Homogeneous Ca2+ stores in rat adrenal chromaffin cells
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, 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], [6], [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 caffeine-induced 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 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 (IP3R) and Ry receptor (RyR) were distributed in the same ER Ca2+ storage sites at the perinuclear region and at cell periphery.
Section snippets
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
Distribution of Ca2+store site-associated proteins
To identify caffeine-sensitive sites, 0.5 μM 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
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 IP3R and RyR with SERCA pumps. This notion is consistent with the
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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