Modulation of the hyperdynamic circulation of cirrhotic rats by nitric oxide inhibition

GASTROENTEROLOGY

1992;103:1909-1915

Modulation of the Hyperdynamic Circulation of Cirrhotic Rats by Nitric Oxide Inhibition PILAR PIZCUETA, JOSEP M. PIQUE, MERCEDES FERNANDEZ, JAIME BOSCH, JOAN RODRS, BRENDAN J. R. WHITTLE, and

SALVADOR

MONCADA

Hepatic Hemodynamics Laboratory, Liver Unit, Hospital Clinic, University of Barcelona, Spain; and Wellcome Research Laboratories, Beckenham, Kent, England

The effects of A7c-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide (NO) biosynthesis on the splanchnic and systemic circulation, were investigated in rats with cirrhosis induced by carbon tetrachloride. Portal hypertension in these rats was accompanied by decreased arterial blood pressure and peripheral vascular resistance as well as by splanchnic vasodilation with increased portal venous inflow and decreased splanchnic resistance. Intravenous bolus administration of L-NMMA (25 mg/kg) significantly increased systemic blood pressure and decreased cardiac output. L-NMMA also significantly increased systemic and splanchnic vascular resistance; whereas blood flow to the stomach, small intestine, colon, pancreas, mesentery, spleen, and kidney was decreased significantly. LNMMA did not alter the portal pressure or portosystemic shunting in these cirrhotic rats, yet portal vascular resistance increased, suggesting effects on the intrahepatic and collateral circulation. Pretreatment with L-arginine (300 mg/kg) prevented the hemodynamic changes induced by L-NMMA. These findings support the concept that local excess formation of NO contributes to changes in splanchnic circulation associated with portal hypertension in cirrhosis.

P

ortal hypertension such as that observed in patients with hepatic cirrhosis is associated with hyperdynamic vascular circulation.‘*2 This is manifested by increased blood flow and decreased vascular resistance in the systemic circulation and splanchnic vascular beds and can also be observed in experimental models of cirrhosis and portal hypertension.3-6 The mechanisms of the splanchnic vasodilatation associated with such portal hypertension have not been clarified, but it has been proposed that the increased systemic delivery of vasodilator mediators such as glucagon to the splanchnic beds may contrib-

ute.7-g However, it is also possible that the enhanced formation and release of local vasodilator mediators in the splanchnic and systemic circulation may be involved. One such potential mediator is nitric oxide (NO), the labile endogenous nitrovasodilator’0-‘2 formed from its substrate amino acid, L-arginine, by vascular tissue.13 Studies with NG-monomethyl+arginine (L-NMMA), an inhibitor of vascular NO biosynthesis,14s15 have implicated NO in the regulation of resting systemic arterial blood pressure (BP) in the rabbit, rat, and guinea piglB-‘* and of blood flow in the gastric, mesenteric, and renal vascular beds in the rat.1g-23Likewise, studies with L-NMMA have indicated a role of NO in the modulation of peripheral vascular tone in humans.24*25 It has recently been proposed that the circulatory abnormalities associated with cirrhosis may be a consequence of an inappropriate excessive synthesis of N0.26 Furthermore, in previous studies, it has been shown that L-NMMA can dose dependently attenuate the systemic hypotension and splanchnic vasodilation associated with portal hypertension following two-week partial portal vein ligation in the rat.22*23The effects of inhibiting NO biosynthesis by L-NMMA administration on the hyperdynamic systemic and splanchnic hemodynamics in a rat model of cirrhosis were investigated in the present study. Materials and Methods Experimental

Model

This study was performed on male Sprague-Dawley rats with histologically proven liver cirrhosis. Cirrhosis was induced by inhalation of carbon tetrachloride as previously described. 27-2gTo shorten the time required to induce cirrhosis, the rats received 0.3 g/L phenobarbital diluted in the drinking water starting 1 week before Ccl, administration6 Ccl, was administered by inhalation 0 1992by the American Gastroenterological Association 0016-5065/92/$3.00

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twice a week for increasing periods of time (0.5-5 minutes) for 11 weeks. Control rats were treated with phenobarbital for 11 weeks. All rats were allowed free access to food and water until the time of the study. The techniques used for the hemodynamic measurements have been previously described in detai1.5*6*30*31 The rats were anesthetized with ketamine HCl(lO0 mg/kg intramuscularly). The left ventricle was catheterized via the right carotid artery with PE-50 tubing (Portex Ltd., Kent, England). Another PE-50 catheter was placed in the left femoral artery for systemic arterial BP measurements and for blood withdrawal. Through a &cm midline incision, the portal vein was catheterized via the ileocolic vein using tip-chilled tubing. After verification that free back flow of blood was obtained, the catheter was fixed with cyanoacrylate glue and the abdomen wall was closed with silk sutures. This catheter was subsequently used for portal pressure measurements. Another PE-50 catheter was placed in the right atrium via the left external jugular vein and used for atria1 pressure measurement and drug infusion. All catheters were connected to pressure transducers and calibrated before each experiment, andBPs were registered on a multichannel recorder (Lectromed MTG-PX; St. Peters, Jersey, England). The zero reference point was established 1 cm above the operating table. Rectal temperature was maintained at 37 + 0.5’C throughout the study. After obtaining baseline measurements of resting BP, right atria1 pressure, and portal pressure, 1 mL of saline or L-NMMA was administered over 1 minute as a slow bolus injection through the catheter into the internal jugular vein. Ten minutes later, these pressures were measured again, and cardiac output and regional blood flows were measured using a radioactive microsphere technique as described.5,6*8*30-32A reference blood sample was obtained from the femoral artery catheter over a i’5-second period at a rate of 1 mL/min using a continuous-withdrawal pump. Approximately 50,000 microspheres labeled with 141Ce(diameter, 15 + 3 pm; sp act, 10 mCi/g) were injected into the left ventricle 15 seconds after beginning the blood withdrawal. Portal venous inflow (PVI), which represents the total blood flow entering the portal venous system, was calculated as the sum of blood flow to stomach, spleen, small and large intestines, pancreas, and mesentery. Portosystemic shunting (PSS) was estimated as described2g.32*33using ‘ICr-labeled microspheres (diameter, 15 + 3 pm; sp act, 33 mCi/g) injected into the portal vein, as PSS (%) =

Lung Radioactivity

(cpm) x loo

Liver Radioactivity + Lung Radioactivity

At the end of the experiments, saturated KC1 was injected intravenously (IV). The abdominal organs were dissected, blotted, weighed, cut into small pieces, and placed in counting tubes. The radioactivity (cpm) of each organ was determined in a gamma-scintillation counter (Packard 800~; Packard Instrument Company, Downers Grove, IL).

The interference of Cr radioactivity (energy window, 240400 keV) was corrected using Cr and Ce standards. Resistance in each vascular bed was calculated from the ratio between perfusion pressure (P) and blood flow (Q) in each vascular territory. For the calculation of total peripheral vascular resistance, P was the value for mean arterial pressure minus the right atria1 pressure and Q was the cardiac output; in the calculation of splanchnic vascular resistance, P was mean arterial pressure minus portal pressure and Q was the PVI. For the calculation of portal vascular resistance, P was portal pressure minus right atria1 pressure and Q was the PVI. An additional group of cirrhotic rats (n = 6) was studied after IV administration of L-arginine (300 mg/kg) 5 minutes before the infusion of 25 mg/kg of L-NMMA. L-NMMA as the acetate salt was synthetized in the Department of Medicinal Chemistry of Wellcome Research Laboratories (Beckenham, England) and dissolved freshly in saline when required. L-Arginine was obtained from Sigma Chemical Co. (St. Louis, MO). Phenobarbital was obtained from Bayer (Leverkusen, Germany), and Ccl, from Scharlau (Barcelona, Spain). Radiolabeled microspheres were obtained from New England Nuclear (Boston, MA).

Data Analysis All results are expressed as mean + SEM. Student’s t test for paired and nonpaired data, ANOVA, and the regression coefficient were used in the statistical analysis of the results. Significance was taken at P < 0.05.

Results Hemodynamic

Parameters

in Cirrhotic Rats

Following exposure to Ccl, and phenobarbital over the II-week period, the characteristic hemodynamic disturbance of a significant increase in portal pressure and PVI was observed in comparison with that in control nonexposed rats (Table 1). This was associated with a significant decrease in BP and systemic vascular resistance and with an increase in cardiac output and renal blood flow (Table 1). Effect of L-NMMA on Systemic Blood Pressure In preliminary dose-response studies, IV bolus administration of 6.25-100 mg/kg of L-NMMA induced a dose-dependent increase in BP, with an IV dose of 25 mg/kg of L-NMMA producing 50% of the maximal response. This dose was chosen for the subsequent detailed cardiovascular studies in the cirrhotic rats. An increase in BP (40 -t 4.2 mm Hg; n = 9, P c 0.05) following administration of 25 mg/kg of L-NMMA was evident within 1 minute of its IV administration and reached its maximal effects after 10 minutes. This was accompanied by a significant decrease in cardiac output as shown in Figure 1, yet

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NO AND SPLANCHNIC HEMODYNAMICS

1992

IN CIRRHOSIS

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Table 1. Resting Values for the Hemodynamic Parameters in Control and Cirrhotic Rats Cirrhosis induced by CCI,

Controls (n = 8) Mean arterial pressure (mm Hg) Cardiac output (mL - min-’ *100 g body wt-‘) Systemic vascular resistance (mm Hg . mL-' .min? . 100 g body wt-‘) Portal pressure (mm Hg) Portal venous inflow (mL - min-’ . 100 g body wt-‘) Splanchnic vascular resistance (mm Hg . mL-' .mm’ -100 g body wt-‘) Portal vascular resistance (mm Hg .mL-'amin-’ *100 g body wt-‘) Renal blood flow (mL* min-’ . 100 g body ti-‘) Renal vascular resistance (mm HgemL-‘+min-‘a 100 g body wt-‘)

Untreated

cirrhotic (n = 9)

rats

Cirrhotic rats (L-arginine + L-NMMA) (n = 6)

127 + 5

112 f 4.1°

117 -t6.3*

21+ 2

35 + 2.8"

32 f 3.2*

3.6k 0.3' 12.8f 0.9"

5.4+ 1.0* 12.5f 0.8*

6.3f 0.5 6.6k 0.2

6.3f 0.8'=

5.3f 0.8*

42 +7

19 * 2.2O

22.1+ 3.1*

2.3+ 0.4

2.2f 0.3"

2.6+ 0.6*

3.5f 0.3

6.6+ 0.5"

4.7f 0.4*

9.5 f 0.7

4.2 + 0.18”

5.0 f 0.2*

3.4+ 0.5

NOTE: Results are shown as means f SE. “Significantly different from control rats (P < 0.05). bNot significantly different from untreated cirrhotic rats.

heart rate was not significantly affected (328 f 13 and 320 f 12.4 beats/min, respectively). The cardiac output following L-NMMA administration in cirrhotic rats was not significantly different from that observed under control conditions in normal controls, whereas BP (146 + 4 mm Hg, P < 0.05) was significantly higher than that in normal rats not receiving L-NMMA (Table 1). Effects on the Systemic and Splanchnic Circulation As shown in Figures 1 and 2, in cirrhotic rats administration of L-NMMA induced a greater than twofold increase (P < 0.05) in systemic and splanchnit vascular resistance as well as in portal vascular resistance. Though portal pressure was not significantly changed (0.58 + 0.24 mm Hg), a significant reduction in portal venous inflow was observed following L-NMMA administration (Figure 2). PSS of 15-urn microspheres was 4.3% + 1% in the cirrhotic rats under control conditions and was not significantly altered (4.7% + 2%) following administration of L-NMMA. In cirrhotic rats, the values of systemic, splanchnic, and portal vascular resistance and of PVI followings-NMMA administration (10.7 -t 2 mm Hg * mL-’ - mine1 * 100 g body wt-‘, 52.4 f 11

mm Hg - mL-’ . min-’ - 100 g body wt-‘, 4.6 + 1 mm Hg . mL-’ +min- ’- 100 g body wt-‘, and 3.6 + 0.9 mL - mine1 - 100 g body wt-‘, respectively) were not significantly different from those of normal rats not receiving L-NMMA (Table 1). Effects on Regional Blood Flow Renal blood flow was significantly lower following IV administration of L-NMMA, whereas renal vascular resistance markedly increased (Figure 3). Values of renal blood flow and renal vascular resistance following L-NMMA administration were not significantly different from those observed in normal controls not receiving L-NMMA (Figure 3). Likewise, blood flow in the stomach, small intestine, colon, pancreas, mesentery, and spleen was significantly lower following L-NMMA administration (Figure 4). Efiects of L-Arginine on the Hemodynamic Changes Induced by L-NMMA Pretreatment with 300 mg/kg of L-arginine prevented the effects of L-NMMA on systemic and splanchnic hemodynamics in cirrhotic rats (Table 1).

1912

PIZCUETA

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Vol. 103, No. 6

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Figure 3. Effect of L-NMMA (25 mg/kg IV; W n = 9) or saline (0; n = 9) on renal blood flow (A) and renal vascular resistance (B) in cirrhotic rats. Results are mean + SE. *Significant effect compared with saline (P < 0.05).

20 1

Figure 1. Effect of IV bolus administration of L-NMMA (25 mg/kg; W, n = 9) or saline (0; n = 9) on mean systemic arterial blood pressure, cardiac output, and total systemic vascular resistance in cirrhotic rats. Results are mean + SE. *Significant effect compared with saline (P < 0.05).

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Discussion The present study of an experimental model of cirrhosis and portal hypertension in the rat induced by Ccl, inhalation27-2g and accelerated by phenobarbital shows that inhibition of endogenous NO

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by administration of L-NMMA can lessen many of the associated systemic and splanchnic hyperdynamic circulatory characteristics. Therefore, these findings support recent studies in which L-NMMA ameliorated the extensive vasodilation in the splanchnic beds that accompanies the portal hypertension following partial portal vein ligation in the rat. 22,23 IV administration of L-NMMA increased BP in anesthetized cirrhotic rats, as shown in anesthetized or conscious normotensive rats.‘7’20 This was associated with a decrease in cardiac output and an increase in total peripheral, splanchnic, and renal vascular resistances. A substantial decrease in regional blood flow in the stomach, small intestine, colon, pancreas, mesentery, spleen, and kidney was observed. Previous studies in control noncirrhotic rats using similar microsphere techniques have shown differential sensitivities in these vascular beds to a higher dose of L-NMMA (50 mg/kg), with significant increases in vascular resistance being observed in the stomach, mesentery, spleen, and pancreas but

4 2

0

80

lC

.

Intestine

COlOll Pancreab Mebentery

Spleen

Figure 2. Effect of L-NMMA (25 mg/kg IV; ?? ; n = 9) or saline (0; n = 9) on (A) portal pressure (mmHg); (B) portal venous inflow (mL/min 100 g); (Cl splanchnic vascular resistance (mm Hg/ mL . min-’ 100 g); and (0) portal vascular resistance in cirrhotic rats. Results are mean + SE. *Significant effect compared with saline (P < 0.05).

0

I

1'

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I

2

3

1 4 mllmin1002

Figure 4. Effect of L-NMMA (25 mg/kg IV: e n = 9) or saline (0; n = 9) on blood flow in the stomach, intestine, colon, pancreas, mesentery, and spleen on cirrhotic rats. Results are mean + SE. *Significant effect compared with saline (P < 0.05).

December

1992

not in the small intestine and co1on.22 Therefore, the current findings show that all of these vascular territories in cirrhotic rats are susceptible to the actions of L-NMMA. Other studies using L-NMMA in normotensive conscious rats and implanted flow probes have shown a reduction in mesenteric and renal blood flowtzo whereas studies in anesthetized rats have shown a decrease in resting blood flow to the gastric mucosa’g and renal cortex2’ and in the gastric hyperemia associated with pentagastrin stimulation.33*34 In a recent study by Claris et a1.35 in conscious cirrhotic rats with ascites, the renal plasma flow, estimated by paraaminohippurate (PAH) clearance, was not significantly modified by the administration of Nw-nitro+arginine. The discrepancies between these results and our findings may be due to several factors, such as differences in experimental models, differences in the NO inhibitor used, and, more important, the different techniques used to estimate renal blood flow. Though the microsphere technique (as well as electromagnetic flow probes) measures total renal blood flow, the PAH clearance is an estimation of the “effective” renal plasma flow, because not all the PAH is removed from the blood in a single passage through the kidneys. Therefore, this method leads to an underestimation of renal perfusion unless the renal extraction of PAH is measured by sampling renal venous blood in each clearance period. In fact, the PAH technique does not allow for identification of renal hyperemia in cirrhotic rats,35 a common finding reported by several groups using the radioactive-microspheres technique. 36*37Finally, it should be noted that the renal blood flow and vascular resistance were not changed by a low dose of L-NMMA in our previous study of partial portal vein-ligated rats.23 In all of these studies as well as in the present study, the effects of L-NMMA were prevented by concurrent administration of L-arginine. This confirms that the cardiovascular effects of L-NMMA result from inhibition of NO biosynthesis from this precursor and not from any direct vasoconstrictor properties.15 PVI was significantly reduced by L-NMMA, yet portal pressure was unaffected because of the concomitant increase in portal vascular resistance. In intrahepatic models of portal hypertension such as Ccl,-induced cirrhosis, the formation of portosystemic collaterals is not extensive and PSS is low, as confirmed in the present study. The PSS was not altered by L-NMMA administration. Therefore, the observed alterations in portal vascular resistance reflect not only changes in collateral resistance, but also changes in intrahepatic vascular resistance, in-

NO AND SPLANCHNIC

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dicating a role for endogenous NO in the regulation of the hepatic circulation in cirrhosis. Although a calcium-dependent constitutive synthase enzyme is responsible for NO formation by endothelial cells,38,3g an inducible enzyme that forms NO from L-arginine in these cells and that is also inhibited by L-NMMA has been reported.40 An NO synthase that can be induced by endotoxin or cytokines, described in the macrophage,41-44 has also been identified in vascular smooth muscle and hepatic tissue.45,46 Indeed, it has been suggested that induction of the vascular formation of NO by an inducible enzyme as a consequence of increased levels of circulating endotoxin may contribute to the hyperdynamic circulation of cirrhosis2” Thus, the present study using L-NMMA in cirrhotic rats adds support to the suggestion that the changes in the splanchnic vascular perfusion associated with portal hypertension in cirrhosis are brought about at least in part by local excess formation of NO. Glucagon has also been proposed as a candidate mediator for the splanchnic vasodilation. In a previous study in which rats with portal hypertension due to partial portal ligation and normal controls received maximal doses of L-NMMA, the reduced splanchnic vascular resistance in portal-hypertensive animals did not completely return to control levels despite normalizing systemic hemodynamics.23 This is compatible with other factors also contributing to the splanchnic vasodilation in portal hypertension7-’ Whether both local and circulating mediators can interact to bring about such a hyperdynamic circulation warrants further investigation. Furthermore, if the inappropriate formation of NO under these conditions can be brought about by an inducible NO synthase enzyme, its selective modulation may offer a new approach for the specific therapeutic intervention to ameliorate the cardiovascular problems of cirrhosis. References 1. Bosch J, Mastai R, Kravetz D, Bruix J, Gaya J, Rigau J, Rod& J.

Effects of propranolol on azygos venous blood flow and hepatic and systemic hemodynamics in cirrhosis. Hepatology 1984;4:1200-1205. 2. Bosch J, Navasa M, Garcia-Pagan JC, De Lacy AM, Rod& J. Portal hypertension. Med Clin North Am 1989;73:931-953. 3. Vorobioff J, Bredfeldt J, Groszmann RJ. Hyperdynamic circulation in a portal hypertensive rat model: a primary factor for maintenance of chronic portal hypertension. Am J Physiol 1983;244:G52-G57. 4. Benoit JN, Womack WA, Hernandez L, Granger DN. “Forward” and “backward” flow mechanisms of portal hypertension: relative contributions in the rat model of portal vein stenosis. Gastroenterology 1985:1092-1096. 5. Kravetz D, Bosch J, Arderiu MT, Pizcueta MP, Rod&s J. Hemo-

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dynamic 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;9:808-814. Pizcueta MP, De Lacy AM, Kravetz D, Bosch J, Rod& J. Propranolol decreases portal pressure without changing portalcollateral resistance in cirrhotic rats. Hepatology 1989;lO: 953-957. Benoit JN, Barrowman JA, Harper SL, Kvietys PR, Granger DN. Role of humoral factors in the intestinal hyperemia associated with chronic portal hypertension. Am J Physiol 1984;247:G486-G493. Kravetz D, Bosch J, Arderiu MT, Pizcueta MP, Casamitjana R, Rivera F, Rod&J. Effects of somatostatin on splanchnic hemodynamics and plasma glucagon in portal hypertensive rats. Am J Physiol1988;254:G322-G328. Benoit JN, Zimmerman B, Premen AJ, Go VLW, Granger DN. Role of glucagon in splanchnic hyperemia of chronic portal hypertension. Am J Physiol 1986;251:G674-G677. Palmer RMJ, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987;327:524-526. Khan MT, Furchgott RF. Additional evidence that endothelium-derived relaxing factor is nitric oxide. In: Rand, MJ, Raper, C, eds. Pharmacology. New York: Elsevier, 1988:341344. Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G. Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc Nat1 Acad Sci USA 1987;84:9265-9269, Palmer RMJ, Ashton DS, Moncada S. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature 1988; 333:664-666. Palmer RMJ, Rees DD, Ashton DS, Moncada S. L-Arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation. Biochem Biophys Res Commun 1988;153:1251-1256. Rees DD, Palmer RMJ, Hodson HF, Moncada S. A specific inhibitor of nitric oxide formation from L-arginine attenuates endothelium-dependent relaxation. Br J Pharmacol 1989; 96:418-424. Rees DD, Palmer RMJ, Moncada S. Role of endothelium-derived nitric oxide in the regulation of blood pressure. Proc Nat1 Acad Sci USA 1989;86:3375-3378. Whittle BJR, Lopez-Belmonte J, Rees DD. Modulation of the vasodepressor actions of acetylcholine, bradykinin, substance P and endothelin in the rat by a specific inhibitor of nitric oxide formation. Br J Pharmacol 1989;98:646-652. Aisaka K, Gross SS, Griffith OW, Levi R. Nc-Methyl-L-arginine, an inhibitor of endothelium-derived nitric oxide synthesis, is a potent pressure agent in the guinea pig: does nitric oxide regulate blood pressure in vivo? Biochem Biophys Res Commun 1989;160:881-886. Pique JM, Whittle BJR, Esplugues JV. The vasodilator role of endogenous nitric oxide in the rat gastric microcirculation. Eur J Pharmacol 1989;174:293-296. Gardiner SM, Compton AM, Bennett T, Palmer RMJ, Moncada S. Control of regional blood flow by endothelium-derived nitric oxide. Hypertension 1990;15:486-492. Walder E, Thiemermann C, Vane JR. The involvement of endothelium-derived relaxing factor in the regulation of renal cortical blood flow in the rat. Br J Pharmacol 1991;102:967973. Pizcueta P, Pique JM, Bosch J, Fernandez M, Whittle BJR,

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40. Radomski MW, Palmer RMJ, Moncada S. Glucocorticoids inhibit the expression of an inducible, but not the constitutive, nitric oxide synthase in vascular endothelial cells. Proc Nat1 Acad Sci USA 1990;87:10043-10047. 41. Hibbs JR. Jr, Taintor RR, Vavrin Z, Rachlin EM. Nitric oxide: a cytotoxic activated macrophage effector molecule. Biochem Biophys Res Commun 1988;157:87-94. 42. Marletta MA, Yoon PS, Iyengar R, Leaf CD, Wishnok JS. Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate. Biochemistry 1988;27:8706-9711. 43. Stuehr DJ, Nathan CF. Nitric oxide: A macrophage product responsible for cytostasis and respiratory inhibition in tumour target cells. J Exp Med 1989;169:1543-1555, 44. Rees DD, Cellek S, Palmer RMJ, Moncada S. Dexamethasone prevents the induction by endotoxin of a nitric oxide synthase and associated effects on vascular tone: an insight into endotoxin shock. Biochem Biophys Res Commun 1990;173: 541-547.

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45. Busse R, Mulsch A. Induction of nitric oxide synthase by cytokines on vascular smooth muscle cells. FEBS Lett 1990;275: 87-90. 46. Knowles RG, Merrett M, Salter N, Moncada S. Differential induction of brain, lung and liver nitric oxide synthase by endotoxin in the rat. Biochem J 1990;270:833-836.

Received November 21, 1991. Accepted July 14, 1992. Address requests for reprints to: Jaime Bosch, M.D., Hepatic Hemodynamic Laboratory, Liver Unit, Hospital Clinic, Villarroel, 170, 08036Barcelona, Spain. Supported in part by grants from Fundacib Catalana per a 1’Estudi de les Malalties de1 Fetge, Fondo de Investigaciones Sanitarias de la Seguridad Social (FISS 91/0374) and from Direction General de Investigacibn Cientifica y Tecnica (DGICYT PB90/ 0994). Mercedes Fernandez is a recipient of a grant from Formacibn de1 Personal Investigador (FPI AP90/43419059).