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Vol. 295, Issue 2, 601-606, November 2000
Departments of Surgery (B.H.T., S.R.S., V.M.M.), Internal Medicine (R.D.H.), Nicotine Research Center (R.D.H.), and Physiology and Biophysics (V.M.M.), Mayo Clinic and Foundation, Rochester, Minnesota
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Effects of nicotine on arterial endothelium-dependent relaxations
mediated by nitric oxide are controversial. Experiments were designed
to test the hypothesis that nicotine can directly alter activity of
endothelial nitric-oxide synthase (eNOS). NOS from aortic endothelial
cells of untreated dogs and recombinant eNOS, neuronal NOS, and
inducible NOS were used for these experiments. NOS activity was
determined as conversion of L-[3H]arginine to
L-[3H]citrulline in the absence or presence
of nicotine (10
7-103 M) in vitro. In
separate assays, concentrations of cofactors NADPH, FAD, and
tetrahydrobioprotein were reduced by half to assess for possible
interaction with nicotine. With enzyme from aortic endothelial cells,
total and calcium-dependent accumulation of citrulline increased by
30% in the presence of 105 M nicotine. Nicotine dose
dependently also increased citrulline accumulation by recombinant eNOS
and neuronal NOS but not inducible NOS. Effects of nicotine on
accumulation of citrulline by isolated eNOS and recombinant eNOS were
further modulated by changes in the concentration of NADPH in the
incubation solution. Our data demonstrate a significant effect of
nicotine on eNOS-mediated citrulline accumulation. These results
suggest that effects of nicotine on production of nitric oxide may
depend on NADPH or oxygen radical interactions with NOS and thus may
explain, in part, inconsistent findings of changes in production of
endothelium-derived nitric oxide with nicotine administration.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Smoking
is a leading cause of cardiovascular disease, accounting for 30% of
cardiac deaths in the United States each year (Ockene and Miller,
1997
). Although nicotine is the addictive component of cigarette smoke,
it should be recognized that smoking is not equivalent to nicotine. In
fact, nicotine is but one of several thousand components of cigarette
smoke. Given nicotine's widespread use in tobacco products and in
over-the-counter nicotine patches and gum, and the prescription
products nasal spray and nicotine inhaler, it is important to determine
effects of nicotine on the cardiovascular system independent of tobacco smoking.
Effects of nicotine on the cardiovascular system are multifactorial,
reflecting activity of nicotinic receptors centrally and on peripheral
autonomic ganglia (McPhail et al., 1998). In addition, effects of
nicotine on endothelial cells and expression of endothelium-dependent
relaxations are controversial (Li and Duckles, 1993; Li et al., 1994;
Mayhan and Patel, 1997; Mayhan and Sharpe, 1999; Clouse et al., 2000b;
Miller et al., 2000). These conflicting results reflect in part effects
of nicotine on several endothelium-derived factors contributing to
vascular tone, including changes in production of prostaglandins,
nitric oxide, endothelin, bradykinins, and leukotrienes (Toda, 1975; Bull et al., 1988; Toda and Okamura, 1992; Suzuki et al., 1994).
Nitric oxide is a major endothelium-derived relaxing factor of the
arterial circulation; thus, any perturbation in the synthesis of nitric
oxide could potentially have significant effects on blood pressure,
flow, and vascular resistance (Knowles, 1996). Although direct infusion
of nicotine may reduce nitric oxide-mediated relaxations of mesenteric
arteries, inhaled nicotine may maintain circulating nitric oxide in
humans (Mayhan and Patel, 1997; Miller et al., 1998; Mayhan and Sharpe,
1999). Nicotine could alter production of nitric oxide in several ways.
Nicotine could change production of nitric oxide directly through
nicotinic-receptor activation of nitroxidergic nerves (Toda and
Okamura, 1992), endothelial cells (Macklin et al., 1998), or bypass
receptor activation by directly interacting with biochemical pathways
in endothelial cells. Alternatively, nicotine could alter activity of
nitric-oxide synthase (NOS) indirectly through production of
oxygen-derived free radicals (Mayhan and Sharpe, 1999). Therefore,
because nicotine can cross cell membranes, it is possible that nicotine
could directly affect production of nitric oxide through interaction
with the enzyme NOS. Therefore, experiments were designed to test the
hypothesis that nicotine directly affects the enzymatic conversion of
L-arginine to L-citrulline by nitric-oxide synthase.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Isolation of eNOS from Aortic Endothelial Cells. Adult male mongrel dogs (20-30 kg) were maintained in accordance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Institutes of Health (NIH Publication 86-23, revised 1996).
A 5-cm segment of descending thoracic aorta was removed from anesthetized (30 mg/kg pentobarbital i.v.) male mongrel dogs and placed in cold, modified Krebs-Ringer-bicarbonate solution of the following millimolar composition: 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.0 NaHCO3, 0.026 calcium disodium edetate, 11.1 glucose. Endothelial cells were then immediately scraped from each aorta's surface and placed in 1 ml of homogenate buffer (50 mM Tris-HCl, 320 mM sucrose, 0.1 mM EDTA, two tablets Complete protease inhibitor, pH 7.8) and snap frozen in liquid nitrogen. Samples were stored at
70°C.
To prepare NOS-containing extract, 100 µg/ml phenylmethlysulfonyl
fluoride was added to the aortic cells that were then homogenized for
10 s using a tissue homogenizer (Tekmar, Cincinnati, OH). Homogenates were centrifuged at 2000g for 10 min at 4°C to
remove cellular debris. To collect the crude NOS extract, the
supernatant was run through a desalting column (Bio-Rad, Hercules, CA).
NOS extract from two to four individual animals was pooled to provide adequate protein concentration for each assay. A small aliquot from
pooled samples was set aside to measure protein concentration using the
bicinchonic acid protein assay reagent kit (Pierce, Rockford, IL) with
a SPECTRAmax spectrophotometer (Molecular Devices, Sunnydale, CA).
Samples were frozen overnight at 70°C.
NOS Assay.
NOS activity was measured by the stoichiometric
conversion of L-[3H]arginine to
L-[3H]citrulline using a method
modified from that of Myatt et al. (1993) and previously published by
our group (Wang et al., 1997; Jeppsson et al., 1998). The standard
incubation buffer consisted of 5 µM L-arginine plus 14.7 nM [3H]L-arginine, 54 mM
L-valine, 1.2 mM MgCl2, 1.0 mM NADPH,
10 µM BH4, 2.0 µM FAD, 50 U/ml calmodulin,
and 50 mM Tris buffer. When nicotine was added to an assay, 20 µl of
freshly diluted (S)-nicotine (Sigma Chemical, St. Louis, MO)
replaced a volume of Tris in the incubation buffer to give final
nicotine concentrations of 107 to
103 M. In some experiments, standard molar
concentration of NADPH, FAD, or BH4 were reduced
in the incubation buffer. Each incubation buffer was subdivided into
three tubes, adding either 0.83 mM CaCl2, 1.0 mM
EGTA, or 2.0 mM
NG-monomethyl-L-arginine
to assess total, calcium-independent, and nonspecific NOS activity,
respectively. The reaction was started by adding NOS enzyme to each
tube and allowed to proceed for 1 h at 37°C. The reaction was
terminated by adding 1.5 ml of ice-cold HEPES buffer (20 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 8 mM EDTA, pH 5.5). Columns (Poly-Prep chromatography columns; Bio-Rad) were prepared by adding Dowex suspension, which retains the
highly charged
L-[3H]arginine, but
allows L-[3H]citrulline
to pass through. The elute was collected into Opti-Fluor solution
(Packard, Meriden, CT) in scintillation vials.
L-[3H]Citrulline activity
was measured using a Beckman 6800 liquid scintillation counter. Knowing
the specific activity ratio (150,960 dpm/pmol), NOS activity was
expressed as picomoles of
L-[3H]citrulline produced
per milligram of protein per hour. Calcium-dependent activity was
measured as total minus calcium-independent activity, correcting for
nonspecific activity.
Assays Using Recombinant eNOS, nNOS, and iNOS.
NOS assays
were repeated as described above using recombinant bovine eNOS (gift
from Dr. William Sessa, Yale University, or purchased from Caymen
Chemical, Inc., Ann Arbor, MI), rat nNOS, and murine iNOS (Caymen
Chemical Inc.) with the following modifications. Recombinant nNOS (14.8 and 1.4 µg of enzyme/tube), eNOS (0.9 µg of enzyme/tube), and iNOS
(5.9 µg of enzyme/tube) were incubated in the absence and presence of
nicotine (107, 105, and
103 M) with standard cofactor conditions for 3, 10, and 30 min. In a separate experiment, recombinant eNOS (0.9 µg of
enzyme/tube) was incubated with varying concentrations of NADPH (0.5-1
mM) for 30 min. For experiments using recombinant enzymes, a single assay was performed in triplicate.
Statistical Analysis. All assays using nitric-oxide synthase isolated from canine aortic endothelial cells were performed in duplicate and results were averaged. Data are expressed as mean ± standard error; n refers to the number of separate assays. To provide adequate amounts of enzyme, each assay consisted of combined enzymes isolated from cells of two to four dogs. Assays using recombinant enzyme were performed in triplicate. Results for each assay were calculated as picomoles of L-[3H]citrulline produced per milligram of protein per time. Unless stated otherwise, NOS activity was normalized to that obtained in the absence of nicotine (100%). For assays using NOS extracts from endothelial cells, one-way ANOVA was used to compare more than two means. Significance was determined by P < .05. If overall significance was detected, a post hoc Newman-Keuls test for paired comparisons was applied.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Effects of Nicotine on NOS Isolated from Aortic Endothelial Cells
NOS isolated from canine aortic endothelial cells was incubated
with nicotine for 60 min at 37°C. Total citrulline accumulation increased with increasing concentrations of nicotine, reaching statistical significance at 105 M nicotine
(Fig. 1). Calcium-dependent citrulline
accumulation in the presence of 105 M nicotine
was 28.4% greater than activity in the absence of nicotine (control)
(Table 1). Calcium-independent citrulline accumulation and nonspecific radioactivity were similar among treatment
groups. Calcium-dependent citrulline accumulation significantly exceeded that of calcium-independent accumulation in all assays using
isolated enzyme (P < .001, Table 1).
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Effects of Nicotine on Recombinant NOS
Citrulline accumulation increased linearly with incubation time
for recombinant eNOS and iNOS (Fig. 2).
Nicotine dose dependently increased nNOS-mediated accumulation of
citrulline after 10 min of incubation, reaching about 20 and 43%
increase with 107 and
105 M nicotine, respectively, at this time
point (Fig. 2). Citrulline accumulation with recombinant eNOS increased
between 10 and 16% after a 30-min incubation with all three
concentrations of nicotine. Notably, no significant effect of nicotine
was observed at earlier time points. Accumulation of citrulline with
recombinant iNOS did not increase with nicotine and in contrast to
accumulation of citrulline with nNOS and eNOS decreased by 13% with
103 M nicotine (Fig. 2).
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Possible Mechanism of Nicotine-NOS Interaction
Enzyme Isolated from Aortic Endothelial Cells.
To identify
possible sites where nicotine might affect NOS, assays were performed
using half the standard assay concentrations of NADPH, FAD, or
BH4. Decreasing the concentration of NADPH from 1.0 to 0.5 mM decreased total citrulline accumulation by 25% (Fig. 3). Reducing FAD (2.0-1.0 µM) or
BH4 (10.0-5 µM) did not reduce NOS citrulline
accumulation significantly compared with that obtained using standard
assay conditions (data not shown; n = 4 assays with
each condition). Under conditions of reduced NADPH, but not FAD or
BH4, nicotine caused a statistically significant
dose-dependent decrease in total and calcium-dependent citrulline
accumulation (Fig. 4).
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Recombinant eNOS.
In the presence of
105 M nicotine, accumulation of citrulline by
recombinant eNOS increased with concentrations of NADPH from 0.3 to 0.5 mM during a 30-min incubation (Fig. 5,
top). After a 30-min incubation with NADPH (1 mM), citrulline
accumulation by recombinant eNOS was increased by 50% with
105 M but remained at control levels with
103 M nicotine. However, in the presence of
103 M nicotine, accumulation of citrulline was
60% less than control (absence of nicotine) when NADPH was 0.3 mM
(Fig. 5, bottom).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Results from these experiments demonstrate for the first time that
nicotine may modulate citrulline accumulation from
L-arginine by eNOS. This modulation in vitro is
dose-related over a concentration range of 107
to 103 M nicotine. Concentrations of nicotine
in blood of human smokers or abstinent smokers using nicotine products
for smoking cessation or nonsmokers using nicotine treatments for
ulcerative colitis range from 107 to
106 M (Bannon et al., 1989; Palmer et al.,
1992; Hurt et al., 1993; Sandborn, 1999). Stimulatory effects of
nicotine on NOS activity were observed consistently with
calcium-dependent isoforms of the enzyme, i.e., eNOS and nNOS. This
stimulatory effect of nicotine was associated with "protection"
against enzyme inactivation during the in vitro assay, but was not
apparent during the initial phase of enzyme activity. Furthermore,
stimulatory effects with eNOS were observed with three different
sources of enzyme: crude enzyme from canine aortic endothelial cells
and recombinant enzyme from two different sources.
The mechanism by which nicotine affects NOS cannot be determined from
these experiments. However, some inferences can be derived. Effects of
nicotine on citrulline accumulation by eNOS were influenced by
concentrations of NADPH in the incubation solution. The apparent discrepancy between the dose effect of NADPH and nicotine on citrulline accumulation for NOS from aortic endothelial cells and recombinant enzyme (Figs. 4 and 5a) may relate to the purity of the enzyme preparation. It is unclear as to whether interactions among nicotine, NADPH, and NOS are direct or result from interactions associated with
the production of oxygen radicals (Miller et al., 1998; Vasquez-Vivar et al., 1998; Mayhan and Sharpe, 1999).
Differences in absolute concentrations of citrulline accumulation among
preparations may reflect differences in the purity of the enzymes. It
is unclear as to what other factors may be present in enzyme prepared
from aortic endothelial cells that affect NOS activity. Protein yield
was low when enzyme was extracted from canine endothelial cells and it
was necessary to combine isolates from several dogs to yield sufficient
protein for each assay. Enzyme activity under control conditions for
extracted eNOS was within the range of that reported using the same
extraction method (Myatt et al., 1993) where specific activity ranged
from about 10 to 831 pmol/mg/45 min. And, as would be expected for eNOS
isolated from nonarteriosclerotic or noninfected animals, iNOS or
calcium-independent conversion was low.
What, then, might be the physiological implications of these
observations? Modulation of citrulline accumulation and by implication nitric oxide by nicotine in these in vitro experiments ranged from 25 to 50% of control assay conditions. Although changes within this range
may not seem dramatic for assays optimized for substrate and cofactor
conditions, similar changes in vivo where such parameters are also
modulated may be of consequence. Nicotine applied directly to
endothelial cells alters production of a variety of vasoactive factors
(Bull et al., 1988; Suzuki et al., 1994). However, effects of nicotine
on endothelium-dependent responses in blood vessels is variable and may
be related to mode of administration of nicotine (infusion compared
with transdermal patches or osmotic minipump), dose, and duration of
treatment (Li and Duckles, 1993; Li et al., 1994; Mayhan and Patel,
1997; Mayhan and Sharpe, 1999; Clouse et al., 2000b; Miller et al.,
2000). Results of the present experiments support a biphasic effect of
nicotine on NOS, which is dependent on concentrations of NADPH. These
observations would be consistent with the in vivo literature and
provide a possible mechanistic explanation for the in vivo results. In
vivo, nicotine effects on cells independent of nicotinic receptor
activation are poorly understood and would depend on the concentration
gradient for diffusion of nicotine into the cells. The concentration
gradient would be influenced by dose, duration of treatment, and
metabolism of nicotine. Effects of nicotine on NOS in vivo are the
summation of nicotine's effects on numerous other target sites in the
central and peripheral nervous system. When administered acutely,
nicotine acts as a vasoconstrictor in vivo. However, when administered chronically, vasodilatation may be observed (Bassenge et al., 1988).
Potentially, nicotine could exert opposing effects on the same vessel.
Alternatively, effects of nicotine in vivo either could be potentiated
or masked by effects of metabolites, such as cotinine (Carty et al.,
1996, 1997; Vainio et al., 1998; Rama Sastry et al., 1999). In spite of
the complexity of these interactions, results of the present study
showing in vitro effects of nicotine on NOS could explain, in part, the
biphasic time and dose dependence of changes in nitric oxide-mediated,
endothelium-dependent relaxations of coronary arteries, saphenous
veins, and coronary artery bypass grafts from dogs treated with varying
doses of transdermal nicotine (Clouse et al., 2000a,b; Miller et al.,
2000).
Direct modulation of nNOS and iNOS enzymes by nicotine has not been
demonstrated in vivo. However, nicotine is being tested experimentally
as a possible treatment for Alzheimer's disease, Parkinson's disease,
sleep apnea, and ulcerative colitis (Davila et al., 1994; Fagerstrom et
al., 1994; Pullan et al., 1994; Snaedal et al., 1996; Sandborn et al.,
1997; Sandborn, 1999). Modulation of nNOS and iNOS by nicotine and
associated changes in neuronal or immunologically derived NO may
provide a possible explanation for the effectiveness of
nicotine-treatment for these diseases. Interaction of nicotine with
nNOS and iNOS may be possible given the structural and functional
homology among the NOS enzymes. Effects of nicotine on iNOS were not as
great as with nNOS or eNOS and very modest inhibition, rather than
activation, was observed. This difference was not related to the
kinetics of action because shorter incubation periods did not
demonstrate activation of iNOS. Rather, differences in activation may
relate to the binding characteristics of the NOS isoforms for cofactors
or sensitivity to inhibition by oxygen free radicals.
Nicotine is available over-the-counter for use in smoking cessation.
The use of transdermal nicotine has been proven safe in clinical
smoking cessation trials for humans with coronary arterial disease
(Joseph et al., 1996). In typical doses achieved by smokers, nicotine
causes elevated alertness, mild elevation in blood pressure and heart
rate, and gastrointestinal and urinary stimulation; these are all
effects to which tolerance has been demonstrated. Results of this study
establish a new mechanism by which nicotine may affect these functions
through receptor-independent regulation of NOS. The observation that
effects of nicotine on NOS may be related to cofactor interactions may
explain inconsistent findings when nicotine is applied to endothelial
cells in culture, or to different vascular beds in situ or in vivo. In
addition, interactions of nicotine with nNOS and iNOS may provide
insights into how this drug may alter neurological and immunological functions.
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Footnotes |
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Accepted for publication July 25, 2000.
Received for publication March 28, 2000.
1 This study was funded by grants from the American Heart Association (AHA 96-010290), the Mayo Graduate School of Medicine, and the Mayo Foundation.
2 Current address: Department of Surgery, Alton Ochsner Foundation Hospital, 1516 Jefferson Hgwy., New Orleans, LA 70121.
Send reprint requests to: Virginia M. Miller, Ph.D., Professor of Surgery and Physiology, Department of Surgery, Mayo Clinic and Foundation, 200 First St. SW, Rochester, MN 55905. E-mail: miller.virginia{at}mayo.edu
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Abbreviations |
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NOS, nitric-oxide synthase; eNOS, endothelial NOS; BH4, tetrahydrobioprotein; nNOS, neuronal NOS; iNOS, inducible NOS.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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; 10
; 10
;
10
. Data are expressed as
percentage of accumulation in the absence of nicotine
(L-[3H]citrulline: nNOS = 88 nmol/mg of protein/30 min; eNOS = 18.1 nmol/mg of protein/30 min;
iNOS = 34.3 nmol/mg of protein/30 min). Each data point represents
the mean of triplicate assays from one experiment.
