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 (Km) of 19.7 microM], 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 microM). 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 Km. 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.

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 microM), and by the ability of the structural analogs lumiflavin and lumichrome to inhibit the uptake process [inhibition constant (K) of 1.84 and 6.32 microM, 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.

Uptake of L-carnitine by a human intestinal epithelial cell line, Caco-2.

BACKGROUND & AIMS: The mechanism of intestinal uptake of L-carnitine is controversial. The aim of this study was to clarify the mechanism and regulation of L-carnitine uptake. METHODS: Uptake of [3H]-L-carnitine was measured across the apical membrane of confluent monolayers of Caco-2 cells. RESULTS: [3H]-L-carnitine uptake was linear and appreciable for up to 7 minutes with minimal metabolic alteration, was temperature- and Na(+)-(but not pH-) dependent, and included a saturable component with an apparent Michaelis constant of 45.5 +/- 6.5 mumol/L and a maximum velocity of 83.5 +/- 5.6 nmol.mg protein-1.5 min-1. Unlabeled L-carnitine and its structurally related analogues significantly (P < 0.01) inhibited [3H]-L-carnitine uptake, whereas unrelated compounds were ineffective. L-Carnitine uptake was also energy-dependent, being significantly (P < 0.01) inhibited by metabolic inhibitors. Our results also suggested that a calmodulin- but not a protein kinase C- or protein kinase A-mediated pathway plays a role in regulating L-carnitine uptake by Caco-2 cells. CONCLUSIONS: L-carnitine uptake by intestinal epithelial cells (Caco-2) involves a carrier-mediated system that is temperature-, Na(+)-, and energy-dependent and seems to be under the regulation of a calmodulin-mediated pathway.

Riboflavin uptake by rat liver basolateral membrane vesicles.

The present study examined riboflavin (RF) uptake by purified rat liver basolateral membrane vesicle (BLMV). Uptake of RF was found to be Na(+)- and pH-independent in nature. Studies on RF uptake by BLMV as a function of incubation medium osmolarity have indicated that the uptake is the result of transport (66.5%) into the intravesicular space as well as binding (33.5%) to membrane surfaces. The process of RF uptake by BLMV was saturable as a function of substrate concentration with an apparent Km of 3.55 +/- 0.70 microM and Vmax of 39.89 +/- 3.24 pmol/mg protein/5 s, respectively. cis-Addition of unlabeled RF and its structural analogs lumaflavin and lumichrome inhibited the uptake of [3H]RF while trans-addition of unlabeled RF stimulated the efflux of [3H]RF from preloaded vesicles. No effect on RF uptake was found by the membrane transport inhibitors probenecid, 4,4-diisothiocyanotostilbene-2,2-disulfonic acid (DIDS) and 4-acetamido-4-isothiocyanatostilbene-2,2'-disulfonic acid (SITS). Induction of a transient positive intravesicular space led to a slight stimulation of RF uptake, while induction of a negative intravesicular space led to a slight inhibition in RF uptake. These results demonstrate the existence of a membrane-associated carrier system for RF uptake by liver BLMV. This system appears to be Na(+)- and pH-independent and is influenced to a certain degree by changes in transmembrane electrical potential.

Intestinal uptake of uridine in suckling rats: mechanism and ontogeny.

Nucleosides, essential substrates for a variety of intracellular metabolic reactions, are obtained from dietary and endogenous sources. Nucleotides (which dephosphorylate to nucleosides prior to intestinal absorption) are present in milk and have trophic effects on the developing gastrointestinal tract. The mechanism of transport of nucleosides in the developing intestine of suckling rats is unknown. To address this issue, we therefore examined uridine uptake in rat everted intestinal sacs. In suckling rats (15-17 days old), tissue uptake of low (5-microM) and high (60 microM) concentrations of [3H]-uridine was linear for up to 2 min of incubation. Initial rate of uptake of [3H]-uridine was (i) not significantly different in the jejunum and the ileum; (ii) greater in the presence of Na+, than other cations; (iii) saturable as a function of concentration with a Vmax of 21,044 +/- 2,302 pmol/g tissue wet wt/30 sec and an apparent Km of 33.8 +/- 10.1 microM; (iv) inhibited by high concentration (500 microM) of unlabeled uridine and other nucleosides; (v) temperature-dependent; (vi) energy-dependent; and (vii) pH-sensitive. Developmental maturation was associated with a progressive decrease in the Vmax of the uridine transport process (21,044 +/- 2,302, 14,651 +/- 1,679, and 8,461 +/- 1,369 pmol/g tissue wet wt/30 sec for suckling, weanling, and adult rats, respectively) and a progressive increase in the apparent Km of the uptake system (33.8 +/- 10.1, 55.6 +/- 13.1, and 61.7 +/- 14.5 microM for suckling, weanling, and adult rats, respectively). We concluded that uptake of uridine by the developing intestine of suckling rats involves a carrier-mediated system, which is energy- and temperature-dependent, and requires extracellular sodium. Furthermore, the uptake process was found to undergo clear ontogenic changes with maturation.

Stratospheric ozone depletion and plantinsect interactions: Effects of UVB radiation on foliage quality ofCitrus jambhiri forTrichoplusia ni.

Projected decreases in stratospheric ozone may result in increases in shortwave ultraviolet (UVB) irradiation at the earth's surface. Furanocoumarins, phototoxic compounds found inCitrus jambhiri foliage, increase in concentration when these plants are grown under enhanced UVB. Survivorship schedules ofTrichoplusia ni (Lepidoptera: Noctuidae) caterpillars reared on plants in the presence and absence of enhanced UVB regimes differ significantly; larvae develop more slowly in early life when reared on plants exposed to increased UVB. This same developmental pattern is observed whenT. ni larvae are reared on artificial diets amended with ecologically appropriate amounts of furanocoumarins. Thus, anthropogenically derived changes in stratospheric ozone and concomitant changes in UV light quality at the earth's surface may influence ecological interactions between insects and their host plants by altering secondary metabolism and hence foliage quality for herbivores.

Mechanism of transport of riboflavin in rabbit intestinal brush border membrane vesicles.

Uptake of luminal riboflavin (RF) into the absorptive cells of rabbit small intestine was examined using purified brush border membrane vesicle (BBMV) preparations. These preparations were used in order to eliminate the interference of intracellular metabolism that occurs to the RF molecule during absorption. Uptake of RF by BBMV was found to be mainly (> 76%) the result of transport of the vitamin into the intracellular space with less binding to membrane surfaces. All 3H radioactivity that appeared in the intravesicular space after incubation with [3H]RF was found to be in the form of intact RF. Uptake of RF with time was independent of the presence or absence of a Na+ or a K+ gradient (out > in) and occurred without transaccumulation of the substrate in the intravesicular space. Furthermore, changing the incubation buffer pH showed minimal effect on RF uptake. When examined as a function of concentration, the initial rate of RF uptake was found to be saturable both in jejunal and ileal BBMV with an apparent Km of 7.24 +/- 1.06 and 8.88 +/- 0.90 microM and Vmax of 24.31 +/- 1.48 and 34.24 +/- 1.55 pmol/mg protein/5 sec, respectively. Unlabeled RF and the related compounds lumiflavin, 8-aminoriboflavin, isoriboflavin, and lumichrome all inhibited (but to different degrees) the uptake of physiologic concentration of [3H]RF. On the other hand, 8-hydroxyriboflavin, lumazine, and D-ribose all failed to inhibit [3H]RF uptake. Similarly, the membrane transport inhibitors DIDS, SITS, and furosemide all failed to inhibit [3H]RF uptake. The uptake of RF was found to be insensitive to changes in the transmembrane electrical potential, as shown by studies with anion substitution and valinomycin K(+)-induced negative or positive intravesicular potential methodologies. These results indicate that RF uptake by rabbit intestinal BBMV occurs via a carrier-mediated system that is Na+ independent in nature and transports the substrate by an electroneutral process. The role of this system in the overall absorption process of RF is discussed.

Phototoxicity ofCitrus jambhiri to fungi under enhanced UV-B radiation: Role of furanocoumarins.

Extracts ofCitrus jambhiri foliage exposed to and shielded from UV-B radiation were assayed for phytochemical changes and phototoxicity against four fungal pathogens, two of which (Fusarium solani andF. oxysporum) are causative agents of root rots and two of which (Penicillium italicum andP. digitatum) are associated with fruit rots. Conidial pigment mutants of these four fungal species were assayed to determine whether pigments play a role in protecting fungi against plant photosensitizers. Exposure to 10.2 kJ/ day UV-B radiation for 95 days significantly reduced phototoxicity of leaf extracts to fungi. Although furanocoumarin levels were reduced by UV-B, analysis of covariance revealed that variation in phototoxicity of the extracts cannot be attributed entirely to variation in furanocoumarin content; thus, the possibility exists that nonfuranocoumarin phototoxic constituents, as yet unidentified, respond to UV-B exposure and contribute to overall phototoxic defense ofC. jambhiri against pathogens. Root rot fungi were substantially more sensitive to furanocoumarin phototoxicity than were fruit rot fungi, a pattern consistent with the amount of light exposure normally experienced by these fungi when associated with phototoxic plants. Although pigmented strains of all four species displayed greater resistance to phototoxicity of pure furanocoumarins, no strain differences were detected in assays of foliar extracts; this finding also suggests that nonfuranocoumarin constituents may be involved in the phototoxic defense ofC. jambhiri against pathogens.

Furanocoumarin content and phototoxicity of rough lemon (Citrus jambhiri) foliage exposed to enhanced ultraviolet-B (UVB) irradiation.

Rooted cuttings ofC. jambhiri were grown under enhanced levels of UVB radiation for 95 days. Bacterial phototoxicity and furanocoumarin content were determined in extracts made from various tissues from the aboveground biomass. Young, newly expanded leaves contained significantly higher concentrations of furanocoumarins than older leaves and stems. Additionally, the proportional concentration of psoralen was higher in young leaves than in old leaves. While treatment with UVB did not result in a change in the overall level of furanocoumarins, it did cause an increase in the ratio of psoralen to bergapten. Bacterial phototoxicity paralleled the distribution of furanocoumarin content among tissue types; analysis of covariance suggested that the phototoxic properties of the extracts could be accounted for on the basis of furanocoumarin content alone.