| Summary: | 碩士 === 國立臺灣大學 === 動物學研究研究所 === 94 === Na+/Ca2+ exchanger is an antiporter on plasma membrane which transports Ca2+ out (forward mode) of or into (reverse mode) the cell depending on the electrochemical gradients of Na+ and Ca2+. The exchanger can be either K+-dependent (NCKX) or -independent (NCX). However, the roles of the reverse mode activity in the stimulus-secretion coupling are not clear in rat embryonic neuronal cells. Therefore, we monitored the intracellular Ca2+ concentration ([Ca2+]i) response by fura-2 fluorescence dye. The reverse mode activity can be activated by treating the cell with ouabain to increase intracellular Na+ concentration and replacing extracellular Na+ by NMG. This elevation in [Ca2+]i was inhibited by KB-R7943 and K+ free NMG.. Furthermore, we confirmed NCX1~3, and NCKX2~4 were expressed in rat embryonic neurons by RT-PCR technique. These results show that both NCX and NCKX are involved in this elevation. We also found this elevation in [Ca2+]i was inhibited by thapsigargin, 2-APB or ryanodine to 30.94 ± 5.23, 31.89 ± 5.47, or 53.78 ± 5.47 % of control cells, respectively. This elevation in [Ca2+]i could elevate [Ca2+]i in downstream postsynaptic neurons but blocked by CNQX. About 35 % of the elevation is also suppressed by U73122. These results suggest that the elevation in [Ca2+]i is facilitated by intracellular Ca2+ stores and can induce neurotransmitter release When neuron was stimulated by AMPA, the elevation in [Ca2+]i was significantly inhibited by thapsigargin; on the contrary, the elevation in [Ca2+]i elicited by high K+ stimulation was slightly enhanced by thapsigargin. We propose that Na+ influx through ionotropic receptors may not only depolarize the cell but also activate the reverse mode of Na+/Ca2+ exchanger to induce Ca2+ release from the intracellular stores and in turn release neurotransmitters to facilitate synaptic transmission.
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