Abstract:In cultured bovine adrenal chromaffin cells, our [ 3 H]saxitoxin ([ 3 H]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-␣, novel (n)PKC-⑀, and atypical (a)PKC-. 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 (Ͻ15 min) and sustained (Ͼ15 h) translocation of PKC-␣ and -⑀ (but not -) from cytosol to membranes, whereas a biologically inactive 4␣-TPA had no effect. Thymeleatoxin (TMX), an activator of cPKC, produced similar membrane association of only PKC-␣ 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 [ 3 H]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, Gö 6976, a specific inhibitor of cPKC, completely blocked TMX-induced decrease of [ 3 H]STX binding while preventing by merely 57% TPA-induced decrease of [ 3 H]STX binding. Treatment with 100 nM TPA lowered the Na channel ␣-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 ␤ 1 -subunit mRNA level at 24 h. Gö 6976 failed to prevent TPA-induced reduction of the ␣-subunit mRNA level; TMX did not change the ␣-and ␤ 1 -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 [ 3 H]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-␣ promotes Na channel internalization, whereas nPKC-⑀ decreases the ␣-subunit mRNA level by shortening the half-life of ␣-subunit mRNA without changing its gene transcription.

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Treatment (Ն6 h) of cultured bovine adrenal chromaffin cells with geldanamycin (GA) or herbimycin A (HA), an inhibitor of the 90-kDa heat-shock protein (Hsp90) family, decreased cell surface 125

In cultured bovine adrenal chromaffin cells expressing Na v