HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 288/3/G507    most recent 00385.2004v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (6) Citing Articles via Google Scholar Google Scholar Articles by Morales, S. Articles by Pozo, M. J. Search for Related Content PubMed PubMed Citation Articles by Morales, S. Articles by Pozo, M. J.

NEUROREGULATION AND MOTILITY

Characterization of intracellular Ca²⁺ stores in gallbladder smooth muscle

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

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. 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 (T_1/2) of 4.43 ± 0.40 s, and had a mean amplitude of 0.22 ± 0.02 F₃₄₀/F₃₈₀ (n = 14). The steady-state level, which reflects Ca²⁺ entry activated by store depletion, had an amplitude of 0.060 ± 0.005 F₃₄₀/F₃₈₀. In the absence of extracellular Ca²⁺, CCK caused a transient increase in [Ca²⁺]_i (peak: 0.21 ± 0.03 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 T_1/2 (2.94 ± 0.26 s) and the steady level (0.017 ± 0.003 F₃₄₀/F₃₈₀) were significantly smaller (P < 0.01 and P < 0.001 for T_1/2 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 F₃₄₀/F₃₈₀; 30 µM 2-APB: 0.07 ± 0.01 F₃₄₀/F₃₈₀; 50 µM 2-APB: 0.003 ± 0.002 F₃₄₀/F₃₈₀, n = 9–10, P < 0.001 for both concentrations). These results indicate that CCK releases intracellular Ca²⁺ by selectively activating IP₃R.

View larger version (16K): [in this window] [in a new window]   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.

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 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 F₃₄₀/F₃₈₀, n = 6). After a 15-min perfusion with Ca²⁺-free solution, the response to caffeine was practically eliminated (0.05 ± 0.02 F₃₄₀/F₃₈₀, n = 5), and there was no response after 20 min (0.002 ± 0.001 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.

View larger version (14K): [in this window] [in a new window]   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 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 F₃₄₀/F₃₈₀ time course along the treatment with CCK and caffeine. When the stores were depleted by CCK (peak amplitude: 0.34 ± 0.05 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.

View larger version (15K): [in this window] [in a new window]   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).

View larger version (19K): [in this window] [in a new window]   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).

View larger version (20K): [in this window] [in a new window]   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

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

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS 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.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

1. Adebiyi A and Adaikan PG. Effect of caffeine on response of rabbit isolated corpus cavernosum to high K+ solution, noradrenaline and transmural electrical stimulation. Clin Exp Pharmacol Physiol 31: 82–85, 2004.[CrossRef][ISI][Medline]
2. Alcon S, Morales S, Camello PJ, and Pozo MJ. Tyrosine kinases regulate basal tone and responses to agonists in the guinea pig gallbladder through modulation of Ca²⁺ mobilization. Anal Pharmacol 2: 48–56, 2001.
3. Alcon S, Morales S, Camello PJ, and Pozo MJ. Contribution of different phospholipases and arachidonic acid metabolites in the response of gallbladder smooth muscle to cholecystokinin. Biochem Pharmacol 64: 1157–1167, 2002.[CrossRef][ISI][Medline]
4. Apaydin S, Gonen C, and Guven H. The probable role of nitric oxide on the relaxations obtained by caffeine and aminophylline in rat uterus. Pharmacol Res 38: 387–392, 1998.[CrossRef][ISI][Medline]
5. Boittin FX, Dipp M, Kinnear NP, Galione A, and Evans AM. Vasodilation by the calcium-mobilizing messenger cyclic ADP-ribose. J Biol Chem 278: 9602–9608, 2003.
6. Bolton TB and Lim SP. Properties of calcium stores and transient outward currents in single smooth muscle cells of rabbit intestine. J Physiol 409: 385–401, 1989.[Abstract/Free Full Text]
7. Bourreau JP, Kwan CY, and Daniel EE. Distinct pathways to refill ACh-sensitive internal Ca2+ stores in canine airway smooth muscle. Am J Physiol Cell Physiol 265: C28–C35, 1993.[Abstract/Free Full Text]
8. Burdyga TV and Wray S. The effect of inhibition of myosin light chain kinase by Wortmannin on intracellular [Ca2+], electrical activity and force in phasic smooth muscle. Pflügers Arch 436: 801–803, 1998.[CrossRef][ISI][Medline]
9. Camello-Almaraz C, Pariente JA, Salido G, and Camello PJ. Differential involvement of vacuolar H(+)-ATPase in the refilling of thapsigargin- and agonist-mobilized Ca(2+) stores. Biochem Biophys Res Commun 271: 311–317, 2000.[CrossRef][ISI][Medline]
10. Chowdhury JU, Pang YW, Huang SM, Tsugeno M, and Tomita T. Sustained contraction produced by caffeine after ryanodine treatment in the circular muscle of the guinea-pig gastric antrum and rabbit portal vein. Br J Pharmacol 114: 1414–1418, 1995.[ISI]
11. Eggermont JA, Vrolix M, Wuytack F, Raeymaekers L, and Casteels R. The (Ca2+-Mg2+)-ATPases of the plasma membrane and of the endoplasmic reticulum in smooth muscle cells and their regulation. J Cardiovasc Pharmacol 12, Suppl 5: S51–S55, 1988.
12. Ehrlich BE and Watras J. Inositol 1,4,5-trisphosphate activates a channel from smooth muscle sarcoplasmic reticulum. Nature 336: 583–586, 1988.[CrossRef][Medline]
13. Flynn ER, Bradley KN, Muir TC, and McCarron JG. Functionally separate intracellular Ca2+ stores in smooth muscle. J Biol Chem 276: 36411–36418, 2001.[Abstract/Free Full Text]
14. Golovina VA and Blaustein MP. Spatially and functionally distinct Ca2+ stores in sarcoplasmic and endoplasmic reticulum. Science 275: 1643–1648, 1997.[Abstract/Free Full Text]
15. Gordienko DV, Zholos AV, and Bolton TB. Membrane ion channels as physiological targets for local Ca2+ signalling. J Microsc 196: 305–316, 1999.[ISI][Medline]
16. Janssen LJ and Sims SM. Emptying and refilling of Ca2+ store in tracheal myocytes as indicated by ACh-evoked currents and contraction. Am J Physiol Cell Physiol 265: C877–C886, 1993.[Abstract/Free Full Text]
17. Lomax RB, Camello C, Van Coppenolle F, Petersen OH, and Tepikin AV. Basal and physiological Ca(2+) leak from the endoplasmic reticulum of pancreatic acinar cells. Second messenger-activated channels and translocons. J Biol Chem 277: 26479–26485, 2002.[Abstract/Free Full Text]
18. McCarron JG, Bradley KN, MacMillan D, and Muir TC. Sarcolemma agonist-induced interactions between InsP3 and ryanodine receptors in Ca2+ oscillations and waves in smooth muscle. Biochem Soc Trans 31: 920–924, 2003.[ISI][Medline]
19. Morales S, Camello PJ, Alcon S, Salido GM, Mawe G, and Pozo MJ. Coactivation of capacitative calcium entry and L-type calcium channels in guinea pig gallbladder. Am J Physiol Gastrointest Liver Physiol 286: G1090–G1100, 2004.[Abstract/Free Full Text]
20. Nelson MT, Cheng H, Rubart M, Santana LF, Bonev AD, Knot HJ, and Lederer WJ. Relaxation of arterial smooth muscle by calcium sparks. Science 270: 633–637, 1995.[Abstract/Free Full Text]
21. Pacaud P and Loirand G. Release of Ca2+ by noradrenaline and ATP from the same Ca2+ store sensitive to both InsP3 and Ca2+ in rat portal vein myocytes. J Physiol 484: 549–555, 1995.[Abstract/Free Full Text]
22. Papp B, Paszty K, Kovacs T, Sarkadi B, Gardos G, Enouf J, and Enyedi A. Characterization of the inositol trisphosphate-sensitive and insensitive calcium stores by selective inhibition of the endoplasmic reticulum-type calcium pump isoforms in isolated platelet membrane vesicles. Cell Calcium 14: 531–538, 1993.[CrossRef][ISI][Medline]
23. Perez GJ, Bonev AD, Patlak JB, and Nelson MT. Functional coupling of ryanodine receptors to KCa channels in smooth muscle cells from rat cerebral arteries. J Gen Physiol 113: 229–238, 1999.[Abstract/Free Full Text]
24. Pozo MJ, Perez GJ, Nelson MT, and Mawe GM. Ca(2+) sparks and BK currents in gallbladder myocytes: role in CCK-induced response. Am J Physiol Gastrointest Liver Physiol 282: G165–G174, 2002.[Abstract/Free Full Text]
25. Renzetti LM, Wang MB, and Ryan JP. Contribution of intracellular calcium to gallbladder smooth muscle contraction. Am J Physiol Gastrointest Liver Physiol 259: G1–G5, 1990.[Abstract/Free Full Text]
26. Rosado JA, Lopez JJ, Harper AG, Harper MT, Redondo PC, Pariente JA, Sage SO, and Salido GM. Two pathways for store-mediated calcium entry differentially dependent on the actin cytoskeleton in human platelets. J Biol Chem 279: 29231–29235, 2004.[Abstract/Free Full Text]
27. Ryan JP. Calcium and gallbladder smooth muscle contraction in the guinea pig: effect of pregnancy. Gastroenterology 89: 1279–1285, 1985.[ISI][Medline]
28. Salapatek AM, Lam A, and Daniel EE. Calcium source diversity in canine lower esophageal sphincter muscle. J Pharmacol Exp Ther 287: 98–106, 1998.[Abstract/Free Full Text]
29. Sanders KM. Invited review: mechanisms of calcium handling in smooth muscles. J Appl Physiol 91: 1438–1449, 2001.[Abstract/Free Full Text]
30. Shaffer EA, Bomzon A, Lax H, and Davison JS. The source of calcium for CCK-induced contraction of the guinea-pig gall bladder. Regul Pept 37: 15–26, 1992.[CrossRef][ISI][Medline]
31. Sugita M, Tokutomi N, Tokutomi Y, Terasaki H, and Nishi K. The properties of caffeine- and carbachol-induced intracellular Ca2+ release in mouse bladder smooth muscle cells. Eur J Pharmacol 348: 61–70, 1998.[CrossRef][ISI][Medline]
32. Toescu EC and Petersen OH. The thapsigargin-evoked increase in [Ca2+]i involves an InsP3-dependent Ca2+ release process in pancreatic acinar cells. Pflügers Arch 427: 325–331, 1994.[CrossRef][ISI][Medline]
33. Wray S, Kupittayanant S, and Shmigol T. Role of the sarcoplasmic reticulum in uterine smooth muscle. Novartis Found Symp 246: 6–18, 2002.[Medline]
34. Wu C, Sui G, and Fry CH. The role of the L-type Ca(2+) channel in refilling functional intracellular Ca(2+) stores in guinea-pig detrusor smooth muscle. J Physiol 538: 357–369, 2002.[Abstract/Free Full Text]
35. Xu L, Lai FA, Cohn A, Etter E, Guerrero A, Fay FS, and Meissner G. Evidence for a Ca(2+)-gated ryanodine-sensitive Ca2+ release channel in visceral smooth muscle. Proc Natl Acad Sci USA 91: 3294–3298, 1994.[Abstract/Free Full Text]
36. Young SH, Ennes HS, McRoberts JA, Chaban VV, Dea SK, and Mayer EA. Calcium waves in colonic myocytes produced by mechanical and receptor-mediated stimulation. Am J Physiol Gastrointest Liver Physiol 276: G1204–G1212, 1999.[Abstract/Free Full Text]
37. Yu P, Chen Q, Xiao Z, Harnett K, Biancani P, and Behar J. Signal transduction pathways mediating CCK-induced gallbladder muscle contraction. Am J Physiol Gastrointest Liver Physiol 275: G203–G211, 1998.[Abstract/Free Full Text]
38. Zhang L, Bonev AD, Nelson MT, and Mawe GM. Ionic basis of the action potential of guinea pig gallbladder smooth muscle cells. Am J Physiol Cell Physiol 265: C1552–C1561, 1993.[Abstract/Free Full Text]

This article has been cited by other articles:

				J. J. Diez, J. L. Miguel, R. Codoceo, P. Iglesias, M. A. Bajo, C. Sanchez, G. del Peso, F. Gil, J. Martinez-Ara, P. G. Gancedo, et al. Effects of cinacalcet on gastrointestinal hormone release in patients with secondary hyperparathyroidism undergoing dialysis Nephrol. Dial. Transplant., April 1, 2008; 23(4): 1387 - 1395. [Abstract] [Full Text] [PDF]

				O. B. Balemba, A. C. Bartoo, M. T. Nelson, and G. M. Mawe Role of mitochondria in spontaneous rhythmic activity and intracellular calcium waves in the guinea pig gallbladder smooth muscle Am J Physiol Gastrointest Liver Physiol, February 1, 2008; 294(2): G467 - G476. [Abstract] [Full Text] [PDF]

				O. B. Balemba, T. J. Heppner, A. D. Bonev, M. T. Nelson, and G. M. Mawe Calcium waves in intact guinea pig gallbladder smooth muscle cells Am J Physiol Gastrointest Liver Physiol, October 1, 2006; 291(4): G717 - G727. [Abstract] [Full Text] [PDF]

				O. B. Balemba, M. J. Salter, T. J. Heppner, A. D. Bonev, M. T. Nelson, and G. M. Mawe Spontaneous electrical rhythmicity and the role of the sarcoplasmic reticulum in the excitability of guinea pig gallbladder smooth muscle cells Am J Physiol Gastrointest Liver Physiol, April 1, 2006; 290(4): G655 - G664. [Abstract] [Full Text] [PDF]

				P. J. Gomez-Pinilla, S. Morales, C. Camello-Almaraz, R. Moreno, M. J. Pozo, and P. J. Camello Changes in guinea pig gallbladder smooth muscle Ca2+ homeostasis by acute acalculous cholecystitis Am J Physiol Gastrointest Liver Physiol, January 1, 2006; 290(1): G14 - G22. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 288/3/G507    most recent 00385.2004v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (6) Citing Articles via Google Scholar Google Scholar Articles by Morales, S. Articles by Pozo, M. J. Search for Related Content PubMed PubMed Citation Articles by Morales, S. Articles by Pozo, M. J.