RESUMO
OBJECTIVE: 12-LOX (12-lipoxygenase) produces a number of bioactive lipids including 12(S)-HETE that are involved in inflammation and platelet reactivity. The GPR31 (G-protein-coupled receptor 31) is the proposed receptor of 12(S)-HETE; however, it is not known whether the 12(S)-HETE-GPR31 signaling axis serves to enhance or inhibit platelet activity. Approach and Results: Using pepducin technology and biochemical approaches, we provide evidence that 12(S)-HETE-GPR31 signals through Gi to enhance PAR (protease-activated receptor)-4-mediated platelet activation and arterial thrombosis using both human platelets and mouse carotid artery injury models. 12(S)-HETE suppressed AC (adenylyl cyclase) activity through GPR31 and resulted in Rap1 (Ras-related protein 1) and p38 activation and low but detectable calcium flux but did not induce platelet aggregation. A GPR31 third intracellular (i3) loop-derived pepducin, GPR310 (G-protein-coupled receptor 310), significantly inhibited platelet aggregation in response to thrombin, collagen, and PAR4 agonist, AYPGKF, in human and mouse platelets but relative sparing of PAR1 agonist SFLLRN in human platelets. GPR310 treatment gave a highly significant 80% protection (P=0.0018) against ferric chloride-induced carotid artery injury in mice by extending occlusion time, without any effect on tail bleeding. PAR4-mediated dense granule secretion and calcium flux were both attenuated by GPR310. Consistent with these results, GPR310 inhibited 12(S)-HETE-mediated and PAR4-mediated Rap1-GTP and RASA3 translocation to the plasma membrane and attenuated PAR4-Akt and ERK activation. GPR310 caused a right shift in thrombin-mediated human platelet aggregation, comparable to the effects of inhibition of the Gi-coupled P2Y12 receptor. Co-immunoprecipitation studies revealed that GPR31 and PAR4 form a heterodimeric complex in recombinant systems. CONCLUSIONS: The 12-LOX product 12(S)-HETE stimulates GPR31-Gi-signaling pathways, which enhance thrombin-PAR4 platelet activation and arterial thrombosis in human platelets and mouse models. Suppression of this bioactive lipid pathway, as exemplified by a GPR31 pepducin antagonist, may provide beneficial protective effects against platelet aggregation and arterial thrombosis with minimal effect on hemostasis.
Assuntos
Plaquetas/metabolismo , Trombose das Artérias Carótidas/sangue , Hemostasia , Agregação Plaquetária , Receptores Acoplados a Proteínas G/sangue , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/sangue , Animais , Células CHO , Trombose das Artérias Carótidas/prevenção & controle , Cricetulus , Modelos Animais de Doenças , Feminino , Fibrinolíticos/farmacologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/sangue , Humanos , Masculino , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores de Trombina/sangue , Transdução de Sinais , Trombina/metabolismoRESUMO
Background and objectives: Alzheimer's disease (AD) is a progressive neurodegenerative disease that results in severe dementia. Having ischemic strokes (IS) is one of the risk factors of the AD, but the molecular mechanisms that underlie IS and AD are not well understood. We thus aimed to identify common molecular biomarkers and pathways in IS and AD that can help predict the progression of these diseases and provide clues to important pathological mechanisms. Materials and Methods: We have analyzed the microarray gene expression datasets of IS and AD. To obtain robust results, combinatorial statistical methods were used to analyze the datasets and 26 transcripts (22 unique genes) were identified that were abnormally expressed in both IS and AD. Results: Gene Ontology (GO) and KEGG pathway analyses indicated that these 26 common dysregulated genes identified several altered molecular pathways: Alcoholism, MAPK signaling, glycine metabolism, serine metabolism, and threonine metabolism. Further protein-protein interactions (PPI) analysis revealed pathway hub proteins PDE9A, GNAO1, DUSP16, NTRK2, PGAM2, MAG, and TXLNA. Transcriptional and post-transcriptional components were then identified, and significant transcription factors (SPIB, SMAD3, and SOX2) found. Conclusions: Protein-drug interaction analysis revealed PDE9A has interaction with drugs caffeine, γ-glutamyl glycine, and 3-isobutyl-1-methyl-7H-xanthine. Thus, we identified novel putative links between pathological processes in IS and AD at transcripts levels, and identified possible mechanistic and gene expression links between IS and AD.
Assuntos
Doença de Alzheimer/sangue , Biomarcadores/sangue , Isquemia Encefálica/sangue , 3',5'-AMP Cíclico Fosfodiesterases/análise , 3',5'-AMP Cíclico Fosfodiesterases/sangue , Doença de Alzheimer/complicações , Biomarcadores/análise , Isquemia Encefálica/complicações , Fosfatases de Especificidade Dupla/análise , Fosfatases de Especificidade Dupla/sangue , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/análise , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/sangue , Humanos , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/sangue , Fosfatases da Proteína Quinase Ativada por Mitógeno/análise , Fosfatases da Proteína Quinase Ativada por Mitógeno/sangue , Glicoproteína Associada a Mielina/análise , Glicoproteína Associada a Mielina/sangue , Receptor trkB/análise , Receptor trkB/sangue , Transdução de Sinais/fisiologia , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/complicações , Proteínas de Transporte Vesicular/análise , Proteínas de Transporte Vesicular/sangueRESUMO
BACKGROUND: Inherited platelet function disorders (PFDs) are heterogeneous, and identification of the underlying genetic defects is difficult when based solely on phenotypic and clinical features of the patient. OBJECTIVE: To analyze 329 genes regulating platelet function, number, and size in order to identify candidate gene defects in patients with PFDs. PATIENTS/METHODS: Targeted analysis of candidate PFD genes was undertaken after next-generation sequencing of exomic DNA from 18 unrelated index cases with PFDs who were recruited into the UK Genotyping and Phenotyping of Platelets (GAPP) study and diagnosed with platelet abnormalities affecting either Gi signaling (n = 12) or secretion (n = 6). The potential pathogenicity of candidate gene defects was assessed using computational predictive algorithms. RESULTS: Analysis of the 329 candidate PFD genes identified 63 candidate defects, affecting 40 genes, among index cases with Gi signaling abnormalities, while 53 defects, within 49 genes, were identified among patients with secretion abnormalities. Homozygous gene defects were more commonly associated with secretion abnormalities. Functional annotation analysis identified distinct gene clusters in the two patient subgroups. Thirteen genes with significant annotation enrichment for 'intracellular signaling' harbored 16 of the candidate gene defects identified in nine index cases with Gi signaling abnormalities. Four gene clusters, representing 14 genes, with significantly associated gene ontology annotations were identified among the cases with secretion abnormalities, the most significant association being with 'establishment of protein localization.' CONCLUSION: Our findings demonstrate the genetic complexity of PFDs and highlight plausible candidate genes for targeted analysis in patients with platelet secretion and Gi signaling abnormalities.
Assuntos
Transtornos Plaquetários/genética , Análise Mutacional de DNA , Testes Genéticos/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Polimorfismo de Nucleotídeo Único , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Algoritmos , Transtornos Plaquetários/sangue , Transtornos Plaquetários/diagnóstico , Plaquetas/metabolismo , Criança , Análise por Conglomerados , Biologia Computacional , Exoma , Feminino , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/sangue , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Estudos de Associação Genética , Marcadores Genéticos , Predisposição Genética para Doença , Hereditariedade , Heterozigoto , Homozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Valor Preditivo dos Testes , Transdução de Sinais/genética , Reino Unido , Adulto JovemRESUMO
This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the prognosis of idiopathic scoliosis in asymptomatic and affected children. The assay consists of the evaluation of the functional status of Gi and Gs proteins in peripheral blood mononuclear cells (PBMCs) by cellular dielectric spectroscopy (CDS), using an automated CDS-based instrument, and the classification of children into three functional groups (FG1, FG2, FG3) with respect to the profile of imbalance between the degree of response to Gi and Gs proteins stimulation. The classification is further confirmed by the differential effect of osteopontin (OPN) on response to Gi stimulation among groups and the severe progression of disease is referenced by FG2. Approximately, a volume of 10 ml of blood is required to extract PBMCs by Ficoll-gradient and cells are then stored in liquid nitrogen. The adequate number of PBMCs to perform the assay is obtained after two days of cell culture. Essentially, cells are first incubated with phytohemmaglutinin (PHA). After 24 hr incubation, medium is replaced by a PHA-free culture medium for an additional 24 hr prior to cell seeding and OPN treatment. Cells are then spectroscopically screened for their responses to somatostatin and isoproterenol, which respectively activate Gi and Gs proteins through their cognate receptors. Both somatostatin and isoproterenol are simultaneously injected with an integrated fluidics system and the cells' responses are monitored for 15 min. The assay can be performed with fresh or frozen PBMCs and the procedure is completed within 4 days.
Assuntos
Escoliose/diagnóstico , Análise Espectral/métodos , Estudos de Casos e Controles , Criança , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/sangue , Subunidades alfa Gs de Proteínas de Ligação ao GTP/sangue , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/patologia , Osteopontina/farmacologia , Prognóstico , Escoliose/sangue , Escoliose/patologiaRESUMO
BACKGROUND: Takotsubo cardiomyopathy is an acute heart failure syndrome characterized by myocardial hypocontractility from the mid left ventricle to the apex. It is precipitated by extreme stress and can be triggered by intravenous catecholamine administration, particularly epinephrine. Despite its grave presentation, Takotsubo cardiomyopathy is rapidly reversible, with generally good prognosis. We hypothesized that this represents switching of epinephrine signaling through the pleiotropic ß(2)-adrenergic receptor (ß(2)AR) from canonical stimulatory G-protein-activated cardiostimulant to inhibitory G-protein-activated cardiodepressant pathways. METHODS AND RESULTS: We describe an in vivo rat model in which a high intravenous epinephrine, but not norepinephrine, bolus produces the characteristic reversible apical depression of myocardial contraction coupled with basal hypercontractility. The effect is prevented via G(i) inactivation by pertussis toxin pretreatment. ß(2)AR number and functional responses were greater in isolated apical cardiomyocytes than in basal cardiomyocytes, which confirmed the higher apical sensitivity and response to circulating epinephrine. In vitro studies demonstrated high-dose epinephrine can induce direct cardiomyocyte cardiodepression and cardioprotection in a ß(2)AR-Gi-dependent manner. Preventing epinephrine-G(i) effects increased mortality in the Takotsubo model, whereas ß-blockers that activate ß(2)AR-G(i) exacerbated the epinephrine-dependent negative inotropic effects without further deaths. In contrast, levosimendan rescued the acute cardiac dysfunction without increased mortality. CONCLUSIONS: We suggest that biased agonism of epinephrine for ß(2)AR-G(s) at low concentrations and for G(i) at high concentrations underpins the acute apical cardiodepression observed in Takotsubo cardiomyopathy, with an apical-basal gradient in ß(2)ARs explaining the differential regional responses. We suggest this epinephrine-specific ß(2)AR-G(i) signaling may have evolved as a cardioprotective strategy to limit catecholamine-induced myocardial toxicity during acute stress.
Assuntos
Modelos Animais de Doenças , Epinefrina/sangue , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Receptores Adrenérgicos beta 2/fisiologia , Cardiomiopatia de Takotsubo/sangue , Animais , Antiarrítmicos/administração & dosagem , Antiarrítmicos/sangue , Células Cultivadas , Epinefrina/administração & dosagem , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/agonistas , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/sangue , Humanos , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
PI3Ks (phosphoinositide 3-kinases) play a critical role in platelet functional responses. PI3Ks are activated upon P2Y12 receptor stimulation and generate pro-aggregatory signals. P2Y12 receptor has been shown to play a key role in the platelet aggregation and thromboxane A2 generation caused by co-stimulation with Gq or Gz, or super-stimulation of Gi pathways. In the present study, we evaluated the role of specific PI3K isoforms alpha, beta, gamma and delta in platelet aggregation, thromboxane A2 generation and ERK (extracellular-signal-regulated kinase) activation. Our results show that loss of the PI3K signal impaired the ability of ADP to induce platelet aggregation, ERK phosphorylation and thromboxane A2 generation. We also show that Gq plus Gi- or Gi plus Gz-mediated platelet aggregation, ERK phosphorylation and thromboxane A2 generation in human platelets was inhibited by TGX-221, a PI3Kbeta-selective inhibitor, but not by PIK75 (a PI3Kalpha inhibitor), AS252424 (a PI3Kgamma inhibitor) or IC87114 (a PI3Kdelta inhibitor). TGX-221 also showed a similar inhibitory effect on the Gi plus Gz-mediated platelet responses in platelets from P2Y1-/- mice. Finally, 2MeSADP (2-methyl-thio-ADP)-induced Akt phosphorylation was significantly inhibited in the presence of TGX-221, suggesting a critical role for PI3Kbeta in Gi-mediated signalling. Taken together, our results demonstrate that PI3Kbeta plays an important role in ADP-induced platelet aggregation. Moreover, PI3Kbeta mediates ADP-induced thromboxane A2 generation by regulating ERK phosphorylation.
Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/sangue , Fosfatidilinositol 3-Quinases/sangue , Agregação Plaquetária/fisiologia , Tromboxano A2/sangue , Difosfato de Adenosina/farmacologia , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Humanos , Técnicas In Vitro , Isoenzimas/antagonistas & inibidores , Isoenzimas/sangue , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Knockout , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Agregação Plaquetária/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Pirimidinonas/farmacologia , Receptores Purinérgicos P2/deficiência , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2Y1 , Transdução de Sinais/efeitos dos fármacosRESUMO
In non-erythroid cells, insulin stimulates a signal transduction pathway that results in the activation of phosphoinositide 3-kinase (PI3K) and subsequent phosphorylation of phosphodiesterase 3 (PDE3). Erythrocytes possess insulin receptors, PI3K and PDE3B. These cells release adenosine triphosphate (ATP) when exposed to reduced O(2) tension via a signaling pathway that requires activation of the G protein, Gi, as well as increases in cAMP. Although insulin inhibits ATP release from human erythrocytes in response to Gi activation by mastoparan 7 (Mas 7), no effect on cAMP was described. Here, we investigated the hypothesis that insulin activates PDE3 in human erythrocytes via a PI3K-mediated mechanism resulting in cAMP hydrolysis and inhibition of ATP release. Incubation of human erythrocytes with Mas 7 resulted in a 62 +/- 7% increase in cAMP (n = 9, P < 0.05) and a 306 +/- 69% increase in ATP release (n = 9, P < 0.05), both of which were attenuated by pre-treatment with insulin. Selective inhibitors of PDE3 (cilostazol) or PI3K (LY294002) rescued these effects of insulin. These results support the hypothesis that insulin activates PDE3 in erythrocytes via a PI3K-dependent mechanism. Once activated, PDE3 limits Mas 7-induced increases in intracellular cAMP. This effect of insulin leads, ultimately, to decreased ATP release in response to Mas 7. Activation of Gi is required for reduced O(2) tension-induced ATP release from erythrocytes and this ATP release has been shown to participate in the matching of O(2) supply with demand in skeletal muscle. Thus, pathological increases in circulating insulin could, via activation of PDE3 in erythrocytes, inhibit ATP release from these cells, depriving the peripheral circulation of one mechanism that could aid in the regulation of the delivery of O(2) to meet tissue metabolic need.
Assuntos
Trifosfato de Adenosina/sangue , AMP Cíclico/sangue , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/sangue , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Insulina/farmacologia , Fosfatidilinositol 3-Quinases/sangue , Adulto , Cromonas/farmacologia , Cilostazol , Inibidores Enzimáticos/farmacologia , Feminino , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/sangue , Humanos , Técnicas In Vitro , Insulina/sangue , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Morfolinas/farmacologia , Oxigênio/sangue , Peptídeos/farmacologia , Inibidores da Fosfodiesterase 3 , Inibidores de Fosfoinositídeo-3 Quinase , Transdução de Sinais/efeitos dos fármacos , Tetrazóis/farmacologiaRESUMO
Second messenger-mediated inside-out activation of integrin alphaIIbbeta3 is a key step in platelet aggregation. We recently showed strongly impaired but not absent alphaIIbbeta3-mediated aggregation of CalDAG-GEFI-deficient platelets activated with various agonists. Here we further evaluated the roles of CalDAG-GEFI and protein kinase C (PKC) for alphaIIbbeta3 activation in platelets activated with a PAR4 receptor-specific agonist, GYPGKF (PAR4p). Compared with wild-type controls, platelets treated with the PKC inhibitor Ro31-8220 or CalDAG-GEFI-deficient platelets showed a marked defect in aggregation at low (< 1mM PAR4p) but not high PAR4p concentrations. Blocking of PKC function in CalDAG-GEFI-deficient platelets, how-ever, strongly decreased aggregation at all PAR4p concentrations, demonstrating that CalDAG-GEFI and PKC represent separate, but synergizing, pathways important for alphaIIbbeta3 activation. PAR4p-induced aggregation in the absence of CalDAG-GEFI required cosignaling through the Galphai-coupled receptor for ADP, P2Y12. Independent roles for CalDAG-GEFI and PKC/Galphai signaling were also observed for PAR4p-induced activation of the small GTPase Rap1, with CalDAG-GEFI mediating the rapid but reversible activation of this small GTPase. In summary, our study identifies CalDAG-GEFI and PKC as independent pathways leading to Rap1 and alphaIIbbeta3 activation in mouse platelets activated through the PAR4 receptor.
Assuntos
Plaquetas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/sangue , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Proteína Quinase C/sangue , Difosfato de Adenosina/sangue , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Degranulação Celular/efeitos dos fármacos , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/sangue , Fatores de Troca do Nucleotídeo Guanina/deficiência , Fatores de Troca do Nucleotídeo Guanina/genética , Cinética , Camundongos , Camundongos Knockout , Modelos Biológicos , Oligopeptídeos/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/fisiologia , Proteína Quinase C/antagonistas & inibidores , Receptores de Trombina/sangue , Receptores de Trombina/efeitos dos fármacos , Transdução de Sinais , Proteínas rap1 de Ligação ao GTP/sangueRESUMO
Human erythrocytes, by virtue of their ability to release ATP in response to physiological stimuli, have been proposed to participate in the regulation of local blood flow. A signal transduction pathway that relates these stimuli to ATP release has been described and includes the heterotrimeric G protein G(i) and adenylyl cyclase (AC). In this cell, G(i) activation results in increases in cAMP and, ultimately, ATP release. It has been reported that G(i) expression is decreased in animal models of diabetes and in platelets of humans with type 2 diabetes. Here, we report that G(i2) expression is selectively decreased in erythrocytes of humans with type 2 diabetes and that this defect is associated with reductions in cAMP accumulation and ATP release in response to incubation of erythrocytes with mastoparan 7 (10 micromol/l), an activator of G(i). Importantly, this defect in ATP release correlates inversely with the adequacy of glycemic control as determined by levels of HbA(1c) (A1C). These results demonstrate that in erythrocytes of humans with type 2 diabetes, both G(i) expression and ATP release in response to mastoparan 7 are impaired, which is consistent with the hypothesis that this defect in erythrocyte physiology could contribute to the vascular disease associated with this clinical condition.
Assuntos
Trifosfato de Adenosina/sangue , AMP Cíclico/sangue , Diabetes Mellitus Tipo 2/sangue , Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/sangue , Hemoglobinas Glicadas , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos/farmacologia , Valores de ReferênciaRESUMO
Major depression is associated with the upregulation of alpha(2A)-adrenoceptors in brain tissue and blood platelets. The homologous regulation of these receptors by G-protein-coupled receptor kinases (GRKs) might play a relevant role in the pathogenesis and treatment of depression. This study was designed to assess the status of the complex alpha(2A)-adrenoceptor/Galphai/GRK 2 in the platelets of depressed patients (n=22) before and after treatment with the antidepressant mirtazapine, an antagonist at alpha(2A)-adrenoceptors (30-45 mg/day for up to 6 months). A second series of depressed suicide attempters (n=32) were also investigated to further assess the status of platelet GRK 2 and GRK 6. Platelet alpha(2A)-adrenoceptors and Galphai protein immunoreactivities were increased in depressed patients (49 and 35%) compared with matched controls. In contrast, GRK 2 content was decreased in the two series of depressed patients (27 and 28%). GRK 6 (a GRK with different properties) was found unchanged. In drug-free depressed patients, the severity of depression (behavioral ratings with two different instruments) correlated inversely with the content of platelet GRK 2 (r=-0.46, n=22, p=0.032, and r=-0.55, n=22, p=0.009). After 4-24 weeks of treatment, mirtazapine induced downregulation of platelet alpha(2A)-adrenoceptors (up to 34%) and Galphai proteins (up to 28%), and the upregulation of GRK 2 (up to 30%). The results indicate that major depression is associated with reduced platelet GRK 2, suggesting that a defect of this kinase may contribute to the observed upregulation of alpha(2A)-adrenoceptors. Moreover, treatment with mirtazapine reversed this abnormality and induced downregulation of alpha(2A)-adrenoceptor/Galphai complex. The results support a role of supersensitive alpha(2A)-adrenoceptors in the pathogenesis and treatment of major depression.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/sangue , Transtorno Depressivo Maior/sangue , Transtorno Depressivo Maior/tratamento farmacológico , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/sangue , Mianserina/análogos & derivados , Mianserina/uso terapêutico , Receptor A2A de Adenosina/sangue , Adulto , Análise de Variância , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Distribuição de Qui-Quadrado , Transtorno Depressivo Maior/psicologia , Feminino , Humanos , Masculino , Mianserina/farmacologia , Pessoa de Meia-Idade , Mirtazapina , Tentativa de Suicídio/estatística & dados numéricos , Quinases de Receptores Adrenérgicos betaRESUMO
The serine-threonine kinase Akt has been established as an important signaling intermediate in regulating cell survival, cell cycle progression, as well as agonist-induced platelet activation. Stimulation of platelets with various agonists including thrombin results in Akt activation. As thrombin can stimulate multiple G protein signaling pathways, we investigated the mechanism of thrombin-induced activation of Akt. Stimulation of platelets with a PAR1-activating peptide (SFLLRN), PAR4-activating peptide (AYPGKF), and thrombin resulted in Thr308 and Ser473 phosphorylation of Akt, which results in its activation. This phosphorylation and activation of Akt were dramatically inhibited in the presence of AR-C69931MX, a P2Y12 receptor-selective antagonist, or GF 109203X, a protein kinase C inhibitor, but Akt phosphorylation was restored by supplemental Gi or Gz signaling. Unlike wild-type mouse platelets, platelets from Galphaq-deficient mice failed to trigger Akt phosphorylation by thrombin and AYPGKF, whereas Akt phosphorylation was not affected by these agonists in platelets from mice that lack P2Y1 receptor. However, ADP caused Akt phosphorylation in Galphaq- and P2Y1-deficient platelets, which was completely blocked by AR-C69931MX. In contrast, ADP failed to cause Akt phosphorylation in platelets from mice treated with clopidogrel, and thrombin and AYPGKF induced minimal phosphorylation of Akt, which was not affected by AR-C69931MX in these platelets. These data demonstrate that Gi, but not Gq or G12/13, signaling pathways are required for activation of Akt in platelets, and Gi signaling pathways, stimulated by secreted ADP, play an essential role in the activation of Akt in platelets.
Assuntos
Plaquetas/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/sangue , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/sangue , Ticlopidina/análogos & derivados , Difosfato de Adenosina/farmacologia , Animais , Plaquetas/efeitos dos fármacos , Clopidogrel , Ativação Enzimática/efeitos dos fármacos , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/deficiência , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Humanos , Técnicas In Vitro , Camundongos , Camundongos Knockout , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Fosforilação , Proteína Quinase C/sangue , Proteínas Proto-Oncogênicas c-akt , Receptores Purinérgicos P2/sangue , Receptores Purinérgicos P2/deficiência , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2Y1 , Transdução de Sinais , Trombina/farmacologia , Ticlopidina/farmacologiaRESUMO
Alterations of cellular G proteins have been implicated in the pathophysiology of some psychiatric disorders. So far, no study assessed G protein function in anorexia nervosa (AN) and bulimia nervosa (BN). Therefore, we measured immunoreactive levels of G(alpha s), G(alpha i), G(alpha q/11) and G(beta) protein subunits in mononuclear leukocytes of 71 drug-free women, including 25 subjects with AN, 26 individuals with BN and 20 healthy controls. As compared to healthy women, anorexic patients exhibited significantly increased levels of G(alpha i) and G(beta) proteins, while bulimic patients had significantly increased levels of G(alpha s), G(alpha i) and G(beta) proteins. Immunoreactive levels of peripheral G protein subunits were not significantly correlated with demographic or nutritional parameters. These findings, although obtained in peripheral blood cells, may suggest a derangement of G protein-mediated signal transduction in the pathophysiology of eating disorders.
Assuntos
Anorexia Nervosa/sangue , Bulimia/sangue , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/sangue , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/sangue , Subunidades alfa Gs de Proteínas de Ligação ao GTP/sangue , Subunidades beta da Proteína de Ligação ao GTP/sangue , Leucócitos Mononucleares/química , Adolescente , Adulto , Anorexia Nervosa/fisiopatologia , Western Blotting , Bulimia/fisiopatologia , Feminino , Humanos , Índice de Gravidade de Doença , Transdução de SinaisRESUMO
We have examined platelet functional responses and characterized a novel signaling defect in the platelets of a patient suffering from a chronic bleeding disorder. Platelet aggregation responses stimulated by weak agonists such as adenosine diphosphate (ADP) and adrenaline were severely impaired. In comparison, both aggregation and dense granule secretion were normal following activation with high doses of collagen, thrombin, or phorbol-12 myristate-13 acetate (PMA). ADP, thrombin, or thromboxane A2 (TxA2) signaling through their respective Gq-coupled receptors was normal as assessed by measuring either mobilization of intracellular calcium, diacylglycerol (DAG) generation, or pleckstrin phosphorylation. In comparison, Gi-mediated signaling induced by either thrombin, ADP, or adrenaline, examined by suppression of forskolin-stimulated rise in cyclic AMP (cAMP) was impaired, indicating dysfunctional Galphai signaling. Immunoblot analysis of platelet membranes with specific antiserum against different Galpha subunits indicated normal levels of Galphai2,Galphai3,Galphaz, and Galphaq in patient platelets. However, the Galphai1level was reduced to 25% of that found in normal platelets. Analysis of platelet cDNA and gDNA revealed no abnormality in either the Galphai1 or Galphai2 gene sequences. Our studies implicate the minor expressed Galphai subtype Galphai1 as having an important role in regulating signaling pathways associated with the activation of alphaIIbbeta3 and subsequent platelet aggregation by weak agonists.
Assuntos
Transtornos Plaquetários/etiologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Transdução de Sinais , Difosfato de Adenosina/farmacologia , Adulto , Ácido Araquidônico/sangue , Transtornos Plaquetários/genética , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Cálcio/sangue , Radioisótopos de Carbono , Membrana Celular/química , Colforsina/farmacologia , Colágeno/farmacologia , AMP Cíclico/sangue , DNA Complementar/química , Diglicerídeos/sangue , Epinefrina/farmacologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/sangue , Humanos , Masculino , Monócitos/química , Radioisótopos de Fósforo , Agregação Plaquetária/efeitos dos fármacos , Análise de Sequência de DNA , Serotonina/sangue , Trombina/farmacologia , Tromboxano B2/sangue , TrítioRESUMO
Glycoprotein (GP) VI is a critical platelet collagen receptor, yet the steps involved in GPVI-mediated platelet activation remain incompletely understood. Because activation of Rap1, an abundant small guanosine triphosphatase (GTPase) in platelets, contributes to integrin alpha(IIb)beta(3) activation, we asked whether and how GPVI signaling activates Rap1 in platelets. Here we show that platelet Rap1 is robustly activated upon addition of convulxin, a GPVI-specific agonist. Using a reconstituted system in RBL-2H3 cells, we found that GPVI-mediated Rap1 activation is dependent on FcRgamma but independent of another platelet collagen receptor, alpha(2)beta(1). Interestingly, GPVI-mediated Rap1 activation in human platelets is largely dependent on adenosine diphosphate (ADP) signaling through the P2Y(12) and not the P2Y(1) receptor. However, experiments with specific ADP receptor antagonists and platelets from knockout mice deficient in P2Y(1) or the P2Y(12)-associated G-protein, Galphai(2), indicate that human and murine platelets also have a significant P2Y(12)-independent component of GPVI-mediated Rap1 activation. The P2Y(12)-independent component is dependent on phosphatidylinositol 3-kinase and is augmented by epinephrine-mediated signaling. P2Y(12)-dependent and -independent components are also observed in GPVI-mediated platelet aggregation, further supporting a role for Rap1 in aggregation. These results define mechanisms of GPVI-mediated platelet activation and implicate Rap1 as a key signaling protein in GPVI-induced platelet signaling.
Assuntos
Plaquetas/fisiologia , Lectinas Tipo C , Proteínas de Membrana , Glicoproteínas da Membrana de Plaquetas/fisiologia , Receptores Purinérgicos P2/fisiologia , Proteínas rap1 de Ligação ao GTP/sangue , Difosfato de Adenosina/metabolismo , Animais , Plaquetas/enzimologia , Venenos de Crotalídeos/farmacologia , Ativação Enzimática/efeitos dos fármacos , Epinefrina/fisiologia , Subunidade alfa Gi2 de Proteína de Ligação ao GTP , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/sangue , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/deficiência , Humanos , Integrina alfa2beta1/sangue , Camundongos , Camundongos Knockout , Fosfatidilinositol 3-Quinases/sangue , Agregação Plaquetária , Proteínas Proto-Oncogênicas/sangue , Proteínas Proto-Oncogênicas/deficiência , Antagonistas do Receptor Purinérgico P2 , Receptores de IgG/sangue , Receptores Purinérgicos P2/sangue , Receptores Purinérgicos P2/deficiência , Receptores Purinérgicos P2Y1 , Receptores Purinérgicos P2Y12 , Transdução de SinaisRESUMO
Chronic use of beta2-agonists and increased production of inflammatory mediators during the late allergic reaction after the antigen challenge result in the desensitization of beta-adrenoceptors in the airways with an accompanying rise in non-specific airway hyperresponsiveness. Several proinflammatory cytokines, including interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), play a significant role in orchestrating and perpetuating the inflammatory response and induce the decreased response to bronchodilators in vitro. However, the underlying mechanisms are unknown. In this study, we examined the effect of two cytokines, IL-1beta and TNF-alpha, on the expression of guanine nucleotide binding regulatory proteins (G-proteins), Gs alpha and Gi alpha-3, by Western blotting in the CD4+ cells of nonatopic nonasthmatic (NANA), atopic nonasthmatic (ANA), and atopic asthmatic (AA) subjects. In the purified CD4+ cells, the basal expression of Gs alpha was higher in the ANA group, and significantly lower in the AA group as compared to the NANA group. The basal expression of Gi alpha-3 was significantly greater (3-15 fold) than Gs alpha, with no significant difference between any of the three groups. Both cytokines IL-1beta and TNF-alpha significantly decreased the expression of Gs alpha in the CD4+ cells of the NANA and ANA groups, with no effect in the AA group. However, these cytokines increased the expression of Gi alpha-3, proteins in the AA group, but had no effect in the CD4+ cells of the NANA and ANA groups. These data suggest that a decreased response to beta2-agonists in the late allergic response in allergic asthmatic subjects could be due to the release of inflammatory cytokines, which induce a decrease in the stimulatory G-proteins and an increase in the inhibitory G-proteins.
Assuntos
Asma/sangue , Linfócitos T CD4-Positivos/metabolismo , Proteínas de Ligação ao GTP/sangue , Hipersensibilidade/sangue , Interleucina-1/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Adulto , Idoso , Asma/etiologia , Feminino , Subunidade alfa Gi2 de Proteína de Ligação ao GTP , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/sangue , Subunidades alfa Gs de Proteínas de Ligação ao GTP/sangue , Humanos , Hipersensibilidade/complicações , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas/sangue , Valores de ReferênciaRESUMO
OBJECTIVE: We have previously demonstrated an augmented activation of Gialpha proteins in heart and aorta from spontaneously hypertensive rats (SHRs), which was attributed to an enhanced expression of Gialpha proteins. Since immortalized lymphoblasts derived from lymphocytes of hypertensive patients have been shown to have enhanced Gi activation, the present studies were undertaken to investigate if lymphocytes from SHRs also exhibit enhanced Gi activation and whether this activation is related to enhanced expression of Gi proteins. METHODS: The levels of G-proteins and mRNA were determined by immunoblotting and Northern blotting techniques, using specific antibodies and cDNA probes, respectively. Adenylyl cyclase activity stimulated or inhibited by agonists was determined to examine the functions of G-proteins. RESULTS: The levels of Gialpha-2, Gialpha-3, Gbeta but not of Gs(alpha45) and Gs(alpha47) were significantly increased in lymphocytes from SHRs as compared to their control Wistar Kyoto (WKY) rats. Similarly the mRNA levels of Gialpha-2 and Gialpha-3 were significantly augmented in SHRs as compared to their age-matched WKYs. The increased levels of Gialpha were reflected in increased functions of Gi in SHRs as indicated by increased inhibition of forskolin-stimulated adenylyl cyclase activity by GTPgammaS. The activity of adenylyl cyclase stimulated by GTPgammaS, isoproterenol, NECA, NaF and forskolin was significantly decreased in SHRs as compared to their age-matched WKY rats. On the other hand, inhibitory hormones, atrial natriuretic peptide and angiotensin II inhibited adenylyl cyclase activity to a greater extent in SHRs as compared to their age-matched WKY rats. CONCLUSIONS: These results indicate that lymphocytes from spontaneously hypertensive rats exhibit enhanced Gi activation (function) which may be attributed to the enhanced expression of Gi proteins. It may be suggested that enhanced Gi expression and associated signaling may be one of the factors responsible for enhanced lymphoblasts proliferation observed in hypertension.
Assuntos
Adenilil Ciclases/sangue , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/sangue , Hipertensão/sangue , Linfócitos/metabolismo , Transdução de Sinais/fisiologia , Inibidores de Adenilil Ciclases , Animais , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/sangue , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Masculino , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKYRESUMO
Low platelet membrane alpha2-adrenergic receptor (alpha2AR) density and low basal and forskolin-stimulated cyclic adenosine monophosphate responses, which have been reported in post-traumatic stress disorder (PTSD), suggest either abnormal alpha2AR coupling to G(i) protein or dysregulation in post-receptor signal transduction mechanisms. alpha2AR density in the high- and low-conformational states, agonist affinity in both states and coupling to G(i) protein were investigated in 23 drug-free combat PTSD patients and 25 normal controls. alpha2AR coupling measures were not different between PTSD patients and controls. Total alpha2AR density was higher in PTSD patients than controls, due to a higher density of the receptor in the high-conformational state. There were no differences in agonist affinity to the receptor in either conformational state. Results rule out dysregulation in alpha2AR coupling to G(i) protein. Studies of post-receptor signal transduction mechanisms are warranted.
Assuntos
Plaquetas/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/sangue , Receptores Adrenérgicos alfa 2/sangue , Transtornos de Estresse Pós-Traumáticos/sangue , Adulto , Idoso , Humanos , Cinética , Ligantes , Masculino , Pessoa de Meia-Idade , Ligação Proteica , Escalas de Graduação Psiquiátrica , Transtornos de Estresse Pós-Traumáticos/psicologiaRESUMO
Various studies suggest alpha 2-adrenergic receptor (alpha 2AR) dysregulation in panic disorder (PD). Platelet alpha 2-AR exist in high- and low-conformational states as a function of their coupling to Gi protein. alpha 2AR coupling is important in signal transduction and is modulated by antidepressants. alpha 2AR density in the high- and low-conformational states, agonist affinity, and coupling efficiency were investigated in 21 healthy controls, 21 drug-free PD patients, and eight imipramine-treated patients using norepinephrine displacement of 3H-yohimbine binding. Percentage of receptors in the high-conformational state (%RH) and the ratio of the agonist dissociation constant to the receptor in the low-/high-conformational state (KL/KH), calculated from displacement experiments, were used as coupling indices. Patients had high alpha 2AR density in both conformational states. %RH and KL/KH ratio were significantly different, particularly in patients with Hamilton scale for depression (HAMD) scores > or = 15. Imipramine treatment (29 weeks) had no effect on alpha 2AR density or coupling, despite improvement in anxiety ratings. High pretreatment alpha 2AR density and coupling predicted low severity of anxiety after treatment. Increased alpha 2AR density and abnormal coupling may represent an adaptive mechanism or trait marker in PD.
Assuntos
Antidepressivos Tricíclicos/uso terapêutico , Plaquetas/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/sangue , Imipramina/uso terapêutico , Transtorno de Pânico/sangue , Transtorno de Pânico/tratamento farmacológico , Receptores Adrenérgicos alfa 2/sangue , Adulto , Agorafobia/sangue , Agorafobia/tratamento farmacológico , Agorafobia/psicologia , Antidepressivos Tricíclicos/efeitos adversos , Plaquetas/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Humanos , Imipramina/efeitos adversos , Masculino , Escalas de Graduação Psiquiátrica , Ensaio Radioligante , Resultado do TratamentoRESUMO
Studies suggest alpha2A-adrenoceptors (alpha(2A)AR) dysregulation in major depressive disorder (MDD). Platelet alpha(2A)ARs exist in high- and low-conformational states that are regulated by Gi protein. Although alpha(2A)AR coupling to Gi protein plays an important role in signal transduction and is modulated by antidepressants, it has not been previously investigated. Alpha2AR density in the high- and low-conformational states, agonist affinity and coupling efficiency were investigated in 27 healthy control subjects, 23 drug-free MDD patients and 16 patients after imipramine treatment using [3H]yohimbine saturation and norepinephrine displacement of [3H]yohimbine binding experiments. Coupling measures were derived from NE-displacement experiments. Patients had significantly higher alpha(2A)AR density, particularly in the high-conformational state, than control subjects. Coupling indices were normal in patients. High pre-treatment agonist affinity to the receptor in the high-conformational state and normal coupling predicted positive treatment outcome. Decreased coupling to Gi predicted a negative treatment outcome. Imipramine induced uncoupling (-11%) and redistribution of receptor density in treatment responders only, but had no effect on alpha(2A)AR coupling or density in treatment non-responders. Increased alpha(2A)AR density may represent a trait marker in MDD. The results provide indirect evidence for abnormal protein kinase A (PKA) and protein kinase C (PKC) in MDD which may be pursued in future investigations.
Assuntos
Inibidores da Captação Adrenérgica/uso terapêutico , Antidepressivos Tricíclicos/uso terapêutico , Plaquetas/metabolismo , Transtorno Depressivo Maior/sangue , Transtorno Depressivo Maior/tratamento farmacológico , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/sangue , Imipramina/uso terapêutico , Receptores Adrenérgicos alfa 2/sangue , Inibidores da Captação Adrenérgica/farmacologia , Adulto , Antidepressivos Tricíclicos/farmacologia , Biomarcadores/sangue , Plaquetas/efeitos dos fármacos , Estudos de Casos e Controles , Transtorno Depressivo Maior/psicologia , Humanos , Imipramina/farmacologia , Masculino , Pessoa de Meia-Idade , Norepinefrina/metabolismo , Ligação Proteica , Proteínas Quinases/metabolismo , Escalas de Graduação Psiquiátrica , Receptores Adrenérgicos alfa 2/efeitos dos fármacos , Resultado do Tratamento , Ioimbina/metabolismoRESUMO
BACKGROUND: Abnormal alpha 2-adrenergic receptor (AR) function is implicated in anxiety and depressive disorders. Premenstrual dysphoric disorder (PMDD) is characterized by anxiety and depressive symptoms, which may be associated with changes in alpha 2AR function. Previous studies on alpha 2AR function during phases of the menstrual cycle in controls and PMDD patients are inconsistent. METHODS: alpha 2AR function was examined in 16 PMDD patients and 15 controls during the follicular phase, and in 10 PMDD patients during late luteal phase. Antagonist-measured maximum binding capacity, agonist-measured receptor density in high- and low-conformational states, and agonist affinity to both states were measured. Coupling efficiency to Gi protein was estimated. RESULTS: There were no significant differences in coupling efficiency. PMDD patients had significantly low antagonist affinity; there were no differences in other binding parameters. There were no changes in alpha 2AR binding parameters between phases of menstrual cycle in PMDD women. alpha 2AR density and symptom severity were inversely related during the follicular phase in controls and patients. During luteal phase, alpha 2AR density correlated positively with symptom severity in patients. High follicular alpha 2AR density predicted more severe luteal symptoms in PMDD patients. CONCLUSIONS: These findings are discussed in view of the molecular biology of alpha 2AR, and their role in PMDD, anxiety, and depressive disorders.