Hemodynamic Effects of Terbutaline, a P,-Adrenoceptor Agonist, in Conscious Rats with Secondary Biliary Cirrhosis JORGE L. POO,
hAIN
BRAILLON, ANTOINE HADENGUE, CHRISTOPHE GAUDIN AND DIDIERLEBREC
Laboratoire d 'Hhodynamique Splanchnique, Unite' de Recherches de Physiopathologie Hkpatique (INSERM U-24), H6pital Beaujon, 92118 Clichy, France
The hemodynamic responses to terbutaline- a selective p,-adrenoceptor agonist-were studied in conscious normal rats and in conscious rats with secondary biliary cirrhosis. Compared with those of normal rats, dose-response curves in cirrhotic rats indicated significantly decreased reactivity in arterial pressure and heart rate. Half-maximal effective dose was not significantlydifferent between the two groups. Terbutaline induced significant, dose-dependent decreases in portal pressure in both normal rats (9.3%) and cirrhotic rats (13.8%).In normal rats, terbutaline administration(32 pg * min- * kg-l bodywt) increased both cardiac output and portal tributary blood flow, thus mimicking hemodynamic changes in cirrhotic rats. In cirrhotic rats, despite a significant increase in portal tributary blood flow (from 19.9 f 1.7 mllmin to 22.7 & 1.5 ml/min), terbutaline decreased portal pressure from 17.4 k 1.0 mm Hg to 15.0 2 0.8 mm Hg. This study indicates that increased p,-adrenoceptor stimulation in cirrhotic rats may be involved in hyperdynamic circulation. The association of a decreased portal pressure and increased splanchnic blood flow suggests that p,-adrenoceptor stimulation may modulate hepatic and portal collateral vascular resistance. (HEPATOLOGY 1992;15:459-463.)
p-blockers reduce portal pressure by the combination of p,-antagonism which decreases cardiac output and p,-antagonism, which induces splanchnic vasoconstriction. However, the specific role of P,-adrenoceptors in portal hypertension has not yet been elucidated. Moreover, the hemodynamic effects of P,-adrenergic stimulation on splanchnic circulation are unknown. Thus, to elucidate the mechanisms of action of p-adrenergic antagonists in portal hypertension, we evaluated the hemodynamic effects of terbutaline, a p,-adrenergic agonist, in conscious cirrhotic rats. MATERIALS AND METHODS
Animals. Fifty adult male Sprague-Dawley rats (Laboratoires Charles River, Saint Aubin-les-Elbeuf, France) were divided into two groups. Rats in the first group were rendered cirrhotic and portal hypertensive by sectioning and ligating the common bile duct (6). Rats in the second group underwent sham operation. Hemodynamic studies were performed 4 t o 5 wk after surgery. Body weight at the time of the hemodynamic studies was similar between biliary cirrhotic (327 2 8 gm) and normal rats (316 10 g m ) . Studies performed in this laboratory were approved by the French Agricultural Office. Methods. Rats were allowed free access to food and water until 14 to 16 hr before the study, when food was withdrawn. p-adrenergic antagonists reduce the risk of gas- Animals were anesthetized with ether. Catheters were placed trointestinal bleeding in patients with cirrhosis (1-3). in the left jugular vein for infusion of terbutaline or saline, in This beneficial effect depends at least in part on the left ventricle (in the second set of experiments, for reduction of portal pressure and reduction of blood flow radioactive microsphere injection), in the portal vein for portal into the superior portal systemic circulation (4).The pressure measurements, and in the left femoral artery for mechanisms of action of P-blockers on the splanchnic measurement of arterial pressure. All measurements were circulation have not been, however, entirely elucidated. performed approximately 2 hr after rats awakened, while they Nonselective p-blockers, however, are known to induce rested in a restraining apparatus. Pressure and Chronotropic Dose-response Curves. The more profound splanchnic effects than cardioselective and chronotropic dose responses were performed in p-blockers (5). Thus it is thought that nonselective pressure eight sham-operated rats and eight cirrhotic rats. Heart rate, mean arterial pressure and portal pressure were measured during infusion of increasing doses of terbutaline ( 2 , 4, 8, 16, Received July 11, 1991; accepted October 10, 1991. 32 and 64 yg.min-'.kg-' body wt) through a venous Dr. Po0 held a fellowship from the Fondation pour la Recherche Medicale and catheter. Each dilution of terbutaline was perfused for 10 min; from the CONACYT (Mexico). total infusion volume was 900 pyanimal. No injection was This work was presented in part at the 24th Meeting of the European performed until the effects on arterial pressure and heart rate Association for Study of the Liver, August 30 to September 2, 1989, in Munich, of the previous dose had disappeared. This period lasted 10 to and was published in abstract form (J Hepatol 1989;9(suppl 1):575). 15 min. Preliminary studies showed that the responses were Address reprint requests to: Dr. D. Lebrec, INSERM U-24, HBpital Beaujon, similar between terbutaline injected in increasing doses and 92118 Clichy Cedex, France. doses of terbutaline injected at random. 3111134384
459
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,
Changes in portal pressure (mmHg) I
Changes in mean arterial pressure (mmHg)
a 71. o
Normal rats Cirrhotic rats
0
Normal rats Cirrholie rals
-20 4
, I
I
I
2
8
16
32
64 1
2
Terbulatine [ pg.kg .min )
-40
FIG.2. Response of portal pressure to terbutaline in normal and cirrhotic rats. No significant difference was seen between the two groups.
I
2 8
16
32
64
Changes in heart rate (beats I rnin) 200 1
OYi-16
32
64
Terbutaline (pg.kg'. min' )
FIG.1. Responses of arterial pressure and heart rate to terbutaline in normal and cirrhotic rats. *Significantly different between the two groups.
Maximal chronotropic and pressure responses (Em=) and the doses required to obtain half-maximal blood pressure reduction and half-maximal heart rate increase (ED,,) were calculated using the double reciprocal plot (l/dose vs. Uresponse). In two cirrhotic rats, heart rate and mean arterial pressure were measured during infusion of similar increasing doses of terbutaline. Ten minutes before terbutaline administration, however, they received a &-selective adrenoceptor antagonist (ICI 118,551;30 kg. kg-l -body wt, intravenously). Hemodynamic Measurements. The effect of the intravenous administration of terbutaline (32 pg min- kg- body wt for 10 min) or placebo (saline solution, 0.05 ml/min) was measured in 16 normal rats and 16 rats with secondary biliary cirrhosis. Heart rate and arterial and portal pressures were recorded on a square-wave electromagnetic manometer (CM130; Phillips, Eindhoven, The Netherlands) before and 10 min after administration of saline solution or terbutaline infusion. Regional blood flows and cardiac output were determined by the radioactive microsphere method (15 -+ 3 pm diameter; Du Pont-New England Nuclear, Boston, MA) as described previously ( 6 ) .ll3Sn-1abeledmicrospheres were injected into the left ventricle for basal measurements; 141 Ce-labeled microspheres were used for the second measurement, which was performed 10 min after the onset of terbutaline or saline infusion. The reference blood sample was drawn from the catheter in the femoral artery into a motor-driven syringe at 1 ml/min for 1 min. The animals were then killed with an overdose of pentobarbital sodium. Individual organs were dissected and placed in individual tubes for counting in a gamma-counter (Compteur G4000, Intertechnique, Plaisir, France) at energy settings of 280 to 1,000 keV and 70 to 210 keV for l13Sn and
-
141Ce, respectively. Adequate microsphere mixing was assumed to have occurred when the difference between right and left kidneys was less than 10%. Portal tributary blood flow was considered the sum of blood flows from the stomach, spleen, intestine, colon and mesentery-pancreas. Portal territory vascular resistance (dyn * sec * cm-5 x lo3) = (mean arterial pressure [mm Hg] - portal pressure [mm Hgl) x 80iportal tributary blood flow (ml/min).Portal-collateraland hepatic vascular resistance (dyn * sec cm-5 x lo3) = portal pressure (mm Hg) x 80/portal tributary blood flow (ml/min) (in normal rats that had no portal-collateral circulation, hepatic vascular resistance was used.) Statistical Analysis. Results are expressed as mean 2 S.E.M. Comparisons were performed by one-way ANOVA. A Bonferroni correction for multiple comparisons was used. A p value < 0.05 was considered statistically significant.
RESULTS Pressure and Chronotropic Dose-response Curves.
Dose-response curves are summarized in Fig. 1.At doses of 2 to 32 pg min- kg-' body wt, terbutaline induced dose-dependent increases in heart rate in both normal and cirrhotic rats. Em, was significantly different between the two groups (153 ? 24 beatsimin and 103 f 25 beats/min, respectively), but ED,, was not (8.4 & 2.5 p g - m i n - l - k g - l and 8.5 .t 5.2 pg-min-'. kg- body wt, respectively) significantly different. At doses of 2 to 32 pg * min- kg-I body wt, terbutaline also induced dose-dependent decreases in mean arterial pressure in normal and cirrhotic rats. Em, was significantly higher in normal rats (24.4 2 7.6 mm Hg) than in cirrhotic rats (18.1 +- 3.9 mm Hg). ED,, for arterial pressure was not significantly different between the two groups (12.1 f 5.0 p g - m i n - l - k g body wt and 11.6 k 3.3 p g . m i n - l - k g - l body wt, respectively). In cirrhotic rats that received ICI 118 551, arterial pressure was not affected during the four first doses of terbutaline, but decreased by 7% and 15%for the two final doses, respectively. Heart rate increased by approxkg- body imately 20% with 16, 32 and 64 kg * min wt terbutaline. Terbutaline induced a dose-dependent decrease in portal pressure without any significant difference between the two proum, but maximal effect was not obtained (Fig. 2);
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46 1
P,-ADRENOCEPTOR STIMULATION IN CIRRHOSIS
Vol. 15, No. 3, 1992
TABLE 1. Effect of terbutaline on systemic and splanchnic hemodynamics in conscious normal and cirrhotic rats Normal (n = 8 ) Parameters
Cirrhotic (n = 8)
Basal
Terbutaline
Basal
Terbutaline
393 t 5" 106 t 8 35.4 ? 1.8 84.9 t 6.5 6.5 3- 0.5 14.5 3- 1.3 5.30 0.38 15.2 ? 1.0 537 c 49 34.9 t 4.0
572 i 10* 80 i 3* 51.7 i 3.2' 44.3 i 3.5* 5.9 % 0.5b 18.4 & 1.7' 6.16 i 0.59* 12.1 i 1.12* 363 i 437' 26.9 % 3.5'
395 i 11 97 i 4 46.6 % 2.1' 51.7 i 3.1' 17.4 i 1.F 19.9 i 1.7' 6.06 & 0.43' 13.0 t 1.0 337 i 32' 72.1 i 5.7'
529 t 4' 82 3- 4* 57.8 3- 2.0' 34.7 i 1.6' 15.0 f 0.8' 22.7 2 1.5* 6.95 t 0.43' 12.1 t 0.9* 241 3- 21* 55.9 2 5.3h
8.7 ? 0.7 2.98 2 0.21 8.7 t 0.7 18.4 t 1.0 6.35 f 0.34 18.1 f 1.1
7.9 f 0.60 2.65 i 0.26 5.3 i 0.6' 21.6 i 1.76 7.42 f 0.69' 14.9 i 1.2
14.4 t 1.6" 4.50 i 0.62' 9.5 1.2 32.1 i 2.6' 9.82 ? 0.73' 20.8 i 1.0
16.1 t 26 5.05 t 0.90 8.5 t 1.3 40.4 t 2.5' 12.45 ? 0.86' 21.3 f 1.2
~~
Heart rate (beatsimin) Mean arterial pressure (mm Hg) Cardiac index (ml min-' . 100 gm-l body wt) Systemic vascular resistance (lo3 dyn . sec cmg5) Portal pressure (mm Hgj Portal tributary blood flow (mlimin) ml. m i n - l . 100 g m - l body wt % Cardiac output Portal territory vascular resistance (lo3 dyn sec cm-5) Portal collateral and hepatic vascular resistance (lo3 dyn . sec cm-S) Hepatic arterial blood flow (mlimin) ml . min-'. 100 gm-l body wt Cardiac output Renal blood flow (mumin) m l . m i n g ' . 100 gm-'bodywt % Cardiac output
*
*
Rats were administered a dose of 32 kg. kgg' . min-' "Data expressed as mean t S.E.M. 'Significantly different from basal value (p < 0.05). 'Significantly different from basal value of normal rats (p < 0.05).
TABLE 2. Lack of effect of saline solution on systemic and splanchnic hemodynamics in conscious normal and cirrhotic rats Normal (n
=
8)
Cirrhotic (n = 8 )
Parameters
Basal
Saline solution
Basal
Saline solution
Heart rate (beatsimin) Mean arterial pressure (mm Hg) Cardiac index (ml . min - ' . l o 0 gm - ' body wt) Systemic vascular resistance (lo3 dyn . sec . cm-5) Portal pressure (mm Hg) Portal tributary blood flow (mlimin) ml , m i n - l . 100 g n - l body wt Portal territory vascular resistance (lo3dyn . sec . c m - 9 Portal collateral and hepatic vascular resistance (lo3 dyn. sec. c m - 9 Hepatic arterial blood flow (mlimin) ml . m i n - ' . 100 gm-'body wt Renal blood flow (mlimin) ml m i n g l . 100 gm-' body wt
384 f 5" 104 i 3 34.4 2 1.6 80.4 f 6.5 6.1 i 0.5 13.4 i 1.2 4.32 f 0.36 529 ? 73 36.3 i 5.2
380 i 6.3 105 ? 9 33.6 ? 1.5 83.4 t 17.1 6.2 i 0.5 15.1 i 1.1 4.91 ? 0.38 506 % 73 31.7 i 2.9
403 t 3.7 97 i 3 44.8 i 3.3 54.0 i 4.8 17.5 t 0.8 21.2 i 2.5 6.23 t 0.79 336 i 43 71.5 t 6.9
400 f 5.9 97 t 3 44.9 3- 3.4 54.6 t 6.2 17.3 i 0.8 21.5 f 2.7 6.38 ? 0.82 343 t 52 72.3 ? 9.7
9.9 i 0.8 3.13 ? 0.18 17.6 f 1.4 5.64 f 0.37
8.8 i 0.9 2.83 i 0.29 19.2 F 1.2 6.19 t 0.34
12.5 i 2.5 3.66 t 0.70 26.1 t 2.6 7.28 i 0.74
15.4 i 4.0 4.21 t 1.12 27.1 ? 3.6 7.98 i 0.95
"Data expressed as mean
f
S.E.M.
Hemodynamic Measurements. In both groups of rats, terbutaline significantly increased heart rate, cardiac index and portal tributary blood flow. It significantly decreased mean arterial pressure, systemic vascular resistance, portal pressure and vascular resistance in the splanchnic bed (Table 1).Terbutaline induced significantly more marked changes in normal rats than in cirrhotic rats for cardiac index (47.5% and 24.7%, respectively) and systemic vascular resistance ( - 47.8% and - 31.1%, respectively). The increase in portal tributary blood flow was not significantly different between the two groups (19.2%and 16.1%,respectively). Distribution of cardiac output was significantly decreased in the portal territory and in liver in normal rats,
whereas distribution of cardiac output was not significantly affected by terbutaline in cirrhotic rats. Terbutaline significantly increased renal blood flow in both groups of rats (Table 1). Distribution of cardiac output was significantly decreased in the kidneys of normal rats, but not in cirrhotic rats. The placebo had no significant effect in normal and cirrhotic rats (Table 2). DISCUSSION In this study, to clarify the mechanisms of the portal hypotensive effects of P-blockers in portal hypertension, we used terbutaline to analyze the hemodynamic effects of P,-adrenoceptor stimulation in normal and cirrhotic rats. The lack of response of arterial pressure for low
462
P O 0 ET AL.
doses of terbutaline in rats that received a @,-selective antagonist confirms the specificity of @,-adrenergic agonist of terbutaline. This type of drug is generally used to treat asthma because of its bronchodilator effect, but it also dilates blood vessels (7). Moreover, it has been demonstrated that the P-adrenoceptors in the microcirculation of rat liver are of type P, (8). In this study, the dose-response curves show that terbutaline induces a significant decrease in arterial pressure in both normal and cirrhotic rats. Vasodilatation began with low doses of terbutaline and reached a plateau at a dose of 30 pg * min kg body w t in both groups. The maximum arterial response, however, was significantly more marked in normal rats than in cirrhotic rats. This result indicates that rats with secondary biliary cirrhosis have lesser vascular reactivity to @,-adrenergic stimulation than do normal rats. In both groups, reflex tachycardia occurred; in normal rats, however, the increased heart rate was significantly more marked than in cirrhotic rats. In patients with cirrhosis, similar blunted chronotropic responses to nonspecific @-adrenoceptorstimulation by isoprenaline have been observed (9). The significant maximal response of arterial pressure and heart rate and the lack of difference in half-maximal response between normal and cirrhotic rats indicate that noncompetitive inhibition is likely to occur in uiuo. This result supports previous findings, which show a decreased number of P,-adrenoceptor binding sites in both the mononuclear cells in patients with cirrhosis and severe ascites (10) and in the myocardial P,-receptors of rats with secondary biliary cirrhosis (11). In both the dose-response and blood-flow measurement studies, terbutaline had similar effects on arterial pressure and heart rate. In normal rats, terbutaline induced a marked increase in cardiac output and portal tributary blood flow. These findings were expected, and they resulted from significant systemic and splanchnic vasodilatation. The hyperdynamic circulation observed in normal rats receiving terbutaline mimics the hemodynamic changes of cirrhotic rats. Therefore P-adrenoceptor stimulation may play a role in the hyperkinetic circulation observed in portal hypertension by causing sympathoadrenomedular activation. In this series using normal rats, hepatic arterial blood flow was not affected by terbutaline administration. This lack of response to increased portal tributary blood flow supports the washout hypothesis described by Lautt ( 121. In cirrhotic rats, terbutaline exaggerated splanchnic and systemic hyperkineticism. Changes in cardiac output, however, were less marked in cirrhotic rats than in controls. This might suggest down-regulation of peripheral P,-adrenoceptors. On the other hand, the splanchnic hemodynamic changes induced by terbutaline were similar in cirrhotic and normal rats. Similar findings have previously been observed in rats with portal-vein stenosis receiving dobutamine, a @,adrenoceptor agonist (13). In both groups of rats, terbutaline decreased the proportion of cardiac output ~
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HEPATOLOGY
reaching the splanchnic bed. In both groups of rats, the decrease in portal pressure occurred even though portal tributary blood flow increased significantly. Because portal collateral and hepatic vascular resistance decreased significantly, the decreased portal pressure might have resulted from a reduction of vascular resistance in the liver, in the collateral circulation or both. In normal rats, however, only intrahepatic vascular resistance could decrease. This indicates that P,-adrenoceptors are present in the liver and play a role in the regulation of liver circulation. Our findings in normal rats confirm a previous study showing that @,-adrenoceptors are present in liver sinusoids and counteract constrictor responses produced by a-adrenoceptor activity (8). In cirrhotic rats, the decrease in portal pressure may also depend on collateral vasodilatation induced by terbutaline. This hypothesis is supported by the presence of adrenoceptors in both the portal vein and vessels of the portal circulation and by the role of P,-adrenoceptors in the modulation of venous tone (14, 15). The fact that drugs can alter hepatic vascular resistance (16, 17) or portal collateral vascular resistance (18, 19) has been established in earlier studies. In conclusion, terbutaline decreases portal pressure despite a concommitant increase in portal tributary blood flow in conscious rats with secondary biliary cirrhosis. These findings emphasize the role of hepatic and portal collateral vascular resistance in the modulation of portal hypertension.
Acknowledgments: We are indebted to Laurent Font of the Animal Care Unit for his help, to St6phane Cailmail for technical assistance and to Philippe Letteron for statistical analysis. REFERENCES 1. Pagliaro L, Burroughs AK, Sorensen TIA, Lebrec D, Morabito A, D’Amico G, Tine F. Therapeutic controversies and randomised controlled trials (RCTs): prevention of bleeding and rebleeding in cirrhosis. Gastroenterol Intern 1989;2:71-84. 2. Hayes PC, Davis JM, Lewis JA, Bouchier IAD.Meta-analysis of value of propranolol in prevention of variceal haemorrhage. Lancet 1990;336:153-156. 3. Poynard T, Cales P, Pasta L, Pagliaro L, Ideo G, Pascal J-P, Pagliaro L, et al. Beta-adrenergic-antagonistdrugs in the prevention of gastrointestinal bleeding in patients with cirrhosis and esophageal varices: an analysis of data and prognostic factors in 589 patients from four randomized clinical trials. N Engl J Med 1991;324:1532-1538. 4. Cales P, Braillon A, Jiron MI, Lebrec D. Superior portosystemic collateral circulation estimated by azygos blood flow in patients with cirrhosis: lack of correlation with oesophageal varices and gastrointestinal bleeding: effect of propranolol. J Hepatol 1984;1: 37-46. 5. Hillon P, Lebrec D, Mudoz C, Jungers M, Goldfarb G, Benbamou J P . Comparison of the effects of a cardioselective and a nonselective @-blockeron portal hypertension in patients with cirrhosis. HEPATOLOGY 1982;2:528-531. 6. Lee SS, Girod C, Braillon A, Hadengue A, Lebrec D. Hemodynamic characterization of chronic bile duct-ligated rats: effect of pentobarbital sodium. Am J Physiol 1986;251:G176-G180. 7. Koo A. Vasodilator effect of terbutaline: in uiuo evidence for the existence of @,-adrenoceptors in the microcirculation of rats,
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hamsters, and guinea pigs. J Cardiovasc Pharmacol 1984;6: 897-901. 8. Koo A, Liang NS. P,-adrenoceptors in rat liver microcirculation. Clin Exp Pharmacol Physiol 1979;6:403-407. 9. Ramond MJ, Comoy E, Lebrec D. Alterations in isoprenaline sensitivity in patients with cirrhosis: evidence of abnormality of the sympathetic nervous activity. Br J Clin Pharmacol 1986;21: 191-196. 10. Gerbes AL, Remien J , Jiingst D, Sauerbruch T, Paumgartner G. Evidence for down-regulation of beta-2-adrenoceptors in cirrhotic patients with severe ascites. Lancet 1986;1:1409-1410. 11. Lee SS, Marty J, Mantz J, Samain E, Braillon A, Lebrec D. Desensitization of myocardial P-adrenergic receptors in cirrhotic rats. HEPATOLOGY 1990;12:481-485. 12 Lautt WW. Mechanism and role of intrinsic regulation of hepatic arterial blood flow: hepatic arterial buffer response. Am J Physiol 1985:249:G549-G556. 13. Braillon A, Gales P, Girod C, Lebrec D. Alteration in response of the portal tributary vascular bed to the P-agonist dobutamine in rats with extrahepatic portal hypertension. J Hepatol 1986;2: 267-275.
14. Kaumann AJ, Groszmann RJ. Catecholamines relax portal and mesenteric veins from normal and portal hypertensive rats. Am J Physiol 1989;257:G977-G981. 15. Jensen LS, Juhl GO, Mulvany MJ. Mechanical, morpholopcal and pharmacological properties of oesophageal varices and small mesenteric veins in portal hypertensive rabbits. Acta Physiol Scand 1987;130:649-656. 16. Blei AT. Vasodilator therapy of portal hypertension: focus on the liver. HEPATOLOGY 1989;9:896-899. 17. Reichen J, Le M. Verapamil favorably influences hepatic microvascular exchange and function in rats with cirrhosis of the liver. J Clin Invest 1986;78:448-455. 18. Kroeger RJ, Groszmann RJ. Increased portal venous resistance hinders portal pressure reduction during the administration of p-adrenergic blocking agents in a portal hypertensive model. HEPATOLOGY 1985;5:97-101. 19. Koshy A, Girod C, Lee SS, Hadengue A, Cerini R, Lebrec D. Discrepancy between portal pressureand systemic hemodynamic changes after incremental doses of propranolol in awake portal hypertensive rats. HEPATOLOGY 1989;9:269-273.
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463
hAIN
BRAILLON, ANTOINE HADENGUE, CHRISTOPHE GAUDIN AND DIDIERLEBREC
Laboratoire d 'Hhodynamique Splanchnique, Unite' de Recherches de Physiopathologie Hkpatique (INSERM U-24), H6pital Beaujon, 92118 Clichy, France
The hemodynamic responses to terbutaline- a selective p,-adrenoceptor agonist-were studied in conscious normal rats and in conscious rats with secondary biliary cirrhosis. Compared with those of normal rats, dose-response curves in cirrhotic rats indicated significantly decreased reactivity in arterial pressure and heart rate. Half-maximal effective dose was not significantlydifferent between the two groups. Terbutaline induced significant, dose-dependent decreases in portal pressure in both normal rats (9.3%) and cirrhotic rats (13.8%).In normal rats, terbutaline administration(32 pg * min- * kg-l bodywt) increased both cardiac output and portal tributary blood flow, thus mimicking hemodynamic changes in cirrhotic rats. In cirrhotic rats, despite a significant increase in portal tributary blood flow (from 19.9 f 1.7 mllmin to 22.7 & 1.5 ml/min), terbutaline decreased portal pressure from 17.4 k 1.0 mm Hg to 15.0 2 0.8 mm Hg. This study indicates that increased p,-adrenoceptor stimulation in cirrhotic rats may be involved in hyperdynamic circulation. The association of a decreased portal pressure and increased splanchnic blood flow suggests that p,-adrenoceptor stimulation may modulate hepatic and portal collateral vascular resistance. (HEPATOLOGY 1992;15:459-463.)
p-blockers reduce portal pressure by the combination of p,-antagonism which decreases cardiac output and p,-antagonism, which induces splanchnic vasoconstriction. However, the specific role of P,-adrenoceptors in portal hypertension has not yet been elucidated. Moreover, the hemodynamic effects of P,-adrenergic stimulation on splanchnic circulation are unknown. Thus, to elucidate the mechanisms of action of p-adrenergic antagonists in portal hypertension, we evaluated the hemodynamic effects of terbutaline, a p,-adrenergic agonist, in conscious cirrhotic rats. MATERIALS AND METHODS
Animals. Fifty adult male Sprague-Dawley rats (Laboratoires Charles River, Saint Aubin-les-Elbeuf, France) were divided into two groups. Rats in the first group were rendered cirrhotic and portal hypertensive by sectioning and ligating the common bile duct (6). Rats in the second group underwent sham operation. Hemodynamic studies were performed 4 t o 5 wk after surgery. Body weight at the time of the hemodynamic studies was similar between biliary cirrhotic (327 2 8 gm) and normal rats (316 10 g m ) . Studies performed in this laboratory were approved by the French Agricultural Office. Methods. Rats were allowed free access to food and water until 14 to 16 hr before the study, when food was withdrawn. p-adrenergic antagonists reduce the risk of gas- Animals were anesthetized with ether. Catheters were placed trointestinal bleeding in patients with cirrhosis (1-3). in the left jugular vein for infusion of terbutaline or saline, in This beneficial effect depends at least in part on the left ventricle (in the second set of experiments, for reduction of portal pressure and reduction of blood flow radioactive microsphere injection), in the portal vein for portal into the superior portal systemic circulation (4).The pressure measurements, and in the left femoral artery for mechanisms of action of P-blockers on the splanchnic measurement of arterial pressure. All measurements were circulation have not been, however, entirely elucidated. performed approximately 2 hr after rats awakened, while they Nonselective p-blockers, however, are known to induce rested in a restraining apparatus. Pressure and Chronotropic Dose-response Curves. The more profound splanchnic effects than cardioselective and chronotropic dose responses were performed in p-blockers (5). Thus it is thought that nonselective pressure eight sham-operated rats and eight cirrhotic rats. Heart rate, mean arterial pressure and portal pressure were measured during infusion of increasing doses of terbutaline ( 2 , 4, 8, 16, Received July 11, 1991; accepted October 10, 1991. 32 and 64 yg.min-'.kg-' body wt) through a venous Dr. Po0 held a fellowship from the Fondation pour la Recherche Medicale and catheter. Each dilution of terbutaline was perfused for 10 min; from the CONACYT (Mexico). total infusion volume was 900 pyanimal. No injection was This work was presented in part at the 24th Meeting of the European performed until the effects on arterial pressure and heart rate Association for Study of the Liver, August 30 to September 2, 1989, in Munich, of the previous dose had disappeared. This period lasted 10 to and was published in abstract form (J Hepatol 1989;9(suppl 1):575). 15 min. Preliminary studies showed that the responses were Address reprint requests to: Dr. D. Lebrec, INSERM U-24, HBpital Beaujon, similar between terbutaline injected in increasing doses and 92118 Clichy Cedex, France. doses of terbutaline injected at random. 3111134384
459
460
PO0 ET AL.
,
Changes in portal pressure (mmHg) I
Changes in mean arterial pressure (mmHg)
a 71. o
Normal rats Cirrhotic rats
0
Normal rats Cirrholie rals
-20 4
, I
I
I
2
8
16
32
64 1
2
Terbulatine [ pg.kg .min )
-40
FIG.2. Response of portal pressure to terbutaline in normal and cirrhotic rats. No significant difference was seen between the two groups.
I
2 8
16
32
64
Changes in heart rate (beats I rnin) 200 1
OYi-16
32
64
Terbutaline (pg.kg'. min' )
FIG.1. Responses of arterial pressure and heart rate to terbutaline in normal and cirrhotic rats. *Significantly different between the two groups.
Maximal chronotropic and pressure responses (Em=) and the doses required to obtain half-maximal blood pressure reduction and half-maximal heart rate increase (ED,,) were calculated using the double reciprocal plot (l/dose vs. Uresponse). In two cirrhotic rats, heart rate and mean arterial pressure were measured during infusion of similar increasing doses of terbutaline. Ten minutes before terbutaline administration, however, they received a &-selective adrenoceptor antagonist (ICI 118,551;30 kg. kg-l -body wt, intravenously). Hemodynamic Measurements. The effect of the intravenous administration of terbutaline (32 pg min- kg- body wt for 10 min) or placebo (saline solution, 0.05 ml/min) was measured in 16 normal rats and 16 rats with secondary biliary cirrhosis. Heart rate and arterial and portal pressures were recorded on a square-wave electromagnetic manometer (CM130; Phillips, Eindhoven, The Netherlands) before and 10 min after administration of saline solution or terbutaline infusion. Regional blood flows and cardiac output were determined by the radioactive microsphere method (15 -+ 3 pm diameter; Du Pont-New England Nuclear, Boston, MA) as described previously ( 6 ) .ll3Sn-1abeledmicrospheres were injected into the left ventricle for basal measurements; 141 Ce-labeled microspheres were used for the second measurement, which was performed 10 min after the onset of terbutaline or saline infusion. The reference blood sample was drawn from the catheter in the femoral artery into a motor-driven syringe at 1 ml/min for 1 min. The animals were then killed with an overdose of pentobarbital sodium. Individual organs were dissected and placed in individual tubes for counting in a gamma-counter (Compteur G4000, Intertechnique, Plaisir, France) at energy settings of 280 to 1,000 keV and 70 to 210 keV for l13Sn and
-
141Ce, respectively. Adequate microsphere mixing was assumed to have occurred when the difference between right and left kidneys was less than 10%. Portal tributary blood flow was considered the sum of blood flows from the stomach, spleen, intestine, colon and mesentery-pancreas. Portal territory vascular resistance (dyn * sec * cm-5 x lo3) = (mean arterial pressure [mm Hg] - portal pressure [mm Hgl) x 80iportal tributary blood flow (ml/min).Portal-collateraland hepatic vascular resistance (dyn * sec cm-5 x lo3) = portal pressure (mm Hg) x 80/portal tributary blood flow (ml/min) (in normal rats that had no portal-collateral circulation, hepatic vascular resistance was used.) Statistical Analysis. Results are expressed as mean 2 S.E.M. Comparisons were performed by one-way ANOVA. A Bonferroni correction for multiple comparisons was used. A p value < 0.05 was considered statistically significant.
RESULTS Pressure and Chronotropic Dose-response Curves.
Dose-response curves are summarized in Fig. 1.At doses of 2 to 32 pg min- kg-' body wt, terbutaline induced dose-dependent increases in heart rate in both normal and cirrhotic rats. Em, was significantly different between the two groups (153 ? 24 beatsimin and 103 f 25 beats/min, respectively), but ED,, was not (8.4 & 2.5 p g - m i n - l - k g - l and 8.5 .t 5.2 pg-min-'. kg- body wt, respectively) significantly different. At doses of 2 to 32 pg * min- kg-I body wt, terbutaline also induced dose-dependent decreases in mean arterial pressure in normal and cirrhotic rats. Em, was significantly higher in normal rats (24.4 2 7.6 mm Hg) than in cirrhotic rats (18.1 +- 3.9 mm Hg). ED,, for arterial pressure was not significantly different between the two groups (12.1 f 5.0 p g - m i n - l - k g body wt and 11.6 k 3.3 p g . m i n - l - k g - l body wt, respectively). In cirrhotic rats that received ICI 118 551, arterial pressure was not affected during the four first doses of terbutaline, but decreased by 7% and 15%for the two final doses, respectively. Heart rate increased by approxkg- body imately 20% with 16, 32 and 64 kg * min wt terbutaline. Terbutaline induced a dose-dependent decrease in portal pressure without any significant difference between the two proum, but maximal effect was not obtained (Fig. 2);
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TABLE 1. Effect of terbutaline on systemic and splanchnic hemodynamics in conscious normal and cirrhotic rats Normal (n = 8 ) Parameters
Cirrhotic (n = 8)
Basal
Terbutaline
Basal
Terbutaline
393 t 5" 106 t 8 35.4 ? 1.8 84.9 t 6.5 6.5 3- 0.5 14.5 3- 1.3 5.30 0.38 15.2 ? 1.0 537 c 49 34.9 t 4.0
572 i 10* 80 i 3* 51.7 i 3.2' 44.3 i 3.5* 5.9 % 0.5b 18.4 & 1.7' 6.16 i 0.59* 12.1 i 1.12* 363 i 437' 26.9 % 3.5'
395 i 11 97 i 4 46.6 % 2.1' 51.7 i 3.1' 17.4 i 1.F 19.9 i 1.7' 6.06 & 0.43' 13.0 t 1.0 337 i 32' 72.1 i 5.7'
529 t 4' 82 3- 4* 57.8 3- 2.0' 34.7 i 1.6' 15.0 f 0.8' 22.7 2 1.5* 6.95 t 0.43' 12.1 t 0.9* 241 3- 21* 55.9 2 5.3h
8.7 ? 0.7 2.98 2 0.21 8.7 t 0.7 18.4 t 1.0 6.35 f 0.34 18.1 f 1.1
7.9 f 0.60 2.65 i 0.26 5.3 i 0.6' 21.6 i 1.76 7.42 f 0.69' 14.9 i 1.2
14.4 t 1.6" 4.50 i 0.62' 9.5 1.2 32.1 i 2.6' 9.82 ? 0.73' 20.8 i 1.0
16.1 t 26 5.05 t 0.90 8.5 t 1.3 40.4 t 2.5' 12.45 ? 0.86' 21.3 f 1.2
~~
Heart rate (beatsimin) Mean arterial pressure (mm Hg) Cardiac index (ml min-' . 100 gm-l body wt) Systemic vascular resistance (lo3 dyn . sec cmg5) Portal pressure (mm Hgj Portal tributary blood flow (mlimin) ml. m i n - l . 100 g m - l body wt % Cardiac output Portal territory vascular resistance (lo3 dyn sec cm-5) Portal collateral and hepatic vascular resistance (lo3 dyn . sec cm-S) Hepatic arterial blood flow (mlimin) ml . min-'. 100 gm-l body wt Cardiac output Renal blood flow (mumin) m l . m i n g ' . 100 gm-'bodywt % Cardiac output
*
*
Rats were administered a dose of 32 kg. kgg' . min-' "Data expressed as mean t S.E.M. 'Significantly different from basal value (p < 0.05). 'Significantly different from basal value of normal rats (p < 0.05).
TABLE 2. Lack of effect of saline solution on systemic and splanchnic hemodynamics in conscious normal and cirrhotic rats Normal (n
=
8)
Cirrhotic (n = 8 )
Parameters
Basal
Saline solution
Basal
Saline solution
Heart rate (beatsimin) Mean arterial pressure (mm Hg) Cardiac index (ml . min - ' . l o 0 gm - ' body wt) Systemic vascular resistance (lo3 dyn . sec . cm-5) Portal pressure (mm Hg) Portal tributary blood flow (mlimin) ml , m i n - l . 100 g n - l body wt Portal territory vascular resistance (lo3dyn . sec . c m - 9 Portal collateral and hepatic vascular resistance (lo3 dyn. sec. c m - 9 Hepatic arterial blood flow (mlimin) ml . m i n - ' . 100 gm-'body wt Renal blood flow (mlimin) ml m i n g l . 100 gm-' body wt
384 f 5" 104 i 3 34.4 2 1.6 80.4 f 6.5 6.1 i 0.5 13.4 i 1.2 4.32 f 0.36 529 ? 73 36.3 i 5.2
380 i 6.3 105 ? 9 33.6 ? 1.5 83.4 t 17.1 6.2 i 0.5 15.1 i 1.1 4.91 ? 0.38 506 % 73 31.7 i 2.9
403 t 3.7 97 i 3 44.8 i 3.3 54.0 i 4.8 17.5 t 0.8 21.2 i 2.5 6.23 t 0.79 336 i 43 71.5 t 6.9
400 f 5.9 97 t 3 44.9 3- 3.4 54.6 t 6.2 17.3 i 0.8 21.5 f 2.7 6.38 ? 0.82 343 t 52 72.3 ? 9.7
9.9 i 0.8 3.13 ? 0.18 17.6 f 1.4 5.64 f 0.37
8.8 i 0.9 2.83 i 0.29 19.2 F 1.2 6.19 t 0.34
12.5 i 2.5 3.66 t 0.70 26.1 t 2.6 7.28 i 0.74
15.4 i 4.0 4.21 t 1.12 27.1 ? 3.6 7.98 i 0.95
"Data expressed as mean
f
S.E.M.
Hemodynamic Measurements. In both groups of rats, terbutaline significantly increased heart rate, cardiac index and portal tributary blood flow. It significantly decreased mean arterial pressure, systemic vascular resistance, portal pressure and vascular resistance in the splanchnic bed (Table 1).Terbutaline induced significantly more marked changes in normal rats than in cirrhotic rats for cardiac index (47.5% and 24.7%, respectively) and systemic vascular resistance ( - 47.8% and - 31.1%, respectively). The increase in portal tributary blood flow was not significantly different between the two groups (19.2%and 16.1%,respectively). Distribution of cardiac output was significantly decreased in the portal territory and in liver in normal rats,
whereas distribution of cardiac output was not significantly affected by terbutaline in cirrhotic rats. Terbutaline significantly increased renal blood flow in both groups of rats (Table 1). Distribution of cardiac output was significantly decreased in the kidneys of normal rats, but not in cirrhotic rats. The placebo had no significant effect in normal and cirrhotic rats (Table 2). DISCUSSION In this study, to clarify the mechanisms of the portal hypotensive effects of P-blockers in portal hypertension, we used terbutaline to analyze the hemodynamic effects of P,-adrenoceptor stimulation in normal and cirrhotic rats. The lack of response of arterial pressure for low
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P O 0 ET AL.
doses of terbutaline in rats that received a @,-selective antagonist confirms the specificity of @,-adrenergic agonist of terbutaline. This type of drug is generally used to treat asthma because of its bronchodilator effect, but it also dilates blood vessels (7). Moreover, it has been demonstrated that the P-adrenoceptors in the microcirculation of rat liver are of type P, (8). In this study, the dose-response curves show that terbutaline induces a significant decrease in arterial pressure in both normal and cirrhotic rats. Vasodilatation began with low doses of terbutaline and reached a plateau at a dose of 30 pg * min kg body w t in both groups. The maximum arterial response, however, was significantly more marked in normal rats than in cirrhotic rats. This result indicates that rats with secondary biliary cirrhosis have lesser vascular reactivity to @,-adrenergic stimulation than do normal rats. In both groups, reflex tachycardia occurred; in normal rats, however, the increased heart rate was significantly more marked than in cirrhotic rats. In patients with cirrhosis, similar blunted chronotropic responses to nonspecific @-adrenoceptorstimulation by isoprenaline have been observed (9). The significant maximal response of arterial pressure and heart rate and the lack of difference in half-maximal response between normal and cirrhotic rats indicate that noncompetitive inhibition is likely to occur in uiuo. This result supports previous findings, which show a decreased number of P,-adrenoceptor binding sites in both the mononuclear cells in patients with cirrhosis and severe ascites (10) and in the myocardial P,-receptors of rats with secondary biliary cirrhosis (11). In both the dose-response and blood-flow measurement studies, terbutaline had similar effects on arterial pressure and heart rate. In normal rats, terbutaline induced a marked increase in cardiac output and portal tributary blood flow. These findings were expected, and they resulted from significant systemic and splanchnic vasodilatation. The hyperdynamic circulation observed in normal rats receiving terbutaline mimics the hemodynamic changes of cirrhotic rats. Therefore P-adrenoceptor stimulation may play a role in the hyperkinetic circulation observed in portal hypertension by causing sympathoadrenomedular activation. In this series using normal rats, hepatic arterial blood flow was not affected by terbutaline administration. This lack of response to increased portal tributary blood flow supports the washout hypothesis described by Lautt ( 121. In cirrhotic rats, terbutaline exaggerated splanchnic and systemic hyperkineticism. Changes in cardiac output, however, were less marked in cirrhotic rats than in controls. This might suggest down-regulation of peripheral P,-adrenoceptors. On the other hand, the splanchnic hemodynamic changes induced by terbutaline were similar in cirrhotic and normal rats. Similar findings have previously been observed in rats with portal-vein stenosis receiving dobutamine, a @,adrenoceptor agonist (13). In both groups of rats, terbutaline decreased the proportion of cardiac output ~
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HEPATOLOGY
reaching the splanchnic bed. In both groups of rats, the decrease in portal pressure occurred even though portal tributary blood flow increased significantly. Because portal collateral and hepatic vascular resistance decreased significantly, the decreased portal pressure might have resulted from a reduction of vascular resistance in the liver, in the collateral circulation or both. In normal rats, however, only intrahepatic vascular resistance could decrease. This indicates that P,-adrenoceptors are present in the liver and play a role in the regulation of liver circulation. Our findings in normal rats confirm a previous study showing that @,-adrenoceptors are present in liver sinusoids and counteract constrictor responses produced by a-adrenoceptor activity (8). In cirrhotic rats, the decrease in portal pressure may also depend on collateral vasodilatation induced by terbutaline. This hypothesis is supported by the presence of adrenoceptors in both the portal vein and vessels of the portal circulation and by the role of P,-adrenoceptors in the modulation of venous tone (14, 15). The fact that drugs can alter hepatic vascular resistance (16, 17) or portal collateral vascular resistance (18, 19) has been established in earlier studies. In conclusion, terbutaline decreases portal pressure despite a concommitant increase in portal tributary blood flow in conscious rats with secondary biliary cirrhosis. These findings emphasize the role of hepatic and portal collateral vascular resistance in the modulation of portal hypertension.
Acknowledgments: We are indebted to Laurent Font of the Animal Care Unit for his help, to St6phane Cailmail for technical assistance and to Philippe Letteron for statistical analysis. REFERENCES 1. Pagliaro L, Burroughs AK, Sorensen TIA, Lebrec D, Morabito A, D’Amico G, Tine F. Therapeutic controversies and randomised controlled trials (RCTs): prevention of bleeding and rebleeding in cirrhosis. Gastroenterol Intern 1989;2:71-84. 2. Hayes PC, Davis JM, Lewis JA, Bouchier IAD.Meta-analysis of value of propranolol in prevention of variceal haemorrhage. Lancet 1990;336:153-156. 3. Poynard T, Cales P, Pasta L, Pagliaro L, Ideo G, Pascal J-P, Pagliaro L, et al. Beta-adrenergic-antagonistdrugs in the prevention of gastrointestinal bleeding in patients with cirrhosis and esophageal varices: an analysis of data and prognostic factors in 589 patients from four randomized clinical trials. N Engl J Med 1991;324:1532-1538. 4. Cales P, Braillon A, Jiron MI, Lebrec D. Superior portosystemic collateral circulation estimated by azygos blood flow in patients with cirrhosis: lack of correlation with oesophageal varices and gastrointestinal bleeding: effect of propranolol. J Hepatol 1984;1: 37-46. 5. Hillon P, Lebrec D, Mudoz C, Jungers M, Goldfarb G, Benbamou J P . Comparison of the effects of a cardioselective and a nonselective @-blockeron portal hypertension in patients with cirrhosis. HEPATOLOGY 1982;2:528-531. 6. Lee SS, Girod C, Braillon A, Hadengue A, Lebrec D. Hemodynamic characterization of chronic bile duct-ligated rats: effect of pentobarbital sodium. Am J Physiol 1986;251:G176-G180. 7. Koo A. Vasodilator effect of terbutaline: in uiuo evidence for the existence of @,-adrenoceptors in the microcirculation of rats,
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P,-ADRENOCEPTOR STIMULATION IN CIRRHOSIS
hamsters, and guinea pigs. J Cardiovasc Pharmacol 1984;6: 897-901. 8. Koo A, Liang NS. P,-adrenoceptors in rat liver microcirculation. Clin Exp Pharmacol Physiol 1979;6:403-407. 9. Ramond MJ, Comoy E, Lebrec D. Alterations in isoprenaline sensitivity in patients with cirrhosis: evidence of abnormality of the sympathetic nervous activity. Br J Clin Pharmacol 1986;21: 191-196. 10. Gerbes AL, Remien J , Jiingst D, Sauerbruch T, Paumgartner G. Evidence for down-regulation of beta-2-adrenoceptors in cirrhotic patients with severe ascites. Lancet 1986;1:1409-1410. 11. Lee SS, Marty J, Mantz J, Samain E, Braillon A, Lebrec D. Desensitization of myocardial P-adrenergic receptors in cirrhotic rats. HEPATOLOGY 1990;12:481-485. 12 Lautt WW. Mechanism and role of intrinsic regulation of hepatic arterial blood flow: hepatic arterial buffer response. Am J Physiol 1985:249:G549-G556. 13. Braillon A, Gales P, Girod C, Lebrec D. Alteration in response of the portal tributary vascular bed to the P-agonist dobutamine in rats with extrahepatic portal hypertension. J Hepatol 1986;2: 267-275.
14. Kaumann AJ, Groszmann RJ. Catecholamines relax portal and mesenteric veins from normal and portal hypertensive rats. Am J Physiol 1989;257:G977-G981. 15. Jensen LS, Juhl GO, Mulvany MJ. Mechanical, morpholopcal and pharmacological properties of oesophageal varices and small mesenteric veins in portal hypertensive rabbits. Acta Physiol Scand 1987;130:649-656. 16. Blei AT. Vasodilator therapy of portal hypertension: focus on the liver. HEPATOLOGY 1989;9:896-899. 17. Reichen J, Le M. Verapamil favorably influences hepatic microvascular exchange and function in rats with cirrhosis of the liver. J Clin Invest 1986;78:448-455. 18. Kroeger RJ, Groszmann RJ. Increased portal venous resistance hinders portal pressure reduction during the administration of p-adrenergic blocking agents in a portal hypertensive model. HEPATOLOGY 1985;5:97-101. 19. Koshy A, Girod C, Lee SS, Hadengue A, Cerini R, Lebrec D. Discrepancy between portal pressureand systemic hemodynamic changes after incremental doses of propranolol in awake portal hypertensive rats. HEPATOLOGY 1989;9:269-273.
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