HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by McCarron, J. G. Articles by Muir, T. C. Search for Related Content PubMed PubMed Citation Articles by McCarron, J. G. Articles by Muir, T. C.

Two Ca²⁺ entry pathways mediate InsP₃-sensitive store refilling in guinea-pig colonic smooth muscle

John G. McCarron, Elaine R. M. Flynn, Karen N. Bradley and Thomas C. Muir

MS 0434 Received 13 December 1999; accepted after revision 23 February 2000.

	ABSTRACT

Top Abstract Introduction Methods Results Discussion References

1. Sarcolemma Ca²⁺ influx, necessary for store refilling, was well maintained, over a wide range (-70 to + 40 mV) of membrane voltages, in guinea-pig single circular colonic smooth muscle cells, as indicated by the magnitude of InsP₃-evoked Ca²⁺ transients.

2. This apparent voltage independence of store refilling was achieved by the activity of sarcolemma Ca²⁺ channels some of which were voltage gated while others were not. At negative membrane potentials (e.g. -70 mV), Ca²⁺ influx through channels which lacked voltage gating provided for store refilling while at positive membrane potentials (e.g. +40 mV) voltage-gated Ca²⁺ channels were largely responsible.

3. Sarcolemma voltage-gated Ca²⁺ currents were not activated following store depletion.

4. Removal of external Ca²⁺ or the addition of the Ca²⁺ channel blocker nimodipine (1 µM) inhibited store refilling, as assessed by the magnitude of InsP₃-evoked Ca²⁺ transients, with little or no change in bulk average cytoplasmic Ca²⁺ concentration. One hypothesis for these results is that the store may refill from a high subsarcolemma Ca²⁺ gradient.

5. Influx via channels, some of which are voltage gated and others which lack voltage gating, may permit the establishment of a subsarcolemma Ca²⁺ gradient. Store access to the gradient allows InsP₃-evoked Ca²⁺ signalling to be maintained over a wide voltage range in colonic smooth muscle.

	INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

In both excitable and non-excitable cells, activation of G-protein-coupled or tryosine-kinase-linked receptors releases Ca²⁺ from an internal store by generating inositol 1,4,5-trisphosphate (InsP₃). In non-excitable cells, such as lymphocytes and epithelial cells, depletion of this InsP₃-sensitive store triggers a Ca²⁺ influx across the sarcolemma in a process known as 'store-operated Ca²⁺ entry' (Putney, 1986; Clapham, 1995). This process, it is generally agreed, replenishes the store. The signalling pathway that relays the Ca²⁺ status of the internal store remains unclear (Randriamampita & Tsien, 1993; Kiselyov et al. 1998; Boulay et al. 1999; Hofmann et al. 1999; Csutora et al. 1999).

The precise nature of the sarcolemma channel responsible for store-operated Ca²⁺ entry is also unclear. A number of channels differing from each other both in conductance and in ion selectivity have been implicated (Fasolato et al. 1994; Lewis, 1999); different channels may underlie store refilling in different cell types. Notwithstanding, all sarcolemma store-operated channels lack voltage gating, at least over the wide membrane potential range of -60 to +50 mV (Hoth & Penner, 1993; Luckhoff & Clapham, 1994; Krause et al. 1996; Hofmann et al. 1999; Kerschbaum & Cahalan, 1999).

Despite the absence of voltage gating, changes in membrane potential produce substantial alterations in the Ca²⁺ influx which takes place via store-operated channels (Hoth & Penner, 1993; Luckhoff & Clapham, 1994). This occurs both because of the changes in the electrochemical driving force on the Ca²⁺ ion which accompany changes in membrane potential and because of the inward rectification of some store-operated channels, which will increase Ca²⁺ influx at negative membrane potentials. For example, the unitary current amplitude of the Ca²⁺ release-activated Ca²⁺ channel (I_CRAC; with 20 mM external Ca²⁺ as the charge carrier) decreased from -20 pA at -70 mV to about 0 pA at +40 mV (Kerschbaum & Cahalan, 1999). Not surprisingly, membrane depolarisation can inhibit store refilling in non-excitable cells (Mogami et al. 1997).

In excitable cells, such as smooth muscle, although membrane potential varies widely and almost continuously, store refilling is completely dependent on the influx of Ca²⁺ across the sarcolemma. In some excitable cells, store-operated sarcolemma currents have been proposed to account for this influx (Pacaud & Bolton, 1991; Villalobos & Garciasancho, 1995; Wayman et al. 1996; Bennett et al. 1998; Broad et al. 1999; VanGoor et al. 1999). Perhaps the best known proposed store-operated sarcolemma channels are proteins encoded by some members of the trp gene family (transient receptor potential protein, TRP; Hardie & Minke, 1992; Phillip et al. 1996; Zhu et al. 1996; Zitt et al. 1996; Vannier et al. 1999; Warnat et al. 1999) found in many mammalian excitable cells, such as heart and brain, as well as in non-excitable cells (Wes et al. 1995). Although TRP's relationship to other store-operated channels (such as that conducting I_CRAC) is not fully understood (Clapham, 1996; Zitt et al. 1996; Zhu et al. 1998; Kamouchi et al. 1999), assembly of TRP into homotetrameric or heterotetrameric channels with or without TRPL (transient receptor potential protein like) creates diverse Ca²⁺-permeable pathways that are activated by agonists that generate InsP₃. Since TRP proteins lack the positive amino acid sequence thought to act as voltage sensors in other channels, the open probability of TRP channels does not change over physiological voltages - a characteristic of store-operated channels (Montell, 1997; Xu et al. 1997; Kiselyov et al. 1998; Hofmann et al. 1999).

In addition to its reduction of the driving force on the Ca²⁺ ion, depolarisation also increases the open probability of voltage-gated Ca²⁺ channels in excitable cells. In some excitable cells, voltage-gated Ca²⁺ channels maintain the Ca²⁺ store content (Stojikovic et al. 1992; Kukuljan et al. 1994; Jaffe & Brown, 1994). For example, in hippocampal neurons, trains of action potentials, which activate voltage-gated Ca²⁺ channels, refill the store (Jaffe & Brown, 1994). However, at negative membrane potentials (e.g. -70 mV) where voltage-gated Ca²⁺ channels are largely closed, store refilling via voltage-gated channels may be limited and this restriction could inhibit InsP₃-mediated Ca²⁺ signalling (Jaffe & Brown, 1994; Kukuljan et al. 1994).

The mechanisms that control Ca²⁺ entry into the InsP₃-sensitive internal store in colonic smooth muscle, we now report, involve Ca²⁺ channels some of which are voltage gated and others which are not. Inhibition of Ca²⁺ influx reduces the InsP₃ transient with little or no alteration in bulk average cytosolic Ca²⁺ concentration ([Ca²⁺]_c). The influx of Ca²⁺ via these two pathways, we propose, generates a high subsarcolemma Ca²⁺ concentration to which the store has access and which allows refilling over a wide membrane potential range. A preliminary account of these observations has been given (McCarron et al. 1999).

	METHODS

Top Abstract Introduction Methods Results Discussion References

From male guinea-pigs, stunned by a blow to the head and killed by exsanguination, single smooth muscle cells were dissociated enzymatically from strips of colonic circular muscle (McCarron & Muir, 1999). Membrane currents were measured using conventional tight seal whole-cell recording techniques. The extracellular solution contained (mM): sodium glutamate 80, NaCl 40, tetraethylammonium chloride 20, MgCl₂ 1·1, CaCl₂ 3, Hepes 10, and glucose 10 (pH 7·4 with 1 M NaOH). Unless otherwise stated, 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, guanosine triphosphate 0·5, Hepes 30, and either 0·15 mM fluo-3 penta-ammonium salt and 0·050 mM caged InsP₃ trisodium salt or 0·05 mM fura-2. Whole-cell currents were amplified by an Axopatch-1D amplifier (Axon Instruments, Foster City, CA, USA), low-pass filtered at 500 Hz (8-pole bessel filter; Frequency Devices, Haverhill, MA, USA), digitally sampled at 1·5 kHz using a digidata interface, pCLAMP software (version 6.0.1, Axon Instruments) and Axotape (Axon Instruments), and stored for analysis.

[Ca²⁺]_c was measured using the membrane-impermeable dyes fluo-3 (penta-ammonium salt) or fura-2 (pentapotassium salt) introduced into the cell from the patch pipette. Fluorescence measurements were made using a microfluorimeter which consisted of an inverted fluorescence microscope (Nikon diaphot) and a photomultiplier tube with a bi-alkali photocathode as described previously (Kamishima & McCarron, 1996; McCarron & Muir, 1999). To photolyse caged InsP₃ the output of a xenon flash lamp (Optoelektronik, Hamburg, Germany) was passed through a UG-5 filter to select for ultraviolet light and merged into the excitation light path. The fluorescence changes which occurred with fluo-3 varied with cell contraction or movement; these rendered the small or slow fluorescence changes difficult to interpret. To obtain accurate [Ca²⁺]_c measurements fura-2 (0·05 mM) was used (McCarron & Muir, 1999). Background was determined with the electrode in the 'on-cell' configuration and was subtracted from the fluorescence counts obtained during the experiments. The K_d for fura-2 was determined to be 280 nM from an in vitro calibration. R_min and R_max were also determined from in vitro calibrations and decreased by 15 % to adjust for cell viscosity (Poenie, 1990). No filtering, smoothing or averaging was carried out on the original recordings. Unless otherwise stated all experimental procedures were carried out at room temperature (18-22°C).

Fluo-3 fluorescence signals were expressed as ratios (F/F₀ or F/F₀) of fluorescence counts (F) relative to control values before stimulation (F₀). The statistical analysis used Student's t test applied to the raw data. The results are expressed as means ± S.E.M. of n cells with a value of P < 0·05 considered significant. Averages were derived from single transients each from a minimum of four different cells.

Fura-2 pentapotassium salt and fluo-3 penta-ammonium salt were purchased from Molecular Probes, Inc. (Eugene, OR, USA). Caged Ins(1,4,5)P₃-trisodium salt and thapsigargin were purchased from Calbiochem-Novabiochem Ltd. All other reagents were purchased from Sigma UK.

	RESULTS

Top Abstract Introduction Methods Results Discussion References

Increases in [Ca²⁺]_c were evoked either by depolarisation (-70 to 0 mV) or by flash photolysis of caged InsP₃ (Fig. 1A). In a further series of experiments the InsP₃-evoked (but not the depolarisation-evoked) increase in [Ca²⁺]_c was almost abolished by thapsigargin (1 µM), an inhibitor of the sarcoplasmic reticulum Ca²⁺-ATPase pump (not shown), indicating that InsP₃ liberated Ca²⁺ from the internal store. Thus, flash release of InsP₃ evoked an averaged increase, over baseline, of 1·53 ± 0·2 F/F₀ units before and 0·06 ± 0·01 F/F₀ units after thapsigargin (n = 4) while a depolarisation to 0 mV (from -70 mV) increased F/F₀ to 1·3 ± 0·15 F/F₀ units before and 1·0 ± 0·2 F/F₀ units after thapsigargin in the same four cells.

		View larger version [in this window] [in a new window]
Figure 1. Ca2+ entry and InsP3-evoked increases in [Ca2+]c A, a depolarising pulse to 0 mV (holding potential -70 mV, b) evoked an inward ICa (c) and increased [Ca2+]c (a). In contrast, no inward current (c, left-hand side) accompanied the increase in [Ca2+]c (a, left-hand side) evoked previously by release of InsP3 by a UV flash (arrow in this and subsequent figures). B, Ca2+ transients of approximately equal magnitude (a) accompanied the release of InsP3 by UV flashes (arrows) in 3 mM external Ca2+. Removal of external Ca2+ (Ca2+-free bathing solution containing 1 mM EGTA and 3 mM MgCl2, within vertical dotted lines) markedly reduced the magnitude of the InsP3-evoked Ca2+ transients, which was restored following re-introduction of external Ca2+. b, a summary of results from 11 experiments under identical conditions. C, as the cell was depolarised (c) from -70 mV, the basal [Ca2+]c increased and peaked at -20 mV (a). However, the InsP3-evoked Ca2+ transients (a) were not reduced in magnitude despite the reduction in electrochemical driving force on the Ca2+ ion. This suggests that Ca2+ entry is independent of membrane voltage over this range (-70 to +40 mV). b, a summary of results from 10 identical experiments.

Inward currents rarely accompanied the flash release of InsP₃ (Fig. 1A). Indeed, they were noted in only 3 out of >200 cells examined and, in two of those, the current 'ran down' during the course of the experiment without any alteration in the InsP₃-evoked Ca²⁺ transient. These latter currents, therefore, were not apparently involved in store refilling. Although inward currents were rarely detected, Ca²⁺ influx across the sarcolemma was essential for store refilling since removal of external Ca²⁺ caused a rapid and reversible loss of the InsP₃-evoked Ca²⁺ transient (Fig. 1B). Consequently, store refilling, which took about 40 s to complete (not shown), relied on Ca²⁺ influx across the sarcolemma rather than recycling of store-released Ca²⁺.

The magnitude of the Ca²⁺ influx is reportedly influenced by changes in membrane potential - a major determinant of the driving force on the ion. For example, depolarisation prevents store refilling in non-excitable cells (e.g. Mogami et al. 1997). However, in contrast, despite its dependence on Ca²⁺ influx, the InsP₃-evoked Ca²⁺ transient, in the present experiments, was well maintained over a wide voltage range (-70 to +40 mV; Fig. 1C). This apparent voltage independence of the InsP₃-evoked Ca²⁺ transient implies, surprisingly, that sarcolemma Ca²⁺ influx itself is apparently independent of changes in membrane potential between -70 and +40 mV. One hypothesis that could account for this finding is that Ca²⁺ influx may involve both voltage-gated and non-voltage-gated channels. With this in mind and to determine the extent of the contribution of each channel to Ca²⁺ store refilling, the relationship between the InsP₃-evoked Ca²⁺ transient and the membrane voltage was examined. The protocol for these experiments was to use a number (14) of UV flashes sufficiently large to examine the role of voltage-gated Ca²⁺ channels in store refilling without depleting the caged InsP₃ or causing cell 'run-down'. The protocol began with a depolarisation (330 ms) from -70 to 0 mV which evoked a voltage-dependent Ca²⁺ current (I_Ca; -311 ± 138 pA, n = 5) and a rise in [Ca²⁺]_c (Fig. 2A). Thereafter, UV flashes were applied at 50 s intervals and evoked reproducible increases in [Ca²⁺]_c. Depolarisation to +40 mV, from -70 mV, initially produced a large rise in [Ca²⁺]_c which subsequently declined towards resting levels as the voltage-gated Ca²⁺ channels inactivated. Indeed, using fura-2 as the indicator, [Ca²⁺]_c at +40 mV (130 ± 20 nM, n = 6) was similar to that at -70 mV (99 ± 21 nM, n = 6; not shown). The magnitude of the InsP₃-evoked Ca²⁺ transient at +40 mV was also similar to that at -70 mV. However, after nimodipine (1 µM), which blocks voltage-gated Ca²⁺ channels (Ferry et al. 1985; McCarthy & Cohen, 1989), the InsP₃-evoked Ca²⁺ transient was reduced significantly (P < 0·05) to a level smaller (38 ± 10%; n = 7) than that seen at -70 mV (Fig. 2A). These results show that voltage-gated Ca²⁺ channels at +40 mV maintain the InsP₃-sensitive Ca²⁺ store content. On repolarisation to -70 mV, the InsP₃-evoked Ca²⁺ transient recovered despite the continued block of voltage-gated Ca²⁺ channels as evidenced by the absence of I_Ca (Fig. 2A, -14 ± 17 pA, n = 5). Therefore, at -70 mV, voltage-gated Ca²⁺ channels do not contribute significantly to the maintenance of the Ca²⁺ content of the store.

		View larger version [in this window] [in a new window]
Figure 2. The effect of nimodipine and of altering membrane potential on ICa and InsP3-evoked Ca2+ transients A and B, depolarisation to 0 mV from -70 mV (c) triggered ICa (d; expanded time axes) and Ca2+ transients (a and b). Aa and Ba are summaries of the increases in Ca2+ over baseline evoked by depolarisation (n = 5) and InsP3 (arrows; A, n = 7; B, n = 6) at different membrane potentials. Subsequent liberation of InsP3 (Ab and Bb, arrows), produced approximately reproducible InsP3-evoked Ca2+ transients. After a prolonged depolarisation to +40 mV (Ac; within vertical dotted lines) or -20 mV (Bc; within vertical dotted lines) the [Ca2+]c initially increased then declined, but the InsP3-evoked Ca2+ transients were not reduced compared to values at -70 mV. In the presence of nimodipine (1 µM), however, and during prolonged depolarisation (Ac, +40 mV; Bc, -20 mV), the InsP3-evoked Ca2+ transients (Ab and Bb) were inhibited after 5 UV flashes by approximately 50 % (Ab) or 25 % (Bb) of the control values at -70 mV. On restoration of the membrane potential to -70 mV (Ab and Bb) , in the continued presence of nimodipine, the InsP3-evoked Ca2+ transients recovered to control values at -70 mV, but importantly, ICa remained inhibited (Ad and Bd). C, nimodipine (1 µM) had little effect on the InsP3-evoked Ca2+ transients when the holding potential was maintained at -70 mV (c) but significantly inhibited ICa (P < 0·05; n = 10).

The protocol was next applied to examine the effects of depolarisation to -20 mV on InsP₃-evoked Ca²⁺ increases. The protocol again began with a depolarisation (330 ms) from -70 to 0 mV evoking I_Ca (-168 ± 54 pA; n = 5) and a rise in [Ca²⁺]_c (Fig. 2B). UV flashes were applied at 50 s intervals and again evoked reproducible increases in [Ca²⁺]_c. When the membrane potential was depolarised from -70 to -20 mV (Fig. 2B; the peak of the steady-state [Ca²⁺]_c-voltage relationship with an averaged steady-state [Ca²⁺]_c was 276 ± 32 nM as measured using fura-2; n = 6, not shown), the [Ca²⁺]_c again increased then declined. The InsP₃-evoked transient was not reduced at -20 mV, compared to that at -70 mV. However, once again the InsP₃-evoked transient was inhibited by nimodipine (1 µM; Fig. 2B). The InsP₃-evoked Ca²⁺ transient presumably decreased as Ca²⁺ was lost from the store but not replenished by influx since voltage-gated Ca²⁺ channels were blocked by nimodipine. After five UV flashes, in the presence of nimodipine (Fig. 2B), the Ca²⁺ transient was significantly smaller than that at -70 mV (Fig. 2B) but the extent of the block by nimodipine was smaller than that seen at +40 mV (Fig. 2A). This was consistent with the larger driving force on Ca²⁺ entry at -20 mV than at +40 mV and suggests that the dihydropyridine-insensitive channel lacks voltage gating. Thus the fifth InsP₃-evoked transient at -20 mV in the presence of nimodipine was 58 ± 10% of the first InsP₃-evoked transient at -70 mV (P < 0·05, n = 6, Fig. 2B). On repolarisation (Fig. 2B), in the continued presence of nimodipine, the transient recovered despite the block of I_Ca (+2 ± 7 pA after nimodipine, n = 5, Fig. 2B d).

At -70 mV, nimodipine (1 µM) had little effect on the InsP₃-evoked Ca²⁺ transient but significantly reduced I_Ca (Fig. 2C; -215 ± 36 pA before and -38 ± 13 pA after nimodipine; n = 10; P > 0·05). This is further evidence that at -70 mV dihydropyridine-sensitive voltage-gated Ca²⁺ channels do not contribute significantly to the maintenance of the Ca²⁺ store content.

Failure of nimodipine to inhibit store refilling at -70 mV may arise because of a reduced effectiveness of the drug at negative membrane potentials where the probability of channel opening and/or inactivation is reduced (Bean, 1984; McCarthy & Cohen, 1989; see Discussion). This possibility requires that nimodipine's block of voltage-dependent channels is very rapid (because I_Ca evoked by depolarisation to 0 mV is inhibited; Fig. 2A-C) and implies that voltage-dependent Ca²⁺ channels alone may replenish the store. To examine this possibility cadmium chloride (100 µM) was used. At -70 mV cadmium blocked the voltage-dependent Ca²⁺ current evoked by depolarisations to 0 mV (-92 ± 32 pA to +46 ± 22 pA; n = 4) but did not reduce the InsP₃-evoked Ca²⁺ transient. Thus before the addition of cadmium the InsP₃-evoked Ca²⁺ transient averaged a 1·4 ± 0·6 F/F₀ ratio increase above baseline (n = 4). In the same four cells, the fifth InsP₃-evoked Ca²⁺ transient after cadmium (100 µM) averaged a 1·4 ± 0·6 F/F₀ ratio increase above baseline. This result supports the proposal that a channel other than the voltage-activated Ca²⁺ channel is involved in store refilling at negative membrane potentials. Together these results suggest the involvement of two Ca²⁺ influx pathways in store refilling.

Since activation of store-operated channels (which lack voltage gating) in the sarcolemma stems from depletion of the InsP₃-sensitive Ca²⁺ store (Berridge, 1995), the question arises as to whether voltage-gated Ca²⁺ channels are also activated by store depletion. That this could be so was suggested by the observation that voltage-gated Ca²⁺ channels replenish the store at depolarised membrane potentials (Fig. 2). The effect of store depletion on the magnitude of I_Ca evoked by depolarisation was, therefore, examined. The magnitude of I_Ca was not increased but, rather, significantly (P < 0·05) decreased during store refilling (Fig. 3A). Thus in six experiments I_Ca was -260 ± 59 pA before and -191 ± 38 pA after InsP₃, a reduction of 24 ± 4 %. I_Ca recovered after an additional 60 s to -229 ± 43 pA. Since I_Ca itself was not activated by store depletion, the depolarisation-evoked Ca²⁺ influx, at -20 and +40 mV, presumably provided the Ca²⁺ necessary for store refilling.

		View larger version [in this window] [in a new window]
Figure 3. The effect of InsP3-evoked Ca2+ release on ICa and the action of BAPTA A, depolarisation from -70 to 0 mV (c) triggered ICa (b and on expanded time axis in d) and raised [Ca2+]c (a). Following an InsP3-evoked Ca2+ transient (b, arrow), ICa evoked by an identical depolarisation (-70 to 0 mV) was reduced (b and d). ICa had fully recovered after about 60 s. Therefore, ICa is not activated but is rather inhibited by a reduction in the store Ca2+ content. B, following addition of the Ca2+ chelator BAPTA (10 mM) to the patch pipette filling solution, the ICa (b) triggered by a depolarisation from -70 to 0 mV (c) was not reduced by a preceding InsP3 release (b, arrow). This suggests that inhibition of ICa (Ad) which follows InsP3-mediated Ca2+ release is Ca2+ dependent. Capacity transients on Ab and Bb have been blanked for clarity.

The inhibition of I_Ca by InsP₃ occurred at a time when the bulk average [Ca²⁺]_c had returned to resting levels but during the period of store refilling (Fig. 3A). Thus just before the first depolarisation, F/F₀ was 1·01 ± 0·02 while after InsP₃, but prior to the second depolarisation, it was 1·03 ± 0·06 and just before the third depolarisation it averaged 1·17 ± 0·04 (n = 6; Fig. 3A). An elevation in bulk average [Ca²⁺]_c seems an unlikely explanation, therefore, for the inhibition of I_Ca. On the other hand, inhibition of I_Ca could have been triggered by the prior elevation in [Ca²⁺]_c produced by InsP₃ or indeed by InsP₃ itself or both. To distinguish among these possibilities, cells were dialysed with the Ca²⁺ chelator BAPTA (10 mM) included in the patch pipette filling solution. In the presence of BAPTA, flash release of InsP₃ did not increase [Ca²⁺]_c nor reduce I_Ca (P > 0·05; Fig. 3B). In six such experiments I_Ca was -223 ± 57 pA before and -217 ± 60 pA 12 s after release of InsP_3. and after a further 60 s I_Ca was -228 ± 56 pA. Thus, although the inhibition of I_Ca by the release of InsP₃ occurred at a time when bulk [Ca²⁺]_c was not elevated, the inhibition is nonetheless a Ca²⁺-dependent process. Perhaps the transient increase in [Ca²⁺]_c by InsP₃ triggered a more persistent signalling pathway which inhibited I_Ca. One possible mechanism could be by activation of a Ca²⁺-dependent protein kinase. However, the InsP₃-triggered, Ca²⁺-dependent inhibition of I_Ca was unaffected by the broad spectrum kinase inhibitor H-7 (10 µM). Thus, before H-7, InsP₃ release significantly (P < 0·05) inhibited I_Ca by 19 ± 2 % while after H-7, I_Ca was significantly (P < 0·05) inhibited by 22 ± 10% in the same four cells.

The reason for the decreased InsP₃-evoked Ca²⁺ transient seen either in Ca²⁺-free bathing solution (Fig. 1B) or in the presence of the Ca²⁺ channel blocker nimodipine (Fig. 2A and B) was next investigated. Store refilling reportedly occurs directly from the cytosol and not via structures linking the store to the extracellular space (Hofer et al. 1998; Mogami et al. 1998). Since the store refills from the cytosol, prevention of Ca²⁺ entry into the cytosol could have inhibited store refilling if a sufficient degree of Ca²⁺ entry was required to maintain the bulk average [Ca²⁺]_c at a level that may operate the Ca²⁺ pump. To examine this possibility, the [Ca²⁺]_c was compared before and after treatment with nimodipine, using fura-2 as the [Ca²⁺]_c indicator. At +40 mV in the presence of nimodipine, [Ca²⁺]_c was little different from that at -70 mV in the absence of the drug (Fig. 4A; P > 0·05). Thus at -70 mV resting [Ca²⁺]_c was 111 ± 13 nM while at +40 mV in the presence of nimodipine it was 112 ± 13 nM (n = 4). This indicates that, at +40 mV, nimodipine prevented store refilling (Fig. 2A) even though bulk average [Ca²⁺]_c remained at the level which occurred at -70 mV and at which store refilling happened. Similarly, removal of external Ca²⁺ produced only a modest, though significant (P < 0·05), fall in [Ca²⁺]_c of 15 ± 2 nM (n = 4; Fig. 4B). In this latter series of experiments, resting [Ca²⁺]_c was 91 ± 11 nM, and in the virtual absence of external Ca²⁺ (i.e. in a Ca²⁺-free bathing solution containing 3 mM Mg²⁺ and 1 mM EGTA) was 76 ± 12 nM. Readmission of external Ca²⁺ (Fig. 4B) resulted in an overshoot of [Ca²⁺]_c to above resting levels, to 166 ± 22 nM (P < 0·05), as found during store-operated Ca²⁺ entry (Berridge, 1995). If it is accepted that store refilling occurs directly from the cytosol, and not through structures linking it to the extracellular space (Hofer et al. 1998; Mogami et al. 1998), the present results indicate that the store has access to a cytosolic Ca²⁺ reservoir which is separate from bulk [Ca²⁺]_c values. One hypothesis to explain this observation is that the store has access to a high subsarcolemma Ca²⁺ concentration.

		View larger version [in this window] [in a new window]
Figure 4. The effects of nimodipine and of Ca2+ removal on depolarisation-evoked increases in [Ca2+]c A, depolarisation (from -70 to +40 mV, b) increased [Ca2+]c (a); this increase was abolished by nimodipine (1 µM; dotted vertical line), which blocks Ca2+ influx via voltage-dependent Ca2+ channels. At +40 mV, in the presence of nimodipine, the [Ca2+]c was not less than that at -70 mV in the absence of nimodipine. On the other hand, when Fig. 3A is compared, the InsP3-evoked Ca2+ transient at +40 mV in the presence of nimodipine was reduced to ~50 % of that at -70 mV in its absence. The reduction in the InsP3-evoked transient therefore appears to be independent of bulk average [Ca2+]c. B, depolarisation from -70 to 0 mV (b) triggered a corresponding ICa (c) and increased [Ca2+]c (a). Removal of external Ca2+ (Ca2+-free bathing solution with 1 mM EGTA and 3 mM MgCl2) caused a modest decrease in [Ca2+]c and abolished ICa and the evoked Ca2+ transient. Restoration of 3 mM Ca2+ to the bathing medium virtually restored both responses. An elevation in [Ca2+]c accompanied this, consistent with the activation of a store-operated Ca2+ influx by the Ca2+-free bathing solution.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

In colonic smooth muscle, refilling of the InsP₃-sensitive store was well maintained over a wide voltage range, despite its dependence on sarcolemma Ca²⁺ influx. This contrasts with the situation in non-excitable cells where depolarisation reduced store refilling (Mogami et al. 1997). The apparent voltage independence of store refilling was achieved by contributions from two overlapping, yet separate, Ca²⁺ influx pathways. These pathways operate so that at negative membrane potentials a dihydropyridine-insensitive channel (which lacks voltage gating) in the sarcolemma supports the Ca²⁺ entry necessary for store refilling, while at positive membrane potentials a dihydropyridine-sensitive, voltage-gated Ca²⁺ channel is responsible. At negative membrane potentials, voltage-gated channels are largely closed but, due to the large driving force on Ca²⁺ entry, influx through channels which lack voltage gating could be substantial. As the cell is depolarised, the driving force on Ca²⁺ decreases and the channel which lacks voltage gating can no longer support store refilling. However, because of the increased open probability of the voltage-gated Ca²⁺ channel which accompanies depolarisation, influx via these channels provides the Ca²⁺ required for store refilling. Despite the requirement for Ca²⁺ influx, a store-operated current was not detected. However, small and difficult to resolve Ca²⁺ currents (estimated to be around 0·5 pA) can evoke increases in bulk average [Ca²⁺]_c in other smooth muscles (Fleischmann et al. 1994; Kamishima & McCarron, 1996). Direct measurement has confirmed the small size of such steady-state Ca²⁺ currents (Langton & Standen, 1993; Rubart et al. 1996). Similarly, store replenishing currents, such as I_CRAC, are also known to be small (1 pA) (Penner et al. 1988; Lewis & Cahalan, 1989; reviewed by Lewis, 1999). The existence of a small store-operated Ca²⁺ current that fell below the detection threshold in the present experiments remains a possibility, albeit in the absence of direct evidence, hypothetical.

In the absence of detectable sarcolemma currents, evidence for store-operated Ca²⁺ entry in this smooth muscle preparation is the observation that, following its removal, readmission of external Ca²⁺ often resulted in a [Ca²⁺]_c overshoot, a feature of store-operated Ca²⁺ entry (Berridge, 1995). The view that channels responsible for store-operated Ca²⁺ entry in colonic myocytes lack voltage gating is supported by the observation that the extent of block (by nimodipine) of the InsP₃-evoked Ca²⁺ transient was smaller at -20 than at +40 mV. The larger driving force on Ca²⁺ entry, via a channel that lacks voltage gating, at -20 mV compared with that at +40 mV could account for this finding.

The basis for the view that dual influx pathways are involved in store refilling rests, in part, on the varying extent of the block produced by the dihydropyridine nimodipine at different membrane potentials. Dihydropyridine block of voltage-activated Ca²⁺ channels is voltage dependent (Bean, 1984; McCarthy & Cohen, 1989). At depolarised membrane potentials, the effectiveness of the drug is increased. To offset the possible complicating effects of depolarisation on the inhibitory effect of nimodipine, in the present experiments, the concentration used was much greater than its dissociation constant for the channel (Ferry et al. 1985; McCarthy & Cohen, 1989). Under these conditions it would be anticipated that the voltage-activated Ca²⁺ channels would be largely blocked. Additionally, the effectiveness of nimodipine in blocking I_Ca was directly monitored. Thus it is unlikely that the failure of nimodipine to reduce InsP₃-evoked Ca²⁺ transients at -70 mV could be explained by the reduced effectiveness of the drug at negative membrane potentials. Additional evidence that Ca²⁺ entry pathways, other than the voltage-dependent Ca²⁺ channel, underlie store refilling at negative membrane potentials comes from the failure of cadmium to prevent store refilling at -70 mV while blocking I_Ca. Cadmium at the concentration used in the present study is a potent blocker of I_Ca but would be expected to have little effect on store-operated currents (Hoth & Penner, 1993; Krause et al. 1996).

Another study carried out in vascular smooth muscle on the refilling of the noradrenaline-sensitive Ca²⁺ store produced results similar to those in the present investigation (Casteels & Droogmans, 1981). The voltage-dependent Ca²⁺ channel antagonists D600 and nicardipine were ineffective in blocking store refilling in normal solutions containing 5·9 mM K⁺, as assessed by the contractile response to noradrenaline. However, after depolarising the tissue, in a high K⁺ bathing solution, the Ca²⁺ channel antagonists reduced the extent of store refilling to a level below that seen in normal K⁺ bathing solution. The authors proposed two pathways for store refilling, (1) a direct pathway from the extracellular space to the store that dominated in normal K⁺ bathing solution and (2) a pathway from the cytoplasm to the store that accounted for refilling in the depolarising high K⁺ bathing solution. It is tempting to propose that Casteels & Droogmans' results could be interpreted as follows: at negative membrane potentials Ca²⁺ influx through channels which lack voltage gating accounts for store refilling while influx via voltage-gated Ca²⁺ channels provides the Ca²⁺ required when the tissue is depolarised. The level of store refilling in the high K⁺ depolarising solution, in the presence of the Ca²⁺ channel blockers, was below that in normal K⁺ bathing solution because of the sole reliance on influx via the channels that lack voltage gating and because of the reduced driving force on Ca²⁺ entry. Studies from an excitable cell line (PC12 cells) have also raised the possibility that store refilling involved both store-operated Ca²⁺ entry and voltage-gated Ca²⁺ entry (Bennett et al. 1998).

While necessary for store refilling at positive membrane potentials, the dihydropyridine-sensitive I_Ca was not activated, but rather inhibited, following the liberation of InsP₃ and during store refilling. The InsP₃-triggered inhibition of I_Ca was Ca²⁺ dependent and abolished by a Ca²⁺ chelator in the patch pipette filling solution. Despite its Ca²⁺ dependence, inhibition of I_Ca persisted even after the bulk average [Ca²⁺]_c had returned to resting levels. Relief of Ca²⁺-dependent inactivation of I_Ca is rapid (1 s), in smooth muscle, at negative membrane potentials (Ganitkevich et al. 1987; Matsuda et al. 1990; Giannattasio et al. 1991) suggesting that other mechanisms may be responsible for the reduction in I_Ca. Several other mechanisms could also account for the reduction in I_Ca. For instance, the increased [Ca²⁺]_c could trigger a more persistent signalling pathway perhaps by activation of a Ca²⁺-dependent protein kinase. In the present experiments this seems unlikely as the broad spectrum kinase inhibitor H-7 failed to reduce the inhibition of I_Ca. This suggests that the inhibition of I_Ca did not involve a Ca²⁺-activated kinase though the involvement of another Ca²⁺-sensitive pathway is not ruled out. Other possibilities come from the observation that InsP₃-generating agonists (ACh and carbachol) reduce I_Ca in other smooth muscles, results attributed to G-protein activation and/or release of Ca²⁺ from the internal store (Unno et al. 1995; Wade et al. 1996; Pucovsky et al. 1998). These mechanisms would also seem unlikely explanations for the inhibition of I_Ca in the present study, because caged InsP₃ rather than membrane receptor interactants was used, and since [Ca²⁺]_c was restored apparently to resting levels at a time when I_Ca was inhibited. Since, in the present experiments, inhibition of I_Ca occurred during the time of store refilling the possibility exists that store-operated Ca²⁺ entry, triggered by the InsP₃-evoked depletion of the store, may contribute to the reduction in I_Ca perhaps by generating a high subsarcolemma Ca²⁺ concentration and partially inactivating I_Ca. The extent of such an inhibition of I_Ca would be expected to decrease with depolarisation as store-operated Ca²⁺ entry decreases.

The route of Ca²⁺ flux across the sarcolemma to the store is unclear. The presence of a 'privileged pathway' for Ca²⁺ entry which connected the lumen of the store to the exterior of the cell (Casteels & Droogmans, 1981; Putney, 1986; Cabello & Schilling, 1993) is now held to be less likely and the current belief is that refilling occurs directly from the cytosol (Hofer et al. 1998; Mogami et al. 1998). Indeed, when the [Ca²⁺]_c was clamped around resting levels, using Ca²⁺ chelators, store refilling occurred even in the absence of external Ca²⁺. On this basis, specialised structures directly linking Ca²⁺ influx to store refilling appear unnecessary (Blatter, 1995; Hofer et al. 1998; Mogami et al. 1998). The present results show that store refilling can be inhibited strongly by Ca²⁺ removal or by nimodipine (at +40 mV) with little or no change in bulk average [Ca²⁺]_c. If Ca²⁺ must traverse the cytosol before entering the store, the present results suggest that the store is sensitive to a cytosolic Ca²⁺ component which is separate from the bulk average [Ca²⁺]_c. One hypothesis is that the store may have access to a subsarcolemma Ca²⁺ concentration, generated during store refilling, which is higher than the bulk average [Ca²⁺]_c. If such a high subsarcolemma Ca²⁺ concentration existed, then inhibition of Ca²⁺ influx could antagonise the gradient without significantly changing the bulk average [Ca²⁺]_c. Increasingly, physiological events are shown to be regulated by the subsarcolemma Ca²⁺ concentration independently of the bulk average [Ca²⁺]_c. Thus subsarcolemma ion concentrations may rise more quickly and to higher levels than those of the bulk cytosol (e.g. Llinas et al. 1992; Etter et al. 1994) giving rise to sarcolemma Ca²⁺-regulated currents that do not follow the averaged [Ca²⁺]_c values (e.g. Vogalis et al. 1992; Ganitkevich & Isenberg, 1996; Imaizumi et al. 1998; Machaca & Hartzell, 1999).

In smooth muscle, regions of the sarcoplasmic reticulum closely appose the sarcolemma (Devine et al. 1972; Moore et al. 1993; Nixon et al. 1994) and may provide a tortuous route for Ca²⁺ diffusion so permitting high subsarcolemma Ca²⁺ concentrations to develop and could facilitate Ca²⁺ uptake into the store (vanBreemen et al. 1995). Peripheral sarcoplasmic reticulum components near the sarcolemma reportedly form a single compartment with the central sarcoplasmic reticulum (Devine et al. 1972; Moore et al. 1993; Nixon et al. 1994). The continuity of peripheral and central sarcoplasmic reticulum may enable replenishment of the entire store from Ca²⁺ entry into a few regions of the store. Consistent with such a structural arrangement, albeit in pancreatic acinar cells (a non-excitable cell type), loading of the internal Ca²⁺ store through store-operated Ca²⁺ entry from a small basal region of the cell permitted an InsP₃-evoked release of Ca²⁺ from the apical region of the cell (Mogami et al. 1997). Thus Ca²⁺ loading of the sarcoplasmic reticulum near the sarcolemma may fully replenish the internal store in smooth muscle.

Presumably, at physiological membrane potentials, in these excitable, spontaneously active cells, the particular Ca²⁺ influx pathway involved in refilling the InsP₃-sensitive store is dependent on the extent and duration of membrane depolarisation. At rest, refilling is largely accomplished by Ca²⁺ influx via channels that lack voltage gating while during excitation, when the membrane is depolarised, voltage-gated Ca²⁺ channels are largely operative. Store refilling over such a wide range of membrane voltages, it is proposed, requires the existence of a Ca²⁺ gradient within the cell which, in turn, may depend on the close structural apposition of cellular organelles (sarcoplasmic reticulum and sarcolemma). The availability of an adequate Ca²⁺ store for normal cell function will therefore depend upon the interplay of membrane potential, sarcolemma Ca²⁺ channels and Ca²⁺ gradients within the cell.

	REFERENCES

Top Abstract Introduction Methods Results Discussion References

Bean, B. P. (1984). Nitrendipine block of cardiac calcium channels: High-affinity binding to the inactivated state. Proceedings of the National Academy of Sciences of the USA 81, 6388-6392	[Medline]
Bennett, D. L., Bootman, M. D., Berridge, M. J. & Cheek, T. R. (1998). Ca2+ entry into PC12 cells initiated by ryanodine or inositol 1,4,5-trisphosphate receptors. Biochemical Journal 329, 349-357	[Medline]
Berridge, M. J. (1995). Capacitative calcium entry. Biochemical Journal 312, 1-11	[Medline]
Blatter, L. A. (1995). Depletion and filling of intracellular calcium stores in vascular smooth muscle. American Journal of Physiology 268, C503-512	[Medline]
Boulay, G., Brown, D. M., Qin, N., Jiang, M., Dietrich, A., Zhu, M. X., Chen, Z., Birnbaumer, M., Mikoshiba, K. & Birnbaumer, L. (1999). Modulation of Ca2+ entry by polypeptides of the inositol 1,4,5-trisphosphate receptor (IP3R) that bind transient receptor potential (TRP): Evidence for roles of TRP and IP3R in store depletion-activated Ca2+ entry. Proceedings of the National Academy of Sciences of the USA 96, 14955-14960	[Abstract/Full Text]
Broad, L. M., Cannon, T. R. & Taylor, C. W. (1999). A non-capacitative pathway activated by arachidonic acid is the major Ca2+ entry mechanism in rat A7r5 smooth muscle cells stimulated with low concentrations of vasopressin. The Journal of Physiology 517, 121-134	[Abstract/Full Text]
Cabello, O. A. & Schilling, W. P. (1993). Vectorial Ca2+ flux from the extracellular space to the endoplasmic reticulum via a restricted cytoplasmic compartment regulates inositol 1,4,5-trisphosphate-stimulated Ca2+ release from the internal stores in endothelial cells. Biochemical Journal 295, 357-366	[Medline]
Casteels, R. & Droogmans, G. (1981). Exchange characteristics of the noradrenaline-sensitive calcium store in vascular smooth muscle cells of the rabbit ear artery. The Journal of Physiology 317, 263-279	[Abstract]
Clapham, D. A. (1995). Intracellular calcium. Replenishing the stores. Nature 375, 634-635	[Medline]
Clapham, D. A. (1996). TRP is cracked but is CRAC TRP? Neuron 16, 1069-1072	[Medline]
Csutora, P., Su, Z., Kim, H. Y., Bugrim, A., Cunningham, K. W., Nuccitelli, R., Keizer, J. E., Hanley, M. R., Blalock, J. E. & Marchase, R. B. (1999). Calcium influx factor is synthesised by yeast and mammalian cells depleted of organellar calcium stores. Proceedings of the National Academy of Sciences of the USA 96, 121-126	[Abstract/Full Text]
Devine, C. E., Somlyo, A. V. & Somlyo, A. P. (1972). Sarcoplasmic reticulum and excitation-contraction coupling in mammalian smooth muscles. Journal of Cell Biology 52, 690-718	[Medline]
Etter, E. F., Kuhn, M. A. & Fay, F. S. (1994). Detection of changes in near-membrane Ca2+ concentration using a novel membrane associated Ca2+ indicator. Proceedings of the National Academy of Sciences of the USA 269, 10141-10149.
Fasolato, C., Innocenti, B. & Pozzan, T. (1994). Receptor-activated Ca2+ influx: how many mechanisms for how many channels. Trends in Pharmacological Sciences 15, 77-83	[Medline]
Ferry, D. R., Glossmann, H. & Kaumann, A. J. (1985). Relationship between the stereoselective negative inotropic effects of verapamil enantiomers and their binding to putative calcium channels in human heart. British Journal of Pharmacology 84, 811-824	[Medline]
Fleischmann, B. K., Murray, R. K. & Kotlikoff, M. I. (1994). Voltage window for sustained elevation of cytoplasmic calcium in smooth muscle cells. Proceedings of the National Academy of Sciences of the USA 91, 11914-11918	[Medline]
Ganitkevich, V. Ya. & Isenberg, G. (1996). Dissociation of subsarcolemmal from global [Ca2+] in myocytes from guinea-pig coronary artery. The Journal of Physiology 490, 305-318	[Abstract]
Ganitkevich, V. Ya., Shuba, M. F. & Smirnov, S. V. (1987). Calcium-dependent inactivation of potential-dependent calcium inward current in single smooth muscle cells of the guinea-pig taenia caeci. The Journal of Physiology 392, 431-449	[Abstract]
Giannattasio, B., Jones, S. W. & Scarpa, A. (1991). Calcium currents in the A7r5 smooth muscle-derived cell line: calcium-dependent and voltage-dependent inactivation. Journal of General Physiolgy 98, 987-1003.
Hardie, R. C. & Minke, B. (1992). The trp gene is essential for a light-activated Ca2+ channel in Drosophila photoreceptors. Neuron 8, 643-651	[Medline]
Hofer, A. M., Landolfi, B., Debellis, L., Pozzan, T. & Curci, S. (1998). Free [Ca2+] dynamics measured in agonist-sensitive stores of single intact cells: a new look at the refilling process. EMBO Journal 17, 1986-1998	[Full Text]
Hofmann, T., Obukhov, A. G., Schaefer, M., Harteneck, C., Gudermann, T. & Schultz, G. (1999). Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol. Nature 397, 259-263	[Medline]
Hoth, M. & Penner, R. (1993). Calcium release-activated calcium current in rat mast cells. The Journal of Physiology 465, 359-386	[Abstract]
Imaizumi, Y., Torii, Y., Ohi, Y., Nagano, N., Atsuki, K., Yamamura, H., Muraki, K., Watanabe, M. & Bolton, T. B. (1998). Ca2+ images and K+ current during depolarization in smooth muscle cells of guinea-pig vas deferens and urinary bladder. The Journal of Physiology 510, 705-719	[Abstract/Full Text]
Jaffe, D. B. & Brown, T. H. (1994). Metabotropic glutamate receptor activation induces calcium waves within hippocampal dendrites. Journal of Neurophysiology 72, 471-474	[Medline]
Kamishima, T. & McCarron, J. G. (1996). Depolarization-evoked increases in cytosolic calcium concentration in isolated smooth muscle cells of rat portal vein. The Journal of Physiology 492, 61-74	[Abstract]
Kamouchi, M., Philipp, S., Flockerzi, V., Wissenbach, U., Mamin, A., Raeymaekers, L., Eggermont, J., Droogmans, G. & Nilius, B. (1999). Properties of heterologously expressed hTRP3 channels in bovine pulmonary artery endothelial cells. The Journal of Physiology 518, 345-358	[Abstract/Full Text]
Kerschbaum, H. H. & Cahalan, M. D. (1999). Single channel recording of a store-operated Ca2+ channel in Jurkat T lymphocytes. Science 283, 836-839	[Abstract/Full Text]
Kiselyov, K., Xu, X., Mozhayeva, G., Kuo, T., Pessah, I., Mignery, G., Zhu, X., Birnbaumer, L. & Muallem, S. (1998). Functional interaction between InsP3 receptors and store-operated Htrp3 channels. Nature 396, 478-482	[Medline]
Krause, E., Pfeiffer, F., Schmid, A. & Schulz, I. (1996). Depletion of intracellular stores activates a calcium conducting nonselective cation current in mouse pancreatic acinar cells. Journal of Biological Chemistry 271, 32523-32528	[Abstract/Full Text]
Kukuljan, M., Rojas, E., Catt, K. J. & Stojilkovic, S. S. (1994). Membrane potential regulates inositol 1,4,5-trisphosphate-controlled cytoplasmic Ca2+ oscillations in pituitary gonadotrophs. Journal of Biological Chemistry 269, 4860-4865	[Abstract]
Langton, P. D. & Standen, N. B. (1993). Calcium currents elicited by voltage steps and steady voltages in myocytes isolated from the rat basilar artery. The Journal of Physiology 469, 535-548	[Abstract]
Lewis, R. S. (1999). Store-operated calcium channels. Advances in Second Messenger and Phosphoprotein Research 33, 279-307.	[Medline]
Lewis, R. S. & Cahalan, M. D. (1989). Mitogen-induced oscillations of cytosolic Ca2+ and transmembrane Ca2+ current in human leukemic T cells. Cell Regulation 1, 99-112	[Medline]
Llinas, R. R., Sugimori, M. & Silver, R. B. (1992). Microdomains of high calcium concentration in a presynaptic terminal. Science 256, 677-679	[Medline]
Luckhoff, A. & Clapham, D. E. (1994). Calcium channels activated by depletion of internal calcium stores in A431 cells. Biophysical Journal 67, 177-182	[Abstract]
McCarron, J. G., Flynn, E. R. M., Bradley K. N. & Muir, T. C. (1999). InsP3-sensitive Ca2+ store refilling in colonic smooth muscle. The Journal of Physiology 521.P, 4S.
McCarron, J. G. & Muir, T. C. (1999). Mitochondrial regulation of the cytosolic Ca2+ concentration and the InsP3-sensitive store in guinea-pig colonic smooth muscle. The Journal of Physiology 516, 149-161	[Abstract/Full Text]
McCarthy, R. T. & Cohen, C. J. (1989). Nimodipine block of calcium channels in rat vascular smooth muscle cell lines. Journal of General Physiology 94, 669-692	[Abstract]
Machaca, K. & Hartzell, H. C. (1999). Reversible Ca gradients between the subplasmalemma and cytosol differentially activate Ca-dependent Cl currents. Journal of General Physiology 113, 249-266	[Abstract/Full Text]
Matsuda, J. J., Volk, K. A. & Shibata, E. F. (1990). Calcium currents in isolated rabbit coronary arterial smooth muscle myocytes. The Journal of Physiology 427, 657-680	[Abstract]
Mogami, H., Nakano, K., Tepilin, A. V. & Petersen, O. H. (1997). Ca2+ flow via tunnels in polarised cells: recharging of apical Ca2+ stores by focal Ca2+ entry through basal membrane patch. Cell 88, 49-55	[Medline]
Mogami, H., Tepikin, A. V. & Petersen, O. H. (1998). Termination of cytosolic Ca2+ signals: Ca2+ reuptake into intracellular stores is regulated by free Ca2+ concentration in the store lumen. EMBO Journal 17, 435-442	[Abstract/Full Text]
Montell, C. (1997). New light on TRP and TRPL. Molecular Pharmacology 52, 755-763	[Abstract/Full Text]
Moore, E. D. W., Etter, E. F., Phillipson, K. D., Carrington, W. A., Fogarty, K. E., Lifshitz, L. M. & Fay, F. S. (1993). Coupling of the Na+/Ca2+ exchanger, Na+/K+ pump and the sarcoplasmic reticulum in smooth muscle. Nature 365, 657-660	[Medline]
Nixon, G. F., Mignery, G. A. & Somlyo, A. V. (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. Journal of Muscle Research and Cell Motility 15, 682-700	[Medline]
Pacaud, P. & Bolton, T. B. (1991). Relation between muscarinic receptor cation current and internal calcium in guinea-pig jejunal smooth muscle cells. The Journal of Physiology 441, 477-499	[Abstract]
Penner, R., Matthews, G. & Neher, E. (1988). Regulation of calcium influx by second messengers in rat mast cells. Nature 334, 499-504	[Medline]
Phillip, S., Cacalie, A., Freuchel, M., Wissenbach, U., Zimmer, S., Trost, C., Marquart, A., Murakami, M. & Flockerzi, V. (1996). A mammalian capacitative calcium entry channel homologous to Drosophila TRP and TRPL. EMBO Journal 15, 6166-6171	[Abstract]
Poenie, M. (1990). Alteration of intracellular fura-2 fluorescence by viscosity. A simple correction. Cell Calcium 11, 85-91	[Medline]
Pucovsky, V., Zholos, A. V. & Bolton, T. B. (1998). Muscarinic cation current and suppression of Ca2+ current in guinea-pig ileal smooth muscle cells. European Journal of Pharmacology 346, 323-330	[Medline]
Putney, J. W. (1986). A model for receptor-regulated calcium entry. Cell Calcium 7, 1-12	[Medline]
Randriamampita, C. & Tsien, R. Y. (1993). Emptying of intracellular Ca2+ stores releases a novel small messenger that stimulates Ca2+ influx. Nature 364, 809-814	[Medline]
Rubart, M., Patlack, J. B. & Nelson, M. T. (1996). Ca2+ currents in cerebral artery smooth muscle cells of rat at physiological Ca2+ concentrations. Journal of General Physiology 107, 459-472	[Abstract]
Stojikovic, S. S, Kukuljan, M., Iida, T., Rojas, E. & Catt, K. J. (1992). Integration of cytoplasmic calcium and membrane potential oscillations maintains calcium signalling in pituitary gonadotrophs. Proceedings of the National Academy of Sciences of the USA 89, 4081-4085	[Abstract]
Unno, T., Komori, S. & Ohashi, H. (1995). Inhibitory effect of muscarinic receptor activation on Ca2+ channel current in smooth muscle cells of guinea-pig ileum. The Journal of Physiology 484, 567-581	[Abstract]
vanBreemen, C., Chen, Q. & Laher, I. (1995). Superficial buffer barrier function of smooth muscle sarcoplasmic reticulum. Trends in Pharmacological Sciences 16, 98-105	[Medline]
VanGoor, F., Krsmanovic, L. Z., Catt, K. J. & Stojilkovic, S. S. (1999). Coordinate regulation of gonadotropin-releasing hormone neuronal firing patterns by cytoplasmic calcium and store depletion. Proceedings of the National Academy of Sciences of the USA 96, 4101-4106	[Abstract/Full Text]
Vannier, B., Peyton, M., Boulay, G., Brown, D., Qin, N., Jiang, M., Zhu, X. & Birnbaumer, L. (1999). Mouse trp2, the homologue of the human trpc2 pseudogene, encodes mTrp2, a store depletion-activated capacitative Ca2+ entry channel. Proceedings of the National Academy of Sciences of the USA 96, 2060-2064	[Abstract/Full Text]
Villalobos, C. & Garciasancho, J. (1995). Capactitative Ca2+ entry contributes to the Ca2+ influx induced by thyrotropin-releasing hormone (TRH) in GH3 pituitary cells. Pflügers Archiv 430, 923-935	[Medline]
Vogalis, F., Publicover, N. G. & Sanders, K. M. (1992). Regulation of calcium current by voltage and cytoplasmic calcium in canine gastric smooth muscle. American Journal of Physiology 262, C691-700	[Medline]
Wade, G. R., Barbera, J. & Sims, S. M. (1996). Cholinergic inhibition of Ca2+ current in guinea-pig gastric and tracheal smooth muscle cells. The Journal of Physiology 491, 307-319	[Abstract]
Warnat, J., Philipp, S., Zimmer, S., Flockerzi, V. & Cavalie, A. (1999). Phenotype of a recombinant store-operated channel: highly selective permeation of Ca2+. The Journal of Physiology 518, 631-638	[Abstract/Full Text]
Wayman, C. P., McFadzean, I., Gibson, A. & Tucker, J. F. (1996). Two distinct membrane currents activated by cyclopiazonic acid-induced calcium store depletion in single smooth muscle cells of the mouse anoccygeus. British Journal of Pharmacology 117, 566-572	[Medline]
Wes, P. D., Chevesich, J., Jeromin, A., Rosenberg, C. & Stetten, G. (1995). TRPC1, a human homolog of a Drosophila store-operated channel. Proceedings of the National Academy of Sciences of the USA 92, 9652-9656	[Abstract]
Xu, X. Z., Li, H. S., Guggino, W. B. & Montell, C. (1997). Coassembly of TRP and TRPL produces a distinct store-operated conductance. Cell 89, 1155-1164	[Medline]
Zhu, S., Jiang, M., Peyton, M., Boulay, G., Hurst, R., Stefani, E. & Birnbaumer, L. (1996). trp, a novel mammalian gene family essential for agonist-activated capacitative Ca2+ entry. Cell 85, 661-671	[Medline]
Zhu, X., Jiang, M. & Birnbaumer, L. (1998). Receptor-activated Ca2+ influx via human trp3 stably expressed in human embryonic kidney (HEK)293 cells. Journal of Biological Chemistry 273, 133-142	[Abstract/Full Text]
Zitt, C., Zobel, A., Obukhov, A. G., Harteneck, C., Kalkbrenner, F., Luckhoff, A. & Schultz, G. (1996). Cloning and functional expression of a human Ca2+-permeable cation channel activated by calcium store depletion. Neuron 16, 1189-1196	[Medline]

Acknowledgements

This work was funded by the Wellcome Trust (054328/Z/98/Z) and the British Heart Foundation.

Corresponding author

J. G. McCarron: Institute of Biomedical and Life Sciences, Neuroscience and Biomedical Systems, West Medical Building, University of Glasgow, Glasgow G12 8QQ, UK.

Email: j.mccarron{at}bio.gla.ac.uk

This article has been cited by other articles:

				J. G. McCarron and M. L. Olson A Single Luminally Continuous Sarcoplasmic Reticulum with Apparently Separate Ca2+ Stores in Smooth Muscle J. Biol. Chem., March 14, 2008; 283(11): 7206 - 7218. [Abstract] [Full Text] [PDF]

				A. M. Langager, B. E. Hammerberg, D. L. Rotella, and H. M. Stauss Very low-frequency blood pressure variability depends on voltage-gated L-type Ca2+ channels in conscious rats Am J Physiol Heart Circ Physiol, March 1, 2007; 292(3): H1321 - H1327. [Abstract] [Full Text] [PDF]

				D. MacMillan, S. Chalmers, T. C. Muir, and J. G. McCarron IP3-mediated Ca2+ increases do not involve the ryanodine receptor, but ryanodine receptor antagonists reduce IP3-mediated Ca2+ increases in guinea-pig colonic smooth muscle cells J. Physiol., December 1, 2005; 569(2): 533 - 544. [Abstract] [Full Text] [PDF]

				P. B. Helli, E. Pertens, and L. J. Janssen Cyclopiazonic acid activates a Ca2+-permeable, nonselective cation conductance in porcine and bovine tracheal smooth muscle J Appl Physiol, November 1, 2005; 99(5): 1759 - 1768. [Abstract] [Full Text] [PDF]

				N. Kotecha and M. A. Hill Myogenic contraction in rat skeletal muscle arterioles: smooth muscle membrane potential and Ca2+ signaling Am J Physiol Heart Circ Physiol, October 1, 2005; 289(4): H1326 - H1334. [Abstract] [Full Text] [PDF]

				J. G. McCarron, K. N. Bradley, D. MacMillan, S. Chalmers, and T. C. Muir The Sarcoplasmic Reticulum, Ca2+ Trapping, and Wave Mechanisms in Smooth Muscle Physiology, June 1, 2004; 19(3): 138 - 147. [Abstract] [Full Text] [PDF]

				S. Morales, P. J. Camello, S. Alcon, G. M. Salido, G. Mawe, and M. J. Pozo Coactivation of capacitative calcium entry and L-type calcium channels in guinea pig gallbladder Am J Physiol Gastrointest Liver Physiol, June 1, 2004; 286(6): G1090 - G1100. [Abstract] [Full Text] [PDF]

				J. G. McCarron, D. MacMillan, K. N. Bradley, S. Chalmers, and T. C. Muir Origin and Mechanisms of Ca2+ Waves in Smooth Muscle as Revealed by Localized Photolysis of Caged Inositol 1,4,5-Trisphosphate J. Biol. Chem., February 27, 2004; 279(9): 8417 - 8427. [Abstract] [Full Text] [PDF]

				K. N. Bradley, S. Currie, D. MacMillan, T. C. Muir, and J. G. McCarron Cyclic ADP-ribose increases Ca2+ removal in smooth muscle J. Cell Sci., November 1, 2003; 116(21): 4291 - 4306. [Abstract] [Full Text] [PDF]

				J. G. McCarron, J. W. Craig, K. N. Bradley, and T. C. Muir Agonist-induced phasic and tonic responses in smooth muscle are mediated by InsP3 J. Cell Sci., May 15, 2002; 115(10): 2207 - 2218. [Abstract] [Full Text] [PDF]

				E. R. M. Flynn, K. N. Bradley, T. C. Muir, and J. G. McCarron Functionally Separate Intracellular Ca2+ Stores in Smooth Muscle J. Biol. Chem., September 21, 2001; 276(39): 36411 - 36418. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Services Email this article to a friend Similar articles in this journal