Dense core secretory vesicles revealed as a dynamic Ca(2+) store in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera

Abstract

The role of dense core secretory vesicles in the control of cytosolic-free Ca(2+) concentrations ([Ca(2+)](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 beta-cells: (a) increases in [Ca(2+)](c) cause a prompt increase in intravesicular-free Ca(2+) concentration ([Ca(2+)]SV), which is mediated by a P-type Ca(2+)-ATPase distinct from the sarco(endo) plasmic reticulum Ca(2+)-ATPase, but which may be related to the PMR1/ATP2C1 family of Ca(2+) pumps; (b) steady state Ca(2+) 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 Ca(2+); (c) inositol (1,4,5) trisphosphate has no impact on [Ca(2+)](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 [Ca(2+)](SV). Thus, secretory vesicles represent a dynamic Ca(2+) 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 Ca(2+)-induced Ca(2+) release from vesicles docked at the plasma membrane could participate in triggering exocytosis.

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.

Figure 2.

Measurement of intravesicular pH (A) and Ca ²+ 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 Prism^TM. (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.

Figure 3.

Ca ²+ 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 <10⁻⁹ M free [Ca²+] (buffered with 0.2 mM EGTA, 1 mM HEDTA; free [Mg²⁺] = 0.5 mM) and then at 400 nM free [Ca²+], as indicated. Where present (closed symbols) ATP was 1 mM. Note that accumulation of Ca²+ at zero ATP is likely to be due to synthesis of small amounts of ATP by mitochondria. (B) Dose–response for the inhibition of vesicular [Ca²+] increases by orthovanadate. Cells expressing VAMP.Aq were permeabilized and perifused at 400 nM Ca²+ in the presence of 1 mM ATP, plus the indicated concentrations of NaVO₄. The initial rates of [Ca²+]_SV increase upon the stepped increase in perifusate-free [Ca²+] from <1 to 400 nM were calculated by fitting time course data to a simple first order rate equation by nonlinear regression analysis (Microsoft Excel^TM). (C and E) After Ca²+ depletion and aequorin reconstitution, Ca²+ uptake into intact cells was initiated by replacing KRB containing 1 mM EGTA with EGTA-free KRB at 1.5 mM CaCl₂, as indicated. Closed symbols, cells were incubated with thapsigargin (TG; 1 μM) during the final 10 min of reconstitution, and the perifusion medium was supplemented with CPA (10 μM). Data are representative of three separate experiments in each case.

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 [Ca²+] was increased from <1 to 400 nM as indicated, either in the absence (A, C, E, and F, open symbols) or in the presence of the additions (closed symbols) as follows: A, 10 μM monensin; B, when indicated, 10 μM ionomycin (triangles), or ionomycin plus 10 μM monensin (circles); C, 300 nM bafilomycin; E, 1 μM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP); F, 140 mM NaCl. Trace (D) shows the effect of bafilomycin on vesicular acridine orange fluorescence in 10 individual cells imaged simultaneously (Materials and methods). Data are representative of at least three separate experiments or are the means ± SEM of three (B).

Figure 5.

Effect of IP₃ on [Ca²+]_SV and [Ca²+]_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 [Ca²+], 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 [Ca²+] <1 nM). HEDTA and CaCl₂ were added to give a calculated free [Ca²+] of 200 nM, as indicated. After the achievement of steady state [Ca²+]_SV or [Ca²+]_ER, IP₃ (5 μM) was introduced (filled symbols) as shown (B and F). Mean steady state [Ca²+]_SV and [Ca²+]_ER in three separate experiments in the presence or absence of IP₃ are shown in C and G, respectively. Double asterisk indicates P < 1% for the effect of IP₃ on [Ca²+]_ER. D shows cells cotransfected with VAMP.GFP (pH.fluorin(r); Miesenbock et al., 1998) and mGluR5 cDNAs and depleted of Ca²+, as in A. Localization of vesicles (a) after 1 h of Ca²+- depletion, (b) 120 s after readdition of CaCl₂, and (c) 60 s after DHPG addition.

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 [Ca²+] 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.

Figure 7.

Effect of caffeine and 4-CEP on cytosolic [Ca²+]. (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 [Ca²+]_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 [Ca²+]_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.

Figure 8.

Effect of nutrient secretagogues on steady state [Ca²+] 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 < 5%; double asterisk indicates P < 1% for the effect of 20 mM glucose or mixed nutrients, respectively. In B, cells expressing VAMP.GFP (pH.fluorin(e)) were Ca²+ depleted as in A, before (a and c) or after (b and d) reintroduction of CaCl₂ in the presence of 3 mM (a and b) or 30 mM (c and d) glucose. Bar, 5 μm.

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 [Ca²+] 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 [Ca²+]_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 (<0.5% of total; Rorsman, 1997) located close to the plasma membrane and primed for exocytosis (primed vesicles), larger local [Ca²+]_c increases (e.g., at the mouth of activated plasma membrane Ca²+ channels; Neher, 1998) may activate vesicular RyRs and provoke net Ca²+ release.