Biotransformation of the MPTP analog trans-1-methyl-4-[4-dimethylaminophenylethenyl]-1,2,3,6-tetra- hydropyridine to a fluorescent pyridinium metabolite by intact neuroblastoma cells.
The possibility of imaging monoamine oxidase (MAO) containing neurons through the MAO-mediated conversion of the nonfluorescent tetrahydropyridine compound trans-1-methyl-4-[4-dimethylaminophenylethenyl]-1,2,3,6-tetrahydro pyridine (t-THP) to the corresponding fluorescent trans-1-methyl-4-[4-dimethylaminophenylethenyl]pyridinium species (t-P+) was examined with the aid of human neuroblastoma cells (SH-SY5Y). Fluorescence microscopy and fluorescence measurements established the intracellular formation of a fluorescent species with maximal excitation/emission wavelengths of 485/620 and 530/620 nm corresponding to the fluorescence characteristics of synthetic t-P+. An independent assay confirmed the presence of both MAO-A and MAO-B in these cells. As expected, the development of the fluorescence was inhibited by both clorgyline (an MAO-A inhibitor) and deprenyl (an MAO-B inhibitor). Cytotoxic effects, as determined by trypan blue dye exclusion for viability and by the MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] assay for mitochondrial integrity, were not observed in cells incubated with concentrations of t-THP as high as 10(-3) M for 4 hr. The results from these studies with a neuronal cell line of human origin suggest: (1) that SH-SY5Y cells metabolize and, therefore, can be used for study of tetrahydropyridine compounds in vitro, and (2) that t-THP may be a useful agent to monitor neurodegenerative processes in MAO-rich neurons, including the dopaminergic nigrostriatal neurons that are damaged by the parkinsonian-inducing tetrahydropyrridine MPTP. The potential advantage of using t-THP over related imaging techniques is the possibility of assessing neuronal function by an in vivo processing of the reporter molecule rather than by postmortem immunofluorescent or formaldehyde-based procedures.
Song
X
X
Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, Virginia 24061, USA.
Ehrich
M
M
Flaherty
D
D
Wang
Y X
YX
Castagnoli
N
N
D015632:1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics*; D001711:Biotransformation; D005456:Fluorescent Dyes / Q000493:pharmacokinetics* / Q000633:toxicity; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D020267:MPTP Poisoning; D008856:Microscopy, Fluorescence; D008995:Monoamine Oxidase / Q000378:metabolism; D009447:Neuroblastoma / Q000201:enzymology / Q000378:metabolism*; D011743:Pyrimidines / Q000493:pharmacokinetics / Q000633:toxicity; D013050:Spectrometry, Fluorescence; D014407:Tumor Cells, Cultured
C101344:1-methyl-4-(4-dimethylaminophenylethenyl)-1,2,3,6-tetrahydropyridine; D005456:Fluorescent Dyes; D007527:Isoenzymes; D011743:Pyrimidines; D015632:1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; D008995:Monoamine Oxidase