Phosphorylation of Endothelial Nitric Oxide Synthase in Response to Fluid Shear Stress
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.
Endothelial cells in normal blood vessels secrete NO tonically and increase NO production dynamically in response to increased shear stress. NO contributes to vessel homeostasis by inhibiting vascular smooth muscle tone1 and growth,2 platelet aggregation,3 and leukocyte binding to endothelium.4 With exercise, an increase in cardiac output is transduced into increased regional tissue perfusion via flow-induced enhancement of ecNOS (also known as type III NOS) activity. Although blood flow is a critical regulator of endothelial NO production in the physiological context of the blood vessel, the signaling mechanisms used by endothelial cells to produce NO in response to flow-mediated increases in shear stress remain incompletely defined.
Previous investigations from our laboratories5 6 and others7 have established that exposure of endothelium to increased fluid shear stress may stimulate the release of Ca2+ from intracellular stores, with consequent elevation of [Ca2+]i. It is also generally accepted that ecNOS is regulated by Ca2+ through its interaction with calmodulin, with the binding of the Ca2+-calmodulin complex to a specific region of ecNOS increasing its enzymatic activity.8 Recent experimental results, however, question the concept that ecNOS is solely regulated through Ca2+-dependent mechanisms, as it is also posttranslationally modified by myristoylation9 and palmitoylation,10 and its cellular activity may be modulated by alkalinization,11 translocation,12 and the availability of cofactors.13 These multiple regulatory mechanisms may be used by endothelial cells to modulate ecNOS activity precisely in response to humoral and physical stimuli.
Increased shear stress imposes tension that must be accommodated by the endothelial cytoskeleton through rearrangement of actin and intermediate filament proteins at sites of cell-matrix contact, designated focal adhesion complexes.14 Focal adhesion complexes have been found to contain proteins (such as pp125FAK, paxillin, and tensin) that are tyrosine-phosphorylated after endothelial cell plating and adhesion, suggesting that these phosphorylation events are associated with cytoskeletal modifications.15 Because cytoskeletal changes are common to both adhesion and the response to shear stress, tyrosine phosphorylation may regulate both processes. Indeed, a recent study demonstrated attenuation of flow-dependent NO production in intact blood vessels after inhibition of tyrosine kinases.16 These proximal tyrosine kinase events may be transduced to downstream serine-threonine regulatory phosphorylations by kinases such as the MAP kinase family. We have recently observed that the MAP kinases Erk 1 and 2 are rapidly activated in response to increased shear stress in endothelial cells.17 Although many phosphorylation events are regulated by Ca2+-dependent signal pathways, we found that flow-dependent Erk 1/2 activation does not require an increase in [Ca2+]i. ecNOS contains consensus sequences for phosphorylation by several protein kinases,18 but its regulation by Ca2+-dependent or Ca2+-independent phosphorylation has not been previously determined under physiological conditions.
On the basis of these considerations, we hypothesized that phosphorylation might be an important modulator of ecNOS activity after exposure of BAECs to shear stress.19 Therefore, we compared the changes in NO production, ecNOS phosphorylation, and [Ca2+]i after exposure of BAECs to increased shear stress and Ca2+-mobilizing agents. We demonstrate that (1) increased shear stress stimulates NO production more potently than do Ca2+-mobilizing agents, (2) shear stress, but not Ca2+-mobilizing agents, increases ecNOS phosphorylation in association with increased NO production, and (3) NO production and ecNOS phosphorylation increase without an increase in [Ca2+]i, when BAECs under chronic low shear stress are exposed to an acute increase in shear stress. These results suggest that phosphorylation of ecNOS constitutes a discrete signaling mechanism associated with its activation by fluid shear stress.
Materials and Methods
BAEC cultures were established by collagenase digestion, as previously described,20 and maintained in medium 199 supplemented with 10% FCS. Confluent BAECs at passages 5 to 11 were seeded at high density onto plastic rectangular tissue culture plates or 60-mm dishes and used the following day at 90% to 95% confluence for functional or biochemical studies. For studies of [Ca2+]i, BAECs were plated onto the coverslip of a confocal flow chamber.
Exposure to Fluid Shear Stress and Agonists for NO and ecNOS Phosphorylation Measurements
BAECs were exposed to fluid shear stress in a parallel-plate flow chamber. The rectangular plates were gently rinsed in warmed Krebs' buffer containing (mmol/L) NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, dextrose 11, and NaHCO3 25 at pH 7.40 and secured in the chamber below a Mylar gasket and a glass cover with inflow and outflow slots. Shear stress was calculated from the dimensions of the chamber, gasket thickness, and the buffer perfusion rate and its viscosity.20 Krebs' buffer was stored in a heated oxygenated (95% O2/5% CO2) reservoir and was delivered through tubing wrapped with heating tape to maintain a buffer temperature of 37°C within the chamber. Two different flow stimulus protocols were used. For the protocol designated “static step,” BAECs taken from the static condition in culture were exposed to a rapidly (<2 seconds) imposed level of shear stress. The buffer was recirculated using a peristaltic pump and a closed loop system. Alternatively, a protocol designated “steady step” was used, in which BAECs were exposed to a low level of steady shear stress, followed by a rapid ramp to a higher level. For these experiments, the Krebs' buffer superfused the chamber in a single pass. For agonist stimulation, BAECs in 60-mm culture dishes were gently rinsed with warmed Krebs' buffer, and stimulation was performed by addition of buffer containing agonists at the appropriate dilutions. ATP, A23187, thapsigargin, and genistein were obtained from Sigma Chemical Co.
NO released by BAECs in response to shear stress or agonists was measured as its NOx metabolites, using a chemiluminescence detector.21 Samples of effluent were withdrawn from the culture dishes or a port just downstream from the outflow slot of the flow chamber and stored on ice for assay later that day. Aliquots were injected into a reflux vessel containing vanadium(III) in HCl at 90°C, where the stable NOx nitrate and nitrite are reduced to NO• gas. Released NO• is carried into the NO analyzer, where its reaction with ozone yields light that is detected by a photomultiplier tube and displayed as an analog signal. For each experiment, a standard curve was constructed using 50 to 250 nmol NaNO3 for calculation of nanomolar NOx content per sample. The background signal in the Krebs' buffer was subtracted from each measured value, and the result was normalized for volume and flow rate as described in the figure legends.
Analysis of ecNOS Phosphorylation
BAECs were metabolically labeled for 4 hours with [32P]orthophosphate in PO4-free DMEM supplemented with 10% FCS, rinsed three times with warmed Krebs' buffer, and placed in the flow chamber. After application of fluid shear stress for the described time periods, the chamber was placed in an ice bath; an ice-cold lysis solution containing (mmol/L) NaCl 50, Na2H2P2O7 50, NaF 50, EGTA 5, EDTA 5, Na3VO4 0.1, phenylmethylsulfonyl fluoride 50, HEPES 10, and leupeptin 0.021, along with 1% Triton X-100, at pH 7.4, was injected through the chamber; and extracts were collected into prechilled tubes. To assay agonist-stimulated ecNOS phosphorylation, BAECs in 60-mm dishes were labeled and exposed to agonist in Krebs' buffer. Lysates were collected by scraping, and cellular debris was removed by centrifugation for 30 minutes at 12 000 rpm at 4°C. ecNOS was immunoprecipitated from the supernatants by incubation with an affinity-purified rabbit polyclonal antibody (P459) raised to ecNOS amino acids 628 to 63818 and protein A Sepharose. ecNOS in 10% SDS PAGE gels was identified by Coomassie staining and autoradiography. Fractions of each sample were also run on the same gels and processed for Western analysis using Ab P459 with visualization of ecNOS by ECL. The density of autoradiographic and ECL bands was quantified using an LKB laser scanner and NIH Image 1.51 software. ecNOS phosphorylation was obtained by normalizing the autoradiographic to the ECL band densities and expressing the results as percent control for that experimental time course. To assay for tyrosine phosphorylation of ecNOS, blots were stripped and reprobed with anti-phosphotyrosine antibody 4G10 (UBI).
Exposure to Fluid Shear Stress for Measurement of [Ca2+]i by Confocal Microscopy
For measurement of [Ca2+]i, BAECs were exposed to fluid shear stress in a parallel-plate flow chamber similar to that used for measurements of NOx and ecNOS phosphorylation, with adaptations necessary for confocal microscopy.5 Briefly, the polycarbonate chamber consisted of a base and a cover plate, separated by a Mylar gasket. The Mylar had a rectangular opening, which defined the width, length, and thickness of the channel. Shear stress was determined from the perfusion rate, the fluid viscosity, and the dimensions of the channel as described previously. Krebs' buffer containing probenecid (2.5 mmol/L), to inhibit transport of the dye from the cell,22 flowed from a 200-mL reservoir through a short heating loop immersed in a 38°C water bath and into the confocal flow chamber mounted on the microscope stage. The temperature of the flow chamber was maintained using an air stream heater. Downstream from the flow chamber was a flowmeter (FL-102/1, Omega Engineering) and a lower glass reservoir from which buffer was pumped back to the upper reservoir.
For confocal Ca2+ experiments, the cells were grown on glass coverslips precoated with collagen (collagen type 1, Collaborative Biomedical Products) recessed into the base of the flow chamber cover plate. Before dye loading, the flow chamber was secured by attachment of the base with the Mylar gasket to the cover plate with its monolayer of cells along the coverslip. The cells were rinsed by gentle injection of 1 mL of flow medium into the flow chamber through a three-way tap. One milliliter of Krebs' buffer containing 1.5 μmol/L fluo 3-AM (Molecular Probes), 0.1% bovine serum albumin, and 5 mmol/L probenicid (Sigma) was injected and incubated on the cells for 15 minutes at 20°C. The cells were rinsed by injection of 2 mL of flow medium before the chamber was mounted on the microscope stage for exposure to flow. For some experiments, ATP was injected to give a final concentration of 1.8 μmol/L in the flowing Krebs' buffer at 10 minutes, instead of the step in shear to 25 dyne/cm2.
Measurement of [Ca2+]i by Confocal Microscopy
The confocal imaging system (MRC-600, Biorad) had an Argon-ion laser (excitation wavelengths, 488 nm and 514 nm) coupled to an upright microscope (Nikon, Optiphot-2) with a ×60 objective (Nikon, Plan Apo; numerical aperture, 1.4; oil). A neutral density filter (ND 2) was used to maintain the excitation energy at 0.3 mW for all experiments, and emitted fluorescence was detected using a 540±15-nm filter. The width of the confocal aperture, the background level setting, and the gain were maintained constant (aperture, 5/15; background, 500/1000; and gain, 800/1000) for all studies. These settings eliminated all background fluorescence in the presence of unlabeled cells. The scan area corresponded to a quarter-screen region of 384×256 pixels, which measured 204×136 μm.
At the beginning of each study, a region of the monolayer was selected for imaging during the initiation of flow, and a “before-flow” image was collected. A time series of 20 scans at 3-second intervals was collected, with initiation of flow to 5 dyne/cm2 at the end of the fifth scan. At the termination of this time series, while flow at 5 dyne/cm2 continued, additional images were collected. In order to limit bleaching of the dye, the regions were scanned at 30-second intervals until 4 minutes after the initiation of flow and, subsequently, at 1-minute intervals up to 10 minutes. The flow was then ramped rapidly to 25 dyne/cm2, with collection of 20 images at 3-second intervals, as described for the initial onset of flow. Further images were collected at 30-second and then 1-minute intervals, as described previously, for the succeeding 5 minutes of exposure to flow at 25 dyne/cm2.
The series of gray scale images was analyzed using the computer program Spyglass Transform (Spyglass). For this analysis, a smoothing filter was applied to an image of the cells collected before flow started in order to smooth the outlines of the cells in this image. Thresholding of this image at an intensity of 10 (maximal intensity, 255 pixel units) created a binary image for differentiation of the cells from the background region. The average pixel intensity of the cell region selected by this binary image was then determined for the ensuing time sequence, providing quantification of the average pixel intensity of all of the cells within the image over the time of the sequence. Average monolayer intensities were normalized to the average pixel intensity of the monolayer immediately before the onset of flow.
Results of NO, ecNOS phosphorylation, and [Ca2+]i measurements were expressed as mean±SE. The Super ANOVA software package was used to calculate measures of significance (P<.05).
The effect of fluid shear stress, compared with Ca2+-mobilizing agents, on NOx production by BAECs was studied. After culture under static conditions, sudden-onset laminar shear stress (static step) at 25 dyne/cm2 caused a rapid and potent increase in the production of NOx, followed by a slower sustained phase (Fig 1⇓). Relative to static buffer-treated control samples, shear stress increased cumulative NOx production by 13-fold after 60 minutes of exposure. In contrast, stimulation of static cultured cells with agonists that increase [Ca2+]i, such as the Ca2+ ionophore A23187 or thapsigargin (not shown), failed to elicit the rapid early (<5-minute) NOx production seen with shear stress. Rather, they stimulated a monophasic increase in NOx production that reached sixfold over control at 60 minutes. These differences in the time course and magnitude of NOx production suggested that shear stress rapidly increased ecNOS activity through an additional signal mechanism(s) beyond that due to an increase in cytoplasmic [Ca2+]i.
We hypothesized that a protein kinase cascade mediates mechanotransduction of shear stress to NOx production. To determine whether ecNOS is phosphorylated after exposure to fluid flow, we metabolically labeled BAECs with [32P]orthophosphate and exposed the cells to 25 dyne/cm2 shear stress for 1 minute. Fig 2⇓ shows that ecNOS was rapidly phosphorylated in response to shear stress. Depicted are the results of ecNOS immunoprecipitation from cell lysates, followed by size fractionation using 10% SDS-PAGE and ecNOS imaging by Coomassie stain (Fig 2A⇓), autoradiography (Fig 2B⇓), and Western blot (Fig 2C⇓). A predominant 133-kD ecNOS band is seen, obtained in equal amounts from control cells in static culture and cells exposed to 25 dyne/cm2 shear stress. Quantitative specific immunoprecipitation is indicated by the absence of ecNOS immunoreactivity in the supernatant fractions despite the detection of other proteins by Coomassie stain and autoradiography. ecNOS phosphorylation, normalized to ecNOS protein on Western blots, increased 210% (n=3, P<.02) after 1 minute of exposure to 25 dyne/cm2 shear stress (Fig 3A⇓). To determine whether a [Ca2+]i increase in the absence of flow is sufficient to increase ecNOS phosphorylation, BAECs were labeled with [32P]orthophosphate and exposed to the Ca2+ ionophore A23187 in Krebs' buffer for 0 to 60 minutes. There was no significant increase in ecNOS phosphorylation after stimulation with A23187 (1 μmol/L) for short (≤5-minute) or long (20- to 60-minute) periods of exposure. Fig 3B⇓ shows the cumulative results of A23187-stimulated ecNOS phosphorylation.
To determine the nature of the upstream protein kinase cascade activated by shear stress, cells were pretreated with the tyrosine kinase inhibitor genistein (20 μmol/L for 1 hour). Fig 4⇓ shows that genistein attenuated the rapid early phase of flow-dependent NOx production, resulting in a slow monophasic increase. In contrast, genistein did not affect A23187-dependent NOx production (not shown). These results suggested that the early rapid phase of NOx production is mediated by a tyrosine kinase–dependent pathway and that ecNOS itself might be tyrosine-phosphorylated. However, ecNOS phosphorylation likely occurred on serine and/or threonine residues, because stripping and reprobing of Western blots (as in Fig 2⇑) with an anti-phosphotyrosine antibody revealed no ecNOS phosphotyrosine content (not shown).
To further assess the relationships among flow-dependent NOx production, [Ca2+]i increase, and ecNOS phosphorylation, we used a steady-step protocol in which shear stress was rapidly ramped from a low to high level. Fig 5 shows the steady-step protocol used for the remaining experiments, in which cells were initially exposed for 10 minutes to low (5 dyne/cm2) shear stress, followed by a rapid increase to high shear stress (25 dyne/cm2). To better correlate flow-dependent increases in NOx production and ecNOS phosphorylation, we measured NOx production rate at 0.5- to 2-minute intervals. Low shear stress was associated with a minimal increase in NOx production rate. After a rapid increase to high shear stress, the rate of NOx production maximally increased 470% (P<.01) 1 minute after the flow increase. The increased production rate was sustained, as there was a 240% increase 5 minutes after the onset of high flow (P<.04 relative to 5 dyne/cm2 baseline).
The [Ca2+]i increase during this steady-step protocol was also measured using confocal microscopy of cells loaded with the fluorescent dye fluo 3-AM. As can be seen in a representative experiment (Fig 6⇓), the initial exposure to low (5 dyne/cm2) shear stress increased [Ca2+]i by ≈40% at 20 seconds, followed by a return to baseline by 30 seconds. In contrast, the rapid increase to 25 dyne/cm2 after 10 minutes of low flow failed to increase [Ca2+]i further. To ensure that the lack of a second [Ca2+]i increase was not the consequence of depletion of intracellular Ca2+ stores or deterioration of the cells, we performed experiments in which shear stress was maintained at 5 dyne/cm2 for 15 minutes. Exposure to ATP (1.8 μmol/L) 10 minutes after initiation of low flow resulted in a robust [Ca2+]i increase (Fig 7⇓). In the presence of low shear stress, this relatively low concentration of ATP potently stimulated NOx production, presumably as a consequence of purinergic receptor activation. Cumulative results of these studies are shown in Fig 8⇓, demonstrating that the cells retained the capacity to increase [Ca2+]i, in the presence of low shear stress, when appropriately stimulated.
Having determined the kinetics of NOx production and [Ca2+]i increase, we then characterized the changes in ecNOS phosphorylation using the steady-step protocol. Cells were lysed after 10 minutes of exposure to low shear stress (5 dyne/cm2) or after an additional 0.5-, 1-, or 5-minute exposure to high shear stress (25 dyne/cm2). Phosphorylation of ecNOS was analyzed for each time point as described before and expressed as percent control for that experiment. Fig 9⇓ displays the cumulative results of four experiments, showing that ecNOS was rapidly phosphorylated after a step from low to high shear stress. ecNOS phosphorylation increased 130% (P<.01) 0.5 minute after a step from low to high shear stress and was maintained as a 60% increase at 5 minutes (P<.08). Thus, a steady-step increase in fluid shear stress increased NOx production rate and ecNOS phosphorylation but was not associated with a significant increase in [Ca2+]i.
The major finding of these studies is that NO production following increased shear stress correlated more closely with phosphorylation of ecNOS than with [Ca2+]i. Fluid shear stress potently stimulated a biphasic increase in BAEC NO production, consisting of an early (<5-minute) transient phase and a late (>5-minute) sustained phase. The early phase was associated with a significant increase in ecNOS phosphorylation, which was detected using either the static-step or the steady-step protocol. In contrast, agents that increase [Ca2+]i stimulated a monotonic increase in NO production but did not significantly increase ecNOS phosphorylation. The close temporal correlation between ecNOS phosphorylation and NO production rate suggests that phosphorylation is one of the mechanisms used by endothelial cells to regulate the activity of ecNOS in response to increased shear stress.
The present study is one of the first to closely examine the regulation of NO production with a steady-step flow paradigm, which likely resembles the changes in shear stress occurring in the intact circulation. Our data suggest that the static-step onset of 25 dyne/cm2 shear stress (Figs 1 through 4⇑⇑⇑⇑) stimulated NOx production and ecNOS phosphorylation (210% increase at 1 minute) through both Ca2+-dependent and Ca2+-independent pathways. The confocal microscopic studies indicate that the steady-step increase in shear stress (Figs 5 through 9⇓⇓⇓⇓⇓) stimulated only Ca2+-independent ecNOS phosphorylation, but the latter was still temporally correlated with the NO production rate. Thus, under conditions likely to be applicable to the in vivo situation, the flow-dependent increase in ecNOS phosphorylation and NOx production was largely independent of [Ca2+]i.
The results of these studies are consistent with those of O'Neill,23 who demonstrated [Ca2+]i-independent NO production after exposure of BAECs on microcarrier beads to nonlaminar shear stress. Kuchan and Frangos24 also reported mixed Ca2+ dependence and independence of the early phase, and Ca2+ independence of the chronic phase, of NO production during exposure of human umbilical vein endothelium to prolonged shear stress. Further support for the applicability of the present results to the mechanisms regulating ecNOS in vivo is suggested by a recent report from Busse's laboratory,16 where flow-dependent NO production was detected in vessels at basal levels of [Ca2+]i via a mechanotransduction cascade involving tyrosine kinase(s). Our recent studies indicate that activation of the MAP kinases Erk 1 and 2 by shear stress is mediated in part by a novel Ca2+-independent isoform of PKC.17 Such a signaling event could be involved in the Ca2+-independent component of flow-mediated ecNOS activation. Because many MAP kinase and PKC isoforms exist, these kinases could interact in multiple combinations to effect ecNOS phosphorylation.
The role of phosphorylation in the regulation of NOS is controversial and appears to vary in an isoform- and kinase-specific manner.25 For example, using COS cells stably transfected with ncNOS, Bredt et al26 showed that activation of endogenous PKC or PKA increased ncNOS phosphorylation. Phosphorylation by PKC markedly reduced Ca2+ ionophore–stimulated NO production, but phosphorylation by PKA did not. The PKC and PKA phosphorylations were shown to occur on different phosphopeptides, indicating that phosphorylation of specific ncNOS residues leads to different functional consequences. It appears that the effects of phosphorylation on the activity of ecNOS are also stimulus specific. Michel et al12 reported that ecNOS in cultured BAECs is phosphorylated in response to bradykinin and other neurohumoral agonists that regulate NO production. The bradykinin-stimulated increase in ecNOS phosphorylation was slower in onset, more prolonged in duration, but lesser in magnitude than the rapid increase that we observed in BAECs exposed to laminar fluid shear stress. An alternative explanation of our findings is that shear stress activates protein kinase(s) other than those stimulated by neurohumoral agonists. Activation of different kinases and/or phosphatases may result in phosphorylation of different ecNOS residues in response to shear stress relative to agonists. Regulation of ecNOS by phosphorylation at multiple sites is suggested by its many consensus sequences for PKC and proline-dependent kinases such as the MAP kinases.18
Phosphorylation appears to be one of several mechanisms that regulate ecNOS enzymatic activity. Work from Michel and colleagues9 10 27 suggests that other posttranslational modifications of ecNOS, such as myristoylation9 and palmitoylation,10 also participate in the regulation of its cellular localization and state of enzymatic activation.27 On the basis of the finding that a mutant ecNOS lacking the N-terminal myristoylation site could still be phosphorylated in a cytoplasmic location, these investigators proposed that phosphorylation follows rather than mediates this translocation.10 Our results do not rule out the possibility that flow-stimulated ecNOS phosphorylation is a consequence of ecNOS activation that is primarily mediated by some other mechanism. If so, ecNOS phosphorylation is regulated coordinately with whatever primary process rapidly augments NO production in response to the critical physiological stimulus, fluid shear stress. We have recently observed that anionic phospholipids potently inhibit ecNOS activity via interaction with the calmodulin-binding region.28 It is conceivable that phosphorylation may modulate the relative affinities of the calmodulin-binding region for calmodulin versus membrane phospholipids. Subsequent studies will be focused on defining the ecNOS sites phosphorylated, the kinases involved, and their relative roles in regulating early versus late ecNOS phosphorylation and NO production.
Selected Abbreviations and Acronyms
|BAEC||=||bovine aortic endothelial cell|
|ecNOS||=||endothelial constitutive NOS|
|ncNOS||=||neuronal constitutive NOS|
|PKA, PKC||=||protein kinase A and C|
This study was supported by grants HL-32717, HL-39006, and HL-48867 to Dr Harrison, National Science Foundation grant BES-9412010 to Dr Nerem, and an American Heart Association Grant-in-Aid to Dr Berk. Dr James was supported by an Overseas Research Fellowship of the National Heart Foundation of Australia. The experimental assistance of Sara MacKellar, Santhini Rasasamy, and Amy Wiencken is gratefully acknowledged.
This manuscript was sent to John T. Shepherd, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Presented in part in abstract form at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 14-17, 1994.
- Received July 9, 1996.
- Accepted July 24, 1996.
- © 1996 American Heart Association, Inc.
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