Intracellular mechanisms of nitric oxide plus hydrogen peroxide-mediated neutrophil adherence to cultured human endothelial cells

Abstract.

Objective and Design: We investigated signal-transduction in nitric oxide/hydrogen peroxide (NO/H₂O₂) mediated neutrophil-endothelial adhesion and P-selectin mobilization.¶Materials and Methods: Human endothelial monolayers (HUVEC) were exposed to 0.1 mM H₂O₂ plus an NO donor, 0.5 mM spermine-NONOate, and second message inhibitors and neutrophil adhesion and P-selectin expression measured.¶Results: Neutrophil adherence induced by NO/H₂O₂ was blocked by a PKG inhibitor, (KT5823, 0.5 μM), a PKC inhibitor, (Gö6976, 10 nM), a calcium chelator, TMB-8 (0.1 mM) and a K⁺ channel blocker, glibenclamide, (10 μM), but not by a PKA inhibitor, (H-89, 0.1 μM) or a tyrosine kinase inhibitor, (genistein, 1 μM). P-selectin expression induced by NO/H₂O₂ was blocked by KT5823 and Gö6976, but not by TMB-8 or glibenclamide.¶Conclusions: These data demonstrate that NO/ H₂O₂ promotes neutrophil-endothelial adhesion through PKG, PKC, calcium, and K⁺ channels, but not PKA or tyrosine kinase. Conversely, P-selectin mobilization requires only PKG and PKC.