The rate of Mn2+-induced fluorescence quenching (RFQ) was used as a relative measure of plasma membrane Ca2+ permeability (Pca) in fura-2-loaded cultured hippocampal neurons and cerebellar granule cells during and after protracted (15–30 min) glutamate (GLU) treatment. Some limitations of this method were evaluated using a kinetic model of a competitive binding of Mn2+ and Ca2+ to fura-2 in the cell. In parallel experiment a contribution of Ca2+ influx to the cytoplasinic Ca2+ ([Ca2+]i) was repeatedly examined during and following a prolonged GLU challenge by short-duration “low-Ca2+ trials” (50 pM EGTA) and by measurements of 45CaL2+ uptake. Experiments failed to reveal a putative persistent increase in Pca that earlier was thought to underlie Ca2+ overload of the neuron caused by its toxic GLU treatment. By contrast. a sustained increase of [Ca2+], was found to be associated with a progressive decrease in Pca and Ca2+ influx both in the period of GLU application and after its termination. These findings give new evidence in favour of the hypothesis that the CLU-induced Ca2+ overload of the neuron results mainly from an impairment of its Ca2+ extrusion.
- fluorescence quenching
- membrane permeability