinity (IC50: 8.4 +/- 2.5 x 10(-6) M). Sarafotoxin S6c, a selective agonist for the ETB receptor, could not compete for 125I-ET-1 binding to meningiomas. Endothelin-1 significantly stimulated deoxyribonucleic acid (DNA) synthesis in a dose-dependent manner in cultured human meningioma cells. In contrast, no significant stimulation of DNA synthesis occurred with an S6c concentration up to 10(-7) M. Pretreatment of the meningioma cells with pertussis toxin, a bacterial toxin that adds adenosine 5'-diphosphate-ribose to the alpha subunit of guanine nucleotide binding (G) proteins such as Gi or G(o), induced a concentration-dependent reduction in ET-stimulated DNA synthesis in meningioma cells, but did not affect the epidermal growth factor-induced DNA synthesis. These observations suggest that the ETA receptor is predominantly expressed in human meningioma tissue and that ET may act as a growth factor on the meningioma cells by interacting with the ETA receptor and by pertussis toxin-sensitive mechanisms.