Concerted enhancement of calcium influx. Neurotransmitter release and protein phosphorylation by a phorbol ester in cultured brain neurons

We have recently shown that the phorhol 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, last 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 Ca⁺². Various phorbol derivatives known to activate the Ca⁺² 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, Ca⁺² dependent increase in the amount of³²Pi incorporated into a 43,000 dalton protein and decrease in the amount incorporated into a 55,000 dalton protein. These changes mimicked the Ca⁺² dependent changes in protein phosphorylation which occur upon stimulation of the neurons. Kinetic studies of depolarization induced Ca⁺² uptake by the neurons indicated that 12-O-tetradecanoyl-phorbol-13-acetate enhanced the maximal influx of Ca⁺² through the voltage sensitive Ca⁺² channels by 40%. The results indicate that 12-O-tetradecanoyl-phorbol-13-acetatc acts primarily on the regulation of stimulated Ca²⁴ entry into the cells. Consequently neurotransmitter release at suhmaximal extracellular [Ca⁺²] is enhanced.