Analyses of Gnai3-iresGFP reporter mice reveal unknown Gαi3 expression sites.

Inhibitory G proteins (Gi proteins) are highly homologous but play distinct biological roles. However, their isoform-specific detection remains challenging. To facilitate the analysis of Gαi3 expression, we generated a Gnai3- iresGFP reporter mouse line. An internal ribosomal entry site (IRES) was inserted behind the stop-codon of the Gnai3 gene to initiate simultaneous translation of the GFP cDNA together with Gαi3. The expression of GFP was confirmed in spleen and thymus tissue by immunoblot analysis. Importantly, the GFP knock-in (ki) did not alter Gαi3 expression levels in all organs tested including spleen and thymus compared to wild-type littermates. Flow cytometry of thymocytes, splenic and blood cell suspensions revealed significantly higher GFP fluorescence intensities in homozygous ki/ki animals compared to heterozygous mice (+/ki). Using cell-type specific surface markers GFP fluorescence was assigned to B cells, T cells, macrophages and granulocytes from both splenic and blood cells and additionally blood-derived platelets. Moreover, immunofluorescent staining of the inner ear from knock-in mice unraveled GFP expression in sensory and non-sensory cell types, with highest levels in Deiter's cells and in the first row of Hensen's cells in the organ of Corti, indicating a novel site for Gαi3 expression. In summary, the Gnai3- iresGFP reporter mouse represents an ideal tool for precise analyses of Gαi3 expression patterns and sites.

Serotonin 5-HT2A receptor expression and functionality in postmortem frontal cortex of subjects with schizophrenia: Selective biased agonism via Gαi1-proteins.

Serotonin 5-HT2A receptors (5-HT2ARs) have been implicated in schizophrenia. However, postmortem studies on 5-HT2ARs expression and functionality in schizophrenia are scarce. The 5-HT2AR mRNA and immunoreactive protein expression were evaluated in postmortem tissue from dorsolateral prefrontal cortex (DLPFC) of antipsychotic-free (n = 18) and antipsychotic-treated (n = 9) subjects with schizophrenia, and matched controls (n = 27). Functional coupling of 5-HT2AR to G-proteins was tested by measuring the activation induced by the agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride ((±)DOI) in antibody-capture [35S]GTPγS scintillation proximity assays (SPA). In antipsychotic-free schizophrenia subjects, 5-HT2AR mRNA expression and protein immunoreactivity in total homogenates was similar to controls. In contrast, in antipsychotic-treated schizophrenia subjects, lower mRNA expression (60±9% vs controls) and a trend to reduced protein immunoreactivity (86±5% vs antipsychotic-free subjects) just in membrane-enriched fractions was observed. [35S]GTPγS SPA revealed a significant ~6% higher stimulation of Gαi1-protein by (±)DOI in schizophrenia, whereas activation of the canonical Gαq/11-protein pathway by (±)DOI remained unchanged. Expression of Gαi1- and Gαq/11-proteins did not differ between groups. Accordingly, in rats chronically treated with clozapine, but not with haloperidol, a 30-40% reduction was observed in 5-HT2AR mRNA expression, 5-HT2AR protein immunoreactivity and [3H]ketanserin binding in brain cortical membranes. Overall, the data suggest a supersensitive 5-HT2AR signaling through inhibitory Gαi1-proteins in schizophrenia. Together with previous results, a dysfunctional pro-hallucinogenic agonist-sensitive 5-HT2AR conformation in postmortem DLPFC of subjects with schizophrenia is proposed. Atypical antipsychotic treatment would contribute to counterbalance this 5-HT2AR supersensitivity by reducing receptor expression.

Pediatric ependymoma: GNAO1, ASAH1, IMMT and IPO7 protein expression and 5-year prognosis correlation.

OBJECTIVE: The aim of this work was to evaluate a pediatric ependymoma protein expression that may be useful as a molecular biomarker candidate for prognosis, correlated with clinical features such as age, gender, histopathological grade, ependymal tumor recurrence and patient survival. PATIENTS AND METHODS: Immunohistochemistry assays were performed for GNAO1, ASAH1, IMMT, IPO7, Cyclin D1, P53 and Ki-67 proteins. Kaplan-Meier and Cox analysis were performed for age, gender, histopathological grade, relapse and survival correlation. RESULTS: We found that three proteins correlate with histopathological grade and relapse; two proteins correlate with survival; one protein does not correlate with any clinical feature. CONCLUSION: Our results suggest that, out of the proteins analyzed, five may be considered suitable prognostic biomarkers and one may be considered a predictive biomarker for response to treatment of pediatric ependymoma.

DNMT1 mediated promoter methylation of GNAO1 in hepatoma carcinoma cells.

Hepatocellular carcinoma (HCC) is one of the most lethal and prevalent cancers worldwide and has recently become the second most common cause of cancer-related deaths in men of developing countries. Guanine nucleotide-binding protein (G protein) has been reported to be associated with the early process of HCC. In our previous study, GNAO1, one of members of G protein, was found to be down-regulated in HCC. Thus, the present study aimed to throw light upon the mechanism of the abnormal expression of GNAO1 in HCC. First, qPCR results from two HCC cell lines (SMMC-7721 and QGY-7703) confirmed the down-expression of GNAO1, followed by the validation of the methylation status of the promoter region by bisulfite sequence PCR (BSP). Moreover, 5-Aza-2'-deoxycytidine (DAC) with Trichostatin A (TSA) treatment made it much clear that GNAO1 transcription was inhibited by promoter hypermethylation, contributing to its low expression. It was further revealed that the silencing effect was regulated by methyltransferase 1 (DNMT1), and was further enhanced by transforming growth factor ß (TGF-ß). In addition, the up-regulation of GNAO1 with the help of recombinant plasmid was also found to accelerate cell apoptosis, confirmed by flow cytometry and western blotting analysis. All these results above indicated that the promoter hypermethylation of GNAO1 might play an important role in HCC, suggesting that it might be used as a promising biomarker for HCC diagnosis and targeted therapy.

Inhibition of overexpression of Giα proteins and nitroxidative stress contribute to sodium nitroprusside-induced attenuation of high blood pressure in SHR.

We earlier showed that vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) exhibit enhanced expression of Giα proteins which was attributed to the decreased levels of nitric oxide (NO), because elevation of the intracellular levels of NO by NO donors; sodium nitroprusside (SNP) and S-Nitroso-N-acetyl-DL-penicillamine (SNAP), attenuated the enhanced expression of Giα proteins. Since the enhanced expression of Giα proteins is implicated in the pathogenesis of hypertension, the present study was undertaken to investigate if treatment of SHR with SNP could also attenuate the development of high blood pressure (BP) and explore the underlying molecular mechanisms. Intraperitoneal injection of SNP at a concentration of 0.5 mg/kg body weight twice a week for 2 weeks into SHR attenuated the high blood pressure by about 80 mmHg without affecting the BP in WKY rats. SNP treatment also attenuated the enhanced levels of superoxide anion (O2- ), hydrogen peroxide (H2 O2 ), peroxynitrite (ONOO- ), and NADPH oxidase activity in VSMC from SHR to control levels. In addition, the overexpression of different subunits of NADPH oxidase; Nox-1, Nox-2, Nox-4, P22phox , and P47phox , and Giα proteins in VSMC from SHR were also attenuated by SNP treatment. On the other hand, SNP treatment augmented the decreased levels of intracellular NO, eNOS, and cGMP in VSMC from SHR. These results suggest that SNP treatment attenuates the development of high BP in SHR through the elevation of intracellular levels of cGMP and inhibition of the enhanced levels of Giα proteins and nitroxidative stress.

Antidepression action of BDNF requires and is mimicked by Gαi1/3 expression in the hippocampus.

Stress-related alterations in brain-derived neurotrophic factor (BDNF) expression, a neurotrophin that plays a key role in synaptic plasticity, are believed to contribute to the pathophysiology of depression. Here, we show that in a chronic mild stress (CMS) model of depression the Gαi1 and Gαi3 subunits of heterotrimeric G proteins are down-regulated in the hippocampus, a key limbic structure associated with major depressive disorder. We provide evidence that Gαi1 and Gαi3 (Gαi1/3) are required for the activation of TrkB downstream signaling pathways. In mouse embryonic fibroblasts (MEFs) and CNS neurons, Gαi1/3 knockdown inhibited BDNF-induced tropomyosin-related kinase B (TrkB) endocytosis, adaptor protein activation, and Akt-mTORC1 and Erk-MAPK signaling. Functional studies show that Gαi1 and Gαi3 knockdown decreases the number of dendrites and dendritic spines in hippocampal neurons. In vivo, hippocampal Gαi1/3 knockdown after bilateral microinjection of lentiviral constructs containing Gαi1 and Gαi3 shRNA elicited depressive behaviors. Critically, exogenous expression of Gαi3 in the hippocampus reversed depressive behaviors in CMS mice. Similar results were observed in Gαi1/Gαi3 double-knockout mice, which exhibited severe depressive behaviors. These results demonstrate that heterotrimeric Gαi1 and Gαi3 proteins are essential for TrkB signaling and that disruption of Gαi1 or Gαi3 function could contribute to depressive behaviors.

Reserpine requires the D2-type receptor, dop-3, and the exoribonuclease, eri-1, to extend the lifespan in C. elegans.

Lifespan extension is an all systems encompassing event. Involvement of reduced insulin/IGF1 signalling is well worked out, first in the model organism Caenorhbaditis elegans followed by other systems including humans. But the role of neuronal component in lifespan extension is not well understood due to the refractory nature of neurons to small RNA interference (sRNAi) in C. elegans. Earlier, we have demonstrated that an antihypertensive drug, reserpine, extends lifespan through modulation of neurotransmitter release, especially, acetylcholine, in C. elegans. Intriguingly, the reserpine mediated lifespan extension (RMLE) does not happen through the known longevity pathways. Here, we report that the D2-type dopamine receptor (DOP-3), which acts through the inhibitory Gprotein coupled (G alpha i) pathway mediated signalling is partly required for RMLE. In the dop-3 loss of function mutant RMLE is shortened. DOP-3 acts through Gαo (goa-1). One of the downstream targets of G protein signalling is the transcription factor, jun-1. MRP-1, an ATP binding cassette transporter, belonging to the multidrug resistance protein family is one of the genes turned on by JUN-1. RMLE is shortened in dop-3-->goa-1-->jun1-->mrp-1 loss of function mutants, elucidating the contribution of dop-3 signalling. The dop-3 receptor system is known to inhibit acetylcholine release. This suggests dopamine receptor, dop-3 could be contributing to the modulation of acetylcholine release by reserpine. ERI-1 is a 3'-5' exoribonuclease, one of the negative regulators of sRNAi, whose loss of function makes neurons amenable to siRNA. In the absence of eri-1, RMLE is shortened. In the dop-3 loss-of-function background, lack of eri-1 completely abolishes RMLE. This suggests that dop-3 and eri-1 act in independent parallel pathways for RMLE and these two pathways are essential and sufficient for the longevity enhancement by reserpine in C. elegans.

Identification of dually acylated proteins from complementary DNA resources by cell-free and cellular metabolic labeling.

To establish a strategy to identify dually fatty acylated proteins from cDNA resources, seven N-myristoylated proteins with cysteine (Cys) residues within the 10 N-terminal residues were selected as potential candidates among 27 N-myristoylated proteins identified from a model human cDNA resource. Seven proteins C-terminally tagged with FLAG tag or EGFP were generated and their susceptibility to protein N-myristoylation and S-palmitoylation were evaluated by metabolic labeling with [(3)H]myristic acid or [(3)H]palmitic acid either in an insect cell-free protein synthesis system or in transfected mammalian cells. As a result, EEPD1, one of five proteins (RFTN1, EEPD1, GNAI1, PDE2A, RNF11) found to be dually acylated, was shown to be a novel dually fatty acylated protein. Metabolic labeling experiments using G2A and C7S mutants of EEPD1-EGFP revealed that the palmitoylation site of EEPD1 is Cys at position 7. Analysis of the intracellular localization of EEPD1 C-terminally tagged with FLAG tag or EGFP and its G2A and C7S mutants revealed that the dual acylation directs EEPD1 to localize to the plasma membrane. Thus, dually fatty acylated proteins can be identified from cDNA resources by cell-free and cellular metabolic labeling of N-myristoylated proteins with Cys residue(s) close to the N-myristoylated N-terminus.

Valproic acid mediates miR-124 to down-regulate a novel protein target, GNAI1.

Valproic acid (VPA) is an anti-convulsant drug that is recently shown to have neuroregenerative therapeutic actions. In this study, we investigate the underlying molecular mechanism of VPA and its effects on Bdnf transcription through microRNAs (miRNAs) and their corresponding target proteins. Using in silico algorithms, we predicted from our miRNA microarray and iTRAQ data that miR-124 is likely to target at guanine nucleotide binding protein alpha inhibitor 1 (GNAI1), an adenylate cyclase inhibitor. With the reduction of GNAI1 mediated by VPA, the cAMP is enhanced to increase Bdnf expression. The levels of GNAI1 protein and Bdnf mRNA can be manipulated with either miR-124 mimic or inhibitor. In summary, we have identified a novel molecular mechanism of VPA that induces miR-124 to repress GNAI1. The implication of miR-124→GNAI1→BDNF pathway with valproic acid treatment suggests that we could repurpose an old drug, valproic acid, as a clinical application to elevate neurotrophin levels in treating neurodegenerative diseases.

Gαi2- and Gαi3-deficient mice display opposite severity of myocardial ischemia reperfusion injury.

G-protein-coupled receptors (GPCRs) are the most abundant receptors in the heart and therefore are common targets for cardiovascular therapeutics. The activated GPCRs transduce their signals via heterotrimeric G-proteins. The four major families of G-proteins identified so far are specified through their α-subunit: Gαi, Gαs, Gαq and G12/13. Gαi-proteins have been reported to protect hearts from ischemia reperfusion injury. However, determining the individual impact of Gαi2 or Gαi3 on myocardial ischemia injury has not been clarified yet. Here, we first investigated expression of Gαi2 and Gαi3 on transcriptional level by quantitative PCR and on protein level by immunoblot analysis as well as by immunofluorescence in cardiac tissues of wild-type, Gαi2-, and Gαi3-deficient mice. Gαi2 was expressed at higher levels than Gαi3 in murine hearts, and irrespective of the isoform being knocked out we observed an up regulation of the remaining Gαi-protein. Myocardial ischemia promptly regulated cardiac mRNA and with a slight delay protein levels of both Gαi2 and Gαi3, indicating important roles for both Gαi isoforms. Furthermore, ischemia reperfusion injury in Gαi2- and Gαi3-deficient mice exhibited opposite outcomes. Whereas the absence of Gαi2 significantly increased the infarct size in the heart, the absence of Gαi3 or the concomitant upregulation of Gαi2 dramatically reduced cardiac infarction. In conclusion, we demonstrate for the first time that the genetic ablation of Gαi proteins has protective or deleterious effects on cardiac ischemia reperfusion injury depending on the isoform being absent.

Overexpression of GNAO1 correlates with poor prognosis in patients with gastric cancer and plays a role in gastric cancer cell proliferation and apoptosis.

Guanine nucleotide-binding protein (G protein), alpha activating activity polypeptide O (GNAO1) encodes an alpha subunit of the heterotrimeric guanine nucleotide-binding proteins (Gαo), which plays a significant role in the development of various types of cancer, including breast cancer and hepatocellular carcinoma. However, its role in gastric cancer (GC) has not yet been elucidated. In the current study, the expression of Gαo was analyzed by immunohistochemistry in paraffin-embedded tissue sections from 70 patients with GC and its cellular role was examined by small interfering RNA (siRNA)-mediated downregulation. The overexpression of GNAO1 (scores of 2 or 4) was observed in 44 of the 70 patients with GC (62.9%) and the expression of Gαo significantly correlated with the overall survival of the patients with GC after surgery. The median survival rate of patients with a negative or positive expression of Gαo was 61 or 27 months, respectively (P=0.033). The silencing of GNAO1 in GC cells by RNA interference markedly inhibited the proliferation of GC cells and promoted apoptosis by increasing the accumulation of the pro-apoptotic proteins, Puma and Bim, possibly mediated by extracellular signal-regulated kinase 1 and 2 (ERK1/2). This further confirmed the clinical significance of GNAO1, which was overexpressed in GC tissues. To the best of our knowledge, the present study is the first to demonstrate that GNAO1 is overexpressed in GC and that its overexpression correlates with poor prognosis, as it promotes GC cell viability.

Proteomic profiling of the hypothalamus in a mouse model of cancer-induced anorexia-cachexia.

BACKGROUND: Anorexia-cachexia is a common and severe cancer-related complication but the underlying mechanisms are largely unknown. Here, using a mouse model for tumour-induced anorexia-cachexia, we screened for proteins that are differentially expressed in the hypothalamus, the brain's metabolic control centre. METHODS: The hypothalamus of tumour-bearing mice with implanted methylcholanthrene-induced sarcoma (MCG 101) displaying anorexia and their sham-implanted pair-fed or free-fed littermates was examined using two-dimensional electrophoresis (2-DE)-based comparative proteomics. Differentially expressed proteins were identified by liquid chromatography-tandem mass spectrometry. RESULTS: The 2-DE data showed an increased expression of dynamin 1, hexokinase, pyruvate carboxylase, oxoglutarate dehydrogenase, and N-ethylmaleimide-sensitive factor in tumour-bearing mice, whereas heat-shock 70 kDa cognate protein, selenium-binding protein 1, and guanine nucleotide-binding protein Gα0 were downregulated. The expression of several of the identified proteins was similarly altered also in the caloric-restricted pair-fed mice, suggesting an involvement of these proteins in brain metabolic adaptation to restricted nutrient availability. However, the expression of dynamin 1, which is required for receptor internalisation, and of hexokinase, and pyruvate carboxylase were specifically changed in tumour-bearing mice with anorexia. CONCLUSION: The identified differentially expressed proteins may be new candidate molecules involved in the pathophysiology of tumour-induced anorexia-cachexia.

cAMP attenuates the enhanced expression of Gi proteins and hyperproliferation of vascular smooth muscle cells from SHR: role of ROS and ROS-mediated signaling.

We previously showed that angiotensin II (ANG II)-induced overexpression of inhibitory G proteins (Gi) was attenuated by dibutyryl-cAMP (db-cAMP) in A10 vascular smooth muscle cells (VSMC). Since enhanced levels of endogenous ANG II contributed to the overexpression of Gi protein and hyperproliferation of VSMC from spontaneously hypertensive rats (SHR), the present study was therefore undertaken to examine if cAMP could also attenuate the overexpression of Gi proteins and hyperproliferation of VSMC from SHR and to explore the underlying molecular mechanisms responsible for this response. The enhanced expression of Giα proteins in VSMC from SHR and Nω-nitro-L-arginine methyl ester hypertensive rats was decreased by db-cAMP. In addition, enhanced inhibition of adenylyl cyclase by inhibitory hormones and forskolin-stimulated adenylyl cyclase activity by low concentration of GTPγS in VSMC from SHR was also restored to Wistar-Kyoto (WKY) levels by db-cAMP. Furthermore, db-cAMP also attenuated the hyperproliferation and the increased production of superoxide anion, NAD(P)H oxidase activity, overexpression of Nox1/Nox2/Nox4 and p47phox proteins, increased phosphorylation of PDGF-receptor (R), EGF-R, c-Src, and ERK1/2 to control levels. In addition, the protein kinase A (PKA) inhibitor reversed the effects of db-cAMP on the expression of Nox4 and Giα proteins and hyperproliferation of VSMC from SHR to WKY levels, while stimulation of the exchange protein directly activated by cAMP did not have any effect on these parameters. These results suggest that cAMP via PKA pathway attenuates the overexpression of Gi proteins and hyperproliferation of VSMC from SHR through the inhibition of ROS and ROS-mediated transactivation of EGF-R/PDGF-R and MAPK signaling pathways.

Role of epidermal growth factor receptor transactivation in endothelin-1-induced enhanced expression of Gi protein and proliferation in A10 vascular smooth muscle cells.

We have recently shown that vasoactive peptides such as angiotensin II (Ang II) and endothelin-1 (ET-1) increase the expression of Gi proteins and the proliferation of A10 vascular smooth muscle cells (VSMC) through mitogen-activated protein (MAP) kinase-phosphoinositide (PI) 3-kinase pathways. This study was intended to examine the implication of epidermal growth factor receptor (EGFR) activation in ET-1-induced enhanced expression of Gi proteins and proliferation of A10 VSMC, and to further investigate the underlying mechanisms responsible for these increases. Cell proliferation was determined by [(3)H]thymidine incorporation and the expression of Gi proteins; extracellular signal-regulated kinases 1 and 2 (ERK1/2) and EGFR phosphorylation was determined by Western blotting. Treatment of A10 VSMC with ET-1 enhanced the expression of Gi proteins, which was attenuated by BQ123 and BQ788, antagonists of ET(A) and ET(B) receptor respectively. In addition, ET-1 enhanced the phosphorylation of EGFR in A10 VSMC, which was restored to the control levels by EGFR inhibitor and ETA and ETB receptor antagonists. Furthermore, ET-1 also augmented the proliferation and ERK1/2 phosphorylation of A10 VSMC, which were restored to the control levels by inhibition of EGFR. These data suggest that ET-1 transactivates EGFR, which, through MAP kinase signaling, may contribute to the enhanced expression of Gi proteins and thus increased proliferation of A10 VSMC.

Implication of multiple signaling pathways in the regulation of angiotensin II induced enhanced expression of Giα proteins in vascular smooth muscle cells.

We have previously shown that A10 vascular smooth muscle cells (VSMC) exposed to angiotensin II (Ang II) exhibited overexpression of Giα proteins. In the present study, we examined the involvement of different signaling pathways in regulating Ang II induced enhanced expression of Giα proteins in VSMC by using pharmacological inhibitors. Ang II induced increased expression of Giα proteins in A10 VSMC was markedly attenuated by actinomycin D, losartan (an AT(1) receptor antagonist), dibutyryl cAMP, phospholipase C (PLC) inhibitor U73122, protein kinase C (PKC) inhibitors staurosporine and GP109203X, but not by PD123319 (an AT(2) receptor antagonist). In addition, BAPTA-AM and TMB-8 (chelators of intracellular Ca(2+)); and nifedipine (a blocker of L-type Ca(2+) channels) significantly inhibited Ang II induced enhanced expression of Giα proteins. On the other hand, extracellular Ca(2+) chelation using EGTA did not affect the Ang II evoked enhanced levels of Giα proteins. Furthermore, pretreatment of A10 VSMC with calmidazolium (an inhibitor of calmodulin), or KN93 (an inhibitor of CaM kinase), or genistein (an inhibitor of protein tyrosine kinase, PTK), also attenuated the increased levels of Giα proteins induced by Ang II. These results suggest that Ang II induced enhanced expression of Giα proteins may be regulated by different signaling pathways through AT(1) receptors in A10 VSMC.

Goα expression in the vomeronasal organ and olfactory bulb of the tammar wallaby.

The vomeronasal organ (VNO) detects pheromones via 2 large families of receptors: vomeronasal receptor 1, associated with the protein Giα2, and vomeronasal receptor 2, associated with Goα. We investigated the distribution of Goα in the developing and adult VNO and adult olfactory bulb of a marsupial, the tammar wallaby. Some cells expressed Goα as early as day 5 postpartum, but by day 30, Goα expressing cells were distributed throughout the receptor epithelium of the VNO. In the adult tammar, Goα appeared to be expressed in sensory neurons whose nuclei were mostly basally located in the vomeronasal receptor epithelium. Goα expressing vomeronasal receptor cells led to all areas of the accessory olfactory bulb (AOB). The lack of regionally restricted projection of the vomeronasal receptor cell type 2 in the tammar was similar to the uniform type, with the crucial difference that the uniform type only shows expression of Giα2 and no expression of Goα. The observed Goα staining pattern suggests that the tammar may have a third accessory olfactory type that could be intermediate to the segregated and uniform types already described.

Hydrogen peroxide enhances the expression of Giα proteins in aortic vascular smooth cells: role of growth factor receptor transactivation.

Oxidative stress has been shown to increase the expression of G(i)α proteins in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats. The present study was undertaken to examine if H(2)O(2), which induces oxidative stress, could also enhance the expression of G(i)α proteins in VSMC and to further explore the underlying signaling pathways responsible for this response. Treatment of VSMC with H(2)O(2) increased the expression of G(i)α proteins and not of G(s)α protein in a concentration- and time-dependent manner. A maximal increase of ∼40-50% was observed at 100 µM and 1 h and was restored to control levels by AG1295 and AG1478, inhibitors of epidermal growth factor receptor (EGF-R) and platelet-derived growth factor receptor (PDGF-R), respectively, and PD98059 and U126, inhibitors of extracellular signal-regulated kinase (ERK1/2), and wortmannin and AKT inhibitor VIII, inhibitors of PKB/AKT, respectively. In addition, H(2)O(2) also increased the phosphorylation of EGF-R, PDGF-R, ERK1/2, and AKT, which was attenuated by the respective inhibitors, whereas the inhibitors of EGF-R and PDGE-R also inhibited the enhanced phosphorylation of ERK1/2 and AKT. Furthermore, transfection of cells with short interfering RNA of EGF-R and PDGF-R restored the H(2)O(2)-induced enhanced expression of G(i)α proteins to control levels. The increased expression of G(i)α proteins was reflected in enhanced G(i) functions as demonstrated by enhanced inhibition of adenylyl cyclase by inhibitory hormones and forskolin-stimulated adenylyl cyclase activity by a low concentration of GTPγS, whereas G(s)α-mediated stimulations of AC were significantly decreased. Furthermore, H(2)O(2)-induced enhanced proliferation of VSMC was attenuated by dibutyryl-cAMP. These results suggest that H(2)O(2) increases the expression of G(i)α proteins in VSMC through the transactivation of EGF-R/PDGF-R and ERK1/2 and phosphatidylinositol-3 kinase signaling pathways.

Desensitization of adenosine A(1) receptors in rat immature cortical neurons.

Adenosine plays an important neuroprotective role in brain, usually mediated by the activation of adenosine A1 receptors. Prolonged activation of a G-protein-coupled receptor generally leads to the partial loss of the responsiveness of receptor-mediated transduction pathways (desensitization). Rat immature cortical neurons were treated with 100 nM⁻N6-phenylisopropyladenosine (R-PIA), a selective A1 receptor agonist, and the effect on adenosine A1 receptor/adenylyl cyclase pathway was studied. Incubation with R-PIA for 6, 12, 24 and 48 h elicited a time-dependent decrease in adenosine A1 receptors in plasma membranes (92, 58, 43 and 26% of control, respectively), which was associated with variations in microsomal fraction (21, 56, 124 and 233% of control, respectively), suggesting the internalization and down-regulation of adenosine A1 receptors. Moreover, real-time PCR assays showed a significant increase in mRNA levels coding adenosine A1 receptor after the longest treatment period (48 h). In addition, αGi1₋2 protein levels detected in microsomes and mRNA levels coding αGi1 protein were increased after 48 h of treatment with R-PIA, suggesting the synthesis of new αGi1 proteins. Finally, adenylyl cyclase inhibition elicited by 2-Chloro-N6-cyclopentyladenosine (CPA), a selective adenosine A1 receptor agonist, was significantly reduced after 12, 24 and 48h of treatment (37, 24 and 23%, respectively) as compared to controls (54%), suggesting the desensitization of adenosine A1 receptor/adenylyl cyclase pathway. These results suggest that adenosine A1 receptors desensitize slowly after prolonged receptor activation in immature cortical neurons, showing mechanisms of desensitization similar to those described not only in fetal but also in adult rat brain.

Enhanced levels of endogenous endothelin-1 contribute to the over expression of Giα protein in vascular smooth muscle cells from SHR: Role of growth factor receptor activation.

We earlier showed that vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) exhibit increased expression of Gi proteins. Since the levels of endothelin-1 (ET-1) are enhanced in VSMC from SHR, we undertook the present study to examine the implication of endogenous ET-1 and the underlying mechanisms in the enhanced expression of Giα proteins in VSMC from SHR. The enhanced expression of Giα-2 and Giα-3 proteins in VSMC from SHR was inhibited by ET(A) and ET(B) receptor antagonists, BQ123 and BQ788 respectively. In addition, these antagonists also attenuated the enhanced inhibition of forskolin-stimulated adenylyl cyclase activity by low concentrations of GTPγS and by inhibitory hormones in VSMC from SHR compared to WKY. Furthermore, AG1295, AG1024 and PP2, inhibitors of platelet derived growth factor receptor (PDGFR), insulin-like growth factor 1 receptor (IGF-1R) and c-Src respectively, inhibited the enhanced expression of Giα protein and the enhanced phosphorylation of PDGFR and IGF-1R in VSMC from SHR to WKY levels. In addition, NAD(P)H oxidase inhibitor DPI and N-acetylcysteine (NAC), a scavenger of superoxide anion (O2⁻) also inhibited the enhanced phosphorylation of PDGFR and IGF-1R and c-Src in VSMC from SHR to control levels. Furthermore, the augmented phosphorylation of ERK1/2 in VSMC from SHR was attenuated by BQ123 and BQ788, growth factor receptors inhibitors and PP2. These results suggest that the enhanced levels of endogenous ET-1 in VSMC from SHR increase oxidative stress, which through c-Src-mediated activation of growth factor receptors and associated MAP kinase signaling, contribute to the enhanced expression of Giα proteins.