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Am J Physiol Gastrointest Liver Physiol 291: G717-G727, 2006. First published May 18, 2006; doi:10.1152/ajpgi.00035.2006
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LIVER AND BILIARY TRACT

Calcium waves in intact guinea pig gallbladder smooth muscle cells

Onesmo B. Balemba,1 Thomas J. Heppner,2 Adrian D. Bonev,2 Mark T. Nelson,2 and Gary M. Mawe1,2

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

Submitted 20 January 2006 ; accepted in final form 21 April 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Intracellular Ca2+ waves and spontaneous transient depolarizations were investigated in gallbladder smooth muscle (GBSM) whole mount preparations with intact mucosal layer [full thickness (FT)] by laser confocal imaging of intracellular Ca2+ and voltage recordings with microelectrodes, respectively. Spontaneous Ca2+ waves arose most often near the center, but sometimes from the extremities, of GBSM cells. They propagated regeneratively by Ca2+-induced Ca2+ release involving inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptors and were not affected by TTX and atropine (ATS). Spontaneous Ca2+ waves and spontaneous transient depolarizations were more prevalent in FT than in isolated muscularis layer preparations and occurred with similar pattern in GBSM bundles. Ca2+ waves were abolished by the Ins(1,4,5)P3 receptor inhibitors 2-aminoethoxydiphenyl borate and xestospongin C and by caffeine and cyclopiazonic acid. These events were reduced by voltage-dependent calcium channels (VDCCs) inhibitors diltiazem and nifedipine, by PLC inhibitor U-73122, and by thapsigargin and ryanodine. ACh, CCK, and carbachol augmented Ca2+ waves and induced Ca2+ flashes. The actions of these agonists were inhibited by U-73122. These results indicate that in GBSM, discharge and propagation of Ca2+ waves depend on sarco(endo)plasmic reticulum (SR) Ca2+ release via Ins(1,4,5)P3 receptors, PLC activity, Ca2+ influx via VDCCs, and SR Ca2+ concentration. Neurohormonal enhancement of GBSM excitability involves PLC-dependent augmentation and synchronization of SR Ca2+ release via Ins(1,4,5)P3 receptors. Ca2+ waves likely reflect the activity of a fundamental unit of spontaneous activity and play an important role in the excitability of GBSM.

motility; sarcoplasmic reticulum; calcium transients; transient depolarizations; pacemaker; slow waves; action potentials

SMOOTH MUSCLE CAN BE DIVIDED into two types, phasic or tonic, based on the presence or absence of spontaneous rhythmic activity (17). Whereas the gallbladder is a tonic organ, gallbladder smooth muscle (GBSM) cells exhibit rhythmic spontaneous action potentials (APs) that are shorter in duration and occur at higher frequency than the slow waves of the gut (50). In contrast to gastrointestinal smooth muscle, which is arranged in sheets of smooth muscle fibers, GBSM is arranged in interdigitated bundles that are oriented in multiple directions. Rhythmic excitation can be detected in individual GBSM cells by intracellular recording or in bundles by Ca2+ imaging, but the intact organ maintains a steady level of tone rather than undergoing coordinated phasic contractions. It is possible that net tone in the gallbladder results from asynchronous, multifocal contractions of bundles throughout the wall of the organ. This concept is supported by our recent finding that Ca2+ flashes, which likely reflect Ca2+ entry in association with APs, occur simultaneously among muscle cells in a given bundle, but flash patterns among nonintersecting bundles in a given microscopic field are typically asynchronous (1, 13, 14). Ca2+ flashes are rhythmic, rapidly occurring Ca2+ transients that are caused by Ca2+ influx via voltage-gated calcium channels. These events are rapidly followed by muscle contraction (1, 13, 14). In GBSM cells, Ca2+ flashes occur almost simultaneously in all of the smooth muscle cells in a given bundle and spread within a muscle bundle at a velocity of ~1,900.0 µm/s (1).

A second form of Ca2+ transient commonly observed in intact GBSM preparations, and that is asynchronous within a given GBSM bundle, propagates within individual muscle cells at a velocity of ~68.0 µm/s (1). In other types of smooth muscle, similar Ca2+ transients are referred to as intracellular Ca2+ waves. These events are caused by the sarcoplasmic reticulum (SR) Ca2+ release via 1,4,5-trisitol trisphosphate [Ins(1,4,5)P3] receptors [Ins(1,4,5)P3Rs] (22, 24) or by localized Ca2+ release via Ins(1,4,5)P3Rs that is then amplified by SR Ca2+ release via ryanodine receptors (RyRs) during Ca2+-induced Ca2+ release (CICR) (6, 13). Ins(1,4,5)P3 receptor-mediated SR Ca2+ release is thought to play a role in the origin and propagation of excitability (26, 40, 44–46) and cause tonic contractions or smooth muscle relaxation (7). Furthermore, excitatory agonists may induce smooth muscle contractions by initiating or augmenting Ca2+-wave activity (7, 36).

A third type of Ca2+ transient that is observed in smooth muscle is called the Ca2+ spark. Ca2+ sparks are focal, nonpropagating Ca2+ transients that are caused by SR Ca2+ release via ryanodine-sensitive receptors (32, 38). In GBSM, Ca2+ sparks activate large-conductance Ca2+-activated K+ (BK) channels to reduce GBSM excitability (38).

The origin and propagation of excitation in GBSM is not well understood. To elucidate myogenic and neurogenic contractile activity of the gallbladder, it is important to gain a thorough understanding of the Ca2+ release events that could contribute to myogenic contractions and how excitatory agonists affect these events. The goal of this study was to characterize Ca2+ waves in GBSM, examine cellular mechanisms underlying their generation and propagation, and determine how they are related to Ca2+ flashes and membrane potential. We also sought to understand how Ca2+ waves are modulated by PLC and excitatory agonists to gain a better understanding of their possible role in excitation-contraction coupling. The results presented here suggest that SR Ca2+ release via Ins(1,4,5)P3 receptors plays a critical role in the elementary events leading to Ca2+ waves, Ca2+ flashes, spontaneous transient depolarizations (STDs), and APs. The generation and propagation of Ca2+ waves depend on SR Ca2+ release via Ins(1,4,5)P3 -sensitive receptors, and these events are augmented by excitatory agonists. These findings indicate that Ca2+ release via Ins(1,4,5)P3 receptors in the form of Ca2+ waves promotes excitability of GBSM.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals, sampling, and dissections. Adult (250–350 g) guinea pigs of either sex were used for this study. Guinea pigs were killed ethically by using halothane or isoflurane anesthesia and exsanguinations in accordance to the guidelines of the Animal Care and Use Committee of the University of Vermont. The abdominal cavity was opened by midline incision, the gallbladder was removed and put into ice-chilled modified Krebs solution that was 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. The gallbladders were cut open from the end of the cystic duct to the base of the organ and subdivided into two approximately equal pieces (~2 x 2.5 cm). 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). Dissections were undertaken in ice-chilled Krebs solution, which was changed every 8–10 min. Whole mounts used in the present investigation either contained an intact mucosal layer [full thickness (FT)] or were without mucosal layer [muscularis propria (MP)]. Whole mounts of MP were obtained by teasing off mucosal layer with sharp forceps under stereoscopic microscopic observation. Tissues used for laser confocal imaging to study Ca2+ waves were ~1 x 1.5 cm. In some cases, data related to Ca2+ waves that are presented in this study were acquired with tissue samples from animals that were used to collect data pertaining to Ca2+ flashes presented in a previous publication (1), but different tissue samples were used for the two studies.

Confocal imaging of Ca2+ events. Gallbladder whole mounts were rinsed with HEPES buffer [(in mM) 110 NaCl, 5.4 KCl, 1.8 CaCl2, 1.0 MgCl2, 20 HEPES, 5 glucose, 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 (30 min to 1 h) were done at room temperature. Whole mounts 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; (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. Tissues were then equilibrated for 15–20 min during which multiple fields of observation (n ≥ 20) were examined by using an inverted Nikon TMD microscope with a x60 water-immersion objective lens (1.2 numerical aperture) to qualitatively assess Ca2+-wave activity. Tissues were considered for drug application either if they exhibited globally distributed events or if the number of fields with events was at least greater than 10 fields and allowed for selection of a field displaying sharp and clear Ca2+ waves in at least five different GBSM cells in the bundle(s). Scanning was done using Noran Oz laser confocal system (Noran Instruments, Middleton, WI). Images were acquired using Intervision software (Noran Instruments, WI) on an Indy work station (Silicon Graphics, Mountain View, CA) to record oscillations of fluorescence signals caused by fluctuations of cytosolic Ca2+ concentration ([Ca2+]c) during 20 s of acquisition. Thirty images (frames) were acquired per second to create movie files of 600 images (the online version of this article contains supplemental data). Tissues were continuously superfused with drugs immediately after recording a movie file for control data, and after that imaging was done, every 5th min for a period of 25 min. After the recording, multiple fields of observation were again qualitatively assessed for the effect(s) of the compound tested.

Analysis of movie files. Movie files were analyzed off line for the frequency of Ca2+ waves using custom software written in our laboratory (Dr. A. D. Bonev). Briefly, rectangular boxes of ~1.1–3.2 µm empirically determined to minimize the signal-noise ratio were put on images of GBSM cells (Figs. 1 and 2) that were in sharp focus and displayed intense Ca2+ waves and or Ca2+ flashes. Measurements of Ca2+-wave frequency were taken at a fixed position of a given GBSM cell (Figs. 1 and 2). Baseline fluorescence (F0) was established by averaging 10 images (of the 600 in a movie) that did not have any Ca2+ activity. Movies were visually assessed when normalized ratio images were being reconstructed as traces of F/F0 (Figs. 1 and 2) and tables of frequency, amplitude, image number, and decay maxima being generated. Data were imported into Microsoft Office Excel, and where applicable, variability between frequencies of Ca2+ waves (Hz) was reduced by normalizing each data set. Data were normalized by dividing the frequency obtained at each of the specified 5-min time intervals during drug exposure by the basal (control) frequency value of Ca2+ waves obtained before applying the drug. The usual basal Ca2+ wave frequency ranged between 0. 2 and 0.5 Hz. Normalization of basal Ca2+-wave frequency resulted into a value of 1. Similarly, the normalized frequency values had higher than the normal frequency values when Ca2+-wave activity increased like after application of excitatory agonists, and they are lower where the events were inhibited or abolished. The normalized frequency data are ratios and therefore have no units.


Figure 1
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  		Fig. 1. Images demonstrating the origin and propagation of Ca2+ waves and traces of Ca2+ fluorescence ratios (F/F0) from a movie file of 600 images acquired by Ca2+ imaging. Traces were generated from gallbladder smooth muscle (GBSM) cells in areas indicated by a boxes and a circle. Ca2+ waves originating near the center a smooth muscle cell (picture at 5.400 s) propagated in opposite directions as shown by subsequent pictures. Notice the back of the wave in a picture after 7.433 s. Traces of F/F0 from areas indicated by a box and a circle showing that spontaneous Ca2+ waves in GBSM bundle are multiple and asynchronous events.

 

Figure 2
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  		Fig. 2. Demonstration of Ca2+ waves and localized Ca2+ transients in GBSM cells in intact preparations. The top panels demonstrate how Ca2+ waves propagate slowly. Notice the time difference and distance covered by Ca2+ wave between points i and ii. The bottom panels demonstrate generation of Ca2+ waves and localized nonpropagating Ca2+ transients at sites indicated by boxes 1 and 2. Ca2+ waves at these sites are synchronized (arrows; traces) because these sites are very close to each other. Nonpropagating Ca2+ transients (*) discharged at these sites are asynchronous. These events are shown in movie 1.

 
Intracellular recording. Gallbladder preparations were stretched on Sylgard in a recording chamber serosal side up and placed on the stage of an inverted microscope (Nikon, TMD). Smooth muscle bundles were visualized by x10 objective using Hoffman modulation contrast optics (Modulation Optics, Greenvale, NY). The preparations were continuously superfused at a rate of 8–10 ml/min with the modified aerated (95% O2-5% CO2) Krebs solution. Temperature in the recording chamber was maintained between 35°C and 36°C. Glass microelectrodes used for intracellular recording were filled with 2 M KCl and had input tip resistances of 70–150 M{Omega}. A negative-capacity compensation amplifier (Axoclamp 2A, Axon Instruments, CA) with bridge circuitry was used to record electrical activity. Traces of membrane potential were recorded, saved, and analyzed to determine changes of the frequency and membrane potential by using PowerLab/4SP and Chart 5, V.5.01 software (AD instruments). Measurements of APs (amplitude, slope, duration, membrane potential) were done as described earlier (50).

In these experiments, the extent of stretch was not measured. Samples that were not immediately loaded with fluo 4-AM or used for intracellular recording were kept in ice-chilled HEPES or Krebs buffers, respectively, for 3–6 h.

Statistical analysis (Student's t-test) was done using GraphPad Prism 4. Data were expressed as the means ± SE, and the difference was assumed statistically significant at P ≤ 0.05; n indicates the number of experiments using preparations from different animals.

Chemicals and drugs. Chemicals and drugs used in the present study include fluo-4 AM, and pluronic acid (F-127) (Molecular Probes-Invitrogen, Eugene, OR); thapsigargin, ryanodine (Calbiochem, San Diego, CA); PLC inhibitor {1-[6-(17beta-3-methoxyestra-1,3,5(10)-trien-17-ylamino hexyl)]-1H-pyrrole-2,5-dione (U-73122)}, xestospongin C (Cayman, Ann Arbor, MI), and CCK octapeptide (Bachem, Torrance, CA). Other drugs were 2-aminoethoxydiphenyl borate (2-APB), acetylthiocholine iodide (ACh), caffeine, diltiazem hydrochloride, EDTA, MgCl2·6H202, sucrose, glucose, KCl, NaHCO3, sodium phosphate monobasic, potassium phosphate monobasic, HEPES, dimethyl sulphoxide (DMSO), cyclopiazonic acid (Sigma-Aldrich, St. Louis, MO), NaCl, and CaCl2 (Fischer Scientific-Acros Organic, Oxford, PA). Caffeine, ACh, and diltiazem were dissolved in double-distilled water. Nifedipine was dissolved in ethanol. All other drugs were dissolved in DMSO.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Properties and patterns of spontaneous Ca2+ waves in GBSM. Ca2+ waves and membrane potential were studied in 201 whole mounts (n = 160 FT and n = 41 MP from 127 animals). Unless otherwise noted, all measurements reported here were made from FT whole mounts. In GBSM, multiple, asynchronous Ca2+ waves occurred in an individual GBSM cell. Ca2+ waves were spontaneously discharged most often from a location near the center of a given muscle cell and propagated toward opposite extremities (Figs. 1, 2, and 3, A–C, and movies 1 and 2). Ca2+ waves were sometimes induced by similar events in adjacent cells, and in these cases, they propagated from the extremities toward the center. Ca2+ waves propagated slowly within a given GBSM cell. We have previously reported that the velocity of Ca2+ waves is ~70 µm/s (1). In the current study, we found that the amplitude of Ca2+ waves did not diminish after propagating as far as ~40–50 µm from the origin (2.2 ± 0.1 F/F0 vs. 2.2 ± 0.1 F/F0; n = 11, P = 0.9), indicating that these events were regenerative. Within a given cell, the extent of the propagation of Ca2+ waves was variable, with some Ca2+ waves propagating along the entire cell and others propagating shorter distances. Propagation of Ca2+ waves was instantaneously obliterated by collisions between converging Ca2+ waves.


Figure 3
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  		Fig. 3. Illustration of properties of Ca2+ waves in GBSM. A: schematic drawing demonstrating that Ca2+ waves originate near the center, and each discharge gives rise to 2 Ca2+ waves that propagate regeneratively in the opposite directions, or sometimes Ca2+ waves originate from the extremities. Band C: summary data showing that the velocity and amplitude of Ca2+ waves do not change during propagation.

 
Ca2+ waves occurred asynchronously among muscle cells within a given smooth muscle bundle. Ca2+-wave activity was either multifocal, i.e., involving few bundles, or widespread among GBSM bundles in a given preparation. Within GBSM bundles, Ca2+ waves could occur without Ca2+ flashes. In other bundles, Ca2+ waves occurred asynchronously along with synchronized Ca2+ flashes (movie 2). Unlike Ca2+ flashes, the frequency, intensity, and prevalence of Ca2+ waves varied between GBSM cells in a given muscle bundle. Qualitative assessment showed that Ca2+ waves occurring among Ca2+ flashes had higher intensity and propagated faster. The increase of Ca2+-wave frequency that took place during equilibration from room temperature to 36°C and during the exposure to excitatory agonists gave rise to Ca2+ flashes.

Similar to spontaneous Ca2+ flashes and APs, the generation of spontaneous Ca2+ waves did not appear to involve a neural mechanism because they were not affected by application of a solution containing the Na2+ receptor blocker TTX (2 µM) and muscarinic receptor antagonist ATS (1 µM; vehicle: 1.15 ± 0.2, n = 6 vs. TTX-ATS: 0.9 ± 0.07, n = 5; P = 0.2; 25 min; Fig. 4A). Interestingly, the frequencies of Ca2+ waves and Ca2+ flashes (0.18 ± 0.01 Hz, n = 49 vs. 0.21 ± 0.03 Hz, n = 18; P = 0.4) were comparable (Fig. 4B).


Figure 4
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  		Fig. 4. Summary data showing that inhibition of neural activity with TTX and atropine (ATS) do not affect Ca2+ waves (A). The frequency of Ca2+ waves and Ca2+ flashes is comparable (B); however, Ca2+ waves propagate slower than Ca2+ flashes (C), and leaving the mucosal layer on the preparations increased prevalence of Ca2+ waves (D). FT, full-thickness preparations; MP, muscularis propria preparations. **P ≤ 0.01, and ***P ≤ 0.001.

 
Ca2+ waves and STDs are most prevalent in preparations with intact mucosa. In our initial studies of Ca2+ waves in GBSM, we observed that Ca2+ waves were more frequently encountered in preparations with intact mucosa compared with MP alone. Therefore, we compared Ca2+ waves and transmembrane potential in GBSM whole mounts with or without the mucosal layer intact (FT vs. MP whole mounts). Spontaneous Ca2+ waves were abundant and widespread in FT whole mount preparations, and the frequency of Ca2+ waves was higher in these preparations compared with MP alone (FT: 0.2 ± 0.01 Hz, n = 23 vs. MP: 0.15 ± 0.01 Hz, n = 21; P ≥ 0.004; Fig. 4D).

In intracellular recording studies, STDs were observed in FT and MP and occurred irregularly between APs with a pattern similar to that of Ca2+ waves and Ca2+ flashes (Fig. 5A; see also GoFig. 7B). STDs in GBSM comprised all spontaneous subthreshold depolarizations that did not trigger a regenerative spike on the plateu of the AP.


Figure 5
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  		Fig. 5. Summary comparing the frequency of spontaneous transient depolarizations (STDs; * in A and B), amplitude (C), and duration (D) of APs and the resting membrane potential (E) in FT and MP preparations. **P ≤ 0.01, and ***P ≤ 0.001.

 

Figure 6
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  		Fig. 6. The effects of voltage-dependent calcium channels (VDCCs) inhibitors diltiazem (Dzm; 50 µM) and nifedipine (Nifed; 10 µM; C) on Ca2+ waves. The VDCCs reduced the frequency of Ca2+ waves after prolonged (25 min) exposure (A and B; 25 min). *P ≤ 0.05, and ***P ≤ 0.001. PSS, physiological saline solution.

 

Figure 7
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  		Fig. 7. Traces of fluorescence ratios (F/F0) for Ca2+ waves (A–D) and summary data (E) showing how sarcoplasmic reticulum (SR)-Ca2+ release and inhibition of SR-Ca2+ uptake affect Ca2+ waves in GBSM cells. Inositol 1,4,5-trisphosphate receptor [Ins(1,4,5)P3R] blocker 2-APB (A and F) and xestospongin C (Xest C; B) dramatically reduced Ca2+-wave activity, abolishing them as early as 5 min after application (A). B: inhibition of Ca2+ flashes (synchronized traces) and Ca2+ waves (*) by Xest C. Ca2+-wave frequency was reduced by prolonged (25 min) inhibition of Ca2+ release via ryanodine receptors (RyR; C), whereas, unlike RyR, caffeine abolished Ca2+ waves (D). The SERCA pump inhibitor cyclopiazonic acid (CPA) abolished Ca2+ waves (F; 25 min); thapsigargin (Thapsi) reduced the frequency (E and F) but did not abolish Ca2+ waves. **P ≤ 0.01, and ***P ≤ 0.001.

 
STDs ranged in amplitude from just above the background nose level (~1 mV) to ~15 mV, and similar to Ca2+ waves, STDs were most frequently encountered in FT compared with MP preparations (FT: 0.1 ± 0.02 Hz vs. MP: 0.01 ± 0.01 Hz, n = 12; P < 0.0002). The duration of STDs did not differ between FT and MP preparations (FT: 1.0 ± 0.06 s vs. MP: 1.2 ± 0.1 s, n = 12; P = 0.1). In an effort to determine the type of Ca2+ events displayed in tissues that exhibited both STDs and APs, Ca2+ imaging studies were conducted in bundles in which STDs and APs had been detected by intracellular recording and injected with Lucifer yellow or and 1,1'-didodecyl 3,3,3',3'-indocarbocyanine perchlorate as a means of relocalization. GBSM bundles exhibiting APs with intermittent STDs displayed both Ca2+ flashes and Ca2+ waves.

There appears to be a relationship between the STDs and APs in GBSM cells, and depolarizations leading to suprathreshold events appeared to result from entrained STDs. The average slope (STDs: 0.005 ± 0.0007 vs. AP: 0.007 ± 0.001 mV/s, n = 12; P = 0.1) and duration (STDs: 1.1 ± 0.09 s vs. AP 1.0 ± 0.06 s, n = 12; P = 0.3) of the upstroke depolarization of the STDs and APs were comparable. Furthermore, large STDs with amplitudes of >10 mV often gave rise to small spikes (13.7 ± 0.6 mV; n = 10; Fig. 5A).

We also compared APs in FT and MP preparations. APs had a larger amplitude in FT preparations (FT: 69.1 ± 2.0 mV vs. MP: 58.7 ± 1.9 mV, n = 12; P = 0.0009) as well as a larger overshoot (FT: 18.9 ± 1.6 mV vs. MP: 11.5 ± 2.0 mV, n = 12; P = 0.01; Fig. 5, A–D). Furthermore, the duration of the AP was shorter in FT preparations (FT: 1.2 ± 0.09 s vs. MP: 1.9 ± 0.09 s, n = 12; P < 0.0001), and the resting membrane potential was higher (FT: –52.6 ± 0.8 mV vs. MP: –46.7 ± 1.6 mV, n = 12; P = 0.003; Fig. 5E). Despite these differences, the frequency (FT: 0.3 ± 0.02 Hz vs. MP: 0.3 ± 0.02 Hz, n = 12; P = 0.5) and the average slope of the upstroke depolarization of the AP (FT: 0.005 ± 0.0006 mV/s vs. MP: 0.007 ± 0.001 mV/s, n = 12; P = 0.2) were not significantly different. Taken together, these findings suggest that the electrical activity of MP and its associated Ca2+ mobilization events are influenced by chemical agents and/or current spreading from cells in the inner layers of the gallbladder wall.

Inhibiting voltage-dependent Ca2+ channels reduces Ca2+ wave activity. In GBSM, Ca2+ influx via voltage-dependent Ca2+ channels (VDCCs) determines the level of cytosolic Ca2+ and the Ca2+ content of the SR (1, 29). We evaluated the response of Ca2+ waves to VDCCs blockers diltiazem (Dzm; 50 µM) and nifedipine (10 µM). Both Dzm (vehicle: 1.1 ± 0.06, n = 5 vs. Dzm: 0.6 ± 0.2, n = 5; P = 0.03; 25 min) and nifedipine (vehicle: 1.4 ± 0.2, n = 6 vs. nifedipine: 0.4 ± 0.2, n = 4; P = 0.01; 25 min) reduced the frequency of Ca2+ waves, respectively (Fig. 6, A and B). These results are consistent with Ca2+ influx maintaining the source of Ca2+ waves (SR Ca2+ content).

The roles of Ins(1,4,5)P3R and RyR-mediated SR Ca2+ release on Ca2+-wave activity. Our understanding of Ins(1,4,5)P3R-mediated SR Ca2+-release events has been advanced by the use of Ins(1,4,5)P3R inhibitors, namely 2-APB, xestospongin C, and heparin (9, 24, 44, 45). In GBSM, blocking Ins(1,4,5)P3Rs with 2-APB abolished CCK-induced elevation of intracellular Ca2+ (27), and both 2-APB and xestospongin C abolished Ca2+ flashes (1). 2-APB (100 µM) and xestospongin C (5 µM) were used to evaluate the effect of Ins(1,4,5)P3R inhibition on Ca2+ waves in GBSM. Compared with vehicle, 2-APB rapidly reduced the frequency of Ca2+ waves (vehicle: 1.4 ± 0.2, n = 6 vs. 2-APB: 0.2 ± 0.09, n = 10; P < 0.0001; 10 min) and abolished these events 5–20 min after exposure. Likewise, xestospongin C rapidly reduced the frequency of Ca2+ waves and then abolished these events (n = 3; see Fig. 7, A, B, and F).

In other types of smooth muscle, propagation of Ca2+ waves by CICR involves SR Ca2+ release via RyRs (6, 9, 13) or Ins(1,4,5)P3Rs (6, 9, 13, 23, 24). The cross-talk between the Ins(1,4,5)P3 and RyR receptors is crucial for G protein agonist-mediated actions (22, 25). In GBSM, Ins(1,4,5)P3 and RyR receptors share the same store (27), and Ca2+ sparks (RyRs) have an inhibitory action on excitability (38). Because Ca2+ waves have not been directly studied, we sought to investigate whether RyRs play a role in the generation and/or the propagation Ca2+ waves in GBSM by using ryanodine that blocks these receptors. Prolonged (25 min) incubation with ryanodine (20 µM) reduced frequency (vehicle 1.4 ± 0.2, n = 6 vs. RyR: 0.5 ± 0.1, n = 10; P = 0.002; 25 min) of Ca2+ waves (Fig. 7, C and F).

Caffeine sensitizes RyRs to Ca2+, causing increased intracellular Ca2+ release via RyRs (10, 20). Unlike ryanodine, which locks RyRs in an open or closed state (20), caffeine (3 mM) abolished Ca2+ waves (vehicle: 1.1 ± 0.06, n = 5 vs. caffeine: 0.09 ± 0.05, n = 4; P < 0.0001; 5 min) shortly after exposure (Fig. 7, D and F). These findings suggest that in GBSM, the generation and propagation of Ca2+ waves by CICR depend on Ca2+ release via Ins(1,4,5)P3Rs and require functional RyRs in addition to P3Rs. RyRs could either participate in the Ca2+ waves or in the maintenance of SR Ca2+ to be released by Ins(1,4,5)P3Rs.

Depleting SR Ca2+ stores by using SR Ca2+ ATPase pump inhibitors abolishes Ca2+ waves. In GBSM cells, intracellular Ca2+ depletion with SR Ca2+ ATPase (SERCA) pump inhibitors thapsigargin and cyclopiazonic acid (CPA) induces capacitative Ca2+ entry via the Ins(1,4,5)P3Rs, VDCC, and Gd3-sensitive pathway (29). We tested the effect of SERCA pump inhibitors thapsigargin (2 µM) and CPA (10 µM) on Ca2+ waves in intact GBSM. Thapsigargin augmented Ca2+-wave frequency and induced Ca2+ flashes (≥50% of the preparations) 2–5 min after application in four of six preparations. Compared with vehicle, prolonged (25 min) exposure of thapsigargin reduced the frequency of Ca2+ waves (vehicle: 1.4 ± 0.2, n = 6 vs. thapsigargin: 0.5 ± 0.08, n = 10; P = 0.0007; 25 min). CPA augmented Ca2+-wave frequency in three of five preparations 2–5 min after application but, in contrast to thapsigargin, CPA reduced the frequency of Ca2+ waves rapidly and abolished these events after 15–25 min (vehicle: 1.4 ± 0.2, n = 6 vs. CPA: 0.02 ± 0.02; n = 5; P = 0.0004; 25 min). The difference between the actions of thapsigargin and CPA was significant (thapsigargin: 0.5 ± 0.08, n = 10 vs. CPA 0.02 ± 0.02, n = 5; P = 0.0005; Fig. 7, E and F). Ca2+ flashes were seen in two of 5 preparations treated with CPA after 15–25 min when all Ca2+ waves had been inhibited. These findings suggest differences in the mechanisms of action between thapsigargin and CPA and that SR Ca2+ uptake via SERCA pumps is critical for optimal Ca2+-wave activity.

Excitatory agonists augment Ca2+ waves via PLC dependent generation of Ins(1,4,5)P3. PLC-dependent production of Ins(1,4,5)P3 plays a pivotal role in the generation of Ca2+ waves in muscle and nonmuscle cells (4, 9, 30). Inhibiting PLC with U-73122 abolished CCK-induced contractions in the cat gallbladder (48) but did not affect basal APs in the guinea pig gallbladder (1). We tested the effects of the PLC inhibitor U-73122 (10–50 µM) on Ca2+ waves and on agonist-induced responses in intact GBSM. U-73122 (10 µM; n = 4) did not affect the frequency of Ca2+ waves in the absence of agonists. At 25 µM, U-73122 inhibited Ca2+ waves (vehicle: 1.4 ± 0.2, n = 6 vs. U-73122: 0.4 ± 0.1, n = 5; P < 0.005; 25 min; Fig. 8, A and B). A higher concentration (50 µM; n = 5) induced Ca2+ flashes in GBSM that originally had Ca2+ waves alone.


Figure 8
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  		Fig. 8. Traces of F/F0 for Ca2+ waves (A) obtained by Ca2+ imaging and summary data (B) showing that U-73122 (25 µM) decreased the frequency of Ca2+ waves after 25 min. Demonstration of the effects of excitatory agonists: ACh, 50 (µM), carbachol (3 µM), and CCK (100 nM) on Ca2+ waves in GBSM cells. Control data were obtained immediately before exposure of drugs as shown by the dotted line and an arrow in C. Excitatory agonists augmented the frequency of Ca2+ waves immediately after exposure; however, the frequency of Ca2+ waves decreased with time after ~10 min (C and D). Compared with carbachol (3 µM) alone, pretreatment of tissues with U-73122 for 20 min abolished the augmentation of Ca2+ waves by carbachol (E). *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.

 
We previously demonstrated that in isolated GBSM cells, CCK stimulates excitability by inhibiting Ca2+ sparks (38) and that carbachol and CCK augment Ca2+ flashes (1). In the present study, we tested the effect of carbachol (3 µM), ACh (50 µM), and CCK (10 nM) on Ca2+ waves. Compared with vehicle, all compounds rapidly augmented the frequency of Ca2+ waves during the first 2–5 min (vehicle: 1.1 ± 0.1, n = 4 vs. carbachol: 4.9 ± 1.0, n = 4; P < 0.05; vehicle 1.1 ± 0.1, n = 4 vs. ACh: 2.2 ± 0.3, n = 4; P = 0.008 and vehicle: 1.1 ± 0.1, n = 4 vs. CCK: 2.2 ± 0.1, n = 3; P < 0.0001; Fig. 8, C and D). In most preparations, excitatory agonists also caused synchronization of Ca2+ waves in a given bundle and changed the pattern of occurrence from Ca2+ events to either Ca2+ waves and Ca2+ flashes occurring together or Ca2+ flashes without apparent Ca2+ waves. The transition from Ca2+ wave predominant form to Ca2+ waves and Ca2+ flashes or Ca2+ flashes alone always followed an initial increase in the frequency of Ca2+ waves. U-73122 inhibited the augmentation of Ca2+ waves by carbachol (carbachol: 4.9 ± 1.0, n = 4 vs. U-73122 + carbachol: 1.2 ± 0.2, n = 5; P = 0.006; 5 min; Fig. 8E). These results suggest that excitatory agonists augment Ca2+-wave activity via activation of PLC and subsequent generation of Ins(1,4,5)P3 and increased Ins(1,4,5)P3 -induced SR Ca2+ release.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The aims of the present investigation were to determine whether Ca2+ waves play a role in the excitability of GBSM and to elucidate the cellular mechanisms modulating these events. We also evaluated the influence of the presence or absence of the mucosal layer on Ca2+ waves and membrane potential fluctuations. The data obtained in this study demonstrate that in GBSM cells, intracellular Ca2+ waves arise most often near the center, but sometimes from the extremities, of GBSM cells and propagate regeneratively by Ins(1,4,5)P3R-mediated CICR. Basal and agonist-induced Ca2+-wave activity depends on PLC activation, Ca2+ influx via VDCC, SR Ca2+ content, and Ca2+ release via Ins(1,4,5)P3 receptors. Ca2+ waves correlate with STDs, and excitatory agonists enhance excitability by augmenting Ca2+ waves. These findings suggest that Ca2+ waves (CICR) contribute to the fundamental unit of rhythmic electrical activity in GBSM and play a role in the maintenance of gallbladder tone interprandially and contractile activity during gallbladder emptying.

Ca2+ waves link with Ca2+ flashes and APs to augment excitability of GBSM. We have previously shown that SR Ca2+ release via RyRs (Ca2+ sparks) reduces excitability of GBSM (38). Recently, we reported that we can detect Ca2+ flashes, Ca2+ waves, and Ca2+ sparks in intact GBSM and that inhibition of SR Ca2+ mobilization via Ins(1,4,5)P3Rs abolishes Ca2+ flashes and APs (1). One goal of the present investigation was to determine the relationship between Ca2+ waves and Ca2+ flashes. Results obtained in this study indicate that Ca2+ waves and Ca2+ flashes (or APs) have many properties in common: 1) similar to Ca2+ flashes or APs, constitutive Ca2+ waves are not initiated via a neural mechanism; 2) both Ca2+ waves and Ca2+ flashes are abolished by inhibiting SR Ca2+ release via Ins(1,4,5)P3Rs, by high concentration of caffeine, and they are reduced by the RyR blocker ryanodine; 3) the temporal pattern of Ca2+ waves and Ca2+ flashes are comparable as the frequency of Ca2+ waves and Ca2+ flashes are comparable; 4) both basal Ca2+ waves and basal Ca2+ flashes (1) are augmented or induced and synchronized in a similar fashion by excitatory agonists involving a PLC-P3-dependent mechanism with an agonist-induced increase of Ca2+-wave frequency preceding augmentation and/or induction of Ca2+ flashes; and 5) there is a relationship between increased frequency of Ca2+ waves and induction of Ca2+ flashes.

A relationship also appears to exist between Ca2+ waves and STDs. Ca2+ waves and STDs occur with a similar pattern and prevalence in FT and MP preparations. Furthermore, GBSM bundles exhibiting APs with intermittent STDs also display both Ca2+ flashes and Ca2+ waves, and Ca2+ waves and STDs appear to be equally affected by hyperpolarization caused by mucosal agents. Taken together, these observations suggest that although Ca2+ waves and STDs are not exactly the same, they are interrelated and Ca2+ waves may underlie STDs, supporting the proposition that Ca2+ release via Ins(1,4,5)P3Rs promotes excitability of GBSM cells (1). These findings suggest that STDs may arise from Ca2+-mediated interaction among pacemaker units comprising the SR, mitochondria, and plasma membrane as shown in intestinal interstitial cells of Cajal (ICC) and smooth muscle cells (18, 39). They indicate that, similar to ICC (47), SR Ins(1,4,5)P3Rs couples to VDCC in GBSM.

The duration and the rate of the upstroke depolarization of STDs and APs are comparable indicating that in GBSM, APs may arise from the summation of STDs. A similar phenomenon has been reported for slow-wave APs in the ICC and smooth muscle cells in the gastrointestinal (GI) tract (18, 44, 45) and guinea pig urethra smooth muscle (11). These new findings in the gallbladder suggest a tight association of Ca2+ waves and STDs with Ca2+ flashes and APs, respectively.

The data reported here indicate that there is a strong correlation among Ca2+ waves, Ca2+ flashes, STDs, and APs, and these findings suggest that Ca2+ mobilization via Ins(1,4,5)P3Rs contributes to the rhythmic depolarization of GBSM. In the ICC in the GI tract, Ca2+ release from internal stores initiates pacemaker currents. Ca2+ influx via dihydropyridine-resistant channels occurring during the upstroke depolarization causes sustained plateau of the slow wave action potentials (18, 40). It is likely that Ins(1,4,5)P3R-mediated Ca2+ release at multiple sites corresponds with primary pacemaker currents and that the summation of asynchronous Ca2+ waves corresponds to STDs. Furthermore, it is likely that Ca2+ influx via VDCC is involved in the entrainment of STDs (50). The findings from this study correspond with those of the ICC-smooth muscle model of myogenic activity in the GI tract (18, 40, 45, 46). However, the question of whether pacemaker activity in GBSM involves a specialized pacmaker cell, such as ICC, or whether it is an intrinsic property of GBSM has not been resolved.

In other types of smooth muscle and ICC, Ca2+ influx via sodium-calcium exchanger has been proposed to be responsible for triggering spontaneous depolarizations underlying the origin of rythmic activity in smooth muscle (26) by causing SR Ca2+ release. It has been proposed that SR Ca2+ mobilization triggers spontaneous inward currents to cause depolarization (11, 12, 18, 26, 44–46) via activation of chloride channels (12, 15, 16, 26, 42) or nonselective cation channels (NSCC) (10, 34, 35). We recently reported the existence of a steady-state nonselective Na+ receptors in GBSM (37) and that activation of store depletion-activated channels may play a role in the excitability of GBSM (1, 29). Whether in GBSM, Ca2+-wave activity involves chloride channels or NSCC or a different mechanism to enhance excitability or couple to STDs remains to be examined.

Cellular mechanisms modulating Ca2+ waves in GBSM. The excitability of GBSM cells is modulated by Ca2+ influx via VDCCs (1, 50) and intracellular Ca2+ mobilization (1, 27–29, 38). We evaluated the role of VDCCs in Ca2+-wave activity to determine the association among Ca2+ waves, Ca2+ flashes, or APs. Unlike Ca2+ flashes or APs or associated ionic currents, which are rapidly abolished by VDCCs inhibitors (1, 50), Ca2+ waves were gradually reduced by VDCCs inhibitors supporting the concept that Ca2+ influx via VDCCs is crucial for replenishing SR Ca2+ stores and Ca2+-wave activity.

We also examined the role of Ins(1,4,5)P3Rs and RyRs in GBSM wave activity because both Ins(1,4,5)P3Rs (22, 24) and RyRs (6, 9, 13) are involved in propagation of Ca2+ waves in other types of smooth muscle cells. Inhibiting Ca2+ release via Ins(1,4,5)P3Rs abolished Ca2+ waves and GBSM contractions. These observations are similar to observations in GI smooth muscle cells (10, 22, 24) and support the view that SR Ca2+ release via Ins(1,4,5)P3Rs augments excitability of GBSM and thus gallbladder contractility (1). They also suggest that SR Ca2+ release via Ins(1,4,5)P3Rs may be involved in the pacemaker activity of GBSM, as proposed for GI smooth muscle and ICC (18, 26, 40, 45, 46).

Inhibition of RyR gradually reduced Ca2+ waves, suggesting that RyRs modulate Ca2+ waves through store depletion, as observed in previous studies (1, 29). The difference between ryanodine and caffeine, which sensitizes RyRs, may be due to caffeine-mediated inhibition of Ins(1,4,5)P3Rs (20) or caffeine-mediated depletion of SR stores. As demonstrated in the study of Ca2+-spark activity in isolated GBSM cells (38), inhibition of SERCA pumps with thapsigargin and CPA reduced or abolished SR Ca2+ release, respectively, indicating that SR Ca2+ uptake via SERCA pumps is critical for optimal SR Ca2+ mobilization via both Ins(1,4,5)P3Rs and RyRs. The differences between thapsigargin and CPA in their effectiveness to abolish Ca2+ waves is likely due the ability of thapsigargin to rapidly depolarize GBSM, causing Ca2+ influx and subsequent refill of the SR Ca2+ stores before tangible inhibition of SERCA pumps (1, 29).

Excitatory agonists augment excitability via Ca2+ waves. PLC activation is crucial for Ca2+-wave activity in smooth muscle and nonmuscle cells (9, 23, 30). The results of the present study are consistent with this view that basal and agonist-evoked augmentations of Ca2+ waves are sensitive to PLC inhibition. This is different from our previous findings that constitutive Ca2+ flashes and AP activity do not require PLC activation (1). This study shows that PLC activity via generation of Ins(1,4,5)P3 plays a fundamental role in generating and sustaining basal spontaneous CICR and in agonist-evoked augmentation of CICR to enhance excitability of GBSM. It is possible that agonist-induced Ca2+-wave activity requires a global elevation of intracellular Ins(1,4,5)P3, as shown in other types of smooth muscle cells (8, 23, 24, 30), that is probably caused by depolarization because Ins(1,4,5)P3 production is voltage dependent (8). Agonist-induced augmentation of Ca2+ waves in GBSM decreased with time as reported in other smooth muscle cells (2). This observation has been attributed to an increased intracellular Ca2+ concentration (3) or a decreased Ins(1,4,5)P3 generation (33).

Mucosal factors reduce excitability without affecting Ca2+ wave and STD activity. In vascular smooth muscle, endothelium-derived factors such as nitric oxide inhibit intracellular Ca2+ transients activity (41). Similarly, mucosal agents, including ATP and nitric oxide from the urinary bladder (5, 21) and ureter (19) epithelium and prostanoids and other agents from tracheal mucosa (43), exert inhibitory actions to smooth muscle excitability. In the current study, preparations with mucosal layer intact had reduced excitability as well as prevalent Ca2+ waves and STDs. At this time, we do not know what mucosal factors contributed to this phenomenon or how they affected Ca2+ mobilization and excitability of GBSM. It is possible that gallbladder epithelial cells release ATP that is metabolized to adenosine, which reduces the excitability of GBSM cells (31, 49). It is also possible that physiological differences between the preparations are related to tissue damage during microdissection and/or the presence of mucosal tissue reduces the extent of stretch to GBSM bundles in intact preparations compared with MP.

In conclusion, the results of the present study have revealed that in GBSM cells, the discharge and propagation of Ca2+ waves depend on SR Ca2+ release via Ins(1,4,5)P3Rs, SR Ca2+ content, as well as PLC activity. SR Ca2+ release via Ins(1,4,5)P3Rs couples to VDCC receptors to enhance excitability of GBSM, and it may be crucial in the pacemaking activity in GBSM. Ca2+ waves likely play an important role in maintaining the basal tone as well as neurohumoral-induced stimulation of gallbladder motility and emptying. Finally, agents from gallbladder mucosa, which have not yet been identified, appear to modulate GBSM excitability.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was funded by National Institutes of Health (NIH) Grants NS-26995 (to Dr. G. M. Mawe) and DK-53832 and DK-65947 (to Dr. M. T. Nelson). The Noran Microscope is housed in the COBRE imaging-Physiology core facility funded by NIH Grant NCRR-P20-RR-16435.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. S. Locknar for assistance with imaging, Dr. G. Petkov, Dr. J. Thompkins, L. A. Meriam, E. Krauter, and B. Young for help with sampling. We thank Dr. D. Eckman for developing the Ca2+-imaging techniques and Dr. B. Lavoie for comments on the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. M. Mawe, D-406, Given Bldg., Dept. of Anatomy and Neurobiology, 89 Beaumont Ave., Univ. of Vermont, 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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Balemba OB, Salter MJ, Heppner TJ, Bonev AD, Nelson MT, and Mawe GM. 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 290: G655–G664, 2006.[Abstract/Free Full Text]
  2. Bayguinov O, Hagen B, and Sanders KM. Muscarinic stimulation increases basal Ca2+ and inhibits spontaneous Ca2+ transients in murine colonic myocytes. Am J Physiol Cell Physiol 280: C689–C700, 2001.[Abstract/Free Full Text]
  3. Bayguinov O, Hagen B, and Sanders KM. Substance P modulates localized calcium transients and membrane current responses in murine colonic myocytes. Br J Pharmacol 138: 1233–1243, 2003.[CrossRef][Web of Science][Medline]
  4. Berridge MJ. Elementary and global aspects of calcium signalling. J Exp Biol 200: 315–319, 1997.[Abstract]
  5. Buckner SA, Milicic I, Daza AV, Coghlan MJ, and Gopalakrishnan M. Spontaneous phasic activity of the pig urinary bladder smooth muscle: characteristics and sensitivity to potassium channel modulators. Br J Pharmacol 135: 639–648, 2002.[CrossRef][Web of Science][Medline]
  6. Collier ML, Ji G, Wang Y, and Kotlikoff MI. Calcium-induced calcium release in smooth muscle: loose coupling between the action potential and calcium release. J Gen Physiol 115: 653–662, 2000.[Abstract/Free Full Text]
  7. Dai JM, Kuo KH, Leo JM, van Breemen C, and Lee CH. Mechanism of ACh-induced asynchronous calcium waves and tonic contraction in porcine tracheal muscle bundle. Am J Physiol Lung Cell Mol Physiol 290: L459–L469, 2006.[Abstract/Free Full Text]
  8. Ganitkevich V and Isenberg G. Membrane potential modulates inositol 1,4,5-trisphosphate-mediated Ca2+ transients in guinea-pig coronary myocytes. J Physiol 470: 35–44, 1993.[Abstract/Free Full Text]
  9. Gordienko DV and Bolton TB. Crosstalk between ryanodine receptors and IP(3) receptors as a factor shaping spontaneous Ca(2+)-release events in rabbit portal vein myocytes. J Physiol 542: 743–762, 2002.[Abstract/Free Full Text]
  10. Gordienko DV and Zholos AV. Regulation of muscarinic cationic current in myocytes from guinea-pig ileum by intracellular Ca(2+) release: a central role of inositol 1,4,5-trisphosphate receptors. Cell Calcium 36: 367–386, 2004.[CrossRef][Web of Science][Medline]
  11. Hashitani H and Edwards FR. Spontaneous and neurally activated depolarizations in smooth muscle cells of the guinea-pig urethra. J Physiol 514: 459–470, 1999.[Abstract/Free Full Text]
  12. Hennig GW, Hirst GD, Park KJ, Smith CB, Sanders KM, Ward SM, and Smith TK. Propagation of pacemaker activity in the guinea-pig antrum. J Physiol 556: 585–599, 2004.[Abstract/Free Full Text]
  13. Hennig GW, Smith CB, O'Shea DM, and Smith TK. Patterns of intracellular and intercellular Ca2+ waves in the longitudinal muscle layer of the murine large intestine in vitro. J Physiol 543: 233–253, 2002.[Abstract/Free Full Text]
  14. Heppner TJ, Bonev AD, and Nelson MT. Elementary purinergic Ca2+ transients evoked by nerve stimulation in rat urinary bladder smooth muscle. J Physiol 564: 201–212, 2005.[Abstract/Free Full Text]
  15. Hirst GD and Ward SM. Interstitial cells: involvement in rhythmicity and neural control of gut smooth muscle. J Physiol 550: 337–346, 2003.[Abstract/Free Full Text]
  16. Hollywood MA, Sergeant GP, McHale NG, and Thornbury KD. Activation of Ca2+-activated Cl– current by depolarizing steps in rabbit urethral interstitial cells. Am J Physiol Cell Physiol 285: C327–C333, 2003.[Abstract/Free Full Text]
  17. Horowitz B, Ward SM, and Sanders KM. Cellular and molecular basis for electrical rhythmicity in gastrointestinal muscle. Annu Rev Physiol 61: 19–43, 1999.[CrossRef][Web of Science][Medline]
  18. Kito Y, Ward SM, and Sanders KM. Pacemaker potentials generated by interstitial cells of Cajal in the murine intestine. Am J Physiol Cell Physiol 288: C710–C720, 2005.[Abstract/Free Full Text]
  19. Knight GE, Bodin P, De Groat WC, and Burnstock G. ATP is released from guinea pig ureter epithelium on distension. Am J Physiol Renal Physiol 282: F281–F288, 2002.[Abstract/Free Full Text]
  20. Laporte R, Hui A, and Laher I. Pharmacological modulation of sarcoplasmic reticulum function in smooth muscle. Pharmacol Rev 56: 439–513, 2004.[Abstract/Free Full Text]
  21. Levin RM, Wein AJ, Krasnopolsky L, Atta MA, and Ghoniem GM. Effect of mucosal removal on the response of the feline bladder to pharmacological stimulation. J Urol 153: 1291–1294, 1995.[CrossRef][Web of Science][Medline]
  22. McCarron JG, Bradley KN, MacMillan D, Chalmers S, and Muir TC. The sarcoplasmic reticulum, Ca2+ trapping, and wave mechanisms in smooth muscle. News Physiol Sci 19: 138–147, 2004.[Abstract/Free Full Text]
  23. McCarron JG, Craig JW, Bradley KN, and Muir TC. Agonist-induced phasic and tonic responses in smooth muscle are mediated by InsP(3). J Cell Sci 115: 2207–2218, 2002.[Abstract/Free Full Text]
  24. McCarron JG, MacMillan D, Bradley KN, Chalmers S, and Muir TC. Origin and mechanisms of Ca2+ waves in smooth muscle as revealed by localized photolysis of caged inositol 1,4,5-trisphosphate. J Biol Chem 279: 8417–8427, 2004.[Abstract/Free Full Text]
  25. McGeown JG. Interactions between inositol 1,4,5-trisphosphate receptors and ryanodine receptors in smooth muscle: one store or two? Cell Calcium 35: 613–619, 2004.[CrossRef][Web of Science][Medline]
  26. McHale N, Hollywood M, Sergeant G, and Thornbury KD. Origin of spontaneous rhythmicity in smooth muscle. J Physiol 570: 23–28, 2006.[Abstract/Free Full Text]
  27. Morales S, Camello PJ, Mawe GM, and Pozo MJ. Characterization of intracellular Ca2+ stores in gallbladder smooth muscle. Am J Physiol Gastrointest Liver Physiol 288: G507–G513, 2005.[Abstract/Free Full Text]
  28. Morales S, Camello PJ, Mawe GM, and Pozo MJ. Cyclic AMP-mediated inhibition of gallbladder contractility: role of K+ channel activation and Ca2+ signaling. Br J Pharmacol 143: 994–1005, 2004.[CrossRef][Web of Science][Medline]
  29. Morales SCP, Alcon S, Salido GM, Mawe G, 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]
  30. Murthy KS, Zhou H, Huang J, and Pentyala SN. Activation of PLC-delta1 by Gi/o-coupled receptor agonists. Am J Physiol Cell Physiol 287: C1679–C1687, 2004.[Abstract/Free Full Text]
  31. Naughton P, Baer HP, Clanachan AS, and Scott GW. Adenosine and ATP effects on isolated guinea pig gallbladder. Pflügers Arch 399: 42–45, 1983.[CrossRef][Web of Science][Medline]
  32. Nelson MT and Quayle JM. Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol Cell Physiol 268: C799–C822, 1995.[Abstract/Free Full Text]
  33. Okamoto H, Unno T, Arima D, Suzuki M, Yan HD, Matsuyama H, Nishimura M, and Komori S. Phospholipase C involvement in activation of the muscarinic receptor-operated cationic current in Guinea pig ileal smooth muscle cells. J Pharm Sci 95: 203–213, 2004.[CrossRef]
  34. Pacaud P and Bolton TB. Relation between muscarinic receptor cationic current and internal calcium in guinea-pig jejunal smooth muscle cells. J Physiol 441: 477–499, 1991.[Abstract/Free Full Text]
  35. Parsons SP and Bolton TB. Localised calcium release events in cells from the muscle of guinea-pig gastric fundus. J Physiol 554: 687–705, 2004.[Abstract/Free Full Text]
  36. Perez JF and Sanderson MJ. The frequency of calcium oscillations induced by 5-HT, ACH, and KCl determine the contraction of smooth muscle cells of intrapulmonary bronchioles. J Gen Physiol 125: 535–553, 2005.[Abstract/Free Full Text]
  37. Petkov GV, Balemba OB, Nelson MT, and Mawe GM. Identification of a spontaneously active, Na+-permeable channel in guinea pig gallbladder smooth muscle. Am J Physiol Gastrointest Liver Physiol 289: G501–G507, 2005.[Abstract/Free Full Text]
  38. Pozo MJ, Perez GJ, Nelson MT, and Mawe GM. Ca2+ 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]
  39. Rottingen J and Iversen JG. Ruled by waves? Intracellular and intercellular calcium signalling. Acta Physiol Scand 169: 203–219, 2000.[CrossRef][Web of Science][Medline]
  40. Sanders KM, Koh SD, and Ward SM. Interstitial cells of Cajal as pacemakers in the gastrointestinal tract. Annu Rev Physiol 68: 307–343, 2006.[CrossRef][Web of Science][Medline]
  41. Sell M, Boldt W, and Markwardt F. Desynchronising effect of the endothelium on intracellular Ca2+ concentration dynamics in vascular smooth muscle cells of rat mesenteric arteries. Cell Calcium 32: 105–120, 2002.[CrossRef][Web of Science][Medline]
  42. Sergeant GP, Hollywood MA, McCloskey KD, McHale NG, and Thornbury KD. Role of IP3 in modulation of spontaneous activity in pacemaker cells of rabbit urethra. Am J Physiol Cell Physiol 280: C1349–C1356, 2001.[Abstract/Free Full Text]
  43. Ullman A, Lofdahl CG, Svedmyr N, and Skoogh BE. Nerve stimulation releases mucosa-derived inhibitory factors, both prostanoids and nonprostanoids, in isolated ferret trachea. Am Rev Respir Dis 141: 748–751, 1990.[Web of Science][Medline]
  44. Van Helden DF and Imtiaz MS. Ca2+ phase waves: a basis for cellular pacemaking and long-range synchronicity in the guinea-pig gastric pylorus. J Physiol 548: 271–296, 2003.[Abstract/Free Full Text]
  45. Van Helden DF, Imtiaz MS, Nurgaliyeva K, von der Weid P, and Dosen PJ. Role of calcium stores and membrane voltage in the generation of slow wave action potentials in guinea-pig gastric pylorus. J Physiol 524: 245–265, 2000.[Abstract/Free Full Text]
  46. Ward SM, Dixon RE, Defaoite A, and Sanders KM. Voltage dependent calcium entry underlies propagation of slow waves in canine gastric antrum. J Physiol 561: 793–810, 2004.[Abstract/Free Full Text]
  47. Yoneda S, Takano H, Takaki M, and Suzuki H. Properties of spontaneously active cells distributed in the submucosal layer of mouse proximal colon. J Physiol 542: 887–897, 2002.[Abstract/Free Full Text]
  48. 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]
  49. Zhang L, Bonev AD, Mawe GM, and Nelson MT. Protein kinase A mediates activation of ATP-sensitive K+ currents by CGRP in gallbladder smooth muscle. Am J Physiol Gastrointest Liver Physiol 267: G494–G499, 1994.[Abstract/Free Full Text]
  50. 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]



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