Abstract 1166: Hypoxic Induction of RAMP2 Alters Regulation of Pulmonary Endothelin-1 by Adrenomedullin: Induction Under Normoxia versus Inhibition under Hypoxia
Adrenomedullin (AM) is a pulmonary vasodilator counteracting the effects of endothelin-1 (ET-1). In pulmonary hypertension, gene expression of AM is increased and, in patients, inhaled AM is beneficial. We investigated the effects of AM on pulmonary ET-1 under normoxia and hypoxia. In isolated rat lungs (IRL) perfused over 5 h under normoxia, the calcitonin gene-related peptide type-1 receptor (CGRP1R) antagonist hCGRP(8–37) decreased vascular secretion of ET-1, whereas the AM receptor antagonist hAM(22–52) had no effect. Exogenous AM, at 1 − 10 pM, increased levels of ET-1, which was abolished by hCGRP(8 –37) as well as by the PKA inhibitors H-89 and KT-5720. At 50 and 100 pM, exogenous AM decreased pulmonary vascular ET-1 - an effect that was sensitive to hAM(22–52), the NO synthase inhibitor L-NOARG, and the PKG inhibitor KT-5823. In rat pulmonary artery endothelial cells (RPAEC) cultured over 6 and 12 h under normoxia, these results were confirmed and attributed to changes in ET-1 gene expression. In addition, low exogenous AM promoted activity of the endothelin-converting enzyme (ECE), whereas high AM increased the number of ETB receptor sites. Under normobaric hypoxia, vascular levels of AM and ET-1 were significantly heightened in IRL perfused over 5 h. In the presence of hAM(22–52), L-NOARG, or KT-5823, we observed a further rise of ET-1. In RPAEC, hypoxia, over 6 and 12 h, increased gene expression and secretion of AM and ET-1, and the number of ETB sites. Application of hAM(22–52), L-NOARG, or KT-5823 further elevated ET-1 gene expression and secretion, but downregulated ETB receptor sites. Under hypoxia, CGRP1R blockade or PKA inhibition had no effect, and pretreatment with L-NOARG or KT-5823 abolished the effect of hAM(22–52). In RPAEC, both 100 pM of exogenous AM and hypoxia downregulated RAMP1 protein and CGRP1R binding sites but upregulated RAMP2 protein and AM receptor sites. In conclusion, endogenous pulmonary AM upregulates ET-1 and ECE activity under physiological conditions, via CGRP1R and PKA. In contrast, hypoxia-induced high levels of AM, via AM receptor and NO/PKG, downregulate ET-1 gene expression and promote expression of ETB receptor. This hypoxic switch of AM signaling can be attributed to upregulation of the RAMP2/AM receptor system.