Harold I. Magazine

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-gamma (IFN-gamma) 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.

Morphine stimulates nitric oxide release from invertebrate microglia.

Morphine stimulates nitric oxide (NO) release in human endothelial cells. To determine whether this mechanism also occurs in invertebrates, the mussel Mytilus edulis was studied. Exposure of excised ganglia to morphine for 24 h resulted in a significant dose-dependent decrease in microglial egress that was naloxone sensitive. In coincubating the excised ganglia with morphine and the nitric oxide synthase inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME), an increase in microglial egress was observed, suggesting that morphine may stimulate microglia to release NO. Morphine exposure to these cells in vitro resulted in NO release (39.4 +/- 4.9 nM), a phenomenon found to be naloxone sensitive (10(-6) M; NO level = 5.9 +/- 2.6 nM) and L-NAME sensitive (10(-4) M; NO level = 2.8 +/- 1.8 nM). Opioid peptides did not stimulate NO release, indicating that the process was mediated by the opiate alkaloid selective mu 3 receptor. Coincubation of microglia with L-arginine or the superoxide scavenger, superoxide dismutase, resulted in significantly higher NO levels observed following morphine stimulation. Taken together, the data demonstrate that morphine can stimulate NO release in cells obtained from an invertebrate that represents an animal 500 million years divergent in evolution from man, underscoring the significance of this process and further substantiating the critical importance of morphine as a naturally occurring signal molecule.

Cryopreserved Veins in Myocardial Revascularization: Possible Mechanism for Their Increased Failure

BACKGROUND Cryopreserved veins are used as conduits for myocardial revascularization. However, a high failure rate associated with their use has been reported anecdotally. METHODS To find an explanation for the poor performance of cryopreserved vein grafts, we conducted a retrospective 5-year study on all patients at a single institution in whom cryopreserved vein grafts were used. We further performed in vitro studies measuring cell adhesion, nitric oxide production, and contractile capacity of saphenous vein, internal thoracic artery, and cryopreserved veins. RESULTS; Forty-one patients were identified in whom one or more cryopreserved veins were used as a last resort. Sixteen had events (death or recatheterization). Seven deaths occurred (17%). Event-free survival was 50% at 12 months. Activated granulocyte/monocyte endothelial adherence could be lowered in internal thoracic arteries and saphenous veins with morphine incubation (50% and 57%, respectively), but not in cryopreserved veins. Simultaneous increases in nitric oxide release were also found in internal thoracic arteries and saphenous veins, but not cryopreserved veins. In addition, cryopreserved veins showed a diminished contractile capacity under experimental conditions. CONCLUSIONS In this highly select group of patients, cryopreserved veins had a high early failure rate, which may be partially due to the inability of the endothelium to participate in immunovascular processes.

Detection of endothelial cell-derived nitric oxide: current trends and future directions.

The vascular endothelium is a significant site of NO release that inhibits cellular adhesion and maintains a non-thrombogenic surface. Use of newly described technology suggests for the first time that the maximal release of NO induced by cNOS and iNOS activation may be quite similar, implying that it is the duration of NO release and not the concentration of NO produced from stimulated endothelial cells that accounts for the different biological activities of the enzymes. The respective roles of cNOS and iNOS must be carefully evaluated since both enzymes may have potent biological effects at local sites of production.

Protease activated receptors modulate aortic vascular tone

The effect of agonists of the known protease activated receptors (PAR), the thrombin and the PAR-2 receptors, on vasoactive mediator release and vascular tone were studied using rings of rat aorta. Stimulation of aortic rings with the thrombin receptor agonist, Trap-14, or the PAR-2 agonist, SLIGRL, resulted in a rapid release of nitric oxide. Trap-14 and SLIGRL-induced nitric oxide release was reduced by pre-treatment with BQ-788, an ETB endothelin receptor-specific antagonist. Consistent with a role for endothelin-1 receptor activation in Trap-14 and SLIGRL-induced nitric oxide release, endothelin-1 levels were increased significantly following 5 min treatment of aortic rings with Trap-14 or SLIGRL. Cumulative addition of Trap-14 to aortic rings denuded of endothelium resulted in dose-dependent contraction with an EC50 value of 23 +/- 5 microM, whereas SLIGRL addition failed to induce aortic contraction. These data suggest that the known protease activated receptors are functionally coupled to nitric oxide release. In addition, the thrombin receptor appears to modulate both vasodilator and contractile responses, whereas the PAR-2 receptor is linked only to vasodilation.