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Modulated hemodynamic response to clonidine in bile duct-ligated rats: The role of nitric oxide
European Journal of Pharmacology 542 (2006) 148 – 153 www.elsevier.com/locate/ejphar
Modulated hemodynamic response to clonidine in bile duct-ligated rats: The role of nitric oxide Sina Tavakoli a , Amir Reza Hajrasouliha a , Pejman Jabehdar-Maralani a , Farzad Ebrahimi a , Hamed Sadeghipour a , Mehdi Dehghani a , Hamed Shafaroodi b , Ahmad Reza Dehpour a,⁎ a
Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, P.O. Box 13145-784, Tehran, Iran b Department of Pharmacology, Tehran Medical Unit, Islamic Azad University, Tehran, Iran Received 25 August 2005; received in revised form 11 April 2006; accepted 28 April 2006 Available online 7 May 2006
Abstract Despite the well-known involvement of the peripheral sympathetic abnormalities in the development of cardiovascular complications of cholestasis, the role of the central sympathetic system is still elusive. The goal of this study was to evaluate the effects of central sympathetic tone reduction, through clonidine administration, on hemodynamic parameters of 7-day bile duct-ligated rats. The contributions of nitric oxide and endogenous opioids were also examined by acute intravenous (10 min before clonidine) or chronic daily subcutaneous administrations of N(ω)nitro-L-arginine methyl ester (L-NAME, 3 mg/kg) or naltrexone (20 mg/kg). Seven days after bile duct ligation or sham operation, animals were anesthetized with sodium pentobarbital. After hemodynamic stabilization, clonidine (10 μg/kg) was injected intravenously, which elicited an initial hypertension (the peripheral effect) followed by persistent hypotension and bradycardia (the central effects). Cholestatic rats demonstrated significant basal bradycardia (P < 0.001) and hypotension (P < 0.05), which were corrected by chronic naltrexone but not L-NAME treatment. While the peripheral effect of clonidine was blunted, the central effects were exaggerated in cholestatic rats (P < 0.01). Acute L-NAME treatment accentuated the hypertensive phase in sham-operated and cholestatic rats (P < 0.05). However, the difference between the two groups was preserved (P < 0.01). This treatment attenuated the central effects in both sham-operated and cholestatic rats to the same level (P < 0.001). Chronic L-NAME treatment resulted in exaggeration of the peripheral response in cholestatic and central responses in sham-operated rats (P < 0.05), and abolished the difference between the groups. Naltrexone treatment had no significant effect on either the central or the peripheral responses to clonidine. This study shows that both central and peripheral hemodynamic responses to clonidine are altered in cholestasis. It also provides evidence that nitric oxide contributes to the development of these abnormalities. © 2006 Elsevier B.V. All rights reserved. Keywords: Adrenergic hyporesponsiveness; Bile duct ligation; Cholestasis; Clonidine; Endogenous opioids; Nitric oxide; (Rat)
1. Introduction Cholestatic liver disease is associated with widespread derangements in the cardiovascular system, such as bradycardia (Gaskari et al., 2002; Mani et al., 2002; Nahavandi et al., 2001), hypotension (Bomzon et al., 1990; Jacob et al., 1993), peripheral vasodilation and susceptibility to postoperative renal failure or hemorrhagic shock (Green et al., 1984). Hyporesponsiveness of peripheral tissues to inotropic (Green et al., 1984) and vasopressor agents (Finberg et al., 1981) has been suggested to contribute to the development of these ⁎ Corresponding author. Tel.: +98 21 6611 2802; fax: +98 21 6640 2569. E-mail address: [email protected] (A.R. Dehpour). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.04.052
complications. Our recent reports of the diminished responsiveness of isolated atria (Gaskari et al., 2002; Mani et al., 2002; Nahavandi et al., 2001), papillary muscles (Ebrahimi et al., 2005) and vascular beds (Namiranian et al., 2001) to adrenergic stimulation propose that peripheral responsiveness to sympathetic system may contribute to bradycardia, cardiomyopathy and hypotension of cholestatic rats. Although altered central sympathetic tone has also been reported in cholestatic liver disease (Dabagh et al., 1999), its impact on hemodynamic complications is not clear. Clonidine has long been used as a blood pressure lowering agent. This drug stimulates α2-adrenoceptors in the brain stem and results in prolonged hypotension and bradycardia as a result of reduced sympathetic outflow from the central nervous
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system. Clonidine can also activate α2-adrenoceptors on vascular smooth muscle cells, which accounts for the transient vasoconstriction and hypertension noticed following the intravenous drug administration (Hoffman, 2001). We have recently reported that responsiveness of isolated peripheral tissues to clonidine is attenuated in cholestatic animals (Borhani et al., 2005; Demehri et al., 2003). In addition, it has been reported that clonidine administration reduces norepinephrine spillover and hepatic vein wedge pressure in cirrhotic patients, which suggest it as a potential therapy in advanced liver diseases (Esler and Kaye, 1998; Esler et al., 1992). However, the effect of clonidine administration on hemodynamic abnormalities of acute cholestasis has not yet been evaluated. Endogenous opioids and nitric oxide (NO) can modulate sympathetic nervous system. Their contributions to the development of cardiovascular abnormalities have been reported in several pathologic states (Chowdhary et al., 2002; Massion et al., 2003; Patel et al., 2001; Paton et al., 2002; Pugsley, 2002; Whalen et al., 1999). NO overproduction and accumulation of endogenous opioids have also been reported in the cholestatic liver disease and suggested to be involved in the development of bradycardia (Gaskari et al., 2002; Mani et al., 2002; Nahavandi et al., 2001), hypotension (Hajrasouliha et al., 2004, 2005), peripheral vasodilation (Namiranian et al., 2001), and hyporesponsiveness to adrenergic agents (Gaskari et al., 2002; Mani et al., 2002; Nahavandi et al., 2001; Namiranian et al., 2001). We have also recently reported that these compounds contribute to the abnormal responsiveness to clonidine in isolated tissues of cholestatic animals (Borhani et al., 2005; Demehri et al., 2003). However, there is no data of their role in the modulation of the central response to clonidine administration. The main goal of this study is to evaluate the effect of clonidine administration, and subsequent reduction in the central sympathetic tone, on hemodynamic parameters of 7day bile duct-ligated (BDL) rats, as a known model of acute cholestasis. Contributions of NO and endogenous opioid peptides are also examined in this study. 2. Materials and methods 2.1. Animal manipulation The animals were handled in accordance with the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” (NIH US publication 85-23 revised 1996). Male Sprague–Dawley rats weighing 200–250 g were used. Animals were housed in groups of 3–4 in a room controlled at 22 ± 1 °C and maintained in an alternating 12-h light/12-h dark cycles, and were allowed free access to food and water. Bile duct ligation was performed as described previously (Hajrasouliha et al., 2004, 2005). Laparotomy was performed under general anesthesia induced by an intraperitoneal injection of ketamine HCl (50 mg/kg; Gedoon Richter, Budapest, Hungary) and chlorpromazine HCl (10 mg/kg; Daroupakhsh, Tehran, Iran). In BDL rats, bile duct was identified and doubly ligated. In the sham-operated rats, the bile duct was identified, manipulated and
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one untied loose tie was left in place. Then, the abdominal wall was closed in two layers. One day after laparotomy, BDL rats showed manifestations of cholestasis (jaundice and dark urine). 2.2. Experimental groups The animals were randomly divided into five groups. Each group consisted of two subgroups of six to eight sham-operated or BDL rats. The first group of animals was treated with daily subcutaneous administration of isotonic sterile saline solution (1 ml/kg/day) and served as the control group. The role of NO overproduction was evaluated by acute intravenous (3 mg/kg) or chronic subcutaneous (3 mg/kg/d) injections of N(ω)-nitro-Larginine methyl ester (L-NAME, NO synthase inhibitor, Sigma, Louis, MO, USA), in the second and third groups. The last two groups of animals were treated by acute intravenous (20 mg/kg) or chronic subcutaneous (20 mg/kg/d) injections of naltrexone HCl (Iran Darou, Tehran, Iran). Acute drug administrations were performed 10 min before clonidine injection. Chronic drug administrations were performed for six consecutive days. The first dose was injected the day after surgery, and the last dose was injected 24 h before the experimental protocol, so little drug or drug action was expected to exist at the time of the experiment (Ebrahimi et al., 2005; Namiranian et al., 2001). Treatment regimens were selected based on our previous studies, shown to effectively reduce NO production, antagonize the effects of accumulated endogenous opioids and reverse complications of acute cholestasis (Borhani et al., 2005; Ebrahimi et al., 2005; Gaskari et al., 2002; Hajrasouliha et al., 2004, 2005; Mani et al., 2002; Nahavandi et al., 2001). 2.3. Experimental protocol Seven days after bile duct ligation or sham operation, animals were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg, Merck, Darmstadt, Germany). Through a ventral midline cervical incision, trachea was exposed and intubated with a cannula connected to a rodent ventilator. Rats were ventilated with room air. The right carotid artery was cannulated to measure arterial blood pressure using a pressure transducer (Pressure Transducer Model P-1000-A, Narco Biosystem, Houston, TX, USA). Mean arterial pressure was calculated as diastolic blood pressure plus one third of the differential pressure. The left jugular vein was cannulated for drug injection. After a steady state was achieved, acute administrations (drugs or vehicles) were performed. Clonidine (10 μg/kg, Iran Darou, Tehran, Iran) was injected intravenously, 10 min later. 2.4. Statistical analysis Baseline parameters are expressed as absolute values. Responses to clonidine are expressed as percentages of the baseline values. Data are presented as mean ± S.E.M and were analyzed by analysis of variance (ANOVA) followed by Tukey's HSD as post hoc test to compare the means. P < 0.05 was considered as the significance level between groups.
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3. Results 3.1. Baseline heart rate and mean arterial pressure Baseline heart rate and mean arterial pressure (Table 1) were significantly lower in BDL rats compared to sham-operated animals (P < 0.001 and P < 0.05, respectively). Acute L-NAME treatment had no significant effect on heart rate (from 445 ± 7 to 436 ± 9 beats/min, P > 0.05) and mean arterial pressure (from 109 ± 6 to 118 ± 5 mm Hg, P > 0.05) in sham-operated rats. On the other hand, it corrected baseline hypotension (from 89 ± 4 to 112 ± 4 mm Hg, P < 0.05), but not bradycardia (from 390 ± 7 to 371 ± 8 beats/min, P > 0.05), in BDL animals. Chronic L-NAME administration had no significant effect on hemodynamic parameters in sham-operated and BDL rats (Table 1). Acute naltrexone administration had also no significant effect on heart rate or mean arterial pressure (data not shown). Chronic naltrexone treatment, without any significant effect in sham-operated animals, corrected bradycardia (Table 1; P < 0.001) and hypotension (Table 1; P < 0.05) in BDL rats. 3.2. Response to clonidine administration Clonidine elicited a typical biphasic effect, with an initial transient hypertension followed by persistent hypotension (Fig. 1). The latter reached its plateau 30 min after clonidine administration, which was used for comparison among the groups. While the early hypertensive response (Fig. 2) was significantly blunted in BDL rats (P < 0.01), the late hypotension and bradycardia (Fig. 3) were exaggerated (P < 0.01). Acute L-NAME treatment accentuated the hypertensive phase (Fig. 2) in both sham-operated (P < 0.05) and BDL (P < 0.05) rats, but the difference between the two groups was preserved (P < 0.01). However, the central phase (Fig. 3) was attenuated to the same level in both sham-operated (P < 0.001) and BDL (P < 0.001) animals by this treatment. Chronic LNAME administration, without a significant effect on shamoperated rats, enhanced the hypertensive response (Fig. 2) in BDL rats (P < 0.05) to the level of sham-operated group. On the other hand, it accentuated hypotension and bradycardia (Fig. 3)
Fig. 1. Recordings of the effect of intravenous clonidine administration (10 μg/ kg) on arterial blood pressure (mm Hg) in anesthetized (A) sham-operated and (B) bile duct-ligated rats.
in sham-operated rats (P < 0.05), but had no significant effect on BDL animals. Acute or chronic naltrexone treatment did not result in a significant change in the hemodynamic responses to clonidine in BDL or sham-operated animals (Figs. 2 and 3). 4. Discussion 4.1. Altered baseline hemodynamics and response to clonidine in cholestasis In this study heart rate and mean arterial pressure were significantly lower in BDL rats compared to sham-operated animals, which is consistent with previous reports (Gaskari et al., 2002; Hajrasouliha et al., 2004, 2005; Mani et al., 2002;
Table 1 Baseline heart rate (beats/min) and mean arterial pressure (mm Hg) in shamoperated and BDL rats treated with normal saline, acute (3 mg/kg)/chronic (3 mg/kg/d) N(ω)-nitro-L-arginine methyl ester (L-NAME) or acute (20 mg/kg)/ chronic (20 mg/kg/d) naltrexone Treatment
Saline Acute L-NAME Chronic L-NAME Acute naltrexone Chronic naltrexone
Heart rate (beats/min)
Mean arterial pressure (mm Hg)
Sham
BDL
P-value Sham
441 ± 8 445 ± 7 437 ± 7 433 ± 7 443 ± 9
385 ± 6 390 ± 7 374 ± 8 379 ± 6 447 ± 8a
<0.001 <0.001 <0.001 <0.001 >0.05
BDL
111 ± 4 91 ± 5 109 ± 6 89 ± 4 117 ± 5 103 ± 6 113 ± 6 87 ± 4 114 ± 5 110 ± 6b
BDL = bile duct-ligated, L-NAME = N(ω)-nitro-L-arginine methyl ester. a P < 0.001 compared to the saline-treated BDL group. b P < 0.05 compared to the saline-treated BDL group.
P-value <0.05 <0.05 >0.05 <0.05 >0.05
Fig. 2. The early hypertensive effect of intravenous clonidine administration (10 μg/kg) in sham-operated and bile duct-ligated (BDL) rats treated with normal saline, acute (3 mg/kg)/chronic (3 mg/kg/d) N(ω)-nitro-L-arginine methyl ester (L-NAME) or acute (20 mg/kg)/chronic (20 mg/kg/d) naltrexone. ⁎P < 0.05, ⁎⁎P < 0.01 compared to corresponding groups.
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(Sannajust et al., 1989), this finding along with the evidence of sympathetic overactivity, such as elevated levels of norepinephrine (Dabagh et al., 1999), suggests that cholestatic subjects are more dependent on the central sympathetic tone for the maintenance of hemodynamic stability. This could be a compensatory response to the peripheral cardiovascular hyporesponsiveness to adrenergic agents, a phenomenon which has also been described in advanced liver diseases and cirrhosis (Esler and Kaye, 1998; Esler et al., 1992). 4.2. The roles of NO
Fig. 3. The central hemodynamic responses, (A) hypotension and (B) bradycardia, to intravenous clonidine administration (10 μg/kg) in shamoperated and bile duct-ligated (BDL) rats treated with normal saline, acute (3 mg/kg)/chronic (3 mg/kg/d) N(ω)-nitro-L-arginine methyl ester (L-NAME) or acute (20 mg/kg)/chronic (20 mg/kg/d) naltrexone. ⁎P < 0.05, ⁎⁎P < 0.01, ⁎⁎⁎P < 0.001 compared to corresponding groups.
Nahavandi et al., 2001). In our recent studies we have shown that despite baseline in vivo bradycardia, spontaneous beating rate of isolated atria of BDL rats is not different from shamoperated animals. However, chronotropic responsiveness to adrenergic stimulation is significantly blunted in BDL rats (Gaskari et al., 2002; Mani et al., 2002; Nahavandi et al., 2001). Studies on papillary muscles (Ebrahimi et al., 2005) and vascular beds (Namiranian et al., 2001) have also provided similar results, which support the role of peripheral hyporesponsiveness to adrenergic stimulation in the development of the cardiovascular complications of cholestasis. Intravenous administration of clonidine causes an acute rise in the blood pressure, apparently because of activation of postsynaptic α2-adrenoceptors in vascular smooth muscles (Hoffman, 2001). This transient vasoconstriction is followed by more prolonged hypotension and bradycardia that result from the decreased central outflow of impulses in the central sympathetic nervous system. This phase seems to result from activation of α2-adrenoceptors in the lower brain stem region, possibly in the nucleus tractus solitarius (Hoffman, 2001). According to the findings of this study, the transient hypertensive phase was attenuated in BDL rats. This finding can be explained based on the reports of the hyporesponsiveness of peripheral tissues to clonidine (Borhani et al., 2005; Demehri et al., 2003), in cholestasis. On the other hand, the prolonged central effects of clonidine were significantly exaggerated in BDL rats. Since the central effects of α2-adrenoceptor agonists appear to be influenced by the pre-existing sympathetic tone
NO contributes to the regulation of the sympathetic nervous system, both centrally and peripherally (Chowdhary et al., 2002; Massion et al., 2003; Patel et al., 2001; Paton et al., 2002; Whalen et al., 1999). Its involvement in the development of cardiovascular complications has also been reported in several pathological states (Chowdhary et al., 2002; Massion et al., 2003), including cholestasis (Demehri et al., 2003; Hajrasouliha et al., 2004, 2005; Mani et al., 2002; Nahavandi et al., 2001; Namiranian et al., 2001). Acute, but not chronic, administration of L-NAME, at the dosage used in the present study, was able to correct hypotension, but not bradycardia, in BDL rats. Our findings demonstrate that acute L-NAME administration accentuates the early hypertensive response to clonidine administration in both sham-operated and BDL rats. However, the difference between the two groups was still preserved following acute L-NAME treatment. On the other hand, this treatment blunted the late central response to clonidine in both sham-operated and BDL animals to the same level. It is thought that the acute hemodynamic changes following L-NAME administration is mostly the result of blockade of the tonic peripheral NO production (Sander et al., 1997). Since clonidineinduced vasoconstriction is modulated by endothelial NO production (Figueroa et al., 2001), it could be postulated that the exaggerated early phase following acute L-NAME administration is due to the blockade of this peripheral modulatory mechanism. Data regarding the effects of acute administration of NO synthase inhibitors on the central hypotensive phase are controversial. While Soares de Moura et al. (2000) have shown that peripheral administration of NO synthase inhibitors blocks the hypotensive response to clonidine, Sy et al. (2001) reported that their central administration had no effect on this phase. The increased central sympathetic tone has been suggested to be the dominant mechanism of hemodynamic changes following chronic inhibition of NO synthesis (Sander et al., 1997). Despite the trend towards increase in mean arterial pressure, chronic L-NAME was not able to induce significant hypertension in either sham-operated or BDL animals, as expected by the increased sympathetic tone. However, our finding of the exaggerated central effects of clonidine in chronic L-NAME treated rats is an evidence for the increased dependence of the hemodynamic parameters on the central sympathetic tone even without the development of hypertension. It should be mentioned that this response was blunted in BDL animals compared to sham-operated rats, which might be due to the higher basal sympathetic tone (Dabagh et al., 1999).
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4.3. The roles of endogenous opioids Endogenous opioid peptides are involved in the regulation of the cardiovascular system through both central and peripheral receptors (Pugsley, 2002). They have been shown to reduce the heart rate, cardiac output, peripheral vascular resistance (Champion and Kadowitz, 1998) and modulate the autonomic nervous system (Pugsley, 2002). Abnormalities of the opioid system have been reported in several pathophysiological conditions in both human and animal models of cardiovascular diseases (Pugsley, 2002). The role of accumulation of endogenous opioid peptides in the development of the cardiovascular complications of the cholestatic liver disease has also been extensively investigated (Demehri et al., 2003; Gaskari et al., 2002; Hajrasouliha et al., 2004, 2005; Namiranian et al., 2001). The precise reason for the increased opioid activity is not yet completely understood, but it is likely that both overproduction of endogenous opioids and protection of these peptides from degradation may contribute to the elevation of total opioid activity in cholestasis (Bergasa et al., 1992; Swain et al., 1992). The interaction of opioids and central α2-adrenoceptor agonists has been examined previously, which suggest the existence of an opioidergic component in the hemodynamic effects of central α2-adrenoceptor agonists in spontaneous hypertensive rats, but not in normotensive ones (Mastrianni and Ingenito, 1987). According to the results of the present study while the chronic availability of endogenous opioids is involved in the development of bradycardia and hypotension of BDL rats, naltrexone administration had no effect on the clonidine-induced hemodynamic changes. Therefore, other mechanisms, such as modulation of the parasympathetic or the peripheral sympathetic systems might be involved in the opioid-mediated hemodynamic alterations in BDL rats. 4.4. Conclusion In summary, this study confirms that 7-day BDL is associated with bradycardia and hypotension in the rat model of cholestasis. It also demonstrates that while the peripheral phase of clonidine response is blunted in BDL rats, the central inhibitory effect of clonidine on the sympathetic nervous system results in more profound hypotension and bradycardia in cholestasis. Therefore, it seems that cholestatic rats are more dependent on the central sympathetic tone for the maintenance of the hemodynamic stability. This study also provides evidence for the contribution of NO to the development of these abnormalities. References Bergasa, N.V., Rothman, R.B., Vergalla, J., Xu, H., Jones, E.A., Swain, M.J., 1992. Central mu opioid receptors are down regulated in a rat model of cholestasis. J. Hepatol. 15, 220–224. Bomzon, A., Weinbroum, A., Kamenetz, L., 1990. Systemic hypotension and pressor responsiveness in cholestasis: a study in 3-day bile duct ligated rats. J. Hepatol. 11, 70–76.
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Modulated hemodynamic response to clonidine in bile duct-ligated rats: The role of nitric oxide Sina Tavakoli a , Amir Reza Hajrasouliha a , Pejman Jabehdar-Maralani a , Farzad Ebrahimi a , Hamed Sadeghipour a , Mehdi Dehghani a , Hamed Shafaroodi b , Ahmad Reza Dehpour a,⁎ a
Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, P.O. Box 13145-784, Tehran, Iran b Department of Pharmacology, Tehran Medical Unit, Islamic Azad University, Tehran, Iran Received 25 August 2005; received in revised form 11 April 2006; accepted 28 April 2006 Available online 7 May 2006
Abstract Despite the well-known involvement of the peripheral sympathetic abnormalities in the development of cardiovascular complications of cholestasis, the role of the central sympathetic system is still elusive. The goal of this study was to evaluate the effects of central sympathetic tone reduction, through clonidine administration, on hemodynamic parameters of 7-day bile duct-ligated rats. The contributions of nitric oxide and endogenous opioids were also examined by acute intravenous (10 min before clonidine) or chronic daily subcutaneous administrations of N(ω)nitro-L-arginine methyl ester (L-NAME, 3 mg/kg) or naltrexone (20 mg/kg). Seven days after bile duct ligation or sham operation, animals were anesthetized with sodium pentobarbital. After hemodynamic stabilization, clonidine (10 μg/kg) was injected intravenously, which elicited an initial hypertension (the peripheral effect) followed by persistent hypotension and bradycardia (the central effects). Cholestatic rats demonstrated significant basal bradycardia (P < 0.001) and hypotension (P < 0.05), which were corrected by chronic naltrexone but not L-NAME treatment. While the peripheral effect of clonidine was blunted, the central effects were exaggerated in cholestatic rats (P < 0.01). Acute L-NAME treatment accentuated the hypertensive phase in sham-operated and cholestatic rats (P < 0.05). However, the difference between the two groups was preserved (P < 0.01). This treatment attenuated the central effects in both sham-operated and cholestatic rats to the same level (P < 0.001). Chronic L-NAME treatment resulted in exaggeration of the peripheral response in cholestatic and central responses in sham-operated rats (P < 0.05), and abolished the difference between the groups. Naltrexone treatment had no significant effect on either the central or the peripheral responses to clonidine. This study shows that both central and peripheral hemodynamic responses to clonidine are altered in cholestasis. It also provides evidence that nitric oxide contributes to the development of these abnormalities. © 2006 Elsevier B.V. All rights reserved. Keywords: Adrenergic hyporesponsiveness; Bile duct ligation; Cholestasis; Clonidine; Endogenous opioids; Nitric oxide; (Rat)
1. Introduction Cholestatic liver disease is associated with widespread derangements in the cardiovascular system, such as bradycardia (Gaskari et al., 2002; Mani et al., 2002; Nahavandi et al., 2001), hypotension (Bomzon et al., 1990; Jacob et al., 1993), peripheral vasodilation and susceptibility to postoperative renal failure or hemorrhagic shock (Green et al., 1984). Hyporesponsiveness of peripheral tissues to inotropic (Green et al., 1984) and vasopressor agents (Finberg et al., 1981) has been suggested to contribute to the development of these ⁎ Corresponding author. Tel.: +98 21 6611 2802; fax: +98 21 6640 2569. E-mail address: [email protected] (A.R. Dehpour). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.04.052
complications. Our recent reports of the diminished responsiveness of isolated atria (Gaskari et al., 2002; Mani et al., 2002; Nahavandi et al., 2001), papillary muscles (Ebrahimi et al., 2005) and vascular beds (Namiranian et al., 2001) to adrenergic stimulation propose that peripheral responsiveness to sympathetic system may contribute to bradycardia, cardiomyopathy and hypotension of cholestatic rats. Although altered central sympathetic tone has also been reported in cholestatic liver disease (Dabagh et al., 1999), its impact on hemodynamic complications is not clear. Clonidine has long been used as a blood pressure lowering agent. This drug stimulates α2-adrenoceptors in the brain stem and results in prolonged hypotension and bradycardia as a result of reduced sympathetic outflow from the central nervous
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system. Clonidine can also activate α2-adrenoceptors on vascular smooth muscle cells, which accounts for the transient vasoconstriction and hypertension noticed following the intravenous drug administration (Hoffman, 2001). We have recently reported that responsiveness of isolated peripheral tissues to clonidine is attenuated in cholestatic animals (Borhani et al., 2005; Demehri et al., 2003). In addition, it has been reported that clonidine administration reduces norepinephrine spillover and hepatic vein wedge pressure in cirrhotic patients, which suggest it as a potential therapy in advanced liver diseases (Esler and Kaye, 1998; Esler et al., 1992). However, the effect of clonidine administration on hemodynamic abnormalities of acute cholestasis has not yet been evaluated. Endogenous opioids and nitric oxide (NO) can modulate sympathetic nervous system. Their contributions to the development of cardiovascular abnormalities have been reported in several pathologic states (Chowdhary et al., 2002; Massion et al., 2003; Patel et al., 2001; Paton et al., 2002; Pugsley, 2002; Whalen et al., 1999). NO overproduction and accumulation of endogenous opioids have also been reported in the cholestatic liver disease and suggested to be involved in the development of bradycardia (Gaskari et al., 2002; Mani et al., 2002; Nahavandi et al., 2001), hypotension (Hajrasouliha et al., 2004, 2005), peripheral vasodilation (Namiranian et al., 2001), and hyporesponsiveness to adrenergic agents (Gaskari et al., 2002; Mani et al., 2002; Nahavandi et al., 2001; Namiranian et al., 2001). We have also recently reported that these compounds contribute to the abnormal responsiveness to clonidine in isolated tissues of cholestatic animals (Borhani et al., 2005; Demehri et al., 2003). However, there is no data of their role in the modulation of the central response to clonidine administration. The main goal of this study is to evaluate the effect of clonidine administration, and subsequent reduction in the central sympathetic tone, on hemodynamic parameters of 7day bile duct-ligated (BDL) rats, as a known model of acute cholestasis. Contributions of NO and endogenous opioid peptides are also examined in this study. 2. Materials and methods 2.1. Animal manipulation The animals were handled in accordance with the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” (NIH US publication 85-23 revised 1996). Male Sprague–Dawley rats weighing 200–250 g were used. Animals were housed in groups of 3–4 in a room controlled at 22 ± 1 °C and maintained in an alternating 12-h light/12-h dark cycles, and were allowed free access to food and water. Bile duct ligation was performed as described previously (Hajrasouliha et al., 2004, 2005). Laparotomy was performed under general anesthesia induced by an intraperitoneal injection of ketamine HCl (50 mg/kg; Gedoon Richter, Budapest, Hungary) and chlorpromazine HCl (10 mg/kg; Daroupakhsh, Tehran, Iran). In BDL rats, bile duct was identified and doubly ligated. In the sham-operated rats, the bile duct was identified, manipulated and
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one untied loose tie was left in place. Then, the abdominal wall was closed in two layers. One day after laparotomy, BDL rats showed manifestations of cholestasis (jaundice and dark urine). 2.2. Experimental groups The animals were randomly divided into five groups. Each group consisted of two subgroups of six to eight sham-operated or BDL rats. The first group of animals was treated with daily subcutaneous administration of isotonic sterile saline solution (1 ml/kg/day) and served as the control group. The role of NO overproduction was evaluated by acute intravenous (3 mg/kg) or chronic subcutaneous (3 mg/kg/d) injections of N(ω)-nitro-Larginine methyl ester (L-NAME, NO synthase inhibitor, Sigma, Louis, MO, USA), in the second and third groups. The last two groups of animals were treated by acute intravenous (20 mg/kg) or chronic subcutaneous (20 mg/kg/d) injections of naltrexone HCl (Iran Darou, Tehran, Iran). Acute drug administrations were performed 10 min before clonidine injection. Chronic drug administrations were performed for six consecutive days. The first dose was injected the day after surgery, and the last dose was injected 24 h before the experimental protocol, so little drug or drug action was expected to exist at the time of the experiment (Ebrahimi et al., 2005; Namiranian et al., 2001). Treatment regimens were selected based on our previous studies, shown to effectively reduce NO production, antagonize the effects of accumulated endogenous opioids and reverse complications of acute cholestasis (Borhani et al., 2005; Ebrahimi et al., 2005; Gaskari et al., 2002; Hajrasouliha et al., 2004, 2005; Mani et al., 2002; Nahavandi et al., 2001). 2.3. Experimental protocol Seven days after bile duct ligation or sham operation, animals were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg, Merck, Darmstadt, Germany). Through a ventral midline cervical incision, trachea was exposed and intubated with a cannula connected to a rodent ventilator. Rats were ventilated with room air. The right carotid artery was cannulated to measure arterial blood pressure using a pressure transducer (Pressure Transducer Model P-1000-A, Narco Biosystem, Houston, TX, USA). Mean arterial pressure was calculated as diastolic blood pressure plus one third of the differential pressure. The left jugular vein was cannulated for drug injection. After a steady state was achieved, acute administrations (drugs or vehicles) were performed. Clonidine (10 μg/kg, Iran Darou, Tehran, Iran) was injected intravenously, 10 min later. 2.4. Statistical analysis Baseline parameters are expressed as absolute values. Responses to clonidine are expressed as percentages of the baseline values. Data are presented as mean ± S.E.M and were analyzed by analysis of variance (ANOVA) followed by Tukey's HSD as post hoc test to compare the means. P < 0.05 was considered as the significance level between groups.
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3. Results 3.1. Baseline heart rate and mean arterial pressure Baseline heart rate and mean arterial pressure (Table 1) were significantly lower in BDL rats compared to sham-operated animals (P < 0.001 and P < 0.05, respectively). Acute L-NAME treatment had no significant effect on heart rate (from 445 ± 7 to 436 ± 9 beats/min, P > 0.05) and mean arterial pressure (from 109 ± 6 to 118 ± 5 mm Hg, P > 0.05) in sham-operated rats. On the other hand, it corrected baseline hypotension (from 89 ± 4 to 112 ± 4 mm Hg, P < 0.05), but not bradycardia (from 390 ± 7 to 371 ± 8 beats/min, P > 0.05), in BDL animals. Chronic L-NAME administration had no significant effect on hemodynamic parameters in sham-operated and BDL rats (Table 1). Acute naltrexone administration had also no significant effect on heart rate or mean arterial pressure (data not shown). Chronic naltrexone treatment, without any significant effect in sham-operated animals, corrected bradycardia (Table 1; P < 0.001) and hypotension (Table 1; P < 0.05) in BDL rats. 3.2. Response to clonidine administration Clonidine elicited a typical biphasic effect, with an initial transient hypertension followed by persistent hypotension (Fig. 1). The latter reached its plateau 30 min after clonidine administration, which was used for comparison among the groups. While the early hypertensive response (Fig. 2) was significantly blunted in BDL rats (P < 0.01), the late hypotension and bradycardia (Fig. 3) were exaggerated (P < 0.01). Acute L-NAME treatment accentuated the hypertensive phase (Fig. 2) in both sham-operated (P < 0.05) and BDL (P < 0.05) rats, but the difference between the two groups was preserved (P < 0.01). However, the central phase (Fig. 3) was attenuated to the same level in both sham-operated (P < 0.001) and BDL (P < 0.001) animals by this treatment. Chronic LNAME administration, without a significant effect on shamoperated rats, enhanced the hypertensive response (Fig. 2) in BDL rats (P < 0.05) to the level of sham-operated group. On the other hand, it accentuated hypotension and bradycardia (Fig. 3)
Fig. 1. Recordings of the effect of intravenous clonidine administration (10 μg/ kg) on arterial blood pressure (mm Hg) in anesthetized (A) sham-operated and (B) bile duct-ligated rats.
in sham-operated rats (P < 0.05), but had no significant effect on BDL animals. Acute or chronic naltrexone treatment did not result in a significant change in the hemodynamic responses to clonidine in BDL or sham-operated animals (Figs. 2 and 3). 4. Discussion 4.1. Altered baseline hemodynamics and response to clonidine in cholestasis In this study heart rate and mean arterial pressure were significantly lower in BDL rats compared to sham-operated animals, which is consistent with previous reports (Gaskari et al., 2002; Hajrasouliha et al., 2004, 2005; Mani et al., 2002;
Table 1 Baseline heart rate (beats/min) and mean arterial pressure (mm Hg) in shamoperated and BDL rats treated with normal saline, acute (3 mg/kg)/chronic (3 mg/kg/d) N(ω)-nitro-L-arginine methyl ester (L-NAME) or acute (20 mg/kg)/ chronic (20 mg/kg/d) naltrexone Treatment
Saline Acute L-NAME Chronic L-NAME Acute naltrexone Chronic naltrexone
Heart rate (beats/min)
Mean arterial pressure (mm Hg)
Sham
BDL
P-value Sham
441 ± 8 445 ± 7 437 ± 7 433 ± 7 443 ± 9
385 ± 6 390 ± 7 374 ± 8 379 ± 6 447 ± 8a
<0.001 <0.001 <0.001 <0.001 >0.05
BDL
111 ± 4 91 ± 5 109 ± 6 89 ± 4 117 ± 5 103 ± 6 113 ± 6 87 ± 4 114 ± 5 110 ± 6b
BDL = bile duct-ligated, L-NAME = N(ω)-nitro-L-arginine methyl ester. a P < 0.001 compared to the saline-treated BDL group. b P < 0.05 compared to the saline-treated BDL group.
P-value <0.05 <0.05 >0.05 <0.05 >0.05
Fig. 2. The early hypertensive effect of intravenous clonidine administration (10 μg/kg) in sham-operated and bile duct-ligated (BDL) rats treated with normal saline, acute (3 mg/kg)/chronic (3 mg/kg/d) N(ω)-nitro-L-arginine methyl ester (L-NAME) or acute (20 mg/kg)/chronic (20 mg/kg/d) naltrexone. ⁎P < 0.05, ⁎⁎P < 0.01 compared to corresponding groups.
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(Sannajust et al., 1989), this finding along with the evidence of sympathetic overactivity, such as elevated levels of norepinephrine (Dabagh et al., 1999), suggests that cholestatic subjects are more dependent on the central sympathetic tone for the maintenance of hemodynamic stability. This could be a compensatory response to the peripheral cardiovascular hyporesponsiveness to adrenergic agents, a phenomenon which has also been described in advanced liver diseases and cirrhosis (Esler and Kaye, 1998; Esler et al., 1992). 4.2. The roles of NO
Fig. 3. The central hemodynamic responses, (A) hypotension and (B) bradycardia, to intravenous clonidine administration (10 μg/kg) in shamoperated and bile duct-ligated (BDL) rats treated with normal saline, acute (3 mg/kg)/chronic (3 mg/kg/d) N(ω)-nitro-L-arginine methyl ester (L-NAME) or acute (20 mg/kg)/chronic (20 mg/kg/d) naltrexone. ⁎P < 0.05, ⁎⁎P < 0.01, ⁎⁎⁎P < 0.001 compared to corresponding groups.
Nahavandi et al., 2001). In our recent studies we have shown that despite baseline in vivo bradycardia, spontaneous beating rate of isolated atria of BDL rats is not different from shamoperated animals. However, chronotropic responsiveness to adrenergic stimulation is significantly blunted in BDL rats (Gaskari et al., 2002; Mani et al., 2002; Nahavandi et al., 2001). Studies on papillary muscles (Ebrahimi et al., 2005) and vascular beds (Namiranian et al., 2001) have also provided similar results, which support the role of peripheral hyporesponsiveness to adrenergic stimulation in the development of the cardiovascular complications of cholestasis. Intravenous administration of clonidine causes an acute rise in the blood pressure, apparently because of activation of postsynaptic α2-adrenoceptors in vascular smooth muscles (Hoffman, 2001). This transient vasoconstriction is followed by more prolonged hypotension and bradycardia that result from the decreased central outflow of impulses in the central sympathetic nervous system. This phase seems to result from activation of α2-adrenoceptors in the lower brain stem region, possibly in the nucleus tractus solitarius (Hoffman, 2001). According to the findings of this study, the transient hypertensive phase was attenuated in BDL rats. This finding can be explained based on the reports of the hyporesponsiveness of peripheral tissues to clonidine (Borhani et al., 2005; Demehri et al., 2003), in cholestasis. On the other hand, the prolonged central effects of clonidine were significantly exaggerated in BDL rats. Since the central effects of α2-adrenoceptor agonists appear to be influenced by the pre-existing sympathetic tone
NO contributes to the regulation of the sympathetic nervous system, both centrally and peripherally (Chowdhary et al., 2002; Massion et al., 2003; Patel et al., 2001; Paton et al., 2002; Whalen et al., 1999). Its involvement in the development of cardiovascular complications has also been reported in several pathological states (Chowdhary et al., 2002; Massion et al., 2003), including cholestasis (Demehri et al., 2003; Hajrasouliha et al., 2004, 2005; Mani et al., 2002; Nahavandi et al., 2001; Namiranian et al., 2001). Acute, but not chronic, administration of L-NAME, at the dosage used in the present study, was able to correct hypotension, but not bradycardia, in BDL rats. Our findings demonstrate that acute L-NAME administration accentuates the early hypertensive response to clonidine administration in both sham-operated and BDL rats. However, the difference between the two groups was still preserved following acute L-NAME treatment. On the other hand, this treatment blunted the late central response to clonidine in both sham-operated and BDL animals to the same level. It is thought that the acute hemodynamic changes following L-NAME administration is mostly the result of blockade of the tonic peripheral NO production (Sander et al., 1997). Since clonidineinduced vasoconstriction is modulated by endothelial NO production (Figueroa et al., 2001), it could be postulated that the exaggerated early phase following acute L-NAME administration is due to the blockade of this peripheral modulatory mechanism. Data regarding the effects of acute administration of NO synthase inhibitors on the central hypotensive phase are controversial. While Soares de Moura et al. (2000) have shown that peripheral administration of NO synthase inhibitors blocks the hypotensive response to clonidine, Sy et al. (2001) reported that their central administration had no effect on this phase. The increased central sympathetic tone has been suggested to be the dominant mechanism of hemodynamic changes following chronic inhibition of NO synthesis (Sander et al., 1997). Despite the trend towards increase in mean arterial pressure, chronic L-NAME was not able to induce significant hypertension in either sham-operated or BDL animals, as expected by the increased sympathetic tone. However, our finding of the exaggerated central effects of clonidine in chronic L-NAME treated rats is an evidence for the increased dependence of the hemodynamic parameters on the central sympathetic tone even without the development of hypertension. It should be mentioned that this response was blunted in BDL animals compared to sham-operated rats, which might be due to the higher basal sympathetic tone (Dabagh et al., 1999).
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4.3. The roles of endogenous opioids Endogenous opioid peptides are involved in the regulation of the cardiovascular system through both central and peripheral receptors (Pugsley, 2002). They have been shown to reduce the heart rate, cardiac output, peripheral vascular resistance (Champion and Kadowitz, 1998) and modulate the autonomic nervous system (Pugsley, 2002). Abnormalities of the opioid system have been reported in several pathophysiological conditions in both human and animal models of cardiovascular diseases (Pugsley, 2002). The role of accumulation of endogenous opioid peptides in the development of the cardiovascular complications of the cholestatic liver disease has also been extensively investigated (Demehri et al., 2003; Gaskari et al., 2002; Hajrasouliha et al., 2004, 2005; Namiranian et al., 2001). The precise reason for the increased opioid activity is not yet completely understood, but it is likely that both overproduction of endogenous opioids and protection of these peptides from degradation may contribute to the elevation of total opioid activity in cholestasis (Bergasa et al., 1992; Swain et al., 1992). The interaction of opioids and central α2-adrenoceptor agonists has been examined previously, which suggest the existence of an opioidergic component in the hemodynamic effects of central α2-adrenoceptor agonists in spontaneous hypertensive rats, but not in normotensive ones (Mastrianni and Ingenito, 1987). According to the results of the present study while the chronic availability of endogenous opioids is involved in the development of bradycardia and hypotension of BDL rats, naltrexone administration had no effect on the clonidine-induced hemodynamic changes. Therefore, other mechanisms, such as modulation of the parasympathetic or the peripheral sympathetic systems might be involved in the opioid-mediated hemodynamic alterations in BDL rats. 4.4. Conclusion In summary, this study confirms that 7-day BDL is associated with bradycardia and hypotension in the rat model of cholestasis. It also demonstrates that while the peripheral phase of clonidine response is blunted in BDL rats, the central inhibitory effect of clonidine on the sympathetic nervous system results in more profound hypotension and bradycardia in cholestasis. Therefore, it seems that cholestatic rats are more dependent on the central sympathetic tone for the maintenance of the hemodynamic stability. This study also provides evidence for the contribution of NO to the development of these abnormalities. References Bergasa, N.V., Rothman, R.B., Vergalla, J., Xu, H., Jones, E.A., Swain, M.J., 1992. Central mu opioid receptors are down regulated in a rat model of cholestasis. J. Hepatol. 15, 220–224. Bomzon, A., Weinbroum, A., Kamenetz, L., 1990. Systemic hypotension and pressor responsiveness in cholestasis: a study in 3-day bile duct ligated rats. J. Hepatol. 11, 70–76.
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