Importance of protein kinase C for normal development of transmitter release properties in embryonic chick sympathetic neurons in culture.

Abstract

The hypothesis that multiple trophic inputs are essential for normal development of transmitter release properties in sympathetic neurons was tested using two supportive agents (excess KCl and phorbol 12,13-dibutyrate which produce marked activation of protein kinase C and also support survival of chick sympathetic neurons in culture) in addition to nerve growth factor, ciliary neurotrophic factor and neurotrophin-3. Basal and electrically evoked (10 pulses at 1 Hz and 10 Hz) release of [(3)H]norepinephrine from neurons supported by nerve growth factor was very high (1.5 to 2% of total [(3)H]norepinephrine content) and relatively insensitive to facilitation by tetraethylammonium as compared to release in neuroeffector organs, and the frequency-release response was negative. In K+-supported neurons, basal [(3)H]norepinephrine release was almost four-fold lower, evoked release was four- to eight-fold lower, the frequency response was flat to positive, and tetraethylammonium increased evoked release up to four-fold. Inclusion of nerve growth factor in culture did not modify the effects of K+ on basal or evoked release, and nerve growth factor plus ciliary neurotrophic factor and/or neurotrophin-3 did not produce the changes observed in K+-supported neurons. Neurons supported by phorbol ester had a low background release, low evoked release, a positive frequency-release response, and 10- to 30-fold facilitation by tetraethylammonium of release evoked by 1 Hz or 1 pulse stimulation. Thus, physiological and pharmacological behavior of transmitter release of sympathetic neurons supported by excess KCl or phorbol ester was very similar to their counterparts growing in the body. Neurons supported by nerve growth factor showed an immediate rise in stimulated [Ca(2)+]i that was three- to five-fold above basal levels with either 1 Hz or 10 Hz stimulation. However, in phorbol supported neurons, [Ca(2)+]i rose gradually to about 1.5 times basal levels during 1 Hz stimulation and increased further with 10Hz stimulation. Tetraethylammonium had little effect on stimulated [Ca(2)+]i in nerve growth factor-supported neurons, but greatly facilitated the stimulated rise in [Ca(2)+]i in phorbol-supporte neurons. The data show that multiple trophic inputs distinct from nerve growth factor, neurotrophin-beta or ciliary neurotrophic factor are required for normal physiological function of sympathetic neurons.