© 1997 American Heart Association, Inc.
Articles |
Dual Role for Nitric Oxide in the Regulation of Plasma Volume and Albumin Escape During Endotoxin Shock in Conscious Rats
From the Research Center, Maisonneuve-Rosemont Hospital, Department of Medicine, University of Montréal, Québec, Canada.
Correspondence to János G. Filep, MD, Research Center, Maisonneuve-Rosemont Hospital, Department of Medicine, University of Montréal, 5415 boulevard de l'Assomption, Montréal, Québec, Canada H1T 2M4.
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Abstract To assess the role of nitric oxide (NO) produced by the constitutive (cNOS) and inducible NO synthase (iNOS) in the regulation of vascular functions, we compared the effects of aminoguanidine, a relatively selective inhibitor of iNOS, and NG-nitro-L-arginine methyl ester (L-NAME), a nonselective NOS inhibitor on blood pressure, plasma volume, and albumin escape during the early and delayed phases of endotoxin shock in conscious, chronically catheterized rats. Red blood cell volume and plasma volume were determined by using chromium-51–tagged erythrocytes and iodine-125–labeled albumin, respectively. Injection of lipopolysaccharide (LPS) 10 mg/kg IV resulted in a fall in blood pressure, hemoconcentration, and increased total-body albumin escape, which is reflected by a 25% reduction in plasma volume. When LPS was injected into animals pretreated with L-NAME (7.4 µmol/kg IV 15 minutes before LPS), losses in plasma volume and albumin escape were significantly greater than in rats that received LPS alone, despite that L-NAME attenuated the hypotensive action of LPS. Aminoguanidine pretreatment (162 µmol/kg) had no effect on the early responses to LPS, whereas it was as potent as L-NAME in reversing hypotension when injected 70 minutes after LPS. Aminoguanidine treatment also prevented further losses in plasma volume and markedly attenuated total-body and organ albumin escape rates elicited by LPS. L-NAME produced only a slight attenuation of LPS-induced losses in plasma volume and albumin escape in most organs studied, whereas it potentiated albumin extravasation in the lung. These results demonstrate that inhibition of cNOS potentiates, whereas inhibition of iNOS markedly attenuates, losses in plasma volume and albumin escape elicited by LPS, and suggest that selective inhibitors of iNOS may be more effective than nonselective inhibitors of all forms of NOS in the therapy of septic shock.
Key Words: aminoguanidine • NG-nitro-L-arginine methyl ester • vascular permeability • septic shock • nitric oxide
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Introduction |
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An increasing body of evidence suggests that overproduction of nitric oxide (NO) contributes to the hemodynamic changes associated with endotoxin shock, in particular to the severe therapy-refractory hypotension and vascular hyporeactivity to catecholamines.1 The early increases in NO production result from activation of the constitutive endothelial NO synthase (cNOS) within 30 minutes of intravenous administration of Escherichia coli lipopolysaccharide (LPS) to experimental animals.2 3 In later stages of endotoxin shock, cNOS activity decreases,4 5 whereas a distinct inducible isoform of NO synthase (iNOS) is expressed in various cells, resulting in prolonged production of high amounts of NO, which contributes to the delayed cardiovascular deterioration in endotoxin shock.1
While NOS inhibitors can restore vascular hyporeactivity in vitro2 6 and prevent or reverse hypotension in animal models of endotoxin shock7 8 9 as well as in patients with septic shock,10 nonselective blockade of all NOS isoforms may be harmful. Indeed, nonselective NOS inhibitors have been reported to increase the mortality rate in endotoxemic rodents11 and dogs,6 to enhance the degree of microvascular thrombosis and ischemia in the intestine12 and in the kidney,13 and to potentiate liver injury14 after endotoxin administration. On the other hand, selective inhibitors of iNOS were found to attenuate LPS-induced liver dysfunction in rats.15 Although activation of both endothelial cNOS and iNOS occurs in endotoxic shock, it is believed that only prolonged exposure of cells to large amounts of NO, produced by iNOS, may cause cellular damage in a paracrine or autocrine fashion,11 16 17 whereas NO produced via cNOS may protect against cellular dysfunction/damage.11 12
Recent studies have suggested that NO may also play an important role in the regulation of plasma volume and albumin escape.18 19 20 Reduction of circulating blood volume and enhanced microvascular permeability are characteristic features of endotoxin shock21 22 and contribute to the progression of shock to a multiple organ dysfunction syndrome that is associated with a substantial increase in mortality.21 22 The aim of the present study was to compare the effects of aminoguanidine, a selective inhibitor of iNOS activity23 24 25 and NG-nitro-L-argine methyl ester (L-NAME), a nonselective inhibitor of all isoforms of NOS26 27 on plasma volume and albumin escape during the early and delayed phase of endotoxin shock in conscious rats.
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Materials and Methods |
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Materials
Sodium–51-chromate was obtained from DuPont-NEN; 125I-labeled human serum albumin (125I-HSA) was from ICN Pharmaceuticals. L-NAME, aminoguanidine hemisulphate salt, norepinephrine, and E coli LPS (serotype: O111:B4) were purchased from Sigma Chemical Co.
Experimental Protocols
The experiments were performed on conscious, chronically
catheterized male Wistar rats weighing 210 to 300 g. The animals
were housed in individual metabolic cages, and catheters
were implanted into the abdominal aorta and vena cava as described
previously.28 At least 4 days were allowed to complete
recovery from surgery. During the experiments, the animals could move
freely and had free access to food and water. Mean arterial
blood pressure (MABP) was monitored continuously by a blood pressure
analyzer (Micro-Med) with a COBE CDX III pressure
transducer.
On the day of the experiment, after an equilibration period of 30
minutes, baseline cardiovascular parameters
were measured for 20 minutes. Fig 1
depicts the experimental protocols used to study immediate (series 1)
and delayed (series 2) cardiovascular responses to LPS.
The animals received the following intravenous injections:
51Cr-tagged erythrocytes (
0.5 µCi);
125I-HSA (1 µCi in a volume of 100 µL); saline, L-NAME
7.4 µmol/kg (2 mg/kg) or aminoguanidine 162
µmol/kg (20 mg/kg) or LPS (10 mg/kg, over 2
minutes, as indicated. In series 1, the following groups were studied:
group 1 (n=10), saline only (control); group 2 (n=9), L-NAME; group 3
(n=6), aminoguanidine; group 4 (n=7), LPS; group 5 (n=7), L-NAME plus
LPS; group 6 (n=6), aminoguanidine plus LPS; and group 7 (n=6), L-NAME
plus aminoguanidine plus LPS. To avoid interference of LPS with cNOS,
in this series of experiments LPS was injected only after complete
inhibition of cNOS was achieved by L-NAME.26 27 28 In series
2, the animals were divided into four groups: group 1 (n=8), LPS
followed by saline; group 2 (n=8), LPS followed by L-NAME; group 3
(n=8), LPS followed by aminoguanidine; and group 4 (n=6), LPS followed
by L-NAME plus aminoguanidine. Triplicate arterial blood
samples were taken into glass capillary tubes calibrated to 15 µL for
measuring hematocrit and 51Cr and 125I
radioactivities at 5 and 50 minutes after injection of
125I-HSA. Immediately after the last blood sample was
taken, the animals were given an overdose of sodium pentobarbital, and
the thoracic and abdominal viscera were dissected and portions of
selected organs weighed and placed in separate vials for measurement of
51Cr and 125I radioactivities with a Wallac
1470 Wizard Automatic Gamma Counter. The system was programmed to
correct for cross-talk and spillover between detectors and counting
channels. "Large vessel" hematocrit (LVHct) was determined by a
manual hematocrit reader. Erythrocytes were labeled with sodium
51-chromate in saline as previously described29 and were
resuspended in 0.9% NaCl solution to a hematocrit of 45% to 50%. In
the third series of experiments, pressor responses to
norepinephrine 3.1 nmol/kg (1 µg/kg) were
compared 30 minutes before and 30, 60, 120, and 150 minutes after
injection of LPS (10 mg/kg IV, n=6) in rats treated with
aminoguanidine (162 µmol/kg, n=5), L-NAME (7.4
µmol/kg, n=5), or saline (n=5). All procedures were in
accordance with the Guidelines of the Canadian Council of Animal Care
and were approved by the local Animal Care Committee.
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Red Blood Cell, Plasma, and Blood Volumes
In series 1 and 2, for the blood sample taken 5 minutes after
injection of 125I-HSA, red blood cell volume (RCV), plasma
volume (PV), and blood volume (BV) were determined according to the
following formulas: RCV=total 51Cr activity
injectedxLVHct÷blood 51Cr activity concentration;
PV=total 125I activity injectedx(1-LVHct)÷blood
125I activity concentration; and BV=RCV+PV. The ratio of
whole-body hematocrit to LVHct (Fcells ratio) was
calculated as (RCV÷BV)÷LVHct. For the blood sample taken 50 minutes
after injection of 125I-HSA, the following formulas were
used: BV=(51Cr activity injected-sampling loss of
51Cr activity)÷blood 51Cr activity
concentration)÷Fcells;
RCV=RCVfirst-RCVlost, where
RCVfirst and RCVlost are RCV measured during
the first sample and RCV lost through sampling, respectively;
RCVlost=51Cr activity lost through
sampling÷(51Cr activity injected÷RCVfirst);
and PV=BV-RCV.
125I-Albumin Escape Rate
The rate at which 125I-HSA escaped from the
circulation (125I-AERt) was calculated as
125I-AERt=[(net 125I activity
injected-total plasma 125I activity in the second blood
sample)÷net 125I activity injected]÷50 minutesx100,
where net 125I activity injected is the total
125I activity injected less the cumulative radioactivity
removed from the circulation by blood sampling, and total plasma
125I activity=plasma 125I activity
concentrationxplasma volume at 50 minutes after injection of
125I-labeled albumin.
The rate at which 125I-HSA escaped from the circulation of each organ (125I-AERorgan) was determined by using the formula 125I-AERorgan=(tissue 125I-albumin activity÷net 125I activity injected)÷50 minutes÷corrected organ weightx100. Tissue 125I-albumin activity was calculated as the difference in total organ 125I and organ plasma 125I-albumin activity. Organ plasma 125I activity is the product of organ plasma volume and plasma 125I-albumin activity concentration. Organ plasma volume was determined as organ blood volumex(1-LVHct) for heart, lung, liver, and kidney, where organ hematocrit is similar to that of LVHct or as organ blood volumex[1-(FcellsxLVHct)] for gastrointestinal tract, where the ratio of organ hematocrit to LVHct is similar to the ratio of whole body hematocrit to LVHct.29 Organ blood volume was calculated as (organ 51Cr activity÷blood 51Cr activity concentration) for heart, lung, liver, and kidney; and as (organ 51Cr activity÷blood 51Cr activity concentration)÷Fcells for gastrointestinal tract. Organ weight was corrected by subtracting estimated organ blood weight (organ blood volumexblood specific gravity) from wet organ weight.
Statistical Analysis
Results are expressed as mean±SEM. Results were compared by
one-way ANOVA using ranks (Kruskal-Wallis test) followed by Dunn's
multiple contrast hypothesis test when various treatments were compared
with the same control group or by the Wilcoxon signed rank test
and Mann-Whitney U test for paired and unpaired
observations, respectively. A level of P<.05 was considered
significant for all tests.
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Results |
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Effect of L-NAME and Aminoguanidine on Blood Pressure, Blood Volume, Plasma Volume, and Albumin Escape Rate in Normal Rats
Intravenous injection of saline (control group) neither affected MABP (Fig 2A
) nor evoked
any detectable changes in hematocrit (0.471±0.011 versus 0.469±0.010,
n=10, P>.1), total-body blood volume (85.4±4.0 versus
85.3±4.2 mL/kg, P>.1), and plasma volume (54.6±3.6 versus
54.8±3.5 mL/kg, P>.1).
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As expected, intravenous bolus injection of L-NAME
(7.4 µmol/kg) evoked a prolonged increase in MABP (Fig 2A
). This was accompanied by an increase in hematocrit from
0.471±0.011 to 0.508±0.015 (n=9, P<.01). Plasma volume
decreased from 55.0±3.8 to 49.0±2.9 mL/kg (P<.01),
whereas no changes were detected in red blood cell volume (28.9±0.6
versus 28.8±0.5 mL/kg). Total-body blood volume decreased from
85.5±4.2 to 77.7±3.0 mL/kg (P<.01). Fig 3 compares the values derived from the
second blood samples in the different groups. Hematocrit was
significantly higher, whereas blood and plasma volume were
significantly lower in the L-NAME than in the control group. By
contrast, no significant changes could be detected in these
parameters after administration of aminoguanidine (162
µmol/kg) (Fig 2A and Fig 3).
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Fcells ratios were 0.76±0.02, 0.74±0.01, and 0.75±0.01
in animals that received saline (control), L-NAME, and aminoguanidine,
respectively (P>.1). The total-body albumin escape
rate increased on average by 114% after administration of L-NAME (Fig 3). Albumin escape rates increased in the bronchus, heart,
liver, kidney, and duodenum (Fig 4). On
the other hand, aminoguanidine by itself had no significant effects on
tissue albumin escape rates in the organs studied (Fig 4).
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Effects of L-NAME and Aminoguanidine on Immediate
Cardiovascular Responses to LPS
Intravenous administration of LPS caused a dramatic
fall in MABP within 10 minutes. Thereafter, MABP gradually increased
and was 70 mm Hg 30 minutes after injection of LPS (Fig 2A).
L-NAME pretreatment significantly attenuated the maximum decrease in
MABP elicited by LPS, and these animals maintained significantly higher
MABP values when compared with rats treated with LPS alone (Fig 2A). By
contrast, LPS-induced changes in MABP were similar in
aminoguanidine-treated and untreated animals (Fig 2A). Pretreatment
of the animals with L-NAME plus aminoguanidine resulted in similar
attenuation of LPS- induced changes in MABP as that observed in rats
pretreated with L-NAME. LPS produced maximal decreases of 43±4 and
42±5 mm Hg in MABP in L-NAME plus aminoguanidine-treated (n=6)
and L-NAME–treated rats (n=7), respectively, and MABP was 108±3
and 109±5 mm Hg at 30 minutes after injection of LPS,
respectively (both P>.1). Endotoxemia resulted in a
substantial, time-dependent attenuation of the pressor responses
elicited by norepinephrine (Fig 2C). The pressor responses
to norepinephrine at 30 minutes in LPS-rats treated with
L-NAME were significantly greater than in animals injected with LPS
alone, whereas at 60 minutes, significantly greater responses were
observed both in L-NAME–treated and aminoguanidine-treated animals
(Fig 2C).
LPS-induced hypotension was accompanied by a marked
hemoconcentration. Hematocrit increased from 0.475±0.010 to
0.576±0.020 (n=7, P<.01). Plasma volume decreased by
27±3% (P<.01), whereas no changes were detected in red
blood cell volume (32.9±1.5 versus 32.6±1.5 mL/kg, P>.1).
Accordingly, total-body blood and plasma volume were significantly
lower in LPS-treated than in control animals (Fig 3). LPS increased the
total-body albumin escape rate on average by 238% (Fig 3).
Significant increases in tissue albumin escape rate were
detected in the bronchus, liver, kidney, and duodenum, but not in the
pulmonary parenchyma and heart (Fig 4). LPS-induced plasma
volume losses and total-body albumin escape rate were
significantly greater in animals pretreated with L-NAME than in rats
who had received LPS alone (Fig 3). L-NAME potentiated the
albumin escape rate elicited by LPS in the bronchus, heart,
liver, kidney, and duodenum (Fig 4). In the pulmonary
parenchyma, while neither L-NAME nor LPS alone enhanced albumin
escape, their combination resulted in significant increases in
albumin extravasation (Fig 4).
Unlike L-NAME, aminoguanidine pretreatment did not potentiate
LPS-induced hemoconcentration, losses in plasma volume, and total-body
and organ albumin escape rates (Figs 3 and 4). In animals
pretreated with L-NAME plus aminoguanidine, LPS evoked significantly
greater reductions in plasma volume and increases in hematocrit,
total-body, and organ albumin escape rates than in rats who had
received LPS alone (Figs 3 and 4). These changes did not differ
significantly from those observed in L-NAME–treated rats in response
to LPS (Figs 3 and 4). Fcells ratios were 0.76±0.01,
0.77±0.02, 0.78±0.01, and 0.79±0.01 in animals that received LPS
alone, L-NAME plus LPS, aminoguanidine plus LPS, and L-NAME plus
aminoguanidine plus LPS, respectively (P>.1).
Effects of L-NAME and Aminoguanidine on Delayed
Cardiovascular Responses to LPS
After the initial fall, MABP gradually increased and stabilized at
85 mm Hg from 80 to 120 minutes after LPS and fell toward the
end of the experimental period (Fig 2B). Administration of either
L-NAME or aminoguanidine at 70 minutes after the onset of
endotoxemia resulted in a rapid and sustained rise in MABP (Fig 2B).
Thus MABP of LPS rats treated with L-NAME or aminoguanidine was
significantly higher than in the LPS control group at 80 to 150 minutes
(Fig 2B). The time course of changes in MABP of LPS rats treated with
L-NAME plus aminoguanidine did not differ from that observed after
L-NAME treatment. For instance, MABP was similar at 100 minutes (104±5
versus 102±4 mm Hg, P>.1) and 150 minutes (108±6
versus 106±4 mm Hg, P>.1) after injection of LPS.
The pressor responses to norepinephrine at 120 and 150
minutes in LPS rats treated with either L-NAME or aminoguanidine were
not significantly different from the pressor responses evoked by
norepinephrine before LPS administration, whereas they were
markedly reduced in LPS-treated animals (Fig 2C).
Although hematocrit values were higher (LPS, 0.578±0.020, n=8;
control, 0.475±0.010, n=10, P<.01), whereas blood and
plasma volume were markedly lower at 105 minutes after LPS injection
than in control animals (blood volume, 58.9 mL/kg, n=8 versus 85.5
mL/kg, n=10, P<.01; plasma volume, 34.0±2.9 mL/kg, n=8
versus 55.0±3.8 mL/kg, n=10, P<.01), further increases in
hematocrit and decreases in blood and plasma volume were detected in
the next 45 minutes (Fig 5). During this
time period, hematocrit increased by 0.044±0.010 (n=8,
P<.05), whereas plasma and blood volume decreased by
5.7±0.7 and 5.9±0.7 mL/kg, respectively (n=8, P<.01).
Total-body albumin escape rate was 31.9±3.1% net
125I-HSA injected between 100 and 150 minutes after LPS
administration (Fig 5).
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When injected 70 minutes after LPS, aminoguanidine prevented further
losses in blood and plasma volume (
blood volume, -5.9±0.7 mL/kg,
n=8 versus -1.3±0.6 mL/kg, n=8, P<.01; plasma volume,
-5.7±0.7 mL/kg versus -1.1±0.6 mL/kg in LPS rats treated with
saline and aminoguanidine, respectively, P<.01) and
markedly attenuated LPS-induced albumin escape (Fig 5).
Accordingly, albumin escape rates were significantly lower in
all organs studied with the exception of the kidney (Fig 6). On the other hand, L-NAME injected 70
minutes after LPS produced only a small reversal of the effects of LPS
on blood and plasma volume (blood volume, -3.0±0.8 mL/kg,
plasma volume, -2.8±0.8 mL/kg, both P<.05 compared
with LPS) and total-body albumin escape rate (Fig 6).
Aminoguanidine was more potent than L-NAME in reducing LPS-induced
increases in total-body albumin escape (Fig 5). The effects of
L-NAME on LPS-induced albumin escape varied in various vascular
beds. L-NAME treatment attenuated albumin escape in the
duodenum, whereas it did not affect significantly albumin
extravasation in the pulmonary parenchyma, heart, liver, and
kidney, or even potentiated albumin extravasation in the
bronchus (Fig 6). The effects of L-NAME plus aminoguanidine treatment
were similar to those observed with L-NAME. Plasma volume decreased by
3.0 mL/kg and 4.0±0.6 mL/kg, whereas hematocrit increased by
0.020±0.003 and 0.024±0.008 in LPS rats treated with L-NAME (n=8) and
L-NAME plus aminoguanidine (n=6), respectively (both P>.1)
(Fig 5). No significant differences could be detected between the
effects of L-NAME or L-NAME plus aminoguanidine treatment on total-body
and organ albumin escape rates (Figs 5 and 6).
Fcells ratios were 0.75±0.02, 0.75±0.01, 0.75±0.03, and
0.78±0.02 at 105 minutes after administration of LPS in animals that
were treated with saline, L-NAME, aminoguanidine, and L-NAME plus
aminoguanidine, respectively (P>.1).
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Discussion |
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The present results, obtained in conscious chronically catheterized rats, indicate that inhibition of cNOS or iNOS exerts opposing actions on LPS-induced plasma volume losses and albumin escape. Inhibition of cNOS resulted in an augmentation of losses in plasma and blood volume and albumin escape during the early phase of endotoxin shock, despite preventing LPS-induced fall in MABP. The iNOS selective NOS inhibitor aminoguanidine markedly attenuated decreases in blood and plasma volume and increases in total-body albumin escape during the delayed phase of endotoxin shock. These changes were only slightly inhibited by L-NAME, despite L-NAME appearing to be as potent as amino-guanidine in reversing LPS-induced hypotension.
Aminoguanidine has been described as a potent inhibitor of iNOS,23 24 25 but its selectivity for iNOS may be limited in certain experimental conditions.30 However, aminoguanidine did not increase MABP in control rats and did not reverse the vascular hyporeactivity to norepinephrine at 30 minutes after administration of LPS, which is due to an enhanced formation of NO by cNOS.3 Furthermore, aminoguanidine did not prevent or blunt the L-NAME–mediated exacerbation of LPS-induced plasma volume changes and albumin escape. These observations indicate that aminoguanidine can be considered as a selective iNOS inhibitor at the dose used in the present study. Since the earliest time when increases in tissue expression of iNOS can be detected is at 60 minutes after LPS injection in rats,3 the actions of L-NAME can be solely attributed to inhibition of cNOS during the first 60 minutes of LPS administration.
Confirming previous observations, inhibition of cNOS by L-NAME resulted in a profound elevation in MABP,1 hemoconcentration, and decreases in blood volume, resulting almost exclusively from decreases in plasma volume.31 Consistent with previous observations,18 19 28 31 32 L-NAME also enhanced total-body albumin escape and tissue albumin escape rates in all vascular beds studied with the exception of the pulmonary parenchyma. The findings that losses in plasma and blood volume and total-body albumin escape were higher in response to LPS in L-NAME–treated versus saline-treated animals is not entirely surprising, for potentiation of platelet-activating factor or endothelin-1–induced albumin extravasation by L-NAME has already been reported.28 33 Plasma levels of these mediators are markedly elevated after LPS administration.21 Of interest, L-NAME plus LPS treatment augmented albumin escape in the pulmonary parenchyma, whereas neither L-NAME nor LPS by itself induced albumin extravasation.
Increases in whole-body albumin escape from circulation probably reflects fluid transfer, because in most tissues convection appears to be the dominant mechanism for transmicrocirculatory transport of molecules with dimension similar to albumin.34 Inhibition of NO formed via cNOS may increase albumin escape via transmission of increased systemic arterial pressure to the capillaries, thereby increasing capillary hydrostatic pressure or via increasing vascular permeability. Previous results have indicated that albumin extravasation elicited by L-NAME can primarily be attributed to increase in vascular permeability18 31 secondary to formation of interendothelial cell gaps.35 The mechanisms by which blockade of cNOS enhances albumin escape are not fully understood at present. Accumulation of free radicals,36 37 adhesion of leukocytes to the endothelium,18 20 and unmasking the vascular permeability enhancing action of other mediators, such as platelet-activating factor38 and endothelin-1,31 have been implicated to mediate enhanced vascular permeability.
Beginning at about 60 minutes after administration of LPS, iNOS is expressed and NO production is dramatically enhanced.3 Prolonged exposure of endothelial cells to large amounts of NO results in cellular injury.16 17 39 Recent studies have suggested that some of the cytotoxic actions of NO may not be attributed to the effect of NO per se but rather to the effect of peroxynitrite40 formed in a reaction of NO with superoxide.41 Damage to endothelial and smooth muscle cells may not only contribute to the prolonged hypotension, reduced responsiveness to vasoconstrictors, and to redistribution of blood to capacitance vessels42 but could also lead to further reductions in blood volume. Indeed, this study documents further decreases in plasma volume and increases in albumin escape between 100 and 150 minutes after injection of LPS. These were effectively inhibited by aminoguanidine, whereas only a slight attenuation was detected with L-NAME. The differences in the inhibitory actions of aminoguanidine and L-NAME can be attributed to the nonselective inhibition of both cNOS and iNOS by L-NAME. LPS decreases activity of cNOS in human umbilical vein endothelial cells by shortening the half-life of cNOS mRNA.4 5 However, as cNOS mRNA began to decrease only after 4 hours of culture, it seems unlikely that NO production via cNOS was markedly diminished in our animals within 3 hours after LPS administration. Therefore, it is conceivable that L-NAME inhibited both cNOS and iNOS in our experiments. Although L-NAME slightly reduced total-body albumin escape, it exacerbated LPS-induced albumin extravasation in the bronchus. This phenomenon may be explained by the differences in the distribution of cNOS and iNOS expression in the rat lung. Under physiological conditions, both large airways and pulmonary parenchyma contain high amounts of cNOS activity.3 43 After LPS stimulation, iNOS activity was found to be approximately 50-fold less in the rat trachea than in the parenchyma.43 Previous studies have reported that NO produced by cNOS protects against,28 31 32 44 whereas increased NO production via iNOS enhances albumin extravasation in the large airways.43 Thus, even though inhibition of iNOS is expected to decrease albumin escape, inhibition of NO produced by cNOS would reveal the permeability-enhancing action of other mediators (eg, prostaglandins, platelet-activating factor) released by LPS in the bronchus. Enhanced albumin escape in the bronchus leads to edema formation in the airway wall and consequently to narrowing of the airways. This could contribute to the development of acute respiratory failure and therefore to the increased mortality rate.21 22 These results would also imply that L-NAME should not be used in patients with septic shock because of a potential risk of exacerbating edema formation in the large airways. The observations that neither aminoguanidine nor L-NAME affected significantly albumin escape in the kidney during the delayed phase of endotoxemia are consistent with previous findings that the degree of iNOS induction in the kidney of septic animals is relatively small compared with the degree of induction in other organs.3 In addition, there is a marked renal vasoconstriction due to sympathetic stimulation and possibly inhibition of endothelial cNOS.45
The attenuation of albumin escape by aminoguanidine was independent from increases in MABP in LPS rats, since transmission of increased systemic pressure to capillaries is expected to promote rather than to inhibit albumin extravasation. Furthermore, L-NAME and aminoguanidine elicited similar increases in MABP, which were accompanied with different degree of attenuation of changes in plasma volume and albumin escape or even with changes in the opposite direction in organ albumin escape (eg, effects of L-NAME in the large airways). These findings raise the question of whether aiming to reverse hypotension by NOS inhibitors would be sufficient to obtain a beneficial effect in endotoxin shock.
In addition to inhibition of iNOS activity, aminoguanidine can also inhibit polyamine catabolism46 and catalase activity.47 However, inhibition of catalase activity with concomitant increases in H2O2 level would promote rather than decrease albumin extravasation.37 Since aminoguanidine did not produce detectable inhibition of the effects of L-NAME on LPS-induced plasma volume changes and albumin escape, it is conceivable that aminoguanidine exerted its beneficial actions via inhibition of iNOS.
In summary, the present study demonstrates the dual nature of NO in the regulation of blood volume and albumin extravasation in endotoxic shock in conscious rats. NO produced via cNOS protects against endothelial dysfunction, as evidenced by the observations that inhibition of cNOS potentiates LPS-induced albumin escape and reduction of circulating plasma volume during the early phase of endotoxin shock. On the other hand, excessive production of NO via iNOS contributes to losses in plasma volume and enhanced albumin escape in the delayed phase of endotoxin shock. Aminoguanidine selectively inhibited iNOS, and consequently it might have prevented the cytotoxic effect of NO. These results also suggest that selective inhibitors of iNOS activity may be more effective than nonselective inhibitors of NOS in the therapy of septic shock.
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Acknowledgments |
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This work was supported by a grant from the Medical Research Council of Canada (MT-12573).
Received February 11, 1997; accepted August 26, 1997.
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References |
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