Prolonged NO treatment decreases α-adrenoreceptor agonist responsiveness in porcine pulmonary artery due to persistent soluble guanylyl cyclase activation

Abstract

A cultured porcine pulmonary artery (PA) model was used to examine the effects of prolonged nitric oxide (NO) treatment on the response of this vessel to acutely applied NO and to the α-adrenoreceptor agonist phenylephrine. Two-hour treatment with the NO donor ( Z)-1-[ N-(2-aminoethyl)- N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) decreased both NO and phenylephrine responsiveness. Twenty-four-hour treatment with DETA-NO resulted in a further reduction in NO responsiveness but no further reduction in phenylephrine responsiveness. Acute addition of soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one (ODQ) had no effect on phenylephrine responsiveness in PA not treated with DETA-NO. ODQ treatment fully restored phenylephrine responsiveness in PA treated with DETA-NO. sGCβ1subunit protein levels in PA tissue homogenate were 48.6 ± 6.9, 51.6 ± 3.5, and 41.3 ± 2.8 ng/mg total protein for freshly prepared and 2-h and 24-h NO-treated PA, respectively. Steady-state tissue cGMP was not significantly different in control versus NO-treated PA. sGC specific activity in the absence of added NO was measured in PA homogenate and was 0.29 ± 0.02, 1.38 ± 0.12, and 0.53 ± 0.08 μmol cGMP·min−1·mg sGC−1, in freshly prepared and 2-h and 24-h NO treated PA, respectively. Ten-minute Hb treatment completely normalized sGC basal activity in homogenates prepared from DETA-NO-treated PA, which was 0.23 ± 0.02, 0.18 ± 0.03, and 0.25 ± 0.04 μmol cGMP·min−1·mg sGC−1, in freshly prepared and 2-h and 24-h NO-treated PA, respectively. The kinetics of the Hb reversal of NO-mediated sGC persistent activation do not support sGC covalent modification as the activation mechanism. We conclude that prolonged NO exposure results in a persistently increased sGC specific activity, which accounts for the observed α-adrenoreceptor agonist hyporesponsiveness.