RESUMEN
We examined the hypothesis that the potent vasoconstrictor endothelin (ET)-1 regulates both its own production and production of the vasodilator prostaglandins PGE(2) and prostacyclin in sheep peripheral lung vascular smooth muscle cells (PLVSMC). Confluent layers of PLVSMC were exposed to 10 nM ET-1; expression of the prepro (pp)-ET-1, cyclooxygenase (COX)-1, and COX-2 genes was examined by RT-PCR and Western analysis. Intracellular levels of ET-1 were measured by ELISA with and without addition of the protein synthesis inhibitor brefeldin A (50 microg/ml). Prostaglandin levels were measured by gas chromatography-mass spectrometry. Through use of ET(A) and ET(B) antagonists (BQ-610 and BQ-788, respectively), the contribution of the ET receptors to COX-1 and -2 expression and ppET-1 gene expression was examined. The contribution of phosphorylated p38 and p44/42 MAPK on COX-1 and COX-2 expression was also examined with MAPK inhibitors (p38, SB-203580 and p44/42, PD-98056). ET-1 resulted in transient increases in ppET-1, COX-1, and COX-2 gene and protein expression and release of 6-keto-PGF(1alpha) and PGE(2) (P < 0.05). Both internalization of ET-1 and synthesis of new peptide contributed to an increase in intracellular ET-1 (P < 0.05). Although increased ppET-1 was regulated by both ET(A) and ET(B), COX-2 expression was upregulated only by ET(A); COX-1 expression was unaffected by either antagonist. ET-1 treatment resulted in transient phosphorylation of p38 and p44/42 MAPK; inhibitors of these MAPKs suppressed expression of COX-2 but not COX-1. Our data indicate that local production of ET-1 regulates COX-2 by activation of the ET(A) receptor and phosphorylation of p38 and p44/42 MAPK in PLVSMC.
Asunto(s)
Endotelina-1/metabolismo , Isoenzimas/metabolismo , Pulmón/irrigación sanguínea , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/enzimología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Animales , Antihipertensivos/farmacología , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Dinoprostona/metabolismo , Endotelina-1/genética , Endotelina-1/farmacología , Endotelinas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Isoenzimas/genética , Microcirculación/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Músculo Liso Vascular/citología , Oligopéptidos/farmacología , Fosforilación , Piperidinas/farmacología , Prostaglandina-Endoperóxido Sintasas/genética , Precursores de Proteínas/genética , Receptor de Endotelina A , Receptor de Endotelina B , Receptores de Endotelina/metabolismo , Ovinos , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
We examined gene and surface expression and activity of the endothelin (ET)-1 receptors (ETA and ETB) in subendothelial (L1) and inner medial (L2) cells from the main pulmonary artery of sheep with continuous air embolization (CAE)-induced chronic pulmonary hypertension (CPH). According to quantitative real-time RT-PCR, basal gene expression of both receptors was significantly higher in L2 than L1 cells, and hypertensive L2 cells showed significantly higher gene expression of ETB than controls. Expression of both genes in hypertensive L1 cells was similar to controls. Fluorescence-activated cell sorter analysis confirmed the increased distribution of ET(B) in hypertensive L2 cells. Although only the ETA receptors in control L2 cells showed significant binding of [125I]-labeled ET-1 at 1 h, both receptors bound ET-1 to hypertensive cells. Exposure to exogenous ET-1 for 18 h revealed that only the L2 cells internalized ET-1, and internalization by hypertensive L2 cells was significantly reduced when compared with controls. Treatment with ETA (BQ-610) and ETB (BQ-788) receptor antagonists demonstrated that both receptors contributed to internalization of ET-1 in control L2 cells, whereas in hypertensive cells only when both receptor antagonists were used in combination was significant suppression of ET-1 internalization found. We conclude that in sheep receiving CAE, alterations in ETB receptors in cells of the L2 layer may contribute to the maintenance of CPH via alterations in their expression, distribution, and activity.