Protein Kinase C Activation Enhances K+‐Stimulated Endogenous Dopamine Release from Rat Striatal Synaptosomes in the Absence of an Increase in Cytosolic Ca2+

Abstract: The possibility that protein kinase C modulates neurotransmitter release in brain was investigated by examining the effects of 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) on Ca2+ transport and endogenous dopamine release from rat striatal synaptosomes. TPA (0.16 and 1.6 μM) significantly increased dopamine release by 24 and 33%, respectively, after a 20‐min preincubation with TPA followed by 60 s of depolarization with 30 mM KCl. Depolarization‐induced 45Ca2+ uptake, measured simultaneously with dopamine release, was not significantly increased by TPA. Neither 45Ca2+ uptake nor dopamine release was altered under resting conditions. When the time course of K+‐stimulated 45Ca2+ uptake and dopamine release was examined, TPA (1.6 μM) enhanced dopamine release after 15, 30, and 60 s, but not 1, 3, or 5 s, of depolarization. A slight increase in 45Ca2+ uptake after 60 s of depolarization was also seen. The addition of 30 mM KCl to synaptosomes which had been preloaded with the Ca2+‐sensitive fluorophore fura‐2 increased the cytosolic free Ca2+ concentration ([Ca2+]i) from 445 nM to 506 nM after 10 s of depolarization and remained elevated after 60 s. TPA had no effect on [Ca2+]i under depolarizing or resting conditions. Replacing extracellular Ca2+ with 100 μM EGTA reduced K+‐stimulated (60 s) endogenous dopamine release by 53% and decreased [Ca2+]i to 120 nM. In Ca2+‐free medium, 30 mM KCl did not produce an increase in the [Ca2+]i. TPA (1.6 μM) did not alter the [Ca2+]i under resting or depolarizing conditions, but did increase K+‐stimulated dopamine release in Ca2+‐free medium. The inactive 4α‐phorbol had no effect under any of the above experimental conditions. The demonstration that TPA induced an increase in dopamine release without an increase in the [Ca2+]i may suggest that protein kinase C increases the sensitivity of the exocytotic release process to Ca2+.