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American Journal of Respiratory and Critical Care Medicine Vol 165. pp. 1426-1432, (2002)
© 2002 American Thoracic Society

Original Article

Functional Inhibition of Constitutive Nitric Oxide Synthase in a Rat Model of Sepsis

Jeremy A. Scott, Sanjay Mehta, Michele Duggan, Aurelia Bihari and David G. McCormack

A. C. Burton Vascular Biology Laboratory, Departments of Medicine, Pharmacology, and Toxicology, University of Western Ontario and London Health Sciences Centre, London, Ontario, Canada

Correspondence and requests for reprints should be addressed to David G. McCormack, Division of Respiratory Medicine, London Health Sciences Centre-Victoria Campus, 375 South Street, London, ON, N6A 4G5 Canada. E-mail: david.mccormack{at}lhsc.on.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Induction of inducible nitric oxide synthase (iNOS) expression is likely important in the pathogenesis of sepsis. However, the sepsis-mediated induction of iNOS is associated with a decrease in constitutive NO synthase (cNOS) activity (which is reversible following acute but not chronic sepsis). Whether this decreased cNOS activity is due to functional inhibition of cNOS by the high concentrations of NO produced by iNOS or to downregulation of cNOS expression is not clear. Thus, we tested the hypothesis that sepsis produces a reversible iNOS/NO-mediated inhibition of cNOS activity. Using a rat cecal ligation and perforation (CLP) model of sepsis, we examined the time course of the changes in iNOS and cNOS activities in lung and thoracic aortae. Reversibility of the sepsis-induced decrease in cNOS activity was assessed in vitro by enzyme activity determination following selective inhibition of iNOS. iNOS and endothelial cNOS protein concentrations were determined by Western blotting. In all septic tissues, cNOS activity was depressed at 6, 12, 24, and 48 hours post-CLP. Inhibition of the increased iNOS activity with aminoguanidine, in vitro, partially restored cNOS activity following acute (6–12 hours) but not chronic sepsis (24–48 hours post-CLP). Consistent with the irreversible depression of cNOS activities in tissues following chronic sepsis, endothelial NOS protein concentrations declined progressively during the time course of sepsis. We have demonstrated the restoration of cNOS activity following in vitro inhibition of iNOS, early, and the downregulation of endothelial NOS, later, in a rat CLP model of sepsis. This suggests that further study is required before iNOS-selective inhibition can be considered in human sepsis.

Key Words: nitric oxide synthase • sepsis • aminoguanidine • endothelial nitric oxide synthase • inducible


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The production of nitric oxide (NO) by the family of NO synthase (NOS) isozymes (E.C. 1.14.23.-), mediates many homeostatic and pathophysiologic responses (1–3). Regulation of the NOS isozymes has been shown to be at the transcriptional and posttranslational concentrations as well as through end-product inhibition (4, 5). The induction processes that stimulate expression of the inducible NOS isozyme (iNOS) have also been shown to downregulate the expression of the endothelial NOS isozyme (eNOS) (5–7). Posttranslational phosphorylation and subcellular localization of the isozymes also affects the activity of the NOS isozymes (8). Further, NO itself has been shown to inhibit the function of the neuronal NOS isozyme (nNOS) (9, 10), eNOS (11), and iNOS (12). This end-product feedback inhibition of the NOS isozymes suggests a novel self-regulatory function of the enzymes, which allows for discrete control of the normal homeostatic production of NO by the low-output "constitutive" isozymes (nNOS and eNOS) (13) and a mechanism to regulate production of NO by the high-output inducible isozyme (iNOS). Decreased production of NO by the constitutive NOS (cNOS) isozymes in the presence of increased NO production by iNOS may result in specific attenuation of local homeostatic functions of cNOS-derived NO.

Elevated production of NO, through elevated iNOS activity, contributes to the circulatory alterations associated with sepsis (i.e., vascular hypocontractility and mild hypotension) in both the clinical setting (14–16) and in experimental models (17). The sepsis-induced elevation of iNOS activity is associated with a concomitant decrease of (cNOS) activity (18, 19). This decrease may be due to downregulation of cNOS isozymes (7), possibly mediated by decreased mRNA stability (6, 20), which would limit production of new enzyme, or by functional inhibition of cNOS isozymes by the high concentrations of NO produced by iNOS (9, 11, 21, 22). We have shown previously that inhibition of cNOS activity in thoracic aortae following acute exposure to lipopolysaccharide in vitro can be partially restored following selective inhibition of iNOS (23). However, in a more chronic 24-hour model of sepsis (secondary to peritonitis), we have shown that cNOS activity cannot be completely restored following selective in vivo inhibition of the increased iNOS activity (24).

The current investigation examines the hypothesis that the decrease of cNOS activity in sepsis is due to both downregulation and functional inhibition of the enzyme by the high concentrations of NO produced by iNOS. First, the time course of the depression of cNOS activity following the induction of sepsis by cecal ligation and perforation (CLP) was determined. Then, the reversibility of the depression of cNOS activity was tested in vitro using aminoguanidine, at a dose which selectively inhibited iNOS. We also tested the hypothesis that the isozymes of NOS are differentially sensitive to "self-inhibition" by NO. To address this, we used recombinant eNOS and iNOS and isolated rat cerebellar nNOS to examine the degree of inhibition of NOS activity in vitro produced by an NO donor (S-nitroso-N-acetyl penicillamine [SNAP]).


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cecal Ligation and Perforation Model of Sepsis
This experimental protocol was approved by the Animal Ethics Review Committee at the University of Western Ontario. Following a 7- to 14-day acclimatization period in the animal housing facility, male Sprague-Dawley rats (300–450 g) had jugular and carotid intravascular catheters inserted for fluid and analgesic administration and blood pressure monitoring as described previously (24). Induction of sepsis was performed by a CLP as described previously by our laboratory (24, 25). Following surgery, the animals were allowed to recover in their cages with food and water ad libitum. Sham control animals underwent surgery for the insertion of carotid arterial and jugular venous lines, as described previously, with an abdominal incision to control for the opening of the peritoneum in the CLP animals. Animals were administered fluids and analgesia (fentanyl citrate, 2 µg/ml in 0.9% saline, 10 ml/kg/hour) continuously (6, 12, 24 or 48 hours post-surgery) until they were killed and tissues were harvested. Before euthanasia (i.e., at 6, 12, 24, or 48 hours), arterial blood samples were obtained to determine arterial blood gases, white blood cell count, plasma lactate concentration, and NOx- concentration. Thoracic aortae and lungs were harvested for subsequent determination of calcium-dependent and -independent NOS activities and for eNOS and iNOS protein determination with Western blotting.

NOx- Chemiluminescence Detection
The metabolic end products of NO production (NO2- and NO3-, NOx-) were determined in plasma samples using chemiluminescence detection on an NO analyzer (Model 270 B; Sievers Instruments, Boulder, CO), as described previously (26).

NOS Assay
NOS activity was quantitated as the conversion of [3H] L-arginine to [3H] L-citrulline as described previously (23, 24), with the following modification. Samples were assayed under five different conditions: (1) calcium/calmodulin, (2) calcium/calmodulin + aminoguanidine (30 µM), (3) ethylenediamine tetraacetic acid (EDTA)/ethylene glycol-bis(aminoethylether)-tetraacetic acid (EGTA), (4) EDTA/EGTA + aminoguanidine (30 µM), and (5) EDTA/EGTA + L-NAME (1 mM). Consistent with our previous work (23, 24), pilot in vitro investigations demonstrated inhibition of iNOS by aminoguanidine (30 µM) without any significant inhibition of cNOS activity (data not shown).

Calcium-dependent (constitutive) NOS activity was calculated as the difference between the calcium/calmodulin sample (cNOS + iNOS activities) and the EDTA/EGTA sample (iNOS activity only). Nonspecific radioactivity and metabolism of [3H] L-arginine was accounted for by incubating tissue homogenate with L-NAME (1 mM) in the incubation buffer containing EDTA/EGTA. Thus, the calcium-independent (inducible) NOS activity was calculated as the L-NAME–inhibitable portion of the activity in the samples with EDTA and EGTA. The reversible portion of cNOS activities was determined by calculating the difference between the calcium/calmodulin + aminoguanidine (30 µM) and EDTA/EGTA with aminoguanidine (30 µM). Resultant enzyme activities were expressed as pmol L-citrulline evolved per minute per milligram of protein. The protein concentration of the tissue homogenate was determined by a microplate modification of the Bradford method, with bovine serum albumin as the standard and homogenization buffer as the blank (27).

NO Self-Inhibition of NOS Isozymes
Concentration response relations were constructed for the NO donor (SNAP) with each of the isozymes of NOS. The neuronal NOS was isolated from rat cerebellar supernatant (28). Briefly, rat cerebellum were homogenized in 6 vols (wt/vol) of homogenizing buffer (pH 7.4, 10 mM Hepes buffer, 0.1 mM EDTA, 1 mM dithio-threitol, 1 mg/ml phenylmethyl sulphonyl fluoride, 0.32 M sucrose). The homogenate was ultra-centrifuged at 100,000 x g for 60 minutes at 4° C (Beckman Instruments Canada Inc., Mississauga, ON), and the cytosolic neuronal NOS from the supernatant was isolated for subsequent assays. Recombinant bovine endothelial cNOS (20 U/ml) and rat inducible NOS isozymes (250 U/ml) were purchased from Cayman Chemical Co. (Ann Arbor, MI) and were diluted (to 2 and 5 U/ml, respectively) in homogenization buffer. Aliquots (25 µl) of each of the isozymes were incubated for 15 minutes under control conditions (i.e., all cofactors and substrate provided, with no inhibitors), with increasing concentrations of SNAP (10-9 to 10-1 M), or with L-NAME (1 mM) (to control for background nonspecific radioactivity). Total L-citrulline production was determined by liquid scintillation counting and expressed as a percentage of the control (uninhibited) production. A Hill plot was constructed for each inhibition curve, allowing the calculation of the IC50 from the linear-regression of the curves (i.e., the x-intercept value). Pilot investigations demonstrated no effect of N-acetyl-penicillamine (the non–NO-donating backbone of SNAP) on NOS isozyme activities.

eNOS and iNOS Protein Determination
Lung and thoracic aorta homogenates (from septic and control animals, as described previously) provided sufficient protein to determine eNOS and iNOS protein concentrations by Western blotting, as described previously (29, 30). Briefly, tissue homogenate (50 µg lung protein; 10 µg protein from thoracic aorta) was loaded onto a 7.5% sodium dodecyl sulfate (SDS)-tris-glycine polyacrylamide gel and separated by electrophoresis (1.5 hours; 120 V; MiniGell II, Bio-Rad, Mississauga, ON). Protein was transferred to a nitrocellulose membrane overnight (300 mA; 0–5° C). Membranes were blocked with nonfat dry skim milk (5% in phosphate buffered solution) blocking solution and incubated with eNOS or iNOS antibodies (mouse primary monoclonal antibodies; 1:500 and 1:1000, respectively). Membranes were subsequently incubated with a horseradish peroxidase–conjugated anti-mouse secondary antibody (1:1000; 1 hour; Amersham Pharmacia Biotech, Baie d'Urfe, PQ). Blots were developed by enhanced chemiluminescence (coumaric acid [0.4 nM], luminol 2.5 mM in tris(hydroxymethyl)aminomethane [100 mM; pH 8.5] added 1:1 to a solution of H2O2 [0.02%] in tris[hydroxymethyl]aminomethane [100 mM; pH 8.5]; 1-minute exposure to the blot) and exposure of the blot to X-ray film (Hyperfilm ECL; Amersham Pharmacia Biotech). nNOS protein was not evaluated because pilot investigations found extremely low (not detectable) concentrations in the rat tissues studied (data not shown).

Chemicals
Cytoscint Environmentally Safe scintillation fluid, 1,4 dithio-threitol, phenylmethylsulphonyl fluoride and Coomassie Brilliant Blue G250 were purchased from ICN Biomedical Inc. (Mississauga, ON). [3H]L-arginine and Hyperfilm ECL were purchased from Amersham Pharmacia Biotech. Except where noted previously, all reagents were purchased from Sigma Chemical Co. (Sigma-Aldrich Canada, Mississauga, ON).

Statistical Analysis
All data are expressed as the mean ± standard error of the mean and were compared using Factorial ANOVA with Fisher's post hoc analysis. p Values below 0.05 were considered significant. All statistical tests were calculated using the StatView +4.5 program on a Macintosh computer.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mean Arterial Pressure
Septic animals exhibited a progressive decline in mean arterial pressure compared with sham-operated animals, over the 48 hours postsurgery (Figure 1) . The mean arterial pressure in septic animals was significantly lower than that in sham animals throughout the 48 hours of investigation.



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  		Figure 1. Mean arterial pressure of sham (open bars) and septic rats (solid bars) over the course of 48 hours postsurgery (n = 8 animals per group; *p < 0.05 relative to sham animals at the same time point; #p < 0.05 relative to animals of the same group at 6 hours postsurgery).

 
Lactate, Arterial Blood Gases, Leukocytes
Arterial blood samples were taken throughout the investigation at the time points of interest. Arterial lactate concentrations in septic animals progressively increased from 0.5 ± 0.0 mM at 6 hours to 1.4 ± 0.3 mM at 48 hours (Table 1). Mean arterial lactate concentrations in sham-operated animals was never greater than 0.5 µM throughout the investigation. Arterial blood gases were unaffected by sepsis over the time course of the investigation (data not shown). Circulating leukocyte concentrations were significantly lower in septic animals compared with those in sham-operated animals at all the time points investigated (Table 1).


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  		TABLE 1. White blood cell count and lactate in arterialblood and plasma samples, respectively, from sham andseptic (clp) animals over the time course of the study

 
Arterial Plasma NOx- Concentrations
Total NO-metabolites (NO2- and NO3-; NOx-) were quantified in arterial plasma samples by chemiluminescence (Figure 2) . Plasma NOx- concentrations were increased in septic animals by 6 hours post-CLP and remained consistently high up to 48 hours (p < 0.05 to time-matched shams). Plasma NOx- concentrations remained low and stable in sham animals throughout the time course investigated.



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  		Figure 2. Plasma NOx- concentrations of sham and septic rats over the course of 48 hours postsurgery (n); *p < 0.05 relative to sham animals at the same time point; #p < 0.05 relative to animals of the same group at 6 hours postsurgery.

 
NOS Activities in Sham and Septic Rat Thoracic Aorta
Thoracic aortae from sham-operated animals exhibited a high concentration of cNOS activity, which was stable and consistent throughout the investigation (Figure 3A) . cNOS activity was attenuated by 6 hours in septic animals and progressively, but not significantly (compared with 6 hours after CLP), decreased up to 48 hours post-CLP. Induction of sepsis resulted in a significant increase in iNOS activity by 6 hours, which remained elevated up to 48 hours (p < 0.05 compared with sham animals at the same time point; not significant compared with the same treatment group at different time points).



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  		Figure 3. (A) In vitro cNOS and iNOS activities (U) from sham (open bars) and septic (solid bars) rat thoracic aortae over the course of 48 hours postsurgery (n = 8 animals per group; *p < 0.005, **p < 0.0001 relative to sham animals at the same time point); (B) In vitro cNOS and iNOS activities (U) from septic rat thoracic aortae over the course of 48 hours postsurgery before (solid bars) and after (striped bars) selective inhibition of iNOS with aminoguanidine (30 µM in vitro) (n = 8 animals per group; #p < 0.05, ##p < 0.01 relative to uninhibited NOS activity at the same time point; p < 0.05 relative to NOS activity from the same group at 6 hours postsurgery).

 
Inhibition of iNOS activity in vitro with aminoguanidine (30 µM) resulted in a partial restoration of cNOS activity in septic thoracic aortae (Figure 3B). This increase in cNOS activity was more pronounced in thoracic aortae harvested from animals at earlier time points (i.e., 6–12 hours) than at the later time points (p < 0.05 for 24 and 48 hours septic aortae compared with 6 hours septic aortae). iNOS activity was reduced to sham concentrations with aminoguanidine (30 µM) in septic thoracic aortae at all time points investigated.

NOS Activities in Sham and Septic Rat Lungs
cNOS activities were stable and consistent in lungs from all sham-operated animals (Figure 4A) . Pulmonary cNOS activities in septic rats were significantly attenuated at all the time points examined. iNOS activities were low and stable in lungs from all sham-operated animals and were significantly elevated at all time points in septic lungs. In vitro inhibition of iNOS activity with aminoguanidine (30 µM) resulted in significant restoration of cNOS activity in lung tissues harvested from septic animals 6–24 hours post-CLP (Figure 4B). In contrast, there was no restoration of cNOS activity in lung tissues from animals that had been septic for 48 hours.



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  		Figure 4. (A) In vitro cNOS and iNOS activities (U) from sham (open bars) and septic (solid bars) rat lungs over the course of 48 hours postsurgery (n = 8 animals per group; *p < 0.005, **p < 0.0001 relative to sham animals at the same time point). (B) In vitro cNOS and iNOS activities (U) from septic rat lungs over the course of 48 hours postsurgery before (solid bars) and after (striped bars) selective inhibition of iNOS with aminoguanidine (30 µM) (n = 8 animals per group; #p < 0.05, ##p < 0.01 relative to uninhibited NOS activity at the same time point; p < 0.05 relative to NOS activity from the same group at 6 hours postsurgery).

 
NOS Protein Concentrations in Thoracic Aorta and Lung
NOS protein concentrations (eNOS and iNOS) were determined in a subset of thoracic aortae and lungs harvested from sham and CLP animals at the time points examined (6, 12, 24, and 48 hours)(Figures 5 and 6) . Thoracic aortae (Figure 5A) and lungs (Figure 6A) from both sham and CLP animals at the earlier time points (6 and 12 hours) exhibited similar concentrations of eNOS protein. However, thoracic aortae and lungs from the more chronically septic animals (i.e., 24 and 48 hours) exhibited significantly decreased eNOS protein (p < 0.05 compared with both their respective shams and the 6 hour septic animals at 48 hours). Septic animals exhibited significantly increased iNOS protein concentrations in both thoracic aorta (Figure 5B) and lungs (Figure 6B). nNOS protein concentrations were examined in a subset of tissues and were not detectable with Western blot in either control or septic lungs or thoracic aortae (data not shown).



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  		Figure 5. eNOS (A) and iNOS (B) protein concentrations in thoracic aorta from sham (open bars) and septic (solid bars) rats (*p < 0.05 to control at the same time point; note: the blots shown are from representative individual blots for each time point for which densitometry results were assessed).

 


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  		Figure 6. eNOS (A) and iNOS (B) protein concentrations in thoracic aorta (A) and lung (B) from sham (open bars) and septic (solid bars) rats (*p < 0.05 to control at the same time point; note—the blots shown are from representative individual blots for each time point for which densitometry results were assessed).

 
NOS Isozyme Inhibition by NO
To examine the susceptibility of the various NOS isozymes to self-inhibition by NO, inhibition curves were constructed for each isozyme to the NO donor, SNAP, (Figure 7) . IC50 values were calculated from a Hill plot of the enzyme inhibition data. Calculated IC50 values for SNAP were 51, 200, and 1,800 µM for nNOS, eNOS, and iNOS, respectively. As a control, N-acetyl penicillamine demonstrated no significant effect on NOS isozyme activities (data not shown).



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  		Figure 7. Inhibition of eNOS (J), nNOS (E) and iNOS (H) isozymes by S-nitroso-N-acetyl-penicillamine (SNAP) (IC50 values were calculated from the Hill plot of the inhibition curves; all curves were constructed on the basis of three independent determinations for the NO-donor).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have shown previously that iNOS activity is elevated in rat thoracic aorta and lung following induction of sepsis by CLP (24). We now demonstrate that following induction of sepsis, iNOS activity and expression is elevated at 6 hours and remains elevated up to 48 hours. Consistent with this finding, plasma NOx- concentrations remain elevated at all time points. Further, we demonstrate that the cNOS isozyme activities and eNOS protein concentrations decrease progressively over the time course of sepsis. This depression of cNOS activity is reversible with iNOS inhibition early but not later in sepsis. Consistent with this, eNOS protein expression is downregulated at later time points during sepsis. To corroborate the NO self-inhibition of the NOS isozymes, inhibition curves were constructed for the three NOS isozymes to the NO-donor SNAP. SNAP inhibited all isozymes with a rank order of IC50 values of nNOS < eNOS < iNOS.

Hemodynamic Variables
In this rodent model of sepsis, secondary to peritonitis, animals became progressively more hypotensive, and arterial lactate concentrations increased over the time course investigated. Further, all septic animals became leukopenic, compared with control animals at all the time points examined. These hemodynamic and biochemical changes are consistent with previous investigations using this model of sepsis (24, 25), and are comparable to a profile characteristic of clinical sepsis (31, 32).

iNOS and Plasma NOx- Concentrations
Consistent with previous reports by us and other workers, induction of sepsis resulted in increased iNOS protein concentrations, and increased activity and plasma accumulation of the metabolic end-products of increased NO production (nitrite and nitrate; NO2- and NO3-; NOx-) (24, 29, 33, 34). It is generally considered that increased production of NO is responsible for the hypotension associated with sepsis (16). However, in this investigation septic animals did not exhibit a decrease in mean arterial pressure until 12 hours following CLP, whereas the NOS activity and plasma NOx- concentrations were increased at 6 hours following CLP. Consistent with a previous report using endotoxemic iNOS knockout mice (35), in which the endotoxin-induced decrease in blood pressure was attenuated, but not blocked, and other recent investigations using a similar CLP model of sepsis (36, 37), the present data suggest that mechanisms other than NO are involved in the initial decrease of blood pressure associated with sepsis. Although the actual time course of iNOS induction and progression of hypotension may be different in these models (likely due to lower concentrations of fluid resuscitation [36, 37]), the overall finding that NO is not entirely responsible for the hypotension of experimental sepsis is supported.

cNOS
We have corroborated our previous finding that cNOS activity is decreased in this model of sepsis (24). Further, using a concentration of aminoguanidine that demonstrates more selective inhibition of iNOS (versus cNOS) isozymes (23, 30), we have also demonstrated that this decrease of cNOS activity is reversible in the early stages (6–12 hours) of sepsis and becomes irreversible following more chronic sepsis (24–48 hours). This finding is also consistent with our previous work demonstrating that the depression of cNOS activity is reversible following short-term (5 hours) lipopolysaccharide exposure in vitro (23) but that this depression is not reversible following more chronic (24 hours) sepsis in vivo (24). The reversibility of this attenuated cNOS activity in acute sepsis is likely due to the feedback inhibition of the cNOS isozymes (i.e., eNOS and/or nNOS) by the high concentrations of NO produced by iNOS. NO interacts with the ferrous (II) heme moiety of the NOS isozymes, thus inhibiting enzyme activity (9). Using isolated nNOS and recombinant eNOS and iNOS isozymes, we have demonstrated that these isozymes are differentially susceptible to NO self-inhibition or feedback inhibition; with the neuronal and endothelial (cNOS) isozymes being most sensitive to this type of end-product inhibition. Recently, a conserved amino acid in nNOS, tryptophan-409, has been identified as being responsible, in part, for the regulation of the NO-mediated self-inhibition (38, 39). Indeed, tryptophan-409-mutated nNOS purified from E. coli, exhibits hyperactivity, due to the lack of feedback-inhibition (40, 41). This mechanism of NO self-inhibition, in addition to the interaction with the ferrous heme iron, may explain the increased susceptibility of the nNOS isozyme to end-product inhibition. Although an analogous region on the eNOS isozyme has not been determined to date, the accessibility and interaction of NO with the ferrous heme iron core may regulate the differential susceptibility of this isozyme to end-product inhibition. Thus, this differential susceptibility of the NOS isozymes to self-inhibition by NO supports discrete control of eNOS/nNOS activity in homeostasis and suggests iNOS is much less tightly regulated, post-translationally.

The sepsis-induced depression of cNOS activity may be due to functional inactivation and/or downregulation of the constitutive isozymes. Yoshizumi and coworkers (6) have reported an increased instability of endothelial cNOS mRNA, in vitro, following stimulation with tumor necrosis factor-{alpha}. Liu and colleagues (7) have also demonstrated decreased eNOS mRNA in rat tissues following stimulation with lipopolysaccharide. Furthermore, eNOS mRNA has been shown to decrease by 94% in human umbilical vein endothelial cells (42), despite an apparent increase in overall calcium-dependent NOS activity, following 24 hours exposure to cytokines to mimic sepsis; suggestive that turnover of the constitutive isozymes may be rather slow (at least in vitro). In contrast, our findings suggest that the turnover of the cNOS isozymes in vivo, following sepsis, may be more rapid. Our observation of an approximate 50% decrease of eNOS protein in both thoracic aorta and lung following 24–48 hours of sepsis is consistent with the previous demonstration of a half-life for eNOS protein of approximately 20 hours (43, 44). This alteration of cNOS isozymes in response to sepsis may result in altered NO-dependent homeostatic mechanisms, such as discrete control of vascular relaxation. Further support of the beneficial role of the cNOS isozymes (specifically, eNOS) in sepsis has recently been obtained in that transgenic mice overexpressing eNOS demonstrate improved survival and injury markers following endotoxin exposure (45).

In this investigation, depression of cNOS activities in the later stages of sepsis (i.e., 24–48 hours) did not correlate closely with the downregulation of eNOS protein (i.e., cNOS activities were lower than the eNOS protein concentrations). One explanation for this discrepancy is that the feedback inhibition may still occur, albeit to a lesser degree, in response to the NO produced by the cNOS isozymes themselves, resulting in genuine end-product inhibition. Alternatively, the eNOS enzyme may have been inactivated in a manner that did not involve degradation of the protein. Posttranslational modification of the enzyme, such as phosphorylation of the serine residues, which controls eNOS inactivation and recycling (4, 44, 46, 47), may be such a mechanism. The relative contribution of nNOS protein was not evaluated in this investigation, as this technique evaluates NOS activity profiles in whole organ and tissue homogenates. Whereas we have previously demonstrated a role for neuronally-derived NO in the vascular reactivity of the guinea pig (48) and human pulmonary arteries (49), the relative concentrations of nNOS compared with eNOS and iNOS in the lung homogenates would be extremely low (50). Further, preliminary investigations by ourselves have demonstrated no contribution of neuronally-derived NO in the neurogenic vasodilation of the rat thoracic aorta (J. A. Scott and D. G. McCormack, unpublished data). For these reasons, nNOS was not assessed in either lung or thoracic aorta in the present model. However, this does not discount the possibility that nNOS may play a role in the pathology of sepsis in other tissues (i.e., skeletal muscle) (30, 51) or species (52, 53).

In conclusion, these data demonstrate that pulmonary and systemic vascular cNOS activities are depressed in rodent sepsis and that early on this depression is reversible but becomes less reversible as the time course of sepsis progresses. The irreversible depression of cNOS activity in long-standing sepsis is likely due to downregulation of the eNOS isozyme. This suggests that cNOS-dependent homeostatic mechanisms may become dysfunctional in chronic sepsis. Therefore, therapeutic efforts in sepsis directed at abolishing the production of NO by general NOS inhibition or selective iNOS inhibition (16) may result in a state of NO deficiency (i.e., the cNOS is unable to produce sufficient quantities of NO) (which is avoidable under acute [54] but not chronic situations). Given the important homeostatic effects of NO produced by cNOS, adverse consequences, such as enhanced intravascular thrombosis or decreased epithelial viability, are possible. Therefore, we suggest that further study is required before further consideration of NOS inhibition in sepsis.


   Acknowledgments
 
The authors would like to thank Marta Rohan for her assistance in preparing some of the animals for this study.

Supported by a Canadian Institutes for Health Research MRC/CIHR Canada Studentship.

Received in original form November 29, 2000; accepted in final form March 14, 2002


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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