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 Ca plus 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 [Ca plus] sub i and NO release due to shear stress or agonist (ATP or brominated Ca plus 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 equal 100 micro meter 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. Shear stress caused transient NO release 3 to 5 seconds after the initiation of flow and 1 to 3 seconds after the rise in [Ca plus]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 plus minus 9 nmol/L) remained constant. Maintenance of flow resulted in additional transient NO release, with peak-to-peak intervals of 15.5 plus minus 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 Ca plus blocked shear stress but not ATP-dependent NO release. The kinetics of shear stress-induced NO release (2.23 plus minus 0.07 nmol/L per second) closely resembled the kinetics of Ca plus flux but differed markedly from the kinetics of ATP-induced NO release (5.64 plus minus 0.32 nmol/L per second). These data argue that shear stress causes a Ca plus-mediated ATP-independent transient release of NO, w