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¹ Institute of Biomedical and Life Sciences, Neuroscience and Biomedical Systems, West Medical Building, University of Glasgow, Glasgow G12 8QQ, UK

	Abstract

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Smooth muscle responds to IP₃-generating (sarcolemma acting) neurotransmitters and hormones by releasing Ca²⁺ from the sarcoplasmic reticulum (SR) via IP₃ receptors (IP₃Rs). This release may propagate as Ca²⁺ waves. The Ca²⁺ signal emanating from IP₃ generation may be amplified by its activating further Ca²⁺ release from ryanodine receptors (RyRs) in the process of Ca²⁺-induced Ca²⁺ release (CICR). Evidence for this proposal has relied largely on the use of blocking drugs such as ryanodine, tetracaine and dantrolene, reportedly specific inhibitors of RyRs. Here we have examined whether or not Ca²⁺ released via IP₃Rs subsequently activates RyRs. In addition, the specificity of the blocking agents has been assessed by determining the extent of their ability to block IP₃-mediated Ca²⁺ release under conditions in which RyRs were not activated. IP₃-evoked Ca²⁺ release and Ca²⁺ waves did not require or activate RyRs. However, the RyR blocking drugs inhibited IP₃-mediated Ca²⁺ signals at concentrations thought to be selective for RyRs. In single colonic smooth muscle cells, voltage clamped in the whole cell configuration, carbachol (CCh) evoked propagating Ca²⁺ waves which were not inhibited by ryanodine when the sarcolemma potential was –70 mV. At –20 mV, at which potential the SR Ca²⁺ content was increased and RyRs activated, ryanodine inhibited the Ca²⁺ waves. Photolysed caged IP₃ increased [Ca²⁺]_c; ryanodine, by itself, did not reduce the IP₃-evoked [Ca²⁺]_c increase when the sarcolemma potential was maintained at –70 mV. However, after activation of RyRs by caffeine, in the continued presence of ryanodine, the IP₃-evoked [Ca²⁺]_c increase was inhibited. In other experiments, RyRs were activated (as evidenced by the occurrence of spontaneous transient outward currents) by depolarizing the sarcolemma to –20 mV and again ryanodine was effective in inhibiting IP₃-evoked Ca²⁺ increase. Thus while ineffective by itself, ryanodine inhibited IP₃-evoked Ca²⁺ increases, presumably by causing persistent opening of the channel and depleting the SR of Ca²⁺, after RyRs were activated. These experiments establish that IP₃-evoked Ca²⁺ release and Ca²⁺ waves do not activate RyRs; had they done so ryanodine would have inhibited the Ca²⁺ increase. However, under conditions where ryanodine was ineffective against the IP₃-evoked Ca²⁺ transient (i.e. when RyRs were not activated, e.g. at a membrane potential of –70 mV) tetracaine and dantrolene each blocked IP₃-evoked Ca²⁺ increases. The results show that although IP₃-mediated Ca²⁺ release does not activate RyRs, RyR blockers can inhibit IP₃-mediated Ca²⁺ signals.

(Received 12 August 2005; accepted after revision 22 September 2005; first published online 29 September 2005)
Corresponding author J. G. McCarron: Institute of Biomedical and Life Sciences, Neuroscience and Biomedical Systems, West Medical Building, University of Glasgow, Glasgow G12 8QQ, UK. Email: j.mccarron{at}bio.gla.ac.uk

	Introduction

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The cytosolic Ca²⁺ concentration ([Ca²⁺]_c) controls essential and diverse cellular processes, including signal transduction, enzyme regulation, gene expression, protein trafficking and cell proliferation, and is the major trigger for smooth muscle contraction. [Ca²⁺]_c is critically affected by the activity of the intracellular store (the sarcoplasmic reticulum, SR) from which Ca²⁺ release is controlled by two major channel/receptor complexes, the inositol trisphosphate receptor (IP₃R) and the ryanodine receptor (RyR). IP₃Rs are activated by IP₃ generated in response to many G-protein- or tyrosine kinase-linked receptor activators, including drugs, and by certain neuronal Ca²⁺ binding proteins (CaBPs; Yang et al. 2002; Kasri et al. 2004). RyRs, on the other hand, may also be activated pharmacologically (e.g. by caffeine), by Ca²⁺ influx from outside the cell, in the process of Ca²⁺-induced Ca²⁺ release (CICR), or when the SR's Ca²⁺ content exceeds normal physiological values, i.e. in ‘store overload’ (Trafford et al. 1995; Cheng et al. 1996).

Since Ca²⁺ availability in many cases provides the signal for the cellular response, studies of the generation of those signals have largely been concerned, not surprisingly, with the interplay between the IP₃Rs and RyRs, most notably in the production of Ca²⁺ waves and oscillations (Wakui et al. 1989; Goldbeter et al. 1990; Hajnoczky & Thomas, 1997; Boittin et al. 1999; Gordienko & Bolton, 2002; Heppner et al. 2002; McCarron et al. 2004). Indeed, the spatial and temporal characteristics of these Ca²⁺ signals, by targeting specific signalling pathways, establish the nature of the biological response (Berridge, 2005). While the initiation of oscillations and waves is acknowledged to be a response to sarcolemma-acting agonists which release Ca²⁺ from the SR via IP₃Rs, controversy persists concerning the basis for the subsequent upward phase of each Ca²⁺ oscillation and the propagation of the wave.

A major aspect of this controversy centres on whether or not SR Ca²⁺, initially released by IP₃Rs, then activates RyRs to generate a further release of more substantial amounts of Ca²⁺ by regenerative CICR. Thereafter a cycle of release and diffusion of Ca²⁺ throughout the cell ensues to propagate waves. Alternatively, the entire release process may arise from IP₃R activity alone without significant RyR involvement. In support of the former proposal, drugs which block RyRs, such as ryanodine, tetracaine and dantrolene, often abolish Ca²⁺ oscillations and waves initiated by IP₃-generating agonists (e.g. Iino et al. 1994; Hyvelin et al. 1998; Boittin et al. 1999; Ruehlmann et al. 2000). On the other hand, in some cell types which lack a caffeine-sensitive (RyR) CICR mechanism, Ca²⁺ waves and oscillations are still evident (DeLisle & Welsh, 1992; Lechleiter & Clapham, 1992). These Ca²⁺ oscillations or waves therefore apparently do not require RyRs and must rely on the activity of IP₃Rs alone (Iino, 1990; Bezprozvanny et al. 1991), i.e. waves and oscillations originate, it is assumed, from properties inherent in the IP₃Rs themselves which allow the receptors to open and close in the continued presence of a constant concentration of IP₃ (e.g. Wakui et al. 1989; Hajnoczky & Thomas, 1997).

Many studies of the interaction between RyRs and IP₃Rs and the consequences for waves and oscillations have relied heavily, if not exclusively, on drugs such as ryanodine, dantrolene and tetracaine, which are claimed to be specific inhibitors of RyRs. However, some of these drugs may be less selective than previously assumed. For example, tetracaine, a local anaesthetic, blocks both RyRs and other channels such as the N-methyl-D-aspartate (NMDA) receptor (Nishizawa et al. 2002) and potassium channels (Komai & McDowell, 2001). Although the precise mechanism of action of dantrolene, a hydantoin derivative, clinically used in the management of malignant hyperpyrexia, is not fully known, it reportedly inhibits RyRs either directly or indirectly by reducing receptor activation by Ca²⁺ and calmodulin (Krause et al. 2004). However, dantrolene may also be less specific than previously thought and, for example, like tetracaine inhibits NMDA receptors in cultured cerebellar granule neurones (Makarewicz et al. 2003). Ryanodine, a plant alkaloid, is highly selective for RyRs, to the extent of having been used to purify the receptor (Imagawa et al. 1987). However, its effects on the channel itself are complex and it may maintain RyRs either opened or closed depending on the concentration and incubation period used (Pessah & Zimanyi, 1991). Ryanodine at concentrations less than 100 µM causes persistent opening of RyRs (Rousseau et al. 1987; Kanmura et al. 1988; Xu et al. 1994); at concentrations exceeding 100 µM (depending on the incubation time) it may close the channel (Meissner, 1986; Nagasaki & Fleischer, 1988); and at yet higher concentrations (in the millimolar range) it may cause persistent RyR closure (Nagasaki & Fleischer, 1988; Lai & Meissner, 1989; Zucchi & Ronca-Testoni, 1997). As a result of its ability to both open and close the receptor, different interpretations of the results obtained from ryanodine (even though highly selective for RyRs) on Ca²⁺ signals have occurred. For example, in pulmonary artery smooth muscle cells, ryanodine inhibits IP₃-evoked Ca²⁺ release (using noradrenaline to generate IP₃), a result interpreted to indicate that Ca²⁺ released via IP₃Rs activated RyRs in the process of CICR (e.g. Zheng et al. 2005). On the other hand, in porcine coronary, rat and rabbit mesenteric and rat tail arteries (Katsuyama et al. 1991; Itoh et al. 1992; Iino et al. 1994; Lamont & Wier, 2004), ryanodine's inhibitory action on IP₃-mediated Ca²⁺ increases was attributed to its ability to open RyRs and deplete the store of Ca²⁺ (Katsuyama et al. 1991; Itoh et al. 1992; Iino et al. 1994; Lamont & Wier, 2004) rather than arising from IP₃'s activating CICR at RyRs. In rat mesenteric arteries, for example, ryanodine opened RyRs to deplete the SR of Ca²⁺ following addition of caffeine but not of phenylephrine (Lamont & Wier, 2004) demonstrating that caffeine, but not phenylephrine, activated RyRs. After the SR had been depleted of Ca²⁺ by caffeine and ryanodine, phenylephrine failed to evoke Ca²⁺ waves (Lamont & Wier, 2004). Similarly, in guinea-pig taenia caeci myocytes treated with ryanodine, caffeine released Ca²⁺ on the first application and depleted the SR of the ion. A second caffeine application did not release Ca²⁺. However, caffeine-evoked Ca²⁺ transients were not reduced by ryanodine when preceded by IP₃-evoked Ca²⁺ release (evoked by the muscarinic agonist carbachol) (Iino et al. 1993) again suggesting that RyRs were not activated by IP₃-evoked Ca²⁺ release in the process of CICR. Thus while ryanodine could inhibit IP₃-evoked Ca²⁺ release in each of these tissues, the block was indirect as a consequence of the depletion of the SR of Ca²⁺ and required prior activation of RyRs.

The major approach to the study of the RyR's role has been to use the drugs ryanodine, tetracaine and dantrolene the inhibitory effects of which on IP₃-mediated Ca²⁺ increases have been interpreted, usually, to indicate that the IP₃-mediated Ca²⁺ signal requires subsequent RyR activity. However, the drugs may also block IP₃-mediated Ca²⁺ signals themselves (either directly or indirectly) without RyR involvement in the Ca²⁺ increase. In the present investigation, ryanodine's effects on IP₃-mediated Ca²⁺ release and waves, together with an examination of the effects of the RyR inhibitors tetracaine and dantrolene, have been studied to clarify the RyR's role and to determine whether or not the antagonists may also block the IP₃R. Freshly isolated single colonic smooth muscle cells, voltage clamped in the whole cell configuration were used to avoid [Ca²⁺]_c changes that could have occurred via Ca²⁺ influx as a result of membrane potential changes evoked by the drugs. Flash photolysis (caged IP₃) and hydrostatic application (CCh and caffeine) were used to activate the respective receptors, IP₃R and RyR, to permit a clearer understanding of the effect of the drugs. The results show that Ca²⁺ waves and IP₃-mediated Ca²⁺ release proceed without RyR involvement. However, RyR blockers may inhibit IP₃-mediated Ca²⁺ signals without the involvement of RyRs in the Ca²⁺ increase.

	Methods

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Cell isolation

Male guinea pigs (500–700 g) were killed by cervical dislocation with immediate exsanguination in accordance with the UK Animal (Scientific Procedures) Act 1986. Single colonic myocytes were isolated using a two-step enzymatic dissociation protocol (McCarron & Muir, 1999), stored at 4°C and used the same day.

Membrane current recording

Membrane currents were recorded using tight-seal recording in the whole-cell configuration (McCarron & Muir, 1999). The bathing solution contained (mM): sodium glutamate, 80; NaCl, 40; tetraethylammonium chloride, 20; MgCl₂, 1.1; CaCl₂, 3; Hepes, 10; and D-glucose, 30; pH 7.4, adjusted with NaOH. The Ca²⁺ free bathing solution additionally contained (mM): MgCl₂, 3 (substituted for Ca²⁺); and EGTA, 1. The pipette solution contained (mM): Cs₂SO₄, 85; CsCl, 20; MgCl₂, 1; MgATP, 3; pyruvic acid, 2.5; malic acid, 2.5; NaH₂PO₄, 1; creatine phosphate, 5; GTP, 0.5; Hepes, 30; fluo-3 penta-ammonium salt, 0.1; caged IP₃, 0.025; pH 7.2, adjusted with CsOH. In imaging experiments fluo-3 was omitted from the pipette solution.

[Ca²⁺]_c measurement

[Ca²⁺]_c was measured using the membrane-impermeant fluo-3 dye introduced through the patch pipette. Cells were placed in a Perspex chamber (1 ml) on the stage of an inverted fluorescence microscope (Nikon diaphot) and fluo-3 was excited at 488 nm (9 nm bandpass) by a PTI Deltascan (Photon Technology International, London, UK). Emitted light was guided through a 535 nm barrier filter (bandpass 35 nm) to a photomultiplier in photon counting mode (Kamishima & McCarron, 1996). In other experiments, two dimensional [Ca²⁺]_c images were obtained using a wide-field digital imaging system (McCarron et al. 2004). Here, single cells were illuminated at 488 nm (bandpass 14 nm) from a monochromator (Polychrome IV, T.I.L.L. Photonics, Martinsried, Germany). Emitted light was guided through a 535 nm barrier filter (bandpass 45 nm) to an intensified, cooled, frame transfer CCD camera (Pentamax General IV, Roper Scientific, Trenton, NJ, USA). Full frame images (150 pixels x 150 pixels), with a pixel size of 563 nm at the cell, were acquired at 10 frames s^–1. In these experiments, cells were loaded with fluo-3 AM (10 µM) and wortmanin (10 µM; to prevent contraction) for at least 30 min prior to the beginning of the experiment.

Caged IP₃ was photolysed to the uncaged compound by ultraviolet light, which was selected by passing the output of a xenon flashlamp (Rapp Optoelecktronic, Hamburg, Germany) through a UG-5 filter and merging it into the excitation light path of the microfluorimeter using a quartz bifurcated fibre optic bundle (McCarron & Muir, 1999). To alter the concentration of IP₃, to produce maximal and submaximal responses, the output of the xenon flashlamp was reduced from 38 mJ (maximal) to 23 mJ (submaximal) and applied to the caged compound. Caffeine (10 mM) and carbachol (CCh; 100 µM) were applied by hydrostatic pressure (Pneumatic PicoPump PV820, World Precision Instruments, Sarasota, FL, USA). All experiments were carried out at room temperature (18–22°C) and drugs (except CCh and caffeine) perfused into the bathing solution.

Data analysis

Changes in cytosolic Ca²⁺ were expressed as ratios (F/F₀) of the fluorescence counts (F) relative to baseline counts before stimulation (F₀). F/F₀ indicates the magnitude of the change in F/F₀ at the peak of the evoked transient relative to the baseline ratio. Original fluorescence records were not filtered, smoothed or averaged. Background fluorescence was not subtracted. Statistical analyses were performed using Student's paired or unpaired t test (on raw data) where appropriate. Summarized data are shown as means ±S.E.M. and were taken as statistically significant when P < 0.05. n indicates the number of cells.

Drugs and reagents

Fluo-3 penta-ammonium salt and fluo-3 pentaacetoxymethyl ester were obtained from Molecular Probes Inc. (Eugene, OR, USA). Caged IP₃ trisodium salt was obtained from Calbiochem-Novabiochem (Nottingham, UK). Ryanodine (from Calbiochem-Novabiochem or Sigma, UK (Poole, UK)) was dissolved in dimethyl sulphoxide (DMSO), to give a final bath concentration of < 0.1% DMSO. CCh (Sigma) was dissolved in bath solution. Ca²⁺-free Eagle's minimum essential spinner medium (S-MEM) was purchased from Gibco BRL (Paisley, UK) and supplemented with L-glutamine (2 mM) prior to use. Papain and collagenase were obtained from Worthington Biochemical Corp. (Lakewood, NJ, USA). All other reagents were purchased from Sigma, UK.

	Results

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In single colonic myocytes, which were not voltage clamped, the IP₃-generating muscarinic agonist carbachol (CCh, 100 µM by pressure ejection) increased [Ca²⁺]_c; this increase usually began in one region and progressed as a travelling spatial [Ca²⁺]_c gradient (i.e. a Ca²⁺ wave) throughout the cell. Ryanodine (50 µM) either had no effect (n= 5) or decreased (n= 7) the CCh-evoked [Ca²⁺]_c increase. In the former cases the amplitude of the CCh-evoked Ca²⁺ transients in ryanodine remained at 89 ± 4% of controls (0.67 ± 0.19 F/F₀ before and 0.59 ± 0.15 F/F₀ after ryanodine; n= 5; P > 0.05); in the latter they decreased to 61 ± 7% of control values (1.13 ± 0.34 F/F₀ before, compared with 0.67 ± 0.17 F/F₀ after ryanodine; n= 7; P < 0.05; not shown). One explanation for this difference in the effectiveness of ryanodine may be the variation in sarcolemma potential of single cells.

To investigate any sarcolemma potential dependence of block, the effects of ryanodine on CCh-evoked [Ca²⁺]_c increases were examined in single voltage clamped myocytes at different sarcolemma potentials. At –70 mV the CCh-evoked Ca²⁺ wave was unaltered by ryanodine (50 µM) and remained at 90 ± 4% (1.12 ± 0.13 F/F₀ before, compared with 1.01 ± 0.12 F/F₀ after ryanodine; n= 3; P > 0.05) of the control amplitude (Fig. 1A). On the other hand, when the sarcolemma potential was –20 mV ryanodine significantly (P < 0.05) inhibited the CCh-evoked Ca²⁺ wave to 55 ± 11% of control amplitude (Fig. 1B; 0.85 ± 0.50 F/F₀ before, compared with 0.55 ± 0.38 F/F₀ after ryanodine; n= 3; P < 0.05). Thus ryanodine's ability to inhibit CCh-evoked IP₃-mediated Ca²⁺ increases apparently depends on the sarcolemma potential and arises, presumably, from differences in RyR activity at each potential (see below).

View larger version (34K): [in this window] [in a new window]   Figure 1.  Effect of ryanodine on carbachol (CCh)-evoked Ca2+ waves in single voltage clamped colonic myocytes A, at –70 mV(d), CCh (c) increased [Ca2+]c as represented by the colour changes in the frames (a; blue low, red high [Ca2+]c) and fluo-3 fluorescence transients (F/F0; b). [Ca2+]c increases in response to CCh began in the centre of the cell and progressed (a) throughout (a Ca2+ wave). The time points from which the [Ca2+]c images were obtained (a) are indicated by the lines from the fluorescence transients (F/F0; b). Ryanodine (50 µM; applied for 10 min during the break in the record and for the duration of the bar) did not significantly alter the magnitude of two subsequent CCh-evoked increases. B, on the other hand, in a different cell, when the membrane potential was –20 mV (d; at which the SR Ca2+ content is increased, see text) ryanodine inhibited CCh-evoked Ca2+ waves (a and b). Steady depolarization to –20 mV (from –70 mV) increases [Ca2+]c from 99 ± 21 nM to 276 ± 32 nM (McCarron et al. 2000). The F/F0 ratio at –20 mV (above) was normalized to that value occurring at –20 mV giving a value of 1. Thus ryanodine inhibited CCh-evoked Ca2+ waves when the sarcolemma potential was depolarized (–20 mV), but not when it was –70 mV. Scale bars (Aa and Ba, left hand panels) are 20 µm.

View larger version (22K): [in this window] [in a new window]   Figure 2.  Effect of ryanodine on IP3-mediated Ca2+ release A, IP3 () produced approximately reproducible increases in [Ca2+]c in a single voltage clamped (–70 mV) colonic myocyte; ryanodine (50 µM) did not significantly alter the magnitude of this increase indicating the absence of CICR at RyRs. B, despite the absence of CICR at RyRs, ryanodine reduced IP3-mediated Ca2+ increases after RyRs had been activated by caffeine. Again at –70 mV, IP3 () evoked reproducible increases in [Ca2+]c in a single voltage clamped colonic myocyte (a) which were not significantly reduced by ryanodine (50 µM). Activation of RyR by caffeine (10 mM, by pressure ejection, c), in the continued presence of ryanodine, initially increased [Ca2+]c (a). A second and third application of caffeine (c) to the same cell, however, some 80 s later, generated little increase in [Ca2+]c presumably because of SR store depletion. The IP3 response was also subsequently inhibited (; a). Because the IP3-evoked Ca2+ transient was not blocked by ryanodine alone (only after RyR activation with caffeine), IP3-mediated Ca2+release did not activate RyRs. IP3Rs and RyRs may share a common Ca2+ store; this is depleted of Ca2+ by ryanodine after activation of RyRs by caffeine to reduce the Ca2+ available for IP3Rs. The small rise in basal [Ca2+]c could have been due to the activation of store-operated Ca2+ entry. Experiments in A and B are from different cells.

View larger version (29K): [in this window] [in a new window]   Figure 3.  Depolarization increased the SR Ca2+ content to facilitate the activation of RyRs; ryanodine, subsequently administrated, inhibited IP3-evoked [Ca2+]c transients A, IP3 () evoked approximately reproducible [Ca2+]c increases. Depolarization (–70 mV to –20 mV) increased both [Ca2+]c and the SR Ca2+ content as revealed by the increase in amplitude of the IP3-evoked Ca2+ transients. The increased IP3-evoked [Ca2+]c transients arose from an elevated SR Ca2+ content rather than from an elevated [Ca2+]c since the IP3-evoked Ca2+ transients remained elevated for a time when the membrane potential was restored to –70 mV. B, caffeine evoked approximately reproducible increases in [Ca2+]c. The caffeine-evoked [Ca2+]c transient was also increased by depolarization to –20 mV, again indicating an increased SR Ca2+ content. On repolarization to –70 mV the caffeine-evoked [Ca2+]c transient initially remained elevated before being restored to control amplitudes. C, depolarization (–70 mV to –20 mV) activated RyRs as revealed by the occurrence of STOCs. D, at –20 mV, ryanodine (50 µM) initially increased, then inhibited, the frequency of occurrence and amplitude of STOCs, presumably as the SR was progressively depleted of Ca2+. E, at –20 mV, ryanodine also significantly inhibited IP3 ()-evoked Ca2+ transients in contrast to its ineffectiveness at –70 mV (cf. Fig. 2A and B). This indicates that when the SR content is elevated and RyRs activated, ryanodine, by depleting the SR of Ca2+, reduced the Ca2+ available to inhibit IP3-evoked Ca2+ release.

At –20 mV, the IP₃-evoked Ca²⁺ transient was also significantly reduced by ryanodine to on average 68 ± 15% of controls (P < 0.05, n= 8) although on occasion to an even greater extent (Fig. 3E). Thus IP₃ increased [Ca²⁺]_c by F/F₀ 1.9 ± 0.3 above baseline in controls (the sixth control IP₃ release; n= 8); the sixth IP₃-mediated Ca²⁺ release in the presence of ryanodine (50 µM; F/F₀ 1.3 ± 0.4 above baseline; n= 8; P < 0.05) was significantly reduced. This result may be explained by the ability of ryanodine to lock the RyRs in an open subconductance state (Rousseau et al. 1987; Kanmura et al. 1988; Xu et al. 1994) to reduce the SR Ca²⁺ content and subsequently attenuate IP₃-mediated Ca²⁺ release. Together these results confirm that ryanodine blocks IP₃-mediated Ca²⁺ responses only when RyRs have been previously activated. The block is indirect, as a consequence of the depletion of the SR store of Ca²⁺ to which both IP₃Rs and RyRs have common access (Flynn et al. 2001; McCarron et al. 2002).

Clearly, the [Ca²⁺]_c increase following IP₃R activation follows from IP₃R activity alone. The sole involvement of IP₃Rs in this increase allowed an examination of the affinity of other putative RyR blockers for IP₃Rs and the mechanisms involved. Classical competitive inhibitors would be expected to block Ca²⁺ release in a dose-dependent fashion and be overcome by an increased amount of agonist (in this case IP₃). The effects of RyR blockers (dantrolene and tetracaine) were therefore examined on maximal and submaximal responses to IP₃. Maximal responses were evoked in each cell and the lamp energy reduced to obtain reproducible submaximal responses. In the first series of experiments, IP₃ evoked reproducible maximal increases in [Ca²⁺]_c (1.4 ± 0.26 F/F₀) which were reduced (0.65 ± 0.16 F/F₀) to 50 ± 10% when the lamp energy was reduced. Submaximal IP₃-evoked [Ca²⁺]_c increases were blocked by dantrolene (10 µM) by 22 ± 4% (F/F₀ from 0.65 ± 0.16 to 0.49 ± 0.12, P < 0.05, n= 6, Fig. 4A). Maximal IP₃-evoked [Ca²⁺]_c increases were not reduced by dantrolene (10 µM) (F/F₀ from 0.78 ± 0.23 to 0.72 ± 0.25, P > 0.05, n= 5, Fig. 4B) but were inhibited by 50 µM of the drug by 64 ± 10% (F/F₀ from 1.27 ± 0.28 control to 0.39 ± 0.07 in dantrolene (n= 5, P < 0.05, Fig. 4C)).

View larger version (18K): [in this window] [in a new window]   Figure 4.  Dantrolene inhibited IP3-evoked [Ca2+]c transients in a concentration-dependent manner Dantrolene (10 µM) inhibited submaximal (A) but not maximal (B) IP3-evoked [Ca2+]c increases (); the latter were abolished at 50 µM (C). Membrane potential –70 mV throughout.

View larger version (19K): [in this window] [in a new window]   Figure 5.  Tetracaine inhibited IP3-evoked [Ca2+]c transients in a concentration-dependent manner Tetracaine (100 µM) inhibited submaximal (A) but not maximal (B) IP3-evoked [Ca2+]c increases (); the latter were abolished at 1 mM (C). Membrane potential –70 mV throughout.

	Discussion

Top Abstract Introduction Methods Results Discussion References

Our view that RyRs are not involved in the IP₃-evoked Ca²⁺ increase stems from the effects of ryanodine. Ryanodine inhibits IP₃-evoked Ca²⁺ release only after RyRs have first been activated. Thus, ryanodine, by itself, did not reduce either the CCh-evoked Ca²⁺ wave or the IP₃-evoked Ca²⁺ transient while the membrane potential was clamped at –70 mV. Had IP₃-evoked Ca²⁺ release activated RyR, through CICR, ryanodine would have reduced IP₃-mediated [Ca²⁺]_c increases; this was not observed. On the other hand, after RyRs had been activated either by caffeine or by ‘store-overload’ conditions (as occurred at –20 mV), ryanodine reduced the IP₃-mediated Ca²⁺ signals. This inhibition arose, we propose, from ryanodine's depleting the SR of Ca²⁺, as a consequence of the drug's persistent opening of RyRs, so reducing the Ca²⁺ available for release by IP₃ from the commonly accessed SR store. Ryanodine did not persistently close RyRs under the present experimental conditions (see Introduction). Had it done so, its effects on IP₃-evoked Ca²⁺ release (e.g. at –70 mV) would presumably have been equally ineffective in the absence and presence of caffeine. Thus, in the absence of caffeine, IP₃-evoked Ca²⁺ release did not activate RyRs, so ryanodine was ineffective. In the presence of caffeine and ryanodine, RyRs would have been activated and the receptor closed. In these conditions, the SR would not have been depleted and Ca²⁺ would have been available for release via IP₃Rs. In the event, while ryanodine was ineffective on its own, after RyRs were activated no Ca²⁺ was subsequently available for release as revealed by the absence of IP₃-evoked Ca²⁺ transients. Thus while RyRs do not contribute to the [Ca²⁺]_c rise evoked by IP₃, ryanodine can nonetheless inhibit IP₃-evoked Ca²⁺ release, indirectly as a consequence of depleting the SR of Ca²⁺. Our previous studies have also found that RyRs could not be recruited to propagate localized IP₃-mediated Ca²⁺ increases into a Ca²⁺ wave even when RyR activity itself had been confirmed (McCarron et al. 2004).

Under physiological conditions, the [Ca²⁺]_c required to activate RyRs is some 15 µM (e.g. Cannell et al. 1994) and exceeds the average [Ca²⁺]_c which follows IP₃R activation (< 1 µM). Ca²⁺ release via RyRs can occur at a lower (< 1 µM) bulk [Ca²⁺]_c when the SR contains excessively high levels of Ca²⁺– as in ‘store overload.’ This increases RyR sensitivity to Ca²⁺ (Trafford et al. 1995; Cheng et al. 1996; Lukyanenko et al. 1999). Indeed, RyR-mediated Ca²⁺ waves have been observed in store overload conditions in cardiac myocytes (Kass et al. 1978; Berlin et al. 1989; Trafford et al. 1995; Cheng et al. 1996; Lukyanenko et al. 1999) though the relevance of this to normal functioning is unclear. Co-localization of IP₃Rs and RyRs (see McGeown, 2004) may expose RyRs to a [Ca²⁺]_c that exceeds bulk average values, to open the channel. Evidence for this latter proposal comes from considerations of the distribution of the two receptors. Overlap between the IP₃- and the ryanodine-sensitive Ca²⁺ pools occurs in smooth muscle (see inter aliaNixon et al. 1994; Wibo & Godfraind, 1994). In some studies, using fluorescent probes for RyRs and IP₃Rs, overlap in the distribution of fluorescence from the two probes has been interpreted as support for co-localization of the receptors and for a functional interaction between them (see McGeown, 2004). However, many such studies have demonstrated whole cell fluorescence overlap only in ill-defined regions with little information being presented on whether or not the receptors exist in the same z plane. Even under ideal experimental conditions, using a 1.3 NA oil immersion lens and an emission wavelength of 520 nm (as with fluorescein or rhodamine labelled antibodies), the resolution is restricted to approximately 250 nm in x and y directions and 350 nm in the z direction. This space represents the smallest distinguishable volume element (voxel) and the distance between components within it cannot normally be determined. Under such ideal conditions, which are rarely achieved, two separate receptors, present within a single voxel, could be almost 500 nm apart (top to bottom opposite corners), i.e. distanced from each other by about the width of a mitochondrion. Such a distance seems unlikely to justify the receptors being regarded as co-localized. Well-controlled double-labelling of the IP₃Rs and RyRs at electron microscopic resolution or photobleaching fluorescence resonance energy transfer (FRET) experiments are necessary to define the proximity of receptors before they may be considered co-localized.

Other RyR blocking drugs including tetracaine and dantrolene have served to determine the contribution of RyR activity to the overall Ca²⁺ rise initiated by IP₃R opening (e.g. Simpson et al. 1991; Zheng et al. 2005). The absence of any detectable CICR at RyRs evoked by IP₃-mediated Ca²⁺ release, in the present cell type, enabled an examination of the selectivity of these RyR inhibitors. Tetracaine and dantrolene each inhibited IP₃-mediated Ca²⁺ release. Lower concentrations of the inhibitors reduced submaximal but not maximal responses to IP₃; higher concentrations of the inhibitors reduced maximal responses to IP₃. The inhibition occurred despite the absence of a contribution from RyRs to the Ca²⁺ increase, presumably as a result of the drugs' non-specific effects at IP₃Rs.

In addition to these compounds, interest has been focused on the activity of ruthenium red, which blocks RyRs. This drug also has non-specific effects and may inhibit IP₃-mediated Ca²⁺ signals independently of the involvement of RyRs. For example, in avian atria, ruthenium red inhibited the response to IP₃ by an action unrelated to RyRs, since the response to caffeine was potentiated (Vites & Pappano, 1992). Ruthenium red's inhibition of IP₃-mediated Ca²⁺ release under these conditions may arise from a block of the IP₃R itself or from an inhibition of the mitochondrial uniporter to alter local Ca²⁺ feedback near IP₃Rs (Gunter et al. 2000). Ruthenium red's poor membrane permeability precluded its study in the present investigation.

Ca²⁺ availability provides the signal for many cellular responses and several studies of the generation of those signals have largely concerned the interplay between the IP₃Rs and RyRs, most notably in the production of Ca²⁺ waves and oscillations (e.g. Boittin et al. 1999; Gordienko & Bolton, 2002; Heppner et al. 2002; Lamont & Wier, 2004; McCarron et al. 2004). The use of drugs such as tetracaine and dantrolene has provided a useful experimental approach to determining the extent of the interaction between IP₃Rs and RyRs. Our findings demonstrate, however, that these drugs may be less specific than previously thought. Ryanodine is also frequently used. The present results show that an inhibitory effect of ryanodine on IP₃-mediated Ca²⁺ increases may also be difficult to interpret. In cells which are not voltage clamped, ryanodine may either have no effect or inhibit CCh-evoked [Ca²⁺]_c increases. The inhibition presumably arises from store depletion (to limit the Ca²⁺ available for release by CCh) in those cells in which RyRs are more active – perhaps as a consequence of a more depolarized sarcolemma – and not because RyRs contribute to the Ca²⁺ increase evoked by IP₃. In support, in voltage clamped cells ryanodine inhibited CCh-evoked [Ca²⁺]_c increases at –20 mV (where the SR Ca²⁺ content is increased and RyRs active) but not at –70 mV (where RyRs are not active).

Although in the present investigation Ca²⁺ release via IP₃Rs did not activate RyRs, this does not preclude its doing so under different experimental conditions. For example, in rat portal vein RyR antibodies reduced IP₃-mediated Ca²⁺ release suggesting that Ca²⁺ release via IP₃Rs may have activated RyRs (Boittin et al. 1999). Ryanodine inhibited carbachol-induced Ca²⁺ waves in rat gastric myocytes with a 30% reduction in the SR Ca²⁺ content (White & McGeown, 2002). Uridine 5'-triphosphate promoted waves at RyRs in rat cerebral artery (Jaggar & Nelson, 2000) and the localized release of Ca²⁺ via IP₃Rs may activate CICR in neighbouring RyRs in rabbit portal vein (Gordienko & Bolton, 2002). Plasmalemma agonists used to generate IP₃ may activate other second messengers that could sensitize the RyRs to Ca²⁺ and permit IP₃-mediated Ca²⁺ release to activate, in turn, RyRs. Activation could occur by processes such as phosphorylation (Takasago et al. 1991; Yoshida et al. 1992) and/or by other modulators of Ca²⁺ release from RyRs, including Ca²⁺, calmodulin and adenine nucleotides (Meissner, 2002). RyR sensitivity to cytoplasmic Ca²⁺ is increased under conditions of ‘store overload’vide infra (Trafford et al. 1995; Cheng et al. 1996). It is possible that some types of smooth muscle maintain a higher SR Ca²⁺ content which could facilitate the occurrence of CICR at RyRs following IP₃-mediated Ca²⁺ release. While the findings of the present study do not exclude the possibility that IP₃-mediated Ca²⁺ release may activate CICR at RyRs, they demonstrate that a reduction of IP₃-mediated Ca²⁺ signals by the RyR inhibitors ryanodine, dantrolene and tetracaine does not necessarily indicate an involvement of RyRs in the IP₃-mediated Ca²⁺ increase; each drug is also capable of blocking IP₃-mediated Ca²⁺ signals.

	References

Top Abstract Introduction Methods Results Discussion References

 
Benham CD & Bolton TB (1986). Spontaneous transient outward currents in single visceral and vascular smooth muscle cells of the rabbit. J Physiol 381, 385–406.[Abstract/Free Full Text]

Berlin JR, Cannell MB & Lederer WJ (1989). Cellular origins of the transient inward current in cardiac myocytes. Role of fluctuations and waves of elevated intracellular calcium. Circ Res 65, 115–126.[Abstract/Free Full Text]

Berridge MJ (2005). Unlocking the secrets of cell signalling. Annu Rev Physiol 67, 1–21.[CrossRef][Medline]

Bezprozvanny I, Watras J & Ehrlich BE (1991). Bell-shaped calcium-response curves of Ins(1,4,5)P₃- and calcium-gated channels from endoplasmic reticulum of cerebellum. Nature 351, 751–754.[CrossRef][Medline]

Boittin FX, Macrez N, Halet G & Mironneau J (1999). Norepinephrine-induced Ca²⁺ waves depend on InsP₃ and ryanodine receptor activation in vascular myocytes. Am J Physiol 277, C139–C151.[Medline]

Cannell MB, Berlin JR & Lederer WJ (1994). Spatial non-uniformities in [Ca²⁺]_i during excitation-contraction coupling in cardiac myocytes. Biophys J 67, 1942–1956.[Abstract/Free Full Text]

Cheng H, Lederer MR, Lederer WJ & Cannell MB (1996). Calcium sparks and [Ca²⁺]_c waves in cardiac myocytes. Am J Physiol 270, C148–C159.[Medline]

DeLisle S & Welsh MJ (1992). Inositol trisphosphate is required for the propagation of calcium waves in Xenopus oocytes. J Biol Chem 267, 7963–7966.[Abstract/Free Full Text]

Flynn ERM, Bradley KN, Muir TC & McCarron JG (2001). Functionally-separate intracellular Ca²⁺ stores in smooth muscle. J Biol Chem 276, 36411–36418.[Abstract/Free Full Text]

Goldbeter A, Dupont G & Berridge MJ (1990). Minimal model for signal-induced Ca²⁺ oscillations and for their frequency encoding through protein phosphorylation. Proc Natl Acad Sci U S A 87, 1461–1465.[Abstract/Free Full Text]

Gordienko DV & Bolton TB (2002). Crosstalk between ryanodine receptors and IP₃ receptors as a factor shaping spontaneous Ca²⁺-release events in rabbit portal vein myocytes. J Physiol 542, 743–762.[Abstract/Free Full Text]

Gunter TE, Buntinas L, Sparagna G, Eliseev R & Gunter K (2000). Mitochondrial calcium transport: mechanisms and functions. Cell Calcium 28, 285–296.[CrossRef][Medline]

Hajnoczky G & Thomas AP (1997). Minimal requirements for calcium oscillations driven by the IP₃ receptor. EMBO J 16, 3533–3543.[CrossRef][Medline]

Heppner TJ, Bonev AD, Santana LF & Nelson MT (2002). Alkaline pH shifts Ca²⁺ sparks to Ca²⁺ waves in smooth muscle cells of pressurized cerebral arteries. Am J Physiol 283, H2169–H2176.

Hyvelin J-M, Guibert C, Marthan R & Savineau J-P (1998). Cellular mechanisms and role of endothelin-1-induced calcium oscillations in pulmonary arterial myocytes. Am J Physiol 275, L269–L282.[Medline]

Iino M (1990). Biphasic Ca²⁺ dependence of inositol 1,4,5-trisphosphate-induced Ca release in smooth muscle cells of the guinea pig taenia caeci. J General Physiol 95, 1103–1122.[Abstract/Free Full Text]

Iino M, Kasai H & Yamazawa T (1994). Visualization of neural control of intracellular Ca²⁺ concentration in single vascular smooth muscle cells in situ. EMBO J 13, 5026–5031.[Medline]

Iino M, Yamazawa T, Miyashita Y, Endo M & Kasai H (1993). Critical intracellular Ca²⁺ concentration for all-or-none Ca²⁺ spiking in single smooth muscle cells. EMBO J 12, 5287–5291.[Medline]

Imagawa T, Smith JS, Coronado R & Campbell KP (1987). Purified ryanodine receptor from skeletal muscle sarcoplasmic reticulum is the Ca²⁺-permeable pore of the calcium release channel. J Biol Chem 262, 16636–16643.[Abstract/Free Full Text]

Itoh T, Kajikuri J & Kuriyama H (1992). Characteristic features of noradrenaline-induced Ca²⁺ mobilization and tension in arterial smooth muscle of the rabbit. J Physiol 457, 297–314.[Abstract/Free Full Text]

Jaggar JH & Nelson MT (2000). Differential regulation of Ca²⁺ sparks and Ca²⁺ waves by UTP in rat cerebral artery smooth muscle cells. Am J Physiol 279, C1528–C1539.

Kamishima T & McCarron JG (1996). Depolarisation-evoked increases in the cytosolic calcium concentration in isolated smooth muscle of rat portal vein. J Physiol 492, 61–74.[Medline]

Kanmura Y, Missiaen L, Raeymaekers L & Casteels R (1988). Ryanodine reduces the amount of calcium in intracellular stores of smooth-muscle cells of the rabbit ear artery. Pflugers Arch 413, 153–159.[CrossRef][Medline]

Kasri NN, Holmes AM, Bultynck G, Parys JB, Bootman MD, Rietdorf K, Missiaen L, McDonald F, De Smedt H, Conway SJ, Holmes AB, Berridge MJ & Roderick HL (2004). Regulation of InsP₃ receptor activity by neuronal Ca²⁺-binding proteins. EMBO J 23, 312–321.[CrossRef][Medline]

Kass RS, Lederer WJ, Tsien RW & Weingart R (1978). Role of calcium ions in transient inward currents and aftercontractions induced by strophanthidin in cardiac Purkinje fibres. J Physiol 281, 187–208.[Abstract/Free Full Text]

Katsuyama H, Ito S, Itoh T & Kuriyama H (1991). Effects of ryanodine on acetylcholine-induced Ca²⁺ mobilization in single smooth muscle cells of the porcine coronary artery. Pflugers Arch 419, 460–466.[CrossRef][Medline]

Komai H & McDowell TS (2001). Local anesthetic inhibition of voltage-activated potassium currents in rat dorsal root ganglion neurons. Anesthesiology 94, 1089–1095.[Medline]

Krause T, Gerbershagen MU, Fiege M, Weisshorn R & Wappler F (2004). Dantrolene – a review of its pharmacology, therapeutic use and new developments. Anaesthesia 59, 364–373.[CrossRef][Medline]

Lai FA & Meissner G (1989). The muscle ryanodine receptor and its intrinsic Ca²⁺ channel activity. J Bioener Biomem 21, 227–246.[CrossRef][Medline]

Lamont C & Wier WG (2004). Different roles of ryanodine receptors and inositol(1,4,5)-trisphosphate receptors in adrenergically stimulated contractions of small arteries. Am J Physiol 287, H617–H625.

Lechleiter JD & Clapham DE (1992). Molecular mechanisms of intracellular calcium excitability in X. laevis oocytes. Cell 69, 283–294.[CrossRef][Medline]

Lukyanenko V, Subramanian S, Gyorge I, Wiesner TF & Gyorke S (1999). The role of luminal Ca²⁺ in the generation of Ca²⁺ waves in rat ventricular myocytes. J Physiol 518, 173–186.[Abstract/Free Full Text]

Makarewicz D, Zieminska E & Lazarewicz JW (2003). Dantrolene inhibits NMDA-induced ⁴⁵Ca uptake in cultured cerebellar granule neurons. Neurochem Int 43, 273–278.[CrossRef][Medline]

McCarron JG, Craig JW, Bradley KN & Muir TC (2002). Agonist-induced phasic and tonic responses in smooth muscle are mediated by InsP₃. J Cell Sci 115, 2207–2218.[Abstract/Free Full Text]

McCarron JG, Flynn ERM, Bradley KN & Muir TC (2000). Two pathways mediate InsP₃-sensitive store refilling in guinea-pig colonic smooth muscle. J Physiol 525, 113–124.[Abstract/Free Full Text]

McCarron JG, MacMillan D, Bradley KN, Chalmers S & Muir TC (2004). Origin and mechanisms of Ca²⁺ waves in smooth muscle as revealed by localized photolysis of caged inositol 1,4,5-trisphosphate. J Biol Chem 279, 8417–8427.[Abstract/Free Full Text]

McCarron JG & Muir TC (1999). Mitochondrial regulation of the cytosolic Ca²⁺ concentration and the InsP₃-sensitive Ca²⁺ store in colonic smooth muscle. J Physiol 516, 149–161.[Abstract/Free Full Text]

McGeown JG (2004). Interactions between inositol 1,4,5-trisphosphate receptors and ryanodine receptors in smooth muscle: one store or two? Cell Calcium 35, 613–619.[CrossRef][Medline]

Meissner G (1986). Ryanodine activation and inhibition of the Ca²⁺ release channel of sarcoplasmic reticulum. J Biol Chem 261, 6300–6306.[Abstract/Free Full Text]

Meissner G (2002). Regulation of mammalian ryanodine receptors. Front Biosci 7, d2072–d2080.[Medline]

Nagasaki K & Fleischer S (1988). Ryanodine sensitivity of the calcium release channel of sarcoplasmic reticulum. Cell Calcium 9, 1–7.[Medline]

Nishizawa N, Shirasaki T, Nakao S, Matsuda H & Shingu K (2002). The inhibition of the N-methyl-D-aspartate receptor channel by local anesthetics in mouse CA1 pyramidal neurons. Anesth Analg 94, 325–330.[Abstract/Free Full Text]

Nixon GF, Mignery GA & Somlyo AV (1994). Immunogold localization of inositol 1,4,5-trisphosphate receptors and characterization of ultrastructural features of the sarcoplasmic reticulum in phasic and tonic smooth muscle. J Muscle Res Cell Motil 15, 682–700.[CrossRef][Medline]

Pessah IN & Zimanyi I (1991). Characterization of multiple [³H]ryanodine binding sites on the Ca²⁺ release channel of sarcoplasmic reticulum from skeletal and cardiac muscle: evidence for a sequential mechanism in ryanodine action. Mol Pharmacol 39, 679–689.[Abstract]

Rousseau E, Smith JS & Meissner G (1987). Ryanodine modifies conductance and gating behavior of single Ca²⁺ release channel. Am J Physiol 253, C364–C368.[Medline]

Ruehlmann DO, Lee C-H, Poburko D & vanBreemen C (2000). Asynchronous Ca²⁺ waves in intact venous smooth muscle. Circ Res 86, e72–e79.[Abstract/Free Full Text]

Simpson PB, Nahorski SR & Challiss RA (1991). Agonist-evoked Ca²⁺ mobilization from stores expressing inositol 1,4,5-trisphosphate receptors and ryanodine receptors in cerebellar granule neurones. J Neurochem 67, 364–373.

Takasago T, Imagawa T, Furukawa K, Ogurusu T & Shigekawa M (1991). Regulation of the cardiac ryanodine receptor by protein kinase-dependent phosphorylation. J Biochem 109, 163–170.[Abstract/Free Full Text]

Trafford AW, Lipp P, O'Neill SC, Niggli E & Eisner DA (1995). Propagating calcium waves initiated by local caffeine application in rat ventricular myocytes. J Physiol 489, 319–326.[Medline]

Vites AM & Pappano AJ (1992). Ruthenium red selectively prevents Ins(1,4,5)P₃-but not caffeine-gated calcium release in avian atrium. Am J Physiol 262, H268–H277.[Medline]

Wakui M, Potter BVL & Petersen OH (1989). Pulsatile intracellular calcium release does not depend on fluctuations in inositol trisphosphate concentration. Nature 339, 317–320.[CrossRef][Medline]

White C & McGeown JG (2002). Carbachol triggers RyR-dependent Ca²⁺ release via activation of IP₃ receptors in isolated rat gastric myocytes. J Physiol 542, 725–733.[Abstract/Free Full Text]

Wibo M & Godfraind T (1994). Comparative localization of inositol 1,4,5-trisphosphate and ryanodine receptors in intestinal smooth muscle: an analytical subfractionation study. Biochem J 297, 415–423.[Medline]

Xu L, Lai FA, Cohn A, Etter E, Guerrero A, Fay FS & Meissner G (1994). Evidence for a Ca²⁺-gated ryanodine-sensitive Ca²⁺ release channel in visceral smooth-muscle. Proc Natl Acad Sci U S A 91, 3294–3298.[Abstract/Free Full Text]

Yang J, McBride S, Mak DO, Vardi N, Palczewski K, Haeseleer F & Foskett JK (2002). Identification of a family of calcium sensors as protein ligands of inositol trisphosphate receptor Ca²⁺ release channels. Proc Natl Acad Sci U S A 99, 7711–7716.[Abstract/Free Full Text]

Yoshida A, Ogura A, Imagawa T, Shigekawa M & Takahashi M (1992). Cyclic AMP-dependent phosphorylation of the rat brain ryanodine receptor. J Neurosci 12, 1094–1100.[Abstract]

Zheng YM, Wang QS, Rathore R, Zhang WH, Mazurkiewicz JE, Sorrentino V, Singer HA, Kotlikoff MI & Wang YX (2005). Type-3 ryanodine receptors mediate hypoxia-, but not neurotransmitter-induced calcium release and contraction in pulmonary artery smooth muscle cells. J General Physiol 125, 427–440.[Abstract/Free Full Text]

Zucchi R & Ronca-Testoni S (1997). The sarcoplasmic reticulum Ca²⁺ channel/ryanodine receptor: modulation by endogenous effectors, drugs and disease states. Pharmacol Rev 49, 1–51.[Abstract/Free Full Text]

	Acknowledgements

 
The Wellcome Trust (060094/Z/00/Z) and British Heart Foundation (PG/2001079; PG/02/161) funded this work. The authors thank Mr J. W. Craig for his excellent technical assistance.

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