title: A prototypic intracellular calcium antagonist, TMB-8, protects cultured cerebellar granule cells against the delayed, calcium-dependent component of glutamate neurotoxicity.
abstract: The effect(s) of a prototypic intracellular Ca2+ antagonist, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), on glutamate-induced neurotoxicity was investigated in primary cultures of mouse cerebellar granule cells. Glutamate evoked an increase in cytosolic free-Ca2+ levels ([Ca2+]i) that was dependent on the extracellular concentration of Ca2+ ([Ca2+]o). In addition, this increase in [Ca2+]i correlated with a decrease in cell viability that was also dependent on [Ca2+]o. Glutamate-induced toxicity, quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining, was shown to comprise two distinct components, an "early" Na+/Cl(-)-dependent component observed within minutes of glutamate exposure, and a "delayed" Ca(2+)-dependent component (ED50 approximately 50 microM) that coincided with progressive degeneration of granule cells 4-24 h after a brief (5-15 min) exposure to 100 microM glutamate. Quantitative analysis of cell viability and morphological observations identify a "window" in which TMB-8 (at > 100 microM) protects granule cells from the Ca(2+)-dependent, but not the Na+/Cl(-) -dependent, component of glutamate-induced neurotoxic damage, and furthermore, where TMB-8 inhibits glutamate-evoked increases in [Ca2+]i. These findings suggest that Ca2+ release from a TMB-8-sensitive intracellular store may be a necessary step in the onset of glutamate-induced excitotoxicity in granule cells. However, these conclusions are compromised by additional observations that show that TMB-8 (1) exhibits intrinsic toxicity and (2) is able to reverse its initial inhibitory action on glutamate-evoked increases in [Ca2+]i and subsequently effect a pronounced time-dependent potentiation of glutamate responses. Dantrolene, another putative intracellular Ca2+ antagonist, was completely without effect in this system with regard to both glutamate-evoked increases in [Ca2+]i and glutamate-induced neurotoxicity.
authors.affiliation: Neurochemistry Group, Centre for Biomolecular Science, School of Biological and Medical Sciences, University of St. Andrews, Fife, Scotland.
authors.forename: C S
authors.initials: CS
authors.lastname: Malcolm
authors.forename: L
authors.initials: L
authors.lastname: Ritchie
authors.forename: A
authors.initials: A
authors.lastname: Grieve
authors.forename: R
authors.initials: R
authors.lastname: Griffiths
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured / Q000166:cytology / Q000187:drug effects; D002531:Cerebellum / Q000166:cytology*; D003620:Dantrolene / Q000494:pharmacology; D005707:Gallic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D018698:Glutamic Acid / Q000633:toxicity*; D051379:Mice; D008815:Mice, Inbred Strains; D009125:Muscle Relaxants, Central / Q000494:pharmacology; D018696:Neuroprotective Agents / Q000494:pharmacology*; D009498:Neurotoxins / Q000494:pharmacology; D013997:Time Factors
chemical_list: D002121:Calcium Channel Blockers; D009125:Muscle Relaxants, Central; D018696:Neuroprotective Agents; D009498:Neurotoxins; D018698:Glutamic Acid; C006014:8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate; D005707:Gallic Acid; D003620:Dantrolene; D002118:Calcium