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Abstract |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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Cigarette smoking is associated with impaired endothelium-dependent vasodilation and reduced nitric oxide (NO) in the exhaled air of smokers. To explore the mechanism for the impairment of NO-mediated vasodilation, we studied the effect of cigarette smoke extract (CSE) on NO synthase (eNOS) activity and content in pulmonary artery endothelial cells (PAEC). Incubation of PAEC with CSE resulted in a time- and dose-dependent decrease in eNOS activity. The inhibitory effect of CSE on eNOS activity was not reversible. Both gas-phase and particulate-phase extracts of CSE contributed to the inhibition of eNOS activity. The protein kinase c (PKC) inhibitors staurosporine and chelerythrine did not affect the CSE-induced inhibition of eNOS activity. Catalase, superoxide dismutase (SOD), vitamin C, vitamin E, glutathione, and dithiothreitol (DTT) also did not prevent the CSE-induced inhibition of eNOS activity, and incubation of PAEC with 3 mM nicotine did not change the activity of eNOS. Treatment of PAEC with CSE also caused a nonreversible, time-dependent decrease in eNOS protein content detected by Western blot analysis, and in eNOS messenger RNA (mRNA) detected by Northern blot analysis. Treatment of PAEC with CSE had no effect on cell protein or glutathione contents or on lactate dehydrogenase (LDH) release. These results indicate that exposure to CSE causes an irreversible inhibition of eNOS activity in PAEC, and suggest that the decreased activity is secondary to reduced eNOS protein mass and mRNA. The decrease in eNOS activity may contribute to the high risk of pulmonary and cardiovascular disease in cigarette smokers.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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Cigarette smoke has been implicated as a major risk factor in chronic obstructive pulmonary disease (COPD), chronic hypoxic cor pulmonale, atherosclerosis, and related cardiovascular dysfunction (1). The mechanism for the increased risk of vascular dysfunction is not well understood, but it is presumed to be due to the absorption of tobacco smoke constituents that affect endothelial cell function (5). Vascular endothelial cells generate nitric oxide (NO) from L-arginine via a calcium-dependent constitutive NO synthase (eNOS) (10). NO is a major endogenous vasodilator that contributes to the low vascular resistance in the pulmonary circulation (11, 12), and has been implicated in the mediation of hypoxic pulmonary vasoconstriction as well as in the response to hypoxic pulmonary vasoconstriction (13, 14). It has been reported that endothelium-dependent vasorelaxation is diminished in cigarette smokers and in the lungs of individuals with COPD and hypoxic cor pulmonale (6, 15). Cigarette smoking also reduces exhaled NO, suggesting that cigarette smoke inhibits NO production (16). A reduction in NO production by cigarette smoke might be responsible, at least in part, for the increased risk of systemic and pulmonary vascular disease and dysfunction in cigarette smokers. Therefore, in the present study, we examined the effect of cigarette smoke on eNOS expression and activity in pulmonary artery endothelial cells (PAEC) from the pig. Our results indicate that exposure to cigarette smoke extract (CSE) results in a decrease in eNOS protein and eNOS messenger RNA (mRNA) contents, as well as in eNOS activity in PAEC, and that these effects are not reversed for 24 h following cessation of exposure to cigarette smoke.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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Cell Culture
Endothelial cells were obtained from the main pulmonary artery of 6- to 7-mo-old pigs, and were cultured and characterized as previously reported (17). Third- to sixth-passage cells in monolayer culture in 100-mm dishes were used. After confluence, the PAEC were maintained in RPMI 1640 medium plus 4% fetal bovine serum and 100 U/ml penicillin, 100 µg/ml streptomycin, 20 µg/ml gentamicin, and 2 µg/ml amphotericin B (Fungizone). The cells were used 2 or 3 d after confluence.
Preparation of CSE Solutions
The CSE was prepared as described previously (18). Commercial cigarettes (Marlboro, Philip Morris, Inc., Richmond, VA) were smoked continuously by the apparatus
shown in Figure 1. Mainstream smoke was drawn through
30 ml of phosphate-buffered saline (PBS) that was prewarmed to 37°C by application of a vacuum to the vessel containing the PBS. Each cigarette was smoked for 5 min,
and three cigarettes were used per 30 ml of PBS to generate a CSE-PBS solution. To generate a PBS solution of
the particulate-phase extract (PPE) of cigarette smoke,
smoke from three cigarettes was passed through a glass-fiber Cambridge filter (Fisher, Orlando, FL) that retained 99% of all particulate matter in the smoke. The Cambridge filters with the absorbed particulate matter were
then extracted for 15 min at 37°C in PBS (PPE-PBS solution). The smoke passing through the Cambridge filter was
used to prepare a gas-phase extract (GPE)-PBS solution
for the CSE, as described earlier. Control solutions were
prepared with the same protocols used to generate CSE,
PPE, and GPE solutions, except that the cigarettes were
unlit. The CSE, PPE, and GPE solutions were diluted with
RPMI 1640 medium and used immediately as described
subsequently. In some experiments, the CSE, PPE, and
GPE were stored at 4°C for 24 h prior to use. The results
observed with stored solutions were identical to those obtained with solutions used immediately after preparation.
Final concentrations of these solutions are expressed as
percent values, which were calculated with the following
equation: (ml CSE [or PPE or GPE] solution 
total ml) × 100. Total milliliters in this equation are the sum of milliliters of CSE solution and milliliters of RPMI 1640. Solutions ranging from 2.5 to 10% were used in the present
studies, and these approximately correspond to exposures associated with smoking slightly less than 0.5 pack per day
to slightly less than 2 packs per day of cigarettes.
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Effect of CSE, PPE, and GPE Solutions, Nicotine, and Oxidants on eNOS Activity
PAEC monolayers were incubated with or without CSE, PPE, and GPE solutions (2.5 to 10%) for 2 to 24 h. In some experiments, PAEC exposed to CSE solutions for 24 h were returned to control culture media for 24 h to assess the reversibility of CSE-related effects on eNOS activity. To assess whether exposure to CSE had a generalized toxic effect on the PAEC, we measured total glutathione (GSH) content by the method of Tietze (19) as modified by Bhat and coworkers (20); total protein content by the method of Lowry and associates (21); and lactate dehydrogenase (LDH) release by the method of Bhat and coworkers (20). To determine whether the effect of CSE is associated with protein kinase C (PKC), PAEC were incubated with CSE solution for 2 to 24 h in the presence of two different PKC inhibitors, staurosporine (80 nmol/liter) or chelerythrine (1 µM). These concentrations of staurosporine and chelerythrine have been shown to inhibit fully PKC activity in cellular systems (22, 23). Because cigarette-tar extracts contain hydrogen peroxide (H2O2) and nicotine, PAEC were incubated for 24 h in the presence or absence of 1 to 25 µM H2O2 or 0.1 to 3 mM nicotine. Additionally, to investigate whether oxidants are responsible for CSE-mediated changes in eNOS activity, PAEC were incubated with 10% CSE for 24 h in the presence of catalase (15 U/ml), superoxide dismutase (SOD) (30 U/ml), vitamin C (500 µM), vitamin E (500 µM), reduced glutathione (GSH) (500 µM), or dithiothreitol (DTT) (500 µM).
After each of the experimental treatments, the PAEC
monolayers were scraped and homogenized in buffer A
(Tris-HCl 50 mM, pH 7.4, containing 0.1 mM each of ethylenediaminetetraacetic acid [EDTA] and ethyleneglycol-bis-
(
-aminoethyl ether)-N,N'-tetraacetic acid [EGTA], 1 mM
phenylmethylsulfonyl fluoride [PMSF], and 1.0 µg/ml leupeptin). The homogenates were centrifuged at 100,000 × g
for 60 min at 4°C, and the total membrane pellet was resuspended in buffer B (buffer A + 2.5 mM CaCl2). The resulting suspension was used for eNOS determination by
monitoring the formation of [3H]L-citrulline from [3H]L-arginine (24). The total membranes (100 to 120 µg protein) were incubated (total volume = 0.4 ml) in buffer B
containing 1 mM nicotinamide adenine dinucleotide phosphate (NADPH), 100 nM calmodulin, 10 µM BH4, and
5 µM combined L-arginine and purified [3H]L-arginine (0.6 µCi; specific activity 69 Ci/mmol; NEN, Boston, MA) for
30 min at 37°C. Purification of [3H]L-arginine and measurement of [3H]L-citrulline formation were done as described previously (24). The specific activity of eNOS is
expressed as pmol L-citrulline/min/mg protein. Total membrane protein content was determined by the method of
Lowry and associates (21).
Western Blot Analysis of eNOS Protein
To investigate whether CSE affects eNOS protein content, we incubated PAEC with a 10% CSE solution for 2 to 24 h at 37°C, after which total proteins were extracted for Western blot analysis. In some experiments, PAEC that were incubated with CSE for 24 h were transferred to control culture medium for another 24 h to determine whether the effects of CSE were reversible. After the treatments, PAEC were washed with PBS and then lysed in boiled sample buffer (0.06 M Tris-HCl, 2% sodium dodecyl sulfate [SDS], and 5% glycerol, pH 6.8). The lysate was boiled in a water bath for 5 min to remove insoluble materials. The lysate proteins (15 to 20 µg) were separated on a 7.5% SDS-polyacrylamide gel (PAGE) and electrophoretically transferred onto polyvinylidene difluoride (PVDF; Bio-Rad, Melville, NY) membranes as described by Burnette (25). The membranes were incubated in blocking solution (10 mM Tris-HCl; 3% bovine serum albumin [BSA]; 100 mM NaCl; 0.1% Tween 20, pH 7.5) and then hybridized with a 1:5,000 dilution of monoclonal anti-eNOS antibody (Transduction Laboratories, Lexington, KY) at room temperature for 1 h. After a 1-h incubation with the anti-eNOS antibody, the membranes were washed three times for 5 to 10 min each with blocking solution to remove unbound antibody. The membranes were then incubated for 30 min in the blocking solution containing a 1:2,500 dilution of antirabbit immunoglobulin G (IgG) conjugated with horseradish peroxidase (Promega, Madison, WI), washed three times with blocking solution to remove unbound second antibody, and rinsed briefly in two changes of Tris-buffered saline (TBS) (20 mM Tris-HCl, pH 7.5; 150 mM NaCl). Membranes were then transferred into fresh color development solution (TBS containing 0.35 ml H2O2/100 ml and 50 mg 4-chloro-1-naphthol/100 ml) for 5 to 10 min. The density of the bands was quantitated with the Image 1.47 alias software system (Goody Image, Cincinnati, OH).
RNA Isolation and Northern Blot Analysis of eNOS mRNA
Total cellular RNA was isolated as previously reported (24), using an RNA isolation kit (Promega, Madison, WI). Total RNA (20 µg/lane) was fractionated by electrophoresis on 1.2% agarose gels containing formaldehyde, and was transferred to nylon membranes by capillary blotting. The membranes were hybridized at 43°C for 24 h with digoxigenin-labeled human complementary DNA (cDNA) for eNOS in DIG-Easy-Hyb solution (Boehringer Mannheim Biochemicals, Indianapolis, IN). After stringency washing, the hybrids were detected with antidigoxigenin- alkaline phosphatase (AP) and the chemiluminescence substrate CSPD (Boehringer Mannheim Biochemicals), and were exposed to X-ray film for 20 min. To quantitate the total RNA loading, the blots were stained in 0.04% methylene blue in 0.5 M sodium acetate (pH 5.2) for 5 min. The densities of the bands were determined with the Image 1.47 alias system. Variations in RNA loading were internally controlled with 18S ribosomal RNA (rRNA) levels.
Data Analysis
In each experiment, experimental and control endothelial cells were matched for cell line, age, seeding density, number of passages, and number of days postconfluence to avoid variation in tissue culture factors that can influence the measurements of eNOS activity, protein content, and eNOS mRNA level (26). Results are shown as means ± SE for n experiments. Student's paired t test was used to determine the significance of differences between the means of experimental and control cells. A value of P < 0.05 was taken as significant.
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Results |
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Materials and Methods
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CSE-Induced Inhibition of eNOS Activity in PAEC
To investigate whether cigarette smoke affects eNOS activity, PAEC were incubated with CSE solutions for 2 to 24 h. As shown in Figure 2, treatment of PAEC with CSE solutions (2.5% to 10%) caused a dose-dependent decrease in eNOS activity. The CSE-induced inhibition of eNOS activity was also time-dependent. Inhibition was observed after a 4-h incubation but not after a 2-h incubation, and more prolonged exposures to CSE resulted in greater inhibition of eNOS activity (Figure 3). The CSE-induced inhibition of eNOS activity was not reversed by return of the CSE-treated cells to control media for 24 h (Figure 3). Incubation of PAEC with CSE solutions (2.5 to 10%) for 2 to 24 h had no effect on total cell-protein contents or LDH release (Table 1). However, incubation with 10% CSE for 24 h caused a significant increase in GSH content (11.2 ± 0.9 versus 14.8 ± 1.1 nmol/mg protein, P < 0.05). Incubation with lower concentrations of CSE had no effect on GSH content.
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Several studies indicate that eNOS activity is regulated by PKC (i.e., activation of PKC decreases eNOS activity and inhibition of PKC increases eNOS activity) (23, 27- 29), and a recent study has shown that cigarette smoke induces PKC activity in endothelial cells (9). Thus, CSE solution might inhibit eNOS activity by activating PAEC PKC. However, this seems unlikely, because incubation of PAEC with staurosporine or chelerythrine, known inhibitors of PKC, did not affect CSE-induced inhibition of eNOS activity (Figure 3, Table 2).
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Effect of PPE and GPE Solutions, Nicotine, and H2O2 on eNOS Activity in PAEC
To investigate which component of cigarette smoke is responsible for the inhibition of eNOS activity, cigarette smoke was separated into PPE and GPE. Incubation of PAEC with PPE (2.5% to 10%) or GPE (2.5% to 10%) resulted in a dose-dependent inhibition of eNOS activity (Figure 4). For a given dose of PPE or GPE, the magnitudes of the decreases in eNOS activity were similar in PPE- and GPE-treated cells. In contrast, incubation of PAEC with nicotine (0.1 to 3 mM) or H2O2 (1 to 25 µM) for 24 h did not change eNOS activity (Table 3).
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Effect of Antioxidants on CSE-Induced Inhibition of eNOS
To determine whether oxidants are responsible for the CSE-induced inhibition of eNOS activity, PAEC were incubated with 10% CSE in the presence or absence of catalase, Cu-Zn superoxide dismutase (SOD), vitamin C, vitamin E, reduced glutathione, or DTT. None of these antioxidants was found to change the CSE-induced reduction of eNOS activity in PAEC (Table 2).
Effect of CSE on eNOS Protein Content
As shown in Figure 5, incubation of PAEC with a 10% CSE solution for 2 to 24 h caused a time-dependent decrease in eNOS protein content. The decrease in eNOS protein content was detected after a 4-h incubation but not after a 2-h incubation, and progressed with more prolonged incubation. Total cell-protein contents were comparable in control and CSE-treated cells. The time course for loss of eNOS protein content was nearly identical to that for loss of eNOS activity (Figure 3). Moreover, as with eNOS activity, the decrease in eNOS protein content did not reverse after return of PAEC to control media for 24 h (Figure 5). These results suggest that the decrease in eNOS activity in response to CSE is secondary to a loss of eNOS protein mass.
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Effect of CSE on eNOS mRNA Level
Incubation of PAEC with 10% CSE for 24 h caused a significant decrease in eNOS mRNA content (Figure 6). As with eNOS activity and eNOS protein content, the decrease in eNOS mRNA level did not reverse after return of PAEC to control media for 24 h.
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Discussion |
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The present study demonstrates that exposure of PAEC to solutions containing mainstream CSE results in time- and dose-dependent decreases in eNOS activity and protein content in porcine PAEC, and that these decreases do not reverse following a return of the PAEC to control conditions for 24 h. The decreases in eNOS activity and protein content in PAEC exposed to CSE occurred in the absence of changes in LDH release and in cell protein and GSH contents, suggesting that the effects on eNOS do not arise from a generalized toxic effect of cigarette smoke on these cells. The time course and magnitude of the decreases in eNOS activity were comparable to the time course and magnitude of decreases in eNOS protein content, indicating that the decrease in eNOS protein mass is responsible, at least in part, for the loss of activity. The decrease in steady-state eNOS mRNA in CSE-treated cells suggests that CSE is exerting its inhibitory action at the level of gene transcription. However, it is not clear whether the regulation by CSE involves activation of repressor factors or changes in the rate of transcription or stability of the transcript. Although we did not systematically examine each of these potential mechanisms of action of CSE, we did evaluate the effect of two PKC inhibitors, staurosporine and chelerythrine, on CSE-induced inhibition of eNOS activity, because several studies suggest a relationship between PKC-mediated phosphorylation and loss of eNOS activity (23, 27). However, neither staurosporine nor chelerythrine had any effect on the CSE-induced inhibition of eNOS activity in the cells in our study.
Cigarette smoke is a complex medium containing approximately 4,000 different constituents distributed in gas and particulate phases (30). To determine whether the constituent(s) responsible for CSE-induced inhibition of eNOS activity were confined to either the gas or the particulate phase, we used a Cambridge filter to separate CSE into a GPE and a PPE. Both GPE and PPE solutions caused comparable dose-dependent decreases in eNOS activity, suggesting that the constituent(s) in CSE responsible for inhibition of eNOS activity exists in both the gas and particulate phases.
Cigarette smoke contains a variety of oxidants, including nitrogen oxides, hydrogen peroxide, hydrogen cyanide, and acrolein (30), that are capable of affecting eNOS expression and/or activity. However, incubation of PAEC with catalase, SOD, vitamin C, vitamin E, GSH, or DTT did not protect eNOS from the inhibitory effect of CSE. In addition, intracellular GSH contents were either unchanged or increased (10% CSE for 24 h) in PAEC incubated with CSE for up to 24 h. Together, these results suggest that oxidants are unlikely to be responsible for CSE-induced inhibition of eNOS activity. Our results also demonstrate that nicotine is unlikely to be responsible for the inhibition of eNOS by CSE, because nicotine in concentrations as high as 3 mM did not affect eNOS activity in our PAEC. Additionally, nitrite in CSE is also unlikely to be responsible for eNOS inhibition. The highest level of nitrite measured in the CSE used in our studies was 0.5 µM, and we have previously reported that nitrite concentrations up to 2.5 mM have no effect on eNOS activity in PAEC (24).
There is a strong association between vascular endothelial cell injury and cigarette smoking (31, 32). The mechanism responsible for this vascular dysfunction is not well understood. However, it is presumed to result from the absorption of tobacco smoke constituents that interact with and affect endothelial cell function, and numerous studies support the concept that tobacco smoke components diffuse across the alveolar capillary membrane and its lining fluids to enter the bloodstream and interact with endothelial cells and circulating formed elements in the blood (5- 9, 32-34). Because the pulmonary vasculature is the first vascular bed with which these components can interact, it is not surprising to find that CSE has an effect on eNOS activity in PAEC. CSE has previously been reported to inhibit NOS activity in cultured bovine bronchial epithelial cells (35). This, too, is not unexpected, because airway cells are directly exposed to inhaled cigarette smoke.
Pulmonary endothelial cells are an important source of NO. Several recent studies done with histochemical and immunohistochemical assays, in situ hybridization, and/or Northern blot analyses have clearly shown that eNOS is expressed at a high level in the vascular endothelium of all types of vessels in the human lung (36, 37), where it appears to govern basal pulmonary vascular tone (11, 12) and to play a role in hypoxic pulmonary vasoconstriction (13, 14). Inhibition of eNOS activity in pulmonary vascular endothelial cells could account for the decreased endothelium-dependent vasodilatation observed in cigarette smokers with COPD and hypoxic cor pulmonale (6, 15). It could also account for the reduced NO in the exhaled air of cigarette smokers (16). Finally, and most importantly, CSE-induced inhibition of eNOS in lung vascular endothelial cells may predispose smokers, especially those with COPD, to a host of pulmonary vascular complications, including pulmonary hypertension and cor pulmonale.
In summary, the present study is the first to demonstrate that exposure to CSE causes an irreversible (or poorly reversible) inhibition of eNOS activity in PAEC, and suggests that the decreased activity results from reduced transcription of eNOS mRNA. Further studies are needed to identify the precise constituents of CSE that are responsible for this inhibitory effect.
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Footnotes |
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Address correspondence to: Edward R. Block, Research Service (151), VA Medical Center, 1601 S.W. Archer Rd., Gainesville, FL 32608-1197.
(Received in original form July 15, 1997 and in revised form March 3, 1998).
Acknowledgments: The authors thank Mr. Humberto Herrera for tissue culture assistance. They also thank Ms. Janet Wootten for editorial help and Ms. Denise Christian for secretarial support. This work was supported by the Medical Research Service of the Department of Veterans Affairs.
Abbreviations CSE, cigarette smoke extract; DTT, dithiothreitol; eNOS, constitutive nitric oxide synthase; LDH, lactate dehydrogenase; PAEC, pulmonary artery endothelial cells; SOD, superoxide dismutase.
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References |
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1. U.S. Public Health Services, Office of Smoking and Health, Surgeon General. 1982. The health consequences of smoking: cardiovascular disease. U.S. Department of Health and Human Services, Washington, DC. DHHHS(PHS)84-50204.
2. Holbrook, J. S., S. M. Grundy, C. H. Hennekens, W. B. Kannel, and J. P. Strong. 1984. Cigarette smoking and cardiovascular diseases: a statement for health professionals by a task force appointed by the steering committee of the American Heart Association. Circulation 70: 1114A-1117A .
3. Altshuler, B.. 1989. Quantitative models for lung cancer induced by cigarette smoke. Environ. Health Perspect. 81: 107-108 [Medline].
4.
Higgins, M..
1984.
Epidemiology of COPD. State of the art.
Chest
85:
3S-8S
5. Nadler, J. L., J. S. Velasco, and R. Horton. 1983. Cigarette smoking inhibits prostacyclin formation. Lancet 1: 1248-1250 [Medline].
6. Powell, J. T., and D. J. Higman. 1994. Smoking, nitric oxide and the endothelium. Br. J. Surg. 81: 785-787 [Medline].
7. Nitenberg, A., I. Antony, and J. M. Foult. 1993. Acetylcholine-induced coronary vasoconstriction in young, heavy smokers with normal coronary arteriographic findings. Am. J. Med. 95: 71-77 [Medline].
8.
Kiowski, W.,
L. Linder,
K. Stoschitzky,
M. Pfisterer,
D. Burckhardt,
F. Burkart, and
F. R. Buhler.
1994.
Diminished vascular response to inhibition of endothelium-derived nitric oxide and enhanced vasoconstriction to
exogenously administered endothelin-I in clinically healthy smokers.
Circulation
90:
27-34
9. Kalra, V., Y. Ying, K. Deemer, R. Natarajan, J. L. Nadler, and T. D. Coates. 1994. Mechanism of cigarette smoke condensate induced adhesion of human monocytes to cultured endothelial cells. J. Cell. Physiol. 160: 154-162 [Medline].
10. Moncada, S., R. M. J. Palmer, and E. A. Higgs. 1991. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Rev. 43: 109-142 [Medline].
11.
Stamler, J. S.,
E. Loh,
M. Raddy,
K. E. Currie, and
M. A. Creager.
1994.
Nitric oxide regulates basal systemic and pulmonary vascular resistance in
normal human circulations.
Circulation
89:
2035-2040
12. Cremona, G., A. M. Wood, L. W. Hall, E. A. Bower, and T. Higenbottam. 1994. Effect of inhibition of nitric oxide release and action on vascular tone in isolated lungs of pig, sheep, and man. J. Physiol. (Lond.) 481: 183-195 .
13. Adnot, S., B. Raffestin, S. Eddahibi, P. Braquet, and P.-E. Chabrier. 1991. Loss of endothelium dependent relaxant activity in the pulmonary circulation of rats exposed to chronic hypoxia. J. Clin. Invest. 87: 155-162 .
14.
Isaacson, T. C.,
V. Hampl,
K. Weir,
D. P. Nelson, and
S. L. Archer.
1994.
Increased endothelium-derived NO in hypertensive pulmonary circulation
of chronically hypoxic rats.
J. Appl. Physiol.
76:
933-940
15. Dinh-Xuan, A. T., T. W. Higenbottam, A. Colin, M. B. Clelland, J. Pepke-Zaba, G. Cremona, A. Y. Butt, S. P. Large, F. C. Well, and J. Wallwork. 1991. Impairment of endothelium-dependent pulmonary artery relaxation in chronic obstructive lung disease. N. Engl. J. Med. 324: 1539-1547 [Abstract].
16. Kharitonov, S. A., R. A. Robbins, D. Yates, V. Keatings, and P. J. Barnes. 1995. Acute and chronic effects of cigarette smoke on exhaled nitric oxide. Am. J. Respir. Crit. Care Med. 152: 609-612 [Abstract].
17.
Block, E. R.,
J. M. Patel, and
D. A. Edwards.
1989.
Mechanism of hypoxic
injury to pulmonary artery endothelial cell plasma membranes.
Am. J. Physiol.
257:
C223-C231
18. Su, Y. C., and D. X. Wang. 1991. Effect of cigarette smoking, hypoxia and vasoactive mediators on the production of PGI2 and TXA2 in cultured pulmonary artery endothelial cells. J. Tongji Med. Univ. 11: 6-9 [Medline].
19. Tietze, F.. 1969. Enzymatic method for quantitative determination of nanogram amounts of total and oxidized glutathione. Anal. Biochem. 27: 502-522 [Medline].
20. Bhat, G. B., S. B. Tinsley, J. D. Tolson, J. M. Patel, and E. R. Block. 1992. Hypoxia increases the susceptibility of pulmonary artery endothelial cells to hydrogen peroxide injury. J. Cell. Physiol. 151: 228-238 [Medline].
21.
Lowry, O. H.,
N. J. Rosenbrough,
A. L. Farr, and
R. J. Randall.
1951.
Protein measurements with the folin phenol reagent.
J. Biol. Chem.
193:
265-275
22. Tamaoki, T., H. Nomoto, I. Takahashi, Y. Kato, M. Morimoto, and F. Tomita. 1986. Staurosporine, a potent inhibitor of phospholipid/Ca++ dependent protein kinase. Biochem. Biophys. Res. Commun. 135: 397-402 [Medline].
23.
Ohara, Y.,
H. S. Sayegh,
J. J. Yamin, and
D. G. Harrison.
1995.
Regulation
of endothelial constitutive nitric oxide synthase by protein kinase C.
Hypertension
25:
415-420
24. Patel, J. M., J.-L. Zhang, and E. R. Block. 1996. Nitric oxide-induced inhibition of lung endothelial cell nitric oxide synthase via interaction with allosteric thiols: role of thioredoxin in regulation of catalytic activity. Am. J. Respir. Cell Mol. Biol. 15: 410-419 [Abstract].
25. Burnette, W. N.. 1981. Western blotting: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112: 195-203 [Medline].
26.
Arnal, J.-F.,
J. Yamin,
S. Dockery, and
D. G. Harrison.
1994.
Regulation of endothelial nitric oxide synthase mRNA, protein, and activity during cell
growth.
Am. J. Physiol.
267:
C1381-C1388
27.
Michel, T.,
G. K. Li, and
L. Busconi.
1993.
Phosphorylation and subcellular
translocation of endothelium nitric oxide synthase.
Proc. Natl. Acad. Sci.
USA
90:
6252-6256
28.
Hirata, K.-I.,
R. Kuroda,
T. Sakoda,
M. Katayama,
N. Inoue,
M. Suematsu,
S. Kawashima, and
M. Yokoyama.
1995.
Inhibition of endothelial nitric
oxide synthase activity by protein kinase C.
Hypertension
25:
180-185
29. Murphy, T. V., K. M. L. Cross, P. M. Dunning, and C. J. Garland. 1994. Phorbol esters impair endothelium-dependent and independent relaxation in rat aortic rings. Gen. Pharmacol. 25: 581-588 [Medline].
30. Hoffman, D., and E. L. Wynder. 1986. Chemical constituents and bioactivity of tobacco smoke. In Tobacco, A Major International Health Hazard. D. G. Zaridge and R. Peto, editors. International Agency for Research on Cancer, World Health Organization, Oxford University Press, London. 145-165.
31. Doll, R., and R. Peto. 1976. Mortality in relation to smoking: 20 years' observations on male British doctors. Br. Med. J. 2: 1525-1536 .
32. Blann, A. D., and C. N. McCollum. 1993. Adverse influence of cigarette smoking on the endothelium. Thromb. Hemost. 70: 707-711 [Medline].
33. Pre, J., R. LeFloch, R. Vassy, and C. LeNoble. 1989. Increased plasma levels of fluorescent lipid-peroxidation products in cigarette smokers. Med. Sci. Res. 17: 1024-1030 .
34. Blann, A. D.. 1991. Increased circulating levels of von Willebrand factor antigen in smokers may be due to lipid peroxides. Med. Sci. Res. 19: 535-536 .
35. Robbins, R. A., F. G. Hamel, A. A. Floreani, G. L. Gossman, K. J. Nelson, S. Belenky, and I. Rubinstein. 1993. Bovine bronchial epithelial cells metabolize L-arginine to L-citrulline: possible role of nitric oxide synthase. Life Sci. 52: 709-716 [Medline].
36.
Giaid, A., and
D. Saleh.
1995.
Reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension.
N.
Engl. J. Med.
333:
214-221
37. Kobzik, L., D. S. Bredt, C. J. Lowenstein, J. Drazen, B. Gaston, D. Sugarbaker, and J. S. Stamler. 1993. Nitric oxide synthase in human and rat lung: immunocytochemical and histochemical localization. Am. J. Respir. Cell Mol. Biol. 9: 371-377 .
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