Inhaled Nitric Oxide Pretreatment But Not Post-treatment Attenuates Ischemia-Reperfusion-induced Pulmonary Microvascular Leak :

Nitric oxide provokes vasodilation and inhibits platelet aggregation. We examined the effect of nitric oxide on superoxide anion production by three sources: activated intact neutrophils, xanthine oxidase/hypoxanthine, and the NADPH oxidase. Nitric oxide significantly inhibited the generation of superoxide anion by neutrophils exposed to either FMLP (10(-7)M) or PMA (150 ng/ml) (IC50 = 30 microM). To determine whether the effect of nitric oxide on the respiratory burst was due to simple scavenging of O2+, kinetic studies that compared effects on neutrophils and the cell-free xanthine oxidase system were performed. Nitric oxide inhibited O2+ produced by xanthine oxidase only when added simultaneously with substrate, consistent with the short half-life of NO in oxygenated solution. In contrast, the addition of nitric oxide to neutrophils 20 min before FMLP resulted in the inhibition of O2+ production, which suggests formation of a stable intermediate. The effect of nitric oxide on the cell-free NADPH oxidase superoxide-generating system was also examined: The addition of NO before arachidonate activation (t = -6 min) significantly inhibited superoxide anion production. Nitric oxide did not inhibit O2+ when added at NADPH initiation (t = 0). Treatment of the membrane but not cytosolic component of the oxidase was sufficient to inhibit O2+ generation. The data suggest that nitric oxide inhibits neutrophil O2+ production via direct effects on membrane components of the NADPH oxidase. This action must occur before the assembly of the activated complex.

Leukocyte-endothelial cell adhesion and an altered metabolism of endothelial cell-derived nitric oxide (NO) have been implicated in the microvascular dysfunction associated with ischemia/reperfusion (I/R). The objective of this study was to determine whether NO donors can attenuate the reperfusion-induced increase in venular albumin leakage via an effect on leukocyte-endothelial cell adhesion. Leukocyte adherence and emigration as well as albumin extravasation were monitored in single postcapillary venules in rat mesentery subjected to 20 minutes of ischemia followed by 30 minutes of reperfusion. This I/R protocol elicits significant leukocyte adherence and emigration as well as a profound albumin leakage response. Superfusion of the mesenteric microcirculation with the NO donors sodium nitroprusside, spermine-NO, and SIN1 significantly reduced the I/R-induced leukocyte adherence/emigration and albumin leakage in postcapillary venules, whereas neither spermine nor the NO synthase inhibitor NG-nitro-L-arginine methyl ester affected the I/R-induced responses. Platelet-leukocyte aggregation and mast cell degranulation were also observed in the postischemic mesentery, and the responses were also attenuated by the NO donors. Plasma nitrate/nitrite levels in the superior mesenteric vein were significantly reduced by I/R. The results of this study indicate that I/R-induced microvascular dysfunction (albumin leakage) is attenuated by NO and that the protective effect of NO donors may be related to their ability to reduce leukocyte-endothelial cell and leukocyte-platelet interactions and/or mast cell degranulation.

Inhibition of nitric oxide synthesis increases leukocyte and endothelial interaction in mesenteric venules. In this study, the relationship between inhibition of NO and expression of the adhesion molecule P-selectin was examined. The rat mesentery was superfused with the NO inhibitor NG-nitro-L-arginine methyl ester (L-NAME) either alone or in combination with intravenous infusions of L-arginine, D-arginine, a P-selectin-neutralizing monoclonal antibody (PB1.3 [1 mg/kg]), recombinant human superoxide dismutase (hSOD), or 8 bromoguanosine 3',5'-cyclic monophosphate (8-br-cGMP). Leukocyte rolling and adherence were monitored in mesenteric venules via intravital microscopy. Ileal tissue superfused with L-NAME was examined immunohistochemically for P-selectin expression. Superfusion of the rat mesentery with L-NAME resulted in a significant increase in leukocyte rolling and adherence in the mesenteric venule, which was attenuated by administration of L-arginine but not D-arginine. Monoclonal antibody PB1.3 as well as hSOD and 8-br-cGMP administered before initiation of L-NAME superfusion significantly attenuated the increase in leukocyte rolling and adherence. Immunohistochemistry showed a significant increase in P-selectin expression after 60 minutes of superfusion with L-NAME, which was attenuated by L-arginine, hSOD, and 8-br-cGMP. These data indicate an important functional relationship between endothelial-derived NO production and expression of the endothelial adhesion molecule P-selectin.