No 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 4.70Ϯ0.20-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 2.23Ϯ0.14-fold (100 mol/L) and 4.8Ϯ0.66-fold (500 mol/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 583 (1 mol/L) or protein kinase G (PKG) with KT5823 (1 mol/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. Key Words: shear stress Ⅲ nitric oxide Ⅲ hydraulic conductivity Ⅲ endothelial cells E ndothelial cells provide the principal barrier to transport of fluids and solutes between blood and underlying tissue. In arteries, shear-dependent permeability has been hypothesized to play a role in the localization of atherosclerotic lesions in regions of vessel branching and curvatures. 1 The first clear demonstration of a direct effect of shear stress on endothelial permeability was reported by Jo et al. 2 Other investigators 3-5 have provided supporting evidence for flowor shear-dependent endothelial solute permeability. More recently, Sill et al, 6 using the same in vitro model as Jo et al, demonstrated that endothelial monolayer hydraulic conductivity (L p ) is also sensitive to acute changes in shear stress level. This finding is consistent with an earlier whole-artery study reported by Lever et al 7 and with a study involving frog mesenteric venular capillary measurements performed by Williams and Huxley. 4 The only insight into the cellsignaling mechanism underlying the shear stress response of L p was provided by Sill et al, who demonstrated that the response could be blocked by addition of a cAMP analogue or a phosphodiesterase inhibitor, suggesting a role for cAMP in the pathway.It is well established that shear stress stimulates the release of nitric oxide (NO) from cultured endothelial cells. 8,9 Direct infusion of NO synthase (NOS) inhibitors into inta...

The shear stress of flowing blood on endothelial cells increases water transport (hydraulic conductivity, Lp) in several vascular beds in vivo and has been hypothesized to play a role in elevating vascular transport in ocular diseases such as diabetic retinopathy. The purpose of this study is to determine the response of Lp to varying levels of shear stress using an in vitro model of the blood-retinal barrier: bovine retinal endothelial cells (BRECs) grown on polycarbonate filters. The study also addresses the role of nitric oxide (NO) and other downstream effectors in mediating shear-induced changes in water transport. A step change in shear stress of 10 dyn/cm(2) did not produce a significant change in Lp over 3 hours, whereas a 20 dyn/cm(2) step change elevated Lp by 14.6-fold relative to stationary controls at the end of 3h of shear exposure. 20 dyn/cm( 2) of shear stress stimulated the endothelial monolayers to release nitric oxide in a biphasic manner and incubation of the BRECs with a nitric oxide synthase (NOS) inhibitor, L-NMMA, significantly attenuated the shear-induced Lp response. These experiments demonstrate that NO is a key signaling molecule in the pathway linking shear stress and Lp in BRECs. A widely studied pathway downstream of NO involves the activation of guanylate cyclase (GC), guanosine 3', 5' -- cyclic monophosphate (cGMP) and protein kinase G (PKG). It was observed that incubation of BRECs with the GC inhibitor, LY83583 (10 microM) or the PKG inhibitor, KT5823 (1 microM) did not significantly alter the shear-induced Lp response. Also the cGMP analogue, 8-br-cGMP (1mM), did not affect the baseline Lp over 4h. These results demonstrate that shear stress elevates hydraulic conductivity in BRECs through a signaling mechanism that involves NO but not the GC/cGMP/PKG pathway.