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Pharmacology and Clinical Pharmacology, Department of Basic Medical Sciences, St Georges Hospital Medical School, Cranmer Terrace, London, SW17 ORE, UK
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(Received 1 August 2003;
accepted after revision 5 November 2003;
first published online 7 November 2003)
Corresponding author T. B. Bolton: Pharmacology and Clinical Pharmacology, Department of Basic Medical Sciences, St Georges Hospital Medical School, Cranmer Terrace, London, SW17 ORE, UK. Email: t.bolton{at}sghms.ac.uk
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Introduction |
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Hypotheses about the mechanism and coordination of localized events, and thereby the interpretation of experimental data, have been directed by two assertions. Firstly that localized events are ‘discrete’ due to the spatial segregation of discrete ‘clusters’ of calcium release channels on an otherwise continuous sarcoplasmic reticulum. Physical evidence for this has been provided by immunochemistry and electron microscopy (Protasi et al. 2000; Yin & Lai, 2000). However the quantitative interpretation of the spatio-temporal properties of localized events, in terms of channel clustering, has been severely constrained by the method used to characterize them – confocal line scanning – which samples four dimensional events in only two dimensions. The second assertion is that these clusters are not mixed: they consist entirely of either inositol trisphosphate receptors (IP3Rs) or ryanodine receptors (RyRs), but not both together. This view is reflected in the predominate dichotomy between ‘sparks’ (inhibited by ryanodine) and ‘puffs’ (stimulated by IP3 or IP3-generating agonists). Despite this there have been reports of events with a mixed IP3–ryanodine receptor pharmacology (Koizumi et al. 1999; Haak et al. 2001; Gordienko & Bolton, 2002).
In smooth muscle, localized events when they have been seen, have predominantly been characterized as sparks (Gordienko et al. 1998; Jaggar et al. 2000). There are only a few reports of puffs, namely in colonic myocytes (Bayguinov et al. 2000, 2001a,b) and uteric myocytes (Boittin et al. 2000). In this study we describe novel and heterogeneous localized calcium release events in hitherto undescribed single cells from the muscle of guinea-pig fundus. These events were characterized in terms of their pharmacology and quantitative spatio-temporal dynamics. Some of these results have been presented in abstract form (Parsons & Bolton, 2001, 2002).
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Methods |
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Guinea-pigs were killed humanely by instantaneous stunning, followed by exanguination, in accordance with UK guidelines. Both ventral and dorsal aspects of the gastric fundus were removed in situ from the mucosa as single pieces, using scapel and forceps. These were kept in warmed, HEPES-buffered saline (HBS; see below) and cut into smaller pieces, before enzymatic digestion in HBS with 0.5 mM Ca2+, 0.25 mM Mg2+ and 1 mg ml-1 each of collagenase type 1A (clostridiopeptidase A), protease type XIV, type II-S soybean trypsin inhibitor and bovine serum albumin. Digestion was for 20 min at 36°C. The tissue was then washed with warmed HBS and dispersed by tituration with a blunt-ended, fired glass Pasteur pipette. Dispersion was in warmed HBS with 0.5 mM Ca2+, 0.25 mM Mg2+ and the resulting suspension was pipetted on to glass coverslips, attached to experimental chambers. After allowing the cells to settle and attach for 1 h, the dispersion solution was replaced with HBS containing 2.0 mM Ca2+ and 1.0 mM Mg2+. All experiments were conducted in this solution at room temperature.
Confocal microscopy
Confocal microscopy was performed using a Zeiss Axiovert 100M microscope with the LSM 510 laser scanning module. Unless otherwise stated a x 40 oil immersion plan-neofluar objective (NA = 1.3) was used with a 66 µm pinhole (giving an optical slice of < 0.9 µm). To image [Ca2+]i, 5 µM fluo-3AM was included in the dispersion solution and a 488 nm argon laser was used for excitation, with a 505 nm bandpass emission filter. Temporal changes in cell [Ca2+]i were imaged by repeated scanning of either a whole confocal field (an xyt series) or just a single line positioned along the cell's long axis (line scan or xt series). Unless otherwise stated, xt series were taken at 6.59 Hz (3.8 ms to scan a line, with a 148 ms interline delay). Time series were analysed with programmes personally written in the IDL software suite (v5.3). To ratio a time series, the value of each scanned pixel (F) was divided by the mean of its value (F0) over an quiescent time period (i.e. representative of the baseline fluorescence).
Automatic event detection
A computer program was written with IDL software (version 5.3) to analyse the spatio-temporal properties of individual calcium release events in xt scans. There were two main points to this program: firstly to perform event detection automatically, to avoid the subjective bias imposed by human selection (Song et al. 1997; Cheng et al. 1999); secondly to separate out individual peaks (apparent events), which had coalesced spatially or temporally into ‘compound events’. In such a situation the definition of an event was the area around a peak at its half-maximum amplitude (amplitude at half-maximum or AHM). The AHM was defined as follows:
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| (1) |
The fundamental element of the automatic event detection programme was the IDL routine, ‘label_region.pro’. This routine allows for the definition (separation) of regions within an image (xt scan) at any arbitrary threshold (
), a ‘region’ being a contiguous area of an xt series/ image of value greater than (analogous to the area enclosed by a single contour on a map). In the program this process (region definition) is applied iteratively, beginning at a rather low value of (the initial threshold or i) and then continuing at successively higher values of , increasing by steps of ß (the ‘amplitude resolution’). At each successive value of (i, i+ß, i+ 2ß, i+ 3ß....i+nß), certain rules can be applied to each region individually, to determine whether it may be considered as an event. The result of these rules and their operation (as follows; see Fig. 1) is that an ‘event’ is defined as the contiguous region around a peak (the maximum of the region), at the peak's AHM.
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i (say i= 1.25F/F0).
Step 2. For each region found by steps 1 or 3 (at a threshold of ):
, then this region is not considered as an event and is discarded; and +ß (say ß= 0.2F/F0), then the region is considered as an event and is passed to step 4; +ß then the region is passed to step 3.
Step 3. The process of region definition is repeated within the region found in step 2c) (which has been defined at a threshold of ), at a threshold of +ß. This step in effect splits compound events (regions bound at ) into regions separated at +ß. These regions are then passed back through step 2 again, with increased by ß.
Steps 2 and 3 are repeated iteratively (with increasing by ß with each iteration) until no regions are found to have an AHM > +ß (step 2c above).
Step 4. The maximum amplitude (MA), full width and full duration at half-maximum (FWHM and FDHM) of all the events found by step 2b are calculated. The FWHM and FDHM are defined as the two orthogonal lengths which pass through the event's maximum and are bounded by the event's AHM.
As an additional note, at various stages in the program defined regions are checked to see if they surpass a minimum area (the ‘xt resolution’ or XTR). This prevents the consideration of noise. For all the data shown in the Results section, the XTR was set to 3 µm. s, i was 1.25F/F0 and ß was 0.2F/F0.
Analysis of total activity
To give a single quantitative estimate of overall [Ca2+]i release activity (in the presence of antagonists, etc), 90 s periods of xt images were averaged, over the full spatial axis, to give a mean pixel value (MPV; see Fig. 6). Also small rectangular areas of the xt image were selected by eye as representative of the basal [Ca2+]i (Fig. 6). The mean values of these boxes (the ‘baseline’) are compared to the MPV (Figs 6–11).
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| (2) |
Peaks of initial Ca2+ transients in response to carbachol or caffeine (Fig. 12) were calculated as the maximum value obtained by averaging F/F0 values in all pixels along the scan line, less the baseline value for the period preceding the transient.
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HBS had the following composition (mM): 119 NaCl, 6 KCl, 12 glucose, 10 HEPES, adjusted to pH 7.35 with NaOH. Calcium chloride or magnesium chloride was added to this as indicated elsewhere. Ryanodine, U-73122, neomycin, carbachol and caffeine were diluted from aqueous stock solutions. The following were diluted from DMSO stock solutions: 2 aminoethyldiphenyl borate (2-APB, 60 mM), xestospongin C (XeC, 5 mM) and fluo-3AM (0.88 mM). Drug solutions were applied as 1 ml syringe volumes connected by thin tubing to a chamber of 
300 µl volume. Ryanodine, xestospongin C, 2-APB and U-73122 were from Calbiochem (Beeston, UK). Fluo-3AM was from Molecular Probes (Eugene, OR, USA). All other chemicals were from Sigma Chemical Co. (Poole, UK).
Analysis and statistics
Data have been summarized as the mean ± standard error of the mean (S.E.M.), except where indicated. Comparisons were conducted by either paired or unpaired (unpooled) Student's t tests.
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Results |
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In contrast a second class of cell, which represented 2–3% of the dispersed population, had a membrane characterized by bumps, ridges or knurls (Fig. 2), and showed spontaneous localized [Ca2+]i events in >95% of cases. It is only these cells which are considered throughout the rest of this study. Inspection of xyt series indicated that the spatial and temporal characteristics of these localized events varied considerably. Figure 3A shows a typical example of this. In at least four regions of the cell (arrows) there were sustained increases in or plateaux of [Ca2+]i, lasting over tens of frames (20–40 s). These events seemed to originate at a point source close to the cell membrane and then spread out over a period of 1–2 s to fill the full width, and over 20 µm of the length, of the cell (Fig. 3B). In addition smaller, more localized and transient rises in [Ca2+]i occurred (arrowheads in Fig. 3A).
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In addition to xyt (frame) series, line scan or xt series of localized [Ca2+]i events were acquired by positioning the scan line (typically 40–70 µm long) parallel to the long axis of the cell in an active region. A typical example of such a scan is shown in Fig. 4A. As in an xyt scan (Fig. 3), there is a mixture of shorter and longer localized [Ca2+]i events. Of the shorter, transient events, most consisted of a rapidly rising phase followed by an approximately exponential decay (Fig. 4Bi). Of the longer events, some seemed to represent true plateaux in [Ca2+]i (Fig. 4Bii), whilst others were compound events made up of doublets or triplets of shorter transients (e.g. event 05 in Fig. 4A).
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i= 1.25F/F0; see Methods). The frequency distributions of MA (mean = 3.67F/F0, S.D.= 2.0F/F0), FDHM (mean = 5.6 s, S.D.= 5.8 s) and FWHM (mean = 6.7 µm, S.D.= 3.9 µm) all decayed in a roughly exponential manner (Fig. 5Ai, Bi and Ci, respectively). This would be expected from the random sampling of four-dimensional (x, y, z, t) events by a ‘two-dimensional’ method (the xt scan). For amplitude at least, the distribution should be hyperbolic, i.e. of the form n 
(a – B)-1, where n is the amplitude frequency, a is the amplitude and B is a constant of the observation function (which describes the statistical sampling of an event's maximum amplitude at varying distances from its actual origin; Izu et al. 1998). Consequently if the inverse of the amplitude frequency (1/n) is plotted against amplitude, a linear relationship should be obtained. In fact the inverse distribution of MA was marked by two linear trends of different slope (Fig. 5Bii). This change in slope (an ‘inflection’) reflects the apparent ‘break’ in the normal distribution (Fig. 5Bi) at the same value of MA.
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In control experiments with the four period protocol (n= 6), where drug vehicle (0.33% v/v DMSO) was added alone in the first scan, there was no significant change in any of the parameters over the first three periods (Fig. 6B). The vehicle caused no significant inhibition of Ca2+ release as the difference between the baseline and MPV remained significant (by paired t test) over all four periods. Carbachol caused a significant increase in both the MPV (P < 0.01) and baseline (P < 0.05) after the initial scan-wide transient which averaged 4.69 ± 0.85F/F0 (Fig. 12). Caffeine (5 mM) produced a scan-wide transient of similar amplitude (n= 6; Fig. 12) but had no effect on localized release (Fig. 7): there was no significant change in baseline or MPV immediately after the initial transient and the difference between the MPV and baseline remained significant (statistical tests as before; not shown).
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Pharmacology: IP3R inhibitors. Two different antagonists of IP3R-dependent Ca2+ release were tested for their effect on Ca2+ release (Fig. 9). 2 Aminoethoxydiphenylborate (2-APB) has an IC50 of 42 µM on IP3-induced Ca2+ release from rat cerebellar microsomes, but has no effect on caffeine-induced release from skeletal or cardiac microsomal preparations (Maruyama et al. 1997). Recently it has been employed as an IP3R inhibitor in a number of studies of isolated smooth muscle cells up to concentrations of 100 µM (e.g. Sergeant et al. 2001, 100 µM; Gordienko & Bolton, 2002, 30 µM; White & McGeown 2003 100 µM; Lee et al. 2003, 100 µM). Xestospongin C has also gained popularity as a membrane-permeable IP3R inhibitor and has been used at concentrations up to 10 µM with isolated cells (Bayguinov et al. 2000, 5 µM; Gordienko & Bolton, 2002, 10 µM; White & McGeown 2003, 2 µM; Ozaki et al. 2002, 3 µM; Lee et al. 2003, 1 µM). Xestospongin C (XeC) is a ‘macrocyclic’ bis-1-oxaquinolizidine, isolated from Australian sponges of the Xestospongia genus. It has an IC50 of 358 nM on IP3-induced Ca2+ release from rabbit cerebellar microsomes but does not affect the binding of IP3 itself to the receptor (Gafni et al. 1997). Despite their popularity both 2-APB and XeC may have some limitations in regard to their specificity. 2-APB inhibits store-operated channels (SOCs) in hepatocytes and B cells, seemingly independently of any affect on Ca2+ stores (see Discussion). XeC blocks Ca2+ uptake by permeabilized A7r5 smooth muscle cells (De Smet et al. 1999) and may open the IP3Rs at concentrations above 20 µM (Schaloske et al. 2000).
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XeC (10 µM,n= 6) acted much more quickly than 100 µM 2-APB. This probably reflects the greater membrane permeability of XeC. Therefore a three period, single scan protocol was used: XeC was added after 2 min and after a further 2 min this was supplemented with carbachol, splitting the protocol into three periods (Fig. 9B and D). By the second period the MPV was reduced significantly (P < 0.01) and there was no longer any significant difference between it and the baseline. Unlike ryanodine and 2-APB, XeC alone caused no increase in the baseline. Carbachol increased MPV and the baseline slightly but the difference between them remained insignificant. With the three period protocol XeC had no significant effect on the initial carbachol-induced transient (Fig. 12). This was not due to the limited incubation period of XeC, as there was still no significant inhibition of the transient by 10 µM XeC using a four period protocol as for ryanodine and 2-APB (n= 3, mean transient peak minus baseline = 5.33 ± 0.25F/F0).
Effect of lowering external calcium
Changing the external Ca2+ concentration from the standard 2 mM to nominally Ca2+-free immediately abolished all Ca2+ events (n= 10; Fig. 10). MPV was significantly reduced (P < 0.001) and no significant difference remained between it and the baseline. This was fully reversible with readmission of Ca2+ (n= 5; Fig. 10B and C). At 10 µM, carbachol caused a calcium transient in nominally Ca2+-free solution, which was not significantly different in amplitude from controls (n= 5; Figs 10A and 12). However carbachol had no stimulatory effect on localized release (Fig. 10A and D).
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At a concentration of 4 µM U-73122 caused a gradual increase in global Ca2+, consistent with an inhibition of SR uptake (Fig. 11A). At 1 µM, this did not occur (n= 6), but also there was no immediate inhibition. Therefore the four period protocol was used (Fig. 11B) as with ryanodine and 2-APB. After 12 min (at the third period) the baseline and MPV had both fallen significantly (P < 0.001 and P < 0.01, respectively; Fig. 11B and D). There was no significant difference between baseline and MPV, which was not altered by the addition of carbachol. In fact the amplitude of the initial carbachol-induced transient was significantly reduced (P < 0.02), being abolished in all but two of the cells tested (Fig. 12). Instead there was usually a shallow rise in global calcium, similar to that seen with 4 µM U-73122. The effects of U-73343, a weaker analogue of U-73122, were tested with the same protocol. U-73343 (1 µM) caused no significant change in the MPV or baseline over the last three periods (with respect to the first period), except for a decrease in baseline in period 2 (P < 0.05; results not shown). Values of MPV for the four periods were 1.47, 1.12, 1.32 and 2.35F/F0, respectively, and values of baseline were 0.96, 0.77, 1.05 and 1.32F/F0, respectively. However the difference between the MPV and baseline was only significant for the first two periods (P < 0.05 for both; MPV minus baseline = 0.51, 0.34, 0.28 and 1.02F/F0 for the four periods, respectively). There was no significant inhibition of the initial carbachol-induced transient (Fig. 12).
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Contractile properties
The cells studied above had no or a weak contractile response to carbachol. Without this a comparison of localized release before and after carbachol would not have been satisfactory. Of those cells, analysed above, which responded at all to carbachol (with a initial Ca2+ transient; Fig. 12) only 6 out of 45 cells contracted at all (1 cell under control conditions, 2 in the presense of 10 µM ryanodine, 1 in the presence of 50 µM ryanodine and 2 in nominally Ca2+ free). Further, 2/6 cells contracted in response to 5 mM caffeine. In all these cases the degree of contraction was limited to less than 15% of the length of the line scan, and no correlation was noticed between the amplitude of the Ca2+ transient and the strength of contraction.
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Discussion |
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Localized calcium release was reduced both by ryanodine and by blockers of IP3Rs/PLC, suggesting that it may involve calcium store release through both RyRs and IP3Rs. Despite its dependence on store calcium, localized calcium release was acutely dependent on calcium entry. This has a precedent from studies of puffs in colonic myocytes (Kong et al. 2000; Koh et al. 2001; Bayguinov et al. 2001a) and PLC/IP3-linked agonist-induced [Ca2+]i oscillations in a variety of cell types (e.g. Felder et al. 1992; Hashii et al. 1993; Yao et al. 1994; Thorn, 1995; Wu et al. 1995; Komori et al. 1996; Kohda et al. 1998). This entry could regulate Ca2+ release (1) via regulation of the peripheral SR at some subplasmalemmal space (2) via stimulation of a calcium-sensitive PLC (i.e. PLCß) or (3) via direct activation of the IP3Rs and/or RyRs (i.e. calcium-induced calcium release, CICR) (Fig. 13).
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The concentrations of 2-APB, XeC and other drugs used in this study were similar to those used by others in studies of calcium release in various cell types. Ryanodine has been used at concentrations from 10 to 100 µM (Bayguinov et al. 2000; Gordienko & Bolton, 2002; Hollywood et al. 2003; White & McGeown, 2003), 2-APB at concentrations from 30 to 100 µM (Ma et al. 2001; White & McGeown, 2002; Gordienko & Bolton, 2002; Hollywood et al. 2003; Kim et al. 2003; Lee et al. 2003), XeC at concentrations from 0.3 to 20 µM (Bayguinov et al. 2000; Ma et al. 2000; Schaloske et al. 2000; Gordienko & Bolton, 2002; Hollywood et al. 2003; Lee et al. 2003) and U-73122 up to 3 µM (Kim et al. 2003; Lee et al. 2003). Ma et al. (2000) found that 75 µM 2-APB blocked thapsigargin-stimulated (i.e. store-operated) calcium entry in HEK293 cells, with less than a minute's pretreatment. In contrast 20 µM XeC, applied for 20 min before thapsigargin, only reduced entry by half. Similar results were obtained by Bishara et al. (2002), studying calcium entry in aortic endothelial cells; either thapsigargin, ATP or ionomycin caused a initial calcium transient in the absence of external calcium (indicating SR calcium release), followed by a second slower transient upon calcium readmission (to 2.5 mM), indicative of calcium entry. Whilst 100 µM 2-APB blocked both transients with less then 10 s pretreatment, a much longer pretreatment with 10 µM XeC blocked the initial transient but only inhibited the second transient by half. Lee et al. (2003) found that in murine antral myocytes, 100 µM 2-APB blocked the transient inward current in response to 50 µM carbachol after only a minute's pretreatment. In contrast Ozaki et al. (2002) found that in guinea-pig ileal myocytes 3 µM XeC had no effect on the inward current stimulated by 10 µM carbachol (although it did inhibit a voltage-dependent barium inward current). White & McGeown (2003) found that in guinea-pig vas deferens myocytes calcium transients in response to noradrenaline were blocked by 100 µM 2-APB and U-73122, but not by 2 µM XeC. Hildebrandt et al. (1997) found that in NG108-15 cells, the [Ca2+]i transient and inward current, in response to 1 µM bradykinin were blocked by 5 µM U-73122, but neomycin had no effect on either phenomenon at concentrations up to 3 mM.
These studies suggest a difference between the actions of 2-APB and U-73122 on the one hand and XeC and neomycin (which is not very membrane permeant) on the other. Whilst the former are clearly good inhibitors of receptor- (especially muscarinic-) or store-operated calcium entry/currents, the latter are not (XeC acts weakly), although they may very well inhibit voltage-dependent currents in common with U-73122 (see Hildebrandt et al. 1997 for discussion; Ozaki et al. 2003). This dichotomy was reflected in the pharmacology of calcium release in fundus cells: on the one hand 2-APB and U-73122 inhibited both localized release and the initial carbachol-induced calcium transient, while XeC and neomycin on the other hand only inhibited localized calcium release events. It might be suggested from the above that the initial carbachol-induced transient is more dependent on calcium entry as it was only inhibited by 2-APB and U-73122, whereas localized release is dependent on IP3R/PLC as it was inhibited by all four drugs.
However, a more likely explanation is that, since the initial carbachol response was not abolished in nominally calcium-free solution (Fig. 10A), activation of muscarinic receptors by 10 µM carbachol produces a large increase in IP3 production which overwhelms XeC antagonism, and XeC was ineffective at blocking carbachol-induced calcium store release due to copious IP3 production which still releases substantial stored calcium (Figs 9, 12 and 13). Neomycin is poorly membrane permeant and, possibly for this reason, was similarly ineffective, despite being an effective blocker of localized calcium release events. Nevertheless, XeC had a quick effect on localized release, relative to 2-APB, perhaps because (1) 2-APB is an amine which is a cation (protonated) at physiological pH (Bootman et al. 2002) and so enters the cell more slowly, and (2) XeC was used at 28 times its IC50 for release from cerebellar microsomes (IC50= 358 nM with stimulation by 5 µM IP3, Gafni et al. 1997) as opposed to 2 times in the case of 2-APB (IC50= 42 µM with stimulation by 100 nM IP3, Maruyama et al. 1997). Also, it should be noted that the weak inhibition of localized release by U-73343, relative to U-73122, would fit with its correspondingly smaller IC50 for inhibition of phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis by PLC (Bleasdale et al. 1990).
The initial carbachol-induced transient was reduced by ryanodine but not significantly by XeC, so it might appear that RyRs rather than IP3Rs, are involved. However, U-73122 also blocked the carbachol-induced transient and ryanodine releases calcium from stores, so that after several minutes of ryanodine treatment, store calcium may be reduced. However, the inhibition of localized release by ryanodine was alleviated by carbachol, suggesting that, if IP3 production is increased, the residual store calcium released through IP3Rs was sufficient to support localized calcium release events.
The mechanisms proposed for calcium release in fundus cells provide several possible explanations for the heterogeneity of calcium release event dynamics. RyRs within clusters could be ‘recruited’ stochastically by calcium release through IP3Rs to generate events of varying magnitude by either stochastic variations in IP3 levels (activity of PLC) and/or calcium entry (filling the peripheral SR or activating PLCß (see Swillens et al. 1999; Shuai & Jung, 2002 and Falcke, 2003 for theoretical treatments of cluster recruitment) (Fig. 13). Despite this the greater differences in event magnitude seem to reflect release from spatially separate sites which generate relatively stereotyped events, i.e. some sites (‘frequent discharge sites’; Gordienko & Bolton, 2002) consistently generate large events that fill the width of the cell and lasted tens of seconds, whilst other sites consistently generate smaller transient events (Fig. 2).
In relation to the dynamics of channel clusters and how these relate to the visible event kinetics, one cannot disregard the recent theoretical treatments of amplitude distributions (Pratusevich & Balke, 1996; Smith et al. 1998; Izu et al. 1998; Cheng et al. 1999; Swillens et al. 1999; Shuai & Jung, 2002). Izu et al. (1998) showed that an inflection in the inverse plot of a hyperbolically decaying amplitude distribution, would indicate the presence of two modes in the actual distribution of event amplitudes (i.e. when all events were scanned at their origin). The authors modelled these two modes as two event populations with different mean calcium release channel currents (1 and 2 pA). However, Shuai & Jung (2002) suggested that multiple modes in the amplitude distribution could result from increases in IP3 concentration.
In addition to the general properties of elementary release, it is necessary to consider the actual spatio-temporal profiles of individual events. Most of the smaller events (<3 s FDHM) may be described as typical ‘shot’ events, as for puffs and sparks – that is they consisted of a rapid onset followed immediately by a diffusive (exponential) decay. In this respect they are unremarkable, other than by their size, which probably scales with the number of channels open at their inception. In contrast the larger events (>3 s FDHM) displayed some rather unusual characteristics. In general they did not ‘switch off’ after the initial release, suggesting that there must be a mechanism to prevent autoinhibition of the channel by the calcium it releases (traditionally considered as the limiting/terminating factor in CICR). This might involve some protein accessory to the channel itself. ‘Plateau’ events have been noticed on occasion with sparks in skeletal muscle (Shtifman et al. 2000; Gonzalez et al. 2000; Kirsch et al. 2001), ileal mycoytes (Gordienko et al. 1998), vas deferens myocytes (White & McGeown, 2003) and puffs in Xenopus oocytes (Marchant & Parker, 2002). The lack of multiple levels in the fundus plateau events would argue against the presence of release channel subconductance states shown to operate with plateau events in frog skeletal muscle (Gonzalez et al. 2000).
This is the first study to demonstrate the role of calcium signalling at the cellular level in the fundus. The presence of spontaneous calcium release events in the cells studied, and the absence of such events in the archetypal smooth muscle cells, is consistent with the role of the former as drivers of contractile activity in the fundus but support for this possibility will require further investigation.
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