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Effects of ceftriaxone sodium on in vitro gallbladder contractility in guinea pigs1
Journal of Surgical Research 122, 157–161 (2004) doi:10.1016/j.jss.2004.05.020
Effects of Ceftriaxone Sodium on in Vitro Gallbladder Contractility in Guinea Pigs 1 Mehmet Arpacık, M.D.,* Canan Ceran, M.D.,*,2 Tijen Kaya, Ph.D.,† Barıs¸ Karadas, M.D,† Bülent Sarac, M.D.,† and Gökhan Koyluog˘lu, M.D.* *Department of Pediatric Surgery and †Department of Pharmacology, Cumhuriyet University School of Medicine, Sivas, Turkey Submitted for publication December 16, 2003
INTRODUCTION Background/Purpose. It has been reported that ceftriaxone may induce the formation of gallstones. Changes of gallbladder motility may play a role in this phenomenon. The present study was designed to analyze the gallbladder contractility of ceftriaxone sodium-treated guinea pigs in response to different agonists. Materials and methods. Twenty adult guinea pigs were randomly divided into two groups. Ten guinea pigs were treated with ceftriaxone sodium (100 mg/kg/ day) for 10 days, whereas the remaining 10 served as the control group, receiving 1 ml of distilled water during 10 days as placebo. By the end of the experimental period the animals were sacrificed and the gallbladders were removed. The responses to KCl, papaverine, sodium nitroprusside, carbachol, and histamine on gallbladder strips from control and experimental groups were recorded and analyzed. Results. There was no significant difference between the responsiveness to KCl, papaverine, and sodium nitroprusside on tissues isolated from experimental and control groups. Comparison of the two groups revealed that the maximum responses (E max) to carbachol and histamine were significantly reduced in the experimental group, without any change in the pD 2 values. Conclusion. These data indicate that, after ceftriaxone sodium therapy, the decreased maximum contractile response to carbachol and histamine may contribute to the formation of gallstones. © 2004 Elsevier Inc. All rights reserved.
Key Words: gallbladder; ceftriaxone; pseudolithiasis.
Ceftriaxone (CTX), a potent semisynthetic thirdgeneration cephalosporin, has excellent antimicrobial activity against gram-positive and gram-negative bacteria [1]. It is widely used in serious bacterial infections. It has been reported that CTX can induce reversible gallbladder sludge, which can mimic true cholelithiasis in some cases [2, 3]. Clinical and experimental studies were conducted to determine the incidence and mechanism of this phenomenon. It was proposed that situations affecting gallbladder contraction, such as fasting, might be related to this phenomenon [4]. During treatment with CTX sodium, reduced gallbladder motility to various agonists may play a role in the formation of gallstones. The aim of this study was to determine the changes in gallbladder contractile responses to different agonists after CTX sodium administration in guinea pigs. MATERIALS AND METHODS Twenty male guinea pigs each weighing approximately 500 g were maintained in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals. The study was approved by the Ethical Committee of Animal Research of Cumhuriyet University. Guinea pigs were divided randomly into two groups. All animals were fed with same standard chow in two groups. The experimental group was treated with CTX sodium (100 mg/kg/day) administered via the intramuscular route for ten days, whereas the control group received 1 ml of distilled water as placebo (n ⫽ 10 for each group). Guinea pigs in control and CTX groups were fed with standard diet. Tissue samples were taken on 10th days.
Tissue Preparation 1
Supported by the research funding of the Cumhuriyet University. To whom correspondence and reprint requests should be addressed at Cumhuriyet Universitesi Tıp Fakültesi Cocuk Cerrahisi AD. 58140 Sivas, Turkey. E-mail: [email protected]. 2
The subjects were sacrificed by cervical dislocation, followed by a midline abdominal incision to remove the gallbladder. Gallbladder specimens were immersed in a previously aerated (95% O 2 and 5% CO 2) Krebs’ bicarbonate solution (composition in mM: NaCl, 120;
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KCl, 4.6; CaCl 2, 2.5; MgCl 2, 1.2; NaHCO 3, 22; NaH 2PO 4 and glucose 11.5). With a longitudinal incision, two full-thickness muscle strips (2 ⫻ 10 mm) containing mucosa, smooth muscle, and serosa were obtained from each gallbladder specimen. The muscle strips were placed longitudinally in a muscle bath filled with preaerated 10 ml Krebs’ bicarbonate solution at 37°C. The upper end of the preparation was tied to an isometric transducer (Grass FT 03, Quincy, MA) and preloaded with 1–1.5 g. Tissue was allowed to equilibrate for 30 min until a stable baseline was attained.
Isometric Measurements At the beginning of each experiment, 80 mM KCl was added to the tissue bath and the contraction was considered as the reference response. At the end of each experiment, the contractile response to 80 mM KCl was remeasured to verify that the contractile mechanisms are intact. After the first KCl response, tissues were washed twice and after a period of 30 min the concentration–response curves to papaverine (10 ⫺8 to 10 ⫺4 mol/l), sodium nitroprusside (10⫺8 to 10 ⫺4 mol/l), carbachol (10 ⫺8 to 10 ⫺4 mol/l) and histamine (10⫺8 to 10 ⫺4 mol/l) were obtained cumulatively. Isometric tension was recorded on a Grass model 79 E polygraph. The relaxant effect of papaverine, and the contractile effects of sodium nitroprusside, carbachol, and histamine were evaluated. The relaxation responses to papaverine and contraction responses to sodium nitroprusside, carbachol, and histamine were calculated as percentage in relation to the contraction caused by KCl (80 mM). To evaluate the effects of agonists, the maximum responses (Emax) and pD 2 values (i.e., the negative logarithm of the concentration for the half-maximal response, ED50) were calculated. The pD2 values were used instead of ED 50, since the results could be more clearly expressed mathematically with pD 2 values. The concentration response data obtained in each experiment were plotted as the response/concentration against the response, producing a sigmoid curve in each experiment, as predicted from the Scatchard equation for drug-receptor interaction. pD2 values (apparent agonist affinity constants) were calculated from each agonist concentration-response curve by linear regression of the linear median part of the sigmoid curve and taken as a measure of the sensitivity of the tissues to each agonist.
Drugs The chemicals used in the current experiments, papaverine, sodium nitroprusside, carbachol, and histamine, were from Sigma (St. Louis, MO). All chemicals were dissolved in distilled water. The volume that added to the tissue bath never exceeded 5% of its total volume. All solutions were prepared just prior to its use.
Statistical Analysis All data were expressed as mean ⫾ SEM. Data were analyzed by repeated measured analysis of variance and groups were compared statistically using general linear models of analysis of variance followed by Newman Keuls test. Differences were considered to be significant when P ⬍ 0.05.
RESULTS
Macroscopically all animals developed biliary sludge in the CTX group, whereas bile samples were normal in the control group (Fig. 1). The contractions elicited by 80 mM KCl (reference response) were not significantly different in both groups (3.12 ⫾ 0.16 g and 2.96 ⫾ 0.18 g, respectively; P ⬎ 0.05; Fig. 2). In the control and CTX groups, induction of papaverine (10 ⫺8 to 10 ⫺4 M) produced concentration-dependent relaxation in gall-
FIG. 1. Appearance of the gallbladder in control group (A) and CTX group (B). The gallbladder seems normal in the (A) and biliary lithiasis is clearly demonstrated in the (B) macroscopically.
bladder strips. The relaxation response elicited by papaverine was similar in both groups and there was no significant difference between the E max and pD 2 values (P ⬎ 0.05; Fig. 3, Table 1). The cumulative addition of sodium nitroprusside (10 ⫺8 to 10 ⫺4 M) produced concentration-dependent contractions in both groups. The concentration–response curves for sodium nitroprusside was similar in control and CTX groups and there were no significant changes in the E max and pD 2 values (P ⬎ 0.05; Fig. 4, Table 1). Carbachol (10 ⫺8 to 10 ⫺4 M) and histamine (10 ⫺8 to 10 ⫺4 M) elicited concentration-dependent contraction in gallbladder strips in control and CTX groups (Figs. 5 and 6). The concentration–response curves for carbachol and histamine were shifted to the right, with significantly lower E max values in CTX group in comparison to the control group (P ⬍ 0.05). However, there
ARPACIK ET AL.: GALLBLADDER CONTRACTILITY OF CTX-TREATED GUINEA PIGS
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TABLE 1 The Maximum Contractile Response (E max, % of KCl) and pD 2 Values on Exposure to Papaverine, Sodium Nitroprusside, Carbachol, and Histamine in Gallbladder Strips Isolated from Control and CTX Groups
FIG. 2. Mean values obtained when 80 mM KCl was added on gallbladder strips isolated from control group and CTX group. Data are expressed as the means ⫾ SEM of 10 experiments.
Papaverine E max pD 2 Sodium nitroprusside E max pD 2 Carbachol E max pD 2 Histamine E max pD 2
Control group
CTX group
38.52 ⫾ 15.30 5.26 ⫾ 0.10
42.67 ⫾ 13.17 5.18 ⫾ 0.05
77.63 ⫾ 15.20 6.20 ⫾ 0.18
72.35 ⫾ 13.71 6.08 ⫾ 0.14
155.01 ⫾ 27.13 5.98 ⫾ 0.12
120.63 ⫾ 23.55* 5.94 ⫾ 0.16
149.44 ⫾ 20.12 5.52 ⫾ 0.08
117.15 ⫾ 23.62* 5.48 ⫾ 0.03
* P ⬍ 0.05, significantly different compared with the control group.
was no significance among the corresponding pD 2 values (Figs. 5 and 6; Table 1).
Postmortem studies performed in dogs and baboons treated with CTX have shown precipitations of the calcium salt of CTX in the gallbladder after high dose and prolonged administration [5]. Shaad et al. [6] described a case which had sonographically demonstrated precipitations in the gallbladder during CTX therapy. They also reported that 17 of 37 children treated with CTX for serious infections developed transient biliary concrements and ultrasonographic abnormalities, whereas these symptoms resolved gradually within 2 to 63 days after cessation of the CTX treat-
ment [6]. They were unable to find any obvious pathogenic factor related to this condition [2]. Analysis of gallbladder sediments of two adult patients who underwent cholecystectomy for symptomatic CTX associated biliary pseudolithiasis revealed only small amounts of cholesterol and bilirubinate and a trace amount of CTX but a high content of other calcium salts [7]. There are some reports addressing to the pathogenesis of CTX-related pseudolithiasis. The effect of CTX on bile flow and biliary electrolyte secretion was evaluated and it has been shown that, even at high doses, CTX had no effects on the bile flow and the biliary electrolyte secretion in guinea pigs [8]. Shiffman et al. [4] performed an in vitro study and evaluated possible interactions between calcium, bile
FIG. 3. Concentration–response curves of papaverine in isolated guinea pig gallbladder strips precontracted with 80 mM KCl. Each point is expressed as a percentage of the contraction induced by KCl. Data are expressed as the means ⫾ SEM of 10 experiments.
FIG. 4. Concentration–response curves of sodium nitroprusside in isolated guinea pig gallbladder strips. Each point is expressed as a percentage of the contraction induced by 80 mM KCl. Data are expressed as the means ⫾ SEM of 10 experiments.
DISCUSSION
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FIG. 5. Concentration–response curves of carbachol in isolated guinea pig gallbladder strips. Each point is expressed as a percentage of the contraction induced by 80 mM KCl. Data are expressed as the means ⫾ SEM of 10 experiments. * P ⬍ 0.05 denotes significant difference between groups.
salts, and CTX. They speculated that the development of CTX induced biliary sludge is a result of a solubility problem occurring in the patients receiving high-dose treatment (ⱖ2 g). They also predicted that CTXinduced biliary sludge would not develop at low doses in patients with normal gallbladder motility. However, severely ill patients who are not eating and/or receiving total parenteral nutrition, therefore having little stimulus for gallbladder contraction, bear a great risk of developing pseudolithiasis [4]. Kong et al. [9] reported that fasting and age (older than 24 months) were probably the significant risk factors associated with pseudolithiasis and these findings were compatible with a previous study [4]. In contrast, in a previous study, we have observed that the incidence of pseudolithiasis in pediatric surgical patients, who were fasted, was not different from the patients of other age groups [10]. We presumed that, rather than fasting, CTX itself may induce pseudolithiasis by altering the gallbladder contractility. Results of the present study clearly demonstrate that the receptor-mediated contractile responses of gallbladder smooth muscle to different agonists change after CTX sodium therapy in guinea pigs. In our experiments, we compared the relaxant effect of papaverine and the contractile effects of sodium nitroprusside, carbachol and histamine on the guinea pig’s gallbladder strips of CTX-treated and control groups. Comparison of the pD 2 (-log 10 EC 50) values of the control and CTX-treated groups obtained in response to the above agents did not show any significant difference (P ⬎ 0.05). This indicates that the actual EC 50 values (i.e., concentration resulting in 50% of maximal effect) for used drugs are not significantly different in the CTX group compared with controls. However, E max values elicited by carbachol and histamine were significantly diminished in CTX-treated
group, whereas these values did not show any significance with KCl, papaverine, and sodium nitroprusside. The lower E max values obtained with carbachol and histamine in CTX-treated group may be related to an alteration in the regulation of receptor/postreceptor excitation-contraction coupling. It is well known that the KCl-induced contraction in smooth muscle is caused by an increase in Ca 2⫹ influx through voltage operated Ca 2⫹ channels [11]. Contractions of gallbladder strips elicited by SNP depend on extracellular calcium entry through voltage-operated calcium channels [12]. Papaverine reduces the intracellular Ca 2⫹ concentration by decreasing the calcium gradient across the cell membrane, inhibiting the cAMP phosphodiesterase enzyme, decreasing the uptake of adenosine, and produces smooth muscle relaxation [13]. We used KCl, papaverine and sodium nitroprusside responses to find out the differences due to nonreceptor-mediated mechanisms. The pD 2 and E max values for KCl, papaverine, and sodium nitroprusside in gallbladder smooth muscle in both groups did not show any significant difference, suggesting that CTX sodium does not affect nonreceptor-dependent mechanisms. This finding also rules out the possibility of functional and morphological damage to gallbladder smooth muscle by CTX sodium administration. Acetylcholine and histamine are the classic excitatory neurotransmitters in the gallbladder of the guinea pig. Carbachol is a muscarinic agonist that mimics the neurotransmitter acetylcholine in the gallbladder smooth muscle [14]. Histamine causes depolarizationassociated contraction of gallbladder smooth muscle via the activation of both H 1 (excitatory) and H 2 (inhibitory) receptors [15]. In our study, we used carbachol and histamine responses to classify whether observed differences are due to receptor-mediated mechanism.
FIG. 6. Concentration–response curves of histamine in isolated guinea pig gallbladder strips. Each point is expressed as a percentage of the contraction induced by 80 mM KCl. Data are expressed as the means ⫾ SEM of ten experiments. * P ⬍ 0.05 denotes significant difference between groups.
ARPACIK ET AL.: GALLBLADDER CONTRACTILITY OF CTX-TREATED GUINEA PIGS
We presumed that if receptor-meditated mechanisms are responsible in the formation of gallstones after the administration of CTX sodium that contractile responses to carbachol and histamine would change. Our results demonstrated that E max values for carbachol and histamine were significantly decreased in the CTX-treated group in comparison with the control group, with no significant difference of pD 2 values. This decrement indicates a post-receptor defect rather than decreasing affinity of receptors. Results of this study suggests that decreased maximum contractile response to different agonists after ceftriaxone sodium therapy may contribute to the formation of gallstones. REFERENCES 1.
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Richards, D. M., Heel, R. C., Brogden, R. N., Speight, T. M., and Avery, G. S. Ceftriaxone, a review of its antibacterial activity, pharmacological properties and therapeutic use. Drugs 27: 469, 1984. Schaad, U. B., Wedgwood-Krucko, J., and Tschaeppeler, H. Reversible ceftriaxone-associated biliary pseudolithiasis in children. Lancet 17: 1411, 1988. Cometta, A., Gallot-Lavallee-Villars, S., Iten, A., Cantoni, L., Anderegg, A., Gonvers, J. J., and Glauser, M. P. Incidence of gallbladder lithiasis after ceftriaxone treatment. J. Antimicrob. Chemother. 25: 689, 1990. Shiffman, M. L., Keith, F. B., and Moore, E. W. Pathogenesis of ceftriaxone-associated biliary sludge. Gastroenterology 99: 1772, 1990. Teelman, K., Scharer, K., and Udaka, K. Experimentelle toxikologie von ceftriaxone (Ro 13-9904, Rocephin R). Ceftriaxone ein neus parenterales cephalosporin. Proceedings of the Hahnenklee Symposium, September 1981. In: Grieshaber R, ed. Basel, Switzerland: Editiones Roche, 91-111, 1982.
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Schaad, U. B., Tschaeppeler, H., and Lentze, M. J. Transient formation of precipitations in the gallbladder associated with ceftriaxone therapy. Pediatr. Infect. Dis. 5: 708, 1986.
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Anonymous: Report of cases of positive gallbladder sonograms in patients receiving ceftriaxone (Rocephin R). Hoffman La Roche: Scientific update; 1987.
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Purdum P. P. III, Shiffman, M. L., and Moore, E. W. In vivo studies of biliary ceftriaxone excretion and solubility in guinea pig hepatic bile. J. Lab. Clin. Med. 120: 604, 1992.
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Kong, M. S., and Chen, C. Y. Risk factors leading to ceftriaxoneassociated biliary pseudolithiasis in children. Changgeng Yi Xue Za Zhi 19: 50, 1996.
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Ceran, C., Oztoprak, I., and Koyluoglu, G. The incidence and the clinical importance of ceftriaxone induced pseudolithiasis. Presented at the 19th annual meeting of the Turkish Association of Pediatric Surgeons, Antalya, TR, 1– 4 Oct. 2000.
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Cejalvo, D., Calvo, M. A., Lloris, J. M., Cartijo, J., and Morcillo, E. J. Effects of Ca2⫹ channel antagonists in guinea-pig normal and skinned gallbladder. Eur. J. Pharmacol. 234: 23, 1993.
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Alcon, S., Morales, S., Camello, P. J., Hemming, J. M., Jennings, L., Mawe, G. M., and Pozo, M. J. A. redox-based mechanism for the contractile and relaxing effects of NO in the guinea-pig gallbladder. J. Physiol. 532: 793, 2001.
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De Santis, D., Marrazzo, R., Losasso, C., Loffreda, A., De Paola, C., De Novellis, V., Molinario, L., Schiariti, F., and Marmo, E. Pharmacodynamic profile of fenoverine, a novel modulator of smooth muscle motility. Drugs Exp. Clin. Res. 15: 37, 1989.
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Stengel, P. W., and Cohen, M. L. Muscarinic receptor knockout mice: role of muscarinic acetylcholine receptors M(2), M(3), and M(4) in carbamylcholine-induced gallbladder contractility. J. Pharmacol. Exp. Ther. 301: 643, 2002.
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Hemming, J. M., Guarraci, F. A., Firth, T. A., Jennings, L. J., Nelson, M. T., and Mawe, G. M. Actions of histamine on muscle and ganglia of the guinea pig gallbladder. Am. J. Physiol. Gastrointest. Liver Physiol. 279: G622, 2000.
Effects of Ceftriaxone Sodium on in Vitro Gallbladder Contractility in Guinea Pigs 1 Mehmet Arpacık, M.D.,* Canan Ceran, M.D.,*,2 Tijen Kaya, Ph.D.,† Barıs¸ Karadas, M.D,† Bülent Sarac, M.D.,† and Gökhan Koyluog˘lu, M.D.* *Department of Pediatric Surgery and †Department of Pharmacology, Cumhuriyet University School of Medicine, Sivas, Turkey Submitted for publication December 16, 2003
INTRODUCTION Background/Purpose. It has been reported that ceftriaxone may induce the formation of gallstones. Changes of gallbladder motility may play a role in this phenomenon. The present study was designed to analyze the gallbladder contractility of ceftriaxone sodium-treated guinea pigs in response to different agonists. Materials and methods. Twenty adult guinea pigs were randomly divided into two groups. Ten guinea pigs were treated with ceftriaxone sodium (100 mg/kg/ day) for 10 days, whereas the remaining 10 served as the control group, receiving 1 ml of distilled water during 10 days as placebo. By the end of the experimental period the animals were sacrificed and the gallbladders were removed. The responses to KCl, papaverine, sodium nitroprusside, carbachol, and histamine on gallbladder strips from control and experimental groups were recorded and analyzed. Results. There was no significant difference between the responsiveness to KCl, papaverine, and sodium nitroprusside on tissues isolated from experimental and control groups. Comparison of the two groups revealed that the maximum responses (E max) to carbachol and histamine were significantly reduced in the experimental group, without any change in the pD 2 values. Conclusion. These data indicate that, after ceftriaxone sodium therapy, the decreased maximum contractile response to carbachol and histamine may contribute to the formation of gallstones. © 2004 Elsevier Inc. All rights reserved.
Key Words: gallbladder; ceftriaxone; pseudolithiasis.
Ceftriaxone (CTX), a potent semisynthetic thirdgeneration cephalosporin, has excellent antimicrobial activity against gram-positive and gram-negative bacteria [1]. It is widely used in serious bacterial infections. It has been reported that CTX can induce reversible gallbladder sludge, which can mimic true cholelithiasis in some cases [2, 3]. Clinical and experimental studies were conducted to determine the incidence and mechanism of this phenomenon. It was proposed that situations affecting gallbladder contraction, such as fasting, might be related to this phenomenon [4]. During treatment with CTX sodium, reduced gallbladder motility to various agonists may play a role in the formation of gallstones. The aim of this study was to determine the changes in gallbladder contractile responses to different agonists after CTX sodium administration in guinea pigs. MATERIALS AND METHODS Twenty male guinea pigs each weighing approximately 500 g were maintained in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals. The study was approved by the Ethical Committee of Animal Research of Cumhuriyet University. Guinea pigs were divided randomly into two groups. All animals were fed with same standard chow in two groups. The experimental group was treated with CTX sodium (100 mg/kg/day) administered via the intramuscular route for ten days, whereas the control group received 1 ml of distilled water as placebo (n ⫽ 10 for each group). Guinea pigs in control and CTX groups were fed with standard diet. Tissue samples were taken on 10th days.
Tissue Preparation 1
Supported by the research funding of the Cumhuriyet University. To whom correspondence and reprint requests should be addressed at Cumhuriyet Universitesi Tıp Fakültesi Cocuk Cerrahisi AD. 58140 Sivas, Turkey. E-mail: [email protected]. 2
The subjects were sacrificed by cervical dislocation, followed by a midline abdominal incision to remove the gallbladder. Gallbladder specimens were immersed in a previously aerated (95% O 2 and 5% CO 2) Krebs’ bicarbonate solution (composition in mM: NaCl, 120;
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KCl, 4.6; CaCl 2, 2.5; MgCl 2, 1.2; NaHCO 3, 22; NaH 2PO 4 and glucose 11.5). With a longitudinal incision, two full-thickness muscle strips (2 ⫻ 10 mm) containing mucosa, smooth muscle, and serosa were obtained from each gallbladder specimen. The muscle strips were placed longitudinally in a muscle bath filled with preaerated 10 ml Krebs’ bicarbonate solution at 37°C. The upper end of the preparation was tied to an isometric transducer (Grass FT 03, Quincy, MA) and preloaded with 1–1.5 g. Tissue was allowed to equilibrate for 30 min until a stable baseline was attained.
Isometric Measurements At the beginning of each experiment, 80 mM KCl was added to the tissue bath and the contraction was considered as the reference response. At the end of each experiment, the contractile response to 80 mM KCl was remeasured to verify that the contractile mechanisms are intact. After the first KCl response, tissues were washed twice and after a period of 30 min the concentration–response curves to papaverine (10 ⫺8 to 10 ⫺4 mol/l), sodium nitroprusside (10⫺8 to 10 ⫺4 mol/l), carbachol (10 ⫺8 to 10 ⫺4 mol/l) and histamine (10⫺8 to 10 ⫺4 mol/l) were obtained cumulatively. Isometric tension was recorded on a Grass model 79 E polygraph. The relaxant effect of papaverine, and the contractile effects of sodium nitroprusside, carbachol, and histamine were evaluated. The relaxation responses to papaverine and contraction responses to sodium nitroprusside, carbachol, and histamine were calculated as percentage in relation to the contraction caused by KCl (80 mM). To evaluate the effects of agonists, the maximum responses (Emax) and pD 2 values (i.e., the negative logarithm of the concentration for the half-maximal response, ED50) were calculated. The pD2 values were used instead of ED 50, since the results could be more clearly expressed mathematically with pD 2 values. The concentration response data obtained in each experiment were plotted as the response/concentration against the response, producing a sigmoid curve in each experiment, as predicted from the Scatchard equation for drug-receptor interaction. pD2 values (apparent agonist affinity constants) were calculated from each agonist concentration-response curve by linear regression of the linear median part of the sigmoid curve and taken as a measure of the sensitivity of the tissues to each agonist.
Drugs The chemicals used in the current experiments, papaverine, sodium nitroprusside, carbachol, and histamine, were from Sigma (St. Louis, MO). All chemicals were dissolved in distilled water. The volume that added to the tissue bath never exceeded 5% of its total volume. All solutions were prepared just prior to its use.
Statistical Analysis All data were expressed as mean ⫾ SEM. Data were analyzed by repeated measured analysis of variance and groups were compared statistically using general linear models of analysis of variance followed by Newman Keuls test. Differences were considered to be significant when P ⬍ 0.05.
RESULTS
Macroscopically all animals developed biliary sludge in the CTX group, whereas bile samples were normal in the control group (Fig. 1). The contractions elicited by 80 mM KCl (reference response) were not significantly different in both groups (3.12 ⫾ 0.16 g and 2.96 ⫾ 0.18 g, respectively; P ⬎ 0.05; Fig. 2). In the control and CTX groups, induction of papaverine (10 ⫺8 to 10 ⫺4 M) produced concentration-dependent relaxation in gall-
FIG. 1. Appearance of the gallbladder in control group (A) and CTX group (B). The gallbladder seems normal in the (A) and biliary lithiasis is clearly demonstrated in the (B) macroscopically.
bladder strips. The relaxation response elicited by papaverine was similar in both groups and there was no significant difference between the E max and pD 2 values (P ⬎ 0.05; Fig. 3, Table 1). The cumulative addition of sodium nitroprusside (10 ⫺8 to 10 ⫺4 M) produced concentration-dependent contractions in both groups. The concentration–response curves for sodium nitroprusside was similar in control and CTX groups and there were no significant changes in the E max and pD 2 values (P ⬎ 0.05; Fig. 4, Table 1). Carbachol (10 ⫺8 to 10 ⫺4 M) and histamine (10 ⫺8 to 10 ⫺4 M) elicited concentration-dependent contraction in gallbladder strips in control and CTX groups (Figs. 5 and 6). The concentration–response curves for carbachol and histamine were shifted to the right, with significantly lower E max values in CTX group in comparison to the control group (P ⬍ 0.05). However, there
ARPACIK ET AL.: GALLBLADDER CONTRACTILITY OF CTX-TREATED GUINEA PIGS
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TABLE 1 The Maximum Contractile Response (E max, % of KCl) and pD 2 Values on Exposure to Papaverine, Sodium Nitroprusside, Carbachol, and Histamine in Gallbladder Strips Isolated from Control and CTX Groups
FIG. 2. Mean values obtained when 80 mM KCl was added on gallbladder strips isolated from control group and CTX group. Data are expressed as the means ⫾ SEM of 10 experiments.
Papaverine E max pD 2 Sodium nitroprusside E max pD 2 Carbachol E max pD 2 Histamine E max pD 2
Control group
CTX group
38.52 ⫾ 15.30 5.26 ⫾ 0.10
42.67 ⫾ 13.17 5.18 ⫾ 0.05
77.63 ⫾ 15.20 6.20 ⫾ 0.18
72.35 ⫾ 13.71 6.08 ⫾ 0.14
155.01 ⫾ 27.13 5.98 ⫾ 0.12
120.63 ⫾ 23.55* 5.94 ⫾ 0.16
149.44 ⫾ 20.12 5.52 ⫾ 0.08
117.15 ⫾ 23.62* 5.48 ⫾ 0.03
* P ⬍ 0.05, significantly different compared with the control group.
was no significance among the corresponding pD 2 values (Figs. 5 and 6; Table 1).
Postmortem studies performed in dogs and baboons treated with CTX have shown precipitations of the calcium salt of CTX in the gallbladder after high dose and prolonged administration [5]. Shaad et al. [6] described a case which had sonographically demonstrated precipitations in the gallbladder during CTX therapy. They also reported that 17 of 37 children treated with CTX for serious infections developed transient biliary concrements and ultrasonographic abnormalities, whereas these symptoms resolved gradually within 2 to 63 days after cessation of the CTX treat-
ment [6]. They were unable to find any obvious pathogenic factor related to this condition [2]. Analysis of gallbladder sediments of two adult patients who underwent cholecystectomy for symptomatic CTX associated biliary pseudolithiasis revealed only small amounts of cholesterol and bilirubinate and a trace amount of CTX but a high content of other calcium salts [7]. There are some reports addressing to the pathogenesis of CTX-related pseudolithiasis. The effect of CTX on bile flow and biliary electrolyte secretion was evaluated and it has been shown that, even at high doses, CTX had no effects on the bile flow and the biliary electrolyte secretion in guinea pigs [8]. Shiffman et al. [4] performed an in vitro study and evaluated possible interactions between calcium, bile
FIG. 3. Concentration–response curves of papaverine in isolated guinea pig gallbladder strips precontracted with 80 mM KCl. Each point is expressed as a percentage of the contraction induced by KCl. Data are expressed as the means ⫾ SEM of 10 experiments.
FIG. 4. Concentration–response curves of sodium nitroprusside in isolated guinea pig gallbladder strips. Each point is expressed as a percentage of the contraction induced by 80 mM KCl. Data are expressed as the means ⫾ SEM of 10 experiments.
DISCUSSION
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JOURNAL OF SURGICAL RESEARCH: VOL. 122, NO. 2, DECEMBER 2004
FIG. 5. Concentration–response curves of carbachol in isolated guinea pig gallbladder strips. Each point is expressed as a percentage of the contraction induced by 80 mM KCl. Data are expressed as the means ⫾ SEM of 10 experiments. * P ⬍ 0.05 denotes significant difference between groups.
salts, and CTX. They speculated that the development of CTX induced biliary sludge is a result of a solubility problem occurring in the patients receiving high-dose treatment (ⱖ2 g). They also predicted that CTXinduced biliary sludge would not develop at low doses in patients with normal gallbladder motility. However, severely ill patients who are not eating and/or receiving total parenteral nutrition, therefore having little stimulus for gallbladder contraction, bear a great risk of developing pseudolithiasis [4]. Kong et al. [9] reported that fasting and age (older than 24 months) were probably the significant risk factors associated with pseudolithiasis and these findings were compatible with a previous study [4]. In contrast, in a previous study, we have observed that the incidence of pseudolithiasis in pediatric surgical patients, who were fasted, was not different from the patients of other age groups [10]. We presumed that, rather than fasting, CTX itself may induce pseudolithiasis by altering the gallbladder contractility. Results of the present study clearly demonstrate that the receptor-mediated contractile responses of gallbladder smooth muscle to different agonists change after CTX sodium therapy in guinea pigs. In our experiments, we compared the relaxant effect of papaverine and the contractile effects of sodium nitroprusside, carbachol and histamine on the guinea pig’s gallbladder strips of CTX-treated and control groups. Comparison of the pD 2 (-log 10 EC 50) values of the control and CTX-treated groups obtained in response to the above agents did not show any significant difference (P ⬎ 0.05). This indicates that the actual EC 50 values (i.e., concentration resulting in 50% of maximal effect) for used drugs are not significantly different in the CTX group compared with controls. However, E max values elicited by carbachol and histamine were significantly diminished in CTX-treated
group, whereas these values did not show any significance with KCl, papaverine, and sodium nitroprusside. The lower E max values obtained with carbachol and histamine in CTX-treated group may be related to an alteration in the regulation of receptor/postreceptor excitation-contraction coupling. It is well known that the KCl-induced contraction in smooth muscle is caused by an increase in Ca 2⫹ influx through voltage operated Ca 2⫹ channels [11]. Contractions of gallbladder strips elicited by SNP depend on extracellular calcium entry through voltage-operated calcium channels [12]. Papaverine reduces the intracellular Ca 2⫹ concentration by decreasing the calcium gradient across the cell membrane, inhibiting the cAMP phosphodiesterase enzyme, decreasing the uptake of adenosine, and produces smooth muscle relaxation [13]. We used KCl, papaverine and sodium nitroprusside responses to find out the differences due to nonreceptor-mediated mechanisms. The pD 2 and E max values for KCl, papaverine, and sodium nitroprusside in gallbladder smooth muscle in both groups did not show any significant difference, suggesting that CTX sodium does not affect nonreceptor-dependent mechanisms. This finding also rules out the possibility of functional and morphological damage to gallbladder smooth muscle by CTX sodium administration. Acetylcholine and histamine are the classic excitatory neurotransmitters in the gallbladder of the guinea pig. Carbachol is a muscarinic agonist that mimics the neurotransmitter acetylcholine in the gallbladder smooth muscle [14]. Histamine causes depolarizationassociated contraction of gallbladder smooth muscle via the activation of both H 1 (excitatory) and H 2 (inhibitory) receptors [15]. In our study, we used carbachol and histamine responses to classify whether observed differences are due to receptor-mediated mechanism.
FIG. 6. Concentration–response curves of histamine in isolated guinea pig gallbladder strips. Each point is expressed as a percentage of the contraction induced by 80 mM KCl. Data are expressed as the means ⫾ SEM of ten experiments. * P ⬍ 0.05 denotes significant difference between groups.
ARPACIK ET AL.: GALLBLADDER CONTRACTILITY OF CTX-TREATED GUINEA PIGS
We presumed that if receptor-meditated mechanisms are responsible in the formation of gallstones after the administration of CTX sodium that contractile responses to carbachol and histamine would change. Our results demonstrated that E max values for carbachol and histamine were significantly decreased in the CTX-treated group in comparison with the control group, with no significant difference of pD 2 values. This decrement indicates a post-receptor defect rather than decreasing affinity of receptors. Results of this study suggests that decreased maximum contractile response to different agonists after ceftriaxone sodium therapy may contribute to the formation of gallstones. REFERENCES 1.
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