Distinct influx pathways, not calcium load, determine neuronal vulnerability to calcium neurotoxicity

Many forms of neurodegeneration are ascribed to excessive cellular Ca²⁺ loading (Ca²⁺ hypothesis). We examined quantitatively whether factors other than Ca²⁺ loading were determinants of excitotoxic neurodegeneration. Cell survival, morphology, free intracellular Ca²⁺ concentration ([Ca²⁺](i)), and ⁴⁵Ca²⁺ accumulation were measured in cultured cortical neurons loaded with known quantities of Ca²⁺ through distinct transmembrane pathways triggered by excitatory amino acids, cell membrane depolarization, or Ca²⁺ ionophores. Contrary to the Ca²⁺ hypothesis, the relationships between Ca²⁺ load and cell survival, free [Ca²⁺](i), and Ca²⁺-induced morphological alterations depended primarily on the route of Ca²⁺ influx, not the Ca²⁺ load. Notably, Ca²⁺ loading via NMDA receptor channels was toxic, whereas identical Ca²⁺ loads incurred through voltage-sensitive Ca²⁺ channels were completely innocuous. Furthermore, accounting quantitatively for Ca²⁺ loading via NMDA receptors uncovered a previously unreported component of L-glutamate neurotoxicity apparently not mediated by ionotropic or metabotropic glutamate receptors. It was synergistic with toxicity attributable to glutamate-evoked Ca²⁺ loading, and correlated with enhanced cellular ATP depletion. This previously unrecognized toxic action of glutamate constituted a chief excitotoxic mechanism under conditions producing submaximal Ca²⁺ loading. We conclude that (a) Ca²⁺ neurotoxicity is a function of the Ca²⁺ influx pathway, not Ca²⁺ load, and (b) glutamate toxicity may not be restricted to its actions on glutamate receptors.