Flow-induced prostacyclin production is mediated by a pertussis toxin-sensitive G protein.

Fluid flow and several other agonists induce prostacyclin (PGI2) production in endothelial cells. G proteins mediate the response of a large number of hormones such as histamine, but the transduction pathway of the flow signal is unclear. We found that GDP beta S and pertussis toxin inhibited flow-induced prostacyclin production in human umbilical vein endothelial cells. In addition, flow potentiated the histamine-induced production of PGI2. This suggests that flow stimulates prostacyclin production via a pertussis toxin-sensitive G protein and modulates the stimulus-response coupling of other agonists.     

TIMP-1 inhibits apoptosis in breast carcinoma cells via a pathway involving pertussis toxin-sensitive G protein and c-Src

In addition to inhibiting matrix metalloproteinases, tissue inhibitor of metalloproteinase-1 (TIMP-1) is involved in the regulation of cell growth and survival. To determine its mechanism of action, we investigated effects of TIMP-1 on cell proliferation and survival and signaling pathways induced by TIMP-1 in the human breast carcinoma T-47D cell line. Treatment of T-47D cells with TIMP-1 strongly inhibited apoptosis induced by serum deprivation, but did not affect cell proliferation. TIMP-1 induced phosphorylation of Akt and extracellular signal-regulated protein kinases (ERKs), but pertussis toxin and specific inhibitors of Src family tyrosine kinases, protein tyrosine kinases, and phosphatidylinositol-3 kinase (PI3 kinase) blocked the ability of TIMP-1 to activate Akt and ERKs as well as the anti-apoptotic effect of TIMP-1. We found that TIMP-1 enhanced the kinase activities of c-Src and PI3 kinase and that this enhancement was inhibited by pertussis toxin. Inhibition of ERK activation, however, resulted in a slight decrease of the TIMP-1-induced anti-apoptotic effect. These findings demonstrate that the ability of TIMP-1 to inhibit apoptosis in T-47D cells is mediated by the sequential activation of pertussis toxin-sensitive G protein, c-Src, PI3 kinase, and Akt.     

The macrophage-derived lectin, MNCF, activates neutrophil migration through a pertussis toxin-sensitive pathway.

The macrophage-derived neutrophil chemotactic factor (MNCF) is a D-galactose-binding lectin that induces neutrophil migration in vitro and in vivo. Neutrophil recruitment induced by MNCF is resistant to glucocorticoid treatment and is inhibited by the lectin-specific sugar, D-galactose. In the present study, we characterized the binding of MNCF to neutrophils and the responses triggered by this binding. Exposure to MNCF resulted in cell polarization, formation of a lamellipodium, and deep ruffles on the cell surface. By confocal microscopy, we observed that MNCF was evenly distributed on the cell surface after 30 min of incubation. The labeling intensity progressively diminished with longer incubations. Internalization kinetics showed that MNCF/ligand complexes were rapidly internalized, reaching maximum intracellular concentrations at 120 min and then decreased thereafter. The binding and internalization of MNCF were selectively inhibited by D-galactose. MNCF-induced neutrophil chemotaxis was inhibited by pertussis toxin. This fact strongly suggests that the MNCF-ligand on the neutrophil surface is a G-protein-coupled receptor (GPCR), similar to receptors for well-established neutrophil attractants. Our observations on the ability of MNCF to activate neutrophils are consistent with the increasing evidence for the participation of animal lectins in the innate immune response.     

Adrenomedullin inhibits insulin exocytosis via pertussis toxin-sensitive G protein-coupled mechanism

Direct effects of adrenomedullin on insulin secretion from pancreatic beta-cells were investigated using a differentiated insulin-secreting cell line INS-1. Adrenomedullin (1-100 pM) inhibited insulin secretion at both basal (3 mM) and high (15 mM) glucose concentrations, although this inhibitory effect was not observed at higher concentrations of adrenomedullin. The inhibition of glucose-induced insulin secretion by adrenomedullin was restored with 12-h pretreatment with 1 microg/ml pertussis toxin (PTX), suggesting that this effect could be mediated by PTX-sensitive G proteins. Cellular glucose metabolism evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide colorimetric assay was not affected by adrenomedullin at concentrations that inhibited insulin secretion. Moreover, electrophysiological studies revealed that 10 pM adrenomedullin had no effect on membrane potential, voltage-gated calcium currents, or cytosolic calcium concentration induced by 15 mM glucose. Finally, insulin release induced by cAMP-raising agents, such as forskolin plus 3-isobutyl-1-methylxanthine or the calcium ionophore ionomycin, was significantly inhibited by 10 and 100 pM adrenomedullin. In conclusion, adrenomedullin at picomolar concentrations directly inhibited insulin secretion from beta-cells. This effect is likely due to the inhibition of insulin exocytosis through the activation of PTX-sensitive G proteins.     

Evidence for a pertussis toxin-sensitive signalling pathway in the dual action of angiotensin II on growth hormone release in pituitary cell aggregates

In anterior pituitary cell aggregates cultured in the presence of the glucocorticoid dexamethasone (DEX) angiotensin II (AII) had a dual effect on growth hormone (GH) release. The peptide stimulated the release in aggregates from 2-week-old rats, whereas the peptide had an inhibitory effect in cultures from adult rats. Treatment of aggregates from adult rats with pertussis toxin (PT) reversed the inhibitory effect of AII on GH release in a stimulatory effect; PT treatment of aggregates from 18- to 20-day-old rats significantly enhanced the stimulation of GH release by AII. The effect of PT was seen only when DEX was added to the culture medium. The present data suggest that the glucocorticoid-dependent stimulus-effect coupling of AII on GH release involves both a stimulatory and an inhibitory component, the latter being abolished by PT, and that the stimulatory component predominates during immature life while the inhibitory one during adult life.     

Mastoparan, a wasp venom, activates platelets via pertussis toxin-sensitive GTP-binding proteins

Mastoparan induced limited release of serotonin from intact human platelets, while neither intracellular calcium ion elevation nor arachidonic acid mobilization was observed. Cytolysis induced by mastoparan was negligible in the concentration range that induced serotonin release. In digitonin-permeabilized cells, mastoparan induced Ca(++)-independent release of serotonin and Ca(++)-dependent arachidonic acid release. Both serotonin release and arachidonic acid release were reduced by pertussis toxin, suggesting that platelet activation induced by mastoparan is mediated by GTP-binding proteins.     

The interaction of a kainate receptor from goldfish brain with a pertussis toxin-sensitive GTP-binding protein.

Kainate receptors are present in high concentrations in goldfish brain (Henley and Oswald, 1988a and b; Ziegra et al., 1990), possibly in neuronal and glial cells. In a number of systems, the kainate receptor has been assumed to be an integral ion channel (Watkins and Evans, 1981); but, for some kainate receptors, ion channel activity has not been demonstrated (Wada et al., 1989). This study presents evidence that a portion of the [3H]kainate-binding sites in goldfish brain is sensitive to guanine nucleotides, with a loss of high affinity binding in the presence of nonhydrolyzable GTP analogs. Pertussis toxin pretreatment of membranes causes a loss of high affinity [3H]kainate binding and of the guanine nucleotide-sensitive binding. Pertussis toxin catalyzes the specific [32P]ADP-ribosylation of a 40-kDa substrate in a kainate-sensitive manner. In addition, incorporation of [alpha-32P]GTP-gamma-azidoanilide by photoaffinity labeling was enhanced in the presence of kainate. These results indicate that a subpopulation of [3H]kainate-binding sites in goldfish brain may be coupled to G proteins.     

Tyrosine phosphorylation of an SH2-containing protein tyrosine phosphatase is coupled to platelet thrombin receptor via a pertussis toxin-sensitive heterotrimeric G-protein.

SH-PTP1 is a protein tyrosine phosphatase (PTP) predominantly expressed in haematopoietic cells and containing two src homology-2 (SH2) domains. Here we report that SH-PTP1 is phosphorylated on both serine and tyrosine residues in response to thrombin or phorbol myristate acetate (PMA), which increased by 60 and 40%, respectively, SH-PTP1 activity. Thrombin-induced phosphorylation of SH-PTP1 is an early signalling event (maximal within 10 s) involving neither integrin signalling, nor calcium, nor release of ADP or thromboxane A2. Moreover, in contrast with PMA, the effect of thrombin on the tyrosine phosphorylation of SH-PTP1 was hardly affected by GF109203X, a specific protein kinase C (PKC) inhibitor. Finally, phosphorylation of SH-PTP1 could be provoked in permeabilized platelets by thrombin or GTP gamma S. This was abolished by pertussis toxin, the specificity of this effect being verified with the megakaryocytic cell line Dami cell. Our data thus identify SH-PTP1 as an in vivo substrate of a putative protein tyrosine kinase linked to the thrombin receptor by a Gi protein. This might offer some clue to unravel the mechanism of thrombin not only in platelets but also in nucleated cells, where its mitogenic effect is known to involve pertussis toxin-sensitive G-proteins, tyrosine phosphorylation and the ras pathway.     

ATP-induced Ca2+ influx is regulated via a pertussis toxin-sensitive mechanism in a PC12 cell clone.

A PC12 cell clone that responds to ATP with polyphosphoinositide hydrolysis and with a marked, biphasic intracellular free Ca2+ concentration ([Ca2+]i) response (composed by release from intracellular stores accompanied by stimulated influx from the medium), was pretreated with pertussis toxin. In the pretreated cells the responses induced by ATP were differently modified. Polyphosphoinositide hydrolysis and Ca2+ release were moderately inhibited whereas Ca2+ influx was enhanced. Pharmacological experiments revealed the influx enhancement to be sustained by neither voltage-gated nor second messenger-operated Ca2+ channels. Rather, a channel of the receptor-operated type activated by ATP (P2w receptor) appears to work under the negative control of a pertussis toxin-sensitive G protein, acting presumably by direct interaction with the channel in the plane of the plasma membrane.     

Inhibition by glucagon of the cGMP-inhibited low-Km cAMP phosphodiesterase in heart is mediated by a pertussis toxin-sensitive G-protein.

We have recently reported that glucagon activated the L-type Ca2+ channel current in frog ventricular myocytes and showed that this was linked to the inhibition of a membrane-bound low-Km cAMP phosphodiesterase (PDE) (Méry, P. F., Brechler, V., Pavoine, C., Pecker, F., and Fischmeister, R. (1990) Nature 345, 158-161). We show here that the inhibition of membrane-bound PDE activity by glucagon depends on guanine nucleotides, a reproducible inhibition of 40% being obtained with 0.1 microM glucagon in the presence of 10 microM GTP, with GTP greater than GTP gamma S, while GDP and ATP gamma S were without effect. Glucagon had no effect on the cytosolic low-Km cAMP PDE, assayed with or without 10 microM GTP. Glucagon inhibition of membrane-bound PDE activity was not affected by pretreatment of the ventricle particulate fraction with cholera toxin. However, it was abolished after pertussis toxin pretreatment. Mastoparan, a wasp venom peptide known to activate G(i)/G(o) proteins directly, mimicked the effect of glucagon. PDE inhibition by glucagon was additive with the inhibition induced by Ro 20-1724, but was prevented by milrinone. This was correlated with an increase by glucagon of cAMP levels in frog ventricular cells which was not additive with the increase in cAMP due to milrinone. We conclude that glucagon specifically inhibits the cGMP-inhibited, milrinone-sensitive PDE (CGI-PDE). Insensitivity of adenylylcyclase to glucagon and inhibition by the peptide of a low-Km cAMP PDE were not restricted to frog heart, but also occurred in mouse and guinea pig heart. These results confirm that two mechanisms mediate the action of glucagon in heart: one is the activation of adenylylcyclase through Gs, and the other relies on the inhibition of the membrane-bound low-Km CGI-PDE, via a pertussis toxin-sensitive G-protein.     

l-Cysteine-induced up-regulation of the low-density lipoprotein receptor is mediated via a transforming growth factor-alpha signalling pathway

Sulphur-containing amino acids regulate plasma cholesterol levels in animals and humans. However, their mechanism of action remains unclear. Low-density lipoprotein receptor (LDLR) plays an important role in cholesterol metabolism. We therefore investigated the effects of sulphur-containing amino acids on the expression of LDLR in hepatocytes. HepG2 cells were cultured in Dulbecco’s Modified Eagle’s Medium with or without sulphur-containing amino acids and cysteine-containing compounds. We found that l-cysteine increased LDLR mRNA and enhanced LDLR gene promoter activity through the extracellular-signal-related kinase and p38 mitogen-activated protein kinase signalling pathways in HepG2 cells. Moreover, we observed that l-cysteine stimulated the release of transforming growth factor-alpha (TGF-α) and that TGF-α increased the LDLR mRNA levels. This study provides a report of the l-cysteine mediated up-regulation of the LDLR expression via TGF-α signalling pathway. Our findings provide insights into cholesterol homeostasis and amino acid signalling.     

Serotonin-induced deoxyribonucleic acid synthesis in vascular smooth muscle cells involves a novel, pertussis toxin-sensitive pathway

Serotonin-induced DNA synthesis in bovine aortic smooth muscle cells was totally abolished by pretreatment of cultures with 5 ng/ml pertussis toxin. The half maximally effective concentration of toxin was approximately 10 pg/ml. Pertussis toxin did not affect platelet-derived growth factor (PDGF)-stimulated DNA synthesis and actually enhanced the mitogenic effect of the phorbol ester, phorbol 12-myristate 13-acetate. Pertussis toxin did not inhibit serotonin-stimulated inositol phosphate accumulation or increases in intracellular calcium or cAMP concentrations under conditions sufficient to completely inhibit serotonin-induced (3H)thymidine incorporation. These results demonstrate that a novel, pertussis-sensitive pathway is required for serotonin-, but not platelet-derived growth factor-induced DNA synthesis in vascular smooth muscle cells. The pertussis-sensitive step does not involve cAMP, phosphoinositide hydrolysis, mobilization of intracellular calcium, or phorbol ester-sensitive protein kinase C activity.     

Signal transduction pathway for IL-1. Involvement of a pertussis toxin-sensitive GTP-binding protein in the activation of adenylate cyclase

Human Il-1 alpha induces the synthesis of kappa Ig L chains by the pre-B cell line 7OZ/3, IL-2R alpha by the human NK cell line YT, and PGE2 by human rheumatoid synovial cells. Pertussis toxin (PT) markedly inhibited all three IL-1-induced activation events. The inhibition by PT was associated with a decrease in IL-1-mediated cAMP production. PT also inhibited IL-1-stimulated cAMP production in crude membrane fractions from 7OZ/3, YT, and 3T3 fibroblasts. In addition, IL-1 stimulated GTPase activity present in the membranes IL-1-responsive cells. Furthermore, the IL-1-induced GTPase activity was sensitive to PT. PT induced the ADP-ribosylation of a 46-kDa substrate in membrane preparations from IL-1-responsive cells. Cholera toxin also induced the ADP-ribosylation of a 46-kDa substrate in the same membrane preparations. The present findings indicate that the IL-1R is linked to a PT-sensitive G protein that stimulates the activity of adenylate cyclase.     

Toll-like receptor family and signalling pathway

Toll is a Drosophila gene essential for ontogenesis and anti-microbial resistance. Several orthologues of Toll have been identified and cloned in vertebrates, namely Toll-like receptors (TLRs). Human TLRs are a growing family of molecules involved in innate immunity. TLRs are characterized structurally by a cytoplasmic Toll/interleukin-1 receptor (TIR) domain and by extracellular leucine-rich repeats. TLRs characterized so far activate the MyD88/interleukin-1 receptor-associated kinase (IRAK) signalling pathway. Genetic, gene-transfer and dominant-negative approaches have involved TLR family members (TLR2 and TLR4) in Gram-positive and Gram-negative bacteria recognition and signalling. Accumulating evidence suggests that TLR2 is also involved in signalling-receptor complexes that recognize components of yeast and mycobacteria. However, the definitive roles of other TLRs are still lacking. A systematic approach has been used to determine whether different human leucocyte populations selectively or specifically express TLR mRNA. Based on expression pattern, TLR can be classified as ubiquitous (TLR1), restricted (TLR2, TLR4 and TLR5) and specific (TLR3). Expression and regulation of distinct but overlapping ligand-recognition patterns may underlie the existence of a large, seemingly redundant TLR family. Alternatively, the expression of a TLR in a single cell type may indicate a specific role for this molecule in a restricted setting.     

Association of two pertussis toxin-sensitive G-proteins with the D2-dopamine receptor from bovine striatum.

The solubilized D2-dopamine receptor from bovine striatum exhibits high and low affinity states for dopaminergic agonists. Guanine nucleotides and pertussis toxin convert the solubilized receptor from a high affinity state to a low one. A D2-receptor preparation partially purified by affinity chromatography on a haloperidol adsorbent, exhibited agonist-stimulated GTPase activity. [32P]ADP-ribosylation by pertussis toxin of this receptor preparation resulted in the specific labeling of two protein bands corresponding to mol. wts of 39 and 41 kd, in SDS-PAGE. Association of these G-proteins with the receptor was specifically inhibited by Gpp(NH)p. Immunoblot analysis of these G-proteins indicated that the 41- and 39-kd protein bands are analogous to brain Gi and Go respectively. These experiments demonstrate that two distinct pertussis toxin-sensitive G-proteins are functionally associated with bovine striatum D2-dopamine receptor.     

Matrix metalloproteinase-1 activates a pertussis toxin-sensitive signaling pathway that stimulates the release of matrix metalloproteinase-9

The matrix metalloproteinases (MMPs) are a family of structurally related metalloendopeptidases so named due to their propensity to target extracellular matrix (ECM) proteins. Accumulating evidence, however, suggests that these proteases cleave numerous non-ECM substrates including enzymes and cell surface receptors. MMPs may also bind to cell surface receptors, though such binding has typically been thought to mediate internalization and degradation of the bound protease. More recently, it has been shown that MMP-1 coimmunoprecipitates with the alpha2beta1 integrin, a receptor for collagen. This association may serve to localize the enzymatic activity of MMP-1 so that collagen is cleaved and cell migration is facilitated. In other studies, however, it has been shown that integrin engagement may be linked to the activation of signaling cascades including those mediated by Gialpha containing heterotrimers. As an example, alpha2beta1 can form a complex with CD47 that may associate with Gialpha. In the present study we have therefore investigated the possibility that MMP-1 may affect intracellular changes that are linked to the activation of a Gi protein-coupled receptor. We show that treatment of neural cells with MMP-1 is followed by a rapid reduction in cytosolic levels of cAMP. Moreover, MMP-1 potentiates proteinase activated receptor-1 (PAR-1) agonist-linked increases in intracellular calcium, an effect which is often observed when an agonist of a Gi protein-coupled receptor is administered in association with an agonist of a Gq coupled receptor. In addition, MMP-1 stimulates pertussis toxin sensitive release ofMMP-9 both from cultured neural cells and monocyte/macrophages. Together, these results suggest that MMP-1 signals through a pertussis toxin-sensitive G protein-coupled receptor.     

The epidermal growth factor receptor is coupled to a pertussis toxin-sensitive guanine nucleotide regulatory protein in rat hepatocytes.

Activation of epidermal growth factor (EGF) receptors stimulates inositol phosphate production in rat hepatocytes via a pertussis toxin-sensitive mechanism, suggesting the involvement of a G protein in the process. Since the first event after receptor-G protein interaction is exchange of GTP for GDP on the G protein, the effect of EGF was measured on the initial rates of guanosine 5'-O-(3-[35S]thiotriphosphate) [( 35S]GTP gamma S) association and [alpha-32P]GDP dissociation in rat hepatocyte membranes. The initial rate of [35S]GTP gamma S binding was stimulated by EGF, with a maximal effect observed at 8 nM EGF. EGF also increased the initial rate of [alpha-32P]GDP dissociation. The effect of EGF on [35S]GTP gamma S association was blocked by boiling the peptide for 5 min in 5 mM dithiothreitol or by incubation of the membranes with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S). EGF-stimulated [35S]GTP gamma S binding was completely abolished in hepatocyte membranes prepared from pertussis toxin-treated rats and was inhibited in hepatocyte membranes that were treated directly with the resolved A-subunit of pertussis toxin. The amount of guanine nucleotide binding affected by occupation of the EGF receptor was approximately 6 pmol/mg of membrane protein. Occupation of angiotensin II receptors, which are known to couple to G proteins in hepatic membranes, also stimulated [35S]GTP gamma S association with and [alpha-32P]GDP dissociation from the membranes. The effect of angiotensin II on [alpha-32P]GDP dissociation was blocked by the angiotensin II receptor antagonist [Sar1,Ile8]angiotensin II, demonstrating that the guanine nucleotide binding was receptor-mediated. In A431 human epidermoid carcinoma cells, EGF stimulates inositol lipid breakdown, but the effect is not blocked by treatment of the cells with pertussis toxin. In these cells, EGF had no effect on [35S]GTP gamma S binding. Occupation of the beta-adrenergic receptor in A431 cell membranes with isoproterenol did stimulate [35S] GTP gamma S binding, and the effect could be completely blocked by l-propranolol. These results support the concept that in hepatocyte membranes, EGF receptors interact with a pertussis toxin-sensitive G protein via a mechanism similar to other hormone receptor-G protein interactions, but that in A431 human epidermoid carcinoma cells, EGF may activate phospholipase C via different mechanisms.     

Alpha-adrenergic inhibition of rat pancreatic beta-cell replication and insulin secretion is mediated through a pertussis toxin-sensitive G-protein regulating islet cAMP content.

The rate of DNA synthesis, insulin secretion and cAMP content in isolated pancreatic islets were markedly inhibited by long-term exposure to the alpha 1-adrenoceptor agonist phenylephrine, the alpha 2-adrenoceptor agonist clonidine and the beta-adrenoceptor antagonist propranolol. Pertussis toxin or the stimulatory cAMP analog Sp-cAMPS increased DNA synthesis and insulin secretion in the absence of the adrenergic agents. Pertussis toxin blocked the inhibitory actions of these agents on DNA synthesis, insulin secretion and cAMP content, and a similar protection was imposed by Sp-cAMPS. Thus, long-term alpha-adrenergic stimulation interferes with signaling through pertussis toxin-sensitive G-protein(s) and, by decreasing the islet cAMP content, inhibits beta-cell DNA synthesis and insulin secretion.     

Possible coupling of prostaglandin E2 receptor with pertussis toxin-sensitive guanine nucleotide-binding protein in osteoblast-like cells.

In cloned osteoblast-like cells, MC3T3-E1, prostaglandin E2 (PGE2) stimulated the formation of inositol phosphates in a dose-dependent manner in the range between 10 nM and 10 microM. Pertussis toxin inhibited the effect of PGE2 dose-dependently in the range between 1 ng/ml and 1 micrograms/ml. In the cell membranes, pertussis toxin catalyzed ADP-ribosylation of a protein with an Mr of about 40,000. Pretreatment of membranes with 10 microM PGE2 in the presence of 2.5 mM MgCl2 and 100 microM GTP markedly attenuated this pertussis toxin-catalyzed ADP-ribosylation of the protein in a time-dependent manner. G12 was detected in these cells by immunoblotting with purified anti-G12 alpha antibodies. The results indicate the possible coupling of PGE2 signalling with pertussis toxin-sensitive GTP-binding protein, which is probably G12, in osteoblast-like cells.     

U50488 inhibits outwardly rectifying potassium channel in PC12 cells via pertussis toxin-sensitive G-protein

This study was undertaken to determine the effect of U50488, a kappa-opioid receptor agonist, on outwardly rectifying potassium channel (Ik) in undifferentiated PC12 cells. Using whole-cell and on-cell patch-clamp techniques, we found that U50488 decreased Ik amplitude in a time-dependent manner and Ik activation was delayed. Single-channel kinetic analysis provided a two-stage model for us to illuminate the blockage effect induced by U50488. To identify whether U50488 mediates the effect through opioid receptor and G-protein, several specific blockers and activators were used. Not only naloxone but also PTX and GDPbetaS abolished U50488-induced suppression; however, such effect was not observed when cAMP or other adenylyl cyclase activators were used. It is postulated that kappa-opioid receptor and Gi/o protein, but not cAMP, are involved in U50488-induced suppression of Ik.