Ca²⁺-induced noradrenaline release from central noradrenergic neurons promoted by high K⁺ concentration or ionophore A23187

Summary

In slices of rat occipital cortex and hypothalamus preincubated with ³H-noradrenaline and superfused with various Ca²⁺-free solutions, the effect of introduction of CaCl₂ on efflux of total tritium, ³H-noradrenaline and ³H-metabolites was studied.

• 1. When slices of both brain regions were superfused with a Ca²⁺-free buffer containing 30 mM K⁺ and 119 mM Na⁺, introduction of 0.65–2.6 mM CaCl₂ for 2 min induced a concentration-dependent overflow of tritium. Cocaine (100 μM) doubled the overflow of tritium evoked by 1.3 mM CaCl₂. When slices were superfused with physiological salt solution after 100 min of superfusion with the Ca²⁺-free buffer and 2 stimulation periods with 1.3 mM CaCl₂, electrical field stimulation (3 Hz, 13 mA, 2 ms, 2 min duration) still elicited on overflow of tritium which contained 59% ³H-noradrenaline. The CaCl₂-induced overflow was not affected by tetrodotoxin (1 μM), but was inhibited by Mg²⁺ ions (10 mM).

• 2. When cortical slices incubated with Ca²⁺-free solution containing 300 μM ionophore A 23187 (subsequent to preincubation with ³H-noradrenaline) were superfused with Ca²⁺-free solution containing 0.3 mM Mg²⁺ (otherwise composed like physiological salt solution), introduction of 1.3 or 2.6 mM CaCl₂ for 5 min evoked a concentration-dependent overflow of tritium. The evoked overflow of ³H-noradrenaline was similar to that produced by the same Ca²⁺ concentrations after superfusion with K⁺-rich solution. The Ca²⁺-induced tritium overflow from slices preincubated with A 23187 was completely inhibited by 10 mM MgCl₂.

• 3. The Ca²⁺-induced overflow of tritium from cortex slices superfused with 30 mM K⁺ or preincubated with A 23187 was mainly represented by ³H-noradrenaline; the most important degradation products were ³H-dihydroxyphenylglycol and O-methylated deaminated metabolites. Under both conditions 100 μM cocaine increased the proportional ³H-noradrenaline overflow.

It is concluded that combined application of both methods of Ca²⁺-induced stimulation provides a tool for the determination of the site of action of drugs which alter stimulus-release coupling in noradrenergic neurons of the brain.