NITRIC-OXIDE ATTENUATES REOXYGENATION-INDUCED ICAM-1 EXPRESSION IN CORONARY MICROVASCULAR ENDOTHELIUM - ROLE OF NF-KAPPA-B

Enhanced leukocyte adhesion has been shown to occur in postischemic re perfused hearts due to the upregulation of specific cell-surface adhes ion molecules. Therefore, we investigated the influence of 4 h of reox ygenation after 20 h of hypoxia on ICAM-1 induction in primary culture s of rat coronary microvascular endothelial cells (CMEC). ICAM-1 surfa ce expression as well as oxygen free radical formation were measured b y now cytometry. Changes in ICAM-1 mRNA levels were assessed by Northe rn blot and activation of NF kappa B and AP-1 signalling were analysed by electrophoretic mobility shift assays (EMSA) in CMEC lysates. Alth ough hypoxia alone did not affect cell-surface ICAM-1 expression, 4 h of reoxygenation induced a significant upregulation of ICAM-1. ICAM-1 mRNA could not be found after hypoxia alone, but could be detected as early as 1 h following reoxygenation. Unlike AP-1, the activation of w hich could be detected in CMEC lysates following hypoxia alone, NF kap pa B binding activity was induced only following reoxygenation, concur rent with an increase in the formation of reactive oxygen species (ROS ). A proteasome inhibitor, nor-Leu (25 mu M) inhibited NF kappa B acti vation by reoxygenation and ICAM-1 expression. Blockade of endogenous nitric oxide (NO) synthesis in CMEC with L-nitroarginine (10 mu M) acc entuated post-reoxygenation ICAM-1 expression. Finally an exogenous NO donor, s-nitrosoacetyl-penicillamine (SNAP 100 mu M), suppressed the generation of ROS upon reoxygenation, and blocked the activation of NF kappa B and the upregulation of ICAM-1. Thus, ICAM-1 upregulation in CMEC primary cultures is not induced by hypoxia alone, but appears sho rtly after reoxygenation in the absence of exogenous cytokines or infl ammatory cells. Because upregulation of AP-1 through hypoxia alone did not affect ICAM-1 expression, we conclude that redox-sensitive NF kap pa B activation triggers ICAM-1 upregulation. NO inhibits reoxygenatio n-specific ICAM-1 upregulation, most likely by diminishing oxidative s tress that leads-to NF kappa B activation. (C) 1997 Academic Press Lim ited.