Transport of tricarballylate by intestinal brush-border membrane vesicles from steers.

Abstract

Tricarballylic acid is a non-metabolizable rumen bacterial fermentation product of the naturally occurring tricarboxylic acid trans-aconitic acid. The aim of the present study was to investigate intestinal absorption of tricarballylate using brush-border membrane vesicles (BBMVs) isolated from the proximal jejunum of steers by a Ca2+ precipitation method with subsequent differential centrifugation. Transport of tricarballylate was investigated indirectly (influence of tricarballylate on the uptake of 14C-labelled citrate) as well as directly (uptake of 3H-labelled tricarballylate). Citrate as well as tricarballylate uptake (at a concentration of 0.05 mmol l-1) was strongly stimulated by an inwardly directed initial Na+ gradient. Furthermore, transport of both tricarboxylates under Na+ gradient conditions was clearly enhanced by lowering the extravesicular pH from 7.8 to 5.6. The imposition of an inwardly directed H+ gradient (pH(out)/pH(in) = 5.6/7.8) further enhanced the intravesicular accumulation of citrate as well as of tricarballylate compared with pH(out)/pH(in) = 5.6/5.6. Unequivocal evidence for a common transport site for tricarballylate and citrate was obtained from 'cis-inhibition' and 'trans-stimulation' of Na(+)-dependent citrate uptake by tricarballylate. In further experiments the influence of different substances on the uptake of 3H-labelled tricarballylate was evaluated. Unlabelled tricarballylate, citrate, succinate as well as trans- and cis-aconitate significantly inhibited the accumulation of 3H-labelled tricarballylate by BBMVs. Tricarballylate uptake as a function of the tricarballylate concentration revealed a Na(+)-dependent saturable component (apparent kinetic parameters: maximal transport capacity (Vmax) = 119 pmol (mg protein)-1 (3s)-1; affinity constant (Km) = 0.097 mmol l-1) and a Na(+)-independent diffusional component (diffusion constant: 169 nl (mg protein)-1 (3s)-1). It is concluded that tricarballylate and citrate are transported across the intestinal brush-border membrane by a common, Na(+)-dependent transport mechanism. The stimulatory influence of a low extravesicular pH most probably indicates that the protonated forms of tricarboxylates are better transported than the trivalent species.