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NEUROREGULATION AND MOTILITY

Spontaneous electrical rhythmicity and the role of the sarcoplasmic reticulum in the excitability of guinea pig gallbladder smooth muscle cells

Departments of ¹Anatomy and Neurobiology and ²Pharmacology, University of Vermont, Burlington, Vermont

Submitted 7 July 2005 ; accepted in final form 14 November 2005

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Spontaneous action potentials and Ca²⁺ transients were investigated in intact gallbladder preparations to determine how electrical events propagate and the cellular mechanisms that modulate these events. Rhythmic phasic contractions were preceded by Ca²⁺ flashes that were either focal (limited to one or a few bundles), multifocal (occurring asynchronously in several bundles), or global (simultaneous flashes throughout the field). Ca²⁺ flashes and action potentials were abolished by inhibiting sarcoplasmic reticulum (SR) Ca²⁺ release via inositol (1,4,5)-trisphosphate [Ins(1,4,5)P₃] channels with 2-aminoethoxydiphenyl borate and xestospongin C or by inhibiting voltage-dependent Ca²⁺ channels (VDCCs) with nifedipine or diltiazem or nisoldipine. Inhibiting ryanodine channels with ryanodine caused multiple spikes superimposed upon plateaus of action potentials and extended quiescent periods. Depletion of SR Ca²⁺ stores with thapsigargin or cyclopiazonic acid increased the frequency and duration of Ca²⁺ flashes and action potentials. Acetylcholine, carbachol, or cholecystokinin increased synchronized and increased the frequency of Ca²⁺ flashes and action potentials. The phospholipase C (PLC) inhibitor U-73122 did not affect Ca²⁺ flash or action potential activity but inhibited the excitatory effects of acetylcholine on these events. These results indicate that Ca²⁺ flashes correspond to action potentials and that rhythmic excitation in the gallbladder is multifocal among gallbladder smooth muscle bundles and can be synchronized by excitatory agonists. These events do not depend on PLC activation, but agonist stimulation involves activation of PLC. Generation of these events depends on Ca²⁺ entry via VDCCs and on Ca²⁺ mobilization from the SR via Ins(1,4,5)P₃ channels.

calcium transients; gallbladder motility; slow waves; action potentials

Unlike the gut, the gallbladder is a tonic, compliant organ that is capable of storing a large volume of bile that is released postprandially after appropriate neurohumoral stimulations (20, 21). While the gallbladder functions as a tonic organ, the membranes of individual GBSM cells undergo rhythmic depolarizations, consisting of a spike and plateau phase, that are shorter in duration and occur at higher frequency than the slow wave action potentials of GI smooth muscle (12, 45). To date, it is unclear whether spontaneous electrical rhythmicity in GBSM is a global event, which represents synchronized behavior of the entire muscularis, or if it involves asynchronous multifocal excitatory activity in individual muscle bundles throughout the organ. Additionally, cellular mechanisms for the generation and propagation of these events are not well understood.

Calcium fluxes are likely to play a significant role in the activation and propagation of electrical activity in GBSM. Unlike the slow waves of the bowel, GBSM action potentials are completely abolished by voltage-dependent Ca²⁺ channel (VDCC) inhibitors (45). Furthermore, voltage-activated Ca²⁺ currents in GBSM are inhibited by dihydropyridines (30, 45). We have previously investigated Ca²⁺-release events from the sarcoplasmic reticulum (SR) in isolated gallbladder myocytes at room temperature and detected Ca²⁺ sparks in individual GBSM cells. These events are largely dependent on Ca²⁺ release through ryanodine-sensitive Ca²⁺-release channels in the SR, and Ca²⁺ sparks activate nearby large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels (28). We have also shown that in GBSM cells, ryanodine and inositol (1,4,5)-trisphosphate [Ins(1,4,5)P₃] channels are located on the same intracellular stores (23), and, in the absence of extracellular Ca²⁺, depletion of these stores results in capacitative Ca²⁺ entry (24).

The present study was designed to elucidate rhythmic Ca²⁺ fluctuations in GBSM, how they correspond with electrical rhythmicity and the pattern of activity of these events within and among GBSM bundles, and how they are influenced by SR Ca²⁺ mobilization. We used fast-speed laser confocal imaging of fluo-4 (a Ca²⁺ indictor dye)-loaded tissue to evaluate Ca²⁺ transients and standard intracellular recording to evaluate electrical activity in whole mount preparations of guinea pig GBSM at 35–36°C. The results of this study indicate that Ca²⁺ flashes, which represent Ca²⁺ influx via VDCCs, correspond to action potentials in GBSM. These events are multifocal among GBSM bundles, depend on Ca²⁺ mobilization from intracellular stores, and are enhanced by excitatory agonists. They are activated by SR Ca²⁺ depletion and are critical to refilling intracellular Ca²⁺ stores.

	MATERIALS AND METHODS

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Animals, sampling, and dissection. Adult guinea pigs of either sex, weighing 250–350 g, were used for this study. Guinea pigs were anesthetized with halothane or isoflurane and exsanguinated in accordance with Institutional Animal Care and Use Committee of the University of Vermont guidelines. The abdominal cavity was opened by a midline incision, and the gallbladder was removed and put into ice-cold modified Krebs solution composed of (in mM) 121 NaCl, 5.9 KCl, 2.5 CaCl₂, 1.2 MgCl₂, 25 NaHCO₃, 1.2 NaH₂PO₄, and 8 glucose (pH 7.4). Gallbladders were cut open from the end of the cystic duct to the base of the organ and subdivided into two approximately equal pieces. These tissues were rinsed with ice-chilled Krebs solution and pinned stretched mucosal side up in a dish lined with Sylgard 184 elastomer (Dow Corning; Midland, MI). Whole mounts of the muscularis propria were obtained by teasing off the mucosal layer with sharp forceps under stereoscopic microscopic observation. To obtain more samples, each half gallbladder was cut into two pieces across the middle between the base and neck so that each gallbladder gave four small pieces of 1 x 1.5 cm. Dissections were undertaken in ice-chilled Krebs solution, which was changed every 8–10 min.

Confocal imaging of Ca²⁺ flashes. Gallbladder whole mounts were rinsed with HEPES buffer containing (in mM) 110 NaCl, 5.4 KCl, 1.8 CaCl₂, 1.0 MgCl₂, 20 HEPES, 5 glucose, and 60 sucrose (pH 7.4) and pinned stretched serosal surface up between two Sylgard blocks. The preparations were loaded with 10 µM fluo-4 AM in HEPES buffer containing 2.5 µg/ml Pluronic acid and thereafter washed with HEPES buffer to allow for deesterification. Loading (1 h) and washing (0.5–1 h) were done at room temperature. Tissues were gently mounted serosal side facing the coverslip of the recording chamber (2 ml) that was maintained at 35–36°C by continuous superfusion with aerated (95% O₂-5% CO₂) recirculating physiological saline solution (PSS) containing (in mM) 119 NaCl, 7.5 KCl, 1.6 CaCl₂, 1.2 MgCl₂, 23.8 NaHCO₃, 1.2 NaH₂PO₄, 0.023 EDTA, and 11 glucose (pH 7.3) at a rate of 3 ml/min. After 15–20 min of equilibration, tissues were examined using an inverted Nikon TMD microscope with a x60 water-immersion lens (1.2 numerical aperture). Scanning was done using a Noran Oz laser confocal system (Noran Instruments). Images were acquired using Intervision software (Noran Instruments) on an Indy work station (Silicon Graphics; Mountain View, CA) to record oscillations of fluorescence signals caused by fluctuations of the cytosolic Ca²⁺ concentration ([Ca²⁺]_c) during 20 s of acquisition; 30 images (frames) were acquired per second to create movie files of 600 images. Tissues were continuously superfused with drugs immediately after a movie file was recorded for control data, and after that imaging was done every 5th minute for a period of 25 min.

Analysis of movie files. Movie files were analyzed offline for the frequency of Ca²⁺ flashes using custom software written in our laboratory (A. D. Bonev). Briefly, rectangular boxes of 1.6–5 µm empirically determined to minimize the signal-to-noise ratio were put on images of myocytes (Fig. 1, A and B) that were in sharp focus and displayed intense Ca²⁺ flashes and or Ca²⁺ waves. Baseline fluorescence (F₀) was established by averaging 10 images (of the 600 images in a movie) that did not have any Ca²⁺ activity. Movies were visually assessed when normalized ratio images were reconstructed as traces of measured fluorescence (F)/F₀ (Fig. 1, A and B) and tables of frequency, amplitude, image number, and decay maxima were generated. Data were imported into Microsoft XL, and variability between frequencies of Ca²⁺ flashes (Hz) was reduced by normalizing each data set. Normalization was achieved by dividing the frequency data for each individual treatment by the control frequency value of Ca²⁺ flashes obtained before drugs were applied. The frequency values for Ca²⁺ flashes presented as normalized frequency (ratios) are without units and are approximately two to three times larger than the usual frequency.

View larger version (125K): [in this window] [in a new window]   Fig. 1. Propagation of Ca2+ flashes in a gallbladder smooth muscle (GBSM) bundle. A: Ca2+ flashes are instantaneous widespread transients (0.2–0.5 Hz) that are associated with the influx of Ca2+ into muscle cells during an action potential. B: traces of fluorescence ratios (F/F0) depicting synchronized oscillations of cystolic Ca2+ concentration during Ca2+ flashes in two different regions (indicated by and ). Rhythmicity of Ca2+ flashes in a given bundle was always synchronous.

Statistical analysis (Student t-test) was done using Graph Pad Prism 4. Data are expressed as means ± SE; n indicates the number of experiments using preparations from different animals. Differences were assumed statistically significant at P 0.05.

Chemicals and drugs. Chemicals and drugs used in the present study included fluo-4 AM and Pluronic acid (F-127; Molecular Probes); thapsigargin and ryanodine mixture (Calbiochem); the phospholipase C (PLC) inhibitor (1-{6-[17b-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione (U-73122); xestospongin C (Cayman); and cholecystokinin (CCK) octapeptide (Bachem). Other drugs used were 2-aminoethoxydiphenyl borate (2-APB), acetylthiocholine iodide (ACh), caffeine, diltiazem hydrochloride, EDTA, MgCl₂·6H₂O₂, sucrose, glucose, KCl, NaHCO_3, sodium phosphate monobasic, potassium phosphate monobasic, HEPES, DMSO, cyclopiazonic acid (CPA), 1-octanol, carbenoxolone, and 18-glycyrrhetinic acid (Sigma); NaCl (Fischer Scientific); and CaCl₂ (Acros Organic). Caffeine, ACh, and diltiazem were dissolved in double distilled water. Nifedipine was dissolved in ethanol. All other drugs were dissolved in DMSO.

	RESULTS

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Spontaneous Ca²⁺ transients detected in intact GBSM. Ca²⁺ transients and membrane potential recordings were investigated in a total of 203 gallbladder whole mounts from 132 animals. In these studies, three types of spontaneous Ca²⁺ transients were identified. One type consisted of flash-like Ca²⁺ transients (Ca²⁺ flashes) that appeared to take place simultaneously among the smooth muscle cells of a given bundle and occurred in a rhythmic pattern [Fig. 1, A and B, and movie 1 (see supplemental data at http://ajpgi.physiology.org/cgi/content/full/00310.2005/DC1)]. These Ca²⁺ flashes had a velocity of 1,900 ± 308.2 µm/s (n = 10) and lasted 700 ms. The second type of Ca²⁺ transient consisted of intracellular elevations in [Ca²⁺]_c that propagated slowly and asynchronously (68.0 ± 17.0 µm/s, n = 15, range of 26.0–500.0 µm/s) among the cells in a given smooth muscle bundle; these events are referred to as Ca²⁺ waves (movie 2). The frequencies of Ca²⁺ flashes and Ca²⁺ waves (0.21 ± 0.03 Hz, n = 18, vs. 0.18 ± 0.01 Hz, n = 49, P = 0.4) were comparable.

The third type of Ca²⁺ transient was composed of localized, nonpropagating elevations in [Ca²⁺]_c within individual GBSM cells that were without a detectable pattern within or between GBSM cells. These events are comparable to Ca²⁺ sparks that have been described in isolated GBSM cells (28) and Ca²⁺ "puffs" and purinergic transients that have described in isolated smooth muscle cells from the guinea pig colon (2) and urinary bladder smooth muscle (11), respectively. Ca²⁺ flashes and Ca²⁺ waves were differentiated based on the nature of occurrence, propagation patterns and velocity, and the extent of spread in smooth muscle cells. GBSM cells exhibited either Ca²⁺ flashes or Ca²⁺ waves alone, or both Ca²⁺ flashes and Ca²⁺ waves occurred consecutively in the same cell (movies 1 and 2). Compared with DMSO (0.01%) vehicle, exposure of preparations to a combination of TTX (2 µM) and atropine (1 µM) did not affect the Ca²⁺ flashes (vehicle: 0.9 ± 0.07 vs. TTX-atropine: 1.5 ± 0.3, n = 4, P > 0.05, 25 min). As indicated in MATERIALS AND METHODS, the frequencies of Ca²⁺ flashes, which are presented without parameters, are normalized data (frequency ratios).

Propagation of Ca²⁺ flashes in GBSM cells. The regional distribution of rhythmic Ca²⁺ flashes in GBSM was variable. Rhythmic flashes could be limited to a few cells within a bundle, occur throughout an entire bundle, or be more widespread, involving several bundles or an entire preparation (Fig. 1, A and B, and movies 1 and 2). Although we could not identify the cells that triggered Ca²⁺ flashes, these transients propagated from one end to another along the longitudinal axis of a given muscle bundle. Although GBSM cells contracted after a flash, phasic contractions did not necessarily spread along the longitudinal axis of each muscle bundle. Rhythmicity of Ca²⁺ flashes in a given muscle bundle was synchronized (Fig. 1, A and B). Similarly, the action potential obtained by impaling several GBSM cells within the same bundle had the same pattern and frequency. Moreover, confocal Ca²⁺ imaging showed that within a given preparation, different muscle bundles, especially converging bundles, showed synchronized Ca²⁺ flashes. Rhythmicity in overlapping bundles that did not appear to converge was asynchronous.

Effect of VDCC-mediated Ca²⁺ influx on Ca²⁺ flashes and action potentials. In GBSM cells, Ca²⁺ influx via VDCCs is critical for the generation and propagation of spontaneous action potentials (30, 45). In the present study, we tested the effects of the VDCC inhibitor diltiazem (50 µM) as well as nifedipine and nisoldipine (1 µM each) on Ca²⁺ flashes. Inhibition of VDCCs abolished action potentials and Ca²⁺ flashes in GBSM cells after 5–15 min (Fig. 2A), which is similar to earlier observations in the gallbladder (45) and urinary bladder smooth muscle (11), respectively, supporting the view that both Ca²⁺ flashes and action potentials depend on Ca²⁺ influx via VDCCs.

View larger version (30K): [in this window] [in a new window]   Fig. 2. Demonstration that Ca2+ flashes and action potentials are inhibited by voltage-dependent Ca2+ channels (VDCCs) and that sarcoplasmic reticulum (SR) Ca2+ release via inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] channels (A–C) is involved in the generation and regulation of the rhythmicity and synchronicity of Ca2+ flashes and action potentials. A: inhibition of Ca2+ flashes by 1 µM nifedipine. B–D: traces of normalized F/F0 for Ca2+ flashes (B and D) and action potentials (C) showing reduced or abolished Ca2+ flashes (normalized frequency) and action potentials after treatment with the Ins(1,4,5)P3 channel inhibitors 2-aminoethoxydiphenyl borate (2-APB; 100 µM) and xestospongin C (Xest C; 5 µM). 2-APB depolarized GBSM cells, and both compounds markedly reduced and/or abolished action potentials.

View larger version (23K): [in this window] [in a new window]   Fig. 5. Quantitative data demonstrating the effects of 2-APB, Xest C, RyR, CPA, and Thaps on the frequency of Ca2+ flashes (A) and action potentials (B) and on membrane potential (C). Except Xest C (see Fig. 6A) and 2-APB 5 min (see Fig. 6B), the presented data of which are for the 5-min exposure time, all other measurements were done after 25 min. PSS, physiological saline solution (*P 0.05; **P 0.01; and ***P 0.001).

View larger version (16K): [in this window] [in a new window]   Fig. 3. Demonstration of the actions of ryanodine (RyR) on the rhythmicity and synchronicity of Ca2+ flashes and action potentials as well as membrane potential. RyR reduced the frequency of Ca2+ flashes (A) and action potentials (B) and caused clustering of action potentials as well as repetitive spikes on the plateau of action potentials. Like 2-APB and Xest C, RyR disrupted the synchronicity and rhythmicity of flashes and action potentials.

View larger version (29K): [in this window] [in a new window]   Fig. 4. Acute inhibition of SR Ca2+ uptake (store depletion) augments excitability (A–D) of GBSM cells. Intracellular Ca2+ store depletion by cyclopiazonic acid [CPA (10 µM), only shown in C and D] and thapsigargin (Thaps; 2 µM) increased the frequency of Ca2+ flashes (A) and action potentials (B), caused repetitive spikes on the plateau of action potentials, and depolarized GBSM cells (B). Both compounds exhibited time-dependent actions by first increasing the frequency of action potentials before reducing this parameter (C) and increased the duration of action potentials (D; 25 min). The arrow in C indicates the time of drug application (*P 0.05; **P 0.01).

Excitatory agonists stimulate PLC to augment Ca²⁺ flashes and action potentials in intact GBSM. In muscle strips and isolated cells from the gallbladder, excitatory agonists activate PLC, which hydrolyzes phosphatidylinositol bisphosphate, leading to the generation of diacylglycerol (DAG) and Ins(1,4,5)P₃. The latter induces Ca²⁺ release from the SR (38, 44). The PLC inhibitor U-73122 inhibits Ins(1,4,5)P₃ production and reduces spontaneous Ca²⁺ transients in GI smooth muscle cells (3, 46). We evaluated the effect of excitatory agonists and U-73122 on Ca²⁺ flashes and membrane potentials in intact GBSM (Figs. 6, A and B, and 7, A–C). Compared with DMSO, U-73122 (20 µM) did not affect the frequency of Ca²⁺ flashes (vehicle: 1.2 ± 0.3, n = 4, vs. U-73122: 0.8 ± 0.3, n = 3, P = 0.4, 20 min), action potentials (vehicle: 0.3 ± 0.007 Hz, n = 6, vs. U-73122: 0.3 ± 0.04 Hz, n = 8, P = 0.6, 15 min), or resting membrane potential (vehicle: –50.9 ± 1.1 mV, n = 6, vs. U-73122: –50.7 ± 2.6 mV, n = 4, P = 0.94, 15 min) of GBSM cells (Fig. 7, B and C). However, U-73122 showed time-dependent actions. U-73122 did not alter the frequency of Ca²⁺ flashes and action potentials (vehicle: 0.35 ± 0.007 Hz, n = 6, vs. U-73122: 0.40 ± 0.05 Hz, n = 7, P = 0.4, 5 min) during the first 5–10 min but subsequently slowly reduced the frequency of Ca²⁺ flashes and action potentials (vehicle: 0.31 ± 0.02 Hz, n = 5, vs. U-73122: 0.25 ± 0.04 Hz, n = 4, P = 0.15, 25 min) (not significant). Compared with vehicle, the muscarinic agonists carbachol (3 µM) and ACh (50 µM) as well as CCK (100 nM) dramatically increased the frequency of Ca²⁺ flashes during the first 2–10 min (Figs. 6A and 7, A and B). After 15 min, the frequency of Ca²⁺ flashes decreased progressively. Excitatory agonists (vehicle vs. ACh in the example) increased the frequency (vehicle: 0.3 ± 0.03 Hz, n = 7, vs. ACh: 0.6 ± 0.04 Hz, n = 7, P = 0.0002, 5 min) and duration (data not shown) of action potentials; induced repetitive spikes on the plateau of action potentials, particularly carbachol; and also depolarized the resting membrane potential (vehicle: –51.9 ± 2.0 mV vs. ACh: –35.7 ± 3.0 mV, n = 8, P < 0.002, 5 min) (Figs. 6A and 7, B and C). After the application of excitatory agonists, Ca²⁺ flashes among the muscle bundles in a given field became synchronized.

View larger version (21K): [in this window] [in a new window]   Fig. 6. The ACh-induced increase in action potential frequency is attenuated by the phospholipase C (PLC) inhibitor U-73122. A: ACh causes a depolarization and increase in the frequency of action potentials. B: in the presence of U-73122, the ACh-induced increase in action potential frequency and depolarization are attenuated.

View larger version (18K): [in this window] [in a new window]   Fig. 7. Enhanced excitability of GBSM induced by G protein-coupled muscarinic receptor activation is attenuated by PLC inhibition. A: summary data demonstrating an increase in the frequency of Ca2+ flashes caused by carbachol and cholecystokinin (CCK). B: summary data demonstrating that the PLC inhibitor U-73122 attenuates the effect of ACh on GBSM action potential frequency. C: summary data demonstrating that the PLC inhibitor U-73122 attenuates the effect of ACh on GBSM membrane potential (*P 0.05; **P 0.01; and ***P 0.001).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
The aim of the present investigation was to gain a better understanding of excitation-contraction coupling in the gallbladder. To accomplish this, we evaluated the spread and synchronicity of spontaneous electrical rhythmicity among GBSM bundles and the cellular mechanisms that modulate these events. In this first study of Ca²⁺ events in intact GBSM tissues, we identified three patterns of spontaneous Ca²⁺ transients in intact GBSM: Ca²⁺ sparks, Ca²⁺ waves, and Ca²⁺ flashes. Because Ca²⁺ flashes have been shown to correspond to action potentials in other visceral smooth muscles (10, 11, 31–34, 36), we investigated the spatiotemporal patterns of Ca²⁺ flashes in intact GBSM. We evaluated the responsiveness of Ca²⁺ flashes and associated action potentials to conditions that affected specific aspects of Ca²⁺ entry and Ca²⁺ mobilization from intracellular stores. The results reported here indicate that Ca²⁺ flashes reflect rhythmic increases in intracellular Ca²⁺ concentration during action potentials in GBSM and that these events are dependent on the release of Ca²⁺ via Ins(1,4,5)P₃ channels and Ca²⁺ entry via VDCCs. Furthermore, these events can be enhanced by excitatory agonists via PLC activation. The pattern of Ca²⁺ flash activity suggests that generation of electrical rhythmicity in GBSM occurs at the level of individual GBSM bundles.

Ca²⁺ flashes, action potentials, and the spatiotemporal pattern of activity in GBSM. A principal objective of the present investigation was to determine whether smooth muscle tone in the gallbladder is generated by synchronized activity of the entire muscularis propria or if tone is generated as the net result of multifocal activity at the level of individual smooth muscle bundles. We have previously used intracellular recording techniques to demonstrate that spontaneous rhythmic action potentials are generated in GBSM and that these events appeared to correspond to rhythmic contractions of smooth muscle in the region of the impaled smooth muscle cell (45). Because these observations were limited to individual cells in individual GBSM bundles, we pursued Ca²⁺ imaging as a means to evaluate the rhythmic excitability of many GBSM cells simultaneously within individual bundles and among many bundles in a given region.

On the basis of the findings reported here, it appears that spontaneous Ca²⁺ flashes correspond to spontaneous action potentials because these events occur at a similar frequency, are dihydropyridine sensitive, and occur via a mechanism that requires Ca²⁺ release via SR Ins(1,4,5)P₃sensitive channels, but does not critically depend on PLC activation. Furthermore, a relationship between action potentials and Ca²⁺ flashes has been described in other types of visceral smooth muscles (10, 11, 31–34, 36). Under basal conditions, rhythmicity of Ca²⁺ flashes was synchronized in muscle cells that resided in a given muscle bundle, whereas flash patterns among muscle bundles in a given field were not necessarily synchronized. These data indicate that basal excitation-contraction coupling in GBSM occurs at the level of individual muscle bundles and suggests that basal gallbladder tone reflects the net result of asynchronous contractions of muscle bundles throughout the lamina propria. Interestingly, a more global pattern of rhythmic activity was observed in the presence of excitatory agonists. It is therefore possible that the increased tone that is generated during gallbladder emptying involves a more coordinated pattern of activity within the muscularis. The mechanisms by which this transformation from multifocal to global activity occurs are not understood at this time.

Cellular mechanisms regulating spontaneous Ca²⁺ flashes and action potentials in GBSM cells. The mechanisms underlying electrical rhythmicity, and thus smooth muscle contractions, in the gallbladder are not well understood. In the GI tract, pacemaking and electrical rhythmicity involves coupling among Ins(1,4,5)P₃-mediated SR Ca²⁺ release, mitochondrial Ca²⁺ uptake, and Ca²⁺ influx vial dihydropyridine-insensitive VDCCs, which facilitate propagation by causing depolarization (14, 35–37, 39–41). To begin to understand the possible roles of Ca²⁺ entry and Ca²⁺ mobilization in Ca²⁺ flashes and the associated excitation of GBSM, we evaluated Ca²⁺ activity and action potentials in the presence of compounds that affect intracellular Ca²⁺ stores mobilization and VDCCs.

The findings reported here indicate that, within a given GBSM cell, the generation of Ca²⁺ flashes and action potentials requires Ca²⁺ entry via VDCCs and Ca²⁺ release via Ins(1,4,5)P₃ channels because these events were abolished by VDCC blockers and by the Ins(1,4,5)P₃ inhibitors 2-APB and xestospongin C. The role of Ins(1,4,5)P₃ channels in GBSM excitability is consistent with previous findings in other types of smooth muscles, including GI (19, 35–37, 39–41) and vascular (6) smooth muscle cells. In the GI tract, Ins(1,4,5)P₃ channels play a fundamental role in generating pacemaker potentials/slow waves action potentials (14, 37, 39). In the gallbladder, the source and mechanisms of pacemaking activity are not yet understood. For example, it is not yet clear whether Ins(1,4,5)P₃ channels are involved simply in the generation of Ca²⁺ flashes and action potentials or if they also play a role in the generation and pacing of rhythmic activity in GBSM.

A voltage-dependent Ca²⁺ current can be detected in isolated GBSM (30, 45), and spontaneous action potentials are abolished in the presence of VDCC blockers (45). Consistent with these findings, the data included in the present study demonstrate that Ca²⁺ flashes are also abolished when VDCCs are inhibited. It is likely that Ca²⁺ entering the cell during the action potential contributes to the rise in intracellular Ca²⁺ during the Ca²⁺ flash, but the interdependence between VDCC and Ins(1,4,5)P₃ channel activity is not yet understood. VDCC activity is thought to play a crucial role in maintaining intracellular Ca²⁺ stores in GBSM (24). Furthermore, depolarization of the GBSM cell directly or indirectly due to Ca²⁺ release via Ins(1,4,5)P₃ channels may contribute to the activation of VDCCs, thus giving rise to the action potential. Clearly, further investigations will be required to resolve this sequence of events.

In various types of smooth muscle, including GBSM (28), Ca²⁺ released via ryanodine-sensitive channels, visualized as Ca²⁺ sparks, leads to the activation of BK_Ca channels (13, 26). Therefore, release of Ca²⁺ via ryanodine channels inhibits the excitability of GBSM (28). In the present study, we found that Ca²⁺ flashes and action potentials persisted in the presence of ryanodine, but the temporal pattern of these events was altered. Ryanodine caused random clustering of Ca²⁺ flashes associated with long quiescent periods as long as 10 s without depolarization. Clustering of action potentials was also observed, and these events were associated with bursts of spikes that were superimposed on a prolonged plateaus that reached up to 3 s in duration [GBSM action potentials normally have a duration of 500 ms (45)]. A similar effect has been reported in smooth muscle of the rat ureter (5), where ryanodine caused a bursting of multiple spikes and prolongation of the plateau phase of the action potential. In contrast, in the GI tract, ryanodine abolishes slow wave action potentials in the longitudinal muscle layer (8, 10, 15–17, 22, 27, 43) but does not affect slow wave action potentials in the circular muscle layer of the intestine (25) or stomach (7, 36) or when circular and longitudinal muscle layers of the small intestine are kept intact (19).

Inhibition of SERCA pumps with CPA or thapsigargin led to a transient increase in the frequency of Ca²⁺ flashes and action potentials. These effects were associated with membrane depolarization and an increase in contractile activity. These results are consistent with previous observations in GBSM cells (24, 29) and other visceral smooth muscle cells (9, 42). The time-dependent decreases of these events caused by prolonged exposure (not significant) are similar to the observations on slow waves in the small intestine (4, 19); however, unlike in the gallbladder, SERCA pump inhibitors did not cause a transient augmentation of slow wave action potential in the small intestine (17) and inhibited slow waves and spontaneous transient depolarizations in the guinea pig gastric pylorus (36, 37).

Like slow wave action potentials (7, 37) and Ca²⁺ transients (17) in the gut, Ca²⁺ flashes and action potentials in GBSM cells were abolished by caffeine, which is consistent with our previous observations on action potentials in GBSM (23). In addition to augmenting Ca²⁺ release via rynaodine channels, these actions could be due to an inhibitory effect of caffeine on Ins(1,4,5)P₃ channels (7, 16–19). The abolition of Ca²⁺ flashes and action potentials caused by caffeine also supports the concept described above: SR Ca²⁺ mobilization plays a role in the generation of Ca²⁺ flashes and action potentials.

Influence of excitatory agonists and PLC activation on Ca²⁺ flashes and action potentials. In the present investigation, we found the the PLC inhibitor U-73122 did not affect basal Ca²⁺ flashes and action potentials. These results indicate that constitutive rhythmic activity of GBSM does not involve PLC activation. Conversely, we found that PLC activation can stimulate Ca²⁺ flashes and action potentials because the stimulatory effect of muscarinic receptor activation on Ca²⁺ flashes and action potentials was attenuated by the PLC inhibitor. The reasons for U-73122 not affecting the basal Ca²⁺ flashes or inducing membrane depolarization are unclear. However, the overall findings are similar to observations in rabbit colonic myocytes (43) as well as ICC from the murine small intestine (19).

In conclusion, Ca²⁺ flashes and action potentials are responsible for rhythmic contractions of individual GBSM bundles in the gallbladder muscularis propria, and our data support the model showing that asynchronous electrical and contractile activity of GBSM bundles throughout the muscularis layer of the gallbladder is responsible for the maintenance of net tone in the organ. Synchronous global electrical rhythms that likely result from excitatory agonist stimulation cause gallbladder emptying. Basal Ca²⁺ flash and action potential activity occurs via a mechanism that depends on VDCCs and Ca²⁺ release via Ins(1,4,5)P₃ channels but does not require PLC activation. However, excitatory agonists act via PLC to activate Ins(1,4,5)P₃ channels and augment rhythmic excitability. Activation of Ins(1,4,5)P₃ channels augments excitability, possibly by depleting Ca²⁺ stores, as this appears to be the mechanism that stimulates Ca²⁺ influx. Release of Ca²⁺ via ryanodine channels increases the latency between flashes and action potentials and decreases the excitability of GBSM. These findings indicate that SR Ca²⁺ handling is critical in the generation, maintaining and modulating spontaneous and agonist-induced excitability of GBSM.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by National Institutes of Health (NIH) Grants NS-26995 (to G. M. Mawe) and DK-53832, DK-065947, and HL44455 (to M. T. Nelson). The Noran Microscope is housed in the Centers of Biomedical Research Excellence Imaging/Physiology Core Facility funded by NIH Grant P20-RR-16435.

	ACKNOWLEDGMENTS

 
The authors are very grateful to Dr. Sarah Lockner for assistance with imaging and to Dr. G. Petkov, Dr. J. Thompkins, Dr. B. Lavoie, Dr. L. Meriam, E. Krauter, and B. Young for help with sampling. We thank Dr. Delrae Eckman for helping develop the Ca²⁺-imaging techniques.

	FOOTNOTES

 

Address for reprint requests and other correspondence: G. M. Mawe, Dept. of Anatomy and Neurobiology, Univ. of Vermont, 89 Beaumont Ave., Given Bldg., D-406, Burlington, VT 05405 (e-mail: gary.mawe{at}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.

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