-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 g/ml). Prostaglandin levels were measured by gas chromatography-mass spectrometry. Through use of ET A and ETB 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-PGF1␣ and PGE2 (P Ͻ 0.05). Both internalization of ET-1 and synthesis of new peptide contributed to an increase in intracellular ET-1 (P Ͻ 0.05). Although increased ppET-1 was regulated by both ETA and ETB, COX-2 expression was upregulated only by ETA; 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 ETA receptor and phosphorylation of p38 and p44/42 MAPK in PLVSMC.prostacyclin; prostaglandin E2; endothelin receptors

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 ET A (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 ET B receptors in cells of the L2 layer may contribute to the maintenance of CPH via alterations in their expression, distribution, and activity.ET A receptor; ETB receptor; smooth muscle cells; pulmonary hypertension; endothelin SINCE ITS DISCOVERY 12 YEARS AGO, endothelin (ET) has been shown to contribute to a wide variety of physiological and pathophysiological processes in various systems (22). The ET family of 21 amino acid peptides exists in three distinct isoforms: ET-1, ET-2, and ET-3. It is an extremely potent vasoconstrictor resulting in slowly developing and sustained contraction. Although the endothelial cell was originally described as the source of ET-1, it is now known that several other cell types, including pulmonary vascular smooth muscle cells, synthesize this peptide (33, 36).The biological effects of ET-1 are mediated by two distinct ET receptor subtypes, ET A and ET B (1, 35). The affinity of the ET A receptor for ET-1 has been shown to be ϳ100 times that for ET-3 (ET-1 Ͼ ET-2 Ͼ ET-3), whereas the affinity of the ET B receptor is equipotent for all three isoforms of ET (34). Early reports indicated that the ET A receptor on smooth muscle was responsible for the vasoconstrictor effect of ET-1, whereas the ET B receptor on endothelial cells modulated ET-1-induced vasodilation. However, recent pharmacological studies suggest that there are two classes of ET B with cell specific effects, ET B1 on endothelial cells mediating vasodilation and ET B2 on smooth muscle cells mediating vasoconstriction (9). The ET B receptor also functions as a "clearance receptor" and is particularly important in the lung, where 50% of ET-1 is retained (22,29).ET-1 has been linked to the development of chronic pulmonary hypertension (CPH). Increased expression of the peptide has been demonstrated in endothelial cells of pulmonary arteries from patients with idiopathic and secondary forms of pulmonary hypertension (15), and arterial-to-venous ratios of ET-1 protein were found to be elevated above the normal range in patients with pulmonary hypertension (40). Several studies have linked increased expression of ET-1 to the development of hypoxia-induced CPH in rats (5, 24), and use of ET-1 receptor antagonists has been shown both to inhibit progression of the disease and to promote recovery (4,10,43).Although these studies support a central role for ET-1 in the pathogenesis of CPH, the picture is likely more complicated than first suspected, in that prepro-ET-1 (ppET-1) mRNA is...

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