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Department of Pediatrics, Medical College of Wisconsin, Milwaukee 53226; and Research Services, Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin 53295
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Previously, our laboratory found that
pulmonary hypertension developed and lung nitric oxide (NO) production
was reduced when piglets were exposed to chronic hypoxia (Fike CD,
Kaplowitz MR, Thomas CJ, and Nelin LD. Am J Physiol Lung
Cell Mol Physiol 274: L517-L526, 1998).
The purposes of this study were to determine whether
L-arginine addition augments NO production and to evaluate whether L-arginine uptake is impaired in isolated lungs of
chronically hypoxic newborn piglets. Studies were performed by using 1- to 3-day-old piglets raised in room air (control) or 10%
O2 (chronic hypoxia) for 10-12 days. Lung NO
production was assessed in isolated lungs from both groups by measuring
the perfusate accumulation of nitrites and nitrates (collectively
termed NO
x) before and after
addition of L-arginine (102 M) to the
perfusate. The rate of perfusate
NOx accumulation increased by
220% (from 0.8 ± 0.4 to 2.5 ± 0.5 nmol/min, P < 0.05)
after L-arginine addition to chronic hypoxic lungs but remained unchanged (3.2 ± 0.8 before vs. 3.3 ± 0.4 nmol/min after L-arginine) in control lungs. In the second series of
studies, L-arginine uptake was evaluated by measuring the
perfusate concentration of
L-[3H]arginine at fixed time
intervals. The perfusate concentration of
L-[3H]arginine at each time point
was less (P < 0.05) in control than in chronic hypoxic lungs.
Thus L-arginine uptake was impaired and may underlie in
part the reduction in lung NO production that occurs when piglets are
exposed to 10-12 days of chronic hypoxia. Moreover, these findings
in isolated lungs lead to the possibility that L-arginine
supplementation might increase in vivo lung NO production in piglets
with chronic hypoxia-induced pulmonary hypertension.
neonatal pulmonary hypertension; L-arginine uptake; nitrites; nitrates
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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L-ARGININE IS METABOLIZED by nitric oxide synthase
(NOS) into nitric oxide (NO) and L-citrulline. Our
laboratory has previously found lower NO production, as measured by the
accumulation of the stable metabolic products of NO, nitrites and
nitrates (collectively termed
NOx), in the perfusate of lungs
from piglets with pulmonary hypertension induced by 10-12 days of
exposure to chronic hypoxia, compared with comparable-age control
piglets (11). This finding leads to the suggestion that decreased lung NO production could contribute to the development of chronic
hypoxia-induced pulmonary hypertension in newborn piglets. In turn, it
is possible that reduced amounts of the enzyme responsible for basal NO
production, endothelial NOS (eNOS), could be a mechanism contributing
to the decreased NO production by lungs of the chronically hypoxic
piglets. This possibility is supported by our laboratory's previous
finding that the amount of eNOS was less in whole lung homogenates from the chronically hypoxic compared with the control piglets (11). However, another possible mechanism for decreased lung NO production may be that the bioavailability of L-arginine is altered
during chronic hypoxia (3, 6, 18, 20). If this were the case, then
additional L-arginine may increase lung NO production by increasing bioavailability of L-arginine to lung eNOS.
Evaluation of this possibility is of particular importance because of
the implications for therapeutic manipulation of the
L-arginine-NO pathway. Therefore, to test the hypothesis
that reduced L-arginine bioavailability contributes to
reduced NO production in the lungs of chronically hypoxic newborn pigs,
we determined whether addition of L-arginine to the
perfusate augmented NO production in lungs from chronically
hypoxic piglets. Because L-arginine is
transported into cells via amino acid transporters (17, 27),
altered uptake is one potential mechanism for reduced
L-arginine bioavailability (3). Thus we also evaluated
L-arginine uptake in isolated lungs of control and
chronically hypoxic newborn piglets.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals.
A total of 13 chronically hypoxic piglets was studied, and a total of
16 control piglets was studied. Different litters were used for control
piglets than for chronically hypoxic piglets. The control piglets were
not littermates; therefore, these piglets came from 16 different
litters. Some of the chronically hypoxic piglets were littermates, such
that these piglets came from nine different litters. For the
chronically hypoxic piglets, newborn pigs (1-3 days old) were
placed in a hypoxic normobaric environment (chronic hypoxia) for
10-12 days. Normobaric hypoxia was produced by delivering
compressed air and nitrogen to an incubator (Thermocare). The
O2 content was regulated at 8-10% O2
(PO2 60-72 Torr), and
PCO2 was maintained at 3-6 Torr
by absorption with soda lime. The chamber was opened three
times a day for cleaning and to weigh the animals. The animals were fed
ad libitum with an artificial sow milk replacer from a feeding device
attached to the chamber. Some (n = 4) of the control pigs were
raised by placing newborn pigs (1-3 days old) in a room-air
environment for 10-12 days and maintaining them as described for
the hypoxic piglets. Because we previously found no difference in the
perfusate accumulation of NOx
between lungs from control piglets raised on the farm and lungs from
control piglets raised in a normoxic chamber (11), most (n = 12) of the control piglets were studied on the day of arrival from the
farm at 11-15 days of age. All piglets were anesthetized for lung
isolation and perfusion as described below (see Lung isolation and
perfusion). Before lung isolation and perfusion, catheters were
placed in some anesthetized control (n = 9) and chronically
hypoxic (n = 7) piglets for determination of in vivo pulmonary
vascular resistance as follows.
Measurements in anesthetized animals. On the day of study, some control (n = 9) and some chronically hypoxic (n = 7) animals were weighed and anesthetized with ketamine (30 mg/kg im) and pentobarbital (10 mg/kg iv). Additional intravenous pentobarbital sodium was given as needed via an ear vein to maintain anesthesia during placement of the catheters. First, the trachea of the piglet was cannulated so that the animal could be ventilated, if necessary. Then a catheter was placed into the right femoral artery for monitoring systemic blood pressure and arterial blood gases. Another catheter was placed through the right external jugular vein into the pulmonary artery to monitor pulmonary arterial pressure. To obtain the pulmonary wedge pressure, we advanced the pulmonary arterial catheter into a distal pulmonary vessel. The zero reference for the vascular pressures was the midthorax. To measure cardiac output by the thermodilution technique (model 9520, thermodilution cardiac output computer, Edwards Laboratory), we placed a thermistor into the aortic arch via the left femoral artery, and we placed a catheter that served as an injection port into the left ventricle via the left carotid artery. Cardiac output was measured at end expiration as the mean of three injections of 3 ml of 0.9% saline (0°C). After blood gases, pulmonary arterial pressure, pulmonary wedge pressure, and cardiac output were measured, the animals were given additional anesthesia (3-5 mg/kg pentobarbital iv) for lung isolation and perfusion as described below.
Lung isolation and perfusion.
All animals were anesthetized as described above, given heparin (1,000 IU/kg iv), and then exsanguinated. For lung isolation and perfusion,
the tracheal cannula of each piglet was attached to a large animal
piston-type ventilator, and the lungs were ventilated with a normoxic
gas mixture (17% O2, 6% CO2, and balance
N2) by using a tidal volume of 15-20 ml/kg and a
respiratory rate of 15-20 breaths/min (mean airway pressure of
3-5 mmHg). A midline sternotomy was performed, and a
clamp was placed across the ductus arteriosus. Saline-filled cannulas
were placed into the pulmonary artery and left atrium through incisions
in the right and left ventricles. The diaphragm and all abdominal
contents were removed. The lungs were either left in situ (for
measurement of perfusate accumulation of
NOx) or removed from the thorax (for measurement of L-[3H]arginine
uptake). In all lungs, the vascular cannulas were connected to a
perfusion circuit that was filled with a Krebs Ringer bicarbonate (KRB)
solution containing 5% dextran, mol. wt. 70,000, at 37°C. Briefly,
in the perfusion circuit a rotary pump continuously circulated the
perfusate from a reservoir through a bubble trap into the pulmonary
arterial cannula, through the lungs to the left atrial cannula, and
back to the reservoir. Pulmonary arterial, left atrial, and airway
pressures were continuously monitored. The most dependent edge of the
lung was used as the zero reference for vascular pressures. The height
of the reservoir was adjusted to maintain left atrial pressure at 0 mmHg.
Measurement of perfusate accumulation of
NOx in isolated
lungs.
Isolated, perfused lungs from 10 of the control piglets and nine of the
chronically hypoxic piglets were used in these studies. Lung perfusion
was initially non-recirculating in these studies. When the effluent
from the lung was nearly free of blood, recirculating perfusion was
initiated such that the perfusate had a Hct <1%. The volume of
recirculating perfusate was adjusted to 130-170 ml, and the flow
rate was adjusted to 50 ml · min1 · kg1.
The lungs were perfused for 30-60 min until a stable pulmonary arterial pressure was achieved. Perfusate samples (3 ml) were then
removed from the left atrial cannula every 10 min for a 60-min baseline
period. Next, L-arginine (102 M) was
added to the perfusate, after which perfusate samples were collected
every 10 min for an additional 60 min (L-arginine period).
The perfusate samples were centrifuged, and the supernatant was stored
at 
70°C for future analysis of
NOx concentrations. At the end of
the perfusion, the volume of perfusate remaining in the circuit and
reservoir was measured.
x concentrations. All reagents
were obtained from Sigma Chemical. Fifty microliters of a stock NADPH
solution (0.8 µg NADPH/ml phosphate buffer) and 10 µl of a stock
nitrate reductase solution (5 units nitrate reductase/ml phosphate
buffer) were added to 500 µl of lung perfusate. After incubation for
3 h at room temperature, Greiss reagent [300 µl; 1%
sulfanilamide, 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride, and 2.5% phosphoric acid] was added to the lung perfusate and incubated for 10 min at room temperature, and the absorbance was measured at 546 nm. A standard curve was prepared by
adding known amounts of NaNO3 to fresh perfusate. Fresh
perfusate with added NADPH, nitrate reductase, and the Greiss reagent
as described for the lung perfusate samples was used as a blank. Duplicate assays were carried out for each sample of lung perfusate.
The perfusate NOx concentration
(nmol/ml) was determined for each collection time as described above.
The amount of NOx in the perfusate
(nmol) at each collection time was calculated by multiplying the
perfusate concentration of NOx at
that sample collection time by the volume of the system (perfusion
circuit plus reservoir) at the sample collection time plus the amount
of NOx removed with all previous
samples. The amount of NOx accumulated (nmol) was the amount of
NOx at each collection time minus
the amount of NOx at time
0. The amount of NOx at
time 0 was determined from the y-intercept of a linear
regression line fit to the amount of
NOx in the perfusate vs. time for
the first 60 min of perfusion.
Measurement of L-[3H]arginine uptake in isolated lungs. Isolated, perfused lungs from six other control piglets and four other chronically hypoxic piglets were used in these studies. The perfusion circuit for these studies was modified to contain two reservoirs in parallel, both containing KRB, such that the perfusate entering the lungs could be rapidly changed from one reservoir system to the other. The lungs were initially connected to the first reservoir and were perfused for 30-60 min until a stable pulmonary arterial pressure was achieved. During this stabilization period, tritium-labeled L-arginine (L-[2,3,4,5-3H]arginine monohydrochloride, specific activity 59 Ci/mmol) and enough unlabeled L-arginine to achieve a final perfusate concentration of 10 µM L-arginine were added to the KRB in the second reservoir (25 µCi). At the end of the stabilization period, a time 0 sample was taken from the second reservoir containing the KRB mixed with L-[3H]arginine. Then the perfusate entering the lung was rapidly switched to this L-[3H]arginine-containing reservoir. The perfusate leaving the lungs was discarded until the volume of perfusate in the lung and the tubing from the lung to the reservoir at the time of the switch were filled with the KRB containing the L-[3H]arginine. At this point, recirculating perfusion from the second reservoir was established, and 200-µl samples of perfusate were collected from the reservoir at 2, 5, 10, 15, 30, and 60 min after switching. To measure the total L-[3H]arginine counts per minute at each sampling time point, we mixed each sample with 5 ml of liquid scintillation counting cocktail and placed them in a liquid scintillation counter. To determine the L-[3H]arginine counts per minute added to the reservoir, two 10-µl aliquots of the L-[3H]arginine that had been added to the perfusate were placed directly into 5 ml of liquid scintillation counting cocktail and were then placed in the liquid scintillation counter.
For each sampling time point, the total L-[3H]arginine counts per minute were corrected for the metabolism of L-arginine to its metabolites, L-ornithine and L-citrulline, by measuring the appearance of 3H in L-ornithine and L-citrulline using TLC. To do this, at each time point an additional sample of the perfusate (40 µl) was collected and streaked directly on a TLC plate (Silica Gel 60 F254, EM Science), which was run in a mobile phase consisting of chloroform-methanol-ammonium hydroxide-water (9:9:4:1). The retardation factor values are 0.18 for L-arginine, 0.31 for L-ornithine, and 0.66 for L-citrulline. Each TLC plate was scraped in 1-cm bands into a scintillation vial to which 10 ml of scintillation fluid were added. Each vial was placed in a sonicator for 30 s and then into the liquid scintillation counter. The amount of L-[3H]arginine in the perfusate for each time point was determined from the ratio of 3H in L-arginine to that in its tritiated metabolites and was normalized to the L-[3H]arginine concentration at time 0.Calculations and statistics.
Data are presented as means ± SE. Unpaired t-tests were used
to compare the hemodynamic measurements between control and chronically hypoxic animals. The NOx
accumulation data were analyzed by first determining the rate of
NOx accumulation (nmol/min) in
each lung by linear regression of each individual lung's accumulated
NOx (nmol) vs. time (min) data for
both the baseline and L-arginine time periods. Then the
individual rates of NOx
accumulation (nmol/min) were meaned and compared between control and
chronic hypoxic groups for both baseline and L-arginine
perfusion periods by using a one-way ANOVA and a post hoc test to
determine the significant differences between the two groups and the
two perfusion periods. The
L-[3H]arginine uptake data were
analyzed by using a one-way ANOVA and a Newman-Keuls post hoc test to
compare between control and chronically hypoxic animals the amount of
L-[3H]arginine in the perfusate at
each time point. P < 0.05 was indicative of statistical significance.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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After 10-12 days of hypoxia, the chronically hypoxic piglets
weighed less than the corresponding control piglets (Table
1). The measured values of blood pH,
PO2, and
PCO2 obtained during hemodynamic
measurements in anesthetized piglets breathing room air did not differ
significantly between control and chronically hypoxic piglets (Table
1). Similarly, measured values of pH,
PO2, and
PCO2 in the perfusate of isolated
lungs from chronically hypoxic piglets were not significantly different
from those of control piglets (Table 1).
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Measurements of pulmonary arterial pressure, pulmonary wedge pressure,
cardiac output, and calculated pulmonary vascular resistance [(pulmonary arterial pressure wedge pressure) /
cardiac output] in the anesthetized piglets are shown in Table
2. Pulmonary arterial pressures and
pulmonary vascular resistances were significantly greater in the
chronically hypoxic than in the control piglets (Table 2).
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In the isolated lungs used in the
NOx accumulation studies, the
baseline pulmonary arterial pressures at the end of the stabilization
period were higher in lungs from chronically hypoxic than from control
piglets (Table 3). With addition of
L-arginine to the perfusate, pulmonary arterial pressure decreased by a greater amount in the hypoxic than in the control lungs
when expressed as an absolute change in pressure but not when expressed
as a percent change in pressure (Table 3).
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The NOx concentration measured in
the perfusate at the first collection time and at both the 60- and
120-min collection times was lower in chronically hypoxic lungs than in control lungs at comparable collection times (Table
4). In both the chronically hypoxic and
control groups, the perfusate NOx concentration measured at the 120-min collection time was greater than
that measured at the first collection time (Table 4); but only in the
chronically hypoxic group was the perfusate
NOx concentration greater at the
120-min collection time than at the 60-min collection time (Table 4).
Figure 1 shows the amount of accumulated
NOx (nmol) in both control and
chronically hypoxic animals at each collection time for the 60-min
baseline period and the 60-min L-arginine period. At all
time points after the first 40 min of the baseline period was the
amount of accumulated NOx less in
the chronically hypoxic than in the control lungs (Fig. 1).
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Figure 2 illustrates the perfusate
NOx accumulation rate (nmol/min)
in control and chronically hypoxic animals. During the baseline period,
the rate of NOx accumulation was
75% less (P < 0.05 compared with control) in chronically
hypoxic lungs (0.8 ± 0.4 nmol/min, Fig. 2) than in the control lungs
(3.2 ± 0.8 nmol/min, Fig. 2). In contrast, during the
L-arginine period, the rate of
NOx accumulation did not differ
between chronically hypoxic (2.5 ± 0.5 nmol/min, Fig. 2) and control
(3.3 ± 0.4 nmol/min, Fig. 2) lungs. Moreover, the rate of
NOx accumulation during the
L-arginine period in the chronically hypoxic lungs
(2.5 ± 0.5 nmol/min, Fig. 2) did not differ from that
during the baseline period in the control lungs (3.2 ± 0.8 nmol/min,
Fig. 2). Most importantly, the rate of
NOx accumulation nearly tripled after addition of L-arginine to the perfusate in
chronically hypoxic lungs (0.8 ± 0.4 nmol/min during baseline period
vs. 2.5 ± 0.5 nmol/min during L-arginine period; Fig. 2;
P < 0.05), whereas there was no change from the baseline rate
of NOx accumulation after addition
of L-arginine to the perfusate in control lungs (3.2 ± 0.8 nmol/min during baseline period vs. 3.3 ± 0.4 nmol/min during
L-arginine period, Fig. 2).
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The data for the measurement of
L-[3H]arginine uptake in isolated
lungs from control and chronically hypoxic piglets are illustrated in
Fig. 3. In both groups of lungs, the
L-[3H]arginine perfusate
concentration for each time point was normalized to the
L-[3H]arginine perfusate
concentration at time 0. Note that the normalized perfusate
concentrations of L-[3H]arginine
were significantly less at each time point in the control lungs
compared with chronically hypoxic lungs. Because a recirculating perfusion system was used, the reduced concentrations of
L-[3H]arginine in the perfusate of
control lungs indicate a greater L-[3H]arginine uptake in the
control than in the chronically hypoxic lungs.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In agreement with our laboratory's previous study (11), in this study
we show a reduced rate of perfusate
NOx accumulation in isolated lungs
of piglets with chronic hypoxia-induced pulmonary hypertension. An
important new finding in this study is that L-arginine
addition augmented the rate of perfusate
NOx accumulation in lungs from
chronically hypoxic piglets but did not change the rate in lungs from
comparable age-control piglets. Another new finding in this study is
that L-arginine uptake was impaired in lungs of piglets
exposed to chronic hypoxia. Altogether, these findings indicate that
the reduction in lung NO production that occurs when newborn piglets
are exposed to 10-12 days of chronic hypoxia may be due, at least
in part, to impaired bioavailability of L-arginine, the
substrate for NO synthesis by the enzyme eNOS.
In addition to reduced substrate availability, a reduction in the
enzyme amount could contribute to the decreased
NOx accumulation rate that we
found in lungs of chronically hypoxic piglets. This possibility is
supported by our laboratory's previous findings of decreased eNOS
amounts in whole lung homogenates of chronically hypoxic piglets (11).
NOS immunoreactivity has been localized in pulmonary vascular
endothelium, nerves, and airway epithelium so that the cellular source
of the decreased eNOS in the whole lung homogenates could include any
or all of these tissues (16). This issue remains of interest because
the lower perfusate NOx
accumulation rate most likely reflects vascular NO production, not
airway NO production. Thus if reduction in enzyme amount contributes to
the lower NO perfusate accumulation, then the cellular source of
reduced eNOS should include vascular endothelium. However, it is
possible that, either in addition to or instead of vascular endothelial
cells, airway epithelial cells are the cellular source of reduced eNOS.
Support for this latter possibility is provided by our laboratory's
previous finding that exhaled NO output is reduced in lungs of
chronically hypoxic piglets (11). To help resolve these issues, the
cellular source of the reduced eNOS amounts in whole lung homogenates
of chronically hypoxic piglets needs to be determined.
Another issue is that, in our laboratory's previous study, we found that whole lung homogenate eNOS amounts were reduced by 40%, whereas lung perfusate accumulation of NO was reduced by a much larger amount, 62% (11). This leads to the hypothesis that a mechanism additional to eNOS abundancy might contribute to impaired NO production in lungs of chronically hypoxic newborn piglets.
One possibility is that reduced enzyme activity might also occur during chronic hypoxia. In fact, our measurements of NO production over time reflect enzyme activity, so that our findings support this possibility. As to the cause of altered enzyme activity, oxygen is the electron acceptor in the NOS-mediated synthesis of NO from L-arginine, so that its depletion during chronic hypoxia could impair enzyme activity (21). This explanation does not seem likely in our studies, as the Michaelis-Menten constant for oxygen for the NOS activity of aortic endothelial cells was recently shown to be ~6 mmHg (24). Furthermore, during the perfusion period, all lungs were ventilated with a gas mixture with a PO2 of 125 Torr.
Instead of oxygen limitation, impaired L-arginine availability may explain why NO production was low during baseline conditions and then increased with addition of L-arginine to lungs of chronically hypoxic piglets. In other words, our finding that the rate of NO production over time was improved by the addition of L-arginine suggests not only that NOS activity was reduced in the lungs of chronically hypoxic piglets but that the reduction in NOS activity is at least partly due to impaired L-arginine availability. There are only a few other studies in pulmonary hypertensive animals in which NO production in response to exogenous L-arginine has been measured. These studies have been performed in adults, and the results have been inconsistent (2, 18). Although the amount of data for pulmonary hypertension is very limited, quite a few studies have shown that L-arginine increases NO production in animals with other vascular diseases (4, 7, 19, 22).
The explanation as to why L-arginine supplementation increases NO production remains unclear. Recently it has been found in porcine pulmonary arterial endothelial cells that the cationic amino acid transporter 1 and eNOS form a complex in the caveolae, which suggests that L-arginine transported from the outside of the cell may be directly delivered to eNOS (17). However, with this as an explanation, one might predict that L-arginine addition would increase NO production in control lungs, which we did not find in this study. Another possibility is that endogenous competitive inhibitors of L-arginine uptake or NOS, such as L-glutamine (1) and/or asymmetric dimethylarginine (4), have accumulated during the disease process. This mechanism is purported to at least partly explain the ability of L-arginine to increase NO production in the atherosclerotic vasculature of cholesterol-fed rabbits (4).
It is also possible that the chronically hypoxic lungs become
L-arginine depleted, which would explain the effect of
exogenous L-arginine on NO production. In our study, all
lungs were cleared of blood before the onset of perfusion with an
L-arginine-free perfusate, after which
L-arginine was added to make a concentration of
102 M. Thus different extracellular
L-arginine amounts cannot explain the different perfusate
NOx accumulation rates in control
compared with chronically hypoxic animals in our study. In regard to
intracellular amounts, the concentration of L-arginine in
normal endothelial cells has been found to be at least 100 µM (1,
15). Because this amount exceeds the Michaelis-Menten constant of the
NOS enzyme, [~5 µM (27)], excess L-arginine would not be predicted to alter NO production in normal lungs. Indeed,
in our study, addition of L-arginine had no effect on the
rate of perfusate NOx accumulation
in the control lungs. However, intracellular depletion of
L-arginine may occur during chronic hypoxia and thereby
contribute to the reduced lung NO production in these animals.
Impaired uptake is one mechanism that could lead to intracellular depletion of L-arginine. This idea is supported by findings of others showing reduced L-arginine content and impaired L-arginine uptake in endothelial cells from pulmonary arteries of 6- to 7-mo-old pigs cultured under hypoxic conditions for 3-5 wk (3). Our findings in chronically hypoxic newborn piglets are of particular importance because they are the first showing impaired pulmonary vascular arginine uptake resultant from an in vivo model of chronic hypoxia, indicating that the findings are not limited to in vitro experimentation.
Our findings are also important because they provide a mechanistic basis to pursue manipulating the L-arginine-NO pathway in chronic hypoxia-induced pulmonary hypertension. For example, if reduced NO production is involved in the development of chronic hypoxia-induced pulmonary hypertension, correcting NO production by L-arginine addition might ameliorate the development of this disease. This hypothesis is consistent with the finding that treatment with L-arginine during hypoxic exposure restored endothelium-dependent vasodilation in lungs from chronically hypoxic rats (6, 9). Furthermore, administration of L-arginine to rats during chronic hypoxic exposure ameliorated pulmonary hypertension and vascular remodeling (20). A limitation of these latter studies is that functional changes were evaluated, but NO production was not assessed. It should also be noted that L-arginine has not always been shown to augment NO function either in pulmonary hypertension (2, 8, 14) or in other vascular diseases (13, 23, 26).
Along these lines, one might predict that L-arginine-induced vasodilation would correlate with NO production. Our findings are not consistent with this latter prediction, as we found the same vasodilatory response to L-arginine in control and hypoxic lungs even though L-arginine increased NO production in the hypoxic but not in the control lungs. It is possible that either instead of or in addition to an effect from NO production, the decrease in pulmonary artery pressure with L-arginine might have been from a nonendothelium-dependent, nonspecific vasodilatory effect, as has been demonstrated by others (5, 25). In contrast to our findings in newborn piglets, other investigators have provided evidence for an enhanced vasodilator response to L-arginine in animals with pulmonary hypertension (12, 18).
The influence of experimental conditions on
NOx production and
L-[3H]arginine uptake should be
commented on. Because of the difference in body weights between control
and chronically hypoxic animals, differences in lung size might be
questioned as a source of both the different baseline
NOx accumulation rates and the
different L-[3H]arginine uptakes
measured in perfused lungs of control compared with chronically hypoxic
animals. However, even though we previously reported smaller lung dry
weights in chronically hypoxic than in control piglets (10), we
recently found no difference in vascular volumes in perfused lungs
between the two groups (11). Hence, differences in perfused surface
areas are not a likely explanation for the markedly different values
for baseline NOx accumulation
rates and L-[3H]arginine uptake
measured in lungs of control compared with chronically hypoxic piglets
in this study.
In summary, our findings are consistent with a defect in basal NO production in lungs of chronically hypoxic piglets that can be overcome by the excess substrate L-arginine. This defect appears to be due, at least in part, to impaired transport of L-arginine into pulmonary vascular cells, which in turn could result in a reduction of intracellular L-arginine availability. These findings do not exclude the possibility that another mechanism, such as reduced enzyme abundance, also contributes to impaired NO production. For instance, it is possible that impaired L-arginine uptake by pulmonary vascular endothelial cells is the primary, if not sole, mechanism responsible for decreased lung vascular NO production, whereas reduced eNOS amounts in lung epithelial cells underlie decreased airway NO production. These possibilities require further study. In addition, whether providing excess L-arginine to living piglets would restore functional impairments of the pulmonary vasculature and prevent or inhibit the development of pulmonary hypertension during exposure to chronic hypoxia is an important issue that needs to be investigated.
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ACKNOWLEDGEMENTS |
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This work was supported in part by a Children's Hospital of Wisconsin Foundation Grant (to C. D. Fike) and by March of Dimes Birth Defects Foundation Research Grants (to L. D. Nelin and C. D. Fike). This work was done during the tenure of an American Heart Association Clinician Scientist Award (to L. D. Nelin).
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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: C. D. Fike, Wake Forest Univ. School of Medicine, Dept. of Pediatrics, Medical Center Blvd., Winston-Salem, NC 27157 (E-mail: cfike{at}wfubmc.edu).
Received 17 December 1999; accepted in final form 4 January 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Munzel T,
Venema RC,
James NL,
Bai CL,
Mitch WE,
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Interactions between L-arginine and L-glutamine change endothelial NO production. An effect independent of NO synthase availability.
J Clin Invest
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2565-2572,
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Baudouin, SV,
Bath P,
Martin JF,
Bois RD,
and
Evans TW.
L-Arginine infusion has no effect on systemic haemodynamics in normal volunteers, or systemic and pulmonary haemodynamics in patients with elevated pulmonary vascular resistance.
Br J Clin Pharmacol
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) and chronically hypoxic (hypox; 
) lungs
at each collection time. A: baseline period. B:
L-arginine period. * Group mean amounts differed between
control and chronically hypoxic animals at indicated time points,
P < 0.05. All values are means ± SE.
