Methods and Findings in Experimental and Clinical Pharmacology
Vol. 24, Suppl. A, 2002, pp. 45
ISSN 0379-0355
Copyright 2002 Prous Science, S.A.
CCC: 0379-0355/2002
http://www.prous.com

Molecular Mechanisms Activated During Veratridine-Induced Chromaffin Cell Death

J. Jordán, M.F. Galindo, D. Tornero, A. Benavides, C. González-García and V. Ceña

Centro Regional de Investigaciones Biomédicas, Universidad de Castilla-La Mancha, Albacete, Spain

Veratridine prevents the inactivation of voltage-dependent Na⁺ channels, opening them for long periods of time and producing an increase in [Na⁺]_i and [Ca²⁺]_i . As a consequence of these changes, veratridine is able to produce Ca²⁺-dependent death in excitable cells, including rat neurons and bovine chromaffin cells. The molecular mechanisms involved in veratridine-induced chromaffin cell death have been explored.

We found that exposure to veratridine (30 mM, 1 h) produces a delayed cellular death that reaches 55% of the cells 24 h after veratridine exposure. This death has the features of apoptosis, as DNA fragmentation can be observed. Calcium ions play an important role in veratridine-induced chromaffin cell death because the cell permeant Ca²⁺ chelator BAPTA-AM and extracellular Ca²⁺ removal completely prevented veratridine-induced toxicity.

Following veratridine treatment, there was a decrease in mitochondrial function and an increase in superoxide anion production. The veratridine-induced increase in superoxide production was blocked by tetrodotoxin (TTX; 10 mM), extracellular Ca²⁺ removal and the mitochondrial permeability transition pore blocker, cyclosporine (10 mM). Veratridine-induced death was prevented by different antioxidant treatments, including catalase (100 IU ml^-1), N-acetyl cysteine (100 mM), allopurinol (100 mM) or vitamin E (50 mM). Veratridine-induced DNA fragmentation was prevented by TTX
(10 mM).

Veratridine also produced a time-dependent increase in caspase activity that was prevented by Ca²⁺ removal and TTX (10 mM). Following veratridine treatment, there was an increase in caspase-like activity, blocked by vitamin E (50 mM) and the mitochondrial permeability transition pore blocker cyclosporine (10 mM). In addition, calpain and caspases inhibitors partially prevented veratridine-induced death.

Mitochondrial mechanisms involved in veratridine-induced chromaffin cell death have been explored. Exposure to veratridine (30 mM, 1h) produces cytochrome c release to the cytoplasm, which seems to be mediated by superoxide anions, and blocked by cyclosporine (10 mM), MnTBAP (10 nM), catalase (100 IU ml^-1) and vitamin E (50 mcM). Superoxide anions open the mitochondrial permeability transition pore in isolated mitochondria, an effect that is blocked by vitamin E (50 mM) and cyclosporine (10 mM), but not by the Ca²⁺ uniporter blocker ruthenium red (5 mM).

Exposure of isolated bovine chromaffin mitochondria to Ca²⁺ results in mitochondrial swelling. This effect was prevented by ruthenium red (5 mM) and cyclosporine
(10 mM), while it was not modified by vitamin E (50 mM). In conclusion, superoxide anions seem to mediate veratridine-induced cytochrome c release and cell death in bovine chromaffin cells.

ACKNOWLEDGMENTS

This work has been supported, in part, by grants SAF99-0060, 1FD97-0500 from CICYT, BFI2001-1565 from the Ministerio de Ciencia y Tecnología; 01050 from the Consejería de Sanidad, JCCM; PAI-02-031 from Consejería de Ciencia y Tecnología, JCCM and from the Fundación Campollano-BSCH to V.C. and SAF98-0140 and BFI2001-1058 from CICYT to C.G.G

Methods and Findings in Experimental and Clinical Pharmacology Vol. 24, Suppl. A, 2002, pp. 45
ISSN 0379-0355 Copyright 2002 Prous Science, S.A. CCC: 0379-0355/2002 http://www.prous.com