Pasteurella multocida toxin activates Gβγ dimers of heterotrimeric G proteins

The mitogenic Pasteurella multocida toxin (PMT) is a major virulence factor of P. multocida, which causes Pasteurellosis in man and animals. The toxin activates the small GTPase RhoA, the MAP kinase ERK and STAT proteins via the stimulation of members of two G protein families, G_q and G_12/13. PMT action also results in an increase in inositol phosphates, which is due to the stimulation of PLCβ via Gα_q. Recent studies indicate that PMT additionally activates Gα_i to inhibit adenylyl cyclase. Here we show that PMT acts not only via Gα but also through Gβγ signaling. Activation of Gβγ by PMT causes stimulation of phosphoinositide 3-kinase (PI3K) γ and formation of phosphatidylinositol-3,4,5-trisphosphate (PIP₃) as indicated by the recruitment of a PIP₃-binding pleckstrin homology (PH) domain-containing protein to the plasma membrane. Moreover, it is demonstrated that Gβγ is necessary for PMT-induced signaling via Gα. Mutants of Gα_q incapable of binding or releasing Gβγ are not activated by PMT. Similarly, sequestration of Gβγ inhibits PMT-induced Gα-signaling.     

Modification of Heterotrimeric G-Proteins in Swiss 3T3 Cells Stimulated with Pasteurella multocida Toxin

Many bacterial toxins covalently modify components of eukaryotic signalling pathways in a highly specific manner, and can be used as powerful tools to decipher the function of their molecular target(s). The Pasteurella multocida toxin (PMT) mediates its cellular effects through the activation of members of three of the four heterotrimeric G-protein families, G_q, G₁₂ and G_i. PMT has been shown by others to lead to the deamidation of recombinant Gα_i at Gln-205 to inhibit its intrinsic GTPase activity. We have investigated modification of native Gα subunits mediated by PMT in Swiss 3T3 cells using 2-D gel electrophoresis and antibody detection. An acidic change in the isoelectric point was observed for the Gα subunit of the G_q and G_i families following PMT treatment of Swiss 3T3 cells, which is consistent with the deamidation of these Gα subunits. Surprisingly, PMT also induced a similar modification of Gα₁₁, a member of the G_q family of G-proteins that is not activated by PMT. Furthermore, an alkaline change in the isoelectric point of Gα₁₃ was observed following PMT treatment of cells, suggesting differential modification of this Gα subunit by PMT. G_s was not affected by PMT treatment. Prolonged treatment with PMT led to a reduction in membrane-associated Gα_i, but not Gα_q. We also show that PMT inhibits the GTPase activity of G_q.     

Phospholipase C activation in rat pituitary adenoma (GH) cells

The presence of the pertussis toxin (PTX) insensitive GTP-binding proteins (G-proteins) G_q and/or G₁₁ has been demonstrated in three different prolactin (PRL) and growth hormone (GH) producing pituitary adenoma cell lines. Immunoblocking of their coupling to hormone receptors indicates that G_q and/or G₁₁ confer throliberin (TRH) responsive phospholipase C (PL-C) activity in these cells. The contention was substantiated by immunoprecipitation analyses snowing that anti G_q/11-sera coprecipitated PL-C activity. In essence, only G_q/11 (but neither G_i2, G_i3 nor G_o) seems to mediate the TRH-sensitive PL-C activity, while G_o may be coupled to a basal or constitutive PL-C activity. Immunoblocking studies imply that the B-complex also, to some extent, may stimulate GH₃ pituitary cell line PL-C activity. Finally, the steady state levels of G_q/11 mRNA and protein were downregulated upon long term exposure of the GH3 cells to TRH (but not to vasoactive intestinal peptide = VIP).     

Characterization of the Membrane-targeting C1 Domain in Pasteurella multocida Toxin

Pasteurella multocida toxin (PMT) is a virulence factor responsible for the pathogenesis of some forms of pasteurellosis. The toxin activates G_q- and G_12/13-dependent pathways through the deamidation of a glutamine residue in the α-subunit of heterotrimeric GTPases. We recently reported the crystal structure of the C terminus (residues 575–1285) of PMT (C-PMT), which is composed of three domains (C1, C2, and C3), and that the C1 domain is involved in the localization of C-PMT to the plasma membrane, and the C3 domain possesses a cysteine protease-like catalytic triad. In this study, we analyzed the membrane-targeting function of the C1 domain in detail. The C1 domain consists of seven helices of which the first four (residues 590–670), showing structural similarity to the N terminus of Clostridium difficile toxin B, were found to be involved in the recruitment of C-PMT to the plasma membrane. C-PMT lacking these helices (C-PMT ΔC1(4H)) neither localized to the plasma membrane nor stimulated the G_q/12/13-dependent signaling pathways. When the membrane-targeting property was complemented by a peptide tag with an N-myristoylation motif, C-PMT ΔC1(4H) recovered the PMT activity. Direct binding between the C1 domain and liposomes containing phospholipids was evidenced by surface plasmon resonance analyses. These results indicate that the C1 domain of C-PMT functions as a targeting signal for the plasma membrane.     

Phospholipid metabolism is required for M<Subscript>1</Subscript> muscarinic inhibition of N-type calcium current in sympathetic neurons

The signal transduction cascade mediating muscarinic receptor modulation of N-type Ca²⁺ channel activity by the slow pathway has remained incompletely characterized despite focused investigation. Recently we confirmed a role for the G-protein G_q and identified phospholipase C (PLC), phospholipase A₂ (PLA₂), and arachidonic acid (AA) as additional molecules involved in N-current inhibition in superior cervical ganglion (SCG) neurons by the slow pathway. We have further characterized this signal transduction cascade by testing whether additional molecules downstream of phosphatidylinositol-4,5-bisphosphate (PIP₂) are required. The L-channel antagonist nimodipine was bath-applied to block L-current. Pretreating cells with pertussis toxin (PTX) minimized M₂/M₄ muscarinic receptor inhibition of N-current by the membrane-delimited pathway. Consistent with our previous studies, pharmacologically antagonizing M₁ muscarinic receptors (M₁Rs), G_q, PLC, PLA₂, and AA minimized N-current inhibition by the muscarinic agonist oxotremorine-M (Oxo-M). When cells were left untreated with PTX, leaving the membrane-delimited pathway intact and the same antagonists retested, Oxo-M decreased whole cell currents. Moreover, inhibited currents displayed slowed activation kinetics, indicating intact N-current inhibition by the membrane-delimited pathway. These findings indicate that the antagonists used to block the slow pathway acted selectively. PLA₂ cleaves AA from phospholipids, generating additional metabolites. We tested whether the metabolite lysophosphatidic acid (LPA) mimicked the inhibitory actions of Oxo-M. In contrast to AA, applying LPA did not inhibit whole cell currents. Taken together, these findings suggest that the slow pathway requires M₁Rs, G_q, PLC, PIP₂, PLA₂, and AA for N-current inhibition.Abbreviations AA arachidonic acid - BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid - BSA bovine serum albumin - DAG diacylglycerol - DEDA 7,7-dimethyleicosadienoic acid - ETYA 5,8,11,14-eicosatetraynoic acid - FPL FPL 64176 - IP₃ inositol-1,4,5-trisphosphate - L-channel L-type calcium channel - L-current L-type calcium current - LPA lysophosphatidic acid - M₁R M₁ muscarinic receptor - N-channel N-type calcium channel - N-current N-type calcium current - NMN nimodipine - OAG 1-(cis-9-octadecenoyl)-2-acetyl-sn-glycerol - OPC oleoyloxyethyl phosphorylcholine - Oxo-M oxotremorine methiodide - PIP₂ phosphatidylinositol-4,5-bisphosphate - PLC phospholipase C - PLA₂ phospholipase A₂ - PTX pertussis toxin - SCG superior cervical ganglion     

Specificity of heterotrimeric G protein regulation by human chorionic gonadotropin and low-molecular agonist of luteinizing hormone receptor

Luteinizing hormone (LH) and its homologue, human chorionic gonadotropin (hCG), are very important regulators of the reproductive system. These hormones stimulate various types of G proteins—primarily, G_s and G_q proteins—by binding to the specific LH-hCG receptor, which leads to the activation of adenylate cyclase (AC) and phospholipase C, respectively. It has been suggested that many side effects of LH and hCG are associated with low selectivity of their effect on G proteins. Low-molecular agonists of LH-hCG receptor developed on the basis of thienopyrimidine derivatives do not cause these side effects, and differences in the interaction with G proteins may be ones of the cause for this. To test this, a comparative study of the effect of hCG and synthesized by us thienopyrimidine derivative, 5-amino-N-tert-butyl-2-(methylsulfanyl)-4-(3-(nicotinamido)phenyl)thieno[2,3-d]pyrimidine-6-carboxamide (TP03) on the AC activity and GTP binding of G proteins in plasma membranes isolated from the rat ovaries and testes was performed. Cholera toxin (CT) and pertussis toxin (PT) were used to selectively switch off the signal transduction via G_s and G_i/o proteins, the peptide corresponding to the C-terminal segment 349–359 of the Gα_q subunit was used to suppress G_q-dependent cascades. It was shown that treatment of ovarian and testicular membranes with CT resulted in suppression of TP03 and hCG stimulatory effects on the AC activity, but in different ways influenced the GTP binding stimulation: it completely blocked the effect of 10^–6 M TP03 and reduced by 45–46% the effect of hCG (10^–8 M). Preincubation of membranes with the peptide 349–359 reduced the hCG stimulatory effect on GTP binding by 34 (ovaries) and 45% (testes), but did not affect the corresponding effect of 10^–6 M TP03. Preincubation with the peptide 349–359 also reduced the GTP stimulatory effect of 10^–4 M TP03, but to a small extent. The obtained data indicate that, in contrast to hCG, the targets of which in the ovaries and testes are G_s and G_q proteins, the action of TP03 is realized mainly via G_s proteins. Only at a concentration that exceeds EC₅₀ by two orders TP03 is capable to relatively weakly activate G_q proteins. The PT treatment of the membranes did not affect the effects of TP03 and hCG, which indicates the lack of their effective interaction with G_i/o proteins. Thus, the selectivity of activation of G_s-dependent cascades responsible for the synthesis and production of steroid hormones is a significant advantage of low-molecular agonists of LH-hCG receptor over gonadotropins.     

Modulating Action of Hyperforin on the P-Type Calcium Channels in the Membranes of Rat Cerebellar Purkinje Neurons

A modulating action of hyperforin (an active compound of the extract from Hypericum perforatum) on a high-threshold component of the calcium current, sensitive to application of 100 nM -Aga-IVA toxin and identified as P current, was studied on freshly isolated Purkinje neurons with the use of a patch-clamp technique in the whole-cell configuration. It was shown that extracellular application of 0.8 M hyperforin caused a shift of the current-voltage (I-V) relationship of P current by -(8 ± 2) mV, slowdown of the activation kinetics, and a decrease in the amplitude of this current. The shift of the I-V relationship and slowdown of activation kinetics developed for less than 10 sec, while the P-current amplitude decreased for a much longer time (several minutes) and depended on the intracellular concentration of Ca²⁺ ions. -Aga-IVA toxin at the concentration of 100 nM completely blocked the recorded inward current in the presence of 0.8 M hyperforin. In experiments with intracellular perfusion of Purkinje neurons, we found that interaction of hyperforin with its binding site occurs at the external side of the cell membrane. The study of the mechanisms involved in the hyperforin-induced P-current modulation revealed that 1 mM GTPS (activating G_Ts proteins, as well as activating or blocking G_Ms proteins) or 1-2 mM GDPS (blocking G_Ts and G_Ms proteins) in the intracellular solution did not affect the hyperforin-induced modulation of P current. Hyperforin-induced Ca²⁺-independent shift of the I-V relationship and slowdown of the activation kinetics of P current were abolished in the presence of 0.5 M calmidazolium in the extracellular medium.     

A Gq-type G protein couples muscarinic receptors to inositol phosphate and calcium signaling in exocrine cells from the avian salt gland

Summary Muscarinic acetylcholine receptor (mAChR) activation in isolated cells from the nasal salt gland of the domestic duck (Anas platyrhynchos) results in a rapid increase in the rate of phosphatidylinositol hydrolysis and pronounced intracellular calcium signals. Both responses can be elicited by treating these cells with fluoroaluminate (AlF ₄ ^– ) indicating the involvement of a heterotrimeric G protein in the transmembrane signaling process. To characterize this G protein, electrophoretically separated membrane proteins were blotted onto nitrocellulose filters and probed with peptide-antibodies raised against portions of different -subunits of mammalian G proteins. We could demonstrate the presence of at least four different G proteins in salt gland cell membranes. Two of these proteins (40 and 41 kD) were ADP-ribosylated by pertussis toxin and were recognized by an antiserum against a common sequence in all G protein -subunits. One protein (46 kD) was a cholera toxin-substrate and was recognized by a G_s-specific antiserum; the other (42 kD) was recognized by G_q-specific antisera and was resistant to ADP-ribosylation. Since the initial inositol phosphate production upon receptor activation with carbachol and the resulting calcium signals were not affected by pertussis toxin-pretreatment of salt gland cells, we conclude that muscarinic receptors are coupled to phospholipase C by a G_q-type G protein.This study was supported by National Institutes of Health Grant GM-40457 (to T.J.S.) and, in part, by a fellowship (Hi 448/1) from the Deutsche Forschungsgemeinschaft (to J.-P.H.). We are grateful to Drs. D.R. Manning and P.C. Sternweis for their kind gifts of antisera.     

Characterization of the G protein coupling of SRIF and β-adrenergic receptors to the maxi KCa channel in insulin-secreting cells

Modulation of the Ca- and voltage-dependent K channel—K_Ca—by receptors coupled to the G proteins G _i /G _o and G _s has been studied in insulin-secreting cells using the patch clamp technique. In excised outside-out patches somatostatin (somatotropin-releasing inhibitory factor; SRIF) caused concentration-dependent inhibition of the K_Ca channel, an effect that was prevented by pertussis toxin (PTX). In inside-out patches, exogenous subunits of either G _i or G _o -type G proteins also inhibited the K_Ca channel (IC₅₀ 5.9 and 5.7 pM, respectively). These data indicate that SRIF suppresses K_Ca channel activity via a membrane-delimited pathway that involves the subunits of PTX-sensitive G proteins G _i and/or G _o . In outside-out patches, activation of G _s either by -agonists or with cholera toxin (CTX) increased K_Ca channel activity, consistent with a membrane-delimited stimulatory pathway linking the -adrenergic receptor to the K_Ca channel via G _s . In outside-out patches, channel inhibition by SRIF suppressed the stimulatory effect of -agonists but not that of CTX, while in inside-out patches CTX reversed channel inhibition induced by exogenous _i or _o . Taken together these data suggest that K_Ca channel activity is enhanced by activation of G _s and blocked by activated G _i and/or G _o . Further, K_Ca channel stimulation by activated G _s may be direct, while inhibition by G _i /G _o may involve deactivation of G _s . In inside-out patches K_Ca channel activity was reduced by an activator of protein kinase C (PKC) and enhanced by inhibitors of PKC, indicating that PKC also acts to inhibit the K_Ca channel via a membrane delimited pathway. In outside-out patches, chelerythrine, a membrane permeant inhibitor of PKC prevented the inhibitory effect of SRIF, and in inside-out patches PKC inhibitors prevented the inhibitory effect of exogenous _i or _o . These data indicate that PKC facilitates the inhibitory effect of the PTX-sensitive G proteins which are activated by coupling to SRIF receptors. To account for these results a mechanism is proposed whereby PKC may be involved in G _i /G _o -induced deactivation of G _s .The authors would like to thank Dr. S. Ciani for many helpful discussions, Dr. A.E. Boyd III for supplying the HIT cells, Drs. J. Codina and L. Birnbaumer for supplying the alpha subunits of the G proteins G _i and G _o , and Mrs. Satoko Hagiwara for preparing and maintaining the cell cultures.This work was supported by grant DCB-8919368 from the National Science Foundation and a research grant (W-P 880513) from the American Diabetes Association to B.R., and by grant RO1-DK39652 from the National Institutes of Health to G.T.E.     

Regional Distribution and Quantitative Measurement of the Phosphoinositidase C-Linked Guanine Nucleotide Binding Proteins G1α and Gqα in Rat Brain

Abstract: Levels of the guanine nucleotide binding proteins G₁₁α and G_qα, which produce receptor regulation of phosphoinositidase C., were measured immunologically in 13 regions of rat central nervous system. This was achieved by immunoblotting membranes from these regions with antisera (CQ series) that identify these two polypeptides equally, following separation of the membranes using sodium dodecyl sulphate-polyacrylamide gel electrophoresis conditions that can resolve G_qα and G₁₁α. In all regions examined, G_qα was more highly expressed than G₁₁α. Ratios of levels of G_qα to G₁₁α varied between the regions from 5:1 to 2:1. Quantitative measurements of the levels of G_qα and G₁₁α in each region were obtained by comparison with known amounts of purified liver G_qα and G₁₁α and with E. coli expressed recombinant G_qα. Areas that expressed G_qα highly included olfactory bulb (930 ng/ mg of membrane protein), frontal cortex (700 ng/mg of membrane protein), parietal occipital cortex (670 ng/mg of membrane protein), caudate putamen (1,003 ng/mg of membrane protein), hippocampus (1,045 ng/mg of membrane protein), hypothalamus (790 ng/mg of membrane protein), and cerebellum (950 ng/mg of membrane protein). More modest levels were observed in thalamus (450 ng/mg of membrane protein), pituitary (480 ng/mg of membrane protein), optic chiasma (330 ng/mg of membrane protein), and spinal cord (350 ng/mg of membrane protein). Gna was more evenly expressed with values ranging from about 170 ng/mg of membrane protein in spinal cord and optic chiasma to close to 300 ng/mg of membrane protein in regions expressing high levels of G_qα. A third polypeptide could be identified by the CQ antisera in all brain regions. The possibility that this polypeptide is the α subunit of G₁₄ is discussed.     

Identification of G-proteins in rat parotid gland plasma membranes and granule membranes: presence of distinct components in granule membranes

We have identified by immunoblotting and ADP-ribosylation by cholera toxin and pertussis toxin the presence of Mr 43 and 46 KDa G_s, and 39 and 41 KDa G_i;.. subunits in rat parotid gland plasma membranes but not in granule membranes. A Mr 28 KDa polypeptide that served as substrate for ADP-ribosylation by both cholera toxin and pertussis toxin was present exclusively in granule membranes. Photoaffinity crosslinking of [-³²P]GTP showed the presence of high molecular weight GTP-binding proteins (Mr 160,100 KDa) in granule membranes. Six low molecular weight GTP-binding proteins (Mr 21–28 KDa) were differentially distributed in both plasma membranes and granule membranes. The present study identifies various GTP-binding proteins in rat parotid gland plasma membranes and granule membranes, and demonstrates the presence of distinct molecular weight GTP-binding proteins in granule membranes. These granule-associated GTP-binding proteins may be involved in secretory processes.     

Cholera Toxin Induces Cyclic AMP-Independent Down-Regulation of Gsα and Sensitization of α2-Autoreceptors in Chick Sympathetic Neurons

Abstract: The role of the stimulatory GTP-binding protein (G_S) in the α₂-autoinhibitory modulation of noradrenaline release was investigated in cultured chick sympathetic neurons. The α₂-adrenoceptor agonist UK 14,304 caused a concentration-dependent reduction of electrically evoked [³H]noradrenaline release with half-maximal effects at 14.0 ± 5.5 nM. In neurons treated with 100 ng/ml cholera toxin for 24 h, the half-maximal concentration was lowered to 3.2 ± 1.4 nM without changes in the maximal effect of UK 14,304. The pretreatment with cholera toxin also increased the inhibitory action of 10 nM UK 14,304 when compared with the inhibition of noradrenaline release in untreated cultures derived from the same cell population. In cultures treated with either 10 µM forskolin or 100 µM 8-bromo-cyclic AMP, neither the half-maximal concentration nor the maximal effect of UK 14,304 was altered. Cholera toxin, forskolin, and 8-bromo-cyclic AMP all induced an increase in spontaneous outflow and a reduction in electrically evoked overflow, effects not observed after a pretreatment with dideoxyforskolin. Exposure of neurons to cholera toxin, but not to forskolin or 8-bromo-cyclic AMP, induced a translocation of α-subunits of G_s (G_sα) from particulate to soluble fractions and led ultimately to a complete loss of G_sα from the neurons. In contrast, no effect was seen on the distribution of either α-subunits of G_i- or G_o-type G proteins or of β-subunits. These results indicate that cholera toxin causes a selective, cyclic AMP-independent down-regulation of G_sα. This down-regulation of G_sα is associated with the sensitization of α₂-autoreceptors.     

Pyrolytic Yields of 2-Amino-9H-pyrido[2,3-b]indole and 3-Amino-1-methyl-5H-pyrido[4,3-b]indole as Matagens from Proteins

An extracellular exo-maltohexaohydrolase [EC 3.2.1.98] from a Klebsiella pneumoniae (Aerobacter aerogenes) mutant produced about 40% maltohexaose (G₆) from short-chain amylose ( =23). Mostly G₆ was produced from maltooligosaccharides larger than G₆ by an exo-mechanism action. It also hydrolyzed G₆ and shorter maltooligosaccharides to give smaller maltooligosaccharides. Its position specificity of action on G₃ through G₈ was studied with maltodextrins specifically labeled at the reducing-end glucose unit with ¹⁴C. The highest frequency of cleavage was at the second bond from the reducing end in G₃ through G₆. For G₇ and G₈, the sixth bond from the nonreducing end of the substrate was cleaved with absolute specificity by the exo-mechanism action.

Kinetic parameters of the exo-maltohexaohydrolase on various substrates were also studied. The Michaelis constant (Km) for short-chain amylose was the smallest among the various substrates examined.

G₆ was also formed from G₄ by a transfer action of the enzyme, with an action pattern dependent on the substrate concentration.     

Phylogeny and evolutionary rates of G protein α subunit genes

Summary Rooted phylogenetic trees for a total of 34 genes encoding the stimulatory (s), inhibitory (i), transducin (t), G_x (x), G_z (z), G₁₁ (11), G₁₂ (12), G₁₃ (13), G₁₆ (16), G_q (q), and other (o) G protein a subunits have been constructed. The analysis shows that the G₁₂ (12 and 13), G_q (11, 16, and q), and G_s (s genes) groups form one cluster, and the G_x (x and z genes), G_i (i genes), G_t (t1 and t2), and G_o (o genes) groups form another cluster. During mammalian evolution, the rates of synonymous substitutions for these genes were estimated to be between 1.77 × 10^–9/site/year and 5.63 × 10^–9/site/year, whereas those of nonsynonymous substitutions were between 0.008 × 10^–9/site/year and 0.067 × 10^–9/site/year. These evolutionary rates are similar to those for histone genes, suggesting equally important biological functions of the G protein a subunits. Offprint requests to: S. Yokoyama     

Regulation of Toll-like receptor 4-mediated immune responses through Pasteurella multocida toxin-induced G protein signalling

Background

Lipopolysaccharide (LPS)-triggered Toll-like receptor (TLR) 4-signalling belongs to the key innate defence mechanisms upon infection with Gram-negative bacteria and triggers the subsequent activation of adaptive immunity. There is an active crosstalk between TLR4-mediated and other signalling cascades to secure an effective immune response, but also to prevent excessive inflammation. Many pathogens induce signalling cascades via secreted factors that interfere with TLR signalling to modify and presumably escape the host response. In this context heterotrimeric G proteins and their coupled receptors have been recognized as major cellular targets. Toxigenic strains of Gram-negative Pasteurella multocida produce a toxin (PMT) that constitutively activates the heterotrimeric G proteins G??_q, G??₁₃ and G??_i independently of G protein-coupled receptors through deamidation. PMT is known to induce signalling events involved in cell proliferation, cell survival and cytoskeleton rearrangement.

Results

Here we show that the activation of heterotrimeric G proteins through PMT suppresses LPS-stimulated IL-12p40 production and eventually impairs the T cell-activating ability of LPS-treated monocytes. This inhibition of TLR4-induced IL-12p40 expression is mediated by G??_i-triggered signalling as well as by G?|?-dependent activation of PI3kinase and JNK. Taken together we propose the following model: LPS stimulates TLR4-mediated activation of the NF?B-pathway and thereby the production of TNF-??, IL-6 and IL-12p40. PMT inhibits the production of IL-12p40 by G??_i-mediated inhibition of adenylate cyclase and cAMP accumulation and by G?|?-mediated activation of PI3kinase and JNK activation.

Conclusions

On the basis of the experiments with PMT this study gives an example of a pathogen-induced interaction between G protein-mediated and TLR4-triggered signalling and illustrates how a bacterial toxin is able to interfere with the host??s immune response.     

A systemic Pasteurella multocida toxin aggravates cardiac hypertrophy and fibrosis in mice

Pasteurella multocida toxin (PMT) persistently activates heterotrimeric G proteins of the Gα_q/11, Gα_12/13 and Gα_i family without interaction with G protein‐coupled receptors (GPCRs). We show that PMT acts on heart tissue in vivo and on cardiomyocytes and cardiac fibroblasts in vitro by deamidation of heterotrimeric G proteins. Increased normalized ventricle weights and fibrosis were detected after intraperitoneal administration of PMT in combination with the GPCR agonist phenylephrine. In neonatal rat cardiomyocytes, PMT stimulated the mitogen‐activated protein kinase pathway, which is crucial for the development of cellular hypertrophy. The toxin induced phosphorylation of the canonical phosphorylation sites of the extracellular‐regulated kinase 1/2 and, additionally, caused phosphorylation of the recently recognized autophosphorylation site, which appears to be important for the development of cellular hypertrophy. Moreover, PMT stimulated the small GTPases Rac1 and RhoA. Both switch proteins are involved in cardiomyocyte hypertrophy. In addition, PMT stimulated RhoA and Rac1 in neonatal rat cardiac fibroblasts. RhoA and Rac1 have been implicated in the regulation of connective tissue growth factor (CTGF) secretion and expression. Accordingly, we show that PMT treatment increased secretion and expression of CTGF in cardiac fibroblasts. Altogether, the data indicate that PMT is an inducer of pathological remodelling of cardiac cells and identifies the toxin as a promising tool for studying heterotrimeric G protein‐dependent signalling in cardiac cells.     

Modulation of islet G-proteins, α-glucosidehydrolase inhibition and insulin release stimulated by various secretagogues

Guanine nucleotide-binding proteins (G-proteins) are known to act as important modulators of insulin release from the islets of Langerhans. We have recently found that the deoxynojirimycin-derivative emiglitate, a recognized inhibitor of intestinal -glucosidehydrolase activity, is a powerful inhibitor of glucose-induced insulin release. With the use of isolated mouse islets the present investigation was performed in a primary attempt to elucidate whether this inhibitory mechanism in some way was linked to the -cell G-protein system. Treatment of freshly isolated islets with pertussis toxin (PTX), which is known to inactivate the G_i-proteins, abolished the inhibitory effect of the ₂-adrenoceptor agonist clonidine on insulin release stimulated by the phosphodiesterase inhibitor IBMX in the presence of the protein kinase C activator TPA and even changed it into an increase. Emiglitate did not display any inhibitory action on insulin release induced by these secretagogues. Similarly, clonidine-induced inhibition of glucose stimulated insulin release was reversed by PTX. However, PTX did not influence the suppressive action of emiglitate on glucose-induced insulin secretion. In contrast, the adenylate cyclase activator forskolin totally abolished the inhibitory effect of emiglitate, but not that of the glucose analogue mannoheptulose, on glucose-induced insulin release. Moreover, the stimulatory effect of forskolin and cholera toxin (CTX) (activator of G_s-proteins) on the secretion of insulin was markedly enhanced in the presence of emiglitate. In conclusion, our results suggest that the inhibitory effect of emiglitate on glucose-induced insulin release is not directly related to the G_j-proteins, but most likely exerted solely through the selective suppression of lysosomal -glucosidehydrolase activity, a step in between the proximal and the distal G_i-proteins, in glucose-induced stimulus-secretion mechanisms. Our data also suggests that the inhibitory action of emiglitate on glucose stimulated insulin release can be compensated for by an increased sensitivity of the cyclic AMP-protein kinase A pathway. Hence, emiglitate might indirectly elicit an increased activity of the G_s-proteins to facilitate the secretory process.     

Dependency of growth yield,maintenance and K s-values on the dissolved oxygen concentration in continuous cultures of Azotobacter vinelandii

Azotobacter vinelandii was grown diazotrophically in sucrose-limited chemostat cultures at either 12, 48, 108, 144 or 192 M dissolved oxygen. Steady state protein levels and growth yield coefficients (Y) on sucrose increased with increasing dilution rate (D). Specific rate of sucrose consumption (q) increased in direct proportion to D. Maintenance coefficients (m) extrapolated from plots of q versus D, as well as from plots of 1/Y versus 1/D exhibited a nonlinear relationship to the dissolved oxygen concentration. Constant maximal theoretical growth yield coefficients (Y ^G) of 77.7 g cells per mol of sucrose consumed were extrapolated irrespective of differences in ambient oxygen concentration. For comparison, glucose-, as well as acetate-limited cultures were grown at 108 M oxygen. Fairly identical m- and Y ^G-values, when based on mol of substrate-carbon with glucose and sucrose grown cells, indicated that both substrates were used with the same efficiency. However, acetate-limited cultures showed significantly lower m- and, at comparable, D, higher Y-values than cultures limited by either sucrose or glucose. Substrate concentrations (K _s) required for half-maximal growth rates on sucrose were not constant, they increased when the ambient oxygen concentration was raised and, at a given oxygen concentration, when D was decreased. Since biomass levels varied in linear proportion to K _s these results are interpreted in terms of variable substrate uptake activity of the culture.Abbreviations D dilution rate - K _s substrate concentration required for half maximal growth rate - m maintenance coefficient - q specific rate of substrate consumption - Y growth yield coefficient - Y ^G maximum theoretical growth yield coefficient     

Reduction in Gh protein expression is associated with cytodifferentiation of vascular smooth muscle cells

G_h, a high molecular weight GTP-binding protein that couples ₁-adrenoceptors in heart and liver to phosphatidylinositol (Ptdlns)-specific phospholipase C (PLC), has recently been shown to be a tissue transglutaminase type 11. Transglutaminases have been suggested to play a role in the maintenance of blood vessel structure, and therefore it is possible that changes in their expression may accompany pathological states which involve phenotypic modulation of smooth muscle. Hence, we investigated the expression of G_h during differentiation of rat aortic smooth muscle cells in culture. G_h content was reduced markedly in cultured smooth muscle cells compared to freshly isolated cells as determined by Western blotting using a G_h-specific monoclonal antibody. In contrast, the level of G_q, a heterotrimeric G-protein that couples ₁-adrenoceptors to PLC, was maintained throughout the culture period. These findings indicate that changes in G, expression accompany phenotypic modulation of vascular smooth muscle cells. These changes in G_h protein expression may be important in the altered responsiveness of vessels in pathological disease states.