EFFECTS OF CA2+ CHANNEL ANTAGONISTS ON CHROMAFFIN CELL-DEATH AND CYTOSOLIC CA2+ OSCILLATIONS INDUCED BY VERATRIDINE

Exposure of bovine chromaffin cells to 30 mu M veratridine for 24 h led to 70-80% cell death as reflected by phase contrast microscopy, trypan blue exclusion, lactate dehydrogenase (LDH) release and cell catecholamine contents. Na+ deprivation, Ca2+ deletion or tetrodotoxin (5 mu M) prevented the veratridine-induced cell damage. Nimodipine and verapamil, but not omega-conotoxin GVIA afforded 20-30% protection. Flunarizine protected the cells by 80% and R56865 by 60%. Stimulation of fura-2-loaded single bovine chromaffin cells with 30 mu M of 1,1-dimethyl-4-phenylpiperazinium (DMPP) or 59 mM K+ caused fast increases in cytosolic Ca2+ concentrations, ([Ca2+](i)). The [Ca2+](i) rose from 0.1 to peaks of 1.9 mu M, which quickly declined to near basal levels with a t(1/2) of around 30 s. In spite of sustained stimulation with these two depolarizing agents, the [Ca2+](i) remained low and did not undergo oscillations. In contrast, veratridine (30 mu M) caused large and frequent oscillatory changes in the [Ca2+](i) which were long-lasting and did not disappear even 30 min after washing out the toxin. The [Ca2+](i) oscillations were reversibly suppressed by Na+ or Ca2+ removal and by 5 mu M tetrodotoxin. Selective L-type Ca2+ channel blockers (10 mu M nimodipine or verapamil) or N-type Ca2+ channel blockers (1 mu M omega-conotoxin GVIA) did not affect the [Ca2+](i) oscillations. In contrast, flunarizine or R56865 (10 mu M each) suppressed the oscillations of [Ca2+](i). The results demonstrate that bovine chromaffin cells have the necessary machinery to develop prolonged and repetitive [Ca2+](i) oscillations in the presence of veratridine; however, 'physiological' depolarizing stimuli did not cause oscillations. These non-inactivatin [Ca2+](i) oscillations may induce Ca2+ overload, thus explaining the well known cytotoxic effects of veratridine in neuronal and chromaffin cell cultures. Drugs such as flunarizine and the novel cytoprotective agent R56865, which prevent such oscillations, avoid Ca2+ overload and cell damage. The results also suggest that external Ca2+ entry through N- or L-type Ca2+ channels can equally be associated with the veratridine-evoked cell damage.