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

Exposure of bovine chromaffin cells to 30 mu M veratridine for 24 h le d to 70-80% cell death as reflected by phase contrast microscopy, tryp an blue exclusion, lactate dehydrogenase (LDH) release and cell catech olamine contents. Na+ deprivation, Ca2+ deletion or tetrodotoxin (5 mu M) prevented the veratridine-induced cell damage. Nimodipine and vera pamil, but not omega-conotoxin GVIA afforded 20-30% protection. Flunar izine protected the cells by 80% and R56865 by 60%. Stimulation of fur a-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 cyto solic 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 t wo depolarizing agents, the [Ca2+](i) remained low and did not undergo oscillations. In contrast, veratridine (30 mu M) caused large and fre quent 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 om ega-conotoxin GVIA) did not affect the [Ca2+](i) oscillations. In cont rast, flunarizine or R56865 (10 mu M each) suppressed the oscillations of [Ca2+](i). The results demonstrate that bovine chromaffin cells ha ve the necessary machinery to develop prolonged and repetitive [Ca2+]( i) oscillations in the presence of veratridine; however, 'physiologica l' depolarizing stimuli did not cause oscillations. These non-inactiva tin [Ca2+](i) oscillations may induce Ca2+ overload, thus explaining t he well known cytotoxic effects of veratridine in neuronal and chromaf fin cell cultures. Drugs such as flunarizine and the novel cytoprotect ive agent R56865, which prevent such oscillations, avoid Ca2+ overload and cell damage. The results also suggest that external Ca2+ entry th rough N- or L-type Ca2+ channels can equally be associated with the ve ratridine-evoked cell damage.