S-nitrosoglutathione inhibits 1-adrenergic receptor-mediated vasoconstriction and ligand binding in pulmonary artery

Nozik-Grayck, Eva, Erin J. Whalen, Jonathan S. Stamler, Timothy J. McMahon, Pasquale Chitano, and Claude A. Piantadosi. S-nitrosoglutathione inhibits 1-adrenergic receptor-mediated vasoconstriction and ligand binding in pulmonary artery. Am J Physiol Lung Cell Mol Physiol 290: L136–L143, 2006. First published August 26, 2005; doi:10.1152/ajplung.00230.2005.—Endogenous nitric oxide donor compounds (S-nitrosothiols) contribute to low vascular tone by both cGMP-dependent and -independent pathways. We have reported that S-nitrosoglutathione (GSNO) inhibits 5-hydroxytryptamine (5-HT)-mediated pulmonary vasoconstriction via a cGMP-independent mechanism likely involving S-nitrosylation of its G proteincoupled receptor (GPCR) system. Because catecholamines, like 5-HT, constrict lung vessels via a GPCR coupled to Gq, we hypothesized that S-nitrosothiols modify the 1-adrenergic GPCR system to inhibit pulmonary vasoconstriction by receptor agonists, e.g., phenylephrine (PE). Rat pulmonary artery rings were pretreated for 30 min with and without an S-nitrosothiol, either GSNO or S-nitrosocysteine (CSNO), and constricted with sequential concentrations of PE (10 –10 6 M). Effective cGMP-dependence was tested in rings pretreated with soluble guanylate cyclase inhibitors {either 1H-[1,2,4]oxadiazolo[4,3a]quinoxalin-1-one (ODQ) or LY-83583} or G kinase inhibitor (KT5823), and a thiol reductant [dithiothreitol (DTT)] was used to test reversibility of S-nitrosylation. Both S-nitrosothiols attenuated the PE dose response. The GSNO effect was not prevented by LY-83583, ODQ, or KT-5823, indicating cGMP independence. GSNO inhibition was reversed by DTT, consistent with S-nitrosylation or other GSNOmediated cysteine modifications. In CSNO-treated lung protein, the 1-adrenergic receptor was shown to undergo S-nitrosylation in vitro using a biotin switch assay. Studies of 1-adrenergic receptor subtype expression and receptor density by saturation binding with I-HEAT showed that GSNO decreased 1-adrenergic receptor density but did not alter affinity for antagonist or agonist. These data demonstrate a novel cGMP-independent mechanism of reversible 1-adrenergic receptor inhibition by S-nitrosothiols.