basal release of nitric oxide in the mesenteric artery in portal hypertension and cirrhosis: role of...

HEPATOLOGY

Basal release of nitric oxide in the mesenteric artery inportal hypertension and cirrhosis: Role of dimethylargininedimethylaminohydrolaseEva Serna,*,‡,1 María Dolores Mauricio,*,‡,1 Paloma Lluch,† Gloria Segarra,*,‡ Belén Cortina,*Salvador Lluch*,‡ and Pascual Medina*,‡

*Department of Physiology, Faculty of Medicine and Odontology, University of Valencia, †Hepatology Unit, and ‡Institute of Health ResearchINCLIVA, Valencia, Spain

Key words

asymmetric dimethylarginine, endothelialfactors, nitric oxide inhibitors, potassiumchannels, splanchnic circulation.

Accepted for publication 21 December 2012.

Correspondence

Dr Pascual Medina, Departamento deFisiología, Facultad de Medicina yOdontología, Universidad de Valencia, AvenidaBlasco Ibañez 15, 46010 Valencia, Spain.Email: [email protected]

Conflicts of interest: No conflicts of interest.

1These authors contributed equally to thiswork and share first authorship.

AbstractBackground and Aim: Increased basal release of nitric oxide (NO) in the splanchniccirculation contributes to elevated plasma levels of NO observed in decompensated cir-rhosis. We evaluated in rat mesenteric arteries whether the differences in basal release ofNO, revealed by asymmetric dimethylarginine (ADMA)- and NG-nitro-L-arginine methylester (L-NAME)-induced contractions, were associated with changes in messenger RNA(mRNA) expression of endothelial NO synthase (eNOS) and dimethylarginine dimethy-laminohydrolases (DDAHs).Methods: Rat small mesenteric arteries from 14 Sham-control, from 14 with partial portalvein ligation (PPVL), and from 14 with bile duct excision (BDE)-induced cirrhosis wereprecontracted under isometric conditions with norepinephrine, and additional contractionswere induced with ADMA and L-NAME. mRNA expression of eNOS, DDAH-1, andDDAH-2 in mesenteric arteries were evaluated by real-time polymerase chain reaction.Results: ADMA and L-NAME caused concentration- and endothelium-dependent con-tractions. pD2 values to L-NAME were similar in all groups. In contrast, pD2 values toADMA were similar in PPVL and BDE but were significantly lower than those of theL-NAME and the Sham groups. Relaxation to acetylcholine was not modified by ADMAor L-NAME but was abolished by charybdotoxin plus apamin. There was an increasedmRNA expression of eNOS, DDAH-1, and DDAH-2 in mesenteric arteries from PPVLand BDE compared with the Sham group.Conclusion: Basal release of NO is increased in mesenteric arteries of PPVL and BDErats. The rise in expression of DDAHs indicates a higher degradation of ADMA. Thiswould result in an increased generation of endothelial NO and mesenteric vasodilation.

IntroductionMesenteric arterial vasodilation is a key process in the pathophysi-ology of the hyperdynamic circulatory syndrome of cirrhosis.1

Nitric oxide (NO), an endothelium-derived relaxing factor, seemsto play a decisive role in the pathogenesis of splanchnic vasodila-tion in the portal hypertension that accompanies cirrhosis.2 PlasmaNO levels, measured as plasma nitrite plus nitrate (NOx concen-tration), increase in human decompensated cirrhosis.3,4 The con-centration of NOx in the portal venous plasma of patients withcirrhosis and portal hypertension is threefold higher than that insystemic venous samples,5,6 thus suggesting that NO release isenhanced in the splanchnic vessels of these patients. Furthermore,an increased NO synthase (NOS) activity was demonstrated insplanchnic vessels of portal hypertensive animals.7 In contrast, theactivity of endothelial NOS (eNOS) in the cirrhotic liver ofhumans and rats is significantly decreased,6,8 which suggests a

different regulation of eNOS in the liver and in the splanchnicvessels.

We reasoned that one mechanism involved in the elevatedplasma levels of NO associated with liver cirrhosis could be theresult of an increased generation of basal NO in the endothelium ofthe splanchnic circulation. Basal release of NO from the vesselwall has been described in humans,9,10 and isometric tension hasbeen used to reveal basal release of NO in isolated human11,12 andanimal13–15 vascular preparations. The basal release of NO isrevealed when endothelium-intact artery rings are precontractedand an additional contraction is induced by NOS inhibitors such asasymmetric dimethylarginine (ADMA). The level of this addi-tional contraction provides a functional indication for NO release.

ADMA is degraded to citrulline and dimethylamine by dimethy-larginine dimethylaminohydrolases (DDAHs), whereas NG-nitro-L-arginine methyl ester (L-NAME), another inhibitor of NOS, isnot degraded by DDAHs.16,17 DDAHs are expressed as types 1 and

doi:10.1111/jgh.12119

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880 Journal of Gastroenterology and Hepatology 28 (2013) 880–886

© 2013 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd

2 isoforms,18 and are widely distributed in various organs andtissues, including blood vessels.18–20

The purpose of the present work was to measure the basalrelease of NO in small mesenteric arteries of rats with secondarybiliary cirrhosis (bile duct excision [BDE]-induced cirrhosis) andrats with portal hypertension without cirrhosis. We attempted toestablish whether the differences in the basal release of NO,revealed by ADMA- and L-NAME-induced contractions, wereassociated with changes in messenger RNA (mRNA) expressionof eNOS and DDAHs in mesenteric arteries. Finally, we deter-mined the contribution of NO and Ca2+-activated K+ channels inacetylcholine-induced endothelium-dependent relaxation.

MethodsMale Sprague-Dawley rats (200–250 g) were acquired fromCharles River and housed according to institutional guidelines(constant room temperature 22°C, 12-h light/dark cycle, 60%humidity, standard rat chow, and water ad libitum). All protocolswere approved by the Institutional Ethics Committee at the Uni-versity of Valencia.

Surgical procedures. After induction of anesthesia by isof-lurane, pre-hepatic portal hypertension induced by partial portalvein ligation (PPVL) was performed by placing a 20-gauge needleon the portal vein. A non-absorbable surgical thread ligature wasplaced around the vein and needle, and the needle was then with-drawn.21 The studies were performed 14–16 days after PPVL,when hyperdynamic circulation accompanying portal hyperten-sion is fully established.22 Secondary biliary cirrhosis was inducedvia BDE.23 At laparotomy, the bile duct was cut between a ligatureclose to the hilum of the liver and one close to the duodenum. Thestudies were performed 28 days after BDE. This delay was neededfor secondary biliary cirrhosis to develop.23 For the Sham opera-tion, the duodenum, portal vein, and bile duct were exposed duringlaparotomy, and the abdomen was closed 15 min later.

Before starting the experiments, mean arterial pressure (MAP)and portal pressure (PP) were measured by catheterization of theright carotid artery and ileocolic vein, respectively. Pressure wastransmitted through a Statham pressure transducer and recordedcontinuously. The external zero reference was placed at the mid-portion of the rat.

Blood drawn from the carotid artery in the anaesthetized rat wascollected after hemodynamic assay. The plasma was separated andfrozen at -20°C until total bilirubin and creatinine levels wereassayed in an autoanalyzer, according to the manufacturer’sinstructions.

Isolated rat mesenteric artery preparation. Toassess vascular reactivity in resistance vessels, tertiary branches ofthe mesenteric arcades (200–300 mm external diameter) were dis-sected. Segments (2 mm) were individually mounted in a small-vessel wire myograph (Model 610 M; Danish MyoTech, Aarhus,Denmark) for measurement of isometric tension and filled with5-mL modified Krebs–Heseleit solution (in mmol/L NaCl 115,KCl 4.6, KH2PO4 1.2, MgCl2 1.2, CaCl2 2.5, NaHCO3 25, glucose11.1, ethylenediaminetetraacetic acid 0.01, pH 7.3–7.4) in thepresence of indomethacin (INDO, 10-5 mol/L) and continuouslygassed with 95% O2–5% CO2 while the temperature was main-

tained at 37°C. The arterial segments were stretched at a tension of5 milliNewtons (mN) equivalent to that generated at 0.9 times thediameter of the vessel at 100 mmHg. In some experiments, theendothelium was removed by rubbing the intima with a hair. Func-tional integrity of the endothelium was confirmed routinely by thepresence of relaxation induced by acetylcholine (10-6 mol/L)during contraction obtained with norepinephrine (10-5 mol/L).

The ability of ADMA or L-NAME to inhibit basal activity ofNO was assessed from its enhancement of norepinephrine-inducedcontraction in endothelium-containing mesenteric rings. This wasperformed by obtaining a low-level of contraction (~2–5 mN) tonorepinephrine (0.3–1 ¥ 10-6 mol/L) and then measuring theenhancement of tone produced by a range of ADMA or L-NAMEconcentrations (10-6 to 10-3 mol/L). The ability of L-arginine(10-3 mol/L) to either protect against or reverse the enhancementby ADMA or L-NAME was also assessed. Additionally, the effectsof ADMA or L-NAME were also examined on norepinephrine-induced tone in endothelium-denuded rings.

Concentration-response curves for acetylcholine (10-9 to10-6 mol/L) and sodium nitroprusside (10-10 to 10-7 mol/L) weredetermined in precontracted segments with norepinephrine (0.3–1 ¥ 10-5 mol/L) in the absence and in the presence of ADMA(3 ¥ 10-4 mol/L) or L-NAME (3 ¥ 10-4 mol/L) that were added tothe organ bath 20 min before starting the concentration-responsecurve. In other experiments, relaxation responses to acetylcholinewere carried out under the following conditions: (i) in the absenceof inhibitors (control response); (ii) in the presence of INDO(10-5 mol/L) to inhibit the release of prostaglandins; (iii) in thepresence of INDO and L-NAME (3 ¥ 10-4 mol/L) to inhibit theproduction of prostaglandins and NO; and (iv) in the presence ofINDO, L-NAME, and charybdotoxin (10-7 mol/L) plus apamin(10-6 mol/L) to inhibit the production of prostaglandins, NO, andCa2+-activated K+ channel activity.

Real-time polymerase chain reaction analyses.Small mesenteric arteries (arteries with a diameter < 300 mm) werecollected from each rat into an RNAlater (Ambion, Austin, TX,USA), an RNA stabilization reagent, following the manufacturer’sinstructions. Total RNA was extracted using Tripure isolationreagent (Roche Molecular Biochemicals, Basel, Switzerland),and concentration and integrity were assessed in RNA 6000Nano Labchips in an Agilent 2100 Bioanalyzer (AgilentTechnologies, Foster City, CA, USA). Ready-to-use primers andprobes from the assay-on-demand service of Applied Biosystemswere used for the quantification of selected target genes: eNOS,DDAH-1, and DDAH-2 (Rn02132634_s1, Rn00574200_m1,and Rn01525775_g1, respectively) and endogenous referencegene glyceraldehyde-3-phosphate dehydrogenase (housekeepingcontrol 4352338E). RNA samples were reverse-transcribed usingrandom hexamers and MultiScribe reverse transcriptase (AppliedBiosystems). After complementary DNA synthesis, real-timepolymerase chain reaction (RT-PCR) was carried out using theABI Prism 7900HT Sequence Detection System (Applied Biosys-tems). Samples were run in triplicate, and fold changes weregenerated for each sample by calculating 2-DDCT.24

Drugs. The following drugs were used: acetylcholine chloride,norepinephrine hydrochloride, L-NAME hydrochloride, ADMA

E Serna et al. Dimethylarginine and portal hypertension

881Journal of Gastroenterology and Hepatology 28 (2013) 880–886


hydrochloride, L-arginine hydrochloride, INDO, sodium nitro-prusside dehydrate, charybdotoxin, and apamin (Sigma ChemicalCo., St Louis, MO, USA). Drugs were prepared and dilutedin distilled water except for INDO, which was dissolvedin absolute ethanol. Stock solutions of the drugs were freshlyprepared everyday.

Statistical analysis. All values are expressed as means �standard error of the mean. The contractile effects of ADMA andL-NAME were determined after evoking submaximal tone withnorepinephrine (0.3–1 ¥ 10-6 mol/L). The change from the pre-existing tension was expressed in mN. Relaxation was expressedas a percentage of the norepinephrine-induced contraction. ThepD2 (negative logarithm of the molar concentration at which half-maximum response occurs) was determined from individualconcentration-response curves by nonlinear regression analysis.All n values are presented as the number of rats. Differencesbetween surgical procedure (i.e. Sham vs PPVL vs BDE) andby experimental treatment (i.e. ADMA vs L-NAME) were ana-lyzed by two-way anova, followed by Bonferroni’s post-test tocompare replicate means. The level of statistical significance wasP < 0.05.

Results

Baseline characteristics. Baseline characteristics, bio-chemical, and hemodynamic parameters of control, PPVL, andBDE groups are summarized in Table 1. As expected, MAP wassignificantly lower, and PP and spleen weights were significantlyhigher in PPVL and BDE compared with Sham-operated rats. InBDE group, weight gain was markedly reduced, and rats devel-oped jaundice by 3 weeks following surgery. Their stool becameacholic, and post-mortem examination confirmed enlarged liverand the presence of ascites (10–15 mL). Sham-operated controlsdisplayed normal post-operative recovery. Total bilirubin wasincreased markedly in BDE rats (P < 0.05, BDE vs Sham orPPVL), suggesting severe liver injuries and hepatocyte damage inBDE group. Creatinine concentrations were within the normalrange in the three groups.

Effects of NOS inhibitors on basal NO. ADMA (10-6

to 10-3 mol/L) and L-NAME (10-6 to 10-3 mol/L) did not show

significant changes in resting tension. Following induction ofa low level of contraction (2.1 � 0.5 mN) in endothelium-containing rings with norepinephrine (0.3–1 ¥ 10-6 mol/L), addi-tion of ADMA (10-6 to 10-3 mol/L) or L-NAME (10-6 to10-4 mol/L) led to concentration-dependent increases in tension(Fig. 1). pD2 values for the concentration-response curves toL-NAME were similar in control, PPVL, and BDE groups

Table 1 Baseline characteristics of the Sham-operated, partial portalvein ligation (PPVL), and bile duct excision (BDE) groups

Characteristic Sham (n = 14) PPVL (n = 14) BDE (n = 14)

Body weight gain (g) 37 � 4 34 � 5 8 � 10*Spleen weight (g) 0.74 � 0.04 1.02 � 0.09* 1.46 � 0.15*Liver weight (g) 12.1 � 0.4 11.9 � 0.7 17.1 � 0.8*Mean arterial pressure

(mmHg)115 � 9 90 � 5* 88 � 6*

Portal pressure (mm Hg) 7 � 1 16 � 3* 20 � 2*Creatinine (mg/dL) 0.7 � 0.03 0.7 � 0.05 0.8 � 0.06Bilirubin (mg/dL) 0.2 � 0.1 0.2 � 0.1 10.6 � 1.8*

*P < 0.05 versus Sham group.n = number of experiments.

BDEPPVLSham

0

2.5

5.0

7.5

Con

trac

tion

(mN

)

(-log mol/L)6 5 4 3 6 5 4 3 6 5 4 3

(-log mol/L) (-log mol/L)

Figure 1 Contractions induced by NG-nitro-L-arginine methyl ester (L-NAME) (n = 8) andasymmetric dimethylarginine (ADMA) (n = 8)on rings of rat mesenteric artery with endot-helium from Sham-operated, partial portalvein ligation (PPVL), and bile duct excision(BDE) groups in the absence and in thepresence of L-arginine (L-arg, 10-3 mol/L,n = 6). Contractions were determined afterevoking submaximal tone with 3 ¥ 10-7 mol/Lnorepinephrine. ( ) L-NAME; ( ) ADMA;( ) L-arg + L-NAME; ( ) L-arg + ADMA.

Table 2 pD2 values and maximal responses elicited by L-NAME andADMA in mesenteric arteries after precontraction with norepinephrine

NOS inhibitor n pD2 Emax (mN)

ShamL-NAME 8 5.40 � 0.10 3.9 � 0.7ADMA 8 4.42 � 0.08† 3.8 � 0.5

PPVLL-NAME 8 5.41 � 0.07 6.6 � 0.9**ADMA 8 3.98 � 0.10**† 6.8 � 0.4**

BDEL-NAME 8 5.20 � 0.14 6.6 � 0.9**ADMA 8 3.86 � 0.07**† 6.3 � 0.6**

*P < 0.05 and **P < 0.01 versus Sham group with the same treatment;†P < 0.05 compared with L-NAME treatment in the same group.pD2, -log mol/L of substance causing 50% of the maximal contraction;Emax, maximal contraction; n = number of rats.ADMA, asymmetric dimethylarginine; BDE, bile duct excision; L-NAME,NG-nitro-L-arginine methyl ester; PPVL, partial portal vein ligation.

Dimethylarginine and portal hypertension E Serna et al.



(Table 2). In contrast, pD2 values of the curves to ADMA weresimilar in PPVL and BDE but significantly lower than those of theL-NAME and the Sham groups, suggesting a decreased sensitivityto ADMA. The maximal responses to ADMA and L-NAME weresignificantly higher in PPVL and BDE groups compared withSham rats (Fig. 1, Table 2). The contractile effect induced byADMA and L-NAME was prevented or reverted by L-arginine10-3 mol/L, precursor of NO synthesis, in all groups studied(Fig. 1). ADMA and L-NAME did not induce contractions inendothelium-denuded rings (results not shown).

Effects of NOS inhibitors on acetylcholine-induced relaxations. Mesenteric arteries with endotheliumshowed a similar relaxation in response to acetylcholine in allgroups studied (Fig. 2, Table 3). No relaxation was observedin arteries without endothelium. Relaxant responses to acetyl-choline were not significantly modified after pre-incubationwith INDO (10-5 mol/L). ADMA (3 ¥ 10-4 mol/L) or L-NAME

(3 ¥ 10-4 mol/L) did not modify the relaxation to acetylcholine inarteries from PPVL and BDE groups (Fig. 2). In Sham-operatedgroup, L-NAME diminished slightly but significantly the maximalrelaxation to acetylcholine. The endothelium-dependent relaxationresistant to INDO and NOS inhibition was abolished by the inhibi-tors of Ca2+-activated K+ channels charybdotoxin (10-7 mol/L) plusapamin (10-6 mol/L) in all groups studied (Fig. 2).

Effects of NOS inhibitors on sodiumnitroprusside-induced relaxations. In endothelium-intact and endothelium-denuded rings, sodium nitroprusside (10-10

to 10-7 mol/L) induced complete (100%) relaxation of precon-tracted artery rings in all groups studied, with a pD2 of 7.9 � 0.5.None of the NO inhibitors (10-4 mol/L) modified the relaxationcurves to sodium nitroprusside (n = 4 for each compound; resultsnot shown).

mRNA expression of eNOS and DDAHs. We per-formed real-time RT-PCR on mesenteric ring segments fromSham-operated, PPVL, and BDE groups (n = 6 per group). Therewas an increased mRNA expression of eNOS in mesenteric arter-ies from PPVL and BDE groups of approximately twofold com-pared with Sham-operated group (Fig. 3). The expression of thetwo isoforms of DDAH mRNA was significantly increased inPPVL and BDE groups. As shown in Figure 3, the level ofDDAH-1 mRNA expression in PPVL and BDE rats was up tothreefold and fivefold, respectively. DDAH-2 mRNA was signifi-cantly higher in PPVL and BDE (~twofold) compared with Sham-operated rats (Fig. 3).

DiscussionThe results of the present study demonstrate that basal release ofNO is increased in small mesenteric arteries of rats with secondarybiliary cirrhosis and in rats with portal hypertension without cir-rhosis. The differences in the contractile effects of ADMA andL-NAME together with mRNA determinations of DDAH indicatethat this enzyme is responsible for increased vascular catabolismof ADMA and increased generation of endothelial NO.

The basal release of NO was determined indirectly by measur-ing the effects of ADMA and L-NAME in precontracted artery

PPVL BDESham

100

0

25

50

75

Acetylcholine (-log mol/L)%

Rel

axat

ion

9 8 7 6 9 8 7 6 9 8 7 6Acetylcholine (-log mol/L)Acetylcholine (-log mol/L)

Figure 2 Concentration-response curves toacetylcholine on rings of rat mesenteric arteryfrom Sham-operated, partial portal vein ligation(PPVL), and bile duct excision (BDE) groups inthe absence (Control, n = 8) and in the pres-ence of asymmetric dimethylarginine (ADMA)(n = 6), L-NAME (n = 6), or NG-nitro-L-argininemethyl ester (L-NAME) plus charybdotoxin andapamin (n = 6). Relaxation is expressed as apercentage of the contraction in response tonorepinephrine. ( ) Control; ( ) L-NAME3 ¥ 10-4 mol/L; ( ) ADMA 3 ¥ 10-4 mol/L;( ) L-NAME 3 ¥ 10-4 mol/L plus charybdotoxin10-7 mol/L and apamin 10-6 mol/L.

Table 3 pD2 and maximal response values for acetylcholine in mesen-teric arteries from Sham, PPVL, and BDE groups in the absence (control)and in the presence of L-NAME or ADMA

Acetylcholine n pD2 Emax (%)

ShamControl 8 8.10 � 0.07 100L-NAME (3 ¥ 10-4 mol/L) 6 7.96 � 0.07 92 � 4*ADMA (3 ¥ 10-4 mol/L) 6 8.21 � 0.14 99 � 1

PPVLControl 8 8.04 � 0.10 100L-NAME (3 ¥ 10-4 mol/L) 6 8.00 � 0.10 99 � 1ADMA (3 ¥ 10-4 mol/L) 6 8.22 � 0.15 100

BDEControl 8 8.06 � 0.09 98 � 1L-NAME (3 ¥ 10-4 mol/L) 6 7.86 � 0.08 97 � 1ADMA (3 ¥ 10-4 mol/L) 6 8.04 � 0.15 99 � 1

*P < 0.05 versus control group.pD2, -log mol/L of acetylcholine causing 50% of the maximal relaxation;Emax, maximal relaxation expressed as a percentage of the contractionin response to norepinephrine; n = number of rats.ADMA, asymmetric dimethylarginine; BDE, bile duct excision; L-NAME,NG-nitro-L-arginine methyl ester; PPVL, partial portal vein ligation.




rings. NO synthesis can be inhibited by using competitive inhibi-tors of NOS.25 Therefore, differences in basal NO generationwould be reflected in the level of contraction in response to ADMAand L-NAME.25 We found that in mesenteric arteries from portalhypertensive and cirrhotic rats, both ADMA and L-NAMEincreased markedly the vascular tone compared with Sham-operated rats, suggesting a greater contribution of basal NOrelease. Consonant with these data, we found significantlyincreased eNOS mRNA levels in mesenteric arteries in both PPVLand BDE groups.

The contractile effects induced by NOS inhibitors wereendothelium-dependent and were reversed by L-arginine, the sub-strate for the enzyme for NO synthesis, thus indicating that ADMAand L-NAME increase arterial tone by inhibiting the basal releaseof endothelial NO.

This study found a decreased sensitivity to ADMA as comparedwith that of L-NAME in the three groups of experiments. Thisobservation is consistent with a high degradation of ADMA by theenzyme DDAH in the vessel wall. Previous experiments haveshown that DDAH catalyzes the conversion of ADMA to citrullineand dimethylamine.16 This enzyme is highly specific for the deg-radation of ADMA but not L-NAME.16 Further studies have showncolocalization of DDAH with NOS and ADMA in endothelial cellsand in several anatomical sites.18–20 Thus, the distribution ofDDAH in NO-generating tissues supports the idea that DDAHregulates ADMA levels and NOS activity.19,26 The present studyshows for the first time that mRNA expression of both DDAHs isupregulated in small mesenteric arteries from PPVL and BDEgroups, thus suggesting a higher level of ADMA degradation andtherefore higher NO generation.

One of the intriguing findings in the study of mesenteric vasodi-lation established during cirrhosis is that the increase in systemicADMA4,27,28 does not inhibit NOS nor the excess NO in thesplanchnic endothelial cells in contrast with the decreased genera-tion of NO in the liver. Most studies have focused on NOS activityand NO overproduction in splanchnic vessels overlooking thelocal effects of ADMA and the role of DDAHs in these vessels.The present functional and molecular analyses in small mesenteric

arteries from portal hypertensive and cirrhotic rats demonstrate forthe first time that the low ability of ADMA to inhibit NOS could berelated, at least in part, to a higher expression of DDAH-1 andDDAH-2, and a greater degradation of ADMA. Potentially, this isa new mechanism involved in an increase in basal release of NOand enhanced mesenteric vasodilation.

Although DDAH activity could not be determined in our experi-ments as a result of the small size of the samples, the differencesin contractile sensitivity between L-NAME and ADMA offer areasonable indication that ADMA, and not L-NAME, is rapidlyhydrolyzed by DDAH in the vessel wall and loses part of theinhibitory effect on NO biosynthesis. The contractile responsecurves to ADMA were significantly displaced to the right in PPVLand BDE as compared with those for control rats; this representsindirect evidence of an increased ability of DDAH to catabolizeADMA.

Interestingly, no significant differences in the contractileresponse to ADMA or L-NAME were observed between PPVLand BDE rats. As BDE and PPVL rats had similar increases in PPand liver damage was only present in the BDE group, it is con-ceivable that in small mesenteric arteries, the increase in PP andthe splanchnic vasodilation rather than liver dysfunction arethe main factors causing the altered responses to the NOSinhibitors.

The precise mechanisms underlying the DDAHs expressionelevation observed in the present study remain to be defined. Thecurrent results, however, provide some interesting insights. First,we have demonstrated a clear association between portal hyper-tension, NOS, and DDAHs expressions. Second, mesenteric arter-ies of rats with BDE-induced cirrhosis exhibited a higher DDAH-1expression than did the arteries of rats with PPVL. This higherexpression could be related to the increase of plasmatic levels ofbile acids in the BDE group. It has been described that in theBDE-induced cirrhosis model, plasmatic bile acids are increasedapproximately 25-fold compared with Sham-operated controls.29

This increment is detected by the farnesoid X receptor, a bileacid-responsive nuclear receptor, inducing a DDAH-1 geneupregulation.30

Figure 3 Changes in messenger RNA (mRNA) expression of endothelial nitric oxide synthase (eNOS), dimethylarginine dimethylaminohydrolase(DDAH)-1 and DDAH-2 in mesenteric arteries reported as fold changes relative to controls (Sham-operated group). Results are representative ofsix independent experiments. *P < 0.05 and **P < 0.01 versus Sham group. #P < 0.05 versus partial portal vein ligation (PPVL) group. ( ) Sham,( ) PPVL, ( ) bile duct excision.




That ADMA and L-NAME did not inhibit acetylcholine-induced relaxation in our study indicates that NO-independentpathways contribute to this effect. Endothelium-derived hyperpo-larizing factor contributes greatly to acetylcholine-mediatedvasodilation of isolated mesenteric arteries and is not influencedby NOS inhibition.31 These factors might contribute to the lack ofinhibitory effects of ADMA on acetylcholine-induced vasodila-tion. This non-NO, non-prostanoid factor causes hyperpolarizationthat has been attributed to an increase in K+ conductance of thesmooth muscle cell membrane.32 Our results showed that charyb-dotoxin and apamin completely inhibited the relaxation inducedby acetylcholine, thus suggesting that Ca2+-activated K+ channelsare involved in the non-NO, non-prostanoid acetylcholine relax-ation. Furthermore, it has been demonstrated that ADMA prefer-entially blocks basal activity of NO but has little effect onacetylcholine-induced relaxation.10,15

In conclusion, basal NO release is increased in small mesentericarteries of portal hypertensive and cirrhotic rats. No effectsof NOS inhibition on acetylcholine-stimulated endothelium-dependent vasodilation were observed. Furthermore, our datashow that both DDAH-1 and DDAH-2 act to protect NOSenzymes from the increased circulating levels of ADMA associ-ated with cirrhosis. The decreased contractile effects of ADMA, ascompared with those of L-NAME, and the increased DDAHexpression in small mesenteric arteries suggest a role of DDAHsin the hyperdynamic circulation and the elevated plasma levels ofNO observed in cirrhosis and portal hypertension.

AcknowledgmentsThis work was supported by the Spanish Ministerio de Ciencia eInnovación and Consellería de Sanidad, Generalitat Valenciana.

References1 Vorobioff J, Bredfeldt JE, Groszmann RJ. Hyperdynamic circulation

in portal-hypertensive rat model: a primary factor for maintenanceof chronic portal hypertension. Am. J. Physiol. 1983; 244: G52–7.

2 Martin PY, Gines P, Schrier RW. Nitric oxide as a mediator ofhemodynamic abnormalities and sodium and water retention incirrhosis. N. Engl. J. Med. 1998; 339: 533–41.

3 Guarner C, Soriano G, Tomas A et al. Increased serum nitrite andnitrate levels in patients with cirrhosis: relationship to endotoxemia.Hepatology 1993; 18: 1139–43.

4 Lluch P, Torondel B, Medina P et al. Plasma concentrations of nitricoxide and asymmetric dimethylarginine in human alcoholic cirrhosis.J. Hepatol. 2004; 41: 55–9.

5 Battista S, Bar F, Mengozzi G, Zanon E, Grosso M, Molino G.Hyperdynamic circulation in patients with cirrhosis: directmeasurement of nitric oxide levels in hepatic and portal veins.J. Hepatol. 1997; 26: 75–80.

6 Sarela AI, Mihaimeed FM, Batten JJ, Davidson BR, Mathie RT.Hepatic and splanchnic nitric oxide activity in patients withcirrhosis. Gut 1999; 44: 749–53.

7 Cahill PA, Redmond EM, Hodges R, Zhang S, Sitzmann JV.Increased endothelial nitric oxide synthase activity in the hyperemicvessels of portal hypertensive rats. J. Hepatol. 1996; 25: 370–8.

8 Rockey DC, Chung JJ. Reduced nitric oxide production byendothelial cells in cirrhotic rat liver: endothelial dysfunction inportal hypertension. Gastroenterology 1998; 114: 344–51.

9 Vallance P, Collier J, Moncada S. Effects of endothelium-derivednitric oxide on peripheral arteriolar tone in man. Lancet 1989; 2:997–1000.

10 Mittermayer F, Schaller G, Pleiner J et al. Asymmetricaldimethylarginine plasma concentrations are related to basal nitricoxide release but not endothelium-dependent vasodilation ofresistance arteries in peritoneal dialysis patients. J. Am. Soc.Nephrol. 2005; 16: 1832–8.

11 Segarra G, Medina P, Ballester RM et al. Effects of some guanidinocompounds on human cerebral arteries. Stroke 1999; 30: 2206–10.

12 Segarra G, Medina P, Vila JM et al. Contractile effects of arginineanalogues on human internal thoracic and radial arteries. J. Thorac.Cardiovasc. Surg. 2000; 120: 729–36.

13 Frew JD, Paisley K, Martin W. Selective inhibition of basal but notagonist-stimulated activity of nitric oxide in rat aorta byNG-monomethyl-L-arginine. Br. J. Pharmacol. 1993; 110: 1003–8.

14 Fleming I, Bauersachs J, Schafer A, Scholz D, Aldershvile J, BusseR. Isometric contraction induces the Ca2+-independent activation ofthe endothelial nitric oxide synthase. Proc. Natl. Acad. Sci. U. S. A.1999; 96: 1123–8.

15 Al-Zobaidy MJ, Craig J, Martin W. Differential sensitivity of basaland acetylcholine-induced activity of nitric oxide to blockade byasymmetric dimethylarginine in the rat aorta. Br. J. Pharmacol.2010; 160: 1476–83.

16 Ogawa T, Kimoto M, Sasaoka K. Purification and properties of anew enzyme, NG,NG-dimethylarginine dimethylaminohydrolase,from rat kidney. J. Biol. Chem. 1989; 264: 10205–9.

17 Jacobi J, Sydow K, von Degenfeld G et al. Overexpression ofdimethylarginine dimethylaminohydrolase reduces tissue asymmetricdimethylarginine levels and enhances angiogenesis. Circulation2005; 111: 1431–8.

18 Leiper JM, Santa MJ, Chubb A et al. Identification of two humandimethylarginine dimethylaminohydrolases with distinct tissuedistributions and homology with microbial arginine deiminases.Biochem. J. 1999; 343: 209–14.

19 Kimoto M, Tsuji H, Ogawa T, Sasaoka K. Detection ofNG,NG-dimethylarginine dimethylaminohydrolase in the nitricoxide-generating systems of rats using monoclonal antibody. Arch.Biochem. Biophys. 1993; 300: 657–62.

20 Tojo A, Welch WJ, Bremer V et al. Colocalization of demethylatingenzymes and NOS and functional effects of methylarginines in ratkidney. Kidney Int. 1997; 52: 1593–601.

21 Blanchet L, Lebrec D. Changes in splanchnic blood flow in portalhypertensive rats. Eur. J. Clin. Invest. 1982; 12: 327–30.

22 Sikuler E, Kravetz D, Groszmann RJ. Evolution of portalhypertension and mechanisms involved in its maintenance in a ratmodel. Am. J. Physiol. 1985; 248: G618–25.

23 Kountouras J, Billing BH, Scheuer PJ. Prolonged bile ductobstruction: a new experimental model for cirrhosis in the rat.Br. J. Exp. Pathol. 1984; 65: 305–11.

24 Livak KJ, Schmittgen TD. Analysis of relative gene expression datausing real-time quantitative PCR and the 2-DDCT Method. Methods2001; 25: 402–8.

25 Calver A, Collier J, Leone A, Moncada S, Vallance P. Effect of localintra-arterial asymmetric dimethylarginine (ADMA) on the forearmarteriolar bed of healthy volunteers. J. Hum. Hypertens. 1993; 7:193–4.

26 MacAllister RJ, Parry H, Kimoto M et al. Regulation of nitricoxide synthesis by dimethylarginine dimethylaminohydrolase.Br. J. Pharmacol. 1996; 119: 1533–40.

27 Laleman W, Omasta A, Van de Casteele M et al. A role forasymmetric dimethylarginine in the pathophysiology of portalhypertension in rats with biliary cirrhosis. Hepatology 2005; 42:1382–90.




28 Mookerjee RP, Malaki M, Davies NA et al. Increasingdimethylarginine levels are associated with adverse clinical outcomein severe alcoholic hepatitis. Hepatology 2007; 45: 62–71.

29 Purucker E, Marschall HU, Geier A, Gartung C, Matern S. Increasein renal glutathione in cholestatic liver disease is due to a directeffect of bile acids. Am. J. Physiol. Renal Physiol. 2002; 283:F1281–9.

30 Hu T, Chouinard M, Cox AL et al. Farnesoid X receptor agonistreduces serum asymmetric dimethylarginine levels through hepatic

dimethylarginine dimethylaminohydrolase-1 gene regulation. J. Biol.Chem. 2006; 281: 39831–8.

31 Hwa JJ, Ghibaudi L, Williams P, Chatterjee M. Comparison ofacetylcholine-dependent relaxation in large and small arteriesof rat mesenteric vascular bed. Am. J. Physiol. 1994; 266:H952–8.

32 Taylor SG, Weston AH. Endothelium-derived hyperpolarizing factor:a new endogenous inhibitor from the vascular endothelium. TrendsPharmacol. Sci. 1988; 9: 272–4.




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