title: Study of the bidirectional transport of choline by blocking choline carriers from outside or inside brain nerve terminals.
abstract: Membrane carriers can operate bidirectionally. We studied, in rat neocortex synaptosomes, the choline carrier by comparing the ability of the transport inhibitor hemicholinium-3, present outside or inside the nerve terminals, to prevent uptake and release of [(3)H]choline. Because hemicholinium-3 is membrane-impermeable, it was previously entrapped into synaptosomes during homogenization of brain tissue. External and internalized hemicholinium-3 produced similar maximal inhibition (80-90%) of [(3)H]choline uptake. Also comparable (approximately 30 nM) are the potency of externally applied hemicholinium-3 and the estimated potency of the entrapped inhibitor. Exposure to ouabain elicited release of both [(3)H]acetylcholine and [(3)H]choline from synaptosomes prelabeled with [(3)H]choline. The ouabain (300 microM)-evoked release of [(3)H]choline only was blocked by externally added (IC(50) approximately 10 nM) or internalized (estimated IC(50) approximately 5 nM) hemicholinium-3. Release of previously taken up [(3)H]choline elicited by 100 microM external choline (homoexchange) was prevented by external (IC(50) approximately 30 microM) or entrapped (estimated IC(50) approximately 20 microM) hemicholinium-3. The results suggest that the choline carriers fit into the alternating-access model proposed for classical transmitter transport. Entrapping nonpermeant ligands into synaptosomes could allow investigation of the inward-facing conformation of native transporters and how cytoplasmic ligands affect the bidirectional transport of neurotransmitters.
authors.affiliation: Dipartimento di Medicina Sperimentale, Sezione di Farmacologia e Tossicologia, Università di Genova, Genova, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Marchi
authors.forename: F
authors.initials: F
authors.lastname: Bergaglia
authors.forename: A
authors.initials: A
authors.lastname: Pedrini
authors.forename: M
authors.initials: M
authors.lastname: Raiteri
mesh_terms: D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D001923:Brain Chemistry / Q000187:drug effects; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002794:Choline / Q000378:metabolism* / Q000493:pharmacokinetics; D004305:Dose-Response Relationship, Drug; D006426:Hemicholinium 3 / Q000494:pharmacology; D008297:Male; D019579:Neocortex / Q000378:metabolism; D014179:Neurotransmitter Uptake Inhibitors / Q000494:pharmacology; D010477:Perfusion; D017729:Presynaptic Terminals; D051381:Rats; D017207:Rats, Sprague-Dawley; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism; D014316:Tritium
chemical_list: D002352:Carrier Proteins; D014179:Neurotransmitter Uptake Inhibitors; D014316:Tritium; D006426:Hemicholinium 3; D002794:Choline; D000109:Acetylcholine