Relationship between membrane depolarization, calcium influx and norepinephrine release in sympathetic neurons maintained in culture
Wakade, A.R.; Wakade, T.D.
Journal of Pharmacology and Experimental Therapeutics 223(1): 125-129
1982
ISSN/ISBN: 0022-3565 PMID: 7120113 Document Number: 185076
Dissociated neurons from paravertebral sympathetic chains of 11- to 13-day-old chick embryos were maintained in culture medium supplemented with 10% (vol/vol) heat-inactivated horse serum and nerve growth factor and used to study changes in resting membrane potential, Ca influx and release of [3H]norepinephrine ([3H]NE) in various K concentrations. Incubation of sympathetic neurons with [3H]NE led to an accumulation of radioactivity, and the accumulation was markedly depressed by the neuronal uptake blocker desipramine or by low temperature. Accumulation in neuronal cultures of [3H]tetraphenylphosphonium ([3H]TPP), a lipophilic agent used to measure resting membrane potential of various cells, increased as the incubation time was increased, and a maximum accumulation occurred in .apprx. 1 h. Reduction in external K concentration from 5.9 to 0 mM caused a 50% increase in [3H]TPP accumulation (over that in 5.9 mM K). Increase in K concentrations to 20, 35 and 70 mM produced a decrease in [3H]TPP accumulation. When K concentration was shifted from 0 to 5.9 mM, which produced a significant reduction in [3H]TPP accumulation, there was neither a change in the influx of 45Ca nor a release of [3H]NE. A further increase in K concentration to 20 and 35 mM caused an increase in 45Ca influx and [3H]NE release. At 60 and 120 mM K, there were no additional increases in 45Ca influx, and [3H]NE release also leveled off at these K concentrations. Treatment of neurons with 0.2 .mu.M tetrodotoxin did not modify 45Ca influx or [3H]NE release. In 20 mM Mg, 45Ca influx and [3H]NE were markedly reduced. Although neurons undergo a substantial change in their membrane potential when K concentration is varied between 0-70 mM, influx of Ca and release of sympathetic transmitter begin to occur only after a critical level of depolarization.