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

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 protein-coupled receptor (GPCR) system. Because catecholamines, like 5-HT, constrict lung vessels via a GPCR coupled to G(q), we hypothesized that S-nitrosothiols modify the alpha(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(-8) - 10(-6) M). Effective cGMP-dependence was tested in rings pretreated with soluble guanylate cyclase inhibitors {either 1H-[1,2,4] oxadiazolo[4,3-a] 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 GSNO-mediated cysteine modifications. In CSNO-treated lung protein, the alpha(1)-adrenergic receptor was shown to undergo S-nitrosylation in vitro using a biotin switch assay. Studies of alpha 1-adrenergic receptor subtype expression and receptor density by saturation binding with I-125- HEAT showed that GSNO decreased alpha 1-adrenergic receptor density but did not alter affinity for antagonist or agonist. These data demonstrate a novel cGMP-independent mechanism of reversible alpha 1-adrenergic receptor inhibition by S-nitrosothiols.