Alvaro Ortiz

Biotin uptake by human colonic epithelial NCM460 cells: a carrier-mediated process shared with pantothenic acid

Previous studies showed that the normal microflora of the large intestine synthesizes biotin and that the colon is capable of absorbing intraluminally introduced free biotin. Nothing, however, is known about the mechanism of biotin absorption in the large intestine and its regulation. To address these issues, we used the human-derived, nontransformed colonic epithelial cell line NCM460. The initial rate of biotin uptake was found to be 1) temperature and energy dependent, 2) Na+ dependent (coupling ratio of 1:1), 3) saturable as a function of concentration [apparent Michaelis constant ( K m) of 19.7 μM], 4) inhibited by structural analogs with a free carboxyl group at the valeric acid moiety, and 5) competitively inhibited by the vitamin pantothenic acid (inhibition constant of 14.4 μM). Pretreatment with the protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA) and 1,2-dioctanoyl- sn-glycerol significantly inhibited biotin uptake. In contrast, pretreatment with the PKC inhibitors staurosporine and chelerythrine led to a slight, but significant, increase in biotin uptake. The effect of PMA was mediated via a marked decrease in maximal uptake velocity and a slight increase in apparent K m. Pretreatment of cells with modulators of the protein kinase A-mediated pathway, on the other hand, showed no significant effect on biotin uptake. These results demonstrate, for the first time, the functional existence of a Na+-dependent, specialized carrier-mediated system for biotin uptake in colonic epithelial cells. This system is shared with pantothenic acid and appears to be under the regulation of an intracellular PKC-mediated pathway.

Transport of thiamine in human intestine: mechanism and regulation in intestinal epithelial cell model Caco-2

The present study examined the intestinal uptake of thiamine (vitamin B1) using the human-derived intestinal epithelial cells Caco-2 as an in vitro model system. Thiamine uptake was found to be 1) temperature and energy dependent and occurred with minimal metabolic alteration; 2) pH sensitive; 3) Na+ independent; 4) saturable as a function of concentration with an apparent Michaelis-Menten constant of 3.18 ± 0.56 μM and maximal velocity of 13.37 ± 0.94 pmol ⋅ mg protein-1 ⋅ 3 min-1; 5) inhibited by the thiamine structural analogs amprolium and oxythiamine, but not by unrelated organic cations tetraethylammonium, N-methylnicotinamide, and choline; and 6) inhibited in a competitive manner by amiloride with an inhibition constant of 0.2 mM. The role of specific protein kinase-mediated pathways in the regulation of thiamine uptake by Caco-2 cells was also examined using specific modulators of these pathways. The results showed possible involvement of a Ca2+/calmodulin (CaM)-mediated pathway in the regulation of thiamine uptake. No role for protein kinase C- and protein tyrosine kinase-mediated pathways in the regulation of thiamine uptake was evident. These results demonstrate the involvement of a carrier-mediated system for thiamine uptake by Caco-2 intestinal epithelial cells. This system is Na+ independent and is different from the transport systems of organic cations. Furthermore, a CaM-mediated pathway appears to play a role in regulating thiamine uptake in these cells.The present study examined the intestinal uptake of thiamine (vitamin B(1)) using the human-derived intestinal epithelial cells Caco-2 as an in vitro model system. Thiamine uptake was found to be 1) temperature and energy dependent and occurred with minimal metabolic alteration; 2) pH sensitive; 3) Na(+) independent; 4) saturable as a function of concentration with an apparent Michaelis-Menten constant of 3.18 +/- 0.56 microM and maximal velocity of 13.37 +/- 0.94 pmol. mg protein(-1). 3 min(-1); 5) inhibited by the thiamine structural analogs amprolium and oxythiamine, but not by unrelated organic cations tetraethylammonium, N-methylnicotinamide, and choline; and 6) inhibited in a competitive manner by amiloride with an inhibition constant of 0.2 mM. The role of specific protein kinase-mediated pathways in the regulation of thiamine uptake by Caco-2 cells was also examined using specific modulators of these pathways. The results showed possible involvement of a Ca(2+)/calmodulin (CaM)-mediated pathway in the regulation of thiamine uptake. No role for protein kinase C- and protein tyrosine kinase-mediated pathways in the regulation of thiamine uptake was evident. These results demonstrate the involvement of a carrier-mediated system for thiamine uptake by Caco-2 intestinal epithelial cells. This system is Na(+) independent and is different from the transport systems of organic cations. Furthermore, a CaM-mediated pathway appears to play a role in regulating thiamine uptake in these cells.

Mechanism and regulation of riboflavin uptake by human renal proximal tubule epithelial cell line HK-2

Riboflavin (RF), a water-soluble vitamin, is essential for normal cellular functions, growth, and development. Normal RF body homeostasis depends on intestinal absorption and recovery of the filtered vitamin in renal tubules. The mechanism and cellular regulation of the RF renal reabsorption process, especially in the human situation, are poorly understood. The aim of this study was therefore to address these issues, using a recently established human normal renal epithelial cell line, HK-2, as a model. Uptake of RF by HK-2 cells was found to be 1) linear with time for 5 min of incubation and occurring with minimal metabolic alterations, 2) temperature dependent, 3) Na+ independent, 4) saturable as a function of concentration [apparent Michaelis constant ( K m) of 0.67 ± 0.21 μM and maximal velocity ( V max) of 10.05 ± 0.87 pmol ⋅ mg protein-1 ⋅ 3 min-1], 5) inhibited by structural analogs and anion transport inhibitors, and 6) energy dependent. Protein kinase C-, protein kinase A-, and protein tyrosine kinase-mediated pathways were found to have no role in regulating RF uptake. On the other hand, a Ca2+/calmodulin-mediated pathway appeared to play a role in the regulation of RF uptake by HK-2 cells via an effect on the V max, as well as on the apparent K m of the RF uptake process. The uptake process of RF was also found to be adaptively regulated by the level of the substrate in the growth medium, with the effect being mediated through changes in the apparent K m and the V max of the uptake process. These results demonstrate that RF uptake by the human-derived renal epithelial cell line HK-2 is via a carrier-mediated system that is temperature and energy dependent and appears to be under the regulation of a Ca2+/calmodulin-mediated pathways and substrate level in the growth medium.

Riboflavin uptake by the human-derived liver cells Hep G2: Mechanism and regulation

The water‐soluble vitamin riboflavin (RF) plays a critical role in many metabolic reactions, and thus, is essential for normal cellular functions and growth. The liver plays a central role in normal RF metabolism and is the site of maximal utilization of the vitamin. The mechanism of liver uptake of RF has been studied in animals, but no information is available describing the mechanism of the vitamin uptake in the human situation and its cellular regulation. In this study, we used the human‐derived liver cells Hep G2 as an in vitro model system to address these issues. Uptake of RF by Hep G2 cells was found to be temperature‐ and energy‐dependent but Na+‐independent in nature. Uptake seemed to involve a carrier‐mediated process as indicated by the saturation as a function of substrate concentration (apparent Km 0.41 ± 0.08 μM), and by the ability of the structural analogs lumiflavin and lumichrome to inhibit the uptake process [inhibition constant (Ki) of 1.84 and 6.32 μM, respectively]. RF uptake was energy dependent, and was inhibited by the ‐SH group blocker p‐chloromercuriphenylsulfonate (p‐CMPS) (Ki of 0.10 mM). Specific modulators of intracellular protein kinase A (PKA)‐, protein kinase C (PKC)‐, and protein tyrosine kinase (PTK)‐mediated pathways did not affect RF uptake by Hep G2 cells. On the other hand, specific inhibitors of Ca2+/calmodulin‐mediated pathway significantly inhibited the uptake process; this effect seemed to be mediated through a decrease in the Vmax of the substrate uptake process. Maintaining Hep G2 cells in a RF‐deficient growth medium was associated with a significant up‐regulation in the substrate uptake; this effect was specific for RF and was mediated mainly by means of an increase in the Vmax of the uptake process. These results describe, for the first time, the mechanism and cellular regulation of RF uptake by a human‐derived liver cellular preparation, and shows the involvement of a carrier‐mediated system in the uptake process. Furthermore, the uptake process seems to be regulated by an intracellular Ca2+/calmodulin‐mediated pathway and by extracellular substrate levels. J. Cell. Physiol. 176:588–594, 1998.

Cellular and molecular aspects of thiamin uptake by human liver cells: studies with cultured HepG2 cells

The liver is an important site for thiamin metabolism, utilization, and storage. Little is known about the mechanism of thiamin uptake by the human liver. In this study, we examined cellular and molecular aspects of the human liver thiamin uptake process using the human-derived liver HepG2 cells as a model system. Our studies showed that the initial rate of thiamin uptake to be: (1) Na(+)-independent and occurs with no detectable metabolic alterations in the transported substrate, (2) highly pH-dependent with diminished uptake upon decreasing incubation buffer pH from 8.0 to 5.0, (3) higher following cell acidification compared to unacidified control cells, (4) saturable as a function of concentration with an apparent K(m) of 7.7+/-1.6 microM, (5) inhibited by the thiamin structural analogues oxythiamin and amprolium but not by the unrelated organic cations tetraethylammonium (TEA) and N-methylnicotinamide (NMN), and (6) inhibited in a concentration-dependent manner by the membrane transport inhibitor amiloride. Both of the recently cloned human thiamin transporters, i.e., SLC19A2 and SLC19A3, were found to be expressed in liver HepG2 cells with the former being the predominant form. High promoter activity of the predominant form, i.e., SLC19A2, was detected in HepG2 cells, and the minimal region of the SLC19A2 promoter required for its basal activity in these cells was found to be encoded in a sequence between -356 and -36 and has multiple putative cis-regulatory elements. Mutation of a number of these putative cis-elements diminished promoter activity of the SLC19A2 minimal region. These results show the involvement of a specialized carrier-mediated mechanism for thiamin uptake by human liver HepG2 cells. In addition, SLC19A2 was found to be the predominant thiamin uptake carrier expressed in these cells and its promoter displays a high level of activity in them.