Abnormal Regulation of Aortic NOS2 and NOS3 Activity and Expression From Portal Vein-Stenosed Rats After Lipopolysaccharide Administration

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1 Abnormal Regulation of Aortic NOS2 and NOS3 Activity and Expression From Portal Vein-Stenosed Rats After Lipopolysaccharide Administration JÖRG HELLER, PHILIPPE SOGNI, KHALID A. TAZI, CARINE CHAGNEAU, ODILE POIREL, RICHARD MOREAU, AND DIDIER LEBREC Hyporeactivity to vasoconstrictors in aortae from portal vein-stenosed rats is associated with an increased activity of endothelial NO synthase (NOS3). In contrast, during sepsis, which is common in cirrhosis, vascular hyporeactivity is associated with an induction of inducible NOS2. The aim of this study was to investigate the in vitro reactivity to phenylephrine and the regulation of NOS2 and NOS3 in aortae from portal vein-stenosed rats after lipopolysaccharide (LPS) administration. Aortic vascular reactivity for phenylephrine, aortic NOS activity, and NOS2 and NOS3 protein expression were determined 5 hours after intravenous LPS or saline administration. Moreover, aortic NOS activity was measured after 5-hour in vitro incubation in LPS. LPS induced a significantly smaller decrease in aortic tension in portal vein-stenosed than in sham-operated rats. Under baseline conditions, aortic NOS activity and NOS3 protein expression were higher in portal vein-stenosed than in sham-operated rats, and NOS2 protein expression was not detected in aortae from either group. After LPS administration, NOS activity and NOS2 protein expression increased significantly less in portal vein-stenosed than in sham-operated rat aortae. Similar results were obtained after in vitro incubation with LPS. Endothelium removal or NOS3 inhibition with the calmodulin inhibitor, W7, increased NOS activity in the aortae of portal vein-stenosed rats after LPS incubation. In conclusion, in aortae of portal vein-stenosed rats exposed to LPS, no further decrease in aortic reactivity to phenylephrine was observed, and the induction of NOS2 was down-regulated. Endothelium removal or calmodulin inhibition inhibits NOS3 overactivity and leads to normalized NOS2 activation after LPS in aortae from portal vein-stenosed rats. (HEPATOLOGY 1999;30: ) Abbreviations: NOS2, inducible nitric oxide synthase; NOS3, endothelial nitric oxide synthase; LPS, lipopolysaccharide; NOS, nitric oxide synthase; EDTA, ethylenediaminetetraacetic acid; L-NNA, N -nitro-l-arginine; EGTA, ethylene glyco-bis-amino-ethylether- N-tetraacetic acid; W7, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide; SDS, sodium dodecyl sulfate; SNAP, S-nitroso-N-acetylpenicillamine; pec 50, negative log10 of the molar agonist concentration required to elicit 50% of the maximum response; E max, maximal agonist-induced contraction. From the Laboratoire d Hémodynamique Splanchnique et de Biologie Vasculaire, INSERM U-481, Hôpital Beaujon, Clichy, France. Received March 29, 1999; accepted June 21, Supported in part by Ferring SA France. Dr. Heller was supported by Fondation Nationale Alfred Kastler and Ernst und Berta Grimmke-Stiftung. Address reprint requests to: Dr. Lebrec, INSERM, Hôpital Beaujon, F Clichy, France. lebrec@bichat.inserm.fr; fax: Copyright 1999 by the American Association for the Study of Liver Diseases /99/ $3.00/0 In cirrhosis, the occurrence of sepsis and the morbidity and mortality of septic shock are increased in humans and animal models. 1-4 Vasodilation in septic shock or during endotoxemia is caused part by an overproduction of nitric oxide (NO), which in this case is synthesized by the inducible NO synthase isoform (NOS2) On the other hand, a hyperdynamic syndrome that results in systemic and splanchnic vasodilation, a decreased responsiveness to vasoconstrictors, and an increased cardiac output is also observed in portal hypertension with or without liver disease. In contrast to the hypothesis by Vallance and Moncada, 17 who suggested that circulating bacterial endotoxins induced vascular NOS2 in patients with cirrhosis, there is increasing evidence that the overproduction of NO in aortae of rats with extrahepatic portal hypertension and cirrhotic rats without ascites is caused by increased endothelial, constitutive NO synthase (NOS3) expression and activity without NOS2 induction In fact, because bacterial infections and sepsis are frequent in cirrhosis, NO overproduction in relation to both NOS3 overactivity and NOS2 induction may occur in patients with advanced liver disease and animal models such as CCl 4 - induced cirrhosis with ascites During endotoxin exposure, vascular NOS2 and NOS3 are regulated differentially. 29,30 Lipopolysaccharide (LPS) induces vascular NOS2 mrna and protein, 6-8 and this induces vascular hyporeactivity to vasoconstrictors during endotoxemia or septic shock. On the other hand, NOS3 of vascular endothelial cells has been shown to be down-regulated 29,31 or up-regulated 30 by LPS. Furthermore, NO has been shown to inhibit NOS2 induction 32 or the catalytic activity of NOS2 33,34 in vitro. It is not clear how aortic nitric oxide synthases (NOSs) of portal vein-stenosed rats with baseline NO overproduction are regulated after LPS challenge. Furthermore, it is not clear how aortic reactivity to vasoconstrictors is regulated after endotoxin administration in aortae of portal veinstenosed rats with a preexisting hyporesponsiveness to vasoconstrictors. Because vascular NOS2 expression during septic shock could be cytoprotective, 35 and because NOS3 is essential for the perfusion of essential organs during septic shock, 31 the regulation of vascular NOS2 and NOS3 might influence the response of portal vein-stenosed rats to endotoxins or bacterial infections. Thus, the aim of this study was to assess the in vitro reactivity to phenylephrine and to assess the regulation of NOS2 and NOS3 in the aortae of portal vein-stenosed rats after in vivo LPS administration and in vitro LPS incubation. For this purpose, vascular reactivity, the effect of several NOS inhibitors, NOS activity, and NOS2 and NOS3 protein expression were determined in aortae from 698

2 HEPATOLOGY Vol. 30, No. 3, 1999 HELLER ET AL. 699 portal vein-stenosed rats a model of extrahepatic portal hypertension and sham-operated rats 5 hours after intravenous LPS administration. Moreover, aortae of portal veinstenosed and sham-operated rats were incubated in vitro with LPS, and the effects of endothelium denudation and NOS3 inhibition by calmodulin inhibition were investigated. MATERIALS AND METHODS Animals One hundred thirteen male Sprague-Dawley rats (Charles River Laboratories, Saint-Aubin-lès-Elbeuf, France) were used in the present study. Animals were divided into 2 groups. The first group contained 52 rats that underwent portal vein-stenosis to induce portal hypertension. 36 Briefly, under pentobarbital anesthesia, the abdomen was opened and the portal vein exposed. A polyethylene catheter of 0.96-mm diameter was passed along the portal vein, and a 3.0 silk was used to ligate both the catheter and the portal vein. The catheter was then removed, leaving a calibrated stenosis of the portal vein. Portal hypertension was considered present at 14 days after surgery. The second group included 61 rats that underwent sham operation. In sham-operated animals, 14 days before the study, the portal vein was isolated, but no ligature was placed. All rats were allowed free access to food and water until 14 hours before the study. The protocol was approved by the French Agriculture Office with European legislation for research involving animals. Experiment 1: Vascular Reactivity After In Vivo LPS Administration. Twenty-four hours before the experiments, rats were anesthetized with sodium pentobarbital (100 mg/kg intraperitoneally). The left femoral vein was cannulated (catheter PE-10, Clay Adams, MD) for drug administration. Rats were then deprived of food, but not water. The following day, conscious and unrestrained rats with partial portal vein ligation or with sham operation received 3 mg/kg Escherichia coli LPS (serotype 0111:B4) dissolved in 500 µl of isotonic saline intravenously. Both groups receiving 500 µl isotonic saline served as controls. Five hours later, the animals were anesthetized, and the thoracic aorta was removed. Thereafter, vascular reactivity was compared between aortae from rats with portal vein stenosis and sham-operated rats after LPS or saline administration. The dose of LPS and the 5-hour delay between the injection and the investigation of vessels has been shown to be sufficient to induce NOS2-related vascular hyporeactivity with low mortality. 37,38 The thoracic aorta was placed in a Petri dish containing prewarmed (37 C) modified Krebs-Henseleit salt solution (in mmol/l: NaCl, 4.7 KCl, 2.5 CaCl 2, 1.17 MgSO 4, 1.18 KH 2 PO 2, 25.0 NaHCO 3, ethylenediaminetetraacetic acid [EDTA], 11.1 glucose), cleaned of loose connective tissue and then cut into 3-mm rings. Three rings of each aorta were simultaneously investigated in 3 individual organ chambers. To evaluate vascular reactivity, rings were suspended between 2 stainless-steel stirrups in individual organ chambers filled with 10 ml of modified Krebs-Henseleit solution. The solution was continuously bubbled with a mixture of 95% O 2-5% CO 2 and maintained at 37 C with an outer water jacket and a circulating heat pump. One of the stirrups was anchored to the organ chamber, and the other was connected to a strain gauge to record isometric tension ( , Harvard Apparatus, South Natick, MA). Contractions and relaxations were recorded on a multichannel polygraph ( , Gould Electronics, Blainvilliers, France) and expressed as absolute values (grams). A baseline force of 1.5 g was applied by changing the position of the transducer. Tension was identified in preliminary studies to provide optimal vasoactive responses. Stimulations with different drugs were performed after 45 minutes of equilibration. The presence of functional endothelium was determined by the relaxation evoked by acethylcholine (10 5 mol/l) in aortic rings precontracted by phenylephrine (10 6 mol/l). When relaxation to acethylcholine was maximal, the organ chambers were rinsed 3 times with warm modified Krebs-Henseleit solution. After re-equilibration, the first aortic ring was incubated in Krebs-Henseleit solution alone, the second ring was incubated in Krebs-Henseleit solution containing the nonselective NOS inhibitor, N -nitro-l-arginine (L-NNA) (10 4 mol/l), and the third ring was incubated in Krebs-Henseleit solution containing the preferentially NOS2 inhibitor, aminoguanidine (10 4 mol/l). After 30 minutes of incubation, cumulative dose-response curves were measured for phenylephrine ( mol/l). Experiment 2: NOS Activity After In Vivo LPS Administration. NOS activity was determined in aortic tissue 5 hours after LPS or saline were injected intravenously as described in Experiment 1. NOS activity was measured by determining the conversion of L[ 14 C] arginine to L[14-C] citrulline based on the modified method from Bredt et al. 39 and Cahill et al. 18,19 Rats were anesthetized with pentobarbital. The thoracic aorta was resected, and adherent fat was removed. The vessels were immediately placed in ice-cold buffer containing 50 mmol/l Tris-HCl and 1 mmol/l EDTA (ph 7.4) minced with scissors and homogenized mechanically (Bioblock Scientific, Illkirch, France), followed by sonication (Vibracell, Bioblock) for 20 seconds on ice. The homogenates were centrifugated at 1,000g for 5 minutes at 4 C. The protein concentration of the supernatant was measured as described by Bradford 40 with bovine serum albumin as a standard. Fifty microliters of the supernatant (50 to 100 µg protein) were incubated in a total volume of 160 µl Tris-HCl containing 0.1 mmol/l EDTA, 3 µmol/l tetrahydrobiopterin, 1 mmol/l nicotinamide adenine dinucleotide phosphate, 2.5 mmol/l CaCl 2, 5 mmol/l valine, and 1 µci/ml L[ 14 C] arginine. To determine calcium-independent NOS activity, a parallel reaction was performed containing an additional 5 mmol/l of ethylene glyco-bis-aminoethylether-n-tetraacetic acid (EGTA). To determine NOS-independent arginine-citrulline conversion, another parallel reaction was performed containing an additional 10 4 mol/l L-NNA. The reaction was continued for 1 hour at 25 C and was stopped by adding 1 ml ice-cold phosphate-buffered saline and 3 mmol/l EGTA (ph 5.5). L[ 14 C] citrulline was separated by applying the samples to columns containing pre-equilibrated DOWEX AGW-X8 and eluting them with 1 ml of 1 mmol/l citrulline. The amount of radioactivity was measured by scintillation counting (Beckmann, LS 3801, Irvine, CA). Enzyme activity is expressed as picomoles of citrulline formed per milligram of protein per hour. The relationship between protein concentrations and L[ 14 C] citrulline formation was linear between 35 and 125 µg of protein (data not shown). In the presence of 10 4 mol/l L-NNA, the arginine-citrulline conversion was below 0.1 pmol/mg/hr in all experiments (data not shown). Experiment 3: NOS Protein Expression After In Vivo LPS Administration. NOS protein expression was determined in aortic tissue 5 hours after LPS or saline were injected intravenously as described in Experiment 1. Thoracic aortae were homogenized in buffer containing 20 mmol/l Tris-HCl (ph 7.4), 2.5 mmol/l EDTA, 0.1% sodium dodecyl sulfate (SDS), 1% phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µmol/l pepstatin A, and 5 µg/ml aprotinin. Samples were sonicated with 3 bursts of 10 seconds (45W Vibracell, Bioblock Scientific). Homogenates were centrifugated at 12,000g for 5 minutes at 4 C to remove tissue debris. The supernatant was used to determine NOS protein. Total protein concentration was determined, 40 and an aliquot of homogenate was suspended in SDS sample buffer (0.5 Tris-HCl [ph 6.8], 10% glycerol, 3% SDS, 2% -mercaptoethanol, 30 mmol/l EDTA, and % bromophenol blue). Equal amounts of the denaturated protein per lane were subjected to SDS-polyacrylamide gel electrophoresis (7.5%). The optimal protein concentration was between 50 and 100 µg per lane, which was previously determined by a standard curve using increasing protein concentrations (data not shown). Gels were calibrated using prestained molecular-weight standards and transferred to nitrocellulose by electroblotting. Nitrocellulose filters were blocked by incubation in 5% nonfat dry milk, 1% bovine serum albumin, 0.1 mol/l NaCl, 0.01 mol/l Tris-HCl (ph 7.5), and 0.1% Tween-20 overnight at 4 C. Blots were then incubated for 1 hour with specific antisera against NOS2 or NOS3 (1/2,000) purchased

3 700 HELLER ET AL. HEPATOLOGY September 1999 from Transduction Laboratories (Lexington, KY) in the same buffer. Blots were washed 3 times for 10 minutes in 0.1 mol/l NaCl, 0.01 mol/l Tris-HCl (ph 7.5), 0.1% Tween-20, and thereafter incubated for 1 hour at 25 C with a secondary antibody (anti-rat Ig, peroxidaselinked species-specific whole antibody). Blots were again washed 3 times for 10 minutes in 0.1 mol/l NaCl, 0.01 mol/l Tris-HCl (ph 7.5), and 0.1% Tween-20. Immunoreactivity was assessed using an enhanced Western blot detection system according to the manufacturer s protocol. Western blots included positive and negative controls. Mouse macrophage lysate prepared from the RAW cell line stimulated with interferon gamma (10 ng/ml) and LPS (1 mg/ml) for 12 hours and human endothelial lysate derived from an aortic endothelium cell line in Dulbec s Modified Eagle Medium (DMEM) 10% as provided by Transduction Laboratories served as positive controls for NOS2 and NOS3 antibodies, respectively. In the present study as well as in previously published studies, 18,21,26 no cross-reactivity of NOS2 antibodies against NOS3 protein and vice versa has been observed. Experiment 4: Aortic NOS Activity After In Vitro Incubation in LPS. Rats were anesthetized with sodium pentobarbital, and the thoracic aorta was removed and cleaned of adherent connective tissue. The endothelium was removed in one half of the aortae by gently rubbing with a rough steel rod. In our laboratory, it has previously histologically and functionally been shown that this technique completely eliminates aortic endothelial cells. 20,41 Thereafter, aortae with and without endothelium were incubated for 5 hours in 20 ml sterile, 37 C warm Krebs-Henseleit solution, bubbled with 95% O 2 and 5% CO 2. According to the different protocols, aortae were incubated in: 1) Krebs-Henseleit solution alone; 2) 0.1 mg/ml LPS; 3) 10 µmol/l N- (6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7), a potent calmodulin inhibitor; 4) 0.1 mg/ml LPS and 10 µmol/l W7; and 5) 0.1 mg/ml LPS and 600 µmol/l S-nitroso-Nacetylpenicillamine (SNAP), a NO donor. The LPS concentration has been previously shown to induce NOS-related vascular hyporeactivity within 5 hours. 42,43 The concentration used for W7 has previously been shown to inhibit calmodulin-dependent NOS3 activity. 20,44 The concentration used for S-nitroso-N-acetylpenicillamine has previously been shown to inhibit in vitro NOS2 activation by LPS during 18 hours. 32 NOS activity was measured in aortic tissue as described in Experiment 2. Substances The Western blot detection system, Hyperfilm-ECL, and L[ 14 C] arginine were purchased from Amersham (Arlington Heights, IL). Antibodies to NOS2 and NOS3 were purchased from Transduction Laboratories. All other substances were purchased from Sigma Chemical Co. (St. Louis, MO). Statistical Analysis Results are expressed as mean SEM. E max and pec 50 were calculated with a linear regression method after double-reciprocal transformation. Comparisons between groups were made using two-factor ANOVA for repeated measures or Student s t test as appropriate. P.05 was considered significant. RESULTS Experiment 1: Vascular Reactivity. No significant differences were observed between portal vein-stenosed and shamoperated rats for body weight or length and diameter of aortic rings. Dose-response curves to phenylephrine in portal veinstenosed rats and sham-operated rats with and without LPS administration are shown in Fig. 1. Portal vein-stenosed rats had a significantly impaired maximal response to phenylephrine compared with sham-operated rats (Table 1). The vascular hyporeactivity of portal vein-stenosed rat aortae was corrected by L-NNA but not by aminoguanidine, whereas in sham-operated rats, L-NNA and aminoguanidine had no A FIG. 1. Protein expression of NOS2 in portal vein-stenosed and shamoperated rat aortae. Protein was measured, and equal amounts were applied to 10% SDS-polyacrylamide gels. The protein was resolved by electrophoresis and transferred to nitrocellulose. Blots were probed with a specific NOS2 antibody. Immunoreactivity was visualized by autoradiography after incubation with a secondary antibody. (A) Autoradiography of 1 representative experiment. M, mouse macrophage lysate prepared from the RAW cell line stimulated with interferon gamma (10 ng/ml) and LPS (1 mg/ml) for 12 hours (positive control); E, human endothelial lysate derived from an aortic endothelium cell line. 1, aorta of sham-operated rat 5 hours after saline administration; 2, aorta of portal vein-stenosed rat 5 hours after saline administration; 3, aorta of sham-operated rat 5 hours after LPS administration; 4, aorta of portal vein-stenosed rat 5 hours after LPS administration. (B) NOS2 was not detected in aortae from portal vein-stenosed or sham-operated rats in aortae from rats after saline administration. Autoradiographs were scanned by laser densitometry, and aortic NOS2 protein expression in portal vein-stenosed rats after LPS administration was expressed as the percentage of aortic NOS2 expression in sham-operated rats after LPS administration. significant effect on vascular reactivity (Table 1). In portal vein-stenosed rats, LPS administration did not induce a significant decrease in E max to phenylephrine, whereas in sham-operated rats, E max decreased significantly after LPS administration (Table 1). After LPS administration in portal vein-stenosed rats, L-NNA as well as aminoguanidine cor-

4 HEPATOLOGY Vol. 30, No. 3, 1999 HELLER ET AL. 701 TABLE 1. E max and pec 50 Values to Phenylephrine of Rat Aortic Rings 5 Hours After Intravenous LPS (3 mg/kg) or Saline Administration and Effects of Incubation With Aminoguanidine (10 4 mol/l) and L-NNA (10 4 mol/l) Sham-Operated Portal Vein Stenosed A Baseline AG L-NNA Baseline AG L-NNA E max Saline * * LPS pec 50 Saline * LPS NOTE. Results are expressed as means SEM. Abbreviation: AG, aminoguanidine. *P.05 vs. sham-operated rats. P.05 vs. baseline. P.05 vs. saline (Student s t test). rected vascular reactivity. In contrast, after LPS administration in sham-operated rats, L-NNA completely corrected, while aminoguanidine only partially corrected, vascular hyporeactivity. Experiment 2: Aortic NOS Activity After LPS or Saline Administration. Results of total and calcium-dependent NOS activities in portal vein-stenosed or sham-operated rat aortae after LPS or saline administration are shown in Table 2. Total NOS activity was significantly increased in portal vein-stenosed rats compared with sham-operated rats. However, in all saline-treated rats, calcium-independent NOS activity was below 0.8 pmol/mg of proteins/hr and not different between portal vein-stenosed and sham-operated rats. In portal vein-stenosed rats, total and calcium-independent NOS activity increased significantly less than in shamoperated rat aortae after LPS administration. Experiment 3: Aortic NOS2 and NOS3 Protein Expression After Saline or LPS Administration. NOS2 protein expression was not detectable in aortae from portal vein-stenosed or shamoperated rats. After LPS administration, NOS2 protein expression increased significantly in portal vein-stenosed and shamoperated rat aortae. NOS2 protein expression was, however, significantly less marked in aortae from portal vein-stenosed rats than from sham-operated rats (Fig. 1). Under baseline conditions, NOS3 protein expression was significantly more marked in aortae from portal vein-stenosed than in shamoperated rats. In portal vein-stenosed rats, there was no significant change in NOS3 protein expression after LPS administration, whereas in sham-operated rats, NOS3 expression significantly increased (Fig. 2). TABLE 2. Rat Aortic NOS Activity (pmol/mg of proteins/hr) 5 Hours After LPS (3 mg/kg) or Saline Administration Sham-Operated Rats Portal Vein Stenosed Rats Saline LPS Saline LPS Total NOS activity * * Ca 2 -independent NOS activity * NOTE. Results are expressed as means SEM. *P.05 vs. sham-operated rats. P.05 vs. saline (Student s t test). FIG. 2. Protein expression of NOS3 in portal vein-stenosed and shamoperated rat aortae. Protein was measured, and equal amounts were applied to 10% SDS-polyacrylamide gels. The protein was resolved by electrophoresis and transferred to nitrocellulose. Blots were probed with a specific NOS3 antibody. Immunoreactivity was visualized by autoradiography after incubation with a secondary antibody. (A) Autoradiography of 1 representative experiment. M, mouse macrophage lysate prepared from the RAW cell line stimulated with interferon gamma (10 ng/ml) and LPS (1 mg/ml) for 12 hours; E, human endothelial lysate derived from an aortic endothelium cell line (positive control). 1, aorta of sham operated rat 5 hours after intravenous saline administration; 2, aorta of portal vein-stenosed rat 5 hours after intravenous saline administration; 3, aorta of sham-operated rat 5 hours after intravenous LPS administration; 4, aorta of portal vein-stenosed rat 5 hours after intravenous LPS administration. (B) Autoradiographs were scanned by laser densitometry, and results are expressed as percentage of NOS3 protein expression in sham-operated rat aortae after saline administration. Experiment 4: Aortic NOS Activity After In Vitro Incubation With LPS. After 5 hours of incubation in sterile Krebs-Henseleit solution, NOS activity was significantly higher in aortae from portal vein-stenosed than in aortae from sham-operated rats (Table 3). Incubation of endothelium-denuded aortae or intact aortae in Krebs-Henseleit solution containing W7 significantly decreased NOS activity without any statistical difference between portal vein-stenosed and sham-operated

5 702 HELLER ET AL. HEPATOLOGY September 1999 TABLE 3. Rat Aortic NOS Activity (pmol/mg of proteins/hr) After 5 Hours in Krebs-Henseleit or LPS (0.1 mg/ml), Effects of Endothelium, W7 (10 mol/l), and SNAP (600 mol/l) Sham-Operated Portal Vein Stenosed Basal LPS Incubation Basal LPS Incubation With endothelium * * With endothelium W Without endothelium * Without endothelium SNAP NOTE. Results are expressed as means SEM (n 4 rats). *P.05 vs. sham-operated rats. P.05 vs. basal condition. P.05 vs. aortae with endothelium. (Student s t test). P.05 vs. aortae without endothelium. rats. When intact aortae were incubated in Krebs-Henseleit solution containing LPS, NOS activity only increased significantly in sham-operated rats. NOS activity after incubation in LPS-containing Krebs-Henseleit solution was significantly lower in portal vein-stenosed than in sham-operated rats. In contrast, when endothelium-denuded aortae were incubated with LPS, there was a significantly greater increase in total and calcium-independent NOS activity in portal veinstenosed than in sham-operated rats. Similar results were obtained when intact aortae were incubated in Krebs- Henseleit solution containing W7. In this case, NOS activity increased after the addition of LPS, but there was no difference between portal vein-stenosed and sham-operated rats. Furthermore, when endothelium-denuded aortae of both groups were incubated with LPS and the NO donor, SNAP, NOS activity was significantly lower than in endothelium-denuded aortae incubated with LPS alone and even lower than in intact aortae incubated with LPS. DISCUSSION This study confirmed that under baseline conditions, the aortic hyporeactivity to phenylephrine in portal veinstenosed rats was associated with increased vascular NOS3 activity 18,19 and expression 21 without induction of NOS2. 22,24 In portal hypertensive rats, normal vascular reactivity was restored by L-NNA, but not by NOS2 inhibition with aminoguanidine. Furthermore, total, but not calcium-independent, NOS activity was enhanced. NOS3 protein expression was increased without any significant detection of NOS2 in aortic tissue, and finally, NOS activity was inhibited by endothelial denudation or calmodulin inhibition in the aortae of portal vein-stenosed rats. These results are restricted to the thoracic aorta of portal vein-stenosed rats, because in CCl 4 -treated rats with ascites, NOS2 as well as NOS3 overexpression has been reported, especially in the mesenteric vascular bed. 26 In the present study, in contrast to controls, LPS administration did not change aortic ring tension induced by phenylephrine in portal vein-stenosed rats. Moreover, in aortae from portal vein-stenosed rats, LPS administration did not increase total NOS activity and increased NOS2 activity less than in controls. Finally, NOS2 expression increased significantly less in portal vein-stenosed rats than in sham-operated rats, and NOS3 expression did not change significantly after LPS administration, whereas in sham-operated rats, aortic NOS2 and NOS3 protein levels increased significantly. The effects of LPS on aortic NOS3 protein expression have never been investigated, and thus, no increase in NOS3 protein expression has been reported. On the other hand, it has been shown that LPS may destabilize vascular NOS3 mrna and thus decrease NOS3 mrna expression. 29 However, in normal bovine endothelial cells in culture, the combination of LPS and interferon increased NOS3 mrna and activity. 30 These discrepant results may be the result of two different experiments (e.g., dose of LPS) and different delays after LPS exposure and NOS measurements. Furthermore, LPS may have increased vascular shear stress and thus NOS3 expression, 45 because it has been shown that this dose of LPS produces an increase in heart rate without a significant change in mean arterial pressure in the rat. 37 Because altered hepatic LPS metabolism might contribute to diminished NOS2 induction in portal vein-stenosed rats, in vitro incubation of aortae was performed. In vitro, LPS increased NOS activity in sham-operated, but not in portal vein-stenosed, rats. Thus, the reduced NOS2 activation in aortae from portal vein-stenosed rats after LPS challenge is caused by a local vascular defect. After removal of the aortic endothelium, basal NOS activity was markedly reduced without any differences between portal vein-stenosed and sham-operated rats. In shamoperated rats, incubation of endothelium-denuded aortae with LPS resulted in lower NOS activity than incubation of aortae with an intact endothelium. These results confirm the results of Fleming et al., 42 who found an increased sensitivity to LPS in aortae with an intact endothelium compared with endothelium-denuded aortae in normal rats using isolated vessel techniques. Endothelium-denuded aortae from portal vein-stenosed rats, however, exhibited higher NOS activity than aortae with an intact endothelium after LPS exposure. Thus, destroying the endothelium eliminates endothelial NOS3 overactivity, while the ability to induce NOS activity after LPS exposure is increased in aortae from portal hypertensive rats. To further investigate the influence of the endothelium on NOS induction after LPS exposure, intact aortae were incubated with LPS and the calmodulin inhibitor, W7. It has previously been demonstrated that W7 selectively inhibits NOS3 and corrects vascular hyporeactivity in aortae from portal vein-stenosed and cirrhotic rats. 44,46 Indeed, incubation with W7 inhibited basal NOS activity in sham-operated rats and in portal vein-stenosed rats. In sham-operated rats, W7 reduced NOS activity after LPS exposure. Although W7 selectively inhibits NOS3 activity without affecting NOS2 activity, the synthesis of bioactive NOS2 protein might be somewhat inhibited by W7. Calmodulin is tightly bound to NOS2 during enzyme synthesis, 47 and W7 might therefore partly inhibit calmodulin integration in the NOS2 protein. However, when W7 was added to LPS incubation, NOS activity was significantly higher in portal vein-stenosed rats than in sham-operated rats. This suggests that W7 inhibits aortic NOS3 overactivity in portal vein-stenosed rats and that the reduced ability to induce NOS2 after LPS exposure in portal vein-stenosed rat aortae is corrected by this W7- induced NOS3 inhibition. Finally, when endotheliumdenuded aortae were incubated with LPS and the NO donor, SNAP, NOS activity was lower than after incubation of endothelium-denuded or even intact aortae of both groups. This, indeed, proves that NO can inhibit LPS-induced NOS2 activation. The exact mechanisms of this hypothetical inhibi-

6 HEPATOLOGY Vol. 30, No. 3, 1999 HELLER ET AL. 703 tion of endothelium-nos3 derived NO on NOS2 induction in portal vein-stenosed rats are unclear. In vitro, it has been shown that NO donors inhibit LPS-mediated induction 33 and activity 32 of NOS2 in macrophages. NO might inhibit NOS2 induction by stabilizing the NF- B/I B junction 48,49 and thus inhibiting the nuclear translocation of the NOS2 transcription factor, NF- B. 50 In conclusion, this study demonstrated that in contrast to sham-operated rats, aortae from portal vein-stenosed rats did not reduce in vitro vascular reactivity after LPS administration. This was associated with higher aortic NOS3 activity and expression under baseline conditions, but lower induction of aortic NOS2 activity and expression after LPS challenge in portal vein-stenosed rats compared with shamoperated rats. Furthermore, this study showed that the diminished induction of NOS activity after LPS challenge in aortae of portal vein-stenosed rats can be corrected by endothelium removal or by calmodulin inhibition. These results suggest that an endothelium-derived, calmodulindependent factor, most likely the NO produced by activated endothelial NOS3, inhibits induction of aortic NOS2 after LPS in aortae of portal vein-stenosed rats. Acknowledgment: The authors thank Laurent Font for excellent technical assistance. REFERENCES 1. Caly WR, Strauss E. A prospective study of bacterial infection in patients with cirrhosis. J Hepatol 1993;18: Llach J, Rimola A, Navasa M, Gines P, Salmeron JM, Gines A, Arroyo V, et al. Incidence and predictive factors of first episode of spontaneous bacterial peritonitis in cirrhosis with ascites: relevance of ascitic fluid protein concentration. HEPATOLOGY 1992;16: Moreau R, Hadengue A, Soupison T, et al. 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