Protein kinase C and the opposite regulation of sodium channel alpha- and beta1-subunit mRNA levels in adrenal chromaffin cells.
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.
Yanagita
T
T
Department of Pharmacology, Miyazaki Medical College, Kiyotake, Japan.
Kobayashi
H
H
Yamamoto
R
R
Takami
Y
Y
Yokoo
H
H
Yuhi
T
T
Nakayama
T
T
Wada
A
A
D000313:Adrenal Medulla / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D007700:Kinetics; D046911:Macromolecular Substances; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D015222:Sodium Channels / Q000235:genetics* / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic
D046911:Macromolecular Substances; D012333:RNA, Messenger; D015222:Sodium Channels; D015240:Phorbol 12,13-Dibutyrate; D003513:Cycloheximide; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate