RESUMO
The aim of this study was to identify the role of chymase in the conversion of exogenously administered Big endothelin-1 in the mouse in vivo. Real-time polymerase chain reaction analysis detected mRNA of mucosal mast cell chymases 4 and 5, endothelin-converting enzyme 1a, and neutral endopeptidase 24.11 in pulmonary, cardiac, and aorta homogenates derived from C57BL/6J mice, with the latter tissue expressing the highest levels of both chymase isoforms. Furthermore, hydrolysis of a fluorogenic peptide substrate, Suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin, was sensitive to the chymase inhibitors Suc-Val-Pro-Phe(P)(OPh)(2) (200 microM) and chymostatin [(S)-1-carboxy-2-phenylethyl]-carbamoyl-alpha-[2-iminohexahydro-4(S)-pyrimidyl]-(S)-Gly-X-Phe-al, where X can be the amino acid Leu, Val, or Ile) (100 microM) in supernatants extracted from the same tissue homogenates. In anesthetized mice, Big endothelin-1, endothelin-1 (1-31), and endothelin-1 triggered pressor responses (ED(50)s, 0.67, 0.89, and 0.16 nmol/kg) that were all reduced or potentiated by selective endothelin ET(A) or ET(B) receptor antagonists, respectively, BQ-123 (cyclo[D-Asp-Pro-D-Val-Leu-D-Trp]) or BQ-788 (N-[N-[N-[(2,6-dimethyl-1-piperidinyl)carbonyl]-4-methyl-l-leucyl]-1-(methoxycarbonyl)-D-tryptophyl]-d-norleucine sodium salt), each at 1 mg/kg. The pressor responses to big endothelin-1 were significantly reduced by the neutral endopeptidase inhibitor thiorphan (dl-3-mercapto-2-benzylpropanoylglycine) (1 mg/kg) or the endothelin-converting enzyme inhibitor CGS 35066 [alpha-[(S)-(phosphonomethyl)amino]-3-dibenzofuranopropanoic acid] (0.1 mg/kg). In contrast, the responses to endothelin-1 (1-31) were abolished by thiorphan but unaffected by CGS 35066. In addition, Suc-Val-Pro-Phe(P)(OPh)(2) (20-40 mg/kg) reduced, by more than 60%, the hemodynamic response to big endothelin-1 but not to endothelin-1 (1-31) and endothelin-1. Finally, intravenous administration of big endothelin-1 induced Suc-Val-Pro-Phe(P)-(OPh)(2)-sensitive increases in plasma-immunoreactive levels of endothelin-1 (1-31) and endothelin-1. The present study suggests that chymase plays a pivotal role in the conversion and cardiovascular properties of big endothelin-1 in vivo.
Assuntos
Quimases/metabolismo , Endotelina-1/metabolismo , Anestesia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Receptores de Endotelina/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismoRESUMO
We hypothesized that constitutive endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) have opposite effects on the regulation of endothelin and its receptors. We therefore sought to determine whether deletions of iNOS or eNOS genes in mice modulate pressor responses to endothelin and the expression of ETA and ETB receptors in a similar fashion. Despite unchanged baseline hemodynamic parameters, anesthetized iNOS-/- mice displayed reduced pressor responses to endothelin-1, but not to that of IRL-1620, a selective ETB agonist. Protein content of cardiac ETA receptors was reduced in iNOS-/- mice compared with wild-type mice, but that of ETB receptors was unchanged. Anesthetized eNOS-/- mice presented a hypertensive state, accompanied by an enhanced pressor response to intravenous endothelin-1, whereas the pressor response to IRL-1620 was reduced. Protein levels were also found to be increased for ETA receptors, but reduced for ETB receptors, in cardiac tissues of eNOS-/- mice. In conscious animals, both strains responded equally to the hypotensive effect of an ETA antagonist, ABT-627, whereas orally administered A-192621, an ETB antagonist, increased MAP to a greater extent in eNOS-/- than in wild-type mice. Furthermore, significant levels of immunoreactive endothelin were found in mesenteric arteries in eNOS-/- but not in iNOS-/- or wild-type congeners. Our study shows that repression of iNOS or eNOS has differential effects on endothelin-1 and its receptors. We have also shown that the heart is the main organ in which iNOS or eNOS repression induces important alterations in protein content of endothelin receptors in adult mice.
Assuntos
Endotelina-1/fisiologia , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo II/genética , Transdução de Sinais/fisiologia , Animais , Primers do DNA , DNA Complementar/biossíntese , DNA Complementar/genética , Antagonistas do Receptor de Endotelina A , Antagonistas do Receptor de Endotelina B , Endotelina-1/genética , Endotelina-1/farmacologia , Endotelinas/farmacologia , Camundongos , Camundongos Knockout , Nitratos/metabolismo , Óxido Nítrico Sintase Tipo II/fisiologia , Óxido Nítrico Sintase Tipo III/fisiologia , Nitritos/metabolismo , Fragmentos de Peptídeos/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptor de Endotelina A/agonistas , Receptor de Endotelina B/agonistas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Superóxidos/metabolismo , TelemetriaRESUMO
BACKGROUND: Recombinant human erythropoietin (rhEPO) increases blood pressure (BP) and the vascular production of endothelin-1 in renal failure rats. This study was designed to investigate the effect of rhEPO on BP and on the ET-1/ET(B)R system in rats with normal renal function. To further characterize the effect of rhEPO on the ET-1/ET(B)R system, we also studied heterozygous (+/-) ET(B)R knockout (KO) mice. METHODS: The animals received either the vehicle or rhEPO (100 U/kg subcutaneously three times per week). ET(B)R(+/-) mice were compared with ET(A)R(+/-) and wild-type (WT) mice. In rats, the ET(B)R mRNA expression was assessed in blood vessels as well as the vascular ET(B)R density using immunohistochemistry. In mice, ET-1 concentration was measured in the thoracic aorta. RESULTS: RhEPO administration increased hematocrit levels in all treated animals. This therapy had no effect on BP in normal rats, but it did increase vascular and renal cortex ET(B)R mRNA expression. Immunohistochemistry confirmed that the ET(B)R density was increased in blood vessel endothelium in these normal rats. In contrast, rhEPO increased BP in ET(B)R(+/-) mice and this pressor effect was associated with higher ET-1 concentrations in the thoracic aorta. CONCLUSIONS: RhEPO exerts a pleotropic effect on the endothelial ET-1/ET(B)R system. The increase in endothelial ET(B)R expression may contribute to maintaining normal BP during rhEPO administration in normal animals. Conversely, conditions with deficient ET(B)R expression, such as in ET(B)R(+/-) mice, may lead to hypertension while receiving the same therapy.
Assuntos
Pressão Sanguínea/efeitos dos fármacos , Endotelina-1/análise , Endotélio Vascular/química , Eritropoetina/farmacologia , Receptor de Endotelina B/análise , Animais , Aorta Torácica/química , Imuno-Histoquímica , Córtex Renal/química , Camundongos , RNA Mensageiro/análise , Receptor de Endotelina A/análise , Receptor de Endotelina B/genética , Proteínas RecombinantesRESUMO
Nitric oxide and other reactive oxygen species generated by nitric-oxide synthases (NOS) modulate, among several other cellular responses, the production of eicosanoids and platelet aggregation. The roles of specific NOS in these two phenomena remain to be determined. Thus, the present study assessed whether inducible NOS (iNOS) and endothelial NOS (eNOS) modulate in a similar manner the production of eicosanoids and platelet aggregation. Mice knocked out for eNOS (eNOS-/-) or iNOS (iNOS-/-) and their wild-type (WT) congeners were used to analyze agonist-induced increases in plasma levels of eicosanoids as well as inhibition of platelet aggregation ex vivo. Systemically administered endothelin-1 (ET-1) triggered an increase in plasma levels of 6-keto prostaglandin F(1alpha) (6-keto PGF(1alpha)) in WT and eNOS-/- but not in iNOS-/- mice. ET-1 (0.01-1 nmol/kg) also induced a dose-dependent inhibition of platelet aggregation in WT and eNOS-/- but not in iNOS-/- mice. Another agonist, bradykinin (10 nmol/kg), triggered the release of 6-keto PGF(1alpha) and inhibited platelet aggregation in all strains of mice studied. In addition, ADP-induced platelet aggregation in vitro was similarly reduced by iloprost (100 nM) in iNOS-/- mice and WT congeners. In another series of experiments, ET-1 (0.1 nmol/kg) significantly increased 8-isoprostane plasma levels in WT but not in iNOS-/- mice. Finally, a 3-week treatment with anti-oxidants inhibited the capacity of ET-1 to significantly increase plasma 6-keto PGF(1alpha) in WT mice. We show for the first time that iNOS is involved in the control of ET-1-induced prostacyclin release and related inhibition of platelet aggregation in the murine model.
Assuntos
Endotelina-1/metabolismo , Epoprostenol/metabolismo , Óxido Nítrico Sintase Tipo II/fisiologia , Agregação Plaquetária/fisiologia , Animais , Antioxidantes/farmacologia , Western Blotting , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Dinoprosta/análogos & derivados , Dinoprosta/sangue , Epoprostenol/sangue , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nitratos/sangue , Óxido Nítrico Sintase Tipo II/sangue , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo III , Nitritos/sangue , Agregação Plaquetária/genética , Contagem de Plaquetas , Tromboxano A2/sangue , Tromboxano A2/metabolismoRESUMO
In the vascular system, endothelin (ET) type B (ET(B)) receptors for ET-1 are located on endothelial and on venous and arterial smooth muscle cells. In the present study, we investigated the hemodynamic effects of chronic ET(B) receptor blockade at low and high doses in the Syrian Golden hamster. After 16 days of gavage with A-192621 (0.5 or 30 mg.kg(-1).day(-1)), a selective ET(B) receptor antagonist, hamsters were anesthetized with a mixture of ketamine and xylazine (87 and 13 mg/kg im, respectively), and basal mean arterial blood pressure (MAP) and pressor responses to exogenous ET-1 were evaluated. The lower dose of A-192621 (0.5 mg.kg(-1).day(-1)) did not modify basal MAP, whereas the higher dose (30 mg.kg(-1).day(-1)) increased MAP and plasma ET levels. Radio-telemetry recordings confirmed the increase in MAP induced by the higher dose of A-192621 in conscious hamsters. On the other hand, although the lower dose of A-192621 was devoid of intrinsic pressor effects, it markedly reduced the transient hypotensive phase induced by intravenously injected IRL-1620, a selective ET(B) receptor agonist. Finally, A-192621 (0.5 mg.kg(-1).day(-1)) alone or A-192621 (30 mg.kg(-1).day(-1)) + atrasentan (6 mg.kg(-1).day(-1)), a selective ET(A) receptor antagonist, potentiated the pressor response to exogenous ET-1. Our results suggest that, in the hamster, ET(B) receptors on vascular smooth muscle cells are importantly involved in the clearance of endogenous ET-1, whereas the same receptor type on the endothelium is solely involved in the vasodilatory responses to the pressor peptide. Blockade of endothelial and vascular smooth muscle cell ET(B) receptors triggers a marked potentiation of ET(A)-dependent increases in systemic resistance.
Assuntos
Antagonistas do Receptor de Endotelina B , Endotélio Vascular/efeitos dos fármacos , Hipertensão/induzido quimicamente , Hipertensão/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Anestesia , Animais , Atrasentana , Pressão Sanguínea/efeitos dos fármacos , Estado de Consciência , Cricetinae , Relação Dose-Resposta a Droga , Endotelina-1/antagonistas & inibidores , Endotelina-1/farmacologia , Endotelinas/farmacologia , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Masculino , Mesocricetus , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Fragmentos de Peptídeos/farmacologia , Pirrolidinas/farmacologia , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/metabolismoRESUMO
Mice with disruption of the kinin B(2) receptor (B(2)KO mice) are sensitive to salt-rich diets, which causes hypertension. The aim of the study was to assess the role of endothelin-1 (ET-1) and angiotensin-II in hypertensive B(2)KO mice on a salt-rich diet. We also wanted to verify if there is an upregulation of the mRNA expression of the precursors or receptors for these hormones. Two groups of B(2)KO mice (20-25 g) were investigated. The first group received an 8% NaCl diet with 1% NaCl in drinking water (HS) and the second was fed with normal food with tap water (NS). The antagonists tested were the ET(A) receptor antagonist BQ-123 (1 and 5 mg/kg), the ET(B) receptor antagonist BQ-788 (0.25 and 1 mg/kg), the angiotensin receptor type 1 antagonist losartan (10 mg/kg) and the angiotensin-converting enzyme inhibitor captopril (3 mg/kg). These were injected intraperitoneally 30 min prior to blood pressure measurement by the tail-cuff method. We also studied the level of expression of preproET-1, ET-1 receptors, angiotensinogen and angiotensin receptors by RNA extraction from the heart and kidneys of these mice followed by reverse transcriptase (RT)-PCR. B(2)KO mice (HS) were hypertensive after 8 weeks compared with B(2)KO mice on normal diet (HS, 93.4+/-1.5 mmHg, n=7; NS, 61.4+/-2.7 mmHg, n=7). In the HS group, the mean arterial blood pressure was significantly reduced by BQ-123 (5 mg/kg) to 61.9+/-1.8 mmHg (n=7), by BQ-788 (1 mg/kg) to 58.8+/-2.6 mmHg (n=6), by losartan (10 mg/kg) to 73.2+/-1.7 mmHg (n=8) and by captopril (3 mg/kg) to 86.0+/-2.3 mmHg (n=8). The expression studied by RT-PCR did not show any difference (either in precursors or receptors expression) between hypertensive and normal mice. The four antagonists used seemed to reverse the hypertension. These results suggest that ET-1 and angiotensin-II are probably involved in the mechanism that leads to hypertension since the effect of these hormones is probably not compensated by kinins in B(2)KO mice. Further studies are necessary to understand the implication of the cross-talk between these hormones in the hypertensive state.