1 Department of Surgery, Endotoxin (Etx) causes excessive
activation of the nuclear repair enzyme poly(ADP-ribose)
synthase (PARS), which depletes cellular energy stores and
leads to vascular dysfunction. We hypothesized that PARS inhibition
would attenuate injury to mechanisms of pulmonary vasorelaxation in
acute lung injury. The purpose of this study was to determine the
effect of in vivo PARS inhibition on Etx-induced dysfunction of
pulmonary vasorelaxation. Rats received intraperitoneal saline or Etx
(Salmonella typhimurium; 20 mg/kg) and
one of the PARS inhibitors, 3-aminobenzamide (3-AB; 10 mg/kg) or
nicotinamide (Nic; 200 mg/kg), 90 min later. After 6 h,
concentration-response curves were determined in isolated pulmonary
arterial rings. Etx impaired endothelium-dependent (response to ACh and
calcium ionophore) and -independent (sodium nitroprusside)
cGMP-mediated vasorelaxation. 3-AB and Nic attenuated Etx-induced
impairment of endothelium-dependent and -independent pulmonary
vasorelaxation. 3-AB and Nic had no effect on Etx-induced increases in
lung myeloperoxidase activity and edema. Lung ATP decreased after Etx
but was maintained by 3-AB and Nic. Pulmonary arterial PARS activity
increased fivefold after Etx, which 3-AB and Nic prevented. The
beneficial effects were not observed with benzoic acid, a structural
analog of 3-AB that does not inhibit PARS. Our results suggest that
PARS inhibition with 3-AB or Nic improves pulmonary vasorelaxation and
preserves lung ATP levels in acute lung injury.
poly(ADP-ribose) synthase; pulmonary artery; 3-aminobenzamide; nicotinamide; myeloperoxidase
ENDOTOXEMIA RESULTS IN the increased production of both
nitric oxide (13, 34) and superoxide anion (35). When both of these
species are present in large amounts, the formation of peroxynitrite (ONOO Endotoxin-induced acute lung injury is characterized by lung edema,
neutrophil sequestration, and increased pulmonary vascular resistance
(7, 9). We and others (9, 16, 18, 25) have found that endotoxin causes
dysfunction of pulmonary vasorelaxation in response to agonists that
require the generation of cGMP. This endotoxin-induced dysfunction of
pulmonary vasorelaxation is mediated, in part, by polymorphonuclear
leukocytes; neutrophil depletion attenuates this acute lung injury in a
rat model (24).
Activation of PARS in response to peroxynitrite-mediated DNA
single-strand breaks may be responsible for the cellular energy depletion and vascular dysfunction associated with endotoxemia. In vivo
administration of a PARS inhibitor attenuated the impairment of
contractility in thoracic aortic rings (32) and improved mean arterial
pressure (41) in endotoxin-treated rats. The dysfunction in
endothelium-dependent relaxant responses in rat thoracic aortic rings
caused by endotoxin was also ameliorated by the PARS inhibitor 3-aminobenzamide (3-AB) (27). PARS inhibition with 3-AB also reduced
neutrophil recruitment and tissue edema in zymosan- and carrageenan-triggered models of local inflammation (29).
We hypothesized that in vivo inhibition of PARS would attenuate
endotoxin-induced impairment of pulmonary vasorelaxation. The purpose
of this study was to determine the effect of the PARS inhibitors 3-AB
and nicotinamide (Nic) on endotoxin-induced, cGMP-mediated pulmonary
vasomotor dysfunction. Endothelium-dependent relaxation was studied
with the receptor-dependent agonist ACh and the
receptor-independent agonist calcium ionophore A-23187.
Endothelium-independent relaxation was examined with direct stimulation
of vascular smooth muscle with the use of the nitric oxide donor sodium
nitroprusside (SNP). We also examined the effect of PARS inhibition
with 3-AB and Nic in endotoxemia on lung ATP levels and pulmonary
arterial PARS activity. A secondary purpose of this study was to
determine the effect of 3-AB and Nic on endotoxin-induced lung edema
and myeloperoxidase activity as a measure of neutrophil accumulation.
The results of this study demonstrate that PARS inhibition with 3-AB
and Nic in endotoxemia 1) attenuates
dysfunction of endothelium-dependent and -independent mechanisms of
pulmonary vasorelaxation, 2) has no
effect on lung myeloperoxidase activity and edema, and
3) maintains lung ATP levels.
Animal care and housing.
All animals received humane care in compliance with the National
Research Council's Guide for the Care and Use of
Laboratory Animals. Male Sprague-Dawley rats weighing
250-300 g were quarantined in quiet, humidified, light-cycled
rooms for 2-3 wk before use. Rats were allowed ad libitum access
to food and water throughout quarantine.
Experimental protocol.
Rats were administered normal saline (NS; 1 ml ip), endotoxin
[Etx; 20 mg/kg ip Salmonella
typhimurium lipopolysaccharide (LPS) in 1 ml of
NS], a PARS inhibitor alone (3-AB, 10 mg/kg ip, or
Nic, 200 mg/kg ip, in 1 ml of NS), or Etx followed by 3-AB or Nic. Rats
received 3-AB or Nic 90 min after Etx to avoid any potential
interference with inducible nitric oxide synthase (iNOS) induction. The
doses of 3-AB and Nic were chosen on the basis of multiple previous in
vivo studies employing them in rats (8, 32, 33, 41). A similar set of
experiments were performed with the use of a structural analog of 3-AB
that does not inhibit PARS [benzoic acid (BA); 10 mg/kg
ip]. Rats were provided chow and water ad libitum during the 6-h
period after initial injection. No rats died during the 6-h
experimental time course. A previous experiment using the same dose of
Etx resulted in 15% mortality at 72 h (unpublished data).
Isolated pulmonary arterial ring
preparation.
Isolated pulmonary arterial rings were harvested and prepared as
previously described (9, 24). Five rats (10 rings) were studied in each
group. Rats were anesthetized with pentobarbital sodium (50 mg/kg ip).
Median sternotomy was performed, and heparin sulfate (500 USP units)
was injected into the right ventricular outflow tract. After removal of
the heart and lungs en bloc, the main pulmonary and the right and left
pulmonary arteries were excised. The right and left main branch
pulmonary arteries were then cut into 3-mm-wide rings; two pulmonary
arterial rings were obtained from each rat. Care was taken during this
process to avoid endothelial injury.
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
), a potent oxidant
and nitrating reagent, is favored (4, 12, 34). Nitric oxide is the only
known endogenous molecule produced in high enough concentrations in
pathological conditions that can effectively compete with superoxide
dismutase for superoxide (5). Peroxynitrite is a strong activator of
DNA single-strand breaks, resulting in excessive stimulation of
poly(ADP-ribose) synthase (PARS) (31). This process depletes
NAD+ (23), leading to inhibition
of glycolysis and decreased ATP formation (21). This mechanism of
cellular injury has been proposed as a major pathway involved in the
vascular dysfunction observed in endotoxic shock (32, 39, 40).
Peroxynitrite formation in acute lung injury, as evidenced by
3-nitrotyrosine formation, has been found in endotoxin-treated rats
(34) and in human autopsy specimens with sepsis-induced diffuse
alveolar damage (15). In nonsepsis models of pulmonary inflammation,
PARS inhibition prevented edema from excitatory amino acid
(N-methyl-D-aspartate) toxicity in the isolated perfused rat lung (22) and improved cellular
energetics in macrophages harvested from rats subjected to
carrageenan-induced pleurisy (8) as well as in a human pulmonary epithelial cell line exposed to peroxynitrite (30).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Pulmonary vasorelaxation: concentration-response
curves to ACh, A-23187, and SNP.
The optimal resting mechanical tension (passive load) for pulmonary
rings was determined to be 750 mg in a prior study (9). Rings were
suspended at 750 mg and allowed to reach a steady state for 1 h, during
which time the Earle's balanced salt solution was changed every 15 min. Rings were preconstricted with phenylephrine (PE) to achieve a
PE-induced ring tension between 275 and 325 mg
(~107 M PE). Cumulative
concentration-response curves were then generated over the
concentration range of 10
9
to 10
6 M (ACh, A-23187, and
SNP). For determination of the concentration-response curve, the ring
was allowed to reach a steady state before advancing to the next higher
concentration. The ring tension remaining in the rings in response to
each dose of vasorelaxing agent was expressed in milligrams of tension.
PE concentration-response curves were also generated to ensure that
endotoxemia did not alter
-adrenergic-mediated vasoconstriction.
Lung harvest for myeloperoxidase assay, wet-to-dry weight determination, and ATP assay. Rats were anesthetized with pentobarbital sodium (50 mg/kg ip). Median sternotomy was performed, and heparin sulfate (500 USP units) was injected into the right ventricular outflow tract. Lungs were then surgically removed, externally rinsed with saline, and blotted dry. Five lungs from five rats were studied in each group.
Lung myeloperoxidase assay.
Segments of lung weighing 400-500 g were snap frozen in liquid
nitrogen for subsequent determination of myeloperoxidase (MPO) activity. Lung tissue was homogenized for 30 s in 4 ml of 20 mM potassium phosphate buffer, pH 7.4. Lung protein was quantified with
the use of the Coomassie plus protein assay (Pierce, Rockford, IL). The
samples were then centrifuged for 30 min at 40,000 g at 4°C (Beckman L-80
Ultracentrifuge; Beckman Instruments, Palo Alto, CA). The pellet was
resuspended in 4 ml of 50 mM potassium phosphate buffer, pH 6.0, containing 0.5 g/dl cetrimonium bromide. The samples were sonicated for
90 s at full power (ultrasonic homogenizer; Cole-Parmer Instrument,
Chicago, IL), incubated in a 60°C water bath for 2 h, and
centrifuged for 10 min at maximum speed (Eppendorf 5415C; Baxter, San
Diego, CA). The supernatant (25 µl) was added to 725 µl of 50 mM phosphate buffer, pH 6.0, containing 0.167 mg/ml o-dianisidine and 5 × 104% hydrogen peroxide.
Absorbance of 460-nm visible light was measured between 1 and 3 min
(Beckman DU7 spectrophotometer; Beckman Instruments, Irvine, CA). MPO
activity (units/mg lung protein) was then calculated as
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Lung wet-to-dry weight. Harvested left or right lungs were weighed for determination of wet weight. In separate specimen containers, they were dried over a desiccant (Drierite, Xenia, OH) at 4°C for 5 days to a constant weight and then reweighed.
Lung ATP assay. Lung ATP levels were determined with the use of a quantitative, enzymatic assay (Sigma Diagnostics, St. Louis, MO). Lungs were harvested as described in Lung harvest for myeloperoxidase assay, wet-to-dry weight determination, and ATP assay, and were immediately frozen with the use of liquid nitrogen. Segments of frozen lung weighing 100-200 mg were ground to a fine powder with the use of mortar and pestle and then sonicated (ultrasonic homogenizer) at full power on ice for 30 s in 1 ml of 12% TCA. (Separate segments of the same lungs were used for quantification of lung protein content as described for lung MPO assay.) The samples were allowed to stand 5 min on ice and were then centrifuged at 750 g for 5 min.
The ATP assay couples a specific phosphorylation reaction requiring ATP (formation of 1,3-diphosphoglycerate) with a dephosphorylation reaction involving the oxidation of NADH. The amount of ATP originally present is equivalent on a molar basis to the amount of NAD formed, which is quantified by measuring the change in absorbance at 340 nm (Beckman DU7 spectrophotometer). The absorbance change is compared with a curve generated from standards of ATP (0, 250, 500, and 1,000 µM) to determine the amount of ATP present. Results are presented as nanomoles of ATP per milligram of lung protein.Pulmonary arterial PARS activity. PARS activity was measured with the use of a commercially available assay (Genzyme Diagnostics, Cambridge, MA). Four or five pairs of pulmonary arteries were studied in all groups. After dissection of the pulmonary arterial rings from the various experimental groups as for the response to Ach, A-23187, and SNP, the samples were placed on ice in 2 ml of buffer containing 50 mM Tris · Cl (pH 8.0), 25 mM MgCl2, and 0.1 mM phenylmethylsulfonyl fluoride. The samples were homogenized for 30 s and then sonicated for 20 s at full power (ultrasonic homogenizer). The suspension was centrifuged at 3,000 g for 5 min at 4°C. The protein concentration of the supernatant was determined as described for MPO assay.
Supernatant (the volume containing 20 µg of protein), PARS buffer (10 µl), 1 mM NAD (10 µl), 2 µCi 32P-labeled NAD (at 1 µCi/µl), and distilled water (the volume required to give a final reaction volume of 100 µl) were mixed in a microcentrifuge tube. The reaction was allowed to proceed at room temperature for exactly 1 min. The reaction was stopped by adding 900 µl of 20% TCA (4°C), and the samples were placed on ice. Enzyme activity was determined by measuring the incorporation of radiolabeled NAD as PARS catalyzed the poly(ADP) ribosylation of proteins. The labeled ADP present was then measured by scintillation counting after TCA precipitation onto a filter. Calculation of PARS activity proceeded as follows
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Reagents. Standard reagents as well as the S. typhimurium Etx were obtained from Sigma Chemical (St. Louis, MO), with the exception of A-23187 (Calbiochem, La Jolla, CA). Fresh solutions were prepared daily with either deionized water or NS as the diluent. Concentrations are expressed as final molar concentrations in the organ chambers.
Statistical analysis. Statistical analyses were performed on a Macintosh Quadra computer with StatView software (Brain Power, Calabasas, CA). Data are presented as means ± SE of the number of rings or lungs studied at each point of data collection. In ring experiments, comparisons between groups were made at the same concentrations. Statistical evaluation utilized standard one-way ANOVA with post hoc Bonferroni-Dunn correction. P < 0.05 was accepted as statistically significant.
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RESULTS |
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Effects of PARS inhibition with 3-AB and Nic on
Etx-induced impairment of cGMP-mediated pulmonary
vasorelaxation.
The vasoconstriction response to the -adrenergic agonist PE is
unchanged after Etx (Fig. 1). Etx
administration significantly impaired endothelium-dependent,
receptor-dependent pulmonary arterial vasorelaxation (response to ACh),
and PARS inhibition with 3-AB or Nic attenuated this injury (Fig.
2A).
Rings from saline-treated rats were preconstricted with PE to 285 ± 16 mg tension and relaxed to 16 ± 4 mg tension at
10
6 M ACh, and rings from
Etx-treated rats were preconstricted to 283 ± 16 mg tension and
relaxed to 168 ± 12 mg tension. In Etx+3-AB and Etx+Nic rats, 106 ± 13 and 66 ± 7 mg PE-induced ring tension remained at
10
6 M ACh, respectively
(P < 0.05 vs. Etx alone,
P < 0.05 vs. control).
|
|
|
Effects of PARS inhibition with 3-AB and Nic on
lung MPO activity in endotoxemia.
Etx increased lung MPO activity more than threefold in comparison with
control rats, and PARS inhibition with 3-AB or Nic did not attenuate
this effect (Fig. 4). Lung MPO in controls
was 0.21 ± 0.09 units/mg protein. Endotoxemia for 6 h resulted in 0.75 ± 0.07 units MPO/mg protein
(P < 0.05 vs. control). Lung MPO
activity after Etx and either 3-AB (0.84 ± 0.05 units MPO/mg protein) or Nic (0.65 ± 0.10 units MPO/mg protein) was not
different from that in Etx-treated rats
(P > 0.05 vs. Etx).
|
Effects of PARS inhibition with 3-AB and Nic on
lung edema after Etx.
Lung wet-to-dry weight ratios (W/D) were significantly greater after
Etx treatment, and the PARS inhibitors did not demonstrate any
beneficial effects (Table 1). Although
endotoxemia resulted in an increase in W/D to 4.76 ± 0.03 from the
control value of 4.43 ± 0.03 (P < 0.05), neither 3-AB (W/D 4.79 ± 0.03) nor Nic (W/D 4.80 ± 0.05) attenuated this injury (P > 0.05 vs. Etx).
|
Effect of PARS inhibition with 3-AB and Nic on lung
ATP levels in endotoxemia.
The Etx-induced decrease in lung ATP levels at 6 h was prevented by
PARS inhibition with 3-AB and Nic (Fig. 5).
Etx reduced lung ATP from the control value of 13.39 ± 0.29 to 8.64 ± 0.31 nmol/mg protein (P < 0.05). Etx+3-AB-treated rats had lung ATP levels similar to those of
control rats (12.47 ± 0.50 nmol/mg protein,
P = 0.20) that were also different
from those of rats treated with Etx alone
(P < 0.05). The lung ATP levels of
Etx+Nic-treated rats were also similar to those of saline-treated
rats (13.65 ± 0.69 nmol/mg protein,
P = 0.74 vs. control) and different
from those of rats treated with Etx alone
(P < 0.05). 3-AB or Nic alone had no
effect on lung ATP levels in saline-treated rats (data not shown). The
lung ATP levels of Etx+BA-treated rats (8.02 ± 0.33 nmol/mg
protein) were not different from those of Etx-treated rats
(P = 0.37).
|
Effect of PARS inhibition with 3-AB and Nic after
Etx on pulmonary arterial PARS activity.
Pulmonary PARS activity increased more than fivefold after Etx
treatment, and the PARS inhibitors 3-AB and Nic prevented this increase
(Fig. 6). Etx increased pulmonary arterial
PARS activity from the control value of 0.188 ± 0.010 pmol · min1 · µl
1
to 1.050 ± 0.047 pmol · min
1 · µl
1
(P < 0.05). The pulmonary
arteries from Etx+3-AB-treated rats had PARS activity similar to those
of control rats (0.176 ± 0.015 pmol · min
1 · µl
1,
P = 0.83) and different from rats
treated with Etx alone (P < 0.05).
Similarly, the pulmonary arterial PARS activity of Etx+Nic-treated rats
(0.236 ± 0.27 pmol · min
1 · µl
1)
was not different from control (P = 0.48) but was different from Etx alone
(P < 0.05). Neither 3-AB nor Nic
alone affected pulmonary arterial PARS activity in saline-treated rats
(data not shown). The pulmonary arterial PARS activity of
Etx+BA-treated rats (0.951 ± 0.148 pmol · min
1 · µl
1)
was not different from that of Etx-treated rats
(P = 0.32).
|
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DISCUSSION |
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We found that PARS inhibition with 3-AB and Nic in endotoxemia attenuates the dysfunction of cGMP-mediated pulmonary vasorelaxation and does not affect lung MPO activity and edema but maintains lung ATP levels. Although other studies have demonstrated beneficial effects of PARS inhibitors on the systemic vascular dysfunction in endotoxic shock (27, 32, 39, 41), this is the first study to our knowledge that examines the effect of in vivo PARS inhibition on the Etx-induced impairment of pulmonary arterial vasorelaxation. Two distinct PARS inhibitors, 3-AB and Nic, ameliorated the Etx-induced impairment of both endothelium-dependent and -independent mechanisms of pulmonary vasorelaxation. The prototypical, competitive PARS inhibitor 3-AB does not directly scavenge peroxynitrite (32) and does not prevent the development of DNA strand breakage (40). The beneficial effects of 3-AB appear to be related to PARS inhibition as opposed to other possible pharmacological properties of this drug. Its inactive analog, benzoic acid, did not affect the Etx-induced dysfunction of pulmonary vasorelaxation. PARS generates Nic in its enzymatic catalyzation of the transfer of ADP-ribose to various proteins. Therefore, Nic can decrease PARS activity through negative feedback. Its beneficial effect may also stem from conversion back to NAD+, thus directly restoring cellular energy levels (41). Although Nic is somewhat less specific than 3-AB, both compounds are potent inhibitors of PARS activity (2). In vivo administration of 3-AB and Nic inhibited the Etx-induced increase in pulmonary arterial PARS activity as measured by a commercially available assay. Both exhibited similar beneficial effects in Etx-induced acute lung injury.
We administered the PARS inhibitors after endotoxemia was established
for two reasons. This model approximates the clinical scenario of
sepsis, and we also minimized any possible effect 3-AB and Nic may have
on iNOS upregulation. Although it has been demonstrated that PARS acts
to enhance iNOS gene transcription in vitro (19) and prevents tumor
necrosis factor--stimulated induction of iNOS in a mouse fibroblast
cell line (10), other investigators have demonstrated minimal or no
effects by PARS inhibitors on in vitro (40) and in vivo (36) nitric
oxide production after LPS. The PARS inhibitors 3-AB and Nic can have additional effects depending on the cell type or system studied (28).
Benzamide analogs scavenge hydroxyl radical but not nitric oxide or
peroxynitrite. These compounds can inhibit the expression of adhesion
molecules, inhibit the cytochrome P-450 enzymes, and either
stimulate or inhibit apoptosis. Nic has also been shown to scavenge
oxyradicals and inhibit adhesion receptor expression (28).
In the current study, we found that PARS inhibition with 3-AB and Nic had no effect on Etx-induced lung MPO activity. Previous work in our laboratory (24) has demonstrated the importance of the neutrophil in this model of Etx-induced dysfunction of pulmonary vasorelaxation. Neutrophil depletion with the use of vinblastine or rabbit anti-rat neutrophil antiserum before Etx attenuated the impairment of the response to cGMP-mediated pulmonary vasorelaxation. However, neutrophil depletion did not totally eliminate the dysfunction of pulmonary arterial vasorelaxation. Thus there exist neutrophil-independent mechanisms of vasomotor dysfunction in Etx-induced acute lung injury, and increased PARS activity may be one of these mechanisms contributing to vascular dysfunction. Our results agree with a recent study that also found no effect of 3-AB on Etx-induced increases in rat lung and ileum MPO activity (26). In contrast to our findings, Szabo et al. (26) found a reduction in pulmonary microvascular leakage in 3-AB-treated rats. These results may be explained by the fact that a pretreatment as well as a posttreatment dose of 3-AB was utilized. Perhaps the Etx-induced increase in the lung wet-to-dry ratio is mediated by an early, PARS-dependent mechanism.
In contrast, PARS inhibition decreased neutrophil recruitment in other
models of non-Etx-mediated inflammation. The PARS inhibitor 3-AB reduced myocardial neutrophil accumulation after
ischemia-reperfusion injury (38), and studies in PARS
/
mice demonstrated a role for this enzyme in the
regulation of the adhesion molecules P-selectin and intercellular
adhesion molecule-1 (42). Szabo et al. (29) recently found that PARS
inhibition with 3-AB prevented both local and systemic inflammation
after carrageenan or zymosan challenge. The protective effects appeared
to be more pronounced in the severe forms and delayed phase of
inflammation, and inhibition of PARS increased the rate of adherent
neutrophil detachment from the endothelium. In these nonendotoxemia
models, PARS inhibitors were administered before the inflammatory
stimulus, suggesting that an early PARS-dependent mechanism is involved
in neutrophil accumulation and edema. The lack of effect by 3-AB and
Nic on the Etx-induced increase in MPO activity in the current study
and in the study by Szabo et al. (26) suggests that lung neutrophil
recruitment in severe endotoxemia may be mediated by a PARS-independent
step. Further investigation is needed to establish the effect of PARS inhibition on neutrophil accumulation and function in endotoxemia.
We further examined the role of PARS in Etx-induced acute lung injury
by measuring lung ATP levels and pulmonary arterial PARS activity. The
maintenance of lung tissue ATP after 3-AB treatment in endotoxemia
suggests that PARS activation may be an important pathway in the
reduction of cellular energy levels in this model of lung injury.
However, lung ATP levels may not accurately represent pulmonary
vascular energy status, but previous studies suggests that endotoxemia
depletes cellular energy levels and that this energy deficit may be
linked to a dysfunction of pulmonary arterial vasorelaxation. In rat
aortic smooth muscle cells, the PARS inhibitors 3-AB, Nic, and
PD-128763 inhibited the reduction in cellular
NAD+ and ATP as well as the
suppression of mitochondrial respiration in response to LPS and
interferon- stimulation (32). Rodman et al. (20) found that
inhibitors of oxidative phosphorylation reduced receptor-dependent
relaxation in both aortic and pulmonary arterial rings. In vivo
administration of both 3-AB and Nic prevented the Etx-induced increase
in pulmonary arterial PARS activity, confirming the action of the
inhibitors in the tissue with which we performed our vasorelaxation
experiments. Furthermore, benzoic acid, a compound that is structurally
similar to 3-AB but does not inhibit PARS, had no effect on lung ATP
levels, pulmonary arterial PARS activity, or the dysfunction of
pulmonary vasorelaxation in endotoxemia.
Peroxynitrite formation is the proposed initial stimulus leading to PARS activation, with subsequent impairment of cellular energetics and vascular function in endotoxemia (32, 39, 40). Although the presence of peroxynitrite in the lung has been demonstrated experimentally in Escherichia coli Etx-treated rats (34) and clinically in autopsy specimens from patients with sepsis-induced pulmonary injury (15), its role in the pulmonary circulation remains unclear. Other investigators have found that peroxynitrite itself is a direct pulmonary arterial vasodilator in the dose range of 10-100 µM (6, 37). However, this observed effect of peroxynitrite occurs at concentrations that are not physiologically relevant because peroxynitrite would remain in the nanomolar range even in disease states (17). Although PARS activity was not measured directly, Chabot et al. (6) found an inhibitory effect of 3-AB on this peroxynitrite-induced vasodilation. However, the effective dose of 3-AB was 10 mM, and a dose of 1 mM 3-AB did not affect the vasodilation to peroxynitrite. The high concentration of 3-AB needed to observe inhibition calls into question the specificity of this dose on PARS activity. The published IC50 of 3-AB is 33 µM, with 88% inhibition of PARS occurring at 1 mM (2). Although peroxynitrite may not be the stimulus for pulmonary arterial PARS activation, hydroxyl radical, a species also present in endotoxemia, can cause PARS activation and subsequent endothelial damage (1, 14). Further work is needed to define the relative roles of peroxynitrite and hydroxyl radical in PARS activation and vascular injury.
Although both pulmonary arterial PARS activity and lung ATP levels returned to control values with 3-AB administration, the Etx-induced dysfunction in cGMP-mediated vasorelaxation was only partially attenuated. PARS-independent mechanisms of vascular dysfunction may also contribute to the injury, as has been observed in other models of inflammation (11, 32). Cellular damage in oxidative stress occurs by parallel and/or synergistic pathways, which may or may not involve peroxynitrite generation and PARS activation. Indeed, inhibition of PARS represents just one potential strategy to reduce nitric oxide- or peroxynitrite-mediated cellular injury. Other agents such as specific inhibitors of iNOS, superoxide dismutase mimetics, and scavengers of peroxynitrite also offer promise in the therapy of shock and other inflammatory diseases.
In summary, we found that the PARS inhibitors 3-AB and Nic in Etx-induced acute lung injury attenuate the dysfunction of pulmonary vasorelaxation and maintain lung ATP levels but do not affect lung MPO activity and edema. The data presented here suggest that PARS activation in endotoxemia contributes to the development of pulmonary vascular dysfunction. PARS inhibition may provide a novel therapeutic approach in ameliorating the vascular dysfunction seen in sepsis and acute lung injury.
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ACKNOWLEDGEMENTS |
---|
This work was supported in part by National Institute of General Medical Sciences Grant GM-49222, National Institute of Child Health and Human Development Grant HD-36256-01 (to D. D. Bensard), and an American College of Surgery Faculty Research Grant (to R. C. McIntyre, Jr.).
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FOOTNOTES |
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E. J. Pulido is the Kiwanis Trauma Research Fellow.
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. C. McIntyre, Jr., Dept. of Surgery, Campus Box C-313, Univ. of Colorado Health Sciences Center, 4200 East Ninth Ave., Denver, CO 80262 (E-mail: robert.mcintyre{at}uchsc.edu).
Received 15 December 1998; accepted in final form 10 June 1999.
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