© 1995 American Heart Association, Inc.
Articles |
Shear Stress Induces ATP-Independent Transient Nitric Oxide Release From Vascular Endothelial Cells, Measured Directly With a Porphyrinic Microsensor
From the Departments of Pharmacology (A.J.K., H.C.S.) and Medicine (H.C.S., A.L.C.), Duke University Medical Center, and the Department of Biomedical Engineering (G.A.T.), Duke University, Durham NC; and the Department of Chemistry (S.G., T.M.), Oakland University, Rochester, Minn.
Correspondence to Dr Anthony J. Kanai, Department of Pharmacology, Box 3845, Durham, NC 27710.
Abstract 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
µL). The microsensor was positioned 
100 µm 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/cm2. 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
µmol/L) or Br-A23187 (10 µmol/L) evoked NO release. Prior
incubation of the cells with exogenous NO or the removal and EGTA (100
µmol/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.
Key Words: ATP • fura 2-AM fluorescence • nitric oxide–selective electrode • porphyrinic microsensor • shear stress
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