Protein kinase C and the opposite regulation of sodium channel alpha- and beta1-subunit mRNA levels in adrenal chromaffin cells

• Published in: Journal of neurochemistry Vol. 73; no. 4; p. 1749
• Main Authors: Yanagita, T, Kobayashi, H, Yamamoto, R, Takami, Y, Yokoo, H, Yuhi, T, Nakayama, T, Wada, A
• Format: Journal Article
• Language: English
• Published: England 01.10.1999
• Subjects: Adrenal Medulla - metabolism; Animals; Cattle; Cell Nucleus - metabolism; Cells, Cultured; Chromaffin Cells - metabolism; Cycloheximide - pharmacology; Down-Regulation - drug effects; Enzyme Activation; Gene Expression Regulation; Kinetics; Macromolecular Substances; Phorbol 12,13-Dibutyrate - pharmacology; Protein Kinase C - metabolism; RNA, Messenger - genetics; Sodium Channels - genetics; Sodium Channels - metabolism; Tetradecanoylphorbol Acetate - pharmacology; Transcription, Genetic
• ISSN: 0022-3042

Our previous [3H]saxitoxin binding and 22Na influx assays showed that treatment of cultured bovine adrenal chromaffin cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) or phorbol 12,13-dibutyrate (PDBu), an activator of protein kinase C (PKC), decreased the number of cell surface Na channels (IC50 = 19 nM) but did not alter their pharmacological properties; Na channel down-regulation developed within 3 h, reached the peak decrease of 53% at 15 h, and was mediated by transcriptional/translational events. In the present study, treatment with 100 nM TPA lowered the Na channel alpha-subunit mRNA level by 34 and 52% at 3 and 6 h, followed by restoration to the pretreatment level at 24 h, whereas 100 nM TPA elevated the Na channel beta1-subunit mRNA level by 13-61% between 12 and 48 h. Reduction of alpha-subunit mRNA level by TPA was concentration-dependent (IC50 = 18 nM) and was mimicked by PDBu but not by the biologically inactive 4alpha-TPA; it was prevented by H-7, an inhibitor of PKC, but not by HA-1004, a less active analogue of H7, or by H-89, an inhibitor of cyclic AMP-dependent protein kinase. Treatment with cycloheximide, an inhibitor of protein synthesis, per se sustainingly increased the alpha-subunit mRNA level and decreased the beta1-subunit mRNA level for 24 h; also, the TPA-induced decrease of alpha-subunit mRNA and increase of beta1-subunit mRNA were both totally prevented for 24 h by concurrent treatment with cycloheximide. Nuclear run-on assay showed that TPA treatment did not alter the transcriptional rate of the alpha-subunit gene. A stability study using actinomycin D, an inhibitor of RNA synthesis, revealed that TPA treatment shortened the t(1/2) of alpha-subunit mRNA from 18.8 to 3.7 h. These results suggest that Na channel alpha- and beta-subunit mRNA levels are differentially down- and up-regulated via PKC; the process may be mediated via an induction of as yet unidentified short-lived protein(s), which may culminate in the destabilization of alpha-subunit mRNA without altering alpha-subunit gene transcription.