J. Biol. Chem., Vol. 260, Issue 30, 16294-16302, 12, 1985
Calcium permeability changes and neurotransmitter release in cultured brain neurons. II. Temporal analysis of neurotransmitter release
M Yarom, N Zurgil and N Zisapel
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 1.5-6-s intervals 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 (t1/2 = 9.6 s) which
comprised about 40% of the neurotransmitter content of the cells and a
subsequent slower efflux (t1/2 = 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 6 s
after the onset of depolarization inhibited the fast release but failed to
do so when added later on.(ABSTRACT TRUNCATED AT 400 WORDS)
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