IP3-mediated Ca2+ increases do not involve the ryanodine receptor, but ryanodine receptor antagonists reduce IP3-mediated Ca2+ increases in
1 Institute of Biomedical and Life Sciences, Neuroscience and Biomedical Systems, West Medical Building, University of Glasgow, Glasgow G12 8QQ, UK
Abstract
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.
, http://www.100md.com
Introduction
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).
, 百拇医药
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.
, 百拇医药
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).
, 百拇医药
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.
, http://www.100md.com
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.
, http://www.100md.com
Methods
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
, 百拇医药
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.
, http://www.100md.com
[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.
, http://www.100md.com
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.
, 百拇医药
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.
, 百拇医药
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.
, 百拇医药
Results
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.
, http://www.100md.com
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).
, 百拇医药
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.
, 百拇医药
To investigate the mechanism by which changes in sarcolemma potential may modulate ryanodine's ability to block IP3-mediated Ca2+ increases, the effect of the drug on the increases in [Ca2+]c evoked by IP3 itself (generated by photolysis of a caged form) was examined. At –70 mV, IP3 evoked approximately reproducible increases in [Ca2+]c the amplitudes of which were not significantly altered by ryanodine (50 μM; 1.98 ± 0.37 F/F0 before, compared with 2.02 ± 0.44 F/F0 after ryanodine; n= 5; P > 0.05) (Fig. 2A). This indicates that the Ca2+ increase initially evoked by IP3 did not activate RyRs subsequently, i.e. there was no CICR at RyRs in these cells; had there been so, the RyR ‘blocker’ ryanodine would have reduced IP3-evoked Ca2+ transients. However, at –70 mV, while ineffective by itself, ryanodine blocked IP3-mediated Ca2+ release after RyRs had been activated by caffeine (Fig. 2B). It was again first confirmed that IP3 evoked approximately reproducible increases in [Ca2+]c (Fig. 2B). Then in the continued presence of ryanodine, caffeine on first application, increased [Ca2+]c but thereafter this increase to caffeine was abolished suggesting that ryanodine binds to RyRs only after the channel had been activated. Significantly, after the response to caffeine was lost, the subsequent responses to IP3 were also blocked (Fig. 2B). In controls in a previous study (Flynn et al. 2001) in this cell type in the absence of ryanodine, IP3-evoked [Ca2+]c increases after caffeine were restored within 60 s. These experiments show that while ryanodine by itself does not inhibit IP3-mediated Ca2+ release it can do so after RyRs have been activated. The block of IP3-mediated Ca2+ responses by ryanodine, after caffeine, may arise from depletion of a SR Ca2+ store to which IP3Rs and RyRs have common access (Flynn et al. 2001) to limit the amount of Ca2+ available for release by IP3.
, 百拇医药
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.
, 百拇医药
To confirm the requirement of prior activation of RyRs for ryanodine to block IP3-mediated Ca2+ release, cells depolarized to –20 mV were examined. At this membrane potential the [Ca2+]c is increased; this in turn increases the SR content as revealed by the increased magnitudes of the IP3- and the caffeine-evoked Ca2+ transients (Fig. 3A and B). The IP3-evoked Ca2+ transient increased from 2.53 ± 0.17 F/F0 (at –70 mV) to 4.06 ± 0.35 F/F0 (at –20 mV) (n= 3; P < 0.05). The caffeine-evoked Ca2+ transient increased from 0.99 ± 0.17 F/F0 (at –70 mV) to 1.87 ± 0.42 F/F0 (at –20 mV) (n= 9; P < 0.05). The increased Ca2+ transients occurred as a result of an increased SR Ca2+ content and not from an increased [Ca2+]c because on repolarization to –70 mV, [Ca2+]c returned towards resting levels, but the IP3- and caffeine-evoked Ca2+ transients (a measure of store content) initially remained significantly increased (IP3 4.56 ± 0.18 F/F0, n= 3; caffeine 1.76 ± 0.3 F/F0; n= 9) before being restored to control values with a delay of 5 min (Fig. 3A and B).
, 百拇医药
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.
, 百拇医药
Interestingly an increased SR Ca2+ content as occurs upon sarcolemma depolarization (from –70 mV to –20 mV) and characterizes ‘store overload’ conditions itself activates RyRs (Fig. 3C) (Trafford et al. 1995; Cheng et al. 1996; Lukyanenko et al. 1999). RyR activity is evident in the occurrence of spontaneous transient outward currents (STOCs, Fig. 3C and D; Benham & Bolton, 1986). In this present study STOCs were significantly reduced in amplitude by ryanodine (Fig. 3D) confirming the need for RyRs in their generation.
, 百拇医药
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).
, http://www.100md.com
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)).
, 百拇医药
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.
Tetracaine, a local anaesthetic, which has also RyR blocking activity, inhibited IP3-mediated Ca2+ release. Again, submaximal responses were examined in each cell after the lamp energy was reduced. The maximal response to IP3 in these cells was 1.63 ± 0.56 F/F0, which was reduced to 55 ± 5% of this value to 0.95 ± 0.36 F/F0 when the lamp energy was reduced. Tetracaine (100 μM) significantly (P < 0.05) decreased submaximal IP3-mediated Ca2+ release by 37 ± 4% (F/F0 from 0.95 ± 0.36 to 0.64 ± 0.29, n= 7, Fig. 5A) but not those responses to maximal IP3 (Fig. 5B). In a separate series of experiments, maximal responses to IP3 were, however, significantly reduced by 77 ± 6% at 1 mM tetracaine from a F/F0 of 1.55 ± 0.54 to 0.23 ± 0.05 in tetracaine (n= 6; P < 0.05; Fig. 5C).
, 百拇医药
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
CCh-evoked Ca2+ waves and the [Ca2+]c increases evoked by IP3, in guinea-pig colonic myocytes, arise from the activity of IP3Rs alone; RyRs, via the process of CICR, do not contribute to the Ca2+ increases evoked by IP3. Nonetheless, acknowledged inhibitors of RyRs (ryanodine, tetracaine and dantrolene) inhibit IP3-mediated [Ca2+]c increases. The inhibition does not arise from the involvement of RyRs in the IP3-evoked Ca2+ increase but, rather, from the drugs' ability to block IP3-mediated Ca2+ release either directly (dantrolene and tetracaine) or indirectly (ryanodine).
, 百拇医药
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).
, 百拇医药
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.
, http://www.100md.com
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.
, 百拇医药
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.
, 百拇医药
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).
, 百拇医药
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.
, http://www.100md.com
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.
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.
Berridge MJ (2005). Unlocking the secrets of cell signalling. Annu Rev Physiol 67, 1–21.
, 百拇医药
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.
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.
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.
, 百拇医药
Cheng H, Lederer MR, Lederer WJ & Cannell MB (1996). Calcium sparks and [Ca2+]c waves in cardiac myocytes. Am J Physiol 270, C148–C159.
DeLisle S & Welsh MJ (1992). Inositol trisphosphate is required for the propagation of calcium waves in Xenopus oocytes. J Biol Chem 267, 7963–7966.
Flynn ERM, Bradley KN, Muir TC & McCarron JG (2001). Functionally-separate intracellular Ca2+ stores in smooth muscle. J Biol Chem 276, 36411–36418.
, http://www.100md.com
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.
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.
Gunter TE, Buntinas L, Sparagna G, Eliseev R & Gunter K (2000). Mitochondrial calcium transport: mechanisms and functions. Cell Calcium 28, 285–296.
, 百拇医药
Hajnoczky G & Thomas AP (1997). Minimal requirements for calcium oscillations driven by the IP3 receptor. EMBO J 16, 3533–3543.
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.
, 百拇医药
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.
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.
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.
, 百拇医药
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.
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.
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.
, 百拇医药
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.
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.
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.
, http://www.100md.com
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.
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.
Komai H & McDowell TS (2001). Local anesthetic inhibition of voltage-activated potassium currents in rat dorsal root ganglion neurons. Anesthesiology 94, 1089–1095.
, 百拇医药
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.
Lai FA & Meissner G (1989). The muscle ryanodine receptor and its intrinsic Ca2+ channel activity. J Bioener Biomem 21, 227–246.
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.
, 百拇医药
Lechleiter JD & Clapham DE (1992). Molecular mechanisms of intracellular calcium excitability in X. laevis oocytes. Cell 69, 283–294.
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.
Makarewicz D, Zieminska E & Lazarewicz JW (2003). Dantrolene inhibits NMDA-induced 45Ca uptake in cultured cerebellar granule neurons. Neurochem Int 43, 273–278.
, 百拇医药
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.
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.
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.
, 百拇医药
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.
McGeown JG (2004). Interactions between inositol 1,4,5-trisphosphate receptors and ryanodine receptors in smooth muscle: one store or twoCell Calcium 35, 613–619.
Meissner G (1986). Ryanodine activation and inhibition of the Ca2+ release channel of sarcoplasmic reticulum. J Biol Chem 261, 6300–6306.
, 百拇医药
Meissner G (2002). Regulation of mammalian ryanodine receptors. Front Biosci 7, d2072–d2080.
Nagasaki K & Fleischer S (1988). Ryanodine sensitivity of the calcium release channel of sarcoplasmic reticulum. Cell Calcium 9, 1–7.
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.
, 百拇医药
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.
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.
, http://www.100md.com
Rousseau E, Smith JS & Meissner G (1987). Ryanodine modifies conductance and gating behavior of single Ca2+ release channel. Am J Physiol 253, C364–C368.
Ruehlmann DO, Lee C-H, Poburko D & vanBreemen C (2000). Asynchronous Ca2+ waves in intact venous smooth muscle. Circ Res 86, e72–e79.
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.
, http://www.100md.com
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.
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.
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.
, 百拇医药
Wakui M, Potter BVL & Petersen OH (1989). Pulsatile intracellular calcium release does not depend on fluctuations in inositol trisphosphate concentration. Nature 339, 317–320.
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.
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.
, 百拇医药
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.
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.
, 百拇医药
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.
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.
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., 百拇医药(Debbi MacMillan, Susan Ch)
Abstract
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.
, http://www.100md.com
Introduction
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).
, 百拇医药
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.
, 百拇医药
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).
, 百拇医药
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.
, http://www.100md.com
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.
, http://www.100md.com
Methods
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
, 百拇医药
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.
, http://www.100md.com
[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.
, http://www.100md.com
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.
, 百拇医药
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.
, 百拇医药
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.
, 百拇医药
Results
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.
, http://www.100md.com
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).
, 百拇医药
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.
, 百拇医药
To investigate the mechanism by which changes in sarcolemma potential may modulate ryanodine's ability to block IP3-mediated Ca2+ increases, the effect of the drug on the increases in [Ca2+]c evoked by IP3 itself (generated by photolysis of a caged form) was examined. At –70 mV, IP3 evoked approximately reproducible increases in [Ca2+]c the amplitudes of which were not significantly altered by ryanodine (50 μM; 1.98 ± 0.37 F/F0 before, compared with 2.02 ± 0.44 F/F0 after ryanodine; n= 5; P > 0.05) (Fig. 2A). This indicates that the Ca2+ increase initially evoked by IP3 did not activate RyRs subsequently, i.e. there was no CICR at RyRs in these cells; had there been so, the RyR ‘blocker’ ryanodine would have reduced IP3-evoked Ca2+ transients. However, at –70 mV, while ineffective by itself, ryanodine blocked IP3-mediated Ca2+ release after RyRs had been activated by caffeine (Fig. 2B). It was again first confirmed that IP3 evoked approximately reproducible increases in [Ca2+]c (Fig. 2B). Then in the continued presence of ryanodine, caffeine on first application, increased [Ca2+]c but thereafter this increase to caffeine was abolished suggesting that ryanodine binds to RyRs only after the channel had been activated. Significantly, after the response to caffeine was lost, the subsequent responses to IP3 were also blocked (Fig. 2B). In controls in a previous study (Flynn et al. 2001) in this cell type in the absence of ryanodine, IP3-evoked [Ca2+]c increases after caffeine were restored within 60 s. These experiments show that while ryanodine by itself does not inhibit IP3-mediated Ca2+ release it can do so after RyRs have been activated. The block of IP3-mediated Ca2+ responses by ryanodine, after caffeine, may arise from depletion of a SR Ca2+ store to which IP3Rs and RyRs have common access (Flynn et al. 2001) to limit the amount of Ca2+ available for release by IP3.
, 百拇医药
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.
, 百拇医药
To confirm the requirement of prior activation of RyRs for ryanodine to block IP3-mediated Ca2+ release, cells depolarized to –20 mV were examined. At this membrane potential the [Ca2+]c is increased; this in turn increases the SR content as revealed by the increased magnitudes of the IP3- and the caffeine-evoked Ca2+ transients (Fig. 3A and B). The IP3-evoked Ca2+ transient increased from 2.53 ± 0.17 F/F0 (at –70 mV) to 4.06 ± 0.35 F/F0 (at –20 mV) (n= 3; P < 0.05). The caffeine-evoked Ca2+ transient increased from 0.99 ± 0.17 F/F0 (at –70 mV) to 1.87 ± 0.42 F/F0 (at –20 mV) (n= 9; P < 0.05). The increased Ca2+ transients occurred as a result of an increased SR Ca2+ content and not from an increased [Ca2+]c because on repolarization to –70 mV, [Ca2+]c returned towards resting levels, but the IP3- and caffeine-evoked Ca2+ transients (a measure of store content) initially remained significantly increased (IP3 4.56 ± 0.18 F/F0, n= 3; caffeine 1.76 ± 0.3 F/F0; n= 9) before being restored to control values with a delay of 5 min (Fig. 3A and B).
, 百拇医药
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.
, 百拇医药
Interestingly an increased SR Ca2+ content as occurs upon sarcolemma depolarization (from –70 mV to –20 mV) and characterizes ‘store overload’ conditions itself activates RyRs (Fig. 3C) (Trafford et al. 1995; Cheng et al. 1996; Lukyanenko et al. 1999). RyR activity is evident in the occurrence of spontaneous transient outward currents (STOCs, Fig. 3C and D; Benham & Bolton, 1986). In this present study STOCs were significantly reduced in amplitude by ryanodine (Fig. 3D) confirming the need for RyRs in their generation.
, 百拇医药
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).
, http://www.100md.com
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)).
, 百拇医药
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.
Tetracaine, a local anaesthetic, which has also RyR blocking activity, inhibited IP3-mediated Ca2+ release. Again, submaximal responses were examined in each cell after the lamp energy was reduced. The maximal response to IP3 in these cells was 1.63 ± 0.56 F/F0, which was reduced to 55 ± 5% of this value to 0.95 ± 0.36 F/F0 when the lamp energy was reduced. Tetracaine (100 μM) significantly (P < 0.05) decreased submaximal IP3-mediated Ca2+ release by 37 ± 4% (F/F0 from 0.95 ± 0.36 to 0.64 ± 0.29, n= 7, Fig. 5A) but not those responses to maximal IP3 (Fig. 5B). In a separate series of experiments, maximal responses to IP3 were, however, significantly reduced by 77 ± 6% at 1 mM tetracaine from a F/F0 of 1.55 ± 0.54 to 0.23 ± 0.05 in tetracaine (n= 6; P < 0.05; Fig. 5C).
, 百拇医药
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
CCh-evoked Ca2+ waves and the [Ca2+]c increases evoked by IP3, in guinea-pig colonic myocytes, arise from the activity of IP3Rs alone; RyRs, via the process of CICR, do not contribute to the Ca2+ increases evoked by IP3. Nonetheless, acknowledged inhibitors of RyRs (ryanodine, tetracaine and dantrolene) inhibit IP3-mediated [Ca2+]c increases. The inhibition does not arise from the involvement of RyRs in the IP3-evoked Ca2+ increase but, rather, from the drugs' ability to block IP3-mediated Ca2+ release either directly (dantrolene and tetracaine) or indirectly (ryanodine).
, 百拇医药
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).
, 百拇医药
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.
, http://www.100md.com
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.
, 百拇医药
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.
, 百拇医药
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).
, 百拇医药
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.
, http://www.100md.com
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.
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.
Berridge MJ (2005). Unlocking the secrets of cell signalling. Annu Rev Physiol 67, 1–21.
, 百拇医药
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.
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.
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.
, 百拇医药
Cheng H, Lederer MR, Lederer WJ & Cannell MB (1996). Calcium sparks and [Ca2+]c waves in cardiac myocytes. Am J Physiol 270, C148–C159.
DeLisle S & Welsh MJ (1992). Inositol trisphosphate is required for the propagation of calcium waves in Xenopus oocytes. J Biol Chem 267, 7963–7966.
Flynn ERM, Bradley KN, Muir TC & McCarron JG (2001). Functionally-separate intracellular Ca2+ stores in smooth muscle. J Biol Chem 276, 36411–36418.
, http://www.100md.com
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.
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.
Gunter TE, Buntinas L, Sparagna G, Eliseev R & Gunter K (2000). Mitochondrial calcium transport: mechanisms and functions. Cell Calcium 28, 285–296.
, 百拇医药
Hajnoczky G & Thomas AP (1997). Minimal requirements for calcium oscillations driven by the IP3 receptor. EMBO J 16, 3533–3543.
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.
, 百拇医药
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.
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.
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.
, 百拇医药
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.
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.
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.
, 百拇医药
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.
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.
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.
, http://www.100md.com
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.
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.
Komai H & McDowell TS (2001). Local anesthetic inhibition of voltage-activated potassium currents in rat dorsal root ganglion neurons. Anesthesiology 94, 1089–1095.
, 百拇医药
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.
Lai FA & Meissner G (1989). The muscle ryanodine receptor and its intrinsic Ca2+ channel activity. J Bioener Biomem 21, 227–246.
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.
, 百拇医药
Lechleiter JD & Clapham DE (1992). Molecular mechanisms of intracellular calcium excitability in X. laevis oocytes. Cell 69, 283–294.
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.
Makarewicz D, Zieminska E & Lazarewicz JW (2003). Dantrolene inhibits NMDA-induced 45Ca uptake in cultured cerebellar granule neurons. Neurochem Int 43, 273–278.
, 百拇医药
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.
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.
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.
, 百拇医药
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.
McGeown JG (2004). Interactions between inositol 1,4,5-trisphosphate receptors and ryanodine receptors in smooth muscle: one store or twoCell Calcium 35, 613–619.
Meissner G (1986). Ryanodine activation and inhibition of the Ca2+ release channel of sarcoplasmic reticulum. J Biol Chem 261, 6300–6306.
, 百拇医药
Meissner G (2002). Regulation of mammalian ryanodine receptors. Front Biosci 7, d2072–d2080.
Nagasaki K & Fleischer S (1988). Ryanodine sensitivity of the calcium release channel of sarcoplasmic reticulum. Cell Calcium 9, 1–7.
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.
, 百拇医药
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.
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.
, http://www.100md.com
Rousseau E, Smith JS & Meissner G (1987). Ryanodine modifies conductance and gating behavior of single Ca2+ release channel. Am J Physiol 253, C364–C368.
Ruehlmann DO, Lee C-H, Poburko D & vanBreemen C (2000). Asynchronous Ca2+ waves in intact venous smooth muscle. Circ Res 86, e72–e79.
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.
, http://www.100md.com
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.
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.
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.
, 百拇医药
Wakui M, Potter BVL & Petersen OH (1989). Pulsatile intracellular calcium release does not depend on fluctuations in inositol trisphosphate concentration. Nature 339, 317–320.
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.
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.
, 百拇医药
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.
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.
, 百拇医药
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.
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.
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., 百拇医药(Debbi MacMillan, Susan Ch)