YANAGITA Toshihiko Miyazaki Medical College, Department of Pharmacology, Assistant Professor, 医学部, 助手 (60295227)
YAMAMOTO Ryuichi Miyazaki Medical College, Department of Pharmacology, Assistant Professor, 医学部, 助手 (10094111)
KOBAYASHI Hideyuki Miyazaki Medical College, Department of Pharmacology, Associate Professor, 医学部, 助教授 (40148953)
|Budget Amount *help
¥3,200,000 (Direct Cost : ¥3,200,000)
Fiscal Year 1998 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1997 : ¥2,400,000 (Direct Cost : ¥2,400,000)
In cultured bovine adrenal medullary chromaffin cells (embryologically derived from the neural crest), we examined the mechanisms that regulate the density/function of cell surface Na channels, and Na channel alpha- and (beta_i-subunit mRNA levels, using ^3H-saxitoxin binding, ^<22>Na influx, Western and Northern blot analyses, nuclear run-on assay, and cytosolic [Ca]_i measurement.
1 . Regulation by protein kinase C (PKC) : Thymeleatoxin (an activator of conventional PKC), Go6976 (an inhibitor of conventional PKC), phorbol esters and brefeldin A (an inhibitor of ADP ribosylation factor-l) were employed.
(1) Translocation of PKC-alpha from cytosol to membranes accelerated endocytosis of cell surface Na channels. (2) Membrane association of PKC-epsilon decreased Na channel alpha-subunit mRNA level, followed by a rise of beta1-subunit mRNA level. (3) Fall of alpha-subunit mRNA level was due to the elevated degradation rate of its mRNA, but not to the reduction of its gene transcriptional r
ate. (4) These effects of PKC-epsilon were dependent on the de novo synthesis of protein(s).
2. Regulation by A-kinase : An activation of A-kinase raised the number of cell surface Na channels, which was dependent on protein synthesis, but not accompanied by changes in Na channel alpha- and beta_1-subunit mRNA levels. Na channels newly-incorporated into plasma membrane exhibited the same allosteric gating mechanism, as did the native Na channels.
3. Regulation by [Ca]_i : A sustained rise of [Ca]_i caused by thapsigargin, an inhibitor of sarco(endo)plasmic reticulum Ca-ATPase, lowered levels of cell surface Na channels, a- and betai-subunit mRNAs, whereas a transient increase of [Ca]_i caused by 2,5-di-(t-butyl)-l, 4-benzohydroquinone (DBHQ) had no effect.
4. Regulation by neuroprotective drugs : Riluzole, and NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy)pyrimidine hydrochloride] bound to the transmembrane segment 6 of domain I in Na channel a-subunit, and inhibited Na channel opening, thereby resulting in the reduction of Ca channel gating and catecholamine secretion. We are now studying whether/how these drugs could alter the density of cell surface Na channels. Less