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1.
Am J Physiol Endocrinol Metab ; 320(2): E270-E280, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33166186

RESUMO

The G-protein subunits Gqα and G11α (Gq/11α) couple receptors to phospholipase C, leading to increased intracellular calcium. In this study we investigated the consequences of Gq/11α deficiency in the dorsomedial hypothalamus (DMH), a critical site for the control of energy homeostasis. Mice with DMH-specific deletion of Gq/11α (DMHGq/11KO) were generated by stereotaxic injection of adeno-associated virus (AAV)-Cre-green fluorescent protein (GFP) into the DMH of Gqαflox/flox:G11α-/- mice. Compared with control mice that received DMH injection of AAV-GFP, DMHGq/11KO mice developed obesity associated with reduced energy expenditure without significant changes in food intake or physical activity. DMHGq/11KO mice showed no defects in the ability of the melanocortin agonist melanotan II to acutely stimulate energy expenditure or to inhibit food intake. At room temperature (22°C), DMHGq/11KO mice showed reduced sympathetic nervous system activity in brown adipose tissue (BAT) and heart, accompanied with decreased basal BAT uncoupling protein 1 (Ucp1) gene expression and lower heart rates. These mice were cold intolerant when acutely exposed to cold (6°C for 5 h) and had decreased cold-stimulated BAT Ucp1 gene expression. DMHGq/11KO mice also failed to adapt to gradually declining ambient temperatures and to develop adipocyte browning in inguinal white adipose tissue although their BAT Ucp1 was proportionally stimulated. Consistent with impaired cold-induced thermogenesis, the onset of obesity in DMHGq/11KO mice was significantly delayed when housed under thermoneutral conditions (30°C). Thus our results show that Gqα and G11α in the DMH are required for the control of energy homeostasis by stimulating energy expenditure and thermoregulation.NEW & NOTEWORTHY This paper demonstrates that signaling within the dorsomedial hypothalamus via the G proteins Gqα and G11α, which couple cell surface receptors to the stimulation of phospholipase C, is critical for regulation of energy expenditure, thermoregulation by brown adipose tissue and the induction of white adipose tissue browning.


Assuntos
Doenças do Sistema Nervoso Autônomo/genética , Metabolismo Energético/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Hipotálamo/metabolismo , Obesidade/genética , Animais , Doenças do Sistema Nervoso Autônomo/metabolismo , Doenças do Sistema Nervoso Autônomo/fisiopatologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/deficiência , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/metabolismo , Obesidade/fisiopatologia , Especificidade de Órgãos/genética , Sistema Nervoso Simpático/metabolismo , Sistema Nervoso Simpático/fisiopatologia
2.
Thromb Haemost ; 120(11): 1536-1547, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32854120

RESUMO

Platelet activation plays a pivotal role in physiological hemostasis and pathological thrombosis causing heart attack and stroke. Previous studies conclude that simultaneous activation of Gi and G12/13 signaling pathways is sufficient to cause platelet aggregation. However, using Gq knockout mice and Gq-specific inhibitors, we here demonstrated that platelet aggregation downstream of coactivation of Gi and G12/13 depends on agonist concentrations; coactivation of Gi and G12/13 pathways only induces platelet aggregation under higher agonist concentrations. We confirmed Gi and G12/13 pathway activation by showing cAMP (cyclic adenosine monophosphate) decrease and RhoA activation in platelets stimulated at both low and high agonist concentrations. Interestingly, we found that though Akt and PAK (p21-activated kinase) translocate to the platelet membrane upon both low and high agonist stimulation, membrane-translocated Akt and PAK only phosphorylate at high agonist concentrations, correlating well with platelet aggregation downstream of concomitant Gi and G12/13 pathway activation. PAK inhibitor abolishes Akt phosphorylation, inhibits platelet aggregation in vitro and arterial thrombus formation in vivo. We propose that the PAK-PI3K/Akt pathway mediates platelet aggregation downstream of Gi and G12/13, and PAK may represent a potential antiplatelet and antithrombotic target.


Assuntos
Agregação Plaquetária , Transdução de Sinais/fisiologia , Quinases Ativadas por p21/fisiologia , Difosfato de Adenosina/farmacologia , Animais , Forma Celular , Relação Dose-Resposta a Droga , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/fisiologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/deficiência , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Humanos , Camundongos , Camundongos Knockout , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/fisiologia , Ratos , Tromboxano A2/farmacologia , Proteína rhoA de Ligação ao GTP/metabolismo
3.
Synapse ; 69(9): 434-45, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25963901

RESUMO

G(αq) -coupled receptors are ubiquitously expressed throughout the brain and body, and it has been shown that these receptors and associated signaling cascades are involved in a number of functional outputs, including motor function and learning and memory. Genetic alterations to G(αq) have been implicated in neurodevelopmental disorders such as Sturge-Weber syndrome. Some of these associated disease outcomes have been modeled in laboratory animals, but as G(αq) is expressed in all cell types, it is difficult to differentiate the underlying circuitry or causative neuronal population. To begin to address neuronal cell type diversity in G(αq) function, we utilized a conditional knockout mouse whereby G(αq) was eliminated from telencephalic glutamatergic neurons. Unlike the global G(αq) knockout mouse, we found that these conditional knockout mice were not physically different from control mice, nor did they exhibit any gross motor abnormalities. However, similarly to the constitutive knockout animal, G(αq) conditional knockout mice demonstrated apparent deficits in spatial working memory. Loss of G(αq) from glutamatergic neurons also produced enhanced sensitivity to cocaine-induced locomotion, suggesting that cortical G(αq) signaling may limit behavioral responses to psychostimulants. Screening for a variety of markers of forebrain neuronal architecture revealed no obvious differences in the conditional knockouts, suggesting that the loss of G(αq) in telencephalic excitatory neurons does not result in major alterations in brain structure or neuronal differentiation. Taken together, our results define specific modulation of spatial working memory and psychostimulant responses through disruptions in G(αq) signaling within cerebral cortical glutamatergic neurons.


Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/deficiência , Neurônios/metabolismo , Telencéfalo/metabolismo , Animais , Cocaína/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Ácido Glutâmico/metabolismo , Immunoblotting , Imuno-Histoquímica , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Telencéfalo/citologia , Telencéfalo/efeitos dos fármacos
4.
Circ Res ; 116(5): e28-39, 2015 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-25605649

RESUMO

RATIONALE: Sustained activation of Gαq transgenic (Gq) signaling during pressure overload causes cardiac hypertrophy that ultimately progresses to dilated cardiomyopathy. The molecular events that drive hypertrophy decompensation are incompletely understood. Ca(2+)/calmodulin-dependent protein kinase II δ (CaMKIIδ) is activated downstream of Gq, and overexpression of Gq and CaMKIIδ recapitulates hypertrophy decompensation. OBJECTIVE: To determine whether CaMKIIδ contributes to hypertrophy decompensation provoked by Gq. METHODS AND RESULTS: Compared with Gq mice, compound Gq/CaMKIIδ knockout mice developed a similar degree of cardiac hypertrophy but exhibited significantly improved left ventricular function, less cardiac fibrosis and cardiomyocyte apoptosis, and fewer ventricular arrhythmias. Markers of oxidative stress were elevated in mitochondria from Gq versus wild-type mice and respiratory rates were lower; these changes in mitochondrial function were restored by CaMKIIδ deletion. Gq-mediated increases in mitochondrial oxidative stress, compromised membrane potential, and cell death were recapitulated in neonatal rat ventricular myocytes infected with constitutively active Gq and attenuated by CaMKII inhibition. Deep RNA sequencing revealed altered expression of 41 mitochondrial genes in Gq hearts, with normalization of ≈40% of these genes by CaMKIIδ deletion. Uncoupling protein 3 was markedly downregulated in Gq or by Gq expression in neonatal rat ventricular myocytes and reversed by CaMKIIδ deletion or inhibition, as was peroxisome proliferator-activated receptor α. The protective effects of CaMKIIδ inhibition on reactive oxygen species generation and cell death were abrogated by knock down of uncoupling protein 3. Conversely, restoration of uncoupling protein 3 expression attenuated reactive oxygen species generation and cell death induced by CaMKIIδ. Our in vivo studies further demonstrated that pressure overload induced decreases in peroxisome proliferator-activated receptor α and uncoupling protein 3, increases in mitochondrial protein oxidation, and hypertrophy decompensation, which were attenuated by CaMKIIδ deletion. CONCLUSIONS: Mitochondrial gene reprogramming induced by CaMKIIδ emerges as an important mechanism contributing to mitotoxicity in decompensating hypertrophy.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Cardiomegalia/enzimologia , Cardiomiopatia Dilatada/etiologia , Insuficiência Cardíaca/etiologia , Mitocôndrias Cardíacas/fisiologia , Acetilcisteína/farmacologia , Animais , Apoptose , Benzilaminas/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/deficiência , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Cardiomegalia/fisiopatologia , Cardiomiopatia Dilatada/fisiopatologia , Cardiomiopatia Dilatada/prevenção & controle , Células Cultivadas , Progressão da Doença , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/deficiência , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Perfilação da Expressão Gênica , Insuficiência Cardíaca/fisiopatologia , Canais Iônicos/biossíntese , Canais Iônicos/genética , Canais Iônicos/fisiologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/fisiologia , Miócitos Cardíacos/metabolismo , Estresse Oxidativo , PPAR alfa/biossíntese , PPAR alfa/genética , Mutação Puntual , Pressão , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/farmacologia , Ratos , Espécies Reativas de Oxigênio , Análise de Sequência de RNA , Sulfonamidas/farmacologia , Transfecção , Proteína Desacopladora 3
5.
Cell Signal ; 25(5): 1136-48, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23415771

RESUMO

Pasteurella multocida toxin (PMT) is a mitogenic protein that hijacks cellular signal transduction pathways via deamidation of heterotrimeric G proteins. We previously showed that rPMT activates mTOR signaling via a Gαq/11/PLCß/PKC mediated pathway, leading in part to cell proliferation and migration. Herein, we show that mTOR and MAPK, but not membrane-associated tyrosine kinases, are activated in serum-starved 3T3 cells by an autocrine/paracrine substance(s) secreted into the conditioned medium following rPMT treatment. Surprisingly, this diffusible factor(s) is capable of activating mTOR and MAPK pathways even in MEF Gαq/11 double knockout cells. Microarray analysis identified connective tissue growth factor (CTGF) mRNA as the most upregulated gene in rPMT-treated serum-starved 3T3 cells relative to untreated cells. These results were further confirmed using RT-PCR and Western blot analyses. In accord with rPMT-induced mTOR activation, upregulation of CTGF protein was observed in WT MEF, but not in Gαq/11 double knockout MEF cells. Although CTGF expression is regulated by TGFß, rPMT did not activate TGFß pathway. In addition, MEK inhibitors U0126 or PD98059, but not mTOR specific inhibitors, rapamycin and Torin 1, inhibited rPMT-induced upregulation of CTGF. Importantly, CTGF overexpression in serum-starved 3T3 cells using adenovirus led to phosphorylation of ribosomal protein S6, a downstream target of mTOR. However, despite the ability of CTGF to activate the mTOR pathway, upregulation of CTGF alone could not induce morphological changes as those observed in rPMT-treated cells. Our findings reveal that CTGF plays an important role, but there are additional factors involved in the mitogenic action of PMT.


Assuntos
Proteínas de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Regulação para Cima/efeitos dos fármacos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Linhagem Celular , Fator de Crescimento do Tecido Conjuntivo/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/deficiência , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Técnicas de Inativação de Genes , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Células Swiss 3T3 , Serina-Treonina Quinases TOR/antagonistas & inibidores , Ativação Transcricional , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia
6.
Mol Endocrinol ; 22(11): 2520-30, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18801931

RESUMO

GnRH acts on its cognate receptor in pituitary gonadotropes to regulate the biosynthesis and secretion of gonadotropins. It may also have direct extrapituitary actions, including inhibition of cell growth in reproductive malignancies, in which GnRH activation of the MAPK cascades is thought to play a pivotal role. In extrapituitary tissues, GnRH receptor signaling has been postulated to involve coupling of the receptor to different G proteins. We examined the ability of the GnRH receptor to couple directly to Galpha(q/11), Galpha(i/o), and Galpha(s), their roles in the activation of the MAPK cascades, and the subsequent cellular effects. We show that in Galpha(q/11)-negative cells stably expressing the GnRH receptor, GnRH did not induce activation of ERK, jun-N-terminal kinase, or P38 MAPK. In contrast to Galpha(i) or chimeric Galpha(qi5), transfection of Galpha(q) cDNA enabled GnRH to induce phosphorylation of ERK, jun-N-terminal kinase, and P38. Furthermore, no GnRH-mediated cAMP response or inhibition of isoproterenol-induced cAMP accumulation was observed. In another cellular background, [35S]GTPgammaS binding assays confirmed that the GnRH receptor was unable to directly couple to Galpha(i) but could directly interact with Galpha(q/11). Interestingly, GnRH stimulated a marked reduction in cell growth only in cells expressing Galpha(q), and this inhibition could be significantly rescued by blocking ERK activation. We therefore provide direct evidence, in multiple cellular backgrounds, that coupling of the GnRH receptor to Galpha(q/11), but not to Galpha(i/o) or Galpha(s), and consequent activation of ERK plays a crucial role in GnRH-mediated cell death.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Receptores LHRH/metabolismo , Animais , Linhagem Celular , Proliferação de Células , AMP Cíclico/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/deficiência , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Fosforilação , Receptores LHRH/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção
7.
J Mol Cell Cardiol ; 44(1): 143-50, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18021799

RESUMO

Platelet aggregation and secretion play a crucial role in acute coronary syndromes. In Galpha(q) knock-out mice (Galpha(q)(-/-)) platelet function is eliminated in terms of aggregation and secretion of cytokines. We investigated whether restricted platelet aggregation and secretion reduces myocardial infarct size in vivo. Thirty minute regional myocardial ischemia was followed by 24 h reperfusion (I/R) in vivo. Infarct size was determined by counterstaining. Left ventricular function was measured by ultrasound. Infarct size to area at risk ratio was significantly smaller in Galpha(q)(-/-) mice (5.6+/-1.6%) compared to wild-type (WT) mice (27.2+/-3.0%, p<0.01). Fractional shortening was improved in Galpha(q)(-/-) mice compared to WT (42.2+/-1.4% versus 30.5+/-1.4%, respectively, p<0.01). WT mice, transplanted with Galpha(q)(-/-) bone marrow showed a significant reduction in infarct size compared to control (7.8+/-2.2% versus 18.4+/-2.7%, respectively, p<0.01). Platelets of Galpha(q)(-/-) mice had an impaired aggregation and secretion phenotype. In the in vivo model of ischemia and reperfusion, beyond impaired platelet aggregation, platelet secretion plays an additional role in myocardial infarct extension. Blocking platelet aggregation in combination with secretion might be a promising supplementary therapeutic strategy in acute myocardial infarction.


Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/deficiência , Infarto do Miocárdio/fisiopatologia , Animais , Transplante de Medula Óssea , Hipóxia Celular/efeitos dos fármacos , Separação Celular , Sobrevivência Celular/efeitos dos fármacos , Citometria de Fluxo , Camundongos , Camundongos Knockout , Contração Miocárdica/efeitos dos fármacos , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Agregação Plaquetária/efeitos dos fármacos , Testes de Função Plaquetária , Trombina/farmacologia , Fatores de Tempo
8.
Neuropharmacology ; 52(8): 1671-7, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17493641

RESUMO

Extensive evidence suggests that 5-HT2 receptors may play a role in mental disorders including schizophrenia. In addition, several studies indicate that G(q)-coupled 5-HT(2A) receptors are likely targets for the initiation of events leading to the hallucinogenic behavior elicited by lysergic acid diethylamide (LSD), (+/-)1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), and related drugs. However, 5-HT(2A) receptors couple to other G proteins in addition to G(q) protein. To evaluate the role of the G(q) signaling pathway in DOI-induced behaviors, we utilized two behavioral models of 5-HT(2A) receptor activation: induction of head-twitches by DOI, a common response to hallucinogenic drugs in rodents, and DOI elicited anxiolytic-like effects in the elevated plus maze. Experimental subjects were genetically modified mice [Galpha(q)(-/-)] in which the G(q) alpha gene was eliminated. Galpha(q)(-/-) mice exhibited a decrease in DOI-induced head-twitches, when compared to wild-type littermates. In addition, the DOI-induced increase in anxiolytic-like behavior was abolished in Galpha(q)(-/-) mice. These results, combined with our finding that DOI-induced FOS expression in the medial prefrontal cortex was also eliminated in Galpha(q)(-/-) mice, suggests a key role for G(q) protein in hallucinogenic drug effects.


Assuntos
Comportamento Animal/efeitos dos fármacos , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Alucinógenos/farmacologia , Indofenol/análogos & derivados , Análise de Variância , Animais , Comportamento Animal/fisiologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Relação Dose-Resposta a Droga , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/deficiência , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Movimentos da Cabeça/efeitos dos fármacos , Indofenol/farmacologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Oncogênicas v-fos/metabolismo , Ensaio Radioligante/métodos
9.
Blood ; 105(1): 178-85, 2005 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-15367435

RESUMO

To test the hypothesis that platelet activation contributes to tumor dissemination, we studied metastasis in mice lacking Galphaq, a G protein critical for platelet activation. Loss of platelet activation resulted in a profound diminution in both experimental and spontaneous metastases. Analyses of the distribution of radiolabeled tumor cells demonstrated that platelet function, like fibrinogen, significantly improved the survival of circulating tumor cells in the pulmonary vasculature. More detailed studies showed that the increase in metastatic success conferred by either platelets or fibrinogen was linked to natural killer cell function. Specifically, the pronounced reduction in tumor cell survival observed in fibrinogen- and Galphaq-deficient mice relative to control animals was eliminated by the immunologic or genetic depletion of natural killer cells. These studies establish an important link between hemostatic factors and innate immunity and indicate that one mechanism by which the platelet-fibrin(ogen) axis contributes to metastatic potential is by impeding natural killer cell elimination of tumor cells.


Assuntos
Plaquetas/fisiologia , Fibrina/metabolismo , Fibrinogênio/metabolismo , Células Matadoras Naturais/imunologia , Neoplasias/imunologia , Neoplasias/patologia , Animais , Sobrevivência Celular , Fibrina/deficiência , Fibrina/genética , Fibrinogênio/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/deficiência , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Knockout , Metástase Neoplásica , Neoplasias/genética , Ativação Plaquetária , Trombose/genética , Trombose/metabolismo , Trombose/patologia
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