N-methyl-D-aspartate increases cytosolic Ca2+ via G proteins in cultured hippocampal neurons

The changes of the cytosolic Ca2+ concentrations ([Ca2+]i) induced by N-methyl-D-aspartate (NMDA) in fura-2-loaded cultured hippocampal neurons from rat embryos were investigated by the fast application method, using a fine pipe under extracellular Mg(2+)-free conditions. In the presence of Ca2+, NMDA, at concentrations in excess of 3 microM, induced a biphasic increase of [Ca2+]i, which consisted of an initial increase with a second rise that occurred after cessation of drug application. Under Ca(2+)-free conditions, NMDA (greater than 100 microM) in the absence of glycine or NMDA (greater than 50 microM) in the presence of glycine (greater than 10 microM) induced intracellular Ca2+ mobilization, which was blocked by 30 microM 2-amino-5-phosphonovaleric acid (APV) and reduced by islet-activating protein. When the neurons were superfused with Ca(2+)-free solution, the application of 3-10 microM NMDA, which had been dissolved in Ca(2+)-containing solution, induced the second phase [Ca2+]i increase, whereas application of kainate, quisqualate, or stimulation by 50 mM K+ did not. Islet-activating protein, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), and D-sphingosine reduced the second phase [Ca2+]i increase. These results suggest that NMDA-induced intracellular Ca2+ mobilization is potentiated by the initial entry of Ca2+ into the cells and is regulated in an islet-activating protein-sensitive manner.