Abstract
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, α- and β-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 α-subunit and Na+ channel α- and β1-subunit mRNAs. In CsA-treated cells, veratridine-induced 45Ca2+ influx via voltage-dependent Ca2+ channels and catecholamine secretion were enhanced, whereas high K+-induced45Ca+ 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.
Footnotes
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Send reprint requests to: Dr. Akihiko Wada, Department of Pharmacology, Miyazaki Medical College, Kiyotake, Miyazaki 889-1692, Japan. E-mail: akihiko{at}fc.miyazaki-med.ac.jp
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This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.
- Abbreviations:
- CsA
- cyclosporin A
- FKBP
- FK506 binding protein
- FRAP
- FKBP- and rapamycin-associated protein
- STX
- saxitoxin
- TTX
- tetrodotoxin
- PbTx-3
- Ptychodiscus brevis toxin-3
- hNE-Na
- TTX/STX-sensitive human neuroendocrine type Na+channel α-subunit
- TGN
- trans-Golgi network
- ARF
- ADP-ribosylation factor
- BFA
- brefeldin A
- PKA
- cyclic AMP-dependent protein kinase
- PKC
- protein kinase C
- GAPDH
- glyceraldehyde 3-phosphate dehydrogenase
- PAGE
- polyacrylamide gel electrophoresis
- KRP
- Krebs-Ringer phosphate
- SDS
- sodium dodecyl sulfate
- SSC
- saline-sodium citrate
- PPIase
- peptidyl prolylcis-trans isomerase
- Received September 6, 2000.
- Accepted January 31, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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