Role of Ca2+ in α‐Amino‐3‐Hydroxy‐5‐Methyl‐4‐Isoxazolepropionic Acid‐Mediated Polyphosphoinositide Turnover in Primary Neuronal Cultures

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Abstract
Excitatory amino acid (EAA) receptors and EAA-mediated stimulation of polyphosphoinositide (poly-PI) turnover were studied in cultured neurons at different days in vitro (DIV). Six main observations have emerged from these studies: (a) Neurons increased their sensitivity to EAAs as a function of time in culture, indicated by increasing EAA-mediated poly-PI turnover. (b) Extracellular Ca2+ concentration played an important role in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-stimulated poly-PI turnover in cells at 4 DIV, whereas poly-PI turnover mediated by L-glutamate and trans-1-amino-cyclopentane-1,3-dicarboxylic acid was not Ca(2+)-dependent. (c) A marked stimulation of poly-PI turnover by AMPA was seen in the cultured neurons at 4 DIV, but not at 17 DIV, suggesting that a distinct EAA receptor sensitive to AMPA is transiently expressed. (d) The Ca2+ ionophore A23187 increased poly-PI turnover in cultured neurons, suggesting that Ca2+ entry is involved in stimulating poly-PI turnover. (e) Stimulation of poly-PI turnover by carbachol was greater in neurons at 17 DIV as compared with 4 DIV, and appeared to be Ca(2+)-dependent across DIV. (f) 6-Cyano-7-nitroquinoxaline-2,3-dione, an antagonist for non-N-methyl-D-aspartate ionotropic EAA receptors, inhibited 100% and 35% of AMPA- and quisqualate-induced poly-PI turnover, respectively, suggesting an involvement of ionotropic AMPA/quisqualate receptors in stimulating poly-PI turnover.