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Protein Kinase C of Sympathetic Neuronal Membrane Is Activated by Phorbol Ester‐Correlation Between Transmitter Release, 45 Ca 2+ Uptake, and the Enzyme Activity
Author(s) -
Malhotra Ravindra K.,
Bhave Sanjiv V.,
Wakade Taruna D.,
Wakade Arun R.
Publication year - 1988
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1988.tb01834.x
Subject(s) - phorbol , protein kinase c , stimulation , medicine , norepinephrine , protein kinase a , endocrinology , biology , chemistry , enzyme , biochemistry , dopamine
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 [ 3 H]norepinephrine, 45 Ca 2+ 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 [ 3 H]‐norepinephrine and the release of [ 3 H]norepinephrine was determined before and after electrical stimulation. Stimulation at 1 Hz for 15 s increased the release of [ 3 H]‐norepinephrine over the nonstimulation period. Stimulation‐evoked release gradually declined with time during subsequent stimulation periods. Incubation of neurons in Ca 2+ ‐free Krebs solution containing 1 m M EGTA completely blocked stimulation‐evoked release of [ 3 H]‐norepinephrine. Stimulation‐evoked release of [ 3 H]‐norepinephrine was markedly facilitated by 3 and 10 n M PDB or TPA. The spontaneous release was also enhanced by PDB and TPA. The net accumulation of 45 Ca 2+ during stimulation of sympathetic neurons was increased by two‐to fourfold in the presence of PDB or TPA. PDB at 1–100 n M produced a concentration‐dependent increase in the activation of protein kinase C. PDB at 30 n M increased the activity of protein kinase C of the paniculate 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 paniculate to cytosolic protein kinase C increased from a control value of 0.62 to 4.39 after treatment with 30 n M PDB. TPA (10 and 30 nM ) also increased protein kinase C activity of the paniculate fraction by six‐ to eightfold. Phorbol 13‐acetate had no effect on protein kinase C activity, [ 3 H]norepinephrine release, and 45 Ca 2+ accumulation. These results provide direct evidence that activation of protein kinase C enhances Ca 2+ accumulation, which in turn leads to the facilitation of transmitter release in sympathetic neurons.