INTRODUCTION

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Septic shock is accompanied by refractory hypotension secondary to systemic inflammation due to severe infection with bacterias, fungi, and other pathogens (1). The morbidity and mortality of septic shock remain high in spite of extensive treatment with antibiotics, steroid hormones, fluid resuscitation, and vasopressors (2). Altered systemic and pulmonary vascular tones in septic shock occur directly or indirectly through the release of numerous mediators, for example, cytokines (tumor necrosis factor, interleukin-1, etc.), histamine, platelet-activating factor, thromboxane, leukotrienes, and nitric oxide (NO) (5). Recent clinical and basic studies therefore suggest NO to be one of the most promising therapeutic targets for septic shock (8, 9).

NO has been found to play an important role as a signal molecule as well as a cytotoxic or regulatory effector molecule of the innate immune response (10). NO is synthesized on demand for short periods of time following the enzymatic activation of constitutively expressed endothelial NO synthase (eNOS; type 3) or neuronal NO synthase (nNOS; type 1) (7, 11). In contrast, inducible NO synthase (iNOS; type 2) has been suggested to be responsible for the profound and long-lasting production of NO. An overproduction of NO may lead to the refractory hypotension often observed in patients with septic shock (11). On the other hand, iNOS gene-deficient mice have been reported to show not only resistance to lethality following the injection of lipopolysaccharide, but also a decreased defense against bacterial inoculation (14, 15). Consequently, these results indicate the complex roles of NO produced by iNOS in sepsis, namely the beneficial effect in defending against infectious pathogens on the one hand and the detrimental effect on vascular responsiveness on the other hand.

To elucidate the role of iNOS in septic state produced by the cecal ligation and puncture (CLP) procedure in rats, we examined the time course of iNOS protein expression, iNOS mRNA expression, and NO synthase activity especially in the lung, which is one of the most important target organs in sepsis. There have already been many studies concerning the effects of selective iNOS inhibition on septic shock using various iNOS inhibitors (12, 16). In these studies, because the inhibitors were used at relatively high doses, it is possible that effects other than iNOS inhibition might influence the results. Consequently, we evaluated the effects of a novel, potent, and selective iNOS inhibitor, a fused piperidine derivative (ONO-1714), on the survival rates, the plasma NO_x concentrations (NO₂ + NO₃), lung wet/dry weight ratios, lung iNOS protein expression, and NO synthase activity, focusing in particular on the time course alterations of these parameters after the CLP.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animals and Experimental Design

All experimental protocols were approved by the institutional animal care and use committee of the School of Medicine, Fukuoka University. Male Wistar King rats, weighing 300-350 g, were purchased from the Kyudou Co. (Fukuoka, Japan) and fed a laboratory diet and water ad libitum for at least 1 wk before being used as subjects in these experiments.

Sepsis was produced in the rats by a cecal ligation and puncture (CLP) procedure (19, 20). The rats were anesthetized with ether and their anterior abdominal wall was shaved and then disinfected. A midline incision approximately 2 cm long was made to expose the cecum and the adjoining intestine. The cecum was devascularized and ligated tightly at its base without obstructing the bowel and then punctured twice with a sterile 18-gauge needle on the antimesenteric border and gentle pressure was applied to the cecum until a small amount of feces exuded in order to ensure that the two puncture holes would not close. Next, the cecum was returned to the peritoneal cavity and the incision was closed by two layers with a 4-0 nylon running suture. In a small number of rats sepsis was produced by puncturing the cecum with a small needle (20 gauge) in order to examine the influence of needle size on survival time. In the sham-operated rats, the cecum was exposed, manipulated, and returned to the peritoneal cavity without puncturing it. Saline (3 ml/100 g body weight) was subcutaneously given to all rats at this time to prevent dehydration. After surgery, the rats were returned to the cages and allowed to recover from anesthesia. The rats were allowed to feed on a standard laboratory diet and to drink water ad libitum.

A novel selective iNOS inhibitor (ONO-1714) (1S,5S,6R,7R)-7-chloro-3-imino-5-methyl-2-azabicyclo[4.1.0]heptane hydrochloride (a fused piperidine derivative), was provided by ONO Pharmaceutical Co. Ltd. (Osaka, Japan), solubilized with saline and used in this study. ONO-1714 inhibited nitrite production from RAW264.7 cells treated with lipopolysaccharide (LPS) (10 ng/ml) with the IC₅₀ (concentration that inhibits 50%) value of 20 nM. ONO-1714 inhibited the partially purified human iNOS and eNOS with K_i values of 1.88 nM and 18.8 nM, respectively. The K_i values of NG-monomethyl-L-arginine monoacetate (L-NMMA) on human iNOS and eNOS were 847 nM and 250 nM, respectively. When the selectivity of ONO-1714 with respect to iNOS was compared with that of L-NMMA, ONO-1714 was found to be approximately 34 times more selective for human iNOS than L-NMMA, according to personal communications from Professor Naoyuki Taniguchi (Department of Biochemistry, Osaka University Medical School, Osaka, Japan) and Dr. Masao Naka (Minase Research Institute of ONO Pharmaceutical Co., Ltd., Osaka, Japan). More detailed data concerning the pharmacological profiles of this compound will be published separately under the title "A cyclic amidine analog, ONO-1714; a novel iNOS inhibitor having a potent inhibitory activity." The CLP rats received a subcutaneous injection of various doses of ONO-1714 (0.01-0.1 mg/kg) every 12 h started at different times (from 0 to 12 h) after the CLP. For the vehicle control, the same volume of saline was subcutaneously injected every 12 h.

Measurement of Nitrite/Nitrate in Plasma

Heparinized venous blood (1,000 U/ml) was drawn by cardiac puncture. The blood sample was immediately kept on ice and centrifuged at 3,000 g for 10 min at 4° C. The plasma was aspirated and stored at 80° C until analysis. The frozen plasma was thawed and mixed with methanol (1:1, vol/vol) to precipitate protein, and then centrifuged at 10⁴ g for 20 min at 4° C. The supernatant was applied to an NO_x analyzing system (ENO-11; EICOM Corp., Kyoto, Japan). This system utilizes the HPLC-Griess method and nitrite and nitrate are measured separately (21).

Western Blotting for the Expression of NOS Protein

The rats were exsanguinated by cutting the abdominal aorta under anesthesia and then the lungs and livers were removed from the rats. The organs were immediately frozen in liquid nitrogen and stored at 80° C until use. The lungs or livers were thawed and suspended in five volumes of a lysis buffer (10 mM Tris-HCl, 5 µg/ml leupeptin, 5 µg/ml pepstatin, 1 µg/ml chymostatin, 1 mM phenylmethylsulfonyl fluoride [PMSF], 5 µg/ml trypsin inhibitor, 1% sodium dodecyl sulfate [SDS], at pH 7.4) and homogenized on ice by a Polytron homogenizer (Kinematica, Steinhofhalde, Switzerland). The homogenates were centrifuged at 10⁴ g for 30 min at 4° C. Protein concentrations of the supernatants were measured by the Bradford method (Pierce, Rockford, IL) and then the equal protein amounts of the supernatants were applied to a 7.5% SDS-PAGE. Ten nanograms of authentic mouse macrophage iNOS (Cayman Chemicals, Ann Arbor, MI) was always applied to one lane. The proteins were transferred to a nitrocellulose membrane (Immobilon; Millipore Corp., Bedford, MA). The membrane was incubated with the rabbit immunoglobulin G (IgG) antibody against iNOS purified from RAW 264.7 cells (Upstate Biotech., Lake Placid, NY) at a 1:1000 dilution in Tris-buffered saline with 0.1% Tween 20. Western blotting for nNOS and eNOS was done by using the rabbit polyclonal antibody against rat brain NOS synthetic peptide (724-739) (Biomol Research Laboratory, Inc., Plymouth Meeting, PA) and the rabbit IgG antibody against human eNOS (Transduction Labo., Lexington, KY), respectively. The membrane was washed and incubated with donkey anti-rabbit IgG antibody coupled to horseradish peroxidase (Amersham, UK) in Tris-buffered saline with 0.1% Tween 20 at a 1:1,000 dilution. The blots were developed with the Amersham ECL system. The bands were scanned with a laser densitometer (Personal densitometer SI; Molecular Dynamics).

Northern Blotting for iNOS mRNA Expression

Total RNA was extracted from the lung homogenate using guanidinium isothiocyanate and phenol extraction. The RNA (30 µg per each lane) was size fractionated by electrophoresis on agarose/formaldehyde gel, transferred to a nylon membrane (Hybond-N; Amersham), and hybridized at 42° C for 16-18 h with a ³²P-labeled mouse macrophage iNOS cDNA probe (Cayman Chemicals, Ann Arbor, MI) or a ³²P-labeled GAPDH probe (Clontech, Palo Alto, CA). After hybridization, the blots were washed three times with 0.1× SCC-0.1% SDS for 15 min at 65° C. The blots were exposed to X-ray film with an intensifing screen at 80° C and were scanned with the laser densitometer.

Measurement of NOS Activity

The NOS activity was measured in the lung homogenates as transformation of [¹⁴C]arginine to [¹⁴C]citrulline by a modified method of Bredt and Snyder (22). Lungs were suspended in 10 volumes of a buffer (25 mM Tris-HCl containing 1 mM ethylenediaminetetraacetic acid [EDTA] and 1 mM ethyleneglycoltetraacetic acid [EGTA] at pH 7.4) and homogenized on ice by a Polytron homogenizer. The homogenates were centrifuged at 10⁴ g for 30 min at 4° C and then the supernatants were used for crude enzyme in the following experiments. [¹⁴C]Arginine was purchased from NEN Life Science Products, Inc. (Boston, MA). The specific activity of [¹⁴C]arginine was 320 mCi/mmole. The reaction mixture contained the 10⁴ g supernatant (less than 10% in volume), 0.2 µCi [¹⁴C]arginine, 50 µM L-arginine, 100 µM NADPH, 25 µM tetrahydrobiopterin (BH₄), 2 µM flavin adenine dinucleotide (FAD) and 2 µM flavin mononucleotide (FMN) and incubated for 30 min at 37° C under two conditions, calcium-dependent and calcium-independent reactions. Whereas the former included 0.6 mM Ca²⁺, corresponding to total NOS activity, the latter omitted calcium but included 1 mM EDTA, which corresponded to the iNOS activity (23). After the reaction was stopped by the addition of 0.5 M perchloric acid, [¹⁴C]citrulline and [¹⁴C]arginine were separated by a cationic ionic-exchange column (Shim-pack ISC-05/S0504; Shimadzu, Kyoto, Japan) on a high-performance liquid chromatography (HPLC) system (Waters Assoc, Milford, MA). The citrulline fraction was collected by a fraction collector (Model 201; Gilson, Villiers-le-Bel, France). After adding a scintillation cocktail, the isotope was counted for 3 min. Specific transformation of [¹⁴C]citrulline was calculated as follows: the isotope counts at the citrulline fraction eluted from the column chromatogram of the incubation mixture with the enzymes were subtracted from the isotope counts eluted from the incubation without enzymes and then the remaining counts were divided by the specific activity (320 mCi/mmole). Next, the specific activity of NO synthase was expressed as picomoles of citrulline formation per milligram protein/30 min.

Measurement of the Lung Wet per Dry Weight Ratios

The wet/dry weight ratios of the lungs were measured to estimate the extent of pulmonary edema. The lungs were excised from the thoracic cages and the wet weights were immediately estimated. After the lungs were lyophilized under reduced pressure using a freeze dryer FD-5 (Eyela, Tokyo), the ratios of the wet per dry weights were calculated by dividing both values.

Histological Examination

The rats were exsanguinated by cutting the abdominal aorta under anesthesia. The trachea was immediately cannulated and joined to a tube with a three-way stopcock connected to a reservoir containing the fixative. The lung was fixed in situ by the intratracheal administration of a 10% formaldehyde solution given at a pressure of 15 cm H₂O. The excised lungs were stained by hematoxylin-eosin. The perivascular leakage and interstitial thickening were morphometrically estimated under a microscope at ×400 magnification, using the NIH image.

Statistical Analysis

The data are expressed as means ± SEM. The statistical analysis was performed using the analysis of variance (ANOVA) with the Dunnett test as the multiple comparison method. A comparison of survival curves between the different groups was made using the log-rank test. All p-values were calculated based on the hour of death for the individual rats in each group. A value of p < 0.05 was considered to be significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Effects of iNOS Inhibition on the Survival Rates of CLP Rats

All CLP rats with saline injection died of severe peritonitis associated with lung edema within 48 h after the operation, based on histological examinations (n = 25). When the CLP rats were subcutaneously administered a selective iNOS inhibitor (ONO-1714) at 0.01, 0.03, or 0.1 mg/kg, every 12 h, starting at 12 h after the CLP, the rats treated with 0.03 mg/kg showed the longest survival time among all CLP-saline and ONO-1714-treated rats, as shown in Figure 1A. The sham-operated rats all survived until 72 h (n = 20). No treatment with other doses (0.01 and 0.1 mg/kg) showed a significant improvement in survival compared with the saline treatment (Figure 1A). A study of the administration schedules at this dose (0.03 mg/kg), which started immediately, 6 h, or 12 h after the intervention, showed the adequate time point for the initiation of ONO-1714 to be 12 h after the CLP (Figure 1B). The start of ONO-1714 administration at 6 h after the CLP tended to hasten animal death in comparison with the saline injection (Figure 1B). Twenty-five rats were used for each group in Figures 1A and 1B. The CLP rats treated with ONO-1714, 0.03 mg/kg, subcutaneously, every 12 h, at 12 h after the CLP intervention survived for a significantly longer period than the CLP-saline or sham-operated rats. Consequently, we used this administration schedule for ONO-1714 in the following experiments. On the other hand, a limited number of rats, in which the cecum was punctured using a small needle (20-gauge), showed a much longer survival after either saline or ONO-1714 treatment than the rats punctured using an 18-gauge needle. Three of six saline-treated CLP rats survived until 78 h, but all ONO-1714-treated CLP rats (n = 5) survived until 120 h.

View larger version (25K): [in this window] [in a new window]   Figure 1.   Effects of a selective iNOS inhibitor (ONO-1714) on the survival rates of CLP rats. (A) Effects of various doses of ONO-1714 on survival rates. Septic rats were administered with various doses of ONO-1714 (n = 25) or saline (n = 25) subcutaneously every 12 h beginning at 12 h after the CLP. The number of sham-operated rats was 20. (B) Effects of lag time for the start of ONO-1714 administration at a dose of 0.03 mg/kg after the CLP (n = 25).

Plasma Nitrite/Nitrate (NO_x) Levels of CLP Rats and Influences of ONO-1714

The plasma NO_x levels were estimated over the courses of time for the CLP saline rats (Figure 2). CLP rats at 12 h after intervention exhibited significantly higher levels of plasma NO_x compared with levels of plasma NO_x at 0 h. The marked elevation of NO_x in plasma was observed from 6 to 12 h after the intervention. Thereafter, the NO_x levels in the plasma gradually increased in parallel with the elapsed time, as shown in Figure 2. Treatment with ONO-1714, which was subcutaneously injected at the dose of 0.03 mg/kg every 12 h starting at 12 h after the intervention, significantly decreased the plasma NO_x concentrations in comparison to the saline control at each time from 18 h to 42 h. The sham-operated rats showed only an insignificant increase in the plasma NO_x, with a maximum level of 28.2 ± 4.2 µM at 42 h.

View larger version (13K): [in this window] [in a new window]   Figure 2.   Time course of plasma NOx (NO2 + NO3) levels after the CLP. The plasma NOx concentrations at various time were compared with the control (at time = 0). The plasma NOx concentrations of ONO-1714-treated (0.03 mg/kg subcutaneously, every 12 h) septic rats (open column) started at 12 h after the CLP were compared with those of the saline-treated CLP rats (closed column) at each time point (n = 15). **p < 0.01 compared with control and #p < 0.05 compared with ONO-1714-treated or saline-treated rats at each time point.

NO Synthase Activity in Lungs

The supernatant after centrifugation at 10⁴ g of lung homogenate was used as a crude enzyme for NO synthase activity. The supernatant was incubated with [¹⁴C]arginine under calcium-dependent and calcium-independent conditions, as described in METHODS. The incubation of [¹⁴C]arginine with lung homogenates increased formation of [¹⁴C]citrulline in a linear fashion with incubation time until 30 min at 37° C and also with protein amounts added (data not shown). As shown in Figure 3, total NOS and iNOS activity significantly increased at 6 h after the CLP and continued to elevate until 24 h, but thereafter decreased until 42 h. Treatment with ONO-1714 (0.03 mg/kg subcutaneously, every 12 h) started at 12 h after the intervention decreased the total NOS activity as well as the iNOS activity. It should be noted that the iNOS activity in the lung homogenates from ONO-1714-treated CLP rats showed extremely low levels compared with the total NOS activity at each time point from 24 h to 42 h (Figure 3).

View larger version (19K): [in this window] [in a new window]   Figure 3.   The time course of NO synthase (NOS) activity in lung homogenates from CLP rats and the effects of ONO-1714. The NOS activity in the homogenates from CLP saline rats was assayed under calcium-dependent (closed column) and calcium-independent (open column) conditions and was expressed as picomoles of citrulline per milligram protein/30 min. The lung homogenates from CLP rats treated with ONO-1714 (0.03 mg/kg subcutaneously, every 12 h, at 12 h after CLP) were similarly incubated under calcium-dependent (hatched column) and calcium-independent (dotted column) conditions. There were four rats in each group. Comparison of the NOS activity from CLP saline at 6-42 h with that from CLP saline at 0 h under the calcium-dependent (**p < 0.01), and calcium-independent (#p < 0.05) conditions and comparison of the NOS activity of CLP rats treated with ONO-1714 and with saline at each time point under the calcium-dependent (p < 0.05), and calcium-independent (p < 0.05) conditions.

NO Synthase Expression in the Lungs and Livers

The supernatants from homogenates of lungs and livers were applied to 7.5% SDS-PAGE and then immunoblotted by using anti-iNOS antibody. As shown in Figure 4A, lung homogenates at 0 h after the CLP did not show any band at iNOS protein (130 kD), but those at 6 h showed the band (as indicated by the arrow) and, thereafter, the iNOS bands were observed until 42 h. The density of the bands, however, tended to decrease at 36 h and 42 h after the intervention. Consequently, the relative densitometrical unit was calculated by dividing the density at each band with the density at the band of 10 ng iNOS standard and these are summarized in Figure 4B (n = 4). The treatment with ONO-1714 (0.03 mg/kg subcutaneously, every 12 h) started at 12 h decreased iNOS protein expression at 24, 36 and 42 h after the CLP. For comparison of another target organ in sepsis, supernatants after the centrifugation of liver homogenates were similarly analyzed with lung homogenates and those are summarized in Figure 4C (n = 4). The iNOS expression of liver homogenates also showed a maximum level at 6-24 h and thereafter declined until death. The lung homogenates from sham-operated rats showed only trace amounts of iNOS expression over the time course after the CLP (data not shown). We confirmed that the iNOS antibody used in this study did not cross-react with the authentic standard of eNOS. Other NO synthase isozymes, eNOS and nNOS, in lung homogenates were constantly expressed over the time course (data not shown).

View larger version (34K): [in this window] [in a new window]   View larger version (18K): [in this window] [in a new window]   Figure 4.   The time course of iNOS protein expression (arrow) examined by Western blotting. (A) A representative Western blotting analysis of the lungs obtained from rats at 0 (lane 7), 6, 12, 18, 24, 36, and 48 h (lanes 1-6 ) after the CLP. (B) A densitometrical analysis of the iNOS levels of the lungs obtained from CLP rats treated with saline (closed column) and treated with ONO-1714 (0.03 mg/kg subcutaneously, every 12 h) started at 12 h after the CLP (open column). (C) A densitometrical analysis of the livers obtained from the CLP rats treated with saline. There were four rats in each group.

Time Course of iNOS mRNA Expression in CLP Rats

Total RNA was extracted from the lungs of CLP rats and then was electrophoresed and hybridized with a cDNA probe for iNOS. As shown in Figure 5A, the messenger RNA (mRNA) expression in lungs was not observed at 0 h, but was detected from 12 h to 42 h after the CLP. Densitometrical analysis showed that the relative densitometrical units of mRNA expression, corrected by that of GAPDH mRNA at each time point, continued to increase until 42 h (Figure 5B). In another blotting, iNOS mRNA was also detected in the lungs at 6 h after the CLP. This result contrasted with the time course alteration in the iNOS protein expression in the lungs (see Figure 4).

View larger version (40K): [in this window] [in a new window]   View larger version (13K): [in this window] [in a new window]   Figure 5.   Time course of the iNOS mRNA expression (arrow) examined by Northern blotting. (A) A representative Northern blotting analysis of the lungs obtained from rats at 0 (6), 12 (1), 18 (2), 24 (3), 36 (4), and 42 h (5) after the CLP. The numbers indicate the lane number, which was done by using the independent duplicate samples at each time. (B) A densitometrical analysis of iNOS mRNA expression, expressed as ratios against GAPDH mRNA expression, at each time.

Pulmonary Histological Findings of CLP Rats and the Influences of ONO-1714

Figure 6 shows the representative histological findings of the lungs obtained from CLP-saline or ONO-1714-treated rats. The lungs obtained at 36 h after CLP intervention showed congestion, edematous change, cellular infiltration, and microthrombi in the interstitium (Figure 6A). As shown in Figure 6B, the treatment with ONO-1714 (0.03 mg/kg subcutaneously every 12 h) started at 12 h after the intervention appeared to reduce the perivascular and peribronchiolar edema and thrombus formation in comparison with the CLP saline-treated rats (n = 3). To evaluate the extent of lung injury, a morphometrical analysis was performed using an NIH image program (Version 1.61). We calculated the relative area ratios of the interstitium against the alveolar plus interstitial area in two fields of each lung at 36 h. These ratios were 0.12 ± 0.01, 0.21 ± 0.02, and 0.14 ± 0.01 in the control, CLP saline-treated, and CLP ONO-1714-treated rats, respectively. The CLP rats showed a significantly broader area of the interstitium than the control rats, and treatment with ONO-1714 significantly reduced the area of interstitium. In contrast, the extent of perivascular edema was not significantly different among the three groups.

View larger version (124K): [in this window] [in a new window]   View larger version (120K): [in this window] [in a new window]   Figure 6.   Representative histological analysis of the lung from CLP rats treated with saline (A) and with ONO-1714 at a dose of 0.03 mg/kg subcutaneously, every 12 h starting at 12 h (B). Both groups of rats were sacrificed at 36 h after the intervention (n = 3). Arrow: microthrombi. Original magnification: ×400, hematoxylin and eosin stained.

These histological findings led us to evaluate an influence of this drug on lung fluid retention in CLP rats. Consequently, lung wet per dry weight ratios were calculated. These ratios of ONO-1714-treated rats did not differ from the saline CLP rats at each time, and were 4.50 ± 0.20, 4.74 ± 0.30, 4.73 ± 0.18, and 5.02 ± 0.25 in the former group (ONO-1714) versus 4.95 ± 0.14, 4.84 ± 0.16, 4.63 ± 0.30, and 4.61 ± 0.09 in the latter group (saline) at 18, 24, 36, and 42 h after intervention, respectively (n = 7). This result suggested that the improvement in the survival time was not due to a decrease in fluid retention by this iNOS inhibitor.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Recent studies have suggested that nitric oxide (NO) plays a critical role in the pathophysiology of sepsis and septic shock (6). However, it remains controversial as to whether NO synthase inhibition is beneficial in the treatment of sepsis. To clarify the role of iNOS in sepsis, we examined the time course studies of plasma NO_x, changes of NO synthase protein expression and activity in lungs and livers and, moreover, the effects of a novel selective iNOS inhibitor on the survival rates and on these parameters in the polymicrobial septic (CLP) model. The rats receiving CLP intervention died within 2 d and showed pathophysiological features consistent with acute respiratory distress syndrome and disseminated intravascular coagulation syndrome often seen in human sepsis cases (24). On the other hand, the newly synthesized iNOS inhibitor (ONO-1714), which is more potent and selective than the previously evaluated iNOS inhibitors, was evaluated for the first time in this study and was found to significantly improve the survival time of CLP rats compared with the saline control rats. However, the results concerning the adequate dose and time point of initiation for this drug suggested the complicated effects on this model of sepsis. When we examined the effects on the survival time by changing the start of the iNOS inhibitor after the CLP, a lag time of 12 h was found to be most effective in improving the survival time, although it was not effective when administered either immediately or 6 h after the CLP. Although the reason for the need of a lag time in the initiation of this drug remains obscure, it is speculated that NO production within 12 h after the intervention may play a role in preventing the pathogen from growing. This result may be consistent with reports that iNOS-deficient mice are more susceptible to bacterial infection than its wild type (14, 15). The findings that the plasma NO_x levels showed an abrupt elevation around12 h after the CLP and a lag time of 12 h seemed to be necessary for the initiation of iNOS inhibitor may support the speculation that NO production may help to defend against infectious pathogens soon after the onset of infectious events but thereafter excessive NO production can become detrimental for the host. On the other hand, the doses of the iNOS inhibitor suggested that a dose of 0.03 mg/kg may be preferable to lower or higher doses, namely 0.01 or 0.1 mg/kg. Consequently, these results suggested that an excessive inhibition of NO production by the high dose of the inhibitor may also be harmful for the host possibly because of the inhibition of constitutive NOS, especially eNOS. The fact that at a dose of 0.03 mg/kg, the assay of the NO synthase activity in lung homogenates showed relatively constant levels of total NO synthase activity but extremely low iNOS activity suggested the iNOS selective inhibition due to this drug in the lungs. As for the relatively minimal effect of ONO-1714 on survival rates, it is speculated that one of the reasons may be related to the lethal model of CLP used in this study, that is, puncturing the cecum with the large (18-gauge) needle and omitting administration of antibiotics. In fact, in a limited number of rats, when the small (20-gauge) needle was used to puncture the cecum, the ONO-1714-treated CLP rats all survived.

Among the three isozymes of NO synthase, the inhibition of eNOS increased blood pressure in normal subjects and did not have any beneficial effect on the survival rates of endotoxemic dogs (25). Since profound and long-lasting production of NO is considered to be derived from iNOS, a more selective and potent inhibitor of iNOS without inhibiting eNOS may have a beneficial effect on vascular dysfunction in a septic state (12, 16, 17). S-Methylisothiourea sulfate (1 mg/kg intraperitoneally) has been shown to improve survival in a murine endotoxemia model (12) and another selective iNOS inhibitor, aminoguanidine (15 mg/kg intraperitoneally), improved the survival rates in rodent model of septic shock (16). S-Methylisothiourea sulfate (5 mg/kg intravenously) prolonged survival in a rat model of antibiotic-treated gram-negative sepsis (17). In contrast, another selective iNOS inhibitor, mercaptoethylguanidine (30 mg/ kg intraperitoneally), did not improve survival in a rat model with cecal ligation and puncture (18). Since ONO-1714 (0.03 mg/kg subcutaneously) significantly improved the survival time in polymicrobial sepsis, the potency of ONO-1714 is considered to be at least 30 times more active than the other iNOS inhibitors, aminoguanidine, S-methylisothiourea sulfate, and mercaptoethylguanidine, based on the doses used in the reports (12, 16). As for iNOS selectivity, ONO-1714 is considered to be approximately 34 times more active against iNOS than eNOS (see METHODS). The administration of ONO-1714 significantly reduced the plasma NO_x levels and NO synthase activity in lungs. However, this drug did not improve the lung wet/dry weight ratios, which is inconsistent with the report using iNOS knockout mice and LPS injection (26). A morphometrical analysis indicated interstitial thickening in the lungs of CLP rats, and treatment with ONO-1714 improved this condition. It is therefore speculated that an improvement in tissue perfusion following the inhibition of excessive NO production with this iNOS inhibitor may result in an improvement of the survival rates in sepsis and septic shock.

The plasma NO_x increased at 12 h after the intervention and thereafter continued to increase in parallel with the time until death. On the other hand, the expression of iNOS protein in the lungs increased and correlated well with NO synthase activity over the time course after the CLP. Later than 24 h, however, both the iNOS protein and NO synthase activity decreased until death (at 42 h). Because the iNOS mRNA expression in the lung homogenates continued until 42 h, the reduction of iNOS protein expression was considered to be caused by a mechanism of posttranscriptional regulation (27, 28). Such an alteration of iNOS protein expression was also observed in the liver, which is another target organ of sepsis. The decrease in iNOS protein expression is speculated to be due to a decrease of de novo protein synthesis and/or exaggerated degradation in these organs in association with the progression to multiple organ failure. In contrast, other isoforms of NO synthase, eNOS and nNOS, were constantly observed in the lung homogenates over the time course after the CLP. Although the eNOS expression may not significantly change at the organ level, this result does not rule out an alteration of local eNOS expression in the lung tissue depending on the pathophysiology of sepsis. In fact, eNOS has been reported to be downregulated in the aortic endothelial cells of the CLP rats examined by histochemical techniques (29). On the other hand, this study clarified the dissociation between plasma NO_x levels and iNOS protein expression or iNOS activity in lung and liver homogenates. This dissociation may be related to the decreased scavenger activity for plasma NO_x following deteriorated liver function at a late phase of sepsis, and may also point out a clinically important problem as estimating the plasma NO_x does not exactly predict NOS activity in tissues. Furthermore, since a self-limited mechanism may exist for the regulation in iNOS in a severe septic state, iNOS is suggested to play a limited role in sepsis and septic shock (18). In conclusion, a potent and selective iNOS inhibitor may prove to be beneficial in the treatment of sepsis and septic shock if administered at appropriate doses and time points.