1 Department of Physiology, University of Extremadura, 10071 Cáceres, Spain; and 2 Department of Pharmacology and 3 Department of Anatomy and Neurobiology, University of Vermont, Burlington, Vermont 05405
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ABSTRACT |
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We sought to elucidate the regulation of gallbladder smooth muscle (GBSM) excitability by localized Ca2+ release events (sparks) and large-conductance Ca2+-dependent (BK) channels by determining whether sparks exist in GBSM and, if so, whether they activate BK channels. Sparks were identified in isolated GBSM loaded with fluo 4. Each spark was associated with a transient outward current, suggesting communication of ryanodine receptor (RyR) channels with BK channels. This was confirmed by the inhibition of outward currents with iberiotoxin (100 nM), thapsigargin (200 nM), and ryanodine (10 µM). In current clamp mode, the transient BK currents were associated with brief membrane hyperpolarizations (10.9 ± 1.3 mV). Because transient BK currents could dampen GBSM excitability, we tested whether CCK attenuates these events. CCK (10 nM) reduced the amplitude and frequency of transient BK currents, and subsequent caffeine application restored transient BK current activity. These results support the concept that RyRs and BK channels contribute to the regulation of GBSM excitability and that CCK can act in part by inhibiting this pathway.
guinea pig; smooth muscle; sarcoplasmic reticulum; ryanodine receptor; thapsigargin; L-type calcium channels; caffeine
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INTRODUCTION |
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INCREASES IN GLOBAL INTRACELLULAR Ca2+ concentration ([Ca2+]i) regulate cellular processes from growth to apoptosis. In smooth muscle cells, elevations in [Ca2+]i in response to neurotransmitters or hormones initiate the contractile process. Receptor activation in the plasma membrane causes either activation of nonselective cation channels or G protein-dependent stimulation of phospholipase C and generation of inositol 1,4,5-trisphosphate (IP3) or both, depending on the neurotransmitter or hormone binding the receptor (7). The opening of nonselective cation channels induces depolarization and the consequent activation of voltage-operated Ca2+ channels (VOCCs) (5), which causes Ca2+ influx that collaborates to increase [Ca2+]i. IP3 binds to its receptor in the sarcoplasmic reticulum (SR), a Ca2+ channel, and induces Ca2+ release from the SR (6). Intracellular Ca2+ stimulates calmodulin-dependent activation of myosin light chain kinase to initiate contraction (39).
Despite this procontractile role for [Ca2+]i
increases, in arterial smooth muscle, localized Ca2+
release events from ryanodine receptors (RyRs), termed Ca2+
sparks, oppose vasoconstriction by activating large-conductance Ca2+-dependent (BK) channels (32). Thus,
unlike cardiac muscle, in which Ca2+ sparks combine to
cause a global Ca2+ transient that promotes contraction
(10), smooth muscle sparks activate BK channels in the
nearby plasma membrane (35), resulting in a membrane
hyperpolarization. This hyperpolarization closes voltage-dependent
Ca2+ channels and therefore leads to a reduction of global
[Ca2+]i and relaxation (29). The
relevance of this mechanism is supported by the observation that
inhibition of BK channels with iberiotoxin leads to marked membrane
depolarization and vasoconstriction (29, 32) and reduces
the actions of a variety of smooth muscle relaxants (see Ref.
25 for review). Recently, we (9) have shown
that the targeted deletion of the gene for the 1-subunit
of the BK channel leads to a decrease in the Ca2+
sensitivity of BK channels, a reduction in functional coupling of
Ca2+ sparks to BK channel activation, and increases in
arterial tone and blood pressure.
In the gastrointestinal tract, in which receptive relaxations of smooth muscles actively contribute to the movement of luminal contents, the presence of Ca2+ sparks coupled to activation of outward currents has been reported (2, 3, 20, 27). Inhibitory neurotransmitters, such as ATP, cause the activation of apamin-sensitive K+ channels that is mediated by localized Ca2+ sparks, which could provide a mechanism for coupling ATP to hyperpolarization responses (inhibitory junction potentials; Ref. 2). In addition, inhibition of resting spark activity and/or outward currents by excitatory neurotransmitters in the gastrointestinal tract (3, 4) induces a decrease in spark events and/or outward currents, which could lead to contraction. The gallbladder shows both relaxing and contractile behaviors during bile storage and bile flow phases. This motor activity is the result of changes in gallbladder smooth muscle (GBSM) contractility in response to excitatory or inhibitory neurotransmitters and hormones (37). However, it is not yet known whether Ca2+ spark activity plays a role in the regulation of gallbladder motility.
In this study, we provide the first evidence of Ca2+ sparks in GBSM cells, and we demonstrate functional coupling between Ca2+ sparks and BK channel activation. Our data indicate that RyRs are the main source of spontaneous Ca2+ transients and that basal Ca2+ influx through L-type Ca2+ channels is necessary for full activation of BK channels. CCK, a contractile agonist of GBSM, causes inhibition of spontaneous transient BK currents, suggesting that Ca2+ sparks play a physiological role in the regulation of gallbladder excitability.
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MATERIALS AND METHODS |
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Cell isolation. All procedures were reviewed and approved by the Office of Animal Care Management at the University of Vermont. Guinea pigs (250-350 g) were euthanized by halothane overdose and then exsanguinated. GBSM cells were dissociated enzymatically using a method based on that described previously for the guinea pig gallbladder (15). Briefly, the gallbladder was removed and placed in a Sylgard-coated petri dish containing cold Krebs-Henseleit solution (KHS; for composition, see Solutions and drugs). After the mucosa and the connective tissue that attaches the gallbladder to the liver were removed, 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 0.5 mg/ml dithioerythritol. The tissue was then transferred to fresh ES containing 1 mg/ml BSA, 1 mg/ml collagenase, and 100 µM CaCl2 and incubated in this solution for 9 min at 37°C. After the digestion was finished, the tissue was washed three times using ES, and single cells were isolated by passing the muscle pieces through the tip of a fire-polished glass Pasteur pipette several times. The resultant cell suspension was kept in ES at 4°C until use, generally within 6 h. All experiments were performed at room temperature (22°C).
Local [Ca2+] measurements and confocal microscopy. Confocal images of GBSM cells were obtained using a laser scanning confocal system (Oz, Noran Instruments, Middleton, WI) interfaced with an Indy workstation (Silicon Graphics, Mountainview, CA) and Intervision software. The confocal system was mounted in an inverted Diaphot microscope with a ×60 water-immersion objective (NA 1.2; Nikon). Isolated myocytes were plated in the recording chamber (vol, ~1 ml) and loaded with the Ca2+-sensitive fluorophore fluo 4-AM by incubation in ES containing 5 µM fluo 4 and 2.5 µg/ml pluronic acid for 30 min in the dark at room temperature. Cells were subsequently washed for 30 min with fresh physiological Ca2+ bath solution (BS; for composition, see Solutions and drugs) to remove fluo 4 from the extracellular fluid and to allow for fluo 4 deesterification. Cells were illuminated with a krypton-argon laser at 488 nm, and emitted light was collected with the confocal photomultiplier tube at wavelengths >515 nm. Images were typically acquired at 120 Hz (320 × 240 pixels or 64 × 48 µM, 8.33 ms/image) for 20 s. Experimental data were stored on compact discs for later analysis.
Image data were analyzed with custom software written by Dr. Adrian Bonev (University of Vermont) using Interactive Data Language version 5.0.2 (Research Systems, Boulder, CO). Baseline fluorescence (Fo) was determined by averaging 20 images containing no discernable Ca2+ transients. Ratio images (F/Fo, where F is instantaneous fluorescence at a given time point) were then constructed and analyzed for areas of 2.2 × 2.2 µm in which F/Fo increased rapidly. F/Fo vs. time traces were generated and analyzed for fluorescence increases using Origin software (Microcal Software, Northhampton, MA). Ca2+ sparks were defined as local increases in fluorescence of 1.2 F/Fo.Electrophysiology. Ionic currents were measured in isolated muscle cells using the whole cell perforated-patch configuration of the patch-clamp technique (23). The cell suspension was transferred to an experimental chamber made with a glass coverslip and mounted on the stage of an inverted microscope (Nikon). After cell sedimentation, the chamber was perfused, using a gravity-fed system, with BS (for composition, see Solutions and drugs). The pipette solution (for composition, see Solutions and drugs) also contained amphotericin B (100 µg/ml). Cells were perfused with BS throughout the experiment.
For cells used in this study, cell capacitance was 29.0 ± 0.7 pF, and series resistance was 27.6 ± 0.8 MSimultaneous current and Ca2+
measurements.
To examine the temporal relationship between Ca2+ sparks
and BK channel activation, we measured Ca2+ sparks and
whole cell currents, at a holding potential of 20 mV, using the
methods described above. A trigger source output on the confocal
microscope was used to align the fluorescence and electrical records.
These data were analyzed as described above.
Dissection and contraction recording of guinea pig GBSM strips. Gallbladders were isolated from 300- to 450-g male guinea pigs following deep halothane anesthesia and cervical dislocation and immediately placed in cold KHS (for composition, see Solutions and drugs). Animals were handled in accordance with the guidelines laid down by the Animal Care and Use Committee of the University of Extremadura. The gallbladder was opened by cutting along the longitudinal axis and trimmed of any adherent liver tissue. After being washed with the nutrient solution to remove any biliary component, the mucosa was scraped off and the gallbladder was cut into strips along the longitudinal axis. Each strip measured ~3 × 10 mm. On average, four strips were obtained from each guinea pig gallbladder. Each strip was placed vertically in a 10-ml organ bath filled with the nutrient solution maintained at 37°C and gassed with 95% O2-5% CO2. Isometric contractions were measured using force-displacement transducers connected to a MacLab system consisting of a MacLab hardware unit and software application that runs on the Macintosh computer. The strips were placed under an initial resting tension equivalent to a 1.5-g load and allowed a 60-min period for equilibration, during which time the nutrient solution was changed every 20 min. After that period, the length of each strip was increased at a rate of 1 mm each time until a maximal response to ACh occurred. The muscle length corresponding to the optimal preload was then determined by increasing the length of each strip in increments of 1 mm until a maximal response to ACh (10 µM) was achieved. The optimal preload length was maintained throughout the duration of the experiments.
The effects of thapsigargin (1 µM) and tetraethylammonium chloride (TEA; 1 mM) on the resting tone were assayed by adding these agents at the stated concentrations to the organ bath. To check the effects of 2-aminoethoxydiphenylborate (2-APB) on Ca2+ entry through L-type Ca2+ channels, KCl (60 mM) was assayed in the absence or presence of 50 µM of 2-APB.Solutions and drugs. The KHS used in this study 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% CO2-5% O2. 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 BS used in patch-clamp studies and in simultaneous recording studies contained (in mM) 134 NaCl, 6 KCl, 1 MgCl2, 2 CaCl2, 10 D-glucose, and 10 HEPES, with pH adjusted to 7.4 with NaOH. The pipette solution used in patch-clamp experiments contained (in mM) 110 potassium aspartate, 30 KCl, 10 NaCl, 1 MgCl2, 10 HEPES, and 0.05 EGTA, with pH adjusted to 7.2 with NaOH.
Drugs were obtained from the following sources. ACh chloride, amphotericin B, caffeine, dithioerythritol, EGTA, nifedipine, methoxyverapamil hydrochloride (D-600), thapsigargin, and TEA were from Sigma Chemical (St. Louis, MO). 2-APB was from Calbiochem (La Jolla, CA), and fluo 4 and pluronic acid were from Molecular Probes (Leiden, The Netherlands). Collagenase was from Fluka (Madrid, Spain), papain was from Worthington Biochemical (Lakewood, NJ), and ryanodine was from L.C. Laboratories. Stock solutions of 2-APB and thapsigargin were prepared in DMSO. The solutions were diluted so that the final concentration of DMSO in the recording chamber wasStatistics. Contractile responses are expressed in absolute values (in mN) and/or as the percentage of the maximal response elicited by ACh (10 µM). Values are expressed as means ± SE where applicable. Data were compared using two-tailed Student's t-tests, where appropriate. P < 0.05 was considered statistically significant.
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RESULTS |
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Identification of Ca2+ sparks in
GBSM.
Gallbladder myocytes loaded with the Ca2+-sensitive
indicator fluo 4 produced spontaneous transient elevations in
[Ca2+]i. The spatial and temporal
characteristics of these events suggested that they correspond to
Ca2+ sparks that have been described in other types of
smooth muscle (25, 32), as well as skeletal
(28) and cardiac muscle (10). Spark frequency
was 0.9 ± 0.3 Hz with an average of 1.8 ± 0.4 spark
sites/cell. Increases in the relative fluorescence (F/Fo, see MATERIALS AND METHODS) of 1.55 ± 0.02 were
detected (n = 127 spark events, from 9 cells). Figure
1 shows pseudocolor images corresponding
to the life cycle of a spark in a GBSM cell and the trace of the
fluorescence measurements corresponding to the boxed region.
Frequently, the Ca2+ transients were clustered into groups
consisting of multiple events (Figs. 1 and
2).
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Characterization of transient outward currents.
In smooth muscle cells, Ca2+ increases corresponding to
spark events have been shown (25) to activate transient
outward currents, facilitating a feedback mechanism that opposes
contraction. Therefore, we next characterized the transient outward
currents in GBSM and their dependence on Ca2+ release as
spark events. The holding potential for these studies was 20 mV.
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Ca2+ dependence of BK currents.
To verify that Ca2+ sparks lead to the activation of BK
channels in GBSM, we next investigated whether SR Ca2+
release is necessary for spontaneous transient BK currents and which SR
channels are responsible for this Ca2+ release. First,
gallbladder myocytes were treated with thapsigargin (200 nM), which
blocks the SR Ca2+-ATPase, depleting SR stores. Figure
5A shows an original record of
BK currents from a cell before and after treatment with thapsigargin. Thapsigargin reduced the BK current frequency by 96% (from 1.82 ± 0.53 to 0.03 ± 0.03 Hz, n = 4, P < 0.05, Fig. 5B), indicating that BK
channels are activated by SR Ca2+ release.
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Voltage dependence of BK currents.
To investigate the voltage dependence of transient BK currents,
GBSM cells were held at different potentials using the perforated-patch configuration of whole cell voltage clamp (see MATERIALS AND
METHODS). As shown in Fig. 6, the
frequency and amplitude of BK current were voltage dependent, which is
consistent with the voltage dependence previously demonstrated for BK
channel activity in other smooth muscle cell types (4, 12, 13,
21). Membrane potential depolarization from 40 to
20 mV
increased transient BK current frequency by ~3.7-fold, from 0.37 ± 0.08 to 1.4 ± 0.31 Hz (P < 0.05;
n = 1,429 events from 11 cells), and increased current
amplitudes of these events by ~2.5-fold, from 14.6 ± 0.7 to
36.7 ± 4.6 pA (P < 0.01; Fig. 6). When the
holding potential was increased to
10 mV, the frequency of current
transients was similar to that recorded at
20 mV (1.41 ± 0.30 Hz; n = 1,324 events from 11 cells, Fig. 6), but the
amplitude increased (58.3 ± 6.1 pA; P < 0.01 vs.
20 mV; Fig. 6).
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Ca2+ entry through L-type Ca2+ channels is
critical to maintaining BK currents.
In cardiac muscle, Ca2+ influx through voltage-activated
Ca2+ channels activates RyRs, allowing
Ca2+-induced Ca2+ release (as spark events),
which is essential for muscle contraction (30). The
communication between these Ca2+ channels and RyRs is less
clear in smooth muscle, where either dependence or independence of
Ca2+ sparks or BK currents on Ca2+ entry
through L-type Ca2+ channels has been reported (2, 4,
26). The increase in frequency and amplitude observed in the BK
currents at more depolarized holding potentials, as described above, is
consistent with a role for VOCCs. To examine the role of VOCCs, we
evaluated the effects of the L-type channel blocker nifedipine (0.5 and
1 µM) on transient BK currents in GBSM cells held at 20 mV.
Nifedipine at 0.5 and 1 µM caused similar reductions in the frequency
of current transients (2.50 ± 1.21 to 0.87 ± 0.61 Hz for
0.5 µM nifedipine, 79% inhibition, n = 4, P < 0.05; and 2.45 ± 0.89 to 0.42 ± 0.15 Hz for 1 µM nifedipine, 81% inhibition, n = 5, P < 0.05 vs. control, Fig.
7B). Nifedipine did not modify
current amplitude (46.2 ± 13.8 vs. 47.4 ± 15.2 pA for 0.5 µM nifedipine, n = 4, P = 0.702; and
51.2 ± 5.6 vs. 53.4 ± 7.2 pA for 1 µM nifedipine,
n = 5, P = 0.649). When the effect of
nifedipine reached steady state, the application of caffeine (250 µM)
induced an increase in both the frequency and amplitude of the currents
(Fig. 7A), indicating that the stores still had sufficient
Ca2+ to induce BK channel activation.
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CCK inhibits BK currents.
In the gallbladder, CCK-induced contraction involves the release of
Ca2+ from intracellular stores as well as Ca2+
influx through L-type Ca2+ channels (1, 34,
38). Because the mechanisms responsible for Ca2+
influx in GBSM have not yet been explored and increased protein kinase
C activity suppresses BK currents in vascular smooth muscle (8), we tested whether CCK altered transient BK currents
in GBSM. As demonstrated in Fig. 8, CCK
(10 nM) reduced both the frequency and amplitude of these currents
(3.2 ± 0.8 vs. 1.2 ± 0.5 Hz, 62% of inhibition,
P < 0.005 for frequency; and 42.2 ± 5.4 vs.
30 ± 4.1 pA, 23% of inhibition, P < 0.01 for
amplitude; n = 13 cells for both). In cases in which
CCK caused a complete inhibition of transient BK currents, the currents
did not reappear until 12.5 ± 3.2 min (n = 3)
after washout of CCK. Caffeine (1 mM) added after CCK treatment induced
a burst of transient currents, indicating that the Ca2+
stores were not depleted (n = 3).
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DISCUSSION |
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The purpose of this investigation was to establish whether localized increases in [Ca2+]i occur in GBSM and, if so, how they are generated and whether they lead to the activation of transient K+ currents. In smooth muscle, Ca2+ sparks are primarily caused by the coordinated opening of a cluster of RyRs in the SR (19, 41) and activate a number of BK channels to cause macroscopic BK currents (32). A link between Ca2+ sparks and transient BK currents has been confirmed by simultaneous optical and electrical measurements in these cells (35). Localized Ca2+ release events, termed Ca2+ puffs, that are mediated by IP3 receptor-operated channels, have been reported (3) in colonic smooth muscle, where these events also regulate membrane Ca2+-dependent K+ channels (2, 3).
In the current study, we provide the first demonstration that localized increases in [Ca2+]i occur in GBSM. These increases in [Ca2+]i are transient and do not lead to an elevation in global [Ca2+]i (Ca2+ sparks). Ca2+-dependent conductances are likely targets for localized [Ca2+]i elevations. The Ca2+ sparks in GBSM are caused by the opening of ryanodine-sensitive Ca2+ release channels (RyRs) in the SR.
Ca2+ sparks activate BK currents in GBSM cells. In GBSM, BK channels appeared to be the primary K+ channel type activated by Ca2+ sparks, since the transient outward currents were inhibited 98% by iberiotoxin, the specific blocker of BK channels (17, 33). However, in colonic myocytes, local Ca2+ transients stimulate both BK and SKCa channels (2). In the gallbladder, we have found that apamin, the selective inhibitor of SKCa channels, has no effect on muscle strip tension, resting membrane potential, or action potential properties (Pozo, Nelson, and Mawe, unpublished observations), suggesting that SKCa channels do not play a prominent role in the regulation of gallbladder tone.
The Ca2+ spark-activated transient BK current in GBSM caused a transient hyperpolarization up to 37 mV, similar to that described previously for arterial myocytes (18). Taking into account the high frequency of BK currents, even in resting conditions, spontaneous Ca2+ sparks and the resultant hyperpolarizations could decrease GBSM excitability by decreasing the open-state probability of VOCCs. Consistent with this hypothesis, inhibition of BK currents with the K+ channel blocker TEA, at a dose that inhibits BK channels (1 mM) (17), induced a methoxyverapamil-sensitive contraction of gallbladder strips. In the current study, although a temporal coupling between Ca2+ and spontaneous transient BK currents was observed, the correlation between spark and BK current amplitude was not very high. In other systems, including the urinary bladder and vascular and colonic smooth muscle, a strong correlation between spark and BK current amplitudes has been reported (2, 21, 36). However, a weak correlation between outward current and spark amplitude has been reported in cells from the stomach muscularis of Bufo marinus (41) and feline esophageal smooth muscle (27). It has been proposed that if [Ca2+] in the spark microdomain rapidly reaches steady state, the open probability for K+ channels will be 1. Therefore, many of the BK channels would be saturated with Ca2+, and correlation between spark and BK amplitudes would not be observed. Alternatively, the density of BK channels could be quite variable, which would also affect the apparent correlation. We found that the transient BK current amplitude increased with membrane potential depolarization in a manner consistent with an increase in the K+ driving force. If, during a spark, the BK channels are not maximally activated with spark Ca2+, then the elevation in transient BK current amplitude with membrane potential depolarization should increase more than expected for simple changes in the driving force for K+. The amplification of the effect of membrane depolarization on transient BK current amplitude is caused by the increase in the apparent Ca2+ sensitivity of the BK channel caused by membrane depolarization (2). Therefore, our results are consistent with the model that Ca2+ sparks maximally activate nearby BK channels in GBSM.Voltage dependence of BK currents. Consistent with the voltage dependence that has been established for single BK channels (12, 13), the amplitude of transient BK currents in GBSM was enhanced at depolarized voltages. This increase probably involves the increased K+ driving force at these voltages. Membrane potential depolarization also increased Ca2+ spark frequency, which was dependent on Ca2+ entry through VOCC. This elevation in Ca2+ entry increases cytoplasmic and SR Ca2+, both of which elevate Ca2+ spark frequency (21, 26).
L-type Ca2+ channels are critical to maintaining BK currents in GBSM. Our data suggest that Ca2+ influx through L-type Ca2+ channels is essential to maintaining BK currents in the gallbladder, since nifedipine and membrane potential hyperpolarization reduced the frequency of these currents. Activation of L-type voltage channels by depolarization increases the frequency and amplitude of Ca2+ sparks in vascular (26) and urinary bladder smooth muscle (21), which increases the amplitude and frequency of BK currents. The increase in spark activity could be due to elevations in local [Ca2+] and the concomitant increase in RyR open probability (22) or increases in SR load (42). In cardiac muscle, Ca2+ sparks are induced by membrane potential-dependent entry of Ca2+ through sarcolemmal L-type Ca2+ channels at the transverse tubules (30). RyR channels are positioned in junctional SR elements within short distances (~20 nM) of voltage-dependent Ca2+ channels in the transverse tubules, and high local [Ca2+] is in the level required for significant RyR channel activation (for review, see Ref. 16). Although smooth muscle cells lack the transverse tubular membrane system, L-type Ca2+ channels colocalize with junctional SR (14), and Ca2+ entry through these channels would increase in the microdomain of RyRs, increasing their probability of opening. However, Collier et al. (11) have provided evidence that VOCC and RyRs are distant since there is a considerable time lag between activation of VOCC and subsequent increases in spark probability. It is also possible that the rise in average cytoplasmic Ca2+ is sufficient to activate Ca2+ sparks. On the other hand, the reduction of BK current amplitude exerted by nifedipine pretreatment could be due to a decrease in SR luminal [Ca2+].
CCK inhibits BK currents. In many types of smooth muscle, excitatory agonists have been shown to act at least in part by inhibiting K+ conductances. In vascular and gastrointestinal smooth muscle, excitatory agonists that, like CCK, mediate their responses through phospholipase C and protein kinase C activation reduce the amplitude and frequency of Ca2+ sparks (24), and activators of protein kinase C decrease the activity of Ca2+ sparks and BK currents through an inhibitory effect on RyR channels (3, 8). In the present study, we found that CCK caused a reduction in the amplitude and frequency of transient BK currents. These findings indicate that reducing the activation of BK channels, which are presumably active under resting tone conditions, contributes to the excitatory effect of CCK.
The CCK-induced reduction of BK currents in the gallbladder could reflect a reduction in Ca2+ sparks associated with a decrease in the Ca2+ sensitivity of RyR channels caused by protein kinase C. It is unlikely that a reduction in SR load decreases BK current activity, because subsequent application of caffeine immediately restored transient BK currents, indicating that SR maintains enough Ca2+ to generate Ca2+ sparks through RyR channels in the presence of CCK. In the colon, agonist-induced inhibition of transient BK currents is thought to involve an inhibitory effect of Ca2+ entry through receptor-operated cation channels on IP3 receptor-operated Ca2+ events (3). This mechanism is unlikely to be involved in the GBSM response to CCK, because transient BK currents are RyR mediated and CCK-induced gallbladder contraction is mediated in part by Ca2+ influx through L-type Ca2+ channels (1, 1, 34, 38). Moreover, in our study, suppression of Ca2+ influx by nifedipine caused a reduction in BK currents, indicating a significant role for Ca2+ influx in spark activity and BK currents, whereas in the colon no effects were reported (3) for nicardipine when tested under control conditions or during ACh stimulation.Concluding remarks. In summary, this study provides the first evidence of the presence of local Ca2+ transients or Ca2+ sparks in GBSM cells. These Ca2+ sparks, which are mainly due to the activation of RyR in the SR, are potentially coupled to activation of BK channels. The spontaneous transient activation of BK currents causes transient membrane hyperpolarizations, providing a Ca2+-mediated mechanism to decrease excitability. Inhibition of these events by the excitatory agonist CCK indicates that decoupling between Ca2+ sparks and BK channels can also contribute to the increased excitability in response to these agonists.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health Grants NS-26995, DK-45410 (G. M. Mawe), HC-44455, HL-63722, and DK-53832 (M. T. Nelson) and Ministry of Education and Science of Spain Grant DGES-PB97-0370 (M. J. Pozo).
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FOOTNOTES |
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First published October 24, 2001;10.1152/ajpgi.00326.2002
Address for reprint requests and other correspondence: G. M. Mawe, Given Bldg. C423, Dept. of Anatomy and Neurobiology, Univ. of Vermont, Burlington, VT 05405 (E-mail: gmawe{at}zoo.uvm.edu).
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.
Received 27 July 2001; accepted in final form 17 September 2001.
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