Functionally Separate Intracellular Ca2+ Stores in Smooth Muscle
2001; Elsevier BV; Volume: 276; Issue: 39 Linguagem: Inglês
10.1074/jbc.m104308200
ISSN1083-351X
AutoresElaine R. M. Flynn, Karen N. Bradley, T C Muir, John G. McCarron,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoIn 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. 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. sarcoplasmic reticulum 4,5)P3, inositol 1,4,5-trisphosphate 4,5)P3R, Ins(1,4,5)P3 receptor ryanodine receptor Ca2+-induced Ca2+release cytosolic Ca2+concentration sarcoplasmic reticulum Ca2+ ATPase the ratio of fluorescence counts (F) relative to baseline counts before stimulation (Fo) the magnitude of the change in F/Fo 3-isobutyl-1-methylxanthine cytosolic concentration of cAMP cyclopiazonic acid 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 (1Somlyo A.V. Bond M. Somlyo A.P. Scarpa A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5231-5235Crossref PubMed Scopus (318) Google Scholar) and indirectly either via modulation of the plasmalemmal membrane potential (2Nelson M.T. Cheng H. Rubart M. Santana L.F. Bonev A.D. Knot H.J. Lederer W.J. Science. 1995; 270: 633-637Crossref PubMed Scopus (1208) Google Scholar) or by activation of store-operated Ca2+ entry (3Fasolato C. Innocenti B. Pozzan T. Trends Pharmacol. Sci. 1994; 15: 77-82Abstract Full Text PDF PubMed Scopus (440) Google Scholar). 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 (4Somlyo A.P. Circ. Res. 1985; 57: 497-507Crossref PubMed Scopus (165) Google Scholar, 5Golovina V.A. Blaustein M.P. Science. 1997; 275: 1643-1648Crossref PubMed Scopus (425) Google Scholar), 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 (6Lee C. Chen L.B. Cell. 1988; 54: 37-46Abstract Full Text PDF PubMed Scopus (354) Google Scholar,7Terasaki M. Jaffe L.A. J. Cell Biol. 1991; 114: 929-940Crossref PubMed Scopus (190) Google Scholar). In Purkinje neurons, for example, Ins(1,4,5)P3R-expressing regions may detach from other internal store elements (8Yamamoto A. Otsu H. Yoshimori T. Maeda N. Mikoshiba K. Tashiro Y. Cell Struct. Funct. 1991; 16: 419-432Crossref PubMed Scopus (40) Google Scholar, 9Takei K. Mignery G.A. Mugnaini E. Sudhof T.C. De Camilli P. Neuron. 1994; 12: 327-342Abstract Full Text PDF PubMed Scopus (127) Google Scholar). Indeed, different Ca2+concentrations have been found within the lumen of the SR (10Montero M. Alvarez J. Scheenen W.J.J. Rizzuto R. Meldolesi J. J. Cell Biol. 1997; 139: 601-611Crossref PubMed Scopus (98) Google Scholar) 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 (17Young S.H. Ennes H.S. MacRoberts J.A. Chaban V.V. Dea S.K. Mayer E.A. Am. J. Physiol. 1999; 276: G1204-G1212PubMed Google Scholar). 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 (18Iino M. Kobayashi T. Endo M. Biochem. Biophys. Res. Commun. 1988; 152: 417-422Crossref PubMed Scopus (218) Google Scholar, 19Iino M. Biochem. Biophys. Res. Commun. 1987; 142: 45-52Crossref Scopus (77) Google Scholar, 20Yamazawa T. Iino M. Endo M. FEBS Lett. 1992; 301: 181-184Crossref PubMed Scopus (48) Google Scholar). Conversely, one store expressing both RyR and Ins(1,4,5)P3R and a second separate store RyR alone have also been suggested (21Baró I. Eisner D.A. J. Physiol. (Lond.). 1995; 482: 247-258Crossref Scopus (34) Google Scholar). 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 (5Golovina V.A. Blaustein M.P. Science. 1997; 275: 1643-1648Crossref PubMed Scopus (425) Google Scholar, 16Janiak R. Wilson S.M. Montague S. Hume J.R. Am. J. Physiol. 2001; 280: C22-C33Crossref PubMed Google Scholar, 22Tribe R.M. Borin M.L. Blaustein M.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5908-5912Crossref PubMed Scopus (109) Google Scholar). 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 (16Janiak R. Wilson S.M. Montague S. Hume J.R. Am. J. Physiol. 2001; 280: C22-C33Crossref PubMed Google Scholar, 22Tribe R.M. Borin M.L. Blaustein M.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5908-5912Crossref PubMed Scopus (109) Google Scholar). The situation has been complicated further by the proposed existence in murine bladder smooth muscle cells (23Sugita M. Tokutomi N. Tokutomi Y. Terasaki H. Nishi K. Eur. J. Pharmacol. 1998; 348: 61-70Crossref PubMed Scopus (16) Google Scholar) 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 (24Missiaen L. Parys J.B. De Smedt H. Himpens B. Casteels R. Biochem. J. 1994; 300: 81-84Crossref PubMed Scopus (54) Google Scholar, 25Bezprozvanny I. Bezprozvanny S. Ehrlich B.E. Mol. Biol. Cell. 1994; 5: 97-103Crossref PubMed Scopus (86) Google Scholar). In some studies (e.g. 12Bolton T.B. Lim S.P. J. Physiol. (Lond.). 1989; 409: 385-401Crossref Scopus (93) Google Scholar, 21Baró I. Eisner D.A. J. Physiol. (Lond.). 1995; 482: 247-258Crossref Scopus (34) Google Scholar) 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.28Jaggar J.H. Nelson M.T. Am. J. Physiol. 2000; 279: C1528-C1539Crossref PubMed Google Scholar, 29Schubert R. Noack T. Serebryakov V.N. Am. J. Physiol. 1999; 276: C648-C658Crossref PubMed Google Scholar). 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 (27Komori S. Bolton T.B. Br. J. Pharmacol. 1989; 97: 973-982Crossref PubMed Scopus (30) Google Scholar)), 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 (11Boittin F.X. Macrez N. Halet G. Mironneau J. Am. J. Physiol. 1999; 277: C139-C151Crossref PubMed Google Scholar, 28Jaggar J.H. Nelson M.T. Am. J. Physiol. 2000; 279: C1528-C1539Crossref PubMed Google Scholar), 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. 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 (30McCarron J.G. Muir T.C. J. Physiol. (Lond.). 1999; 516: 149-161Crossref Scopus (81) Google Scholar), stored at 4 °C, and used the same day. 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 (Vm) 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; andd-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. [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. Changes in cytosolic Ca2+ were expressed as a ratio (F/Fo) of the fluorescence counts (F) relative to baseline counts before stimulation (Fo). ΔF/Fo indicates the magnitude of the change in F/Fo 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 whenp < 0.05. n indicates numbers of cells used. 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). Depolarization from a membrane potential (Vm) 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/Fo units above baseline (ΔF/Fo; 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 ΔF/Fo (n = 59;p < 0.001; Fig. 1A). Caffeine (Fig.1B) elevated [Ca2+]c by 2.05 ± 0.18 ΔF/Fo (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. 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 ΔF/Fo; n = 5; Fig.2, A and B) as did caffeine (Fig. 2C, 3.12 ± 0.2 ΔF/Fo, 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 ΔF/Fo; 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 ΔF/Fo; 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. 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, Vm = −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 (11Boittin F.X. Macrez N. Halet G. Mironneau J. Am. J. Physiol. 1999; 277: C139-C151Crossref PubMed Google Scholar, 28Jaggar J.H. Nelson M.T. Am. J. Physiol. 2000; 279: C1528-C1539Crossref PubMed Google Scholar). The plasmalemma agonists used in these experiments to generate Ins(1,4,5)P3could 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" (31Cheng H. Lederer M.R. Lederer W.J. Cannell M.B. Am. J. Physiol. 1996; 270: C148-C159Crossref PubMed Google Scholar, 32Trafford A.W. Lipp P. O'Neill S.C. Niggli E. Einser D.A. J. Physiol. 1995; 489: 319-326Crossref PubMed Scopus (50) Google Scholar). Such store overload conditions could conceivably arise in some smooth muscle types, facilitating CICR. 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 (24Missiaen L. Parys J.B. De Smedt H. Himpens B. Casteels R. Biochem. J. 1994; 300: 81-84Crossref PubMed Scopus (54) Google Scholar, 25Bezprozvanny I. Bezprozvanny S. Ehrlich B.E. Mol. Biol. Cell. 1994; 5: 97-103Crossref PubMed Scopus (86) Google Scholar) 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. 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+ (33McCarron J.G. Flynn E.R.M. Bradley K.N. Muir T.C. J. Physiol. (Lond.). 2000; 525: 113-124Crossref Scopus (36) Google Scholar), and removing it reduced the response to Ins(1,4,5)P3 to 5 ± 2% of controls (n = 6; p < 0.05;Vm = −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. 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, Aand 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+. Caffeine inhibits phosphodiesterase activity and so may elevate the intracellular concentration of cAMP ([cAMP]c) (34Butcher T.W. Sutherland E.W. J. Biol. Chem. 1962; 237: 1244-1250Abstract Full Text PDF PubMed Google Scholar). 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 (35Raeymaekers L. Eggermont J.A. Wuytack F. Casteels R. Cell Calcium. 1990; 11: 261-268Crossref PubMed Scopus (68) Google Scholar) 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 ΔF/Fo(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 ΔF/Fo; 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 ΔF/Fo; 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; Vm = −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. Ca2+ stores have been differentiated on the basis of their sensitivity to the SERCA inhibitors cyclopiazonic acid (CPA) and thapsigargin (5Golovina V.A. Blaustein M.P. Science. 1997; 275: 1643-1648Crossref PubMed Scopus (425) Google Scholar, 36Bian J. Ghosh T.K. Wang J.-C. Gill D.L. J. Biol. Chem. 1991; 266: 8801-8806Abstract Full Text PDF PubMed Google Scholar, 37Robinson I.M. Burgoyne R.D. J. Neurochem. 1991; 56: 1587-1593Crossref PubMed Scopus (63) Google Scholar). 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+]cfrom 1.07 ± 0.05 F/Fo to 1.73 ± 0.12 F/Fo 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 andB). Ins(1,4,5)P3-evoked Ca2+transients were reduced, from an average of 4.19 ± 0.40 ΔF/Fo in control to 0.23 ± 0.06 ΔF/Fo 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 ΔF/Fo in control to −0.04 ± 0.06 ΔF/Fo in the presence of thapsigargin (n = 10; p < 0.001; Fig.8A). CPA (10 μm) also increased resting [Ca2+]c from 1.18 ± 0.09F/Fo immediately prior to CPA to 1.46 ± 0.11 F/Fo 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, Dand E). On average, the response to Ins(1,4,5)P3was reduced from 1.70 ± 0.32 ΔF/Fo to 0.09 ± 0.0
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