The coupling between depolarization-induced calcium entry and neurotransmitter release was studied in rat brain neurons in culture. The endogenous dopamine content of the cells was determined by high performance liquid chromatography utilizing electrochemical detection. The amount of dopamine in unstimulated cells was found to be about 16 ng/mg of protein. Depolarization of the neurons by elevated K+ caused a Ca2+-dependent release of dopamine from the cells. Following 1 min of depolarization, the cellular dopamine content and the amount of [3H]dopamine in cells preloaded with the radioactive transmitter were reduced by 35%. The release of [3H]dopamine by the neurons was measured at 1.5-6intervals by a novel rapid dipping technique. Depolarization in the presence of Ca2+ (1.8 mM) enhanced the rate of neurotransmitter release by 90-fold (0.072 ± 0.003 s-1) over the basal release in the presence of Ca2+. The evoked release consisted of a major rapidly terminating phase (t(1/2) = 9.6 s) which comprised about 40% of the neurotransmitter content of the cells and a subsequent slower efflux (t(1/2) = 575 s) which was observed during following prolonged depolarization. Predepolarization of the cells in the absence of extracellular Ca2+ did not affect the kinetics of the evoked release. The fast evoked release could be re-elicited in the cells after 20 min 'rest' in reference low K+ buffer. The effects of varying the extracellular Ca2+ concentrations on the kinetic parameters of the evoked release were measured. The amount of neurotransmitter released during the fast kinetic phase was very sensitive to the external Ca2+ (from 0% in the absence of Ca2+ to 40% of the neurotransmitter content at Ca2+ 0.3 mM). The rate constant of the fast release did not depend on the extracellular Ca2+, whereas the rate constant of the slow release increased from 0.0004 ± 0.0001 s-1 at 0.4 mM Ca2+ to 0.0012 ± 0.0002 s-1 at 0.8 mM Ca2+. The fast evoked release was inhibited by verapamil in a concentration-dependent manner. By contrast, verapamil enhanced the basal and the slow release independent of the presence of Ca2+. Both fast and slow phases of the evoked release were blocked by Co2+. Addition of Co2+ within the first 6s after the onset of depolarization inhibited the fast release but failed to do so when added later on. The results indicate that the amount of neurotransmitter which is released during the fast phase is determined by the amount of Ca2+ entering the cells during the initial stages of stimulation and that subsequently Ca2+ is responsible for the cessation of neurotransmitter release. The possibility that inactivation of the voltage-dependent Ca2+ channel limits the fast phase of neurotransmitter release is discussed.
|Number of pages||9|
|Journal||Journal of Biological Chemistry|
|State||Published - 1985|