A PROTOTYPIC INTRACELLULAR CALCIUM-ANTAGONIST, TMB-8, PROTECTS CULTURED CEREBELLAR GRANULE CELLS AGAINST THE DELAYED, CALCIUM-DEPENDENT COMPONENT OF GLUTAMATE NEUROTOXICITY

The effect(s) of a prototypic intracellular Ca2+ antagonist, 8-(N,N-di ethylamino) octyl-3,4,5-trimethoxybenzoate (TMB-8), on glutamate-induc ed neurotoxicity was investigated in primary cultures of mouse cerebel lar granule cells. Glutamate evoked an increase in cytosolic free-Ca2 levels ([Ca2+](i)) that was dependent on the extracellular concentrat ion of Ca2+ ([Ca2+](o)). In addition, this increase in [Ca2+](i) corre lated with a decrease in cell viability that was also dependent on [Ca 2+](o). Glutamate-induced toxicity, quantified by (4,5-dimethylthiazol -2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining, was shown to co mprise two distinct components, an ''early'' Na+/Cl--dependent compone nt observed within minutes of glutamate exposure, and a ''delayed'' Ca 2+-dependent component (ED(50) similar to 50 mu M) that coincided with progressive degeneration of granule cells 4-24 h after a brief (5-15 min) exposure to 100 mu M glutamate. Quantitative analysis of cell via bility and morphological observations identify a ''window'' in which T MB-8 (at >100 mu M) protects granule cells from the Ca2+-dependent, bu t not the Na+/Cl--dependent, component of glutamate-induced neurotoxic damage, and furthermore, where TMB-8 inhibits glutamate-evoked increa ses in [Ca2+](i). These findings suggest that Ca2+ release from a TMB- 8-sensitive intracellular store may be a necessary step in the onset o f glutamate-induced excitotoxicity in granule cells. However, these co nclusions are compromised by additional observations that show that TM B-8 (1) exhibits intrinsic toxicity and (2) is able to reverse its ini tial inhibitory action on glutamate-evoked increases in [Ca2+](i) and subsequently effect a pronounced time-dependent potentiation of glutam ate responses. Dantrolene, another putative intracellular Ca2+ antagon ist, was completely without effect in this system with regard to both glutamate-evoked increases in [Ca2+](i) and glutamate-induced neurotox icity.