Ca2+-induced Ca2+ Release via Inositol 1,4,5-trisphosphate Receptors Is Amplified by Protein Kinase A and Triggers Exocytosis in Pancreatic β-Cells
2004; Elsevier BV; Volume: 279; Issue: 44 Linguagem: Inglês
10.1074/jbc.m407673200
ISSN1083-351X
Autores Tópico(s)Cellular transport and secretion
ResumoHormones, such as glucagon and glucagon-like peptide-1, potently amplify nutrient stimulated insulin secretion by raising cAMP. We have studied how cAMP affects Ca2+-induced Ca2+ release (CICR) in pancreatic β-cells from mice and rats and the role of CICR in secretion. CICR was observed as pronounced Ca2+ spikes on top of glucose- or depolarization-dependent rise of the cytoplasmic Ca2+ concentration ([Ca2+]i). cAMP-elevating agents strongly promoted CICR. This effect involved sensitization of the receptors underlying CICR, because many cells exhibited the characteristic Ca2+ spiking at low or even in the absence of depolarization-dependent elevation of [Ca2+]i. The cAMP effect was mimicked by a specific activator of protein kinase A in cells unresponsive to activators of cAMP-regulated guanine nucleotide exchange factor. Ryanodine pretreatment, which abolishes CICR mediated by ryanodine receptors, did not prevent CICR. Moreover, a high concentration of caffeine, known to activate ryanodine receptors independently of Ca2+, failed to mobilize intracellular Ca2+. On the contrary, a high caffeine concentration abolished CICR by interfering with inositol 1,4,5-trisphosphate receptors (IP3Rs). Therefore, the cell-permeable IP3R antagonist 2-aminoethoxydiphenyl borate blocked the cAMP-promoted CICR. Individual CICR events in pancreatic β-cells were followed by [Ca2+]i spikes in neighboring human erythroleukemia cells, used to report secretory events in the β-cells. The results indicate that protein kinase A-mediated promotion of CICR via IP3Rs is part of the mechanism by which cAMP amplifies insulin release. Hormones, such as glucagon and glucagon-like peptide-1, potently amplify nutrient stimulated insulin secretion by raising cAMP. We have studied how cAMP affects Ca2+-induced Ca2+ release (CICR) in pancreatic β-cells from mice and rats and the role of CICR in secretion. CICR was observed as pronounced Ca2+ spikes on top of glucose- or depolarization-dependent rise of the cytoplasmic Ca2+ concentration ([Ca2+]i). cAMP-elevating agents strongly promoted CICR. This effect involved sensitization of the receptors underlying CICR, because many cells exhibited the characteristic Ca2+ spiking at low or even in the absence of depolarization-dependent elevation of [Ca2+]i. The cAMP effect was mimicked by a specific activator of protein kinase A in cells unresponsive to activators of cAMP-regulated guanine nucleotide exchange factor. Ryanodine pretreatment, which abolishes CICR mediated by ryanodine receptors, did not prevent CICR. Moreover, a high concentration of caffeine, known to activate ryanodine receptors independently of Ca2+, failed to mobilize intracellular Ca2+. On the contrary, a high caffeine concentration abolished CICR by interfering with inositol 1,4,5-trisphosphate receptors (IP3Rs). Therefore, the cell-permeable IP3R antagonist 2-aminoethoxydiphenyl borate blocked the cAMP-promoted CICR. Individual CICR events in pancreatic β-cells were followed by [Ca2+]i spikes in neighboring human erythroleukemia cells, used to report secretory events in the β-cells. The results indicate that protein kinase A-mediated promotion of CICR via IP3Rs is part of the mechanism by which cAMP amplifies insulin release. Glucose is the most important physiological stimulator of insulin secretion from pancreatic β-cells. A major signal transduction pathway involves metabolism of glucose with increase of the ATP/ADP ratio, depolarization caused by closure of ATP/ADP-sensitive K+ (KATP) channels, and opening of L-type Ca2+ channels with influx of the ion. The resulting elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i) 1The abbreviations used are: [Ca2+]i, cytoplasmic Ca2+ concentration; ER, endoplasmic reticulum; IP3, inositol 1,4,5-trisphosphate; IP3R, inositol 1,4,5-trisphosphate receptor; RyR, ryanodine receptor; CICR, Ca2+-induced Ca2+ release; PKA, protein kinase A; AM, acetoxymethyl ester; 8-pCPT-2′-O-Me-cAMP, 8-(4-chlorophenylthio)-2′-O-methyl-cAMP; 8-pMeOPT-2′-O-Me-cAMP, 8-(4-methoxyphenylthio)-2′-O-methyl-cAMP; 5,6-DCl-cBIMPS, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole-3′,5′-cyclic monophosphorothioate; 8-Br-cAMPS, 8-bromoadenosine-3′,5′-cyclic monophosphorothioate; 8-CPT-cAMPS, 8-(4-chlorophenylthio)adenosine-3′,5′-cyclic monophosphorothioate; GLP-1, glucagon-like peptide-1; 8-Br-cAMP, 8-bromo-cAMP; Epac, cAMP-regulated guanine nucleotide exchange factor; IBMX, 3-isobutyl-1-methylxanthine; 2-APB, 2-aminoethoxydiphenyl borate; SERCA, sarco(endo)plasmic reticulum ATPase 3; Rp, Rp isomer; Sp, Sp isomer. triggers exocytosis of the insulin-containing granules (1Ashcroft F.M. Rorsman P. Prog. Biophys. Mol. Biol. 1989; 54: 87-143Crossref PubMed Scopus (955) Google Scholar). By stimulating Ca2+ sequestration in the endoplasmic reticulum (ER) (2Gylfe E. Pfluegers Arch. Eur. J. Physiol. 1991; 419: 639-643Crossref PubMed Scopus (40) Google Scholar, 3Tengholm A. Hellman B. Gylfe E. J. Biol. Chem. 1999; 274: 36883-36890Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 4Tengholm A. Hellman B. Gylfe E. J. Physiol. (Lond.). 2001; 530: 533-540Crossref Scopus (46) Google Scholar), glucose also has an important role in preparing the β-cell to respond to hormones and neurotransmitters, which act by mobilizing Ca2+ from the ER (5Biden T.J. Prentki M. Irvine R.F. Berridge M.J. Wollheim C.B. Biochem. J. 1984; 223: 467-473Crossref PubMed Scopus (128) Google Scholar, 6Hellman B. Gylfe E. Wesslén N. Biochem. Int. 1986; 13: 383-389PubMed Google Scholar, 7Ahren B. Diabetologia. 2000; 43: 393-410Crossref PubMed Scopus (701) Google Scholar). The latter effects are in most cases caused by activation of phospholipase C, catalyzing the formation of inositol 1,4,5-trisphosphate (IP3). The IP3 receptor (IP3R) is a Ca2+ channel in the ER membrane (8Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6188) Google Scholar). Another putative pathway for Ca2+ release from the ER is via ryanodine receptors (RyRs). Although RyRs are expressed in β-cells (9Islam M.S. Leibiger I. Leibiger B. Rossi D. Sorrentino V. Ekström 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, 10Holz G.G. Leech C.A. Heller R.S. Castonguay M. Habener J.F. J. Biol. Chem. 1999; 274: 14147-14156Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 11Varadi A. Rutter G.A. Diabetes. 2002; 51: S190-S201Crossref PubMed Google Scholar, 12Beauvois M.C. Arredouani A. Jonas J.C. Rolland J.F. Schuit F. Henquin J.C. Gilon P. J. Physiol. (Lond.). 2004; 559: 141-156Crossref Scopus (25) Google Scholar), their physiological role remains controversial (12Beauvois M.C. Arredouani A. Jonas J.C. Rolland J.F. Schuit F. Henquin J.C. Gilon P. J. Physiol. (Lond.). 2004; 559: 141-156Crossref Scopus (25) Google Scholar, 13Cancela J.M. Petersen O.H. Diabetes. 2002; 51: S349-S357Crossref PubMed Google Scholar, 14Johnson J.D. Kuang S. Misler S. Polonsky K.S. FASEB J. 2004; 18: 878-880Crossref PubMed Scopus (67) Google Scholar). Ca2+-induced Ca2+ release (CICR) is a mechanism by which any local rise of [Ca2+]i becomes further amplified by Ca2+ release from stores. The heart is a classic example of CICR, where it provides a link between depolarization-dependent influx of "triggering" Ca2+ and release of contraction-inducing Ca2+ from the sarcoplasmic reticulum (15Fabiato A. Fabiato F. Nature. 1979; 281: 146-148Crossref PubMed Scopus (78) Google Scholar). In heart cells, CICR is caused by activation of RyRs. However, in many other types of cells, IP3Rs are equally competent in mediating CICR because they display a similar autocatalytic Ca2+ release mechanism (16Roderick H.L. Berridge M.J. Bootman M.D. Curr. Biol. 2003; 13: R425Abstract Full Text Full Text PDF PubMed Google Scholar). The binding of IP3 thus sensitizes the IP3Rs to the stimulatory effect of Ca2+ (17Iino M. J. Gen. Physiol. 1990; 95: 1103-1122Crossref PubMed Scopus (496) Google Scholar, 18Bezprozvanny I. Watras J. Ehrlich B.E. Nature. 1991; 351: 751-754Crossref PubMed Scopus (1441) Google Scholar). As in the heart, CICR in the β-cell may provide a link between influx of Ca2+ and release from intracellular stores, resulting in amplification of the Ca2+ signal triggering insulin secretion (19Lemmens R. Larsson O. Berggren P.O. Islam M.S. J. Biol. Chem. 2001; 276: 9971-9977Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Several studies propose that CICR in β-cells is mediated by RyRs (10Holz G.G. Leech C.A. Heller R.S. Castonguay M. Habener J.F. J. Biol. Chem. 1999; 274: 14147-14156Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 19Lemmens R. Larsson O. Berggren P.O. Islam M.S. J. Biol. Chem. 2001; 276: 9971-9977Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 20Kang G. Chepurny O.G. Holz G.G. J. Physiol. (Lond.). 2001; 536: 375-385Crossref Scopus (173) Google Scholar, 21Bruton J.D. Lemmens R. Shi C.L. Persson-Sjögren S. Westerblad H. Ahmed M. Pyne N.J. Frame M. Furman B.L. Islam M.S. FASEB J. 2002; 17: 301-303Crossref PubMed Scopus (56) Google Scholar, 22Kang G. Joseph J.W. Chepurny O.G. Monaco M. Wheeler M.B. Bos J.L. Schwede F. Genieser H.G. Holz G.G. J. Biol. Chem. 2003; 278: 8279-8285Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). Critical experiments in the latter studies rely on the use of tumor-transformed clonal β-cells, and we have recently confirmed the expression of functional RyRs in rat insulinoma cells (23Dyachok O. Tufveson G. Gylfe E. Cell Calcium. 2004; 36: 1-9Crossref PubMed Scopus (34) Google Scholar). However, our study also showed that CICR in primary β-cells from mice, rats, and human subjects is caused by activation of IP3Rs rather than RyRs. Agents raising cAMP have been found to promote intracellular Ca2+ mobilization in insulin-releasing cell lines and pancreatic β-cells, and this action was proposed to be mediated by sensitization of either IP3Rs (24Liu Y.J. Grapengiesser E. Gylfe E. Hellman B. Arch. Biochem. Biophys. 1996; 334: 295-302Crossref PubMed Scopus (79) Google Scholar, 25Liu Y.J. Tengholm A. Grapengiesser E. Hellman B. Gylfe E. J. Physiol. (Lond.). 1998; 508: 471-481Crossref Scopus (104) Google Scholar, 26Tsuboi T. da Silva Xavier G. Holz G.G. Jouaville L.S. Thomas A.P. Rutter G.A. Biochem. J. 2003; 369: 287-299Crossref PubMed Scopus (0) Google Scholar) or RyRs (9Islam M.S. Leibiger I. Leibiger B. Rossi D. Sorrentino V. Ekström 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, 10Holz G.G. Leech C.A. Heller R.S. Castonguay M. Habener J.F. J. Biol. Chem. 1999; 274: 14147-14156Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 20Kang G. Chepurny O.G. Holz G.G. J. Physiol. (Lond.). 2001; 536: 375-385Crossref Scopus (173) Google Scholar, 21Bruton J.D. Lemmens R. Shi C.L. Persson-Sjögren S. Westerblad H. Ahmed M. Pyne N.J. Frame M. Furman B.L. Islam M.S. FASEB J. 2002; 17: 301-303Crossref PubMed Scopus (56) Google Scholar, 22Kang G. Joseph J.W. Chepurny O.G. Monaco M. Wheeler M.B. Bos J.L. Schwede F. Genieser H.G. Holz G.G. J. Biol. Chem. 2003; 278: 8279-8285Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 26Tsuboi T. da Silva Xavier G. Holz G.G. Jouaville L.S. Thomas A.P. Rutter G.A. Biochem. J. 2003; 369: 287-299Crossref PubMed Scopus (0) Google Scholar, 27Holz G.G. Diabetes. 2004; 53: 5-13Crossref PubMed Scopus (296) Google Scholar). Phosphorylation of the RyRs by the cAMP-dependent protein kinase A (PKA) was assumed to be a prerequisite for CICR (9Islam M.S. Leibiger I. Leibiger B. Rossi D. Sorrentino V. Ekström 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, 10Holz G.G. Leech C.A. Heller R.S. Castonguay M. Habener J.F. J. Biol. Chem. 1999; 274: 14147-14156Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). However, in later studies, a PKA-independent pathway involving cAMP-regulated guanine nucleotide exchange factor (Epac) has been suggested (20Kang G. Chepurny O.G. Holz G.G. J. Physiol. (Lond.). 2001; 536: 375-385Crossref Scopus (173) Google Scholar, 22Kang G. Joseph J.W. Chepurny O.G. Monaco M. Wheeler M.B. Bos J.L. Schwede F. Genieser H.G. Holz G.G. J. Biol. Chem. 2003; 278: 8279-8285Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 27Holz G.G. Diabetes. 2004; 53: 5-13Crossref PubMed Scopus (296) Google Scholar). Because important evidence for this concept was obtained with clonal β-cells, we have now studied the mechanisms by which cAMP promotes CICR in primary mouse and rat β-cells. We show that cAMP-facilitated CICR is caused by PKA-dependent activation of IP3Rs. Moreover, our data indicate that CICR is part of the mechanism by which cAMP amplifies insulin release. Materials—Reagents of analytical grade and deionized water were used. Fura-2 and its acetoxymethyl ester (fura-2/AM), fluo-4 acetoxymethyl ester (fluo-4/AM), and ryanodine were from Molecular Probes Inc. (Eugene, OR). Biolog Life Science Institute (Bremen, Germany) was the source of 8-(4-chlorophenylthio)-cGMP, 8-(4-chlorophenylthio)-2′-O-methyl-cAMP (8-pCPT-2′-O-Me-cAMP), 8-(4-methoxyphenylthio)-2′-O-methyl-cAMP (8-pMeOPT-2′-O-Me-cAMP) as well as the Sp and Rp isomers of 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole-3′,5′-cyclic monophosphorothioate (Sp-5,6-DCl-cBIMPS), 8-bromoadenosine-3′,5′-cyclic monophosphorothioate (Rp-8-Br-cAMPS), and 8-(4-chlorophenylthio)adenosine-3′,5′-cyclic monophosphorothioate (Rp-8-CPT-cAMPS). Sigma Chemical Co. provided bovine serum albumin (fraction V), carbachol, EGTA, HEPES, caffeine, porcine glucagon, human glucagon-like peptide-1 amide fragment 7–36 (GLP-1), 8-bromo-cAMP (8-Br-cAMP), 8-bromo-cGMP, and 3-isobutyl-1-methylxanthine (IBMX). Cyclopiazonic acid was from Alexis Corp. (Lausen, Switzerland), and 2-aminoethoxydiphenyl borate (2-APB) was from Aldrich (Gillingham, UK). Fetal calf serum was bought from Invitrogen, and collagenase was from Roche Molecular Biochemicals GmbH. Diazoxide, methoxyverapamil, and forskolin were kindly donated by Schering-Plough Int. (Kenilworth, NJ), Knoll AG (Ludwigshafen, Germany), and Aventis (Stockholm, Sweden) respectively. Membrane polycarbonate filters (25-mm diameter, 25 μm thick with 3-μm pores with a density of 3000/cm2) were from Osmonics Inc. (Livermore, CA). Preparation and Culture of Cells—Islets of Langerhans were collagenase-isolated from pieces of pancreas from ob/ob mice or Wistar rats. Free cells were prepared by shaking the islets in a Ca2+-deficient medium (28Lernmark Å. Diabetologia. 1974; 10: 431-438Crossref PubMed Scopus (290) Google Scholar). The cells were suspended in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin, 100 μg/ml streptomycin and 30 μg/ml gentamicin and allowed to attach to circular 25-mm cover slips during 1–3 days in culture at 37 °C in a humidified atmosphere of 5% CO2. The ob/ob mouse islets contain more than 90% β-cells (29Hellman B. Ann. N. Y. Acad. Sci. 1965; 131: 541-558Crossref PubMed Scopus (303) Google Scholar), which respond normally to glucose and other regulators of insulin release (30Hahn H.J. Hellman B. Lernmark Å. Sehlin J. Täljedal I.B. J. Biol. Chem. 1974; 249: 5275-5284Abstract Full Text PDF PubMed Google Scholar). The selection of β-cells for analysis was based on their large size and low nuclear/cytoplasmic ratio compared with the cells secreting glucagon, somatostatin (31Hellman B. Acta Endocrinol. (Copenh). 1959; 32: 92-112Crossref PubMed Google Scholar, 32Berts A. Gylfe E. Hellman B. Biochem. Biophys. Res. Commun. 1995; 208: 644-649Crossref PubMed Scopus (72) Google Scholar), and pancreatic polypeptide (33Liu Y.J. Hellman B. Gylfe E. Endocrinology. 1999; 140: 5524-5529Crossref PubMed Scopus (13) Google Scholar). Human erythroleukemia 92.1.7 (HEL) cells were obtained from Prof. K. E. O. Åkerman (Uppsala, Sweden) and cultured in suspension in RPMI 1640 medium (34Shariatmadari R. Lund P.E. Krijukova E. Sperber G.O. Kukkonen J.P. Åkerman K.E. Pfluegers Arch. Eur. J. Physiol. 2001; 442: 312-320Crossref PubMed Scopus (13) Google Scholar). Image Analysis of Cytoplasmic Ca2+—In most experiments, loading of cells with the indicator fura-2 was performed during 30-min incubation at 37 °C in a HEPES-buffered medium (25 mm; pH 7.4) containing 0.5 mg/ml bovine serum albumin, 138 mm NaCl, 4.8 mm KCl, 1.2 mm MgCl2, 1.28 mm CaCl2, 20 mm glucose, 250 μm diazoxide, 50 μm methoxyverapamil, and 0.25 μm fura-2/AM. In the experiment shown in Fig. 1A, diazoxide and methoxyverapamil were omitted, and the glucose concentration was 3 mm. Methoxyverapamil was also omitted in the experiments shown in Figs. 1, B–D, and 6A. Testing the effect of ryanodine, 100 μm of this compound was present during loading and throughout the experiment. The cover slips with attached cells were used as exchangeable bottoms of a modified open Sykes-More chamber (35Sykes J.A. Moore E.B. Proc. Soc. Exp. Biol. Med. 1959; 100: 125-127Crossref PubMed Scopus (80) Google Scholar). The chamber profile was defined by a 4-mm wide, 7-mm long oval hole in a 1-mm thick silicon rubber gasket with a 25-mm outer diameter. A thin 25-mm diameter stainless steel plate with an identical central opening pressed the rubber gasket to the coverslip by the threaded Sykes-More chamber mount. Inlet and outlet cannulas fixed to the stainless steel plate allowed laminar flow superfusion. The chamber was placed on the stage of an inverted microscope (Eclipse TE2000U; Nikon, Kanagawa, Japan). The chamber holder and the CFI S Fluor 40 × 1.3 numerical aperture oil immersion objective (Nikon) were maintained at 37 °C by custombuilt thermostats. The chamber was superfused at a rate of 0.3 ml/min with the loading medium lacking indicator.Fig. 6CICR is resistant to ryanodine. Mouse pancreatic β-cells were loaded for 30 min with 0.25 μm fura-2/AM in medium containing 20 mm glucose, 250 μm diazoxide, 100 μm ryanodine (A–C), and 50 μm methoxyverapamil (B and C). The cells were then rinsed and superfused with the same medium lacking indicator. As indicated by bars, the cells were exposed to 90 mm KCl, 50 μm methoxyverapamil, 100 μm ryanodine, 10 nm glucagon, 250 μm Sp-5,6-DCl-cBIMPS (Sp-BIMPS), and 20 mm caffeine. KCl depolarization induced CICR spiking in 15 of 27 cells (A; p < 0.001), glucagon in 46 of 48 cells (B; p < 0.001), and Sp-5,6-DCl-cBIMPS in 35 of 50 cells (C; p < 0.001). The presence of caffeine abolished CICR spiking in all 35 Sp-5,6-DCl-cBIMPS-responsive cells (C; p < 0.001).View Large Image Figure ViewerDownload (PPT) The microscope was equipped with an epifluorescence illuminator (Cairn Research Ltd, Faversham, UK) connected through a 5-mm diameter liquid light guide to an Optoscan monochromator (Cairn Research Ltd) with rapid grating and slit width adjustment and a 150-watt xenon arc lamp. The monochromator provided excitation light at 340 nm (1.7 nm half-bandwidth) and 380 nm (1.4 nm half-bandwidth). Emission was measured at 510 nm (40 nm half-bandwidth) using a 400-nm dichroic beam splitter and a cooled OrcaER-1394 Firewire digital charge-coupled device camera (Hamamatsu Photonics, Hamamatsu City, Japan) equipped with a C8600–2 image intensifier (Hamamatsu Photonics). The Metafluor software (Universal Imaging Corp. Downingtown, PA) controlled the monochromator and the charge-coupled device camera, acquiring pairs of images at 340 and 380 nm every 2 s with integration for 60–80 ms at each wavelength and 510 nm (long-pass filter) using a 495 nm dichroic beam splitter. Fura-2 fluorescence from the HEL cells was excited at 340 and 380 nm and fluo-4 fluorescence from the β-cells at 470 nm. Images were obtained every 2 s with integration for 100 ms at each wavelength and <1 ms for changing wavelength and slits. Because of the stronger fluorescence from fluo-4, the image intensifier gain was reduced during excitation at 470 nm to balance the signals. [Ca2+]i in the HEL cells was calculated as described above. The fluo-4 fluorescence intensity indicating [Ca2+]i variations in the β-cells was expressed as the ratio between deviation from the basal fluorescence and the basal fluorescence (ΔF/F0). Statistical Analysis—Only recordings from isolated individual β-cells were included in the analyses. Statistical evaluations of the proportion of cells with a certain response were made with Fishers exact test or χ2 test with Yates' correction using SigmaStat software (SPSS Inc., Chicago, IL). Wilcoxon signed rank test was used to compare the frequency of Ca2+ spikes. Statistical significance was set at a p value of < 0.05. CICR Is Promoted by Glucagon, GLP-1, and other cAMP Agonists—In accordance with earlier data (37Grapengiesser E. Gylfe E. Hellman B. Biochem. Biophys. Res. Commun. 1988; 151: 1299-1304Crossref PubMed Scopus (138) Google Scholar, 38Grapengiesser E. Gylfe E. Hellman B. Arch. Biochem. Biophys. 1989; 268: 404-407Crossref PubMed Scopus (82) Google Scholar), increase of the glucose concentration from 3 to 20 mm induced initial lowering of [Ca2+]i in β-cells followed by slow, large amplitude oscillations of [Ca2+]i (Fig. 1A). The use of low indicator concentrations facilitated the detection of rapid [Ca2+]i spikes, which were superimposed on top of the large amplitude oscillations. Addition of diazoxide, which hyperpolarizes the β-cells by opening KATP channels, immediately abolished the slow [Ca2+]i oscillations as well as the spikes. In the presence of diazoxide, the membrane potential is near the equilibrium potential for K+. Under these conditions, depolarization with 90 mm KCl induced a rapid rise of [Ca2+]i with superimposed [Ca2+]i spikes, indicating that the depolarization-dependent influx of Ca2+ triggers CICR. Most subsequent experiments were performed in the presence of 20 mm glucose to stimulate Ca2+ sequestration in the ER (2Gylfe E. Pfluegers Arch. Eur. J. Physiol. 1991; 419: 639-643Crossref PubMed Scopus (40) Google Scholar, 3Tengholm A. Hellman B. Gylfe E. J. Biol. Chem. 1999; 274: 36883-36890Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 4Tengholm A. Hellman B. Gylfe E. J. Physiol. (Lond.). 2001; 530: 533-540Crossref Scopus (46) Google Scholar) and diazoxide to keep the membrane potential close to the equilibrium potential for K+. Fig. 1B illustrates that the modest elevation of [Ca2+]i obtained with 17 mm KCl only triggered occasional CICR spikes. After return to the physiological K+ concentration addition of a low concentration of the adenylyl cyclase activator forskolin evoked occasional CICR spikes (not shown). However, combining 17 mm KCl with forskolin increased the frequency of CICR spiking 4-fold. The ability of cAMP to uncover CICR in response to depolarization-dependent rise of [Ca2+]i was not restricted to mouse β-cells. Fig. 1, C and D, illustrates [Ca2+]i spiking in rat β-cells depolarized with 30 mm KCl in the presence of the adenylyl cyclase-activating hormones GLP-1 and glucagon, respectively. However, the mouse β-cells were more sensitive, and the same concentrations of glucagon and GLP-1 caused repetitive CICR spiking even in the absence of depolarization-dependent elevation of [Ca2+]i. This is illustrated in Fig. 2, which like most subsequent experiments was performed in the presence of methoxyverapamil to block depolarization-dependent Ca2+ entry and keep [Ca2+]i at basal levels. Apart from the physiological activation of adenylyl cyclase with glucagon (Fig. 2A) and GLP-1 (Fig. 2B) CICR spiking from the baseline was also obtained after direct activation of adenylyl cyclase with forskolin (Fig. 2C), by inhibition of cAMP degradation with the phosphodiesterase inhibitors IBMX (Fig. 2D) or caffeine (Fig. 2E), and by cell membrane-permeable 8-Br-cAMP (Fig. 2F). However, 8-bromo-cGMP or the more selective protein kinase G activator 8-(4-chlorophenylthio)-cGMP failed to trigger CICR in β-cells subsequently responding to cAMP agonists (not shown). PKA Rather Than Epac Mediates the cAMP Effect on CICR— Occasional Ca2+ spikes were observed in 2 of 64 individual islet cells exposed to the Epac-specific activator 8-pCPT-2′-O-Me-cAMP and in 1 of 57 cells exposed to the even more potent 8-pMeOPT-2′-O-Me-cAMP (data not shown). Epac activator-induced Ca2+ spikes were sometimes seen also in islet cell clusters. These data indicate that the response represents a different cell type than the dominating β-cells. However, 111 of 174 cells (64%; p < 0.001) reacted to the PKA-specific activator Sp-5,6-DCl-cBIMPS with generation of repetitive [Ca2+]i spikes. Fig. 3, A and B, illustrate lack of response to the Epac activators 8-pCPT-2′-O-Me-cAMP and 8-pMeOPT-2′-O-Me-cAMP in cells subsequently reacting to the PKA activator. Further evidence for a PKA mechanism was obtained from the observation that the frequencies of the Ca2+ spiking induced by glucagon and forskolin were reduced by about 70% by the competitive PKA antagonists Rp-8-Br-cAMPS (Fig. 3C) and Rp-8-CPT-cAMPS (Fig. 3D). cAMP-promoted CICR Is Independent of RyRs and Prevented by IP3R Inhibition—Low concentrations of caffeine have been used extensively as phosphodiesterase inhibitor raising cAMP. We now found that 2 mm caffeine mimics other cAMP agonists in inducing CICR (Figs. 2E and 4D). However, high caffeine concentrations interfere with both IP3 production (39Toescu E.C. O'Neill S.C. Petersen O.H. Eisner D.A. J. Biol. Chem. 1992; 267: 23467-23470Abstract Full Text PDF PubMed Google Scholar) and IP3Rs (40Ehrlich B.E. Kaftan E. Bezprozvannaya S. Bezprozvanny I. Trends Pharmacol. Sci. 1994; 15: 145-149Abstract Full Text PDF PubMed Scopus (423) Google Scholar), which explains why 20 mm immediately inhibited CICR promoted by glucagon (Fig. 4A), GLP-1 (Fig. 4B), IBMX (Fig. 4C), and 2 mm caffeine (Fig. 4D). In addition, high concentrations of caffeine activate RyRs (41Sitsapesan R. Williams A.J. J. Physiol. (Lond.). 1990; 423: 425-439Crossref Scopus (236) Google Scholar, 42Solovyova N. Veselovsky N. Toescu E.C. Verkhratsky A. EMBO J. 2002; 21: 622-630Crossref PubMed Scopus (167) Google Scholar), but 20 mm caffeine never induced an acute mobilization of intracellular Ca2+ typically observed in cells with functional RyRs (12Beauvois M.C. Arredouani A. Jonas J.C. Rolland J.F. Schuit F. Henquin J.C. Gilon P. J. Physiol. (Lond.). 2004; 559: 141-156Crossref Scopus (25) Google Scholar, 23Dyachok O. Tufveson G. Gylfe E. Cell Calcium. 2004; 36: 1-9Crossref PubMed Scopus (34) Google Scholar). Because the caffeine data indicate that cAMP-promoted CICR involves the IP3 signaling pathway, we tested the effect of the membrane-permeable IP3R inhibitor 2-APB (43Maruyama T. Kanaji T. Nakade S. Kanno T. Mikoshiba K. J. Biochem. (Tokyo). 1997; 122: 498-505Crossref PubMed Scopus (781) Google Scholar). The inhibitory effect of 2-APB is incomplete (44Bootman M.D. Collins T.J. Mackenzie L. Roderick H.L. Berridge M.J. Peppiatt C.M. FASEB J. 2002; 16: 1145-1150Crossref PubMed Scopus (611) Google Scholar), and we found that 50 μm prevented Ca2+ signaling induced by 10 μm carbachol in 35% of the cells but never the respons
Referência(s)