Activity and subcellular trafficking of the sodium-coupled choline transporter CHT is regulated acutely by peroxynitrite.
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 IC(50) = 0.9 +/- 0.14 mM and t((1/2)) = 4 min. SIN-1 significantly reduced V(max) of choline uptake without altering the K(m). 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.
Pinthong
Metta
M
Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada.
Black
Stefanie A G
SA
Ribeiro
Fabiola M
FM
Pholpramool
Chumpol
C
Ferguson
Stephen S G
SS
Rylett
R Jane
RJ
D000818:Animals; D019921:Biotinylation; D018929:Cell Culture Techniques; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002794:Choline / Q000037:antagonists & inhibitors / Q000378:metabolism; D018678:Cholinergic Agents / Q000378:metabolism / Q000494:pharmacology; D003470:Culture Media; D003627:Data Interpretation, Statistical; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D006426:Hemicholinium 3 / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D020128:Inhibitory Concentration 50; D007668:Kidney / Q000166:cytology; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D049449:Luminescence; D008564:Membrane Potentials / Q000187:drug effects; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D009447:Neuroblastoma / Q000473:pathology; D009584:Nitrogen / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D030421:Peroxynitrous Acid / Q000096:biosynthesis / Q000378:metabolism*; D021381:Protein Transport; D051381:Rats; D012964:Sodium / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
D018678:Cholinergic Agents; D003470:Culture Media; D004791:Enzyme Inhibitors; D026901:Membrane Transport Proteins; C065637:choline transporter; D030421:Peroxynitrous Acid; D006426:Hemicholinium 3; D014443:Tyrosine; C002385:linsidomine; D012964:Sodium; D008981:Molsidomine; D007770:L-Lactate Dehydrogenase; D009584:Nitrogen; D002794:Choline