Protein kinase C-alpha and -epsilon down-regulate cell surface sodium channels via differential mechanisms in adrenal chromaffin cells.
In cultured bovine adrenal chromaffin cells, our [3H]saxitoxin ([3H]STX) binding, immunoblot, and northern blot analyses specified protein kinase C (PKC) isoform-specific posttranscriptional and posttranslational mechanisms that direct down-regulation of cell surface Na channels. Immunoblot analysis showed that among 11 PKC isoforms, adrenal chromaffin cells contained only conventional (c)PKC-alpha, novel (n)PKC-epsilon, and atypical (a)PKC-zeta. Treatment of adrenal chromaffin cells with 100 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) or 100 nM phorbol 12,13-dibutyrate (PDBu) caused a rapid (<15 min) and sustained (>15 h) translocation of PKC-alpha and -epsilon (but not -zeta) from cytosol to membranes, whereas a biologically inactive 4alpha-TPA had no effect. Thymeleatoxin (TMX), an activator of cPKC, produced similar membrane association of only PKC-alpha at 100 nM, with the potency of TMX being comparable with those of TPA and PDBu. Treatment with either 100 nM TPA or 100 nM TMX reduced cell surface [3H]STX binding to a comparable extent at 3, 6, and 12 h, whereas TPA lowered the binding to a greater extent than TMX at 15, 18, and 24 h; at 15 h, Gö6976, a specific inhibitor of cPKC, completely blocked TMX-induced decrease of [3H]STX binding while preventing by merely 57% TPA-induced decrease of [3H]STX binding. Treatment with 100 nM TPA lowered the Na channel alpha-subunit mRNA level between 3 and 12 h, with its maximum 52% fall at 6 h, and it was accompanied by a subsequent 61 % rise of the beta1-subunit mRNA level at 24 h. Gö6976 failed to prevent TPA-induced reduction of the alpha-subunit mRNA level; TMX did not change the alpha- and beta1-subunit mRNA levels throughout the 24-h treatment. Brefeldin A, an inhibitor of vesicular exit from the trans-Golgi network, augmented TPA- and TMX-induced decrease of [3H]STX binding at 1 and 3 h. Our previous and present studies suggest that PKC down-regulates cell surface Na channels without altering the allosteric gating of Na channels via PKC isoform-specific mechanisms; cPKC-alpha promotes Na channel internalization, whereas nPKC-epsilon decreases the alpha-subunit mRNA level by shortening the half-life of alpha-subunit mRNA without changing its gene transcription.
Yanagita
T
T
Department of Pharmacology, Miyazaki Medical College, Japan.
Kobayashi
H
H
Yamamoto
R
R
Kataoka
H
H
Yokoo
H
H
Shiraishi
S
S
Minami
S
S
Koono
M
M
Wada
A
A
D000311:Adrenal Glands / Q000166:cytology; D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D002227:Carbazoles / Q000494:pharmacology; D002273:Carcinogens / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000737:chemistry* / Q000166:cytology / Q000201:enzymology*; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D015151:Immunoblotting; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010703:Phorbol Esters / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011493:Protein Kinase C / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D012530:Saxitoxin / Q000378:metabolism / Q000494:pharmacology; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013779:Tetrodotoxin / Q000494:pharmacology; D014316:Tritium
D002227:Carbazoles; D002273:Carcinogens; D004791:Enzyme Inhibitors; D007211:Indoles; D007527:Isoenzymes; D010703:Phorbol Esters; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D015222:Sodium Channels; D014316:Tritium; C081021:Go 6976; D020126:Brefeldin A; D012530:Saxitoxin; D013779:Tetrodotoxin; C080287:thymeleatoxin; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D013755:Tetradecanoylphorbol Acetate