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J Physiol. Aug 1, 2002; 542(Pt 3): 743–762.
PMCID: PMC2290443

Crosstalk between ryanodine receptors and IP3 receptors as a factor shaping spontaneous Ca2+-release events in rabbit portal vein myocytes

Abstract

In smooth muscle cells freshly isolated from rabbit portal vein, there was only one site discharging the majority of spontaneous Ca2+-release events; the activity of this single site was studied using laser scanning confocal imaging after loading the cells with the fluorescent Ca2+ indicator fluo-4 acetoxymethyl ester. Localised spontaneous Ca2+-release events visualised by line-scan imaging revealed two predominant spatiotemporal patterns: (i) small-amplitude, fast events similar to Ca2+ sparks in cardiomyocytes and (ii) larger and slower events. The sum of two Gaussian profiles was well fitted to the amplitude histogram (peak frequencies at 1.8 and 3.2 F/F0) and spatial spread (full width at half-maximal amplitude) histogram (peak frequencies at 2 and 3.8 μm) for the 230 localised Ca2+-release events analysed. The existence of two populations of Ca2+-release events was also supported by the histograms of the rise times and half-decay times, which revealed modes at 38 and 65 ms, respectively. Shifting the scan line along the z-axis during imaging from a single discharge site suggested that the appearance of two populations of Ca2+-release events is not due to out-of-focus imaging. Both small and large events persisted upon 3–5 min exposure to 1–5 μm nicardipine, but were abolished after 10–15 min exposure to 50–100 μm ryanodine, 0.1 μm thapsigargin or 10 μm cyclopiazonic acid. Only small-amplitude, fast events persisted in the presence of inhibitors of inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release, 10 μm xestospongin C or 30 μm 2-aminoethoxy-diphenylborate (2-APB), or in the presence of 2.5 μm U-73122 (a phospholipase C (PLC) inhibitor). Coupling between neighbouring Ca2+-release domains giving rise to spontaneous [Ca2+]i waves was abolished in the presence of 2-APB. Examination of the saltatory propagation of the waves suggested that the critical factor that determines propagation between domains is a time-dependent change in the sensitivity of ryanodine receptors and/or IP3 receptors to Ca2+, which can give rise to ‘loose coupling’ between release sites. These results suggest that activation of IP3 receptors (due to the tonic activity of PLC and ongoing production of IP3) recruits neighbouring domains of ryanodine receptors, leading to larger Ca2+ releases and saltatory propagation of [Ca2+]i waves in portal vein myocytes.

Ionised calcium plays an important role in the control of diverse activities in excitable and non-excitable cells through regulation of numerous enzymes, membrane ion channels and contractile proteins. In general, the spatiotemporal patterns of global Ca2+ signalling in intact cells reveal a high degree of complexity. An abrupt rise in cytosolic Ca2+ concentration ([Ca2+]i) in response to a stimulus may arise by Ca2+ entering the cell through Ca2+-permeable membrane ion channels (voltage- or mechanosensitive and receptor- or store-operated) and/or by Ca2+ released from intracellular stores, especially the endoplasmic/ sarcoplasmic reticulum (ER/SR). The latter occurs through Ca2+-release channels, which consist of ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs).

Despite the fact that RyRs and IP3Rs are both Ca2+ sensitive and can activate each other via a Ca2+-induced Ca2+-release (CICR) mechanism, leading to an all-or-none regenerative response, nevertheless graded Ca2+ signals evoked by increasing stimuli targeting RyRs (Isenberg & Han, 1994) and IP3Rs (Bootman et al. 1994) can be observed. The resolution of this paradox has been suggested (Berridge, 1997) to lie in the local control of microdomains that function and are regulated autonomously, and a number of theoretical (Keizer et al. 1998; Leighton et al. 2001) and experimental (Cheng et al. 1993, 1996; Klein et al. 1996; Berridge, 1997; Bridge et al. 1999; Wier & Balke, 1999) studies have addressed this question in striated muscle, where arrays of multiple Ca2+-release sites are the rule. Using laser-scanning fluorescence confocal imaging and Ca2+-sensitive indicators, localised Ca2+-release events produced by functional RyR units in locally controlled microdomains were demonstrated directly in cardiac (Cheng et al. 1993) and skeletal muscles (Klein et al. 1996) and were referred to as ‘Ca2+ sparks’.

Smooth muscle cells (SMCs) utilise both CICR, which is involved when voltage-dependent Ca2+ entry triggers Ca2+ store release (Bolton & Gordienko, 1998; Imaizumi et al. 1998; Ohi et al. 2001) or when a Ca2+ wave propagates (Gordienko et al. 1998), and IP3-induced Ca2+ release (IICR), which follows activation of a wide variety of G-protein coupled receptors (Boittin et al. 1998, 1999, 2000; Gordienko et al. 1999; Bayguinov et al. 2000; Mauban et al. 2001). There is growing evidence that the RyRs and IP3Rs are functionally coupled, at least in some SMCs. Functional studies suggest that RyRs and IP3Rs share the same Ca2+ pool in the SMCs of rabbit jejunum (Komori & Bolton, 1991), guinea-pig ileum (Zholos et al. 1994; Komori et al. 1995), guinea-pig colon (Flynn et al. 2001), rat portal vein (Pacaud & Loirand, 1995), rat mesenteric artery (Baro & Eisner, 1992) and canine renal artery (Janiak et al. 2001). In addition to Ca2+ release from IP3R-operated stores, RyRs may be recruited to amplify the signal in response to agonist stimulation of some SMCs (Boittin et al. 1999; Bayguinov et al. 2000). The structural basis for functional coupling is co-localisation of RyRs and IP3Rs in both the peripheral and central SR, as shown in intestinal, vas deferens, aortic and portal vein myocytes (Wibo & Godfraind, 1994; Lesh et al. 1998; Boittin et al. 1999; Tasker et al. 2000). In some types of SMCs, however, ryanodine-sensitive and IP3-sensitive Ca2+ stores may be organised into spatially separate compartments (Golovina & Blaustein, 1997; Janiak et al. 2001) and SMCs that possess exclusively one type (ryanodine-sensitive or IP3-sensitive) of Ca2+ store have been reported (Burdyga et al. 1998; Boittin et al. 2000). Events of localised Ca2+ release mediated by RyRs (Ca2+ sparks: Nelson et al. 1995; Mironneau et al. 1996; Bolton & Gordienko, 1998; Gordienko et al. 1998, 1999, 2001; ZhuGe et al. 1998, 1999; Löhn et al. 2000; Mauban et al. 2001; Ohi et al. 2001) or by IP3Rs (Ca2+ puffs: Bayguinov et al. 2000; Boittin et al. 2000) have been directly demonstrated in SMCs using fluorescence confocal imaging. Sites of spontaneous Ca2+ spark discharge may coincide with sites of initiation of IP3-induced Ca2+ release, thus suggesting possible intercommunication between RyRs and IP3Rs in functional microdomains (Gordienko et al. 1999; Bolton et al. 2002).

In the present study we investigated the mechanisms responsible for the variability of spontaneous Ca2+ release in rabbit portal vein myocytes using line-scan confocal imaging, which allows the acquisition of high-resolution spatial information (although only in one dimension) at a high rate. We have demonstrated recently that in these SMCs the majority of spontaneous Ca2+-release events occur at a single site, a frequent discharge site (FDS), within the cell (Gordienko et al. 2001). This provides an opportunity to avoid the complications caused by Ca2+ release from numerous out-of-focus sites such as would occur in striated muscles where Ca2+-release sites are packed at regular intervals in a dense three-dimensional array.

Methods

Cell preparation

Experiments were performed on SMCs freshly isolated from rabbit portal vein. Male New Zealand White rabbits (2-3 kg) were killed by an overdose of pentobarbitone injected into the ear vein, as approved under Schedule 1 of the UK Animals (Scientific Procedures) Act 1986. The portal vein was dissected, and after removal of fat and the adventitial layer it was cut into small pieces that were placed in Ca2+-free physiological salt solution (PSS, see below). After a 10 min rinse, the pieces of the tissue were incubated at 36 °C for 5 min in the same solution supplemented with protease type 8 (0.3 mg ml−1) followed by 10 min in 100 μm Ca2+ PSS containing collagenase type 1A (1 mg ml−1). The pieces of the tissue were then rinsed at room temperature for 20 min in enzyme-free solution and triturated with a wide-bore pipette. Several cycles of trituration, each followed by transfer to fresh solution with gradually increasing concentrations of Ca2+ (from 0.125 to 1.25 mm), facilitated the removal of debris and damaged cells from the suspension and generally improved the yield of relaxed cells. Small aliquots of the cell suspension in the highest [Ca2+]o were placed in experimental chambers filled with PSS of the following composition (mm): NaCl 120, KCl 6, CaCl2 2.5, MgCl2 1.2, glucose 12, Hepes 10; pH adjusted to 7.4 with NaOH. The chambers were then kept for 40 min at 4 °C to allow the cells to attach to the glass coverslip forming the bottom of the chamber. The myocytes were loaded with the fluorescent Ca2+-sensitive indicator fluo-4 acetoxymethyl ester (fluo-4 AM) by exposure to 5 μm fluo-4 AM (diluted from a stock containing 2 mm fluo-4 AM and 0.025 % (w/v) pluronic acid in DMSO) for 20 min followed by a 40 min wash in PSS to allow time for de-esterification. To minimise spontaneous contraction of the myocytes, 10 μm wortmannin was added to the bathing solution 10 min before imaging commenced. Experiments were performed at room temperature (20-25 °C) and cells were used within 8 h of isolation.

Confocal microscopy

Experimental chambers containing cells were placed on the stage of an Axiovert 100M inverted microscope attached to a LSM 510 laser-scanning unit (Zeiss, Oberkochen, Germany). Confocal imaging was performed using a Zeiss plan-Apochromat × 63 1.4 NA oil-immersion objective. To optimise signal quality, the pinhole was set to provide a confocal optical section below 1.2 μm (measured with 0.2 μm fluorescent beads). To improve temporal resolution, [Ca2+]i imaging was performed in the line-scan mode, where a single scan line was set close (within 1.5 μm) and parallel to the cell membrane in the central region of the myocyte and scanned at 200–500 Hz. The line-scan images presented here were formed by aligning (from left to right) the successive fluorescence images of the scan line so that the horizontal dimension of the images is time (increasing from left to right) and the vertical dimension is the position along the scan line. The fluorescence intensity in the images was normalised to the average fluorescence intensity during the first 100–300 lines, and colour coded.

Fluo-4 AM fluorescence was excited by the 488 nm line of a 200 mW Argon ion laser (Laser-Fertigung, Hamburg, Germany) and the illumination intensity was set with acousto-optical tuneable filter. The emitted fluorescence was captured with the confocal detector at wavelengths above 505 nm. The SCSi interface of the confocal microscope was hosted by a Pentium PC (32-bit Windows NT 4.0 operating system) running LSM510 software (Zeiss, Oberkochen, Germany). Image processing was carried out using an Indy workstation (Silicon Graphic, Mountain View, CA, USA) with custom routines written in IDL (Research Systems, Boulder, CO, USA). The final figures were produced using MicroCal Origin (MicroCal Software, Northampton, Massachusetts, USA) and CorelDraw 7.0 (Corel, Canada). Where appropriate, data are expressed as mean values ± s.e.m. for the number of cells (n) analysed.

Reagents

Protease (type 8), collagenase (type 1A), Hepes, DMSO, 1,4-dihydro-2,6-dimethyl-4-[3-nitrophenyl]methyl-2[methyl(phenylmethyl)amino]-3,5-pyridinedicarboxylic acid ethyl ester (nicardipine) and wortmannin were obtained from Sigma-Aldrich (Poole, Dorset, UK). Fluo-4 AM and pluronic F-127 were obtained from Molecular Probes (Eugene, OR, USA). Ryanodine, xestospongin C, 2-aminoethoxy-diphenylborate (2-APB), cyclopiazonic acid (CPA) and thapsigargin were obtained from Calbiochem (Beeston, Nottingham, UK). 1-[6-[[(17β)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5,-dione (U-73122) and 1-[6-[[(17β)-3-methoxyestra-1,3,5 (10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidinedione (U-73343) were obtained from Research Biomedical International/Sigma-Aldrich (Gillingham, Dorset, UK).

Results

Amplitudes and spatio-temporal characteristics of spontaneous Ca2+-release events in portal vein myocytes

We have recently demonstrated that spontaneous Ca2+-release events, visualised in fluo-4-loaded rabbit portal vein myocytes, occurred in the majority of cases at a single site (frequent discharge site, FDS) which was associated with a prominent portion of the SR enriched with RyRs, as judged from the high level of BODIPY TR-X ryanodine binding (Gordienko et al. 2001). This portion of the superficial SR network was usually located within 1–2 μm of the nuclear envelope in the central region of the myocyte. The aim of the present study was to characterise the Ca2+-release properties of this FDS; therefore, the fluo-4 fluorescence signal from this region of the myocyte was acquired at the highest possible rate and spatial resolution, depending upon the protocol of the experiment. To achieve this, [Ca2+]i imaging was performed in the line-scan mode, where a single scan line was set over the FDS (within 1.5 μm of plasmalemma) and parallel to the cell membrane in the central region of the myocyte and scanned at 200–500 Hz. With this approach, spontaneous Ca2+-release events were observed in about 95 % of cells tested (n = 208).

In the majority of line-scan images, the events occurred as temporally and spatially isolated sporadic discharges (Fig. 1) and their amplitude, kinetics and spatial spread varied substantially, even in the case where Ca2+ release was initiated at the same site (within the resolution of the confocal microscope) within the myocyte, as emphasised by the white dashed line on the image (Fig. 1A). The smaller events, seen in the image, had amplitude and spatio-temporal characteristics similar to those of the Ca2+ sparks reported in other tissues, and are therefore henceforth referred to as ‘Ca2+ sparks’. Sparks alternated with much larger and longer [Ca2+]i transients (Fig. 1A), where the rise in [Ca2+]i still remained localised, only spreading over a distance of 8–12 μm, and failing to propagate as a [Ca2+]i wave. The difference between Ca2+ sparks and the larger Ca2+-release events is emphasised in Fig. 1B. The Ca2+ spark was characterised by a more rapid upstroke (the normalised fluorescence signal reached a peak of F/F0 = 1.8 within 16 ms) than the larger [Ca2+]i transient (peak signal of F/F0 = 3.1 within 28 ms) and was restricted to a much smaller area (full width at half-maximal amplitude (FWHM) = 1.4 μm) than the larger event (FWHM = 3.5 μm). The declining phase of the Ca2+ spark was well fitted by a single exponential with the time constant τ = 27 ms (τ = 20.5 ± 1.6 ms for 29 Ca2+ sparks detected in 13 randomly selected line-scan images), while the decay of the larger [Ca2+]i transient was best fitted by two exponentials with time constants τ1 = 40 ms and τ2 = 461 ms (τ1 = 37.6 ± 4.2 ms and τ2 = 323.8 ± 42.9 ms for 25 events detected in 13 randomly selected line-scan images).

Figure 1
Variation in the amplitude and spatio-temporal profile of local spontaneous [Ca2+]i transients detected by line-scan confocal imaging of fluo-4-loaded portal vein myocytes

It seems unlikely that such heterogeneity of spatio-temporal properties of discrete spontaneous Ca2+-release events observed in portal vein myocytes arises from a variation in the distance between the points of the release and the scan line. An out-of-focus large Ca2+-release event, having an apparent amplitude similar to that of Ca2+ sparks, would exhibit a much longer half-decay time. This is illustrated by the blue trace on the plot (Fig. 1Bb), where the kinetics of an out-of-focus large Ca2+-release event is imitated by the time course of the fluorescence signal at the edge of an in-focus large [Ca2+]i transient. Although an in-focus Ca2+ spark (Fig. 1Ba) has a peak amplitude similar to that of a fluorescence signal produced by Ca2+ diffusion from a larger out-of-focus Ca2+ release (blue trace on the plot in Fig. 1Bb), its time course is much faster (see also Pratusevich & Balke, 1996).

The results of experiments where the effect of z-displacements of the scan line on the apparent amplitude and spatio-temporal properties of Ca2+-release events was tested (n = 20) led to a similar conclusion. This is illustrated by the example shown in Fig. 2. The line-scan image obtained with best focusing onto the site of origin of Ca2+ release (Fig. 2A) revealed that both Ca2+ spark (depicted by purple bars) and the larger local [Ca2+]i transient (depicted by green bars) originated at the same (within optical resolution of our system) x-position (depicted by red bars on the images, Fig. 2). It might be argued that the difference in size of the two events could be due to the difference in the distance between their sites of origin and the scan line in the z-dimension. In this case, the z-displacement of the scan line towards a more distant Ca2+-release site would increase the apparent amplitude of the event originating from this site (Pratusevich & Balke, 1996). This, however, was not observed. Instead, the amplitudes of the Ca2+ spark and the larger [Ca2+]i transient were both reduced in line-scan images obtained following 1 μm displacement of the objective below (Fig. 2B) and above (Fig. 2C) its initial position. Furthermore, the out-of-focus larger Ca2+-release event (depicted by green bars, Fig. 2B and C) still had a much slower time course than the in-focus Ca2+ spark (depicted by purple bars in Fig. 2A), even though its apparent amplitude was smaller than that of the in-focus spark. Thus, the Ca2+ spark and the larger [Ca2+]i transient are likely to arise from two different types of Ca2+ release, which may be discharged at different times from the same domain (accepting the limits of resolution of the confocal microscope) within an FDS.

Figure 2
The effect of z-displacement of the scan line on characteristics of the local spontaneous [Ca2+]i transients in portal vein myocytes

Unlike striated muscles where Ca2+-release sites are packed in close proximity at regular intervals corresponding to sarcomeric structures, the release sites in rabbit portal vein myocytes are few and in the majority of cases only one FDS was observed (Gordienko et al. 2001). This provides an opportunity to discriminate between in- and out-of-focus Ca2+-release events (see Fig. 2). Characteristics of spontaneous discrete Ca2+-release events were therefore evaluated from the line-scan images, where the events occurred as temporally and spatially isolated sporadic discharges, after best possible focusing (e.g. Fig. 1 and Fig. 2A). The quantitative properties of discrete spontaneous Ca2+-release events observed at the single FDS in a number of rabbit portal vein myocytes (n = 47) are further summarised in Fig. 3. For 230 individual Ca2+-release events the peak amplitude (F/F0), spatial spread (FWHM), time to peak and time of decay to half-maximal amplitude were measured and are presented as histogram plots. The amplitude histogram (Fig. 3A) and FWHM histogram (Fig. 3B) were both well fitted by the sum of two Gaussian profiles (shown as shaded regions on the plots). These were characterised by a mean ± s.d. of 1.8 ± 0.13 F/F0 and 3.2 ± 0.7 F/F0 (Fig. 3A), and 2 ± 0.5 μm and 3.8 ± 1 μm (Fig. 3B), respectively. The smaller mean values in each pair are consistent with parameters of Ca2+ sparks described in other preparations (Cheng et al. 1993; Nelson et al. 1995; Mironneau et al. 1996; Gordienko et al. 1998; ZhuGe et al. 1999), while the second modes in the distributions are characteristic of larger [Ca2+]i transients (cf. Fig. 1 and Fig. 2). The histograms of the rise times (Fig. 3C) and half-decay times (Fig. 3D) are also bimodal, although these distributions may be affected by failure to detect very brief events due to a limited sampling rate (Lacampagne et al. 1999); in addition, the bi-exponential decay of the larger transients (Fig. 1Bb) complicates the interpretation of half-decay times as plotted. The positive correlation between amplitude, spatial spread and temporal parameters of spontaneous Ca2+-release events (see insets in Fig. 3BD) further confirms that variation in the characteristics of the release events is real and cannot be explained by just a difference in the position of their initiating point relative to the scan line (Cheng et al. 1999). Thus, fluorescence signals associated with discrete spontaneous Ca2+-release events at a single FDS in portal vein myocytes are characterised by a bi-modal, non-monotonic distribution of their amplitudes and spatio-temporal parameters. This rules out a reversible gating of a single two-state Ca2+-release channel as a mechanism underlying these events and favours, instead, channel groups that could co-operate allosterically or through their Ca2+ sensitivity, giving rise to the events of stereotyped amplitude and spatio-temporal characteristics (Ríos et al. 2001).

Figure 3
Distribution of amplitudes and spatio-temporal parameters of local [Ca2+]i transients in portal vein myocytes

On occasion, line-scan imaging revealed complex patterns of the fluorescence signal arising due to a short latency between several discrete Ca2+ releases at the same site (Fig. 4A and B), from near-synchronous release of Ca2+ at several discrete, adjacent sites (Fig. 4C) or from propagation of Ca2+ release along the scan line in ‘fire-diffuse-fire’ (saltatory) manner (Fig. 4D and Fig. 7A). Taking into account that in both cases (shown in Fig. 4A and B) Ca2+ sparks and larger Ca2+-release events were initiated at the same site within the myocyte (within the resolution of the confocal microscope), it seems unlikely that the termination of Ca2+ sparks is due to either local depletion of Ca2+ from the SR (since larger Ca2+ release was observed within several tens of milliseconds following the peak of Ca2+ spark indicating that not all available Ca2+ is released from the SR during spark discharge; Fig. 4A) or to inactivation of RyRs by a local increase in [Ca2+]i (since a Ca2+ spark was observed on the descending phase of a larger Ca2+-release event when local [Ca2+]i was higher than the level achieved during an isolated spark discharge; Fig. 4B). Close packing of Ca2+-release domains may allow Ca2+ spreading from one domain to induce Ca2+ release (via CICR) at a closely adjacent domain. Spatio-temporal summation of several Ca2+ sparks discharged in quick succession at different domains in close proximity was observed to give a rise to a [Ca2+]i transient of longer duration and wider spatial spread (Fig. 4C) or to waves of elevated [Ca2+]i propagating in a saltatory manner (Fig. 4D and Fig. 7A). There was a notable lag (up to 100 ms) between the initial elevation of [Ca2+]i at one domain or site (due to Ca2+ diffusing from the adjacent domain or site) and the subsequent Ca2+ release induced by this elevation (Fig. 4D). This lag time varied widely (compare Fig. 4D and Fig. 7A) and probably depends upon a number of factors (e.g. existing SR luminal [Ca2+], basal [Ca2+]i level in the microdomain near the RyRs, the distance between RyR functional units and the receptor isoforms involved). These observations also suggest that the single FDS in a rabbit portal vein myocyte consists of several closely associated domains, each of which can discharge a Ca2+ spark, and that if conditions are favourable, one domain can activate other adjacent domains. This would give rise to a larger [Ca2+]i event that consists of several sparks discharged closely in time and summating either incompletely (Fig. 4C) or completely, such that the individual components are irresolvable (e.g. large events in Fig. 1 and Fig. 2). A submicron ‘jitter’ in the initiating point of subsequent Ca2+-release events recently reported in these myocytes (Gordienko et al. 2001) also suggests that several Ca2+-release domains constitute a single FDS.

Figure 4
Complex patterns of spontaneous Ca2+ release observed in rabbit portal vein myocytes
Figure 7
Effect of inositol 1,4,5-trisphosphate receptor (IP3R) antagonists on spontaneous local Ca2+-release events in rabbit portal vein myocytes

RyR-mediated Ca2+ release is involved in the genesis of all localised spontaneous [Ca2+]i transients in portal vein myocytes

Since the smooth muscle of portal vein is known to exhibit spontaneous electrical activity (Sutter, 1990) and utilise both RyRs and IP3Rs to mediate Ca2+ release (Pacaud & Loirand, 1995; Boittin et al. 1999), it is possible that the heterogeneity of the discrete spontaneous [Ca2+]i transients observed in our experiments may arise from variation in the mechanisms involved in their genesis. The contribution of RyR-mediated Ca2+ release to spontaneous [Ca2+]i transients observed in portal vein myocytes was tested with ryanodine which, with increasing concentrations, is known first to increase the probability of the channel opening, then to lock Ca2+-release channels into subconductance states (eventually leading to depletion of the caffeine-sensitive store) at submicromolar concentrations, and ultimately (at micromolar concentrations) to inhibit completely the channel opening without store depletion (Cheng et al. 1993; Sutko et al. 1997; Janiak et al. 2001).

Exposure of portal vein myocytes for 10–15 min to a very high (50-100 μm) concentration of ryanodine completely abolished the spontaneous [Ca2+]i transients observed in control (n = 46), as illustrated by Fig. 5. As can be seen, both sparks and larger spontaneous [Ca2+]i transients were observed in control conditions (Fig. 5A), but both were completely inhibited after a 10 min incubation with 100 μm ryanodine (Fig. 5B). To ensure that the observed effect of ryanodine was not due to Ca2+ depletion from the store, 10 mm caffeine was applied following incubation with 100 μm ryanodine. In the presence of ryanodine, caffeine still triggered Ca2+ release, which appeared as an initial [Ca2+]i transient at the site of initiation of spontaneous Ca2+-release events and then propagated to adjacent sites (Fig. 5C). The amplitude of caffeine-induced Ca2+ release was not affected by 10–15 min exposure of the myocyte to 100 μm ryanodine (n = 9; see inset Fig. 5C). Taking into account that ryanodine is a highly selective agent and does not directly affect either sarcolemmal Ca2+ channels (Balke & Wier, 1991) or IP3Rs (Bayguinov et al. 2000; Boittin et al. 2000; Janiak et al. 2001), these results suggest strongly that the genesis of both Ca2+ sparks and the larger spontaneous Ca2+-release events in portal vein myocytes is intimately associated with activation of RyRs.

Figure 5
Small and large local [Ca2+]i transients both result from Ca2+ release involving ryanodine receptor (RyR) activation

Experiments with nicardipine (1-5 μm), an inhibitor of L-type Ca2+ channels, further confirmed that neither sparks nor the larger spontaneous [Ca2+]i transients observed in portal vein myocytes were caused by Ca2+ entering the cell though sarcolemmal voltage-gated Ca2+ channels. Both the Ca2+ sparks and larger Ca2+-release events observed in control conditions (Fig. 6A) persisted after 3–5 min incubation with the Ca2+ channel blocker (see Fig. 6B), and their frequency, amplitudes and spatiotemporal characteristics were similar to those observed in the control (n = 20). Furthermore, no detectable decrease in basal [Ca2+]i was observed upon this short-term exposure of the myocytes to nicardipine. Thus, it seems likely that Ca2+ entry through sarcolemmal voltage-gated Ca2+ channels is neither a factor involved in short-term modulation of spontaneous activity of RyR functional units nor responsible for the variability of spontaneous Ca2+-release events observed, at least under these experimental conditions.

Figure 6
Ca2+ sparks and larger local [Ca2+]i transients both persisted in the presence of nicardipine

Effect of the blockers of IP3R-mediated Ca2+ release on spontaneous Ca2+-release events in portal vein myocytes

Xestospongin C (Bayguinov et al. 2000) and 2-APB (Maruyama et al. 1997; Ascher-Landsberg et al. 1999) were recently reported to be effective and selective blockers of the IP3R Ca2+-release channels in smooth muscle. We therefore employed these agents to examine whether Ca2+ sporadically ‘leaking’ through IP3Rs could be a factor affecting spontaneous activity of neighbouring RyR domains by sensitising them and thereby modulating the amplitude, duration and spatial spread of spontaneous [Ca2+]i transients observed. A 10 min exposure of a myocyte to 10 μm xestospongin C decreased the frequency of spontaneous Ca2+-release events and significantly reduced the variability in the amplitude and spatiotemporal profile of the [Ca2+]i transients, abolishing larger events (Fig. 7A). The Ca2+-release events persisting in the presence of xestospongin C had an amplitude of 1.59 ± 0.05 F/F0, a spatial spread (FWHM) of 1.98 ± 0.08 μm and very brief kinetics with a time to peak of 17.5 ± 1 ms and a half-decay time of 18.7 ± 0.9 ms (n = 38), and therefore closely resembled the Ca2+ sparks described in other preparations (Cheng et al. 1993; Nelson et al. 1995; Mironneau et al. 1996; Gordienko et al. 1998; ZhuGe et al. 1999). In the presence of xestospongin C, these were abolished by subsequent application of 50–100 μm ryanodine (n = 9).

Incubation of the myocyte for 10 min in 30 μm 2-APB (Fig. 7B) had an effect similar to that of xestospongin C. Only Ca2+ sparks persisted in the presence of 30 μm 2-APB, and these had an amplitude of 1.66 ± 0.04 F/F0, a spatial spread (FWHM) of 1.74 ± 0.06 μm, time to peak of 18.8 ± 1.1 ms and a half-decay time of 19.6 ± 1.1 ms (n = 38). In the presence of 2-APB they were blocked by subsequent application of 50–100 μm ryanodine (n = 5). It should be emphasised that in both cases shown in Fig. 7, Ca2+ sparks persisting in the presence of xestospongin C or 2-APB were observed at the sites (depicted by red bars on the line-scan images in Fig. 7) where the larger [Ca2+]i transients were observed in the control. This observation strongly suggests that Ca2+ leaking from the neighbouring IP3Rs at these sites modulates (probably via CICR) the number of RyRs recruited upon spontaneous Ca2+ release, thereby affecting the amplitude and spatio-temporal profiles of the spontaneous [Ca2+]i transients observed.

Furthermore, depending on the scale of the leakage through IP3R-coupled Ca2+-release channels and the spatial arrangement of RyRs and IP3Rs, crosstalk between these receptors might not only modulate local discrete Ca2+ release, but could also facilitate propagation of the waves of elevated [Ca2+]i. This is illustrated by Fig. 8. Under control conditions, spontaneous propagating [Ca2+]i waves, seen near the beginning of the line-scan image (Fig. 8Aa), were initiated at virtually the same site depicted by the white arrow and white dashed line. This is seen more clearly on the enlarged fragment of the image (Fig. 8Ab). After enlargement, non-uniformities at the leading edge of the [Ca2+]i wave become apparent (these are depicted by white arrowheads in Fig. 8Ab). This is further emphasised by plotting spatial profiles (Fig. 8Ac) of the normalised fluorescence signal at different times for the distances shown by black lines on the image (the first Ca2+ wave after rotation by 90 deg) below the plot. Note that black lines on the image are displaced in time such that each of them crosses the local peak of the fluorescence signal. Discrete local regions of elevated [Ca2+]i that were activated sequentially in time and in space give rise to multiple peaks on the plot. The saltatory nature of the [Ca2+]i waves and their high velocity (80-100 μm s−1) both support the idea that [Ca2+]i wave propagation is a regenerative process of sequential recruitment (via CICR) of discrete, spatially separated Ca2+-release domains (Cheng et al. 1993, 1996; Berridge, 1997; Bolton & Gordienko, 1998; Gordienko et al. 1998; Keizer et al. 1998; Bolton et al. 1999; Bridge et al. 1999; Wier & Balke, 1999; Leighton et al. 2001). Incubation of the myocyte for 12 min with 30 μm 2-APB completely abolished propagating [Ca2+]i waves, while fast localised events, Ca2+ sparks, persisted (Fig. 8B). It should be emphasised that the majority of these Ca2+ sparks were discharged at the site of initiation of the spontaneous [Ca2+]i waves observed in the control (depicted by the white arrow and white dashed line on the line-scan images in Fig. 8A and B). Thus, 2-APB, an inhibitor of IP3R-mediated Ca2+ release, demolished the coupling between neighbouring RyR functional units that underpins saltatory [Ca2+]i wave propagation.

Figure 8
2-APB abolishes spontaneous [Ca2+]i waves in rabbit portal vein myocytes

It was reported recently that in the A7r5 SMC line, xestospongin C (De Smet et al. 1999) and 2-APB (Missiaen et al. 2001) could both inhibit SR/ER calcium ATPase (SERCA), thus preventing Ca2+ uptake into the SR. We therefore compared the effects of the well-established specific SERCA inhibitors, thapsigargin and CPA, with the effects of blockers of IP3Rs to establish whether the effects of the latter could be mimicked by the inhibition of SR Ca2+ uptake. After 10 min incubation with 0.1 μm thapsigargin (Fig. 9A) or with 10 μm CPA (Fig. 9B), both the Ca2+ sparks and larger [Ca2+]i transients were abolished and, in the case of CPA, they recovered after drug washout (Fig. 9Bc). It should be noted that an increase in global [Ca2+]i resulting from SERCA inhibition cannot be seen in the line-scan images presented (Fig. 9Ab and Bb), since the images were self-ratioed. However, a slight increase in the global fluorescence signal associated with SERCA inhibition by either thapsigargin or CPA was observed in raw data images (not shown), but was never observed in the case of xestospongin C or 2-APB. Thus, in our hands, 10 min exposure of the myocytes to either thapsigargin (n = 8) or CPA (n = 10) completely abolished all spontaneous Ca2+-release events, unlike the inhibitors of IP3R-mediated Ca2+ release. Furthermore, a brief (2-3 min) exposure of the myocytes to SERCA inhibitors (0.1 μm thapsigargin or 10 μm CPA), leading to partial depletion of the Ca2+ store, decreased the frequency of spontaneous Ca2+-release events, but unlike the IP3R blockers, failed to selectively inhibit the larger Ca2+-release events (data not shown, n = 5). It must therefore be concluded that the effect of xestospongin C and 2-APB on spontaneous [Ca2+]i transients in rabbit portal vein myocytes is unlikely to be mediated through SERCA inhibition, but arises from the blockade of IP3R-mediated Ca2+ release. Further evidence supporting the involvement of IP3Rs in the modulation of spontaneous RyR-mediated Ca2+ release was obtained when the effect of the inhibition of phospholipase C (PLC) on spontaneous Ca2+-release events was examined.

Figure 9
Effect of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitors on spontaneous local Ca2+-release events in rabbit portal vein myocytes

Effect of PLC inhibition on spontaneous Ca2+-release events in portal vein myocytes

To establish whether Ca2+ leakage through IP3R-coupled release channels is a result of the spontaneous basal activity of PLC resulting in ongoing production of IP3 (Prestwich & Bolton, 1991), the effect of PLC inhibition on spontaneous Ca2+-release events was examined. A 5 min incubation of the myocyte with 2.5 μm U-73122, an effective inhibitor of PLC in smooth muscle (Bayguinov et al. 2000), decreased the frequency of spontaneous Ca2+-release events and abolished large [Ca2+]i transients (Fig. 10A). Ca2+-release events persisting in the presence of U-73122 (Fig. 10Ab) were similar to the Ca2+ sparks that persisted in the presence of xestospongin C (Fig. 7Ab) or 2-APB (Fig. 7Bb). They had an amplitude of 1.81 ± 0.04 F/F0, a spatial spread (FWHM) of 1.8 ± 0.06 μm and very brief kinetics with a time to peak of 17.9 ± 0.8 ms and a half-decay time of 15.3 ± 1 ms (n = 28). In contrast, U-73343, an inactive analogue of U-73122, had no effect on spontaneous [Ca2+]i transients (n = 10; Fig. 10B). Thus, inhibition of PLC had the same effect on spontaneous Ca2+-release events as the inhibitors of IP3R-mediated Ca2+ release. We therefore conclude that in rabbit portal vein myocytes, Ca2+ leakage through IP3Rs is activated by the ongoing production of IP3 arising from the spontaneous basal activity of PLC. This IP3R-mediated Ca2+ leakage, in turn, sensitises neighbouring RyR domains and thereby gives rise to larger [Ca2+]i-release events that are probably composed of multiple discharges of closely related domains within a single FDS; in extreme cases spontaneous [Ca2+]i wave propagation is evoked.

Figure 10
Large spontaneous [Ca2+]i transients require the ongoing production of IP3 in portal vein myocyte

Discussion

In this study we have demonstrated that spontaneous Ca2+ release in freshly isolated rabbit portal vein myocytes occurs as discrete events of two types: Ca2+ sparks and larger [Ca2+]i transients. The small thickness (below 4 μm) of the preparation in the region of interest and the rather low frequency of discharges at only a single FDS (Gordienko et al. 2001) composed of several RyR domains (see below) allowed us to discriminate between in- and out-of-focus events (Fig. 1 and Fig. 2). The conclusion was reached that variability in the Ca2+-release event parameters was due rather to variation in the number of RyRs recruited than to differences in the distance between the scan line and the site of origin of the event.

Our results indicate that crosstalk between RyRs and IP3Rs is a major factor in the modulation of spontaneous Ca2+ release in freshly isolated rabbit portal vein myocytes. The functional basis for this crosstalk appears to be a spontaneous basal activity of PLC leading to ongoing production of IP3 and IP3-induced Ca2+ release, which in turn may affect via CICR the neighbouring RyRs and thereby modulate the extent of spontaneous Ca2+ releases. The structural basis for intercommunication between RyRs and IP3Rs is their co-localisation in portal vein myocytes (Boittin et al. 1998, 1999). Indeed, immunostaining of portal vein myocytes with anti-type 1 IP3R antibodies revealed that this IP3 receptor isoform, which is known to have a high sensitivity to both IP3 and Ca2+, was well expressed in the subplasmalemmal SR in the central region of the myocyte (Tasker et al. 1999, 2000). Using BODIPY TR-X ryanodine staining we have recently demonstrated that this element of the superficial SR network in portal vein myocytes is enriched with RyRs and represents the origin of the majority of spontaneous discrete Ca2+-release events. It is also the initiating site of the propagating [Ca2+]i waves and myocyte contraction (Gordienko et al. 2001).

Basal activity of PLC in murine colonic myocytes was reported to result in spontaneous localised Ca2+-release events that were insensitive to ryanodine and mediated by IP3Rs (Bayguinov et al. 2000). These were referred to as ‘Ca2+ puffs’ by analogy to the IP3R-mediated Ca2+-release events observed in Xenopus oocytes (see Berridge, 1997). Both Ca2+ puffs discharged through IP3-gated channels in response to low concentrations of IP3, either photoreleased from its caged precursor or produced by low doses of acetylcholine, and spontaneous Ca2+ puffs potentiated by Ca2+ overload were observed in rat ureteric myocytes (Boittin et al. 2000). In rabbit portal vein myocytes, discrete Ca2+-release events arising from co-ordinated opening of IP3R clusters were never observed. In contrast, all spontaneous Ca2+-release events observed in these myocytes were completely abolished by high (50-100 μm) concentrations of ryanodine (Fig. 5). If RyRs and IP3Rs share the same Ca2+ store, the depletion of this store should abolish all Ca2+-release events in the myocytes. It should be noted, however, that the effect of ryanodine we have observed could not be attributed to ryanodine-induced depletion of the Ca2+ store, since subsequent application of caffeine triggered Ca2+ release (similar to that in the absence of ryanodine pre-treatment) at the site where spontaneous Ca2+-release events were observed in control conditions (Fig. 5C). It is therefore concluded that similar to the majority of smooth muscles where spontaneous Ca2+ sparks have been reported (Nelson et al. 1995; Mironneau et al. 1996; Bolton & Gordienko, 1998; Gordienko et al. 1998, 1999; ZhuGe et al. 1998, 1999; Löhn et al. 2000; Mauban et al. 2001), spontaneous Ca2+-release events observed in rabbit portal vein myocytes arise from co-ordinated opening of RyR clusters. The ability of caffeine to induce Ca2+ release from the SR after ryanodine pre-treatment has been demonstrated previously in various types of SMCs, e.g. rat portal vein myocytes (Boittin et al. 1999), canine pulmonary artery and renal artery myocytes (Janiak et al. 2001), guinea-pig colonic myocytes (Flynn et al. 2001) and guinea-pig ileal myocytes (Komori et al. 1995). Two different mechanisms may underlie this phenomenon: (1) caffeine-induced increase of the open probability of ryanodine-modified Ca2+-release channels relieving the channel block (Du et al. 2001) and/or (2) preferential binding of ryanodine to Ca2+-release channels in the activated state (Sutko et al. 1997) leading to preferential blockade (during ryanodine pre-treatment) of only RyRs involved in genesis of spontaneous Ca2+-release events. If the latter is the case, then only a very small portion of RyRs is involved in the genesis of spontaneous Ca2+-release events in rabbit portal vein myocytes (even within a single FDS), since the amplitude of caffeine-induced Ca2+ release was not affected by exposure of the myocyte to 100 μm ryanodine (see inset Fig. 5C).

Since short (3-5 min) exposure of the myocytes to 1–5 μm nicardipine (a blocker of plasmalemmal voltage-gated Ca2+ channels) had no effect on either spontaneous Ca2+-release events or basal [Ca2+]i (Fig. 6), it seems likely that it is SR-luminal Ca2+ rather than Ca2+ entry through plasmalemmal voltage-gated Ca2+ channels that regulates RyR clusters, giving rise to spontaneous Ca2+-release events (see Bolton et al. 1999). However, activation of the voltage-gated Ca2+ current by depolarising voltage steps under whole-cell voltage-clamp conditions caused an elevation of basal [Ca2+]i and increased the frequency of localised Ca2+-release events in rabbit portal vein myocytes (data not shown; see also Arnaudeau et al. 1997; Bolton et al. 2002).

The idea that the Ca2+-release events observed in portal vein myocytes arise from the co-ordinated openings of groups of Ca2+-release channels rather than from openings of single channels is further supported by the occurrence of modes in the distributions of their amplitudes, spatial spreads and temporal parameters (Fig. 3). Indeed, if Ca2+-release events were produced by random openings of independent single channels, they should not have preferred amplitudes and spatio-temporal parameters. Channel groups, however, could co-operate (e.g. via CICR) giving rise to the Ca2+-release events of preferred amplitudes and spatio-temporal characteristics (Ríos et al. 2001). IP3R-mediated Ca2+ release would potentiate CICR and thereby increase the number of RyRs involved in Ca2+-release events, leading to a modal distribution of the event parameters. This idea is supported by the observation that the agents blocking IP3Rs-mediated Ca2+ release significantly reduced the variability in amplitude and spatio-temporal profile of the spontaneous [Ca2+]i transients by abolishing larger events (Fig. 7).

In our previous studies, spontaneous Ca2+-release events were not scattered randomly within SMCs, but there were areas, or FDSs, giving rise to the majority of the release events (Gordienko et al. 1998, 1999, 2001). FDSs, therefore, were defined by mapping the occurrence of discrete Ca2+-release events in the time series of x-y images. The computing was performed by setting a certain threshold (usually 50 % above the background signal) to discriminate between the event and the background noise. It should be emphasised that the low acquisition rate employed in these experiments (usually 2 Hz) did not allow identification of the position of the initiating point of an individual release event, and therefore information about the ‘microarchitecture’ of FDSs was lost. The extensive use in this study of line-scan confocal imaging, which allows acquisition of high-resolution spatial information (although only in one dimension) at a high rate (200-500 Hz), revealed that there was more than one RyR domain within a single FDS in rabbit portal vein myocytes. Indeed, in a number of line-scan images Ca2+ sparks originated at several positions (Figs 5, ,6,6, ,7,7, ,88 and and9),9), while the position of the initiating point of the larger [Ca2+]i transients varied slightly from one release to another (Fig. 4A and Fig. 9Aa; see also Gordienko et al. 2001). Similar ‘jitter’ on a submicron scale in the precise origin of Ca2+ sparks was recently reported in guinea-pig mesenteric artery myocytes (Pucovsky et al. 2002). Furthermore, on some occasions spatiotemporal summation of several Ca2+ sparks discharged in quick succession in different domains at close proximity were observed to give a rise to [Ca2+]i transients of longer duration and wider spatial spread (Fig. 4C) or to waves of elevated [Ca2+]i propagating in a saltatory manner (Fig. 4D and Fig. 8A).

The absence of discrete Ca2+-release events arising from the coordinated opening of the groups of IP3Rs could be explained by the lack of clustered arrangement of IP3Rs in rabbit portal vein myocytes. Indeed, immunostaining experiments revealed that rat ureteric myocytes discharging IP3-induced Ca2+ puffs exhibited a clustered distribution of IP3Rs (Boittin et al. 2000), while rat portal vein myocytes, where IP3Rs are not organised into clustered units, failed to discharge IP3-evoked Ca2+ puffs (Boittin et al. 1998, 1999). Furthermore, if the local increase in [Ca2+]i resulting from IP3-induced Ca2+ release could trigger neighbouring RyRs to release Ca2+ but was insufficient to activate neighbouring IP3Rs to give rise to a Ca2+ puff, then RyRs should be more sensitive to regenerative feedback by Ca2+ than IP3Rs, at least at resting [IP3]i. This might be expected from the allosteric regulation of IP3Rs by cytoplasmic Ca2+ (at submicromolar [Ca2+]i) and IP3, recently reported in guinea-pig portal vein myocytes (Hirose et al. 1998), and from the rather low [Ca2+]i threshold (which could be as low as 75 nm, see Arnaudeau et al. 1997) needed to trigger the RyR-mediated Ca2+ release, giving rise to a Ca2+ spark. Thus, it is possible that despite being below the resolution limits of our imaging system, Ca2+ release mediated by IP3Rs could be yet large enough to affect neighbouring RyRs and thereby modulate the number of RyR domains involved in local Ca2+ release. However, direct physical coupling between RyRs and IP3Rs cannot be completely ruled out. Furthermore, since inhibition of PLC in rabbit portal vein myocytes (Fig. 10) mimicked the effect of inhibitors of IP3Rs on spontaneous [Ca2+]i transients (Fig. 7), the ongoing production of IP3 arising from the spontaneous basal activity of PLC (Prestwich & Bolton, 1991; Bayguinov et al. 2000) rather than intrinsic spontaneous activity of IP3R-coupled Ca2+-release channels is responsible for the coupling between IP3Rs and RyRs, which leads to the variability of spontaneous Ca2+-release events in rabbit portal vein myocytes. Since both IP3 and Ca2+ augment the activity of IP3Rs in an allosteric manner (Hirose et al. 1998), it is also possible that Ca2+ released through RyRs in conjunction with basal activity of PLC (resulting in ongoing IP3 production) may potentiate the activity of IP3Rs. This results in a large increase in local [Ca2+]i, giving rise to the regenerative Ca2+ release from RyRs observed either as a large spontaneous Ca2+-release event or as a propagating calcium wave.

Another important finding of this study is the demonstration of the involvement of IP3R-mediated Ca2+ release in the genesis of spontaneous propagating Ca2+ waves. In rabbit portal vein myocytes, saltatory [Ca2+]i waves were observed consisting of clearly identifiable sparks or larger Ca2+-release events (Fig. 4D and Fig. 8A). This indicates that the waves arose from the sequential activation of spatially segregated fundamental Ca2+-release units, and that CICR is a major mechanism of the progressive recruitment of neighbouring Ca2+-release channels leading to wave formation (Cheng et al. 1993, 1996; Berridge, 1997; Bolton & Gordienko, 1998; Gordienko et al. 1998; Keizer et al. 1998; Bolton et al. 1999; Bridge et al. 1999; Wier & Balke, 1999; Leighton et al. 2001). Inhibition of IP3R-mediated Ca2+ release by 2-APB abolished [Ca2+]i waves but did not affect Ca2+ sparks, which continued to appear at the wave-initiation site more frequently than at the other sites, but failed to recruit adjacent RyR domains to release Ca2+ (Fig. 8B). Thus, potentiation of CICR by IP3R-mediated Ca2+ release is crucial for the coupling between neighbouring RyR domains. The precise mechanism of this potentiation is unclear. One possibility could be that IP3-induced Ca2+ release would increase CICR sensitivity by changing the basal [Ca2+]i level in the microdomain near the RyRs and thereby decrease the lag (Fig. 4D) between the initial elevation of [Ca2+]i at a certain site (due to Ca2+ diffusing from an adjacent site) and the subsequent Ca2+ release induced by this elevation. However, there are a number of other factors that could affect this lag, for example existing SR luminal [Ca2+], the distance between RyR domains, receptor isoforms involved and the rate at which [Ca2+]i increases.

It was recently demonstrated that positive co-operativity between IP3Rs and RyRs in rat portal vein and in duodenal myocytes modulates the Ca2+ responses initiated by IP3-generating neurotransmitters (Boittin et al. 1999). Ca2+ signals triggered by the stimulation of P2Y receptors in murine colonic myocytes were shown to be sensitive not only to inhibitors of PLC or IP3Rs, but also to ryanodine, suggesting that RyRs are also recruited to amplify Ca2+ release in response to agonist stimulation in these cells (Bayguinov et al. 2000). We have recently demonstrated that carbachol-induced stimulation of muscarinic receptors in guinea-pig ileal myocytes, leading to activation of IP3Rs, increases the frequency of spontaneous Ca2+ sparks (discharged by RyRs) and gives rise to periodic propagating [Ca2+]i waves (Gordienko et al. 1999). Stimulation of adrenoceptors (which is an IP3-generating process) in rabbit portal vein myocytes increased the frequency of discrete Ca2+-release events at the FDS, eventually leading to their summation, triggering a [Ca2+]i wave and myocyte contraction (Bolton et al. 2002). It is likely, therefore, that this FDS-related SR element is a store from which Ca2+ is first released upon EC coupling in these myocytes.

Finally, the physiological relevance of the variability in spontaneous Ca2+ release arising from the spontaneous basal activity of PLC and co-operativity between IP3Rs and RyRs may involve a differential targeting at the level of the cell membrane ion channels (Gordienko et al. 1999). Indeed, the smooth muscle of the portal vein is known to exhibit spontaneous electrical activity (Sutter, 1990), and under voltage-clamp conditions the myocytes isolated from rabbit portal vein revealed spontaneous transient inward currents (STICs), carried through Ca2+-activated Cl channels, and spontaneous transient outward currents (STOCs), carried through Ca2+-activated K+ channels (Greenwood et al. 1999). Both STICs and STOCs are known to be triggered by Ca2+ release from the SR (Nelson et al. 1995; Mironneau et al. 1996; Bolton & Gordienko, 1998; ZhuGe et al. 1998, 1999; Gordienko et al. 1999; Ohi et al. 2001) and, once activated, cause transient changes in the cell membrane potential. Thus, local changes in [Ca2+]i in the vicinity of the plasma membrane produced by discrete Ca2+-release events are transduced by Ca2+-dependent membrane ion channels into changes in cell membrane potential. If different types of Ca2+-dependent ion channels sense differences in the spatio-temporal pattern of Ca2+ release (e.g. STOCs are activated by Ca2+ sparks while STICs require larger [Ca2+]i transients; see Mironneau et al. 1996), then spontaneous activity of PLC in rabbit portal vein myocytes leading to variability in spontaneous Ca2+-release events would modulate the cell membrane potential and so the spontaneous electrical activity of these cells (see Sergeant et al. 2001). This hypothesis, however, requires further testing in patch-clamp experiments combined with confocal imaging.

Acknowledgments

We are grateful to Dr A. Albert for assistance with cell preparation. D. V. Gordienko is a Wellcome Trust Senior Lecturer (060659) and is on leave from A. A. Bogomoletz Institute of Physiology, Kiev, Ukraine. T. B. Bolton is supported by The Wellcome Trust (042293).

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