Basal synthesis of acetylcholine in hippocampal synaptosomes is not dependent upon membrane-bound choline acetyltransferase activity.
Choline acetyltransferase, the enzyme which catalyses the formation of acetylcholine within cholinergic nerve terminals, exists in both cytosolic and membrane-associated subcellular pools. In the present study, alteration in nerve terminal Cl- homeostasis was used as an experimental tool to elucidate the role of membrane-bound choline acetyltransferase in regulation of the biosynthesis of acetylcholine in rat hippocampal synaptosomes under basal or resting conditions. Reduction of extracellular Cl- concentration from 131 to 48 mM through iso-osmotic replacement with isethionate ions produced a selective decrease, to approximately 50% of control, of nerve terminal membrane-associated choline acetyltransferase activity. Under these experimental conditions, there were no changes in the activity of cytosolic enzyme or high-affinity choline uptake, or in acetylcholine synthesis. Replacement of medium Cl- with Br- supported maintenance of synaptosomal membrane-bound choline acetyltransferase activity better than did I- or isethionate ions; high-affinity choline uptake activity and acetylcholine synthesis were affected similarly. Incubation of synaptosomes with low concentrations of the Cl- channel blockers 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (50 microM) and niflumic acid (100 microM) selectively decreased activity of the membrane-bound enzyme, with no effect on cytosolic choline acetyltransferase or high-affinity choline uptake activities. Acetylcholine synthesis was unchanged, even though membrane-bound choline acetyltransferase activity was decreased in some samples (250 microM 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid) to about 10% of control. Experimental manipulations designed to alter neuronal Cl- homeostasis resulted in selective changes in membrane-bound choline acetyltransferase activity, thereby allowing the first direct examination of its physiological role in regulation of acetylcholine synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
Schmidt
B M
BM
Department of Physiology, University of Western Ontario, London, Canada.
Rylett
R J
RJ
D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology; D000109:Acetylcholine / Q000096:biosynthesis*; D000818:Animals; D018118:Chloride Channels; D002712:Chlorides / Q000378:metabolism; D002795:Choline O-Acetyltransferase / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D005260:Female; D006624:Hippocampus / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D006706:Homeostasis / Q000187:drug effects; D066298:In Vitro Techniques; D007700:Kinetics; D008565:Membrane Proteins / Q000037:antagonists & inhibitors; D009419:Nerve Tissue Proteins / Q000096:biosynthesis; D009544:Niflumic Acid / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013347:Subcellular Fractions / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D013570:Synaptic Membranes / Q000187:drug effects / Q000201:enzymology*; D013574:Synaptosomes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*
D018118:Chloride Channels; D002712:Chlorides; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D009544:Niflumic Acid; D002795:Choline O-Acetyltransferase; D000109:Acetylcholine