SHEAR-STRESS INDUCES ATP-INDEPENDENT TRANSIENT NITRIC-OXIDE RELEASE FROM VASCULAR ENDOTHELIAL-CELLS, MEASURED DIRECTLY WITH A PORPHYRINIC MICROSENSOR

Shear stress causes the vascular endothelium to release nitric oxide ( NO), which is an important regulator of vascular tone. However, direct measurement of NO release after the imposition of laminar flow has no t been previously accomplished because of chemical (oxidative degradat ion) and physical (diffusion, convection, and washout) complications. Consequently, the mechanism, time course, kinetics, and Ca2+ dependenc e of NO release due to shear stress remain incompletely understood. In this study, we characterized these parameters by using fura 2 fluores cence and a polymeric porphyrin/Nafion-coated carbon fiber microsensor (detection limit, 5 nmol/L; response time, 1 millisecond) to directly measure changes in [Ca2+](i) and NO release due to shear stress or ag onist (ATP or brominated Ca2+ ionophore [Br-A23187]) from bovine aorti c endothelial cells. The cells were grown to confluence on glass cover slips, loaded with fura 2-AM, and mounted in a parallel-plate flow cha mber (volume, 25 mu L). The microsensor was positioned approximate to 100 mu m above the cells with its long axis parallel to the direction of flow. Laminar flow of perfusate was maintained from 0.04 to 1.90 mL /min, which produced shear stresses of 0.2 to 10 dyne/cm(2). Shear str ess caused transient NO release 3 to 5 seconds after the initiation of flow and 1 to 3 seconds after the rise in [Ca2+](i), which reached a plateau after 35 to 70 seconds. Although the amount (peak rate) of NO release increased as a function of the shear stress (0.08 to 3.80 pmol /s), because of the concomitant increase in the flow rate, the peak NO concentration (133+/-9 nmol/L) remained constant. Maintenance of flow resulted in additional transient NO release, with peak-to-peak interv als of 15.5+/-2.5 minutes. During this 13- to 18-minute period, when t he cells were unresponsive to shear stress, exogenous ATP (10 mu mol/L ) or Br-A23187 (10 mu mol/L) evoked NO release. Prior incubation of th e cells with exogenous NO or the removal and EGTA (100 mu mol/L) chela tion of extracellular Ca2+ blocked shear stress but not ATP-dependent NO release. The kinetics of shear stress-induced NO release (2.23+/-0. 07 nmol/L per second) closely resembled the kinetics of Ca2+ flux but differed markedly from the kinetics of ATP-induced NO release (5.64+/- 0.32 nmol/L per second). These data argue that shear stress causes a C a2+-mediated ATP-independent transient release of NO, where the peak r ate of release but not the peak concentration depends on the level of shear stress. The transient nature of this response may be due to NO-i nduced inhibition of Ca2+ influx via a mechanism yet to be determined.