Characterization of intracellular Ca2+ stores in gallbladder smooth muscle

Sara Morales,1 Pedro J. Camello,1 Gary M. Mawe,2 and María J. Pozo1

1Department of Physiology, Nursing School, University of Extremadura, Cáceres, Spain; 2Department of Anatomy and Neurobiology, University of Vermont, Burlington, Vermont

Submitted 25 August 2004 ; accepted in final form 21 October 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The existence of functionally distinct intracellular Ca2+ stores has been proposed in some types of smooth muscle. In this study, we sought to examine Ca2+ stores in the gallbladder by measuring intracellular Ca2+ concentration ([Ca2+]i) in fura 2-loaded isolated myocytes, membrane potential in intact smooth muscle, and isometric contractions in whole mount preparations. Exposure of isolated myocytes to 10 nM CCK caused a transient elevation in [Ca2+]i that persisted in Ca2+-free medium and was inhibited by 2-aminoethoxydiphenylborane (2-APB). Application of caffeine induced a rapid spike-like elevation in [Ca2+]i that was insensitive to 2-APB but was abolished by pretreatment with 10 µM ryanodine. These data support the idea that both inositol trisphosphate (IP3) receptors (IP3R) and ryanodine receptors (RyR) are present in this tissue. When caffeine was applied in Ca2+-free solution, the [Ca2+]i transients decreased as the interval between Ca2+ removal and caffeine application was increased, indicating a possible leakage of Ca2+ in these stores. The refilling of caffeine-sensitive stores involved sarcoendoplasmic reticulum Ca2+-ATPase activation, similar to IP3-sensitive stores. The moderate Ca2+ elevation caused by CCK was associated with a gallbladder contraction, but caffeine or ryanodine failed to induce gallbladder contraction. Nevertheless, caffeine caused a concentration-dependent relaxation in gallbladder strips either under resting tone conditions or precontracted with 1 µM CCK. Taken together, these results suggest that, in gallbladder smooth muscle, multiple pharmacologically distinct Ca2+ pools do not exist, but IP3R and RyR must be spatially separated because Ca2+ release via these pathways leads to opposite responses.

inositol trisphosphate receptor; ryanodine receptor; refilling; caffeine; thapsigargin


STORAGE OF CA2+ in intracellular organelles provides the potential for release of Ca2+ during physiological signaling in several cell types, including smooth muscle. In smooth muscle cells, the sarcoplasmic reticulum (SR) plays a major role in storing Ca2+, maintaining low intracellular Ca2+ concentration ([Ca2+]i) and regulating Ca2+ release in response to appropriate stimuli (29). Ca2+ release from SR involves the participation of two receptor/channel complexes. One class, termed IP3R, is sensitive to D-myo-inositol 1,4,5-trisphosphate (IP3) produced in response to hormones and neurotransmitters binding to G protein-coupled receptors (12). A second class of complexes, referred to as RyR, is sensitive to caffeine and ryanodine, and it appears to be involved in the regenerative control of [Ca2+]i via Ca2+-induced Ca2+ release (35). To serve as a Ca2+ source for regulating intracellular function, and taking into account that the SR membrane is not permeable to Ca2+, specialized, active Ca2+-ATPases, known as SERCA pumps, exist in the SR membrane (11). These pumps generate and maintain a 10,000-fold Ca2+ gradient between the SR lumen and the cytoplasm and can be blocked by selective inhibitors, such as thapsigargin and cyclopiazonic acid.

The SR appears to be a continuous, interconnected network of tubules in close apposition to plasma membrane (14) where Ca2+ extrusion mechanisms are present (11). The existence of different intracellular SR Ca2+ stores has been proposed in some types of cells, including smooth muscle (6). These Ca2+ stores are classified on the basis of the arrangement of RyR and IP3R. According to this model, a single store can exist involving both receptors (21), but it may also be independent stores that express only one, RyR or IP3R (36), two Ca2+ stores with one containing both RyR and IP3R and a second only RyR (13), and three Ca2+ stores with one containing RyR and IP3R, another expressing only IP3R, and the third containing only RyR (31). The precise locations of these receptors in the stores and their contributions in the regulation of smooth muscle physiology greatly vary among the types of smooth muscle considered. Typically, IP3R-evoked Ca2+ release is associated with Ca2+ oscillations, Ca2+ waves, and smooth muscle contraction (18). However, the role of RyR in Ca2+ handling and smooth muscle function appears to be more diverse. For example, in rabbit portal vein and guinea pig gastric antrum smooth muscle cells, caffeine, an RyR agonist, induces sustained contractions (10), whereas in other tissues, like mouse bladder or rabbit corpus cavernosum, it evokes relaxation (1, 31). The relaxing effect can be explained by the theory that unloading of SR through RyR can be preferentially directed to a restricted subplasmalemmal space, thus ensuring efficient extrusion (33). In addition to this, elevations of [Ca2+]i near the plasma membrane, where Ca2+-dependent conductances are located, can lead to changes in transmembrane currents (e.g., Ca2+ sparks activating transient outward K+ currents) and even to alterations in the excitability of the tissue (5, 20). Thus SR Ca2+ release is not always related to smooth muscle contraction.

To date, characterization of the intracellular Ca2+ store in the gallbladder smooth muscle (GBSM) is lacking. As in other smooth muscle preparations, GBSM function depends, at least in part, on intracellular Ca2+ release (2, 25, 27, 30, 37). Thus the aim of this investigation was to clarify how ryanodine-sensitive and IP3-dependent Ca2+ release occur and interact with each other in guinea pig GBSM cells. To accomplish this, we analyzed the [Ca2+]i elevation and contractile responses to caffeine and CCK and characterized the intracellular Ca2+ mobilization compartments involved in the responses. We show here that, in GBSM cells, there is a single SR Ca2+ store containing both types of receptors, but the localization of those receptors in the stores must be different because of the distinct functional roles that are associated with these mechanisms of Ca2+ release.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tissue preparation. Gallbladders, isolated from 300- to 500-g male guinea pigs after deep halothane anesthesia and cervical dislocation, 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 trimmed of any adherent liver tissue. After the preparation was washed with the nutrient solution to remove any biliary component, the mucosa was carefully dissected away. All of the experiments were carried out according to the guidelines of the Animal Care and Use Committees of the University of Extremadura and the University of Vermont.

Contraction recording of guinea pig GBSM strips. Typically, four strips (measuring ~3 x 10 mm) were obtained from a single guinea pig gallbladder. Each strip was placed 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, caffeine, or ryanodine on gallbladder tone were studied by addition of these agents, at known concentrations, to the organ bath. We also studied the effects of caffeine on CCK-induced contractions. Preparations were first precontracted with 1 µM CCK. Once the steady state of contraction was reached, cumulative doses of caffeine were added either in the presence or absence of 100 µM isobutyl methylxanthine (IBMX).

Intracellular recording from smooth muscle. The methods to be used for intracellular electrophysiological recording were similar to those previously described (38). The gallbladder whole mount preparation was pinned, serosal side up, in a 3-ml tissue chamber and placed on the stage of an inverted microscope (Diaphot T300; Nikon). Smooth muscle bundles were visualized at x200 magnification with Hoffman Modulation Contrast optics (Modulation Optics, Greenvale, NY). The preparations were continuously perfused at a rate of 10–12 ml/min with modified Krebs solution (for composition see Solutions and drugs) aerated with 95% O2-5% CO2. Temperature was maintained between 36 and 37°C at the recording site.

Glass microelectrodes were filled with 2.0 M KCl and had resistances in the range of 50–110 M{Omega}. A negative-capacity compensation amplifier (Axoclamp 2A; Axon Instruments, Foster City, CA) with bridge circuit was used to record membrane potentials, and outputs were displayed on an oscilloscope (Hitachi VC-6050). Electrical signals were recorded using the computer program Chart (ADInstruments). Wortmannin (100–400 nM), which inhibits myosin light chain kinase activity without altering electrical properties or Ca2+ transients in smooth muscle (8), was added to the Krebs solution to inhibit tissue contractions so that intracellular impalements could be maintained. Experimental compounds were applied by addition to the superfusing solution.

Cell isolation. GBSM cells were dissociated enzymatically using a previously described method (24). Briefly, after preparing the tissue as indicated above, the gallbladder was cut into small pieces and incubated for 35 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. 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 coverslip (0.17 mm thick) filled with Na+-HEPES solution (for composition see Solutions and drugs) and mounted on the stage of an inverted microscope (Diaphot T200; 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. For deesterification of the dye, ≥20 min were allowed to elapse before Ca2+ measurements were started.

Cells were illuminated at 340 and 380 nm by a computer-controlled filter wheel (Lambda-2; Sutter Instruments) at 0.3–1 cycles/s, and the emitted fluorescence was selected by a 500-nm bandpass filter. The emitted fluorescence images were captured with a cooled digital charge-coupled device camera (model C-4880–91; Hamamatsu Photonics) and recorded using dedicated software (Argus-HisCa; Hamamatsu Photonics). 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+] was not performed in view of the many uncertainties related to the binding properties of fura 2 with Ca2+ inside of smooth muscle cells.

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 composition (in mM) of the modified Krebs solution used in the intracellular recordings was: 121 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 NaH2PO4, 1.2 MgCl, 25 NaHCO3, and 8 D-glucose. 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.

Drug concentrations are expressed as final bath concentrations of active species. Drugs and chemicals were obtained from the following sources: caffeine, CCK-(26–33) (CCK-8) sulfated, 1,4-dithio-DL-threitol, thapsigargin, and wortmannin were from Sigma Chemical (St. Louis, MO); IBMX was from Calbiochem (La Jolla, CA); 2-aminoethoxydiphenylborane (2-APB) and TTX were from Tocris (Bristol, UK); fura 2-AM and ryanodine were 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, IBMX, ryanodine, thapsigargin, and wortmannin were prepared in DMSO. The solutions were diluted such that the final concentrations of DMSO were ≤0.1% vol/vol. These concentrations of solvents did not themselves affect the mechanical activity of the tissue or interfere with fura 2 fluorescence.

Quantification and statistics. Results are expressed as means ± SE of n cells or gallbladder strips. Gallbladder tension is given in millinewtons (mN). Basal active tension and changes in tension were obtained after subtracting passive tension from all measurements. Passive tension was obtained at the end of the experiments after the tissues were exposed to Ca2+-free K-HS containing 2.5 mM EGTA. Gallbladder relaxation was expressed as the percent reduction of the basal active tension. When caffeine was tested on CCK-induced contractions, relaxations were expressed as a percentage of the maximal response to the hormone. 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
 
Ca2+ release mechanisms through IP3R and RyR are present in GBSM cells. CCK was used to stimulate IP3R, since it has been previously shown to act through activation of phospholipase C and inositol phosphate cascade (3). The response to CCK (10 nM) consisted of a transient elevation followed by a steady-state level slightly above the resting level (Fig. 1A). The transient peak, which is associated with store depletion, was reached in 6.21 ± 0.43 s, with a half decay time (T1/2) of 4.43 ± 0.40 s, and had a mean amplitude of 0.22 ± 0.02 {Delta}F340/F380 (n = 14). The steady-state level, which reflects Ca2+ entry activated by store depletion, had an amplitude of 0.060 ± 0.005 {Delta}F340/F380. In the absence of extracellular Ca2+, CCK caused a transient increase in [Ca2+]i (peak: 0.21 ± 0.03 {Delta}F340/F380, time to peak: 5.82 ± 0.52 s, n = 17, P > 0.05, Fig. 1B) that was similar to the initial response in the presence of extracellular Ca2+, but both the T1/2 (2.94 ± 0.26 s) and the steady level (0.017 ± 0.003 {Delta}F340/F380) were significantly smaller (P < 0.01 and P < 0.001 for T1/2 and steady level, respectively), since there was not Ca2+ influx in response to depletion of the stores. When 10 nM CCK was applied in the presence of 2-APB, an inhibitor of IP3R, there was an almost complete abolition of the CCK-induced Ca2+ peak (control: 0.23 ± 0.04 {Delta}F340/F380; 30 µM 2-APB: 0.07 ± 0.01 {Delta}F340/F380; 50 µM 2-APB: 0.003 ± 0.002 {Delta}F340/F380, n = 9–10, P < 0.001 for both concentrations). These results indicate that CCK releases intracellular Ca2+ by selectively activating IP3R.



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Fig. 1. Effects of CCK and caffeine in resting intracellular Ca2+ concentration ([Ca2+]i) in single gallbladder smooth muscle (GBSM) cells. A: when 10 nM CCK was used to induce inositol trisphosphate (IP3)-dependent Ca2+ release, the response consisted of a transient elevation followed by a steady-state level (n = 14). B: a similar [Ca2+]i peak was obtained when CCK was assayed in Ca2+-free medium containing 1 mM EGTA (n = 13). C: caffeine (10 mM) induced a rapid, spike-like elevation in [Ca2+]i followed by a small steady-state level (n = 10). D: a similar peak was recorded when 10 mM caffeine was tested in Ca2+-free medium containing 1 mM EGTA (n = 17). F340/F380, ratio of fluorescence at 340 nm to that at 380 nm.

 
Caffeine was used in the current study to activate RyR. As shown in Fig. 1C, acute application of 10 mM caffeine caused a Ca2+ transient that had an amplitude of 0.61 ± 0.07 {Delta}F340/F380, a time to peak of 8.1 ± 0.55 s, and a T1/2 of 4.16 ± 0.44 s (n = 10). Application of caffeine did not cause a sustained steady level (0.012 ± 0.013 {Delta}F340/F380), which may indicate that capacitative Ca2+ entry was slightly activated by store depletion through RyR. Lower concentrations of caffeine also mobilized Ca2+ from the stores, with similar kinetics (300 µM: 0.034 ± 0.034 {Delta}F340/F380; 1 mM: 0.25 ± 0.05 {Delta}F340/F380; 3 mM: 0.53 ± 0.11 {Delta}F340/F380, n = 8–10). Ca2+ transient in response to 10 mM caffeine remained unchanged when caffeine was applied in Ca2+-free Na+-HEPES buffer (peak: 0.59 ± 0.06 {Delta}F340/F380; time to peak: 7.94 ± 0.38 s; T1/2: 3.82 ± 0.24 s, n = 17, P > 0.05 vs. control; Fig. 1D). The presence of 50 µM 2-APB did not affect the caffeine-induced Ca2+ transient (0.04% inhibition, n = 9, P > 0.05), but 10 µM ryanodine almost completely abolished the caffeine-induced increase in [Ca2+]i (caffeine: 0.61 ± 0.12 {Delta}F340/F380; caffeine plus ryanodine: 0.0021 ± 0.0014 {Delta}F340/F380, n = 7, P < 0.001). These data suggest that smooth muscle cells from guinea pig gallbladder contain a ryanodine- and caffeine-sensitive internal store for Ca2+.

RyR containing stores are "leaky." We have previously demonstrated that IP3-sensitive Ca2+ stores in the gallbladder have a rapid turnover and that exposure to Ca2+-free medium depletes the IP3-sensitive intracellular Ca2+ stores and leads to capacitative Ca2+ entry activation (19). To test whether this is a characteristic shared by the stores gated by RyR, we designed a similar protocol to assess the temporal course of intracellular RyR-sensitive Ca2+ store depletion. In this protocol, caffeine was applied at different time points after withdrawal of external Ca2+. As represented in Fig. 2, application of 10 mM caffeine immediately after Ca2+ removal induced a transient [Ca2+]i peak followed by a rapid recovery toward resting levels (peak amplitude: 0.59 ± 0.002 {Delta}F340/F380, n = 17). This Ca2+ transient progressively decayed as the delay between Ca2+ removal and caffeine application was increased (peak amplitude after 4 min in Ca2+-free medium: 0.43 ± 0.12 {Delta}F340/F380, n = 6). After a 15-min perfusion with Ca2+-free solution, the response to caffeine was practically eliminated (0.05 ± 0.02 {Delta}F340/F380, n = 5), and there was no response after 20 min (0.002 ± 0.001 {Delta}F340/F380, n = 13), indicating that the intracellular ryanodine-sensitive Ca2+ stores were depleted because of the presence of Ca2+ leak in nonstimulated GBSM cells.



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Fig. 2. Removal of extracellular Ca2+ depletes ryanodine-sensitive stores. GBSM cells were perfused with Ca2+-free medium during 0, 4, 15, or 20 min before stimulation with 10 mM caffeine to release ryanodine-sensitive internal stores, resulting in progressive loss of the response to caffeine. The traces are typical of 5–17 cells.

 
IP3R- and RyR-containing stores refill through SERCA pump. Different Ca2+ stores have also been characterized according to the dependence of the refilling of the stores on extracellular Ca2+ (13) or on their sensitivity to Ca2+ transport systems (9, 22). We have demonstrated above that both IP3R- and RyR-gated stores are depleted in the absence of extracellular Ca2+, so their refilling would need the influx of this ion. The effects of thapsigargin on CCK- and caffeine-induced Ca2+ transients were evaluated to examine whether the refilling of these stores also depends on the functionality of thapsigargin-sensitive SERCA pumps. After testing the response to 10 nM CCK or 10 mM caffeine, cells were treated with 1 µM thapsigargin for 30 min, and CCK or caffeine was applied again. Thapsigargin almost completely abolished the CCK-induced Ca2+ transient (control: 0.16 ± 0.04 {Delta}F340/F380; thapsigargin: 0.05 ± 0.01 {Delta}F340/F380, 96.9% inhibition, n = 12, P < 0.001). Similarly, thapsigargin significantly reduced the caffeine-induced Ca2+ transients (0.56 ± 0.11 vs. 0.011 ± 0.007 {Delta}F340/F380, 98.3% inhibition, n = 10, P < 0.001), indicating that both IP3R- and RyR-containing stores refill through activation of thapsigargin-sensitive SERCA pumps.

IP3R- and RyR-containing stores overlap. According to our results, stores containing IP3R and RyR share common features. To determine whether or not these receptors share the same Ca2+ store, we depleted IP3-sensitive stores with CCK in a Ca2+-free medium, and then we washed out CCK and challenged the cells with 10 mM caffeine, also in a Ca2+-free medium. The time lapse between removing Ca2+ from the extracellular solution and caffeine application was always 4 min, to prevent the total depletion of the stores. Figure 3A shows a representative trace of the {Delta}F340/F380 time course along the treatment with CCK and caffeine. When the stores were depleted by CCK (peak amplitude: 0.34 ± 0.05 {Delta}F340/F380, n = 9), caffeine failed to release Ca2+. We also tested CCK in cells in which their ryanodine-sensitive stores had been previously depleted by exposure to 10 µM ryanodine and 10 mM caffeine. Figure 3B shows a trace of the responses to caffeine plus ryanodine and to CCK that is representative of 12 experiments. Under these conditions, CCK also failed to cause a detectable Ca2+ transient. These results indicate that both receptors share the same Ca2+ store.



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Fig. 3. IP3 receptor (IP3R) and ryanodine receptor (RyR) containing stores overlap in GBSM cells. A: caffeine-induced increase in [Ca2+]i is almost abolished after the depletion of IP3-sensitive stores by 10 nM CCK in Ca2+-free solution (n = 9). The time lapse between CCK and caffeine applications was 4 min. B: CCK induced negligible increases in [Ca2+]i after the depletion of ryanodine (Rya)-sensitive stores by 10 mM caffeine and 10 µM ryanodine (n = 12).

 
Ca2+ release through RyR does not cause gallbladder contraction. We and others have demonstrated that Ca2+ release from IP3R induces contraction (3, 30, 37). Figure 4A shows the typical CCK-induced increase in the tension of a gallbladder strip. Considering the caffeine-induced Ca2+ transient we describe in this paper, a contractile response to caffeine might be expected. However, when caffeine was applied to gallbladder strips under basal tone conditions, a concentration-dependent reduction in the active tension was detected with a maximum effect of 58.0 ± 11.9% reduction for 10 mM caffeine, which was not neurogenic in origin since it remained unchanged in the presence of 1 µM TTX (Fig. 4B). Furthermore, when caffeine was added to strips during a contraction induced by 1 µM CCK, a concentration-dependent relaxation was observed (Fig. 4C). Because it could be argued that caffeine was causing relaxation as the result of its side effects as an inhibitor of the phosphodiesterase activity in GBSM cells, we inhibited the phosphodiesterase activity with 100 µM IBMX and found that the response to caffeine remained the same (Fig. 4D), suggesting that this inhibitory effect was different in origin. In agreement with this, ryanodine also induced a concentration-dependent miogenic relaxation of gallbladder strips (1 µM ryanodine: 0.54 ± 0.28 mN, 4% reduction of active tension; 5 µM ryanodine: 1.91 ± 0.49 mN, 16% reduction of active tension; 10 µM ryanodine: 3.02 ± 0.52 mN, 25% reduction of active tension; n = 5). To test whether the relaxing effect of caffeine was related to changes in the excitability of the tissue, we evaluated the effect of caffeine on the electrical activity with intracellular microelectrodes. As represented in Fig. 5, addition of 10 mM caffeine to the bathing solution caused hyperpolarization of GBSM (6.6 ± 0.9 mV, P < 0.01) that was associated with a loss of the spontaneous action potentials.



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Fig. 4. Ca2+ release through IP3R and RyR have different effects on gallbladder tone. A: original recording of gallbladder tone showing the 10 nM CCK-induced contraction (n = 8). B: application of increasing concentrations of caffeine (100 µM-10 mM) caused a relaxation of gallbladder strips that was insensitive to 1 µM TTX (n = 10 and 5). C: when caffeine was tested on 100 nM CCK-induced contractions, a concentration-dependent relaxing response was recorded (n = 7). D: concentration-dependent curves for the relaxing effects of caffeine on CCK-induced contraction in the absence and presence of 100 µM isobutyl methylxanthine (IBMX; n = 7).

 


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Fig. 5. Caffeine causes hyperpolarization of GBSM. Representative experimental trace of records of membrane potential showing that, when 10 mM caffeine was added to the bathing solution, there was membrane hyperpolarization that was associated with an abolition of the spontaneous action potentials (n = 5). This cell had a resting membrane potential of –45 mV.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Ca2+ release from internal stores is of great importance in smooth muscle function. It is well established that, in smooth muscle, intracellular Ca2+ release can occur via IP3R and RyR, but the distribution of these receptors in the intracellular stores is variable and largely unknown. To date, there are no functional correlations for heterogeneity of intracellular stores in smooth muscle, and, in the case of gallbladder, even the characterization of intracellular stores is lacking. Therefore, in this study, we investigated this issue and discovered the coexistence of RyR and IP3R in the same Ca2+ pool in native GBSM cells. This common store has a rapid turnover and needs the activation of SERCA pumps to maintain intrastore Ca2+ levels. These Ca2+ release receptors are apparently distributed separately within the SR, since Ca2+ release through IP3R and RyR has different effects on gallbladder contractility.

The pattern for Ca2+ release through both types of receptors is slightly different. Thus, for caffeine-induced Ca2+ transient, there is a small steady-state level above the resting [Ca2+]i after the Ca2+ peak compared with that caused by CCK, indicating that there is little activation of Ca2+ influx as a consequence of Ca2+ release through RyR. This suggests that there must not be a direct cross talk between RyR and capacitative Ca2+ channels, although a capacitative behavior has been shown after total depletion of the stores and Ca2+ restoration (19).

Three main lines of evidence in our results indicate the presence of a single Ca2+ pool in GBSM cells. First, dependence of intracellular stores on external Ca2+ has been used to characterize the stores (13). We have previously demonstrated that IP3-sensitive stores in GBSM are leaky in that they are depleted when Ca2+ is removed from the extracellular solution (19). In the current study, we found that ryanodine-sensitive Ca2+ stores in GBSM cells show a high dependence on extracellular Ca2+ to maintain the releasable Ca2+ pool even in the absence of any triggered Ca2+ release. Thus just the exposure of the cells to Ca2+-free medium for ~15 min causes the caffeine-mobilizable stores to be depleted, in keeping with our previous observation for the CCK-mobilizable pool (19). The similarity in the temporal pattern of the Ca2+ leakage in both IP3R- and RyR-containing stores is one indication that these receptors could share a continuous store. In addition, these observations suggest that, in GBSM cells, the exchange of Ca2+ between the extracellular space, the sarcoplasmic pool, and the SR lumen are in dynamic equilibrium. The channels or mechanisms responsible for this leak have not been identified, although, in other cells, resting levels of IP3 (32) and the translocons (17) seem to participate in this passive release. We have shown that, in GBSM cells, there is a continuous and spontaneous Ca2+ release through RyR in the form of sparks, which could also account for the depletion of the stores when they cannot refill, as occurs in the absence of extracellular Ca2+ (24).

Another finding supporting the presence of IP3R and RyR in the same pool is the effect of SERCA inhibitors. Some types of muscles have been reported to refill the stores independently of SERCA pumps: in canine tracheal myocytes, a preferred pathway exists whereby Ca2+ enters stores directly via L-type Ca2+ channels, bypassing the sarcoplasm (16), and in tracheal and esophageal smooth muscle the ACh-sensitive Ca2+ store is not sensitive to the SERCA-specific inhibitor cyclopiazonic acid (7, 28). In GBSM, however, sarcoplasmic Ca2+ enters the SR mainly via the SERCA pumps, since thapsigargin abolished subsequent Ca2+ release by caffeine and CCK. Our results are in agreement with other studies in smooth muscle cells showing the dependence of Ca2+ store refilling on activation of SERCA pumps (13, 34). On the other hand, differences in the sensitivity to SERCA pump inhibitors have also been used to differentiate intracellular Ca2+ pools (22, 26). In our study, the refilling of both IP3- and ryanodine-sensitive stores required the activation of thapsigargin-sensitive pumps, implying that Ca2+ enters the SR probably through stimulation of the 100-kDa Ca2+ pump isoform (22).

A third result in our study showing communication between IP3- and ryanodine-mobilized stores is the finding that, in a Ca2+-free solution, depletion of the stores with ryanodine and caffeine totally abolished the CCK-induced Ca2+ transients, and depletion of the IP3-sensitive stores with CCK eliminated the caffeine-induced Ca2+ transient. In addition, we have previously demonstrated that depletion of IP3-sensitive stores with CCK reduces spontaneous transient outward current (STOC) activity, and STOCs are activated by Ca2+ release via RyR channels (24). Taken together, these data indicate that IP3- and ryanodine-sensitive receptors overlap in GBSM cells and share a common pool. This model is consistent with known similarity in the distributions of IP3R and RyR in some types of smooth muscle (6, 21) but differs from other studies where, in addition to a common Ca2+ pool for IP3R and RyR, discrete and small sarcoplasmic and endoplasmic reticulum compartments containing only RyR are also present (13, 14).

Our study could lead to the conclusion that Ca2+ signals that arise from Ca2+ mobilized from intracellular stores in GBSM are more simple than in other smooth muscle types, since there is only a pool responsible for Ca2+ release, whereas in other cell types different Ca2+ stores collaborate in the complexity of Ca2+ signaling. However, our study demonstrates a different role in the control of smooth muscle contractility for Ca2+ release through both type of receptors despite being in the same store. Thus Ca2+ release through IP3R is linked to gallbladder contraction and caffeine- and ryanodine-induced Ca2+ release reduce basal active tension and cause relaxation on CCK-induced active tension. Similar relaxing responses to caffeine in precontracted preparations have also been reported in other smooth muscle preparations (1, 4, 31). The apparent discrepancy in our results could be explained by a different location of both types of receptors within the store. Thus RyR could form microdomaines facing the plasma membrane where they can mainly affect ionic conductances. The coupling between sarcoplasmic RyR and plasmalemma Ca2+-dependent BK channels has largely been shown in smooth muscle cells (15, 23). Ca2+ release through RyR could be aimed to control excitability more than have a direct effect on contractile machinery (20). In fact, we have previously shown the presence of such a coupling between Ca2+ release through RyR channels and BK channel activation in the gallbladder (24). Activation or Ca2+ release through RyR could cause a hyperpolarization of GBSM as a consequence of the increase in hyperpolarizing currents. Our present finding of caffeine-induced hyperpolarization is consistent with this hypothesis.

A critical point in the functional dissociation between CCK- and ryanodine-mobilized pools is how to explain that the [Ca2+]i increase evoked by caffeine and ryanodine does not contract the muscle. The argument that RyR-evoked Ca2+ release could be accumulated in the narrow space between the SR and plasma membrane where it is extruded before reaching the contractile machinery (33) cannot apply here given that our [Ca2+]i measurement are global and not limited to the subplasmalemmal domain. Ca2+-independent signals activated by CCK (3) are also unlikely to explain the difference, since the simple activation of plasma membrane L-type Ca2+ channels contracts these cells (2). The most likely explanation is that sustained contraction is not linked to the initial [Ca2+]i peak but to Ca2+ influx through capacitative and L-type Ca2+ channels (19), and only a pool associated with both components of the Ca2+ signal, like CCK, would induce a significant contraction.

In conclusion, our data suggest that the SR in GBSM cells comprise a Ca2+ store that is shared by both IP3R and RyR, but located in different domains. This store is leaky and depends on the influx of extracellular Ca2+ and thapsigargin-sensitive SERCA pumps to refill. In addition, different spatial location of IP3R and RyR within the store associated with different effect on smooth muscle contraction likely leads to the heterogeneity to the Ca2+ signaling, allowing a more versatile and complex role of intracellular Ca2+ release in this cellular model.


    GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
This work was supported by the Spanish MCyT Grants SAF-2001-0295 and BFU2004-00637/BFI (to M. J. Pozo) and National Institute of Neurological Disorders and Stroke Grant NS-26995 (to G. M. Mawe). S. Morales was supported by a Ministry of Education Predoctoral Research Grant.


    ACKNOWLEDGMENTS
 
We thank M. P. 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.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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