A Dynamic Pool of Calcium in Catecholamine Storage Vesicles

Chromaffin vesicles contain very high concentration of Ca2+ (∼20–40 mm total), compared with ∼100 nm in the cytosol. Aequorin, a jellyfish photoprotein with Ca2+-dependent luminescence, measures [Ca2+] in specific subcellular compartments wherein proteins with organelle-specific trafficking domains are fused in-frame to aequorin. Because of the presence of vesicular trafficking domain within CgA we engineered sorting of an expressed human CgA-Aequorin fusion protein (hCgA-Aeq) into the vesicle compartment as confirmed by sucrose density gradients and confocal immunofluorescent co-localization studies. hCgA-Aeq and cytoplasmic aequorin (Cyto-Aeq) luminescence displayed linear functions of [Ca2+] in vitro, over >5 log10 orders of magnitude (r > 0.99), and down to at least 10–7 m sensitivity. Calibrating the pH dependence of hCgA-Aeq luminescence allowed estimation of [Ca2+]ves at granule interior pH (∼5.5). In the cytoplasm, Cyto-Aeq accurately determined [Ca2+]cyto under both basal ([Ca2+]cyto = 130 ± 35 nm) and exocytosis-stimulated conditions, confirmed by an independent reference technique (Indo-1 fluorescence). The hCgA-Aeq chimera determined vesicular free [Ca2+]ves = 1.4 ± 0.3 μm under basal conditions indicating that >99% of granule total Ca2+ is in a “bound” state. The basal free [Ca2+]ves/[Ca2+]cyto ratio was thus ∼10.8-fold, indicating active, dynamic Ca2+ uptake from cytosol into the granules. Stimulation of exocytotic secretion revealed prompt, dynamic increases in both [Ca2+]ves and [Ca2+]cyto, and an exponential relation between the two (y = 0.99 × e(1.53x), r = 0.99), reflecting a persistent [Ca2+]ves/[Ca2+]cyto gradient, even during sharp increments of both values. Studies with inhibitors of Ca2+ translocation (Ca2+-ATPase), Na+/Ca+-exchange, Na+/H+-exchange, and vesicle acidification (H+-translocating ATPase), documented a role for these four ion transporter classes in accumulation of Ca2+ inside the vesicles.

Catecholamine storage vesicles (chromaffin granules) contain quite substantial concentrations of catecholamines (ϳ0.6 M), adenosine triphosphate (ATP, ϳ150 mM), Ca 2ϩ (ϳ20 -40 mM), and chromogranins (ϳ2-4 mM) in their soluble cores (1)(2)(3). The granule accumulation of Ca 2ϩ , at ϳ20 -40 mM total concentration, is in striking contrast to the ϳ100 nM Ca 2ϩ concentration in the cytosol, suggesting that a Ca 2ϩ concentration gradient of up to ϳ10 5 -fold might occur across the granule membrane. However, little is known about the processes that might underlie Ca 2ϩ accumulation in secretory granules, the fraction of granule Ca 2ϩ , which is free (versus bound) is not well understood, and no tools currently exist to measure [Ca 2ϩ ] in situ within chromaffin granules in living cells.
Changes in cytoplasmic [Ca 2ϩ ] can be monitored by introducing Ca 2ϩ -chelating fluorescent dyes such as Indo-1 (5,14) or Fura-2 (15,16) into the cytosol. These fluorescent dyes can quantify [Ca 2ϩ ] in the cytosol, but their intracellular diffusion precludes their use to measure [Ca 2ϩ ] in particular organelles. The jellyfish (Aequoria victoria) green fluorescent protein (GFP) 1 has been engineered to detect cytoplasmic [Ca 2ϩ ] by fluorescent resonant energy transfer (FRET) of chimeric donor/ acceptor GFP modules in response to Ca 2ϩ binding (17), but severe fluorescence intensity quenching of one or the other of the donor/acceptor GFP modules at the very acidic chromaffin granule interior pH of ϳ5.5 (18,19) precludes use of this method in the granule (17).
The jellyfish photoprotein apoaequorin (ϳ21-22 kDa) forms a complex with its cofactor coelenterazine and molecular oxygen; binding of calcium at three specific aequorin sites permits oxidation of colenterazine to coelenteramide, yielding a photon at ϳ466 nm by luminescence (20). This aequorin luminescence can be quantified by a photomultiplier tube in the dark and its strict calcium dependence has been used to measure local calcium concentration in particular subcellular depots to which aequorin has been steered by exploiting the intracellular trafficking information in appropriately selected fusion proteins (21); successful examples include targeting to endoplasmic reticulum (22), mitochondria (23), Golgi apparatus (24), or nucleus (25). Such targeted recombinant aequorins provide the most specific means of monitoring free [Ca 2ϩ ] in particular subcellular compartments.
Chromogranin A (CgA) is the major protein in the soluble core of chromaffin granules (1,3) and its N terminus has been suggested to contain the targeting signal for trafficking to chromaffin granules (26,27). Therefore, we reasoned that a CgA-Aequorin chimera would be trafficked to the chromaffin granules by using the N-terminal targeting information of CgA, and could then monitor free vesicular [Ca 2ϩ ] under basal or secretion-stimulated conditions. Such a CgA-Aequorin chimera should be useful to probe questions about chromaffin cell Ca 2ϩ that have remained elusive for decades, such as: (i) What is the free [Ca 2ϩ ] in the core of chromaffin granules? (ii) Are chromaffin granule Ca 2ϩ stores predominantly free or bound? (iii) How do chromaffin granule Ca 2ϩ stores respond to secretory stimuli? (iv) What transporters underlie the accumulation of granular Ca 2ϩ ? Our studies suggest that chromaffin granules actively accumulate Ca 2ϩ against a substantial concentration gradient (vesicle/cytosol), that Ͼ99% of Ca 2ϩ inside the vesicle remains in bound form, that vesicular [Ca 2ϩ ] changes promptly in response to secretory stimulation, and that several discrete ion transporters in the granule membrane mediate these processes.
Human Chromogranin A-Aequorin In-frame Chimera (hCgA-Aeq)-The entire human CgA (hCgA) coding DNA (open reading frame), excluding the stop codon (1371 bp), was first amplified by PCR from the pGEM-hCgA plasmid (from Lee Helman, NCI, National Institutes of Health, Ref. 28) using these forward (5Ј-CTTCGAATTCTGACCGC-CATGCGCTCCGCCGCTGTCCTG-3Ј, containing the underlined EcoRI site) and backward (5Ј-CCCGCGGTACCGTGCCCCGCCGTAGTGC-CTGCAG-3Ј, containing the underlined KpnI site) primers. The aequorin-coding region (588 bp) was also amplified by PCR from the Cyto-Aeq plasmid using these forward (5Ј-CGGGGTACCTATGAC-CAGCGAACAATACTCAGTC-3Ј, containing the underlined KpnI site) and backward (5Ј-ATAAGAATGCGGCCGC GAGTTTCTTAGGGGA-CAGCTCCACCGTA-3Ј, containing the underlined NotI site) primers. Following digestion with EcoRI and KpnI, the hCgA PCR product was subcloned between the EcoRI and KpnI sites of the eukaryotic expression vector pEGFP-N1 (containing the CMV immediate-early gene promoter; Clontech, Palo Alto, CA). The KpnI/NotI-digested aequorin PCR product was subsequently ligated between the KpnI and NotI sites of the pEGFP-N1/hCgA plasmid, thereby replacing EGFP, resulting in the hCgA-Aeq plasmid wherein the human CMV promoter drives expression of the fused/in-frame hCgA-Aequorin chimera, with hCgA at the N terminus and aequorin at the C terminus. The correct in-frame fusion of aequorin at the downstream end of hCgA (hCgA-Aeq) was confirmed by sequencing the plasmid construct. Plasmids were grown in Escherichia coli, and supercoiled plasmid DNA molecules were purified using Qiagen columns (Qiagen Inc., Chatsworth, CA).

Subcellular Localization of the Vesicular hCgA-Aeq Chimera:
Sucrose Gradient Fractionation and Immunoblotting PC12 cells grown in 10-cm plates were transfected with hCgA-Aeq. 16 h after transfection, cells were incubated with 1 Ci of [ 3 H]-norepinephrine (PerkinElmer Life Sciences, Inc.) and 5 M coelenterazine (Molecular Probes, Eugene, OR) for 3 h at 37°C. Cells were then subjected to three washes with DME/high glucose medium and two washes with PBS, each for 5 min at 37°C. Cells from three plates were pooled in 3 ml of ice-cold 10 mM HEPES, pH 7.4, 1 mM dithiothreitol, 0.3 M sucrose, and briefly (10 -15 s) homogenized by a Tissuemizer (Tekmar, Cincinnati, OH) set at output 50%. The suspension was then layered over a 40 ml continuous sucrose density gradient (0.3-2.0 M) and centrifuged at 30,000 rpm, 4°C for 2.5 h (30). 24 samples from different fractions of the sucrose gradient were collected and each fraction was subjected to: (i) [ 3 H]norepinephrine assay (by liquid scintillation counting), (ii) determination of sucrose concentration (by refractometry), and (iii) measurement of aequorin luminescence (to 300 l of a gradient fraction, 100 l of 10 mM HEPES, pH 7.4, 400 mM KCl, 40 mM CaCl 2 , 0.4% Triton X-100 was added by injection, and luminescence was recorded) by a luminometer (Model LB 953, EG&G Berthold. 10 l of sucrose density gradient fractions were electrophoresed on a 9% SDS-polyacrylamide gel, and the proteins were blotted onto a nitrocellulose membrane. The membrane was incubated overnight at 4°C with a rat anti-jellyfish aequorin primary antibody (a gift from Larry Ruben, Dallas, TX; 1:500) and for 1 h at room temperature with goat anti-rat IgG conjugated to horseradish peroxidase (1:5000), followed by chemiluminescent detection using an ECL system (Pierce).

Subcellular Localization: Immunolocalization of the Vesicular hCgA-Aeq Chimera
Neurite-differentiated (NGF; 100 ng/ml for 2 days) (31) and control PC12 cells grown on 4 well slides (LabTek, Chamber slides) were transfected with hCgA-Aeq. Neurite-differentiated transfected cells were maintained in NGF (100 ng/ml). The medium was removed 16 h after transfection, washed twice with PBS, and fixed with 2% paraformaldehyde for 1 h at room temperature. PBS-washed cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min and incubated with blocking buffer (0.1% bovine serum albumin, 8 mM glycine in PBS) for 15 min at room temperature. This was followed by incubation with rat anti-jellyfish aequorin primary antibody (1:100) and/or mouse antidopamine ␤-hydroxylase (DBH; 1:150) (BD Pharmingen, San Diego, CA) overnight at 4°C. Cells were washed twice with PBS (5 min each) and incubated for 1 h at room temperature either with Alexa Fluor® 594 (red) conjugated to goat anti-rat IgG (Molecular Probes, 1:150) to detect hCgA-Aeq, and/or Alexa Fluor® 488 (green) conjugated goat anti-mouse IgG (Molecular Probes, 1:150) to detect DBH. The slides were washed thrice with PBS (10 min each), mounted with ProLong® antifade reagent (Molecular Probes), kept at room temperature for 2 h and stored at 4°C before viewing under a Zeiss LSM 510 laser scanning confocal microscope.

Secretagogue-stimulated Release of Norepinephrine
Norepinephrine secretion was assayed as described previously (5). In brief, PC12 cells labeled with 1 Ci of L-[ 3 H]norepinephrine for 3 h at 37°C, washed twice with release buffer (150 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 10 mM HEPES pH 7), and incubated for 20 min (at 37 or 8°C) in release buffer with or without a secretagogue (60 M nicotine, 100 M ATP, 1 M ionomycin, 2 mM BaCl 2 or 55 mM KCl). In depolarization experiments, the secretion buffer contained 55 mM KCl and 100 mM NaCl. Precooled (8°C) release buffer with or without secretagogue was used in low temperature experiments. In some experiments, the release buffer was supplemented with 400 mM sucrose. Supernatant was collected 20 min after treatment, and then the cells were exposed to lysis buffer (150 mM NaCl, 5 mM KCl, 2 mM CaCl 2, 10 mM HEPES pH 7, 0.1% (v/v) Triton X-100). Release medium and cell lysates were assayed for [ 3 H]norepinephrine by liquid scintillation counting, and results were expressed as % secretion: (amount released/(amount released ϩ amount in cell lysate)) ϫ 100. Net secretion is secretagogue-stimulated release minus basal release.
The cells received secretagogues (60 M nicotine, 60 mM KCl, 100 M ATP, or 1 M ionomycin) in KRB or calcium-free KRB through softwarecontrolled injector ports. For measurement of aequorin luminescence under basal conditions KRB only was injected to the cell suspension.
In experiments to inhibit exocytosis by low temperature, coelenterazine-reconstituted hCgA-Aeq-expressing PC12 cells were precooled to 8°C before secretagogue exposure. In experiments to inhibit exocytosis by hyperosmolarity, hCgA-Aeq-expressing PC12 cells were exposed to secretagogues in KRB buffer supplemented with 400 mM sucrose. Luminescence before and after injection of a secretagogue was recorded by the PMT and saved in a Kaleidagraph spreadsheet. At the conclusion of the experiment, activated (unoxidized) aequorin still remaining inside the cells was determined by cell lysis in the presence of 10 mM CaCl 2 and 0.05% Triton X-100.
For in vitro calibration of aequorin luminescence with respect to free [Ca 2ϩ ], Cyto-Aeq-or hCgA-Aeq-expressing PC12 cells were reconstituted with coelenterazine (5 M) in calcium-free KRB, washed with calcium-free KRB, and lysed in calcium-free KRB with 0.05% Triton X-100. The cell lysates (100 l) were mixed with 100 l of EGTAbuffered Ca 2ϩ solutions (using the on-line MaxChelator program www.stanford.edu/ϳcpatton/maxc.html), to achieve a range of specific, free [Ca 2ϩ ] in the final solution), followed by recording of the light emission. The rate of light emission (photon counts per second) was recorded just after addition of a Ca 2ϩ buffer (L). After 1 min, the cell lysate was exposed to a saturating calcium solution (10 mM CaCl 2 in 0.05% Triton X-100) and the recording was continued until the light emission returned to baseline. The luminescence signal at each point of the experiment was normalized by L max, the integral of photon counts from that point to the end of the experiment (32).

Independent Measurement of Cytosolic [Ca 2ϩ ] by Indo-1 Fluorescence
Indo-1/AM loading solution was freshly prepared as follows: A 50-g aliquot of Indo-1/AM (Molecular Probes) was mixed with 1.5 l of a 20% (w/v) Pluronic® F127 solution (low toxicity dispersing agent; Molecular Probes) and 14.5 l of Me 2 SO, yielding a 3 mM solution of Indo-1/AM. This was diluted 1:1000 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. To load the cells with Indo-1, PC12 cells grown on LabTek 4-chamber slides having 1 German borosilicate cover glass floor (Nunc) were incubated with 500 l of loading solution per well at 37°C in a 5% CO 2 atmosphere for 30 min. After rinsing the cells with KRB, the LabTek chamber was mounted on the stage of a Nikon Diaphot microscope that was interfaced to a Solamere Technologies dual emission photometry system. Excitation was provided by a 100 W mercury lamp and an Indo-1 excitation filter set from Chroma, which features an excitation filter centered at ex ϭ 365 nm with a band-pass of 5 nm. A 380 nm dichroic long-pass beam splitter was used under the nose cone of the microscope to separate the excitation and emission fluorescence light pathways. The emission path was split and monitored using separate photomultiplier tubes with band pass emission filters centered at em ϭ 405/30 nm or em ϭ 485/25, for channel 1 or channel 2, respectively. Data were collected at room temperature at an acquisition rate of 4 Hz with equal gain settings on each channel via a MacLab interface and Power MacIntosh computer using Chart 3.5 software.
For typical experimental runs, the cells were placed in 300 l of buffer per well. Basal recordings of Indo-1 fluorescence were made from the field of view afforded by a 100ϫ oil-immersion objective. The fields were ϳ50% confluent and the signals corresponded to data obtained simultaneously from several cells (ϳ20 to 40 per field). Chemicals (e.g. nicotine, ATP) were typically added as 300 l of a 2ϫ stock solution. Following determinations of basal and secretagogue-induced changes in Indo-1 fluorescence, the cells were placed in buffer containing no added calcium with 3 mM EGTA. After ϳ3 min, this was replaced with a buffer containing no added calcium, with 3 mM EGTA and 10 M ionomycin. Stable R min values (Indo-1 emission signals at 405 and 485 nm) were obtained within 20 min. During the equilibration with 3 mM EGTA and ionomycin, excitation was intermittent to prevent excessive photobleaching. This buffer was then replaced by a buffer containing 10 mM calcium and 10 M ionomycin to achieve the R max (equilibration with this buffer was typically achieved within 2 min). Following determination of R min and R max , the buffer was replaced by a buffer containing 10 mM MnCl 2 , which quenches all Indo-1 fluorescence, resulting in a determination of autofluorescence and background illumination for the field; photomultiplier outputs in the presence of MnCl 2 were subtracted from the previously obtained data for each run, post-hoc.
The fluorescence emission (F 405 /F 485 ) Indo-1 ratio was calibrated to [Ca 2ϩ ] cyto by the equation:

Data Presentation and Statistics
Data are reported as the mean value Ϯ one S.E. One representative result from at least three independent experiments is shown. When only two conditions (e.g. control and experimental) were compared, the data were evaluated by unpaired Student's t tests. Statistical significance was concluded at p Ͻ 0.05. Statistics were computed with the programs InStat (GraphPad Software; San Diego, CA), or Kaleidagraph (Synergy/Abelbeck Software; Reading, PA).

Construction and Subcellular Localization of the hCgA-Aeq
Chimera-The structure of the hCgA-Aeq chimera is shown in Fig. 1A. The construct encodes full-length hCgA and aequorin proteins such that the photoprotein aequorin is fused in-frame at the C terminus of hCgA. The human CMV promoter drives expression of the hCgA-Aeq chimeric protein.
To detect the subcellular localization of the chimera, sucrose density gradient centrifugation was performed on homogenates of PC12 cells transfected with hCgA-Aeq and labeled with [ 3 H]norepinephrine. After centrifugation, we measured sucrose concentration, norepinephrine radioactivity, and aequorin luminescence in different fractions of the gradient. [ 3 H]norepinephrine and aequorin luminescence co-localized in the same gradient fractions with peaks at 1.2 M sucrose ( Fig. 1B), suggesting that the hCgA-Aeq chimera is trafficked into chromaffin granules in PC12 cells. Consistent with this observation, SDS-PAGE followed by anti-aequorin immunoblotting of the gradient fractions also detected a protein of ϳ80 kDa (approximately the combined molecular weights of CgA, at ϳ49 kDa, and aequorin, at ϳ21-22 kDa) peaking in the same gradient fractions at ϳ1.2 M sucrose, as shown in Fig. 1B.
To confirm appropriate targeting of the hCgA-Aeq fusion protein to chromaffin granules, confocal immunofluorescence microscopy was performed. The hCgA-Aeq fusion protein was visualized with red Alexa Fluor® 594 whereas DBH (chosen as a marker protein of the chromaffin granules) was detected with green Alexa Fluor® 488. The hCgA-Aeq chimera displayed a peripheral pattern typical of secretory granules ( Fig. 2A, red), and co-localized with the distribution pattern of DBH (Fig. 2B,  green); indeed, simultaneous visualization of hCgA-Aeq (red) and DBH (green) yielded nearly complete overlap co-fluorescence (Fig. 2C, yellow), suggesting co-localization in the same subcellular particle: the chromaffin granule. The immuno-colocalization experiment was also performed with neuron-differentiated PC12 cells; the results demonstrate trafficking of the hCgA-Aeq chimera to DBH-containing granules at the neurite termini (Fig. 2, D-F; arrows).  KCl, pH 7.0), and plotted the log(L/L max ) versus log[Ca 2ϩ ], where L is the rate of light emission just after addition of a particular calcium buffer, and L max is total remaining light emission at that moment. The calcium response curves are shown in Fig. 3, A and B. For both Cyto-Aeq and hCgA-Aeq, log 10 (L/L max ) linearly predicted log 10 [Ca 2ϩ ] over several orders of magnitude, and down to as low as 10 Ϫ7 M [Ca 2ϩ ].

Calibration of Aequorin Luminescence with [Ca 2ϩ ] in Vitro-
Because the chromaffin granule interior is acidified to pH ϳ5.5 (18,19), we studied whether aequorin luminescence is influenced by pH, using a series of buffers with different pH values (pH ϭ 5.42-9.40) but a fixed amount of calcium (10 mM), and recombinant hCgA-Aeq. We found that the L/L max values at pH 5.42 were 3.3-fold lower than those at pH 7.2 (Fig. 3C). Therefore, to estimate [Ca 2ϩ ] at pH ϭ 5.5 (approximating the chromaffin granule interior), in vivo L/L max values for transfected/expressed hCgA-Aeq were multiplied by the factor 3.3. In vivo L/L max values for transfected/expressed Cyto-Aeq were not, of course, so corrected, since physiological cytoplasmic pH is typically ϳ7.0 -7.4.
Based on the [Ca 2ϩ ] (Fig. 3, A and B) and pH (Fig. 3C , where x ϭ log 10 (L/L max ); and for the vesicular free calcium in molarity, Comparison of the Cytosolic [Ca 2ϩ ] Obtained from Cyto-Aeq Luminescence to an Independent Method: Indo-1 Fluorescence-To assure that the equations employed to convert aequorin luminescence to [Ca 2ϩ ] provide reasonable estimates of free calcium concentration, PC12 cells (same passage) were used to estimate cytoplasmic calcium in two independent ways: by measuring Indo-1 fluorescence (Fig. 4A) as well as aequorin luminescence (Fig. 4C) Table I) as obtained using Cyto-Aeq luminescence.
We also measured the [Ca 2ϩ ] cyto in the same batch of live PC12 cells after stimulation with a purinergic P 2x agonist (100 M ATP) (5) or a nicotinic cholinergic agonist (60 M nicotine). After ATP stimulation, the cytosolic [Ca 2ϩ ] increased to ϳ1.3 M (calculated from Indo-1 fluorescence) or ϳ1.4 M (calculated from aequorin luminescence) (Fig. 4). Similarly, both techniques could detect an elevation of cytosolic [Ca 2ϩ ] to ϳ400 nM after nicotinic stimulation (data not shown). Thus, both basal and peak [Ca 2ϩ ] cyto values obtained by two different reagents are quite close, suggesting that the aequorin technique bears substantial fidelity to a well established technique (Indo-1 fluorescence) in the cytosol.
Free [Ca 2ϩ ] in Chromaffin Granules Estimated by hCgA-Aeq Luminescence in Live PC12 Cells-To estimate free intragranular [Ca 2ϩ ], PC12 cells were transfected with the plasmid hCgA-Aeq, aequorin was reconstituted with coelenterazine 24 h after transfection, and luminescence was recorded continuously in the live cells (Fig. 5, A and C), with algebraic conversion of luminescence to an estimate of [Ca 2ϩ ] ves (Fig. 5, [Ca 2ϩ ] ves is substantially higher than [Ca 2ϩ ] cyto (Table I) How might chromaffin granule interior pH influence our results? Although chromaffin granule pH is reportedly ϳ5.5 (18,19), we did not directly estimate granule pH in these studies. The pH dependence of hCgA-Aeq luminescence (   (Fig. 8A). The absolute increments (M, stimulated Ϫ basal) in local [Ca 2ϩ ] during secretion (Table I) (Table I).
These results demonstrate that the calcium pools in both the cytosol and chromaffin granules are dynamic in nature, responding to secretory stimuli; importantly, during secretion there is a prompt and substantial influx of calcium from the cytosol into the granules.
In another set of experiments, after aequorin reconstitution,

FIG. 5. Measurement of free calcium concentration inside the chromaffin granules by the hCgA-Aeq chimeric protein.
PC12 cells were transiently transfected with hCgA-Aeq, aequorin was reconstituted by incubation with coelenterazine, cells were suspended in KRB, luminescence was monitored in a luminometer, and the luminescence values were converted to vesicular free calcium concentration as detailed under "Materials and Methods." A, aequorin luminescence profile of PC12 cells before and after stimulation with nicotine (60 M) followed by cell lysis. The inset shows an increase in the hCgA-Aeq luminescence upon addition of nicotine with an expanded scale. B, calculated vesicular free calcium concentration before and after nicotinic stimulation. C, aequorin luminescence profile of PC12 cells before and after stimulation with ATP (100 M) followed by cell lysis. D, calculated vesicular free calcium concentration before and after ATP stimulation. PC12 cells transfected with Cyto-Aeq and hCgA-Aeq were subjected to secretory stimulation in the absence of extracellular free CaCl 2 . No change in the [Ca 2ϩ ] cyto or [Ca 2ϩ ] ves was observed after injection of the secretagogues (data not shown); thus, an influx of extracellular calcium seems to be the ultimate source for the increments of both cytoplasmic and vesicular calcium during exocytosis.  (Figs. 9  and 10). The suggestion is that calcium is accumulated from the extracellular space into cytosol and vesicles even in the basal (unstimulated) state.

Effect of Blockade of Sarcoendoplasmic Reticulum Ca 2ϩtranslocating ATPases (SERCAs) on Cytosolic and Vesicular
Free [Ca 2ϩ ]-Since basal free [Ca 2ϩ ] in the chromaffin vesicle was substantially (ϳ10.8-fold) higher than that in the cytosol (Figs. 4 and 5), we explored the role in vesicular calcium accumulation by a Ca 2ϩ -translocating ATPase (SERCA pump) on the vesicle membrane, in analogy with SERCA accumulation of calcium in other organelles, such as endoplasmic reticulum (22). We therefore tested the effect of the SERCA inhibitor thapsigargin on cytosolic and vesicular free [Ca 2ϩ ]. PC12 cells transfected with Cyto-Aeq or hCgA-Aeq were coelenterazinetreated for aequorin reconstitution, washed, suspended in calcium-free KRB, and incubated with mock stimulation versus 10 M thapsigargin for 20 min at room temperature, followed by injection of KRB with calcium (final concentration 2 mM). Transient Ca 2ϩ uptake into the chromaffin vesicle was significantly reduced by SERCA inhibition: peak free [Ca 2ϩ ] ves diminished from 5. ϳ36.3% decrement upon SERCA inhibition (Fig. 9A). On the other hand, the [Ca 2ϩ ] cyto remained almost unchanged (Ͻ10% inhibition) after treatment with thapsigargin (Fig. 9B). As a control, injection of KRB without calcium had no effect on either cytoplasmic or vesicular calcium concentrations (data not shown). Thus, a SERCA is likely to contribute specifically to calcium accumulation by chromaffin vesicles.
Probing the Role of a Chromaffin Granule Na ϩ /Ca 2ϩ Exchanger (NCX) in Vesicular Calcium Storage-In addition to the plasma membrane, chromaffin granule membranes are also reported to have transmembrane Na ϩ /Ca 2ϩ exchanger (NCX; Na ϩ /Ca 2ϩ antiporter or countertransporter) isoforms (37,38). To probe the role of NCX, we used the NCX inhibitor KB-R7943, which potently and selectively blocks calcium influx (39). We treated hCgA-Aeq or Cyto-Aeq transfected (aequorinreconstituted) PC12 cells with KB-R7943 (10 M) in calciumfree KRB for 20 min prior to injection of calcium (2 mM final concentration). We found that transient Ca 2ϩ influx into chromaffin granules was inhibited by ϳ34.5%, such that peak [Ca 2ϩ ] ves decreased from 5.8 Ϯ 0.3 M to 3.8 Ϯ 0.2 (n ϭ 4 experiments) M in the presence of KB-R7943 (Fig. 9C). The [Ca 2ϩ ] cyto peak increment also decreased, from 1. (n ϭ 4 experiments; Fig. 9D). Thus, NCXs seem to mediate calcium influx from the extracellular space in the basal state, ultimately contributing to calcium pools in both the vesicles and the cytoplasm.
Effect of Alkalinization of the Vesicle Core on Cytosolic and Vesicular Free [Ca 2ϩ ]-Because the chromaffin vesicle is an acidic (pH ϳ5.5; (18,19)) compartment, and the low vesicular pH seems to be crucial for vesicular accumulation of cations such as catecholamines (2), we questioned whether there was also a role of pH in control of vesicle [Ca 2ϩ ]. We probed the role of pH by vesicular alkalinization (with the weak bases NH 4 Cl or tyramine), as well as inhibition of the H ϩ -translocating ATPase by the inhibitor bafilomycin A1 (40).
It should be noted that in these experiments with alkalinizing agents, we used the same algebraic equation employed for estimation of [Ca 2ϩ ] ves at pH 5.5 (viz. [Ca 2ϩ ] ves ϭ ͌(10 [y Ϫ 8.6] ), where y ϭ log 10 (3.3 ϫ L/L max ) because of the lack of our knowledge on the exact pH upon vesicular alkalinization. However, our pH response curve (Fig. 3C) suggests that the calculated [Ca 2ϩ ] ves values after vesicular alkalinization are overestimated because the multiplication factor at pH Ͼ5.5 should then be Ͻ3.3. Thus, the limitation of estimating the exact pH upon vesicular alkalinization does not qualitatively affect our finding of lowering of [Ca 2ϩ ] ves under such conditions.
Role of a Na ϩ /H ϩ Exchanger (NHE) in Granular Ca 2ϩ Uptake-Because vesicular pH is important in calcium accumulation (Fig. 10, A and B), and because H ϩ may enter or leave chromaffin granules via a Na ϩ /H ϩ antiporter (41,42), we evaluated the possibility that such NHEs might influence calcium accumulation by vesicles. We probed involvement of NHEs using specific inhibitors (41,42), either the weak inhibitor amiloride (1 mM) or the more potent inhibitor ethyl isopropyl amiloride (10 M). After aequorin reconstitution in calcium-free KRB, hCgA-Aeq-or Cyto-Aeq-transfected PC12 cells were incubated with amiloride for 20 min, followed by injection of 2 mM calcium-containing KRB. In the presence of amiloride, granular Ca 2ϩ uptake was inhibited such that the [Ca 2ϩ ] ves peak decreased by ϳ19.0%, from 5.8 Ϯ 0.3 M to 4.7 Ϯ 0.1 M (n ϭ 4 experiments; Fig. 10C). The more potent amiloride derivative ethyl-isopropyl amiloride caused more pronounced inhibition of vesicular Ca 2ϩ uptake, decreasing peak [Ca 2ϩ ] ves by ϳ34.5% to 3.8 Ϯ 0.3 M (n ϭ 4 experiments; Fig. 10C). However, cytosolic [Ca 2ϩ ] remained unchanged after treatment with either amiloride or ethyl-isopropyl amiloride (data not shown). These results suggest that a vesicular Na ϩ /H ϩ antiporter assists in accumulation of [Ca 2ϩ ] into the vesicle core, a conclusion in accord with studies in chromaffin granule "ghosts" (42).
Are plasma membrane), at the same time leaving the initial stages in the secretory signaling cascade untouched. We utilized two methods to block the final stage of exocytosis: hyperosmolarity or low temperature (43).
To inhibit exocytosis by hyperosmolarity, PC12 secretion was triggered in Ca 2ϩ -containing release buffer supplemented with 400 mM sucrose (43). Such extracellular hyperosmolarity led to substantial decreases in stimulus-triggered net [ 3 H]norepinephrine secretion: by 60% for nicotine, 53% for ATP, 70% for KCl, 47% for ionomycin, or 58% for BaCl 2 (Fig. 11A). The same batch of PC12 cells was transfected with hCgA-Aeq and after aequorin reconstitution the cells were suspended in KRB versus the hyperosmolar buffer (KRB with calcium, plus 400 mM sucrose), followed by determination of the in vivo hCgA-Aeq luminescence profile before and after exocytotic secretory stimulation (injection of 100 M ATP). During exocytosis, the increment in CgA-Aeq luminescence was not affected by hyperosmolarity of the extracellular buffer (Fig. 11C), suggesting that secretion-induced changes in granular free [Ca 2ϩ ] were not simply due to exocytotic exposure of the chromaffin granule interior to the extracellular space, and that the increment in [Ca 2ϩ ] ves was likely seen in all chromaffin granules, and not just those docked at the plasma membrane awaiting exocytosis.
To inhibit the final stages of exocytosis by hypothermia, we lowered the temperature of the experiment to 8°C. Previous studies reported blockade of exocytosis at 12°C (43). However, we found more effective blockade of exocytosis at 8°C as compared with 12°C. Secretion from PC12 cells was substantially reduced at 8°C as compared with that at 37°C (by 53% for nicotine, 72% for ATP, 67% for KCl, 64% for ionomycin, or 68% for BaCl 2 ; Fig. 11B). After aequorin reconstitution, hCgA-Aeq transfected PC12 cells were suspended in KRB with calcium and cooled to 8°C, followed by stimulation with 100 M ATP. No diminution of hCgA-Aeq peak luminescence during secretory stimulation was observed during hypothermic blockade of terminal exocytosis (Fig. 11D), once again suggesting that the increment in vesicular calcium concentration during secretion was not simply the result of exocytotic exposure of hCgA-Aeq to the extracellular space.

DISCUSSION
Trafficking the hCgA-Aeq Chimera into Chromaffin Granules-The chromaffin granule Ca 2ϩ pool has been suggested to play important roles in several cellular events (44,45), but no reagent to monitor local [Ca 2ϩ ] in real time in this acidic compartment in vivo has heretofore been developed. However, in the recent years, the jellyfish (Aequorea victoria) luminescent photoprotein aequorin has been used to measure free [Ca 2ϩ ] in particular subcellular locations in living cells (21,22,46).
Since CgA is the major soluble protein in the interior of chromaffin granules, we reasoned that an expressed hCgA-Aeq chimera would be trafficked into chromaffin granules using CgA targeting signal(s): the well-characterized N-terminal hydrophobic signal peptide, plus additional sequences within the N-terminal domain of the mature protein, which steer it into the regulated secretory pathway (26,27). Indeed, when PC12 cells were transfected with our expression plasmid hCgA-Aeq, FIG. 9. Effect of inhibition of Ca 2؉ transporters on the cytosolic and vesicular calcium uptake. PC12 cells were transfected with Cyto-Aeq or hCgA-Aeq, and aequorin was reconstituted by incubation with coelenterazine in the absence of extracellular Ca 2ϩ (with 1 mM EGTA), cells were suspended in calciumfree KRB and incubated with mock solution versus a transport inhibitor (the SERCA inhibitor thapsigargin [10 M], or the NCX inhibitor KB-R7943 [10 M]) for 20 min, followed by injection of calciumfree KRB or regular KRB containing 2 mM Ca 2ϩ . Luminescence was monitored, and photon counts were converted to cytosolic or vesicular free [Ca 2ϩ ] as detailed under "Materials and Methods." Decrements in vesicular Ca 2ϩ uptake were noted after the SERCA inhibitor thapsigargin (A), or the NCX inhibitor KB-R7943 (C). Inhibition of cytosolic Ca 2ϩ uptake was also noted after SERCA inhibition by thapsigargin (B), or NCX inhibition by KB-R7943 (D). the chimeric protein was correctly trafficked into chromaffin granules, as evidenced by sucrose-density gradient co-sedimentation with catecholamines ( Fig. 1), as well as by confocal immunofluorescence microscopic co-localization with the chromaffin granule reference protein dopamine ␤-hydroxylase (Fig. 2).
Choice of Chimera and Validation of Aequorin Method-Since CgA binds with calcium at high capacity (2) it is most likely that the hCgA-Aeq chimeric protein would bind to calcium. In that case, the hCgA-Aeq may act as a low affinity aequorin probe. Indeed, the L/L max values and hence log (L/ L max ) values for hCgA-Aeq over a broad range of [Ca 2ϩ ] are substantially lower than those for the wild-type Cyto-Aeq (Fig.  3, A and B). Thus, the hCgA-Aeq probe acting like a low affinity aequorin may be advantageous for monitoring [Ca 2ϩ ] in the chromaffin granules.
Aequorin luminescence in vitro was a linear estimator of free [Ca 2ϩ ] over several orders of magnitude (Fig. 3), and in the cytosol in vivo free [Ca 2ϩ ] ([Ca 2ϩ ] cyto ) estimates by aequorin luminescence paralleled those obtained by the fluorescent dye Indo-1 (Fig. 4); thus, the aequorin method was validated by an independent criterion. Free versus Bound Ca 2ϩ in Vesicles-We observed that basal free Ca 2ϩ concentration in chromaffin granules in the PC12 cells was 1.4 Ϯ 0.3 M (Figs. 4, 5, and 7). This figure represents only ϳ0.004 -0.007% of the total granular Ca 2ϩ concentration, if the total calcium is estimated to be ϳ20 -40 mM (2). Thus, the great majority (Ͼ99.9%) of Ca 2ϩ within vesicles would appear to be bound, and thus inaccessible to the local hCgA-Aeq photoprobe for free [Ca 2ϩ ] ves . Haigh et al. (41) also investigated Ca 2ϩ binding within the chromaffin granule matrix, and concluded that only ϳ0.03% of granule Ca 2ϩ is free (unbound). How is calcium sequestered within the granule core? Our previous in vitro studies indicate that CgA itself binds Ca 2ϩ electrostatically, at low affinity (K d ϭ 1.3 ϫ 10 Ϫ4 M) though high capacity (17 mol Ca 2ϩ /mol CgA), and might account for binding of as much as ϳ94% of Ca 2ϩ within the granule core, at the very high prevailing local CgA concentration (2). ATP is also present at substantial concentrations (ϳ150 mM) within chromaffin granules (2); considering that the formation (stability) constants for association of Ca 2ϩ with triphosphate are k 1 ϭ 10 5.8 and k 2 ϭ 10 3.7 (47) active calcium accumulation by this organelle.

Estimation of Free [Ca 2ϩ ] in Chromaffin Granules within
The Chromaffin Granule Ca 2ϩ Pool Is Dynamic during Secretion-We observed that free [Ca 2ϩ ] in chromaffin granules increased sharply along with an increase of free [Ca 2ϩ ] in the cytosol, upon stimulation of the cells with exocytotic secretagogues such as nicotine, ATP, KCl, or ionomycin in the presence of extracellular Ca 2ϩ (Fig. 8). However, in the absence of extracellular calcium there was no change in granular or cytosolic free [Ca 2ϩ ] after challenge with these agonists (data not shown), suggesting that an influx of Ca 2ϩ from the extracellular medium first into the cytosol and in turn from the cytosol into the granules is required for the secretion-associated granular Ca 2ϩ increment. Thus, chromaffin granules seem to remove Ca 2ϩ from the cytosol during secretion, rather than releasing Ca 2ϩ into the cytosol during this process. A modest general decline in the vesicle/cytosol [Ca 2ϩ ] ratios during secretion (from 10.8 down to 3.95 to 6.71; Table I 7). B, hCgA-Aeq-expressing PC12 cells labeled with L-[ 3 H]norepinephrine were incubated at 37°C or exocytosis-inhibiting low temperature (8°C) for 20 min with or without a secretagogue in regular secretion buffer. Secretion buffer for experiments involving KCl as secretagogue contained 100 mM NaCl to maintain isotonicity. After 20 min, secretion was terminated by aspirating the secretion buffer, and lysing cells into 150 mM NaCl, 5 mM KCl, 2 mM CaCl 2, 10 mM HEPES pH 7, 0.1% (v/v) Triton X-100. Secretion medium and cell lysates were assayed for [ 3 H]norepinephrine secretion by liquid scintillation counting, and results were expressed as % secretion as detailed under "Materials and Methods." Bottom panels, C, PC12 cells expressing hCgA-Aeq were aequorin-reconstituted and suspended in KRB (125 mM NaCl, 5 mM KCl, 1 mM Na 3 PO 4 , 1 mM MgSO 4 , 2 mM CaCl 2 , 5.5 mM glucose, 20 mM HEPES, pH 7.4) or KRB supplemented with exocytosis-inhibiting 400 mM sucrose followed by injection of ATP (100 M). D, PC12 cells expressing hCgA-Aeq were aequorin-reconstituted and suspended in KRB and were incubated at 37°C or exocytosis-inhibiting low temperature (8°C) prior to and during stimulation with ATP. Aequorin luminescence was monitored continuously before and after addition of ATP.
in M, and x ϭ [Ca 2ϩ ] cyto in M) (Fig. 8B) indicates that the granule is able to maintain a Ca 2ϩ concentration gradient over rather extreme ranges.
Thus, the chromaffin granule was able to maintain a substantial [Ca 2ϩ ] storage gradient over cytosol during stimulated secretion (Table I); indeed, during the most extreme stimulation (by ATP; Table I ]. Hence, the active accumulation mechanism(s) for calcium into chromaffin granules follows kinetics comparable to those for cytosolic Ca 2ϩ transients during secretion (Fig. 8). By contrast, Haigh and Phillips (42) found that Ca 2ϩ uptake into isolated chromaffin granule ghosts in vitro was relatively slow; the isolated membranes might lack participating components of the intact transport system in vivo.
Ca 2ϩ Influx into Chromaffin Granules: Dependence on a Ca 2ϩ -translocating ATPase (SERCA) and Na ϩ /Ca 2ϩ Exchanger (NCX)-To explore the role of particular Ca 2ϩ transporters in Ca 2ϩ influx into granules, we blocked SERCAs by thapsigargin (48) (Fig. 9A) and NCXs by KB-R7943 (39) (Fig.  9C). Both the inhibitors substantially decreased Ca 2ϩ uptake into the granules, and in each case the effects were more pronounced for granular than cytoplasmic Ca 2ϩ (Fig. 9, B  and D).
The presence of NCXs on chromaffin granule membranes has been suggested by both functional evidence (42,43,49) and identification of specific NCX1 transcript isoforms in chromaffin cells (38,50), and Haigh and Phillips (42) reported indirect evidence for a role of NCXs in Ca 2ϩ accumulation by chromaffin granule ghosts. Our results with the "influx" mode inhibitor KB-R7943 (Fig. 9C) are consistent with such a role for NCX in vesicular Ca 2ϩ uptake.
Diminution of peak Ca 2ϩ uptake by granules in the presence of the SERCA inhibitor thapsigargin (Fig. 9A) suggests a SERCA on the granule membrane. While SERCAs are widely established as mediators of calcium uptake in the endoplasmic reticulum of chromaffin cells (51), SERCAs on the chromaffin granule membrane are not conclusively established by our pharmacologic data (Fig. 9A) and await molecular confirmation.
Role of Granule H ϩ Transporters and pH-Since the acidic pH compartment within chromaffin granules is crucial to accumulation of catecholamines (2), we evaluated the effects of H ϩ gradient (Fig. 10, A and B) or H ϩ transport (Fig. 10C) disruption on vesicular Ca 2ϩ storage. Indeed, disruption of the acidic pH of the granule core (Fig. 10, A and B) impaired the ability of granules to accumulate Ca 2ϩ , whether the granule pH gradient was diminished by weak bases (Fig. 10A) or H ϩ pump inhibition (Fig. 10B); lack of effect of these procedures on cytosolic [Ca 2ϩ ] reinforces their specificity for granule events. The implication is that the chromaffin granule membrane H ϩtranslocating ATPase plays an active role in Ca 2ϩ accumulation by the granule.
Similarly, inhibition of Na ϩ /H ϩ exchange (Fig. 10C) impaired the ability of granules to accumulate Ca 2ϩ , even without changes in cytosolic [Ca 2ϩ ]. Indirect (pharmacologic) evidence (41) has previously been presented for a functional Na ϩ /H ϩ exchanger (NHE) on the chromaffin granule membrane (though the particular NHE isoform is not established), and the Na ϩ /H ϩ antiporter may assist in coupling Ca 2ϩ transport to H ϩ translocation into chromaffin granule ghosts (42).
Role of Exocytotic Exposure of the Luminophore-The luminescence increments of vesicular hCgA-Aeq during stimulation of secretion were not blunted during blockade of the final stage of exocytosis, using the tools of hyperosmolarity or low temperature (Fig. 11); hence, the vesicular [Ca 2ϩ ] increments observed are authentic responses to cytosolic transients early in the secretory process, and do not simply represent increments in luminescence consequent upon late exocytotic exposure to the higher [Ca 2ϩ ] in the extracellular space.
Comparison to Other Studies of Ca 2ϩ Transport in Pancreatic Insulin Secretory Granules or Chromaffin Granules-In a series of studies of Ca 2ϩ storage in insulin secretory granules of calcium influx into the granules, their relative contributions remain to be explored. It is conceivable that one of these carriers play a predominant role over the others; indeed, while passive ion transporters such as the NCX or the NHE catalyze ion movements in response to concentration gradients, it is likely that active, energy-consuming generators of vesicular ion gradients, such as the SERCA and the H ϩ -ATPase, would determine the initial or set point for calcium fluxes. Within the realm of active transporters, SERCA inhibition achieved the greatest inhibition of calcium flux (by ϳ36.3%; Fig. 9A). This novel model for Ca 2ϩ accumulation within the chromaffin granule should prove useful in design of future experiments to confirm or refute the presence of specific transport carriers in the underlying such processes in the granule membrane.