Concerted enhancement of calcium influx, neurotransmitter release and protein phosphorylation by a phorbol ester in cultured brain neurons.
We have recently shown that the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate enhances the depolarization induced, calcium dependent release of [3H]dopamine from cultured brain neurons in the rat. In the present study the effects of 12-O-tetradecanoyl-phorbol-13-acetate on the kinetic parameters of depolarization induced calcium influx and on Ca2+ dependent neurotransmitter release and protein phosphorylation were investigated. Depolarization induced neurotransmitter release from the neurons occurs in two phases: an initial, fast release and a subsequent slow release. At low extracellular Ca2+, 12-O-tetradecanoyl-phorbol-13-acetate enhanced the quantity of fast release and in addition, increased the rate constant of the slow release. These effects mimicked the effects of increasing the extracellular Ca2+. Various phorbol derivatives known to activate the Ca2+ activated phospholipid dependent protein kinase (protein kinase C) were also able to enhance the stimulated release of [3H]dopamine from the neurons. 12-O-tetradecanoyl-phorbol-13-acetate induced the incorporation of 32Pi into a protein with an apparent molecular weight of 45,000 daltons regardless of depolarization or of the presence of Ca2+. In addition, 12-O-tetradecanoyl-phorbol-13-acetate induced in unstimulated neurons, Ca2+ dependent increase in the amount of 32Pi incorporated into a 43,000 dalton protein and decrease in the amount incorporated into a 55,000 dalton protein. These changes mimicked the Ca2+ dependent changes in protein phosphorylation which occur upon stimulation of the neurons. Kinetic studies of depolarization induced Ca2+ uptake by the neurons indicated that 12-O-tetradecanoyl-phorbol-13-acetate enhanced the maximal influx of Ca2+ through the voltage sensitive Ca2+ channels by 40%. The results indicate that 12-O-tetradecanoyl-phorbol-13-acetate acts primarily on the regulation of stimulated Ca2+ entry into the cells. Consequently neurotransmitter release at submaximal extracellular [Ca2+] is enhanced.
D000818:Animals; D001921:Brain / Q000187:drug effects* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004298:Dopamine / Q000378:metabolism*; D004622:Embryo, Mammalian; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D013268:Stimulation, Chemical; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D009419:Nerve Tissue Proteins; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D004298:Dopamine