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1.
Mol Cell Endocrinol ; 399: 60-8, 2015 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-25150623

RESUMO

GPER (aka GPR30) has been identified as an important mechanism by which estrogen mediates its effects. Previous studies from our laboratories and those of others have demonstrated that GPER activation mediates a range of vascular contractile and growth regulatory responses. However, the importance of GPER in mediating the actions of estradiol (E2) in rat aortic endothelial cells is unclear. Therefore, we sought to determine the importance of GPER vs. the "classical" estrogen receptor (ER) in mediating the endothelial growth regulatory effects of E2. To do this we assessed the effect of E2 in regulating phosphoERK content and apoptotic rates in rat aortic endothelial cells and the role of GPER in mediating these effects. E2 mediated a concentration-dependent inhibition of both ERK phosphorylation and serum deprivation-induced apoptosis with a maximal effect at a concentration of 10 nM. Pretreatment with the ER antagonist ICI 182780 abolished E2-mediated inhibition of both ERK phosphorylation and apoptosis. In contrast, pretreatment with GPER antagonist G15 had no significant effect on E2-mediated inhibition of ERK phosphorylation or on apoptosis. Further, downregulation of GPER expression with a GPER shRNA adenovirus did not block E2-mediated inhibitory effects on ERK phosphorylation and apoptosis. In fact, these inhibitory effects of E2 were further enhanced by GPER downregulation. Downregulation of ERα expression reversed the E2-mediated inhibitory effects to stimulatory effects. E2's phosphoERK and apoptosis stimulatory effects seen with ERα downregulation are attenuated by pretreatment with G15. In conclusion, in rat aortic endothelial cells, E2-mediated endothelial effects are predominantly driven by ER and not by GPER.


Assuntos
Apoptose/efeitos dos fármacos , Células Endoteliais/metabolismo , Estradiol/farmacologia , Estrogênios/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Animais , Aorta , Células Cultivadas , Células Endoteliais/citologia , Receptor alfa de Estrogênio/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fosforilação/efeitos dos fármacos , Ratos
2.
Hypertension ; 35(1 Pt 1): 38-42, 2000 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10642272

RESUMO

Impaired receptor-stimulated adenylyl cyclase activation has been observed in lymphocytes from hypertensive subjects and has been linked to an increase in lymphocyte G-protein receptor kinase-2 (GRK-2) protein expression. However, whether the increase in lymphocyte GRK-2 reflected an increase in vascular GRK-2 was unknown. Therefore, we compared GRK-2 protein expression in lymphocytes and aortas obtained from normotensive Wistar rats, Wistar-Kyoto rats (WKY), and spontaneously hypertensive rats (SHR) and from aortas of Dahl rats. Impaired beta-adrenergic responsiveness was observed in lymphocytes and vascular tissues obtained from hypertensive SHR (10 and 15 weeks old) but not in those obtained from prehypertensive SHR (5 weeks old). Immunodetectable lymphocyte GRK-2 protein expression was increased in 10-week-old SHR (143+/-10% of the expression in 10-week-old Wistar rats and 131+/-11% of the expression in 10-week-old WKY, n=5 in each group). Immunodetectable vascular smooth muscle cell GRK-2 was comparably increased (169+/-14% of the expression in Wistar rats and 138+/-7% of the expression in WKY, n=5 in each group). Also, in hypertensive Dahl salt-sensitive rats, vascular GRK-2 protein expression was increased (185+/-14% of the expression in Dahl salt-resistant rats, n=5 in each group) compared with Dahl salt-resistant controls. These studies support a generalized defect in vascular GRK-2 protein expression in hypertension, which could be an important factor in the impairment of beta-adrenergic-mediated vasodilation, characteristic of the hypertensive state.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Hipertensão/enzimologia , Linfócitos/enzimologia , Músculo Liso Vascular/enzimologia , Adenilil Ciclases/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Aorta/efeitos dos fármacos , Aorta/fisiopatologia , Quinase 2 de Receptor Acoplado a Proteína G , Humanos , Hipertensão/fisiopatologia , Técnicas In Vitro , Isoproterenol/farmacologia , Masculino , Músculo Liso Vascular/fisiopatologia , Ratos , Ratos Endogâmicos Dahl , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia , Quinases de Receptores Adrenérgicos beta
3.
Clin Pharmacol Ther ; 66(3): 275-81, 1999 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10511063

RESUMO

BACKGROUND: Beyond their mitogenic effects, hormones such as insulin, which activate receptor tyrosine kinases, regulate vascular tone. Further, we have demonstrated that receptor tyrosine kinase activation enhances adenylyl cyclase activation, a prominent mechanism that mediates vasodilation. However, whether tyrosine kinase-mediated human vascular responses parallel tyrosine kinase-mediated cellular effects on adenylyl cyclase activity is unknown. METHODS AND RESULTS: To assess tyrosine kinase-mediated vascular responses, vascular sensitivity to insulin was assessed with the dorsal hand vein linear variable differential transformer technique. Insulin infusion resulted in a dose-dependent relaxation in all subjects. Cellular responses were assessed by means of the insulinomimetic vanadate-mediated sensitization of vascular adenylyl cyclase activity. Vanadate stimulated a tyrosine kinase-dependent enhancement of adenylyl cyclase function in human and rat aortic vascular smooth muscle cells, human lymphocytes, and human aortic endothelial cells. Further, maximal insulin-mediated vasodilation was significantly positively correlated with maximal vanadate-mediated enhancement of human lymphocyte adenylyl cyclase activity. CONCLUSION: Insulin-mediated vasodilation is positively correlated with vanadate-mediated enhancement of adenylyl cyclase activity. Vanadate-mediated enhancement of adenylyl cyclase activity in lymphocytes may represent an index of tyrosine kinase-mediated vascular effects.


Assuntos
Adenilil Ciclases/metabolismo , Linfócitos/enzimologia , Proteínas Tirosina Quinases/metabolismo , Vanadatos/farmacologia , Vasodilatação/efeitos dos fármacos , Adenilil Ciclases/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Colforsina/metabolismo , AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Feminino , Proteínas de Ligação ao GTP/metabolismo , Mãos/irrigação sanguínea , Humanos , Infusões Intravenosas , Insulina/administração & dosagem , Linfócitos/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Proteínas Tirosina Quinases/efeitos dos fármacos , Vasodilatadores/administração & dosagem
4.
Clin Pharmacol Ther ; 65(5): 545-51, 1999 May.
Artigo em Inglês | MEDLINE | ID: mdl-10340920

RESUMO

In human hypertension we have recently identified an increase in lymphocyte G-protein receptor kinase-2 (GRK-2) protein expression, the key protein regulating the interaction between G-protein-coupled receptors and activation of adenylyl cyclase. However, it was not known whether this increase in GRK-2 protein expression was attributable to regulation at the level of translation. Furthermore, the relationship between extent of GRK-2 expression, receptor activation of adenylyl cyclase, and blood pressure was unclear. We therefore studied lymphocytes from 7 young subjects with borderline hypertension and 14 young normotensive subjects. Immunodetectable GRK-2 protein expression in lymphocytes from subjects with hypertension was increased (155%+/-7% of normotensive subjects; P < .05). In addition, GRK-2 protein expression was positively correlated with blood pressure (r = 0.53; P = .013) and inversely correlated with beta-adrenergic-mediated adenylyl cyclase activity (r = -0.54, P = .012). However, lymphocyte GRK-2 messenger ribonucleic acid (mRNA) content was not altered (110%+/-13% of that observed in normotensive control subjects). Increased GRK-2 protein expression may be an important factor in the impairment of beta-adrenergic-mediated vasodilation, characteristic of the hypertensive state.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/biossíntese , Hipertensão/sangue , Adulto , Pressão Sanguínea , Northern Blotting , Regulação da Expressão Gênica , Humanos , Linfócitos/metabolismo , Masculino , Quinases de Receptores Adrenérgicos beta
5.
Hypertension ; 29(1 Pt 2): 422-7, 1997 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9039137

RESUMO

A functional impairment in vasodilator tone may be important in the pathogenesis and/or maintenance of elevated peripheral vascular resistance in hypertension. Previous studies of hypertensive subjects have demonstrated impaired beta-adrenergic-mediated vasodilation paralleling a reduction in lymphocyte beta-adrenergic-stimulated adenylyl cyclase activity. We have suggested that this impairment is related to a defect in G-protein function. To determine whether this defect alters the coupling between the G-protein complex and adenylyl cyclase, we performed [3H]forskolin binding studies in lymphocytes from hypertensive subjects, older normotensive subjects, and younger normotensive control subjects. Maximal specific [3H]forskolin binding was used as an index of adenylyl cyclase binding sites. Gpp(NH)p-, NaF/AlCl3-, and isoproterenol-stimulated binding were used as indices of G-protein/adenylyl cyclase coupling. In the absence of other stimulators, maximal [3H]forskolin binding was not significantly different among groups. However, both Gpp(NH)p- and isoproterenol-stimulated [3H]forskolin binding were significantly decreased in lymphocytes from hypertensive subjects. Overall, Gpp(NH)p- and isoproterenol-stimulated [3H]forskolin binding were significantly inversely correlated with blood pressure. No differences in NaF/AlCl3-stimulated [3H]forskolin binding were detected between groups. These studies indicate that G-protein/adenylyl cyclase coupling is impaired in lymphocytes from younger hypertensive subjects and may contribute to the blood pressure-related defect in beta-adrenoceptor-stimulated adenylyl cyclase activity.


Assuntos
Colforsina/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Hipertensão/metabolismo , Linfócitos/metabolismo , Adenilil Ciclases/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Adulto , Fatores Etários , Humanos , Pessoa de Meia-Idade
6.
Hypertension ; 26(5): 725-32, 1995 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-7591010

RESUMO

Defective vasodilator function could be important in the pathogenesis and/or maintenance of the hypertensive state and the predisposition of the elderly to hypertension. Impaired beta-adrenergic-mediated vasodilation and reduced lymphocyte beta-adrenergic activation of adenyl cyclase have been demonstrated both in aging and with hypertension. The cellular mechanisms responsible for these alterations remain unclear. To determine if these defects may be due to alterations in guanine nucleotide regulatory proteins (G proteins) that link receptor activation with effector function, we assessed (1) human lymphocyte adenyl cyclase activity, (2) stimulatory G proteins by cholera toxin-mediated [32P]ADP ribosylation and, in hypertensive subjects, with alpha s-specific and beta-subunit antisera, and (3) inhibitory G proteins by pertussis toxin-mediated [32P]ADP ribosylation and, in older subjects, with alpha i,1,2- and beta-subunit-specific antisera. Lymphocytes from older subjects and from hypertensive subjects demonstrated a comparable reduction in isoproterenol-stimulated adenyl cyclase. However, aluminum fluoride-stimulated activity was reduced only in lymphocytes from hypertensive subjects. Furthermore, aluminum fluoride-stimulated activity was inversely correlated with mean arterial pressure. In lymphocytes from younger hypertensive subjects, cholera toxin-mediated labeling was significantly increased. In contrast, inhibitory G protein labeling by immunodetection was unaltered. In lymphocytes from older subjects, cholera toxin-mediated labeling was not altered; however, pertussis toxin-mediated labelling was significantly increased. In contrast, inhibitory G protein labeling by immunodetection was unaltered. Overall, the study suggests alterations of G protein function of adenyl cyclase is impaired. However, these defects are associated with divergent alterations in stimulatory and inhibitory G proteins.


Assuntos
Adenilil Ciclases/metabolismo , Envelhecimento/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Hipertensão/fisiopatologia , Linfócitos/fisiologia , Toxina Adenilato Ciclase , Agonistas Adrenérgicos beta/farmacologia , Adulto , Compostos de Alumínio/farmacologia , Anticorpos , Toxina da Cólera , Ativação Enzimática , Fluoretos/farmacologia , Humanos , Hipertensão/sangue , Isoproterenol/farmacologia , Pessoa de Meia-Idade , Toxina Pertussis , Fatores de Virulência de Bordetella
7.
Acta Biochim Pol ; 34(4): 421-30, 1987.
Artigo em Inglês | MEDLINE | ID: mdl-3450104

RESUMO

The native dimeric form of enolase from pig muscle was immobilized on Sepharose 4B activated with cyanogen bromide. The amount of matrix-bound enolase, its specific activity and kinetic properties depend on the extent of gel activation with CNBr. Only on the Sepharose activated with small quantities of CNBr the amount of protein which remained after treatment with Gdn.HCl was about 50% of the initially bound enolase, indicating that the enzyme was bound covalently to the matrix through a single subunit. The matrix-bound monomers obtained in this way were inactive and were unable to reassociate to dimers on addition of free subunits. The matrix-bound monomers obtained after KBr treatment were inactive but retained the ability to reassociate into active dimers after addition of free subunits. The results indicate that single matrix-bound subunits of pig muscle enolase are enzymatically inactive and dimeric structure is essential for catalytic activity.


Assuntos
Músculos/enzimologia , Fosfopiruvato Hidratase , Animais , Estabilidade Enzimática , Enzimas Imobilizadas , Suínos
8.
Comp Biochem Physiol B ; 71(2): 259-63, 1982.
Artigo em Inglês | MEDLINE | ID: mdl-6977430

RESUMO

1. Binding activities of hepatic extracts of the rat, chicken, frog and fish for 125I-labeled asialo-, agalacto-, and aN-acetylglucosamino-derivatives of orosomucoid and haptoglobin, were determined. 2. Hepatic receptors of the rat were found to bind preferably asialo-derivatives, those of the chicken agalacto-ones. 3. Hepatic receptors of lower vertebrates showed much lower affinity, in the case of the frog for all the modified glycoproteins whereas in the case of the fish with the exception of aN-acetylglucosamino-derivatives which were not bound at all. 4. Specific binding of derivatives of orosomucoid was higher than that of haptoglobin but equal in terms of molarity.


Assuntos
Glicoproteínas/metabolismo , Fígado/metabolismo , Animais , Assialoglicoproteínas , Carboidratos/sangue , Galinhas , Peixes , Haptoglobinas/metabolismo , Orosomucoide/análogos & derivados , Orosomucoide/metabolismo , Rana esculenta , Ratos , Ratos Endogâmicos , Especificidade da Espécie
9.
Acta Biochim Pol ; 28(2): 147-56, 1981.
Artigo em Inglês | MEDLINE | ID: mdl-7324699

RESUMO

Experiments were performed on rats injected with a malignant hepatoma (evoked by aflatoxin B1) or with inflammation induced by turpentine injection. On the 4th or 8th day after the injection, the following determinations were made: concentration of seromucoid, haptoglobin, sialic acid and fucose in blood serum; binding of asialo-glycoproteins by liver extracts; elimination of asialo-[125I]haptoglobin and asialo-[125I]orosomucoid from circulation. It was found that hepatoma and inflammation affected neither the activity of liver receptors for asialo-glycoproteins, nor the rate of asialo-glycoprotein elimination from circulation.


Assuntos
Glicoproteínas/metabolismo , Inflamação/metabolismo , Neoplasias/metabolismo , Animais , Assialoglicoproteínas , Feminino , Haptoglobinas/metabolismo , Fígado/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Masculino , Orosomucoide/metabolismo , Ratos
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