IMPORTANCE OF PROTEIN-KINASE-C FOR NORMAL DEVELOPMENT OF TRANSMITTER RELEASE PROPERTIES IN EMBRYONIC CHICK SYMPATHETIC NEURONS IN CULTURE

The hypothesis that multiple trophic inputs are essential for normal d evelopment of transmitter release properties in sympathetic neurons wa s tested using two supportive agents (excess KCl and phorbol 12,13-dib utyrate which produce marked activation of protein kinase C and also s upport survival of chick sympathetic neurons in culture) in addition t o nerve growth factor, ciliary neurotrophic factor and neurotrophin-3. Basal and electrically evoked (10 pulses at 1 Hz and 10 Hz) release o f [H-3]norepinephrine from neurons supported by nerve growth factor wa s very high (1.5 to 2% of total [H-3]norepinephrine content) and relat ively insensitive to facilitation by tetraethylammonium as compared to release in neuroeffector organs, and the frequency-release response w as negative. In K+-supported neurons, basal [H-3]norepinephrine releas e was almost four-fold lower, evoked release was four- to eight-fold l ower, the frequency response was flat to positive, and tetraethylammon ium increased evoked release up to four-fold. Inclusion of nerve growt h factor in culture did not modify the effects of K+ on basal or evoke d release, and nerve growth factor plus ciliary neurotrophic factor an d/or neurotrophin-3 did not produce the changes observed in K+-support ed neurons. Neurons supported by phorbol ester had a low background re lease, 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 ex cess KCl or phorbol ester was very similar to their counterparts growi ng in the body. Neurons supported by nerve growth factor showed an imm ediate rise in stimulated [Ca2+](i) that was three- to five-fold above basal levels with either 1 Hz or 10 Hz stimulation. However, in phorb ol supported neurons, [Ca2+](i) rose gradually to about 1.5 times basa l levels during 1 Hz stimulation and increased further with 10 Hz stim ulation. Tetraethylammonium had little effect on stimulated [Ca2+](i) in nerve growth factor-supported neurons, but greatly facilitated the stimulated rise in [Ca2+](i) in phorbol-supported neurons. The data sh ow that multiple trophic inputs distinct from nerve growth factor, neu rotrophin-3 or ciliary neurotrophic factor are required for normal phy siological function of sympathetic neurons.