U18666A inhibits regulation of intracellular cholesterol metabolism and neurotransmitter release in human neuroblastoma cells

Niemann-Pick type C disease (NPC) is an autosomal recessive, cholesterol storage disorder which leads to severe neurological impairment and death usuall)' within the second decade. In the present study, we have shown that 3-B-[(2-diethylamino)ethoxy] androst-5-en-17-one (U18666A) mimics many of the biochemical features of NPC in the human neuroblastoma cell line SKN-SH and its neuronal subclone SH-SY5Y. U18666A (0.2 pg/ml) specifically inhibited cholesterol esterification stimulated by low density lipoprotein (LDL) and sphingomyelinase, but not 25-hydroxycholesterol. U18666A also caused lysosomal accumulation of LDL-derived cholesterol and blocked LDLmediated suppression of LDL receptor levels. Treatment of phorbol esterdifferentiated SH-SYSY cells with 0.2 μg U18666A/ml for 18h inhibited potassium depolarization-induced release of [³H]norepinephrine by 75%, while neurotransmitter release evoked by the calcium ionophore A23187 was only inhibited by 30%. No effect of U18666A on cell morphology or levels of protein kinase C isoforms (α,δ) implicated in neurotransmitter release were observed. These results suggest that U18666A alters plasma membrane cholesterol which in turn may affect norepinephrine release primarily at the level of a voltage-gated calcium channel. By extension, impaired neurotransmitter signalling associated with altered cholesterol metabolism may be a feature of NPC.