Thiamine Uptake by Hymenolepis diminuta

Abstract

Over the concentration range from 0.005 to 100 mM the uptake of thiamine (vitamin B1) by Hymenolepis diminuta was linear with respect to concentration (product-moment correlation coefficient > 0.99). Radiothiamine uptake was inhibited by thiamine, thiamine monophosphate (TMP), oxythiamine, and neopyrithiamine. At 0.1 mM substrate, maximum inhibition was 57% at 800:1 inhibitor/substrate ratio. Iodoacetate (1 mM) inhibited thiamine uptake only if worms were preincubated in the inhibitor for 5 min. Thiamine uptake was not inhibited by thiamine pyrophosphate (TPP), 2,4-dinitrophenol, alanine, arginine, glutamic acid, histidine, methionine, glucose, galactose, glycerol, uracil, cytosine, thymine, riboflavin, folic acid, or nicotinamide. After a 30-sec incubation in labeled thiamine, 93% of the radioactivity (extractable as thiamine, TMP, and TPP) was present as thiamine; 3% was present as TMP, and 4% at TPP. After 30 min incubation, the relative amounts of labeled thiamine, TMP, and TPP were 61, 7, and 32%, respectively. Thiamine uptake appeared to take place by combination of both mediated and nonmediated mechanisms, and the mediated transport system is relatively specific. Hymenolepis diminuta absorbs numerous compounds when incubated in vitro. These include amino acids (Read, Rothman, and Simmons, 1963), monosaccharides (Laurie, 1957; Phifer, 1960), purines and pyrimidines (MacInnis et al., 1965; MacInnis and Ridley, 1969), and fatty acids (Arme and Read, 1968; Chappell et al., 1969). Although it appears that some vitamins (of either host or exogenous origin) are important in the developmental physiology of cestodes (Hager, 1941; Addis and Chandler, 1944, 1946; Platzer and Roberts, 1969, 1970), and that H. diminuta absorbs thiamine (vitamine B1) from the digestive tract of its host (Chandler et al., 1950), no information is available concerning the mechanism(s) of vitamin absorption in this parasite. This paper reports the results of experiments in which thiamine uptake by H. diminuta was studied. MATERIALS AND METHODS Young, male Sprague-Dawley rats (Holtzman Co.) were infected with 30 H. diminuta cysticercoids and the worms removed 11 days postinfection. After removal, worms were rinsed in 3 changes of Krebs-Ringer solution containing 25 mM Tris-(hydroxymethyl)-aminomethane-maleate buffer (pH 7.4) (KRT of Read et al., 1963), randomized into groups of 5 worms, and preincubated Received for publication 30 September 1971. * This work was supported in part by a grant from the NIH (AI-01384). t U. S. Public Health Service Postdoctoral Fellow, 1-FO2-AI-45108-01. at 37 C for 15 min prior to incubations. Worms were then transferred to an incubation medium (in a shaking water bath at 37 C) consisting of 5 ml of KRT to which 4C-thiamine (thiamine [thiazole-2-14C]) or 5S-thiamine (14 mCi/mM and 65 mCi/mM, respectively; Amersham Serale Corp., Arlington Heights, Illinois) had been added. The concentration of thiamine in the incubation medium was adjusted by the addition of unlabeled thiamine'HC1 (Sigma Chemical Co., St. Louis). All incubations were of 2-min duration and consisted of 5 replicates, unless otherwise noted. After incubation, worms were removed and rinsed in 2 changes of fresh KRT, blotted on filter paper, and placed in 5 ml of 70% ethanol for 24 hr (ethanol extracted 98%o+ of the absorbed radioactivity). Radioactivity in the ethanol extracts was determined with a Nuclear-Chicago Gas-Flow Counter and the worms dried for 24 hr at 95 C before weighing. Data were converted to ,umoles thiamine absorbed/g alcohol extracted dry wt/hr unless otherwise noted. Inhibitor studies were conducted as described above, except that single inhibitors were added to the incubation media. The following inhibitors were used: Thiamine HCl, thiamine monophosphoric acid chloride HCl (thiamine monophosphate, TMP), thiamine pyrophosphate chloride (TPP, cocarboxylase), oxythiamine (5-[2-hydroxyethyl ] 3[ ( 4-hydroxy -2-methyl5-pyrimidinyl) methyl]-4-methylthiazolium chloride), neopyrithiamine (1-[(4-amino-2-methyl)-5-pyrimidylmethyl]2-methyl-3-[P,-hydroxyethyl] pyridinium bromide hydrobromide), histidine, alanine, methionine, glutamic acid, arginine, glucose, galactose, glycerol, cytosine, uracil, thymine, nicotinamide, riboflavin, folic acid, 2,4-dinitrophenol (DNP), and iodoacetate (all obtained from Sigma Chemical or CalBiochem, Los Angeles). The concentration of all inhibitors was 5 mM, with the following exceptions: iodoacetate and DNP were 1 mM, and