Endothelial cells release nitric oxide (NO) more potently in response to increased shear stress than to agonists which elevate intracellular free calcium concentration ([Ca2+]i). To determine mechanistic differences in the regulation of endothelial constitutive NO synthase (ecNOS), we measured NO production by bovine aortic endothelial cells exposed to shear stress in a laminar flow chamber or treated with Ca2+ ionophores in static culture. The kinetics of cumulative NO production varied strikingly: shear stress (25 dyne/cm2) stimulated a biphasic increase over control that was 13-fold at 60 minutes, whereas raising [Ca2+]i caused a monophasic 6-fold increase. We hypothesized that activation of a protein kinase cascade mediates the early phase of flow-dependent NO production. Immunoprecipitation of ecNOS showed a 210% increase in phosphorylation 1 minute after flow initiation, whereas there was no significant increase after Ca2+ ionophore treatment. Although ecNOS was not tyrosine-phosphorylated, the early phase of flow-dependent NO production was blocked by genistein, an inhibitor of tyrosine kinases. To determine the Ca2+ requirement for flow-dependent NO production, we measured [Ca2+]i with a novel flow-step protocol. [Ca2+]i increased with the onset of shear stress, but not after a step increase. However, the step increase in shear stress was associated with a potent biphasic increase in NO production rate and ecNOS phosphorylation. These studies demonstrate that shear stress can increase NO production in the absence of increased [Ca2+]i, and they suggest that phosphorylation of ecNOS may importantly modulate its activity during the imposition of increased shear stress.

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The effect of extracellular L-arginine and L-glutamine on nitric oxide (NO) release was studied in cultured bovine aortic endothelial cells and in rabbit aortic rings. Increasing L-arginine (0.01 to 10 mM) did not alter NO release from cultured endothelial cells or modify endothelium-dependent relaxation to acetylcholine in isolated vessels. L-Glutamine (0.6 and 2 mM) inhibited NO release from cultured cells (in response to bradykinin) and from aortic rings (in response to acetylcholine or ADP). L-Arginine (0.1-10 mM) dosedependently reversed the L-glutamine inhibition of receptorstimulated NO release in both models. In contrast to its inhibitory response to receptor-mediated stimuli, glutamine alone slightly potentiated NO release in both models when the calcium ionophore, A23187, was added. Furthermore, cultured cells incubated with L-arginine (0.01-10 mM), in the presence or absence of glutamine, released similar amounts of NO in response to A23187. L-Glutamine did not affect intracellular L-arginine levels. Neither D-glutamine nor D-arginine affected NO release or endothelium-dependent vascular relaxation. L-Glutamine had no effect on the activity of endothelial NOS assessed by L-arginine to L-Citrulline conversion. These findings show that in the absence of L-glutamine, manipulating intracellular L-arginine levels over a wide range does not affect NO release. L-Glutamine in concentrations circulating in vivo may tonically inhibit receptor-mediated NO release by interfering with signal transduction. One mechanism by which L-arginine may enhance NO release is via reversal of the inhibitory effect of L-glutamine, but apparently independently of enhancing NO synthase substrate. (J. Clin. Invest. 1995. 95:2565-2572

Polyetheretherketone (PEEK) is an attractive material for the encapsulation of active medical implants. PEEK, however, shows hydrophobic surface properties which are not favorable for protein absorption and cell adhesion. We show that oxygen rich nanofilms “sticky thin film,” deposited on PEEK surfaces from plasma using a plasma immersion ion implantation and deposition technique with a (CH4/O2) gas mixture greatly improved cell adhesion (up to 75%) and spreading (up to 81%). Strong correlations were found between cell adhesion and the water contact angle, the polar component of surface energy, and to a lesser extent oxygen concentration of the PEEK surfaces. Surface polarity of the plasma deposited “sticky thin film” was deemed to be the predominant factor in influencing cell adhesion.

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The VentrAssist implantable rotary blood pump (IRBP) is an implantable centrifugal blood pump with a hydrodynamically suspended impeller; optimal efficiency requires small running clearances (70-300 microm). The effect of running clearance and polish on hemolysis was evaluated in vitro. Three different human blood suspensions were compared: phosphate buffered saline (PBS), plasma volume expander (Hemaccel), and whole blood. The test conditions were: blood hematocrit 30%, flow rate 5 L/min, pressure across pump 100 mm Hg, 6 h flow period, and 37 degrees C. Normalized Index of Hemolysis (NIH) for the Biomedicus BP-80, used as a control, was: 0.0040 +/- 0.0023 (n = 9; x +/- SD) and 0.00014 +/- 0.00009 (n = 5) for pooled blood suspensions in PBS and Hemaccel respectively, and 0.00053 +/- 0.0002 (n = 3) in whole blood. Hemolysis was reduced by improved surface finish and unaffected by running clearance. NIH for the VentrAssist IRBP with 0.2 microm Ra surface finish was 0.000167 +/- 0.00007 (n = 4) g/100 L in whole human blood, demonstrating minimal hemolysis.

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The purpose of this study was to characterize the effect of various shear conditions on endothelial cell intracellular calcium ([Ca2+]i). Bovine aortic endothelial cells (BAEC) were loaded with Fluo-3 and exposed to flow in a parallel plate flow chamber designed for confocal microscopy. The flow medium was medium 199 (M-199), which was prepared with and without adenosine triphosphate (ATP). In the presence of ATP, initiation of flow at a shear stress of 2.5 dyn/cm2 evoked a strong, sustained elevation of [Ca2+]i that gradually returned to baseline levels over 10 to 15 min. By contrast, in the absence of ATP, initiation of flow at 2.5 dyn/cm2 produced only transient increases in [Ca2+]i in a small proportion of the cells. As shear rate was increased from 2.5 to 15 dyn/cm2 in this medium, both the relative fluorescence of the monolayer and the proportion of cells across the monolayer that displayed calcium transients increased in a dose-dependent fashion. In conclusion, the response of an endothelial cell monolayer to increasing levels of shear is not only to increase [Ca2+]i within individual cells, but to increase the duration of response and the number of cells responding at the onset of shear. This recruitment of larger numbers of cells at higher levels of shear may represent a novel signaling mechanism within the endothelium.