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Biomedical subjects

H M Said

Publications and source records attributed to H M Said.

At least 19 recordsLinked to original sources
PubMed2001

Expression of the aromatase cytochrome P450 encoding gene in cattle and sheep.

During this report the tissue-specific expression and promoter usage of the aromatase cytochrome P450 encoding gene, Cyp19, are compared between cattle and sheep. In addition, data will be presented on the identification of cis-acting regulatory sequences located in the bovine placenta-specific promoter 1.1. In cattle and sheep Cyp19 is mainly expressed in the foetal placental layer and ovarian granulosa cells but also in other organs as brain or testis. Differently spliced transcripts of Cyp19 which include an invariable coding region but a variable 5'-untranslated region could be detected in tissues of both species. However, in contrast to ovary and brain which express homologous transcript variants, different transcripts are present in placentae suggesting that also different placenta-specific promoter regions are active in cattle and sheep. The analysis of the chromatin structure of the main placental promoter 1.1 in different bovine tissues revealed that hypomethylation and the occurrence of DNaseI hypersensitive sites (HS) within this region are associated with promoter activity. Active regulatory elements were identified in reporter gene studies in JEG-3 choriocarcinoma cells. The co-localisation of an E-box element within one of the placenta-specific HS suggests that this element is important for Cyp19 expression in the bovine placenta.

Alternative Splicing↗
PubMed2001

Chromatin structure of the bovine Cyp19 promoter 1.1. DNaseI hypersensitive sites and DNA hypomethylation correlate with placental expression.

Expression of the Cyp19 gene, encoding aromatase cytochrome P450, is driven by several tissue-specific promoters. The underlying mechanisms of this complex regulation have not yet been elucidated in detail. In the present report we investigate a possible link between chromatin structure and tissue-specific regulation of the bovine Cyp19 gene. We analysed the DNA methylation status and mapped DNaseI hypersensitive sites in the region encompassing the Cyp19 promoter 1.1 (P1.1) which controls Cyp19 expression in the bovine placenta. We show that P1.1 is hypomethylated in placental cotyledons (foetal layer) whereas it is methylated in placental caruncles (maternal layer), testis and corpus luteum. Furthermore, two placenta-specific DNaseI hypersensitive sites, HS1 and HS2, were observed within P1.1. Both DNA hypomethylation and the presence of DNaseI hypersensitive sites correlate with transcriptional activity of P1.1. Sequence analysis of hypersensitive sites revealed potential cis-regulatory elements, an E-box in HS1 and a trophoblast-specific element-like sequence in HS2. It could be demonstrated by electrophoretic mobility shift assays that both sequence motifs are specific targets for placenta-derived nuclear factors. In conclusion, observed tissue-specific differences of the chromatin structure which correlate with tissue-specific promoter activity suggest that chromatin might be an important regulator of aromatase expression in cattle.

Animals↗
PubMed2001

Molecular characterization of the 5' regulatory region of rat sodium-dependent multivitamin transporter gene.

Previous studies have demonstrated the involvement of a specialized, Na(+)-dependent carrier-mediated system for biotin uptake in mammalian intestine. The molecular identity of the carrier protein, the Na(+)-dependent multivitamin transporter (SMVT), has recently been identified. Upon characterization of transcript expression in the rat intestine, four distinct transcript variants (I-IV) due to heterogeneity at the 5'-untranslated region were found (Chatterjee NS, Kumar CK, Ortiz A, Rubin SA, and Said HM. Am J Physiol Cell Physiol 277: C605-C613, 1999). This finding raised the possibility that multiple promoters may be involved in driving the transcription of the SMVT gene. To test this possibility, we cloned the 5' regulatory region of the SMVT gene by genome walking. A 6.5-kb genomic DNA fragment was identified and sequenced. Three putative promoters (P1, P2, and P3) that were separated by exons of the four previously identified transcript variants were, indeed, found. P1 was found to contain multiple putative regulatory regions like GATA-1, AP-1, AP-2, and C/EBP, including several repeats of purine-rich regions and two TATA-like elements. P2 and P3 were GC rich and also revealed the presence of many putative regulatory elements including several SP-1 consensus sequences. The functional identity of each promoter and the minimal regions required for its function were established by the luciferase assay following transfection of rat-derived cultured intestinal epithelial IEC-6 cells. The highest functional activity of the cloned promoters was found to be in the order of P1 > P2 > P3. These findings represent the first characterization of the 5' regulatory region of any mammalian SMVT gene and should assist in the understanding of transcriptional regulation of this important gene.

5' Untranslated Regions↗
PubMed2001

Mechanism of thiamine uptake by human jejunal brush-border membrane vesicles.

Thiamine, a water-soluble vitamin, is essential for normal cellular functions, growth and development. Thiamine deficiency leads to significant clinical problems and occurs under a variety of conditions. To date, however, little is known about the mechanism of thiamine absorption in the native human small intestine. The objective of this study was, therefore, to characterize the mechanism of thiamine transport across the brush-border membrane (BBM) of human small intestine. With the use of purified BBM vesicles (BBMV) isolated from the jejunum of organ donors, thiamine uptake was found to be 1) independent of Na(+) but markedly stimulated by an outwardly directed H(+) gradient (pH 5.5(in)/pH 7.5(out)); 2) competitively inhibited by the cation transport inhibitor amiloride (inhibitor constant of 0.12 mM); 3) sensitive to temperature and osmolarity of the incubation medium; 4) significantly inhibited by thiamine structural analogs (amprolium, oxythiamine, and pyrithiamine), but not by unrelated organic cations (tetraethylammonium, N-methylnicotinamide, or choline); 5) not affected by the addition of ATP to the inside and outside of the BBMV; 6) potential insensitive; and 7) saturable as a function of thiamine concentration with an apparent Michaelis-Menten constant of 0.61 +/- 0.08 microM and a maximal velocity of 1.00 +/- 0.47 pmol. mg protein(-1). 10 s(-1). Carrier-mediated thiamine uptake was also found in BBMV of human ileum. These data demonstrate the existence of a Na(+)-independent, pH-dependent, amiloride-sensitive, electroneutral carrier-mediated mechanism for thiamine absorption in native human small intestinal BBMV.

Adenosine Triphosphate↗
PubMed2001

Mechanism of thiamine uptake by human colonocytes: studies with cultured colonic epithelial cell line NCM460.

Thiamine (vitamin B(1)) is essential for normal cellular functions and growth. Mammals cannot synthesize thiamine and thus must obtain the vitamin via intestinal absorption. The intestine is exposed to a dietary thiamine source and a bacterial source in which the vitamin is synthesized by the normal microflora of the large intestine. Very little is known about thiamine uptake in the large intestine. The aim of this study was, therefore, to address this issue. Our results with human-derived colonic epithelial NCM460 cells as a model system showed thiamine uptake to be 1) temperature- and energy dependent, 2) Na(+) independent, 3) increased with increasing buffer pH from 5 to 8 and after cell acidification but inhibited by amiloride, 4) saturable as a function of concentration, 5) inhibited by thiamine structural analogs but not by unrelated organic cations, and 6) inhibited by modulators of a Ca(2+)/calmodulin-mediated pathway. NCM460 cells and native human colonic mucosa expressed the recently cloned human thiamine transporter THTR-1 (product of the SLC19A2 gene) at both mRNA and protein levels. These results demonstrate for the first time that human NCM460 colonocytes possess a specific carrier-mediated system for thiamine uptake that appears to be under the regulation of an intracellular Ca(2+)/calmodulin-mediated pathway. It is suggested that bacterially synthesized thiamine in the large intestine may contribute to thiamine nutrition of the host, especially toward cellular nutrition of the local colonocytes.

Carrier Proteins↗
PubMed2001

Mechanism of folate transport across the human colonic basolateral membrane.

Previous studies from our laboratory have demonstrated the existence of a folate transporter in the human colonic apical membranes. The current studies were undertaken to examine the possible presence and function of a folate carrier in the human colonic basolateral membrane vesicles (BLMV). BLMV were purified from mucosal scrapings of colons of organ donors by a Percoll-density gradient centrifugation technique, and uptake studies were performed using a rapid filtration technique. Our results on [(3)H]Pte-Glu uptake are summarized as follows: 1) uptake was sensitive to osmolarity of the incubation medium; 2) Na(+) removal from the incubation medium did not affect folate uptake into BLMV; 3) uptake was significantly increased with decreasing incubation buffer pH from 8 to 4; 4) uptake demonstrated saturation kinetics with an apparent Michaelis constant of 9.6 +/- 0.48 microM and a maximal velocity of 8.10 +/- 0.36 pmol x mg protein(-1) x 10 s(-1); 5) uptake was markedly inhibited by the structural analog methotrexate (inhibitory constant = 8.28 +/- 1.0 microM); 6) uptake into BLMV demonstrated a trans-stimulation phenomenon; 7) anion exchange inhibitors DIDS and SITS significantly inhibited folate uptake; and 8) uptake was potential-insensitive, as voltage clamping of vesicles or making them inside positive with K(+)/valinomycin failed to influence folate uptake. Western blot analysis using purified human colonic basolateral membrane preparations and specific polyclonal antibodies against the human reduced folate carrier (hRFC) has shown expression of the hRFC protein at this membrane domain. These data demonstrate the existence of a pH-dependent, DIDS-sensitive, electroneutral, carrier-mediated mechanism for folate transport across the human colonic basolateral membranes.

Adult↗
PubMed2001

Intracellular trafficking/membrane targeting of human reduced folate carrier expressed in Xenopus oocytes.

The major cellular pathway for uptake of the vitamin folic acid, including its absorption in the intestine, is via a plasma membrane carrier system, the reduced folate carrier (RFC). Very little is known about the mechanisms that control intracellular trafficking and plasma membrane targeting of RFC. To begin addressing these issues, we used Xenopus oocyte as a model system and examined whether the signal that targets the protein to the plasma membrane is located in the COOH-terminal cytoplasmic tail or in the backbone of the polypeptide. We also examined the role of microtubules and microfilaments in intracellular trafficking of the protein. Confocal imaging of human RFC (hRFC) fused to the enhanced green fluorescent protein (hRFC-EGFP) showed that the protein was expressed at the plasma membrane, with expression confined almost entirely to the animal pole of the oocyte. Localization of hRFC at the plasma membrane was not affected by partial or total truncation of the COOH-terminal tail of the polypeptide, whereas a construct of the cytoplasmic tail fused to EGFP was not found at the plasma membrane. Disruption of microtubules, but not microfilaments, prevented hRFC expression at the plasma membrane. These results demonstrate that the molecular determinant(s) that directs plasma membrane targeting of hRFC is located within the backbone of the polypeptide and that intact microtubules, but not microfilaments, are essential for intracellular trafficking of the protein.

Actin Cytoskeleton↗
PubMed2000

Riboflavin uptake by human-derived colonic epithelial NCM460 cells.

Normal microflora of the large intestine synthesize a number of water-soluble vitamins including riboflavin (RF). Recent studies have shown that colonic epithelial cells possess an efficient carrier-mediated mechanism for absorbing some of these micronutrients. The aim of the present study was to determine whether colonic cells also possess a carrier-mediated mechanism for RF uptake and, if so, to characterize this mechanism and study its cellular regulation. Confluent monolayers of the human-derived nontransformed colonic epithelial cells NCM460 and [(3)H]RF were used in the study. Uptake of RF was found to be 1) appreciable and temperature and energy dependent; 2) Na(+) independent; 3) saturable as a function of concentration with an apparent K(m) of 0.14 microM and V(max) of 3.29 pmol x mg protein(-1) x 3 min(-1); 4) inhibited by the structural analogs lumiflavin and lumichrome (K(i) of 1.8 and 14.1 microM, respectively) but not by the unrelated biotin; 5) inhibited in a competitive manner by the membrane transport inhibitor amiloride (K(i) = 0.86 mM) but not by furosemide, DIDS, or probenecid; 6) adaptively regulated by extracellular RF levels with a significant and specific upregulation and downregulation in RF uptake in RF-deficient and oversupplemented conditions, respectively; and 7) modulated by an intracellular Ca(2+)/calmodulin-mediated pathway. These studies demonstrate for the first time the existence of a specialized carrier-mediated mechanism for RF uptake in an in vitro cellular model system of human colonocytes. This mechanism appears to be regulated by extracellular substrate level and by an intracellular Ca(2+)/calmodulin-mediated pathway. It is suggested that the identified transport system may be involved in the absorption of bacterially synthesized RF in the large intestine and that this source of RF may contribute toward RF homeostasis, especially that of colonocytes.

Biological Transport↗
PubMed2000

Riboflavin transport by isolated perfused rabbit renal proximal tubules.

Rabbit renal proximal tubular transport of riboflavin (RF) was examined by using the in vitro isolated tubule perfusion technique. We found that proximal tubules actively reabsorbed (J(lb)) and secreted (J(bl)) RF. At 0.1 microM RF concentration, J(bl) was significantly higher than J(lb), resulting in a net secretion. This net secretion of RF was decreased at 0.01 microM RF concentration and increased at 1 microM RF concentration. Both J(lb) and J(bl) were inhibited by lowering temperature or by adding iodoacetate, a metabolic inhibitor, and lumichrome, an RF analog, suggesting the involvement of carrier-mediated transport mechanisms. J(bl) was inhibited by probenecid, an anion transport inhibitor, and by para-aminohippuric acid, an organic anion, suggesting the relevance of RF secretion to renal organic anion transport. J(bl) was also inhibited by alkaline pH (8.0) and by the calmodulin inhibitor trifluoperazine, indicating the influence of pH and Ca(2+)/calmodulin-dependent pathway on RF secretion. Finally, we found that addition of chlorpromazine, a phenothiazine derivative, inhibited both J(lb) and J(bl), raising the concern about the nutritional status in patients receiving such a type of medication.

Animals↗
PubMed2000

Adaptive regulation of intestinal folate uptake: effect of dietary folate deficiency.

Folate is an essential micronutrient that, in mammals, must be obtained from exogenous sources via intestinal absorption. Previous studies have characterized different aspects of the mechanism of the intestinal folate uptake process. Much less, however, is known about regulation of this process. In this study, we examined the effect of dietary folate deficiency on intestinal folate uptake using the rat as an animal model. The results showed that dietary folate deficiency leads to a significant (P < 0.01) and specific upregulation in the transepithelial transport of folic acid. The upregulation in transepithelial folate transport 1) was found to be due to an induction in carrier-mediated folate uptake across the brush-border membrane (BBM) and was mediated via a significant (P < 0.01) increase in the maximal velocity but not the apparent Michaelis constant of the uptake process, 2) was associated with a marked increase in the steady-state mRNA level of reduced folate carrier-1 and in the level of the expressed protein at the intestinal BBM, and 3) was associated with a marked (>10-fold) increase in the activity of the intestinal BBM form of folate hydrolase. Results of this study demonstrate, for the first time, that dietary folate deficiency leads to a marked upregulation in intestinal folate uptake and in the activity of folate hydrolase. Furthermore, the upregulation in folate uptake is associated with an increase in mRNA and protein levels of folate carrier, suggesting possible involvement of a transcriptional regulatory mechanism(s) in the upregulation.

Adaptation, Physiological↗
PubMed1999

Cellular uptake of biotin: mechanisms and regulation.

This review describes our knowledge of biotin transport in the small intestine of humans and other mammals and presents recent findings in the area. Previous studies have shown that biotin transport across the brush border membrane of the small intestinal absorptive cells occurs via a carrier-mediated, Na+ gradient-dependent, electroneutral mechanism. Exit of biotin out of the enterocyte, i.e., transport across the basolateral membrane, also occurs via a carrier-mediated process, but the process is Na+ independent and electrogenic. Recent studies from our laboratory have shown that the uptake process of biotin in Caco-2 cells, a human-derived cultured intestinal epithelial cell line, are under the cellular regulation of both a protein kinase C- and a Ca/calmodulin-mediated pathway. In addition, the uptake process is shared by another water-soluble vitamin, pantothenic acid. For the first time, other recent studies have detected the existence of a Na+-dependent, carrier-mediated mechanism for biotin uptake at the apical membrane of colonocytes, which could theoretically mediate absorption of the biotin synthesized by colonic microflora. This system was again found to be shared by pantothenic acid, which is also synthesized by the normal microflora of the large intestine.

Animals↗
PubMed1999

Intestinal absorption of vitamins.

This article provides an overview of advances in understanding the cellular and molecular mechanisms and regulation of intestinal absorption processes of vitamins. The vitamins covered are the water-soluble vitamins folic acid, cobalamin (vitamin B12), biotin, pantothenic acid, and thiamine (vitamin B1) and the lipid-soluble vitamin A. For folate, significant advances have been made in regard to i) digestion of dietary folate polyglutamates to folate monoglutamates by the cloning of the responsible enzyme; ii) identification of the cDNA responsible for the intestinal folate transporter; iii) delineation of intracellular mechanisms that regulate small intestinal folate uptake; and iv) identification and characterization of a specific, pH-dependent, carrier-mediated system for folate uptake at the luminal (apical) membrane of human colonocytes. Studies on cobalamine have focused on cellular and molecular characterization of the intrinsic factor and its receptor. Studies on biotin transport in the small intestine have shown that the uptake process is shared by another water-soluble vitamin, pantothenic acid. Furthermore, a Na-dependent, carrier-mediated biotin uptake system that is also shared with pantothenic acid has been identified at the apical membrane of human colonocytes. This carrier is believed to be responsible for the absorption of the bacterially synthesized biotin and pantothenic acid in the large intestine. Also, preliminary studies have reported the cloning of a biotin transporter from the small intestine. As for thiamine intestinal transport, a study has shown thiamine uptake by small intestinal biopsy specimens to be via a carrier-mediated, Na-independent mechanism, which appears to be up-regulated in thiamine deficiency. Studies on vitamin A intestinal absorption have shown the existence of a receptor-mediated mechanism for the uptake of retinol bound to retinol-binding protein in the small intestine of suckling rats. Another study has shown that retinoic acid increases the mRNA level of the cellular retinol binding protein II and the rate of retinol uptake by Caco-2 intestinal epithelial cells. The study suggested that retinoids may play a role in the regulation of vitamin A intestinal absorption.

Journal Article↗
PubMed1999

Molecular mechanism of the intestinal biotin transport process.

Previous studies have characterized different aspects of the cellular/membrane mechanism and regulation of the intestinal uptake process of the water-soluble vitamin biotin. Little, however, is known about the molecular mechanisms of the uptake process. In this study, we have identified a cDNA from rat small intestine that appears to be involved in biotin transport. The open reading frame of this cloned cDNA consisted of 1,905 bases and was identical to that identified for the vitamin transporter in placental tissue. Significant heterogeneity, however, was found in the 5' untranslated region of this clone, with three distinct variants (II, III, IV) being identified in the small intestine; the placental variant (variant I), however, was not present in the small gut. Variant II was found to be the predominant form expressed in the rat small and large intestines. Functional identity of the cloned intestinal cDNA was confirmed by stable expression in COS-7 cells, which showed a four- to fivefold increase in biotin uptake in transfected COS-7 cells compared with controls. The induced biotin uptake in transfected COS-7 cells was found to be 1) Na(+) dependent, 2) saturable as a function of concentration with an apparent K(m) of 8. 77 microM and a V(max) of 779.7 pmol. mg protein(-1). 3 min(-1), and 3) inhibited by unlabeled biotin and pantothenic acid and their structural analogs. The distribution of complementary mRNA transcripts of the cloned cDNA along the vertical and longitudinal axes of the intestinal tract was also determined. Results of this study describe the molecular characteristics of the intestinal biotin absorption process and report the identification of a cDNA that encodes a Na(+)-dependent biotin uptake carrier that appears to exist in the form of multiple variants.

5' Untranslated Regions↗
PubMed1999

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 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.

Biological Transport↗
PubMed1998

Riboflavin transport by rabbit renal basolateral membrane vesicles.

The present study examined riboflavin (RF) uptake by isolated rabbit renal basolateral membrane (BLM). RF uptake was linear during the initial 10 seconds and leveled off thereafter with longer incubation. Studies on RF uptake as a function of incubation medium osmolarity indicated that the BLM RF uptake was the results of transport (approximately 45%) into the intravesicular space as well as binding (approximately 55%) to membrane surfaces. The RF binding to BLM was Na+-dependent so that replacement of Na+ by other cations eliminated the binding component of RF uptake. The process of BLM RF uptake was saturable as a function of substrate concentration and was significantly inhibited by cis-addition of its structural analogs, lumiflavin and lumichrome, indicating the involvement of a carrier-mediated process. The BLM RF uptake was affected by changes in extravesicular pH so that, as compared to pH 7.5, RF uptake was lower at pH 6.5 and higher at pH 8.5. The effect of extravesicular pH persisted when the transmembrane H+ gradient was dissipated by FCCP, indicating the direct effect of pH on BLM RF uptake. The BLM RF uptake was not affected by alterations of the transmembrane electrical potential, induced by either the presence of anions with different membrane permeability (Cl-=NO-3>SO-4>gluconate-) or using nigericin (10 microg/mg protein) with an outwardly or inwardly directed transmembrane K+ gradient. The BLM RF uptake was, however, inhibited by probenecid and p-aminohippurate, and was enhanced by trans-RF. In summary, these results demonstrate the existence of a Na+-dependent BLM binding of RF and a membrane-associated carrier system for RF uptake by renal BLM.

Animals↗
PubMed1998

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.

Antiemetics↗
PubMed1998

Comparison of intestinal folate carrier clone expressed in IEC-6 cells and in Xenopus oocytes.

We recently identified a cDNA clone from mouse small intestine, which appears to be involved in folate transport when expressed in Xenopus oocytes. The open reading frame of this clone is identical to that of the reduced folate carrier (RFC) (K. H. Dixon, B. C. Lanpher, J. Chiu, K. Kelley, and K. H. Cowan. J. Biol. Chem. 269: 17-20, 1994). The characteristics of this cDNA clone [previously referred to as intestinal folate carrier 1 (IFC-1)] expressed in Xenopus oocytes, however, were found to be different from the characteristics of folate transport in native small intestinal epithelial cells. To further study these differences, we determined the characteristics of RFC when expressed in an intestinal epithelial cell line, IEC-6, and compared the findings to its characteristics when expressed in Xenopus oocytes. RFC was stably transfected into IEC-6 cells by electroporation; its cRNA was microinjected into Xenopus oocytes. Northern blot analysis of poly(A)+ RNA from IEC-6 cells stably transfected with RFC cDNA (IEC-6/RFC) showed a twofold increase in RFC mRNA levels over controls. Similarly, uptake of folic acid and 5-methyltetrahydrofolate (5-MTHF) by IEC-6/RFC was found to be fourfold higher than uptake in control sublines. This increase in folic acid and 5-MTHF uptake was inhibited by treating IEC-6/RFC cells with cholesterol-modified antisense DNA oligonucleotides. The increase in uptake was found to be mainly mediated through an increase in the maximal velocity (Vmax) of the uptake process [the apparent Michaelis-Menten constant (K(m)) also changed (range was 0.31 to 1.56 microM), but no specific trend was seen]. In both IEC-6/RFC and control sublines, the uptake of both folic acid and 5-MTHF displayed 1) pH dependency, with a higher uptake at acidic pH 5.5 compared with pH 7.5, and 2) inhibition to the same extent by both reduced and oxidized folate derivatives. These characteristics are very similar to those seen in native intestinal epithelial cells. In contrast, RFC expressed in Xenopus oocytes showed 1) higher uptake at neutral and alkaline pH 7.5 compared with acidic pH 5.5 and 2) higher sensitivity to reduced compared with oxidized folate derivatives. Results of these studies demonstrate that the characteristics of RFC vary depending on the cell system in which it is expressed. Furthermore, the results may suggest the involvement of cell-or tissue-specific posttranslational modification(s) and/or the existence of an auxiliary protein that may account for the differences in the characteristics of the intestinal RFC when expressed in Xenopus oocytes compared with when expressed in intestinal epithelial cells.

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