Role of pertussis toxin-sensitive G protein in metabolic vasodilation of coronary microcirculation

We have previously demonstrated that pertussis toxin (PTX)-sensitive G protein (G(PTX)) plays a major role in coronary microvascular vasomotion during hypoperfusion. We aimed to elucidate the role of G(PTX) during increasing metabolic demand. In 18 mongrel dogs, coronary arteriolar diameters were measured by fluorescence microangiography using a floating objective. Myocardial oxygen consumption (MVO(2)) was increased by rapid left atrial pacing. In six dogs, PTX (300 ng/ml) was superfused onto the heart surface for 2 h to locally block G(PTX). In eight dogs, the vehicle (Krebs solution) was superfused in the same way. Before and after each treatment, the diameters were measured during control (130 beats/min) and rapid pacing (260 beats/min) in each group. Metabolic stimulation before and after the vehicle treatment caused 8.6 +/- 1. 8 and 16.1 +/- 3.6% dilation of coronary arterioles <100 microm in diameter (57 +/- 8 microm at control, n = 10), respectively. PTX treatment clearly abolished the dilation of arterioles (12.8 +/- 2. 5% before and 0.9 +/- 1.6% after the treatment, P < 0.001 vs. vehicle; 66 +/- 8 microm at control, n = 11) in response to metabolic stimulation. The increases in MVO(2) and coronary flow velocity were comparable between the vehicle and PTX groups. In four dogs, 8-phenyltheophylline (10 microM, superfusion for 30 min) did not affect the metabolic dilation of arterioles (15.3 +/- 2.0% before and 16.4 +/- 3.8% after treatment; 84.3 +/- 11.0 microm at control, n = 8). Thus we conclude that G(PTX) plays a major role in regulating the coronary microvascular tone during active hyperemia, and adenosine does not contribute to metabolic vasodilation via G(PTX) activation.     

Regulation of acetylated low density lipoprotein uptake in macrophages by pertussis toxin-sensitive G proteins

Class A scavenger receptors (SR-A) mediate the uptake of modified low density lipoprotein (LDL) by macrophages. Although not typically associated with the activation of intracellular signaling cascades, results with peritoneal macrophages indicate that the SR-A ligand acetylated LDL (AcLDL) promotes activation of cytosolic kinases and phospholipases. These signaling responses were blocked by the treatment of cells with pertussis toxin (PTX) indicating that SR-A activates G(i/o)-linked signaling pathways. The functional significance of SR-A-mediated G(i/o) activation is not clear. In this study, we investigated the potential role of G(i/o) activation in regulating SR-A-mediated lipoprotein uptake. Treatment of mouse peritoneal macrophages with PTX decreased association of fluorescently labeled AcLDL with cells. This inhibition was dependent on the catalytic activity of the toxin confirming that the decrease in AcLDL uptake involved inhibiting G(i/o) activation. In contrast to the inhibitory effect on AcLDL uptake, PTX treatment did not alter beta-VLDL-induced cholesterol esterification or deposition of cholesterol. The ability of polyinosine to completely inhibit AcLDL uptake, and the lack of PTX effect on beta-VLDL uptake, demonstrated that the inhibitory effect is specific for SR-A and not the result of non-specific effects on lipoprotein metabolism. Despite having an effect on an SR-A-mediated lipoprotein uptake, there was no change in the relative abundance of SR-A protein after PTX treatment.These results demonstrate that activation of a PTX-sensitive G protein is involved in a feedback process that positively regulates SR-A function.     

Quantitative determination of pertussis toxin-sensitive G proteins using [32P]ADP-ribosylation in human platelet membranes: negative correlation with ages.

The optimum condition to quantitate the [32P]ADP-ribosylation catalyzed by pertussis toxin (islet-activating protein, IAP) in human platelet membranes was investigated. Autoradiography indicated the incorporation of 32P into the band corresponding to the molecular weight of 40-41 kDa, which was augmented by the addition of GTP in the presence of 10 mM MgCl2. On the other hand, non-hydrolyzable GTP analogue, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) enhanced the IAP-catalyzed [32P]ADP-ribosylation only in the absence of MgCl2. The amounts of IAP-catalyzed [32P]ADP-ribosylation in the presence of 100 microM GTP and 10 mM MgCl2 were linear in proportion to the protein concentrations within the limited range of protein concentrations, indicating that this simple quantitative method could be adequately used to evaluate the IAP-sensitive G proteins. Data from fifteen healthy volunteers (7 males and 8 females ranging 24 to 60 years old) indicate that the amounts of IAP-sensitive G proteins in platelet membranes are significantly negatively correlated with ages.     

WNT-5A stimulates the GDP/GTP exchange at pertussis toxin-sensitive heterotrimeric G proteins

The lipoglycoproteins of the WNT family act on seven transmembrane-spanning Class Frizzled receptors. Here, we show that WNT-5A evokes a proliferative response in a mouse microglia-like cell line (N13), which is sensitive to pertussis toxin, thus implicating the involvement of heterotrimeric G proteins of the G_i/o family. We continue to show that WNT-5A stimulation of N13 membranes and permeabilized cells evokes the exchange of GDP for GTP of pertussis toxin-sensitive G proteins employing [γ-³⁵S]GTP assay and activity state-specific antibodies to GTP-bound G_i proteins. Our functional analysis of the PTX-sensitivity of WNT-induced G protein activation and PCR analysis of G protein and FZD expression patterns suggest that WNT-5A stimulation leads to the activation of G_i2/3 proteins in N13 cells possibly mediated by FZD₅, the predominant FZD expressed. In summary, we provide for the first time molecular proof that WNT-5A stimulation results in the activation of heterotrimeric G_i2/3 proteins in mammalian cells with physiological protein stochiometry.     

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.     

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.     

Phorbol ester-induced changes in cytoplasmic Ca2+ in human neutrophils. Involvement of a pertussis toxin-sensitive G protein

Activation of neutrophils by most soluble stimuli is associated with a marked increase in intracellular free Ca2+ ([Ca2+]i). However, under physiological conditions (Na+-rich media), the potent activator 12-O-tetradecanoylphorbol-13-acetate (TPA) causes no change or a decrease in [Ca2+]i. We report here that the [Ca2+]i response to phorbol esters varies depending on the ionic composition of the medium. A marked increase in [Ca2+]i was detected in Na+-free solutions. Maximal effects were observed when N-methyl-D-glucammonium+ or choline+ were substituted for Na+, whereas an intermediate response was recorded in K+ medium. The increase in [Ca2+]i was substantially (approximately 65%) inhibited by removal of external Ca2+. A [Ca2+]i increase was also elicited by other beta-phorbol diesters and by diacylglycerol, but not by unesterified phorbol or by alpha-phorbol diesters, indicating involvement of protein kinase C. The increase in [Ca2+]i observed in Na+-free media is not due to inhibition of Na+/Ca2+ exchange, since no change in [Ca2+]i in response to TPA was observed in: 1) cells suspended in Li+, which is not countertransported for Ca2+; 2) cells preloaded with Na+ to eliminate the driving force for Na+/Ca2+ exchange; and 3) cells treated with 3',4'-dichlorobenzamyl, an inhibitor of Na+/Ca2+ exchange. Similarly, the [Ca2+]i increase in Na+-free media is not linked to the absence of Na+/H+ exchange and the associated cytoplasmic acidification since: 1) it was not observed in Na+ media in the presence of inhibitors of the Na+/H+ antiport and 2) it was not mimicked by inducing acidification with nigericin. Pretreatment with pertussis toxin largely inhibited the phorbol ester-induced change in [Ca2+]i, while activation of protein kinase C under these conditions was unaffected. It is concluded that in the absence of extracellular Na+ (or Li+), activation of protein kinase C leads to a net Ca2+ influx into the cytoplasm through a process mediated by a GTP-binding or G protein. Opening of a Na+-sensitive Ca2+ channel could partially explain these observations. Alternatively, the nature of the monovalent cation could conceivably affect the conformation of a G protein or of an associated receptor, inducing the appearance of a site susceptible to an activating phosphorylation by protein kinase C.     

Enhanced IPC by activation of pertussis toxin-sensitive and -insensitive G protein-coupled purinoceptors

Extracellular ATP plays an important role in ischemic preconditioning (IPC) through the activation of P(2y) purinoceptors. This study examined whether ATP-stimulated P(2y) purinoceptors are coupled to pertussis toxin (PTX)-insensitive G protein and whether activation of this pathway enhances myocardial protection afforded by IPC. The rat was treated with PTX for 48 h, and the heart was then isolated and buffer perfused. The heart underwent IPC by three cycles of 5-min ischemia and 5-min reperfusion before 25 min of global ischemia. Isovolumic left ventricular function was measured, and functional recovery at 30 min after reperfusion was taken as an end point of myocardial protection. PTX pretreatment partially inhibited functional protection by IPC. Treatment with 100 microM 8-(p-sulfophenyl) theophylline (SPT) during IPC had no further effect on PTX-induced inhibition of functional protection by IPC, whereas suramin (300 microM) or reactive blue (RB) (10 microM) completely abolished myocardial protection in the preconditioned heart pretreated with PTX. Supplementation with adenosine (30 microM), ATP (30 microM), or UTP (50 microM) significantly enhanced IPC-induced functional protection, although preconditioning with these nucleotides without IPC had no protective effect. Adenosine-enhanced IPC was inhibited by pretreatment with PTX and SPT but not by suramin or RB, whereas ATP-enhanced IPC was inhibited by suramin or RB in combination with PTX pretreatment. On the other hand, UTP-enhanced IPC was not affected by PTX pretreatment but was inhibited by suramin or RB. Adenosine supplemented IPC without PTX pretreatment and ATP supplemented IPC with PTX pretreatment were not affected by nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (100 microM). Although the protein kinase C inhibitor Ro318425 (0.3 microM) or tyrosine kinase inhibitor genistein (50 microM) had no significant effect on the functional protection afforded by adenosine-supplemented IPC without PTX pretreatment and ATP-supplemented IPC with PTX pretreatment, the combination of Ro318425 and genistein attenuated functional protection afforded by both the purinoceptor agonist-supplemented IPC. These results suggest the crucial involvement of PTX-sensitive and -insensitive G protein coupled purinoceptors in enhanced IPC by supplementation with adenosine, ATP, and UTP.     

Neuropeptide Y inhibits cardiac adenylate cyclase through a pertussis toxin-sensitive G protein

Neuropeptide Y, a major neuropeptide and potent vasoconstrictor, inhibited isoproterenol-stimulated adenylate cyclase activity in cultured rat atrial cells as well as in atrial membranes. Prior treatment of the cells with pertussis toxin blocked the inhibitory action of neuropeptide Y. Pertussis toxin is known to uncouple the receptors for other inhibitors of adenylate cyclase by ADP-ribosylation of the alpha-subunit of Gi, the inhibitory guanine nucleotide binding component of adenylate cyclase. The toxin specifically catalyzed the ADP-ribosylation of a 41-kilodalton atrial membrane protein which corresponded to the Gi subunit. These results suggest that neuropeptide Y may mediate some of its physiological effects through specific receptors linked to the inhibitory pathway of adenylate cyclase.     

Purified epithelial Na+ channel complex contains the pertussis toxin-sensitive G alpha i-3 protein.

We have recently demonstrated that the amiloride-sensitive Na+ channel in the apical membrane of the renal epithelial cell line, A6, is modulated by the alpha i-3 subunit of the Gi-3 protein. We also showed that a 700-kDa protein complex can be purified from the membranes of A6 epithelia which (a) can reconstitute the amiloride-sensitive Na+ influx in liposomes and planar bilayer membranes and (b) consists of six major protein bands observed on reducing sodium dodecyl sulfate-polyacrylamide gels with molecular masses ranging from 35 to 320 kDa. The present study was undertaken to determine if the alpha i-3 subunit was a member of this Na+ channel complex. G alpha i structure and function were identified by Western blotting with specific G alpha i subunit antibodies and Na+ channel antibodies, through ADP-ribosylation with pertussis toxin, and by immunocytochemical localization of the Na+ channel and G alpha i proteins. We demonstrate that two protein substrates are ADP-ribosylated in the 700-kDa complex in the presence of pertussis toxin and are specifically immunoprecipitated with an anti-Na+ channel polyclonal antibody. One of these substrates, a 41-kDa protein, was identified as the alpha i-3 subunit of the Gi-3 protein on Western blots with specific antibodies. Na+ channel antibodies do not recognize G alpha i-3 on Western blots of Golgi membranes which contain alpha i-3 but not Na+ channel proteins, nor do they immunoprecipitate alpha i-3 from solubilized Golgi membranes; however, alpha i-3 is coprecipitated as part of the Na+ channel complex from A6 cell membranes by polyclonal Na+ channel antibodies. Both alpha i-3 and the Na+ channel have been localized in A6 cells by confocal imaging and immunofluorescence with specific antibodies and are found to be in distinct but adjacent domains of the apical cell surface. In functional studies, alpha i-3, but not alpha i-2, stimulates Na+ channel activity. These data are therefore consistent with the localization of Na+ channel activity and modulatory alpha i-3 protein at the apical plasma membrane, which together represent a specific signal transduction pathway for ion channel regulation.     

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.     

Involvement of pertussis toxin-sensitive and -insensitive GTP-binding proteins in luteinizing hormone exocytosis distal to second messenger generation.

Inhibition of luteinizing hormone (LH) exocytosis by guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) in permeabilized pituitary cells has indicated the involvement of one or more GTP-binding proteins in the exocytotic mechanism distal to second messenger generation. We now report that two inhibitory sites of action of GTP gamma S can be distinguished by their dependence on GTP gamma S concentration and their sensitivity to pertussis toxin. Ca(2+)-stimulated exocytosis was half-maximally inhibited by 6.8 microM GTP gamma S, a six-fold higher concentration than that required for inhibition of exocytosis stimulated by phorbol ester plus cAMP. In addition, GTP gamma S inhibition of Ca(2+)-stimulated exocytosis was insensitive to pertussis toxin, in contrast to the inhibition of exocytosis stimulated by phorbol ester plus cAMP, which was abolished by pretreatment with pertussis toxin. These results indicate that at least two stimulus-specific GTP-binding proteins are involved in regulating LH exocytosis distal to second messenger generation.     

Angiotensin II negatively modulates L-type calcium channels through a pertussis toxin-sensitive G protein in adrenal glomerulosa cells.

In bovine adrenal glomerulosa cells, angiotensin II and extracellular K+ stimulate aldosterone secretion in a calcium-dependent manner. In these cells, physiological concentrations of extracellular potassium activate both T-type (low threshold) and L-type (high threshold) voltage-operated calcium channels. Paradoxically, the cytosolic calcium response to 9 mM K+ is inhibited by angiotensin II. Because K+-induced calcium changes observed in the cytosol are almost exclusively due to L-type channel activity, we therefore studied the mechanisms of L-type channel regulation by angiotensin II. Using the patch-clamp method in its perforated patch configuration, we observed a marked inhibition (by 63%) of L-type barium currents in response to angiotensin II. This effect of the hormone was completely prevented by losartan, a specific antagonist of the AT1 receptor subtype. Moreover, this inhibition was strongly reduced when the cells were previously treated for 1 night with pertussis toxin. An effect of pertussis toxin was also observed on the modulation by angiotensin II of the K+ (9 mM)-induced cytosolic calcium response in fura-2-loaded cells, as well as on the angiotensin II-induced aldosterone secretion, at both low (3 mM) and high (9 mM) K+ concentrations. Finally, the expression of both Go and Gi proteins in bovine glomerulosa cells was detected by immunoblotting. Altogether, these results strongly suggest that in bovine glomerulosa cells, a pertussis toxin-sensitive G protein is involved in the inhibition of L-type channel activity induced by angiotensin II.     

Participation of pertussis toxin-sensitive GTP-binding regulatory proteins in the suppression of baroreceptor reflex by neurotensin in the rat.

We evaluated the molecular mechanism that may underlie the suppressive effect of neurotensin (NT) on the baroreceptor reflex (BRR), using Sprague-Dawley rats that were anesthetized with sodium pentobarbital (50 mg/kg, i.p.). Intracerebroventricular (i.c.v.) application of NT (15 nmol) significantly inhibited the BRR response. Such an inhibition was appreciably antagonized by pretreating animals with i.c.v. injection of pertussis toxin (10 or 20 pmol), N-ethylmaleimide (1 or 2 nmol), forskolin (30 or 60 nmol) or phorbol 12-myristate 13-acetate (2 or 4 nmol), but not by cholera toxin (15 or 30 pmol). More specifically, pretreatments with bilateral microinjection into the nucleus tractus solitarius (NTS) of pertussis toxin (80 or 160 fmol), N-ethylmaleimide (80 pmol), forskolin (480 pmol) or phorbol 12-myristate 13-acetate (16 or 32 pmol) also blunted the NT-induced suppression of BRR, although cholera toxin (120 or 240 fmol), or 1,9-dideoxyforskolin (480 pmol) had no appreciable effect. These results suggest that a pertussis toxin-sensitive guanine nucleotide-binding regulatory protein(s), which is not likely to be Gs, possibly Gi or Gp, may be involved in the transmembrane signaling process that underlies the suppression of BRR response by NT at the NTS.     

Involvement of a pertussis toxin-sensitive G protein in the action of gastrin on gastric parietal cells

The mechanism whereby gastrin triggers phosphoinositide breakdown was investigated in an enriched preparation of isolated rabbit parietal cells (approx. 75%). In a permeabilized preparation of myo-[3H]inositol-labelled cells, GTP[S], a non-hydrolysable GTP analogue, enhanced [3H]inositol trisphosphate ([3H]InsP3 accumulation in a dose-dependent manner; submaximal concentrations of GTP[S] (less than 10 microM), potentiated gastrin-induced [3H]InsP3 release; preincubation for 5 min with GDP[S], a non-hydrolysable GDP analogue, dose-dependently reduced [3H]InsP3 accumulation stimulated by gastrin even in presence of GTP[S]. Exposure of intact parietal cells for 3 h to pertussis toxin (PTx) (200 ng/ml) led to a 15-50% reduction in gastrin-induced [14C]aminopyrine [(14C]AP) uptake (an index of in vitro acid secretion) and [3H]inositol phosphate ([3H]InsP) accumulation. A decrease in the accumulation of the different [3H]inositol phosphate occurred in gastrin-stimulated parietal cells treated with PTx. A rightward shift of gastrin dose-response curves in the presence of PTx was observed for [14C]AP uptake (EC50 values: 0.125 +/- 0.045 nM without PTx and 1.05 +/- 0.63 nM with PTx), for [3H]InsP accumulation (EC50 values: 0.16 +/- 0.08 nM without PTx and 1.56 +/- 0.58 nM with PTx) and [125I]gastrin binding (IC50 values: 0.247 +/- 0.03 nM without PTx and 2.38 +/- 0.56 nM with PTx). In contrast, cholera toxin (CTx) treatment (100 ng/ml) for 3 h was without effect on gastrin-induced [3H]InsP accumulation. CTx induced a pronounced potentiation of gastrin-stimulated [14C]AP uptake; this effect can be mimicked by IBMX (a phosphodiesterase inhibitor) and by forskolin (an activator of adenylyl cyclase). We conclude that: (i) one or more than one G protein appeared to be involved in gastrin receptor coupling to phospholipase C (PL-C); (ii) these G proteins are not substrates for CTx; (iii) one of these appeared to be a PTx-sensitive 'Gi-like' protein which could be involved in hormone-induced acid secretion, (iiii) the potentiating effect of CTx observed on AP uptake stimulated by gastrin suggests the existence of a cooperative effect between cAMP pathway (CTx) and the gastrin-induced phosphoinositide breakdown in acid secretory activity of parietal cells.     

Collagen-binding motif peptide, a cleavage product of osteopontin, stimulates human neutrophil chemotaxis via pertussis toxin-sensitive G protein-mediated signaling

The collagen-binding motif (CBM) peptide, a cleavage product of osteopontin (OPN), stimulated intracellular calcium increase in human neutrophils. CBM peptide-stimulated calcium was inhibited by pertussis toxin (PTX), suggesting the influence of PTX-sensitive G-proteins. In addition CBM peptide stimulated the chemotactic migration of human neutrophils and human monocytes. CBM peptide-induced neutrophil chemotaxis was completely inhibited by PTX, once again indicating the influence of Gi proteins. CBM peptide was also found to induce mitogen activated protein kinase activation. CBM peptide-induced neutrophil chemotaxis was mediated by p38 kinase as well as an extracellular signal-regulated protein kinase. Taken together, the results suggest that a cleavage product of OPN, CBM peptide, initiates immune responses by inducing neutrophil trafficking via certain PTX-sensitive cell surface receptors.     

Selective regulation of eosinophil degranulation by interleukin 1 beta.

Recent evidence confirms that cytokines such as IL-1, IL-4, IL-5, and GM-CSF may enhance or inhibit eosinophil function. Functions that are susceptible to modulation include eosinophil-mediated antibody-dependent damage of helminthic parasites, oxidative metabolism and degranulation. We have employed IgG and IgE-coated Sepharose beads to investigate selective modulation of IgG and IgE-mediated enzyme release by IL-1 beta. Both IgG and IgE-coated beads induced release of granular enzymes beta-glucuronidase and arylsulfatase. Enzyme release from IgG-stimulated eosinophils was inhibited by preincubation with IL-1 beta (100 pg/ml, P less than or equal to 0.05). In contrast, enzyme release by IgE-stimulated eosinophils was enhanced by IL-1 beta (100 pg/ml, P less than or equal to 0.05). These studies support the hypothesis that IL-1 beta has specific selective actions on eosinophil function. Furthermore, these actions on particle-stimulated enzyme release suggest that IgG and IgE mediated processes in eosinophils are differentially regulated.