Antagonism of LPS and IFN-γ induction of iNOS in human saphenous vein endothelium by morphine and anandamide by nitric oxide inhibition of adenylate cyclase

Nitric oxide (NO) production regulates vasodilation in many blood vessels. Additionally, constitutive NO release is being associated with positive biomedical phenomena, whereas inducible NO synthase (iNOS)- associated NO release with detrimental consequences in regard to endothelial inflammatory activities. As yet, an important link demonstrating why one is activated over the other is not available. Previous studies have demonstrated that morphine and anandamide effector processes are coupled to NO release in human endothelial cells (ECs). This study now extends this observation in that these endogenous signaling molecules may use NO directly to inhibit adenylate cyclase activity. Activation of human ECs, obtained from the saphenous vein, with morphine- or anandamide-stimulated NO release (35 nM and 28 nM, respectively) that peaked within 5 min and returned to basal levels within 10 min of agonist stimulation, consistent with constitutive NO synthase (cNOS) activation. Significant release of NO from ECs stimulated with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) occurred after 2 h after exposure and remained significantly increased over basal levels for 24- 48 h (28 nM), consistent with iNOS activation. Preincubation of ECs with morphine or anandamide before, but not after, the addition of LPS + IFN, blocked iNOS activity. Exposure of ECs to the NO donor, SNAP, before the addition of LPS + IFN, blocked iNOS induction, whereas preincubation of ECs with inhibitors of NOS, before morphine or anandamide exposure, restored LPS + IFN induction of iNOS, suggesting a direct impact of NO on the regulation of iNOS activity. Morphine and anandamide stimulation of ECs did not stimulate cyclic adenosine monophosphate (cAMP) accumulation, whereas a marked increase in cAMP was observed in ECs treated with LPS + IFN (8.2 to 33 pmol/mg protein). Treatment of ECs with LPS + IFN did not induce cAMP accumulation in ECs treated with morphine, anandamide, or SNAP before LPS + IFN exposure. These data suggest that cAMP is required for the induction of iNOS in ECs and that NO may directly impair adenylate cyclase activity, preventing iNOS activation.