Vol. 297, Issue 2, 657-665, May 2001

Up-Regulation of Cell Surface Sodium Channels by Cyclosporin A, FK506, and Rapamycin in Adrenal Chromaffin Cells

Seiji Shiraishi , Toshihiko Yanagita, Hideyuki Kobayashi, Yasuhito Uezono, Hiroki Yokoo, Shin-Ichi Minami, Mayumi Takasaki and Akihiko Wada

Departments of Pharmacology (S.S., T.Y., H.K., Y.U., H.Y., S.M., A.W.) and Anesthesiology (S.S., M.T.), Miyazaki Medical College, Kiyotake, Miyazaki, Japan

Treatment of cultured bovine adrenal chromaffin cells with cyclosporin A (CsA) increased cell surface [³H]saxitoxin ([³H]STX) binding by 56% in a time (t_1/2 = 15.2 h)- and concentration (EC₅₀ = 2.9 µM)-dependent manner but did not change the K_d value. In CsA-treated cells, veratridine-induced ²²Na⁺ influx was augmented with no change in the EC₅₀ of veratridine; also, - and -scorpion venom and Ptychodiscus brevis toxin-3 enhanced veratridine-induced ²²Na⁺ influx in a more than additive manner, as in nontreated cells. CsA treatment for 1 to 24 h inhibited calcineurin activity, measured by the in vitro assay, with the IC₅₀ of 0.6 µM but did not alter cellular level of calcineurin. FK506 or rapamycin elevated [³H]STX binding by 36 or 25%, whereas GPI-1046, an immunophilin ligand incapable to inhibit calcineurin, or okadaic acid, an inhibitor of protein phosphatases 1 and 2A, had no increasing effect. The rise of [³H]STX binding by CsA was attenuated by the coincident treatment with brefeldin A (BFA), an inhibitor of vesicular exit from the trans-Golgi network. The internalization rate of cell surface Na⁺ channels, as determined in the presence of BFA, was decreased in CsA (but not rapamycin)-treated cells (t_1/2 = 20.3 h), compared with nontreated cells (t_1/2 = 13.7 h). CsA treatment, however, did not elevate cellular levels of Na⁺ channel -subunit and Na⁺ channel - and ₁-subunit mRNAs. In CsA-treated cells, veratridine-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels and catecholamine secretion were enhanced, whereas high K⁺-induced ⁴⁵Ca⁺ influx was not. Thus, the inhibition of calcineurin or rapamycin-binding protein causes up-regulation of cell surface functional Na⁺ channels via modulating externalization and internalization of Na⁺ channels, thus enhancing Ca²⁺ channel gating and catecholamine secretion.