Sites of transmitter release and relation to intracellular Ca 2+ in cultured sympathetic neurons

Abstract

Fluorescence imaging of indo-1 loaded cells was used to monitor influx and distribution of Ca 2+ in cell bodies, neurites and growth cones of sympathetic neurons cultured from embryonic chick. Similar experiments on release of tritiated noradrenaline were performed to assess the relationship between intracellular Ca 2+ concentration ([Ca 2+]) and transmitter release. Effects of Ca 2+ channel antagonists on electrically stimulated rise in [Ca 2+] i were dependent on the neuronal region examined. Cadmium and verapamil blocked Ca 2+ entry in cell bodies but were less effective in neurites and growth cones. Nifedipine partially inhibited Ca 2+ entry in cell bodies and was less effective in neurites and growth cones. Combination of cadmium and nifedipine blocked [Ca 2+] i rise in all neuronal regions. Omega-conotoxin was an effective Ca 2+ channel blocker in all regions. Ca 2+ channel blockers had effects on [3H]noradrenaline release which paralleled effects on [Ca 2+] i in neurites (and growth cones) but not cell bodies. Cadmium, verapamil and nifedipine each caused a partial, reversible block of the evoked release. Combination of cadmium and nifedipine completely blocked evoked [ 3+H]noradrenaline release. Omegaconotoxin caused complete, irreversible block of electrically evoked release. During prolonged depolarization with 125 mM K + Krebs solution, elevation of [Ca 2+] i was maintained in cell bodies but was transient in neurites and growth cones. The amplitude and time course of [ 3H]noradrenaline release paralleled [Ca 2+] i in neurites and growth cones, but not the cell body under the above conditions. A new method is described to study localized uptake and release of [ 3H]noradrenaline in cell bodies versus neurites of sympathetic neurons. Incubation of these modified cultures with [ 3H]noradrenaline showed that cell bodies had very low [ 3H]noradrenaline uptake (0.23 × 10 −6 c.p.m./mg protein), whereas neurites contained approximately 20 times more radioactivity. Depolarization of neurites by excess K + and field stimulation caused a large increase in the net release of [ 3H]noradrenaline. The release was unaffected by removal of cell bodies. Neurites remained functionally viable for more than 2 h after separation from their cell bodies. [ 3H]Noradrenaline release could be evoked repeatedly over this time. [ 3H]Noradrenaline release from isolated neurites was partially blocked by nifedipine and fully blocked by combination of cadmium and nifedipine or by omega-conotoxin. The uptake and release of [ 3H]noradrenaline by neurites alone (expressed per mg protein) accounted for the total [ 3H]noradrenaline in intact cultures with neurites and cell bodies. Therefore, we conclude that neurites (and growth cones) are the prominent sites of uptake, storage and release of sympathetic transmitter. The regulation of Ca 2+ influx and distribution is distinctly different in different neuronal regions and [Ca 2+], of neurites and growth cones only are related to [ 3H]noradrenaline release. Partial effects of cadmium or nifedipine indicate the involvement of more than one Ca 2+ channel type in Ca 2+ entry and [ 3H]noradrenaline release. Regional variation in the effects of these agents suggests regional differences in the types of Ca 2+ channel. The complete block of [ 3H]noradrenaline release and Ca 2+ entry in all regions by omega-conotoxin indicates effective block of all neuronal Ca 2+ channel types.