Effect of peritonitis on gallbladder smooth muscle contractility in guinea pigs

Journal of Surgical Research 120, 219 –224 (2004) doi:10.1016/j.jss.2003.12.010

Effect of Peritonitis on Gallbladder Smooth Muscle Contractility in Guinea Pigs Tijen Temiz Kaya, Assoc. Prof., Ph.D.,* Gokhan Koyluoglu, Assoc. Prof., M.D.,† ,1 Barıs Karadas, M.D.,* Turan Yıldız, M.D.,† Ihsan Bagcıvan, M.D.,* Canan Ceran, M.D.,† and Sehsuvar Go¨kgo¨z, Assoc. Prof., M.D.‡ *Department of Pharmacology, †Department of Pediatric Surgery, and ‡Department of Surgery, Faculty of Medicine, Cumhuriyet University, Sivas, Turkey Submitted for publication October 16, 2003

Background. The mechanisms involved in the impaired gallbladder contractile response in peritonitis are unknown. The aim of this study was to determine the effect of peritonitis on the contraction and relaxation responses to different agonists in gallbladder smooth muscle in guinea pig. Materials and methods. Peritonitis was induced by cecal ligation and puncture (CLP) in 10 guinea pigs. Another group of 10 guinea pigs underwent a sham operation and acted as controls. Twenty-four hours after the operation, the guinea pigs were killed, and gallbladder strips were placed in organ bath. The contraction responses to KCl, carbachol, and histamine, and relaxation responses to cyclooxygenase inhibitors (indomethacin, nimesulide, and DFU) on KCl-induced contractions were recorded. Results. There was no significant difference between the contractile responsiveness to KCl, but maximum contractile responses (E max) to carbachol and histamine were significantly reduced. Indomethacin, nimesulide, and DFU concentration dependently inhibited on KCl-induced contractions of gallbladder smooth muscle. E max values of indomethacin, nimesulide, and DFU were significantly reduced in the peritonitis group compared with controls (P < 0.05). The inhibitor effects of nimesulide and DFU were considerably similar, but inhibitor effect of indomethacin was significantly less than that measured for nimesulide and DFU in both control and peritonitis groups (P < 0.05). Conclusions. The contraction responses to carbachol and histamine and relaxation responses to COX inhibitors on gallbladder smooth muscle are significantly decreased by peritonitis. Although the mechanism of 1 To whom correspondence and reprint requests should be ad¨ rtu¨lu¨pınar mah, Hoca Ahmet Yesevi sok, Ziyabey apt. dressed at O No. 2/9, 58030 Sivas, Turkey. Fax: ⫹90.346.219 12 84. E-mail: [email protected].

the decrease in contraction and relaxation responses in CLP-induced peritonitis is completely unknown, we speculate that impaired smooth muscle responses may be related to an alteration in the regulation of receptor/postreceptor excitation-response coupling and/or through changes on Ca 2ⴙ influx. © 2004 Elsevier Inc. All rights reserved.

Key Words: peritonitis; gallbladder; smooth muscle; in vitro. INTRODUCTION

Gallbladder dysfunction is a common accompaniment to multiple organ failure [1]. Impaired gallbladder emptying is being diagnosed with increasing frequency in critically ill, septic patients and typically occurs in conditions associated with biliary stasis, including major surgery, severe trauma and sepsis, longterm total parenteral nutrition, and prolonged fasting. The pathogenesis of gallbladder hypomotility is unclear. Peritonitis (sepsis) causes severe local damage to intraabdominal organs due to overproduction of various proinflammatory mediators such as tumor necrosis factor, interleukin-1␤, interleukin-6, and cyclooxygenase (COX) products. COX metabolites, the prostanoids, play a significant role in gallbladder physiology and disease [2, 3]. Cyclooxygenases are the enzymes responsible for the conversion of arachidonic acid to prostaglandins. There are two isoforms of cyclooxygenase described, cyclooxygenase-I (COX-1) and -II (COX-2). COX-1 is constitutively expressed and is thought to be responsible for the synthesis of physiological prostaglandins that protect the gastric mucosa and which are involved in renal tubular function. COX-2, which is an inducible

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form, was first described in 1991 [4] and is induced during tissue damage by several factors, including endotoxin, interleukin-1, and hypoxia [5]. Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit both isoform activities [6]. Indomethacin is a nonselective inhibitor of both COX-1 and -2, but nimesulide and DFU more selectively inhibit COX-2 than COX-1 [6 –10]. Nimesulide (N-(4-nitro-2-phenoxyphenyl)-methanesulfanilamide) shows 30- to 100-fold selectivity for inhibition of COX-2 compared to COX-1 [8]. DFU (5,5-dimethyl-3(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone was identified as a novel highly selective COX-2 inhibitor. It shows 1000-fold selectivity for inhibition of COX-2 compared to COX-1 [9]. COX metabolites, the prostanoids, have been shown to be involved in gallbladder muscle contraction [10]. It has been shown that COX inhibitors relax various smooth muscles, such as trachea, uterus, aorta, and upper urinary tract [11–14]. Carbachol, a muscarinic agonist, and histamine are the classic excitatory agents in guinea pig gallbladder. Carbachol and histamine contract gallbladder smooth muscle due to the activation of M 21 and H 1 receptors, respectively [15–16]. The aim of our study was to determine the effect of experimental peritonitis on the contraction and relaxation responses to different agonists in gallbladder smooth muscle in guinea pigs. We hypothesized that contractile responses to different agonists of gallbladder smooth muscle could change after peritonitis.

MATERIALS AND METHODS

Twenty male guinea pigs each weighing approximately 550 g were maintained in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals and the experiments were approved by the Cumhuriyet University Medical Faculty, Animal Care Committee. The experimental peritonitis model used in this study was based on findings of the previous report by Martin et al. [15]. This model has previously been shown to induce hyperdynamic normotensive sepsis within a 24 h period.

Peritonitis Model (Cecal Ligation and Puncture, CLP) Each guinea pig was anesthetized intramuscularly with 3 mg/kg xylazine and 90 mg/kg ketamine. Once adequate anesthesia was attained, the animal was placed in a supine position. A 2-cm abdominal incision was made to expose the cecum. Then we ligated the cecum just below the ileocecal valve with 4-0 silk ligature, so that intestinal continuity was maintained. Using an 18-gauge needle, the cecum was perforated in three locations, 1 cm apart, on the antimesenteric surface of the cecum, and the cecum was gently compressed until feces were extruded. The cecum was replaced into the peritoneal cavity, and the incision was closed in two layers. Afterward, we observed the guinea pigs in a recovery cage for 24 h. The guinea pigs had free access to food and water after the operation. The control group (sham group) underwent laparotomy, and the cecum was manipulated but not ligated or punctured.

Experimental Design The day before the surgical procedures, animals were fasted overnight but allowed ad libitum access to water. Guinea pigs were assigned randomly to two groups: (a) the control group (sham operated): guinea pigs were anesthetized and their cecum was manipulated but not ligated or punctured (n ⫽ 10); (b) peritonitis group: guinea pigs made septic by cecal ligation and puncture (n ⫽ 10).

Tissue Preparation Twenty-four hours later, the guinea pigs were killed by cervical dislocation. The abdomen was opened with a midline incision and the gallbladder was removed and placed in previously aerated (95% O 2 and 5% CO 2) Krebs-bicarbonate solution (composition in mM: NaCl, 120; KCl, 4.6; CaCl 2, 2.5; MgCl 2, 1.2; NaHCO 3, 22; NaH 2PO 4 and glucose, 11.5). The gallbladder was opened with a longitudinal incision and full-thickness muscle strips (2 ⫻ 10 mm) containing mucosa, smooth muscle, and serosa were cut along the longitudinal axis of the gallbladder. The muscle strips were placed in a longitudinal direction in a 10-ml muscle bath, filled with pre-aerated Krebsbicarbonate solution (KBS) at 37°C. The upper end of the preparation was tied to an isometric transducer (Grass FT 03, Quincy, MA, USA) 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 bath, and the contraction was considered as reference response (RR). At the end of the experiment, the contractile response to 80 mM KCl was measured again. Following KCl response, the contractile responses to carbachol (10 ⫺8–10 ⫺4 M) and histamine (10 ⫺8–10 ⫺4 M), and the relaxation responses to indomethacin (10 ⫺8–10 ⫺4 M), nimesulide (10 ⫺8–10 ⫺4 M), and DFU (10 ⫺8–10 ⫺4 M) precontracted with KCl (80 mM) tissues were obtained by adding one of those agents to the bath in a cumulative manner. Isometric tension was recorded on a Grass model 79 E polygraph. All experiments were performed in a paired way. The contraction and relaxation responses to carbachol, histamine, and COX inhibitors were calculated as a percentage of the contraction caused by KCl (80 mM) on the gallbladder strips isolated from the control and peritonitis groups (number of gallbladder strips ⫽ 20 per group, two strips from each animal). Contractile responses in 10 strips and relaxation responses in 10 strips were examined. To evaluate the effects of agonists, the maximum responses (E max) and pD 2 values (i.e., the negative logarithm of the concentration for the half-maximal response, ED 50) were calculated. 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. pD 2 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 Chemicals used in the current experiments were carbachol, histamine, indomethacin, and nimesulide from Sigma (St. Louis, MO, USA), and DFU from Merck, Sharp, & Dohme (Sivas, NJ, USA). All chemicals were dissolved in distilled water, except for indomethacin, nimesulide, and DFU, which were dissolved in DMSO and diluted with distilled water. Concentration added to the organ bath of DMSO never exceeded 1% of the total volume. All drugs were freshly prepared on the day of the experiments.

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nimesulide and DFU in the control and peritonitis groups (P ⬍ 0.05) (Table 1). DMSO (used as a solvent vehicle of indomethacin, nimesulide, and DFU) has no relaxant effect on gallbladder strips precontracted with 80 mM KCl in the control and peritonitis groups (data not shown). DISCUSSION

The results from this study clearly demonstrate that the contraction responses to carbachol and histamine and the relaxation responses to COX inhibitors (indomethacin, nimesulide, and DFU) are significantly decreased by peritonitis in guinea pig gallbladder smooth muscle. The pD 2 (⫺log 10 EC 50) values of the contractile and relaxant drugs in control and peritonitis groups were not significantly different (P ⬎ 0.05). This indiFIG. 1. Mean contraction elicited by KCl (80 mM) on gallbladder smooth muscle strips isolated from control (sham-operated) and peritonitis guinea pigs. Data are expressed as the means ⫾ SEM of 20 experiments.

Data Analysis All data are expressed as mean ⫾ SEM. Groups were compared statistically using general linear models of ANOVA followed by Newman–Keuls test and t test when appropriate. Differences were considered to be significant when P was less 0.05.

RESULTS

The contractions elicited by 80 mM KCl (RR) were not significantly different in the peritonitis group than in the control (sham-operated) group (2.98 ⫾ 0.21 g, 3.03 ⫾ 0.12 g, respectively; P ⬎ 0.05) (Fig. 1) (n ⫽ 20, in two groups). Carbachol (10 ⫺8–10 ⫺4 M) and histamine (10 ⫺8–10 ⫺4 M) elicited concentration-dependent contraction in gallbladder smooth muscle strips isolated from guinea pigs in control and peritonitis groups (Figs. 2A and 2B) (n ⫽ 10, in two groups). The E max values for carbachol and histamine were significantly lower in the peritonitis group than in the control group (P ⬍ 0.05), but there was no difference in the corresponding pD 2 values (Table 1). Indomethacin (10 ⫺8–10 ⫺4 M), nimesulide (10 ⫺8–10 ⫺4 M), and DFU (10 ⫺8–10 ⫺4 M) concentration dependently inhibited the KCl (80 mM)-induced contractions of gallbladder smooth muscle strips isolated from guinea pigs in control and peritonitis groups (Figs. 3A, 3B, and 3C) (n ⫽ 10, in two groups). E max values to indomethacin, nimesulide, and DFU were significantly reduced in the peritonitis group compared with the control group (P ⬍ 0.05). There was no significant difference between pD 2 of indomethacin, nimesulide, and DFU in both groups (P ⬎ 0.05). However, the inhibitor effect of indomethacin was significantly less than that measured for both

FIG. 2. Contractile responses to carbachol (A) and histamine (B) in gallbladder smooth muscle strips from guinea pigs in control (sham-operated) and peritonitis groups. Contractions were decreased in muscle strips from gallbladder with peritonitis compared to controls. Data are expressed as the means ⫾ SEM of 10 experiments. *P ⬍ 0.05 denotes significant difference from control.

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TABLE 1 E max (% of KCl) and pD 2 Values of Carbachol, Histamine, Indomethacin, Nimesulide, and DFU in Gallbladder Smooth Muscle Strips from Guinea Pigs in Control (Sham-Operated) and Peritonitis Groups Drugs Carbachol E max pD 2 Histamine E max pD 2 Indomethacin E max pD 2 Nimesulide E max pD 2 DFU E max pD 2

Control

Peritonitis

150.34 ⫾ 11.21 6.02 ⫾ 0.03

115.53 ⫾ 10.50* 6.06 ⫾ 0.04

145.09 ⫾ 8.82 5.75 ⫾ 0.02

114.89 ⫾ 9.88* 5.64 ⫾ 0.03

58.50 ⫾ 6.15 6.05 ⫾ 0.03†

tory neurotransmitters in guinea pig gallbladder. Carbachol is a muscarinic agonist that mimics the neurotransmitter acetylcholine in the gallbladder smooth

39.6 ⫾ 3.52* 6.02 ⫾ 0.02†

82.12 ⫾ 4.83 6.08 ⫾ 0.02

67.90 ⫾ 4.14* 6.10 ⫾ 0.04

84.80 ⫾ 4.11 6.09 ⫾ 0.01

70.56 ⫾ 4.94* 6.12 ⫾ 0.03

Note. Data expressed as a mean ⫾ SEM. * P ⬍ 0.05, statistically different from E max values in the control group. † P ⬍ 0.05, statistically different from pD 2 values of nimesulide and DFU in control and peritonitis groups.

cates that the actual EC 50 values (i.e., concentration resulting in 50% of maximal effect) for used drugs are not significantly different in the peritonitis group compared with controls. The E max values for carbachol, histamine, and COX inhibitors were significantly diminished in the peritonitis group, while the E max value for KCl was not different. The lower E max values for the contractile and relaxant drugs in the peritonitis group may be related to an alteration in the regulation of receptor/postreceptor excitation-response coupling. In addition, the E max values for DFU and nimesulide were significantly greater than those calculated for indomethacin in both control and peritonitis groups. There was no significant difference between the pD 2–log 10 EC 50 values of DFU, nimesulide, and indomethacin in all tissues. This indicates that the efficacy of DFU and nimesulide are significantly higher than that in indomethacin, but potency for the three drugs used are not significantly different in control and peritonitis groups. It is well known that the KCl-induced contraction in smooth muscle is due to an increase in Ca 2⫹ influx through voltage-operated Ca 2⫹ channels [17]. We used the KCl responses to classify observed changes as due to nonreceptor-mediated mechanisms in peritonitis. The contractile responses for KCl in gallbladder smooth muscle in both groups were not different. This result rules out the possibility of functional and morphological damage in gallbladder smooth muscle after peritonitis. Acetylcholine and histamine are the classic excita-

FIG. 3. Relaxation responses of indomethacin (A), nimesulide (B), and DFU (C) in KCl-contracted gallbladder strips from guinea pigs in control (sham-operated) and peritonitis groups. Relaxation responses were decreased in muscle strips from gallbladder with peritonitis compared to controls. Data are expressed as the means ⫾ SEM of 10 experiments. *P ⬍ 0.05 denotes significant difference from control.

KAYA ET AL.: EFFECT OF PERITONITIS ON GALLBLADDER

muscle [16]. The effect of histamine as depolarization associated contraction of gallbladder smooth muscle represents the activation of both H 1 (excitatory) and H 2 (inhibitory) receptors [18]. In our study, we used carbachol and histamine responses to classify observed differences as due to receptor-mediated mechanism. Our results demonstrate that E max values for carbachol and histamine were significantly decreased, with no difference in the pD 2 values in peritonitis group compared to control group. A decrease in maximal response to carbachol and histamine in the peritonitis group found in this study indicates a postreceptor defect rather than a decrease in the affinity of receptors. Another possibility is desensitization of gallbladder smooth muscle cells to carbachol and histamine after peritonitis. The prostanoids have been shown to be involved in gallbladder muscle contraction [10]. The prostanoids produce smooth muscle contractions primarily by stimulating intracellular sites to release Ca 2⫹ by activating their membrane receptors [19]. Prostanoids have been shown to mediate gallbladder inflammatory responses [17] and NSAIDs have been shown to decrease gallbladder inflammation [20]. In our study, the pD 2 values for indomethacin, nimesulide, and DFU on KCl-contracted tissues in both groups were not changed. The decreased E max values for indomethacin, nimesulide, and DFU after peritonitis may be related to decreased response of gallbladder smooth muscle cells to cyclooxygenase inhibitors after high levels of prostanoids in peritonitis. In addition, inhibitor effects of nimesulide and DFU, selective COX-2 inhibitors, KCl-contracted gallbladder smooth muscle, were significantly greater than indomethacin, nonselective COX inhibitor, in control and peritonitis. These findings suggest that selective COX-2 inhibitors in guinea pig gallbladder smooth muscle are stronger relaxant agents than nonselective COX inhibitors. It is possible that COX-1 and COX-2 inhibitors exert their smooth muscle relaxant effect by mechanisms other than inhibition of PG synthesis [21]. In addition to suppressing the arachidonic cascade, indomethacin could inhibit uterus smooth muscle through other mechanisms, although for most other effects, the median effective dose is generally much higher than for cyclooxygenase inhibition [21, 22]. It was suggested that Ca2⫹ antagonism may be one of several mechanisms of action for cyclooxygenase-1 and cyclooxygenase-2 inhibitors, including the inhibition of prostaglandin synthesis [21]. In summary, this model of CLP-induced peritonitis in guinea pig produces a marked decrease in contraction and relaxation responses to different agonists in gallbladder smooth muscle. We speculate that the impaired smooth muscle responses is, in part, related to an alteration in the regulation of receptor/postreceptor

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