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H M Said

Publications and source records attributed to H M Said.

At least 37 records · Page 2Linked to original sources
PubMed1998

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.

Alkaloids↗
PubMed1998

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 microM and maximal velocity (Vmax) 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 Vmax, 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 Vmax 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.

1-Methyl-3-isobutylxanthine↗
PubMed1997

Riboflavin transport by rabbit renal brush border membrane vesicles.

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 Km of 25.7 +/- 7.6 microM and Vmax of 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- = NO3- > SO4- > gluconate-) or by using nigericin (10 microg/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.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1997

A protein-tyrosine kinase-regulated, pH-dependent, carrier-mediated uptake system for folate in human normal colonic epithelial cell line NCM460.

A significant proportion of the bacterially synthesized folate in the large intestine exists in the form of folate monoglutamate. Recent studies in our laboratory using human colonic apical membrane vesicles have shown the existence of an efficient carrier-mediated system for folate uptake. Nothing, however, is known about the cellular regulation of the colonic uptake process. In this study, we used a recently established human normal colonic epithelial cell line NCM460 to address this issue. Uptake of folic acid by NCM460 cells was: 1) linear with time for 4 min of incubation and occurred with minimal metabolic alterations, 2) temperature- and pH- (but not Na+) dependent, 3) saturable as a function of concentration (apparent Km of 1.4 microM), 4) inhibited by structural analogs and anion transport inhibitors, and 5) energy-dependent. These characteristics of folic acid uptake by NCM460 cells are similar to those seen with apical membrane vesicles derived from human native colonic tissue. Using these cells, we found that protein kinase C- and Ca2+/calmodulin-mediated pathways have no role in regulating folic acid uptake. On the other hand, cAMP (through a mechanism independent of protein kinase A) and protein-tyrosine kinase-mediated pathways were found to play a role in the regulation of folic acid uptake by these cells. These results establish the suitability of NCM460 cells as an in vitro model system for investigating the details of the mechanism of colonic folate uptake and its regulation. Folic acid uptake by these cells appears to involve a carrier-mediated system, which is temperature-, pH-, and energy-dependent and appears to be under the regulation of cAMP and protein tyrosine kinase.

Cell Line↗
PubMed1997

Human intestinal folate transport: cloning, expression, and distribution of complementary RNA.

BACKGROUND & AIMS: Despite intensive investigations, very little is known about the molecular identity(ies) of the intestinal folate transport system(s), especially in humans. The aim of this study was to isolate a functional human intestinal folate carrier complementary DNA (cDNA) clone and determine the distribution of complementary RNA at the tissue and cellular levels. METHODS: Hybridization screening, modified Marathon cDNA amplification, expression in Xenopus oocytes, Northern analysis, and in situ hybridization were used. RESULTS: The hIFC-1 cDNA contains an open reading frame for 591 amino acids (relative molecular mass = 64,826, pI = 9.4, 12 transmembrane domains, three protein kinase C phosphorylation sites, and one N-glycosylation site) with 74% DNA and 66% amino acid sequence homologies with the mouse cDNA counterpart. Xenopus oocytes injected with hIFC-1 cRNA show induced folate uptake that was (1) saturable with substrate concentration (apparent Michaelis constant = 0.71 +/- 0.06 micromol/L; maximum velocity = 128 +/- 3 fmol x h(-1) x oocyte(-1)), (2) inhibited by methotrexate, folinic acid, and folic acid (Ki = 0.84 micromol/L, 0.71 micromol/L, and 10 micromol/L, respectively), and (3) sensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (Ki = 0.29 mmol/L). Northern analysis showed wide distribution of hIFC1-complementary messenger RNA species in various human tissues. In situ hybridization on sections of human jejunum showed preferential hIFC-1 expression in epithelial cells, especially in the upper half of the villi. CONCLUSIONS: These results represent the first molecular characterization of a human small intestinal folate carrier.

Amino Acid Sequence↗
PubMed1997

Intracellular regulation of intestinal folate uptake: studies with cultured IEC-6 epithelial cells.

Although the mechanism of folate uptake in the small intestine has been well characterized, very little is known about the intracellular regulation of the uptake process. Using mature confluent monolayers of the intestinal epithelial cell line IEC-6 as an in vitro intestinal epithelial cell model, we have found the uptake of folic acid to be similar to that of the native small intestine in that it is 1 ) temperature, energy, and pH dependent, 2) Na+ independent, 3) inhibited by structural analogs and anion transport inhibitors, and 4) saturable as a function of substrate concentration [apparent Michaelis constant (Km) = 0.45 +/- 0.06 microM; maximal velocity (Vmax) = 3.08 +/- 0.14 pmol x mg protein(-1) x 5 min(-1)]. Furthermore, IEC-6 cells were found by Northern blot analysis to lack the expression of the membrane folate-binding protein. Pretreatment of IEC-6 monolayers with specific protein tyrosine kinase (PTK) inhibitors genistein and tyrphostin A25 caused a significant inhibition in folic acid uptake. On the other hand, their negative controls, genistin and tyrphostin A1, respectively, had no effect. The inhibitory effect of genistein was mediated through inhibition in the Vmax of the folate uptake process with no change in the apparent Km. Pretreatment of IEC-6 monolayers with compounds that increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP) level (e.g., dibutyryl cAMP) also resulted in a significant (though modest) inhibition in folic acid uptake; however, specific inhibitors of protein kinase A did not affect the uptake process. Specific modulators of protein kinase C and Ca2+/calmodulin-mediated pathways did not significantly affect folic acid uptake. These results demonstrate the suitability of IEC-6 monolayers as an intestinal epithelial model to study folate transport and demonstrate for the first time that uptake of folic acid is regulated by a PTK- and a cAMP-mediated pathway.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1997

Evidence for the existence of a carrier-mediated folate uptake mechanism in human colonic luminal membranes.

Colonic bacteria synthesize significant amounts of folate. The current studies were undertaken to examine the presence of a possible folate transporter and its characterization in apical membranes of the human colon. Apical membrane vesicles were purified from mucosal scrapings of proximal organ donor colons using a differential centrifugation and divalent cation precipitation technique. [3H]folate (PteGlu) uptake was measured by a rapid filtration technique. Our results demonstrate that [3H]PteGlu uptake into these vesicles 1) was significantly increased with decreasing pH of the incubation buffer, 2) was markedly inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid but not by acetazolamide, amiloride, bumetanide, furosemide, and diphenyl 2-carboxylic acid, 3) was sensitive to temperature and osmolarity of the incubation medium, 4) was inhibited by structural analogs methotrexate and 5-methyltetrahydrofolate, 5) exhibited trans-stimulation phenomenon, 6) was potential insensitive, and 7) exhibited saturation kinetics with an apparent Michaelis constant for PteGlu of 8.2 +/- 2.4 microM and a maximal enzyme reaction rate of 19.8 +/- 2.9 pmol.mg protein-1.10 s-1. Studies on distal colonic membranes also demonstrated the presence of a folate transporter with similar kinetic characteristics. These results demonstrate the existence of a pH-dependent, DIDS-sensitive, electroneutral carrier-mediated mechanism for folate absorption in the human colon.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1996

Intestinal folate transport: identification of a cDNA involved in folate transport and the functional expression and distribution of its mRNA.

Although the mechanism of folate intestinal transport has been the subject of intensive studies, very little is known about the molecular identity of the transport system(s) involved. In this investigation, we screened a mouse intestinal cDNA library using as probe the cDNA clone of a reduced folate carrier (RFC1) of mouse leukemia L1210 cells, and identified a positive clone, IFC1(RFC1). The cloned cDNA consisted of 2274 base pairs with an open reading frame that encodes a putative polypeptide of 512 amino acids with a predicted molecular mass of 58,112 daltons and 12 putative transmembrane domains. The polypeptide appears to carry a net positive charge (pI = 8.6) which may be important for its interaction with the negatively charged substrate. Functional identity of the IFC1(RFC1) clone was established by expression in Xenopus oocytes. An 11-fold increase in 5-methyltetrahydrofolate (5-MTHF) uptake was observed in oocytes injected with 10 ng IFC1(RFC1) cRNA compared to water-injected controls. The expressed folate uptake in the cRNA injected oocyte was (1) 4,4'-diisothiocyanatosilbene-2,2'-disulfonic acid (DIDS)-sensitive; and (2) saturable with an apparent Km of 1.99 +/- 0.32 micrometers and a V(max) of 3782 +/- 188 fmol/oocyte per h. The distribution of mRNA species complementary to IFC1(RFC1) in different mouse tissues was examined by Northern blot analysis. In addition to the small intestine, expression of such mRNA species were also found in the kidney, large intestine, brain, heart and liver. Furthermore, mRNA species complementary to IFC1(RFC1) were also detected by Northern blot analysis in the small intestine of human and other animal species (rat and rabbit). Expression of mRNA complementary to IFC1(RFC1) was markedly higher in rat intestinal villus cells than in crypt cells. These results represent the first identification of a folate transporter in mammalian intestine.

Animals↗
PubMed1996

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.

Biological Transport↗
PubMed1995

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.

Animals↗
PubMed1995

Riboflavin uptake by native Xenopus laevis oocytes.

The existence of a membrane-associated uptake carrier for riboflavin (RF) is demonstrated in Xenopus oocytes. Uptake of low (0.017 microM) and high (3 microM) concentrations of RF was linear with time for up to 2 hours, and occurred with little initial binding to oocytes, and little metabolism. Uptake of RF was found to be independent of extracellular pH and Na+. The initial rate of RF uptake was saturable as a function of concentration with an apparent Km of 0.41 +/- 0.02 microM and a Vmax of 2.86 +/- 0.04 fmol/oocyte per h. Uptake of 3H-RF was inhibited by unlabeled RF and by the structural analogs lumiflavin, isoriboflavin (iso-RF), 8-aminoriboflavin (8-NH2-RF), 8-hydroxyriboflavin (8-OH-RF), and lumichrome, but was not affected by flavin adenine dinucleotide (FAD), D-ribose or lumazine. Uptake of RF was significantly retarded by the metabolic inhibitor 2,4-dinitrophenol. The sulfhydryl group-modifying reagents p-chloromercuriphenylsulfonate (pCMPS), p-chloromercuribenzoate (pCMB), N-ethylmaleimide and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) all caused significant inhibition in RF uptake. The inhibitory effect of pCMPS was completely reversed by treatment of pCMPS-pretreated cells with reducing agents. While the transmembrane transport inhibitors 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and furosemide had no effect on RF uptake, amiloride and probenecid suppressed RF uptake in a dose-dependent fashion. Closer examination of the inhibition mediated by amiloride showed that it was competitive in nature with an apparent Ki of approximately 1.8 mM, whereas the inhibition induced by probenecid was nonspecific. Together, these findings indicate that Xenopus oocytes possess an endogenous, specific, membrane-associated carrier-mediated uptake system for RF. The results also demonstrate the usefulness of Xenopus oocytes as a model system with which to study the RF transport event across biological membranes, which should further out present understanding of RF uptake by various vertebrate cells.

Amiloride↗
PubMed1994

Human intestinal cell line Caco-2: a useful model for studying cellular and molecular regulation of biotin uptake.

The mechanisms of enterocyte and molecular regulation of biotin uptake are poorly understood. An intestinal cell line processing the transport characteristics of native intestinal cells is highly desirable to investigate the finer details of the cellular processing and molecular regulation of biotin transport. In the present study, we investigated the uptake of the water-soluble vitamin biotin by a human intestinal cell line Caco-2. Uptake of both low (4 nM) and high (20 microM) concentrations of biotin by confluent monolayers of Caco-2 cells was appreciable and linear for up to 10 min of incubation. Replacement of Na+ in the incubation medium with other monovalent cations--K+, choline, Li+ and NH4(+)--caused a significant inhibition of biotin uptake; a relatively lesser inhibition was seen with Li+. Initial rate of uptake of biotin was temperature-dependent and saturable as a function of concentration at 37 degrees C but not at 4 degrees C. The Vmax and apparent Km of the temperature-dependent saturable process were 520 pmol/mg protein per min and 9.5 microM, respectively. The addition of unlabeled biotin and the structural analogue desthiobiotin to the incubation media caused a significant inhibition of the uptake of [3H]biotin. The inhibitory effect of desthiobiotin was competitive in nature with an inhibition constant (Ki) of 41 microM. Biocytin, on the other hand, was a weak inhibitor and biotin methyl ester and diaminobiotin did not have any effect. Pretreatment of Caco-2 cells with the monovalent cation ionophore gramicidin and the Na+, K+(-)ATPase inhibitor ouabain caused significant inhibition of biotin uptake. Pretreatment with the K+ ionophore valinomycin did not affect biotin uptake. Using the 'Activation Method', the stoichiometric ratio of biotin- to Na+ coupling was found to be 1:1. Growing confluent Caco-2 cells in a biotin-deficient environment resulted in rapid up-regulation of biotin transport with a marked increase (258%) in the Vmax of biotin uptake. These findings demonstrate that biotin uptake by Caco-2 cells is via a carrier-mediated system. This system is temperature-dependent, driven by Na(+)-gradient and is regulated by the substrate level. These in-vitro findings are very similar to and further confirm previous findings in human and animal studies and dispute other findings previously reported for Caco-2 cells; the present study also demonstrates the suitability of this system for further characterization of the cellular and molecular regulation of biotin uptake.

Biological Transport↗
PubMed1994

Uptake of biotin by human hepatoma cell line, Hep G2: a carrier-mediated process similar to that of normal liver.

Little is known about the cellular and molecular regulation of the uptake process of the water-soluble vitamin biotin into liver cells, the major site of biotin utilization and metabolism. Such studies are best done using a highly viable and homogeneous cellular system that allows examination of prolonged exposure to an agent(s) or a particular condition(s) on the uptake process. Isolated hepatocytes when maintained in primary culture lose their ability to transport biotin by the specialized carrier system. The aim of the present study was, therefore, to examine the mechanism(s) of biotin uptake by the cultured human-derived liver cells, Hep G2. Uptake of biotin by Hep G2 cells was appreciable and linear for up to 10 min of incubation. The uptake process was Na+ gradient-dependent as indicated by studies of Na+ replacement and pretreatment of cells with gramicidin and ouabain. Biotin uptake was also dependent on both incubation temperature and intracellular energy. Unlabeled biotin and the structural analogs with free carboxyl groups (thioctic acid, desthiobiotin) but not those with blocked carboxyl group (biocytin, biotin methyl ester, and thioctic amide) caused significant inhibition of 3H-biotin uptake at 37 degrees C but not 4 degrees C. Initial rate of biotin uptake was saturable as a function of concentration at 37 degrees C but was lower and linear at 4 degrees C. Pretreatment of Hep G2 cells with sulfhydryl group inhibitors (e.g., p-chloromercuribenzene sulfonate) led to a significant inhibition in biotin uptake; this inhibition was effectively reversed by reducing agents (e.g., dithiothreitol). Biotin uptake was also inhibited by the membrane transport inhibitors probenecid (noncompetitively), DIDS and furosemide but not by amiloride. Pretreatment of Hep G2 cells with valinomycin did not alter biotin uptake. The stoichiometric ratio of biotin to Na+ uptake in Hep G2 cells was also determined and found to be 1:1. These findings demonstrate that biotin uptake by these cultured liver cells is mediated through a specialized carrier system that is dependent on Na(+)-gradient, temperature, and energy and transports the vitamin by an electroneutral process. These findings are similar to those seen with native liver tissue preparations and demonstrate the suitability of Hep G2 cells for in-depth investigations of the cellular and molecular regulation of biotin uptake by the liver.

Biological Transport↗
PubMed1994

Retinyl ester hydrolytic activity associated with human intestinal brush border membranes.

Hydrolysis of retinyl esters in the lumen of the small intestine is required before absorption. Previously, rat brush border membranes (BBMs) were found to contain a pancreatic-derived esterase preferring retinyl esters with short fatty acyl chains and an intrinsic esterase preferring esters with long fatty acyl chains. Here, similar activities were found for preparations of human BBMs. Long-chain ester hydrolysis was stimulated best by deoxycholate, a dihydroxy bile salt, whereas short-chain ester hydrolysis was stimulated best by taurocholate, a trihydroxy bile salt. A 10-fold difference in KM values for retinyl palmitate (0.53 mumol/L) and caproate (5.5 mumol/L) also indicated distinguishable long-chain and short-chain activities. Differences between retinyl butyrate and retinyl caproate hydrolysis suggested the possible presence of two short-chain esterase activities associated with both rat and human BBMs, in addition to the long-chain activity. Similarities between human BBM retinyl ester hydrolytic activities and those observed for rat BBMs suggest that the rat is a good model for humans in this step of vitamin A metabolism.

Adult↗
PubMed1994

Expression of a rabbit renal ascorbic acid transporter in Xenopus laevis oocytes.

We examined the expression of renal ascorbic acid transporter(s) in Xenopus laevis oocytes after microinjection of cells with poly(A)+ RNA extracted from rabbit kidney cortex. Concomitant expression of the Na+-glucose cotransporter served as a control in these studies. Injection of poly(A)+ RNA into oocytes produced over a fivefold increase in the uptake of [14C]ascorbic acid (570 microM) compared with water-injected cells. Size fractionation of the kidney cortex mRNA by sucrose gradient revealed that the mRNA species that induced ascorbic acid transporter expression in oocytes was present in a fraction centered around 2.0 kilobases (kb) and had a size range of 1.8-3.1 kb. Injection of the active fraction into oocytes produced a > 40-fold increase in ascorbic acid uptake compared with water-injected controls. Expression of ascorbic acid transporter(s) was noticeable as early as 2 days after injection and was maximal after 7 days; it was also dependent on the amount of mRNA injected into oocytes. The induced uptake of [14C]ascorbic acid after injection of mRNA into oocytes was 1) Na+ dependent, as indicated by the almost complete lack of transport on removal of Na+ from the incubation medium; 2) significantly inhibited by unlabeled ascorbic acid and its structural analogue isoascorbic acid but not by D-glucose; and 3) saturable as a function of increasing the substrate concentration in the incubation medium (100-1,000 microM), with an apparent Km of 258 +/- 72.5 microM and a maximum velocity of 29.6 +/- 2.8 pmol.oocyte-1.2 h-1. These data demonstrate that X. laevis oocytes are a suitable system to functionally express the mammalian renal ascorbic acid transporter.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Mechanism of riboflavine uptake by Caco-2 human intestinal epithelial cells.

The cellular and molecular regulation of intestinal absorption of the water-soluble vitamin riboflavine (RF) is poorly understood. The availability of a suitable in vitro cultured system that possesses the transport characteristics of the native intestinal absorptive cells would provide a powerful means to address this issue. In this study, we examined RF uptake by the human-derived cultured Caco-2 intestinal epithelial cells. RF uptake was Na+ and pH independent and occurred without metabolic alterations of the transported RF. Initial rate of RF uptake was temperature dependent and saturable as a function of concentration at 37 degrees C but not at 4 degrees C (apparent Michaelis constant = 0.30 +/- 0.03 microM, maximal velocity = 209.90 +/- 24.40 pmol.mg protein-1.3 min-1). Unlabeled RF, lumiflavine, 8-amino-riboflavine, isoriboflavine, and lumichrome in the incubation solution caused significant inhibition of RF uptake. RF uptake was also energy dependent and was sensitive to the inhibitory effect of sulfhydryl group reagents. The membrane transport inhibitor amiloride, but not 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, 4-acetamide-4'-isothiocyanostilbene-2,2'-disulfonic acid, furosemide, or probenecid, inhibited RF uptake in a competitive (inhibitory constant = 0.48 mM) and reversible manner. Growing Caco-2 monolayers in a RF-deficient and oversupplemented media caused significant up- and downregulation of RF uptake, respectively. These results demonstrate the existence of a carrier-mediated system for RF uptake by Caco-2 cells and provide new information regarding amiloride sensitivity, involvement of sulfhydryl groups, and up- and downregulation by the substrate level and clarify the controversy regarding the role of Na+ in the uptake process.(ABSTRACT TRUNCATED AT 250 WORDS)

2,4-Dinitrophenol↗
PubMed1994

Regulation of riboflavin intestinal uptake by protein kinase A: studies with Caco-2 cells.

Although the mechanism of riboflavin (RF) intestinal uptake has been the subject of many studies, virtually nothing is known about the cellular regulation of the uptake process. In the present study, we investigated the role of protein kinase A (PKA)- and C (PKC)-mediated pathways in the regulation of RF intestinal uptake using the confluent Caco-2 monolayers. Treatment of Caco-2 cells with 3-isobutyl-1-methylxanthine (IBMX), forskolin, cholera toxin, or dibutyryl adenosine 3',5'-cyclic monophosphate caused a significant inhibition in RF uptake. The inhibitory effect of IBMX was reversible and resulted from a significant decrease in the maximal velocity of the RF uptake process with no change in its apparent Michaelis constant. The IBMX-induced inhibition in RF uptake was not mediated via inhibition in the synthesis of the RF carrier protein or through inhibition in the recruitment of preexisting carrier protein into the plasma membrane. Calyculin A also inhibited RF uptake when added alone and further potentiated the inhibitory effect of IBMX when added together. Phorbol 12-myristate 13-acetate or chelerythrine, on the other hand, showed no significant effect on RF uptake. These results demonstrate for the first time that compounds that increase intracellular cAMP levels downregulate RF intestinal uptake and that this effect is mediated via a decrease in the activity of the RF uptake carrier. It is suggested that a PKA-mediated pathway(s) plays an important role in regulating RF intestinal uptake.

1-Methyl-3-isobutylxanthine↗