Reverse mode Na+/Ca2+ exchangers trigger the release of Ca2+ from intracellular Ca2+ stores in cultured rat embryonic cortical neurons

The importance of Na+/Ca2+ exchangers in the regulation of the physiological and pathological functions of the nervous system has been widely recognized. In this study, we used primary cultured E14.5 cortical neurons as a model system to study the possible roles of the reverse mode Na+/Ca2+ exchange activity in neurotransmission. Using RT-PCR, several exchanger isoforms, ncx1, ncx3 and nckx2–4 were found to be expressed in freshly isolated and cultured cortical neurons. Expression of ncx2 was undetectable in freshly isolated neurons but increased with time in culture. Neurons were treated with ouabain to increase the intracellular Na+ concentration and the extracellular Na+ was replaced by N-methyl-d-glucamine to activate reverse mode Na+/Ca2+ exchange. During the maturation of the neurons, the exchange activity shifted from mostly K+-dependent exchange to both K+-dependent and K+-independent exchange. The [Ca2+]i rises were mostly suppressed by ryanodine and thapsigargin treatments, indicating contributions from the intracellular Ca2+ stores. This [Ca2+]i elevation could propagate to the axon terminal and resulted in elevated [Ca2+]i at the postsynaptic neurons based on the fact that the elevation in the postsynaptic neuron was inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione and tetanus toxin. When neurons were stimulated by AMPA to increase the intracellular Na+ concentration, the [Ca2+]i elevations were significantly inhibited by thapsigargin pretreatment and by KB-R7943. These results demonstrate that, in cultured cortical neurons, the influx of Na+ through the ionotropic glutamate receptor activates reverse Na+/Ca2+ exchange, which then triggers the release of Ca2+ from intracellular Ca2+ stores to enhance Ca2+ signaling and neurotransmitter release.