Shear-induced increase in hydraulic conductivity in endothelial cells is mediated by a nitric oxide-dependent mechanism

This study addresses the role of nitric oxide (NO) and its downstream mecha nism in mediating the shear-induced increase in hydraulic conductivity (L-p ) of bovine aortic endothelial cell monolayers grown on porous polycarbonat e filters. Direct exposure of endothelial monolayers to 20-dyne/cm(2) shear stress induced a 4.70+/-0.20-fold increase in L-p at the end of 3 hours. S hear stress (20 dyne/cm(2)) also elicited a multiphasic NO production patte rn in which a rapid initial production was followed by a less rapid, sustai ned production. In the absence of shear stress, an exogenous NO donor, S-ni troso-N-acetylpenicillamine, increased endothelial L-p 2.23+/-0.14-fold (10 0 mu mol/L) and 4.8+/-0.66-fold (500 mu mol/L) at the end of 3 hours. In se parate experiments, bovine aortic endothelial cells exposed to NO synthase inhibitors, N-G-monomethyl-L-arginine and N-G-nitro-L-arginine methyl ester , exhibited significant attenuation of sheer-induced increase in L-p in a d ose-dependent manner. Inhibition of guanylate cyclase (GC) with LY-83,583 ( 1 mu mol/L) or protein kinase G (PKG) with KT5823 (1 mu mol/L) failed to at tenuate the shear-induced increase in L-p. Furthermore, direct addition of a stable cGMP analogue, 8-bromo-cGMP, had no effect in altering baseline L- p, indicating that the GC/cGMP/PKG pathway is not involved in shear stress- NO-L-p response. Incubation with iodoacetate (IAA), a putative inhibitor of glycolysis, dose-dependently increased L-p. Addition of IAA at levels that did not affect baseline L-p greatly potentiated the response of L-p to 20- dyne/cm(2) shear stress. Finally, both shear stress-induced and IAA-induced increases in L-p could be reversed with the addition of dibutyryl cAMP. Ho wever, additional metabolic inhibitors, 2 deoxyglucose (10 mmol/L) and olig omycin (1 mu mol/L), or reactive oxygen species scavengers, deferoxamine (1 mmol/L) and ascorbate (10 mmol/L), failed to alter shear-induced increases in L-p. Our results show that neither the NO/cGMP/PKG pathway nor a metabo lic pathway mediates the shear stress-L-p response. An alternate mechanism downstream from NO that is sensitive to IAA must mediate this response.