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J. Biol. Chem., Vol. 276, Issue 39, 36411-36418, September 28, 2001

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Functionally Separate Intracellular Ca²⁺ Stores in Smooth Muscle^*

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

From Neuroscience and Biomedical Systems, Institute of Biomedical and Life Sciences, West Medical Bldg., University of Glasgow, Glasgow G12 8QQ, United Kingdom

Received for publication, May 11, 2001, and in revised form, July 16, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In smooth muscle, release via the inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃R) and ryanodine receptors (RyR) on the sarcoplasmic reticulum (SR) controls oscillatory and steady-state cytosolic Ca²⁺ concentrations ([Ca²⁺]_c). The interplay between the two receptors, itself determined by their organization on the SR, establishes the time course and spatial arrangement of the Ca²⁺ signal. Whether or not the receptors are co-localized or distanced from each other on the same store or whether they exist on separate stores will significantly affect the Ca²⁺ signal produced by the SR. To date these matters remain unresolved. The functional arrangement of the RyR and Ins(1,4,5)P₃R on the SR has now been examined in isolated single voltage-clamped colonic myocytes. Depletion of the ryanodine-sensitive store, by repeated application of caffeine, in the presence of ryanodine, abolished the response to Ins(1,4,5)P₃, suggesting that Ins(1,4,5)P₃R and RyR share a common Ca²⁺ store. Ca²⁺ release from the Ins(1,4,5)P₃R did not activate Ca²⁺-induced Ca²⁺ release at the RyR. Depletion of the Ins(1,4,5)P₃-sensitive store, by the removal of external Ca²⁺, on the other hand, caused only a small decrease (~26%) in caffeine-evoked Ca²⁺ transients, suggesting that not all RyR exist on the common store shared with Ins(1,4,5)P₃R. Dependence of the stores on external Ca²⁺ for replenishment also differed; removal of external Ca²⁺ depleted the Ins(1,4,5)P₃-sensitive store but caused only a slight reduction in caffeine-evoked transients mediated at RyR. Different mechanisms are presumably responsible for the refilling of each store. Refilling of both Ins(1,4,5)P₃-sensitive and caffeine-sensitive Ca²⁺ stores was inhibited by each of the SR Ca²⁺ ATPase inhibitors thapsigargin and cyclopiazonic acid. These results may be explained by the existence of two functionally distinct Ca²⁺ stores; the first expressing only RyR and refilled from [Ca²⁺]_c, the second expressing both Ins(1,4,5)P₃R and RyR and dependent upon external Ca²⁺ for refilling.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Release of Ca²⁺ from the sarcoplasmic reticulum (SR)¹ store, a mechanism that regulates smooth muscle contractile activity, involves the participation of two receptor/channel complexes, the ryanodine receptor (RyR) and the inositol 1,4,5-trisphosphate receptor (Ins(1,4,5)P₃R). Release from this store regulates the bulk average [Ca²⁺]_c both directly (1) and indirectly either via modulation of the plasmalemmal membrane potential (2) or by activation of store-operated Ca²⁺ entry (3). The magnitude, time course, and frequency of the SR Ca²⁺ signal depend on the functional interaction, localization, and arrangement of the Ins(1,4,5)P₃R and RyR on the SR store(s).

Although, morphologically, the SR appears as an interconnected network of tubules (4, 5), it may adopt different configurations within the cell and components may detach and reattach thereby influencing the pattern and distribution of the RyR and Ins(1,4,5)P₃R (6, 7). In Purkinje neurons, for example, Ins(1,4,5)P₃R-expressing regions may detach from other internal store elements (8, 9). Indeed, different Ca²⁺ concentrations have been found within the lumen of the SR (10) suggesting that discontinuities may exist within the structures surrounding the lumen itself. This provides a morphological basis for the existence of various arrangements of Ca²⁺ stores.

The SR Ca²⁺ stores in smooth muscle are classified on the basis of the arrangement of Ins(1,4,5)P₃R and RyR; yet conflicting evidence exists regarding their number. A single store, containing both RyR and Ins(1,4,5)P₃R, has been proposed, based on the observation that caffeine (which activates RyR) prevented Ins(1,4,5)P₃-mediated Ca²⁺ release (e.g. 11-16). Two separate Ca²⁺ stores have also been proposed, one that expresses only RyR, the other only Ins(1,4,5)P₃R. In support of this latter view, depletion of the RyR-sensitive store failed to abolish agonist-evoked Ins(1,4,5)P₃-mediated Ca²⁺ release and vice versa (17). More elaborate arrangements of SR Ca²⁺ stores have also been proposed. In some smooth muscles (e.g. taenia coli, pulmonary artery, myometrium) one store may express both RyR and Ins(1,4,5)P₃R, whereas a second, in the same cell, may express Ins(1,4,5)P₃R alone (18-20). Conversely, one store expressing both RyR and Ins(1,4,5)P₃R and a second separate store RyR alone have also been suggested (21).

Further support for the existence of two separate stores has come from studies on the response of each of the receptors to inhibitors of the SR Ca²⁺ pump, thapsigargin and cyclopiazonic acid (CPA), each of which depletes the stores of Ca²⁺. Differences in the sensitivity to the pump inhibitors of the Ca²⁺ release evoked by either caffeine or Ins(1,4,5)P₃ have been interpreted as evidence for the existence of separate stores for each receptor (5, 16, 22). For example, in arterial myocytes the ryanodine/caffeine-sensitive store was not sensitive to either thapsigargin or CPA, whereas the Ins(1,4,5)P₃-sensitive Ca²⁺ store was depleted by each (16, 22). The situation has been complicated further by the proposed existence in murine bladder smooth muscle cells (23) of three Ca²⁺ stores, one containing RyR and Ins(1,4,5)P₃R, another expressing only Ins(1,4,5)P₃R, and a third containing only RyR.

Whereas the proposed arrangements of RyR and Ins(1,4,5)P₃R may reflect the complexity of the underlying biology, differences in experimental approaches may also have contributed to the variety of views expressed. For example, caffeine, commonly used to activate RyR, also inhibits Ins(1,4,5)P₃R (24, 25). In some studies (e.g. 12, 21) caffeine remained present while an Ins(1,4,5)P₃-generating agonist was applied. Invariably, such experiments demonstrated an inhibition of the Ins(1,4,5)P₃-mediated response and the results have been taken as evidence for the existence of a common Ca²⁺ store. Inhibition of the Ins(1,4,5)P₃ receptor by caffeine, rather than depletion of a common store, could have accounted for the absence of response to an Ins(1,4,5)P₃-generating agonist.

Additional difficulties in the classification of SR Ca²⁺ stores, i.e. their location and number, have followed the use of plasmalemmal agonists and the multiple, yet separate, biochemical pathways so activated. Two particular aspects of such difficulties are evident. First, when membrane currents are used as indicators of [Ca²⁺]_c, agonists may modify these currents independently of SR Ca²⁺ release (e.g. 28, 29). Second, regulation of the RyR and Ins(1,4,5)P₃R by Ca²⁺ derived from agonist activation of several different biochemical pathways may occur with misleading consequences. For example, in rabbit portal vein, depletion of the Ins(1,4,5)P₃-sensitive store, by norepinephrine, abolished the response to caffeine (which acts on the RyR (27)), consistent with both receptors residing on a common Ca²⁺ store. On the other hand, Ca²⁺ released from the SR via Ins(1,4,5)P₃R activation may have triggered a regenerative Ca²⁺-induced Ca²⁺ release (CICR) at the RyR (11, 28), which could have amplified the Ins(1,4,5)P₃-evoked Ca²⁺ transient. If so, two outcomes could be anticipated (a) the continued presence of Ins(1,4,5)P₃ could deplete the RyR-sensitive Ca²⁺ pool; (b) depletion of the RyR-sensitive Ca²⁺ pool would reduce the response to Ins(1,4,5)P₃. Either of these results could be misinterpreted as support for the existence of a common Ca²⁺ store.

Notwithstanding these difficulties, it is important to determine the arrangement of Ca²⁺ stores in smooth muscle to help clarify the precise mechanisms of Ca²⁺ release, a vital ingredient in our understanding of contractility. This problem has been addressed in the current investigation by seeking answers to the following questions: 1) Are Ins(1,4,5)P₃R and RyR present on the same store or on separate stores of the SR? 2) Does Ca²⁺ released from the Ins(1,4,5)P₃-sensitive store trigger CICR via activation of the RyR? 3) Are there differences between the refilling mechanisms of Ins(1,4,5)P₃-sensitive and ryanodine-sensitive intracellular Ca²⁺ stores? In this study freshly isolated single smooth muscle cells rather than multicellular preparations were used, removing the difficulty of there being different store characteristics existing in different cells or of store reorganization, which may accompany the use of cell culture preparations. Ca²⁺ influx was controlled under voltage clamp conditions and directly measured in this investigation. Flash photolysis of caged Ins(1,4,5)P₃ (Ins(1,4,5)P₃) and caffeine were each used to minimize activation of second messenger systems so that a clearer understanding of the control of Ca²⁺ release from the receptors could be obtained. From the results of the present study it is proposed that two functionally distinct SR Ca²⁺ stores exist in colonic myocytes; one expressing both Ins(1,4,5)P₃R and RyR and dependent upon an external Ca²⁺ source for replenishment and a second store containing only RyR, which can be refilled form [Ca²⁺]_c.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Isolation-- From male guinea pigs (500-700 g) stunned by a blow to the head and immediately killed by exsanguination, a segment of distal colon (~5 cm) was removed. The circular muscle was separated from the longitudinal layer, and single cells were prepared from the former using a two-step enzymatic process (30), stored at 4 °C, and used the same day.

Membrane Current Recording-- Cells were voltage-clamped in the dialyzed whole cell configuration. Currents were amplified by an Axopatch 1D (Axon Instruments, Union City, CA), low pass filtered at 500 Hz (8-pole Bessel filter, Frequency Devices, Haverhill, MA), and 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. Cells were held at a membrane potential (V_m) of 70 mV unless otherwise indicated. 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 contained MgCl₂ (3 mM) and EGTA (1 mM). 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 Ins(1,4,5)P₃, 0.025; pH 7.2 adjusted with CsOH. The access afforded by the whole cell patch electrode allowed entry into the cell of the membrane-impermeant fluo-3 and caged Ins(1,4,5)P₃.

Cytosolic Ca²⁺ Concentration ([Ca²⁺]_c) Measurement-- [Ca²⁺]_c was measured using the membrane-impermeable fluo-3 (penta-ammonium salt). Fluorescence measurements were made using a microfluorometer consisting of an inverted fluorescence microscope (Nikon Diaphot) and a photomultiplier tube with a bi-alkali photocathode. Fluo-3 was excited at 488 nm (bandpass 9 nm) from a PTI Delta Scan (Photon Technology International Inc., East Sheen, London, UK) through the epi-illumination port of the microscope (using one arm of a bifurcated quartz fiber optic bundle). Excitation light was passed through a field stop diaphragm, to reduce background fluorescence, and reflected off a 505-nm long-pass dichroic mirror; emitted light was guided through a 535-nm barrier filter (bandpass 35 nm) to a photomultiplier in photon-counting mode. Longer wavelengths, from bright field illumination with a 610-nm Shott glass filter, were reflected onto a charge-coupled device camera (Sony model XC-75) mounted onto the viewing port of the Delta Scan thus allowing the cell to be monitored during experiments. Interference filters and dichroic mirrors were obtained from Glen Spectra (London, UK). To photolyze caged Ins(1,4,5)P₃ the output of a xenon flashlamp (Rapp OptoElektronic, Hamburg, Germany) was passed though a UG-5 filter to select ultraviolet light and merged into the excitation light path of the microfluorometer using the second arm of the quartz bifurcated fiber optic bundle. The nominal flash lamp energy was 57 mJ, measured at the output of the fiber optic bundle. The flash duration was about 1 ms.

Caffeine (10 mM) was applied by hydrostatic pressure (Pneumatic PicoPump PV820, World Precision Instruments, Inc., Sarasota, FL). All experiments were carried out at room temperature (18-22 °C), and drugs were applied either hydrostatically via a pipette or into the bathing solution as indicated in the text.

Data Analysis-- Changes in cytosolic Ca²⁺ were expressed as a ratio (F/F_o) of the fluorescence counts (F) relative to baseline counts before stimulation (F_o). F/F_o indicates the magnitude of the change in F/F_o 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 either Mann-Whitney tests (on normalized data) or paired Student's t tests (on raw data). Summarized data are shown as means ± S.E. and taken to be statistically significant when p < 0.05. n indicates numbers of cells used.

Drugs and Reagents-- fluo-3 penta-ammonium salt was obtained from Molecular Probes, Inc. (Eugene, OR). Caged Ins(1,4,5)P₃ trisodium salt, thapsigargin, cyclopiazonic acid (CPA), and ryanodine were obtained from Calbiochem-Novabiochem Ltd. Thapsigargin, forskolin CPA, and ryanodine were each dissolved in dimethyl sulfoxide, to give a final bath concentration <0.1% dimethyl sulfoxide. Ca²⁺-free Eagle's minimum essential spinner medium was purchased from Life Technologies, Inc. (Paisley, UK). Papain and collagenase were obtained from Sigma Chemical Co., UK or Worthington Biochemical Corp. (Lakewood, NJ). All other reagents were purchased from Sigma, UK (Poole, UK).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Depolarization from a membrane potential (V_m) of 70 mV to 0 mV (Fig. 1C) activated a voltage-dependent Ca²⁺ current averaging 160 ± 18 pA (Fig. 1D) and a transient increase in [Ca²⁺]_c, which averaged 1.83 ± 0.15 F/F_o units above baseline (F/F_o; n = 59; p < 0.001;Fig. 1A). Flash photolysis of caged Ins(1,4,5)P₃ (Ins(1,4,5)P₃, upward-pointing arrows) increased [Ca²⁺]_c by an average of 2.26 ± 0.19 F/F_o (n = 59; p < 0.001; Fig. 1A). Caffeine (Fig. 1B) elevated [Ca²⁺]_c by 2.05 ± 0.18 F/F_o (n = 59; p < 0.001) through activation of RyR. Ins(1,4,5)P₃ and caffeine each evoked reproducible increases in [Ca²⁺]_c when applied at ~50-s intervals.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Ca2+ influx, InsP(1,4,5)P3, and caffeine each increased [Ca2+]c. Depolarization from a membrane potential (Vm) of 70 mV to 0 mV (C) activated a voltage-dependent Ca2+ current (D, and on an expanded time base in E) and elevated [Ca2+]c (A). Ins(1,4,5)P3 ('s in this and following figures) also produced approximately reproducible increases in [Ca2+]c (A), as did caffeine (caff.; B).

Are All InsP(1,4,5)P₃ Receptors Present on the Ca²⁺ Store Containing RyR?-- To test this, the response to Ins(1,4,5)P₃, following depletion of the ryanodine-sensitive store by caffeine, was examined. At 70 mV, Ins(1,4,5)P₃ evoked approximately reproducible increases in [Ca²⁺]_c (3.28 ± 0.35 F/F_o; n = 5; Fig. 2, A and B) as did caffeine (Fig. 2C, 3.12 ± 0.2 F/F_o, n = 5, Fig. 2, A and B). Caffeine-evoked Ca²⁺ transients were inhibited to 6 ± 3% of controls by ryanodine (50 µM; 0.14 ± 0.08 F/F_o; n = 5; p < 0.001). Significantly, after this inhibition of the caffeine-evoked Ca²⁺ transient, the Ins(1,4,5)P₃-evoked Ca²⁺ transient was reduced to 7 ± 2% of control values (0.19 ± 0.04 F/F_o; n = 5; p < 0.001; Fig. 2). These results are compatible with the view that Ins(1,4,5)P₃R and RyR exist on a common SR Ca²⁺ store.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Depletion of the caffeine-sensitive store inhibited the response to InsP(1,4,5)P3. Ins(1,4,5)P3 () and caffeine (C) each produced reproducible rises in [Ca2+]c (A and B). In the presence of ryanodine (50 µM) the response to caffeine (C) decreased (A and B) as did the response to Ins(1,4,5)P3 (; A and B). Summary data from five cells are shown in A. *, significant inhibition of the Ins(1,4,5)P3-evoked Ca2+ transient; **, significant inhibition of the caffeine-evoked Ca2+ transient. 1st, 2nd, etc. refers to the order of responses after treatment in this and other figures.

Does CICR from the RyR Contribute to the InsP(1,4,5)P₃-evoked Ca²⁺ Transient?-- If on the other hand two separate stores exist, i.e. one for Ins(1,4,5)P₃R and another for RyR, release of a small amount of Ca²⁺ from the Ins(1,4,5)P₃-sensitive store could trigger a further, larger release of Ca²⁺ from the separate ryanodine-sensitive store by CICR. If so, depletion of the ryanodine-sensitive store, by caffeine and ryanodine, would reduce the Ins(1,4,5)P₃-evoked response. If Ca²⁺, released through the Ins(1,4,5)P₃R, triggered CICR at the RyR, ryanodine alone would reduce Ins(1,4,5)P₃-evoked Ca²⁺ transients. This was not observed (Fig. 3). Ins(1,4,5)P₃ evoked reproducible increases in [Ca²⁺]_c of similar magnitude in the presence (50 µM) and absence of ryanodine (n = 5, Fig. 3, V_m = 70 mV). Thus Ca²⁺ released by Ins(1,4,5)P₃ did not subsequently trigger CICR from the RyR, and this provides further evidence for the existence of a common Ca²⁺ store. In other investigations, reduction, by ryanodine, of the Ca²⁺ transient evoked by Ins(1,4,5)P₃-generating agents was interpreted as evidence that Ins(1,4,5)P₃-evoked Ca²⁺ activates CICR at the RyR (11, 28). The plasmalemma agonists used in these experiments to generate Ins(1,4,5)P₃ could also have activated other second messengers that in turn sensitized the RyR to Ca²⁺ enabling Ins(1,4,5)P₃-evoked Ca²⁺ release to activate CICR at the RyR. Alternatively, Ca²⁺ release from the SR store may activate further Ca²⁺ release under conditions of "store overload" (31, 32). Such store overload conditions could conceivably arise in some smooth muscle types, facilitating CICR.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Ca2+ released through InsP(1,4,5)P3 receptors did not activate RyR. Ins(1,4,5)P3 () produced reproducible increases in [Ca2+]c (A and B). Ryanodine (50 µM) did not significantly alter the magnitude of Ins(1,4,5)P3-evoked transients, which averaged 1.98 ± 0.37 F/Fo before and 2.02 ± 0.44 F/Fo after ryanodine (n = 5; p > 0.05). A, summarizes data from five cells (mean ± S.E.).

To ensure that the absence of an Ins(1,4,5)P₃-evoked Ca²⁺ transient following depletion of the ryanodine/caffeine-sensitive store (Fig. 2) was due neither to inactivation of the Ins(1,4,5)P₃R by caffeine (24, 25) nor to allocation of an inadequate period for store refilling after caffeine, the time course of recovery of the response to Ins(1,4,5)P₃ after caffeine was examined at 70 mV. The magnitude of the Ins(1,4,5)P₃-evoked transient was reduced to 3 ± 1% 10 s after caffeine (n = 5; Fig. 4, A and G). Recovery was time-dependent; 50 s after exposure to caffeine the Ins(1,4,5)P₃-evoked Ca²⁺ transient had returned to 92 ± 5% of controls (n = 6; Fig. 4, E and G). These results suggest that, at the time intervals used (50-60 s), neither inactivation of the Ins(1,4,5)P₃R by caffeine nor an insufficient time period for store refilling accounted for the inhibition of the Ins(1,4,5)P₃-evoked Ca²⁺ transient by caffeine and ryanodine (Fig. 2). Collectively, the data (Figs. 2, 3, and 4) suggest that all Ins(1,4,5)P₃R exist on a store that also contains RyR.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Time dependence of the recovery of the InsP(1,4,5)P3-evoked Ca2+ transient after caffeine. The magnitude of the Ins(1,4,5)P3-evoked () Ca2+ transient was virtually abolished 10 s after caffeine (A) from 1.89 ± 0.72 F/Fo to 0.06 ± 0.04 F/Fo, (p < 0.05, n = 5). Recovery of the response to Ins(1,4,5)P3 was time-dependent (A-F). The amplitude of the Ins(1,4,5)P3-evoked transient had fully recovered to 3.01 ± 0.9 F/Fo compared with 3.37 ± 1.07 F/Fo before caffeine (p > 0.05; n = 6) 50 s after caffeine (F). The amplitude of both Ins(1,4,5)P3- and caffeine-evoked transients also increased proportionately with time, presumably as the SR Ca2+ content increased. G shows summary data from six identical experiments showing the time course of recovery of the Ins(1,4,5)P3-evoked Ca2+ transient after caffeine. *, significant inhibition of the Ins(1,4,5)P3-evoked transient (p < 0.05).

Not All RyR Are Present on the Store, Which Contains InsP(1,4,5)P₃R-- To determine whether or not all RyR were present on the store that contained Ins(1,4,5)P₃R, the Ins(1,4,5)P₃-sensitive store was depleted by removal of external Ca²⁺ and the ability of caffeine to activate the RyR and evoke a Ca²⁺ transient was examined. Refilling of the Ins(1,4,5)P₃-sensitive store is dependent on external Ca²⁺ (33), and removing it reduced the response to Ins(1,4,5)P₃ to 5 ± 2% of controls (n = 6; p < 0.05; V_m = 70 mV, Fig. 5). However, after the almost complete loss of the Ins(1,4,5)P₃-evoked transient, caffeine evoked a Ca²⁺ transient that averaged 74 ± 25% of control values (n = 6; p < 0.05; Fig. 5). These results are consistent with there being a second separate Ca²⁺ store that contains only RyR.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Depletion of the InsP(1,4,5)P3-sensitive store reduced but did not abolish the response to caffeine. Ins(1,4,5)P3 () and caffeine (C) each evoked approximately reproducible increases in [Ca2+]c (A and B; Vm = 70 mV). Removal of external Ca2+ (and addition of 1 mM EGTA) reduced the Ins(1,4,5)P3-evoked Ca2+ transient to 5 ± 2% of controls after five UV flashes (from 1.91 ± 0.11 F/Fo to 0.08 ± 0.02 F/Fo; n = 6; p < 0.05). Following depletion of the Ins(1,4,5)P3-sensitive store, caffeine (caff.; C) produced a Ca2+ transient that averaged 74 ± 25% of the control amplitude (1.18 ± 0.4 F/Fo compared with 1.57 ± 0.13 F/Fo prior to depletion of the Ins(1,4,5)P3-sensitive store; n = 6; p < 0.05). Data from six cells are summarized in A; *, significant inhibition of the Ins(1,4,5)P3-evoked Ca2+ transient; **, significant inhibition of the caffeine-evoked Ca2+ transient.

Refilling of the Ca²⁺ Stores-- The above results (Fig. 5) raised the possibility that the degree of dependence of the two stores on external Ca²⁺ for Ca²⁺ release may differ. This was examined following withdrawal of external Ca²⁺ by investigating the refilling of the RyR- and Ins(1,4,5)P₃-sensitive stores after either caffeine or Ins(1,4,5)P₃. The caffeine-evoked Ca²⁺ transient (via RyR; Fig. 6A) was reduced, on average, to some 87 ± 9% of controls (n = 5; p = 0.5; Fig. 6, A and B). In contrast, the Ins(1,4,5)P₃-evoked Ca²⁺ transient (acting through Ins(1,4,5)P₃R) was reduced to 6 ± 2% of controls (Fig. 6B; see Fig. 5). These results suggest that, unlike the situation with the Ins(1,4,5)P₃-sensitive Ca²⁺ store (Fig. 5), Ca²⁺ release from the RyR by caffeine may be recycled so that refilling is largely independent of external Ca²⁺.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   The amplitude of the caffeine-evoked response was reduced but not abolished in the absence of external Ca2+. Caffeine (caff.; A (iii)) produced reproducible increases in cytosolic Ca2+ (A (ii)). Removal of external Ca2+ reduced but did not abolish the amplitude of the caffeine-evoked response (3.35 ± 0.47 F/Fo compared with 2.89 ± 0.47 F/Fo; n = 5; p = 0.5). A (i), summary data from five cells (mean ± S.E.). In comparison, refilling of the Ins(1,4,5)P3 ()-sensitive store was prevented following removal of external Ca2+ (B; from 1.81 ± 0.13 to 0.09 ± 0.02 F/Fo; n = 7; p < 0.05). B (i) (summary data from seven cells) demonstrates that the amplitude of the Ins(1,4,5)P3-evoked Ca2+ transient (B (ii)) was inhibited (mean ± S.E.). Note the different time bars in A and B.

Effects of Elevation of cAMP on InsP(1,4,5)P₃-evoked Ca²⁺ Transients-- Caffeine inhibits phosphodiesterase activity and so may elevate the intracellular concentration of cAMP ([cAMP]_c) (34). The persistence of the store Ca²⁺ content, in the absence of external Ca²⁺, as indicated by the maintained amplitude of the caffeine-evoked Ca²⁺ transient, could have arisen from stimulation of SERCA by an elevated [cAMP]_c due to caffeine (35) rather than to a difference in the refilling mechanism. To examine this possibility, dependence of Ins(1,4,5)P₃ store refilling on external Ca²⁺ was examined when [cAMP]_c had been increased (a) by the phosphodiesterase inhibitor IBMX (500 µM) and (b) by forskolin (1 µM), which stimulates adenylate cyclase thereby raising [cAMP]_c. In the absence of either drug, Ins(1,4,5)P₃-evoked Ca²⁺ transients of approximately reproducible amplitude that averaged 1.89 ± 0.12 F/F_o (n = 6). Following incubation (10 min) with either IBMX or forskolin, Ins(1,4,5)P₃-evoked transients of approximately reproducible amplitude (2.18 ± 0.67 F/F_o; n = 6; Fig. 7, for IBMX), which were not significantly different from controls. Upon removal of external Ca²⁺, in the continued presence of either IBMX or forskolin, repeated application of Ins(1,4,5)P₃ depleted the Ins(1,4,5)P₃-sensitive store as evidenced by the decline in the amplitude of the Ca²⁺ transient. With IBMX, after the fourth Ins(1,4,5)P₃ challenge, the Ca²⁺ increase averaged 15 ± 3% of the Ins(1,4,5)P₃-evoked Ca²⁺ transients observed in IBMX in the presence of external Ca²⁺ (0.56 ± 0.31 F/F_o; p < 0.01; n = 6; Fig. 7). Qualitatively similar results were obtained with forskolin. Removal of external Ca²⁺ again inhibited the amplitude of the Ins(1,4,5)P₃-evoked transient significantly to 8 ± 3% of controls (p < 0.05 by Mann-Whitney test; data not shown; V_m = 70 mV). In these same cells only 3 ± 2% of the Ins(1,4,5)P₃-evoked transient remained in the presence of forskolin (1 µM) following the removal of external Ca²⁺ (n = 3; p < 0.05 by Mann-Whitney test). Together the results with IBMX and forskolin indicated that elevation of [cAMP]_c is unlikely to offset the effect of external Ca²⁺ withdrawal on store content.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Elevated [cAMP]c does not maintain SR store Ca2+ content in the absence of external Ca2+. Ins(1,4,5)P3 () produced approximately consistent increases in [Ca2+]c (A and B). IBMX (500 µM) did not significantly alter the magnitude of the Ins(1,4,5)P3-evoked Ca2+ transients (p > 0.05). The break in the record (//) indicates an interval of 6 min to allow IBMX to take effect. Removal of external Ca2+ inhibited the response to Ins(1,4,5)P3, such that upon the fourth challenge with Ins(1,4,5)P3, the evoked Ca2+ was 15 ± 3% of control values (p < 0.05). Summary data (mean ± S.E.) from six cells (A) showed that IBMX did not maintain Ins(1,4,5)P3-evoked Ca2+ transients in the absence of external Ca2+.

Effects of SR Ca²⁺ Pump Inhibitors-- Ca²⁺ stores have been differentiated on the basis of their sensitivity to the SERCA inhibitors cyclopiazonic acid (CPA) and thapsigargin (5, 36, 37). The ability of CPA and thapsigargin to each inhibit Ins(1,4,5)P₃- and caffeine-evoked Ca²⁺ transients was therefore examined. Cells were once again held at a membrane potential of 70 mV. Ins(1,4,5)P₃ and caffeine (Fig. 8, C and F) each produced reproducible increases in [Ca²⁺]_c at ~50-s intervals (Fig. 8, B and E). Thapsigargin (500 nM) increased resting [Ca²⁺]_c from 1.07 ± 0.05 F/F_o to 1.73 ± 0.12 F/F_o after 5 min (n = 10; p < 0.001; Fig. 8B), and inhibited the responses to both Ins(1,4,5)P₃ and caffeine (Fig. 8 A and B). Ins(1,4,5)P₃-evoked Ca²⁺ transients were reduced, from an average of 4.19 ± 0.40 F/F_o in control to 0.23 ± 0.06 F/F_o in the presence of thapsigargin (n = 10; p < 0.001; Fig. 8A). The caffeine-evoked Ca²⁺ transient was also reduced from 3.81 ± 1.38 F/F_o in control to 0.04 ± 0.06 F/F_o in the presence of thapsigargin (n = 10; p < 0.001; Fig. 8A). CPA (10 µM) also increased resting [Ca²⁺]_c from 1.18 ± 0.09 F/F_o immediately prior to CPA to 1.46 ± 0.11 F/F_o after 5 min in the drug (n = 16; p < 0.001; Fig. 8E). CPA inhibited both the Ins(1,4,5)P₃-evoked and caffeine-evoked Ca²⁺ transients (Fig. 8, D and E). On average, the response to Ins(1,4,5)P₃ was reduced from 1.70 ± 0.32 F/F_o to 0.09 ± 0.04 F/F_o in CPA (n = 16; p < 0.001; Fig. 8D) and that to caffeine from 1.65 ± 0.34 F/F_o to 0.07 ± 0.06 F/F_o (n = 16; p < 0.001; Fig. 8D). Thus, refilling of both the Ins(1,4,5)P₃-sensitive and caffeine-sensitive stores is prevented by each of the SERCA inhibitors thapsigargin and CPA; use of SERCA inhibitors is unlikely to be of value in differentiating between Ca²⁺ stores in these cells.

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Thapsigargin and CPA each inhibited the refilling of both InsP(1,4,5)P3-sensitive and caffeine-sensitive Ca2+ stores. Ins(1,4,5)P3 () and caffeine (C and F) each produced Ca2+ transients of approximately consistent amplitude (A, B and D, E). Thapsigargin (500 nM; A-C) and CPA (10 µM; D-F) each elevated resting [Ca2+]c and inhibited both the Ins(1,4,5)P3-evoked and caffeine-evoked Ca2+ transients (n = 10 and n = 16, respectively). Summary data showing the mean ± S.E. of Ins(1,4,5)P3-evoked and caffeine-evoked Ca2+ transients, and their inhibition by thapsigargin and CPA, is summarized in A and D, respectively. *, significant inhibition of the Ins(1,4,5)P3-evoked Ca2+ transient; **, significant inhibition of the caffeine-evoked Ca2+ transient compared with control values (p < 0.05).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ca²⁺ release from and uptake into internal SR Ca²⁺ stores plays a central role in the activity of most cells, including that of excitation-contraction coupling in smooth muscle. Although it is conceded that release occurs via Ins(1,4,5)P₃R and RyR, the interrelationship between these two receptors in their access to stored Ca²⁺ is unclear. A diversity of store and receptor arrangements has been proposed. This study has demonstrated the presence of two Ca²⁺ stores in colonic smooth muscle, one expressing both Ins(1,4,5)P₃R and RyR the other only RyR. Support for this view comes from two key observations in which the stores accessed by either Ins(1,4,5)P₃R or RyR were depleted of Ca²⁺. First, the store accessed by RyR was depleted of Ca²⁺ (by repetitive activation with caffeine in the presence of ryanodine to maintain the RyR in the open state), and under these circumstances the response to Ins(1,4,5)P₃ was virtually abolished. Second, when the store accessed by Ins(1,4,5)P₃R was depleted, by withdrawal of external Ca²⁺, and the Ins(1,4,5)P₃ response was effectively abolished, significantly, the Ca²⁺ response to RyR activation by caffeine persisted. The observation that the Ins(1,4,5)P₃-evoked Ca²⁺ response was almost abolished after caffeine (in the presence of ryanodine) could not be attributed to inhibition of an amplification of the Ins(1,4,5)P₃-evoked response by CICR, because Ins(1,4,5)P₃-evoked Ca²⁺ release did not activate this mechanism. Nor could the observation be explained by inactivation of the Ins(1,4,5)P₃R by caffeine because the Ins(1,4,5)P₃-evoked Ca²⁺ transient had fully recovered at the time intervals between application of caffeine and Ins(1,4,5)P₃ used in the present study. Finally, the results of the present study demonstrated differences in the refilling mechanisms of the two stores. The store expressing both Ins(1,4,5)P₃R and RyR was dependent on external Ca²⁺ for replenishment whereas the store with only RyR was not.

The precise number and arrangement of Ca²⁺ stores, among different cell types, is a matter of debate. In cerebellar Purkinje neurons, as in the present study, ryanodine inhibited the Ins(1,4,5)P₃-evoked Ca²⁺ transient, in keeping with the view that both Ins(1,4,5)P₃R and RyR were present on a common store. However, whether or not an additional separate store with only RyR involved was not determined (38). One store with both Ins(1,4,5)P₃R and RyR and an additional separate store, with only RyR similar to the present view, have also been proposed in vascular smooth muscle (21). Other studies had initially raised the possibility of there being two stores but with different receptor arrangements from those presently proposed. For example, from experiments in smooth muscle, of the two Ca²⁺ stores proposed, one contained both RyR and Ins(1,4,5)P₃R, the other only Ins(1,4,5)P₃R (19, 20). Alternatively, separate stores have been proposed for both RyR and Ins(1,4,5)P₃ receptors (e.g. 39-41). A commonly employed method with which to study the receptors on the Ca²⁺ stores has been to inhibit store Ca²⁺ pumps by thapsigargin or cyclopiazonic acid (CPA) and to observe the responses to activation of Ins(1,4,5)P₃R and RyR. In various cell types, thapsigargin and CPA each abolished Ca²⁺ release in response to activation of either RyR or Ins(1,4,5)P₃R but not to activation of both. This supported the idea of there being more than one store (36, 37, 42-44). Studies in smooth muscle and astrocytes suggested that, despite an apparently uniform Ca²⁺ content throughout most of the store, CPA and the RyR activator caffeine released Ca²⁺ from seemingly separate compartments (5, 22), i.e. there were in effect two stores. However, the relationship between SR Ca²⁺ pumps on the one hand and the Ins(1,4,5)P₃R and RyR on the other could not be presently differentiated by the use of Ca²⁺ pump inhibitors. SR Ca²⁺ pumps presumably on both the common Ins(1,4,5)P₃R/RyR store and the RyR-only store were each inhibited by thapsigargin and CPA (see also 45, 46). Differences in the sensitivity of the Ca²⁺ pumps on the internal SR Ca²⁺ store to the inhibitors (47, 48), which may also vary among different tissues, were presumably responsible for these observations.

Differences in SR Luminal Ca²⁺ Regulation of the Receptors Does Not Account for the Differences in Response to InsP(1,4,5)P₃ and Caffeine-- On the basis of the two-store system proposed, the observation that a substantial caffeine-evoked Ca²⁺ transient persisted, after depletion of the Ins(1,4,5)P₃-sensitive store, could be explained if the opening of the Ins(1,4,5)P₃R and RyR was each regulated differently by the SR luminal Ca²⁺ concentration (49-52). Thus, as luminal Ca²⁺ decreased, the Ins(1,4,5)P₃ sensitivity to Ins(1,4,5)P₃ could have diminished and the receptor could no longer be able to release Ca²⁺, even though the SR itself had not been depleted. The sensitivity of the RyR to caffeine could have been affected to a lesser degree than that of the Ins(1,4,5)P₃R by decreases in luminal Ca²⁺, and caffeine could have remained effective in evoking Ca²⁺ release. However, if a single common store for both Ins(1,4,5)P₃R and RyR existed (rather than the presently proposed two stores), luminal regulation of the receptors with the accompanying differences in sensitivity between RyR and Ins(1,4,5)P₃R could also serve as a model in which the SR could function, in effect, as two stores. If luminal regulation was to account for the present results, a 25% decrease in the SR Ca²⁺ content would be sufficient to prevent Ca²⁺ release via the Ins(1,4,5)P₃R, significantly different from previously published values (70-95% (53-55)). This arises from the observation that some 75% of the Ca²⁺ transient evoked by caffeine persisted when the Ins(1,4,5)P₃-evoked transient was abolished. SR luminal Ca²⁺ regulation of the Ins(1,4,5)P₃R thus seems unlikely to account for the present observations. Nor is luminal regulation responsible for differences between the two stores in their dependence upon external Ca²⁺ for replenishment. Thus the response to caffeine is maintained whereas that to Ins(1,4,5)P₃ disappears after the removal of external Ca²⁺. Together, these findings support the presence of two discrete SR Ca²⁺ stores; one able to refill from [Ca²⁺]_c, the other from extracellular Ca²⁺ entry.

Mechanisms of Store Refilling-- Depletion of Ins(1,4,5)P₃-sensitive stores in all cell types so far examined activates a store-operated Ca²⁺ entry pathway, which is necessary for its replenishment (reviewed in Refs. 56-58) as is depletion of the ryanodine-sensitive store, although not in all cell types (59-63). In the present study, replenishment of the store containing RyR alone did not require external Ca²⁺, suggesting that plasmalemmal store-operated Ca²⁺ entry is unnecessary for the maintenance of the Ca²⁺ content of this store. In the store containing both RyR and Ins(1,4,5)P₃R, on the other hand, external Ca²⁺ entry, presumably via store-operated channels, is essential for store refilling in colonic myocytes (33). Perhaps differences in store location within the cell determines the external Ca²⁺ dependence of the refilling mechanisms. The store containing both RyR and Ins(1,4,5)P₃R may be positioned closer to the plasmalemma than that containing only RyR and may be functionally more closely linked to Ca²⁺ entry via store-operated channels (63). Different isoforms of SERCA, with different Ca²⁺ binding affinities, e.g. K_0.5 0.31 ± 0.02 µM and 0.17 ± 0.01 µM Ca²⁺ for SERCA2a and SERCA2b, respectively (Ref. 42 for review; see also Refs. 65-67), may be associated with the separate stores and could explain the ability of the SR to refill in the presence of different [Ca²⁺]_c. Ca²⁺ uptake into stores by SERCA2b may also be modulated by calmodulin (68), phospholamban (64, 67), and calreticulin and calnexin (26) so that differential modulation of Ca²⁺ pumps may also help to explain differences in store refilling in the absence of external Ca²⁺.

The present study emphasizes the complexity of the organization of the SR Ca²⁺ stores. It proposes the existence of two in the control of Ca²⁺ release; one containing both RYR and Ins(1,4,5)P₃R and a second only RyR. The results also highlight the possibility of structural components within the SR, each with their unique content of receptors/channels permitting local control of the SR Ca²⁺ signal in response to receptor modulation.

	ACKNOWLEDGEMENT

We thank J. W. Craig for excellent technical assistance.

	FOOTNOTES

^* This work was funded in part by the Wellcome Trust (0554328/Z/98/Z) and British Heart Foundation (PG/2001079).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

^§ A Caledonian Research Foundation Fellow. To whom correspondence should be addressed: Tel.: 44-141-330-5143; Fax: 44-141-330-6610; E-mail: j.mccarron@bio.gla.ac.uk.

Published, JBC Papers in Press, July 26, 2001, DOI 10.1074/jbc.M104308200

	ABBREVIATIONS

The abbreviations used are: SR, sarcoplasmic reticulum; Ins(1, 4,5)P₃, inositol 1,4,5-trisphosphate; Ins(1, 4,5)P₃R, Ins(1,4,5)P₃ receptor; RyR, ryanodine receptor; CICR, Ca²⁺-induced Ca²⁺ release; [Ca²⁺]_c, cytosolic Ca²⁺ concentration; SERCA, sarcoplasmic reticulum Ca²⁺ ATPase; F/F_o, the ratio of fluorescence counts (F) relative to baseline counts before stimulation (F_o); F/F_o, the magnitude of the change in F/F_o; IBMX, 3-isobutyl-1-methylxanthine; [cAMP]_c, cytosolic concentration of cAMP; CPA, cyclopiazonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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