Homogeneous Ca2+ stores in rat adrenal chromaffin cells

Cell Calcium 33 (2003) 19–26

Homogeneous Ca2+ stores in rat adrenal chromaffin cells Masumi Inoue a,∗ , Yasuji Sakamoto a , Naoji Fujishiro a , Issei Imanaga a , Shoichiro Ozaki b , Glenn D. Prestwich b , Akira Warashina c b

a Department of Physiology, Fukuoka University School of Medicine, Fukuoka 814-0180, Japan Department of Medical Chemistry, University of Utah, 419 Wakara Way, Suite 205, Salt Lake City, UT 84108, USA c Department of Physiology, Niigata University School of Medicine, Asahimachi-dori, Niigata 951-8510, Japan

Received 12 August 2002; accepted 12 September 2002

Abstract The localization and function of Ca2+ stores in isolated chromaffin cells of rat adrenal medulla were investigated using confocal laser microscopy and amperometry. Binding sites for BODIPY-inositol 1,4,5-trisphosphate (IP3 ), -ryanodine (Ry), and -thapsigargin (Thap) were both perinuclear and at the cell periphery. The endoplasmic reticulum (ER), which was identified by ER Tracker dye, took up fluorescent Ry and IP3 , and the majority of BODIPY-Ry-binding area was bound by fluorescent IP3 . Under Ca2+ -free conditions, the amount of caffeine-induced catecholamine secretion was 33% of that of muscarine-induced secretion, but muscarine induced little or no secretion after exposure to caffeine. Muscarine-induced Ca2+ increases, as observed with fluo-3, lasted for a few tens of seconds under Ca2+ -free conditions, whereas a caffeine-induced Ca2+ transient diminished rapidly with a half decay time of 3 s and this spike-like Ca2+ transient was then followed by a sustained increase with a low level. These results indicate that IP3 receptors and Ry receptors (RyRs) are present in common ER Ca2+ storage and the lower potency of caffeine for secretion may be due to a rapid decrease in RyR channel activity to a low level. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Ca2+ stores; Adrenal chromaffin cells; Confocal laser microscopy; Amperometry; IP3 ; Ryanodine

1. Introduction An increase in intracellular Ca2+ concentration ([Ca2+ ]) plays a pivotal role for many cellular functions, such as secretion, contraction, cell division, and gene transcription [1], and a variety of external signals are transmitted into the cell as an increase in [Ca2+ ]. This increase in [Ca2+ ] is attributed to either the membrane depolarization and the subsequent activation of voltage-dependent Ca2+ channels, or to the mobilization of Ca2+ ions from intracellular storage sites. Intracellular Ca2+ ions are mobilized from these stores in response to inositol 1,4,5-trisphosphate (IP3 ) or cyclic ADP ribose [2]. In renal smooth muscle cells, IP3 and caffeine-sensitive store sites were overlapped, whereas in pulmonary smooth muscle cells these two sites are different [3]. Similarly, in astrocytes, the majority of IP3 -sensitive Ca2+ stores differed from caffeine-sensitive sites, and Ca2+ sequestration by the former and the latter was thapsigargin (Thap) sensitive and insensitive, respectively [4]. Thus, ∗ Corresponding author. Tel.: +81-92-801-1011x3550; fax: +81-92-865-6032. E-mail address: [email protected] (M. Inoue).

Ca2+ store sites are heterogeneous with respect to releasing and sequestering mechanisms of Ca2+ ions in many types of cells. Furthermore, Ca2+ store sites are structurally heterogeneous, and this variety of Ca2+ storage sites may help to transmit the Ca2+ signals locally in the cell. In addition to the nucleus and the endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) [5–7], accumulating evidence indicates that secretory granules in some exocrine and endocrine cells can function as Ca2+ stores [8,9]. In bovine adrenal chromaffin cells, angiotensin I mobilized Ca2+ ions from sites close to the nucleus and caffeineinduced increase in [Ca2+ ] occurred diffusely in the cytosol [10]. Based on these results, adrenal chromaffin cells were suggested to have two different Ca2+ store sites: one was IP3 sensitive and localized in the vicinity of the nucleus; the other was caffeine sensitive and present diffusely in the cytoplasm. In guinea pig adrenal chromaffin cells, however, the muscarinic receptor-mediated Ca2+ mobilization was markedly suppressed by the preceding exposure to caffeine, suggesting that the majority of IP3 -sensitive Ca2+ store sites overlap caffeine-sensitive ones [11,12]. In the present experiment, IP3 - and caffeine-sensitive Ca2+ storage sites in rat adrenal chromaffin cells were elucidated using the

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fluorescence technique and laser confocal microscopy, and effects on catecholamine secretion of Ca2+ mobilized from these sites were investigated using amperometry. We found that IP3 receptor (IP3 R) and Ry receptor (RyR) were distributed in the same ER Ca2+ storage sites at the perinuclear region and at cell periphery.

2. Materials and methods 2.1. Amperometry Experiments were performed on chromaffin cells enzymatically isolated from rat adrenal medullae [13]. The adrenal medulla was cut into two to three pieces and incubated for 30 min with 0.25% collagenase dissolved in Ca2+ -deficient balanced salt solution. After incubation, tissues were washed three times in Ca2+ -deficient solution and left in this solution at 23–25 ◦ C until commencement of the experiments. Before the start of the experiments, one or two pieces of tissue were placed in the bath apparatus on an inverted microscope and chromaffin cells were dissociated mechanically using fine needles. The bath apparatus was constantly perfused with saline and chemicals were bath applied. Catecholamine release from dissociated cells was measured using amperometry [14]. The current due to oxidation of catecholamine at the tip of the carbon-fiber electrode was fed to a brush recorder after low-pass filtering at 5 Hz. For quantitative analysis, the signals were low-pass filtered at 100 Hz, then digitized at a sampling interval of 5 ms, and the total charge of evoked currents were measured. The agonist-induced secretion was obtained by subtraction of the basal secretion before stimulation from that observed for 30–60 s stimulation. The standard saline contained (mM): 137 NaCl, 5.4 KCl, 1.8 CaCl2 , 0.5 MgCl2 , 0.53 NaH2 PO4 , 5 d-glucose, 5 HEPES, and 4 NaOH (pH 7.4). In a Ca2+ -deficient saline, 1.8 mM CaCl2 in the standard saline was replaced with 3.6 mM MgCl2 .

was measured in the section with the brightest fluorescence (one central and/or one peripheral site for each cell were measured), then expressed as a fraction of background intensity in the cytoplasm. To visualize IP3 -binding sites, fixed cells were exposed to 30 ␮M of BODIPY-IP3 or IP3 conjugated with 7-nitrobenz-2-oxa-1,3-diazole (NBD-IP3 ) or with 6-(tetramethylrhodamino-5-carboxamido)hexanoic acid (TMRh-IP3 ) [16,17]. The fluorescence was observed as FITC fluorescence for NBD-IP3 and BODIPY-IP3 or as rhodamine (543 nm illumination and emission above 570 nm) for TMRh-IP3 . To localize the ER, fixed cells were exposed to 1 ␮M ER Tracker Blue–White DPX (Molecular Probe) for 30 min and then the fluorescence was observed with a 365 nm illumination and emission above 395 nm. 2.3. Measurement of Ca2+ concentration Dissociated cells were loaded with 10 ␮M fluo-3 AM (Molecular Probe) [13]. The cells were illuminated with the 488 nm laser and emission was monitored between 515 and 565 nm. Fluorescence images were acquired every second with an FWHM of 1.79 or 2.35 ␮m. To study the effects of caffeine or muscarine, half the 2 ml solution in the dish was replaced with a test solution containing the chemical, unless otherwise noted, and the administration was completed within 10 s. The intensity of fluo-3 fluorescence usually decreased with each illumination. Thus, 20 frames of fluorescence were obtained prior to application of chemicals and the extent of photobleaching was estimated by a curve-fitting of the intensities in the frames with a linear function. Intensity in each frame was then corrected for photobleaching. The actual intensity in an 80th frame was 4.2 ± 3.1% (n = 10) larger than the value estimated in this manner. An increase in the fluorescence intensity in response to muscarine or caffeine was expressed as a fraction of a prestimulus level. All data were expressed as mean ± S.E.M. and statistical significance was determined using Student’s t-test.

2.2. Fluorescent image analysis

3. Results

Dissociated adrenal chromaffin cells were fixed in 2% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.2) for 2 h at 4 ◦ C. The fluorescence was observed using laser confocal microscopy with a full-width at half-maximal intensity (FWHM) of 0.652 ␮m, unless otherwise noted [15]. Whole cell images were acquired with illumination of a 488 nm laser and emission of all wavelengths. BODIPY FL ryanodine (BODIPY-Ry) at 0.5 ␮M (Molecular Probe) or 1 ␮M BODIPY FL thapsigargin (BODIPY-Thap) (Molecular Probe) was applied to fixed cells and the binding was observed as fluorescein isothiocyanate (FITC) fluorescence (the 488 nm laser illumination and emission of 510–525 nm). The fluorescent Ry bindings were observed from the bottom to the top of the cell with a step of 0.7 ␮m, and the intensity of the fluorescent Ry binding

3.1. Distribution of Ca2+ store site-associated proteins To identify caffeine-sensitive sites, 0.5 ␮M BODIPY-Ry was applied to fixed adrenal chromaffin cells and binding regions were visualized using laser confocal microscopy. The BODIPY-Ry binding or “hot” sites, which were visible as FITC fluorescence, were localized not only at the area adjacent to the nucleus, but also at cell periphery (Fig. 1A). The fluorescence intensities of such hot areas at the central place and cell periphery were 2.57 ± 0.24 (n = 17) and 2.88 ± 0.33 (n = 8) times background intensity in the cytoplasm. These fluorescence intensities were markedly diminished by the prior addition of 10 ␮M Ry (Fig. 1B; relative intensities of brighter sites at the central place and cell periphery, 1.55 ± 0.25 (n = 8) and 1.53 ± 0.33 (n = 6) times

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Fig. 1. Distribution of Ca2+ mobilization-related proteins in adrenal chromaffin cells. (A and B) Confocal fluorescence images of BODIPY-Ry bindings in absence and presence of Ry, respectively. Fixed chromaffin cell was exposed to 0.5 ␮M BODIPY-Ry. Ten micromoles of Ry was applied for 30 min before and during application of BODIPY-Ry. (C and D) Confocal fluorescence images of NBD-IP3 and BODIPY-Thap bindings, respectively. Thirty micromoles of NBD-IP3 and 1 ␮M BODIPY-Thap were applied to fixed cells. Arrowheads represent bindings at cell periphery. Asterisks in this and the following figures indicate the nucleus. Fluorescent images of BODIPY-Ry, BODIPY-Thap, and NBD-IP3 were acquired with a 488 nm laser illumination and emission of 510–525 nm. FWHM was 0.652 ␮m.

cytoplasmic background), indicating that the hot area reflects specific binding of Ry and not of the fluorescent BODIPY moiety. The peripheral Ry-binding regions were present underneath 11.6 ± 1.3% (n = 13) of the plasma membrane in cells (one optical section with clear fluorescent reactions was analyzed for each cell). We next investigated the distribution of 30 ␮M NBD-IP3 and 1 ␮M BODIPY-Thap-binding regions in cells. As shown in Fig. 1C and D, fluorescent IP3 and Thap were also localized perinuclearly and peripherally. The peripheral IP3 -binding sites were juxtaposed to 15.4 ± 1.5% (n = 33) of the plasma membrane, a value which was comparable to that of the peripheral Ry-binding site. Similar distributions of fluorescent IP3 , Ry, and Thap suggest that Ca2+ ions are taken up into storage by sarco/endoplasmic reticulum Ca2+ (SERCA) pumps and IP3 R and RyR are localized on common sites, possibly the ER [5]. The colocalization of IP3 R and RyR was directly examined using a double staining technique. Fig. 2A shows that the majority of BODIPY-Thap-binding sites, which were localized mainly at the perinuclear region, were stained with ER Tracker, which selectively binds to the ER membrane

[18]. Furthermore, perinuclear and peripheral IP3 -binding regions were also stained with ER Tracker, and the majority of BODIPY-Ry bindings exhibited rhodamine fluorescence due to TMRh-IP3 bindings (Fig. 2E and F). These results suggest that the majority of RyRs and IP3 Rs were localized in common ER. 3.2. Catecholamine secretion in response to muscarine and caffeine To elucidate functionally the coincidence of IP3 - and caffeine-sensitive storage sites, effects of muscarine- and caffeine-induced Ca2+ mobilization on catecholamine secretion were investigated using amperometry (Fig. 3). Application of 30 ␮M muscarine in the absence of external Ca2+ ions induced transient secretion, which usually lasted for a few tens of seconds, whereas that of 15 mM caffeine evoked secretion of shorter durations (less than 10 s). The total amount of the caffeine-induced secretion was 33.0±6.8% (n = 7) of that of the muscarine-induced secretion. Despite this potency difference in inducing secretion, only in

Fig. 2. Presence of Ry- and IP3 -binding sites in endoplasmic reticulum identified with ER Tracker. (A and B) Confocal fluorescence images of 0.5 ␮M BODIPY-Ry and 1 ␮M ER Tracker bindings in the same optical section of a fixed chromaffin cell. ER Tracker fluorescence was observed with illumination of a 365 nm laser and emission of above 395 nm. (C and D) Confocal fluorescence images of 30 ␮M BODIPY-IP3 and 1 ␮M ER Tracker bindings in the same section of fixed chromaffin cells, respectively. (E and F) Confocal fluorescence images of 0.5 ␮M BODIPY-Ry and 30 ␮M TMRh-IP3 bindings in the same section of a fixed chromaffin cell, respectively. TMRh-IP3 bindings were visualized with a 543 nm illumination and emission of >570 nm. Arrows in (C)–(F) images indicate peripheral binding sites.

Fig. 3. Catecholamine secretion in response to muscarine and caffeine in the absence of external Ca2+ . (A–C) Amperometric records of catecholamine secretion from the same cell. Thirty micromoles of muscarine and 15 mM caffeine were applied to a cell during the indicated period (double line for muscarine; single line for caffeine) in the absence of external Ca2+ (dotted line Ca(−)). The traces were interrupted for 5 min.

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two of the six cells tested, muscarine induced a tiny amount of secretion after application of caffeine, whereas in all seven cells caffeine failed to induce secretion after muscarine stimulation. These results further support our notion that the muscarine-sensitive Ca2+ store sites almost overlap the caffeine-sensitive sites. 3.3. Measurement of Ca2+ change in response to muscarine and caffeine The difference in amount and time course of muscarineand caffeine-induced secretions led us to examine changes in [Ca2+ ] using 10 ␮M fluo-3 AM. The intensity of fluo-3 fluorescence was heterogeneous at the resting state in the cell, the nuclear intensity being higher than the cytoplasmic (Fig. 4Ab). The extent of an increase in [Ca2+ ] in response to 30 ␮M muscarine was also heterogeneous (Fig. 4B). To compare extents of Ca2+ increase in different areas of the cell, the extent of an increase was expressed as a fraction of a control intensity before stimulation. In Fig. 4C, such relative increases in [Ca2+ ] were plotted against time. The time

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courses of Ca2+ changes did not differ among the areas, but the extent of the increase varied. The largest increase in fluorescence intensity consistently occurred in the nucleus (the ratio of the relative increase in the nucleus to that in the cytoplasm, 1.77 ± 0.10, n = 20). This larger Ca2+ response in the nucleus may not be due to the apparent absence of SERCA pump in the nuclear envelope, since the relative Ca2+ increase in the nucleus in response to 2.5 ␮M Thap was 2.11 ± 0.26 (n = 4) times that in the cytoplasm in the absence of external Ca2+ ions (not shown). The area numbered 4 in Fig. 4Aa showed the largest increase in fluorescence in the cytoplasm, suggesting that the majority of Ca2+ store sites were present in this region. In 20 cells where the fluorescence intensity was measured separately in three to four areas of the cytoplasm, the maximum difference among the relative increases in local [Ca2+ ] was 0.47±0.07 (n = 20), which corresponded to 29% of the largest increase in local [Ca2+ ]. We next investigated a change in [Ca2+ ] in response to caffeine under Ca2+ -free conditions. In eight cells, bath application of 7.5 mM caffeine produced Ca2+ transients with

Fig. 4. Heterogeneous Ca2+ increase in response to muscarine in the absence of external Ca2+ . (A, a) Cell image acquired with illumination of a 488 nm laser and emission of all wavelengths. (A, b and c) Fluo-3 fluorescence images before and after addition of 30 ␮M muscarine. The intensity of fluo-3 fluorescence was expressed in artificial color (low to high intensity corresponds to red to green). FWHM was 2.38 ␮m. (B) Intensities of fluorescence in five areas of the cell are plotted against time. The 1 ml of 60 ␮M muscarine-containing solution (final concentration, 30 ␮M) was added to 1 ml dish solution during the indicated period. The numbers in (A, a) correspond to those in (B); b and c in (A) correspond to b and c in (B). (C) Relative change in fluorescence intensity of each area is plotted against time. Intensity in each area was corrected for photobleaching and a change in intensity was expressed as a fraction of a prestimulus level (see Section 2).

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Fig. 5. Ca2+ transient in response to caffeine in the absence of external Ca2+ . (A) Relative increases in [Ca2+ ] in response to 7.5 mM caffeine are plotted against time. The symbols (䊉) and (䊊) were obtained from different cells. The 1 ml of caffeine-containing solution was added to 1 ml dish solution during the indicated periods. (B) Relationship between half decay time (T1/2 ) and relative peak of Ca2+ transient. The symbols (䊊) and (䊉) represent Ca2+ mobilization in response to 7.5 mM caffeine and 30 ␮M muscarine in the absence of external Ca2+ ions, respectively.

>1.9 of peak values, which diminished rapidly with a half decay time (T1/2 ) of 2.9 ± 0.3 s (Fig. 5A and B), and this spike-like Ca2+ increase was then followed by a sustained increase with a low level. In six of the eight cells, the sustained increase followed the spike-like increase immediately without a temporary return to a prestimulus level, whereas in one cell shown in Fig. 5A, there was a short gap between these Ca2+ responses. On the other hand, in the remaining seven cells, caffeine-induced Ca2+ transients with peak amplitudes of 0.84 ± 0.13 decayed slowly with T1/2 of 18.0 ± 3.9 s. These results differed from those for muscarine-induced Ca2+ transients: T1/2 (15.8 ± 1.7, n = 24) of the latter with peak amplitudes >1.9 did not differ significantly from T1/2 (12.7 ± 1.4, n = 22) with those <1.9. The results suggest that inactivation of the RyR in chromaffin cells is facilitated with an increase in Ca2+ efflux through channels.

4. Discussion 4.1. Homogeneous Ca2+ store sites The main findings in the present study are that IP3 -, Ry-, and Thap-binding areas were located at cell periphery and in the perinuclear region and these areas were also stained with ER Tracker dye, which selectively binds to the ER membrane [18]. In addition, the majority of fluorescent IP3 -binding areas were bound by Ry. These results indicate that rat adrenal chromaffin cells have one pool of ER Ca2+ storage, which has both IP3 R and RyR with SERCA pumps. This notion is consistent with the results obtained with the functional analysis of Ca2+ store sites. Muscarine induced little or no catecholamine secretion after caffeine stimulation and vice versa under Ca2+ -free conditions. Furthermore, prior exposure to a SERCA pump inhibitor abolished an in-

crease in [Ca2+ ] in response to caffeine in rat [19] and guinea pig [20] adrenal chromaffin cells. Our conclusion differs from that in bovine [10] and guinea pig [20] chromaffin cells. In the latter, muscarine-induced Ca2+ mobilization was more effective in evoking secretion than the caffeine-induced one, as noted in the present experiment. On the other hand, the former was less efficient in producing Ca2+ -dependent K+ currents than the latter. Based on these results, IP3 -sensitive Ca2+ store sites were suggested to be distributed differently from caffeine-sensitive sites. This notion was not supported by the present morphological findings. The different effects of the muscarine- and caffeine-induced Ca2+ mobilizations on secretion may be explained by the different properties of IP3 R and RyR channels involved. The 7.5 mM caffeine-induced Ca2+ transients with >1.9 of peak values decreased rapidly with a T1/2 of 2.9 s, whereas muscarine-induced Ca2+ transients with similar peaks diminished slowly with a T1/2 of 15.8 s. Thus, the rapid diminution of the former may not be due to the uptake by mitochondria, which were reported to be responsible for the rapid decay of depolarization-evoked Ca2+ transients in rat adrenal chromaffin cells [21]. Rather, it may be ascribed to inactivation or adaptation of RyR. The RyRs isolated from cardiac and skeletal myocytes were shown to inactivate in the order of a few seconds after the open probability was markedly enhanced by depolarization, a step increase in [Ca2+ ], or caffeine [22]. As shown in Fig. 5A, spike-like Ca2+ increases in response to caffeine were always followed by sustained Ca2+ increases with low levels. Thus, the open probability of RyR in adrenal chromaffin cells might diminish rapidly from a high to low level after stimulation with high concentrations of caffeine. Since the Ca2+ store sites can be assumed to be almost empty after exposure to a high concentration of caffeine, the sustained Ca2+ increase with a low level, which is not sufficient for secretion or activation of Ca2+ -dependent K+ currents [23],

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may be due to slow release from caffeine-sensitive store sites. The different mobilizations of Ca2+ from common store sites by IP3 R and RyR suggest that the IP3 R and the RyR in adrenal chromaffin cells are involved in different functions. 4.2. Heterogeneous Ca2+ increases The extent of muscarine-induced Ca2+ increase in the nucleus was higher than that in the cytoplasm. This nuclear Ca2+ response might be mediated by IP3 R present in the nuclear envelope. In the nucleus isolated from mouse liver, Ca2+ ions were taken up in an ATP-dependent manner by the nuclear envelope and an increase in nuclear [Ca2+ ] occurred in response to IP3 and cADP ribose [24]. In adrenal chromaffin cells, however, exposure to fluorescent Ry, IP3 or Thap did not result in a continuous staining around the nucleus. This result suggests that the nuclear envelope of adrenal chromaffin cells has no machinery involved in Ca2+ mobilization. Thus, the larger fluorescence increase in the nucleus, compared with that in the cytoplasm, is likely due to interference of the nucleoplasm with the fluo-3 fluorescence [25]. The irregular changes in the nuclear [Ca2+ ] in response to muscarine completely agreed with those in the cytoplasmic [Ca2+ ] (Fig. 4). This result indicates that Ca2+ transients in the cytoplasm efficiently propagated to the nucleus. In HeLa cells, the Ca2+ increase occurring near the nucleus propagated into the nucleus more efficiently than in the cytoplasm, and the IP3 R-containing ER present near the nucleus was suggested to play a pivotal role for transmission of a Ca2+ mobilizing receptor signal into the nucleus [26]. Thus, the IP3 R in the ER near the nucleus in adrenal chromaffin cells may also be involved in such an efficient transmission. This efficient transmission of the Ca2+ signal to the nucleus might be responsible for the induction of tyrosine hydroxylase, a rate-limiting enzyme for synthesis of catecholamine, by increased neuronal activity in the splanchnic nerve [27] and for the transcription of the proenkephalin gene by activation of the histaminergic H1 receptor, which results in increased phosphoinositide breakdown [28].

Acknowledgements This study was supported by a grant-in-aid from Japan Society for the Promotion of Science (M.I.: 13670050) and by a grant from the National Institutes of Health (G.D.P.: NS 29632).

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