PC12 variants deficient in catecholamine transport.

Abstract

We have isolated PC12 cell variants deficient in transporter-mediated uptake of 3,4-dihydroxyphenylethylamine (dopamine). The variants either were obtained nonselectively, or they were selected by resistance to guanethidine or N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Dopamine uptake into guanethidine-resistant cells occurred with a decreased Vmax; the Km for dopamine and inhibition by guanethidine were normal. MPTP-resistant cells lacked the capacity to take up dopamine. Most of the variants resembled wild-type PC12 in their response to nerve growth factor and the storage and secretion of dopamine. MPTP-resistant cells exhibited several deficiencies in addition to dopamine transport, i.e., no measurable storage of dopamine or acetylcholine and no observable response to nerve growth factor. Wild-type and variant cells were compared with respect to the labeling of cell proteins with [3H]xylamine, which binds covalently to certain proteins apparently only after entering PC12 via the catecholamine transporter. When intact variant cells were used, there was markedly reduced labeling of the proteins by [3H]xylamine. Almost all of these proteins were readily labeled when cell homogenates were exposed to [3H]xylamine. However, MPTP-resistant cells were missing three of these proteins. Northern blot analysis with cDNA clones revealed that the MPTP-resistant cells had markedly reduced levels of several specific mRNA species.