Studies on accumulation and metabolic fate of [ N-Me 3H]choline in human term placenta fragments

Abstract

Fragments from human term placenta accumulated [ N-Me 3H]choline against a concentration gradient. Uptake was linearly related to incubation time and temperature. Analysis of the kinetics of choline accumulation revealed the concurrent existence of a diffusional component occuring both at low temperature as well as high (50 mM) choline concentration and a carrier-mediated transport which had characteristics predicted by the Michaelis-Menten equation showing a K m = 3.46 × 10 −4 M and a V max of 75 nmoles/ml intracellular water × min −1. Net concentration ratios, corrected for diffusion and extracellular water, were larger than 1.0 within 5 min and about 4.0 after 15 min. Hemicholinium-3 was a competitive inhibitor of choline uptake with a K i of 0.45 mM. [ 3H]Choline accumulation was decreased by conditions known to lower intracellular ATP levels. Thus, 2,4-dimitrophenol (1 mM), sodium cyanide (5 mM) and anaerobic incubation reduced [ 3H]choline accumulation 36, 54 and 33 per cent respectively. Ouabain (0.1 mM) also decreased the concentration ratios by 50 per cent. Modification of the ionic environment led to an increase of 36 per cent in the amount of tritium in intracellular water when Na + was reduced to one half of the usual 145 mM or 150 per cent when it was completely omitted and replaced by an osmotically equivalent amount of sucrose. Li + was without effect, while high K + (> 25mM), Rb + and Cs + (145 mM) depressed [ 3H]choline accumulation. The metabolic fate of [ 3H]choline was studied. Following a 5-min incubation with 5 μM [ 3H]choline 95 per cent of the radioactivity was acid-soluble and 5 per cent remained in the acid-insoluble fraction. After 30 min the distribution was 88 and 12 per cent, respectively. Paper high voltage electrophoretic analysis of the acid-soluble material showed that after 5 min 55 per cent of the 3H had a mobility equal to authentic choline, 35 per cent equal to acetylcholine, 6 per cent to phosphorylcholine and 1 per cent to betaine. After 20 min it was 25 per cent in choline, 60 per cent in acetylcholine, 10 per cent in phosphorylcholine and 2 per cent in betaine. A chloroform-methanol extract from the acid-insoluble residue revealed a linear increase of 3H-content suggesting incorporation of [ 3H]choline into phospholipids.