Pertussis Toxin Substrate Research Articles

The Chaperonin TRiC/CCT Inhibitor HSF1A Protects Cells from Intoxication with Pertussis Toxin.

Pertussis toxin (PT) is a bacterial AB5-toxin produced by Bordetella pertussis and a major molecular determinant of pertussis, also known as whooping cough, a highly contagious respiratory disease. In this study, we investigate the protective effects of the chaperonin TRiC/CCT inhibitor, HSF1A, against PT-induced cell intoxication. TRiC/CCT is a chaperonin complex that facilitates the correct folding of proteins, preventing misfolding and aggregation, and maintaining cellular protein homeostasis. Previous research has demonstrated the significance of TRiC/CCT in the functionality of the Clostridioides difficile TcdB AB-toxin. Our findings reveal that HSF1A effectively reduces the levels of ADP-ribosylated Gαi, the specific substrate of PT, in PT-treated cells, without interfering with enzyme activity in vitro or the cellular binding of PT. Additionally, our study uncovers a novel interaction between PTS1 and the chaperonin complex subunit CCT5, which correlates with reduced PTS1 signaling in cells upon HSF1A treatment. Importantly, HSF1A mitigates the adverse effects of PT on cAMP signaling in cellular systems. These results provide valuable insights into the mechanisms of PT uptake and suggest a promising starting point for the development of innovative therapeutic strategies to counteract pertussis toxin-mediated pathogenicity.

The Inhibitory G Protein G<sub>i</sub> Identified as Pertussis Toxin-Catalyzed ADP-Ribosylation

Pertussis toxin (PTX) produced by Bordetella pertussis was first introduced by Ui and his colleagues in research on signal transduction under the name islet-activating protein in 1979, when the mechanism of toxin-induced stimulation of insulin release from pancreatic islets was reported in the rat. The stimulatory effect of PTX in vivo results from the blockage of α(2)-adrenergic receptor-mediated inhibition of insulin release. The receptor-induced inhibition of cAMP formation was also abolished in pancreatic islets isolated from PTX-treated rats, suggesting that the toxin caused uncoupling of adenylyl cyclase inhibition from receptor stimulation. The action of PTX on isolated membranes required a cytosolic factor, nicotinamide adenine dinucleotide (NAD), and the uncoupling induced by PTX was shown to be due to the toxin-catalyzed ADP-ribosylation of a 41-kDa protein with NAD as another substrate. The 41-kDa PTX substrate was soon identified and purified as the α-subunit of the inhibitory G protein that transmits an inhibitory signal from membrane receptors to adenylyl cyclase. After demonstration of the molecular mechanism of PTX, the toxin was widely utilized as a probe for identifying and analyzing major αβγ-trimeric G proteins. Thus, PTX-sensitive G proteins appeared to carry positive and negative signals from many membrane receptors to a variety of effectors other than adenylyl cyclase.

The role of G-protein in matrix-mediated motility of highly and poorly invasive melanoma cells

Membranes from 2 K1735 murine melanoma clones of high invasive capacity show increased amounts of pertussis toxin (PT) substrate when compared to a weakly invasive cellular counterpart. Using a panel of specific G-protein antibodies, we identified Gi alpha 2 as the PT-sensitive G-protein uniquely abundant in highly invasive cells. In addition, RNA hybridization results confirm the immunoblot observations that Gi alpha 2 is present at higher levels in strongly invasive cells. This result suggests that the elevated expression of Gi alpha 2 in highly invasive cells is not entirely due to differences in either translational efficiency or protein degradation but is related to altered RNA transcriptional initiation, processing and/or degradation. ADP-ribosylation of Gi alpha-subunits by PT inhibited the fibronectin, laminin and collagen type-IV-stimulated motility of the 2 highly invasive clones, while PT treatment of cells from a poorly invasive clone resulted in little or no reduction of the fibronectin, laminin or collagen type-IV-stimulated lower motility. Furthermore, PT treatment of highly or poorly invasive K1735 clones does not result in any alteration in cellular cAMP accumulation, suggesting that the PT substrate is not linked with the adenylyl cyclase enzyme complex. The data suggest that a PT-sensitive G-protein, probably Gi alpha 2 regulates second messenger pathways that contribute to elevated motility in highly invasive K1735 cells.

Inhibition of Gαi2 Activation by Gαi3 in CXCR3-mediated Signaling

G protein-coupled receptors (GPCRs) convey extracellular stimulation into dynamic intracellular action, leading to the regulation of cell migration and differentiation. T lymphocytes express G alpha(i2) and G alpha(i3), two members of the G alpha(i/o) protein family, but whether these two G alpha(i) proteins have distinguishable roles guiding T cell migration remains largely unknown because of a lack of member-specific inhibitors. This study details distinct G alpha(i2) and G alpha(i3) effects on chemokine receptor CXCR3-mediated signaling. Our data showed that G alpha(i2) was indispensable for T cell responses to three CXCR3 ligands, CXCL9, CXCL10, and CXCL11, as the lack of G alpha(i2) abolished CXCR3-stimulated migration and guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) incorporation. In sharp contrast, T cells isolated from G alpha(i3) knock-out mice displayed a significant increase in both GTPgammaS incorporation and migration as compared with wild type T cells when stimulated with CXCR3 agonists. The increased GTPgammaS incorporation was blocked by G alpha(i3) protein in a dose-dependent manner. G alpha(i3)-mediated blockade of G alpha(i2) activation did not result from G alpha(i3) activation, but instead resulted from competition or steric hindrance of G alpha(i2) interaction with the CXCR3 receptor via the N terminus of the second intracellular loop. A mutation in this domain abrogated not only G alpha(i2) activation induced by a CXCR3 agonist but also the interaction of G alpha(i3) to the CXCR3 receptor. These findings reveal for the first time an interplay of G alpha(i) proteins in transmitting G protein-coupled receptor signals. This interplay has heretofore been masked by the use of pertussis toxin, a broad inhibitor of the G alpha(i/o) protein family.

A novel G protein alpha subunit in embryo of the ascidian, Halocynthia roretzi.

A cDNA clone encoding a novel G protein alpha subunit, HrGalpha(n) was isolated from the larvae of ascidian, Halocynthia roretzi. In contrast with overall amino acid identity (63%) with G protein alpha subunit of G(i) or G(o) subclass, HrGalpha(n) has a unique amino acid sequence, which lacks a residue for pertussis toxin substrate, but retains for cholera toxin substrate for ADP-ribosylation. The sequence characteristics and molecular phylogenetic analysis suggest that HrGalpha(n) defines a novel subclass within G(i) class of G protein alpha subunits. The zygotic expression of HrGalpha(n) was first detected at the 64-cell stage and observed in all blastomeres except for B7.4, B7.5 and B7.6 cells till the 110-cell stage. As progress of the developmental stages, the expression of HrGalpha(n) became restricted and was observed in the muscle, mesenchyme and a part of trunk lateral cells in tailbud embryos. With HrGalpha(n)-GFP fusion-gene construct it was showed that the genomic fragment containing 2674 bp upstream of the putative translation start site of HrGalpha(n) contained the regulatory sequence responsible for the expression in the muscle and mesenchyme cells, and that the regulatory sequence functioned also in Ciona intestinalis. Our results suggest a possible involvement of HrGalpha(n) in the signaling system regulates the cell fate during the embryogenesis of the ascidian.

Activation of Rat Frizzled-1 Promotes Wnt Signaling and Differentiation of Mouse F9 Teratocarcinoma Cells via Pathways That Require Gαq and Gαo Function

The frizzled gene family of putative Wnt receptors encodes proteins that have a seven transmembrane-spanning motif characteristic of G-protein-linked receptors, although no loss-of-function studies have demonstrated a requirement for G-proteins for Wnt signaling by the gene product of frizzled-1. Medium conditioned by mouse F9 teratocarcinoma stem cells stably transfected to express either Xenopus Wnt-5a or Wnt-8 was used to test primitive endoderm formation of F9 stem cells. F9 stem cells expressing the rat Frizzled-1 receptors demonstrated endoderm formation in response to conditioned medium containing Wnt-8 but not to medium containing Wnt-5a. Primitive endoderm formation stimulated by Wnt-8 acting on the rat Frizzled-1 receptor was blocked by treatment with pertussis toxin by depletion of either Galpha(o) or Galpha(q) via antisense oligodeoxynucleotides, as well as by inhibitors of protein kinase C (bisindoylmaleimide) and of mitogen-activated protein kinase kinase (PD98059). Our results demonstrate the requirement for G-protein subunits Galpha(o) (a pertussis toxin substrate) and Galpha(q) for signaling by Frizzled-1, and an obligate role for the protein kinase C (likely mediated through stimulation of Galpha(q)) and mitogen-activated protein kinase network at the level of mitogen-activated protein kinase kinase.

Chimeric Gα q mutants harboring the last five carboxy-terminal residues of Gα i2 or Gα o are resistant to pertussis toxin-catalyzed ADP-ribosylation

Three widely-used Gα q chimeras harboring the last five residues of Gα i, Gα o and Gα z (qi5, qo5 and qz5) were examined for their ability to serve as substrates for pertussis toxin (PTX)-catalyzed ADP-ribosylation. In COS-7 cells coexpressing one of the three opioid receptors (μ, δ, and κ) and a Gα q chimera, agonist-induced stimulation of phosphoinositide-specific phospholipase C (PI-PLC) was largely insensitive to PTX treatment. Only the qi5-mediated stimulation of PI-PLC by κ-opioids was partially inhibited by PTX. In βγ-release assays, PTX treatment did not affect the ability of opioid receptors to activate these chimeras. [ 32P]ADP-ribosylation labeled Gα i/o but not qi5 or qo5, although the expression of these chimeras was confirmed by immunodetection. Thus, Gα q chimeras with a Gα i/o-like tail are insensitive to PTX treatment.

Selective activation of a chimeric Gi1/Gs G protein alpha subunit by the human IP prostanoid receptor: analysis using agonist stimulation of high affinity GTPase activity and [35S]guanosine-5'-O-(3-thio)triphosphate binding.

A FLAG-tagged form of the human IP prostanoid receptor was expressed stably in HEK 293 cells. This bound [3H]iloprost with high affinity and stimulated cAMP production when exposed to agonist. Iloprost produced weak stimulation of GTPase activity and [35S]guanosine-5'-O-(3-thio)triphosphate binding in membranes of these cells. Pretreatment of cells with pertussis toxin did not modify iloprost-mediated stimulation, but this was blocked by cholera toxin. The effects of iloprost were not increased by coexpression of either Gsalpha or Gi1alpha. In contrast, coexpression of a chimeric G protein alpha subunit in which the carboxyl-terminal six amino acids of Gi1alpha were altered to those of Gsalpha resulted in robust stimulation by iloprost. Because the chimeric G protein alpha subunit (Gi1/Gs6alpha) is not a substrate for either pertussis or cholera toxin, pretreatment of cells coexpressing the IP prostanoid receptor and Gi1/Gs6alpha with a mixture of these toxins resulted in resolution of the signal derived from activation of the chimeric G protein. Agonist-stimulated [35S]guanosine-5'-O-(3-thio)triphosphate binding and GTPase activity assays are the most commonly used strategies to examine interactions between G protein-coupled receptors and G proteins. These usually are not appropriate for receptors such as the IP prostanoid receptor that interact with G proteins with low rates of guanine nucleotide exchange and hydrolysis. Chimeric G proteins such as Gi1/Gs6alpha that allow appropriate receptor contacts to be converted to the higher nucleotide turnover rates typical of the Gi family G proteins can overcome this and offer a novel means to examine agonist function at such receptors.

A Heterotrimeric G Protein of the Gi Family Is Required for cAMP-triggered Trafficking of Aquaporin 2 in Kidney Epithelial Cells

Vasopressin is the key regulator of water homeostasis in vertebrates. Central to its antidiuretic action in mammals is the redistribution of the water channel aquaporin 2 (AQP2) from intracellular vesicles to the apical membrane of kidney epithelial cells, an event initiated by an increase in cAMP and activation of protein kinase A. The subsequent steps of the signaling cascade are not known. To identify proteins involved in the AQP2 shuttle we exploited a recently developed cell line (CD8) derived from the rabbit cortical collecting duct and stably transfected with rat AQP2 cDNA. Treatment of CD8 cells with pertussis toxin (PTX) inhibited both the vasopressin-induced increase in water permeability and the redistribution of AQP2 from an intracellular compartment to the apical membrane. ADP-ribosylation studies revealed the presence of at least two major PTX substrates. Correspondingly, two alpha subunits of PTX-sensitive G proteins, Galphai2 and Galphai3, were identified by Western blotting. Introduction of a synthetic peptide corresponding to the C terminus of the Gi3 alpha subunit into permeabilized CD8 cells efficiently inhibited the cAMP-induced AQP2 translocation; a peptide corresponding to the alpha subunits of Gi1/2 was much less potent. Thus a member of the Gi family, most likely Gi3, is involved in the cAMP-triggered targeting of AQP2-bearing vesicles to the apical membrane of kidney epithelial cells.

Concomitant regulation of Ca2+ mobilization and G13 expression in human erythroleukemia cells.

In human erythroleukemia (HEL) cells, stimulation of alpha2-adrenoceptors by adrenaline or neuropeptide Y Y1 receptors by neuropeptide Y, concomitantly inhibit cAMP accumulation and stimulate mobilization of Ca2+ from intracellular stores via pertussis toxin-sensitive G-proteins. Treatment of HEL cells in chemically-defined, serum-free medium with 1.25% dimethylsulfoxide (DMSO) for 4 days, increased alpha2-adrenoceptor number by 120%, while the neuropeptide Y receptor number was not significantly changed. In DMSO-treated HEL cells, Ca2+ elevations by adrenaline or neuropeptide Y were significantly reduced by 28% and 57%, respectively, while basal Ca2+ and elevations by thrombin or thapsigargin were not significantly altered. Adrenaline and neuropeptide Y-induced inhibition of forskolin-stimulated cAMP accumulation was not significantly altered upon DMSO treatment. While immunodetectable alpha-subunits of Gi2 were not significantly changed by DMSO treatment, those of Gi3 were reduced by 27%. Inactivation of pertussis toxin substrates by pertussis toxin treatment and inhibition of adrenaline or neuropeptide Y stimulated Ca2+ elevations were linearly correlated. These data are compatible with the idea that, in HEL cells, alpha2-adrenoceptors and neuropeptide Y receptors couple to inhibition of adenylyl cyclase via Gi2 while they couple to Ca2+ elevations via Gi3.

Characterization of mastoparan-induced histamine release from RBL-2H3 cells

Mastoparan (5–30 μM), a tetradecapeptide isolated from wasp venom, caused histamine release from RBL-2H3 cells in a concentration- and time-dependent manner. Mastoparan-induced histamine release remained after removing the extracellular Ca 2+, whereas the antigen-induced one disappeared. Pertussis toxin did not inhibit mastoparan-induced histamine release from the cells, and mastoparan did not stimulate phosphoinositide hydrolysis. In agreement with the results, RBL-2H3 cells had a small amount of ADP-ribosylation substrates for pertussis toxin. Neomycin (1–5 mM) suppressed mastoparan-induced histamine release and phospholipase D activation. However, butanol slightly inhibited mastoparan-induced histamine release. Moreover, 2,3-diphosphoglycerate inhibited mastoparan-induced phospholipase D activation, but not it's histamine release. On the other hand, mastoparan caused the leakage of lactate dehydrogenase from the cells in a similar concentration range to the histamine release. This leakage was also suppressed by neomycin. These results suggest that mastoparan enhances the membrane permeability, resulting in histamine release in a pertussis toxin-insensitive manner, and that mastoparan-induced phospholipase D activation may not relate to histamine release.

Expression of GTP-binding proteins and protein kinase C isozymes in platelet-like particles derived from megakaryoblastic leukemia cells (MEG-01)

The expression of the various GTP-binding proteins and protein kinase C (PKC) in the platelet-like particles produced by MEG-01 cells was analyzed by RT-PCR and immunoblotting. We selected 14 human low Mr GTPbinding proteins (LMW-GPs) and nine PKCs expressed in platelets and/or megakaryocytes, and designed specific primer pairs for the proteins. RT-PCR analysis revealed that the particles express the mRNAs of many LMW-GPs such as rap 1A, rap 1B, rap 2B, ral A, rho A, rac 1, rac 2, Cdc 42, rab 1, rab 3B, rab 6, ram/rab 27 and ran . By immunoblotting analysis, Rap1, RhoA, Rac, Cdc42, Rab6 and Rab8 were identified in the particles. As for PKCs, the particles were observed to express the mRNAs of PKC- alpha ,- beta I,- beta -II,- delta , epsilon , eta and theta , but not- gamma and zeta . Using immunoblot analysis, PKC- beta I,- beta II and zeta were shown to exist in the particles, although the contents were lower than those in platelets. Furthermore, the presence of Gi2- alpha , a heterotrimeric G protein that is the major pertussis toxin substrate in human platelets, and beta subunits was observed in the particles. Taken together, the particles possess some similarity to human platelets based on the expression profiles of GTP-binding proteins and PKCs.

Pneumocystis carinii Infection Alters GTP-Binding Proteins in the Lung

The GTP-binding regulatory proteins (G proteins) in the membranes of the lung parenchyma from normal, uninfected ferrets were compared to those from immunosuppressed animals with and without Pneumocystis carinii pneumonitis. In lung membranes, pertussis toxin (PT) catalyzed ADP ribosylation of a 41-kDa protein; treatment with cholera toxin (CT) led to ribosylation of a 44-kDa polypeptide. Compared to that in the normal ferrets, the level of the 44-kDa protein was dramatically suppressed in the P. carinii-infected animals. Western blot analysis with specific antibodies to alpha s (which recognized CT substrates), alpha common (which reacted to PT substrates), the alpha q/11 epitope, and the beta subunit demonstrated that these proteins were decreased in animals with P. carinii pneumonitis (PCP). Western blotting of PCP-free membranes with a pan-Ras antibody revealed a 21-kDa polypeptide (corresponding to small G proteins). The level of the 21-kDa protein in membranes from PCP-affected animals was only 30% of that in membranes from PCP-free animals. Finally, analogous studies performed with rat lung membranes revealed similar findings. These data suggest that, independent of its exacerbation of immunosuppression, PCP leads to extensive changes in the GTP-binding proteins in the lung.

Effects of triiodothyronine administration on the adenylyl cyclase system in brown adipose tissue of rat.

This study was undertaken to investigate the effect of triiodothyronine (T3) administration to euthyroid rats on beta 3-adrenoceptor (beta 3-AR) expression and on the different components of the adenylyl cyclase (AC) system in brown adipose tissue (BAT). In rats treated with T3, the beta 3-AR density (assessed by the binding of [3H]CGP-12177) showed a decrease of 50%, as did their mRNA, as analyzed by reverse transcriptase-polymerase chain reaction. In hyperthyroid rats, compared with control rats, there was a 40% increase in G alpha s activity (stimulated by NaF or GTP gamma S) and a fourfold increase in the protein concentration (Western blotting). In contrast, the level of the pertussis toxin substrate Gi declined by 35% in response to T3. Analysis of dose-response curves for isoproterenol and CGP-12177 revealed that neither basal nor stimulated AC activities nor 50% stimulatory concentration for these agonists was changed by T3 administration. In conclusion, these results suggest that downregulation of the beta 3-AR by T3 was counter-balanced by changes in other components of the AC cascade (i.e., Gs and Gi), so no change occurred in the capacity of BAT to generate adenosine 3',5'-cyclic monophosphate.

Purification and G Protein Subunit Regulation of a Phospholipase C-β from Xenopus laevis Oocytes

Xenopus oocytes exhibit both pertussis toxin-sensitive and -insensitive inositol lipid signaling responses to G protein-coupled receptor activation. The G protein subunits Galphai, Galphao, Galphaq, Galphas, and Gbetagamma all have been proposed to function as activators of phospholipase C in oocytes. Ma et al. (Ma, H.-W., Blitzer, R. D., Healy, E. C., Premont, R. T., Landau, E. M., and Iyengar, R. J. Biol. Chem. 268, 19915-19918) cloned a Xenopus phospholipase C (PLC-betaX) that exhibits homology to the PLC-beta class of isoenzymes. Although this enzyme was proposed to function as a signaling protein in the pertussis toxin-sensitive inositol lipid signaling pathway of oocytes, its regulation by G protein subunits has not been directly assessed. As such we have utilized baculovirus-promoted overexpression of PLC-betaX in Sf9 insect cells and have purified a recombinant 150-kDa isoenzyme. PLC-betaX catalyzes hydrolysis of phosphatidylinositol(4,5)bisphosphate and phosphatidylinositol(4)monophosphate, and reaction velocity is dependent on Ca2+. Recombinant PLC-betaX was activated by both Galphaq and Gbetagamma. PLC-betaX exhibited a higher apparent affinity for Galphaq than Gbetagamma, and Galphaq was more efficacious than Gbetagamma at lower concentrations of PLC-betaX. Relative to other PLC-beta isoenzymes, PLC-betaX was less sensitive to stimulation by Galphaq than PLC-beta1 but similar to PLC-beta2 and PLC-betaT. PLC-betaX was more sensitive to stimulation by Gbetagamma than PLC-beta1 but less sensitive than PLC-beta2 and PLC-betaT. In contrast PLC-betaX was not activated by the pertussis toxin substrate G proteins Galphai1, Galphai2, Galphai3, or Galphao. These results are consistent with the idea that PLC-betaX is regulated by alpha-subunits of the Gq family and by Gbetagamma and do not support the idea that alpha-subunits of pertussis toxin-sensitive G proteins are directly involved in regulation of this protein.

Multiple G proteins and phospholipase C isoforms in human myometrial cells: implication for oxytocin action.

Although a physiological role for oxytocin during parturition is well accepted, the mechanisms by which it activates myometrial contractility during labor have not been completely elucidated. We have previously shown the presence of Gq and two pertussis toxin (PT) substrates of the Gi family in human myometrial cells. In the present study, we have identified by Western blotting the G protein and phospholipase C (PLC) isoforms present in these cells and investigated their implication in oxytocin signaling by measuring the formation of inositol phosphates (IPs) and mobilization of intracellular calcium. We found G protein subunits alpha(q), alpha(11), alpha(i1), alpha(i2), alpha(i3), alpha(z), and two splice variants of alpha(s)- and beta-subunits. We have also detected the presence of five PLC isoforms: beta 1, beta 2, beta 3, gamma 1, and gamma 2. Oxytocin-induced IPs formation and intracellular Ca2+ mobilization were inhibited to approximately 50% after pretreatment of the cells with PT, suggesting that oxytocin activates PLC beta by interacting with at least two types of G proteins: a member of the Gq family (PT resistant) and a member of the Gi family (PT sensitive). The tyrosine phosphatase inhibitor pervanadate stimulated IPs formation in myometrial cells. Using the protein kinase inhibitors staurosporine, phenylarsine oxide, and Ro 31-8220 and the protein kinase C activator phorbol dibutyrate, we have shown that pervanadate and oxytocin activate PLC by different mechanisms. Furthermore, oxytocin did not activate tyrosine phosphorylation in human myometrial cells, as measured with an antiphosphotyrosine antibody, indicating that it does not activate a PLC gamma isoform. We conclude that oxytocin activates human myometrium by interacting with at least two G proteins and possibly three PLC beta isoforms.

Ca2+ signalling in K562 human erythroleukaemia cells: effect of dimethyl sulphoxide and role of G-proteins in thrombin- and thromboxane A2-activated pathways.

The human leukaemic cell line K562 is a pluripotent stem cell with the potential to mature along a megakaryocytic or erythroid line. In these cells, thrombin and U46619 (9,11-dideoxy-9 alpha, 11 alpha-methanoepoxy prostaglandin F2 alpha), a thromboxane A2 analogue, increased intracellular Ca2+ in a rapid and concentration-dependent manner. The peak transient observed with both thrombin and U46619 was preserved upon stimulation in the absence of extracellular calcium and blunted with phorbol myristate acetate, suggestive of activation of phospholipase C. Short-term treatment with leupeptin abolished the calcium response to thrombin, but did not alter that to U46619. Both pertussis toxin (PT) and DMSO pretreatment inhibited thrombin- but not U46619-stimulated intracellular calcium elevation, indicating that these agonists signal through different G-proteins. Western blot analysis of crude membranes from K562 cells revealed the presence of G12 alpha and G13 alpha; the other known PT-substrates, Gi1 alpha and G0 alpha, were not detected. Consistent with this observation, ADP-ribosylation experiments revealed the presence of two PT substrates which co-migrated with human erythrocyte G12 alpha and G13 alpha. An antibody raised against Gq/11 alpha, a subfamily of G-protein alpha subunits unmodified by PT, specifically recognized 42 kDa protein(s) in K562 cells. PCR amplification of reverse-transcribed K562 RNA followed by DNA sequencing showed that these cells express messages for both Gq alpha and G11 alpha. Treatment of K562 cells with DMSO reduced the levels of thrombin receptor mRNA, without simultaneous changes in the expression of G12 alpha and G13 alpha. We have thus identified Ca(2+)-mobilizing agonists and related G-proteins in K562 cells, together with changes induced by DMSO in this signalling pathway.

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

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. 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. 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. 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.

Increase of myocardial inhibitory G-proteins in catecholamine-refractory septic shock or in septic multiorgan failure: Am J Med 1995; 98/2: 183–186

Purpose The aim of the study was to investigate the mechanisms of myocardial catecholamine refractoriness in septic shock. Methods The inhibitory guanine nucleotidebinding proteins (Gia) were studied with pertussis toxin labeling and radioimmunologically in myocardium from patients who died while in catecholamine-refractory septic shock and from patients who died of noncardiac disease. Results An increase by 62% (immunological Gia) and 221% (pertussis toxin substrate) of myocardial Gia was observed in patients with catecholamine-refractory shock compared with controls. The increases of Gia were greater than those found in chronic heart failure reported earlier. Conclusions An increase in the expression of Gia could also be important in conditions other than chronic heart failure, eg, septic shock. An increase of Gia could play a pathophysiologically relevant role in catecholamine refractoriness in septic shock and could provide a target for pharmacologic treatment in this condition.

Cloning of a Molluscan G Protein alpha Subunit of the Gq Class which is Expressed Differentially in Identified Neurons

Through molecular cloning we have identified a molluscan G protein alpha subunit which belongs to the G alpha q family and is expressed in the central nervous system (CNS) of the pond snail, Lymnaea stagnalis. The deduced protein product shares a very high degree of amino sequence identity with vertebrate and invertebrate G alpha q/G alpha 11 subunits (80-82% and 76-77%, respectively). Large parts of the protein have been completely conserved, among which are residues 25-58, including the nucleotide-binding A domain. Especially the C-terminal half (amino acids 195-353), implicated in receptor and effector interactions, is highly conserved (94% sequence identity with murine sequences). This region includes the nucleotide-binding C, G, and I domains, which are identical to cognate motifs of vertebrate G alpha q/11. Like the latter proteins, the Lymnaea G alpha q C-terminus lacks a cysteine that could serve as a substrate for pertussis toxin. In situ hybridization reveals G alpha q-encoding mRNA(s) to be present throughout the CNS. Interestingly, however, close inspection of two identified cell types in the cerebral ganglia, the light-green cells, involved in the regulation of growth and metabolism and the anterior lobe cells which are involved in the control of male aspects of reproduction, indicates that they express the mRNA(s) at significantly different levels. Even within the heterologous cluster of light-green cells there appears to be differential expression of the pertinent mRNA. Such observations have hitherto not been reported for specific cell types occurring in vivo.