![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Department of Physiology, Nursing School, University of Extremadura, Caceres, Spain
Received March 22, 2007; accepted July 3, 2007.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
-conotoxin, indicating that acute cholecystitis affects action potential propagation in the intrinsic nerves. Nitric oxide (NO)-mediated neurotransmission was reduced by inflammation, which also increased the reactivity of sensitive fibers. Melatonin treatment prevented qualitative changes in gallbladder neurotransmission, but it did not improve EFS-induced contractility. The hormone recovered gallbladder neuromuscular function once the biliary obstruction was resolved, even when the treatment was started after the onset of gallbladder inflammation. These findings show for the first time the therapeutic potential of melatonin in the recovery of gallbladder neuromuscular function during acute cholecystitis.
). Acetylcholine (ACh) released from cholinergic neurons induces contraction of the gallbladder smooth muscle through muscarinic receptors (Parkman et al., 1999b), and it has neuromodulatory functions, promoting or inhibiting the release of other neurotransmitters (Parkman et al., 1999b). Cholinergic neurons coexpress other neurotransmitters such as NO and several neuropeptides (Talmage et al., 1992). Afferent nerve fibers containing calcitonin gene-related peptide and tachykinins have also been described in the ganglionated plexus of the gallbladder (Mawe and Gershon, 1989).
Acute acalculous cholecystitis (AC) is characterized by gallbladder inflammation in the absence of gallstones. Although its pathogenesis is unknown, gallbladder stasis is always present, probably as the result of the deleterious neural and muscular actions of inflammatory mediators such as reactive oxygen species and prostaglandins (Pozo et al., 2004). In animal models, it has been described that cholecystitis reduces gallbladder contractile responses to agonists that act directly on smooth muscle cells (Parkman et al., 1999a; Xiao et al., 2001) and that it also causes alterations in calcium signaling and contractile machinery (Gomez-Pinilla et al., 2006b). In addition, EFS-induced contractions are also impaired in inflamed gallbladder, mainly due to the reduction in the function of cholinergic nerves and the up-regulation of the inhibitory nitrergic component (Parkman et al., 2000). The effect of cholecystitis on afferent fibers has not yet been explored.
Melatonin (MEL), the main product of pineal gland, is a potent free radical scavenger, and it activates a broad group of antioxidant cellular mechanisms (Tan et al., 2002). These properties made melatonin efficacious against different diseases where oxidative stress is the main cause (Karasek, 2004). The gastrointestinal tract is an important source of melatonin (Kvetnoy et al., 2002). The liver and the gallbladder are especially exposed to high levels of the hormone, because hepatic metabolism is the major pathway for deactivation of melatonin (Lane and Moss, 1985), which is also present in active form in bile and concentrated in the gallbladder (Tan et al., 1999). In the gastrointestinal tract, melatonin has a gastroprotective function (Konturek et al., 1997) and therapeutic effects against malignancy associated with irritable bowel syndrome (Head and Jurenka, 2003).
The aims of this study were to investigate the effects of acute cholecystitis in the neuromuscular transmission and to evaluate the impact of melatonin treatment. Our results indicate that melatonin restores neuromuscular function in inflamed gallbladder, which can be of importance to recover gallbladder contractility in this pathological condition.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). This method was approved by the Animal Care and Ethical Committees of the University of Extremadura (Caceres, Spain). In brief, after anesthesia with 20 mg/kg i.p. ketamine hydrochloride and 5 mg/kg i.p. xylacine, a laparotomy was performed, and the distal end of the common bile duct was ligated. Two days after, the animals were sacrificed for tissue harvest (n = 28). In the model used in this study, the gallbladder is stretched as the result of bile duct ligation and the continuous bile output. Taking this into account, it would be difficult to see any improvement in the neuromuscular function by keeping the bile duct ligated, which represents a remarkably extreme pathological condition. To solve this dilemma, in a group of animals 2 days later, CBDL, the common bile duct, was deligated (DL) under anesthesia with microsurgical scissors, and 2 days later, the animals were sacrificed (n = 28). For both experimental models, a group of guinea pigs were sham-operated (n = 4), which included all of the surgical steps, with the exception of common bile duct ligation. Melatonin and Tempol Administration. Guinea pigs were treated orally with melatonin (2.5 or 30 mg/kg/day). Melatonin was dissolved in glucose solution (1.5%), and it was placed in the oropharynx by using a syringe. This treatment was applied daily at the same time, just before the light in the animal house was switched off (7:00 PM). Melatonin was administered 14 days before the sacrifice of the animals in both experimental groups, AC and DL. In a group of animals subjected to DL, melatonin treatment (30 mg/kg) started 12 h after CBDL was performed, and it continued until the sacrifice of the animal. Tempol was administered in the drinking water at 1 mM for 14 days before the animal was sacrificed.
Functional Studies. At the appropriated time, the animals were killed with deep halothane anesthesia and cervical dislocation. Gallbladders were removed, and they were immediately placed in ice-cold Krebs-Henseleit solution (for composition, see "Solutions and Drugs") at pH 7.35. The gallbladder was cut in longitudinal full-thickness strips (3 x 10 mm) that were placed vertically in a 10-ml organ bath filled with Krebs-Henseleit solution maintained at 37°C and gassed with 95% O2, 5% CO2. Isometric contractions were measured using force displacement transducers that were interfaced with a Macintosh computer using a MacLab hardware unit and software (ADInstruments, Colorado Springs, CO). The muscle strips were placed under an initial resting tension equivalent to a 1.5-g load. Intrinsic nerves were activated by EFS with a pair of external platinum ring electrodes connected to a square-wave stimulator (CS9/3BO; Cibertec, Madrid, Spain). Trains of stimuli (0.3-ms duration; 5–40 Hz; 350-mA current strength) were delivered for 10 s at 3-min intervals. After construction of a frequency-response curve and to pharmacologically characterize the neurogenic responses, antagonists/inhibitors were added to the organ bath for 20 min, and then the EFS protocols were repeated.
Malondialdehyde and Reduced Glutathione Assays. Gallbladder fragments of approximately 10 mg were placed in an ice-cold phosphate buffer at a proportion of 1:5 (w/v), homogenized with an homogenizer (Ika-Werke, Staufen, Germany) for 2 min, and centrifuged at 10,000 rpm for 15 min at 4°C. The protein concentration was then quantified with a commercial kit (TPRO-562; Sigma-Aldrich, St. Louis, MO), and the rest of homogenate was treated with ice-cold perchloric acid (7%, v/v) to eliminate proteins, and it was kept at –80°C until analysis. Malondialdehyde (MDA) level, an index of lipidic peroxidation, was determined based on colorimetric Recknagel's method (Waller and Recknagel, 1977). In brief, the samples were incubated with 0.4% thiobarbituric acid at 80°C for 20 min, and then the sample absorbance at 550 nm was measured. Reduced glutathione determination was carried out following the Hissin and Hilf (1976) method. Samples were incubated with 0.005% orthophthaldehyde in darkness at room temperature for 45 min, and the fluorescent complex that was formed, indicative of reduced glutathione (GSH) level, was measured with a fluorimeter (excitation, 350 nm; emission, 425 nm).
Solutions and Drugs. The Krebs-Henseleit solution contained 113 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, and 11.5 mM D-glucose. This solution had a final pH of 7.35 after equilibration with 95% O2, 5% CO2. The phosphate buffer used to homogenize the tissue contained 20 mM NaCl, 2.7 mM KCl, 16 mM Na2HPO4, and 4 mM NaH2PO4, pH 7.4. Drug concentrations are expressed as final bath concentrations of active species. Drugs and chemicals were obtained from the following sources: atropine, L-NAME, melatonin, and tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl or 4-hydroxy-tempo) were from Sigma-Aldrich. -Conotoxin GVIA, (E)-capsaicin, and tetrodotoxin citrate were from Tocris Cookson Inc. (Bristol, UK). Ketamine was from Merial (Lyon, France). Xylacine was supplied by Bayer AG (Kiel, Germany). Other chemicals used were of analytical grade from Panreac (Barcelona, Spain). Stock solutions of atropine, capsaicin, and -conotoxin GVIA were prepared in DMSO. The solutions were diluted such that the final concentration of DMSO was 
0.1% (v/v). This concentration of DMSO did not have effects on gallbladder tone.
Data Analysis. Results are expressed as means ± S.E.M. of n gallbladder strips from at least five different animals. Gallbladder tension is given in millinewtons per milligram of tissue. Statistical differences between multiple groups or the effects of inhibitor treatments were tested using appropriate analysis of variance (ANOVA). Differences were considered significant at P < 0.05.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
-conotoxin GVIA, an N-type calcium channel blocker, there was a reduction (63% inhibition at 25 Hz) in the contractile response evoked by EFS (Fig. 2B). These results indicate that in inflamed gallbladder, the transmission of the action potential along neural fibers is impaired and that EFS stimulates neurotransmitter release directly from nervous terminals.
|
To elucidate the neurotransmitters involved in the EFS-induced contraction, we tested several antagonists/inhibitors on this neural response. In control animals, we found that 1 µM atropine reduced the EFS-elicited contractile response (82% inhibition at 25 Hz; Fig. 3A), but the strips from cholecystitic animals were less sensitive to atropine blockade (30% inhibition at 25 Hz; Fig. 3D). The impact of inflammation on the contribution of NO was tested by using the inhibitor of the nitric-oxide synthase, L-NAME, at 100 µM. This inhibitor enhanced EFS-induced contraction in strips from control guinea pigs, especially at the lowest frequencies assayed (90% enhancement at 5 Hz; Fig. 3B), but it had little effect in inflamed strips (17% enhancement at 5 Hz; Fig. 3E).
|
). When we induced sensory nerve desensitization by treatment with a high concentration of capsaicin (10 µM), we found no effect in control strips (Fig. 3C), but this treatment induced an inhibition of EFS-elicited contractile responses in cholecystitic strips (56% inhibition; Fig. 3F).
Effects of Melatonin on Neuromuscular Function in Acute Cholecystitis. We have reported previously that melatonin treatment was able to restore gallbladder neuromuscular function in aging (Gomez-Pinilla et al., 2006a). To determine whether this hormone had beneficial effects in the alterations described above, we treated the animals with 2.5 and 30 mg/kg melatonin (MEL 2.5; MEL 30) as described under Materials and Methods. Under these conditions, none of melatonin doses used enhanced the amplitude of the contractile responses evoked by EFS (Fig. 4A), but the contractions partially recovered the sensitivity to TTX (85 and 77% inhibition for MEL 2.5 and MEL 30, respectively, at 25 Hz; Fig. 4B). Although the treatment dose-dependently increased the inhibitory effects of atropine and it decreased the inhibitory effects of capsaicin significantly at some frequencies (Fig. 4, C and E), these changes were small. However, melatonin was able to protect nitrergic nerves, because when L-NAME was added to the organ bath, the EFS-evoked contractile responses were enhanced in similar proportions to those found in control strips (91 and 85% enhancement for MEL 2.5 and MEL 30, respectively, at 5 Hz; Fig. 4D). These results indicate that melatonin had some effects on inflammation of the gallbladder, but there are some contractile disabilities related to the experimental method used in this study, as indicated under Materials and Methods, that melatonin treatment cannot restore.
|
Thus, we tested melatonin effects in animals that underwent the deligation protocol. When deligation was performed in animals that were not treated with melatonin, the neuromuscular function worsened, as indicated by the reduction in the EFS-induced contraction (Fig. 5, A and B). Taking into account the small amplitude of these contractions, we did not apply antagonists/inhibitors of the neurotransmitters to determine the nature of this response. However, when the animals were treated with melatonin 10 days before performing the surgical procedures and until the animal was sacrificed, there was a very noticeable improvement in gallbladder neuromuscular function. As shown in Fig. 5C, melatonin treatment increased the gallbladder neurogenic responses in a dose-dependent way. In the strips from animals treated with melatonin, the EFS-elicited responses recovered the sensitiveness to TTX (70 and 73% inhibition for MEL 2.5 and MEL 30, respectively, at 25 Hz; Fig. 6A) and atropine (73 and 76% inhibition for MEL 2.5 and MEL 30, respectively, at 25 Hz; Fig. 6B) to a level comparable with that seen in control tissue. Although capsaicin still induced a small inhibition of EFS-induced responses (5 and 10% inhibition for MEL 2.5 and MEL 30, respectively, at 25 Hz %; Fig. 6D), the reduction was significantly smaller than that found in inflamed tissue; at the highest frequencies, this effect was not different from that registered in control tissue. In this experimental group, 30 mg/kg melatonin also re-established the sensitivity to L-NAME (Fig. 6C), but this was not the case for 2.5 mg/kg melatonin, suggesting that the effects of CBDL in the nitrergic function are exacerbated by the deligation procedure.
|
|
These effects of melatonin could be related to its antioxidant and scavenger properties as indicated by the reduction in the lipidic peroxidation and the increase in the levels of GSH induced by melatonin treatment (Table 1).
|
Effects of Tempol on Neuromuscular Function in Acute Cholecystitis. To test whether other antioxidants/scavengers also have beneficial effects on the impaired neuromuscular transmission in acute cholecystitis, we also tested the effects of tempol, a membrane-permeable superoxide dismutase mimetic (Krishna et al., 1996). Administration of tempol in the drinking water for 14 days to guinea pigs that underwent the protocol of deligation prevented the functional impairment of EFS-induced contraction, although to a lesser extent than 30 mg/kg melatonin (63 and 26% recovery for MEL 30 and tempol, respectively; Fig. 7, A and B; P < 0.01). The recovery was accompanied by the normalization of the neurotransmission, because TTX, atropine, L-NAME, and capsaicin had similar effects in tempol-treated animals as in control animals (Fig. 7, C–F). These prophylactic effects also correlated with a decrease in the MDA levels and an increase in the GSH content that were altered by the AC and DL protocols (Table 1).
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Our results indicate that EFS evokes a gallbladder response by the release of different inhibitory and excitatory neurotransmitters. The smaller contractile responses to EFS in cholecystitic strips suggest the existence of an inflammation-induced impairment in the gallbladder intrinsic nerves, in agreement with previous results in human and animal models (McKirdy et al., 1994a; Parkman et al., 2000). However, the reduced gallbladder smooth muscle contractility to ACh found in cholecystitis (Parkman et al., 1999a, 2000; Gomez-Pinilla et al., 2006b) could also contribute to the impaired neuromuscular function in inflamed tissue. The most striking finding in our study was the lack of sensitivity shown by cholecystitic strips to TTX, which could be explained by a direct release of neurotransmitter from nervous terminal. This was confirmed by the sensitivity of the EFS-induced responses to -conotoxin GVIA, a blocker of N-type calcium channel located in the presynaptic membrane whose activation is necessary for neurotransmitter release. These results suggest that inflammation evokes a functional denervation in the gallbladder that avoids the genesis or propagation of action potential through efferent fibers. Alterations in the properties and/or expression levels of voltage-dependent Na+ channels have been implicated in a variety of pathological states, including inflammation of the viscera (Yoshimura et al., 2001; Stewart et al., 2003; Beyak et al., 2004). In this regard, the more common effect of inflammation on Na+ channels is the up-regulation of TTX-resistant slow (Nav1.8) type (Yoshimura et al., 2001; Beyak et al., 2004). Alterations in the pharmacological profile of Na+ channels could also explain the TTX-resistant contractions reported in this study.
Classically, ACh released in response to EFS is the main excitatory component of the gallbladder contraction (Yau and Youther, 1984; Parkman et al., 1997). Here, we show that in control conditions, atropine abolished EFS-induced contraction, whereas in inflamed tissue, it just reduced EFS-elicited contraction approximately 50%, indicative of a functional denervation of the cholinergic component, similar to results described previously in inflamed gallbladder (Parkman et al., 2000).
Nonadrenergic noncholinergic neurotransmission in guinea pig gallbladder was described more than a decade ago (Mourelle et al., 1993), and NO is the main nonadrenergic noncholinergic neurotransmitter involved (McKirdy et al., 1994b; Alcón et al., 2001). Inflammation evokes a functional impairment in gallbladder nitrergic innervation as demonstrated by the lack of effects of L-NAME in cholecystitic strips compared with control tissue. This result does not support the study from Parkman et al. (2000), where L-NAME only had an effect in inflamed tissue, indicating that normal gallbladder does not release NO from the intrinsic plexus. This is in conflict with the presence of nitrergic nerves described in guinea pig gallbladder (Mawe et al., 2006) and with the functional data reported above.
Neurotransmitters released from sensory nerves evoked contraction or relaxation of the gallbladder (Maggi et al., 1989). In our study, sensory denervation with capsaicin had no effect in control conditions, whereas it reduced EFS-elicited contractile response in inflammation, suggesting excitatory neurotransmitter release from sensory nerves in inflamed gallbladder. The major participation of the sensory innervation is a common finding in neurogenic inflammation (Sann et al., 1996). In the gallbladder, we have shown that aging, which is also related to increased oxidative stress, is associated with over-reactivity of sensory fibers (Gomez-Pinilla et al., 2006a).
The most important finding of our study is that melatonin has prophylactic and therapeutic effects on inflammation-induced impairment in gallbladder neuromuscular function. Thus, with 14-day melatonin treatment, the EFS-induced contractile response recovered the sensitiveness to TTX, indicating that melatonin protects the voltage-dependent Na+ channels involved in the neural transmission of the action potential. Furthermore, the nitrergic innervation recovered its functionality and sensory fibers became less sensitive to EFS. However, melatonin itself did not improve the contractile response to EFS unless the obstruction of the bile duct was relieved. Under these conditions, melatonin reversed the impairment in contractility in a dose-dependent manner, and it fully recovered the different neural components stimulated by EFS. It must be pointed out that 2.5 mg/kg melatonin had no effects on the nitrergic innervation after deligation, although this treatment was efficacious in increasing the participation of these inhibitory nerves with the bile duct-ligated animals. Deligation itself worsened gallbladder contractility, as consequence probably of an increase in oxidative stress insult due to reperfusion of the organ once the mechanical stretch was alleviated. This is supported by the increase in the MDA levels indicative of lipidic peroxidation and oxidative stress injury found after deligation. On this basis, it seems that nitrergic innervation is especially sensitive to the enhanced oxidative stress after deligation. In agreement with this, we have recently reported a minor participation of nitrergic nerves in neuromuscular transmission in aging and its recovery after melatonin treatment (Gomez-Pinilla et al., 2006a). Furthermore, melatonin has been shown to have neurally mediated actions in the gut, regulating either cholinergic, nitrergic, and/or sensory innervation (Barajas-López et al., 1996; Reiter et al., 2003).
According to our results, melatonin not only protects against inflammation but also resolves the inflammation-induced impairment of neuromuscular function. Thus, when melatonin treatment started after the onset of gallbladder inflammation, there was an enhancement of the contractile response to EFS that also recovered the neurotransmission pattern. However, the prophylactic administration of melatonin was more effective than the therapeutic administration, which could be related to the increase in the antioxidant defenses induced by the administration of melatonin before the oxidative insult.
It is well accepted that melatonin administration at pharmacological doses decreases free radical formation and leads to a substantial recovery of the major antioxidant enzymes (Reiter, 1998). Recent evidence has shown that melatonin has protective effects on liver and hepatic injury after extrahepatic bile duct ligation in rats (Shiesh et al., 2000; Esrefoglu et al., 2005; Ohta et al., 2005). In addition to liver and hepatic damage, free radical accumulation associated with bile duct ligation has been implicated in the genesis of gallstone (Eder et al., 1996). In this regard, antioxidant treatment with melatonin not only reversed the increased oxidative stress but also prevented gallstone formation (Shiesh et al., 2000). In our preparation, either prophylactic or therapeutic melatonin treatments were effective in reducing MDA levels and in increasing the endogenous antioxidant defense GSH, indicating that melatonin antioxidant effects can be responsible for the improvement in the neuromuscular function. In fact, the treatment of the animals with the membrane-permeant superoxide dismutase mimetic tempol also induced a significant improvement in the neuromuscular function of inflamed gallbladder, which is in agreement with other reports showing that tempol reduces the dysfunctions associated to oxidative stress insult (Chatterjee et al., 2000; Mehta et al., 2004). Collectively, our data suggest a prophylactic and therapeutic role of melatonin in experimental acute cholecystitis, a remarkable finding due to the lack of an effective pharmacological treatment for acute cholecystitis.
In conclusion, the results obtained in the present study indicate that inflammation impairs gallbladder neuromuscular function as the result of changes in the neural inputs to smooth muscle. These changes can be summarized as a denervation of efferent nerves together with a hyperactivity of afferent fibers. Melatonin significantly ameliorated the inflammation-related changes in gallbladder neuromuscular transmission, indicating its potential to combat inflammation-induced gallbladder damage.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
P.J.G.-P. is recipient of a doctoral fellowship from Junta de Extremadura.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: ACh, acetylcholine; AC, acute acalculous cholecystitis; MEL, melatonin; CBDL, common bile duct ligation; DL, deligation; EFS, electrical field stimulation; MDA, malondialdehyde; GSH, glutathione; L-NAME, N-nitro-L-arginine methyl ester; DMSO, dimethyl sulfoxide; ANOVA, analysis of variance; TTX, tetrodotoxin.
Address correspondence to: Dr. María J. Pozo, Department of Physiology, Nursing School, Avda Universidad s/n, 10071 Cáceres, Spain. E-mail: mjpozo{at}unex.es
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Alcón S, Morales S, Camello PJ, Salido GM, Miller SM, and Pozo MJ (2001) Relaxation of canine gallbladder to nerve stimulation involves adrenergic and nonadrenergic non-cholinergic mechanisms. Neurogastroenterol Motil 13: 555–566.[CrossRef][Medline]
Barajas-López C, Peres AL, Espinosa-Luna R, Reyes-Vazquez C, and Prieto-Gomez B (1996) Melatonin modulates cholinergic transmission by blocking nicotinic channels in the guinea-pig submucous plexus. Eur J Pharmacol 312: 319–325.[CrossRef][Medline]
Beyak MJ, Ramji N, Krol KM, Kawaja MD, and Vanner SJ (2004) Two TTX-resistant Na+ currents in mouse colonic dorsal root ganglia neurons and their role in colitis-induced hyperexcitability. Am J Physiol Gastrointest Liver Physiol287: G845–G855.
Chatterjee PK, Cuzzocrea S, Brown PA, Zacharowski K, Stewart KN, Mota-Filipe H, and Thiemermann C (2000) Tempol, a membrane-permeable radical scavenger, reduces oxidant stress-mediated renal dysfunction and injury in the rat. Kidney Int 58: 658–673.[CrossRef][Medline]
Eder MI, Miquel JF, Jongst D, Paumgartner G, and Von Ritter C (1996) Reactive oxygen metabolites promote cholesterol crystal formation in model bile: role of lipid peroxidation. Free Radic Biol Med 20: 743–749.[CrossRef][Medline]
Esrefoglu M, Gul M, Emre MH, Polat A, and Selimoglu MA (2005) Protective effect of low dose of melatonin against cholestatic oxidative stress after common bile duct ligation in rats. World J Gastroenterol 11: 1951–1956.[Medline]
Gomez-Pinilla PJ, Camello-Almaraz C, Moreno R, Camello PJ, and Pozo MJ (2006a) Melatonin treatment reverts age-related changes in guinea pig gallbladder neuromuscular transmission and contractility. J Pharmacol Exp Ther 319: 847–856.
Gomez-Pinilla PJ, Morales S, Camello-Almaraz C, Moreno R, Pozo MJ, and Camello PJ (2006b) Changes in guinea pig gallbladder smooth muscle Ca2+ homeostasis by acute acalculous cholecystitis. Am J Physiol Gastrointest Liver Physiol 290: G14–G22.
Head KA and Jurenka JS (2003) Inflammatory bowel disease Part 1: ulcerative colitis–pathophysiology and conventional and alternative treatment options. Altern Med Rev 8: 247–283.[Medline]
Hissin PJ and Hilf R (1976) A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochem 74: 214–226.[CrossRef][Medline]
Karasek M (2004) Melatonin, human aging, and age-related diseases. Exp Gerontol 39: 1723–1729.[CrossRef][Medline]
Konturek PC, Konturek SJ, Brzozowski T, Dembinski A, Zembala M, Mytar B, and Hahn EG (1997) Gastroprotective activity of melatonin and its precursor, L-tryptophan, against stress-induced and ischaemia-induced lesions is mediated by scavenge of oxygen radicals. Scand J Gastroenterol 32: 433–438.[Medline]
Krishna MC, Russo A, Mitchell JB, Goldstein S, Dafni H, and Samuni A (1996) Do nitroxide antioxidants act as scavengers of 
or as SOD mimics? J Biol Chem 271: 26026–26031.
Kvetnoy IM, Ingel IE, Kvetnaia TV, Malinovskaya NK, Rapoport SI, Raikhlin NT, Trofimov AV, and Yuzhakov VV (2002) Gastrointestinal melatonin: cellular identification and biological role. Neuro Endocrinol Lett 23: 121–132.[Medline]
Lane EA and Moss HB (1985) Pharmacokinetics of melatonin in man: first pass hepatic metabolism. J Clin Endocrinol Metab 61: 1214–1216.[Abstract]
Maggi CA, Santicioli P, Renzi D, Patacchini R, Surrenti C, and Meli A (1989) Release of substance P- and calcitonin gene-related peptide-like immunoreactivity and motor response of the isolated guinea pig gallbladder to capsaicin. Gastroenterology 96: 1093–1101.[Medline]
Mawe GM and Gershon MD (1989) Structure, afferent innervation, and transmitter content of ganglia of the guinea pig gallbladder: relationship to the enteric nervous system. J Comp Neurol 283: 374–390.[CrossRef][Medline]
Mawe GM, Saccone GT, and Pozo MJ (2006) Neural control of the gallbladder and sphincter of Oddi, in Physiology of the Gastrointestinal Tract (Johnson LR, Barret KE, Ghishan FK, Merchant JL, Said HM, and Wood JD eds) pp 841–849, Elsevier Academic Press, San Diego, CA.
McKirdy ML, Johnson CD, and McKirdy HC (1994a) Inflammation impairs neurally mediated responses to electrical field stimulation in isolated strips of human gallbladder muscle. Dig Dis Sci 39: 2229–2234.[CrossRef][Medline]
McKirdy ML, McKirdy HC, and Johnson CD (1994b) Non-adrenergic non-cholinergic inhibitory innervation shown by electrical field stimulation of isolated strips of human gall bladder muscle. Gut 35: 412–416.
Mehta SH, Webb RC, Ergul A, Tawfik A, Dorrance AM, and Tawak A (2004) Neuroprotection by tempol in a model of iron-induced oxidative stress in acute ischemic stroke. Am J Physiol Regul Integr Comp Physiol 286: R283–R288.
Mourelle M, Guarner F, Molero X, Moncada S, and Malagelada JR (1993) Regulation of gall bladder motility by the arginine-nitric oxide pathway in guinea pigs. Gut 34: 911–915.
Ohta Y, Imai Y, Matsura T, Yamada K, and Tokunaga K (2005) Successively postadministered melatonin prevents disruption of hepatic antioxidant status in rats with bile duct ligation. J Pineal Res 39: 367–374.[CrossRef][Medline]
Parkman HP, Bogar LJ, Bartula LL, Pagano AP, Thomas RM, and Myers SI (1999a) Effect of experimental acalculous cholecystitis on gallbladder smooth muscle contractility. Dig Dis Sci 44: 2235–2243.[CrossRef][Medline]
Parkman HP, James AN, Bogar LJ, Bartula LL, Thomas RM, Ryan JP, and Myers SI (2000) Effect of acalculous cholecystitis on gallbladder neuromuscular transmission and contractility. J Surg Res 88: 186–192.[CrossRef][Medline]
Parkman HP, Pagano AP, Martin JS, and Ryan JP (1997) Electric field stimulation-induced guinea pig gallbladder contractions: role of calcium channels in acetylcholine release. Dig Dis Sci 42: 1919–1925.[CrossRef][Medline]
Parkman HP, Pagano AP, and Ryan JP (1999b) Subtypes of muscarinic receptors regulating gallbladder cholinergic contractions. Am J Physiol 276: G1243–G1250.[Medline]
Pozo MJ, Camello PJ, and Mawe GM (2004) Chemical mediators of gallbladder dysmotility. Curr Med Chem 11: 1801–1812.[Medline]
Reiter RJ (1998) Cytoprotective properties of melatonin: presumed association with oxidative damage and aging. Nutrition 14: 691–696.[CrossRef][Medline]
Reiter RJ, Tan DX, Mayo JC, Sainz RM, Leon J, and Bandyopadhyay D (2003) Neurally-mediated and neurally-independent beneficial actions of melatonin in the gastrointestinal tract. J Physiol Pharmacol 54 (Suppl 4): 113–125.[Medline]
Sann H, Dux M, Schemann M, and Jancso G (1996) Neurogenic inflammation in the gastrointestinal tract of the rat. Neurosci Lett 219: 147–150.[CrossRef][Medline]
Shiesh SC, Chen CY, Lin XZ, Liu ZA, and Tsao HC (2000) Melatonin prevents pigment gallstone formation induced by bile duct ligation in guinea pigs. Hepatology 32: 455–460.[CrossRef]
Stewart T, Beyak MJ, and Vanner S (2003) Ileitis modulates potassium and sodium currents in guinea pig dorsal root ganglia sensory neurons. J Physiol 552: 797–807.
Talmage EK, Pouliot WA, Cornbrooks EB, and Mawe GM (1992) Transmitter diversity in ganglion cells of the guinea pig gallbladder: an immunohistochemical study. J Comp Neurol 317: 45–56.[CrossRef][Medline]
Tan D, Manchester LC, Reiter RJ, Qi W, Hanes MA, and Farley NJ (1999) High physiological levels of melatonin in the bile of mammals. Life Sci 65: 2523–2529.[CrossRef][Medline]
Tan DX, Reiter RJ, Manchester LC, Yan MT, El Sawi M, Sainz RM, Mayo JC, Kohen R, Allegra M, and Hardeland R (2002) Chemical and physical properties and potential mechanisms: melatonin as a broad spectrum antioxidant and free radical scavenger. Curr Top Med Chem 2: 181–197.[CrossRef][Medline]
Waller RL and Recknagel RO (1977) Determination of lipid conjugated dienes with tetracyanoethylene-14C: significance for study of the pathology of lipid peroxidation. Lipids 12: 914–921.[CrossRef][Medline]
Xiao ZL, Chen Q, Biancani P, and Behar J (2001) Abnormalities of gallbladder muscle associated with acute inflammation in guinea pigs. Am J Physiol Gastrointest Liver Physiol 281: G490–G497.
Yau WM and Youther ML (1984) Modulation of gallbladder motility by intrinsic cholinergic neurons. Am J Physiol Gastrointest Liver Physiol 247: G662–G666.
Yoshimura N, Seki S, Novakovic SD, Tzoumaka E, Erickson VL, Erickson KA, Chancellor MB, and de Groat WC (2001) The involvement of the tetrodotoxin-resistant sodium channel Na(v)1.8 (PN3/SNS) in a rat model of visceral pain. J Neurosci 21: 8690–8696.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
, P < 0.05 MEL30 versus AC; and 
, P < 0.01 MEL 30 versus AC; ANOVA).
