Protein kinase C of sympathetic neuronal membrane is activated by phorbol ester--correlation between transmitter release, 45Ca2+ uptake, and the enzyme activity.
The effects of phorbol esters [phorbol 12,13-dibutyrate (PDB), 12-O-tetradecanoylphorbol 13-acetate (TPA), and phorbol 13-acetate] were investigated on the release of [3H]norepinephrine, 45Ca2+ accumulation, and protein kinase C activity in cultured sympathetic neurons of the chick embryo. Sympathetic neurons derived from 10-day-old chick embryo were cultured in serum-free medium supplemented with insulin, transferrin, and nerve growth factor. After 3 days, neurons were loaded with [3H]-norepinephrine and the release of [3H]norepinephrine was determined before and after electrical stimulation. Stimulation at 1 Hz for 15 s increased the release of [3H]-norepinephrine over the nonstimulation period. Stimulation-evoked release gradually declined with time during subsequent stimulation periods. Incubation of neurons in Ca2+-free Krebs solution containing 1 mM EGTA completely blocked stimulation-evoked release of [3H]-norepinephrine. Stimulation-evoked release of [3H]-norepinephrine was markedly facilitated by 3 and 10 nM PDB or TPA. The spontaneous release was also enhanced by PDB and TPA. The net accumulation of 45Ca2+ during stimulation of sympathetic neurons was increased by two- to fourfold in the presence of PDB or TPA. PDB at 1-100 nM produced a concentration-dependent increase in the activation of protein kinase C. PDB at 30 nM increased the activity of protein kinase C of the particulate fraction from 0.09 to 0.58 pmol/min/mg protein. There was no significant change in protein kinase C activity of the cytosolic fraction (0.14 pmol/min/mg versus 0.13 pmol/min/mg protein). The ratio of the particulate to cytosolic protein kinase C increased from a control value of 0.62 to 4.39 after treatment with 30 nM PDB. TPA (10 and 30 nM) also increased protein kinase C activity of the particulate fraction by six- to eightfold. Phorbol 13-acetate had no effect on protein kinase C activity, [3H]norepinephrine release, and 45Ca2+ accumulation. These results provide direct evidence that activation of protein kinase C enhances Ca2+ accumulation, which in turn leads to the facilitation of transmitter release in sympathetic neurons.
Malhotra
R K
RK
Department of Pharmacology, State University of New York, Brooklyn.
Bhave
S V
SV
Wakade
T D
TD
Wakade
A R
AR
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002132:Calcium Radioisotopes; D002462:Cell Membrane / Q000201:enzymology; D004558:Electric Stimulation; D004789:Enzyme Activation; D009474:Neurons / Q000201:enzymology* / Q000648:ultrastructure; D009638:Norepinephrine / Q000378:metabolism*; D010703:Phorbol Esters / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism*; D013564:Sympathetic Nervous System / Q000166:cytology / Q000201:enzymology* / Q000648:ultrastructure
D002132:Calcium Radioisotopes; D010703:Phorbol Esters; D011493:Protein Kinase C; D002118:Calcium; D009638:Norepinephrine