(Hypertension. 1995;25:415-420.)
© 1995 American Heart Association, Inc.
Articles |
Regulation of Endothelial Constitutive Nitric Oxide Synthase by Protein Kinase C
From the Division of Cardiology, Emory University School of Medicine, and the Atlanta Veterans Administration Medical Center, Atlanta, Georgia.
Correspondence to David G. Harrison, MD, Division of Cardiology, Emory University School of Medicine, PO Drawer LL, Atlanta, GA 30322.
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Abstract |
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Abstract Protein kinase C (PKC) plays a key role in a variety of signal transduction processes. The promoter region of the endothelial constitutive nitric oxide synthase (ecNOS) gene contains a transcriptional factor AP-1 binding element. In the present study, we sought to determine the effect of PKC inhibition on the expression of ecNOS in cultured bovine aortic endothelial cells (BAEC). The PKC inhibitor staurosporine (10 to 100 nmol/L) increased the expression of ecNOS mRNA, assessed by Northern analysis, in a dose-dependent manner. A newly developed, more specific PKC inhibitor, chelerythrine (1 to 3 µmol/L), also increased the level of ecNOS mRNA. Incubation of BAEC with phorbol 12-myristate 13-acetate (100 nmol/L) for 24 hours, which downregulates PKC, increased ecNOS mRNA expression. The protein content of ecNOS, assessed by Western analysis, was also increased in staurosporine-treated or chelerythrine-treated BAEC. The release of nitrogen oxides from staurosporine-treated or chelerythrine-treated cells both under basal conditions and in response to calcium ionophore A23187 was significantly increased (P<.05). In conclusion, the present study suggests that regulation of ecNOS is mediated by PKC. The increased release of nitric oxide induced by PKC inhibition may play a protective role against atherogenic process.
Key Words: endothelium • nitric oxide • phorbol esters • protein kinase C
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Introduction |
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Protein kinase C (PKC) is a family of serine-threonine–specific kinases, consisting of at least eight isoforms1 that play a key role in a range of signal transduction processes.2 3 4 Activation of PKC induces phosphorylation of many proteins in cells, causing alterations to various biological systems thought to underlie several pathological states, including hypertension,5 6 7 cardiac hypertrophy,8 ischemia,9 10 and atherosclerosis.11 12 Besides cAMP-dependent and Ca2+-calmodulin–dependent protein kinases, endothelial cells also contain PKC.13 However, the role of PKC in these cells has been studied in less detail than in other tissues.
The endothelium-derived vasorelaxing factor14 has been identified as nitric oxide (NO)15 or a closely related compound16 derived from the amino acid L-arginine.17 The endothelial constitutive nitric oxide synthase (ecNOS) has been purified and cloned and its cDNA sequence determined.18 19 20 21 22 Endothelial release of NO is acutely stimulated by receptor-dependent agonists such as acetylcholine, ADP, bradykinin, histamine, substance P, and receptor-independent calcium ionophores and by fluid shear stress.23 Activation of PKC by phorbol esters inhibits the endothelium-dependent vasodilatory responses evoked by acetylcholine, substance P, isoproterenol, or histamine.24 25 26 27 28 29 Hecker et al30 recently reported that preincubation with the PKC activator phorbol 12-myristate 13-acetate (PMA) acutely attenuated the release of NO from bovine aortic endothelial cells (BAEC) stimulated with bradykinin30 and that calphostin C, a specific PKC inhibitor, augmented the bradykinin-stimulated release of NO from endothelial cells.30
While many stimuli acutely regulate ecNOS activity, few factors
are known to regulate ecNOS mRNA expression. Shear stress increases
ecNOS mRNA and protein.21 Tumor necrosis factor-
decreases ecNOS mRNA posttranscriptionally.31 Exercise
training has been reported to increase aortic ecNOS
mRNA.32 Thus, although the ecNOS was initially termed a
constitutive enzyme, it appears that its expression is regulated by a
variety of stimuli.
The transcriptional factor AP-1 recognizes a specific DNA sequence that functions as a cis element, mediating a transcriptional response to the PKC activator PMA.33 34 Recently, AP-1 sites have been found in both the human35 and bovine ecNOS promoter.36
On the basis of these considerations, we hypothesized that ecNOS mRNA expression may be regulated by PKC. Therefore, the purpose of the present study was to determine the effect of PKC inhibition on ecNOS mRNA expression.
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Methods |
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Cultured Cell Preparation
BAEC were obtained as described previously37 and cultured in M199 medium with 25 mmol/L HEPES buffer supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin, and 100 mg/mL streptomycin. The cells used in this study were between passages 5 and 8.
Incubation With Reagents
Confluent BAEC received fresh media just before application of
PKC activator or inhibitor. PKC inhibitor staurosporine (10 to 100
nmol/L) or chelerythrine (1 to 3 µmol/L) or PKC activator
(downregulator) PMA (100 nmol/L) was applied at time zero, and samples
were obtained for RNA analysis from various cultures at 1, 3, 6, 9,
12, and 24 hours. At each time point, samples were also obtained from
cells not exposed to the intervention as a time control. Staurosporine
was dissolved in dimethyl sulfoxide, and chelerythrine was dissolved in
distilled water. These stock solutions were then diluted with 50 mmol/L
phosphate buffer (pH 7.8) and directly added to the medium. Each
intervention was applied on only one occasion.
Northern Blotting Analysis
Endothelial cells were grown in 100-mm dishes (Corning Glass
Works), and on the day of study, they were washed twice with 10 mL
phosphate-buffered saline (PBS) and then lysed in guanidinium
isothiocyanate solution. Total cellular RNA was isolated with an acid
guanidinium thiocyanate–phenol-chloroform according to the method
described by Chomczynski and Sacchi.38 Total RNA (20 µg)
was size-fractionated on a 1.0% agarose–3% formaldehyde gel in the
presence of 1 µg/mL ethidium bromide. The RNA was transferred to a
nitrocellulose filter (GIBCO BRL) and covalently linked by UV
irradiation with a UV cross-linker (Stratagene Cloning Systems).
Hybridizations were performed at 42°C for 18 hours with a
[32P]dCTP-labeled, random primed, 2.1-kb cDNA fragment of
bovine endothelial NOS obtained by Sst-I digestion of the full-length
bovine NOS cDNA21 in 5x standard saline citrate (SSC),
1% sodium dodecyl sulfate (SDS), 5x Denhardt solution, 50%
formamide, 10% dextran sulfate, and 100 µg/mL sheared salmon sperm
DNA. Approximately 1.5x106 cpm/mL of labeled probe
was used per hybridization. After hybridization, filters were washed
twice with 2x SSC, 1% SDS at 55°C for 30 minutes and then with
0.2x SSC, 0.1% SDS at 55°C for 30 minutes. Autoradiography was
performed with an intensifying screen at -70°C. Laser densitometry
and digital analysis of scanned images were used for quantification
of autoradiograms (UltraScan XL, Pharmacia LKB Biotechnology). In all
studies, the nitrocellulose filters were stripped and subsequently
hybridized with a cDNA probe for human ß-actin (Clontech
Laboratories). Variation in RNA loading was internally controlled by
use of a ratio to the ß-actin mRNA levels.
Western Blotting Analysis
BAEC were grown in 6-well plates (Corning Glass Works).
The cells were exposed to staurosporine 10 nmol/L or chelerythrine 3
µmol/L for 24 hours. At the end of the incubation period, the cells
were washed twice with cold PBS, scraped with a rubber policeman,
collected in centrifuge tubes, and spun at 500g for 5
minutes. The supernatant was then discarded, and the pelleted cells
were homogenized by an ultrasonic cell disrupter (Kontes) in 200 µL
of 50 mmol/L phosphate buffer (pH 7.8) containing 5 µg/mL
phenylmethylsulfonyl fluoride. The homogenates were then
centrifuged for 10 minutes at 20 000g, and the supernatant
was used for Western blotting analysis. Protein concentrations were
determined with a modified Bradford assay39 with bovine
serum albumin used for standards. Protein (30 µg) was
size-fractionated electrophoretically with 7.5% SDS–polyacrylamide
gel (SDS-PAGE). Prestained SDS-PAGE standards were used as a molecular
weight marker in blotting applications. The proteins were transferred
to a nitrocellulose membrane (Hybond ECL, Amersham Corp) and blocked
with 6% casein Tris-buffered saline solution (pH 7.6) containing 0.1%
Tween-20 (TBS-T) for 2 hours at room temperature. The membranes were
then incubated with a 1:5000 dilution of the murine monoclonal antibody
mAb H3240 directed against BAEC NOS. The membranes were
subsequently incubated with a goat anti-mouse secondary antibody
conjugated to horseradish peroxidase. After incubation with each
antibody, the membranes were washed four times for 5 minutes with TBS-T
at each step to minimize the background. Signals were detected with the
ECL detection system (Amersham Corp) on special autoradiography films
(Hyperfilm ECL, Amersham Corp).
Measurement of NOS Activity by Quantification of the NO Release
NO production was evaluated by measurement of nitrite
(NO2-) and nitrate
(NO3-), the stable degradation products of
NO
, with a modification of previously described
techniques.37 41 BAEC were grown in 6-well plates (Corning
Glass Works). The cells were exposed to staurosporine 10 nmol/L or
chelerythrine 3 µmol/L for 24 hours, washed gently three times with
modified Krebs'-HEPES buffer (composition [mmol/L]: NaCl 99.0; KCl
4.69; CaCl2 1.87; MgSO4 1.2; NaHCO3
25; K2HPO4, Na-HEPES 20; and D-glucose
11.1, pH 7.4), and then incubated in 1 mL Krebs'-HEPES buffer with or
without 1 µmol/L calcium ionophore A23187 at 37°C for 1 hour.
Buffer (100 µL) was then injected into a reflux chamber containing
Vanadium III dissolved in 3N HCl heated to
>85°C.42 These conditions reduce both nitrite and
nitrate stoichiometrically to NO. The released
NO was purged with a stream of nitrogen gas directed by
vacuum into the reaction chamber of a chemiluminescence NO analyzer
(model 2108, Dasibi Environmental Corp). The chemiluminescence analyzer
was calibrated daily with nitrate standards. The amount of nitrogen
oxides released was normalized to the protein content in the respective
culture dish.
Materials
All reagents were purchased from Sigma Chemical Co except where
specified. The murine monoclonal antibody H32 directed against BAEC NOS
was kindly provided by Drs Jennifer Pollock and Ulrich
Förstermann, Vascular Biology Group, Abbott Laboratories.
Staurosporine was obtained from Kamiya Biomedical Co. Chelerythrine
chloride was obtained from LC Services Corp. [32P]dCTP
and a goat anti-mouse secondary antibody conjugated to horseradish
peroxidase were obtained from Amersham Corp. Sst-I, M199 cell culture
medium, penicillin, and streptomycin were obtained from GIBCO BRL. FCS
was obtained from Hyclone Laboratories, and prestained SDS-PAGE
standards and Tween-20 were obtained from Bio-Rad Laboratories.
Statistical Analysis
For each experiment, the ratios of ecNOS mRNA to the
corresponding ß-actin mRNA were calculated. To analyze dose
responses, these values are presented as a percent of control
values. For the time course studies, data are presented as a
percent of the time control for both ecNOS and ß-actin; furthermore,
the ratio of ecNOS to ß-actin was normalized to the respective time
control value. The data are expressed as mean±SEM. Comparisons of data
between different groups were made by ANOVA followed by a Fisher's
test of least significant difference when significance was indicated.
For comparison of two variables where paired data were available,
paired t tests were used, and when applicable, a Bonferroni
correction was applied. Probability values <.05 were considered
significant.
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Results |
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In the initial experiments, ecNOS mRNA levels were measured at different time points, up to 24 hours, after the addition of staurosporine (10 nmol/L) to the culture medium. Incubation of BAEC with staurosporine increased ecNOS mRNA expression (Fig 1, top). Staurosporine had minimal effects on the steady-state levels of mRNA for ß-actin, a constitutively expressed housekeeping gene (Fig 1, middle). The ecNOS message started to increase after 3 hours of exposure to 10 nmol/L staurosporine (Fig 1, middle). The peak response normalized as a ratio of ecNOS message to ß-actin message was observed after 6 hours of exposure to staurosporine (Fig 1, bottom). Thus, in further experiments with Northern analysis, this 6-hour incubation period was used to obtain maximal effect on ecNOS mRNA expression.
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In the next experiments, we tested the effects of different concentrations of staurosporine, varying from 10 to 100 nmol/L, on the expression of ecNOS in BAEC. Incubation of BAEC with staurosporine for 6 hours increased the ecNOS message in a dose-dependent manner (Fig 2). After correction of ß-actin mRNA levels, ecNOS mRNA abundance at 6 hours of incubation with 10, 30, and 100 nmol/L staurosporine was increased by 99±16%, 150±44%, and 220±70%, respectively, compared with time control (P<.05, n=5 for each concentration) (Fig 2, bottom).
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P<.05 between two different concentrations.Incubation of BAEC with 1 to 3 µmol/L chelerythrine43 44 for 6 hours also increased the ecNOS message in a dose-dependent manner (Fig 3). The ecNOS mRNA abundance at 6 hours of incubation with 1 and 3 µmol/L chelerythrine was increased by 83±11% and 160±24%, respectively (P<.05, n=4 for each concentration) (Fig 3, bottom).
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Prolonged exposure to phorbol ester PMA serves to downregulate endogenous PKC.45 46 Incubation of BAEC with PMA (100 nmol/L) for 24 hours increased the ecNOS message in a manner similar to that observed with the inhibitors of PKC (Fig 4, top). The ecNOS mRNA abundance at 24 hours of incubation with 100 nmol/L PMA was increased by 160±70% (P<.05, n=4) (Fig 4, bottom).
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Staurosporine (10 nmol/L) and chelerythrine (3 µmol/L) also increased ecNOS protein content as assessed by Western analysis after incubation for 24 hours by 110±5 and 130±15%, respectively (P<.05, n=4) (Fig 5).
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Finally, ecNOS activity was measured by examination of the production of nitrogen oxides (nitrite and nitrate, NOx), the stable degradation products of nitric oxide by BAEC. BAEC incubated with 10 nmol/L staurosporine for 24 hours released significantly more NOx per hour per 1 mg protein than control cells (1.72±0.20 versus 1.23±0.09 nmol · h-1 · mg-1 protein, P<.05, n=6). NOx release in response to the calcium ionophore A23187 (1 µmol/L) was also increased by incubation with 10 nmol/L staurosporine (10.1±0.66 versus 7.30±0.66 nmol · h-1 · mg-1 protein, P<.05, n=6) (Fig 6). Furthermore, BAEC incubated with 3 µmol/L chelerythrine for 24 hours also released significantly more NOx per hour per 1 mg protein than control cells (2.87±0.22 versus 2.17±0.16 nmol · h-1 · mg-1 protein, P<.05, n=6). NOx release in response to the calcium ionophore A23187 (1 µmol/L) was also increased by incubation with 3 µmol/L chelerythrine (6.79±0.17 versus 5.35±0.36 nmol · h-1 · mg-1 protein, P<.05, n=6) (Fig 7).
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Discussion |
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In the present study, we have shown that PKC inhibition induces mRNA and protein expression of ecNOS and ecNOS activity measured by examination of nitrate and nitrite release. Incubation of endothelial cells with phorbol ester PMA (100 nmol/L) for 24 hours (which downregulates PKC)45 46 also induced ecNOS mRNA expression. The results of these experiments suggest that basal PKC activity in cultured endothelial cells grown in serum containing media tonically inhibits expression of the endothelial cell NOS.
Members of the PKC family of isoenzymes can be readily inhibited by many chemicals, but the validity of the use of any one of these is frequently questioned. All these inhibitors lack potency, have poor selectivity, or exhibit non–kinase-related biological activities. To overcome these problems, we used two structurally unrelated PKC inhibitors (staurosporine and chelerythrine) and attempted to downregulate PKC by prolonged incubation with phorbol ester PMA. Importantly, all three approaches yielded similar experimental results.
Staurosporine is the most potent protein kinase inhibitor (IC50 value, 2.7 nmol/L47 ), inhibiting the catalytic domain of PKC, and has a wide range of research applications. Staurosporine, however, lacks selectivity for PKC over several other kinases such as cAMP-dependent protein kinase and myosin light-chain kinase.48 It is of note that the PKC inhibitor chelerythrine43 44 may be particularly useful in studies such as these. Chelerythrine (IC50 value, 0.66 µmol/L43 ) is reported to have a higher degree of selectivity for PKC than the other available antagonists.43 In addition, it inhibits the catalytic domain of PKC whether or not the regulatory domain is bound.43 Chelerythrine exhibits competitive kinetics with the substrate histone IIIS for phosphorylation even in the presence of ATP.43 This is useful in experiments with intact cells, where the intracellular concentration of ATP is high.
In these studies, the degree of increase in NOS mRNA and protein expression after inhibition of PKC was modest, on the order of a twofold to threefold increase in mRNA and protein and a 30% to 40% increase in activity as judged by nitrite and nitrate production. While this may on first inspection seem inconsequential, it is important to note that depending on initial vascular tone and the ambient amount of nitric oxide present, small amounts of additional nitric oxide may cause very pronounced vasodilatation.16 Thus, even modest induction of the ecNOS may have important physiological consequences. More intense induction of the enzyme may produce undesirable effects such as those encountered with the inducible NOS in the setting of septic shock.49 50 51
Certain cytokines encountered in the setting of inflammation may activate the endothelial cell PKC and increase endothelial cell superoxide anion production.52 In other cell types, activation of PKC may occur in the setting of a variety of disease states.5 6 7 8 9 10 11 12 It is interesting to speculate that if endothelial cell PKC activity is increased in these situations, this may decrease endothelial cell NOS expression and perhaps decrease endothelial cell production of nitric oxide. Because the endothelium-derived nitric oxide has a number of important roles in maintenance of vascular homeostasis, such a phenomenon might predispose to platelet aggregation and leukocyte adhesion and impair endothelium-dependent vascular relaxation.
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health (HL-39006, HL-32717, HL-15696, HL-48667, and DK-45215) and a merit grant from the Veterans Administration. We appreciate Cynthia M. Curry for assistance in preparation of this manuscript.
Received April 22, 1994; first decision July 6, 1994; accepted November 1, 1994.
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  N. Sud, S. Wedgwood, and S. M. Black Protein kinase C{delta} regulates endothelial nitric oxide synthase expression via Akt activation and nitric oxide generation Am J Physiol Lung Cell Mol Physiol, March 1, 2008; 294(3): L582 - L591. [Abstract] [Full Text] [PDF]
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 A. Miyazaki-Akita, T. Hayashi, Q. F. Ding, H. Shiraishi, T. Nomura, Y. Hattori, and A. Iguchi 17beta-Estradiol Antagonizes the Down-Regulation of Endothelial Nitric-Oxide Synthase and GTP Cyclohydrolase I by High Glucose: Relevance to Postmenopausal Diabetic Cardiovascular Disease J. Pharmacol. Exp. Ther., February 1, 2007; 320(2): 591 - 598. [Abstract] [Full Text] [PDF]
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 C. Rask-Madsen and G. L. King Proatherosclerotic Mechanisms Involving Protein Kinase C in Diabetes and Insulin Resistance Arterioscler Thromb Vasc Biol, March 1, 2005; 25(3): 487 - 496. [Abstract] [Full Text] [PDF]
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 H. Aramoto, J. W. Breslin, P. J. Pappas, R. W. Hobson II, and W. N. Duran Vascular endothelial growth factor stimulates differential signaling pathways in in vivo microcirculation Am J Physiol Heart Circ Physiol, October 1, 2004; 287(4): H1590 - H1598. [Abstract] [Full Text] [PDF]
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S. Olson, R. Oeckler, X. Li, L. Du, F. Traganos, X. Zhao, and T. Burke-Wolin Angiotensin II stimulates nitric oxide production in pulmonary artery endothelium via the type 2 receptor Am J Physiol Lung Cell Mol Physiol, September 1, 2004; 287(3): L559 - L568. [Abstract] [Full Text] [PDF]
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M. M. Tickerhoof, P A. Farrell, and D. H. Korzick Alterations in rat coronary vasoreactivity and vascular protein kinase C isoforms in Type 1 diabetes Am J Physiol Heart Circ Physiol, December 1, 2003; 285(6): H2694 - H2703. [Abstract] [Full Text] [PDF]
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 A. K. Trauernicht, H. Sun, K. P. Patel, and W. G. Mayhan Enalapril Prevents Impaired Nitric Oxide Synthase-Dependent Dilatation of Cerebral Arterioles in Diabetic Rats Stroke, November 1, 2003; 34(11): 2698 - 2703. [Abstract] [Full Text] [PDF]
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J.-C. Schneider, D. El Kebir, C. Chereau, S. Lanone, X.-L. Huang, A. S. De Buys Roessingh, J.-C. Mercier, J. Dall'Ava-Santucci, and A. T. Dinh-Xuan Involvement of Ca2+/calmodulin-dependent protein kinase II in endothelial NO production and endothelium-dependent relaxation Am J Physiol Heart Circ Physiol, June 1, 2003; 284(6): H2311 - H2319. [Abstract] [Full Text] [PDF]
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J.-C. Schneider, D. El Kebir, C. Chereau, J.-C. Mercier, J. Dall'Ava-Santucci, and A. T. Dinh-Xuan Involvement of Na+/Ca2+ exchanger in endothelial NO production and endothelium-dependent relaxation Am J Physiol Heart Circ Physiol, August 1, 2002; 283(2): H837 - H844. [Abstract] [Full Text] [PDF]
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H. G. Bohlen and G. P. Nase Obesity lowers hyperglycemic threshold for impaired in vivo endothelial nitric oxide function Am J Physiol Heart Circ Physiol, July 1, 2002; 283(1): H391 - H397. [Abstract] [Full Text] [PDF]
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I. Spyridopoulos, C. Luedemann, D. Chen, M. Kearney, D. Chen, T. Murohara, N. Principe, J. M. Isner, and D. W. Losordo Divergence of Angiogenic and Vascular Permeability Signaling by VEGF: Inhibition of Protein Kinase C Suppresses VEGF-Induced Angiogenesis, but Promotes VEGF-Induced, NO-Dependent Vascular Permeability Arterioscler Thromb Vasc Biol, June 1, 2002; 22(6): 901 - 906. [Abstract] [Full Text] [PDF]
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 J. E. Freedman, C. Parker III, L. Li, J. A. Perlman, B. Frei, V. Ivanov, L. R. Deak, M. D. Iafrati, and J. D. Folts Select Flavonoids and Whole Juice From Purple Grapes Inhibit Platelet Function and Enhance Nitric Oxide Release Circulation, June 12, 2001; 103(23): 2792 - 2798. [Abstract] [Full Text] [PDF]
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K. Bove, P. Neumann, N. Gertzberg, and A. Johnson Role of ecNOS-derived NO in mediating TNF-induced endothelial barrier dysfunction Am J Physiol Lung Cell Mol Physiol, May 1, 2001; 280(5): L914 - L922. [Abstract] [Full Text] [PDF]
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J. A. Beckman, A. B. Goldfine, M. B. Gordon, and M. A. Creager Ascorbate Restores Endothelium-Dependent Vasodilation Impaired by Acute Hyperglycemia in Humans Circulation, March 27, 2001; 103(12): 1618 - 1623. [Abstract] [Full Text] [PDF]
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 R. A. P. Skyrme-Jones, R. C. O'Brien, K. L. Berry, and I. T. Meredith Vitamin E supplementation improves endothelial function in type I diabetes mellitus: a randomized, placebo-controlled study J. Am. Coll. Cardiol., July 1, 2000; 36(1): 94 - 102. [Abstract] [Full Text] [PDF]
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B.-W. Hochleitner, E.-O. Hochleitner, P. Obrist, T. Eberl, A. Amberger, Q. Xu, R. Margreiter, and G. Wick Fluid Shear Stress Induces Heat Shock Protein 60 Expression in Endothelial Cells In Vitro and In Vivo Arterioscler Thromb Vasc Biol, March 1, 2000; 20(3): 617 - 623. [Abstract] [Full Text] [PDF]
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 H. Li and U. Förstermann Structure-Activity Relationship of Staurosporine Analogs in Regulating Expression of Endothelial Nitric-Oxide Synthase Gene Mol. Pharmacol., March 1, 2000; 57(3): 427 - 435. [Abstract] [Full Text]
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K. Kuboki, Z. Y. Jiang, N. Takahara, S. W. Ha, M. Igarashi, T. Yamauchi, E. P. Feener, T. P. Herbert, C. J. Rhodes, and G. L. King Regulation of Endothelial Constitutive Nitric Oxide Synthase Gene Expression in Endothelial Cells and In Vivo : A Specific Vascular Action of Insulin Circulation, February 15, 2000; 101(6): 676 - 681. [Abstract] [Full Text] [PDF]
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 R. D Rakhit, R. J Edwards, and M. S Marber Nitric oxide, nitrates and ischaemic preconditioning Cardiovasc Res, August 15, 1999; 43(3): 621 - 627. [Full Text] [PDF]
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H. Ando, T. Kubin, W. Schaper, and J. Schaper Cardiac microvascular endothelial cells express alpha -smooth muscle actin and show low NOS III activity Am J Physiol Heart Circ Physiol, May 1, 1999; 276(5): H1755 - H1768. [Abstract] [Full Text] [PDF]
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A. Bouloumie, V. B. Schini-Kerth, and R. Busse Vascular endothelial growth factor up-regulates nitric oxide synthase expression in endothelial cells Cardiovasc Res, March 1, 1999; 41(3): 773 - 780. [Abstract] [Full Text] [PDF]
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 Y. Su, W. Han, C. Giraldo, Y. De Li, and E. R. Block Effect of Cigarette Smoke Extract on Nitric Oxide Synthase in Pulmonary Artery Endothelial Cells Am. J. Respir. Cell Mol. Biol., November 1, 1998; 19(5): 819 - 825. [Abstract] [Full Text]
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U. Förstermann, J.-p. Boissel, and H. Kleinert Expressional control of the `constitutive' isoforms of nitric oxide synthase (NOS I and NOS III) FASEB J, July 1, 1998; 12(10): 773 - 790. [Abstract] [Full Text]
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 A. F. Castro, C. Amorena, A. Muller, G. Ottaviano, M. T. Tellez-Inon, and A. C. Taquini Extracellular ATP and bradykinin increase cGMP in vascular endothelial cells via activation of PKC Am J Physiol Cell Physiol, July 1, 1998; 275(1): C113 - C119. [Abstract] [Full Text] [PDF]
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F. E. Sieber, R. J. Traystman, P. R. Brown, L. J. Martin, and F. M. Faraci Protein Kinase C Expression and Activity After Global Incomplete Cerebral Ischemia in Dogs • Editorial Comment Stroke, July 1, 1998; 29(7): 1445 - 1453. [Abstract] [Full Text] [PDF]
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S. B. Williams, A. B. Goldfine, F. K. Timimi, H. H. Ting, M.-A. Roddy, D. C. Simonson, and M. A. Creager Acute Hyperglycemia Attenuates Endothelium-Dependent Vasodilation in Humans In Vivo Circulation, May 5, 1998; 97(17): 1695 - 1701. [Abstract] [Full Text] [PDF]
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H. Li, S. A. Oehrlein, T. Wallerath, I. Ihrig-Biedert, P. Wohlfart, T. Ulshöfer, T. Jessen, T. Herget, U. Förstermann, and H. Kleinert Activation of Protein Kinase Calpha and/or epsilon Enhances Transcription of the Human Endothelial Nitric Oxide Synthase Gene Mol. Pharmacol., April 1, 1998; 53(4): 630 - 637. [Abstract] [Full Text]
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K. Hishikawa, B. S. Oemar, Z. Yang, and T. F. Luscher Pulsatile Stretch Stimulates Superoxide Production and Activates Nuclear Factor-{kappa}B in Human Coronary Smooth Muscle Circ. Res., November 19, 1997; 81(5): 797 - 803. [Abstract] [Full Text]
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Q. Huang and Y. Yuan Interaction of PKC and NOS in signal transduction of microvascular hyperpermeability Am J Physiol Heart Circ Physiol, November 1, 1997; 273(5): H2442 - H2451. [Abstract] [Full Text] [PDF]
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Z. Zuo and R. A. Johns Inhalational Anesthetics Up-Regulate Constitutive and Lipopolysaccharide-Induced Inducible Nitric Oxide Synthase Expression and Activity Mol. Pharmacol., October 1, 1997; 52(4): 606 - 612. [Abstract] [Full Text]
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 G. Garcia-Cardena, R. Fan, D. F. Stern, J. Liu, and W. C. Sessa Endothelial Nitric Oxide Synthase Is Regulated by Tyrosine Phosphorylation and Interacts with Caveolin-1 J. Biol. Chem., November 1, 1996; 271(44): 27237 - 27240. [Abstract] [Full Text] [PDF]
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J. A. Beckman, A. B. Goldfine, M. B. Gordon, L. A. Garrett, and M. A. Creager Inhibition of Protein Kinase C{beta} Prevents Impaired Endothelium-Dependent Vasodilation Caused by Hyperglycemia in Humans Circ. Res., January 11, 2002; 90(1): 107 - 111. [Abstract] [Full Text] [PDF]
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