Uptake of ( 3 H-methyl)choline by microsomal, synaptosomal, mitochondrial and synaptic vesicle fractions of rat brain. The effects of hemicholinium.

Abstract

Abstract— Microsomal, mitochondrial, synaptosomal and synaptic vesicle fractions of rat brain took up [3H‐methyl]choline by a similar carrier‐mediated transport system. The apparent Km for the uptake of [3H‐methyl]choline in these subcellular fractions was about 5 × 10−5 M. Choline uptake was also observed in microsomal fractions prepared from liver and skeletal muscle. Virtually identical kinetic properties for [3H‐methyl]choline transport were found in the synaptosomal fractions prepared from the whole brain, cerebellum or basal ganglia. Countertransport of [3H‐methyl]choline from the synaptosomal fraction was demonstrated against a concentration gradient. HC‐3 was a competitive inhibitor of the uptake of [3H‐methyl]choline in brain microsomal, synaptosomal and mitochondria] fractions with respective values for Ki of 4.0, 2.1 and 2.3 × 10−5 M. HC‐15 was a competitive inhibitor of the transport of [3H‐methyl]choline in the synaptosomal fraction, with a Ki of 1.7 × 10−4 M. Upon entry into the microsomal fraction, 74 per cent of the radioactivity could be recovered as unaltered choline, 10 per cent as phosphorylcholine, 1.5 per cent as acetylcholine and 2.5 per cent as phospholipid.Choline acetyltransferase (EC 2.3.1.6) was assayed with [14C]acetylCoA in synaptosomal fractions prepared from basal ganglia and cerebellum, and in the 31,000 g supernatant fraction of a rat brain homogenate. Enzyme activity was 11‐fold greater in the synaptosomal fraction from the basal ganglia than in that from the cerebellum. HC‐3 did not inhibit choline acetyltransferase and there was no evidence for acetylation of HC‐3.Our findings suggest that choline uptake is a ubiquitous property of membranes in the CNS and cannot serve to distinguish cholinergic nerve endings and their synaptic vesicles.