Nitric oxide, a vasodilator molecule, plays a central

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1 GASTROENTEROLOGY 2003;125: Mesenteric Vasoconstriction Triggers Nitric Oxide Overproduction in the Superior Mesenteric Artery of Portal Hypertensive Rats MING HUNG TSAI,*, YASUKO IWAKIRI,*,, GREGORY CADELINA,* WILLIAM C. SESSA, and ROBERTO J. GROSZMANN*, *Hepatic Hemodynamic Laboratory, Veterans Administration Medical Center, West Haven; Section of Digestive Diseases, Department of Internal Medicine, and Boyer Center for Molecular Medicine and Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut Background & Aims: Vasoconstriction of the superior mesenteric artery (SMA)is the earliest hemodynamic event occurring after partial portal vein ligation (PVL). We tested the hypothesis that this early vasoconstriction of the SMA may initiate enos up-regulation in PVL. Methods: Portal hypertension with or without mesenteric vasoconstriction was induced by differentially calibrated stenosis of the portal vein (PVL-20G and PVL- 18G, respectively). In a separate group of rats, mesenteric vasoconstriction was achieved by renal artery ligation. Sham-operated rats were used as controls. Effects of vasoconstriction of the SMA in PVL and RAL rats were evaluated by measuring perfusion pressure changes in isolated SMA beds in response to methoxamine, nitric oxide synthase activity, and enos protein expression. Mean arterial pressure, portal pressure, and SMA blood flow were measured by catheterization and Doppler flowmetry. SMA vascular resistance was calculated from arterial pressure, portal pressure, and SMA flow. Results: There was a significant increase in SMA vascular resistance in PVL-20G ( vs mm Hg/% flow; P < 0.05)and RAL ( vs mm Hg/% flow; P < 0.05)but not in PVL-18G, showing mesenteric vasoconstriction in both PVL-20G and RAL groups. The mesenteric vasculature of PVL-20G and RAL animals showed hyporeactivity to methoxamine (P < 0.01). Whereas both PVL groups were portal hypertensive (P < 0.01), RAL rats were not. The SMA hyporeactivity of PVL-20G and RAL rats was corrected by N G -monomethyl-l-arginine, and nitric oxide synthase enzyme activity was significantly higher in PVL- 20G and RAL rats (P < 0.05). Conclusions: Mesenteric arterial vasoconstriction plays a triggering role in upregulation of enos catalytic activity in the SMA of portal hypertensive rats. Nitric oxide, a vasodilator molecule, plays a central role in the regulation of vascular tone. 1 In portal hypertension, excessive NO production in arteries of the systemic and particularly of the splanchnic circulation contributes to the vasodilatation and the hyperdynamic circulatory syndrome, 2 5 a hallmark of portal hypertension in liver cirrhosis. 6 Currently, however, little is known about the mechanism(s) behind the initiation of this splanchnic NO overproduction in portal hypertension. The hemodynamic disarray of systemic and splanchnic circulation evolves with different timing and to a greater extent in the splanchnic vasculature of portal hypertensive rats. 4,7,8 In fact, we have recently shown that endothelial nitric oxide synthase (enos)-derived NO overproduction in the superior mesenteric artery (SMA) is evident 1 day after the induction of portal hypertension by partial portal vein ligation (PVL). 9 In this PVL model, the hyperdynamic circulation becomes evident on the fourth or fifth postoperative day, 10 suggesting that enos up-regulation and subsequent NO overproduction occurs before the development of the hyperdynamic circulation. Mesenteric vasoconstriction is the initial event observed in response to an acute increase in portal pressure in PVL animals. 8,11 Furthermore, a decreased portal venous inflow, possibly due to mesenteric vasoconstriction, has also been observed in the natural history of portal hypertension in schistosomiasis Mansoni-infected hamsters, a chronic model of portal hypertension. 12 Because vasoconstriction of various origins has been documented to trigger enos up-regulation, we tested the hypothesis that SMA vasoconstriction observed in the early phase of portal hypertension in PVL animals may trigger the initial enos up-regulation and NO overproduction. Abbreviations used in this paper: enos, endothelial nitric oxide synthase; inos, inducible nitric oxide synthase; L-NMMA, N G -monomethyl-l-arginine; MAP, mean arterial pressure; NOS, nitric oxide synthase; PVL, portal vein ligation; RAL, renal artery ligation; R SMA, superior mesenteric artery resistance; SMA, superior mesenteric artery by the American Gastroenterological Association /03/$30.00 doi: /s (03)01360-x

2 November 2003 enos IN ACUTE PORTAL HYPERTENSION 1453 To test this hypothesis, SMA vasoconstriction should be induced without producing portal hypertension. Renal artery stenosis is known to trigger mesenteric vasoconstriction. 16 It has also been shown that renal vasoconstriction takes place in the setting of portal hypertension, probably through different mechanisms Therefore, we induced SMA vasoconstriction by renal artery ligation (RAL) without producing portal hypertension. On the other hand, to further establish the causal relationship between mesenteric vasoconstriction and enos up-regulation, we also created portal hypertensive states with and without SMA vasoconstriction by performing differentially calibrated stenosis of the portal vein. These models allowed us to examine the role of vasoconstriction in enos up-regulation in the SMA. We found that the SMA vasoconstriction, not portal hypertension in itself, increased NOS catalytic activity and leads to SMA hyporeactivity to vasoconstrictors. Overall, our findings suggest the potential role of SMA vasoconstriction in triggering the initial NO overproduction in the SMA of portal hypertensive animals. Materials and Methods The investigation was performed in male Sprague- Dawley rats (Harlan Sprague-Dawley Laboratories, Indianapolis, IN) weighing g. All experimental procedures in this study were performed in accordance with the standard procedures indicated in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, DHHS publication, 1985). Surgical Procedures Implantation of SMA flow probes. Pulsed-Doppler flow probes were sterilized with ethylene oxide gas. The animals were fasted for 8 hours before surgery but allowed free access to water. All of the animals were anesthetized with ketamine hydrochloride 100 mg/kg body wt (Ketalar; Parke- Davis, Avon, CT). Survival surgery was performed under sterile conditions as described previously. 20 In brief, the abdomen and middorsal neck were shaved and cleansed with povidone iodine solution. A midline laparotomy incision was made, and the small and large intestines were wrapped in sterile salinesoaked gauze and gently displaced to the left side of the animal. The SMA was identified at its aortic origin and a 5-mm segment gently dissected free from surrounding tissues. A pulsed-doppler cuff-type flow transducer (ID, 1 mm; frequency, 20 mhz; Iowa Doppler, Iowa City, IA) was placed around the SMA and secured with a 4-0 silk tie threaded through the cuff. The leads were tunneled to the middorsal region of the back and fixed to the skin with buttons. Partial PVL and sham operation. A prehepatic portal hypertensive animal model extensively studied in our laboratory and others 7,11 was used. This model mimics the hyperdynamic splanchnic and systemic circulation observed in cirrhosis. 7,11 Portal hypertension was induced surgically in aseptic conditions. Briefly, the portal vein was freed from surrounding tissue after a midline abdominal incision. In the traditional PVL model, a ligature (silk gut 3-0) was placed around a 20-gauge (PVL-20G) blunt-tipped needle lying along the portal vein. In a separate group, in which portal hypertension without mesenteric vasoconstriction was desired, a larger 18-gauge blunt-tipped needle was used instead (PVL- 18G; n 19). Subsequent removal of the needle yielded a calibrated stenosis of the portal vein. In sham-operated rats, the same operation was performed with the exception that, after the portal vein was isolated, no ligature was placed. In the experiments to study the SMA flow, the baseline measurement (in kilohertz) of SMA flow was always obtained before PVL and sham operation. The range of the pulsed-doppler probe was adjusted until the maximal kilohertz shift for phasic flow was obtained; thereafter, the range was kept constant until the final experiment. Partial RAL. A midline laparotomy was performed. Small and large intestines were gently retracted to the right side of the animals. The intestines were wrapped in warm, sterile, saline-soaked gauze. The left renal artery was localized and a 5-mm segment dissected with thin, curved forceps. A ligature (silk gut 3-0) was made around a 30-gauge, blunttipped needle lying along the left renal artery. Subsequent withdrawal of the needle yielded a calibrated stenosis of the left renal artery. The efficacy of RAL was validated by preliminary study (renal blood flow decreased to 45.2% 2.56% of baseline by using pulsed-doppler flowmetry; n 4) and by visualization of wedged ischemic patches in the experiments 10 hours after surgery. The baseline flow of SMA was documented before RAL as previously described. In vitro perfusion system. The in vitro perfusion technique used was a modification of that originally described by McGregor 21 and previously used in our laboratory. 22,23 Briefly, the SMA was cannulated with a PE-60 catheter and gently perfused with 15 ml of warm Krebs solution to eliminate blood. After the SMA was isolated with its mesentery, the gut was sharply dissected near its mesenteric border. The SMA with its associated mesenteric tissue was placed into a 37 C water-jacketed container and perfused at a constant rate (4 ml/min) with oxygenated 37 C Krebs solution (95% O 2, 5% CO 2 ) through a roller pump (Masterflex; Cole-Parmer Co., Barrington, IL). The Krebs solution had the following composition (in mmol/l): 118 NaCl, 4.7 KCl, 1.2 KH 2 PO 4, 1.2 MgSO 4, 2.5 CaCl 2, 25 NaHCO 3, edetate calcium disodium, and 11 glucose, ph 7.4. Under these conditions, this preparation has been shown to be viable over several hours with unchanged perfusate NO concentration under basal conditions 22,23 and unaltered pressor responsiveness. 23 The effluent from the perfused tissue was removed continuously from the perfusion chamber to prevent exogenous exposure of the tissue to perfusate-containing drugs. The tissue preparation was covered lightly with a piece of Parafilm (American National Can, Norwalk, CT) to prevent drying. Polyvinyl chloride free circulation tubing (Abbott Laboratories, Abbott Park, IL)

3 1454 TSAI ET AL. GASTROENTEROLOGY Vol. 125, No. 5 was used to avoid NO absorption by the tubes. Pressure was transmitted through a Hewlett-Packard (Cupertino, CA) transducer and recorded on a Macintosh (Andover, MA) desktop computer. Experimental Design Rats were divided into study protocols as described in the following text. This study was divided into 2 parts. The first part was aimed to determine the temporal evolution of enos in the SMA of the traditional PVL (PVL-20G) before the establishment of the hyperdynamic circulation. The second part was aimed to test the role of mesenteric vasoconstriction in initiating enos up-regulation. After surgery, animals were housed in individual cages and allowed free access to normal rat diet and water. The experiments were performed while the rats were under ketamine anesthesia (100 mg/kg intramuscularly) in a nonfasting state. In the first study, experiments were performed 1 hour, 10 hours, 16 hours, 1 day, and 2 days after surgery. In the second study, experiments were performed at 10 hours and 16 hours for the RAL and PVL groups, respectively. In vitro perfusion study. In all experiments, baseline perfusion at 4 ml/min was established for 30 minutes before methoxamine-containing Krebs solution was used to challenge the preparation. After this period, vessel preparation in this system is maximally dilated. 21 Two doses of methoxamine (30 and 100 mol/l; Sigma Chemical Co., St. Louis, MO) were administrated noncumulatively by constant infusion to the mesenteric vasculature of PVL rats at different time points (n 35 for PVL-20G; n 6 for PVL-18G; n 7 for RAL) and corresponding sham-operated rats (n 37) for 2 minutes. The role of NO was examined by additional experiments. Vessel preparations (PVL-20G, n 18; RAL, n 5; corresponding sham rats, n 22) were preincubated with N G -monomethyl-l-arginine (L-NMMA) ( mol/l; Alexis Co., San Diego, CA) for 50 minutes before a vasopressor was added. L-NMMA was present at the same molar concentration over the whole study period. Western blotting. SMA vessels were tested for the presence of enos and inducible nitric oxide synthase (inos) protein. SMAs were harvested after freeing the artery from the surrounding tissue over a length of 3 4 cm, starting at its aortic origin. Vessels were washed in phosphate-buffered saline and homogenized in a lysis buffer described previously. 10 Protein supernatants were quantified using the Lowry assay, and equal amounts of protein from each sample were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose membranes. Membranes were probed with an antibody recognizing enos (Transduction Laboratories, Lexington, KY) and -actin (Sigma Chemical Co.) and a monoclonal antibody recognizing inos (Transduction Laboratories). The specificity of the enos and inos antibodies for rat tissue was established previously by using quiescent and activated rat sinusoidal endothelial cells, respectively, as positive controls. 10 Enhanced chemiluminescence was used for protein detection. The intensity of the bands corresponding to the proteins of interest was measured using a densitometer. To ensure an equal loading of protein, expression of -actin was used for standardization. Nitric oxide synthase activity assay. Nitric oxide synthase (NOS) activity was measured by determining the conversion of 14 C-labeled L-arginine to 14 C-labeled L-citrulline. Briefly, mesenteric tissue was harvested as previously described and then minced with fine scissors and homogenized in lysis buffer. The tissue homogenates were centrifuged, and supernatant was used for the assay. Samples were incubated with a reaction buffer containing 1 mmol/l reduced nicotinamide adenine dinucleotide phosphate, 3 mol/l Tetrahydrobiopterin (BH 4 ), 100 nmol/l calmodulin, 2.5 mmol/l CaCl 2, 1 mol/l flavin adenine dinucleotide, 1 mol/l flavin mononucleotide (FMN), and 1.4 mol/l L-[ 14 C]-arginine (25.2 nci) at 37 C. To determine NOS activity, duplicate samples were incubated for 30 minutes in the presence and absence of 2 mmol/l N G -nitro-l-arginine methyl ester. The reaction was terminated by the addition of 1 ml of cold stop buffer (20 mmol/l HEPES, 2 mmol/l ethylenediaminetetraacetic acid, and 2 mmol/l ethylene glycol-bis[ -aminoethyl ether]- N,N,N,N -tetraacetic acid, ph 5.5), and the reaction mix was passed over a Dowex AG 50WX-8 (Bio-Rad Laboratories, Hercules, CA) resin column into a vial and analyzed using a liquid scintillation counter. Radiolabeled counts per minute of L-citrulline generation were measured and used to determine N G -nitro-l-arginine methyl ester inhibitable NOS activity. Hemodynamic Measurements Pulsed-Doppler flowmetry. As previously mentioned (see Surgical Procedures), a baseline measurement of SMA flow was taken at the time of surgery (after Doppler flow probe implantation but before PVL or RAL). Later, during the final experiment, the flow probe leads were reconnected to measure SMA flow at the various time points following PVL or RAL. The value of the mean flow was recorded and used for calculations. Pulsatile velocity tracing was also obtained to ensure the quality of the Doppler shift signal. The pulsed-doppler technique does not measure absolute flow. Pulsed-Doppler flowmetry quantitates blood cell velocity, which is directly and linearly proportional to volumetric flow. 24 In previous work, we have validated the relationship between flow velocity as a Doppler shift (in kilohertz) and absolute flow (in milliliters per minute). 25 To overcome the inherent variation among probes, we expressed the SMA flow as a percentage of baseline flow (in kilohertz) as we previously described. 8,20 Mean arterial pressure, portal pressure, and SMA resistance. Mean arterial pressure (MAP) and portal pressure were measured by catheterization of the right femoral artery and ileocolic vein, respectively. Pressure was transmitted through a Hewlett-Packard transducer and recorded on a Macintosh desktop computer. SMA resistance (R SMA ) was calculated from the formula described in our previous report 8 : R SMA (mm Hg/% flow) (MAP Portal Pressure)/% SMA Flow.

4 November 2003 enos IN ACUTE PORTAL HYPERTENSION 1455 Statistical Analysis Results are expressed as mean SE. Statistical analysis was performed using 2-way analysis of variance with repeated measurements, one-way analysis of variance followed by Scheffe test, and unpaired Student t test when appropriate. A 2-tailed P value of 0.05 was considered statistically significant. Results There were no significant differences in body weight among experimental groups. Time Course Experiment Although induction of portal hypertension by partial ligation of the portal vein (PVL-20G) is a commonly used method to study the hemodynamic characteristics associated with portal hypertension, it is not known how early the SMA hyporesponse to vasoconstrictors occurs in this model. To determine the initial factor that induces enos activation in the SMA, it is critical to know the point at which SMA becomes hyporesponsive to vasoconstrictors in this model. Thus, we first performed an in vitro perfusion pressure study to confirm Figure 1. The perfusion pressure changes in response to methoxamine in isolated mesenteric arterial beds at different times. (A) The vasopressor responses of the PVL-20G animals were compared with the sham-operated animals at different times. n 5 8 per group per each time point. *P 0.01, # P compared with the corresponding sham rats. (B) The vasopressor responses to methoxamine after NOS inhibitor (L-NMMA) treatment in the isolated SMA beds. n 4 6 per group per each time point. MTx30, methoxamine 30 mol/l; MTx100, 100 mol/l. Figure 2. Total NOS activity in rat SMA at different times. NOS activity was significantly enhanced in the SMA of the PVL-20G rats after 10 hours compared with the sham-operated rats. n 3 6 per group per each time point. *P 0.05 compared with corresponding shamoperated rats. the point at which SMA becomes hyporesponsive to vasopressor in PVL. We also determined enos enzyme activity and enos protein expression associated with SMA vasodilatation at the different time points after PVL. Baseline perfusion pressures were not significantly different in vascular preparations of PVL-20G and sham groups (data not shown). Hyporeactivity in response to methoxamine became significant in the isolated SMA beds of PVL-20G rats compared with sham-operated rats beginning 10 hours postoperatively and thereafter (P 0.01; Figure 1A). To test whether this decreased contractile response to methoxamine in the SMA of PVL- 20G rats was due to NO, we used the NOS inhibitor L-NMMA. The treatment of vessels with L-NMMA significantly increased the perfusion pressures in both sham and PVL-20G rats (P 0.01; Figure 1B) and abolished the perfusion pressure difference between the 2 groups. We determined NOS activity of SMA isolated from the PVL-20G and sham-operated rats at different times (Figure 2). NOS activity became significantly higher in PVL-20G rats beginning 10 hours postoperatively and thereafter compared with the sham group (P 0.05). We also performed Western blot analysis to determine the protein expression of enos and inos in SMA. In agreement with previous studies, 2,4,10,22,26 inos protein was not detected in SMA. Interestingly, there was no significant difference in enos protein expression between the sham and PVL-20G groups (data not shown), suggesting that up-regulation of enos catalytic activity, not the protein expression, is the initial event responsible for early NO overproduction in the SMA of PVL-20G animals. 9 Collectively, these observations suggest that

5 1456 TSAI ET AL. GASTROENTEROLOGY Vol. 125, No. 5 Figure 3. The perfusion pressure changes in response to methoxamine in isolated mesenteric arteries. (A) The comparison of contractile responses with methoxamine in the isolated mesenteric arterial beds of PVL-20G, PVL-18G, and sham rats. MTx30, methoxamine 30 mol/l; MTx100, 100 mol/l. n 6 for sham, n 8 for PVL-20G, and n 6 for PVL-18G. *P 0.05 compared with sham; **P 0.05 compared with PVL-18G. (B) The comparison of contractile responses to methoxamine in mesenteric arterial beds isolated from the RAL and sham rats (n 7 in each group; *P 0.01). The SMA beds were isolated at 10 hours after RAL or sham operation. (C) Effects of NOS inhibition by L-NMMA on the contractile response to methoxamine in isolated mesenteric arterial beds. Preincubation with L-NMMA abolished the differences in the pressure response to methoxamine between the PVL-20G (n 4) and sham (n 5) groups. (D) Preincubation with L-NMMA abolished the differences in the pressure response to methoxamine between the RAL (n 5) and sham (n 4) groups. NO overproduction occurs as early as 10 hours, a much earlier time than the development of the hyperdynamic circulation, which is usually seen 4 days after the induction of portal hypertension. Comparisons Between RAL and PVL (20G and 18G) In vitro perfusion study. As shown in the time course experiment, SMA beds of PVL-20G rats developed hyporesponsiveness to the vasoconstrictor methoxamine as early as 10 hours after PVL and thereafter. Based on this observation, we determined the vasopressor response to methoxamine in SMA beds isolated from RAL rats. Baseline perfusion pressures of SMA beds were not significantly different between RAL and sham-operated rats (data not shown). Similar to PVL-20G rats (P 0.01; Figure 3A), RAL rats showed hyporesponsiveness to methoxamine at 10 hours postoperatively (P 0.01; Figure 3B). However, unlike PVL-20G and RAL rats, PVL-18G rats did not exhibit hyporeactivity to methoxamine (P 0.222; Figure 3A). To test whether this decreased response to methoxamine in RAL rats was due to NO overproduction, we perfused an NO inhibitor (L- NMMA) first and then determined the contractile response to methoxamine in the SMA of RAL rats. Similar to the PVL-20G rats (Figure 3C), L-NMMA corrected the hyporeactivity of the SMA to methoxamine in RAL rats (Figure 3D). This correction of the vasoreactivity differences between the RAL and sham-operated rats by L-NMMA indicates that NO mediates the hyporeactivity to methoxamine observed in PVL-20G and RAL rats. NOS catalytic activity. Similar to the PVL-20G group (P 0.05; Figure 4A), NOS activity of RAL rats was significantly higher than that of the sham group (P 0.05; Figure 4B). Unlike PVL-20G and RAL rats,

6 November 2003 enos IN ACUTE PORTAL HYPERTENSION 1457 Figure 4. Total NOS activity in homogenized SMA beds. (A) Total NOS activity in the SMA of the PVL (n 5 for PVL-20G; n 4 for PVL-18G) and the sham (n 5) groups. *P Total NOS activity was significantly enhanced in the SMA of PVL-20G rats compared with PVL-18G and sham rats. (B) Total NOS activity in the SMA isolated 10 hours after RAL (n 6) and sham (n 6) operation. RAL exhibited significantly higher NOS activity compared with the sham rats. *P there was no significant difference in NOS activity between PVL-18G and sham-operated rats (Figure 4A). This further confirms the involvement of NO in SMA hyporeactivity to methoxamine observed in PVL-20G and RAL rats. Western blot analysis. To determine the NOS isoform responsible for the NO overproduction in SMA beds, enos and inos protein expression were determined in SMA beds isolated from PVL-20G, PVL-18G, RAL, and sham animals (Figure 5A and B). The densitometric analysis suggested that there was no significant difference in enos protein levels among groups. inos protein was not detected in any groups. Cell lysate of macrophages challenged with lipopolysaccharide was used as a positive control for inos protein. Hemodynamic studies. Hemodynamic characteristics were determined in PVL-20G, PVL-18G, and RAL rats as well as their corresponding sham-operated rats. As shown in Figure 6A, both of the PVL groups were portal hypertensive compared with the sham-operated group (PVL-20G, ; PVL-18G, ; sham, mm Hg; P 0.001). MAP was significantly lower in both PVL groups compared with the sham-operated group (PVL-20G, ; PVL-18G, ; sham, mm Hg; P 0.01). The SMA flow in the PVL-20G group was significantly lower compared with the PVL-18G and sham-operated groups (PVL-20G, 37.42% 0.86%; PVL-18G, 78.6% 2.38%; sham, 99.44% 3.43%; P 0.001). The PVL-20G group exhibited significantly higher R SMA compared with the PVL-18G and sham groups (PVL-20G, ; PVL-18G, ; sham, mm Hg/% flow; P 0.05), whereas the PVL-18G group did not display an increase in R SMA (P 0.991). Hemodynamic data of the RAL group and the corresponding sham animals are shown in Figure 6B. MAP was significantly higher in the RAL group at 10 hours compared with the sham group ( vs mm Hg; P 0.05). Similar to the PVL group, the SMA flow was significantly lower in the RAL group compared with the sham group (59.05% 3.94% vs % 2.56%; P 0.05). The RAL rats also exhibited significantly higher R SMA than that of the sham group ( vs mm Hg/% flow; P 0.05), suggesting the presence of vasoconstriction in the SMA of RAL rats. There was no difference between the RAL and sham groups in portal pressure. Discussion In this study, we observed that SMA vasoconstriction induced by RAL leads to NO-mediated SMA hyporeactivity similar to that observed in portal hypertensive rats. On the other hand, we also showed that, in the early stage, a portal hypertensive state without mesenteric vasoconstriction is not associated with enos up-regulation. These findings suggest a possible contribution of early vasoconstriction of the SMA to the initial NO overproduction observed in the SMA of portal hypertensive animals. Vasoconstriction mediated by a myogenic reflex in PVL rats 27 is the initial response of the SMA to an acute elevation in portal pressure. 8,28 This is followed by a vascular escape phenomenon characterized by restitution of blood flow. NO has been reported to ameliorate vaso-

7 1458 TSAI ET AL. GASTROENTEROLOGY Vol. 125, No. 5 Figure 5. enos and inos protein expression in SMA beds. (A) enos and inos protein expression in the SMA of the PVL (n 4 for PVL-18G; n 3 for PVL-20G) and the sham (n 3) groups. Bovine aorta endothelial cell lysate was used as a positive control for enos. Lysate of peritoneal macrophages stimulated with lipopolysaccharide 100 ng/ml for 16 hours was used for the positive control for inos protein. (B) enos and inos protein expression in renal artery harvested 10 hours after RAL (n 3) and sham (n 3) operation. There was no significant difference in SMA enos expression in PVL-20G, PVL-18G, and RAL rats compared with the corresponding sham-operated rats. constriction of various origins in different vascular beds, 13 15,29 thus contributing to vascular escape. 30 SMA blood flow was markedly decreased in PVL and RAL rats as previously described. 8,16 This reduction in SMA blood flow is the result of SMA vasoconstriction, which was triggered by a myogenic response in the PVL rats and probably by vasoconstrictors such as the reninangiotensin system in the RAL rats. A unique characteristic of enos is the production of NO in response to an increase in blood flow and vasoconstriction. 31 In this stage of the PVL model, vasoconstriction seems to be the stimulus for enos up-regulation. Increased production of NO has been shown in various animal models of chronic liver diseases and portal hypertension. 6 It plays a pivotal role in the vasodilatation and the development of the hyperdynamic state in cirrhotic animals and patients, thus contributing to morbidity and mortality in chronic liver disease. 32 Vascular hyporeactivity to vasoconstrictors is one of the major pathophysiologic hallmarks accompanying chronic portal hypertension, which is mediated largely by NO. 23 Despite the well-established role of NO in the hemodynamic impairment of chronic portal hypertension, the mechanisms behind the NOS up-regulation remain poorly understood and open to speculation. In the first part of the present study, we showed that enos up-regulation of the SMA with impaired response to vasoconstrictors takes place within a few hours after the induction of portal hypertension. Therefore, the up-regulation of enos-derived NO overproduction of the splanchnic vasculature occurs during the period in which there is reflex vasoconstriction of the SMA in response to portal vein constriction. The decreased SMA flow in the initial stages of portal hypertension is not unique to the PVL model. We have also shown similar change in a schistosomiasis Mansoni model of portal hypertension. 12 Interestingly, in Schistosoma-infected hamsters, the portal inflow gradually decreases, while the portal pressure slowly increases in the absence of portosystemic shunting. This evidence might substantiate our speculation that chronic liver disease may share some common mechanisms with PVL to initiate NOS up-regulation at a different pace and to a different extent. It is possible that a minimal portal pressure threshold is necessary to trigger a myogenic vasoconstriction of the SMA and that this threshold is reached late in chronic liver diseases. Whether this is also true in cirrhosis remains to be studied. In the early phase of portal hypertension (as early as 10 hours after PVL), our data suggested that the catalytic activity of enos, not enos protein expression, was up-regulated to induce NO-mediated hyporeactivity to the vasopressor methoxamine in the SMA, the main site of the pathophysiologic abnormalities in portal hypertension. In contrast, it has been suggested that both enos protein expression and enzyme activity are upregulated in chronic portal hypertension. 9 These lines of evidence suggest that posttranslational modification of enos and a subsequent increase in enzyme activity play

8 November 2003 enos IN ACUTE PORTAL HYPERTENSION 1459 Figure 6. Hemodynamic characteristics of PVL-20G, PVL-18G, and RAL and the sham-operated rats. (A) Hemodynamic study of the PVL and sham groups. PP, portal pressure; Q SMA, SMA blood flow. *P 0.05 compared with the sham group. Sham, n 5; PVL-20G, n 6; PVL-18G, n 5. (B) Hemodynamic study of the RAL and sham groups. *P Sham, n 6; RAL, n 5. One-way analysis of variance followed by Scheffe test.

9 1460 TSAI ET AL. GASTROENTEROLOGY Vol. 125, No. 5 a role in the early enos up-regulation in the SMA of portal hypertensive animals. In fact, we recently reported that enos is posttranslationally up-regulated by phosphorylated Akt in early portal hypertension. 9 Additionally, no inos protein was detected, hence ruling out a significant role of this isoform in NO overproduction in the SMA of acute portal hypertensive rats. NOS activity tends to be higher in PVL at 1 hour. However, the difference is not statistically significant until 10 hours after the induction of portal hypertension. This may reflect a process of an accumulation of a vasodilator to reach a physiologic significance. Actually, it is consistent with the observation that the magnitude of reactive dilatation depends on the duration of increased intraluminal pressure. 33 The mechanisms behind the vasoconstriction triggering NO overproduction have not been clearly understood. It might initiate either from the smooth muscle by presenting signals to the endothelium 34 and/or from the endothelium per se by sensing a deformation in cellular shape. 35 Considering the vasoconstrictive nature of the available drugs aimed at hyperdynamic circulation and the contribution of NO to this hemodynamic aberration, further investigation into the interplay is necessary. In conclusion, enos-mediated vascular hyporesponsiveness to methoxamine takes place during mesenteric vasoconstriction in PVL rats. It is sustained along the early course of PVL and precedes plasma volume expansion, portosystemic shunting, and hyperdynamic circulation. Mesenteric vasoconstriction, the first hemodynamic event after PVL, contributes to initiation of enos up-regulation in the SMA of PVL rats (20G). enos, not inos, is the isoform of NOS that is responsible for NO overproduction in the SMA of portal hypertensive animals. References 1. Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G. Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci U S A 1987;84: Cahill PA, Foster C, Redmond EM, Gingalewski C, Wu Y, Sitzmann JV. Enhanced nitric oxide synthase activity in portal hypertensive rabbits. Hepatology 1995;22: Martin PY, Xu DL, Niederberger M, Weigert A, Tsai P, St John J, Gines P, Schrier RW. Upregulation of endothelial constitutive NOS: a major role in the increased NO production in cirrhotic rats. Am J Physiol 1996;270:F494 F Niederberger M, Gines P, Martin PY, Tsai P, Morris K, McMurtry I, Schrier RW. Comparison of vascular nitric oxide production and systemic hemodynamics in cirrhosis versus prehepatic portal hypertension in rats. Hepatology 1996;24: Niederberger M, Martin PY, Gines P, Morris K, Tsai P, Xu DL, McMurtry I, Schrier RW. Normalization of nitric oxide production corrects arterial vasodilation and hyperdynamic circulation in cirrhotic rats. 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