Abstract
Excess formation of nitric oxide and superoxide by-products (peroxynitrite, reactive oxygen, and reactive nitrogen species) attenuates cholinergic transmission potentially having a role in Alzheimer disease pathogenesis. In this study, we investigated mechanisms by which acute exposure to peroxynitrite impairs function of the sodium-dependent hemicholinium-3 (HC-3)-sensitive choline transporter (CHT) that provides substrate for acetylcholine synthesis. The peroxynitrite generator 3-morpholinosydnonimine (SIN-1) acutely inhibited choline uptake in cells stably expressing FLAG-tagged rat CHT in a dose- and time-dependent manner, with an IC50 = 0.9 ± 0.14 mM and t½ = 4 min. SIN-1 significantly reduced Vmax of choline uptake without altering the Km. This correlated with a SIN-1-induced decrease in cell surface CHT protein, observed as lowered levels of HC-3 binding and biotinylated CHT at the plasma membrane. It is noteworthy that short-term exposure of cells to SIN-1 accelerated the rate of internalization of CHT from the plasma membrane, but it did not alter return of CHT back to the cell surface. SIN-1 did not disrupt cell membrane integrity or cause cell death. Thus, the inhibitory effect of SIN-1 on choline uptake activity and HC-3 binding was related to enhanced internalization of CHT proteins from the plasma membrane to subcellular organelles.
Footnotes
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This research was supported by operating grants to R.J.R. from the Alzheimer Society of Canada and Canadian Institutes for Health Research (CIHR). M.P. was funded by a Scholarship from the Higher Commission on Education, Ministry of Education of Thailand, as part of an exchange program between Mahidol University, Bangkok, Thailand, and the University of Western Ontario, London, Canada. S.A.G.B. is the recipient of a Doctoral Award from CIHR, and S.S.G.F. is a Career Investigator of the Heart and Stroke Foundation of Ontario and holds the Canada Research Chair in Molecular Neurobiology.
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ABBREVIATIONS: ACh, acetylcholine; CHT, high-affinity choline transporter protein; AD, Alzheimer disease; SIN-1, 3-morpholinosydnonimine; HEK, human embryonic kidney; FeTPPS, 5,10,15,20-tetrakissulfonatophenyl porphyrinato iron (III); FBS, fetal bovine serum; DCF, 2′,7′-dichlorofluorescein; DCFH, reduced 2′,7′-dichlorofluorescein; DCFH-DA, 2′,7′-dichlorofluorescein diacetate; SOD, superoxide dismutase; NHS, N-hydroxysuccinimide; RA, all-trans retinoic acid; KRH, Krebs-Ringer-HEPES; LDH, lactate dehydrogenase; MTT, [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]; PBS, phosphate-buffered saline; PBS/CM, phosphate-buffered saline with 0.1 mM CaCl2 and 1 mM MgCl2; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate; PAGE, polyacrylamide gel electrophoresis; MesNa, sodium 2-mercaptoethanosulfonic acid; TfR, transferrin receptor.
- Received August 15, 2007.
- Accepted October 29, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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