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 microM), a PKC inhibitor, (Go6976, 10 nM), a calcium chelator, TMB-8 (0.1 mM) and a K+ channel blocker, glibenclamide, (10 microM), but not by a PKA inhibitor, (H-89, 0.1 microM) or a tyrosine kinase inhibitor, (genistein, I microM). 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.