REGULATION OF EXPRESSION OF CHOLINERGIC NEURONAL PHENOTYPIC MARKERS IN NEUROBLASTOMA LA-N-2

Cholinergic neurons in PNS and CNS are identified by the presence of c holine acetyltransferase and the accumulation of choline by a high-aff inity, sodium-coupled choline transporter to be used for acetylcholine synthesis. It appears that expression of choline acetyltransferase ca n be altered by several physiological conditions, including hormones a nd trophic factors, but little is known about control of expression of the sodium-coupled choline carrier or whether these two phenotypic ma rkers are regulated similarly. In the present study, the cholinergic h uman neuroblastoma LA-N-2 was used to investigate regulation of expres sion of choline acetyltransferase and choline uptake activity associat ed with differentiation and neurite extension. Cells grown in serum-co ntaining basal medium maintained a relatively undifferentiated morphol ogy, expressed low levels of choline acetyltransferase activity, and a ccumulated choline by a sodium-dependent process followed by conversio n to acetylcholine. Transfer of cells to an enriched, serum-free defin ed medium resulted in morphological and neurochemical differentiation, with an enhancement of cholinergic phenotype. Hemicholinium-sensitive choline uptake activity was increased about sixfold over a 4-day peri od, with no change in choline acetyltransferase or acetylcholinesteras e specific activity. Acetylcholine synthesis was increased in parallel with the changes in choline accumulation; choline metabolism in the d ifferentiated cells differed significantly from that observed in the u ndifferentiated cells, with proportionally less converted to phosphory lcholine and proportionally more remaining as unmetabolized choline an d converted to acetylcholine. The enhanced choline accumulation appear ed to be mediated by an increased number of choline carriers, demonstr ated by increased binding of the affinity ligand [H-3]-choline mustard to the transporter and by an increased V(max) for the uptake process. The increased expression of the transport function appeared to be und er transcriptional control, as the enhancement of uptake was blocked b y the RNA polymerase II inhibitor alpha-amanitin as well as by the pro tein synthesis inhibitor cycloheximide. These results show that expres sion of sodium-coupled choline carriers and choline acetyltransferase may be regulated separately in the differentiating neuroblastoma LA-N- 2 and that neurotransmitter synthesis is controlled by provision of pr ecursor rather than at the level of the biosynthetic enzyme.