Effect of shear stress on the hydraulic conductivity of cultured bovine retinal microvascular endothelial cell monolayers

The shear stress of flowing blood on endothelial cells increases water tran sport (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 det ermine the response of Lp to varying levels of shear stress using an in vit ro model of the blood-retinal barrier: bovine retinal endothelial cells (BR ECs) grown on polycarbonate filters. The study also addresses the role of n itric 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) d id 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 t he endothelial monolayers to release nitric oxide in a biphasic manner and incubation of the BRECs with a nitric oxide synthase (NOS) inhibitor, L-NMM A, significantly attenuated the shear-induced Lp response. These experiment s demonstrate that NO is a key signaling molecule in the pathway linking sh ear stress and Lp in BRECs. A widely studied pathway downstream of NO invol ves the activation of guanylate cyclase (GC), guanosine 3', 5' - cyclic mon ophosphate (cGMP) and protein kinase G (PKG). It was observed that incubati on of BRECs with the GC inhibitor, LY83583 (10 muM) or the PKG inhibitor, K T5823 (1 muM) did not significantly alter the shear-induced Lp response. Al so the cGMP analogue, 8-br-cGMP (1mM), did not affect the baseline Lp over 4h. These results demonstrate that shear stress elevates hydraulic conducti vity in BRECs through a signaling mechanism that involves NO but not the GC /cGMP/PKG pathway.