Nitric oxide donor-induced persistent inhibition of cell adhesion protein expression and NFkappaB activation in endothelial cells.

Nitric oxide (NO), applied by inhalation or released from NO donors, has been used to reduce the expression of cell adhesion molecules (CAM) and ameliorate other consequences of ischemia/reperfusion (I/R) injury. In this study, we have assessed the time frames of pretreatment and of the duration of the preconditioned state using human umbilical vein endothelial cells (HUVECs) and the NO donor, SNAP, in combination with cysteine. The induction of vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM) and E-selectin by the cytokines TNFalpha and IL-1beta, and by bacterial lipopolysaccharide (LPS) was reduced by SNAP/Cys preincubation (30 min, 1mM) to less than 10% of controls. This refractory state in respect to cytokine-induced CAM expression persisted for 6h after washout of the NO donor in the combination TNFalpha/VCAM, and a partial block was still observed after 8h. The effect was not mediated by the cGMP pathway, as was demonstrated by using the inhibitor of guanylyl cyclase, ODQ, and the cGMP analogue, 8-Br-cGMP. The TNFalpha-induced expression of CAM was exclusively dependent on the transcription factor NFkappaB since the inhibitor of NFkappaB activation, BAY 11-7082, completely blocked the induction. The TNFalpha-induced phosphorylation and degradation of the inhibitor of kappaB (IkappaBalpha) was suppressed for up to 8h after SNAP/Cys pretreatment. The inhibitory S-nitrosation of IkappaB kinase (IKKbeta), as assessed by the biotin-switch-procedure and immunoprecipitation, was only detectable immediately after SNAP/Cys incubation but not at later time points. In summary, a short preincubation of HUVEC with SNAP/Cys results in a persistent suppression of NFkappaB-dependent expression of CAM. The stabilization of IkappaBalpha over the same time span may be causally related to this effect.