Uptake and Release of Ca2+ by the Endoplasmic Reticulum Contribute to the Oscillations of the Cytosolic Ca2+ Concentration Triggered by Ca2+ Influx in the Electrically Excitable Pancreatic B-cell
1999; Elsevier BV; Volume: 274; Issue: 29 Linguagem: Inglês
10.1074/jbc.274.29.20197
ISSN1083-351X
AutoresPatrick Gilon, Abdelilah Arredouani, Philippe Gailly, Jesper Gromada, J. C. Henquin,
Tópico(s)Photoreceptor and optogenetics research
ResumoThe role of intracellular Ca2+ pools in oscillations of the cytosolic Ca2+ concentration ([Ca2+]c) triggered by Ca2+ influx was investigated in mouse pancreatic B-cells. [Ca2+]c oscillations occurring spontaneously during glucose stimulation or repetitively induced by pulses of high K+ (in the presence of diazoxide) were characterized by a descending phase in two components. A rapid decrease in [Ca2+]c coincided with closure of voltage-dependent Ca2+ channels and was followed by a slower phase independent of Ca2+ influx. Blocking the SERCA pump with thapsigargin or cyclopiazonic acid accelerated the rising phase of [Ca2+]coscillations and increased their amplitude, which suggests that the endoplasmic reticulum (ER) rapidly takes up Ca2+. It also suppressed the slow [Ca2+]c recovery phase, which indicates that this phase corresponds to the slow release of Ca2+ that was taken up by the ER during the upstroke of the [Ca2+]c transient. Glucose promoted the buffering capacity of the ER and amplified the slow [Ca2+]crecovery phase. The slow phase induced by high K+ pulses was not affected by modulators of Ca2+- or inositol 1,4,5-trisphosphate-induced Ca2+ release, did not involve a depolarization-induced Ca2+ release, and was also observed at the end of a rapid rise in [Ca2+]c triggered from caged Ca2+. It is attributed to passive leakage of Ca2+ from the ER. We suggest that the ER displays oscillations of the Ca2+ concentration ([Ca2+]ER) concomitant and parallel to [Ca2+]c. The observation that thapsigargin depolarizes the membrane of B-cells supports the proposal that the degree of Ca2+ filling of the ER modulates the membrane potential. Therefore, [Ca2+]ER oscillations occurring during glucose stimulation are likely to influence the bursting behavior of B-cells and eventually [Ca2+]c oscillations. The role of intracellular Ca2+ pools in oscillations of the cytosolic Ca2+ concentration ([Ca2+]c) triggered by Ca2+ influx was investigated in mouse pancreatic B-cells. [Ca2+]c oscillations occurring spontaneously during glucose stimulation or repetitively induced by pulses of high K+ (in the presence of diazoxide) were characterized by a descending phase in two components. A rapid decrease in [Ca2+]c coincided with closure of voltage-dependent Ca2+ channels and was followed by a slower phase independent of Ca2+ influx. Blocking the SERCA pump with thapsigargin or cyclopiazonic acid accelerated the rising phase of [Ca2+]coscillations and increased their amplitude, which suggests that the endoplasmic reticulum (ER) rapidly takes up Ca2+. It also suppressed the slow [Ca2+]c recovery phase, which indicates that this phase corresponds to the slow release of Ca2+ that was taken up by the ER during the upstroke of the [Ca2+]c transient. Glucose promoted the buffering capacity of the ER and amplified the slow [Ca2+]crecovery phase. The slow phase induced by high K+ pulses was not affected by modulators of Ca2+- or inositol 1,4,5-trisphosphate-induced Ca2+ release, did not involve a depolarization-induced Ca2+ release, and was also observed at the end of a rapid rise in [Ca2+]c triggered from caged Ca2+. It is attributed to passive leakage of Ca2+ from the ER. We suggest that the ER displays oscillations of the Ca2+ concentration ([Ca2+]ER) concomitant and parallel to [Ca2+]c. The observation that thapsigargin depolarizes the membrane of B-cells supports the proposal that the degree of Ca2+ filling of the ER modulates the membrane potential. Therefore, [Ca2+]ER oscillations occurring during glucose stimulation are likely to influence the bursting behavior of B-cells and eventually [Ca2+]c oscillations. The physiological response to a stimulus is often transduced by oscillations of the cytosolic free Ca2+ concentration ([Ca2+]c). In electrically nonexcitable cells, [Ca2+]c oscillations are mainly driven by antiparallel changes of the Ca2+ concentration within intracellular Ca2+ stores. In electrically excitable cells, [Ca2+]c oscillations are generally produced by intermittent influx of Ca2+ through voltage-dependent Ca2+ channels in the plasma membrane. In some of these cells, such as muscle cells and neurons, release of Ca2+ from intracellular stores can also contribute to the changes in [Ca2+]c (1Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2268) Google Scholar, 2Berridge M.J. J. Physiol. Lond. 1997; 499: 290-306Crossref Scopus (916) Google Scholar). The insulin-secreting pancreatic B-cell is electrically excitable. Its main physiological stimulus, glucose, triggers insulin secretion by increasing [Ca2+]c through the following steps. Acceleration of glucose metabolism increases the ATP/ADP ratio, which closes ATP-sensitive K+ channels (KATPchannels) in the plasma membrane (3Ashcroft F.M. Rorsman P. Prog. Biophys. Mol. Biol. 1989; 54: 87-143Crossref PubMed Scopus (952) Google Scholar). This closure decreases the K+ conductance, which allows a yet unknown current to depolarize the plasma membrane, leading to opening of voltage-dependent Ca2+ channels, stimulation of Ca2+ influx, and eventually a rise in [Ca2+]c. In the presence of 10–15 mmglucose, B-cells display [Ca2+]c oscillations that result mainly from intermittent Ca2+ influx (4Gilon P. Henquin J.C. J. Biol. Chem. 1992; 267: 20713-20720Abstract Full Text PDF PubMed Google Scholar, 5Santos R.M. Rosario L.M. Nadal A. Garcia Sancho J. Soria B. Valdeolmillos M. Pfluegers Arch. 1991; 418: 417-422Crossref PubMed Scopus (316) Google Scholar). However, it has been speculated that Ca2+- or inositol 1,4,5-trisphosphate (IP3) 1The abbreviations IP3inositol 1,4,5-trisphosphateAChacetylcholineCPAcyclopiazonic acidERendoplasmic reticulumISOCstore-operated currentPMCAplasma membrane Ca2+-ATPaseSERCAsarco-endoplasmic reticulum Ca2+-ATPaseTGthapsigargin -induced Ca2+ release might contribute to each [Ca2+]c oscillation induced by glucose (6Ämmälä C. Larsson O. Berggren P.O. Bokvist K. Juntti-Berggren L. Kindmark H. Rorsman P. Nature. 1991; 353: 849-852Crossref PubMed Scopus (102) Google Scholar, 7Barker C.J. Nilsson T. Kirk C.J. Michell R.H. Berggren P.O. Biochem. J. 1994; 297: 265-268Crossref PubMed Scopus (15) Google Scholar, 8Gromada J. Frokjaer-Jensen J. Dissing S. Biochem. J. 1996; 314: 339-345Crossref PubMed Scopus (27) Google Scholar, 9Islam M.S. Leibiger I. Leibiger B. Rossi D. Sorrentino V. Ekstrom T.J. Westerblad H. Andrade F.H. Berggren P.O. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6145-6150Crossref PubMed Scopus (93) Google Scholar, 10Liu Y.J. Grapengiesser E. Gylfe E. Hellman B. Arch. Biochem. Biophys. 1996; 334: 295-302Crossref PubMed Scopus (79) Google Scholar). inositol 1,4,5-trisphosphate acetylcholine cyclopiazonic acid endoplasmic reticulum store-operated current plasma membrane Ca2+-ATPase sarco-endoplasmic reticulum Ca2+-ATPase thapsigargin The aim of the present study was to investigate the possible role of intracellular Ca2+ stores in [Ca2+]coscillations induced by Ca2+ influx in normal pancreatic B-cells. Strategies using targeted Ca2+-sensitive proteins (11Rizzuto R. Brini M. Murgia M. Pozzan T. Science. 1993; 262: 744-747Crossref PubMed Scopus (1009) Google Scholar, 12Miyawaki A. Llopis J. Heim R. McCaffery J.M. Adams J.A. Ikura M. Tsien R.Y. Nature. 1997; 388: 882-887Crossref PubMed Scopus (2619) Google Scholar) or trapped fluorescent low-affinity Ca2+indicators (13Chatton J.-Y. Liu H. Stucki J.W. FEBS Lett. 1995; 368: 165-168Crossref PubMed Scopus (61) Google Scholar, 14Mogami H. Tepikin A.V. Petersen O.H. EMBO J. 1998; 17: 435-442Crossref PubMed Scopus (200) Google Scholar, 15Golovina V.A. Blaustein M.P. Science. 1997; 275: 1643-1648Crossref PubMed Scopus (425) Google Scholar) have recently been developed to measure directly the free Ca2+ concentration within intracellular organelles. However, these techniques suffer from drawbacks such as difficult transfection procedures of photoproteins, very low light emission and Ca2+-induced degradation of aequorin, and contamination of the trapped fluorescence of low-affinity Ca2+ indicators by the cytosolic signal, which severely limit their use in intact primary cells. We, therefore, used the classical technique of Ca2+ measurement within the cytosol, which is not invasive and is applicable to single or electrically coupled B-cells. The results demonstrate that [Ca2+]c oscillations occurring spontaneously during stimulation by glucose, or artificially induced by pulses of high K+, are accompanied by cycles of rapid uptake and subsequent slow release of Ca2+ by the endoplasmic reticulum (ER). Thapsigargin-sensitive Ca2+-ATPases (SERCA pumps) are responsible for the sequestration process during the upstroke of the [Ca2+]c transient, whereas the subsequent phase of release does not involve depolarization-, Ca2+- or IP3-mediated processes and likely results from leakage from the ER. This suggests that the Ca2+ concentration within the endoplasmic reticulum ([Ca2+]ER) oscillates. As the filling state in Ca2+ of the ER may modulate the membrane potential of B-cells (16Worley III, J.F. McIntyre M.S. Spencer B. Mertz R.J. Roe M.W. Dukes I.D. J. Biol. Chem. 1994; 269: 14359-14362Abstract Full Text PDF PubMed Google Scholar), it is possible that [Ca2+]ERoscillations play a role in the control of the oscillations of the membrane potential. Except for patch-clamp measurements and the experiments illustrated in Fig. 4 D (see below), the medium used was a bicarbonate-buffered solution that contained 120 mm NaCl, 4.8 mm KCl, 0.5–10 mm CaCl2, 1.2 mm MgCl2, 24 mm NaHCO3and 0–20 mm glucose as indicated. When the concentration of KCl was increased, that of NaCl was decreased accordingly to keep the osmolarity of the medium unchanged. Ca2+-free solutions were prepared by substituting MgCl2 for CaCl2and were supplemented with 0.5 or 2 mm EGTA as indicated in the legends to Figs. 2, 3, and 5.Figure 2Pulses of 45 mm K+induce [Ca2+]c oscillations with a slow recovery phase that is prevented by SERCA pump inhibition in pancreatic B-cells. The medium contained 10 mm glucose and 250 μm diazoxide in all experiments. 30-s pulses of 45 mm K+ were applied as shown by bars. The Ca2+ concentration of the perifusion medium was either 10 mm throughout (A) or was changed as indicated (B–C). A and B, whole islets were used. In A, solid lines show changes in [Ca2+]c, whereas the dotted linesillustrate associated changes of the membrane potential recorded in separate islets. For the traces labeled thapsigargin in Aand B, the islets were incubated with 1 μm TG during the loading procedure with fura-PE3 ([Ca2+]c measurements) or during a 90–120-min preincubation in the culture medium prior the experiments (membrane potential measurements). C, a single pancreatic B-cell was used; it was injected with fura-2 dextran. TG was applied when indicated. Ca2+-free solutions were supplemented with 2 mm EGTA. The traces are representative of results obtained in 12 (A) ([Ca2+]c), 4 (A) (MP), and 7 (B) islets and 4 single cells (C).View Large Image Figure ViewerDownload (PPT)Figure 3Ca2+ is rapidly taken up by the ER during the upstroke of each [Ca2+]coscillation and is released at the end of each oscillation. All solutions contained 10 mm glucose and 250 μmdiazoxide. The Ca2+ concentration of the perifusion medium was changed when indicated. Ca2+-free solutions were supplemented with 2 mm EGTA. 100 μm ACh and 30-s (A–D) or 20-s (E) pulses of 45 mm K+ were applied when indicated. The traces are representative of results obtained in 11 (A), 12 (B), 4 (C), 8 (D), and 4 (E) islets.View Large Image Figure ViewerDownload (PPT)Figure 5The slow [Ca2+]crecovery phase following high K+-induced [Ca2+]c oscillations does not involve depolarization- or IP3-induced Ca2+ release and a similar slow decay is observed after an abrupt rise in [Ca 2+ ]c triggered by uncaging Ca2+ by flashes of UV light. A, whole islets loaded with fura-PE3. B, clusters of islet cells loaded with fura-2 and nitrophenyl-EGTA. C, single cells injected with fura-2, with or without heparin. All perifusion solutions contained 10 mm glucose and 250 μm diazoxide throughout. The Ca2+ concentration of the medium was either constant throughout the whole experiment (B), or it was changed and test agents (100 μm ACh or 1 μmTG) were added when indicated (A and C). [Ca2+]c was increased by uncaging Ca2+ from nitrophenyl-EGTA with two or three flashes of UV light (arrows in B) or by 30-s pulses of 45 mm K+ (bars in A–C). InA and C, Ca2+-free solutions were supplemented with 2 mm EGTA. In B, interruption of the trace corresponds to a period during which 1 μm TG was applied. The traces are representative of results obtained in 10 islets (A), 3 clusters of cells (B), and 4 (control) and 6 (heparin) single cells (C).View Large Image Figure ViewerDownload (PPT) In the experiments illustrated in Fig. 4 D, it was important to minimize changes in the activity of the Na+/Ca2+ exchange between solutions containing various K+ concentrations. Therefore, KCl was not replaced with NaCl but with choline chloride to keep a similar Na+concentration in all solutions. The low K+ solution contained: 79.8 mm NaCl, 4.8 mm KCl, 40.2 mm choline chloride, 2.5 mm CaCl2, 1.2 mm MgCl2, 24 mmNaHCO3, and 0.01 mm atropine, which prevented activation of muscarinic receptors by choline. The solutions containing higher K+ concentrations were prepared by substituting KCl for choline chloride. All solutions were gassed with O2/CO2 (94:6) to maintain a pH of 7.4 at 37 °C. Except for electrophysiological recordings, they were supplemented with 1 mg/ml bovine serum albumin (fraction V; Roche Molecular Biochemicals). Thapsigargin was obtained from Sigma or from Alomone Laboratories (Jerusalem, Israel). Ryanodine was from RBI (Natick, MA) or from Alomone Laboratories, diazoxide was from Schering-Plough Avondale (Rathdrum, Ireland), caffeine was from Merck A.G. (Darmstadt Germany), and ruthenium red was from Alexis Corp. (San Diego, CA). All other chemicals were from Sigma. All experiments were performed with tissue from fed female NMRI mice (25–30 g). Pancreatic islets were isolated aseptically after collagenase digestion of the pancreas, and when needed, they were dispersed into cells as described previously (17Miura Y. Henquin J.C. Gilon P. J. Physiol. Lond. 1997; 503: 387-398Crossref Scopus (75) Google Scholar). Cells were allowed to attach to 22-mm circular coverslips and cultured for 2–3 days. Intact islets were maintained in culture for 1–3 days. When the membrane potential of B-cells was to be measured with an intracellular microelectrode, the islets were allowed to attach to the coverslip by a culture period of at least 2 days. The culture medium was RMPI 1640 medium containing 10 mm glucose, 10% heat-inactivated fetal calf serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin. Cultured islets were loaded with 2 μm fura-PE3/AM (Teflabs, Austin, TX) for 90–120 min at 37 °C in a bicarbonate-buffered solution containing 10 mm glucose. Cultured cells were loaded with 1 μm fura-2/AM (Molecular Probes, Eugene, OR) for 60 min in a similar bicarbonate-buffered medium. The tissue was then transferred into a temperature-controlled (37 °C) perifusion chamber of ∼1 ml (Intracell, Royston, Herts, United Kingdom) with a bottom made of a glass coverslip and mounted on the stage of an inverted microscope. The flow rate of the perifusion was approximately 2 ml/min. When rapid exchange of solutions was required, a ∼250-μl chamber was used and solutions were changed by Iso-Latch valves (Parker Hannifin, Fairfield, NY). [Ca2+]i was directly measured in cells attached to the coverslip or in islets held in place close to the coverslip by gentle suction with a glass micropipette. In some experiments, cultured cells were pressure-injected with an 5242 Eppendorf microinjector (Hamburg, Germany). The injected solution contained either 6–10 mm fura-2 K+ salt or 10 mmfura-dextran K+ salt (molecular weight, 3000) (Molecular Probes) dissolved in H2O, and it was supplemented or not with test substances. The techniques used to monitor [Ca2+]c have been described previously (4Gilon P. Henquin J.C. J. Biol. Chem. 1992; 267: 20713-20720Abstract Full Text PDF PubMed Google Scholar). Clusters of B-cells were incubated with 5 μmnitrophenyl-EGTA AM and 1.5 μm fura-2 AM (Molecular Probes) for 60 min at 37 °C. Photolysis of nitrophenyl-EGTA was performed by two or three consecutive 1-ms UV flashes of 240 J (Xenon flashlamp system XF-10, Hi-Tech, Hamburg, Germany). The islets were mounted in a perifusion chamber (7 ml/min at 37 °C) following attachment to glass coverslips. The membrane potential of a single cell within the islet was continuously measured with a high resistance microelectrode. Voltage-clamp experiments were performed on single B-cells using the perforated patch-whole cell configuration and an EPC-7 patch-clamp amplifier (List Elektronik, Darmstadt, Germany). The holding potential was −70 mV, and the cells were submitted either to 100-ms depolarizations to 0 mV or to bursts of 100-ms depolarizations (2 Hz) from −50 mV to −10 mV for 12 s. The associated changes in [Ca2+]i were measured using an IonOptix fluorescence imaging system (IonOptix, Inc., Milton, MA). The extracellular solution contained 138 mm NaCl, 5.6 mm KCl, 1.2 mm MgCl2, 2.6 mm CaCl2, 5 mm HEPES (pH 7.4 with NaOH), and 10 mm glucose. The pipette solution contained 76 mm Cs2SO4, 10 mm NaCl, 10 mm KCl, 1 mm MgCl2, and 5 mm HEPES (pH 7.35 with CsOH). Electrical contact with the cell interior was established by adding 0.24 mg/ml amphotericin B to the pipette solution, and the voltage-clamp was considered satisfactory when the series conductance (Gseries) was >35–40 nano Siemens. All experiments were performed at 33 °C, and the zero-current potential of the pipette was adjusted with the pipette in the bath solution. The experiments are illustrated by recordings that are averaged or representative traces of results obtained with the indicated number of cells or islets from at least three different cultures. The statistical significance of differences between means was assessed by unpaired Student's t test. B-cells within intact islets display a rhythmic electrical activity when perifused with a medium containing an insulin-releasing glucose concentration (10 mm) and 10 mm Ca2+ (Fig.1 A). These bursts of electrical activity consist of sharp depolarizing waves of the membrane potential with superimposed spikes reflecting Ca2+ influx through voltage-dependent Ca2+ channels (18Henquin J.C. Meissner H.P. Experientia. 1984; 40: 1043-1052Crossref PubMed Scopus (278) Google Scholar). Under these conditions, [Ca2+]c also oscillates, but, in contrast to the fast, monophasic repolarization of the oscillations of membrane potential, the descending phase of each Ca2+ oscillation clearly displays two components (Fig.1 B). Whereas the initial fast one appears to coincide with the closure of voltage-dependent Ca2+ channels following rapid repolarization of the plasma membrane, the second, much slower phase appears to occur during the repolarized intervals. Previous experiments have shown that intracellular Ca2+stores of whole islets are efficiently emptied by thapsigargin (TG), a specific inhibitor of the SERCA pump (19Inesi G. Sagara Y. J. Membr. Biol. 1994; 141: 1-6Crossref PubMed Scopus (161) Google Scholar), but that this emptying requires preincubation of the islets with the drug (17Miura Y. Henquin J.C. Gilon P. J. Physiol. Lond. 1997; 503: 387-398Crossref Scopus (75) Google Scholar). In islets pretreated with 1 μm TG, the amplitude of [Ca2+]c oscillations was much larger than in control islets, and the descending phase of each [Ca2+]c oscillation was surprisingly very fast with no slow second phase (Fig. 1 C). This suggests that the slow phase observed in control islets results from a release of Ca2+ from the ER, rather than from a slow Ca2+extrusion from the cytosol. The effect of intracellular Ca2+ store depletion on [Ca2+]c oscillations was also investigated in clusters of islet cells, a preparation in which the SERCA pump can be blocked by an acute addition of TG or cyclopiazonic acid (CPA). CPA is an inhibitor structurally unrelated to TG (19Inesi G. Sagara Y. J. Membr. Biol. 1994; 141: 1-6Crossref PubMed Scopus (161) Google Scholar) and has also been shown to empty the ER of Ca2+ in pancreatic B-cells (20Miura Y. Gilon P. Henquin J.C. Biochem. Biophys. Res. Commun. 1996; 224: 67-73Crossref PubMed Scopus (48) Google Scholar). In the presence of 15 mm glucose and 2.5 mmCa2+, [Ca2+]c oscillated slowly and regularly (Fig. 1 D). Addition of 50 μm CPA to the medium accelerated the oscillations, which increased in amplitude and frequency and became sharper mainly because of the disappearance of the slow recovery phase. Similar results were obtained in clusters of islet cells treated by TG (not shown). In this series of experiments, glucose-induced [Ca2+]c oscillations were inhibited by diazoxide, which, by opening KATP channels, clamps the membrane potential at a hyperpolarized level. [Ca2+]c oscillations were then reinduced by rhythmically depolarizing the plasma membrane with high K+. Raising the K+ concentration of the perifusion medium from 4.8 to 45 mm rapidly depolarized the plasma membrane from −70 ± 2 mV to −22 ± 3 mV in control islets (n = 4; Fig. 2 A, dotted line). The amplitude of this depolarization was not affected by TG pretreatment of the islets (−71 ± 3 to −22 ± 3 mV, n = 4). The time required to clamp the plasma membrane at a new, stable potential was also similar in both groups, as follows. Controls: 31 ± 2 s from 4.8 to 45 mmK+ (t 12 = 4 ± 0 s) and 34 ± 1 s from 45 to 4.8 mm K+(t 12 = 5.7 ± 0.5 s), n = 4; TG-treated islets: 31 ± 1 s from 4.8 to 45 mmK+ (t 12 = 4.5 ± 0.3 s) and 34 ± 1 s from 45 to 4.8 mm K+(t 12 = 5.7 ± 0.5 s),n = 4. In control islets, high K+ pulses (for 30 s) induced [Ca2+]c oscillations characterized by a descending phase that displayed an initial fast component concomitant with rapid repolarization of the plasma membrane, followed by a slow decline (Fig. 2 A, solid line). In TG-pretreated islets, [Ca2+]c oscillations were higher than in control islets (467 ± 21 versus 352 ± 11 nm,n = 10, p < 0.01) and devoid of a slow recovery phase. Similar results were obtained after pretreatment of the islets with 50 μm CPA. The effects of TG on voltage-dependent Ca2+current were evaluated in single B-cells using the perforated patch configuration. Under control conditions, a 100-ms voltage-step from −70 to 0 mV elicited a peak Ca2+ current of 52 ± 6 pA (n = 8) that was not significantly affected by a 5-min exposure to 1 μm TG (48 ± 3 pA). The integrated whole-cell Ca2+ current was similarly unaffected by TG (data not shown). This excludes the possibility that the larger rise in [Ca2+]c induced by high K+ in TG-treated islets results from an increased Ca2+current. We also verified that the slow [Ca2+]c recovery is not a peculiarity observed only in a medium containing 10 mm Ca2+. To this end, 30-s pulses of 45 mm K+ were applied in the presence of various concentrations of external Ca2+ (0.5–10 mm) (Fig. 2 B). The amplitude of the resulting [Ca2+]c peaks clearly depended on the Ca2+ concentration of the medium, but a slow decaying phase, prevented by TG pretreatment, was observed at all external Ca2+ concentrations tested. The observation that TG suppresses the slow recovery after [Ca2+]coscillations of various amplitude also excludes the possibility that TG might increase the rate of Ca2+ extrusion from the cytosol due to a high Ca2+ signal. The slow [Ca2+]c recovery phase could be artifactual and reflect changes in the Ca2+ concentration within the ER if fura-PE3 is compartmentalized. To exclude this possibility, single B-cells were microinjected with fura-dextran, a Ca2+ probe that is exclusively localized in the cytosol (21Schlatterer C. Knoll G. Malchow D. Eur. J. Cell Biol. 1992; 58: 172-181PubMed Google Scholar). High K+ pulses induced [Ca2+]coscillations with a slow recovery phase (Fig. 2 C). Addition of TG to the medium induced a transient increase in [Ca2+]c reflecting intracellular Ca2+pool emptying. Subsequent depolarization by pulses of high K+ triggered [Ca2+]c oscillations that were of much larger amplitude than before addition of the SERCA pump inhibitor and that lacked a slow [Ca2+]crecovery phase. These observations strongly support the conclusion that the slow decaying [Ca2+]c phase results from release of Ca2+ from the ER. Application of high K+ pulses every 5 min triggered a train of [Ca2+]coscillations with a slow decaying phase, which indicates that the phenomenon is not a transient one (Fig.3 A). The experiments depicted in Fig. 3 B were designed to explore the temporal requirements for refilling the intracellular Ca2+ stores responsible for the slow decay in [Ca2+]c. The islets were repetitively depolarized by 30-s pulses of high K+. Extracellular Ca2+ (10 mm) was present before and during the depolarization (first and last pulses) or only during the depolarization (second to seventh pulses). The slow recovery phase was present and not attenuated by Ca2+omission during the repolarization phases (compare Fig. 3, Aand B). This shows first that it does not result from Ca2+ influx, and second that Ca2+ entry during depolarization is sufficient to refill the pools from which Ca2+ is slowly released. However, no slow recovery phase was observed when high K+pulses were applied in the continuous presence of acetylcholine (ACh), a potent IP3-producing agent in pancreatic B-cells (Fig.3 C). This is likely due to the fact that Ca2+cannot accumulate into the ER because it immediately exits from the ER into the cytosol through IP3 receptors that are maintained opened by the continuous presence of ACh. The ability of the ER to take up Ca2+ rapidly was next tested (Fig. 3 D). Islets perifused with a Ca2+-free medium were submitted to three pulses of 100 μm ACh applied at 12.5-min intervals. A 30-s pulse of high K+/high Ca2+ was applied between the second and the third pulses of ACh. Whereas the first application of ACh triggered a large [Ca2+]c peak, the second one induced only a small rise in [Ca2+]csuggesting that intracellular Ca2+ stores were nearly completely emptied already by the first application of ACh. However, the third application of ACh in a Ca2+-free medium after the short pulse with high K+/high Ca2+ induced a transient rise in [Ca2+]c that was much larger than that seen after the second application of ACh. This indicates further that intracellular Ca2+ pools rapidly refill during the large [Ca2+]c rises triggered by high K+ pulses. If the slow recovery phase reflects release of Ca2+ from the ER, its characteristics should depend on the filling state of the ER. This was tested by emptying the ER with ACh between two series of 3 pulses of high K+/high Ca2+ of 20 s duration (Fig. 3 E). The first three [Ca2+]c oscillations were all characterized by a slow recovery phase. In contrast, the first two oscillations following intracellular Ca2+ pool depletion by ACh were of lower amplitude and displayed a much smaller slow recovery phase than before ACh application. Because the pulses were of constant duration, the lower amplitude of [Ca2+]c oscillations post-ACh is unlikely to result from a decreased Ca2+ influx. It may rather be explained by a more avid sequestration of Ca2+into an emptied than into a filled ER. Because the first high K+/high Ca2 pulse did not carry enough Ca2+ to fully refill the ER, no slow recovery phase could be seen, and three pulses were needed to refill the ER enough to see a slow recovery phase of an amplitude similar to that observed at the end of the first series of [Ca2+]c oscillations. These data demonstrate that the buffering capacity of the ER permits a rapid control of [Ca2+]c and that its ability to release Ca2+ is affected by its filling state. Comparison of the [Ca2+]c changes induced by a pulse of high K+ in control and TG-treated islets permits estimation of the kinetics of Ca2+ uptake and release from the ER (Fig. 4 A). After normalization of resting [Ca2+]c before each [Ca2+]c oscillation the averaged [Ca2+]c oscillation of TG-treated islets was subtracted from the averaged [Ca2+]c oscillation of control islets (Fig. 4 B). The downward deflection of the curve reflects Ca2+ uptake by the ER, whereas the upward deflection reflects release from the ER. This shows that the uptake is very fast, whereas the release is comparably slow and lasts several minutes. The role of the ER during the whole [Ca2+]coscillation is best demonstrated by the comparison of the rates of [Ca2+]c changes as a function of [Ca2+]c in control and TG-treated islets (Fig.4 C). It clearly shows that the ER strongly buffers the rate of [Ca2+]c changes during the whole [Ca2+]c oscillation, thereby preventing any abrupt large change in [Ca2+]c. The above results suggest that the amount of Ca2+ that is taken up by the ER is directly proportional to [Ca2+]c. This was indirectly verified by measuring the amplitude of the [Ca2+]c peak that occurred upon addition of TG to clusters of cells in which [Ca2+]c was clamped artificially at different levels with various concentrations of K+ (4.8–45 mm). The amplitude of the [Ca2+]cpeak directly depended on the steady-state level of [Ca2+]c before TG addition (Fig. 4 D), suggesting that the Ca2+ loading of the ER is directly proportional to the level of [Ca2+]c. This did not result from a K+ effect, as the amplitude of the rise in [Ca2+]c was similar in clusters perifused with a Ca2+-free medium containing 4.8 or 45 mmK+. The large rise in [Ca2+]cproduced by TG in th
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