Increased endothelial nitric oxide synthase activity in the hyperemic vessels of portal hypertensive rats

Journul

of Hepatology

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in Denmark.

Munks~uard

1996; All rights

25:

370-378

reserved

Copyright 0 European Association for the Study of the Liver 1996

Copenhagen

Journal

of Hepatology

ISSN 0168.8278

Increased endothelial nitric oxide synthase activity in the hyperemic vessels of portal hypertensive rats Paul A. Cahill, Eileen M. Redmond, Robert Hodges, Shuangmin Zhang and James V. Sitzmann The Johns Hopkins Medical Institutions, The Department of SurgeryBaltimore, Maryland, USA

Background/Aim: Portal hypertension is characterized by splanchnic hyperemia due to a reduction in mesenteric vascular resistance. Mediators of this hyperemia include nitric oxide. This is based on several reports indicating a marked splanchnic hyporesponsiveness in portal hypertension to vasoconstrictor stimuli both in vitro and in viva, and a subsequent reversal using specific inhibitors of nitric oxide synthase. The objective of this study was to determine firstly whether the functional activity and/or expression of nitric oxide synthase is altered in portal hypertensive vasculature and secondly which isoenxyme form was responsible for the preferential response to nitric oxide blockade in these animals. Methods: We compared nitric oxide synthase functional activity in the hyperemic vasculature of sham and portal hypertensive rats (following partial portal vein ligation). Nitric oxide synthase activities were determined by measuring the conversion of L-argimne to citrulline using ion-exchange chromotagraphy and the amount of immunodetectable nitric oxide synthase in sham and portal hypertensive vessels was determined by Western blot.

C

portal hypertension (PHT) results in increased portal pressure and reduced splanchnic vascular resistance, leading to marked splanchnic hyperemia (14). In addition, there is decreased peripheral vascular response to vasoconstrictor stimuli (511). However, neither the mechanism of the systemic hemodynamic changes nor its relationship to splanchnit vascular reactivity in PHT is well understood. HRONIC

Received 2 November: revised 22 December 199.5; accepted 4 January

Correspondence: Paul A. Cahill, Ph.D., Georgetown University Medical Center, Pasquerilla Healthcare Center, Fourth Floor, 3800 Reservoir Road, NW, Washington DC 20007, USA. Tel. 202-687 1019. Fax 202-687 1045.

370

Results: Ca2’-dependent nitric oxide synthase activity was significantly elevated @X0.05) in portal hypertensive particulate fractions from the superior mesenteric artery, thoracic aorta and portal vein. Vascular tissue cGMP levels and plasma nitrite levels were both significantly elevated in portal hypertension. Immunodetection with speciftc antisera raised against the inducible nitric oxide synthase demonstrated a lack of induction within the hyperemic vascuh~ture.Immunodetection with antisera against endothelial nitric oxide synthase showed a significant increase in portal hypertensive portal vein only. These results demonstrate enhanced calcium-dependent nitric oxide synthase activity in portal hypertension hyperemic vessels concurrent with elevated tissue cGMP levels. Conclusion: We conclude that enhanced endothelial nitric oxide synthesis may in part contribute to the hyperdynamic circulation of portal hypertension.

Key words: Nitric oxide synthase; Portal hypertension; Splanchic hyperemia.

The etiology of the increased mesenteric blood flow includes a decreased responsiveness to and/or production of endogenous vasoconstrictors (5lo), or an excess of vasodilatory substances (11-14). The suggested major vasoactive substances implicated in PHT include: angiotensin-II, vasopressin, a-adrenergics, Ladrenergics, glucagon, nitric oxide (NO), and prostacyclin (PGI,) (1,3,5,7-9,14,15). Moreover, this marked pressor hyporesponsiveness can be attenuated using specific inhibitors of NO synthesis. These reports imply that loss of vascular responsiveness to vasoconstrictive stimuli in the superior mesenteric artery and thoracic aorta of PHT animals may be due to enhanced NO synthesis or release possibly stimu-

Nitric oxide synthase activity in PHT rats

lated by endogenous activators of NO and/or endothelial shear stress (16-19). It was of interest therefore to determine whether the hemodynamic derangements previously described by us (2,15) and others (4,8,9) in PHT could regulate the expression of NOS in vivo, or vice versa. Nitric oxide synthase (NOS) enzymatically converts L-arginine and molecular oxygen to L-citrulline and NO (17,20). NOS has been purified, characterized and cloned from brain, macrophages, and endothelial cells (21-25). Three isoforms have been identified. Type I NOS isolated from rat, porcine, bovine, and human brain (17,20,21), and Type III NOS (ECNOS) isolated from bovine aortic endothelial cells (17,20,26) are both regulated by Ca2’ and calmodulin. In contrast, the inducible Type II NOS (iNOS) found in macrophages and vascular smooth muscle cells following activation by various cytokines is not subject to regulation by Ca2’ (17,20,25). Activation of NOS within endothelial cells and release of EDRF (NO) results in the stimulation of a soluble guanylyl cyclase, leading to a profound increase in intracellular cGMP levels in vascular smooth muscle cells (18,20,27). Finally, NO in the presence of oxygen and water rapidly decomposes to the stable and inactive end products, nitrite and nitrate (18,20,27). The current study was undertaken to directly determine whether the amount and/or functional activity of NOS was regulated in PHT vessels. We sought to define which isoform, if any, was regulated in PHT, the possible vessel distribution of this regulation (arterial or venous), and finally the potential sites for regulation of these enzymes, (endothelial or smooth muscle cells). To this end, we measured NOS activity in the superior mesenteric artery, thoracic aorta, and the portal vein of sham and PHT rats, and correlated these changes with iNOS and ECNOS expression, plasma nitrite and tissues cGMP levels.

Materials and Methods Materials L[14-C]Arginine (specific activity, 33 1 mCi/mmol) and 2’-0-succinyl iodotyrosine [1251]methyl ester guanosine 3’ S-cyclic phosphoric acid (specific activity 2000 Ci/mmol), was purchased from New England Nuclear (Boston, MA). NG-nitro-L-arginine methylester (L-NAME), R-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), nitroblue tetrazolium, Dowex AG50W-X8 (Na+ form), were purchased from Sigma Chemical (St. Louis, MO, USA). The monoclonal iNOS and polyclonal

ECNOS antibodies were purchased from Transduction Laboratories (Lexington, KY, USA). The anticGMP serum was obtained from Immunotech (Marseilles, France). All other chemicals were of the highest purity commercially available. Animals Male Sprague Dawley rats (Charles River Labs, MA, USA, 175-200 g) were used for all studies. All studies were approved by the Johns Hopkins University Animal Care and Use Committee, and adhered to AAALC and federal guidelines for the humane care and treatment of animals. Pre-hepatic PHT was produced by partial portal vein ligation (PVL), as described previously (4). Briefly, a laparotomy was performed under anesthesia. Under aseptic conditions the portal vein was isolated and a loose ligature was placed around the portal vein proximal to the confluence of the right and left branches. A blunt 20-gauge needle was placed beside the portal vein, the ligature was tightened around the needle and vein, and the needle was removed, producing a standard, calibrated stenosis. The abdomen was closed and the animals were allowed to recover for 2 weeks. Sham-operated animals were used as controls. Splenic pulp pressure was used as an index of portal venous pressure to verify the development of portal hypertension. Data from animals in the portal hypertensive group with splenic pulp pressures less than 12 mmHg were discarded. Preparation of particulate and cytosolic fractions Animals (sham=4 and PHT=6) were killed with pentobarbital. The superior mesenteric artery (SMA) was dissected to its tertiary branches, transected from the aorta, harvested, and placed in ice-cold buffer containing 50 mM Tris HCl, and 2 mM EDTA (pH 7.4). The portal vein proximal to the ligature and to the tertiary branches of the superior mesenteric vein, the small intestine, and the thoracic aorta were also removed and placed in buffer. Adherent fat was removed by sharp dissection. Tissues were minced with a fine scissors and homogenized in a Tissumizer (Tekmar Ultra Turrax, Cincinnati, OH, USA) for 10-s periods. The homogenates were centrifuged at 1000 x g for 5 min before centrifugation at 40000 x g for 1 h at 4°C. The supernatants (cytosolic fractions) were collected and stored at -70°C. The pellets (particulate fractions) were resuspended in 50 mM Tris.HCl containing 1 mM EDTA (pH 7.4) and stored at -70°C. Protein was measured by the method of Bradford (31) with bovine serum albumin as a standard before determination of NOS activity. 371

PA. Cahill et al.

Assay of NOS activity NOS activity was measured in both cytosolic and

particulate fractions of the superior mesenteric artery, thoracic aorta and portal vein by determining the conversion of L[14-Clarginine to L[14-C]citrulline based on a method of Bredt & Snyder (22). Briefly, the 40000xg fractions (10-50 l.tg) were incubated in a total volume of 0.16 ml in 50 n&I Tris.HCl containing 0.1 mM EDTA, 3 pM tetrahydrobiopterin, 1 mM NADPH, 2.5 mM CaCl, and 1 pCi/ml of L[14Clarginine (~100000 cpm). The reaction was initiated by the addition of 50 yl of the fraction and continued for 1 h at 25°C. The reaction was terminated by the addition of 2 ml of a stopping buffer containing 30 mM HEPES and 3 mM EDTA (pH 5.5). The reaction mix was passed over a 0.5 ml Dowex AGSOWX8 cation exchange column (Na+ form). The L[14C]citrulline was then eluted with 2x0.5 ml of distilled water, which is collected and counted by liquid scintillation spectrometry. The reproducibility of this assay was greater than 95% for the same sample. Tissue cGMP levels

Tissue cGMP levels were determined in supernatant fractions prepared from homogenates following extraction of the cGMP with O.lM HCl and separation by centrifugation at 3000 rpm for 30 min at 4°C. Concentrations of immunoreactive cGMP in tissue fractions were determined by radioimmunoassay, following acetylation as previously described (32-35). The variability between assays was less than 5% for the same sample. Nitrite assay

Plasma samples were deproteinized with 35% sulfosalicyclic acid (1:5 vol/vol) as previously described (36). The samples were then vortexed every 5 min for 30 min at room temperature before centrifugation at 17000xg for 10 min. The nitrite concentration was determined by diazotization and absorbance reading at 546 nm. Aliquots (500 l.tl) of deproteinized plasma were added to 500 pl of Greiss reagent (0.5% sulfanilamide and 0.05% naphthylethylenediamine dihydrochloride in 2.5% phosphoric acid) for 20 min at room temperature. Concentrations were determined relative to a standard curve using sodium nitrite prepared in phosphate buffered saline and A,,, nm immediately measured using a spectrophotometer (model Du 30, Beckman Instruments, Columbia, MD, USA). The assay variability was less than 10% for each sample. Western blotting

Membrane and cytosolic proteins (SO-100 pg) were 372

separated on 10% SDS-polyacrylamide gel, respectively. After SDS-PAGE, the separated proteins were electrophoretically transferred to nitrocellulose membranes (HYBOND-C, Amersham) using a Transphor electroblotter unit (Hoefer Scientific Instruments, San Francisco, CA, USA) at 100 V for 3 h as previously described (2). After transfer, the membranes were incubated for l-2 h in blocking solution containing 24 mM Tris base (pH 7.6), 0.05% (vol/vol) Tween-20 and 15 mM NaCl (TTBS), supplemented with 1% nonfat dry milk. The membranes were washed once for 5 min with TTBS. The membranes were incubated with specific antisera against the inducible NOS (iNOS) or the constitutive endothelial NOS (ECNOS) diluted in TTBS containing 1% nonfat dry milk for 90 min at room temperature with gentle rocking. After washing the blots 3 times for 10 min each in TTBS, they were incubated with the second antibody solution (horseradish peroxidase conjugated IgG diluted in TTBS (1:3 500-6000) containing 1% nonfat dry milk) for 1 h at room temperature with gentle agitation. The blots were finally washed 3 times for 10 min in TTBS, then incubated with a mixture of equal volume of ECL detection solution A and B (Amersham, Arlington Heights, IL, USA) for 1 min at room temperature. The blots were covered in plastic wrap and placed in a film cassette to expose to Hyperfilm (Amersham, Arlington Heights) film for 5-15 s. Equal protein loading was confirmed by either Coomassie Blue-staining or India ink staining of protein in each lane of the same blot. The signal intensity (integral volume) of the appropriate bands on the autoradiogram was analyzed using a Personal Densitometer (Molecular Dynamics, Sunnyvale, CA, USA) and the Imagequant software package (Biosoft, Indianapolis, IN, USA). Statistics

Results are expressed as mean&SEM. Statistical analysis was performed using the independent Student ttest for two groups of data, and ANOVA for multiple comparison. Probability of 0.05 was accepted as significant. Results PVL-ligated

rat model of PHT

Two weeks after partial portal vein ligation, splenic pulp pressure (SPP), an index of portal venous pressure was significantly increased from values of 7.6ti.51 to 18.WO.8 mmHg. There was no significant difference between the body weights of either group of animals (data not shown).

Nitric oxide synthase activity in PHT rats B. Cytosolic

A. Patiiculnte

0.4

0

(-) L-NAME

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(+) L-NAME WW) (+) EGTA (5 mhl)

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(-) L-NAME (4) L-NAME (IOOpM) (+) EGTA (S mM)

0.2

1-_ 0--0-0.1

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PHI

Fig. 1. The effect of 5 mM EGTA and 100 w L-NAME on NOS activity in the 40000 x g particulate fractions and cytosolic fractions of the superior mesenteric artery prepared from sham (n=4) and PHT (n=6) rats. Data are the mean&SEM of 3 separate experiments petiormed in triplicate. The levels of NOS activity were significant (* ~~0.05) as determined by an independent Student t-test.

NOS activity

Particulate and cytosolic fractions prepared from homogenates of sham and PHT rat superior mesenteric artery, portal vein and thoracic aorta were analyzed for NOS activity by determining the conversion of [14C]arginine to [14C]citrulline. The conversion was linear over a 30-min period, reaching a maximum after 60 min (data not shown). There was also a linear relationship between protein concentration and [14C]citrulline formation up to 50 yg of protein (data not shown). In addition, [14C]citrulline formation was inhibited (>95%) by the specific inhibitor of NOS, L-NAME (100 pM) in cytosolic and particulate fractions from sham and PHT superior mesenteric artery (Fig. 1A and B). The NOS activity was primarily calcium dependent, since in the presence of 5 mM EGTA [14C]citrnlline formation was completely inhibited in both fractions from sham animals (Fig. 1A and B). Similarly, in the presence of 5 mM EGTA, NOS activity was inhibited (>95%) in the PHT superior mesenteric artery particulate and cytosolic fractions, respectively. Hence it would appear that the same pool of NOS (i.e., Ca*‘-dependent) was active in both sham and PHT superior mesenteric artery fractions. To determine whether this pool of NOS activity was differentially regulated in PHT, we compared [14C]citrulline formation in cytosolic and particulate fractions prepared from sham and PHT homogenates of the superior mesenteric artery (Fig. 1 A and B). Particulate NOS activity represented -90% of total

activity in the superior mesenteric artery homogenate, and was significantly inhibited in the presence of 100 pM L-NAME. There was a significant increase (75_+7%, p
A.

0.40 P

r Cl I

B %

6. Cytosolic

(-) L-NAME (+) L-NAME (1OOpM)

0.30 -

(+) L-NAME (1OOpM)

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%

+

&P

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(ZI (+) EGTA (5 mm)

-I-

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2.2 = .g 0.102 Y

-S

O-

1.1 --

--

SHAM

PHI

TA

a.10 0.20 0 i- b

-_

6

SHAM

-_ Ftn

TA

Fig. 2. NOS activity in the 40000 x g particulate fractions and cytosolic fractions of the thoracic aorta prepared from sham (n=4) and PHT (n=6) rats. NOS activity was determined in the absence or presence of 100 mM L-NAME and with or without 5 mM EGTA. Data are the meansEM of 3 separate experiments performed in triplicate. The levels of NOS activity were significant (*p
373

8.

A. Particulate

z

II

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(-) L-NAME

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(+) L-NAME

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(+) EGTA (5 mM)

(1OO~hl)

Cytosolic (-) L-NAME

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(+) L-NAME

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(+) EGTA (5 mM)

(IOOpM)

0.4

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PV

PV

lion of the [“Clcitrulline formation in cytosolic and particulate fractions prepared from sham and PHT portal vein homogenates revealed a significant increase (3.26k.2 fold) in NOS activity within the pa~~icLllatc fraction from PHT rats (Fig. 3B). In contrast to the superior mesenteric artery and thoracic aorta, cytosolic Ca”-dependent NOS activity was also signi~calltly increased in PHT (Fig. 3A and B). Tissue cCMP

ctnci ~~l~~~tn~~ f?ifrite ~~t~rtttiitu~i~~t~~s

Since NOS activity is closely associated with activation of soluble guanylyl cyclase by NO and the subsequent f~~rmation of intracellular cGMP (32,33,45), we determined superior mesenteric artery cGMP levels in sham and PHT animals. The data demonstrate a significant increase (2.3k0.2 fold, p&OS) in superior mesenteric artery tissue cGMP levels in PHT (Fig. 4A). In a similar mal~ner thoracic aorta cGMP levels were significantly elevated (1.410.1 fold, p
significantly elevated (2.1 &O.18 mM in sham as compared to 3.1 l&O.3 MM in PHT) in PHT serum compared to sham plasma. ECNOS md iNOS e.upression in PHT wsdature Using specific antisera against the inducible NOS enzyme (INOS), we tested for the presence of iNOS immunoreactivity in cytosolic and pa~icuIate fractions from sham and PHT superior mesenteric artery, thoracic aorta and portal vein (Fig. 5). Lysates of thoracic aorta from rats which had been treated with 5 mg/kg LPS for 4X h were used as a positive control for detection of iNOS in rat vasculature. Antiserum against iNOS specifically recognized a major protein band with relative molecular weight of 130 kDa in mouse macrophages stimulated with 100 @ml LPS (Fig. 5). Moreover. this same immunoreactive band was detected in our positive control lysate from thoracic aorta of LPS-treated rats. However, iNOS antisera failed to detect specific antigen present in

ECNOS

t

snam PHT rham PHT PV

SMA

Jixrn

140 kDa

PHT

TA

WUVEC

Fig. 6. Western blot of imiI~urlodetect~ble ECNOS in particulate fructions (-100 /_Q) prepared from sham and PHT superior mesenteric artery (SMA), thorucic aorta (TA) and portul vein (PV). Positive ~t~~~tr~~l lames contaitr &xztes prepured ,from humrm endothelial cells (HUVEC). Datu are representative of3 experiments bvith similar re.ur1t.v.

cytosolic fractions prepared from sham and PHT superior mesenteric artery, thoracic aorta and portal vein, respectively (Fig. 5). Likewise there was no labelling of iNOS in particulate fractions prepared from sham or PHT superior mesenteric artery, thoracic aorta and portal vein, respectively (data not shown). These data suggest that the increased NOS activity within the vasculature of PHT animals was not due to an induction of iNOS protein within these vessels. In contrast to iNOS immunoreactivity, western blots prepared from control lysates of human endothelial cells, ECNOS antisera recognized a major protein band with relative n~~~lecular weight of 140 kDa (Fig. 6). ECNOS antisera specifically recognized a similar 140-kDa band in particulate fractions of sham and PHT superior mesenteric artery, thoracic aorta and portal vein (Fig. 6). Analysis of the data demonstrated a marked increase in the total amount of immunoreactive ECNOS in PHT portal vein particulate fractions, as compared to sham. However, in contrast to the functional activity of ECNOS (citrulline data) there was no difference in immunoreactive ECNOS in the particulate fractions prepared from sham and PHT superior mesenteric artery and thoracic aorta (Fig. 6).

Discussion The hyperdynamic circulation following partial portal vein stenosis can be attenuated by specific inhibitors of NO synthesis (5,6,8-10,15). This suggests that an excessive formation of NO could mediate the hemodynamic changes associated with chronic PHT (13). In the present study, we have directly measured NOS activity within the hyperelnic vasculat~lre of PHT rats, and have provided compelling evidence for

the first time of enhanced ECNOS activity. even in the absence of increased protein expression, within specific vascular beds of PHT rats. In order to test the hypothesis that enhanced NOS functional activity and/or expression within the splanchnic vascular bed was responsible for the exaggerated response to L-NAME in PHT as reported by our laboratory (1X15) and others (5,6,8-lOJO), we measured NOS activity by determining 1) the formation of ~‘~C~citrLllline from ~‘~C~argiIline, 2) the formation of cGMP as an indicator of direct activation of soluble guanylyl cyclase by NO: and 3) immunoreactive NOS (ECNOS and iNOS) within the hyperemic vasculature. Our data suggest that in as much as ECNOS is a membrane bound calcium-dependent protein, the enhanced NOS activity evident in the PHT hyperemic vessels supports an upregulation of the endothelial constitutive NOS enzyme activity. While these data do not rule out the possible contribution of other vasodilators in mediating the intestinal vasodilation of PHT ( 1I, 12.14,15), they imply a role for ECNOS in modulating splanchnic hemodynamics in PHT, in as much as specific inhibitors of ECNOS reversed the abnormal hen~odynatnics of PHT (5,6,8,9,29,X1). The factors that regulate the substrate availability and enzyme activity involved in endogenous NO synthesis within these hyperemic vessels are not yet fully understood (17,27). The reason for the enhanced NOS activity within the superior mesenteric artery and thoracic aorta of PHT without a signi~cant change in the amount of ECNOS protein is at present unknown. The functional activity of ECNOS is reported to be regulated following myristoylation, glycosylation or phosphorylation (20,21,27,38.39) and hence these postranslational modifications may contribute to increased functional activity of the enzyme in PHT without any change in ECNOS constitutive expression. Alternatively, recent studies have focussed on the modulation of tetrahydrobiopterin levels which may in turn contribute to the regulation of ECNOS activity in endothelial cells (38.40). It is therefore possible that the elevated cGMP levels within the hyperemic vessel may contribute to tetrahydrobiopterin synthesis through GTP cyclohydrolase and hence contribute to the functional activity of ECNOS without increasing the amount of NOS protein (38,41). ECNOS activity can thus be both increased or decreased by cytokines due to an enhanced supply of endogenous tetrahydrobiopterin or a fall in ECNOS mRNA, respectively (4 1). Finally, the reason for the differential increase in ECNOS protein expression in the PHT portal vein is unclear. 375

/?A. Cahill et al.

Previous studies have demonstrated a marked vascular hypertrophy of the portal venous smooth muscle following partial portal vein ligation (42), which may in turn contribute to enhanced ECNOS protein expression in this vessel due to increased hemodynamically imposed mechanical forces. Interestingly, endothelial cells that tonically generate NO have been shown to alter their NO production following smooth muscle proliferation or hypertrophy, due to mechanical deformation of the vessel wall (4344). It is unclear whether the heterogeneous behavior between arteries and veins in PHT is due to differences in the endothelium, in the smooth muscle, or both (4546). Nevertheless, previous studies have shown that these differences could in part originate from the insensitivity of venous smooth muscle to the vasodilator effect of NO (45,46). In addition, the hyporesponsiveness to vasoconstrictors in the PHT rat portal vein has been shown to be partially mediated by NO (6), whereas several studies have concluded that the marked splanchnic arterial hyporesponsiveness to endogenous vasoconstrictors in PHT was completely reversed following inhibition of NO synthesis (8-10). Therefore, despite containing enhanced NOS activity and protein levels as well as cGMP levels, the portal vein NOS activity may not regulate vascular tone to the same degree as in the superior mesenteric artery. The reason for the EGTA insensitive NOS activity in portal vein preparations is not obvious. However, the percentage of EGTA insensitive NOS activity was similar in both sham and PHT portal vein, ruling out a preferential Ca*‘independent NOS component in PHT vessels. In accordance, we were unable to detect iNOS protein expression in either particulate or cytosolic fractions prepared from sham or PHT animals. There may therefore be a component of the NOS activity in portal vein preparations that is Ca*‘-independent but is not immunodetectable. The source and etiology of the enhancedNO production in PHT remains controversial. Since the vascular endothelium and/or the underlying smooth muscle are both potential sites for enhanced vascular NO production, many studies have focussed on the possible regulation of nitric oxide synthase (NOS) activity in these cells. Moreover, whether increased NO production represents a primary event in PHT or whether it is secondary to increased hemodynamic stress is still unresolved. It is noteworthy that exaggerated NO levels have been shown to preferentially regulate angiotensin II and endothelin-1 receptors in vitro, which in turn may contribute to the response of these pressor hormones in PHT (33,34). Several pos376

sible mediators of iNOS production within the hyperemit vasculature have been proposed. A role for endotoxins in mediating the intestinal vasodilatation associated with portal hypertension and cirrhosis has been reported by several laboratories (3,12,17,30,4749). However, most of these studies did not directly measure NOS activity and/or expression within the hyperemic vasculature of these animals. Despite the initial hypothesis that iNOS mediates the intestinal hyperemia of PHT (17), we contend that the preferential response to NOS inhibition in PHT. animals is a direct result of ECNOS inhibition and not iNOS, at least within the hyperemic vasculature. This current study rules out any induction of iNOS within these vessels at 14 days post-ligation. However, the possibility of iNOS expression occurring earlier cannot be ruled out. Our data would therefore suggest that the preferential hemodynamic response to L-NAME and/ or L-NNA in PHT animals reported by us (13,lS) and others (5,6,8-10,30) is not due to inhibition of iNOS, but rather ECNOS. Our studies agree with the recent study of Fernandez et al. (50) who presented evidence against a role for iNOS in the hyperdynamic circulation of PHT. However, this study failed to detect any differences in ECNOS activity in the nonvascular tissues studied. Receptor-mediated activation of ECNOS by circulating hormones may be involved in the enhanced ECNOS activity (3,12,51). Plasma concentrations of norepinephrine are reported to be elevated in PHT and patients with cirrhosis (3,12). Alpha,,-adrenergic receptor activation is closely coupled to Gi (inhibitory guanine nucleotide regulatory) proteins (52), which are linked to NOS activity in endothelial cells in response to norepinephrine (52-54). We have recently shown increased Gi functional activity within the hyperemic vasculature of PHT animals, which may in turn be associated with a parallel increase in Gi functional activity coupled to ECNOS activation via o+ receptors in the hyperemic vessel (2). Finally, several studies have demonstrated that chronic alterations in local blood flow can exert a regulatory EDRF/NO-mediated influence on responses (19,41). Indeed, increases in local blood flow regulate ECNOS gene expression and enhanced vascular nitrite release in vivo, presumably due to increased endothelial shear stress (16,19). An enhanced activity of ECNOS can reduce the response of a-adrenergic vasoconstriction in arterial segments subjected to increased endothelial shear stress (l&19). This exaggerated NO production is regulated at the level of an inhibitory guanine nucleotide regulatory protein (Gia) (18). Therefore, one might

Nitric oxide synthase activity in PHT rats

expect that the constitutive Ca2’ dependent ECNOS is regulated in PHT as a direct result of the hemodynamically imposed mechanical forces, perhaps at the level of a Gia protein within the hyperemic vasculature. In this regard, hormone-stimulated ECNOS response has been reported to be greater in patients with cirrhosis (55). In conclusion, our data show for the first time regulation of ECNOS activity in the hyperemic vasculature of PHT rats. The enhanced ECNOS activity reflects exaggerated endothelial NO release within the hyperemic vessel, which may contribute to and/or underlie the hyperdynamic state of PHT.

Acknowledgements This work was supported in part by a grant from American Heart Association #92009340, and NIH Grant DK47067 (JVS) and HL08978 (PAC). We wish to thank Dr. M. Clemens for his review of the manuscript and helpful suggestions.

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References 1. Ballet F. Hepatic circulation: potential for therapeutic intervention. Pharmacol Ther 1990; 47: 281-328. 2. Cahill PA, Wu Y, Sitzmann JV. Altered adenylyl cyclase activities and G-protein abnormalities in portal hypertensive rabbits. J Clin Invest 1994; 93: 2691-700. considera3. Reichen J. Liver function and pharmacological tions in the pathogenesis and treatment of portal hypertension. Hepatology 1990; 11: 1066-78. 4. Vorobioff J, Bredfeldt JE, Groszmann RJ. Hyperdynamic circulation in portal hypertensive rat model: a primary factor for maintenance of chronic portal hypertension. Am J Physiol 1983; 244: G52-G27. 5. Castro A, Jimenez W, Claria J, Martinez JM, Bosch J, Arroyo AV, Piulats J, Rivera P, Bosch J. Impaired responsiveness to angiotensin II experimental cirrhosis: role of nitric oxide. Hepatology 1993; 18: 367-72. 6. Kamath S, Hunter LW, Tyce GM, Miller VM, Rorie DK. Hyporesponsiveness to norepinephrine in the portal vein of portal hypertensive rats is partially mediated by nitric oxide. Hepatology 1993; 18: 281A, 900. 7. Kiel JW, Pitts L, Benoit JN, Granger DN, Shepherd AP. Reduced vascular sensitivity to norepinephrine in portal hypertensive rats. Am J Physiol 1985; 248: G192-5. 8. Lee F-Y, Albillos A, Colombato LA, Groszmann RJ. The role of nitric oxide in the vascular hyporesponsiveness to methoxamine in portal hypertensive rats. Hepatology 1992; 16: 1043-8. 9. Lee F-Y, Colombato LA, Albillos A, Groszmann RJ. No-nitro-L-arginine administration corrects peripheral vasodilation and systemic capillary hypotension and ameliorates plasma volume expansion and sodium retention in portal hypertensive rats. Hepatology 1993; 17: 85-90. 10. Sieber CC, Groszmann RJ. Nitric oxide mediates hyporeactivity to vasopressors in mesentetic vessels of portal hypertensive rats. Gastroenterology 1992; 103: 235-9. 11. Sitzmann JV, Li SS, Lin PW. Prostacyclin mediates splanch-

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22.

23.

24.

25.

26.

27.

28.

29.

30.

nit vascular response to norepinephrine in portal hypertension. J Surg Res 1989; 47: 208-11. Benoit JN, Zimmerman B, Premem AJ, Go VLW, Granger DN. Role of glucagon in splanchnic hyperemia of chronic portal hypertension. Am J Physiol1986; 251: G674-7. Cahill PA, Foster CL, Redmond EM, Gingalewski C, Wu Y, Sitzmann JV. Enhanced nitric oxide synthase activity in portal hypertensive rabbits. Hepatology 1995; 22: 598-606. Sitzmann JV, Bulkley GB, Mitchell M, Campbell K. Role of prostacyclin in the splanchnic hyperemia contributing to portal hypertension. Ann Surg 1988; 209: 322-7. Wu Y, Bums RC, Sitzmann JV. Effects of nitric oxide and cycloxygenase inhibition on splanchnic hemodynamics in portal hypertension. Hepatology 1993; 18: 1416-21. Buga GM, Gold ME, Fukuto JIM, Ignarro LJ. Shear stress-induced release of nitric oxide from endothelial cells grown on beads. Hypertension 1991; 17: 187-93. Moncada SR, Palmer M, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 199 1; 43: 109-41. Ohno M, Gibbons GH, Dzau VJ, Cooke JP. Shear stress elevated endothelial cGMP, role of potassium channel and Gprotein coupling. Circulation 1994; 88: 193-7. Rubanyi GM, Romero JC, Vanhoutte PM. Flow-induced release of endothelium-derived relaxing factor. Am J Physiol 1986; 255: H1145-9. Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J 1992; 6: 3051-64. Bredt DS, Huang PM, Glatt GE, Lowenstein C, Reed RR, Snyder S. Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase. Nature 199 1; 351: 714-8. Bredt DS, Snyder SH. Isolation of nitric oxide synthase, a calmodulin-requiring enzyme. Proc Nat1 Acad Sci USA 1990; 87: 682-5. Iida S, Ohshima H, Oguchi S, Hata T, Suzuki H, Kawasaki H, Esum H. Identification of inducible calmodulin-dependent nitric oxide synthase in the liver of rats. J Biol Chem 1992; 267: 25385-8. Radomski MW, Palmer RMJ, Moncada S. Glucocorticoids inhibit the expression of an inducible, but not constitutive, nitric oxide synthase in vascular endothelial cells. Proc Nat1 Acad Sci USA 1990; 87: 10043-7. Xie Q-W, Cho I-II, Calaycay J, Munford RA, Swiderek M, Lee TD, Ding A, Nathan C. Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science 1992; 256: 225-8. Lamas S, Marsden PA, Li GK,Tempst P, Michel T. Endothelial nitric oxide synthase: molecular cloning and characterization of a distinct constitutive enzyme isoform. Proc Nat1 Acad Sci USA 1992; 89: 6348-52. Schmidt HHW, Lohmann SM, Walter U. The nitric oxide and cGMP signal transduction system: regulation and mechanism of action. Biochim Biophys Acta 1993; 1178: 153-75. Wu Y, Li SS, Campbell KA, Sitzmann JV. Modulation of splanchnic vascular sensitivity to angiotensin II. Surgery 1991; 110: 162-8. Iwata F, Joh I, Kawai K, Itoh M. Role of EDRF in splanchnic blood flow of normal and chronic portal hypertensive rats. Am J Physioll992; 263: G149-54. Pizcueta MP, Pique JM, Bosch J, Whittle BJR, Moncada S. Effects of inhibiting nitric oxide biosynthesis on the systemic

377

EA. Cahill et al.

31.

32.

33.

34.

35.

36.

37.

38. 39.

40.

41.

42.

43.

and splanchnic circulation of rats with portal hypertension. Br J Pharmacol 1992; 105: 184-90. Bradford MM. A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein dye binding. Ann Biochem 1976; 72: 248-54. Cailla H L C, Vannier J, Delaage MA. Guanosine 3’ 5 ‘-cyclic monophosphate assay at the 10-i’ mole level. Anal Biochem 1976; 70: 195-202. Cahill PA, Redmond EM, Foster C, Sitzmann JV. Nitric oxide regulates angiotensin II receptors in vascular smooth muscle cells. Eur J Pharmacol 1995; 288: 219-29. Redmond EM, Cahill PA, Hodges R, Zhang S, Sitzmann JV. Regulation of endothelin receptors by nitric oxide in rat vascular smooth muscle cells. J Cell Physiol 1996: in press. Cahill PA, Redmond EM, Keenan AK. Vascular atrial natriuretie factor receptor subtypes are not independently regulated by atria1 peptides. J Biol Chem 1990; 265: 21896-906. Green LC, Wagner J, Glogowski PL, Skipper JS, Wishnok P, Tannenbaum SR. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem 126: 1982: 131-8. Sogni P, Moreau R, Oshuga H, Cailmail S, Olerti F, Hadengue A, Pussard E, Lebrec D. Evidence of normal nitric oxide-mediated vasodilation in conscious rats with cirrhosis. Hepatology 1992; 16: 980-3. Nathan C. Nitric oxide and biopterin: a study in chiaroscuro. J Clin Invest 1994; 93: 1875-6. Sessa WC, Barber CM, Lynch KR. Mutation of N-myristoylation site converts endothelial nitric oxide synthase from a membrane to a cytosolic protein. Circ Res 1993; 72: 921-5. Werner-Felmayer G, Wener ER, Fuchs D, Hausen A, Reibnegger G, Schmidt K, Weiss G, Wachter H. Pteridine biosynthesis in human endothelial cells. Impact on nitric oxide-mediated formation of cGMP. J Biol Chem 1993; 268: 1842-6. Rosenkranz-Weiss P, Sessa WC, Milstein S, Kaufman S, Watson K, Pober JS. Regulation of nitric oxide synthesis by proinflammatory cytokines in human umbilical vein endothelial cells: elevations in tetrahydrobiopterin levels enhance endothelial nitric oxide synthase specific activity. J Clin Invest 1994; 93: 223-3. Johansson B. Structural and functional changes in rat portal vein after experimental portal hypertension. Acta Physiol Stand 1976; 98: 381-3. Amal J F, Yamin J, Sayegh H, Dockery S, Harrison DG. Regulation of endothelial nitric oxide synthase mRNA, pro-

378

44.

45.

46.

47.

48.

49.

50.

5 1.

52.

53. 54.

55.

tein and activity during cell growth. Circulation 1994; 90: A2178. Lamontagne D, Pohl U, Busse R. Mechanical deformation of vessel wall and shear stress determine the basal release of endothelium relaxing factor in the intact rabbit coronary vascular bed. Circ Res 1992; 70: 123-30. Feletou M, Hoeffner U, Vanhoutte PM. Endothelium-dependent relaxing factors do not affect the smooth muscle of portal-mesenteric veins. Blood Vessels 1989; 26: 21-32. Suga T, Itoh H, Shimomura A, Kusagawa M, Ito M, Takase K, Konishi T, Nakano T. Comparison of the effects of various vasodilators on the rat portal vein and mesenteric artery. Eur J Pharmacol 1993; 242: 129-36. Lopez-Talevera JC, Merrill W, Groszmann RJ. Treatment with anti-tumor necrosis factor-a polyclonal antibodies prevents the development of the hyperdynamic circulation and reduces portal pressure in portal hypertensive rats. Hepatology 1993; 18: 14OA, 336. Palmer RMJ. The discovery of nitric oxide in the vessel wall: A unifying concept in the pathogenesis of sepsis. Arch Surg 1993; 128: 396-401. Michielsen PP, Ramon AM, Van Assche RJ, Pelckmans PA, Van Maercke WM. Endotoxinemia in portal hypertension. Hepatology 1993; 18: 284A, 909. Femandez M, Pagan JC, Casadevall M, Bemadich C, Piera C, Whittle B, Pique JM, Bosch J, Rodes J. Evidence for a role for Inducible Nitric Oxide Synthase in the hyperdynamic circulation of portal hypertensive rats. Gastroenterology 1995; 108: 1487-95. Joh T, Granger N, Benoit J. Endogenous vasoconstrictor tone in intestine of normal and portal hypertensive rats. Am J Physiol 1993; 264: H171-7. Brodde O-E, Michel MC. Adrenergic receptors and their signal transduction mechanisms in hypertension. J Hypertens 1992; 10 (suppl7): s13345. Flavahan NA, Vanhoutte PM. G-proteins and endothelial responses. Blood Vessels 1990; 27: 218-29. Flavahan NA, Shimokawa H, Vanhoutte PM. Pertussis toxin inhibits endothelium-dependent relaxations evoked by certain endothelial activators in porcine coronary arteries. J. Physiol 1989; 408: 549-61. Ros J, Jimenez W, Lamas L, Claria J, Arroyo V, Rivera F, Rodes J. Nitric oxide production in arterial vessels of cirrhotic rats. Hepatology 1995; 21: 554-60.