Ryanodine Receptor Type I and Nicotinic Acid Adenine Dinucleotide Phosphate Receptors Mediate Ca2+ Release from Insulin-containing Vesicles in Living Pancreatic beta -Cells (MIN6)*

Kathryn J. Mitchell, F. Anthony LaiDagger , and Guy A. Rutter§

From the Henry Wellcome Laboratories of Integrated Cell Signaling and Department of Biochemistry, School of Medical Sciences, University Walk, University of Bristol, Bristol BS8 1TD and the Dagger  Department of Cardiology, Wales Heart Research Institute, University of Wales College of Medicine, Cardiff CF14 4XN, United Kingdom

Received for publication, October 7, 2002, and in revised form, January 18, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

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 beta -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 beta -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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Increases in cytoplasmic Ca2+ concentration ([Ca2+]c)1 are important for stimulation of neurosecretion in general (1) and for the activation of insulin secretion from pancreatic islet beta -cells (1, 2). In the latter cell type, increases in [Ca2+]c usually occur as a result of either nutrient-induced influx of Ca2+ ions through voltage-gated Ca2+ channels on the plasma membrane (3) or via the release of Ca2+ from intracellular Ca2+ stores (4). The endoplasmic reticulum (ER) (5, 6) and Golgi apparatus (7) probably represent the major Ca2+ stores in beta -cells (8-10) as in other cell types (11). However, we have recently provided evidence that dense core secretory vesicles also play a role in intracellular Ca2+ signaling in beta -cells (9, 12). Importantly, secretory vesicles occupy a substantial proportion of the intracellular volume of beta -cells (13, 14) and may contain close to half the total cellular Ca2+. As such, these organelles potentially provide a huge store of mobilizable Ca2+ ions (15).

Previous studies involving measurements of intravesicular free Ca2+ concentration ([Ca2+]SV) and immunoelectron microscopy (9) indicated that ryanodine, but not inositol 1,4,5-trisphosphate (11), receptors mediate Ca2+ release from secretory vesicles in beta -cells (9, 10, 16). cDNAs encoding three RyR isoforms have so far been identified in mammals. The type I isoform (RyR I) is expressed mainly in skeletal muscle (17), whereas RyR II is abundant in the heart (18). RyR III is present in a variety of tissues and cell types, most notably the brain (19). RyR II has been reported previously to be the most abundantly expressed isoform (at the mRNA level) in wild type (20) and ob/ob mouse islets (20, 21), as well as in rat islets (22) and clonal beta TC3 cells (22). Moreover, the presence of RyR II protein has also been demonstrated in derived INS-1 beta -cells (23). Lower levels of RyR I and RyR III mRNA have also been detected in beta TC3 (22) and HIT-T15 cells (24), respectively. However, the physiological role(s) of RyRs in beta -cells remains unclear, given that RyR II mRNA levels in ob/ob mouse islets are reportedly ~1000-fold less than in the heart (21), whereas RyR II protein levels in INS-1 beta -cells were ~10-fold lower than in brain (23).

Receptors for nicotinic acid adenine dinucleotide phosphate (NAADP), a novel intracellular Ca2+-mobilizing agent (25), may represent an alternative pathway for Ca2+ efflux from dense core secretory vesicles (26). Although other studies (27-30) 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 beta -cell. Although functional NAADP-sensitive Ca2+ stores were recently revealed in human beta -cells (31), NAADP-induced Ca2+ release was not observed in dispersed beta -cells from either normal or ob/ob mouse islets (32). 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 beta -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 beta -cells.

    EXPERIMENTAL PROCEDURES
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Cell Culture and Adenoviral Infection-- MIN6 cells, a well differentiated mouse insulinoma beta -cell line (33) (passages 20-30), were grown in Dulbecco's modified Eagle's medium (Invitrogen) containing 25 mM glucose and 2 mM pyruvate, and supplemented with 15% (v/v) fetal bovine serum, 20 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 50 µM beta -mercaptoethanol, in a humidified atmosphere containing 5% CO2 (9, 34). 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) (35) or aequorin targeted to either the secretory vesicles (VAMP.Aq) (9) or the ER (ER.Aq) (36), 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 (9, 37).

Detection of mRNA for Ryanodine Receptors in beta -Cells and Islets-- 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 (38) 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 µM reverse 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.

Semi-quantitative PCR-- 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 beta -actin primers (39), 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).

Preparation of Cell Lysates and Membrane Fractions-- 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 (40). 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 M NaCl, 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 × g for 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.

Ryanodine Receptor Type I Antiserum Production and Purification-- 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 (41), and antibody specificity was confirmed by enzyme-linked immunosorbent assay and immunoblot analysis with brain, skeletal, and cardiac muscle microsomes, prepared as described previously (41). 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).

Immunoblotting-- 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) (42). 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).

Measurements of Free Ca2+ Concentration with Recombinant Targeted Aequorins-- 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 mM NaH2PO4, 0.5 mM MgSO4, 3 mM glucose, 10 mM Hepes, 2 mM NaHCO3, pH 7.4) supplemented with 1 mM EGTA, for 10 min at 4 °C (9). Aequorin was reconstituted with 5 µM coelenterazine n (43) 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) (44). 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 mM KH2PO4, 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 (9).

Microinjection, Flash Photolysis, and Ca2+ Imaging-- MIN6 cells were seeded onto 24 mm poly-L-lysine-coated coverslips and microinjected, as previously described (45-47), 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 (48) (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 (49). 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.

Assay of Insulin Secretion-- 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 mM glucose 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 (34).

Statistics-- Free [Ca2+] was calculated using METLIG software (50). Data represent the means ± S.E. of at least three separate experiments. Statistical analysis was performed using the paired Student's t test.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Detection of Ryanodine Receptor mRNAs in beta -Cell Lines and Primary Islets-- 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).

Expression of Ryanodine Receptor Protein in MIN6 Cells-- In order to confirm the presence, and identify the intracellular localization, of RyRs in MIN6 beta -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. 1A, 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. 1A, 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 (40). 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. 1B, upper panels), whereas reactivity to RyR III (Fig. 1B, lower panel) was undetectable.


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Fig. 1.   Immunoblot of RyRs in MIN6 beta -cells. A, subcellular fractions of MIN6 cells obtained by differential centrifugation were migrated on a 5% (v/v) polyacrylamide gel, transferred onto an Immobilon-P transfer membrane, and stained with anti-skeletal muscle RyR (type I) or anti-RyR II antibody. The latter antibody is known to also weakly react with RyR I. Lanes were loaded as follows: lane 1, whole cell lysate; lane 2, cytosolic fraction; lane 3, nuclear fraction; lane 4, mitochondria and secretory vesicles; lane 5, ER and Golgi. B, whole cell lysate from MIN6 cells (lane 1), skeletal muscle protein (lane 2), and MIN6 cell vesicle protein (see "Experimental Procedures"; lane 3) were separated on a 5% (v/v) polyacrylamide gel, transferred onto a membrane, and probed with anti-skeletal muscle RyR antibody or isoform-specific antibodies, RyR I and RyR III.

Effect of Ryanodine Receptor Inhibition on Secretory Vesicle and ER Ca2+ Concentrations-- 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 (9), were utilized to monitor free Ca2+ concentrations in each compartment. Concentrations of ryanodine (10 µM) expected to lead to closure of all RyR isoforms (51) 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 (52) significantly increased [Ca2+]SV (from 40.1 ± 6.7 to 90.4 ± 14.8 µM; n = 4, p < 0.01; Fig. 2C), whereas neither [Ca2+]ER (Fig. 2D) nor [Ca2+]c (Fig. 2E) 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 (52-55), suggesting that dantrolene inhibits the type I, but not the type II, RyR in MIN6 beta -cells.


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Fig. 2.   Effect of ryanodine and dantrolene on steady-state Ca2+ concentrations in the secretory vesicle and ER. After Ca2+ depletion and aequorin reconstitution, MIN6 cells infected with VAMP.Aq (A and C), ER.Aq (B and D), or Cyt.Aq (E) encoding adenoviruses were perifused in KRB containing 1 mM EGTA. Where indicated, EGTA was replaced with 1.5 mM CaCl2 in the absence (open symbols) or presence (closed symbols) of ryanodine (Ryan.) (10 µM; A and B) or dantrolene (Dan.) (10 µM; C-E). Data are the means of four separate experiments.

Effect of NAADP on Secretory Vesicle and ER Ca2+ Concentrations-- 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 beta -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. 1A, 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 (25, 56) were therefore examined in permeabilized cells at a concentration of this compound previously shown to be optimal in human beta -cells (31) and other mammalian cell types (26). 100 nM NAADP caused a small but highly significant decrease in [Ca2+]SV (Fig. 3A) but was completely without effect on [Ca2+]ER (Fig. 3B).


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Fig. 3.   Effect of NAADP on secretory vesicle and ER Ca2+ concentrations in permeabilized MIN6 cells. After Ca2+ depletion and aequorin reconstitution, MIN6 cells infected with VAMP.Aq (A and C-E) or ER.Aq (B) encoding adenoviruses were permeabilized and perifused in IB containing 1 mM EGTA. Where indicated, free [Ca2+] was increased from <1 to 400 nM using an EGTA-buffered Ca2+ solution (see "Experimental Procedures"). Cells were perifused with 100 nM NAADP at steady-state [Ca2+]. Ryanodine (C), dantrolene (D), or nimodipine (E) were present throughout the experiment. Data are means of four separate experiments.

To determine whether the effects of NAADP may be mediated by a receptor identical or similar to that described previously (26) in mammalian and other cell systems, we next explored the pharmacology of the observed NAADP-induced changes in secretory vesicle [Ca2+]. Concentrations of ryanodine sufficient to inhibit all RyR isoforms (51), but known to have no effect on NAADP receptor activity (57), failed to alter NAADP-induced [Ca2+]SV changes (Fig. 3C). Similarly, NAADP-induced release of secretory vesicle Ca2+ was unaffected by dantrolene (Fig. 3D). By contrast, nimodipine, an inhibitor of L-type Ca2+ channels shown previously (26) to block NAADP receptors, completely blocked NAADP-induced changes in [Ca2+]SV (Fig. 3E).

Effect of Photorelease of Caged NAADP on [Ca2+]c in Intact MIN6 Cells-- To determine whether (i) NAADP may mediate Ca2+ release selectively from dense core vesicles in living cells, and (ii) to explore the impact of this release of cytosolic Ca2+ concentrations, we next micro-injected an inactive precursor of NAADP ("caged NAADP") (48) into single MIN6 beta -cells, and we monitored the impact of its rapid uncaging by flash photolysis (Fig. 4). Photo-released NAADP provoked an increase in the fluorescence of the co-microinjected Ca2+ reporter, Oregon Green, of 4.3 ± 2.1% with respect to basal fluorescence (n = 7 cells; Fig. 4A), consistent with the mobilization of intracellular Ca2+. The magnitude of this increase was not significantly affected by depletion of ER/Golgi Ca2+ stores with the sarco(endo)plasmic reticulum Ca2+-ATPase inhibitor, thapsigargin (5.6 ± 1.4%; n = 8 cells; Fig. 4B), nor by incubation with ryanodine (3.7 ± 0.9%; n = 7 cells; Fig. 4C).


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Fig. 4.   Effect of uncaging NAADP on cytosolic Ca2+ concentrations in single living MIN6 cells. A, cells were microinjected with Oregon Green 488 bis(2-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid-1 dextran in the absence (control) or presence of 2 µM caged NAADP. Confocal images were acquired at 5-s intervals before and after induction of photolysis with UV light (~360 nm) for 1 s. Microinjected cells were incubated with ryanodine (10 µM; C) or depleted of Ca2+ (see "Experimental Procedures") and incubated with thapsigargin (1 µM; B) for 5 min prior to imaging and photolysis. Data represent the means of normalized fluorescence values for at least seven cells in each case.

Importance of Secretory Vesicle Ca2+ Release for Glucose-stimulated Insulin Secretion-- The above studies indicated that release of Ca2+ from secretory vesicles, mediated by either type I RyR or NAADP receptors, may be important for the triggering of insulin secretion by nutrients. To determine whether Ca2+-induced Ca2+ release via type I RyRs or Ca2+ release through NAADP receptors was qualitatively the more important pathway for Ca2+ efflux in living cells, we blocked the former with dantrolene (see above). In accordance with previous findings in islets (58) and INS1E cells (59), the stimulation of insulin release from MIN6 beta -cells by 30 mM (versus 3 mM) glucose (3.07 ± 0.51-fold; n = 3, p < 0.01) was completely inhibited in the presence of the drug (Fig. 5A). Similarly, glucose-induced insulin secretion was abolished (from 2.02 ± 0.11-fold stimulation to no significant effect; n = 4, p < 0.01) in the presence of 100 µM ryanodine (Fig. 5B). This latter finding is in contrast to previous reports where ryanodine had little or no effect on insulin secretion in mouse beta -cells (60) nor in INS1E cells (59), a result attributed to the poor permeation of ryanodine across the cell membrane in these systems. By contrast, both the present (Fig. 2, A and B) and previous studies (61) indicate that intact MIN6 cells may be more permeable to this drug.


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Fig. 5.   Dantrolene and ryanodine inhibit glucose-induced insulin secretion. MIN6 cells were cultured for 16 h with 3 mM glucose and then incubated with KRB supplemented with 3 mM glucose for 30 min. Cells were then incubated for a further 30 min with 3 or 30 mM glucose, with additions as stated. Released insulin was measured by radioimmunoassay (see "Experimental Procedures"). Data are the means of at least three separate experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ryanodine Receptor mRNA Expression in Pancreatic beta -Cells-- In this study, we found that mRNAs encoding both RyR I and RyR II are expressed in beta -cell lines as well as in rat islets at approximately equal levels. These findings are in apparent contrast with previous reports (20-22) indicating that RyR II mRNA was by far the most abundant RyR isoform, at least in mouse beta -cells. Thus, in these earlier studies, only faint reverse transcriptase-PCR products were generated with RyR I primers in mouse-derived beta TC3 cells, whereas the same primers did not detect any RyR I mRNA in rat islets (22). Similarly, ob/ob mouse islets, which are enriched in beta -cells, were shown to contain ~1000-fold less RyR II mRNA than the heart (21). A number of factors may underlie these apparently discrepant results. First, the primers used to detect RyR I in previous reports may have been less efficient in amplifying the beta -cell RyR I than those used in the present study. Consistent with this view, the primers used by Takasawa et al. (20) scarcely detected RyR I mRNA in the brain, a tissue in which this isoform is abundant (19, 62). Second, the presence and density of RyRs in different beta -cell lines and rodent strains may well differ. Thus, RyR II protein was hardly detectable in RINm5F cells, whereas INS1 cells were shown to express significant amounts (23). Similarly, one group (21) described low levels of RyR II mRNA in ob/ob mice islets, whereas another (20) failed to detect this message in islets from distinct colonies of ob/ob mice. An intriguing possibility is that these differences in RyR II expression in the islet may contribute to the differing severities of diabetes in the different mouse strains.

Ryanodine Receptor Protein Expression and Subcellular Localization in MIN6 beta -Cells-- RyR subtypes are expressed in various combinations in specific tissues and cell types. Thus, RyR II and III are expressed in the heart, RyR I and RyR III in skeletal muscle, and all three subtypes in smooth muscle and brain (62). Although the relevance of multiple isoform expression is not clearly understood, one possibility is that co-expression serves to amplify Ca2+ signals. Indeed, such a mechanism may explain why the presence of both RyR I and RyR II is required for the activation of Ca2+-induced Ca2+ release upon Ca2+ influx in vascular myocytes (63).

The present study revealed the presence of both RyR I and RyR II in MIN6 beta -cells and islets at both the mRNA and protein levels (Fig. 1). Moreover, using subcellular fractionation as well as vesicle immunopurification, we demonstrate a distinct subcellular localization for each RyR isoform in this cell type. Thus, type I RyRs were present on both the ER and secretory vesicles, whereas RyR II immunoreactivity was more abundant in the former. Although we also detected a small amount of RyR II immunoreactivity in the crude mitochondrial/secretory vesicle fraction (Fig. 1A), it should be noted that at least part of this reactivity may result from contamination of this fraction with ER/Golgi fragments and/or cross-reactivity with RyR I, because the anti-type II RyR antibody used also weakly cross-reacts with RyR I.

Supporting the view that Ca2+-induced Ca2+ release from secretory vesicles is mediated principally via RyR I channels, and from the ER via RyR II, blockade of RyR I receptors in whole cells with dantrolene (55) affected steady-state Ca2+ concentrations only in the former compartment (Fig. 2, C versus D). Together, these data therefore provide both structural and functional evidence that RyR I and RyR II are located on distinct organelles in MIN6 beta -cells.

Interestingly, inhibition of type I RyRs, shown here (Fig. 5) and in earlier studies (58) to block glucose-induced insulin secretion, now seems likely to involve largely a blockade of Ca2+ release from secretory vesicles, rather than from the ER (Fig. 2). This result, which accords well with the reported effects of depleting vesicle Ca2+ on insulin release (64), reinforces the view that the release of vesicle Ca2+ plays an important role in triggering or facilitating the exocytotic release of insulin (Fig. 6).


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Fig. 6.   Potential cross-talk between two different but converging messenger pathways that lead to Ca2+ release from dense core secretory vesicles. Exposure of islet beta -cells or MIN6 cells to nutrients causes an increase in intracellular free [ATP], which in turn may modulate the activity of CD38, increasing the intracellular concentrations of NAADP and cADPr. NAADP-induced Ca2+ release from the secretory vesicles (1) could potentially activate Ca2+-induced Ca2+ release from the neighboring type I RyRs (2), previously sensitized to Ca2+ by cADPr (3). This would result in a domain of high Ca2+ concentration underneath the plasma membrane, independent of Ca2+ influx through voltage-gated Ca2+ channels, which may contribute to the stimulation of insulin secretion.

Role of NAADP Receptors in beta -Cells-- An unexpected finding of the present study was that dantrolene, which blocks RyR I channels, affected Ca2+ concentrations in secretory vesicles but not in the ER despite the presence of RyR I receptors on both organelles. One possible explanation for this result is our demonstration that MIN6 beta -cells possess an NAADP-sensitive intracellular Ca2+ store that appears to coincide, at least in part, with secretory vesicles (Figs. 3 and 4). Importantly, these data are consistent with previous findings that have suggested NAADP releases Ca2+ from a non-ER Ca2+ pool at the frog neuromuscular junction (65) and that the NAADP-sensitive Ca2+ pool in sea urchin eggs is thapsigargin-insensitive (66).

We show here that in pancreatic beta -cells, NAADP-induced Ca2+ release is insensitive to ryanodine and dantrolene, confirming previous reports (26) that NAADP acts on a channel distinct from the RyR (Figs. 3, C and D, and 4C). Although the receptor for NAADP is as yet unidentified in molecular terms, in agreement with previous findings in sea urchin eggs (67), brain (28), smooth muscle (68), and heart (57), L-type Ca2+ channel inhibitors were found here to block NAADP-induced Ca2+ release from beta -cell vesicles (Fig. 3E), suggesting that a common or similar receptor is involved in each of these cellular systems. Interestingly, NAADP-mediated Ca2+ release in living beta -cells was not significantly affected by blockade of RyRs (Fig. 4, C versus A), a result also consistent with an action of this messenger via a non-RyR channel.

Two Converging Pathways Are Involved in Ca2+ Release from Secretory Vesicles-- cADPr, the proposed endogenous ligand for RyRs (69, 70), and NAADP are likely to be synthesized in beta -cells by the same enzyme, an ADP ribosyl cyclase termed CD38 (71). At the cell surface, the catalytic domain of CD38 is likely to be exposed to the extracellular space (72). However, internalized catalytically active CD38 is found in non-clathrin-coated vesicles (73). Moreover, endocytotic vesicles have been shown previously to accumulate the precursor for cADPr, beta -NAD, via an unidentified dinucleotide transport system (74). As well as catalyzing the conversion of beta -NAD to cADPr, CD38 itself is thought to be involved in pumping the cyclic nucleotide into the cytosol (75), thus providing a potential mechanism for the accumulation of intracellular cADPr and potentially NAADP.

Interestingly, transgenic mice overexpressing CD38 specifically in beta -cells show enhanced glucose-induced insulin secretion (76), whereas deletion of both alleles of CD38 gives rise to glucose intolerance (77). Because CD38 activity is regulated allosterically by ATP (76), glucose-induced increases in the intracellular free concentration of this nucleotide (47, 78) may lead to increases in the intracellular concentrations of either cADPr, NAADP, or both. Although glucose-dependent increases in cADPr have been reported previously (69), key future goals will be to determine whether glucose is able to increase NAADP levels in beta -cells and whether such increases are altered in models of type 2 diabetes mellitus.

    ACKNOWLEDGEMENTS

We thank Dr. Mark Jepson and Alan Leard (Bristol MRC Imaging Facility) for help with confocal imaging and Dr. Aniko Varadi for help with flash photolysis. We also thank Dr. Judith A. Airey (University of Nevada) for providing the anti-RyR III antibody, Prof. Luigia Santella (University of Naples) for caged NAADP, and Dr. Frederique Diraison (University of Bristol) for the preparation of rat islets.

    FOOTNOTES

* This work was supported by a Biotechnology and Biological Sciences Research Council Studentship (to K. J. M.), Wellcome Trust Program Grant 067081, the Human Frontiers Science Program, the Medical Research Council (UK), and Diabetes UK.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Wellcome Trust Research Leave Fellow. To whom correspondence should be addressed. Tel.: 44-117-954-6401; Fax: 44-117-928-8274; E-mail: g.a.rutter@bris.ac.uk.

Published, JBC Papers in Press, January 21, 2003, DOI 10.1074/jbc.M210257200

    ABBREVIATIONS

The abbreviations used are: [Ca2+]c, free cytosolic Ca2+ concentration; [Ca2+]ER, free ER Ca2+ concentration; [Ca2+]SV, free secretory vesicle Ca2+ concentration; cADPr, cyclic ADP ribose; ER, endoplasmic reticulum; EGFP, enhanced green fluorescent protein; NAADP, nicotinic acid adenine dinucleotide phosphate; RyR, ryanodine receptor; Aq, aequorin; VAMP, vesicle associated membrane protein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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