| Co-Investigator(Kenkyū-buntansha) |
YOKOO Hiroki Miyazaki Medical College, Department of Pharmacology, Assistant Professor, 医学部, 助手 (30332894)
YANAGITA Toshihiko Miyazaki Medical College, Department of Pharmacology, Assistant Professor, 医学部, 助手 (60295227)
KOBAYASHI Hideyuki Miyazaki Medical College, Department of Pharmacology, Associate Professor, 医学部, 助教授 (40148953)
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| Research Abstract |
In cultured bovine adrenal chromaffin cells, we studied this project, by using ^3H-saxitoxin binding, ^<22>Na influx, Western and Northern blot analyses, nuclear run-on assay, and cytoplasmic [Ca^<2+>]i measurement.
(1) Protein kinas C (PKC) isoform-specific mechanisms for Na channel down-regulation : PKC-α accelerated internalization of cell surface Na channels. PKC-ε increased degradation of Na channel α- subunit mRNA (without changing its transcriptional rate) via de novo synthesis of short-lived protein(s), thus lowering α- subunit mRNA level.
(2) Na channel down-regulation by [Ca^<2+>]i rise : its amplitude and duration : (a) Moderate and relatively prolonged rise of [Ca^<2+>]i activated cPKC-α, calcineurin and calpain, thus promoting internalization of Na channels via clathrin-coated vesicles. Large and sustained rise of [Ca^<2+>]i decreased Na channel α- and β_1- subunit mRNA levels. Small and transient rise of [Ca^<2+>]i had no effect on cell surface density of Na channels, (b) Calcineurin, and FKBP- and rapamycin-associated protein (FKBP), a serine/threonine protein kinase, were involved in the suppression of vesicular externalization of newly-synthesized Na channels from the trnas-Golgi network. (C) Allosteric gating of Na channels by veratridine, α- and β-scorpion venom and brevetoxin was not impaired in the down-regulated Na channels.
(3) Neuroprotective NS-7 : gating inhibition and up-regulation of Na channels : NS-7 bound to domain I segment 6 of Na channel a-subunit, and inhibited Na channel gating, thus reducing gating of voltage-dependent Ca channels and exocytic secretion of catecholamines. Long-term treatment with NS-7 accelerated Na channel externalization, while inhibiting Na channel internalization (without changing Na channel subunit mRNA levels), thus up-regulating Na channels.
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