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发表于 2009-3-5 23:32 |只看该作者 |倒序浏览 |打印

1 Departments of Biochemistry, University of Bristol, BS8 1TD Bristol, United Kingdom

2 Experimental and Diagnostic Medicine Section of General Pathology, University of Ferrara, 44100 Ferrara, Italy

3 Biomedical Sciences and CNR Center for Study of Biological Membranes, University of Padova, 35121 Padova 17, Italy3 x/ o* l  }, Q5 j4 Y& X

4 Experimental Medicine, University of Genova Medical School, 16132 Genova, Italy
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Address correspondence to Guy Rutter, Department of Biochemistry, University of Bristol, BS8 1TD Bristol, UK. Tel.: (44) 117-954-6401. Fax: (44) 117-928-8274. E-mail: g.a.rutter@bris.ac.uk% u. K8 p" C0 V( `- w

Abstract

The role of dense core secretory vesicles in the control of cytosolic-free Ca2  concentrations (c) in neuronal and neuroendocrine cells is enigmatic. By constructing a vesicle-associated membrane protein 2–synaptobrevin.aequorin chimera, we show that in clonal pancreatic islet ?-cells: (a) increases in c cause a prompt increase in intravesicular-free Ca2  concentration (SV), which is mediated by a P-type Ca2 -ATPase distinct from the sarco(endo) plasmic reticulum Ca2 -ATPase, but which may be related to the PMR1/ATP2C1 family of Ca2  pumps; (b) steady state Ca2  concentrations are 3–5-fold lower in secretory vesicles than in the endoplasmic reticulum (ER) or Golgi apparatus, suggesting the existence of tightly bound and more rapidly exchanging pools of Ca2 ; (c) inositol (1,4,5) trisphosphate has no impact on SV in intact or permeabilized cells; and (d) ryanodine receptor (RyR) activation with caffeine or 4-chloro-3-ethylphenol in intact cells, or cyclic ADPribose in permeabilized cells, causes a dramatic fall in SV. Thus, secretory vesicles represent a dynamic Ca2  store in neuroendocrine cells, whose characteristics are in part distinct from the ER/Golgi apparatus. The presence of RyRs on secretory vesicles suggests that local Ca2 -induced Ca2  release from vesicles docked at the plasma membrane could participate in triggering exocytosis.

Key Words: calcium; secretory vesicle; insulin; ryanodine receptor; aequorin( b% ]* o4 ~( E' V+ T2 ?  @- A6 b) d
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In most mammalian cells, the ER (Streb et al., 1984; Rizzuto et al., 1993; Brini et al., 1993) and Golgi complex (Pinton et al., 1998) are believed to represent the major mobilizable intracellular Ca2  stores (Rutter et al., 1998). Uptake of Ca2  into these stores is mediated largely by sarco(endo)plasmic reticulum Ca2 –ATPases (SERCAs)* (Moller et al., 1996) and helps to maintain resting cytosolic Ca2  concentrations (c) at levels (10-7 M) some four orders of magnitude lower than in the extracellular space (10-3 M). Release of Ca2  from the ER and Golgi complex is provoked by hormones and other agonists which generate inositol 1,4,5 trisphosphate (IP3) (Berridge, 1993) to open intracellular receptors (Mikoshiba, 1997). In certain cell types, such as skeletal muscle, these stores are also accessed by "Ca2 -induced Ca2  release", via receptors for the insecticide, ryanodine (Lai et al., 1988; Takeshima et al., 1989).
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Secretory vesicles of both the dense core (Hellman et al., 1976; Hutton et al., 1983) and small synaptic types (Andrews and Reese, 1986) possess a substantial proportion of the total Ca2  in cells specialized for peptide secretion. However, despite many studies, the role of secretory vesicles as Ca2  buffers or stores remains controversial (Pozzan et al., 1994; Rutter et al., 1998; Yoo, 2000).

Mobilization of dense core vesicle Ca2  by IP3 was originally suggested as a possibility in chromaffin cells (Yoo and Albanesi, 1990). Later data suggested that IP3 receptors may be present on the vesicle membrane in insulin-secreting cells (Blondel et al., 1994), though subsequent experiments showed the antibody used cross-reacted with insulin (Ravazzola et al., 1996). IP3, as well as the receptor (RyR) agonist cyclic ADP ribose (cADPr) (Galione, 1994) have been reported to release Ca2  from individual acinar cell zymogen granules (Gerasimenko et al., 1996), although contamination of these preparations (e.g., with ER or Golgi apparatus–derived vesicles) is a potential problem (Yule et al., 1997). Finally, evidence for the direct participation of secretory granules in the control of cytoplasmic Ca2  concentration was recently provided in intact goblet cells (Nguyen et al., 1998).

At present, the molecular mechanisms responsible for Ca2  uptake into secretory vesicles are also a matter of controversy. At high Ca2  concentrations (>50 μM), uptake into dense core vesicles in permeabilized hypophysial cells (Troadec et al., 1998; Thirion et al., 1999) and into isolated chromaffin cell granules (Krieger-Brauer and Gratzl, 1982) can occur via Na /Ca2  exchange, while synaptic vesicles also accumulate Ca2  via a Ca2 -H  antiport system (Goncalves et al., 1998). However, the pump/exchange mechanisms active at physiological c are uncertain.
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To examine the pathways by which Ca2  crosses the limiting membrane of the dense core secretory vesicle of living islet ?-cells, we have developed a new approach to monitor the free Ca2  concentration within the secretory vesicle matrix (SV) using recombinant targeted aequorin (Rizzuto et al., 1995). Cloned from the jellyfish Aequorea victoria (Inouye et al., 1985), aequorin is a calcium-sensitive bioluminescent protein (Cobbold and Rink, 1987), previously used to measure free Ca2  concentrations in a variety of subcellular organelles (Rutter et al., 1998). Importantly, aequorin activity is less severely inhibited at low pH values (

Vesicle-associated membrane protein (VAMP)2/synaptobrevin (Sudhof et al., 1989) is a vesicle-specific SNARE with a single transmembrane-spanning region. Expression of chimaeric cDNA encoding a fusion protein between VAMP2 and aequorin (VAMP.Aq) has therefore allowed the intravesicular-free Ca2  concentration to be monitored dynamically in live MIN6 ?-cells. With this approach, we show that Ca2  is actively pumped into dense core vesicles when c increases, and may be released via RyR, but not IP3, receptors. This release may be important at sites of high intracellular Ca2 , including sites of exocytosis at the plasma membrane.
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Results
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Subcellular targeting of recombinant VAMP.aequorin

Chimeric cDNA encoding hemagglutinin (HA)1-tagged aequorin, fused to VAMP2 (Sudhof et al., 1989), was generated as shown in Fig. 1 A.

Figure 1. Localization of VAMP.Aq. (A) Schematic map of VAMP.Aq. VAMP2 and aequorin cDNAs were fused via an HA1 epitope tag linker (Materials and methods) in order to localize mutated aequorin to the secretory vesicle lumen. (B) Confocal immunolocalization of VAMP.Aq. MIN6 cells were transfected with VAMP.Aq and stained with (a) mouse anti-HA1 monoclonal antibody (1:200) and (b) guinea pig antiinsulin antibody (1:150). (c) Extent of colocalization. (C) Immunoelectron microscopic localization of insulin (15-nm gold) or VAMP.Aq (anti-HA tag, 10-nm gold). Morphometric analysis of separate sections from 10 singly labeled cells revealed the following distribution of anti-HA gold particles: dense core vesicles, 36; ER, 2; Golgi apparatus, 0; plasma membrane, 16; endosomes, 19.
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Immunocytochemical analysis of MIN6 cells transfected with VAMP.Aq cDNA revealed close overlap with insulin staining (Fig. 1 B). Explored at a higher resolution by immunoelectron microscopy (Fig. 1 C), VAMP.Aq immunoreactivity was highly enriched in 61 of 148 (41.2%; n = 11 cells) vesicles colabeled for insulin (Fig. 1 C). Analyzed by single labeling for VAMP.Aq, staining of the ER, Golgi apparatus, and small synaptic-like microvesicles (Reetz et al., 1991) was very low, while reactivity was also present on the plasma membrane and in endosomes (see the legend to Fig. 1 and Discussion).

Reconstitution and calibration of secretory vesicle and ER-targeted aequorins' T: ?9 o4 a  D; ~" J
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Given the high total Ca2  content of secretory vesicles (Hutton et al., 1983), we used the approach adopted previously to measure Ca2  in the ER lumen (Montero et al., 1995). Apoaequorin was reconstituted at a low free Ca2  concentration (Montero et al., 1995), achieved by depleting cells of Ca2  (Materials and methods). Depletion of vesicle Ca2  had no marked effect on glucose or K -stimulated insulin secretion, or on vesicle motility (Pouli et al., 1998b; Tsuboi et al., 2000; unpublished data).
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To determine the response of the expressed aequorins to Ca2  in situ, permeabilized cells were incubated at buffered Ca2  concentrations in the presence of ionomycin and monensin (Fig. 2, C and E). The sensitivity to Ca2  (at pH 7.0) of the VAMP.Aq chimaera was similar to that reported previously for mutant (D119A) aequorin (Montero et al., 1995). Intravesicular pH in intact cells was determined using a fusion construct between VAMP2 and a mutated, pH-sensitive GFP (pH.fluorin(e); Miesenbock et al., 1998), and gave a pH value of 6.3 ± 0.02 (n = 85 cells; Fig. 2 A). Confirming that this low intravesicular pH was unlikely to significantly affect VAMP.Aq, near identical calibration data were obtained at pH 5.7 (unpublished data). Therefore, we used the constants obtained in vitro (Montero et al., 1995) to calculate  from the fractional rate of aequorin consumption (F) according to:  = 1.4410(LOG-3.4) (Rutter et al., 1993).% V' `7 A! E+ ?# v7 d/ j7 v
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Figure 2. Measurement of intravesicular pH (A) and Ca2  with cytosolic aequorin (B), VAMP.Aq (C and D), and ER.Aq (E and F). (A) Confocal images were acquired from cells transfected with pH.fluorin(e) (Miesenbock et al., 1998), maintained initially in KRB, pH 7.4, before digitonin-permeabilization and exchange into IB at the specified pH values, and buffered with morpholinosulphonic acid, Hepes, or TRIS (10 mM), plus 10 μM ionomycin, 10 μM monensin, and 1 μM FCCP. Normalized fluorescence ratios before permeabilization were in the range of 0.16–0.18 (arrow); data were fitted using the Graph Pad PrismTM. (C and E) After Ca2  depletion and aequorin reconstitution, transfected cells were permeabilized with digitonin and perifused in the presence of ionomycin, monensin, and CPA (10 μM each) with N-(2-hydroxyethyl)ethylene diamine triacetate (HEDTA)-buffered Ca2  solutions at 0.5 mM calculated free Mg2 . Cells expressing either (B) cytosolic aequorin, (D) VAMP.Aq, or (F) ER.Aq were perifused with KRB, supplemented with 1 mM EGTA (KRB/EGTA) and, where indicated, EGTA was replaced with 1.5 mM CaCl2. Cells were finally lysed in Ca2 -rich hypotonic medium (10 mM CaCl2, 0.1 mM digitonin) for calibration (see Materials and methods and Results). The background count rate (1,200 cps; D and F) was identical during perifusion of untransfected cells and is due to autooxidation of coelenterazine n.% G4 t8 m6 w7 Z1 u% ?

Dynamic measurement of secretory vesicle and ER Ca2  concentrations

To monitor uptake of Ca2  into vesicles in living cells, we provoked rapid Ca2  influx into cells through store-operated channels (Parekh and Penner, 1997). During reintroduction of CaCl2 to cells previously perifused in EGTA, c approached 500 nM, then fell back to 150–200 nM (Fig. 2 B). By contrast, steady state free  in the secretory vesicle matrix was 30–90 μM (51; 7.5 μM; n = 5 separate cultures; Fig. 2 D), whereas free  in the ER lumen was fivefold higher (249; 12.9 μM; n = 3 preparations; Fig. 2 F)." y1 E; w7 a' s, u6 c

Pathways of Ca2  uptake into dense core secretory vesicles% {, Q6 K5 ^6 e! I0 r

Both the rate and extent of the  increases in the secretory vesicles and the ER were strongly dependent on the presence of added ATP in permeabilized cells (Fig. 3, A and D). The increase in SV upon reintroduction of Ca2  ions was completely inhibited by the P-type Ca2  pump inhibitor, orthovanadatate, at >100 μM (Fig. 3 B), but was insensitive to 10 μM orthovanadatate, a concentration which strongly inhibits the plasma membrane Ca2 -ATPase (Carafoli, 1991). Preincubation with the specific SERCA inhibitor, thapsigargin (Thastrup, 1990), and perifusion with cyclopiazonic acid (CPA) (Mason et al., 1991) had no effect on the changes in SV (Fig. 3 C and Table I), but markedly (>85%) inhibited ER  increases (Fig. 3 E).
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Figure 3. Ca2  uptake into secretory vesicles (A–C) or ER (D and E). After Ca2  depletion and aequorin reconstitution, cells were permeabilized with 20 μM digitonin in IB; (A and D; see Materials and methods). Cells were perifused initially at
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Table I. Kinetic paramers for Ca2  uptake into the ER versus secretory vesicles
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Arguing against vesicular Ca2  uptake via Ca2 /H  exchange, monensin, an Na /H  exchanger, had no effect on SV increases in permeabilized cells (Fig. 4 A). Similarly, ionomycin (a Ca2 /H  exchanger) decreased SV marginally (20%; Fig. 4 B), which is consistent with the inability of this ionophore to bind to and transport Ca2  against a prevailing H  gradient, but caused a rapid and complete decline in SV in the additional presence of monensin (Fig. 4 B). Neither the vacuolar H -ATPase inhibitor, bafilomycin (Fig. 4, C and D), nor the protonophore carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), had any effect on vesicular Ca2  uptake (Fig. 4 E).2 T) ^+ b: ?1 A( d1 Y& a
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Figure 4. Effects of collapse of H  and Na  gradients on vesicular Ca2  accumulation in permeabilized cells. Cells were Ca2  depleted and aequorin reconstituted with coelenterazine n before digitonin permeabilization. Cells were perifused in IB (Materials and methods) and free  was increased from

SV increases were also completely unaffected by raising  from 0, 2, or 140 mM (Fig. 4 F), and by simultaneous blockade of Na /Ca2  exchange and Ca2 /H  exchange (zero Na , 300 nM bafilomycin; unpublished data).

Effects of IP3 on SV and ER! G# X- w$ Y' D1 r6 ~$ H6 u! T
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To achieve large changes in intracellular IP3 levels, we coexpressed the metabotropic glutamate receptor, mGluR5, with VAMP.Aq or ER.Aq. Stimulation of mGluR5-transfected cells with the specific agonist, (S)-3,5-dihydroxyphenylglycine (DHPG) (Thomas et al., 2000) caused a significant decrease in ER (Fig. 5 E), but an increase in SV (Fig. 5 A). DHPG had no significant effect on the distribution of vesicles (imaged after expression of VAMP.GFP/pHfluorin (r); Fig. 5 D), indicating that enhanced exocytosis and exposure of the VAMP.Aq to the extracellular medium was unlikely to contribute to the observed increases in SV (Fig. 5 A, and see Discussion).

Figure 5. Effect of IP3 on SV and ER in intact (A and E) and permeabilized (B, C, F, and G) cells. MIN6 cells were cotransfected with (A) VAMP.Aq or (E) ER.Aq plus a plasmid bearing mGluR5 cDNA. After the depletion of intracellular Ca2  stores and aequorin reconstitution, cells were perifused in KRB, which initially contained 1 mM EGTA. Where indicated, EGTA was replaced with 1.5 mM CaCl2. After the achievement of steady state , cells were challenged with the mGluR5 receptor agonist DHPG (30 μM) as indicated. Data are means of five independent experiments. (B, C, F, and G) Cells transfected with plasmids encoding VAMP.Aq (B and C) or ER.Aq (F and G) were Ca2  depleted and aequorin was reconstituted before permeabilization and perifusion in IB buffer which initially contained 0.1 mM EGTA (free
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Similarly, in permeabilized cells, IP3 had no effect on SV (Fig. 5, B and C), while causing a large decrease in ER (Fig. 5, F and G).

RyR activation decreases SV and ER4 R* R" G' p9 G# c  V9 b) c

The RyR agonists caffeine or 4-chloro-3-ethylphenol (4-CEP) (Zorzato et al., 1993) provoked rapid decreases in SV and ER (Fig. 6, A and C) in intact cells. Similarly, cADPr (Galione, 1994) caused clear decreases in both parameters in permeabilized cells (Fig. 6, B and D), and the effects of cADPr were strongly potentiated by palmitoyl CoA (Fig. 6, B and D), a coagonist of RyRs (Chini and Dousa, 1996).% W. @1 V& T7 J: M" g
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Figure 6. Effect of RyR agonists on secretory vesicle and ER Ca2  concentrations. Cells were transfected (A and C) or infected with adenoviruses (B and D) encoding either VAMP.Aq (A and B) or ER.Aq (C and D), then perifused in KRB containing 1 mM EGTA (A and C) or digitonin permeabilized and perifused with IB which initially contained 0.1 mM EGTA (B and D). Replacement of EGTA with 1.5 mM CaCl2 (intact cells, A and C), or an increase in perifusate-free  to 200 nM (permeabilized cells, B and D), as indicated. Other additions were: caffeine, 10 mM; 4-CEP, 500 μM; cADPr, 5 μM; palmitoyl CoA, 50 μM.  w! x" b! N$ B" I

4-CEP mobilizes Ca2  from an acidic, CPA-insensitive store in fura-2–loaded MIN6 cells
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The above results suggested that activation of RyR on vesicles should cause an increase in cytoplasmic c, even after the depletion of the ER Ca2  pool. Added to fura-2–loaded MIN6 cells, 4-CEP caused a substantial increase in c in the absence of external Ca2  (Fig. 7 A). This c increase was partly retained after depletion of SERCA-dependent stores, giving a peak c increase of 30–40% of that in control cells (Fig. 7 B versus A). The c increase elicited by 4-CEP was also partially retained after treatment of cells with ionomycin (Fig. 7 C), but completely abolished after treatment with ionomycin plus monensin, to deplete acidic Ca2  stores (Fig. 7 D). Demonstrating that the effects of 4-CEP on SV were likely mediated by RyR, immunoreactivity to these channels was revealed on vesicle membranes by direct immunoelectron microscopy (Fig. 7 E).. l' _. }/ Y/ c( ^, L/ F$ p' d0 _* L

Figure 7. Effect of caffeine and 4-CEP on cytosolic . (A–D) Cells were loaded with fura-2 and imaged as described (Materials and methods). Where indicated (B–D), cells were preincubated with 1 μM thapsigargin in Ca2 -free KRB (supplemented with 1 mM EGTA for 10 min) and then subsequently perifused in Ca2 -free medium, initially in the absence of thapsigargin or other additions. Trace (A) shows the effect of 500 μM 4-CEP, added as indicated, on c in cells untreated with thapsigargin, and (B) the effect of the drug on cells treated with thapsigargin and subsequently perifused in the presence of 10 μM CPA. Note the abolition of the response to carbachol, which caused a large increase in c in untreated cells (unpublished data). In C, CPA and ionomycin (10 μM each) were added as indicated. Panel D was as C, but with the further addition of monensin (mon, 10 μM) to the perifusate. Data are the means (± SEM) on observations on a total of (A) 58, (B) 35, (C) 28, and (D) 40 single cells, imaged in 3–5 separate experiments. (E) Immunoelectron microscopic localization of RyR (arrows) on dense core vesicles.
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Effects of nutrient secretagogues on ER and SV
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Exposure of islet ?- or MIN6 cells to glucose or other nutrients causes an increase in intracellular-free  (Kennedy et al., 1999) closure of ATP-sensitive K  (KATP) channels (Bryan and AguilarBryan, 1997) and Ca2  entry via voltage-sensitive (L-type) Ca2  channels (Safayhi et al., 1997).
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High (20 mM) glucose, or a combination of nutrient secretagogues (Ashcroft and Ashcroft, 1992), caused a large increase in steady state  both in the ER and in vesicles (Fig. 8, A and C), with no significant change in vesicular distribution (Fig. 8 B), which is consistent with the increases in c seen under these conditions (Grapengiesser et al., 1988).% ^( l: c  \5 w/ h$ r5 k

Figure 8. Effect of nutrient secretagogues on steady state  in secretory vesicles and the ER. Ca2  depletion, and aequorin reconstitution, were carried out as described in Fig. 3 before exposure of cells expressing VAMP.Aq (A) or ER.Aq (C) to the concentrations of nutrients shown for 2 min in complete KRB medium containing 1.5 mM CaCl2. Mixed nutrients, 20 mM glucose, 10 mM glutamine, 10 mM leucine. In all cases, cells were preperifused for 5 min and maintained in the presence of 1 mM 3-isobutyl-1- methylxanthine (IBMX). Asterisk indicates P
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Discussion* c2 Y# X* N3 R
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Aequorin as a reporter of secretory vesicle–free Ca2  concentration1 ~* o9 m9 c3 A: b  c9 \
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We show here that aequorin can be targeted to the lumen of dense core secretory vesicles in living cells, with no incorporation into the ER, Golgi, or trans-Golgi network using the cell's own protein-sorting machinery (Fig. 1). However, a proportion of VAMP.Aq was also present on the plasma membrane and endosomes (Fig. 1 C). Since our recordings were made at either high (1.5 mM, intact cells) or low (400 nM, permeabilized cells) external Ca2  concentrations, the plasma membrane–located photoprotein should be either rapidly exhausted, or inactive, respectively. In any case, it would appear that plasma membrane–targeted aequorin is not reconstituted, since we would have expected to see a "spike" of luminescence upon readdition of high Ca2  concentrations to intact cells (e.g., Fig. 3 C). None was seen, suggesting that the mistargeted protein may be inactivated by unknown mechanisms during recycling between the plasma membrane and endosomes.

Similarly, endosomal aequorin is likely to be inactive under our conditions, since (a) the total endosomal Ca2  content is low in neuroendocrine cells (Pezzati et al., 1997) and (b) free  is particularly low (3.0 μM) in acidic endosomes (Gerasimenko et al., 1998); active VAMP.Aq was located exclusively in an acidic compartment (Fig. 4 B). Simulation of the contribution of endosomal VAMP.Aq reveals that our measurements may slightly underestimate SV (by

Mutated aequorins respond well to Ca2  concentrations over the range (30–90 μM) which pertains within the secretory vesicle, as well as the rest of the secretory pathway (200–300 μM) (Montero et al., 1997; Pinton et al., 1998), without significant interference from the low pH environment. This feature of aequorin contrasts with GFP-based Ca2  probes (Miyawaki et al., 1997; Baird et al., 1999; Emmanouilidou et al., 1999), whose fluorescence is strongly reduced at acidic pHs (Miesenbock et al., 1998; Fig. 2 E). Thus, aequorin may represent the most suitable molecularly targetable Ca2  reporter presently available for the secretory vesicle interior.
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Free Ca2  concentration in the secretory vesicle lumen

Despite possessing a larger total Ca2  content (Andersson et al., 1982; Hutton et al., 1983; Nicaise et al., 1992), secretory vesicles displayed a significantly lower free  than the ER or Golgi apparatus. Our measured values for SV (50 μM) correspond fairly well to measurements using other techniques in isolated chromaffin granules (Krieger-Brauer and Gratzl, 1982; 24 μM; null point titration), respiratory tract goblet cells (24 μM; calcium orange 5 N fluorescence; Nguyen et al., 1998) and platelet -granules (12 μM; null point titration; Grinstein et al., 1983). Thus, free Ca2  represents

The identity of the Ca2  binding proteins (or other molecules) responsible for chelating free Ca2  in these vesicles is unknown. Chromogranins (Yoo and Albanesi, 1990), or the mammalian homologue of Tetrahymena thermophila granule lattice protein 1 (Grlp1) (Chilcoat et al., 1996), are each strong possibilities. In addition, Ca2  chelation by small molecules, such as ATP (Hutton et al., 1983), may also be involved. Finally, it is likely that in islet ?-cells the insulin crystal itself also participates in chelating vesicular Ca2  (Palmieri et al., 1988; see below).

Uptake of Ca2  into secretory vesicles; Q* Z) ~2 S" R3 Q
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The dense core secretory vesicle pool of islet ?-cells has previously been considered relatively inert (Howell et al., 1975; Prentki et al., 1984). We provide evidence here that net uptake of Ca2  into the dense core secretory vesicle population occurs during activated Ca2  influx (Figs. 2 and 3), and in response to a receptor agonist (Fig. 5) or to nutrients (Fig. 8). However, and as discussed below (see also Fig. 9), these measurements do not exclude the possibility that discrete vesicle pools may experience different SV changes.
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Figure 9. Scheme: redistribution of organellar Ca2  in secretory cells in response to G protein–coupled receptors (e.g., acetyl choline, AcCh) or glucose. IP3 generated in response to AcCh releases Ca2  from the endoplasmic reticulum and Golgi apparatus, leading to an increase in cytosolic  and uptake of Ca2  into dense core secretory vesicles distant from the plasma membrane (deep vesicles). Vesicular Ca2  uptake is catalyzed by an undefined Ca2 -ATPase, with properties similar to PMR1/ATP2C1 (see Discussion). Increases in blood glucose lead to (1) the uptake of the sugar via glucose transporters, (2) enhanced ATP synthesis and closure of ATP-sensitive K  channels, and (3) Ca2  influx through L-type Ca2  channels. The resultant increases in c are likely to promote net Ca2  uptake (reflecting the balance of uptake versus release) into vesicles distant from the cell surface (deep vesicles). For those vesicles (, L$ T' v0 t+ Y3 @1 y/ ^% E

We considered the possibility that the increases in SV observed upon challenge of cells with agonists (Fig. 5) or nutrients (Fig. 8) may be due in part to the activation of exocytosis, and thus the exposure of aequorin within the vesicle matrix to the extracellular Ca2  concentrations (1.5 mM). However, this phenomenon is likely to contribute negligibly to the observed signals, since only a tiny fraction of the total vesicle population (the "primed" pool) in ?-cells (5–20/13,000 per min; Rorsman, 1997) undergoes fusion, even during the maximal stimulation of exocytosis. Consistent with this, we could detect no significant net movement of VAMP.GFP fluorescence to the plasma membrane after stimulation with DHPG (Fig. 5 D) or nutrients (Fig. 8 B).

Changes in intravesicular Ca2  concentration detected with vesicle-targeted aequorin seem likely to result largely from the flux of Ca2  ions across the vesicle membrane. However, two caveats are important: (a) Ca2  release from/association with binding sites (or a "polyanionic matrix") (Nguyen et al., 1998) within the vesicle may also occur, especially if the intravesicular concentrations of other ions (e.g., K ) change; (b) the number of Ca2  ions that are transported is unknown, since this depends on the binding to intraorganellar sites. As discussed above, the present studies demonstrate that the ratio of free to bound Ca2  within the secretory vesicles is much lower than that in the ER or Golgi lumen. This would appear at first to suggest that changes in intravesicular Ca2  concentration must involve the flux of a very large number of Ca2  ions. However, an alternative possibility is that there are two (or more) pools of bound Ca2  in the secretory vesicle, one (the larger) comprising Ca2  which is tightly bound (perhaps to a structural component such as the insulin crystal) and the other a more loosely bound and readily exchangeable pool. Depletion of this latter, "labile" pool, may thus lead to a substantial decrease in the free vesicular , while involving the release into the cytosol of relatively few Ca2  ions.
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The present data suggest that Ca2  accumulation into vesicles is catalyzed at physiological c chiefly by a P-type Ca2 -ATPase. Previous studies have indicated that transport of Ca2  into the Golgi apparatus is mediated partly by a SERCA pump, and partly by another, unidentified thapsigargin-insensitive system (Pinton et al., 1998). This second ATP-dependent Ca2  uptake system may be closely related to ATP2C1 (Hu et al., 2000), the mammalian homologue of the yeast Golgi Ca2  transport ATPase, PMR1 (Sorin et al., 1997). Supporting this view, mRNA-encoding ATP2C1 is abundant in MIN6 cells, and polyclonal antibodies raised to ATP2C1 reveal a punctate pattern of intracellular staining (unpublished data). Intriguingly, although patients defective in the ATP2C1 gene develop skin lesions ("Hailey-Hailey" disease; Hu et al., 2000), it is unclear whether these individuals also tend to suffer from neuroendocrine or other disorders (e.g., diabetes mellitus).

Secretory vesicles are an IP3-insensitive, but caffeine/cADPr-sensitive Ca2  store2 o; G, b4 h( ?4 i, ^
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The present results suggest that IP3 is unlikely to stimulate the release of Ca2  from dense core secretory vesicles directly (Fig. 5).7 ]5 b' x, ?8 g, |: }; X* F# p0 j7 r
9 M* Z4 ?' }+ Q, B4 i; H# A. `
By contrast, we now show (Figs. 6 and 7) that functional RyR channels are present on the dense core vesicle membrane of MIN6 cells. Type II RyR mRNA is present in ob/ob mouse islets, mouse ?-TC3 cells (Islam et al., 1998), and in rat islets (Holz et al., 1999). RyR II protein has been detected in INS1 ?-cells (Gamberucci et al., 1999), and ryanodine binding sites revealed in human islets (Holz et al., 1999), mouse islets and MIN6 cells (Varadi and Rutter, 2001). Evidence for functional RyRs on the ER INS-1 ?-cells (Maechler et al., 1999) has also recently been provided. The present data indicate that dense core vesicles may represent a large fraction (30%) of the total RyR–accessible Ca2  pool in MIN6 ?-cells (Fig. 7 B). However, the proportion of the total vesicular Ca2  pool which is mobilizable via RyR is small. Thus, the c increases observed when the total acidic Ca2  pool was emptied with CPA, ionomycin, and monensin (Fig. 7 D) were much larger than those apparent when cells were treated with 4-CEP after CPA and ionomycin (Fig. 7 C).( P1 W" B! O) @

Role of cADPr in ?-cells

In the present studies, cADPr caused an apparent release of Ca2  from both the ER and from the secretory vesicles in permeabilized MIN6 ?-cells. Moreover, we have recently observed (Varadi and Rutter, 2001) that photolysis of caged cADPr increases c in MIN6 cells. These findings were surprising in light of earlier results using ob/ob mouse islets (Islam et al., 1993) or INS-1 ?-cells (Rutter et al., 1994), where cADPr was ineffective in permeabilized cells. However, it should be noted that the present studies may provide greater sensitivity to small Ca2  fluxes by measuring changes in  inside the secretory vesicle or ER.% k/ ]8 ]7 t5 b% X- h3 n
6 Y( S1 E: N. A" q* X; E4 q- @# r3 g
Could changes in intracellular cADPr concentration play a role in the regulation of insulin secretion as proposed by Takasawa et al. (1993)? Supporting this view, mice homozygous for inactivation of the CD38 (ADPribose cyclase) gene display impaired glucose tolerance (Kato et al., 1999). However, no change in the islet cADPr content was observed by others in response to acutely elevated glucose concentrations in vitro (Scruel et al., 1998), suggesting further studies are necessary.2 @# @% k6 m6 \* {0 W

Conclusions

Dense core secretory vesicles rapidly take up Ca2  ions via an ATP-dependent non-SERCA Ca2  pump (see Scheme, Fig. 9). Ca2  accumulated into vesicles may later be released via RyRs once these vesicles reach a high local Ca2  concentration beneath the plasma membrane (Neher, 1998) or elsewhere in the cell. This release may further contribute to the high local c in the vicinity of the submembrane vesicles (Emmanouilidou et al., 1999), and may be important to trigger exocytosis (Scheenen et al., 1998).

Materials and methods
' y# v4 \. Z# D- q. h3 W! h
Materials+ i. }5 ]) G) x6 l) r

Cell culture reagents were obtained from GIBCO BRL or Sigma-Aldrich and molecular biologicals from Roche Diagnostics. Fura-2 and cADPr were from Sigma-Aldrich and IP3 was from Molecular Probes.! r$ F( H3 T8 q4 Z7 Z

Construction of VAMP.Aq cDNA3 d/ F/ U9 K3 i+ t8 x

VAMP2 cDNA was amplified by PCR using specific primers (forward: 5'-AAG CTT ACC ATG TCG GCT ACC GCT GCC ACC-3'; reverse: 5'-AAG CTT AGT GCT GAA GTA AAC GAT GAT-3', HindIII sites are underlined). This fragment was then inserted upstream of the HindIII-EcoRI fragment encoding HA1-tagged D119-A aequorin (Kendall et al., 1992) in plasmid pBS . Chimeric VAMP.Aq cDNA was shuttled as a NotI-XhoI fragment into pcDNAI (Invitrogen; Fig. 1 A).

Construction of adenoviral VAMP.Aq and ER.Aq2 [- n8 {: I7 t

ER.Aq (Montero et al., 1995) and VAMP.Aq cDNAs were transferred into plasmid pShuttleCMV (He et al., 1998) as Kpn1-Xho1 and Xba1-HindIII fragments, respectively. The resultant constructs were recombined with vector pAdEasy-1, and viral particles were generated as described (Ainscow and Rutter, 2001).

Cell culture, transient transfection, and adenoviral infection
; m& c" }, H8 g& m
MIN6 ?-cells (Miyazaki et al., 1990; passages #20 and #30) were cultured in DME, with additions as given previously (Ainscow and Rutter, 2001). Cells cultured on 13 mm poly-L-lysine–coated coverslips were transfected with VAMP.Aq or ER.Aq plasmid DNA (1 μg) using Lipofectamine (Promega) in Optimem I serum-free medium (GIBCO BRL), or infected with adenoviruses as described (Ainscow and Rutter, 2001). The total quantity of reconstituted aequorin was higher in cells transfected with ER.Aq than VAMP.Aq cDNAs due to the inclusion in the former of intronic regulatory elements. Thus, typical integrated counts/culture (Fig. 2) after the expression of VAMP.Aq were 3 x 105 and 1.5 x 106 for conventional and adenoviral expression, respectively, and 2 x 106 and 2 x 107 for ER.Aq.! |6 i( W& f$ w/ O8 S
/ g+ R' S2 q  L) {) p4 C
measurement with expressed aequorins
! w: G) _0 A& m) h2 M# `1 M
Cells were Ca2  depleted by incubation with ionomycin (10 μM), the SERCA inhibitor CPA (10 μM; Mason et al., 1991; Pinton et al., 1998), and monensin (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 5 min at 4°C. Aequorin was reconstituted in 0.1 mM EGTA, 5 μM coelenterazine (cytosolic aequorin) or coelenterazine n (Shimomura et al., 1993; VAMP.Aq, ER.Aq) for 1–2 h at 4°C.8 U* t! P  s3 N6 ~& e
* c( r! p8 A; F5 ^+ u
Intact cells were perifused (2 mL/min-1) with KRB plus additions as stated (37°C) close to a photomultiplier tube (ThornEMI), and emitted light was collected (one data point) with a photon-counting board (ThornEMI; Cobbold and Rink, 1987). Where indicated, cells were permeabilized with digitonin (20 μM, 1 min at 22°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 Na  succinate, 20 mM Hepes, pH 7.05) at 37°C.+ o/ \0 H9 Z5 L2 v/ j- L

imaging with fura-2+ Y0 s" K1 i0 @  v  B
, I: r  I- C7 y* }4 Z4 j7 ~: j" _
Cells were loaded with 5 μM fura-2-AM (Sigma-Aldrich) for 40 min (37°C) in KRB plus 0.05% Pluronic F-127 (BASF). c was monitored in single cells at 37°C as described (Ainscow et al., 2000), using a Leica DM-IRBE inverted microscope (40x objective), and a Hamamatsu C4742-995 charge-coupled device camera driven by OpenLab (Improvision) software.9 Q  W4 B2 t, B: r0 n1 }6 w
- q2 C; I% `! }# A
Imaging acridine orange fluorescence, c5 s6 o( N* `0 |  a' m  `
$ V* x( B% M9 G
Permeabilized cells were loaded with 3 μM acridine orange (Tsuboi et al., 2000) before imaging (37°C) on a Leica DM-IRBETM inverted optics epiflurosecence microscope, at 493 ± 10 excitation, 530 nm emission (filters were from Chroma Techology).
& D9 P+ c' ?+ a! A) N
Immunocytochemistry
- @. l: c0 a) R" \, Y
48 h after transfection or infection, cells were fixed in 3.7% (vol/vol) paraformaldehyde and probed with antibodies essentially as described (Pouli et al., 1998a).: F$ S4 x9 N0 i' Y
* l2 ?4 |1 A" {
Immunoelectron microscopy

Gelatin-embedded, aldehyde-fixed cells were frozen in liquid nitrogen and ultrathin cryosections were obtained with a Reichert-Jung Ultracut E. Immunogold localization was performed as described (Confalonieri et al., 2000). Sections were immunostained either with rabbit anti-HA antibody (Sigma-Aldrich), or with a sheep anti-RyR antibody (Molecular Probes) followed by 10-nm protein A-gold. For double labeling, anti-HA–labeled sections were incubated with 1% glutaraldehyde in 0.1 M Na2PO4, pH 7.0, followed by incubation with a guinea pig antiinsulin antibody (Dako) and 15-nm protein A-gold. Sections were examined with a ZEISS EM 902 electron microscope. No labeling was detected in control sections (unpublished data).$ F& C% ~% U$ H" S

Confocal imaging$ s& F  U/ P; ^9 q) _

Cells were imaged at 37°C on a Leica SP2 confocal spectrophotometer, (488 nm excitation) using a 100x oil immersion objective., e* B0 K5 K; S" C% n' S
4 X& L+ v. G, @
Other methods and statistics
! ~$ `8 n7 Z5 ?+ e, z- Y  O
Free  and  were calculated using "METLIG" (Rutter and Denton, 1988). Data are presented as the mean ± SEM for the number of observations given, and statistical significance calculated using Student's t test.) Y5 m, A" V; q% B! ~& m
  u: y4 x$ o8 t# t
References" @: i$ X% o$ ?
9 Q0 @& G  \* R5 v; i+ ~
Ainscow, E.K., and G.A. Rutter. 2001. Mitochondrial priming modifies Ca2  oscillations and insulin secretion in pancreatic islets. Biochem. J. 353:175–180.
- }$ h8 ]9 s$ [3 K, _% @* z
Ainscow, E.K., C. Zhao, and G.A. Rutter. 2000. Acute overexpression of lactate dehydrogenase-A perturbs ?-cell mitochondrial metabolism and insulin secretion. Diabetes. 49:1149–1155.; W9 h5 Z/ ]. J* i
* M& C! F' a/ L  e
Andersson, T., P.O. Berggren, E. Gylfe, and B. Hellman. 1982. Amounts and distribution of intracellular magnesium and calcium in pancreatic ?-cells. Acta. Physiol. Scand. 114:235–241.* n+ t* n+ i9 J: S6 X  \# L
4 G: I2 k7 W; l$ f
Andrews, S.B., and T.S. Reese. 1986. Intracellular structure and elemental analysis in rapid-frozen neurons. Ann. NY Acad. Sci. 483:284–294.
1 a3 v, R  K  |8 I& J  c
Ashcroft, F.M., and S.J.M. Ashcroft. 1992. Mechanisms of insulin secretion. Insulin, Molecular Biology to Pathology. F.M. Ashcroft and S.J.H. Ashcroft, editors. Oxford University Press, Oxford, NY. 97–150.

Baird, G.S., D.A. Zacharias, and R.Y. Tsien. 1999. Circular permutation and receptor insertion within green fluorescent proteins. Proc. Natl. Acad. Sci. USA. 96:11241–11246.

Berridge, M.J. 1993. Inositol trisphosphate and calcium signalling. Nature. 361:315–325.7 f9 \8 j8 @0 V, T* g* E

Blinks, J.R. 1989. Use of calcium-regulated photoproteins as intracellular Ca2  indicators. Methods Enzymol. 172:164–203.

Blondel, O., M.M. Moody, A.M. Depaoli, A.H. Sharp, C.A. Ross, H. Swift, and G.I. Bell. 1994. Localization of inositol trisphosphate receptor subtype 3 to insulin and somatostatin secretory granules and regulation of expression in islets and insulinoma cells. Proc. Natl. Acad. Sci. USA. 91:7777–7781.' k: `: V# G3 W2 D; Q

Brini, M., M. Murgia, L. Pasti, D. Picard, T. Pozzan, and R. Rizzuto. 1993. Nuclear Ca2  concentration measured with specifically targeted recombinant aequorin. EMBO J. 12:4813–4819.8 J) ]  p( d0 j0 P: m% n2 _2 C

Bryan, J., and L. AguilarBryan. 1997. The ABCs of ATP-sensitive potassium channels: more pieces of the puzzle. Curr. Opin. Cell Biol. 9:553–559." _/ C* h+ e& F+ y7 V9 J
5 T- W2 j+ e' @1 t; H8 D+ \
Carafoli, E. 1991. Calcium pump of the plasma membrane. Physiol. Rev. 71:129–153.3 Q' Y2 \  e4 a' J; ]7 U, o) I# M, \

Chilcoat, N.D., S.M. Melia, A. Haddad, and A.P. Turkewitz. 1996. Granule lattice protein 1 (Grl1p), an acidic, calcium-binding protein in Tetrahymena thermophila dense-core secretory granules, influences granule size, shape, content organization, and release but not protein sorting or condensation. J. Cell Biol. 135:1775–1787." Z: K5 O5 J3 q6 }

Chini, E.N., and T.P. Dousa. 1996. Palmitoyl-CoA potentiates the Ca2  release elicited by cyclic ADP-ribose. Am. J. Physiol. 270:C530–C537.7 K# c  |. o6 b" m) h8 z5 ?, [6 a
! x7 [- o' n8 Y2 w- @
Cobbold, P.H., and T.J. Rink. 1987. Fluorescence and bioluminescence measurment of cytoplasmic free calcium. Biochem. J. 248:313–328.

Confalonieri, S., A.E. Salcini, C. Puri, C. Tacchetti, and P.P. Di Fiore. 2000. Tyrosine phosphorylation of Eps15 is required for ligand-regulated, but not constitutive, endocytosis. J. Cell Biol. 150:905–912.
1 X; h# r# l0 g3 K; w$ s0 `
Davidson, H.W., C.J. Rhodes, and J.C. Hutton. 1988. Intraorganellar calcium and pH control proinsulin cleavage in the pancreatic ? cell via two distinct site-specific endopeptidases. Nature. 333:93–96.% J8 W6 v+ @9 ~7 L+ E- Y, A
4 u5 M5 j( X9 g9 J
Emmanouilidou, E., A. Teschemacher, A.E. Pouli, L.I. Nicholls, E.P. Seward, and G.A. Rutter. 1999. Imaging  changes at the secretory vesicle surface with a recombinant targeted cameleon. Curr. Biol. 9:918.9 i" R; R% D* C# y
, ]7 J, r4 }& s$ f) x* B
Galione, A. 1994. Cyclic ADP-ribose, the ADP-ribosyl cyclase pathway and calcium signalling. Mol. Cell. Endocrinol. 98:125–131.
4 V4 N& ?- I7 [
Gamberucci, A., R. Fulceri, W. Pralong, G. Banhegyi, P. Marcolongo, S.L. Watkins, and A. Benedetti. 1999. Caffeine releases a glucose-primed endoplasmic reticulum Ca2  pool in the insulin secreting cell line INS-1. FEBS Lett. 446:309–312.: D. N6 t) ^% [! _% b
7 V, h, w/ v* \& {! r
Gerasimenko, O.V., J.V. Gerasimenko, P.V. Belan, and O.H. Petersen. 1996. Inositol trisphosphate and cyclic ADP-ribose-mediated release of Ca2  from single isolated pancreatic zymogen granules. Cell. 84:473–480.  y+ a6 {. c5 r2 x& q0 Y# V

Gerasimenko, J.V., A.V. Tepikin, O.H. Petersen, and O.V. Gerasimenko. 1998. Calcium uptake via endocytosis with rapid release from acidifying endosomes. Curr. Biol. 8:1335–1338.

Goncalves, P.P., S.M. Meireles, C. Gravato, and M.G. Vale. 1998. Ca2 -H  antiport activity in synaptic vesicles isolated from sheep brain cortex. Neurosci. Lett. 247:87–90.( @; t2 N7 n7 x6 u- e2 ~

Grapengiesser, E., E. Gylfe, and B. Hellman. 1988. Glucose-induced oscillations of cytoplasmic Ca2  in the pancreatic ?-cell. Biochem. Biophys. Res. Commun. 151:1299–1304.
8 T2 m9 r/ I- d! A1 i9 G9 M- k8 B
Grinstein, S., W. Furuya, M.J. Vander, and R.G. Hancock. 1983. The total and free concentrations of Ca2  and Mg2  inside platelet secretory granules. Measurements employing a novel double null point technique. J. Biol. Chem. 258:14774–14777." D) R8 ?8 M, o: x* y" l
! ^* I. }" a  D9 I2 O) `" w7 O
He, T.C., S. Zhou, L.T. da Costa, J. Yu, K.W. Kinzler, and B. Vogelstein. 1998. A simplified system for generating recombinant adenoviruses. Proc. Natl. Acad. Sci. USA. 95:2509–2514.
& n3 G9 b6 ~! W) U  |
Hellman, B., J. Sehlin, and I.B. Taljedal. 1976. Calcium and secretion: distinction between two pools of glucose-sensitive calcium in pancreatic islets. Science. 194:1421–1423./ S8 ^" l) K% @2 @) M( d
5 b! G( Q! l% f( B+ T
Holz, G.G., C.A. Leech, R.S. Heller, M. Castonguay, and J.F. Habener. 1999. cAMP-dependent mobilization of intracellular Ca2  stores by activation of ryanodine receptors in pancreatic ?-cells. A Ca2  signaling system stimulated by the insulinotropic hormone glucagon-like peptide-1-(7-37). J. Biol. Chem. 274:14147–14156.* B! y8 a0 m. r, }( C

Howell, S.L., W. Montague, and M. Tyhurst. 1975. Calcium distribution in islets of Langerhans: a study of calcium concentrations and of calcium accumulation in B cell organelles. J. Cell Sci. 19:395–409.; L2 {, l& @! T6 c. M) D

Hu, Z., J.M. Bonifas, J. Beech, G. Bench, T. Shigihara, H. Ogawa, S. Ikeda, T. Mauro, and E.H. Epstein, Jr. 2000. Mutations in ATP2C1, encoding a calcium pump, cause Hailey-Hailey disease. Nat. Genet. 24:61–65.
, f! @1 d, ^9 e
Hutton, J.C., E.J. Penn, and M. Peshavaria. 1983. Low-molecular-weight constituents of isolated insulin-secretory vesicles. Bivalent cations, adenine nucleotides and inorganic phosphate. Biochem. J. 210:297–305.

Inouye, S., M. Noguchi, Y. Sakaki, Y. Takagi, T. Miyata, S. Iwanaga, T. Miyata, and F.I. Tsuji. 1985. Cloning and sequence analysis of cDNA for the luminescent protein aequorin. Proc. Natl. Acad. Sci. USA. 82:3154–3158.$ X6 y* Y8 Z/ v& s: b7 X# U8 E
1 J' r1 x' U0 _6 R* S7 U
Islam, M.S., O. Larsson, and P.O. Berggren. 1993. Cyclic ADP-ribose and pancreatic ? cells. Science. 262:1499.

Islam, M.S., I. Leibiger, B. Leibiger, D. Rossi, V. Sorrentino, T.J. Ekstrom, H. Westerblad, F.H. Andrade, and P.O. Berggren. 1998. In situ activation of the type 2 ryanodine receptor in pancreatic ? cells requires cAMP-dependent phosphorylation. Proc. Natl. Acad. Sci. USA. 95:6145–6150.
+ s- P/ _) ^2 Q* d
Kato, I., Y. Yamamoto, M. Fujimura, N. Noguchi, S. Takasawa, and H. Okamoto. 1999. CD38 disruption impairs glucose-induced increases in cyclic ADP-ribose, i, and insulin secretion. J. Biol. Chem. 274:1869–1872.0 u9 ?' \$ r& s: a. M5 R
$ n) r0 L8 |& [. L# i+ B; t
Kendall, J.M., G. SalaNewby, V. Ghalaut, R.L. Dormer, and A.K. Campbell. 1992. Engineering the Ca2  activated photoprotein aequorin with reduced affinity for calcium. Biochem. Biophys. Res. Commun. 187:1091–1097.( [- I# H% K; G! O/ q2 S' y  H

Kennedy, H.J., A.E. Pouli, L.S. Jouaville, R. Rizzuto, and G.A. Rutter. 1999. Glucose-induced ATP microdomains in single islet ?-cells. J. Biol. Chem. 274:13281–13291.
, g8 [" B$ T2 ], a7 x
Krieger-Brauer, H., and M. Gratzl. 1982. Uptake of Ca2  by isolated secretory vesicles from adrenal medulla. Biochim. Biophys. Acta. 691:61–70.% X7 z' S' D6 ^% r4 x

Lai, F.A., H.P. Erickson, E. Rousseau, Q.Y. Liu, and G. Meissner. 1988. Purification and reconstitution of the calcium release channel from skeletal muscle. Nature. 331:315–319.
+ Y/ b2 T( ~+ j) J0 B: M3 `8 x
Maechler, P., E.D. Kennedy, E. Sebo, A. Valeva, T. Pozzan, and C.B. Wollheim. 1999. Secretagogues modulate the calcium concentration in the endoplasmic reticulum of insulin-secreting cells. Studies in aequorin-expressing intact and permeabilized INS-1 cells. J. Biol. Chem. 274:12583–12592.

Mason, M.J., C. Garcia-Rodriguez, and S. Grinstein. 1991. Coupling between intracellular Ca2  stores and the Ca2  permeability of the plasma membrane. Comparison of the effects of thapsigargin, 2,5-di-(tert-butyl)-1,4-hydroquinone, and cyclopiazonic acid in rat thymic lymphocytes. J. Biol. Chem. 266:20856–20862.
: w7 q+ R3 Z3 |  r$ K
Miesenbock, G., D.A. De Angelis, and J.E. Rothman. 1998. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature. 394:192–195.
! g9 \, e: e1 @5 A  X# e% W
Mikoshiba, K. 1997. The InsP3 receptor and intracellular Ca2  signaling. Curr. Opin. Neurobiol. 7:339–345.9 ]: Y' c3 b2 N  K( i1 ~

Miyawaki, A., J. Llopis, R. Heim, J.M. McCaffery, J.A. Adams, M. Ikura, and R.Y. Tsien. 1997. Fluorescent indicators for Ca2  based on green fluorescent proteins and calmodulin. Nature. 388:882–887.& u6 }, T* ~6 L% Q
  N6 x7 J0 y/ K0 d1 h* h* t# k
Miyazaki, J., K. Araki, E. Yamato, H. Ikegami, T. Asano, Y. Shibasaki, Y. Oka, and K. Yamamura. 1990. Establishment of a pancreatic ? cell line that retains glucose inducible insulin secretion: special reference to expression of glucose transporter isoforms. Endocrinology. 127:126–132.

Moller, J.V., B. Juul, and M. le Maire. 1996. Structural organization, ion transport, and energy transduction of P-type ATPases. Biochim. Biophys. Acta. 1286:1–51.
3 @& I" `3 W2 i
Montero, M., M. Brini, R. Marsault, J. Alvarez, R. Sitia, T. Pozzan, and R. Rizzuto. 1995. Monitoring dynamic changes in free Ca2  concentration in the endoplasmic reticulum of intact cells. EMBO J. 14:5467–5475.6 H0 M3 q5 [+ p# g
7 f' U% `: W4 A
Montero, M., J. Alvarez, W.J. Scheenen, R. Rizzuto, J. Meldolesi, and T. Pozzan. 1997. Ca2  homeostasis in the endoplasmic reticulum: coexistence of high and low  subcompartments in intact HeLa cells. J. Cell Biol. 139:601–611.( e1 k- h" o: _/ r# r
% w' e1 u5 p, {) ]4 B1 J
Neher, E. 1998. Vesicle pools and Ca2  microdomains: new tools for understanding their roles in neurotransmitter release. Neuron. 20:389–399.3 j6 v# w$ j! ]7 Y

Nguyen, T., W.C. Chin, and P. Verdugo. 1998. Role of Ca2 /K  ion exchange in intracellular storage and release of Ca2 . Nature. 395:908–912.

Nicaise, G., K. Maggio, S. Thirion, M. Horoyan, and E. Keicher. 1992. The calcium loading of secretory granules. A possible key event in stimulus-secretion coupling. Biol. Cell. 75:89–99.
0 E; ^' X4 i6 {4 B6 \
Orci, L., M. Ravazzola, M. Amherdt, O. Madsen, J.D. Vassalli, and A. Perrelet. 1985. Direct identification of prohormone conversion site in insulin-secreting cells. Cell. 42:671–681.
. ~; h, j. T5 O/ _7 n, j
Palmieri, R., R.W. Lee, and M.F. Dunn. 1988. 1H Fourier transform NMR studies of insulin: coordination of Ca2  to the Glu(B13) site drives hexamer assembly and induces a conformation change. Biochemistry. 27:3387–3397.
/ M* a/ I! x2 T
Parekh, A.B., and R. Penner. 1997. Store depletion and calcium influx. Physiol. Rev. 77:901–930.
- s$ d; j0 x0 H1 S8 P1 C' r
Pezzati, R., M. Bossi, P. Podini, J. Meldolesi, and F. Grohovaz. 1997. High-resolution calcium mapping of the endoplasmic reticulum-Golgi-exocytic membrane system. Electron energy loss imaging analysis of quick frozen-freeze dried PC12 cells. Mol. Biol. Cell. 8:1501–1512.! ^. h0 \" F# X( H
* S( Q4 |" {* D& w4 e9 i
Pinton, P., T. Pozzan, and R. Rizzuto. 1998. The Golgi apparatus is an inositol 1,4,5-trisphosphate-sensitive Ca2  store, with functional properties distinct from those of the endoplasmic reticulum. EMBO J. 17:5298–5308.

Pouli, A.E., H.J. Kennedy, J.G. Schofield, and G.A. Rutter. 1998a. Insulin targeting to the regulated secretory pathway after fusion with green fluorescent protein and firefly luciferase. Biochem. J. 331:669–675.5 {' |% r( |9 x- ^

Pouli, A.E., E. Emmanouilidou, C. Zhao, C. Wasmeier, J.C. Hutton, and G.A. Rutter. 1998b. Secretory-granule dynamics visualized in vivo with a phogrin green fluorescent protein chimaera. Biochem. J. 333:193–199.

Pozzan, T., R. Rizzuto, P. Volpe, and J. Meldolesi. 1994. Molecular and cellular physiology of intracellular calcium stores. Physiol. Rev. 74:595–636.1 I& ~7 D# z4 K5 M

Prentki, M., D. Janjic, T.J. Biden, B. Blondel, and C.B. Wollheim. 1984. Regulation of Ca2  transport by isolated organelles of a rat insulinoma. Studies with endoplasmic reticulum and secretory granules. J. Biol. Chem. 259:10118–10123.

Ravazzola, M., P.A. Halban, and L. Orci. 1996. Inositol 1,4,5-trisphosphate receptor subtype 3 in pancreatic islet cell secretory granules revisited. Proc. Natl. Acad. Sci. USA. 93:2745–2748.7 ?7 Y: k: r. y
" K! t4 L2 W3 u& a
Reetz, A., M. Solimena, M. Matteoli, F. Folli, K. Takei, and P. De Camilli. 1991. GABA and pancreatic B-cells: colocalization of glutamic acid decarboxylase (GAD) and GABA with synaptic-like microvesicles suggests their role in GABA storage and secretion. EMBO J. 10:1275–1284.0 Q" {4 a+ r, w* f+ i0 |

Rizzuto, R., M. Brini, M. Murgia, and T. Pozzan. 1993. Microdomains with high Ca2  close to IP3-sensitive channels that are sensed by neighbouring mitochondria. Science. 262:744–747.$ ~/ g( V' o  e5 C6 U, R+ e$ ]9 |
; z  i5 \$ j" Q% f0 K% r
Rizzuto, R., M. Brini, C. Bastianutto, R. Marsault, and T. Pozzan. 1995. Photoprotein-mediated measurement of calcium ion concentration in mitochondria of living cells. Methods Enzymol. 260:417–428.
2 j7 }  |( V0 Y; S& h) s
Rorsman, P. 1997. The pancreatic ?-cell as a fuel sensor: an electrophysiologist's viewpoint. Diabetologia. 40:487–495.
: v) _, w& K- u' `+ Q% n
Rutter, G.A., and R.M. Denton. 1988. Regulation of NAD -linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2  ions within toluene-permeabilized rat heart mitochondria. Interactions with regulation by adenine nucleotides and NADH/NAD  ratios. Biochem. J. 252:181–189 (erratum published 253:935).
$ B9 g" A5 N$ M( y7 Q
Rutter, G.A., J.-M. Theler, M. Murghia, C.B. Wollheim, T. Pozzan, and R. Rizzuto. 1993. Stimulated Ca2  influx raises mitochondrial free Ca2  to supramicromolar levels in a pancreatic ?-cell line: possible role in glucose and agonist-induced insulin secretion. J. Biol. Chem. 268:22385–22390.

Rutter, G.A., J.-M. Theler, G. Li, and C.B. Wollheim. 1994. Ca2  stores in insulin-secreting cells: lack of effect of cADP ribose. Cell Calcium. 16:71–80.
. T& L0 s" o3 ]9 S- `( d6 Y
Rutter, G.A., C. Fasolato, and R. Rizzuto. 1998. Calcium and organelles: a two-sided story. Biochem Biophys. Res. Commun. 253:549–557.- _0 }! r0 W' B3 N/ b# U/ P

Safayhi, H., H. Haase, U. Kramer, A. Bihlmayer, M. Roenfeldt, H.P. Ammon, M. Froschmayr, T.N. Cassidy, I. Morano, M.K. Ahlijanian, and J. Striessnig. 1997. L-type calcium channels in insulin-secreting cells: biochemical characterization and phosphorylation in RINm5F cells. Mol. Endocrinol. 11:619–629.3 ?3 [( [' D/ S/ o
( S2 U( D( }7 ~* n* K% S+ ~
Scheenen, W.M., C.B. Wollheim, T. Pozzan, and C. Fasolato. 1998. Ca2  depletion from granules inhibits exocytosis. A study with insulin-secreting cells. J. Biol. Chem. 273:19002–19008.& t* o: k! M8 j* ^1 W7 n

Scruel, O., T. Wada, K. Kontani, A. Sener, T. Katada, and W.J. Malaisse. 1998. Effects of D-glucose and starvation upon the cyclic ADP-ribose content of rat pancreatic islets. Biochem. Mol. Biol. Int. 45:783–790.! n' y, @, Z4 {6 ~. S1 O: m

Shimomura, O., B. Musicki, Y. Kishi, and S. Inouye. 1993. Light-emitting properties of recombinant semi-synthetic aequorins and recombinant fluorescein-conjugated aequorin for measuring cellular calcium. Cell Calcium. 14:373–378.

Sorin, A., G. Rosas, and R. Rao. 1997. PMR1, a Ca2 -ATPase in yeast Golgi, has properties distinct from sarco/endoplasmic reticulum and plasma membrane calcium pumps. J. Biol. Chem. 272:9895–9901.! c; M% F7 B! }+ R; b7 i  q% s

Streb, H., E. Bayerdorffer, W. Haase, R.F. Irvine, and I. Schulz. 1984. Effect of inositol-1,4,5-trisphosphate on isolated subcellular fractions of rat pancreas. J. Membr. Biol. 81:241–253.: }0 h. ^" c2 L6 g1 N
$ D0 `2 `) ~1 V8 t
Sudhof, T.C., M. Baumert, M.S. Perin, and R. Jahn. 1989. A synaptic vesicle membrane protein is conserved from mammals to Drosophila. Neuron. 2:1475–1481.

Takasawa, S., K. Nata, H. Yonekura, and H. Okamoto. 1993. Cyclic ADP-ribose in insulin secretion from pancreatic B-cells. Science. 259:370–373.4 J. C4 L9 S" {7 n$ w- n+ X
. U0 u& |; l/ C0 }3 k  X- k6 P4 u
Takeshima, H., S. Nishimura, T. Matsumoto, H. Ishida, K. Kangawa, N. Minamino, H. Matsuo, M. Ueda, M. Hanaoka, T. Hirose, et al. 1989. Primary structure and expression from complementary DNA of skeletal muscle ryanodine receptor. Nature. 339:439–445.' C4 D3 n0 ^% a& B8 R# B- D
" ?2 f+ x$ K) q& V
Thastrup, O. 1990. Role of Ca2 -ATPases in regulation of cellular Ca2  signalling, as studied with selective microsomal Ca2 -ATPase inhibitor, thapsigargin. Agents Actions. 29:8–15.$ g; a' I4 p. Q. ]0 t7 q7 s

Thirion, S., J.D. Troadec, N.B. Pivovarova, S. Pagnotta, S.B. Andrews, R.D. Leapman, and G. Nicaise. 1999. Stimulus-secretion coupling in neurohypophysial nerve endings: a role for intravesicular sodium? Proc. Natl. Acad. Sci. USA. 96:3206–3210.
$ {" T1 c; p3 {3 F% {7 r+ w
Thomas, L.S., D.E. Jane, R. Harris, and M.J. Croucher. 2000. Metabotropic glutamate autoreceptors of the mGlu(5) subtype positively modulate neuronal glutamate release in the rat forebrain in vitro. Neuropharmacology. 39:1554–1566.$ ^( O# d8 }+ x) i' I/ `
- ^* i6 B2 b6 p, g
Troadec, J.D., S. Thirion, J.P. Laugier, and G. Nicaise. 1998. Calcium-induced calcium increase in secretory vesicles of permeabilized rat neurohypophysial nerve terminals. Biol. Cell. 90:339–347.; g$ }- ^0 {* f. T

Tsuboi, T., C. Zhao, S. Terakawa, and G.A. Rutter. 2000. Simultaneous evanescent wave imaging of insulin vesicle membrane and cargo during a single exocytotic event. Curr. Biol. 10:1307–1310.; V" s2 i% E- E3 u; `3 G
  X; G1 d- H6 X) n3 {" F4 e
Varadi, A., and G.A. Rutter. 2001. Dynamic imaging of endoplasmic reticulum  in MIN6 cells using recombinant cameleons: roles of SERCA2 and ryanodine receptors. Diabetes. In press.' J; Z, Y$ V/ W- v4 L2 T

Yoo, S.H. 2000. Coupling of the IP3 receptor/Ca2  channel with Ca2  storage proteins chromogranins A and B in secretory granules. Trends Neurosci. 23:424–428.
1 I) `- o! N( q* d
Yoo, S.H., and J.P. Albanesi. 1990. Inositol 1,4,5-trisphosphate-triggered Ca2  release from bovine adrenal medullary secretory vesicles. J. Biol. Chem. 265:13446–13448.
9 f1 N! O7 O$ P$ [8 y  u
Yule, D.I., S.A. Ernst, H. Ohnishi, and R.J.H. Wojcikiewicz. 1997. Evidence that zymogen granules are not a physiologically relevant calcium pool. Defining the distribution of inositol 1,4,5-trisphosphate receptors in pancreatic acinar cells. J. Biol. Chem. 272:9093–9098.; X# Y8 a7 ]( L. Z" s( ]

Zorzato, F., E. Scutari, V. Tegazzin, E. Clementi, and S. Treves. 1993. Chlorocresol: an activator of ryanodine receptor-mediated Ca2  release. Mol. Pharmacol. 44:1192–1201.(Kathryn J. Mitchell1, Paolo Pinton2,3, A)

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