NO overproduction by eNOS precedes hyperdynamic splanchnic circulation in portal hypertensive rats

Reiner Wiest1, Vijay Shah1,2,3, William C. Sessa2, and Roberto J. Groszmann1,3

1 Hepatic Hemodynamic Laboratory, Veterans Affairs Medical Center, West Haven 06516; and 2 Boyer Center for Molecular Medicine and 3 Department of Medicine, Yale University School of Medicine, New Haven, Connecticut 06510


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chronic high blood flow and the hyperdynamic circulatory syndrome in portal hypertension are associated with endothelial constitutive nitric oxide (NO) synthase (eNOS) upregulation and increased NO release. In portal vein-ligated (PVL) rats the splanchnic circulation is not yet hyperdynamic on day 3 postoperatively. In vitro perfused superior mesenteric arteries (SMAs) of day 3 PVL and sham rats were challenged with increasing flow rates or the alpha -adrenoreceptor agonist methoxamine (30 and 100 µM) before and after incubation with the NO inhibitor, Nomega -nitro-L-arginine (L-NNA, 10-4 M). Perfusate NO metabolite (NOx) concentrations were measured by chemiluminescence. PVL rats expressed a significant hyporesponsiveness to increases in flow rate or methoxamine that was overcome by incubation with L-NNA. The PVL vasculature showed significantly higher slopes of NOx production vs. flow-induced shear stress, higher increases in perfusate NOx concentration in response to methoxamine, and higher eNOS protein levels (Western blot) compared with sham rats. In conclusion, eNOS-upregulation and increased NO release by the SMA endothelium occur before the development of the hyperdynamic splanchnic circulation, suggesting a primary role of NO in the pathogenesis of arterial vasodilatation.

vasodilation; endothelial nitric oxide synthase; superior mesenteric artery


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE HYPERDYNAMIC circulatory syndrome (HCS) associated with decreased systemic and splanchnic vascular resistance, arterial hypotension, and increased cardiac output is a common hemodynamic feature in patients with advanced liver cirrhosis and animal models of portal hypertension (3, 55). The initiating mechanism of these circulatory abnormalities is an arteriolar vasodilatation, which particularly occurs in the splanchnic circulation (11, 45).

Studies using nitric oxide synthase (NOS) inhibitors have demonstrated that inhibition of nitric oxide (NO) formation attenuates the HCS in rats with CCl4-induced cirrhosis or partial portal vein ligation (PVL) (10, 39, 40). Increased NO synthesis has been reported to mediate the impaired vascular reactivity to major endogenous vasopressors (49, 50), a classical expression of arteriolar vasodilatation, in portal hypertension (8, 25). This has been confirmed in patients with liver cirrhosis (7), who also have been demonstrated to exhibit increased concentrations of NO metabolites (NOx) in serum and urine, indicating an increased NO production in these subjects (20, 23). This line of evidences suggests the existence of an excessive vascular production of NO in chronic portal hypertension.

NO is produced from L-arginine upon conversion to L-citrulline by different NOS isozymes of which two have been identified in vasculature: 1) an endothelial constitutively expressed NOS (eNOS), which is continuously present, and 2) an inducible NOS (iNOS), which requires de novo synthesis (35). Classically, eNOS causes NO release in response to physical stimuli, such as an increased blood flow and shear stress (5, 44). This is the normal mechanism mediating vascular dilatation secondary to increases in blood flow (52). Chronic high blood flow has been demonstrated to increase aortic eNOS expression and NO production (37, 47), suggesting that increased NO synthesis is a normal chronic adaptive mechanism of the endothelium in response to chronic increases in flow-induced shear stress.

Recently, several investigations demonstrated a major role for eNOS as NOS isoform responsible for vascular NO overproduction in the HCS in chronic portal hypertension. Martin et al. (32) reported an upregulation of eNOS protein in aortic and mesenteric arteries of cirrhotic rats. These findings have been confirmed by Morales-Ruiz et al. (36), who showed an increase in eNOS mRNA transcripts in arterial vessels of rats with experimental cirrhosis. A similar upregulation of eNOS has also been demonstrated in the rat PVL model, as evidenced by increased eNOS protein levels and enhanced Ca2+-dependent NOS activity in aortic and mesenteric arteries from PVL animals (6, 18, 38). Furthermore, by measuring perfusate NO concentrations in in vitro perfused superior mesenteric arteries (SMAs), we recently provided direct evidence of increased NO synthesis by the SMA of PVL animals in response to shear stress (22). However, because all of these studies were performed in chronic portal hypertensive animals with fully developed HCS, it is not yet known whether NO is the cause or the consequence of the HCS. The answer to this question would be a major step toward understanding the mechanism initiating arteriolar vasodilatation in portal hypertension.

In the PVL model the HCS is not fully developed until 4 days after induction of portal hypertension by portal vein constriction (51). Furthermore, no increase in blood flow in the superior mesenteric vasculature exists during the first 3 days after constriction of the portal vein (11). In fact, blood flow in the SMA as well as total portal venous inflow are not different in PVL rats from that in sham rats on day 3 after PVL or sham operation, respectively (11, 51). The current study was performed in rats on day 3 after surgery, thus at a time when the splanchic circulation is vasodilated but not hyperdynamic (11, 51). The aim of this investigation was to assess in in vitro perfused superior mesenteric vasculature of portal hypertensive rats whether endothelial NO production in response to changes in flow rate or vasoconstrictor stimulus is increased or not before development of a hyperdynamic splanchnic circulation.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The investigation was performed in male Sprague-Dawley rats (Harlan Sprague Dawley Laboratories, Indianapolis, IN), weighing 285-345 g. All experimental procedures in this study were conducted in accordance with the standard procedures indicated in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, DHHS Publication No. 86-23, 1985).

Induction of Portal Hypertension

A prehepatic portal hypertensive animal model extensively studied in our laboratory (9) was used. Portal hypertension was induced surgically in aseptic conditions. Briefly, the rats were anesthetized with ketamine hydrochloride (Ketalar, 100 mg/kg body wt; Parke-Davis, Avon, CT). After a midline abdominal incision, the portal vein was freed from surrounding tissue. A ligature (silk gut 3-0) was placed around a 20-gauge blunt-tipped needle lying alongside the portal vein. 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. After the operation, the animals were housed in plastic cages and allowed free access to rat food and water. All studies were performed in 12- to 18-h-fasted animals 3 days after operation.

SMA Pressure Measurements

In a separate group of animals measurements of the intraluminal pressure in the SMA were performed on days 1, 2, and 3 after PVL or sham operation (n = 3 for each day, respectively). A midline laparotomy was performed, and the small and large intestines were gently retracted to the left side of the animal. The intestines were then wrapped in warm (37°C), sterile, saline-soaked gauze and covered with parafilm (American National Can, Greenwich, CT) to avoid evaporation and heat loss. The SMA was localized, and a 1-cm segment starting about 1 cm from its aortic origin was dissected free. The SMA then was cannulated with a PE-50 catheter connected to a Statham P23 Db strain-gauge transducer (Statham, Oxnard, CA), and the SMA pressure was recorded on a Grass 7D polygraph inscriber (Grass Instrument, Quincy, MA).

In Vitro Perfusion System

The in vitro perfusion technique used was a partial modification of a technique originally described by McGregor (33) and previously used in our laboratory (49, 50). 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% O2-5% CO2) through a roller pump (Masterflex; Cole-Parmer, Barrington, IL). The Krebs solution had the following composition (in mmol/l): 118 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 2.5 CaCl2, 25 NaHCO3, 0.026 edetate Ca2+ disodium (EDTA Ca2+), and 11.0 glucose, pH 7.4. 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) to prevent drying. Polyvinyl chloride-free circulation tubing (Abbott Laboratories, Abbott Park, IL) was used to avoid NOx absorption by the tubing. Pressure was measured using a P23 Db strain-gauge transducer (Statham) on a side arm just proximal to the perfusing cannula and continuously recorded on a Grass 7D polygraph inscriber (Grass Instrument). Under these conditions, this in vitro perfusion preparation is viable for several hours, showing unchanged perfusate NO concentrations under basal conditions (22) and unaltered pressor responsiveness (49, 50).

Experimental Design

In all experiments baseline perfusion at 4 ml/min was established for 30 min before study protocol I or II was performed. After this period, vessel preparations in this system are maximally dilated (49). At these baseline conditions the solution perfused through the mesenteric vasculature was collected into glass test tubes. Each perfusate sample was immediately frozen and stored at -30°C until NOx assay. Experiments for protocols I and II were performed serially before (1st perfusion cycle) and after incubation with Nomega -nitro-L-arginine (L-NNA; 10-4 M; Sigma Chemical, St. Louis, MO) for 20 min (2nd perfusion cycle). After the first perfusion cycle each vessel preparation was perfused at the basal flow rate of 4 ml/min for 50 min (after that time the perfusate NOx concentrations have returned to baseline levels; data not shown) before L-NNA was added. The NOS inhibitor was also present in the perfusion system throughout the second perfusion cycle at the same molar concentration. Before the perfusion study, perfusion pressure was measured through the catheter without mesenteric tissue. Reported perfusion pressures represent the on-line recorded absolute perfusion pressure minus pressure generated by the PE-60 catheter. To determine the background NOx level in each experiment, the Krebs solution passed through the perfusion system including the catheter but without mesenteric tissue was collected.

Study protocol I. Changes in flow. This protocol included a total of 14 rats: 8 rats with PVL and 6 sham-operated rats. The perfusion rate was increased gradually by increasing the rate of revolution of the roller pump (4, 8, 16, 24, 32, 40, and 48 ml/min). Every increment in flow rate was initiated slowly (over 20-30 s). After perfusion pressure was stable and 20 s after increasing flow rate, the mesenteric vasculature perfusate was collected for 30 s. After the final collection of perfusate at 48 ml/min, flow rate was returned to 4 ml/min. For each flow rate an index of shear stress was calculated from the following equations
<IT>R</IT> = <FR><NU>&Dgr;P</NU><DE>Q</DE></FR> = <FR><NU>8&eegr;<IT>L</IT></NU><DE>&pgr;<IT>r</IT><SUP>4</SUP></DE></FR>  solving for  <IT>r</IT> = <FENCE><FR><NU>8&eegr;<IT>L</IT></NU><DE>&pgr;</DE></FR> <FR><NU>Q</NU><DE>&Dgr;P</DE></FR></FENCE><SUP>1/4</SUP> (1)
where R is resistance, Delta P is perfusion pressure, eta  is viscosity, L is vessel length, and Q is flow rate.

Shear stress is also a function of r
&tgr; = <FENCE><FR><NU>4&eegr;Q</NU><DE>&pgr;<IT>r</IT><SUP>3</SUP></DE></FR></FENCE>
Substituting with the expression for r gives
&tgr; = <FR><NU>4&eegr;Q</NU><DE>&pgr; <FENCE><FR><NU>8&eegr;<IT>L</IT></NU><DE>&pgr;</DE></FR></FENCE><SUP>3/4</SUP> <FENCE><FR><NU>Q</NU><DE>&Dgr;P</DE></FR></FENCE><SUP>3/4</SUP></DE></FR> or &tgr; = &Dgr;P<SUP>3/4</SUP>Q<SUP>1/4</SUP><FENCE><FR><NU>&eegr;</NU><DE>2&pgr; <IT>L</IT><SUP>3</SUP></DE></FR></FENCE><SUP>1/4</SUP> (2)
or &tgr; = <IT>K</IT>&Dgr;P<SUP>3/4</SUP>Q<SUP>1/4</SUP>
where
<IT>K</IT> = <FENCE><FR><NU>&eegr;</NU><DE>2&pgr;<IT>L</IT><SUP>3</SUP></DE></FR></FENCE><SUP>1/4</SUP>  or  <IT>K</IT> = <FR><NU>1</NU><DE>2</DE></FR><FENCE><FR><NU>8&eegr;</NU><DE>&pgr;<IT>L</IT><SUP>3</SUP></DE></FR></FENCE><SUP>1/4</SUP>
Assuming L to be the same for each vessel preparation and eta  of the Krebs solution constant for all experiments, then K is considered to be a constant factor. Therefore, the index of shear stress (tau ) as applied to mesenteric vessel preparations in our experiments, is defined by Delta P and Q.

Study protocol II. Administration of methoxamine. Two doses of methoxamine (30 and 100 µM, Sigma Chemical) were administered noncumulatively by constant infusion to the mesenteric vessel preparation of sham (n = 6) and PVL rats (n = 7) for 2 min. Perfusate was collected for 2 min, at each pressor response period starting with the increase in perfusion pressure and each sample collecting for 1 min.

Determination of NOx Concentrations

The NOx concentration of each sample was measured using a Sievers Nitric Oxide Analyzer (Sievers Instruments, Boulder, CO). This assay is based on spectrophotometric analysis following a chemiluminescent reaction between NO and ozone. Then 50 µl of each sample were placed into the purge vessel containing 3 ml of the 0.1 M vanadium chloride (VCl3) in 1 M HCl at 95°C. The NO generated from nitrite and nitrate ions was carried into the analyzer by vacuum through the gas bubbler trap containing 5 ml of 1 M NaOH. This analyzer quantitates dissolved NO and nitrite-derived NO that has been generated by acid and stripped from the solution by nitrogen gas. The NO then reacts with analyzer-generated ozone to form excited NO2, which releases light in the red and near-infrared regions of the spectrum, which is detected by a thermoelectrically cooled, red-sensitive photomultiplier tube. The lower limit of sensitivity for this machine is below 2 pmol NO/s. Perfusate NOx concentrations in protocol II represent the mean of the two perfusate samples obtained during each pressure response period. NO production rate in protocol I is calculated by multiplying the perfusate flow rate by the concentration of NOx in the perfusate and is expressed as nanomoles per minute. All perfusate NOx concentrations and related NOx production rates were corrected by the corresponding background NOx levels assessed in experimental controls perfusing the catheter without the mesenteric tissue.

Western Blotting

SMA vessels were analyzed for the presence of eNOS and iNOS protein. SMAs were harvested in two series, one consisting of PVL and sham rats (n = 8, respectively) and the other of PVL and normal control rats (n = 8, respectively). Vessels were washed in PBS and homogenized in a lysis buffer as previously described (48). Protein supernatants were quantitated using the Lowry assay, and equal amounts of protein from each sample were separated by SDS-PAGE and electroblotted to nitrocellulose membranes. Membranes were probed with monoclonal antibody recognizing eNOS (Transduction Laboratory, Lexington, Kentucky) and a polyclonal antibody recognizing iNOS (Affinity Bioreagents, Golden, Colorado). The specificity of the eNOS and iNOS antibodies for rat tissue was previously established by using quiescent and activated rat sinusoidal endothelial cells, respectively, as positive controls. Enhanced chemiluminescence was used for protein detection.

Statistical Analysis

Results were expressed as means ± SE. Statistical analysis was performed using ANOVA (2-way, with repeated measurements), paired and unpaired Student's t-test. Each relationship of NOx production rate and shear stress index was tested for regression by simple and polynomial regression analysis. Only the curve with the best statistical fitting (highest r2 value) is displayed. Statistical significance was set at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

There were no differences in body weight in the experimental groups. PVL rats showed significantly elevated spleen weights, expressed as percentage of body weight (PVL 2.89 ± 0.79 vs. sham 2.34 ± 0.70 mg/kg body wt, P < 0.0001).

In Vivo: SMA Pressure Measurements

Stenosis of the portal vein resulted in significant lower pressures in the SMA artery on day 1, 2, and 3 after induction of portal hypertension compared with sham operation (PVL 108 ± 5.8, 119.7 ± 2.3, and 121.7 ± 2.8 vs. sham 137.3 ± 4.8, 144.7 ± 2.9, and 142.7 ± 3.5 mmHg, P < 0.05, respectively).

In Vitro: Basal Conditions

Baseline perfusion pressures were not significantly different in vessel preparations of PVL and sham rats in study protocol I and II (PVL 9.0 ± 1.1 and 10.2 ± 1.4 vs. sham 11.2 ± 1.7 and 12.2 ± 1.2 mmHg, respectively). In experiments using the NOS inhibitor L-NNA no significant change in baseline perfusion pressure was observed. Because the chemiluminescence technique is highly sensitive, precautions have to be taken to avoid contamination. In all experiments, background NOx levels of Krebs solution used were found to be low and constant over the experimental procedure, demonstrating that measured changes in perfusate NOx concentrations represent endothelial NO release. Background NOx levels were not significantly different between study protocols and the same for PVL and sham rats. No difference in perfusate NOx concentration and thus NOx production rate at baseline perfusion pressure between PVL and sham rats as well as between study protocol I and II was found (PVL 0.68 ± 0.36 × 10-7 M or 0.27 ± 0.14 nmol/min and 0.72 ± 0.16 × 10-7 M or 0.28 ± 0.06 nmol/min vs. sham 0.49 ± 0.14 × 10-7 M or 0.20 ± 0.13 nmol/min and 0.57 ± 0.17 × 10-7 M or 0.23 ± 0.07 nmol/min, respectively).

Study Protocol I: Responses to Changes in Flow Rate

Mesenteric arterial bed perfusion pressure. Pressure response to stepwise increases in flow rate was significantly lower in PVL rats compared with sham rats (ANOVA, P < 0.01; Fig. 1A). The corresponding values observed at the highest explored flow rate were 49.0 ± 3.9 and 65.8 ± 3.8 mmHg for PVL and sham rats, respectively (P < 0.01).


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Fig. 1.   A: pressure response to flow changes in in vitro perfused mesenteric vessel beds of portal vein-ligated (PVL) and sham rats 3 days after PVL. P < 0.01 (ANOVA with repeated measurements). * P < 0.05 and ** P < 0.01 vs. sham rats. Perfusion pressure in response to increasing flow is significantly smaller in PVL rats. B: relationship between amount of nitric oxide (NO) metabolites (NOx) production and index of shear stress induced by changes in flow rate. Slopes of NOx production rate vs. shear stress index were significantly higher in PVL rats than in sham rats (P < 0.001, ANOVA with repeated measurements). Complete equations and regression coefficients found for displayed data. PVL: 0 + 0.032 · X + (4.168 × 10-3) · X 2, r = 0.89, r2 = 0.78 (not shown). Sham: 0 + 0.024 · X + (7.65 × 10-1) · X 2, r = 0.92, r2 = 0.84 (not shown). C: after Nomega -nitro-L-arginine (L-NNA) incubation, pressure response to changes in flow rate in PVL and sham rats was no longer significantly different, demonstrating NO dependency of observed hyporesponsiveness in PVL rats.

Perfusate NOx concentration and mesenteric arterial NOx production. Perfusate NOx concentrations were significantly elevated in PVL rats compared with sham rats (ANOVA, P < 0.01). Corresponding, mesenteric arterial NOx production rate was significantly higher in PVL rats compared with sham rats (ANOVA, P < 0.01). NOx concentration and NOx production rate increased gradually, as flow rate increased, and they were significantly higher in PVL rats compared with sham rats at flow rates of 24, 32, 40, and 48 ml/min (P < 0.05). At a flow rate of 48 ml/min the corresponding values of NOx concentration and NOx production rate were 2.88 ± 0.40 × 10-7 M and 13.8 ± 1.9 nmol/min for PVL vs. 0.99 ± 0.14 × 10-7 M and 4.8 ± 0.7 nmol/min for sham rats, respectively (P < 0.01). The relationship between the NOx production rate and the calculated index of shear stress was for both PVL and sham rats found to be statistically best described by a second-order polynomial regression analysis and to be highly significant (PVL r = 0.89, P < 0.001, sham r = 0.92, P < 0.001). The slopes of NOx production rate vs. shear stress index were significantly higher in PVL rats than in sham rats (PVL 0.139 ± 0.016 vs. sham 0.049 ± 0.005, P < 0.0001; Fig. 1B).

Effect of blockade of NO synthesis by L-NNA on pressure response to changes in flow rates. L-NNA preincubation resulted in significantly increased mesenteric artery perfusion pressures in PVL rats at flow rates of 16, 24, 32, 40, and 48 ml/min (P < 0.01). In the sham group no significant change was observed. After NO synthesis blockade with L-NNA no statistical difference in pressure response to stepwise increases in flow rates between perfused vessels of sham and PVL rats could be observed (Fig. 1C).

Study Protocol II: Responses to Methoxamine Administration

Vasopressor response to methoxamine in mesenteric arterial beds. Increases in perfusion pressure over baseline (Delta  mmHg) in response to methoxamine were significantly lower in vessel preparations of PVL rats compared with sham rats (ANOVA, P < 0.0001). The corresponding values for 30 and 100 µM of methoxamine administered were 40.9 ± 13.2 and 76.1 ± 16.4 mmHg for PVL and 159.5 ± 13.8 and 184.2 ± 7.1 mmHg for sham rats, respectively (P < 0.0001; Fig. 2A). Therefore, 3 days after inducing portal hypertension, vessel preparations of PVL rats already expressed a significant hyporeactivity to methoxamine.


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Fig. 2.   A: increases in perfusion pressure in response to methoxamine (MT, 30 and 100 µM as MT30 and MT100) in in vitro-perfused superior mesenteric arterial vascular beds of PVL and sham rats 3 days after PVL. P < 0.0001 (ANOVA with repeated measurements). * P < 0.0001 vs. sham rats. B: corresponding NO release during pressure response periods (detected as difference in perfusate NOx concentration between baseline and each pressure response period). P < 0.01 (ANOVA with repeated measurements). * P < 0.01 vs. sham rats. C: preincubation with L-NNA almost completely abolished differences in pressure response to methoxamine administration between the study groups.

Changes in perfusate NOx concentration in response to methoxamine. The increase in perfusate NOx concentration over baseline NOx concentration (at baseline pressure) during administration of 30 and 100 µM methoxamine amounted to 1.67 ± 0.21 × 10-7 M and 2.60 ± 0.28 × 10-7 M for PVL and 0.87 ± 0.08 × 10-7 M and 1.44 ± 0.13 × 10-7 M for sham rats, respectively (P < 0.01; Fig. 2B). Therefore, already at 3 days after partial PVL at each pressure response period NO release was significantly higher in mesenteric arterial preparations of PVL rats than in sham rats (ANOVA, P < 0.01).

Effect of blockade of NO synthesis by L-NNA on pressure response to methoxamine. L-NNA obliterated the significant hyporeactivity in superior mesenteric arterial beds of PVL rats (Fig. 2C). The increases in perfusion pressures over baseline observed at 30 and 100 µM methoxamine were 149.3 ± 25.8 and 165.7 ± 17.5 mmHg for PVL and 159.5 ± 13.9 and 184.2 ± 7.1 mmHg for sham rats, respectively (not significant). Calculating the percentage increase in response to methoxamine in each vessel preparation after L-NNA incubation, blockade of NO formation induced a significant higher increase in perfusion pressure in PVL compared with sham rats (ANOVA, P < 0.01), showing a significantly more pronounced role of NO production in mesenteric vasculature of PVL rats already 3 days after portal-venous constriction.

Western Blot Analysis

To determine the enzymatic source of the increase in NOx concentration detected in the in vitro perfused SMA, we examined eNOS and iNOS protein expression from SMA vessels on day 3 after PVL or sham operation (Fig. 3). eNOS protein levels were markedly increased in vessels of PVL rats. Similar results were also obtained in additional experiments performed in PVL and normal control rats. Western blotting for iNOS demonstrated no detectable iNOS protein in SMA from PVL or sham rats, whereas cell lysates from activated sinusoidal endothelial cells from rat liver were iNOS positive.


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Fig. 3.   Endothelial NO synthase (eNOS) and inducible NOS (iNOS) protein levels in superior mesenteric artery vessels from sham and PVL rats on day 3 after induction of portal hypertension. A: marked increase in eNOS protein level was detected in vessels from PVL animals. B: no induction of iNOS found in vessels from sham or PVL rats (as control a strong iNOS band detected with recombinant iNOS protein can be appreciated).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Several previous reports have demonstrated an increased vascular NO production in different models of portal hypertension at a time when the hyperdynamic circulation is fully developed (6, 10, 18, 32, 38). Chronic high blood flow has been shown to increase endothelial NO release in different vascular beds and animal species (37, 47). At the present time, it is unknown whether vascular NO overproduction in portal hypertension is the primary event that induces vasodilatation or whether NO hypersecretion occurs secondary to the chronically increased blood flow. In the present study, we have provided compelling evidence that NO overproduction in the endothelium of SMAs of acute portal hypertensive animals precedes the development of a hyperdynamic circulation in this vascular bed and therefore is not secondary to a chronic increase in blood flow. In addition, we have demonstrated that this increase in NO production is eNOS derived.

We have previously characterized (11, 51) in the PVL model, the sequence of hemodynamic events in the splanchnic circulation after PVL. This is characterized first by vasodilatation in nonsplanchnic vasculature and vasoconstriction and decreased blood flow in the superior mesenteric vessel bed secondary to a myogenic reflex induced by the acute increase in portal pressure (11). Any reduction in splanchnic arteriolar resistance occurs not before day 4, when plasma volume expansion is increased and collateral beds have opened (51). All experiments were performed on day 3 after PVL or sham operation. At this time, blood flow in the superior mesenteric arterial bed has been clearly demonstrated to be not increased (11, 51). Additionally, our model of acute portal hypertension unlike that of chronic portal hypertension appears free from structural changes of the vessel wall in the splanchnic vasculature, which potentially influence vascular pressor response. Mesenteric vessel preparations have been shown to be maximally dilated after 15 min of in vitro perfusion with Krebs solution and to express no active vascular tone, inasmuch as different vasodilators were found not to influence basal perfusion pressure (49). This indicates that in vitro basal perfusion pressure is determined solely by the flow resistance due to the structure of the vessel wall. In this study, superior mesenteric arterial beds of acute portal hypertensive rats did not differ significantly in in vitro basal perfusion pressures and corresponding NO release from those of sham rats. In contrast, in the PVL model of chronic portal hypertension, usually studied 10 or more days after PVL (6, 18, 23, 25, 40, 49, 50), in vitro basal perfusion pressure is always found reduced (49, 50). This reduction is not reversed by inhibition of NO or prostaglandins (49), as well as removal of the endothelium (unpublished data), and is considered to be due to structural changes of the vessel wall induced by chronic vasodilatation. Increased arterial flow, the characteristic hemodynamic feature of HCS in chronic portal hypertension (3, 11, 55), has been shown to induce arterial wall remodeling with an increase in internal diameter and in the medial cross-sectional area of the vessel (1, 4).

Shear stress as the final tractive force acting on the cell surface has been shown to cause endothelial NO production via a mechanotransduction cascade (5, 44). This includes tyrosine kinases mediating the phosphorylation of specific intracellular proteins involved in the signaling pathway determining shear stress-induced synthesis and release of NO derived from eNOS (53). Shear stress can be generated either by an increase in flow or by vasoconstriction at constant flow (21). In protocol I, shear stress was applied to the vessel preparations using steady laminar flow for 50 to 60 s after a slow change in flow rate (over 20-30 s). In protocol II, methoxamine administration resulted in rapid pressure increase, and during the initial phase of pressure response perfusate samples were collected for 2 min. Recent investigations suggest that shear stress-induced eNOS activation and NO release consist of an initial transient Ca2+-calmodulin-dependent peak and a sustained Ca2+-calmodulin-independent plateau phase (26, 34). The reported kinetics with an increase in intracellular Ca2+ concentration ([Ca2+]i), which is at best transient, indicate that shear stress elicits NO production independent of a continuous increase in [Ca2+]i. Inasmuch as in our experiments perfusate samples were collected under continuous exposure to shear stress, we suspect that the measured NO release in our perfusion studies is at least partly Ca2+-calmodulin independent. Noteworthy, tyrosine kinases appear to be involved in both activation pathways (16), and recently Lopez-Talavera et al. (30) reported that tyrosine kinase inhibition ameliorates the hyperdynamic state and decreases NO production in portal hypertensive rats. However, the precise mechanism of signaling events in endothelial cells in response to shear stress remains to be elucidated (53).

Protocol I showed almost 200% higher slopes of NO production rate vs. flow-induced shear stress in 3-day PVL rats (Fig.1B). The hemodynamic reflection in vivo of this increased vascular NO release in vitro is a reduction in SMA pressure in the presence of an unchanged blood flow (11, 51) and thus a lower vascular tone compared with sham animals at this time point. This is supported by the significantly lower in vitro pressure response to increasing flow rates in 3-day PVL rats. To test the hypothesis whether endothelial NO overproduction is responsible for the observed vascular hyporesponsiveness we used L-NNA. This specific blocker of NO synthesis has been demonstrated to be the most potent NO inhibitor available (54). L-NNA cannot be used in the vanadium-chemiluminescence assay inasmuch as it reacts with the VCl3 solution, thereby emitting light, and thus we were unable to measure NO levels during the second perfusion cycle. However, preincubation with the specific NO blocker L-NNA abolished the significant difference in pressure response. This demonstrates an appropriate inhibition of NO synthesis and reveals the predominant role of endothelial NO production in SMAs of portal hypertensive rats even in the absence of a hyperdynamic circulation in this vessel bed.

Vascular hyporeactivity to vasoconstrictors is one of the major pathophysiological abnormalities accompanying chronic portal hypertension (8, 25). In protocol II, we demonstrated that in vitro perfused SMAs exhibit a significant hyporesponsiveness to methoxamine as early as day 3 after induction of portal hypertension. Again, corresponding pressure-induced NO release was significantly higher in vessel preparations of PVL rats, and L-NNA corrected the vascular hyporeactivity. Taken together, vascular hyporeactivity to methoxamine in the in vitro perfused SMAs of portal hypertensive rats precedes the development of a hyperdynamic splanchnic circulation and is at least partly mediated by NO.

The enzymatic source of enhanced NO production in portal hypertension has been controversial (6, 10, 14, 32, 36). Agents promoting iNOS production include inflammatory cytokines, endotoxin, and lipopolysaccharide (46). Endotoxins are always present in the portal circulation, and bacterial translocation to mesenteric lymph nodes has been reported to be increased, particularly in acute portal hypertension (19). However, the current study rules out any significant induction of iNOS in SMAs 3 days after PVL. NO production mediated by iNOS has been shown to be independent of physical factors such as flow and shear stress (12, 35), and no expression of iNOS protein was detectable in the mesenteric vasculature from 3-day PVL rats. On the contrary, by directly measuring endothelial NO release in response to shear stress we are assessing specifically eNOS-derived NO production. In addition, Western blotting with eNOS antibodies showed a consistent augmentation in the eNOS immunoreactivity demonstrating a clear upregulation of eNOS protein expression in the 3-day PVL rat (Fig. 3).

The mechanisms involved in eNOS upregulation and increased NO production remain to be defined. Chronic high blood flow and shear stress have been shown to increase eNOS mRNA and protein expression (37, 41). In our study, the blood flow in the superior mesenteric vasculature is not elevated at any time point before the experimental conditions are initiated (11, 51). However, besides flow rate, viscosity, vessel length, and pressure define the shear stress level (Eq. 2, MATERIALS AND METHODS). With the plasma volume being increased as early as day 2 after PVL the blood viscosity is either equal to or even less than that of sham rats. Vessel length was assumed to be the same for all animals because this is a parameter that does not change significantly in physiological and/or pathological conditions. Thus the only remaining variable that could affect shear stress in PVL rats compared with sham rats is the intraluminal pressure. We measured the arterial pressure in the SMA of PVL and sham rats on day 1, 2, and 3 after induction of portal hypertension. At each time point, superior mesenteric arterial pressure was lower in portal hypertensive rats compared with sham rats. Therefore factors other than the physical stimulus of shear stress must account for the observed higher eNOS expression and elevated NO release in SMAs of acute portal hypertensive rats. One possible mechanism may be receptor-mediated activation of eNOS by circulating mediators or hormones. Tumor necrosis factor-alpha (TNF-alpha ) has recently been shown to be a major contributor in the development of the HCS, inasmuch as antagonism of TNF-alpha with anti-TNF-alpha antibody or inhibition of TNF-alpha synthesis by thalidomide prevents these changes in PVL rats (29, 31). In fact, TNF-alpha has been reported to enhance endothelial NO synthesis without induction of iNOS expression by increasing eNOS activity (2, 43). Moreover, enhanced estrogen serum levels have been demonstrated in rats with portal bypass (13), and estrogen was recently found to stimulate eNOS activity in cultured fetal endothelial cells (27). In addition, other candidates could be substance P and catecholamines, which have been shown to stimulate eNOS expression or activity and are known to be increased in chronic portal hypertension (15, 24, 42). Oxygen tension has also been shown to play a role in the regulation of the eNOS gene-protein expression and the eNOS activity. The findings in the literature are controversial in nonpulmonary vascular endothelial cells (17). However, in the pulmonary vasculature it has been demonstrated that hypoxia downregulates eNOS-derived NO production (17, 28). Further investigations will be necessary to elucidate the specific molecular mechanisms and individual factors involved in the observed eNOS upregulation.

In summary, in acute portal hypertensive rats without hyperdynamic splanchnic circulation SMA beds already express a significant hyporeactivity to methoxamine and increasing flow rates, and this is at least partly mediated by NO. eNOS-derived NO overproduction in the SMA vascular endothelium precedes the development of a hyperdynamic splanchnic circulation, supporting the hypothesis that eNOS upregulation and subsequently increased NO release in response to the physiological stimulus shear stress are playing a primary role in the pathogenesis of the hyperdynamic circulation in portal hypertension. Finally, these results emphasize the etiopathogenic character of an increased vascular NO synthesis initiating arteriolar vasodilatation in portal hypertension.


    ACKNOWLEDGEMENTS

We gratefully acknowledge D. Groszmann (Dept. of Mechanical Engineering, Tufts Univ., Medford, MA) for reviewing the mathematical derivations used in this manuscript. In addition, we thank Maryann Vergato for secretarial assistance and preparation of the manuscript and Gregory W. Cadelina for excellent technical assistance.


    FOOTNOTES

This study was funded by a Veterans Affairs Merit Review, B. Braun Foundation, Braun-Melsungen, Germany.

Present address of V. Shah: Gastroenterology Research Unit, Alfred 2-435, Mayo Clinic, 200 1st St., SW, Rochester, MN 55905.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R. J. Groszmann, Hepatic Hemodynamic Laboratory/111J, Veterans Administration Medical Center, 950 Campbell Ave., West Haven, CT 06516.

Received 18 August 1998; accepted in final form 18 December 1998.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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