Increased aortic cyclic guanosine monophosphate concentration in experimental cirrhosis in rats: Evidence for a role of nitric oxide in the pathogenesis of arterial vasodilation in cirrhosis

Increased Aortic Cyclic Guanosine Monophosphate Concentration in Experimental Cirrhosis in Rats: Evidence for a Role of Nitric Oxide in the Pathogenesis of Arterial Vasodilation in Cirrhosis MICHEL NIEDERBERGER, 1 PERE GINES, 2 PHOEBE TSAI,3 PIERRE-YVES MARTIN, 3 KENNETH MORRIS, 3 ANDR]~ WEIGERT,3 IVAN MCMURTRY, 3 AND ROBERT W. SCHRIER3

Arterial v a s o d i l a t i o n is c o n s i d e r e d to b e t h e k e y factor in t h e d e v e l o p m e n t o f s o d i u m a n d w a t e r r e t e n t i o n leadi n g to a s c i t e s f o r m a t i o n in cirrhosis. To d e t e r m i n e if nitric o x i d e (NO) is i n v o l v e d in t h e p a t h o g e n e s i s o f arterial v a s o d i l a t i o n in cirrhosis, w e m e a s u r e d t h e c o n c e n t r a t i o n of c y c l i c g u a n o s i n e m o n o p h o s p h a t e (cGMP), t h e seco n d m e s s e n g e r o f NO, in arterial t i s s u e f r o m rats w i t h c a r b o n t e t r a c h l o r i d e - i n d u c e d cirrhosis. Aortic cGMP c o n c e n t r a t i o n w a s m a r k e d l y i n c r e a s e d in c i r r h o t i c rats, p a r t i c u l a r l y in t h o s e w i t h a s c i t e s (ascites, 826 _+ 70; n o ascites, 597 _+ 48; controls, 331 _+ 25 filaol/mg, ANOVA F = 23.1, P < .0001), a n d c o r r e l a t e d i n v e r s e l y w i t h arterial p r e s s u r e (r = - . 5 6 , P < .0001) and s y s t e m i c v a s c u l a r res i s t a n c e (r = - . 6 9 , P = .014) a n d d i r e c t l y w i t h c a r d i a c i n d e x (r = .74, P < .01). The c h r o n i c a d m i n i s t r a t i o n of the NO s y n t h e s i s i n h i b i t o r Na-nitro-L-arginine-methyl e s t e r (L-NAME) (10 m g / k g / d a y for 7 days) i n d u c e d a m a r k e d r e d u c t i o n in aortic cGMP c o n c e n t r a t i o n in cirr h o t i c rats w i t h a s c i t e s to s i m i l a r v a l u e s o b t a i n e d in LN A M E - t r e a t e d c o n t r o l rats (86 _+ 14 vs. 89 _+ 8 fmol/mg, r e s p e c t i v e l y , NS), i n d i c a t i n g t h a t t h e h i g h - a o r t i c cGMP c o n t e n t in c i r r h o t i c rats w a s c a u s e d b y an i n c r e a s e d NO

s y n t h e s i s . M e a n arterial p r e s s u r e after L-NAME treatm e n t i n c r e a s e d to s i m i l a r v a l u e s in b o t h g r o u p s of animals. T h e s e r e s u l t s s u g g e s t that in c i r r h o s i s t h e r e is an i n c r e a s e d v a s c u l a r p r o d u c t i o n o f NO that m a y play a role in t h e p a t h o g e n e s i s o f arterial vasodilation. (HEPATOLOGY 1995;21:1625-1631.)

R e n a l function abnormalities, such as s o d i u m a n d w a t e r r e t e n t i o n l e a d i n g to ascites formation, are comm o n findings in a d v a n c e d cirrhosis. 1 Several lines of evidence s u g g e s t t h a t the k e y p a t h o g e n e t i c factor for s o d i u m a n d w a t e r r e t e n t i o n a n d f u n c t i o n a l r e n a l failu r e in cirrhosis is a n a r t e r i a l v a s o d i l a t i o n t h a t induces a h y p e r d y n a m i c c i r c u l a t o r y s t a t e c h a r a c t e r i z e d by arterial h y p o t e n s i o n a n d h i g h - c a r d i a c o u t p u t Y This arterial v a s o d i l a t i o n would cause a b a r o r e c e p t o r - m e d i ated s t i m u l a t i o n of vasoconstrictor, a n t i n a t r i u r e t i c , a n d a n t i d i u r e t i c s y s t e m s (i.e., r e n i n - a n g i o t e n s i n - a l d o sterone, s y m p a t h e t i c n e r v o u s systems, a n d the nonosmotic secretion of a r g i n i n e v a s o p r e s s i n ) t h a t w o u l d e v e n t u a l l y lead to s o d i u m a n d w a t e r r e t e n t i o n a n d ascites formation. 9~1 The cause of this a r t e r i a l v a s o d i l a t i o n Abbreviations: NO, nitric oxide; cGMP, cyclicguanosine monophosphate; is p r e s e n t l y u n k n o w n . Recently, a s u b s t a n c e w i t h a p o t e n t v a s o d i l a t o r efL-NAME,NG-nitro-L-arginine-methyl-ester;AII, angiotensin II; PGI2,prostacyclin;MAP,mean arterial pressure; HR, heart rate; CO, cardiacoutput; SVR, fect, e n d o t h e l i u m - d e r i v e d r e l a x i n g factor, h a s been desystemic vascular resistance; ANP, atrial natriuretic peptide; CI, cardiac in- scribed. It is c u r r e n t l y accepted t h a t e n d o t h e l i u m - d e dex; NOS, nitric oxide synthase. rived r e l a x i n g factor is e q u i v a l e n t to nitric oxide (NO), From 1the Department of Medicine, University Hospital of Bern, Bern, Switzerland; 2the Liver Unit, Hospital Clinic I Provincial, Barcelona, Spain; a chemical congener, or a n a d d u c t of NO. 12'13 A m o n g a and 3the Department of Medicine, Universityof ColoradoSchoolof Medicine, wide v a r i e t y of biological actions, N O is believed to play Denver, CO. a n i m p o r t a n t role in the r e g u l a t i o n of blood flow a n d ReceivedApril 11, 1994;acceptedDecember31, 1994. a r t e r i a l p r e s s u r e in n o r m a l c i r c u m s t a n c e s a n d also in Supported by a grant from the National Institutes of Health (DK 19928) Bethesda, MD. MNreceiveda grant fromthe SwissNational ScienceFounda- the p a t h o g e n e s i s of a r t e r i a l h y p o t e n s i o n in some p a t h o tion. PG receivedgrants fromthe DirecciSnGeneral de InvestigaciSny T~cnica logical conditions, such as septic shock. ~<15 Vallance (DGICYT92-317) and the AsociaciSnEspafiola para el Estudio del H~gado. a n d M o n c a d a ~6 h a v e r e c e n t l y proposed t h a t a n inP-YMreceiveda grant fromthe UniversityHospital of Geneva, Geneva, Swit- c r e a s e d v a s c u l a r s y n t h e s i s of N O could be the cause of zerland. a r t e r i a l v a s o d i l a t i o n in cirrhosis. B e c a u s e the direct Dr Niederbergerand Dr Gin6swere affiliatedwith the DepartmentofMedicine, Universityof ColoradoSchoolof Medicine when the research presented m e a s u r e m e n t of NO levels is t e c h n i c a l l y difficult, the p r o d u c t i o n of N O h a s been indirectly assessed in r a t s in this article was conducted. Addressreprint requests to: RobertW. Schrier,MD,Chairman,Department w i t h cirrhosis, as well as in o t h e r models of p o r t a l hyof Medicine, Universityof ColoradoHealth Science Center, Box B-178, 4200 pertension, by i n v e s t i g a t i n g t h e effects of N O s y n t h e s i s E Ninth Ave,Denver, CO 80262. Copyright © 1995 by the American Association for the Study of Liver inhibition on systemic h e m o d y n a m i c s . U s i n g this approach, some studies h a v e f o u n d evidence s u g g e s t i n g Diseases. a role for N O in the p a t h o g e n e s i s of a r t e r i a l vasodila0270-9139/95/2106-002153.00/0 1625

1626

NIEDERBERGER ET AL

tion, 17-2° w h e r e a s o t h e r i n v e s t i g a t i o n s do not s u p p o r t the existence of a n i n c r e a s e d p r o d u c t i o n of N O in t h e s e e x p e r i m e n t a l models. 2123 The a p p r o a c h u s e d in this s t u d y w a s to d e t e r m i n e the c o n c e n t r a t i o n of cyclic g u a n o s i n e m o n o p h o s p h a t e (cGMP), the i n t r a c e l l u l a r m e s s e n g e r of N O responsible for v a s c u l a r relaxation, in a r t e r i a l tissue from r a t s w i t h e x p e r i m e n t a l cirrhosis. O u r r e s u l t s indicate t h a t aortic c G M P c o n c e n t r a t i o n is i n c r e a s e d in r a t s w i t h cirrhosis, p a r t i c u l a r l y in t h o s e w i t h ascites, a n d correlates i n v e r s e l y w i t h a r t e r i a l p r e s s u r e a n d s y s t e m i c v a s c u l a r r e s i s t a n c e a n d directly w i t h cardiac index. T h e s e findings s u g g e s t t h e existence of a n i n c r e a s e d a r t e r i a l p r o d u c t i o n of N O t h a t m a y p l a y a role in t h e p a t h o g e n e s i s of a r t e r i a l vasodilation. MATERIALS AND METHODS Animals

The investigation was performed in male Sprague-Dawley rats (Sasco, Omaha, NE) housed in a controlled environment and allowed free access to food and water until the time of study. The protocol was approved by the University of Colorado Health Sciences Center Animal Care and Use Committee. Several groups of rats were included and divided into three protocols. Protocol 1: Effects of NG-Nitro-L-Arginine-Methyl-Ester (LNAME) a n d Sodium Nitroprusside on Aortic cGMP Concentration in Normal Rats. The first part of the study investigated

whether changes in NO content in vivo induced by pharmacological manipulations of NO-soluble guanylate cyclase pathway are associated with parallel changes in arterial cGMP concentration. Three groups of rats (250 to 350 g) were studied: control rats (n = 3), rats chronically treated with LNAME (Bachem, Torrance, CA) in their drinking water for 1 week (10 mg/dL, n = 3, or 50 mg/dL, n = 3) to inhibit NO synthesis, and rats treated with an intravenous infusion of sodium nitroprusside (Sigma Co., St Louis, MO) for 30 minutes (30 #g/kg/min, n = 3, or 150 #g/kg/min, n = 3) to increase tissue NO. To exclude that the effects of L-NAME and sodium nitroprusside on aortic cGMP concentration were changes that were accounted for in arterial pressure and not modifications in NO content, the effect of NO-independent vasoactive substances on aortic cGMP was also investigated. Two groups of rats (250 to 350 g) were studied: rats treated with an intravenous infusion of angiotensin II (AII) (50 ng/kg/min, n = 4, or 200 ng/kg/min, n = 4; Sigma Co.) for 30 minutes to increase arterial pressure, and rats treated with an intravenous bolus of prostacyclin (PGI2) (5 #g/kg, n = 4 or 20 #g/kg, n = 4; Sigma Co.) to reduce arterial pressure. Protocol 2: Aortic cGMP Concentration in Cirrhotic Rats a n d in Phenobarbital-Treated Control Rats. Cirrhosis was

induced by weekly intragastric administration of CC14 and phenobarbital in the drinking water (350 mg/dL) as previously described. 24 A control group of rats treated only with phenobarbital in the drinking water was also studied. Aortic cGMP concentration was measured in a total of 58 rats: phenobarbital-treated control rats (n = 20), cirrhotic rats without ascites (n = 16), and cirrhotic rats with ascites (n = 22). The diagnosis of cirrhosis was confirmed by histological examination and the presence of ascites was confirmed by visual examination at laparotomy. Rats with and without ascites were studied 7 to 19 weeks (mean, 12 _+ I weeks) and 8 to 19

HEPATOLOGYJune 1995 weeks (mean, 12 +_ I weeks), respectively, after starting the cirrhosis-induction program. Phenobarbital-treated control rats were studied after 9 to 18 weeks (mean, 13 _+ 1 weeks) of phenobarbital administration. In cirrhotic rats, the study was performed 9 to 11 days after the last dose of CC14.In 12 rats included in this protocol (6 phenobarbital-treated control rats and 6 cirrhotic rats with ascites), a complete hemodynamic evaluation, including mean arterial pressure (MAP), heart rate (HR), cardiac output (CO), and systemic vascular resistance (SVR), was performed. In the remaining 46 rats only MAP and HR were measured. Protocol 3: Effects of Chronic NO Synthesis Inhibition With L-NAME on Aortic cGMP Concentration in Cirrhotic Rats With Ascites and Phenobarbital-Treated Control Rats. The effects

of chronic inhibition of NO synthesis with L-NAME on aortic cGMP and arterial pressure were investigated in rats with cirrhosis and ascites (n = 6) and phenobarbital-treated control rats (n = 6). In addition, the effect of chronic inhibition of NO synthesis with L-NAME on plasma atrial natriuretic peptide (ANP) concentration in cirrhotic rats with ascites (n = 6) was also examined. L-NAME (10 mg/kg/day) was given by gavage twice a day for 7 days. Experimental Procedures

Arterial pressure was measured with the rats in the conscious state using an intraarterial catheter. Rats were anesthetized with ether, and the right femoral artery was cannulated with a polyethylene catheter (PE10, Intramedic; Clay Adams, Parsippany, NJ). The catheter was led to the back of the neck and exteriorized. Rats were placed in individual cages with no restriction to movement and allowed to recover for 48 hours. The arterial catheter was then connected to a pressure transducer (Viggo-Spectramed, Oxnard, CA) and a multichannel recorder (Grass Instruments Co., Quincy, MA) and MAP and HR were recorded. In rats treated with sodium nitroprusside, AII or PGI2, a femoral catheter (PE50, Intramedic, Clay Adams) was used for the intravenous infusions. Immediately after measurement of MAP and HR, the rats were killed by decapitation, and the thoracic aorta was rapidly excised, rinsed in cold phosphate-buffered saline, and snap-frozen in liquid nitrogen within a period of less than 3 minutes after decapitation. Tissue samples were then stored at -70°C until cGMP determination. Trunk blood was collected for plasma ANP measurement in a subset of animals. CO was measured by a dye dilution methodY Rats were anesthetized with ether, and a carotid artery and the two jugular veins were cannulated with a polyethylene catheter (PE50, Intramedic; Clay Adams). The catheters were tunneled subcutaneously through the back of the neck and exteriorized. After 2 days of recovery from anesthesia, the conscious rats were placed in a small well-ventilated Plexiglas chamber (Narco Bio-systems, Houston, TX) that was fluxed with room air. CO was then measured using the standard indocyanine green dye technique, as previously described. 26'27 In brief, 5 #g of green dye was injected into a jugular catheter, while blood was simultaneously pumped through the shunt from carotid artery to jugular vein at 3 mL/min through a densitometer cuvette. This signal was entered in a computer, physiograph, and oscilloscope, generating a dye curve. Arterial pressure signal was entered in a physiograph and microcomputer. HR was determined from the arterial pressure trace. SVR was calculated by MAP/CO. Cardiac index (CI) was determined by CO/animal weight. After the hemodynamic evaluation, rats were allowed to recover for 24 hours and then killed by decapitation to obtain the thoracic aorta for cGMP determination.

HEPATOLOGY Vol. 21, No. 6, 1995

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NIEDERBERGER ET AL

TABLE 1. MAP, HR, and Aortic cGMP Concentrations in Normal Rats

and Rats Treated With L.NAME and S o d i u m Nitroprusside L-NAME

MAP (mm Hg) HR (beats/n~dn) Aortic cGMP (fmol/mg)

Control, n=3

10 mg/dL,

129 __ 2 427 ___ 15 440 ___47

154 ± 3 300 ± 17 248 ± 59

n=3

Sodium

50 mg/dL,

ALL,

n=3

159 _+ 9 310 _+ 21 152 _+ 16

D e t e r m i n a t i o n o f Aortic cGMP Concentration a n d Plasma ANP A o r t i c c G M P c o n c e n t r a t i o n w a s m e a s u r e d a c c o r d i n g to t h e

method described by Arnal et al. 28'29 The thoracic aorta was homogenized in 1 mL of 0.1 N of hydrochloric acid with an all-glass homogenizer at 4°C. The homogenates were then centrifuged at 3,000g for 60 minutes, and aliquots of the supernatants were stored at -20°C until assayed. The protein concentration was determined in each sample using the Bradford method (Biorad, Richmond, CA). cGMP was determined with a commercial radioimmunoassay kit (Amersham Corp., Arlington Heights, IL) using an acetylation protocol and expressed as fmol/mg of protein. Immunoreactive plasma ANP was determined as previously described. 3° S t a t i s t i c a l Methods Statistical analysis of t h e r e s u l t s w a s p e r f o r m e d u s i n g ANOVA and Newman-Keuls test, paired and unpaired Student's t-tests, and linear regression analysis. Results are expressed a s m e a n _+ S E M a n d w e r e c o n s i d e r e d s i g n i f i c a n t a t P < .05. RESULTS

Effects of L-NAME a n d S o d i u m N i t r o p r u s s i d e on Aortic cGMP Concentration in N o r m a l R a t s . L - N A M E -

treated rats had significantly higher MAP (156 _+ 5 mm Hg, P < .05) and significantly lower HR (305 __ 12 beats/min, P < .0001) t h a n control rats (129 ± 2 mm Hg and 427 _+ 15 beats/min, respectively). The hemodynamic pattern was similar with the two doses of LNAME used (Table 1). Rats treated with sodium nitroprusside showed marked arterial hypotension (74 _+ 8 mm Hg, P < .001 as compared with control rats) with a significant increase in h e a r t rate (512 ± 19 beats/ min, P < .01 as compared with control rats). One rat treated with the highest dose of sodium nitroprusside died during the infusion period and is not included in the results. The hemodynamic changes observed in rats treated with L-NAME and sodium nitroprusside were associated with marked changes in aortic cGMP concentration (Table 1) (Fig. 1). L-NAME-treated rats had significantly lower aortic cGMP concentration t h a n control rats (200 _+ 35 vs. 440 ± 47 fmol/mg, respectively, P < .05), whereas rats treated with sodium nitroprusside had extremely high values of aortic cGMP (9,305 _+ 2,623 fmol/mg, P < .01 as compared with controls) (ANOVA F = 10.5, P < .01). In all animals, a highly significant inverse correlation was found be-

n=6

156 _+ 5 305 _+ 12 200 ± 35

Nitroprusside

30 fcg/kg/min, n=3

150 ttg/kg/min, n=2

85 +_ 9 500 ± 21 9,857 _+ 3,817

58 _+ 2 530 _+ 40 8,479 _+ 4,897

All, n=5

74 _+ 8 512 +_ 19 9,305 + 2,623

tween aortic cGMP and arterial pressure (r -- -.91, P < .0001) (Fig. 1). The administration of AII and PGI2 also induced marked changes in systemic hemodynamics (Table 2). In rats treated with AII MAP increased to values similar to those observed in L-NAME-treated rats, whereas the administration of PGI2 induced arterial hypotension. Despite these marked effects on systemic hemodynamics, aortic cGMP concentration in rats treated with AII or PGI2 was similar to values observed in control rats. No relationship was found between aortic cGMP concentration and MAP in these two groups of rats (r = -.07, P = .79) (Fig. 2). A o r t i c cGMP Concentration in C i r r h o t i c R a t s a n d in P h e n o b a r b i t a l - T r e a t e d Control Rats. C i r r h o t i c r a t s with ascites had significantly lower MAP and significantly higher HR t h a n cirrhotic rats without ascites and phenobarbital-treated control rats (MAP: 96 _+ 3, 112 _+ 2, and 120 _+ 2 mm Hg, respectively; ANOVA: F = 25.4, P < .0001; HR: 399 + 10, 357 + 12, and 352 _+ 9 beats/min, respectively; ANOVA: F = 6.7, P < .01). Aortic cGMP concentration was markedly higher in cirrhotic rats with ascites t h a n in phenobarbitaltreated controls. Cirrhotic rats without ascites showed intermediate values (ascites, 826 _+ 70; no ascites, 597 _+ 48; phenobarbital-treated controls, 331 _+ 25 fmol/ mg; ANOVA, F = 23.1, P < .0001) (Fig. 3). In the whole group of animals there was a significant inverse correlation between aortic cGMP concentration and MAP (r = -.56, P < .0001) (Fig. 4). 100000 ~

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FIG. 1. (Left) Aortic cGMP concentration (mean ± SEM) in control rats (n = 3), rats chronically treated with L-NAME in the drinking water (n = 6), and rats treated with sodium-nitroprusside (n = 5). *P < .05, **P < .01 vs. control rats. (Right) Correlation between aortic cGMP concentration and MAP in the same groups of animals (r = -.91, P < .0001). Aortic cGMP concentration is expressed on a logarithmic scale.

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HEPATOLOGY J u n e 1995

TABLE 2. M A P , H R , a n d A o r t i c c G M P C o n c e n t r a t i o n s i n N o r m a l R a t s a n d R a t s T r e a t e d w i t h AII a n d P G I 2 AII

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109 t 3 511 _+ 7 423 _+ 61

85 _+ 3 515 _+ 15 469 _+ 52

n=3 MAP (ram Hg) HR (beats/min) Aortic cGMP (fmol/mg)

129 +_ 4 427 _+ 15 440 _+ 47

Table 3 shows baseline MAP, HR, CO, CI, and SVR in the cirrhotic rats with ascites and phenobarbitaltreated control rats submitted to a complete hemodynamic evaluation. Compared with phenobarbitaltreated control rats, cirrhotic rats with ascites had significantly reduced MAP and SVR and significantly increased CO, CI, and HR. In these animals, aortic cGMP correlated inversely with MAP and SVR (r = -.59, P < .05 and r = -.69, P = .014, respectively) and directly with CI (r = .74, P < .01) (Fig. 5). Effects o f C h r o n i c N O S y n t h e s i s I n h i b i t i o n With LN A M E on A o r t i c c G M P C o n c e n t r a t i o n in C i r r h o t i c R a t s With A s c i t e s a n d P h e n o b a r b i t a l - T r e a t e d C o n t r o l Rats.

The chronic inhibition of NO synthesis with L-NAME in cirrhotic rats with ascites was associated with a very low aortic cGMP concentration and similar to t h a t observed in phenobarbital-treated control rats studied under the same conditions (86 _ 14 vs. 89 + 8 fmol/ mg, respectively) (Fig. 6). MAP at the end of the 7day t r e a t m e n t with L-NAME was similar in rats with cirrhosis and in control rats (140 + 3 and 137 _+ 2 m m Hg, respectively). To exclude the possibility t h a t changes in aortic cGMP concentration after chronic inhibition of NO synthesis in cirrhotic rats with ascites were accounted for by modifications in ANP levels during treatment, the plasma concentration of ANP was measured in an additional group of cirrhotic rats with ascites chronically treated with L-NAME. The plasma concentration of ANP in these animals was similar to t h a t obtained in a nontreated group of cirrhotic rats with ascites (n = 6) (107 _+ 11 vs. 112 _+ 5 fmol/mL, respectively, NS).

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The Peripheral Arterial Vasodilation Hypothesis of sodium and water retention in cirrhosis is supported by systemic hemodynamic and humoral alterations t h a t are known to occur with primary arterial vasodilation.ll Specifically, systemic vascular resistance and arterial pressure decline as cardiac output rises; these hemodynamic changes are associated with stimulation of the sympathetic, renin-angiotensin-aldosterone, and nonosmotic vasopressin systems. 9'1°'31'32 Moreover, those compensated cirrhotic patients who develop ascites during mineralocorticoid administration have lower systemic vascular resistance and higher cardiac output t h a n compensated cirrhotic patients who show mineralocorticoid escape and thus, no occurrence of ascites. 88 The hepatic venous pressure gradients, as an index of sinusoidal pressure, were no different between these two groups of patients.

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No ascites

ascites

FIG. 3. Aortic cGMP concentration ( m e a n _+ SEM) in p h e n o b a r b i t a l - t r e a t e d control r a t s (n = 20), cirrhotic r a t s w i t h o u t ascites (n = 16), a n d cirrhotic r a t s w i t h ascites (n = 22). *P < .05 a n d ***P < .001 vs. control rats; ##P < .01 vs. cirrhotic r a t s w i t h o u t ascites.

HEPATOLOGY Vol. 21, No. 6, 1995

NIEDERBERGER

ET AL

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FIG. 5. C o r r e l a t i o n b e t w e e n aortic c G M P c o n c e n t r a t i o n a n d b a s e line SVR (r = - . 6 9 , P = .014) (left) a n d CI (r = .74, P < .01) (right) in p h e n o b a r b i t a l - t r e a t e d control r a t s (n = 6) a n d cirrhotic r a t s w i t h a s c i t e s (n = 6).

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140

160

Mean Arterial Pressure (mmHg) FIG. 4. C o r r e l a t i o n b e t w e e n aortic c G M P c o n c e n t r a t i o n a n d m e a n a r t e r i a l p r e s s u r e in p h e n o b a r b i t a l - t r e a t e d control r a t s (n = 20), cirrhotic r a t s w i t h o u t a s c i t e s (n = 16), a n d cirrhotic r a t s w i t h a s c i t e s (n = 22)• r = - . 5 6 , P < .0001.

Secondly, whereas central blood volume expansion with colloid or head-out water immersion m a y improve sodium and water excretion, a4 only the addition of a vasoconstrictor to reverse the arterial vasodilation has been shown to normalize sodium and water excretion in decompensated, ascitic cirrhotic patients. 4 In this study, the improved water excretion directly correlated with the increase in systemic vascular resistance. On this background, the mediators of the systemic vasodilation are very important to delineate. Specifically, reversal of this arterial vasodilation has the potential of reversing ascites accumulation and the renal vasoconstriction, thus reversing the hepatorenal syndrome. In this study, the potential role of vascular NO and its secondary messenger as the mediator of arterial vasodilation was examined in an experimental model of cirrhosis that mimics cirrhosis in humans• In previous studies, the vascular roles of NO in models of cirrhosis and portal hypertension have been conilicting. 172~ The first challenge of this study was to determine if aortic cGMP could be measured and modulated in the normal rat. In that regard, the administration of LNAME, an inhibitor of nitric oxide synthase (NOS),

significantly decreased cGMP, and nitroprusside, a NO donor, significantly increased aortic cGMP (Fig. 1). Because there was a significant correlation between these aortic cGMP concentrations and MAP in these groups of animals, the possibility existed that MAP rather than NO was the determinant of the aortic cGMP concentrations. Comparable MAP changes were therefore induced by pressor (AII) and depressor (PGI2) agents, which are known not to involve NO or cGMP. In these latter studies, no changes were observed in aortic cGMP, and no correlation was found between this parameter and MAP (Fig. 2). Because L-NAME was given for 1 week and AII was administered as an acute intravenous infusion, the possibility still exists that the reduction in aortic cGMP in L-NAME treated animals was, at least partially, caused by the chronic increase in arterial pressure itself, instead of being exclusively caused by the inhibition of NO synthesis induced by the L-arginine analogue. However, this possibility is unlikely, because it has been shown that the acute administration of L-arginine completely normalizes the aortic cGMP concentration in rats with chronic arterial

1000 800 ~ 600 a.

TABLE 3. B a s e l i n e H e m o d y n a m i c D a t a i n P h e n o b a r b i t a l Treated Control Rats and Cirrhotic Rats With Ascites

Cirrhotic R a t s With Ascites (n = 6) M A P ( m m Hg) H R ( b e a t s / m i n 1) CO ( m L / m i n -1) CI ( m L / m i n 1/100 g-l) SVR ( m m H g / m i n / m L 1/ 100 g 1)

102 400 187 51

± 6 _+ 17 ÷ 15 ± 4

2•2 ± 0.3

Control (n = 6) 126 347 138 33

=! O o

400-

._o O

200,

P

__ 5 ± 19 ± 7 _ 2

<.02 .06 <.02 <.01

3.9 ± 0.3

<.01

0 Control L-NAME

As ci tes L-NAME

FIG. 6. Aortic c G M P c o n c e n t r a t i o n ( m e a n _+ SEM) in p h e n o b a r b i t a l - t r e a t e d control r a t s (n = 6) a n d cirrhotic r a t s w i t h a s c i t e s (n = 6), b o t h c h r o n i c a l l y t r e a t e d w i t h L-NAME (10 mg/kg/d) for 7 days.

1630

NIEDERBERGER ET AL

hypertension induced by t r e a t m e n t with L-NAME for 4 weeks. 2s Furthermore, basal concentration of aortic cGMP in spontaneous hypertensive rats has been shown to be not significantly different from that of normal rats. 29 Finally, the determination of vascular cGMP constitutes a direct m e a s u r e m e n t of neither NO production nor NOS activity. Nevertheless, the results of the current study, together with data from other investigations, 2s'35 strongly suggest that arterial cGMP concentration provides a good estimate of the NO synthase/soluble guanylate cyclase p a t h w a y activity in the arterial wall. The next goal was to examine aortic cGMP in cirrhotic animals. In this study, the compensated cirrhotic rats showed higher aortic cGMP content than the control rats. Moreover, the decompensated cirrhotic rats with ascites showed significantly higher aortic cGMP concentrations as compared with the compensated cirrhotic rats (Fig. 3). Furthermore, the potential importance of cGMP in mediating arterial vasodilation in cirrhosis was supported by the finding of significant correlations between MAP, CI, and SVR and aortic cGMP concentrations in these animals (Figs. 4 and 5). Since cGMP is the secondary messenger for vasodilators other than NO, it could be possible that the increase in cGMP in cirrhotic rats was caused by N O independent substances. For example, ANP has been shown to be increased in cirrhosis ~s and although the vascular effect of ANP is NO independent, it has cGMP as its secondary m e s s e n g e r s Therefore, the next group of studies performed was aimed at examining if the increased aortic cGMP in the cirrhotic rats could be decreased to concentrations comparable to those in control rats during NO blockade with L-NAME. If other c G M P - m e d i a t e d , but NO-independent, vasodilators were involved in the increase in aortic cGMP and lower MAP in the cirrhotic animals, L-NAME-treated control and cirrhotic rats would not be expected to show comparable aortic cGMP concentrations and MAP. As shown in Fig. 6, the aortic cGMP concentrations were comparable in L - N A M E - t r e a t e d control and cirrhotic rats; moreover, the MAPs in these two groups of animals were comparable. The absence of changes in ANP levels during L-NAME t r e a t m e n t in cirrhotic rats with ascites further confirmed that the decrease in aortic cGMP in these rats was caused by an inhibition of NO synthesis, not a reduction in ANP levels. Several studies in experimental animals have shown that in cirrhosis there is a marked splanchnic arterial vasodilation. 3s-4° Therefore, it would have been of interest to measure the concentration of cGMP in resistance vessels from the splanchnic circulation. Unfortunately, this was not possible with the method used in this study, because a significant amount of vascular tissue is required for cGMP determination. Therefore, it is not known if the increased concentration of cGMP found in aortic tissue can be extrapolated to other vascular territories, specifically the splanchnic circulation. Despite this limitation, the progressive increase in aortic cGMP in the compensated and decompensated cirrhotic

HEPATOLOGYJune 1995

rats, as well as the correlation between the hemodynamic parameters and aortic cGMP, suggests that an increased vascular production of NO participates in the pathogenesis of the cirrhosis-induced arterial vasodilation. These results are also compatible with previous findings of the restoration of vascular responsiveness to vasoconstrictors in experimental cirrhosis with inhibition of NOS, 41 the greater increase in MAP in experimental cirrhosis with the NO inhibitor (N~-L-nitro-ar ginine), 17 and the increased serum nitrite and nitrite concentrations in patients with cirrhosisY However, there remain important questions about the role of NO in cirrhosis. For example, it is not known if the constitutive or the inducible NOS is involved. Because of the known effect of endotoxin to induce NO production in vascular smooth m u s c l e 14'15'43 and the reported increased plasma endotoxin levels in cirrhosis, 44 it has been proposed that the systemic arterial vasodilation of cirrhosis is secondary to the inducible NOS. 16 However, this possibility is not compatible with the finding that endothelial denudation normalizes the impaired response to vasoconstrictors observed in aortic rings from cirrhotic rats. 4~ Moreover, using a specific cDNA probe for the inducible NOS, exogenous endotoxin b u t not experimental cirrhotic was shown to increase the inducible NOS mRNA concentration in aortic tissue extracts. 45 A vascular role of the constitutive NOS in the endothelium of cirrhotic animals or patients is therefore in need of study. These results provide further support for the role of NO in the progressive arterial vasodilation observed with compensated and decompensated experimental cirrhosis. The reversal of this vascular effect of NO has potential implications for improving the morbidity and mortality observed in cirrhotic patients.

Acknowledgment: We thank Dr W. H a m m o n d for his assistance with the histological study. REFERENCES

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