LAMINAR-FLOW STIMULATES ATP- AND SHEAR STRESS-DEPENDENT NITRIC-OXIDE PRODUCTION IN CULTURED BOVINE ENDOTHELIAL-CELLS

Based on the fact that nitric oxide (NO) production is associated with changes in intracellular cGMP levels and is selectively inhibited by Nω -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 [125I] 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/107 cells; p>0.001), but not in its absence (control, 256.6 ± 60.6; flow, 301.5 ± 91.4 fmol/107 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. © 1994 Academic Press, Inc.