Riboflavin transport by rabbit renal brush border membrane vesicles

• Published in: Biochimica et biophysica acta 1997-12, Vol.1330 (2), p.172-178
• Main Authors: Yanagawa, Norimoto, Jo, Oak D., Said, Hamid M.
• Format: Article
• Language: English
• Subjects: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid - pharmacology; 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid - pharmacology; Animals; Biological Transport - drug effects; Brush border membrane; Carrier Proteins - metabolism; diet-related diseases; human nutrition; Hydrogen-Ion Concentration; In Vitro Techniques; Kidney - drug effects; Kidney - metabolism; Kidney - ultrastructure; Male; Membrane Potentials; Microvilli - drug effects; Microvilli - metabolism; nutrition physiology; Organic anion transport; Rabbits; Riboflavin; Riboflavin - pharmacokinetics; Sodium - metabolism
• ISSN: 0005-2736; 0006-3002; 1879-2642; 1878-2434
• DOI: 10.1016/S0005-2736(97)00153-3

The present study examined riboflavin (RF) uptake by isolated rabbit renal brush border membrane (BBM). RF uptake was linear for up to 30 s and leveled off thereafter reaching an equilibrium with longer incubation. Studies on RF uptake as a function of incubation medium osmolarity indicated that the uptake was the results of transport (61.4%) into the intravesicular space as well as binding (38.6%) to membrane surfaces. The process of RF uptake was saturable as a function of substrate concentration with an apparent K mof 25.7 ± 7.6 μM and V maxof 75.6 ± 14.7 pmol/mg protein/10 s. cis-Addition of unlabeled RF and its structural analogues, lumiflavin and lumichrome, inhibited the uptake of [ 3H]RF significantly, indicating the involvement of a carrier-mediated process in RF uptake by renal BBM. RF uptake by renal BBM was partly Na +-dependent so that when Na + was replaced by potassium, choline, lithium or tetramethylammonium, the RF uptake was reduced to ca. 60% of the control. This Na +-dependency was unlikely to be due to Na +-cotransport mechanism because RF uptake occurred without the characteristic ‘overshoot’ phenomenon as for other Na +-cotransport systems and the elimination of transmembrane Na +-gradient by preloading Na + to the intravesicular space did not affect RF uptake. In contrast, removal of Na + eliminated the binding component of RF uptake, suggesting the requirement of Na + for RF binding to BBM. The RF uptake was not affected when extravesicular pH was varied within the physiological pH range of 6.5 to 8.5. No effect on BBM [ 3H]RF uptake was found when the transmembrane electrical potential was altered by either the presence of anions with different membrane permeability ( Cl − = NO 3 − SO 4 −gluconate −) or by using nigericin (10 μg/mg protein) with an outwardly or inwardly directed transmembrane K + gradient. The uptake of RF by BBM vesicles was, however, inhibited by probenecid and organic anion transport inhibitors, 4,4-diiso-thiocyanatostilbene-2,2-disulfonic acid (DIDS, 1 mM) and 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS, 1 mM). In summary, these results demonstrate the existence of a membrane-associated, and organic anion inhibitor-sensitive, carrier system for RF uptake by renal BBM.