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Entry of Ca2+ through voltage-dependent Ca2+ 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 Ca2+ 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 Ca2(+)-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 Ca2+ concentration ([Ca2+]i) after addition of Ca2+ to Ca(2+)-Mg(2+)-free medium. The increased [3H]noradrenaline release and [Ca2+]i were dependent on external Na+ and Ca2+, but were not affected by the Ca2+ 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 [Ca2+]i declined rapidly and were sensitive to the Ca2+ 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluorescence imaging of indo-1 loaded cells was used to monitor influx and distribution of Ca2+ 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 Ca2+ concentration ([Ca2+]i) and transmitter release. Effects of Ca2+ channel antagonists on electrically stimulated rise in [Ca2+]i were dependent on the neuronal region examined. Cadmium and verapamil blocked Ca2+ entry in cell bodies but were less effective in neurites and growth cones. Nifedipine partially inhibited Ca2+ entry in cell bodies and was less effective in neurites and growth cones. Combination of cadmium and nifedipine blocked [Ca2+]i rise in all neuronal regions. Omega-conotoxin was an effective Ca2+ channel blocker in all regions. Ca2+ channel blockers had effects on [3H]noradrenaline release which paralleled effects on [Ca2+]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 [3H]noradrenaline release. Omega-conotoxin caused complete, irreversible block of electrically evoked release. During prolonged depolarization with 125 mM K+ Krebs solution, elevation of [Ca2+]i was maintained in cell bodies but was transient in neurites and growth cones. The amplitude and time course of [3H]noradrenaline release paralleled [Ca2+]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 x 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.(ABSTRACT TRUNCATED AT 400 WORDS)

The concentration of cytosolic free Ca2+ ([Ca2+]i) and the release of tritiated norepinephrine ([3H]NE) were monitored during Ba2+ stimulation of sympathetic neurons cultured from chick embryos. Ba2+ (2.5 mM in Ca(2+)-free medium) caused a rise in [Ca2+]i in all regions (cell bodies, neurites, and growth cones) of sympathetic neurons and evoked [3H]NE release in the absence of other stimuli. The increase in [Ca2+]i and release of [3H]NE were sustained for up to 30 min in the presence of Ba2+. When Ba(2+)-stimulated cells were immediately washed in Ca(2+)-free Ba(2+)-free EGTA solution, both the elevated [Ca2+]i and [3H]NE release returned to basal levels, with similar, fast, time courses. Ba2+ also blocked Ca2+ efflux from neurons loaded with 45Ca. We conclude from the parallel effects of Ba2+ on [Ca2+]i and [3H]NE release that Ba2+ stimulates exocytosis by a Ca(2+)-dependent mechanism. The Ba(2+)-induced rise in [Ca2+]i is a result of two separate actions: (i) the release of Ca2+ from intracellular sites and (ii) an effective block of Ca2+ extrusion. The ability of Ba2+ to release Ca2+ in growth cones that are insensitive to caffeine suggests that Ba2+ may displace Ca2+ from binding sites other than endoplasmic reticulum.

We studied the effects of lanthanum (La3+) on the release of 3H-norepinephrine (3H-NE), intracellular Ca2+ concentration, and voltage clamped Ca2+ and K+ currents in cultured sympathetic neurons. La3+ (0.1 to 10 microM) produced concentration-dependent inhibition of depolarization induced Ca2+ influx and 3H-NE release. La3+ was more potent and more efficacious in blocking 3H-NE release than the Ca(2+)-channel blockers cadmium and verapamil, which never blocked more than 70% of the release. At 3 microM, La3+ produced a complete block of the electrically stimulated rise in intracellular free Ca2+ ([Ca2+]i) in the cell body and the growth cone. The stimulation-evoked release of 3H-NE was also completely blocked by 3 microM La3+. However, 3 microM La3+ produced only a partial block of voltage clamped Ca2+ current (ICa). Following La3+ (10 microM) treatment 3H-NE release could be evoked by high K+ stimulation of neurons which were refractory to electrical stimulation. La3+ (1 microM) increased the hyperpolarization activated, 4-aminopyridine (4-AP) sensitive, transient K+ current (IA) with little effect on the late outward current elicited from depolarized holding potentials. We conclude that the effective block of electrically stimulated 3H-NE release is a result of the unique ability of La3+ to activate a stabilizing, outward K+ current at the same concentration that it blocks inward Ca2+ current.