MODULATION OF ELECTRICALLY-EVOKED [H-3] NORADRENALINE RELEASE FROM CULTURED CHICK SYMPATHETIC NEURONS

In the present study we attempted a comprehensive characterization of modulation of noradrenaline release from chick sympathetic neurons. To this purpose sympathetic neurons derived from chick lumbosacral parav ertebral ganglia and kept in culture for 7 days were loaded with 0.05 mu mol/l [H-3]-noradrenaline and subjected to electrical field stimula tion (36 pulses/3 Hz). Since the released transmitter was partially re captured, superfusion was usually performed in the presence of (+)-oxa protiline, an inhibitor of noradrenaline re-uptake. [H-3]-Noradrenalin e was released in a manner which was dependent on extracellular Ca2+ a nd sensitive to tetrodetoxin (TTX). omega-Conotoxin (omega-CTX; 100 nm ol/l) abolished [H-3]-noradrenaline release indicating that influx thr ough omega-CTX-sensitive Ca2+-channels was essential for transmitter r elease. -5-nitro-4-[2-(trifluoromethyl)-phenyl]-3-pyridine carboxylic acid methyl ester (( +/-)Bay K 8644) and -dihydro-2,6-dimethyl-3-nitro -5-pyridinecarboxylic acid isopropyl ester ((+)-202-791), agonists at L-type voltage sensitive Ca2+-channels (VSCCs), increased noradrenalin e release and induced, in addition, an overflow of tritium which was C a2+-dependent and prevented by the presence of TTX. The L-type VSCC an tagonists (-)-202-791 and azanyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridin edicar boxylic acid methyl, isopropyl ester) ((+)-PN 200-110) diminish ed [H-3]-noradrenaline release. These data suggest that L-type VSCCs, probably located on the cell body of the neuron, play an additional ro le in modulation of release. The full alpha(2)-adrenoceptor agonists 5 -bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK-14,304) and noradren aline significantly inhibited noradrenaline release, whereas clonidine , a partial alpha(2)-agonist, produced only a slight inhibition even a t 10 mu mol/l. The facilitation of noradrenaline release observed in t he presence of the alpha(2)-adrenoceptor antagonist rauwolscine was ve ry low in comparison to that obtained with brain slices and isolated s mooth muscle tissues. These results corroborate the observation that n oradrenaline release from chick sympathetic neurons is regulated by an alpha(2)-adrenoceptor which needs further subtype characterization. T he experiments were mostly performed at 25 degrees C, since a rise in temperature to 37 degrees C increased the resting outflow, but not the evoked overflow of tritium, approximately 4-fold. In the presence of pargyline to block monoamine oxidase, however, the temperature-depende nt enhancement was diminshed and the release showed properties compara ble to those observed at 25 degrees C (with respect to TTX-sensitivity , Ca2+ dependence and modulation via alpha(2)-adrenoceptors). In addit ion to the alpha(2)-adrenoceptors, we detected inhibitory beta-adrenoc eptors, opioid kappa and delta receptors, and P-2 purinoceptors as wel l as facilitatory prostaglandin (PG) E receptors. No indication was fo und for a functional relevance of 5-hydroxytryptamine (5-HT), opioid m u, PGD, adenosine A(1) or glutamate receptors. In conclusion, electric ally evoked noradrenaline release from cultured chick sympathetic neur ons shows the properties of action-potential-induced transmitter relea se and is bidirectionally regulated by various substances. Therefore, sympathetic neurons in culture offer the possibility to investigate di rectly the mechanisms bringing about receptor-coupled modulation of tr ansmitter release.