| Project/Area Number |
01870011
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B).
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| Allocation Type | Single-year Grants |
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| Research Field |
General pharmacology
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| Research Institution | Keio University |
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Principal Investigator |
KATO Ryuichi Keio University School of Medicine Professor, 医学部, 教授 (40112685)
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| Co-Investigator(Kenkyū-buntansha) |
SASAKAWA Nobuyuki Keio University School of Medicine Research Associate, 医学部, 助手 (20187107)
NAKAKI Toshio Keio University School of Medicine Assistant Professor, 医学部, 講師 (30164148)
YAMAMOTO Satoshi Keio University School of Medicine Associate Professor, 医学部, 助教授 (50138129)
日比 清勝 日本分光工業(株), LC事業部, 課長
真砂 央 日本分光工業(株), 第2事業部
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| Project Period (FY) |
1989 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥10,000,000 (Direct Cost: ¥10,000,000)
Fiscal Year 1990: ¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1989: ¥7,000,000 (Direct Cost: ¥7,000,000)
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| Keywords | Intracellular Ca^<2+> concentration / Catecholamine release / Adrenal chromaffin cells / PC 12 cells / Desensitization / Perfusion / Microcarrier beads / PC12 / 細胞内カルシウム濃度 / 分泌 / 副腎髄質細胞 |
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| Research Abstract |
PC 12 cells, which are cloned cell line derived from rat adrenal pheochromocytoma, and purified bovine adrenal chromaffin cells were attached to microcarrier beads (Cytodex 1^<<ﾟ!>>; Pharmacia) and cultured in Dulbecco's Modified Eagle Medium for 4-7 days. The beads were packed into a perfusion mini qaurtz cuvette (volume : 70 mul). The cuvette was placed in the warmed (37 ^ﾟC) sample chamber of a fluorometer. The cells were perfused with Locke's solution at a flow rate of 1-1.5 ml/min and loaded with Fura-2 by perfusing the cells with Fura-2AM (2 muM) for 15 min. A pulsative stimulation of the cells was performed by an injection of stimulant into the flow stream in a volume of 100 mul through loopinjector. For the determination of intracellular calcium concentration ([Ca^<2+>]i), the fluorescence intensity (excitation 340 and 380 nm, emission 500 nm) and florescence ratio (F_<340>/F_<380>) were monitored. The perfusate from the cuvette was directly introduced into an electrochemical flow cell detector for the detection of catecholamine release. When the PC 12 cells were stimulated with nicotine, high K^+ and ATP, transient increase in [Ca^<2+>]_i and catecholamine release were observed. In cultured adrenal chromaffin cells, carbamylcholine and KCl also induced increase in [Ca^<2+>]_i and catecholamine release in a concentration-dependent manner. These results suggest that the simultaneous monitoring of [Ca^<2+>]_i and catecholamine release in this perfusion system is useful for studies on the regulatory mechanisms of stimulus-secretion coupling.
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