Gi Protein Expression Research Articles

Short-term effects of β2-AR blocker ICI 118,551 on sarcoplasmic reticulum SERCA2a and cardiac function of rats with heart failure.

The study was conducted to examine the effects of ICI 118,551 on the systolic function of cardiac muscle cells of rats in heart failure and determine the molecular mechanism of selective β2-adrenergic receptor (β2-AR) antagonist on these cells. The chronic heart failure model for rats was prepared through abdominal aortic constriction and separate cardiac muscle cells using the collagenase digestion method. The rats were then divided into Sham, HF and HF+ICI 50 nM goups and cultivated for 48 h. β2-AR, Gi/Gs and sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) protein expression levels in the cardiac muscle cells were evaluated by western blotting and changes in the systolic function of cardiac muscle cells based on the boundary detection system of contraction dynamics for individual cells was measured. The results showed that compared with the Sham group, the survival rate, percentage of basic contraction and maximum contraction amplitude percentage of cardiac muscle cells with heart failure decreased, Gi protein expression increased while Gs and SERCA2a protein expression decreased. Compared with the HF group, the maximum contraction amplitude percentage of cardiac muscle cells in group HF+ICI 50 nM decreased, the Gi protein expression level increased while the SERCA2a protein expression level decreased. Following the stimulation of Ca2+ and ISO, the maximum contraction amplitude percentage of cardiac muscle cells in the HF+ICI 50 nM group was lower than that in group HF. This indicated that ICI 118,551 has negative inotropic effects on cardiac muscle cells with heart failure, which may be related to Gi protein. Systolic function of cardiac muscle cells with heart failure can therefore be reduced by increasing Gi protein expression and lowering SERCA2a protein expression.

Expression and characterization of duck enteritis virus gI gene

BackgroundAt present, alphaherpesviruses gI gene and its encoding protein have been extensively studied. It is likely that gI protein and its homolog play similar roles in virions direct cell-to-cell spread of alphaherpesviruses. But, little is known about the characteristics of DEV gI gene. In this study, we expressed and presented the basic properties of the DEV gI protein.ResultsThe special 1221-bp fragment containing complete open reading frame(ORF) of duck enteritis virus(DEV) gI gene was extracted from plasmid pMD18-T-gI, and then cloned into prokaryotic expression vector pET-32a(+), resulting in pET-32a(+)-gI. After being confirmed by PCR, restriction endonuclease digestion and sequencing, pET-32a(+)-gI was transformed into E.coli BL21(DE3) competent cells for overexpression. DEV gI gene was successfully expressed by the addition of isopropyl-β-D-thiogalactopyranoside(IPTG). SDS-PAGE showed that the recombinant protein His6-tagged gI molecular weight was about 61 kDa. Subsequently, the expressed product was applied to generate specific antibody against gI protein. The specificity of the rabbit immuneserum was confirmed by its ability to react with the recombinant protein His6-tagged gI. In addition, real time-PCR was used to determine the the levels of the mRNA transcripts of gI gene, the results showed that the DEV gI gene was transcribed most abundantly during the late phase of infection. Furthermore, indirect immunofluorescence(IIF) was established to study the gI protein expression and localization in DEV-infected duck embryo fibroblasts (DEFs), the results confirmed that the protein was expressed and located in the cytoplasm of the infected cells, intensively.ConclusionsThe recombinant prokaryotic expression vector of DEV gI gene was constructed successfully. The gI protein was successfully expressed by E.coli BL21(DE3) and maintained its antigenicity very well. The basic information of the transcription and intracellular localization of gI gene were presented, that would be helpful to assess the possible role of DEV gI gene. The research will provide useful clues for further functional analysis of DEV gI gene.

Hydrogen Peroxide Enhances the Expression of Gi Protein in Aortic Vascular Smooth Muscle Cells : Relationship with Adenylyl Cyclase

Reactive oxygen species (ROS) play a critical role in the pathogenesis of many diseases including hypertension, atherosclerosis, and diabetes. We have recently shown that ROS contribute to enhanced expression of Giα protein in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR). The current study was undertaken to investigate the effect of H2O2, an experimental mimicker of oxidative stress, on Giα protein expression and the implication of extracellular signal‐regulated kinase (ERK1/2), and phosphatidylinositol‐3 kinase (PI3K) signaling in H2O2‐induced altered expression of Giα protein. Aortic VSMC were treated with different concentrations of H2O2 (50 μM to 250 μM) for different periods of time (30 min to 4 hr). The protein expression was determined by Western Blotting using specific antibodies. H2O2 enhanced the expression of Giα in a concentration and time‐dependent manner with a maximum increase at 100μM for 1hr, whereas Gsα expression remained unchanged. The enhanced expression of Giα was demonstrated by the increased inhibition of adenylyl cyclase by inhibitory hormones such as angiotensin II, oxotremorine, and C‐ANP4–23. We also found that H2O2 induced the phosphorylation of the serine/threonine kinases ERK1/2, and AKT/PKB (protein kinase B). Moreover, MEK, and PI3K inhibitors restored the H2O2–induced enhanced expression of Giα proteins to control levels, suggesting the implication of these proteins kinases in the enhanced level of Giα. In conclusion, the modulation of Gi protein expression by H2O2 in aortic vascular smooth muscle cells involves the ERK1/2, and PI3K signaling pathways.This study was supported by the Canadian Institutes of Health Research grant

Nitric oxide modulates Gi-protein expression and adenylyl cyclase signaling in vascular smooth muscle cells

We have previously shown that treatment of rats with the nitric oxide (NO) synthase inhibitor N 6-nitro-L-arginine methyl ester for 4 weeks resulted in the augmentation of blood pressure and enhanced levels of Giα proteins. The present studies were undertaken to investigate if NO can modulate the expression of Gi proteins and associated adenylyl cyclase signaling. A10 vascular smooth muscle cells (VSMC) and primary cultured cells from aorta of Sprague-Dawley rats were used for these studies. The cells were treated with S-nitroso- N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP) for 24 h and the expression of Giα proteins was determined by immunobloting techniques. Adenylyl cyclase activity was determined by measuring [ 32P]cAMP formation for [α- 32P]ATP. Treatment of cells with SNAP (100 μM) or SNP (0.5 mM) decreased the expression of Giα-2 and Giα-3 by about 25–40% without affecting the levels of Gsα proteins. The decreased expression of Giα proteins was reflected in decreased Gi functions (receptor-independent and -dependent) as demonstrated by decreased or attenuated forskolin-stimulated adenylyl cyclase activity by GTPγS and inhibition of adenylyl cyclase activity by angiotensin II and C-ANP 4–23, a ring-deleted analog of atrial natriuretic peptide (ANP) that specifically interacts with natriuretic peptide receptor-C (NPR-C) in SNAP-treated cells. The SNAP-induced decreased expression of Giα-2 and Giα-3 proteins was not blocked by 1 H[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylyl cyclase, or KT5823, an inhibitor of protein kinase G, but was restored toward control levels by uric acid, a scavenger of peroxynitrite and Mn(111)tetralis (benzoic acid porphyrin) MnTBAP, a peroxynitrite scavenger and a superoxide dismutase mimetic agent that inhibits the production of peroxynitrite, suggesting that NO-mediated decreased expression of Giα protein was cGMP-independent and may be attributed to increased levels of peroxynitrite. In addition, Gsα-mediated stimulation of adenylyl cyclase by GTPγS, isoproterenol, and forskolin was significantly augmented in SNAP-treated cells. These results indicate that NO decreased the expression of Giα protein and associated functions in VSMC by cGMP-independent mechanisms. From these studies, it can be suggested that NO-induced decreased levels of Gi proteins and resultant increased levels of cAMP may be an additional mechanism through which NO regulates blood pressure.

Peroxynitrite Modulates The Expression of Gi Protein And Adenylyl Cyclase Signaling in Vascular Smooth Muscle Cells.

We have further shown that vascular smooth muscle cells(VSMC) exposed to SNAP (NO donor)decreased the levels and functions of Giα proteins, which was restored to control levels by MnTBAP(peroxynitrite(ONOO-) scavenger) and not by ODQ(soluble guanylyl cyclase inhibitor) suggesting that NO-induced decreased Gi protein expression may be cGMP-independent and attributed to the increased levels of ONOO-. The present Studies were undertaken to investigate the role of ONOO- in the modulation of Gi protein expression in VSMC. A10 and aortic VSMC were used and treated with 3-Morpholinosydnonimine hydrochloride (SIN-1) or ONOO- for 24 hrs and the expression of Giα proteins was determined by immunoblotting techniques. SIN-1 and ONOO- decreased the expression of Giα-2 and Giα-3 proteins in a concentration manner. The maximal inhibition of about 30% and 40 % was observed at 0.5mM SIN-1 or at 0.5mM ONOO- in A-10 and VSM cells respectively. The decreased expression of Giα proteins was reflected in decreased Gi functions. Angiotensin II(AngII) and C-ANP4-23– mediated inhibitions of adenylyl cyclase were significantly attenuated by SIN-1 or ONOO- treatments. On the other hand, the expression of Gsα protein was not altered by these treatments in both types of cells, however, the stimulatory effects of isoproterenol, sodium fluoride (NaF) and forskolin (FSK) on adenylyl cyclase were significantly augmented. These results suggest that ONOO- decreased the expression of Gi proteins and associated functions in vascular smooth muscle cells. (supported by grant from Canadian Institutes of Health and Research)

Redox modulation of Gi protein expression and adenylyl cyclase signaling: role of nitric oxide.

Nitric oxide (NO) has been shown to regulate a variety of physiological functions, including vascular tone. The inhibition of NO synthase by N(omega)-nitro-L-arginine methyl ester (L-NAME) has been reported to increase arterial blood pressure. The present studies were undertaken to investigate if the increased blood pressure by L-NAME is associated with enhanced expression of Gi proteins, implicated in the pathogenesis of hypertension. L-NAME was administered orally into Sprague-Dawley rats for a period of 4 weeks. Control rats were given plain tap water only. The systolic blood pressure was enhanced in L-NAME-treated rats as compared with control rats; however, the heart-to-body weight ratio was not different in the two groups. The levels of Gialpha-2 and Gialpha-3 proteins and their mRNA as determined by western and northern blotting, respectively, were significantly augmented in hearts from L-NAME-treated rats, whereas the levels of Gsalpha and Gbeta were unaltered. In addition, the effect of low concentrations of GTPgammaS on forskolin-stimulated adenylyl cyclase activity (receptor-independent functions of Gialpha) was significantly enhanced, whereas the receptor-dependent inhibitions of adenylyl cyclase were completely attenuated in L-NAME-treated rats. Whereas cholera toxin-mediated stimulation of adenylyl cyclase was unaltered in both group of rats, the stimulatory effects of some agonists on adenylyl cyclase activity were diminished in L-NAME-treated rats. These results suggest the implication of NO in the modulation of Gi protein expression and associated adenylyl cyclase signaling.

Rhinovirus elicits proasthmatic changes in airway responsiveness independently of viral infection

Background: Rhinovirus (RV), the principal pathogen responsible for the common cold, is importantly implicated in triggering attacks of asthma secondary to changes in airway responsiveness. Objective: Because the airway histopathologic features of RV infection are relatively modest, we tested the hypothesis that RV can directly elicit proasthmatic-like changes in airway smooth muscle (ASM) responsiveness independently of actual viral infection and its associated cytopathic effects. Methods: Isolated ASM tissues and cultured ASM cells were inoculated with either infectious or noninfectious (UV-irradiated) RV16 and RV2, the latter serotypes belonging to the “major” and “minor” groups of RV subtypes, respectively. ASM constrictor and relaxant responsiveness, Gi protein expression, and proinflammatory cytokine release were subsequently compared under the different treatment conditions. Results: In contrast to RV2, which had no effect, RV16 inoculation elicited enhanced ASM contractility and impaired relaxation to cholinergic and β-adrenergic agonists, respectively, in association with increased ASM membrane Gi protein expression and induced release of the proinflammatory cytokines IL-5 and IL-1β. These proasthmatic-like effects were also observed in ASM exposed to UV-irradiated RV16, wherein viral replication was completely inhibited. In contrast, pretreatment of ASM with a neutralizing antibody directed against ICAM-1, the host receptor for the “major” group of RVs, completely abrogated the proasthmatic effects of RV16. Conclusions: The results demonstrate that (1) RV16 elicits proasthmatic changes in ASM responsiveness that can occur independently of actual viral infection of the ASM and (2) the effects of RV16 are attributed solely to binding of the virus to its host receptor (ICAM-1) on the ASM cell surface. Collectively, these findings support the notion that RV-induced exacerbation of wheezing in asthmatic individuals can occur even in the absence of any cytopathology associated with viral infection. (J Allergy Clin Immunol 2001;108:997-1004.)

Inhibition of alcohol dehydrogenase blocks enhanced Gi-protein expression following ethanol treatment in experimental hepatocellular carcinoma in vitro.

Chronic alcohol abuse is one of the major contributors to the onset and progression of hepatocellular carcinoma (HCC). We have previously identified increased expression and function of inhibitory guanine nucleotide regulatory proteins (Gi-proteins) in primary human and animal models of HCC. Stimulation of Gi-proteins in HCC stimulates cell mitogenesis, an effect not observed in hepatocytes. The aim of this study was to determine the effect of ethanol and ethanol metabolism on Gi-protein expression in an experimental model of HCC. Pharmacological agents that inhibit alcohol metabolism were used in conjunction with ethanol or ethanol metabolites. We were also able to assess the relative contribution of alcohol and acetaldehyde, the major metabolite of alcohol, on Gi-protein expression in HCC and hepatocytes. These studies used the rat hepatic tumorigenic H4IIE cell line in conjunction with isolated rat hepatocytes. Cells were cultured in vitro and exposed to ethanol, ethanol in the presence of an alcohol dehydrogenase (ADH) inhibitor, or acetaldehyde for varying lengths of time. Ethanol metabolism and changes in Gi-protein expression were subsequently determined by assay. Exposure to ethanol alone led to significant dose and time dependent increases in Gialpha1/2 and Gialpha3 protein and mRNA expression in HCC cells. In contrast, ethanol failed to alter Gialpha1/2, and only moderately affected Gialpha3 protein expression in isolated cultured hepatocytes. Pretreatment of HCC cells and hepatocytes with 4-methyl pyrazole (4-MP, 10 microm) significantly inhibited alcohol metabolism. Treatment of HCC cells with 4-MP inhibited changes in Gi-protein expression following exposure to ethanol (25 mm, 24 h). In addition, the increased expression of Gi-proteins observed after exposure to ethanol in HCC were mimicked by direct exposure of HCC cells to acetaldehyde in a dose and time dependent manner. These data suggest that alcohol metabolites, not alcohol, lead to increased Gi-protein expression in HCC in vitro. Ethanol and ethanol metabolites, in contrast, fail to significantly alter Gialpha1/2 protein expression in hepatocytes. These data may have significant implications in HCC progression in vivo.

Inhibition by SR 59119A of Isoprenaline-, Forskolin- and VIP-induced Relaxation of Human Isolated Bronchi

In the human isolated bronchus (HIB) it has been shown that β3-adrenoceptor stimulation fails to induce relaxation of airway smooth muscle. It has however been reported in human ventricular endomyocardial biopsies that β3-adrenoceptor stimulation induced a marked negative inotropic effect which could be linked to Gi protein activation. The aims of this study were: (1) to determine in HIB (internal diameter 1–2 mm) whether the selective β3-adrenoceptor agonist SR 59119A (N[7-methoxy-1,2,3,4-tetrahydronaphthalen-(2R)methyl]-(2R)-2-hydroxy-2-(3-chlorophenyl)ethanamine hydrochloride) was able to inhibit adenylate–cyclase-mediated airway smooth muscle relaxation induced by isoprenaline, forskolin or vasoactive intestinal peptide (VIP) and (2) to investigate the role of the Gi protein in this interaction. SR 59119A (0.1 μM and 1 μM) induced a shift to the right of concentration response curve for isoprenaline (−0.15±0.06 and −0.54±0.21 log unit, P<0.05 and P<0.01 respectively), forskolin (−0.12±0.02 and −0.30±0.05 log unit, P<0.001), and VIP (−0.42±0.12 log unit, P<0.01 with SR59119A 10−6M). The inhibitory effect of SR 59119A was (1) abolished by an incubation of HIB with pertussis toxin (1 μg/ml, during 15 h in Krebs–Henseleit solution, at 21°C), which is known to inactivate the Gi protein and (2) increased after an incubation of HIB with the pro-inflammatory cytokine IL-1β (10 ng/ml, during 15 h in Krebs–Henseleit solution, at 21°C), which is known to up-regulate Gi protein expression. Our results suggest that the selective β3-adrenoceptor agonist SR59119A might inhibit the cAMP-dependent relaxation of human isolated bronchus through Gi protein-mediated inhibition of adenylate cyclase.

Mechanism of Rhinovirus-induced Changes in Airway Smooth MuscleResponsiveness

Hakonarson H, Maskeri N, Carter C, Hodinka RL, Campbell D, Grunstein MMJ Clin Invest.102199817321741Although an important association has been established between specific viral respiratory tract infections and acute exacerbations of asthma, the mechanistic basis of this interplay remains to be elucidated. The aim of this investigation was to address the hypothesis that rhinovirus, the most common viral respiratory pathogen implicated in acute exacerbations of asthma, directly affects airway smooth muscle to produce proasthmatic changes in receptor-coupled airway smooth muscle responsiveness.Adult New Zealand White rabbits were used as the source for airway smooth muscle cells. Human rhinovirus serotype 16 and adenovirus were cultured using standard techniques and inoculated into the prepared cultures of airway smooth muscle cells. The investigators then proceeded to obtain pharmacodynamic measurements of airway smooth muscle responsiveness and determine cyclic adenosine monophosphate (cAMP) accumulation and Gi protein expression. Moreover, the investigators determined ICAM-1 expression in rabbit airway smooth muscle cells by reverse transcriptase polymerase chain reaction and Southern blot analysis, Western blot analysis, and flow cytometry. ICAM-1 expression was also determined in human airway smooth muscle cells by Northern blot analysis.Isolated rabbit and human airway smooth muscle tissue and cultured airway smooth muscle cells were inoculated with human rhinovirus (serotype 16) or adenovirus, each for 6 or 24 hours. As compared with adenovirus, which had no effect, inoculation of airway smooth muscle tissue with rhinovirus induced heightened airway smooth muscle tissue constrictor responsiveness to acetylcholine and attenuated the dose-dependent relaxation of airway smooth muscle to β-adrenoceptor stimulation with isoproterenol. These changes were largely prevented by pretreating the tissues with pertussis toxin or with a monoclonal blocking antibody to ICAM-1, which is the principal endogenous receptor for most rhinoviruses. The investigators also observed that the rhinovirus-induced changes in airway smooth muscle responsiveness were associated with diminished cAMP accumulation in response to dose-dependent administration of isoproterenol, and this effect was accompanied by upregulated expression of a Gi protein subtype in the airway smooth muscle. Finally, rhinovirus-induced effects on airway smooth muscle responsiveness were accompanied by cell surface expression of ICAM-1.This investigation provides new evidence that by the binding of rhinovirus to its ICAM-1 receptor in airway smooth muscle directly induces proasthmatic phenotypic changes in airway smooth muscle responsiveness and that this binding results in upregulation of ICAM-1 and the enhanced expression and activation of Gi protein in the rhinovirus-infected tissue.Over the past decade, we have witnessed an explosion of information regarding the role of certain respiratory viruses, particularly rhinoviruses, in the pathogenesis of asthma and airways hyperresponsiveness. This collective body of evidence supports the concept that the airway hyperresponsiveness observed in asthmatic patients during specific viral respiratory infections results from airway inflammation, cytokine release and, in some cases, specific immunoglobulin E production. This current investigation identifies the important role and molecular mechanism by which airway smooth muscle autologously induces its state of altered responsiveness after infection with rhinovirus. Hopefully, this and related data will “pave the way” for the development of rational, clinically useful therapies to reduce the morbidity and mortality experienced by patients with asthma as a direct consequence of certain viral upper respiratory infections.

Varicella-zoster virus Fc receptor component gI is phosphorylated on its endodomain by a cyclin-dependent kinase.

Varicella-zoster virus (VZV) glycoprotein gI is a type 1 transmembrane glycoprotein which is one component of the heterodimeric gE:gI Fc receptor complex. Like VZV gE, VZV gI was phosphorylated in both VZV-infected cells and gI-transfected cells. Preliminary studies demonstrated that a serine 343-proline 344 sequence located within the gI cytoplasmic tail was the most likely phosphorylation site. To determine which protein kinase catalyzed the gI phosphorylation event, we constructed a fusion protein, consisting of glutathione-S-transferase (GST) and the gI cytoplasmic tail, called GST-gI-wt. When this fusion protein was used as a substrate for gI phosphorylation in vitro, the results demonstrated that GST-gI-wt fusion protein was phosphorylated by a representative cyclin-dependent kinase (CDK) called P-TEFb, a homologue of CDK1 (cdc2). When serine 343 within the serine-proline phosphorylation site was replaced with an alanine residue, the level of phosphorylation of the gI fusion protein was greatly reduced. Subsequent experiments with individually immunoprecipitated mammalian CDKs revealed that the VZV gI fusion protein was phosphorylated best by CDK1, to a lesser degree by CDK2, and not at all by CDK6. Transient-transfection assays carried out in the presence of the specific CDK inhibitor roscovitine strongly supported the prior results by demonstrating a marked decrease in gI phosphorylation while gI protein expression was unaffected. Finally, the possibility that VZV gI contained a CDK phosphorylation site in its endodomain was of further interest because its partner, gE, contains a casein kinase II phosphorylation site in its endodomain; prior studies have established that CDK1 can phosphorylate casein kinase II.

Expression of brain Gi protein in the aging F344 rat following exposure to corticosterone

G protein expression has been shown to be modulated by circulating plasma corticosterone in young animals. A loss of G protein expression regulation by corticosterone in the elderly could explain declines in the function of G protein-coupled receptors and their effective signal transduction processes in the nervous system found in normal and pathological aging. In this study, adrenalectomized 3- and 18-months-old female Fischer 344 rats were exposed to low, moderate or high levels of plasma corticosterone to determine the effect of this hormone on Gi protein expression in the hippocampus and frontal cortex. Basal Gi protein expression, assessed by Western blot analysis, did not vary across age in either brain region. Hippocampal Gi protein levels increased following moderate and high corticosterone administration in the 3-months old animals (125% ; P < 0.05) but not in the 18-months old animals. In contrast, in the frontal cortex, Gi protein expression increased significantly in the 18-months-old group (93% ; P < 0.05) following exposure to high concentrations of corticosterone. These results suggest that steroid hormones, specifically corticosterone, may differentially modulate neurotransmitter-G protein coupling in an age-dependent manner.

Enhanced G-protein-induced relaxation in portal hypertensive rats: Role of nitric oxide

Portal hypertension (PHT) is characterized by splanchnic hyperemia due to a reduction in mesenteric vascular resistance. The reasons for the decreased resistance include an increased responsiveness to a vasodilator substance. Because the activation of an inhibitory guanine nucleotide regulatory (Gi) protein can result in endothelium-dependent relaxation, we tested the hypothesis that exaggerated Gi-protein induced relaxation via a nitric oxide (NO)-dependent pathway partly reflects the enhanced Gi-protein expression in PHT vessels. PHT was created in Sprague-Dawley rats by a partial portal-vein ligation. Control animals were sham operated. Using isolated vascular rings in the absence or presence of an intact endothelium, NG-nitro-l-arginine methyl ester (l-NAME), and pertussis toxin, dose response relationships for sodium fluoride (NaF; range, 0.1-4 mmol/L), a Gi protein activator, were determined in a cumulative manner. Gi-protein expression was determined by Western blotting. NaF caused a dose-dependent relaxation in both sham and portal hypertensive pre-contracted vessels, an effect that was significantly inhibited by pertussis toxin, endothelial denudation, and l-NAME. Concentrations of NaF greater than 4 mmol/L caused contractions, an effect that was unaffected by l-NAME. The NaF-induced relaxation response was significantly greater in PHT vessels as compared with sham concomitant with increased Gi-protein expression in PHT vessels. These data suggest that the enhanced endothelial Gi-protein-induced relaxation in PHT vessels may partly reflect enhanced expression of Gi-proteins in PHT vessels and may, thus, represent an important mechanism for exaggerated NO-dependent relaxation in the PHT vasculature. (Hepatology 1997 Jul;26(1):27-33)

Enhanced G-protein-induced relaxation in portal hypertensive rats: role of nitric oxide.

Portal hypertension (PHT) is characterized by splanchnic hyperemia due to a reduction in mesenteric vascular resistance. The reasons for the decreased resistance include an increased responsiveness to a vasodilator substance. Because the activation of an inhibitory guanine nucleotide regulatory (Gi) protein can result in endothelium-dependent relaxation, we tested the hypothesis that exaggerated Gi-protein induced relaxation via a nitric oxide (NO)-dependent pathway partly reflects the enhanced Gi-protein expression in PHT vessels. PHT was created in Sprague-Dawley rats by a partial portal-vein ligation. Control animals were sham operated. Using isolated vascular rings in the absence or presence of an intact endothelium, N(G)-nitro-L-arginine methyl ester (L-NAME), and pertussis toxin, dose response relationships for sodium fluoride (NaF; range, 0.1-4 mmol/L), a Gi protein activator, were determined in a cumulative manner. Gi-protein expression was determined by Western blotting. NaF caused a dose-dependent relaxation in both sham and portal hypertensive pre-contracted vessels, an effect that was significantly inhibited by pertussis toxin, endothelial denudation, and L-NAME. Concentrations of NaF greater than 4 mmol/L caused contractions, an effect that was unaffected by L-NAME. The NaF-induced relaxation response was significantly greater in PHT vessels as compared with sham concomitant with increased Gi-protein expression in PHT vessels. These data suggest that the enhanced endothelial Gi-protein-induced relaxation in PHT vessels may partly reflect enhanced expression of Gi-proteins in PHT vessels and may, thus, represent an important mechanism for exaggerated NO-dependent relaxation in the PHT vasculature.

Mechanisms of impaired beta-adrenoceptor-induced airway relaxation by interleukin-1beta in vivo in the rat.

We studied the in vivo mechanism of beta-adrenergic receptor (beta-AR) hyporesponsiveness induced by intratracheal instillation of interleukin-1beta (IL-1beta, 500 U) in Brown-Norway rats. Tracheal and bronchial smooth muscle responses were measured under isometric conditions ex vivo. Contractile responses to electrical field stimulation and to carbachol were not altered, but maximal relaxation induced by isoproterenol (10(-6)-10(-5) M) was significantly reduced 24 h after IL-1beta treatment in tracheal tissues and to a lesser extent, in the main bronchi. Radioligand binding using [125I]iodocyanopindolol revealed a 32+/-7% reduction in beta-ARs in lung tissues from IL-1beta-treated rats, without any significant changes in beta2-AR mRNA level measured by Northern blot analysis. Autoradiographic studies also showed significant reduction in beta2-AR in the airways. Isoproterenol-stimulated cyclic AMP accumulation was reduced by IL-1beta at 24 h in trachea and lung tissues. Pertussis toxin reversed this hyporesponsiveness to isoproterenol but not to forskolin in lung tissues. Western blot analysis revealed an IL-1beta-induced increase in Gi(alpha) protein expression. Thus, IL-1beta induces an attenuation of beta-AR-induced airway relaxation through mechanisms involving a reduction in beta-ARs, an increase in Gi(alpha) subunit, and a defect in adenylyl cyclase activity.

Mechanism of cytokine-induced modulation of beta-adrenoceptor responsiveness in airway smooth muscle.

To elucidate the role of specific proinflammatory cytokines in regulating airway responsiveness, we examined the effects and mechanisms of action of IL-1beta, TNF-alpha, and IL-2 on the beta-adrenoceptor- and postreceptor-coupled transmembrane signaling mechanisms regulating relaxation in isolated rabbit tracheal smooth muscle (TSM) segments. During half-maximal isometric contraction of the tissues with acetylcholine, relaxation responses to isoproterenol, PGE2, and forskolin were separately compared in control (untreated) TSM and tissues incubated for 18 h with IL-1beta (10 ng/ml), TNF-(alpha (100 ng/ml), or IL-2 (200 ng/ml). Relative to controls, IL-1beta- and TNF-alpha-treated TSM, but not IL-2-treated tissues, depicted significant attenuation of their maximal relaxation and sensitivity (i.e., -log dose producing 50% maximal relaxation) to isoproterenol (P < 0.001) and PGE2 (P < 0.05); whereas the relaxation responses to direct stimulation of adenylate cyclase with forskolin were similar in the control and cytokine-treated tissues. Further, the attenuated relaxation to isoproterenol and PGE2 was ablated in the IL-1beta-treated TSM that were pretreated with either the muscarinic M2-receptor antagonist, methoctramine (10(-6) M), or pertussis toxin (100 ng/ml). Moreover, Western immunoblot analysis demonstrated that: (a) Gi protein expression was significantly enhanced in membrane fractions isolated from IL-1beta-treated TSM; and (b) the latter was largely attributed to induced enhanced expression of the Gi alpha2 and Gi alpha3 subunits. Collectively, these observations provide new evidence demonstrating that IL-lbeta and TNF-alpha induce impaired receptor-coupled airway relaxation in naive TSM, and that the latter effect is associated with increased muscarinic M2-receptor/Gi protein-coupled expression and function.

Endothelial Gi Protein Expression Is Markedly Low in Human Coronary Microvessels

Gi protein functionally mediates endothelium-dependent relaxations in large epicardial coronary arteries but not in small coronary arteries, which suggests a different involvement of Gi protein in the endothelium-dependent relaxations between large and small coronary arteries. We previously showed that endothelial Gi protein is present in human epicardial coronary artery. In the present study, we examined the expression of endothelial Gi protein in human coronary microvessels. Immunohistochemical staining with a specific antibody against human Gi protein was performed in intramyocardial coronary microvessels and vasa vasorum from 34 autopsy cases. The immunoreactive levels of the endothelial Gi protein were semiquantitated into four grades (none, 0; slight, +1; moderate, +2; high, +3), and the mean value of the ratings of all endothelial cells was then used as an index of the endothelial Gi protein expression of the vessel. The immunoreactive levels of the endothelial Gi protein were extremely low in intramyocardial coronary microvessels and in vasa vasorum, irrespective of the age of the patients, the presence or absence of coronary risk factors, or the influence of medical treatments. These results may therefore explain in part why endothelium-dependent relaxations in coronary microvessels are not functionally mediated by Gi protein.

Endothelial Gi protein in human coronary arteries

Endothelium-dependent relaxations mediated by Gi protein are prominently impaired in atherosclerotic coronary arteries. However, it remains to be determined whether the expression of endothelial Gi protein per se is reduced in coronary atherosclerosis. Thus, in the present study the expression of endothelial Gi protein was examined by immunohistochemical staining using a specific antibody against human Gi protein (alpha-subunits of Gi-1 and Gi-2 proteins) in the proximal segment of the left anterior descending coronary arteries (segment 6) from 40 consecutive autopsy cases. The immunoreactive level of the Gi protein was semi-quantitated in four grades (none, 0; slight, +; moderate, +2; high, +3) and the mean value of the ratings of all endothelial cells was used as an index of the endothelial Gi protein expression of the artery. The immunoreactive level of the Gi protein in human coronary arteries was significantly reduced with ageing and extent of coronary atherosclerosis (both P < 0.05), and was lower in patients with than in those without hypertension (P < 0.01) or hyperlipidaemia (P < 0.05). In addition, the level was significantly lower in the eccentric portions than in the concentric ones in each atherosclerotic coronary artery (P < 0.0001). These alterations in the immunoreactive level of endothelial Gi protein in human coronary arteries may explain, in part, why Gi protein-mediated, endothelium-dependent relaxations are prominently impaired in atherosclerosis.

Beta-adrenoceptor stimulation-induced increase in cardiac Gi-protein expression and in carbachol sensitivity

Increased plasma concentrations of catecholamines are assumed to be responsible for the decreased sensitivity to catecholamines of the failing heart. We investigated in rat heart the influence of a 4-day infusion of isoprenaline (Iso; 2.4 mg.kg-1.d-1), propranolol (Prop; 9.9 mg.kg-1.d-1), Iso + Prop or 0.9% NaCl as control (Ctr) on myocardial Gi-mRNA and Gi-protein levels and on the negative inotropic effect of carbachol in papillary muscles. In Iso-treated rats, hybridization experiments with 32P-cDNAs revealed a 49 +/- 18% (n = 7-8) and 27 +/- 7% (n = 8) increase in Gi alpha-2- and Gi alpha-3-mRNA respectively, and pertussis toxin-catalyzed ADP-ribosylation revealed a 22 +/- 7% (n = 8) increase in Gi-protein as compared to Ctr. These alterations were accompanied by an increased potency of carbachol (mean EC50: 0.04 microM vs. 0.28 microM) in the presence of Iso in isolated electrically driven (1 Hz) papillary muscles. Prop had no effect on Gi-protein expression but antagonized all Iso-induced effects. In conclusion, beta-adrenergic stimulation leads to an increased expression of Gi and to an enhanced negative inotropic potency of muscarinic agonists. An enhanced muscarinic receptor coupling via Gi might play a pathophysiological role in heart diseases with increased plasma catecholamine levels.