phorbol-12-13-didecanoate and safingol

Nerve terminals ("synaptosomes") isolated from rat brain hippocampus were loaded with the fluorescent Ca2+ indicator fura-2 and were subjected to depolarization with an elevated K+ concentration in a stopped-flow spectrophotometer to measure the activity of voltage-gated Ca2+ channels in the presynaptic membrane. Three components of Ca2+ influx were seen, which were tentatively identified as two classes of voltage-dependent Ca2+ channels with different inactivation kinetics (tau of approximately 60 ms and 1 s, respectively) and Na+/Ca2+ exchange working in the "reverse" mode. The activity of both classes of voltage-dependent Ca2+ channels was slightly augmented by the phorbol ester phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), but the effect of PMA was markedly enhanced by the protein phosphatase inhibitor okadaic acid (OKA). The PKC inhibitors calphostin C and dihydrosphingosine (DHS) caused a prompt decrease in voltage-dependent Ca2+ channel activity, but the effect of DHS could be slowed by coaddition of OKA. These results suggest that the activity of presynaptic voltage-dependent Ca2+ channels in the hippocampus is under a dynamic balance between PKC phosphorylation (leading to activation) and protein phosphatase dephosphorylation (leading to inactivation) and that both of these metabolic pathways are tonically active in the nerve terminals.

Topics: Animals; Calcium; Calcium Channels; Fura-2; Hippocampus; Kinetics; Naphthalenes; Phorbol Esters; Phosphorylation; Polycyclic Compounds; Potassium; Presynaptic Terminals; Protein Kinase C; Rats; Sodium; Spectrometry, Fluorescence; Sphingosine; Tetradecanoylphorbol Acetate