Ryanodine Receptor Type I and Nicotinic Acid Adenine Dinucleotide Phosphate Receptors Mediate Ca2+ Release from Insulin-containing Vesicles in Living Pancreatic β-Cells (MIN6)
2003; Elsevier BV; Volume: 278; Issue: 13 Linguagem: Inglês
10.1074/jbc.m210257200
ISSN1083-351X
AutoresKathryn J. Mitchell, F. Anthony Lai, Guy A. Rutter,
Tópico(s)Adenosine and Purinergic Signaling
ResumoWe have demonstrated recently (Mitchell, K. J., Pinton, P., Varadi, A., Tacchetti, C., Ainscow, E. K., Pozzan, T., Rizzuto, R., and Rutter, G. A. (2001)J. Cell Biol. 155, 41–51) that ryanodine receptors (RyR) are present on insulin-containing secretory vesicles. Here we show that pancreatic islets and derived β-cell lines express type I and II, but not type III, RyRs. Purified by subcellular fractionation and membrane immuno-isolation, dense core secretory vesicles were found to possess a similar level of type I RyR immunoreactivity as Golgi/endoplasmic reticulum (ER) membranes but substantially less RyR II than the latter. Monitored in cells expressing appropriately targeted aequorins, dantrolene, an inhibitor of RyR I channels, elevated free Ca2+ concentrations in the secretory vesicle compartment from 40.1 ± 6.7 to 90.4 ± 14.8 μm(n = 4, p < 0.01), while having no effect on ER Ca2+ concentrations. Furthermore, nicotinic acid adenine dinucleotide phosphate (NAADP), a novel Ca2+-mobilizing agent, decreased dense core secretory vesicle but not ER free Ca2+ concentrations in permeabilized MIN6 β-cells, and flash photolysis of caged NAADP released Ca2+ from a thapsigargin-insensitive Ca2+ store in single MIN6 cells. Because dantrolene strongly inhibited glucose-stimulated insulin secretion (from 3.07 ± 0.51-fold stimulation to no significant glucose effect;n = 3, p < 0.01), we conclude that RyR I-mediated Ca2+-induced Ca2+ release from secretory vesicles, possibly potentiated by NAADP, is essential for the activation of insulin secretion. We have demonstrated recently (Mitchell, K. J., Pinton, P., Varadi, A., Tacchetti, C., Ainscow, E. K., Pozzan, T., Rizzuto, R., and Rutter, G. A. (2001)J. Cell Biol. 155, 41–51) that ryanodine receptors (RyR) are present on insulin-containing secretory vesicles. Here we show that pancreatic islets and derived β-cell lines express type I and II, but not type III, RyRs. Purified by subcellular fractionation and membrane immuno-isolation, dense core secretory vesicles were found to possess a similar level of type I RyR immunoreactivity as Golgi/endoplasmic reticulum (ER) membranes but substantially less RyR II than the latter. Monitored in cells expressing appropriately targeted aequorins, dantrolene, an inhibitor of RyR I channels, elevated free Ca2+ concentrations in the secretory vesicle compartment from 40.1 ± 6.7 to 90.4 ± 14.8 μm(n = 4, p < 0.01), while having no effect on ER Ca2+ concentrations. Furthermore, nicotinic acid adenine dinucleotide phosphate (NAADP), a novel Ca2+-mobilizing agent, decreased dense core secretory vesicle but not ER free Ca2+ concentrations in permeabilized MIN6 β-cells, and flash photolysis of caged NAADP released Ca2+ from a thapsigargin-insensitive Ca2+ store in single MIN6 cells. Because dantrolene strongly inhibited glucose-stimulated insulin secretion (from 3.07 ± 0.51-fold stimulation to no significant glucose effect;n = 3, p < 0.01), we conclude that RyR I-mediated Ca2+-induced Ca2+ release from secretory vesicles, possibly potentiated by NAADP, is essential for the activation of insulin secretion. free cytosolic Ca2+concentration free ER Ca2+ concentration free secretory vesicle Ca2+ concentration cyclic ADP ribose endoplasmic reticulum enhanced green fluorescent protein nicotinic acid adenine dinucleotide phosphate ryanodine receptor aequorin vesicle associated membrane protein Increases in cytoplasmic Ca2+ concentration ([Ca2+]c)1are important for stimulation of neurosecretion in general (1Rutter G.A. Mol. Aspects Med. 2001; 22: 247-284Google Scholar) and for the activation of insulin secretion from pancreatic islet β-cells (1Rutter G.A. Mol. Aspects Med. 2001; 22: 247-284Google Scholar,2Wollheim C.B. Blondel B. Trueheart P.A. Renold A.E. Sharp G.W. J. Biol. Chem. 1975; 250: 1354-1360Google Scholar). In the latter cell type, increases in [Ca2+]cusually occur as a result of either nutrient-induced influx of Ca2+ ions through voltage-gated Ca2+ channels on the plasma membrane (3Safayhi H. Haase H. Kramer U. Bihlmayer A. Roenfeldt M. Ammon H.P. Froschmayr M. Cassidy T.N. Morano I. Ahlijanian M.K. Striessnig J. Mol. Endocrinol. 1997; 11: 619-629Google Scholar) or via the release of Ca2+ from intracellular Ca2+ stores (4Wollheim C.B. Biden T.J. J. Biol. Chem. 1986; 261: 8314-8319Google Scholar). The endoplasmic reticulum (ER) (5Streb H. Bayerdorffer E. Haase W. Irvine R.F. Schulz I. J. Membr. Biol. 1984; 81: 241-253Google Scholar, 6Rizzuto R. Brini M. Murgia M. Pozzan T. Science. 1993; 262: 744-747Google Scholar) and Golgi apparatus (7Pinton P. Pozzan T. Rizzuto R. EMBO J. 1998; 17: 5298-5308Google Scholar) probably represent the major Ca2+ stores in β-cells (8Kennedy E.D. Rizzuto R. Theler J.M. Pralong W.F. Bastianutto C. Pozzan T. 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Diabetologia. 1997; 40: 487-495Google Scholar) and may contain close to half the total cellular Ca2+. As such, these organelles potentially provide a huge store of mobilizable Ca2+ ions (15Hutton J.C. Penn E.J. Peshavaria M. Biochem. J. 1983; 210: 297-305Google Scholar). Previous studies involving measurements of intravesicular free Ca2+ concentration ([Ca2+]SV) and immunoelectron microscopy (9Mitchell K.J. Pinton P. Varadi A. Tacchetti C. Ainscow E.K. Pozzan T. Rizzuto R. Rutter G.A. J. Cell Biol. 2001; 155: 41-51Google Scholar) indicated that ryanodine, but not inositol 1,4,5-trisphosphate (11Clapham D.E. Cell. 1995; 80: 259-268Google Scholar), receptors mediate Ca2+release from secretory vesicles in β-cells (9Mitchell K.J. Pinton P. Varadi A. Tacchetti C. Ainscow E.K. Pozzan T. Rizzuto R. Rutter G.A. J. Cell Biol. 2001; 155: 41-51Google Scholar, 10Varadi A. Rutter G.A. Diabetes. 2002; 51 Suppl. 1: 190-201Google Scholar, 16Ravazzola M. Halban P.A. Orci L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2745-2748Google Scholar). cDNAs encoding three RyR isoforms have so far been identified in mammals. The type I isoform (RyR I) is expressed mainly in skeletal muscle (17Marks A.R. Tempst P. Hwang K.S. Taubman M.B. Inui M. Chadwick C. Fleischer S. Nadal-Ginard B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8683-8687Google Scholar), whereas RyR II is abundant in the heart (18Nakai J. Imagawa T. Hakamat Y. Shigekawa M. Takeshima H. Numa S. FEBS Lett. 1990; 271: 169-177Google Scholar). RyR III is present in a variety of tissues and cell types, most notably the brain (19Ledbetter M.W. Preiner J.K. Louis C.F. Mickelson J.R. J. Biol. Chem. 1994; 269: 31544-31551Google Scholar). RyR II has been reported previously to be the most abundantly expressed isoform (at the mRNA level) in wild type (20Takasawa S. Akiyama T. Nata K. Kuroki M. Tohgo A. Noguchi N. Kobayashi S. Kato I. Katada T. Okamoto H. J. Biol. Chem. 1998; 273: 2497-2500Google Scholar) and ob/obmouse islets (20Takasawa S. Akiyama T. Nata K. Kuroki M. Tohgo A. Noguchi N. Kobayashi S. Kato I. Katada T. Okamoto H. J. Biol. Chem. 1998; 273: 2497-2500Google Scholar, 21Islam 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: 6145-6150Google Scholar), as well as in rat islets (22Holz G.G. Leech C.A. Heller R.S. Castonguay M. Habener J.F. J. Biol. Chem. 1999; 274: 14147-14156Google Scholar) and clonal βTC3 cells (22Holz G.G. Leech C.A. Heller R.S. Castonguay M. Habener J.F. J. Biol. Chem. 1999; 274: 14147-14156Google Scholar). Moreover, the presence of RyR II protein has also been demonstrated in derived INS-1 β-cells (23Gamberucci A. Fulceri R. Pralong W. Banhegyi G. Marcolongo P. Watkins S.L. Benedetti A. FEBS Lett. 1999; 446: 309-312Google Scholar). Lower levels of RyR I and RyR III mRNA have also been detected in βTC3 (22Holz G.G. Leech C.A. Heller R.S. Castonguay M. Habener J.F. J. Biol. Chem. 1999; 274: 14147-14156Google Scholar) and HIT-T15 cells (24Taniguchi T. Yamada Y. Yasuda K. Kubota A. Ihara Y. Kagimoto S. Kuroe A. Watanabe R. Inada A. Seino Y. Endocrinol. Metab. 1996; 3: 135-138Google Scholar), respectively. However, the physiological role(s) of RyRs in β-cells remains unclear, given that RyR II mRNA levels inob/ob mouse islets are reportedly ∼1000-fold less than in the heart (21Islam 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: 6145-6150Google Scholar), whereas RyR II protein levels in INS-1 β-cells were ∼10-fold lower than in brain (23Gamberucci A. Fulceri R. Pralong W. Banhegyi G. Marcolongo P. Watkins S.L. Benedetti A. FEBS Lett. 1999; 446: 309-312Google Scholar). Receptors for nicotinic acid adenine dinucleotide phosphate (NAADP), a novel intracellular Ca2+-mobilizing agent (25Lee H.C. Aarhus R. J. Biol. Chem. 1995; 270: 2152-2157Google Scholar), may represent an alternative pathway for Ca2+ efflux from dense core secretory vesicles (26Patel S. Churchill G.C. Galione A. Trends Biochem. Sci. 2001; 26: 482-489Google Scholar). Although other studies (27Cancela J.M. Churchill G.C. Galione A. Nature. 1999; 398: 74-76Google Scholar, 28Bak J. White P. Timar G. Missiaen L. Genazzani A.A. Galione A. Curr. Biol. 1999; 9: 751-754Google Scholar, 29Berg I. Potter B.V. Mayr G.W. Guse A.H. J. Cell Biol. 2000; 150: 581-588Google Scholar, 30Mojzisova A. Krizanova O. Zacikova L. Kominkova V. Ondrias K. Pfluegers Arch. 2001; 441: 674-677Google Scholar) have demonstrated NAADP-induced Ca2+ release in a variety of mammalian cells and cell lines, few data are currently available regarding the role of NAADP in the β-cell. Although functional NAADP-sensitive Ca2+ stores were recently revealed in human β-cells (31Johnson J.D. Misler S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14566-14571Google Scholar), NAADP-induced Ca2+ release was not observed in dispersed β-cells from either normal or ob/ob mouse islets (32Tengholm A. Hellman B. Gylfe E. Cell Calcium. 2000; 27: 43-51Google Scholar). However, in the latter report, neither the RyR agonists caffeine and ryanodine nor cyclic ADP-ribose (cADPr) induced Ca2+ release. In the present study, we show that islets and MIN6 β-cells express two RyR isoforms, RyR I and RyR II, that display distinct subcellular localizations. Thus, whereas type I RyRs are present at approximately equal density in a vesicle/mitochondrial fraction, and in microsomes, RyR II was considerably more abundant on ER membranes. Surprisingly, dantrolene, a selective inhibitor of RyR I, increased steady-state free [Ca2+] in secretory vesicles but not in the ER, suggesting the presence on vesicles of a further activator or channel capable of amplifying the effects of RyRs on Ca2+ release. We provide evidence that receptors for NAADP may serve this role, and we thus demonstrate that secretory vesicles, but not the ER, are an NAADP-responsive Ca2+ store in β-cells. MIN6 cells, a well differentiated mouse insulinoma β-cell line (33Miyazaki J. Araki K. Yamato E. Ikegami H. Asano T. Shibasaki Y. Oka Y. Yamamura K. Endocrinology. 1990; 127: 126-132Google Scholar) (passages 20–30), were grown in Dulbecco's modified Eagle's medium (Invitrogen) containing 25 mm glucose and 2 mmpyruvate, and supplemented with 15% (v/v) fetal bovine serum, 20 mm glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 50 μm β-mercaptoethanol, in a humidified atmosphere containing 5% CO2 (9Mitchell K.J. Pinton P. Varadi A. Tacchetti C. Ainscow E.K. Pozzan T. Rizzuto R. Rutter G.A. J. Cell Biol. 2001; 155: 41-51Google Scholar, 34Ainscow E.K. Zhao C. Rutter G.A. Diabetes. 2000; 49: 1149-1155Google Scholar). For [Ca2+] measurements with recombinant aequorins (see below), cells were seeded onto 13-mm diameter poly-l-lysine-coated glass coverslips and grown to 50–80% confluency. Cells were then infected with Cyt.Aq, VAMP.Aq, or ER.Aq adenoviruses encoding untargeted aequorin (Cyt.Aq) (35Brini M. Marsault R. Bastianutto C. Alvarez J. Pozzan T. Rizzuto R. J. Biol. Chem. 1995; 270: 9896-9903Google Scholar) or aequorin targeted to either the secretory vesicles (VAMP.Aq) (9Mitchell K.J. Pinton P. Varadi A. Tacchetti C. Ainscow E.K. Pozzan T. Rizzuto R. Rutter G.A. J. Cell Biol. 2001; 155: 41-51Google Scholar) or the ER (ER.Aq) (36Montero M. Brini M. Marsault R. Alvarez J. Sitia R. Pozzan T. Rizzuto R. EMBO J. 1995; 14: 5467-5475Google Scholar), at a multiplicity of infection of 30 infectious units per cell. Measurements of aequorin bioluminescence were performed using a purpose-built photomultiplier system 48 h after infection, as described previously (9Mitchell K.J. Pinton P. Varadi A. Tacchetti C. Ainscow E.K. Pozzan T. Rizzuto R. Rutter G.A. J. Cell Biol. 2001; 155: 41-51Google Scholar, 37Rutter G.A. Theler J.M. Murgia M. Wollheim C.B. Pozzan T. Rizzuto R. J. Biol. Chem. 1993; 268: 22385-22390Google Scholar). Total RNA was extracted from cell lines or rat tissue using TRI ReagentTM (Sigma) according to the manufacturer's instructions and reverse-transcribed using Moloney murine leukemia virus-reverse transcriptase (Promega). PCR amplification was performed with primers designed to amplify an isoform-specific region of each of the three RyR subtypes (38Fitzsimmons T.J. Gukovsky I. McRoberts J.A. Rodriguez E. Lai F.A. Pandol S.J. Biochem. J. 2000; 351: 265-271Google Scholar) as follows: RyR I (forward, 5′-GAAGGTTCTGGACAAACACGGG-3′; reverse, 5′-TCGCTCTTGTTGTAGAATTTGCGG-3′); RyR II (forward, 5′-GAATCAGTGAGTTACTGGGCATGG-3′; reverse, 5′-CTGGTCTCTGAGTTCTCCAAAAGC-3′); and RyR III (forward, 5′-CCTTCGCTATCAACTTCATCCTGC-3′; reverse, 5′-TCTTCTACTGGGCTAAAGTCAAGG-3′). The PCR mix consisted of 5 μl of 10× Buffer 3 (Roche Molecular Biochemicals), 6 μl of 25 mm MgCl2, 1 μl of 10 mm dNTPs, 0.25 μl of Taq DNA polymerase (Roche Molecular Biochemicals), 5 μl of reverse transcriptase-PCR cDNA, 0.4 μm forward primer, 0.4 μmreverse primers, and distilled H2O, at a final volume of 50 μl. Amplification conditions are as follows: 94 °C for 2 min and then 94 °C for 45 s, 55.5 °C for 45 s, 72 °C for 1 min for 32 cycles and then 72 °C for 10 min. Negative controls were performed by omission of the reverse transcription step or by exclusion of the template from the PCR. PCR products were separated by migration on a 2% (w/v) agarose gel. Products were excised and purified using a QIAQuickTM gel extraction kit (Qiagen) and subjected to restriction digest analysis and automated sequencing. Total RNA from rat islets, skeletal muscle, or heart was reverse-transcribed, and semi-quantitative PCR was performed in the following mix: 5 μl of 10× Buffer 3 (Roche Molecular Biochemicals), 6 μl of 25 mm MgCl2, 1 μl of 10 mm dNTPs, 0.25 μl of Taq DNA polymerase (Roche Molecular Biochemicals), 5 μl of reverse transcriptase-PCR cDNA, 0.4 μm RyR I or RyR II primers, 0.1 μm β-actin primers (39Miquerol L. Lopez S. Cartier N. Tulliez M. Raymondjean M. Kahn A. J. Biol. Chem. 1994; 269: 8944-8951Google Scholar), and distilled H2O at a final volume of 50 μl. Semi-quantitative PCR was then performed as follows: RyR I primers, 94 °C for 2 min and then 94 °C for 45 s, 55 °C for 45 s, 72 °C for 1 min, for 25 cycles and then 72 °C for 10 min; RyR II primers, 94 °C for 2 min and then 94 °C for 45 s, 58 °C for 45 s, 72 °C for 1 min, for 20 cycles and then 72 °C for 10 min. Amplification in the linear phase was confirmed in each case by trials with 12–30 cycles (data not shown). MIN6 cells or homogenized rat skeletal muscle were extracted into radioimmunoprecipitation assay (RIPA) buffer, consisting of phosphate-buffered saline supplemented with 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, and 0.1% (w/v) SDS. Secretory vesicle protein was obtained by immunoadsorption of phogrin.EGFP-containing vesicles (40Varadi A. Ainscow E.K. Allan V.J. Rutter G.A. J. Cell Sci. 2002; 115: 4177-4189Google Scholar). In brief, MIN6 cells infected with phogrin.EGFP were homogenized, and the nuclear fraction was removed by centrifugation. Pre-cleared homogenate was then incubated with anti-GFP antibody (Roche Molecular Biochemicals) bound to protein A-Sepharose. Immunoadsorbed vesicles were washed then incubated at 99 °C for 10 min in SDS-PAGE loading buffer to dissociate vesicle proteins. To generate MIN6 membrane fractions, cells were harvested in Tris-saline buffer (10 mm Tris-HCl, 0.14 mNaCl, pH 7.4) containing 5% (v/v) Tween 40. Fractions were collected by differential centrifugation using the following spins: 200 ×g for 10 min, nuclear fraction; 15,000 × gfor 5 min, mitochondrial/secretory vesicle fraction; 100,000 ×g for 1 h, plasma membrane, ER, Golgi fraction; supernatant from 100,000 × g spin, cytosolic fraction. Rabbit polyclonal antiserum was raised to a RyR I-specific sequence of 15 amino acids (residues 830–845; RREGPRGPHLVGPSRC) that is 100% conserved in all known mammalian RyRI sequences and absent from the mammalian RyR II and III sequences. Rabbits (New Zealand White) were immunized with the keyhole limpet hemocyanin-conjugated peptide as described previously (41Tunwell R.E. Wickenden C. Bertrand B.M. Shevchenko V.I. Walsh M.B. Allen P.D. Lai F.A. Biochem. J. 1996; 318: 477-487Google Scholar), and antibody specificity was confirmed by enzyme-linked immunosorbent assay and immunoblot analysis with brain, skeletal, and cardiac muscle microsomes, prepared as described previously (41Tunwell R.E. Wickenden C. Bertrand B.M. Shevchenko V.I. Walsh M.B. Allen P.D. Lai F.A. Biochem. J. 1996; 318: 477-487Google Scholar). For immunoblot analysis, microsomes were separated on a 5% (v/v) polyacrylamide gel (30 μg of protein/lane), and proteins were electrophoretically transferred to polyvinylidene difluoride membrane before probing with antibody at a dilution of 1:1000. Affinity-purified antibody (anti-RyR I; number 2142) was prepared by acid elution following incubation of the crude RyR I antisera either with skeletal muscle RyR protein immobilized on polyvinylidene difluoride membrane strips or on protein A-agarose columns (Sigma). Protein samples were resolved by SDS-PAGE on 5% (v/v) polyacrylamide gels and transferred onto Immobilon-P transfer membrane (Millipore) following a standard protocol. Membranes were probed with anti-skeletal muscle RyR antibody (1:2500; Upstate Biotechnology, Inc.), anti-RyR I (1:500; number 2142), anti-RyR II (1:200; Affinity BioReagents), and anti-RyR III antibodies (1:500; number 110E) (42Airey J.A. Beck C.F. Murakami K. Tanksley S.J. Deerinck T.J. Ellisman M.H. Sutko J.L. J. Biol. Chem. 1990; 265: 14187-14194Google Scholar). Immunostaining was revealed with horseradish peroxidase-conjugated anti-rabbit IgG (Sigma; 1:100 000), anti-mouse IgG (Sigma; 1:10,000), or anti-sheep IgG (Dako; 1:4000) using an enhanced chemiluminescence (ECL) detection system (Roche Diagnostics). Cells were depleted of Ca2+ by incubation with ionomycin (10 μm), monensin (10 μm), and cyclopiazonic acid (10 μm) in modified Krebs-Ringer bicarbonate buffer (KRB: 140 mm NaCl, 3.5 mm KCl, 0.5 mmNaH2PO4, 0.5 mm MgSO4, 3 mm glucose, 10 mm Hepes, 2 mmNaHCO3, pH 7.4) supplemented with 1 mm EGTA, for 10 min at 4 °C (9Mitchell K.J. Pinton P. Varadi A. Tacchetti C. Ainscow E.K. Pozzan T. Rizzuto R. Rutter G.A. J. Cell Biol. 2001; 155: 41-51Google Scholar). Aequorin was reconstituted with 5 μm coelenterazine n(43Shimomura O. Kishi Y. Inouye S. Biochem. J. 1993; 296: 549-551Google Scholar) for 1–2 h at 4 °C in KRB supplemented with 1 mm EGTA. Intact cells were perifused with KRB plus additions as stated at 2 ml·min−1 in a thermostatted chamber (37 °C) in close proximity to a photomultiplier tube (ThornEMI) (44Cobbold P.H. Rink T.J. Biochem. J. 1987; 248: 313-328Google Scholar). Where indicated, cells were permeabilized with 20 μm digitonin for 1 min at 37 °C and subsequently perifused in intracellular buffer (IB: 140 mm KCl, 10 mm NaCl, 1 mmKH2PO4, 5.5 mm glucose, 2 mm MgSO4, 1 mm ATP, 2 mm sodium succinate, 20 mm Hepes, pH 7.05). Additions to this buffer were as stated in the figure legends. At the end of all experiments cells were lysed in a hypotonic Ca2+-rich solution (100 μm digitonin, 10 mm Ca2+ in H20) to discharge the remaining aequorin pool for calibration of the aequorin signal (9Mitchell K.J. Pinton P. Varadi A. Tacchetti C. Ainscow E.K. Pozzan T. Rizzuto R. Rutter G.A. J. Cell Biol. 2001; 155: 41-51Google Scholar). MIN6 cells were seeded onto 24 mm poly-l-lysine-coated coverslips and microinjected, as previously described (45Rutter G.A. White M.R. Tavare J.M. Curr. Biol. 1995; 5: 890-899Google Scholar, 46Kennedy H.J. Viollet B. Rafiq I. Kahn A. Rutter G.A. J. Biol. Chem. 1997; 272: 20636-20640Google Scholar, 47Kennedy H.J. Pouli A.E. Ainscow E.K. Jouaville L.S. Rizzuto R. Rutter G.A. J. Biol. Chem. 1999; 274: 13281-13291Google Scholar), with Oregon Green 488 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-1 dextran (Molecular Probes, Eugene, OR; 2.5 mg·ml−1) in Tris-HCl buffer, pH 8.0, in the absence (control) or presence of 2 μm caged NAADP (48Lee H.C. Aarhus R. Gee K.R. Kestner T. J. Biol. Chem. 1997; 272: 4172-4178Google Scholar) (kindly provided by Dr. Luigia Santella, University of Naples). The concentration of caged NAADP in the microinjection pipette was 2 μm, resulting in a final intracellular concentration of 50–150 nm, given an injection volume of 2.5–7.5% of the total cell volume (49Rutter G.A. Kennedy H.J. Wood C.D. White M.R. Tavare J.M. Chem. Biol. 1998; 5: R285-R290Google Scholar). 2–4 h after microinjection, cells were imaged on a Leica TCS-SP2 laser-scanning confocal illumination system attached to a Leica DM IRBE inverted epifluorescence microscope, using a 63× PL Apo 1.4 numerical aperture oil immersion objective. Fluorescence was excited at 488 nm (argon laser), and fluorescence was detected at wavelengths longer than 515 nm using a long pass cut-off filter at this wavelength. A second laser (CoherentTM) provided light for 1 s at wavelengths of 351 and 364 nm (via the objective lens) and was used for photolysis of caged NAADP at selected regions of interest within the cell (corresponding to ∼1% of the surface of the confocal slice). Images were acquired at 5-s intervals before and after the UV pulse. Where indicated, ER/Golgi Ca2+ stores were depleted of Ca2+ by washing the cells in Ca2+-free KRB and incubation in Ca2+-free KRB supplemented with 1 μm thapsigargin, 10 μm cyclopiazonic acid, and 1 mm EGTA for 10 min. Cells were then maintained in Ca2+-free KRB during confocal imaging and photorelease of NAADP. MIN6 cells were incubated in full growth medium (see above) containing 3 mm glucose for 16 h and then incubated in KRB supplemented with 3 mmglucose for 30 min at 37 °C. The medium was removed and retained, and cells were then stimulated with KRB supplemented with 30 mm glucose for a further 30 min at 37 °C. Released and total insulin were measured by radioimmunoassay (34Ainscow E.K. Zhao C. Rutter G.A. Diabetes. 2000; 49: 1149-1155Google Scholar). Free [Ca2+] was calculated using METLIG software (50Rutter G.A. Denton R.M. Biochem. J. 1988; 252: 181-189Google Scholar). Data represent the means ± S.E. of at least three separate experiments. Statistical analysis was performed using the paired Student's t test. Using isoform-specific primers, PCR products corresponding to RyR I and RyR II cDNA were readily amplified from MIN6-, INS1-, or rat islet-derived cDNAs (data not shown). By contrast, RyR III cDNA was not amplified from these sources using the chosen primer pair (see "Experimental Procedures"), although RyR III from brain cDNA was amplified, as expected. The identity of each of the generated PCR products was confirmed by both restriction analysis and by automatic sequencing, which revealed 100% identity with the corresponding mouse (GenBankTM accession numbers X83932 and AF295105 for RyR I and RyR II, respectively) and rat (GenBankTM accession numbers AF130879 and U95157) cDNAs. Semi-quantitative PCR revealed ∼5-fold lower RyR I mRNA levels in rat islet-derived than in skeletal muscle-derived cDNA, and RyR II mRNA levels were ∼8-fold lower in islet than in heart cDNA (data not shown). In order to confirm the presence, and identify the intracellular localization, of RyRs in MIN6 β-cells, subcellular fractionation and immunoblotting (Western) was performed. Probing of crude MIN6 cell fractions with a subtype-specific RyR antibody, which recognizes type I (and III) isoforms, indicated similar levels of RyR immunoreactivity in both mitochondrial/dense core secretory vesicle and microsomal fractions (Fig. 1 A,upper panel). By contrast, RyR II immunoreactivity was much more abundant on the latter fraction, with only weak staining for RyR II in the crude vesicle/mitochondria fraction (Fig. 1 A,lower panel). To demonstrate that RyR I immunoreactivity was present on secretory vesicles in the crude secretory vesicle/mitochondrial fraction examined above, immunoblotting was also performed with immunopurified dense core secretory vesicles (40Varadi A. Ainscow E.K. Allan V.J. Rutter G.A. J. Cell Sci. 2002; 115: 4177-4189Google Scholar). Immunoreactivity toward both anti-RyR I/III, and to a selective anti-RyR I antibody (see "Experimental Procedures"), was clearly evident in these membranes (Fig. 1 B, upper panels), whereas reactivity to RyR III (Fig. 1 B,lower panel) was undetectable. The above fractionation studies indicated that the relative abundance of type I/type II RyRs was higher in secretory vesicles than the ER but provided no information on the relative abundance of the two isoforms on either membrane, considered alone. To determine the relative importance of RyR I and RyR II on each organelle, we therefore used a functional approach in living MIN6 cells. Recombinant aequorins, targeted specifically to either organelle by the addition of appropriate presequences (9Mitchell K.J. Pinton P. Varadi A. Tacchetti C. Ainscow E.K. Pozzan T. Rizzuto R. Rutter G.A. J. Cell Biol. 2001; 155: 41-51Google Scholar), were utilized to monitor free Ca2+ concentrations in each compartment. Concentrations of ryanodine (10 μm) expected to lead to closure of all RyR isoforms (51Meissner G. J. Biol. Chem. 1986; 261: 6300-6306Google Scholar) substantially raised the steady-state concentrations of free Ca2+ in both the secretory vesicle matrix ([Ca2+]SV) and the ER lumen ([Ca2+]ER) (Fig.2, A and B). In contrast, the skeletal muscle relaxant dantrolene (52Van Winkle W.B. Science. 1976; 193: 1130-1131Google Scholar) significantly increased [Ca2+]SV (from 40.1 ± 6.7 to 90.4 ± 14.8 μm; n = 4,p < 0.01; Fig. 2 C), whereas neither [Ca2+]ER (Fig. 2 D) nor [Ca2+]c (Fig. 2 E) was affected by this agent. These findings are in agreement with previous studies where dantrolene was shown to bind directly to and inhibit pig and rabbit skeletal muscle (types I and III), but not cardiac (type II), RyRs (52Van Winkle W.B. Science. 1976; 193: 1130-1131Google Scholar, 53Paul-Pletzer K. Palnitkar S.S. Jimenez L.S. Morimoto H. Parness J. Biochemistry. 2001; 40: 531-542Google Scholar, 54Fruen B.R. Mickelson J.R. Louis C.F. J. Biol. Chem. 1997; 272: 26965-26971Google Scholar, 55Zhao F. Li P. Chen S.R. Louis C.F. Fruen B.R. J. Biol. Chem. 2001; 276: 13810-13816Google Scholar), suggesting that dantrolene inhibits the type I, but not the type II, RyR in MIN6 β-cells. The above results suggest that although ryanodine-sensitive Ca2+ efflux from secretory vesicles is likely to be mediated principally via type I RyRs, this channel subtype apparently plays a minor role, if any, in mediating Ca2+release from the ER in MIN6 β-cells. This result was unexpected given that subcellular fractions enriched with ER membranes apparently contained the same amount or more immunoreactivity to RyR I as a crude secretory vesicle/mitochondria fraction (Fig. 1 A,upper panel). One simple explanation of this observation is that the absolute number of type II RyRs on the ER is very much greater than type I receptors, a difference that may not be apparent given the different antibodies and dilutions used to quantitate each of these isoforms (Fig. 1). However, as an alternative explanation, we next explored the possibility that another Ca2+ release channel may be functional on secretory vesicles, whose presence may stimulate the activity of neighboring RyR I channels. The effects on [Ca2+]SV of the recently identified Ca2+-mobilizing molecule NAADP (25Lee H.C. Aarhus R. J. Biol. Chem. 1995; 270: 2152-2157Google Scholar, 56Chini E.N. Beers K.W. Dousa T.P. J. Biol. Chem. 1995; 270: 3216-3223Google Scholar) were therefore examined in permeabilized cells at a concentration of this compound previously shown to be optimal in human β-cells (31Johnson J.D. Misler S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14566-14571Google Scholar) and other mammalian cell types (26Patel S. Churchill G.C. Galione A. Trends Biochem. Sci. 2001; 26: 482-489Google Scholar). 100 nm NAADP caused a small but highly significant decrease in [Ca2+]SV (Fig.3 A) but was completely without effect on [Ca2+]ER (Fig. 3 B). To determine whether the effects of NAADP may be mediated by a receptor identical or similar to
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