Shear-Induced Increase in Hydraulic Conductivity in Endothelial Cells Is Mediated by a Nitric Oxide–Dependent Mechanism

Abstract

Abstract —This study addresses the role of nitric oxide (NO) and its downstream mechanism in mediating the shear-induced increase in hydraulic conductivity (L p ) of bovine aortic endothelial cell monolayers grown on porous polycarbonate 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. Shear stress (20 dyne/cm 2 ) also elicited a multiphasic NO production pattern in which a rapid initial production was followed by a less rapid, sustained production. In the absence of shear stress, an exogenous NO donor, S -nitroso- N -acetylpenicillamine, increased endothelial L p 2.23±0.14-fold (100 μmol/L) and 4.8±0.66-fold (500 μmol/L) at the end of 3 hours. In separate 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 shear-induced increase in L p in a dose-dependent manner. Inhibition of guanylate cyclase (GC) with LY-83,583 (1 μmol/L) or protein kinase G (PKG) with KT5823 (1 μmol/L) failed to attenuate 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. However, additional metabolic inhibitors, 2 deoxyglucose (10 mmol/L) and oligomycin (1 μmol/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 metabolic pathway mediates the shear stress–L p response. An alternate mechanism downstream from NO that is sensitive to IAA must mediate this response.