1 Department of Pharmacology and Toxicology and 2 Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia 30912
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ABSTRACT |
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It is commonly believed that
the activity of NO synthase (NOS) solely controls NO production from
its substrates, L-Arg and O2. The Michaelis-Menten constant
(Km) of NOS for
L-Arg is in the micromolar
range; cellular levels of L-Arg
are much higher. However, evidence strongly suggests that cellular
supply of L-Arg may become
limiting and lead to reduced NO and increased superoxide anion
(O2·) formation, promoting
cardiovascular dysfunction. Uptake of
L-Arg into cells occurs
primarily (~85%) through the actions of a
Na+-independent, carrier-mediated
transporter (system y+). We have
examined the effects of NOS agonists (substance P, bradykinin, and ACh)
and NO donors
(S-nitroso-N-acetyl-penicillamine and dipropylenetriamine NONOate) on transport of
L-Arg into bovine aortic
endothelial cells (BAEC). Our results demonstrate that NOS agonists
increase y+ transporter activity.
A rapidly acting NO donor initially increases L-Arg uptake; however, after
longer exposure, L-Arg uptake is suppressed. Exposure of BAEC without
L-Arg to substance P and a
Ca2+ ionophore (A-23187) increased
O
2· formation, which was blocked
with concurrent presence of
L-Arg or the NOS antagonist
N
-nitro-L-arginine methyl ester.
We conclude that factors including NO itself control
y+ transport function and the
production of NO and O
2·.
nitric oxide; L-arginine uptake; vascular dysfunction; transporter regulation; endothelial cells
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INTRODUCTION |
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INTRACELLULAR L-ARGININE
(L-Arg) is the substrate
required by NO synthase (NOS) to liberate NO. However, when cellular
supply of L-Arg is limited, NOS
utilizes molecular oxygen as the principal substrate, producing
superoxide anion (O2·) and other
reactive free radicals that can lead to cardiovascular dysfunction and
the pathogenesis of disease (16, 21, 31).
The total intracellular concentration of L-Arg (0.1-1 mM) in endothelial cells (EC) greatly exceeds the Michaelis-Menten constant (Km) of endothelial NOS (eNOS) for L-Arg (~3 µM) (29). This suggests that eNOS is saturated with substrate and that levels of intracellular L-Arg are not limiting for NO production. However, other studies have shown that availability of L-Arg varies greatly within the EC due to intracellular compartmentalization and sequestration in addition to degradation by arginase or the presence of endogenous inhibitors of eNOS (i.e., asymmetrical dimethylarginine; Refs. 36, 11). Recently, it was also shown that concurrent cellular L-Arg transport may be more important than intracellular L-Arg levels in providing L-Arg to eNOS for NO production (23). Therefore, total intracellular concentration of L-Arg may not truly reflect the L-Arg available at the site of eNOS action.
Supply of L-Arg may become limiting and reduce formation of NO in normal and pathological states. Treatment of guinea pigs with L-Arg has been shown to increase the duration of the vasodilatory response to ACh under normal physiological conditions (2); prior stress with norepinephrine infusion accentuates this enhancement process. Gold et al. (14) demonstrated that ACh and a Ca2+ ionophore, which release NO, can induce tolerance in isolated arterial rings. Tolerance was associated with depletion of L-Arg and was reversed with L-Arg repletion. L-Arg may also become limiting under pathological conditions. Endothelial dysfunction in cardiomyopathic hamsters can be reversed by L-Arg (22). In addition, humans with acute hyperglycemia exhibit intense vasoconstriction and impaired endothelial function (4), which can be reversed by intravenous infusions of low concentrations of L-Arg (37). Other diseases in which pathology is attributed to a deficiency of L-Arg include hypertension, atherosclerosis, restenosis/postcoronary angioplasty, and reperfusion injury (8, 10, 15, 24, 38). Similarly, addition of L-Arg in these circumstances also ameliorates the deficit in endothelium-dependent relaxation.
Intracellular L-Arg is derived
from several sources, and determinants of
L-Arg level include the
transport of L-Arg into cells, the amount of intracellular
L-citrulline recycled back to
L-Arg, the rate of degradation
of L-Arg (arginase), the
incorporation of L-Arg into
proteins (compartmentalization), and the amount of
L-Arg utilized upon activation
of intracellular eNOS. Uptake of
L-Arg into EC occurs through two
carrier-mediated transporters and through passive diffusion. The
saturable carrier-mediated transporters include a
Na+-dependent active
transporter (system Bo,+) and a
Na+-independent transporter
(system y+) (Fig.
1). The majority (80%) of
L-Arg delivered into most cells is through the y+ transporter (26,
27). Regulation of L-Arg
transport appears to involve cellular membrane potential. Exposure of
EC to hyperpolarizing agents, including ATP and bradykinin (BK),
increases L-Arg uptake, whereas
a decrease in L-Arg transport
was observed when cells were treated with agents that cause cellular
depolarization (3, 41). In addition, factors that reduce the activity
of the y+ transporter, including
free radicals, may also reduce the
L-Arg available for eNOS
(27).
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We hypothesized that when the balance of transporter regulatory factors
is negative, L-Arg supply
becomes limiting and subsequent production of
O2· may contribute to vascular and
organ pathology. Our purpose was to determine and compare the effects
of eNOS agonists and NO donors on
L-Arg uptake by EC. Effects of
eNOS stimulation on O
2· production
were also assessed in the presence and absence of
L-Arg and the eNOS antagonist
N
-nitro-L-arginine methyl ester
(L-NAME).
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MATERIALS AND METHODS |
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Drugs and Chemicals
Cell culture medium M-199 and penicillin-streptomycin were purchased from GIBCO (Gaithersburg, MD), fetal bovine serum and iron-supplemented calf serum from HyClone (Logan, UT), and Thermanox coverslips from Fisher Scientific (Pittsburgh, PA). The NO donor dipropylenetriamine (DPTA) NONOate was purchased from Calbiochem (La Jolla, CA). L-[2,3,4,5-3H]arginine monohydrochloride (sp act 61 Ci/mmol) and [3H]tetraphenylphosphonium (TPP+; 29 Ci/mmol) were from Amersham (Arlington Heights, IL). Ecoscint-A scintillation fluid was from National Diagnostics (Atlanta, GA). S-nitroso-N-acetyl-penicillamine (SNAP), the eNOS agonists substance P (SP), BK, and ACh, and all other chemical reagents were purchased from Sigma Chemical (St. Louis, MO).Characterization of L-Arg Transport System in BAEC
Confluent cultures of bovine aortic EC (BAEC; passages 2-6) were prepared in 24-well plates. To determine the Na+-independent contribution of the y+ transporter for L-Arg supplied to the cells, BAEC were incubated in an uptake buffer (in mM: 25 HEPES, 1.8 CaCl2, 5.4 KCl, 140 choline chloride, 0.8 MgSO4, and 5 glucose) containing 20 nM L-[3H]Arg for periods of 1, 2.5, 5, 15, 30, and 60 min. In some experiments, 50 µM unlabeled L-Arg was added to this mixture to ensure that the transporters we examined were those that were expected. Uptake of L-Arg was terminated by addition of ice-cold buffer, and cells were washed three times with 1 ml of buffer. After the final washing, cells were lysed by addition of 1 ml 0.5% SDS in 0.1 N NaOH. Cellular lysates were added to 15 ml of Ecoscint-A scintillation fluid. The amount of L-[3H]Arg was determined by scintillation spectroscopy (Beckman Instruments) and represented cellular transport of L-Arg. We also determined the contribution of the bo,+ system, another Na+-independent transporter, and y+L, another Na+-dependent transporter, by adding 10 mM L-leucine to our Na+-containing uptake buffer (12). Addition of leucine did not alter the uptake of L-Arg, indicating that these transport systems are not operative. Cellular uptake of L-Arg via the Bo,+ system was determined by substitution of sodium chloride for choline chloride and incubation as described above. The difference between uptake determined for both transporters (uptake in presence of Na+) and that observed for y+ alone represented the amount of L-Arg uptake contributed by the Na+-dependent Bo,+ system. In addition, experiments were performed in which y+ and Bo,+ transporters were characterized for cellular uptake of L-[3H]Arg for 1, 2.5, 5, 15, 30, and 60 min in the presence of excess (10 mM) unlabeled L-Arg. These results allowed us to determine to what extent nonspecific cellular binding and diffusion contributed to apparent L-Arg uptake; this contribution was subtracted from experimental values. It has been previously reported that passive, nonsaturable diffusion of L-Arg can occur, but only when extracellular levels of L-Arg are in the millimolar range (39). Comparing experiments in which we used only L-[3H]Arg (20 nM) with those utilizing added cold L-Arg (50 µM), we found no difference in contributions of the y+ and Bo,+ transporters and passive diffusion and the degree of enhancement of L-Arg uptake produced by BK. Thus use of only labeled L-Arg was valid for characterization of transport of L-Arg with these systems.Cellular Uptake of L-[3H]Arg in Presence of eNOS Agonists and NO Donors
BAEC were preincubated with the eNOS agonists BK (1 µM), SP (1 µM), and ACh (5 µM) or with the NO donors SNAP (200 µM, equivalent to 0.4 µM NO) and DPTA NONOate (0.01-10 µM, equivalent to 0.02-20 µM NO) for 1, 2, or 4 h. BK, SP, ACh, and SNAP were reapplied every 15 min for the duration of exposure. At the end of these treatment periods, cells were washed with uptake buffer and incubated for 1 h in uptake buffer containing L-[3H]Arg and the treatment agent. Other cells that were not preincubated but rather were acutely exposed to treatments for 2, 15, 30, or 60 min were incubated with uptake buffer containing both L-[3H]Arg and treatments for the duration of acute periods. Uptake of L-Arg was terminated and total cellular transport of L-Arg was determined as described above.Effects of Agents on Cell Membrane Potential
Effects of our test agents on membrane potential were determined by their effects on accumulation of TPP+ in BAEC. This lipophilic, positively charged compound enters the cell more readily when membrane potential is increased. Confluent cultures in 24-well plates were washed twice with uptake buffer. They were then incubated with 1 µM [3H]TPP+ without or with BK (1 µM), SNAP (200 µM), or KCl (140 mM) for 15 min. After the exposure, cells were washed with ice-cold uptake buffer three times. Cells in each well were solubilized by the addition of 1 ml of 0.5% SDS in 0.1 N NaOH. An aliquot (0.8 ml) of the lysate was added into scintillation fluid and counted. Nonspecific absorption to the culture wells was estimated in parallel conditions without cells. This amount was subtracted from total [3H]TPP+ accumulation. Protein content in each well was determined using a micro bicinchoninic acid protein assay reagent kit (Pierce).Cellular O2· Formation
in Presence of eNOS Agonists
Data Analysis
Data are expressed as means ± SE. Comparisons of data between experimental groups and their appropriate controls were made using ANOVA or paired Student's t-tests. P ![]() |
RESULTS |
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Cellular Transport of L-Arg into BAEC
Initial data demonstrated that transport of cellular L-[3H]Arg into BAEC occurs linearly with time for up to 1 h (Fig. 2). We also found that the primary transporter of L-Arg into these BAEC is the y+ transporter, which was responsible for ~85% of the L-[3H]Arg delivered to cells. The Bo,+ transporter system accounted for an average of 10% total transport. Passive diffusion as a percent of total cellular L-Arg uptake was variable and decreased as the period of uptake was increased, accounting for 5 and 1.5% of total cellular L-Arg transport during 15 and 60 min of uptake, respectively. Inclusion of 10 mM L-leucine in the medium did not diminish L-Arg uptake, indicating that the system bo,+ and y+L transporters were not active for L-Arg in these cells.
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Effect of eNOS agonists on cellular uptake of
L-Arg.
L-Arg transporter activity was
augmented after acute exposure to eNOS agonists (Figs.
3, A and
B, and
4). SP enhanced
y+ transport of
L-Arg into cells by 24% after
only 15 min of exposure (Fig. 3A).
This elevated L-Arg uptake was
maintained for exposures of 30 and 60 min, with 24 and 21% increases,
respectively. In addition, the effect of SP on cellular transport of
L-[3H]Arg
was enhanced after pretreatment with SP for more prolonged durations.
After 2 h of exposure of BAEC to SP,
y+ transporter activity was
enhanced as much as 34% over control values. This increase in
transporter activity was also maintained after 3 and 5 h of exposure,
with cellular L-Arg increases of 27 and 21%, respectively. BK was also effective in augmenting cellular
uptake of L-Arg into cells, with
a maximum increase of 42% observed after 15 min of exposure (Fig.
3B) and slightly lower but still
marked increases of 39 and 26% occurring after treatment for 30 and 60 min, respectively. Prolonged exposure of BAEC to BK enhanced cellular
uptake of L-Arg by 38% after 2 h of exposure. A similar magnitude of increase was also observed after
3 and 5 h of exposure, with increases in transport of 19 and 22%,
respectively. Effects of a third eNOS agonist, ACh, on the cellular
uptake of L-[3H]Arg
were similar to those for SP and BK. A 22% increase of
L-[3H]Arg
uptake was observed after 2 min of exposure to ACh. After 15 min of
exposure to ACh, L-Arg uptake
reached a maximum increase of 27%. Treatment with ACh for 30 or 60 min
resulted in 18 and 16% increases of
L-Arg uptake, respectively.
These elevations were statistically significant
(P < 0.05). These results
demonstrate that the eNOS agonists SP, BK, and ACh enhance
y+ transport of
L-Arg into BAEC.
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Effect of NO donors on cellular uptake of L-Arg. Treatment of EC with 200 µM SNAP (0.4 µM NO) markedly increased activity of the y+ transporter by 37% after 15 min of exposure (Fig. 3C). This elevation was not seen after 30 min of exposure. By 1 h, uptake of L-[3H]Arg was reduced by 22%. Inhibition was maintained; 46, 45, and 36% reductions were observed after 2, 3, and 5 h of exposure to NO, respectively. This biphasic action over time differs from the actions of SP and BK (Fig. 3, A and B). To confirm that the reduction in cellular uptake of L-[3H]Arg was due to NO released from SNAP, experiments were performed using another NO donor, DPTA NONOate. Unlike SNAP, which donates large amounts of NO in a short time, DPTA NONOate allows for a slower (half time ~5 h), more sustained release of NO that is constant over time. Exposure to 1 µM DPTA NONOate (2 µM NO) had no significant effect on the y+ system at the earlier periods (15 and 30 min); however, significant inhibitions of 22, 24, and 29% for L-Arg transport were observed after 1, 2, and 4 h of exposure, respectively (data not shown). This repression appeared to be concentration dependent, with maximum inhibition of 20, 24, and 44% occurring after 2 h of exposure to concentrations of 0.01, 1, and 10 µM (0.02, 2, and 20 µM NO), respectively (Fig. 4). Collectively, these results suggest that the effects of a rapidly acting NO donor on L-Arg transport are biphasic, with acute exposure to NO enhancing and prolonged exposure reducing system y+ activity.
Effects of Agents on Cell Membrane Potential
Accumulation of [3H]TPP+ in cells in response to BK (1 µM), SNAP (200 µM), and KCl (100 mM) is depicted in Fig. 5. This lipophilic cation will passively distribute across the plasma membrane in proportion to membrane potential (41). Basal uptake of [3H]TPP+ in normal uptake buffer over 15 min is shown in Fig. 5. Concurrent exposure of cells to BK increased TPP+ accumulation by 39%. Exposure of cells to SNAP did not elicit a change in TPP+ accumulation. Exposure of cells to a high extracellular concentration of KCl caused a 51% reduction in TPP+ accumulation in these cells. These results indicate that acute exposure (15 min) to SNAP does not result in membrane hyperpolarization as occurs with BK.
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Cellular O2· Formation
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DISCUSSION |
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The transport of L-Arg into
cells is critical for maintaining adequate
L-Arg levels such that optimal
coupling of L-Arg with eNOS can
occur (16). Therefore, factors affecting the
y+ transporter system for
L-Arg have the potential to
limit the production of NO (18). Without ample
L-Arg, eNOS will principally utilize O2 to form
O2· (31, 32), which may contribute
to the pathogenesis of disease. As a consequence, there is a need to
determine the exact factors controlling
L-Arg supply and their role in
affecting O
2· production in normal
as well as pathological circumstances.
We have characterized the cellular L-Arg transport system in BAEC. Our data confirm that the primary source of L-Arg supply is through activity of the system y+ transporter (11) and that delivery of L-Arg into cells occurs linearly over 2 h. In addition, we have verified that system Bo,+ transport activity and passive diffusion contribute minimally to the delivery of L-Arg into BAEC under basal conditions. The bo,+ and y+L transporters do not appear to be operative in these cells. Our experimental results were similar to those observed using human umbilical EC (26) and porcine aortic EC (3). These experiments were important to determine which transport mechanism should be studied.
Our data demonstrate that BK causes an increase in cellular uptake of L-Arg. These results are consistent with a study by Bogle et al. (3) in which porcine aortic EC grown on microcarrier beads increased their cellular uptake of L-[3H]Arg in the presence of BK within 10 min. In addition to these findings, we were able to demonstrate that this enhancement of cellular uptake of L-Arg was maintained from 15 min through 2 h of exposure to BK. More importantly, we were able to demonstrate an increase in y+ transporter activity for two other eNOS agonists, SP and ACh. As stated before, cellular membrane potential is thought to be the dominant mechanism by which y+ system activity is controlled. Hyperpolarization associated with stimulation of y+ system is thought to occur by first increasing intracellular Ca2+. This increase in Ca2+ activates Ca2+-dependent K+ channels, resulting in K+ efflux and hyperpolarization (1, 34). Because BK, SP, and ACh have also been shown to induce cellular hyperpolarization (1, 13, 34), these data suggest that the increase in y+ transporter activity observed occurred by a similar mechanism.
Interestingly, our data for the NO donor SNAP show initial stimulation of the y+ transporter within 15 min, followed by no change and then by inhibition of cellular L-Arg uptake with more prolonged exposures to NO. This initial increase of cellular uptake of L-Arg was not associated with a cellular hyperpolarization as occurs with BK. We had expected an EC hyperpolarization with initial NO release by SNAP, as has been observed for NO donors in smooth muscle (7). However, the mechanism of this initial stimulating effect of SNAP on L-Arg uptake does not appear to involve EC hyperpolarization. Longer exposures to SNAP of 1-4 h resulted in a marked reduction of L-Arg transport. A different NO donor that releases NO slowly over time, DPTA, did not acutely alter L-Arg uptake but, like SNAP, suppressed L-Arg uptake over a 1- to 4-h period.
There is evidence that oxidative properties of NO may be responsible for the reduction of cellular L-Arg transport seen with longer exposures. Patel et al. (27) demonstrated that NO, through constant gas infusion and release from SNAP, decreases y+ system transporter activity. The negative effect of NO on y+ transport of L-Arg into cells was determined to be associated with oxidation of sulfhydryl moieties in the transporter proteins, since treatment with the disulfide reducing agent dithiothreitol restored transporter activity. Furthermore, treatment of EC with the sulfhydryl reactive chemicals N-ethylmaleimide and acrolein reduced y+ transporter activity.
The biphasic effect of SNAP on transport function over time was not
observed in cells with prolonged exposure to eNOS agonists. It would be
expected that stimulation of eNOS would also increase NO production and
oxidation of the y+ transporter
system, resulting in inhibition of
L-Arg uptake similar to that
observed with SNAP. One explanation for the lack of biphasic action
with eNOS agonists could be that the amount of NO produced upon eNOS
activation is far less than the amount of NO released from SNAP.
Therefore, levels of eNOS-derived NO never accumulate enough for
significant oxidation of the y+
transporter. Another possibility to explain the lack of inhibition of
L-Arg transport with eNOS
agonists is the fact that upon stimulation with eNOS,
L-Arg is converted to the
intermediate
NG-hydroxyl-L-arginine
(L-HOArg) before formation of
L-citrulline and NO.
L-HOArg is known to be an
antioxidant and an inhibitor of arginase (6, 40). Therefore, the
L-HOArg intermediate may provide
protection from oxidation by newly formed NO. Prevention of the
metabolism of L-Arg in the
ornithine cycle may result in a net increase in the amount of
L-Arg available for eNOS and
lead to a reduction in O2·
formation. Both of these actions should protect the system
y+ transporter from inactivation
(5). Hence the transport of L-Arg into cells via the
y+ transport system may be
unfavorably altered with elevated levels of NO.
High concentrations of NO could occur during circumstances in which
eNOS is constantly stimulated. Pathophysiological conditions associated
with increased eNOS activity include hypoxia (28), hyperglycemia (9),
and hypertensive states mediated by elevations in ANG II (high renin
essential and renovascular hypertension) (33). The combination of
increased eNOS activity (L-Arg
demand) and decreased Arg uptake
(L-Arg supply) has the potential
to create an L-Arg deficiency
("demand-supply mismatch") that can result in the increased
O2· production (31, 16) seen in
states such as ischemia-reperfusion injury (16, 20, 17).
Increased O
2· production and eNOS
activity have also been shown to be associated with hyperglycemia (9).
We have characterized the production of
O2· in BAEC alone and during
treatment with eNOS agonists. In addition, we looked at the effects of
basal and eNOS agonist-induced O
2·
production with concurrent addition of
L-Arg and
L-NAME. Our data demonstrate
that BAEC without L-Arg in the
medium produce O
2·, which
increases with time, and that addition of
L-NAME reduces basal
O
2· production. Because
L-NAME is an eNOS antagonist,
this suggests that a prominent source of basal
O
2· production is eNOS in EC.
Others have noted that L-NAME
can reduce O
2· production in
ischemia-reperfusion injury of skeletal muscle, especially in
the presence of A-23187 (17), in
L-Arg-starved neurons (30), and
in ANG II-stimulated rat aortic EC (33). Addition of SP or A-23187 to
BAEC stimulated increases in O
2· much greater than basal levels. Interestingly, a striking reduction of
O
2· production was observed upon
pretreatment of cells with either
L-Arg or
L-NAME before addition of SP or A-23187. These data suggest that excessive
O
2· formation is associated with
agonist-induced eNOS activation; this can be ameliorated with
L-Arg supplementation. There are other sources of O
2· production in
EC, including membrane-bound NAD(P)H oxidase (25) and xanthine oxidase (19).
Collectively, our findings strongly suggest that, although intracellular L-Arg levels far exceed the concentration of L-Arg required by eNOS for NO production, the amount of L-Arg available for utilization by eNOS can be insufficient, especially in conditions of chronic eNOS stimulation. The explanation for this L-Arg paradox may be provided by the work of McDonald and colleagues (23). Using porcine pulmonary artery EC with antibodies specific for caveolin, eNOS, and the y+ transporter, they demonstrated that all of these proteins are colocalized within the plasma membrane caveolae. This suggests that eNOS associated with this complex is sequestered from overall intracellular L-Arg and relies on the de novo transport of L-Arg into the cell via the y+ transporter within the caveolae for NO production. If the transporter becomes damaged, as seen with oxidation, L-Arg supply could immediately become limiting and may be the basis for endothelial dysfunction. In addition, this eNOS-y+ transporter-caveolae complex may explain why endothelial dysfunction is quickly reversed by increases in extracellular L-Arg. Once transporter function is reduced, the L-Arg concentration gradients increase and delivery of L-Arg into cells is shifted toward passive diffusion. Therefore, extracellular supplementation of L-Arg may be helpful in driving passive diffusion of L-Arg when the integrity of carrier-mediated transporters cannot be maintained (26).
In conclusion, we believe that concurrent
L-Arg supply to eNOS via system
y+, independent of overall
intracellular L-Arg, is critical
in establishing and maintaining vascular function. NOS products,
including NO itself, appear to control
y+ activity, and the summation of
these factors is critical in determining NO and
O2· formation, both of which
contribute to vascular dysfunction and disease.
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ACKNOWLEDGEMENTS |
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We thank Sandra B. Usry for assistance in preparation of the manuscript, Traci A. Taylor for technical assistance, and Dr. Ruth B. Caldwell for critical reading of the manuscript.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. William Caldwell, Dept. of Pharmacology and Toxicology, Medical College of Georgia, 1120 15th St., Augusta, GA 30912-2300 (E-mail: wcaldwel{at}mail.mcg.edu).
Received 3 December 1998; accepted in final form 1 September 1999.
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