Massive exocytosis triggered by sodium-calcium exchange in sympathetic neurons is attenuated by co-culture with cardiac cells

Abstract

Entry of Ca 2+ through voltage-dependent Ca 2+ channels is known to be linked to the exocytotic release of transmitter from sympathetic neurons. In this paper we provide evidence that transmitter release can also be stimulated by Ca 2+ influx via the Na-Ca exchanger. Furthermore, the release linked to Na-Ca exchange is regulated by cardiac target cells. Cultured sympathetic neurons of the chick embryo incubated in Ca 2+-Mg 2+-free Krebs solution for 20 min and then switched to Ca 2+-containing solution exhibited 15–20-fold increases in [ 3H]noradrenaline release over the spontaneous release. Electrophysiologic studies showed that neurons were completely depolarized in Ca 2+-Mg 2+-free medium. Indo-1 fluorescence revealed a large and sustained increase in intracellular free Ca 2+ concentration ([Ca 2+] i) after addition of Ca 2+ to Ca 2+-Mg 2+-free medium. The increased [ 3H]noradrenaline release and [Ca 2+] i were dependent on external Na + and Ca 2+, but were not affected by the Ca 2+ channel blockers lanthanum, cadmium, verapamil or omega-conotoxin. A conventional depolarizing stimulus (125 mM K +) produced a 13-fold increase in [ 3H]noradrenaline release over spontaneous release. However, K +-induced release and rise in [Ca 2+] i declined rapidly and were sensitive to the Ca 2+ channel blockers. When sympathetic neurons were co-cultured with embryonic cardiac cells the release induced by change from Ca 2+-Mg 2+-free to Ca 2+-Krebs solution was dramatically reduced. The change from Ca 2+-Mg 2+-free to Ca 2+-Krebs solution was ineffective in evoking [ 3H]noradrenaline release from sympathetic neurons in situ using perfused hearts of 15-day-old chick embryos. Taken together, our results implicate Na-Ca exchange in the release of sympathetic transmitter in embryonic neurons and suggest that regulation of developing neurons by target cells may affect Ca 2+ handling by the Na-Ca exchanger.