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 not been previously accomplished because of chemical (oxidative degradation) and physical (diffusion, convection, and washout) complications. Consequently, the mechanism, time course, kinetics, and Ca2+ dependence of NO release due to shear stress remain incompletely understood. In this study, we characterized these parameters by using fura 2 fluorescence 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 agonist (ATP or brominated Ca2+ ionophore [Br-A23187]) from bovine aortic endothelial cells. The cells were grown to confluence on glass coverslips, loaded with fura 2-AM, and mounted in a parallel-plate flow chamber (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 stress 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 intervals of 15.5+/-2.5 minutes. During this 13- to 18-minute period, when the 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 the cells with exogenous NO or the removal and EGTA (100 mu mol/L) chelation 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 Ca2+-mediated ATP-independent transient release of NO, where the peak rate 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-induced inhibition of Ca2+ influx via a mechanism yet to be determined.