Ba 2+-induced chromaffin cell death: cytoprotection by Ca 2+ channel antagonists

Abstract

Exposure of bovine adrenal medullary chromaffin cells to Ba 2+ ions (in the absence of Ca 2+ ions) caused their death, measured as lactate dehydrogenase (LDH) release. The concentration of Ba 2+ required to damage the cells by about 65% ranged between 1 and 10 mM (no Ca 2+ added); the required exposure time was rather brief (15 min–4 h). The simultaneous presence of Ca 2+, Mg 2+ or Zn 2+ together with Ba 2+ (2 mM, 4 h) afforded cyprotection (60–80%). Individual selective blockers of Ca 2+ channel subtypes afforded no protection. However, combined nifedipine (3 μM) plus ω-conotoxin MVIIC (3 μM) offered full protection. Substantial protection was also seen with the “wide-spectrum” Ca 2+ channel blockers penfluridol (0.3 μM), lubeluzole (3 μM), dotarizine (3 μM), flunarizine (3 μM), and mibefradil (3 μM). This protection was due to blockade of Ba 2+ entry through Ca 2+ channels because dotarizine (10 μM) inhibited the increase in cytosolic [Ba 2+] seen in fura-2-loaded chromaffin cells. Once Ba 2+ accumulated in the cytosol, it was not extruded by the Na +/Ca 2+ exchanger, as shown by the prolonged and sustained elevation of the fura-2 signal. This contrasts with the fast dissipation of the fura-2 signal generated by [Ca 2+] i elevation. Thus, Ba 2+ overload can cause cell death by mechanisms similar to those reported for Ca 2+ overload and might be used as a novel and convenient tool to search for new cytoprotective compounds.