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Am J Physiol Gastrointest Liver Physiol 290: G14-G22, 2006. First published August 18, 2005; doi:10.1152/ajpgi.00284.2005
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NEUROREGULATION AND MOTILITY

Changes in guinea pig gallbladder smooth muscle Ca2+ homeostasis by acute acalculous cholecystitis

Pedro J. Gomez-Pinilla, Sara Morales, Cristina Camello-Almaraz, Rosario Moreno, María J. Pozo, and Pedro J. Camello

Department of Physiology, Nursing School, University of Extremadura, Caceres, Spain

Submitted 24 June 2005 ; accepted in final form 12 August 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Impaired smooth muscle contractility is a hallmark of acute acalculous cholecystitis. Although free cytosolic Ca2+ ([Ca2+]i) is a critical step in smooth muscle contraction, possible alterations in Ca2+ homeostasis by cholecystitis have not been elucidated. Our aim was to elucidate changes in the Ca2+ signaling pathways induced by this gallbladder dysfunction. [Ca2+]i was determined by epifluorescence microscopy in fura 2-loaded isolated gallbladder smooth muscle cells, and isometric tension was recorded from gallbladder muscle strips. F-actin content was quantified by confocal microscopy. Ca2+ responses to the inositol trisphosphate (InsP3) mobilizing agonist CCK and to caffeine, an activator of the ryanodine receptors, were impaired in cholecystitic cells. This impairment was not the result of a decrease in the size of the releasable pool. Inflammation also inhibited Ca2+ influx through L-type Ca2+ channels and capacitative Ca2+ entry induced by depletion of intracellular Ca2+ pools. In addition, the pharmacological phenotype of these channels was altered in cholecystitic cells. Inflammation impaired contractility further than Ca2+ signal attenuation, which could be related to the decrease in F-actin that was detected in cholecystitic smooth muscle cells. These findings indicate that cholecystitis decreases both Ca2+ release and Ca2+ influx in gallbladder smooth muscle, but a loss in the sensitivity of the contractile machinery to Ca2+ may also be responsible for the impairment in gallbladder contractility.

contractility; calcium stores; calcium channels; F-actin; calcium ion

THE KEY EVENTS THAT MODULATE smooth muscle contraction include increases in the cytosolic Ca2+ concentration ([Ca2+]i) and phosphorylation of the regulatory light chains of myosin II by myosin light chain kinase (32). Changes in [Ca2+]i are regulated through the control of Ca2+ transport mechanisms across plasma and intracellular store membranes (4). Because of the wide spectrum of Ca2+ regulatory actions, alterations of [Ca2+]i signals can have deep impact on cell function. Thus Ca2+ deregulation has been reported to participate in conditions like cell death (21) or inflammation (42). However, information regarding the nature of changes of [Ca2+]i signals during inflammatory processes in smooth muscle is rather scarce. In gastrointestinal smooth muscle, there is evidence that inflammation attenuates Ca2+ signals through changes in plasma membrane ion channel expression (11) and regulation (12). By contrast, there are conflicting reports on the effects of inflammation on Ca2+ release from intracellular stores (7, 26, 29).

Acute inflammation in the absence of gallstones, a pathological condition commonly referred to as acute acalculous cholecystitis, is an increasingly prevalent complication among individuals in the intensive care unit and in patients without predisposing illness (2). The pathogenesis of this disease is unclear. It has been speculated that impaired muscle contractility is secondary to inflammation and may play a role in the clinic pathology of acute acalculous cholecystitis (8, 23, 24). Previous reports using functional methods have described that, in gallbladder smooth muscle, the main targets for inflammation are ion channels and G protein-coupled receptors of the plasma membrane, whereas intracellular pathways [inositol trisphosphate (InsP3) receptors, protein kinase C, and G proteins] are not affected (43). However, the effects of acute acalculous cholecystitis on gallbladder smooth muscle Ca2+ signals have not been tested previously. The aim of this study was to explore the alterations of Ca2+ signals during cholecystitis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Model of acute acalculous cholecystitis. Acute acalculous cholecystitis was induced in male guinea pigs by ligation of the common bile duct for 2 days. This method has been previously shown to reproduce the pathological changes seen in human acute acalculous cholecystitis (20, 23, 24, 43, 44), and it was approved by the Ethical Committee of the University of Extremadura. A laparotomy was performed under anesthesia with ketamine hydrochloride (20 mg/kg ip) and xylacine (5 mg/kg ip). Next, the distal end of the common bile duct was ligated (4–0 silk) at its junction with the duodenum using minimal manipulation of the bile duct and no manipulation of the gallbladder. Under these conditions, there was no interruption of the blood supply to the gallbladder. The surgical incision was then sutured. A group of control animals were sham operated, which included all the surgical steps except for the common bile duct ligation. After the operative procedures, the animals were housed separately, and they were provided with food and water ad libitum. Later (2 days), the animals were killed with deep halothane anesthesia and cervical dislocation for tissue harvest. Gallbladders were immediately placed in cold Krebs-Henseleit solution (K-HS; for composition, see Solutions and drugs) at pH 7.35. The gallbladder was opened from the end of the cystic duct to the base, and the mucosa was dissected away carefully.

Cell isolation. Gallbladder smooth muscle cells were dissociated enzymatically using a previously described method (25). Briefly, after preparing the tissue as indicated above, the gallbladder was cut into small pieces and incubated for 34 min at 37°C in enzyme solution (ES, for composition, see Solutions and drugs) supplemented with 1 mg/ml BSA, 1 mg/ml papain, and 1 mg/ml dithioerythritol. Next, the tissue was transferred to fresh ES containing 1 mg/ml BSA, 1 mg/ml collagenase, and 100 µM CaCl2 and incubated for 9 min at 37°C. The tissue was then washed three times using ES, and the single smooth muscle cells were isolated by several passages of the tissue pieces through the tip of a fire-polished glass Pasteur pipette. The resultant cell suspension was kept in ES at 4°C until use, generally within 6 h. Cell viability was routinely checked by trypan blue staining of cells (85 ± 5 and 83 ± 7% of viability for control and cholecystitic cell suspensions, respectively). All experiments involving isolated cells were performed at room temperature (22°C).

Cell loading and [Ca2+]i determination. [Ca2+]i was determined by epifluorescence microscopy using the fluorescent ratiometric Ca2+ indicator fura 2. Isolated cells were loaded with 4 µM fura 2-AM at room temperature for 25 min. An aliquot of cell suspension was placed in an experimental chamber made with a glass poly-D-lysine-treated cover slip (0.17 mm thick) filled with Na+-HEPES solution (for composition, see Solutions and drugs) and mounted on the stage of an inverted microscope (Eclipse TE2000-S; Nikon). After cell sedimentation, a gravity-fed system was used to perfuse the chamber with Na+-HEPES solution in the absence or presence of experimental agents. Cells were illuminated at 340 and 380 nm by a computer-controlled monochromator (Optoscan; Cairn Research) at 0.3–1 cycles/s, and the emitted fluorescence was selected by a 510/40-nm bandpass filter. The emitted fluorescence images were captured with a cooled digital charge-coupled device camera (ORCAII-ER; Hamamatsu Photonics) and recorded using dedicated software (Metafluor; Universal Imaging). The ratio of fluorescence at 340 nm to fluorescence at 380 nm (F340/F380) was calculated pixel by pixel and used to indicate the changes in [Ca2+]i. A calibration of the ratio for [Ca2+]i was not performed in view of the many uncertainties related to the binding properties of fura 2 with Ca2+ inside of smooth muscle cells.

Contraction recording of guinea pig gallbladder strips. Gallbladder strips (measuring ~3 x 10 mm) were mounted vertically in a 10-ml organ bath filled with K-HS 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 Spring, CO). The muscle strips were placed under an initial resting tension equivalent to 1.5 g load and allowed to equilibrate for 60 min, with solution changes every 20 min. Every strip coming from the same animal was used in a different experimental protocol. The direct effects of CCK, ionomycin, or KCl on gallbladder tone were studied by addition of these agents to the organ bath.

F-actin content measurement. The F-actin content of control and cholecystitic gallbladder smooth muscle cells was determined according to a previously published procedure (17). Briefly, samples of cell suspensions (200 µl) were placed in Na+-HEPES solution and quickly transferred to 200 µl ice-cold 3% (wt/vol) formaldehyde in PBS solution (for composition, see Solutions and drugs) for 10 min. Fixed cells were permeabilized by incubation for 10 min with 0.025% (vol/vol) Nonidet P-40 detergent dissolved in PBS. Cells were then incubated for 30 min with FITC-labeled phalloidin (FITC-phalloidin; 1 µM) in PBS solution supplemented with 0.5% (wt/vol) BSA. After incubation, the cells were collected by centrifugation for 2 min at 10,000 g and resuspended in PBS solution. Staining of actin filaments was measured using a confocal laser-scanning system (model MRC-1024; Bio-Rad) with excitation wavelength of 488 nm and emission at 515 nm. The cellular F-actin content was quantified as arbitrary units of fluorescence using the ImageJ software.

Solutions and drugs. The K-HS contained (in mM): 113 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, and 11.5 D-glucose. This solution had a final pH of 7.35 after equilibration with 95% O2-5% CO2. The ES used to disperse cells was made up of (in mM): 10 HEPES, 55 NaCl, 5.6 KCl, 80 sodium glutamate, 2 MgCl2, and 10 D-glucose, with pH adjusted to 7.3 with NaOH. The Na+-HEPES solution contained (in mM): 10 HEPES, 140 NaCl, 4.7 KCl, 2 CaCl2, 2 MgCl2, and 10 D-glucose, with pH adjusted to 7.3 with NaOH. The Ca2+-free Na+-HEPES solution was prepared by substituting EGTA (1 mM) for CaCl2. The PBS solution used in F-actin studies contained (in mM): 137 NaCl, 2.7 KCl, 5.62 Na2HPO4, 1.09 NaH2PO4, and 1.47 KH2PO4, with pH adjusted to 7.2. Drug concentrations are expressed as final bath concentrations of active species. Drugs and chemicals were obtained from the following sources: (±)BAY K 8644, caffeine, CCK-(26–33) sulfated, 1,4-dithio-DL-threitol, thapsigargin, FITC-phalloidin, nitrendipine, pinacidil, and trypan blue were from Sigma Chemical (St. Louis, MO); 2-aminoethoxydiphenylborane (2-APB) was from Tocris (Bristol, UK); 2,3,6-tri-O-butyryl-myo-inositol-1,4,5-trisphosphate-hexakis(propionoxymethyl)ester [Bt3-Ins(1,4,5)P3-PM] was from SiChem (Bremen, Germany), fura 2-AM was from Molecular Probes (Molecular Probes Europe, Leiden, Netherlands); collagenase was from Fluka (Madrid, Spain); and papain was from Worthington Biochemical (Lakewood, NJ). Other chemicals used were of analytical grade from Panreac (Barcelona, Spain). Stock solutions of fura 2-AM, thapsigargin, ionomycin, pinacidil, and 2-APB were prepared in DMSO, and (±)BAY K 8644, nitrendipine, and FITC-phalloidin were prepared in ethanol. The solutions were diluted such that the final concentrations of DMSO or ethanol were ≤0.1% vol/vol. These concentrations of solvents did not interfere with fura 2 fluorescence.

Quantification and statistics. Results are expressed as means ± SE of n cells or gallbladder strips. All results from [Ca2+]i determinations are given as {Delta}F340/F380. Gallbladder tension is given in millinewtons per milligram of tissue. Statistical differences between means were determined by Student's t-test. Differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
To characterize Ca2+ dynamics in cholecystitis, we challenged gallbladder smooth muscle cells with different types of stimuli: CCK, the natural agonist for gallbladder contraction, and caffeine, an activator of ryanodine receptors (RyR), were used to release Ca2+ from intracellular stores, whereas KCl-rich medium and activation of capacitative Ca2+ entry were used to generate Ca2+ signals via plasma membrane Ca2+ entry.

Cholecystitis-induced changes in Ca2+ release from intracellular stores. CCK (10 nM) induced a fast [Ca2+]i response comprising an initial rise due to Ca2+ release from internal stores (15) and a subsequent plateau, slightly higher than resting [Ca2+]i values (Fig. 1A), which is entirely dependent on influx of extracellular Ca2+ (15, 16). As shown in Fig. 1A, cholecystitis decreased the initial peak by ~50% (control: 0.36 ± 0.05 {Delta}F340/F380, cholecystitis: 0.180 ± 0.044 {Delta}F340/F380, n = 36 and 14 cells from 5 and 3 animals, respectively, P < 0.05). To more directly test whether Ca2+ release through InsP3 receptor was impaired by inflammation, we used 10 µM Bt3-Ins(1,4,5)P3-PM, a membrane permeable analog of InsP3 (27), in a Ca2+-free medium, which caused a Ca2+ transient similar to that reached after CCK treatment (0.426 ± 0.030 {Delta}F340/F380, n = 13 cells from 3 animals; Fig. 1B) without the sustained plateau as Ca2+ influx. This transient was significantly reduced in smooth muscle cells from cholecystitic animals (0.203 ± 0.028 {Delta}F340/F380, n = 15 cells from 3 animals, P < 0.001; Fig. 1B). Caffeine, at millimolar concentration, releases Ca2+ via RyR in several muscle types, including gallbladder smooth muscle (16). Similar to CCK, application of caffeine induced a fast [Ca2+]i peak that returned immediately to the resting value (Fig. 1C). As was the case for CCK, inflammation induced a significant inhibition of caffeine-evoked peak (control: 0.465 ± 0.034 {Delta}F340/F380, cholecystitis: 0.208 ± 0.031 {Delta}F340/F380, n = 11 and 26 cells from 4 and 3 animals, respectively, P < 0.05; Fig. 1C).



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  		Fig. 1. Cholecystitis reduces transient cytosolic Ca2+ concentration ([Ca2+]i) responses to store depletion but does not change Ca2+-releasable pool size. Representative [Ca2+]i responses to 10 nM CCK (A), 10 µM 2,3,6-tri-O-butyryl-myo-inositol-1,,4,5-trisphosphate-hexakis(propionoxymethyl)ester [Bt3-Ins(1,4,5)P3; B], and 10 mM caffeine (C) in fura-loaded gallbladder smooth muscle cells from control (solid line) and bile duct-ligated (dotted line) animals. D: a low concentration of ionomycin (50 nM) was used to estimate the Ca2+ content of intracellular stores. Gallbladder smooth muscle cells were treated with a Ca2+-free solution during 45 s before application of ionomycin. The resulting transient in Ca2+ is the result of Ca2+ release from intracellular stores. No difference was observed between control and acute acalculous cholecystitis cells. Traces are typical of 8–36 cells from 3–5 animals. F340/F380, ratio of fluorescence at 340 and 380 nm.

 
Taken together, the results described above suggest that cholecystitis may reduce the Ca2+ pool that is mobilized by InsP3 and RyR agonists. This impairment could be because of a decrease in the Ca2+ store content or a reduction in InsP3 receptor and RyR sensitivity. To discriminate between these possibilities, we applied a low level of ionomycin in Ca2+-free solution, a treatment that releases the Ca2+ stores while bypassing channels and receptors (18). Figure 1D shows that [Ca2+]i transients induced by 50 nM ionomycin in control and inflamed cells were similar (control: 0.188 ± 0.029 {Delta}F340/F380, cholecystitis: 0.181 ± 0.017 {Delta}F340/F380, n = 14 and 8 cells from 3 animals for both groups, not significant), indicating that the amount of Ca2+ in the stores was not affected by inflammation.

Cholecystitis-induced changes in Ca2+ influx. In gallbladder smooth muscle and other excitable tissues, Ca2+ entry plays a major role in Ca2+ signals. In the case of the gallbladder, extracellular Ca2+ mainly enters via L-type voltage-operated Ca2+ channels (1, 28, 30). Therefore, we explored how cholecystitis affects voltage-activated Ca2+ entry. Application of a depolarizing medium containing 60 mM KCl induced a sustained [Ca2+]i increase (0.28 ± 0.03 {Delta}F340/F380, n = 13 cells from 6 animals; Fig. 2A) that was sensitive to the L-type channel blocker nitrendipine (1 µM; 0.014 ± 0.002 {Delta}F340/F380, 95% inhibition, n = 13 cells from 3 animals, P < 0.001; Fig. 2B). As expected, when cells were hyperpolarized by the treatment with the ATP-sensitive K+ channel opener pinacidil (10 µM) KCl-induced influx was almost abolished (0.031 ± 0.005 {Delta}F340/F380, 89% inhibition, n = 14 cells from 3 animals, P < 0.001; Fig. 2C), indicating that voltage-operated Ca2+ channels were activated by KCl. Inflammation decreased the KCl-evoked Ca2+ response to 57% of control values (0.12 ± 0.01 {Delta}F340/F380, n = 8 cells from 5 animals, P < 0.05; Fig. 2A). This reduction parallels the changes in the phenotype and/or functional state of L-type Ca2+ channels as indicated by the lack of effect of nitrendipine (0.088 ± 0.01 {Delta}F340/F380, n = 12 cells from 3 animals; Fig. 2B) or pinacidil (0.086 ± 0.01 {Delta}F340/F380, n = 7 cells from 3 animals; Fig. 2C) on the KCl-evoked [Ca2+]i signal in inflamed cells. To specifically activate L-type Ca2+ channels, we used 1 µM (±)BAY K 8644, a dihydropyridine that stabilizes the L-type Ca2+ channel in a gating mode with long channel openings and short closings, thus activating selectively Ca2+ influx through these channels (9). Inflammation reduced (±)BAY K 8644-induced Ca2+ influx to 59% of control values (control: 0.192 ± 0.007 {Delta}F340/F380, cholecystitis: 0.102 ± 0.011 {Delta}F340/F380, n = 17 and 12 cells from 3 animals, respectively, P < 0.001). Similar to its effect on KCl-induced Ca2+ influx, nitrendipine did not modify the (±)BAY K 8644-induced Ca2+ increase in inflamed cells but reduced this response in control cells by 83% (control: 0.033 ± 0.003 {Delta}F340/F380, cholecystitis: 0.101 ± 0.014 {Delta}F340/F380, n = 7 cells from 3 animals in each group).



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  		Fig. 2. Cholecystitis reduces Ca2+ influx through L-type Ca2+ channels. A: [Ca2+]i signal in response to a depolarizing solution containing 60 mM KCl and 2 mM Ca2+ in control (solid line) and inflamed (dotted line) cells. As observed, cholecystitis reduced the plateau response. In B and C, cells were pretreated with the inhibitor of L-type Ca2+ channels nitrendipine (1 µM) and the ATP-dependent K+ channel opener pinacidil (10 µM). Note the lack of effect of these inhibitors on cholecystitic cells. Traces are typical of 7–36 cells from 3–6 animals.

 
In addition to working via L-type Ca2+ channels, we have recently demonstrated that capacitative Ca2+ entry also mediates [Ca2+]i signals in response to Ca2+ mobilization from intracellular stores (15), supporting the sustained phase of [Ca2+]i signals in response to agonists in gallbladder smooth muscle cells. This mechanism is more complex in gallbladder smooth muscle than in other systems because depletion of stores initiates Ca2+ influx through the following two pathways: voltage-independent "classical" capacitative Ca2+ channels and coactivation of L-type Ca2+ channels (15). To activate this mechanism and test the effects of inflammation, we followed the protocol shown in Fig. 3A where cells were pretreated with 1 µM thapsigargin, a specific inhibitor of the Ca2+ ATPase of the stores (35), during 30 min in a Ca2+-free medium to fully deplete the stores. The reintroduction of Ca2+ in the bath resulted in a sustained [Ca2+]i plateau because of Ca2+ entry from the extracellular medium, which is frequently used as index of capacitative Ca2+ entry (6, 15, 17). This protocol induces a persistent activation of capacitative Ca2+ entry and allows repeated measurements of Ca2+ entry upon restoration of extracellular [Ca2+] (Fig. 3A). Capacitative Ca2+ entry was clearly impaired by inflammation (control: 0.095 ± 0.005 {Delta}F340/F380, cholecystitis: 0.062 ± 0.005 {Delta}F340/F380, n = 17 and 49 cells from 8 and 11 animals, respectively, P < 0.05; Fig. 3A). In addition, the pharmacology of the remaining capacitative Ca2+ entry was also altered in inflamed cells. Thus, although in normal cells capacitative Ca2+ entry was sensitive to the well-recognized blocker of classical capacitative Ca2+ channels [2-APB (100 µM) and to nitrendipine (51 and 57% of reduction, respectively)], the residual capacitative Ca2+ entry present in cholecystitic cells was insensitive to both of these compounds (0.069 ± 0.012 {Delta}F340/F380 vs. 0.078 ± 0.007 and 0.062 ± 0.009 vs. 0.068 ± 0.016 for nitrendipine and 2-APB, respectively, n = 8–11 cells from 3–6 animals; Fig. 3, B and C). This was also the case when nitrendipine and 2-APB were applied together, a condition that almost blocked capacitative Ca2+ entry in control cells (89% inhibition; Fig. 3D). However, in cholecystitis, nitrendipine- and 2-APB-insensitive capacitative Ca2+ entry was blocked by gadolinium (52% inhibition, 0.059 ± 0.008 {Delta}F340/F380 vs. 0.031 ± 0.007 {Delta}F340/F380, n = 10 cells from 3 animals, P < 0.05), a trivalent cation widely used to block capacitative channels in different cellular types, including excitable tissues (10, 31, 36).



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  		Fig. 3. Capacitative Ca2+ entry is impaired in gallbladder smooth muscle cells from animals with acute cholecystitis. A: after application of a Ca2+-free medium with the Ca2+ pump inhibitor thapsigargin (Tps, 1 µM), 2 mM external Ca2+ was restored to initiate Ca2+ entry, evidenced by a fast-plateau [Ca2+]i. Another similar capacitative plateau was then obtained upon Ca2+ removal and readmission. As shown in the traces on top, capacitative Ca2+ entry was decreased in inflamed cells (dotted line). B-D: changes in [Ca2+]i under the same protocol but after application of 100 µM 2-aminoethoxydiphenylborane (2-APB), 1 µM nitrendipine, and 2-APB plus nitrendipine in control (solid lines) and cholecystitic (dotted lines) cells before the second entry episode. None of these treatments blocked capacitative entry in cholecystitis, but they evoked a significant reduction in control cells. Traces are typical of 8–49 cells from 3–11 animals.

 
Taken together, our results show that both Ca2+ release from the stores and Ca2+ influx from extracellular medium are impaired by acute cholecystitis. Given that inflammation could alter fura 2 behavior because of changes in intracellular parameters (e.g., viscosity, pH,...), we compared the ratio of fura 2 fluorescence in response to a high level of ionomycin (1 µM) in the presence of extracellular Ca2+. At this concentration, ionomycin acts as a Ca2+ ionophore; thus, [Ca2+]i is elevated independently of channels and receptors. Figure 4 shows that the sustained plateau evoked by this treatment was not affected in inflamed cells (control: 0.356 ± 0.050 {Delta}F340/F380, cholecystitis: 0.306 ± 0.075 {Delta}F340/F380, n = 32 and 18 cells from 5 and 3 animals, respectively, not significant), ruling out the possibility that cholecystitis introduces an artifact in [Ca2+]i determinations.



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  		Fig. 4. Cholecystitis does not induce artifacts in Ca2+ determination. Control (solid line) and inflamed (dotted line) cells were treated with 1 µM ionomycin in 2 mM Ca2+ medium to induce a sustained [Ca2+]i increase independent of channel and receptor activation. No difference was observed in response to cholecystitis. Traces are representative of 32 and 18 cells from 5 and 3 animals for control and cholecystitis, respectively.

 
Cholecystitis-induced changes in gallbladder contractility. [Ca2+]i is a major determinant of smooth muscle contraction; therefore, we investigated the effect of cholecystitis on gallbladder contractility to evaluate the association between impairment of [Ca2+]i signals and contractile deficit in inflammation. Figure 5, A -C, shows that, similar to [Ca2+]i signals, the contraction induced by CCK, KCl, and capacitative Ca2+ entry were also impaired, although to a greater extent than their corresponding Ca2+ signals, which could indicate a decrease in the Ca2+ sensitivity of contractile machinery. To check this, we assayed the contractile response to ionomycin since, as described above, inflammation did not change the ionomycin-induced [Ca2+]i signal. However, the same [Ca2+]i elevation induced a much lower contraction in cholecystitic strips than in control tissue (control: 3.12 ± 0.57 mN/mg, cholecystitis: 0.72 ± 0.157 mN/mg, n = 7 and 8 strips from 4 and 5 animals, respectively, P < 0.001; Fig. 5D), which reveals that this disorder can also alter the contractile machinery independent of the Ca2+ signal. To investigate one of the possible causes of this impairment in contractility, we determined the total amount of the contractile protein F-actin labeling gallbladder smooth muscle cells with FITC-stained phalloidin. We found that cholecystitis induced a statistically significant decrease in the actin content (control: 38.93 ± 1.93, cholecystitis: 29.57 ± 3.78, data as arbitrary units of fluorescence, n = 16 and 13 cells from 5 and 3 animals, respectively, P < 0.05).



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  		Fig. 5. Cholecystitis induces a general impairment in gallbladder contractile response. Gallbladder smooth muscle strips from control and cholecystitic animals were challenged with 10 nM CCK (A), 60 mM KCl (B), capacitative Ca2+ entry protocol (C), or 1 µM ionomycin in Ca2+-containing solution (D), and isometric contraction was recorded. Inflammation induced a clear impairment in the contraction for all the treatments. Traces are representative of 37–49 strips from 4–8 animals.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We present here data showing that acute acalculous cholecystitis evoked by bile duct ligation induces loss of Ca2+ signals in response to physiological and pharmacological stimuli, involving a downregulation of Ca2+ release and Ca2+ entry mechanisms.

Bile duct ligation is a commonly used model of acute acalculous cholecystitis (23, 43). This disease is accompanied by a loss of contractile response to agonists and intrinsic nerve stimulation and gallbladder stasis (23, 24). Our results showing that cholecystitis impairs both contraction and [Ca2+]i signals evoked by CCK and depolarization could explain, at least in part, this loss of contractility. The contractile impairment was detected not only in response to agonists and depolarization but also when the Ca2+ increase was achieved by pharmacological release of intracellular Ca2+ stores or by ionophore treatment. However, the contractile impairment likely involves additional mechanisms, as evidenced by the decrease of a high-concentration ionomycin-evoked contraction. A possibility is that deregulation of Ca2+ signals or some inflammatory mediators alter the expression of proteins involved in contraction, as suggested by the reduction in F-actin in cholecystitic cells.

Impairment of Ca2+ signals during inflammation occurs at least at two levels: decrease of Ca2+ release from pools and decrease of Ca2+ influx through store-operated channels and voltage-operated L channels. In the case of stores, our data point to a decrease in RyR and/or InsP3 receptor sensitivity to its agonists, since caffeine and the membrane permeable analog of InsP3, Bt3-Ins(1,4,5)P3-PM, released less Ca2+ from intracellular stores in cholecystitic cells. Given the similar effects of a low concentrations of ionomycin in control and inflamed cells, a reduction in the Ca2+ content of the stores cannot explain the impairment in Ca2+ release. Because the assayed agonists act directly on the RyR and InsP3 receptor and we measure directly the released Ca2+, alterations in the plasma membrane or G proteins could not be responsible for the reduced response to caffeine or to the membrane permeable analog of InsP3. It has been previously suggested that the muscle defect associated with acute inflammation in the gallbladder is located in the plasma membrane, with the signal transduction distal to the membrane receptors unaffected (43). In that study, similar concentration-response curves were obtained when the contractile effect of InsP3 was assayed in control and cholecystitic gallbladder smooth muscle cells, which could suggest that Ca2+ release through InsP3 receptors was totally preserved in inflammatory conditions. However, our study clearly demonstrates that the release of Ca2+ through InsP3 receptor stimulated by either CCK or Bt3-Ins(1,4,5)P3-PM is decreased in inflammation. Although the reduction in Bt3-Ins(1,4,5)P3-PM is only explained by alterations in the InsP3 receptor, as discussed above, the impaired CCK-mediated [Ca2+]i release could also reflect concurrent damage occurring at any place between receptor binding and Ca2+ release mechanisms.

KCl-induced Ca2+ influx in control gallbladder smooth muscle cells is sensitive to nitrendipine blockade, which means that KCl activates mainly L-type Ca2+ channels in the gallbladder. The reduction in KCl-induced Ca2+ transient by cholecystitis could be the result of the decrease in the L-type channel expression as described in inflamed circular smooth muscle of the canine colon (14) or by changes in the channel structure. However, the most striking feature of inflammation-evoked Ca2+ imbalance is the change of functional and pharmacological profiles of plasma membrane Ca2+ entry pathways. Cholecystitis induced not only a loss of voltage-operated L-type Ca2+ influx, as judged by the decrease of KCl- and BAY K 8644-induced [Ca2+]i response, but also a change in the identity or at least the functional state of the channels activated, as evidenced by the insensitivity to nitrendipine. Two different classes of high-voltage-activated Ca2+ channels can be distinguished pharmacologically: L-type Ca2+ channels, which are highly sensitive to dihydropyridine agonists and antagonists, and non-L-type channels (P type, Q type, N type, and R type). The latter, which play a prominent role for fast neurotransmitter release in neurons, do not display dihydropyridine sensitivity but are selectively blocked by different peptide enzymes (5). This difference in dihydropyridine sensitivity is the result of different {alpha}1-subunit isoforms that, together with accessory subunits such as {alpha}2, {delta}, and {beta}, form the Ca2+ channel complexes. The isoforms {alpha}1S and {alpha}1C from Cav1.1 and Cav1.2 L-type channels (expressed in skeletal and cardiac/smooth muscles, respectively) present a dihydropyridine-binding domain that links with high affinity the dihydropyridine agonists and antagonists to promote or block current through the channels. The structural determinants of agonist and antagonist activity is the orientation of the pseudoaxial aryl group in the molecule. Enantiomers having an uporiented pseudoaxial aryl group are Ca2+ channel blockers, whereas downorientation results in channel activation (agonists; for review, see Ref. 34). In addition, individual amino acid residues that form the dihydropyridine pocket are required for agonist and antagonist activity (41). The fact the BAY K 8644 was able to induce Ca2+ influx in cholecystitic cells indicates that L-type Ca2+ channels are still functional in inflammation, although they are activated by the dihydropyridine agonist to a less extent than in control cells (similar to KCl-induced Ca2+ influx). The loss of antagonist sensitiveness could be explained by inflammation-mediated alterations in these specific amino acid residues that renders agonist activity. Another possible explanation for the lack of nitredipine effect is that, in inflammation, Cav1.2 L-type Ca2+ channels change to the Cav1.3 subtype, which, being expressed in many of the cells that express Cav1.2, are less sensitive to dihydropyridine antagonists and require weak depolarization to activate (13). This could explain that, in the presence of pinacidil, which counteracts the KCl-induced depolarization, we still observed KCl-mediated Ca2+ influx in inflamed cells. However, it could also be possible that inflammation induced impairment in ATP-dependent K+ channels and the consequent pinacidil-induced hyperpolarization. Thus, in the presence of pinacidil, KCl-induced depolarization would be the same as in the presence of KCl. Changes in L-type channels can be of particular importance for contraction, given that these channels are not only essential for initiation of contraction in response to neurotransmitters as ACh, but they also participate in the maintenance of contraction once the stores have been depleted by CCK (15). The change in the pharmacological phenotype of L-type Ca2+ channels in smooth muscle induced by inflammation would have important therapeutic implications.

Besides voltage-activated L-type Ca2+ channels, the capacitative Ca2+ route is also altered by cholecystitis. At present, the best molecular candidates for capacitative Ca2+ channels are the TRP proteins (so-called because of their homology with the Transient Receptor Potential protein that underlies phototransduction in Drosophila; see Ref. 37). In this regard, much attention has been focused on the canonical TRP (TRPC) subfamily. Data from our laboratory indicate that at least TRPC1 serves as a capacitative channel in gallbladder smooth muscle cells and that the transcription and expression of TRPC1–4 is modulated by Ca2+-dependent calmodulin kinase II/calcineurin-nuclear factor of activated T cells pathways (Morales, Camello, and Pozo, unpublished observations). The low level of capacitative Ca2+ entry displayed by cholecystitic cells is likely to be the result of the loss of these channels or changes in the subtype of TRP channels, since we observed a reduction in mRNA TRPC transcription and protein expression during cholecystitis (Morales, Camello, and Pozo, unpublished observations). In addition to this, inflammation also alters the nitrendipine-sensitive component of capacitative Ca2+ entry, in agreement with the alteration of KCl- and BAY K 8644-induced Ca2+ influx. The capacitative entry remaining in cholecystitic cells is sensitive to Gd3+. Although Gd3+ has been frequently used to block classical capacitative Ca2+ entry (10, 31, 36), it has also been described that this trivalent metal ion can block low- and high-voltage-activated Ca2+ channels (3). This broad spectrum of blocking multiple channels isoforms could be responsible for the success of Gd3+ on blocking capacitative Ca2+ entry when the possible isoforms or the functional state of the channels is different under inflammatory stress.

Taken together, it is clear that desensitization of Ca2+ pools and changes in the functional status of plasma membrane Ca2+ channels account for the extensive loss of Ca2+ signals and contraction observed in cholecystitis. However, it must be noted that the inflammation somehow alters contraction of cells independent of the [Ca2+]i signal, as evidenced by the reduction of the contractile response to ionomycin. Here, we show for the first time that inflammation can alter the amount of contractile proteins such as actin, which could be responsible for the reduction in the sensitivity of the contractile machinery to Ca2+. This finding could, at least in part, explain the dramatic loss of contractile response compared with the impairment in Ca2+ homeostasis. In agreement with our results, fluorescent staining for the actin cytoskeleton showed distortion and fragmentation in the intestinal epithelium and brush border of a murine dextran sodium sulfate-induced colitis model, which improved by antioxidant treatment (22). It has been recently reported that the content of cytoskeletal protein markers specific for smooth muscle (including actin) was not modified in primary and secondary cultures grown from control and cholecystitic guinea pig gallbladders (19). However, in that study, the analysis of cytoskeletal proteins was performed after at least 7–10 days (primary culture) or 10–14 days (secondary culture) in culture media, an environment far from the in situ inflammatory conditions, which could reverse possible alterations caused by inflammation. In fact, we have previously described in guinea pig smooth muscle short-term changes in F-actin (7–8 min; see Ref. 17).

Inflammation-induced alterations of other mechanisms that mediate Ca2+ sensitization such as RhoA/Rho kinase pathway-mediated inhibition of myosin light-chain phosphatase (33) could also be responsible for the impairment in the contractile response.

During cholecystitis, gallbladder stasis can cause an increase in bile acid concentration that frequently precedes the onset of the inflammation (38). Gallbladder smooth muscle cells are sensitive to hydrophobic bile acids, which increase in the pathogenesis of most acute cholecystitis cases (43, 44). Because during bile duct ligation gallbladder smooth muscle cells are undoubtedly exposed to high concentrations of bile acids, proceeding from the lumen and from the vascular compartment, it is very likely that these acids can exert their deleterious effects on smooth muscle physiology (44). In fact, recent reports have shown that hydrophobic bile acids can activate cationic conductances and Ca2+ signals in pancreatic acinar cells (39, 40). This could lead to a chronic state of depolarization, which can easily account for the genetic and functional changes observed in our experimental conditions. Additional studies are needed to determine the role of bile acids in gallbladder smooth muscle Ca2+ homeostasis.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Ministerio de Educacion y Ciencia Grants SAF 2001–0295 and BFU 2004–0637 and Junta de Extremadura Grant 2PR03A020. P. J Gómez-Pinilla received a Doctoral Fellowship from Junta de Extremadura.


   ACKNOWLEDGMENTS
 
We thank Gary M. Mawe for critical reading of the manuscript and Purificación Delgado for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. J. Pozo, Dept. of Physiology, Nursing School, Avda Universidad s/n, 10071 Cáceres, Spain (e-mail: mjpozo{at}unex.es)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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