Laminar Flow Stimulates ATP- and Shear Stress-Dependent Nitric Oxide Production in Cultured Bovine Endothelial Cells

Abstract Based on the fact that nitric oxide (NO) production is associated with changes in intracellular cGMP levels and is selectively inhibited by N ω -methyl L-arginine (L-NMA), we investigated the shear stress dependency of NO production in endothelial cells (ECs) from its cGMP responses to various shear stress loads. Cultured fetal bovine aortic ECs treated with a phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX; 1 mM), were exposed to a laminar flow of Krebs buffer solution for 5 minutes in a parallel-plate flow chamber and examined for changes in intracellular cGMP levels by radioimmunoassay using an [ 125 I] cGMP kit. Application of flow increased the cGMP levels. The increase was significant in the presence of extracellular ATP (1 μM)(control, 286.1 ± 43.6; flow, 506.5 ± 44.9 fmol/10 7 cells; p>0.001), but not in its absence (control, 256.6 ± 60.6; flow, 301.5 ± 91.4 fmol/10 7 cells; N.S.). The cGMP levels increased significantly as the magnitude of shear stress applied increased. Treatment of ECs with a specific inhibitor of NO production, L-NMA (200 μM), completely inhibited the flow-induced increase in cGMP, and L-arginine reversed the L-NMA-induced inhibition, indicating that the increase in cGMP was due to NO produced by the flow. The flow-induced increase in NO production was markedly suppressed when extracellular Ca ++ was chelated by adding EGTA to the perfusate. These findings suggest that flow stimulates NO production to increase cGMP levels shear stress-dependently in ECs and that extracellular Ca ++ and ATP modulate the effects of flow.