MINIREVIEW Synchronization of Ca 2+ oscillations: a coupled oscillator-based mechanism in smooth muscle Mohammad S. Imtiaz 1 , Pierre-Yves von der Weid 1 and Dirk F. van Helden 2 1 Department of Physiology and Pharmacology, University of Calgary, Alberta, Canada 2 School of Biomedical Sciences, University of Newcastle, Callaghan, NSW, Australia Long-range signaling Biological organs display coordinated activities that can extend over large distances. The spatial extent of signaling required for such long-distance coordination is many orders of magnitude greater than the size of the participating cells; for example, coordinated con- tractions of the intestine can occur over 250 cm lengths [1], whereas smooth muscle cells are small (typical size range 50–200 lm [2]). The problem is further exacerbated when one considers that millions of cells, each with its own intrinsic rhythm, partici- pate in this ‘mob action’, and yet a meaningful global outcome emerges. It is fascinating that in systems such as the gut, even isolated muscle tissue preparations continue to show coordinated rhythmic contractions in the absence of any external neural control [3]; thus, in such systems, the synchronizing mechanism is embedded within the rhythmically oscil- lating cells themselves. In this article, we review a long-range signaling mechanism in smooth muscle that explains global outcomes of local interactions [4– 10]. The main feature of this signaling mechanism is coupled oscillator-based synchronization of Ca 2+ oscillations across cells, which drives membrane potential changes and causes coordinated contrac- tions. The key elements of this mechanism are a Ca 2+ release–refill cycle of endoplasmic reticulum ⁄ Keywords Ca 2+ oscillations; Ca 2+ stores; coupled oscillators; lymphatics; slow waves; synchronization Correspondence M. S. Imtiaz, Department of Physiology & Pharmacology, Faculty of Medicine, University of Calgary, Health Sciences Centre, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada Fax: +1 403 210 8195 Tel: +1 403 210 9838 E-mail: mimtiaz@ucalgary.ca (Received 31 March 2009, revised 11 September 2009, accepted 14 October 2009) doi:10.1111/j.1742-4658.2009.07437.x Entrained oscillations in Ca 2+ underlie many biological pacemaking phe- nomena. In this article, we review a long-range signaling mechanism in smooth muscle that results in global outcomes of local interactions. Our results are derived from studies of the following: (a) slow-wave depolariza- tions that underlie rhythmic contractions of gastric smooth muscle; and (b) membrane depolarizations that drive rhythmic contractions of lymphatic smooth muscle. The main feature of this signaling mechanism is a coupled oscillator-based synchronization of Ca 2+ oscillations across cells that drives membrane potential changes and causes coordinated contractions. The key elements of this mechanism are as follows: (a) the Ca 2+ release– refill cycle of endoplasmic reticulum Ca 2+ stores; (b) Ca 2+ -dependent modulation of membrane currents; (c) voltage-dependent modulation of Ca 2+ store release; and (d) cell–cell coupling through gap junctions or other mechanisms. In this mechanism, Ca 2+ stores alter the frequency of adjacent stores through voltage-dependent modulation of store release. This electrochemical coupling is many orders of magnitude stronger than the coupling through diffusion of Ca 2+ or inositol 1,4,5-trisphosphate, and thus provides an effective means of long-range signaling. Abbreviations [Ca 2+ ] c , cytosolic Ca 2+ concentration; 18-b-GA, 18-b-glycyrrhetinic acid; ICC, interstitial cell of Cajal; Ins(1,4,5)P 3 , inositol 1,4,5-trisphosphate. 278 FEBS Journal 277 (2010) 278–285 ª 2009 The Authors Journal compilation ª 2009 FEBS sarcoplasmic reticulum Ca 2+ stores, Ca 2+ -dependent modulation of membrane currents, voltage-dependent modulation of store release, and cell–cell coupling through gap junctions or other mechanisms. Ca 2+ store-based pacemaking Gastric smooth muscle slow waves Slow waves are rhythmic electrical depolarizations that control the mechanical activity of many smooth mus- cles [1,11–13] (Fig. 1). Slow waves cause entry of Ca 2+ through opening of L-type Ca 2+ channels and contrac- tions of the smooth muscle. Cyclical release of Ca 2+ from inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ]-sensitive endoplasmic Ca 2+ stores underlies the generation of slow waves [12–15]. The store-generated change in cytosolic Ca 2+ concentration ([Ca 2+ ] c ) causes opening of excitatory channels, which allows inward current flow and generates rhythmic pacemaker depolarization [4,16–18]. However, the difficulty with oscillatory Ca 2+ release providing a pacemaker mechanism is that it requires synchronization of large numbers of stores across many cells [4,19]. Gastric smooth muscle cells and associated interstitial cells of Cajal (ICCs) form a syncytium interconnected by gap junctions. Such syn- cytia have low input impedance, and hence require a massive amount of current to cause pacemaker depo- larization. On the basis of experimental and theoretical considerations, we now consider how Ca 2+ oscillations can be synchronized across multiple cells in a syn- cytium. Synchronization of Ca 2+ oscillations One reported means by which stores achieve local syn- chrony is by Ca 2+ waves, a significant form of signal- ing in living organisms [20–22]. Ca 2+ waves are considered to be generated by the release of Ca 2+ from a dominant store, triggering Ca 2+ -induced Ca 2+ release from adjacent stores, and the continuation of this process along the array of stores. However, Ca 2+ waves propagate relatively slowly, typically at < 0.1 mmÆ s )1 . Thus, Ca 2+ waves cannot explain the synchrony of Ca 2+ oscillations underlying slow waves, which appear to be conducted at velocities of many millimeters per second. Coupled oscillators Another means by which stores can synchronize their Ca 2+ release cycle is by coupled oscillator-based interac- tions. The theory of coupled oscillators emerged from a fortuitous observation of pendulum clocks by the Dutch physicist Christiaan Huygens [23]. He noted that clock pendulums could synchronize their oscillations even though they were separated by distances of meters. This synchronization of clock pendulums occurred through coupling between the pendulums by transmission of minute vibrations through the wall. An example of cou- pled oscillators is a group of pendulums that are con- nected to each other by springs. When all pendulums are randomly set to swing, over time, interactions through the springs result in the appearance of a global synchrony pattern involving all the pendulums. Fig. 1. Central interruption of intercellular connectivity decouples slow waves. Pacemaker potentials ⁄ slow waves simulta- neously recorded at two sites along a guinea pig gastric smooth muscle tissue strip before (1), during (2) and after (3) central application of 60 l M 18-b-GA. Decoupling commenced  1.5 min after application of the blocker and was not phase-locked, as more slow waves occurred at site 2 than at site 1. For example, upon commencement of decoupling, four slow waves occurred at site 1 and five at site 2, with delays between the slow waves (site 2 ) site 1) of 0.8, 3.2, 7.9 and 9.5 s for the first five sequential slow waves. Nifedipine (1 l M) was present throughout. V m = )59 mV. Adapted from [8]. M. S. Imtiaz et al. Synchronization by voltage-modulated store release FEBS Journal 277 (2010) 278–285 ª 2009 The Authors Journal compilation ª 2009 FEBS 279 An experiment that illustrates the underlying cou- pled oscillator nature of slow waves involved a single bundle strip of circular smooth muscle dissected from the guinea pig gastric pylorus (Fig. 1). Initially, slow waves occurred synchronously in the strip, as mea- sured with two intracellular microelectrodes. When the gap junction blocker 18-b-glycyrrhetinic acid (18-b- GA; 40 mm) was applied centrally in a narrow stream approximately 0.5 mm wide to this strip, slow waves recorded at the two electrodes continued to occur but were no longer synchronized. When 18-b-GA was removed, slow waves in the two regions resynchro- nized. What is the mechanism of coupling between Ca 2+ stores? Oscillating Ca 2+ stores can interact by altering the phase of adjacent oscillators through Ca 2+ -induced- Ca 2+ release. Here, coupling by exchange of Ca 2+ [and ⁄ or Ins(1,4,5)P 3 for Ins(1,4,5)P 3 receptor-operated stores] through gap junctions could serve as the spring joining the pendulums in the above analogy. However, coupling through release of Ca 2+ results in very weak coupling, as the effective diffusion of Ca 2+ is limited to very short distances ( 5 lm) [24]. The same applies to coupling through diffusion of second messengers such as Ins(1,4,5)P 3 , even though the effective diffu- sion of Ins(1,4,5)P 3 is approximately three times higher than that of Ca 2+ [24]. However, a candidate mecha- nism that could serve as a coupling spring involves electrical membrane potential changes caused by Ca 2+ store-activated inward current flow [5,8,18,25]. Electri- cal coupling can be 100–1000 times stronger than chemical coupling, as the electrical length constant of smooth muscle (i.e. the distance needed for a steady- state voltage resulting from current injection to decrease to  37% of its original size) is typically in the range 2–3 mm [26]. Finding experimental evidence that electrical cou- pling is the key ‘spring’ interlinking the Ca 2+ stores has involved repeating the decoupling experiment of Fig. 1, but inhibiting the oscillators (i.e. the Ca 2+ stores) while leaving the connectivity between cells intact [8]. An example of such an experiment is pre- sented in which caffeine was used to block store Ca 2+ release and resulting slow-wave potentials (Fig. 2A). Application of the caffeine-containing physiological sal- ine solution to the central region of a single bundle strip of guinea pig gastric circular smooth muscle caused decoupling when the store inhibitor was applied in a very wide stream about 5 mm in width, but not when the stream was narrower (e.g. 3 mm; Fig. 2B). These distances are commensurate with coupling being 20 mV 10 mV 2 min F F 0 =1 Ca 3.0 mm 5.0 mm 20 s B A Caffeine Caffeine Control Control el1 el2 Fig. 2. Central interruption of stored Ca 2+ release decouples slow waves. (A) Caffeine (0.5 m M), applied to an Oregon Green- loaded guinea pig gastric smooth muscle tissue strip, blocked slow waves (upper trace) and underlying Ca 2+ release-associ- ated increases in [Ca 2+ ] c (lower trace). F 0 , baseline fluorescence; F, fluorescence; nF ⁄ F 0 , relative change in fluorescence normalized to baseline. (B) Slow waves recorded at two sites 6 mm apart along a strip before, during and after central applica- tion of 1 m M caffeine applied at widths of 3 and 5 mm. The 3 mm stream markedly increased jitter between the delays. By con- trast, the 5 mm stream decoupled the slow waves. Decoupling commenced  1 min after application of the blocker and was not phase-locked, with slow waves at the two recording sites now occurring at significantly different frequencies (P < 0.05; frequencies 3.7 ± 0.1 per min and 4.4 ± 0.1 per min at electrodes 1 and 2, respectively; n = 10). Nifedipine (1 l M) was present throughout in (A) and (B). V m : (A) )56 mV; (B) ) 67 mV. Adapted from [8]. Synchronization by voltage-modulated store release M. S. Imtiaz et al. 280 FEBS Journal 277 (2010) 278–285 ª 2009 The Authors Journal compilation ª 2009 FEBS mediated by intercellular current flow in these strips, which exhibited a length constant of about 3 mm. This and related experiments [8] fit the hypothesis that oscil- lations in stored Ca 2+ couple intercellularly across the syncytial smooth muscle by electrical coupling to gener- ate highly synchronous slow waves. Modeling studies As considered above, electrical conduction is many orders of magnitude stronger than chemical coupling, and this provides the ‘spring’ that underlies entrainment of Ca 2+ stores to pace tissue syncytia. However, cou- pled oscillator interactions also require chemical cou- pling, in that store-generated changes in [Ca 2+ ] c are required to activate inward membrane current, with the resulting membrane depolarization activating or advancing the phase of other Ca 2+ stores. The electrical and chemical transduction pathways are as depicted in Fig. 3. The key mechanisms are as follows: (a) cyclical release of Ca 2+ from stores can occur spontaneously and is modulated by two signals – Ca 2+ and Ins(1,4,5)P 3 ; (b) release of Ca 2+ from stores activates an inward current and depolarizes the membrane [18] – thus, store oscillations are transformed into membrane potential oscillations; (c) membrane potential can mod- ulate store excitability ⁄ oscillations by modulating Ca 2+ and ⁄ or Ins(1,4,5)P 3 concentrations in the cytosol – this provides a pathway for transforming electrical signals into chemical signals to which the stores respond; (d) cells are connected by gap junctions and form a syncy- tium, so stores can now interact across cells through electrical signals; and (e) the effective distance that Ca 2+ and Ins(1,4,5)P 3 can diffuse is very short, in the low micrometer range, whereas electrical coupling is in the order of millimeters – thus, whereas stores are weakly coupled through chemical diffusion, they are strongly interconnected by electrical coupling. We now illustrate the coupling mechanism outlined above with a two-cell model example (Fig. 4). This sys- tem is based on gastric smooth muscle, where depolar- ization of the membrane is modeled to cause an increase in Ins(1,4,5)P 3 concentration in the cytosol [25]. Cytosolic Ca 2+ concentrations of two uncoupled model cells are shown in Fig. 4A. Cell 1 (solid line) is more sensitive to Ins(1,4,5)P 3 , and is therefore oscillat- ing, whereas cell 2 (dashed line) is quiescent, because it is less sensitive to Ins(1,4,5)P 3 . Electrical coupling is then instituted between the two cells, and because of voltage coupling-based interactions, cell 2 begins to oscillate (Fig. 4B). This occurs because the oscillatory Ca 2+ release from cell 1 (Fig. 4C) activates an inward current, which, owing to electrical coupling, now depo- larizes both cells (Fig. 4D). Depolarization in cell 2 causes an increase in cytosolic Ins(1,4,5)P 3 concen- tration through voltage-dependent activation of Ins(1,4,5)P 3 (Fig. 3), with the increased cytosolic Ins(1,4,5)P 3 concentration causing generation of oscil- lations in cell 2. Importantly, although the frequency of the oscillations in cell 2 might be different to that of cell 1, coupled oscillator interactions advance or retard the cycle of each cell so that they remain entrained. Chemical versus electrochemical coupling A similar sequence of events occurs when the above example of two oscillators is extended to a system Cytosol-Ca 2+ Ca 2+ St or e +/ – +/ – Ins(1,4,5)P 3 (V) or Ca 2+ (V) Local oscillato r Ca 2+ V AT Pase Cytosol-Ca 2+ Ca 2+ St or e +/ – +/ – Local oscillato r Ca 2+ V Ins(1,4,5) P 3 R Ins(1,4,5) P 3 R AT Pase Strong electrical couplin g W eak chemical coupling Gap junction Ins(1,4,5)P 3 (V) or Ca 2+ (V) Fig. 3. A schematic representation of the two-cell system. Each cell is a local oscillator composed of a cytosolic store Ca 2+ -excitable sys- tem. The cytosolic Ca 2+ of each oscillator is transformed into membrane potential (V) oscillations by a Ca 2+ -activated inward current. The membrane potentials of the cells are strongly linked. Each local oscillator is weakly linked to the membrane potential by a voltage-dependent feedback loop such as voltage-dependent Ins(1,4,5)P 3 synthesis or voltage-dependent Ca 2+ influx. Ins(1,4,5)P 3 R, Ins(1,4,5)P 3 receptor; ATPase, ATPase pump. Adapted from [37]. M. S. Imtiaz et al. Synchronization by voltage-modulated store release FEBS Journal 277 (2010) 278–285 ª 2009 The Authors Journal compilation ª 2009 FEBS 281 composed of a large number of Ca 2+ store oscillators. In this simulation, the intrinsic frequencies of oscilla- tors are different from each other, and as the [Ins(1,4,5)P 3 ] is increased in the model tissue, a global synchronous rhythm emerges following events that grow from a noisy baseline (Fig. 5A). The above simulation outcome is very similar to what is observed in isolated gastric smooth muscle tis- sue. When gastric smooth muscle is freshly dissected and isolated, it usually remains quiescent, and mem- brane potential recordings display a noisy baseline. Confocal Ca 2+ imaging records obtained during this time reveal asynchronous isolated Ca 2+ events [8] simi- lar to those seen in the simulated voltage recordings of Fig. 5B1. However, over time, these release events begin to synchronize and summate to larger events (Fig. 5B2), and finally a global synchronous rhythm emerges (Fig. 5B3). We tested the potency of electrochemical coupling by running the same simulation but allowing no voltage-dependent modulation of Ca 2+ store release. This was achieved by blocking voltage-dependent syn- thesis of Ins(1,4,5)P 3 . In this case, no global synchrony emerged, and the baseline remained noisy even though the cells were coupled both electrically and by diffu- sion of Ca 2+ and Ins(1,4,5)P 3 (chemical coupling). In fact, the outcome was very similar to what is seen when no coupling exists between the cells (achieved by deleting gap junctions in the simulation) [8,10]. This example indicates that: (a) voltage-dependent modula- tion of store release in electrically coupled cells is a very efficient long-range coupling mechanism; and (b) chemical coupling by itself is not sufficient to synchro- nize Ca 2+ release events. In this regard, we note that a modeling study by Koenigsberger et al. [6] showed that diffusive coupling through Ca 2+ is sufficient to 40 42 44 46 48 50 0 1 2 3 [C a 2+ ] c , Z (µM) [C a 2+ ] c , Z (µM) [C a 2+ ] c , Z (µM) Ti me (min ) 14 0 14 2 14 4 14 6 14 8 15 0 0 1 2 3 Ti me (min ) A B 14 1 14 2 14 3 14 4 145 146 147 0.25 0.3 0.35 Time ( min ) Time (min) Time (min) 14 1 14 2 14 3 14 4 145 146 147 –70 –60 –50 –40 14 1 14 2 14 3 14 4 145 146 147 0.5 1 1.5 2 D C E V (mV) Cell 1 Cell 2 G ap junction Gap junction Cell 1 Cell 2 Cell 1 Cell 2 [Ins(1,4,5)P 3 ] c , (µM) Fig. 4. Synchronization of a cell pair. A two-cell system shows how synchrony can be achieved through voltage-dependent modulation of store release. (A, B) [Ca 2+ ] c plot of cell 1 and cell 2 before (A) and after (B) coupling. (C, E) [Ca 2+ ] c and [Ins(1,4,5)P 3 ] c , respectively, for the two cells after they are coupled. Note that the membrane potentials (D) for both cells are same, owing to large electrical coupling. Note that changes in [Ins(1,4,5)P 3 ] c for both cells follow changes in the membrane potential. Adapted from [10]. Synchronization by voltage-modulated store release M. S. Imtiaz et al. 282 FEBS Journal 277 (2010) 278–285 ª 2009 The Authors Journal compilation ª 2009 FEBS synchronize Ca 2+ oscillations. However, their simula- tion entailed only a small number of cells. Our findings agree with those of Koenigsberger et al. for the case of a small number of cells that have similar intrinsic oscil- latory frequencies and that are not separated by large distances, but their results do not apply to long-range coupling involving large numbers of cells. The electrochemical coupling of intracellular stores is found, with variations, in other systems as well. Below, we present some details that illustrate the same principles of pacemaking and synchronization mecha- nism in lymphatic smooth muscle. Lymphatic pacemaking A rhythmic constriction–relaxation cycle is displayed by blood and lymphatic vessels, a phenomenon known as vasomotion. Lymphatic vessels are divided into chambers by interconnecting valves. Rhythmic constric- tion and relaxation of these chambers propels lymph fluid through the lymphatic vessels. The pacemaking mechanism underlying contractions of lymphatic smooth muscle has been found to be dependent on Ins(1,4,5)P 3 -receptor operated Ca 2+ release from intra- cellular Ca 2+ stores [19]. Spontaneous Ca 2+ releases from Ins(1,4,5)P 3 receptor-operated Ca 2+ stores acti- vate a transient inward current, causing a spontaneous transient depolarization. However, the amount of Ca 2+ released from individual or small groups of stores is small, and results in spontaneous transient depolarizations that do not reach the threshold for opening L-type Ca 2+ channels which underlie action potential and constriction. This mechanism can only be effective if there are cooperative interactions between the release cycles of the Ca 2+ stores, as would be effected by stores interacting as coupled oscillators [4]. Indeed, this is highly likely to be the situation underpin- ning vasomotion in both blood and lymphatic vessels [5,6,9]. The mechanism operates on the same principles as outlined for gastrointestinal smooth muscle, but dif- fers from it in that the ‘springs’ that couple the oscilla- tors now rely on voltage coupling mediated by Ca 2+ entry through L-type Ca 2+ channels rather than volt- age-dependent production of Ins(1,4,5)P 3 . Ca 2+ oscillations in other cell types Gastrointestinal store-based pacemaker activity is, in fact, more complicated than considered so far, in that the pacemaker cells driving the slow waves are the ICCs [27–29]. These cells form networks in regions such as the myenteric plexus (i.e. ICC-MY) and intra- muscularly within the smooth muscle (i.e. ICC-IM), interconnecting with each other and with adjacent smooth muscle. As a consequence, the dominant Ca 2+ stores that underlie pacemaking reside in these cells [8,14]. However, whether this is the case may depend on the tissue. For example, the pacemaker activity that generates vasomotion in blood and lymphatic vessels, although Ca 2+ store-based, may be driven by Ca 2+ stores in the smooth muscle, as a role for ICC-like cells has yet to be confirmed [5,9,19]. In contrast, Ca 2+ store-based pacemaking in the rabbit urethra is generated in ICC-like cells [13,30]. There is now evidence that sinoatrial cells that pace the heart also show Ca 2+ store-based oscillation. This 0 20 40 60 80 100 120 140 160 180 –65 –60 –55 –50 –45 V (mV) V (mV) V (mV) 1 2 3 2 min 10 s 3 20 mV 2 1 3 2 1 50 100 150 200 250 300 –65 –60 –55 Time (min) Time (min) Time (min) 0 20 40 60 80 100 120 140 160 180 –66 –64 –62 A B C D Fig. 5. Synchronization of a cell population. (A) The emergence of synchronized global slow waves in a gap junction-coupled model cell syncytium. (B) The emergence of slow waves in guinea pig pyloric smooth muscle. Nifedipine (1 l M) was present throughout. The voltage scale bar applies to all records. Events marked with labeled arrows are shown on an expanded time scale. The resting membrane potential was )59 mV. Expanded regions 1, 2 and 3 are similar to events similarly marked in the model syncytium mem- brane potential in (A). (C) When voltage-dependent synthesis of Ins(1,4,5)P 3 is blocked, no synchronous events arise in the model syncytium, even though all of the other parameters are the same as in (A). (D) Similarly, no synchronous events arise if gap junctions are blocked in the model syncytium, even though all the parame- ters are the same as in (A). Adapted from [37]. M. S. Imtiaz et al. Synchronization by voltage-modulated store release FEBS Journal 277 (2010) 278–285 ª 2009 The Authors Journal compilation ª 2009 FEBS 283 operates together with the classic membrane oscillator generated by voltage-dependent channels in the cell membrane to drive the heart [31,32]. It differs from the smooth muscle cell store oscillator in that it utilizes ryanodine receptor-operated rather than Ins(1,4,5)P 3 receptor-operated Ca 2+ stores. It remains to be seen whether Ca 2+ stores have a role in the syn- chronization of sinoatrial nodal cells. However, in the heart muscle, increased Ca 2+ store excitability can cause the emergence of unwanted pacemakers that result in pathological waves of contractions known as arrhythmias [33,34]. Indeed, this raises the question of why stores in the atrial and ventricular muscle do not normally synchronize, as they do in the pacemaker node. This is, of course, a very important feature of the heart, as otherwise the muscle systems themselves would have autonomous pacemaker capability. The reason for this needs to be explored, but there is a very interesting analogous circumstance in the stom- ach. Here, only the middle and lower sections of the stomach exhibit slow waves and associated rhythmic contractions; the upper region of the stomach (i.e. the gastric fundus) is nonrhythmic. As has been noted, slow waves are generated by stored Ca 2+ release [14], a mechanism that requires long-range intercellular synchronization of oscillatory stored Ca 2+ release [8]. The gastric fundus should exhibit slow waves, as it has abundant pacemaker cells (i.e. ICCs) that exhibit store Ca 2+ release coupled to membrane depolariza- tion [35]. However, coupling does not happen! The reason for this is that stores in this region lack a key component of their coupling mechanism, namely the feedback by which membrane depolarization causes stored Ca 2+ release [35]. The consequence is that the coupling link that allows long-range store coupling is no longer functional, and hence store pacemaking cannot occur in this smooth muscle. Conclusion and future directions In this article, we have reviewed long-range signaling through Ca 2+ release from intracellular Ca 2+ stores, which is a key determinant of whether stores can pro- duce sufficient synchrony to act as a pacemaker mech- anism. Voltage-dependent coupling between Ca 2+ stores is critical for such signaling, as it is several orders of magnitude stronger than chemical coupling through diffusion of Ca 2+ and ⁄ or Ins(1,4,5)P 3 . In our model, electrochemical coupling was considered to occur by intercellular current flow through presumed gap junctions. However, such electrical coupling could also occur wholly or in part by capacitive coupling, as shown in the study of Yamashita [36] (see accompany- ing review), and it will be interesting to determine the relative role of this mechanism. In summary, store-based pacemaking, whether oper- ated by Ins(1,4,5)P 3 receptors or by ryanodine recep- tors, has a role in a range of tissues where cells are electrically connected. The key for a functional pace- maker mechanism in such cell syncytia is that oscilla- tory store Ca 2+ release generates inward currents and resultant depolarization, that the cellular network readily conducts currents, and that the conducted depolarization in turn leads to activation of other Ca 2+ stores. This latter step could be mediated by depolarization-induced Ca 2+ entry and ⁄ or production of Ins(1,4,5)P 3 [9,25]. 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