Mixed endothelin receptor antagonist, SB209670, decreases portal pressure in biliary cirrhotic rats in vivo by reducing portal venous system resistance

Journal of Hepatology 2000; 32: 43-50 Printed in Denmark All rights reserved Munksgaard Copenhagen

Copyright 8 European Association for the Study of the Liver 2000 Journal of Hepatology ISSN 0168-8278

Mixed endothelin receptor antagonist, SB209670, decreases portal pressure in biliary cirrhotic rats iiz viva by reducing portal venous system resistance Hideyuki

Kojima, Jyunichi Yamao, Tatsuhiro Tsujimoto, Hiroshi Fukui

Masahito

Uemura,

Akira Takaya and

Third Department of Internal Medicine, Nara Medical University, Kashihara, Japan

Background/Aims: This study aimed to evaluate the hemodyuamic effects of endothelin-1 or mixed endothelin receptor antagonist, SB209670 in cirrhotic rats, and to elucidate the role of endothelin in cirrhotic portal hypertension. Methods: Secondary biliary cirrhosis was induced by bile duct ligation. Hemodynamics were studied using the radioactive microsphere technique. Results: Plasma and hepatic endothelin levels in cirrhotic rats were significantly higher than those in normal rats (plasma, 9.021.3 vs. 2.620.5 pglml, p
duction was associated with reduced portal venous system resistance (vehicle, 2.520.2 vs. SB209670, 1.7kO.l mmHg - min - 100 g bwlml, p
P

Endothelin (ET), a potent vasoconstrictive peptide, has been suggested to contribute to the pathogenesis of portal hypertension. This suggestion is based on the observations that plasma and hepatic ET levels are elevated in human and carbon tetrachloride-induced liver cirrhosis (3-5), and ET causes sinusoidal and presinusoidal constriction via ET, and ETn receptors and increases perfusion pressure in the perfused normal and cirrhotic rat liver (6-9). Recently, two studies reported that the mixed ET, and ETn receptor antagonist, bosentan, reduces portal pressure in experimental cirrhosis (10,ll). However, the mechanism of its portal hypotensive effect is controversial. Sogni et al. (10) showed that the reduction in hepatocollateral vascular resistance, representing the sum of resistance in the in-

HYPERTENSION is characterized by a pathological increase in portal pressure, and often leads to the formation of esophageal varices and ascites. It is now considered that an increased intrahepatic vascular resistance is the initial event responsible for portal hypertension, and thereafter the increased portal tributary blood flow due to hyperdynamic circulation contributes to the development and maintenance of portal hypertension (1,2). ORTAL

Received 20 January;

Correspondence:

revised 19 May: accepted 8 July 1999

Hideyuki Kojima, Third Department of Internal Medicine, Nara Medical University, 840 Shijocho Kashihara-shi, Nara 634-8522, Japan. Tel: 81 744 22 3051. Fax: 81 744 24 7122.

Key words: Endothelin; Endothelin receptor antagonist; Hemodynamics; Liver cirrhosis; Portal hypertension; Radioactive microsphere technique.

43

H. Kojima et al.

trahepatic vasculature and collateral circulation, resulted in reduced portal pressure. In contrast, another group reported that the reduced portal pressure was achieved mainly via a decrease in hepatic arterial flow, although the relationship between portal pressure and hepatic arterial flow was not described (11). These controversial results prompted us to further investigate the hemodynamic effects of ET antagonist in cirrhosis. SB209670 is an orally active and extremely potent mixed ETA and ETn receptor antagonist, which may allow us to elucidate a role for endogenous ET in portal hypertension (12). The aims of this study were to evaluate the hemodynamic effects of ET-1 or SB209670 in cirrhotic rats and to elucidate the role of ET in portal hypertension.

Materials and Methods Animalpreparation Male Sprague-Dawley rats weighing 200-250 g were used. Secondary biliary cirrhosis with intrahepatic portal hypertension was induced by bile duct ligation, as previously described (13,14). In brief, the common bile duct was exposed by median laparotomy and doubly ligated with 4-O silk thread. The common bile duct was then resected between two ligatures and the abdominal incision was closed. After 4 weeks of surgery, these rats were used for the following studies. Plasma and hepatic ET levels

Plasma and hepatic ET levels were measured in 6 biliary cirrhotic and 6 normal rats. Trunk blood samples were collected in chiied tubes containing EDTA-2Na (1 mg/ml) and aprotinin (500 KIU/ml) and were centrifuged at 4°C. The plasma obtained was frozen until analyzed. Liver was resected and homogenized for 1 min in 10 volumes of 1 M acetic acid and immediately heated at 100°C for 10 min to inactivate proteases. After chilling, the homogenates were centrifuged at 15000 g for 10 min at 4°C. Supernatants were stored at -20°C until analyzed. ET level was measured by radioimmunoassay (RIA) after extraction, as previously described (3,15). In brief, plasma or supernatants were acidified with trifluoroacetic acid (TFA) and applied to Spe-C, cartridges (Baker Chemical Co., Phillipsburg, NJ, USA) preactivated with methanol, distilled water and 0.09% TFA. The absorbed materials were eluted with 0.09% TFA and 60% acetonitrile in 0.09% TFA. The elute was then evaporated and reconstituted for RIA (Otsuka Assay Institute, Japan). The antiserum did not cross-react with ET-2 and ET-3. Cross-reactivity with big ET-1 was only 2%. The intraassay and interassay variations of this method were both below 10%.

at 1 cm above the operating table. Rectal temperature was maintained at 37”C?O.5”C by heat table throughout the study. Cardiac output (CO) and regional blood flows were measured using a radioactive microsphere technique, as previously described (17,18). A reference sample was obtained from the femoral artery catheter for 75 s at a rate of 1 mYmin using a continuous withdrawal pump (CFV2100; Nihon Kohden, Tokyo, Japan). Approximately 60 000 microspheres labeled with 51Cr (15.5kO.l w diameter, specific activity: 44.71 mCi/g; New England Nuclear, Boston, MA, USA) were injected into the left ventricle 15 s after the start of blood withdrawal. CO was calculated as CO (mVmin)=Injected radioactivity (cpm)XReference blood flow (ml/min)/Reference blood radioactivity (cpm). ‘The cardiac index was expressed per 100 g body weight. For the calculation of regional blood flows, injected radioactivity was replaced by the radioactivity of each organ in the previous equation. Porto-systemic shunting (PSS) was estimated using 57Co-labeled microspheres (15.520.1 pm diameter, specific activity: 16.49 mCi/g) infused slowly via an ileocolic vein catheter as PSS (%)=Lung radioactivity (cpm)X lOO/(Liver+Lung) radioactivity (cpm) (19,20). Portal venous inflow (PVI) was calculated as the sum of the blood flow to stomach, spleen, small and large intestines, pancreas, and mesentery. Collateral blood flow (CBF) was estimated as CBF (ml/min. 100 g bw)=PVIXPSS/lOO. At the end of experiments, the animals were killed with a bolus injection of sodium phenobarbital (50 mg/kg, iv). The abdominal organs, kidneys and lungs were dissected, blotted, cut into small pieces, and placed in counting tubes. The radioactivity (cpm) of each organ was determined in a gamma-scintillation counter (ARC-380; ALOKA, Tokyo, Japan). The interference of 5’Cr radioactivity (energy window, 25&423 keV) into the 57Co channel (energy window, 82-181 keV) was corrected using 51Cr and 57Co standards. Vascular resistances were calculated from the ratio between perfusion pressure (P) and blood flow (Q) of each vascular territory. For the calculation of systemic vascular resistance, P was mean arterial pressure minus right atria1 pressure, and Q was cardiac index; for the splanchnic arterial resistance, P was mean arterial pressure minus portal pressure, and Q was PVI; for the portal venous system resistance, P was portal pressure minus right atria1 pressure, and Q was PVI. Experimental design

1. Six cirrhotic and six normal rats with and the same number without the pretreatment with SB209670 (18 pmollkg) received intraportal administration of ET-l. SB209670, kindly supplied by SmithKline Beecham Pharmaceuticals (King of Prussia, PA, USA), was administered intraportally 5 min before ET-1 administration. ET-l, purchased from the Peptide Institute (Osaka, Japan), was infused at a dosage of 3 nmol/kg during a period of 1 min through a double-lumen catheter

r

wei11-w) 100 r

tP@a

WW

12

r-PO.Wll

r po.001~

Hemodynamic studies

Hemodynamic studies were performed under ketamine anesthesia (100 mg/kg im), which has been shown to approximate most closely the conscious state in terms of cardiac output and regional blood flow (16), although hemodynamic studies are analyzed better in the conscious and unrestrained state and anesthesia affects the splanchnic hemodynamics. A femoral artery was cannulated with a PE-50 catheter for arterial pressure measurement and blood withdrawal. The left ventricle was catheterized via the right carotid artery with PE-50 tubing under pressure monitoring. Further PE-50 catheters were inserted into the right jugular vein for atria1 pressure measurement and the femoral vein for drug infusion. Median laparotomy was performed and the portal vein was cannulated via the ileocolic vein with a PE50 catheter. All catheters except for the femoral vein catheter were connected to pressure transducers (AP-611G; Nihon Kohden, Tokyo, Japan) for blood pressure monitoring. The zero point was established

44

Plasma

Liver

Fig, 1. Plasma and hepatic endothelin levels in normal rats (0) and biliary cirrhotic rats (m). Plasma and hepatic endothelin levels were measured in 6 normal rats and 6 biliary cirrhotic rats as by radioimmunoassay, as described in Materials and Methods. Values are presented as meant-SEA4

Endothelin in portal hypertension (outer diameter 1.0 mm), allowing simultaneous pressure measurement and drug infusion, placed in the ileocolic vein. Mean arterial and portal pressures were recorded before and 0.5, 1, 2, 3, 4 and 5 min after ET-l infusion and at 5-min intervals for 25 min thereafter. 2. Biliary cirrhotic rats and normal rats received vehicle or several doses of SB209670 (1.8, 5.4, 18 pmol/kg; five cirrhotic and five normal rats received 1.8 or 18 pmol/kg, 16 cirrhotic and 10 normal rats received vehicle or 5.4 pal/kg) through the femoral vein catheter. These doses of SB209670 were chosen on the basis of previous studies (12). Portal and arterial pressures were monitored continuously before drug or vehicle injection and for the following 60 min. Thereafter, systemic and splanchnic hemodynamics were determined using the radioactive microsphere technique.

Statistics Data are shown as mean+SEM. Statistical comparisons were performed with appropriate methods (unpaired Student’s t-test for Fig. 1, 2, 3 and 4 and Table 1; one-way analysis of variance followed by Dunnett’s test for Fig. 2 and 3). Results were considered statistically significant at pCO.05.

Results Plasma and hepatic ET levels Figure 1 shows plasma and hepatic ET levels in normal and biliary cirrhotic rats. Plasma ET levels in biliary cirrhotic rats were significantly higher than those in normal rats (9.0t1.3 vs. 2.6205 pg/ml, p
(mmHg)

ca S r”

addition, hepatic ET levels in cirrhotic rats were significantly higher and showed an approximately 6-fold increase as compared with those in normal rats (74.8& 13.3 VS.12.622.5 pg/g wet tissue, p
(mmW

A

80

10

80 0

5

10

15

20

25

30

B

** *- * 3* * * ** -IF-

5 ff 0

a l

WinI 5

10

15

20

25

30

Fig. 2. Effect of endothelin-1 on mean arterial pressure (A, C) andportalpressure

(B, D) in normal rats (A, B) and biliary cirrhotic rats (C, D) with (0) or without (0) the pretreatment with SB209670 (18 pmollkg bw), which was administered intraportally 5 min before ET-l administration. Endothelin-I (3 nmollkg bw) was administered through the ileocolic vein during a period of 1 min. Values are presented as mean?SEM from 6 rats. *p
45

H. Kojima et al.

(mmW 140 -

L 3

t h

A

(mmW 10 -

130.

B

f! 8z z ; 6-

120. ‘i 'E: e 110. I 5 IOO-

It

4-

f 90,,,,,,,,,,,,,, 0 10 20

230

40

50

6O(min)

(mmW 140

2

30

40

50

60 (min)

D

130 g! 16 :

8 Q 120 3

%

8 110 to

14

I! 8 12

E 100 s

.,,,,,,.,.,,I 10 20

(mmH9) 18

C

g!

0

90 40

60

60

min)

10 0

IO

20

30

40

50

Fig. 3. Effect of mixed endothelin receptor antagonist, SB209670, and vehicle (O-O) on mean arterial pressure (A, C) and portal pressure (B, D) in normal rats (A, B) and biliary cirrhotic rats (C, D). Several doses of SB209670 (1.8 pmoll kg bw, O---O; 5.4 ,umollkg bw, a---@; I8 pmollkg bw, a-0) were administered through the femoral vein catheter as a bolus. Values are presented as mean?SEM. #p
in cirrhotic rats vs. 216.3% in normal rats). The pretreatment with SB209670 completely abolished both an initial decrease and a sustained increase in systemic arterial pressure by ET-1 administration (Fig. 2A, C). In addition, the portal hypertensive effect of ET-1 was diminished partly in normal rats and almost completely in cirrhotic rats by the pretreatment with SB209670 (Fig. 2B, D). Effect of SB209670 hemodynamics

on the splanchnic

and systemic

Figure 3 shows the changes in systemic arterial and portal pressure after intravenous adminstration of several doses of SB209670. In cirrhotic rats, a dose of 5.4 ,umollkg of SB209670 caused a significant reduction in portal pressure more than 35 min after intravenous administration, but did not modify the systemic arterial pressure (Fig. 3C, D). A dose of 18 pmol/kg of SB209670 showed a portal hypotensive effect in cirrhotic rats as well, but caused no more reduction in portal pressure than did 5.4 pmollkg of SB209670 and a significant reduction in systemic arterial pressure. On 46

the other hand, any doses of SB209670 failed to modify either systemic arterial or portal pressure in control rats (Fig. 3A, B). Table 1 shows the effect of SB209670 (5.4 pmollkg) on the splanchnic and systemic hemodynamics in normal and biliary cirrhotic rats. The cirrhotic rats which received vehicle had lower mean arterial pressure, systemic vascular and splanchnic arterial resistance than did normal rats, while cardiac index and PVI were higher in cirrhotic rats, thus confirming that the systemic and splanchnic circulations in cirrhotic rats are hyperdynamic. Portal pressure, portal venous system resistance and PSS were far greater in cirrhotic rats than those in normal rats. In cirrhotic rats, SB209670 caused the changes in systemic hemodynamics characterized by a decrease in SVR (vehicle, 3.350.3 vs. SB209670, 2.420.2 mmHg * min. 100 g bwl ml, pcO.05). Although a reduction in mean arterial pressure and an increase in cardiac index were noticed as well, these changes showed no statistical significance (mean arterial pressure, 11524 vs. 107+3 mmHg, p= 0.16; cardiac index, 39.323.1 vs. 48.623.6 ml/min* 100 g bw, p=O.O6). As shown in Fig. 3 and Table 1,

Endothelin in portal hypertension

,-

B

p
0 0

0

El 0

%

t

Vehicle

SB209670

Vehicle

SB209670

12 10 8 6 4 2 0 Vehicle

SB209670

Vehicle

SB209670

Fig. 4. Effects of mixed endothelin receptor antagonist, SB209670, on portal venous inflow (A), portal venous system resistance (B), collateral blood$ow (C) andporto-systemic shunt (D) in biliary cirrhotic rats. SB209670 (5.4 ,umollkg bw) or vehicle were administered through the femoral vein catheter in the 16 cirrhotic rats.

SB209670 significantly reduced portal pressure in cirrhotic rats (vehicle, 14.0~0.7 vs. SB209670, 11.7205 mmHg, p
TABLE

with changes in PVI and CBF, which increased with the administration of SB209670 as well, but showed no statistically significant change (PVI, 6.6k0.6 vs. 7.8k0.7 ml/min* 100 g bw, p=O.18; CBF, 2.320.7 vs. 3.020.7 ml/min * 100 g bw, p=O.45) (Fig. 4, Table 1). PSS and renal blood flow were similar in cirrhotic rats

1

Effects of SB209670

(5.4 ,umol/kg bw) and vehicle on splanchnic

and systemic

hemodynamics

Normal

Mean arterial pressure (mmHg) Cardiac index (ml/min . 100 g bw) Systemic vascular resistance (mmHg . min . 100 g bw/ml) Portal pressure (mmHg) Portal venous inflow (mUmin. 100 g bw) Portal venous system resistance (mmHg . min. 100 g bw/ml) Splanchnic arterial resistance (mmHg . min. 100 g bw/ml) Collateral blood flow (mUmin. 100 g bw) Portosystemic shunting (%) Renal blood flow (ml/min . 100 g bw) Values are presented

mean?SEM

rats

rats and biliary

cirrhotic

rats

Biliary cirrhotic

rats

Vehicle (?I= 10)

SB209670 (n= 10)

Vehicle (n= 16)

SB209670 (n= 16)

12725 29.622.7 4.6kO.5 7.OkO.4 4.1 k-o.5 1.8kO.2 32.0k3.8 0.01~0.003 0.2t0.1 3.220.4

12215 35.023.1 3.8-+0.3 6850.4 4.3-cO.6 1.720.2 31.453.5 0.01 kO.004 0.2kO.l 3.420.4

1 15k4a 39.3k3.1” 3.3k0.3” 14.0k0.7b 6.620.6” 2.550.2” 17.3k1.5b 2.310.7b 33.927.0b 4.620.5

107?3 48.6k3.6 2.4?0.2” 11.7z0.5d 7.8kO.7 1.7kO.ld 13.8kl.2’ 3.020.7 36.51-7.2 S.lkO.6

from 10 normal rats and 16 biliary cirrhotic rats. received vehicle. “p
ap<0.05,bp
in normal

cirrhotic

rats which received vehicle.

47

H. Kojima et al.

which received vehicle and SB209670. In normal rats, SB209670 had no significant effect on systemic or splanchnic hemodynamics (Fig. 3, Table 1).

Discussion This study demonstrates that plasma and hepatic ET levels are elevated in biliary cirrhotic rats and that intraportal administration of exogenous ET-1 increases portal pressure in both cirrhotic and normal rats. In addition, the mixed ET receptor antagonist, SB209670, decreased portal pressure by reducing portal venous system resistance in cirrhotic rats in viva. It is well recognized that exogenous ET increases perfusion pressure in the isolated perfused normal and cirrhotic rat liver (6,8). However, Sogni et al. (10) showed that intravenous administration of ET-1 lowers portal pressure in cirrhotic rats in vivo. Intravenous administration of ET-1 causes vasoconstriction of the splanchnic arteries, leading to the decrease in PVI. Although ET-1 may simultaneously increase the vascular resistance in portal circulation, the depressor effect due to reduced PVI may exceed the pressor effect due to increased vascular resistance in portal circulation and result in the reduction in portal pressure. In contrast, we and other investigators (21) have administered ET’1 into the portal vein and found the portal hypertensive effect in cirrhotic rats in vivo. ET-l infused directly into portal circulation may cause a stronger vasoconstriction in portal circulation, as compared to that in the splanchnic arteries, and increase portal pressure. Interestingly, the magnitude of portal hypertensive effects with ET-1 was lower in cirrhotic rats than normal rats. In addition, plasma and hepatic ET levels were elevated in rats with biliary cirrhosis as well, in agreement with the previous results in human and carbon tetrachloride-induced cirrhosis (3-5). There is a possibility that an overproduced endogenous ET in cirrhotic liver may occupy more intrahepatic ET receptors, resulting in a decrease in unbound receptors and hyporeactivity to exogenous ET-l. Considering that ET derived mainly from sinusoidal endothelial cells and Ito cells in the liver modulates intrahepatic vascular resistance in a paracrine or autocrine manner (22,23), an overproduced endogenous ET in cirrhotic liver may increase portal pressure in the same way as exogenous ET-1 administered via the portal vein. In the present study, exogenous ET-1 did not lower portal pressure in the early stage, in contrast to its hypotensive effect on the systemic arterial pressure. This initial reduction in systemic arterial pressure has been attributed to a vasodilative effect through production of nitric oxide via the ETn receptor on vascular endothelial cells (24,25). Thus, the absence of ET

48

l-induced initial reduction in portal pressure suggests that the net dilator effect of ET-1 on the portal circulation is absent or weak and that ETn receptors in the portal venous system mainly mediate vasoconstriction. Leivas et al. (26) have recently shown that ETA and ETn receptor mRNA are overexpressed in human cirrhotic liver and that the abundance of ETn receptor mRNA as well as that of ETA receptor mRNA directly correlates with portal pressure. Moreover, it is known that the ET..+,receptor antagonist, BQ123, alone does not modify the portal pressure in both of normal and cirrhotic rats (4). These findings suggest the significance of the ETn receptor as well as the ETA receptor in cirrhotic portal hypertension. Thus, a mixed ETA and ETn receptor antagonist such as SB209670 may be useful in the pharmacological treatment of portal hypertension. In this study, SB209670 reduced portal pressure in cirrhotic rats in vivo. This portal hypotensive effect is similar to the previous reports using another mixed ET antagonist, bosentan (lO,ll), but its mechanism has been controversial. One group attributed a portal hypotensive effect of mixed ET antagonist to a reduction in hepatocollateral vascular resistance, representing the sum of resistance in the intrahepatic vasculature and collateral circulation, while another group attributed this effect to a decrease in hepatic arterial flow. To elucidate the mechanism of the portal hypotensive effect of the mixed ET antagonist, we used biliary cirrhotic rat due to bile duct ligation as an experimental model with intrahepatic portal hypertension and investigated the hemodynamic effects of the mixed ET antagonist in cirrhotic portal hypertension. Carbon tetrachloride-induced cirrhosis and partial portal vein ligation have also been used as a rat model of portal hypertension. However, carbon tetrachloride-induced cirrhosis in rats is a model of intrahepatic portal hypertension with minimal extrahepatic portosystemic collateral veins (26). Partial portal vein ligation in rats is a model with prehepatic portal hypertension characterized by extensive portosystemic collateral vein formation (20,27). Unlike these models, the biliary cirrhotic rat is a model of intrahepatic portal hypertension associated with moderate portosystemic collateral vein formation as seen in patients with alcoholic liver cirrhosis (13,14,28,29), and therefore it may be useful in assessing the hemodynamic effect of mixed ET antagonist in cirrhotic portal hypertension. In this study, the portal hypotensive effect of mixed ET antagonist was associated with a reduction in portal venous system resistance, but not with changes in PVI and CBE Portal venous system resistance consists of two resistances against the intrahepatic vasculature and col-

Endothelin in portal hypertension

lateral circulation. The finding that mixed ET antagonist improves the cirrhotic portal hypertension without modifying PVI and CBF may provide indirect evidence that a mixed ET antagonist influences the intrahepatic vascular resistance of cirrhotic liver. However, our results cannot evaluate the separate effect of mixed ET antagonist on intrahepatic and collateral circulation, because we measured CBF and PSS using microspheres injected into the ileocolic vein, which did not allow us to assess splenorenal shunts. Although the sites of intrahepatic vascular resistance in cirrhotic liver are still unclear, recent reports have suggested the significance of Ito cells in the intrahepatic microcirculation and portal hypertension (6,21-23,30). Ito cells are located in the perisinusoidal space of Disse and modulate the sinusoidal blood flow as liver-specific pericytes (30,31). These cells possess both ET, and ETs receptors (6,7), and contract in response to ET, leading to increased intrahepatic vascular resistance (6,30). Moreover, Ito cells undergo “activation” and transform into highly contractile myofibroblasts (30,32,33), which overproduce ET and overexpress ET, and ETn receptors on themselves (21,23,34), during the development of liver cirrhosis. Thus, ETs overproduced in cirrhotic liver may strongly contract Ito cells via both ET, and ETn receptors and may play an important role in cirrhotic portal hypertension. The finding that mixed ET antagonist decreased portal pressure by reducing portal venous system resistance in cirrhotic rats provides further support for a role of endogenous ET in the pathogenesis of cirrhotic portal hypertension. In conclusion, the mixed ET antagonist, SB209670, reduced portal pressure without modifying systemic arterial pressure and renal blood flow in cirrhotic rats. This finding suggests a potential use of mixed ET antagonist in the pharmacological treatment of portal hypertension. Moreover, this portal hypotensive effect was attributed to a reduction in portal venous system resistance. These results, together with the findings that plasma and hepatic ET levels were elevated in cirrhotic rats and that intraportal infusion of exogenous ET-1 increased portal pressure in both normal and cirrhotic rats, provide further support for a role of ET in portal hypertension.

References 1. Reichen J. Liver function and pharmacological considerations in the pathogenesis and treatment of portal hypertension. Hepatology 1990; 11: 106678. 2. Vorobioff J, Bredfedlt JE, Groszmann RJ. Hyperdynamic circulation in portal-hypertensive rat model: a primary factor for maintenance of chronic portal hypertension. Am J Physiol 1983; 244: G52-7. 3. Uchihara M, Izumi N, Sato C, Marumo E Clinical significance

4.

5.

6.

7.

8.

9.

10.

11.

12.

13. 14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

of elevated plasma endothelin concentration in patients with cirrhosis. Hepatology 1992; 16: 95-9. Leivas A, Jimenez W, Lamas S, Bosch-Marce M, Oriola J, Claria J, et al. Endothelin 1 does not play a major role in the homeostasis of arterial pressure in cirrhotic rats with ascites. Gastroenterology 1995; 108: 1842-8. Gandhi CR, Sproat LA, Subbotin VM. Increased hepatic endothelin-1 levels and endothelin receptor density in cirrhotic rats. Life Sci 1996; 58: 55-62. Zhang JX, Bauer M, Clemens MG. Vessel- and target cell-specific actions of endothelin-1 and endothelin-3 in rat liver. Am J Physi01 1995; 269: G269-77. Rockey DC. Characterization of endothelin receptors mediating rat hepatic stellate cell contraction. Biochem Biophys Res Commun 1995; 207: 725-31. Elliot AJ, Vo LT, Grossman VL, Bhathal PS, Grossman HJ. Endothelin-induced vasoconstriction in isolated perfused liver preparations from normal and cirrhotic rats. J Gastroenterol Hepatol 1997; 12: 3148. Zhang B, Calmus Y, Wen L, Sogni P, Lotersztajn S, Houssin D, et al. Endothelin-1 induces liver vasoconstriction through both ET,., and ET, receptors. J Hepatol 1997; 26: 110410. Sogni E Moreau R, Gomola A, Gadano A, Cailmail S, Calmus Y, et al. Beneficial hemodynamic effects of bosentan, a mixed ET, and ETa receptor antagonist, in portal hypertensive rats. Hepatology 1998: 28: 655-9. Reichen J, Gerbes AL, Steiner MJ, Sagesser H, Clozel M. The effect of endothelin and its antagonist Bosentan on hemodynamics and microvascular exchange in cirrhotic rat liver. J Hepato1 1998; 28: 1020-30. Ohlstein EH, Nambi P Douglas SA, Edwards RM, Gellai M, Lago A, et al. SB209670, a rationally designed potent nonpeptide endothelin receptor antagonist. Proc Nat1 Acad Sci USA 1994; 91: 80526. Lebrec D, Blanchet L. Effect of two models of portal hypertension on splanchnic organ blood flow in the rat. Clin Sci 1985; 68: 23-8. Sikuler E, Buchs AE, Yaari A, Keynan A. Hemodynamic characterization of conscious and ketamine-anesthetized bile duct-ligation rats. Am J Physiol 1991; 260: Gl61-6. Ando K, Hirata Y, Shichiri M, Emori T, Marumo E Presence of immunoreactive endothelin in human plasma. FEBS Lett 1989; 245: 1646. Seyde WC, Longnecker DE. Anesthetic influence on regional hemodynamics in normal and hemorrhaged rats. Anesthesiology 1984; 61: 68698. Malik AB, Kaplan JE, Saba TM. Reference sample method for cardiac output and regional blood flow determinations in the rat. J Appl Physiol 1976; 40: 472-5. Ishise S, Pegram BL, Yamamoto J, Kitamura Y, Frohlich ED. Reference sample microsphere method: cardiac output and blood flows in conscious rat. Am J Physiol 1980; 239: H443-9. Choiikier M. Groszmann RJ. Measurement of portal systemic shunting in the rat by using y-labeled microspheres. Am J Physiol 1981; 240: G371-5. Blei AT, O’Reilly DJ, Gottstein J. Portal-systemic shunting and the hemodynamic effects of nitroglycerin in the rat. Gastroenterology 1984; 86: 1428-36. Gandhi CR, Nemoto EM, Watkins SC, Subbotin VM. An endothelin receptor antagonist TAK-044 ameliorates carbon tetrachloride-induced acute liver injury and portal hypertension in rats. Liver 1998; 18: 3948. Housset C, Rockey DC, Bissel DM. Endothelin receptors in rat liver: lipocytes as a contractile target for endothelin 1. Proc Nat1 Acad Sci USA 1993; 90: 926670. Rockey DC, Fouassier L, Chung JJ, Carayon A, Vallee P, Rey C, et al. Cellular localization of endothelin-1 and increased production in liver injury: potential for autocrine and paracrine effects on stellate cells. Hepatology 1998; 27: 472-80. Ishikawa K, Ihara M, Noguchi K, Mase T, Mino N, Saeki T, et al. Biochemical and pharmacological profile of a potent and

49

H. Kojima et al.

25.

26.

27.

28.

50

selective endothelin B-receptor antagonist, 84-788. Proc Nat1 Acad Sci USA 1994; 91: 489226. De Nucci G, Thomas R, D’Orleans-Juste P, Antunes E, Walder C, Warner T, et al. Pressor effects of circulating endothelin are limited by its removal of in the pulmonary circulation and by the release of prostacyclin and endothelium-derived relaxing factor. Proc Nat1 Acad Sci USA 1988; 85: 9797-800. Leivas A, Jimenez W, Bruix J, Boix L, Bosch J, Arroyo V, et al. Gene expression of endothelin-1 and ETA and ETs receptors in human cirrhosis: relationship with hepatic hemodynamics. J Vast Res 1998; 35: 18693. Kravetz D, Bosch J, Arderiu M, Pizcueta MP Rodes J. Hemodynamic effects of blood volume restitution following a hemorrhage in rats with portal hypertension due to cirrhosis of the liver: influence of the extent of portal-systemic shunting. Hepatology 1989; 9: 808814. Groszmann RJ, Kotelanski B, Cohn JN, Khatri IM. Quantitation of portosystemic shunting from the splenic and mesenteric beds in alcoholic liver disease. Am J Med 1972; 53: 715-22.

29. Lebrec D, Kotelanski B, Cohn JN. Splanchnic hemodynamics in cirrhotic patients with esophageal varices and gastrointestinal bleeding. Gastroenterology 1976; 70: 1108-l 1. 30. Rockey DC, Weisiger RA. Endothelin induced contractility of stellate cells from normal and cirrhotic rat liver: implications for regulation of portal pressure and resistance. Hepatology 1996; 24: 23340. 31. Pinzani M, Failli P Ruocco C, Casini A, Milani S, Baldi E, et al. Fat-storing cells as liver-specific pericytes. J Clin Invest 1992; 90: 642-6. 32. Friedman SL. The cellular basis of hepatic fibrosis. N Engl J Med 1993; 328: 1828-35. 33. Irle C, Kocher 0, Gabbiani G. Contractility of myofibroblasts during experimental liver cirrhosis. J Submicrosc Cytol Path01 1980; 12: 209-17. 34. Pinzani M, Milani S, De Franc0 R, Grappone C, Caligiuri A, Gentilini A, et al. Endothelin 1 is overexpressed in human cirrhotic liver and exerts multiple effects on activated hepatic stellate cells. Gastroenterology 1996; 110: 53448.