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)
Department of Physiology, University of Western Ontario, London, Canada.
B M
BM
Schmidt
R J
RJ
Rylett
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