Sequence, tissue distribution, and functional characterization of the rat fructose transporter GLUT5

cDNA clones encoding rat GLUT5-small intestinal facilitative hexose transporter were isolated from a jejunum library by cross-hybridization with a human GLUT5 cDNA probe. The cDNA sequence indicates that rat GLUT5 is composed of 502 amino acids and has 81.5% amino acid identity and 87.3% similarity with the sequence of human GLUT5. Expression of synthetic rat GLUT5 mRNA in Xenopus oocytes showed that rat GLUT5 was able to mediate the uptake of fructose and, to a lesser extent, of glucose. RNA blotting studies showed that GLUT5 mRNA was present in rat small intestine, kidney, and brain. Although GLUT5 mRNA is expressed in human testis, adipose tissue, and skeletal muscle, it could not be detected by RNA blotting in these rat tissues. Developmental studies showed low levels of GLUT5 mRNA in rat small intestine and kidney during the prenatal period with a rapid induction of GLUT5 expression occurring postnatally. In situ hybridization studies of GLUT5 mRNA expression in the small intestine revealed differential expression along the crypt-villus axis with the highest levels of mRNA being in the midvillus region. In addition, there was quantitatively more GLUT5 mRNA detected in the proximal as opposed to the distal small intestine.

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Functional characterization of PCFT/HCP1 as the molecular entity of the carrier-mediated intestinal folate transport system in the rat model

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00309.2007 ◽

2008 ◽

Vol 294 (3) ◽

pp. G660-G668 ◽

Cited By ~ 67

Author(s):

Katsuhisa Inoue ◽

Yasuhiro Nakai ◽

Sayaka Ueda ◽

Shunsuke Kamigaso ◽

Kin-ya Ohta ◽

...

Keyword(s):

Small Intestine ◽

Transport System ◽

Functional Characterization ◽

Cellular Membrane ◽

Molecular Entity ◽

Xenopus Laevis Oocytes ◽

Rat Small Intestine ◽

Distribution Profile ◽

Folate Transport

Proton-coupled folate transporter/heme carrier protein 1 (PCFT/HCP1) has recently been identified as a transporter that mediates the translocation of folates across the cellular membrane by a proton-coupled mechanism and suggested to be the possible molecular entity of the carrier-mediated intestinal folate transport system. To further clarify its role in intestinal folate transport, we examined the functional characteristics of rat PCFT/HCP1 (rPCFT/HCP1) expressed in Xenopus laevis oocytes and compared with those of the carrier-mediated folate transport system in the rat small intestine evaluated by using the everted tissue sacs. rPCFT/HCP1 was demonstrated to transport folate and methotrexate more efficiently at lower acidic pH and, as evaluated at pH 5.5, with smaller Michaelis constant ( Km) for the former (2.4 μM) than for the latter (5.7 μM), indicating its characteristic as a proton-coupled folate transporter that favors folate than methotrexate as substrate. rPCFT/HCP1-mediated folate transport was found to be inhibited by several but limited anionic compounds, such as sulfobromophthalein and sulfasalazine. All these characteristics of rPCFT/HCP1 were in agreement with those of carrier-mediated intestinal folate transport system, of which the Km values were 1.2 and 5.8 μM for folate and methotrexate, respectively, in the rat small intestine. Furthermore, the distribution profile of the folate transport system activity along the intestinal tract was in agreement with that of rPCFT/HCP1 mRNA. This study is the first to clone rPCFT/HCP1, and we successfully provided several lines of evidence that indicate its role as the molecular entity of the intestinal folate transport system.

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Role of 5-HT in cholera toxin-induced mucin secretion in the rat small intestine

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1996.270.6.g1001 ◽

1996 ◽

Vol 270 (6) ◽

pp. G1001-G1009 ◽

Cited By ~ 11

Author(s):

B. A. Moore ◽

K. A. Sharkey ◽

M. Mantle

Keyword(s):

Small Intestine ◽

Receptor Antagonist ◽

Cholera Toxin ◽

Receptor Subtypes ◽

Rat Small Intestine ◽

Mucin Secretion ◽

Common Pathway ◽

Distal Small Intestine ◽

Intestinal Segments

We examined the role of 5-hydroxytryptamine (5-HT) in cholera toxin (CT)-induced mucin secretion in the proximal and distal regions of the rat small intestine. Neither the 5-HT2 receptor antagonist ketanserin nor the cyclooxygenase inhibitor indomethacin was capable of inhibiting choleraic mucin secretion. However, in the presence of the mixed 5-HT3/4 receptor antagonist tropisetron at doses that block both receptor subtypes, the secretory response was reduced to baseline levels in the proximal and distal small intestine. The selective 5-HT3 receptor antagonist ondansetron had no significant effect. These findings suggest that choleraic mucin secretion is mediated primarily through the activation of a 5-HT4-like receptor. Mucin secretion in response to the exogenous application of 5-HT occurs via two pathways: one is mediated by a 5-HT4-like receptor and is capsaicin sensitive but tetrodotoxin (TTX) insensitive, and one lacks the capsaicin-sensitive 5-HT4-mediated response but is TTX sensitive. Both converge on a common pathway that is cholinergic. No significant differences were observed between proximal and distal intestinal segments.

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Neural mediation of cholera toxin-induced mucin secretion in the rat small intestine

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1993.265.6.g1050 ◽

1993 ◽

Vol 265 (6) ◽

pp. G1050-G1056 ◽

Cited By ~ 6

Author(s):

B. A. Moore ◽

K. A. Sharkey ◽

M. Mantle

Keyword(s):

Small Intestine ◽

Cholera Toxin ◽

Fold Increase ◽

Open Loop ◽

Sensory Nerves ◽

Loop Model ◽

Rat Small Intestine ◽

Mucin Secretion ◽

Distal Small Intestine

We examined the role of enteric nerves in cholera toxin (CT)-induced mucin secretion in proximal and distal regions of rat small intestine. Stimulation of intestinal loops with 120 micrograms (1.5 mumol) CT using an in vitro open-loop model resulted in an approximately four-fold increase in luminal mucin content over unstimulated controls in both regions of the gut. Prior treatment of loops with tetrodotoxin had no effect on the amount of mucin released in response to CT. However, permanent destruction of primary sensory afferent nerves by neonatal treatment of rats with capsaicin reduced the mucin response to CT to baseline levels in both regions. In normal animals, atropine resulted in approximately 40% inhibition of mucin secretion in both the proximal and distal small intestine. The atropine-sensitive secretory response appears to be a component of the capsaicin-sensitive response. These results suggest that choleraic mucin secretion is mediated primarily by a capsaicin-sensitive neurogenic pathway involving local activation of sensory nerves, which may then elicit mucin secretion through interaction with cholinergic nerves.

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Cloning and functional characterization of the human GLUT7 isoform SLC2A7 from the small intestine

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00396.2003 ◽

2004 ◽

Vol 287 (1) ◽

pp. G236-G242 ◽

Cited By ~ 65

Author(s):

Qiang Li ◽

Andrei Manolescu ◽

Mabel Ritzel ◽

Sylvia Yao ◽

Melissa Slugoski ◽

...

Keyword(s):

Small Intestine ◽

Brush Border Membrane ◽

Functional Characterization ◽

Northern Blotting ◽

Rat Tissues ◽

Amino Acid Residues ◽

Terminal Sequence ◽

Structural Determinants ◽

Close Sequence

Facilitated glucose transporters (GLUTs) mediate transport of sugars across cell membranes by using the chemical gradient of sugars as the driving force. Improved cloning techniques and database analyses have expanded this family of proteins to a total of 14 putative members. In this work a novel hexose transporter isoform, GLUT7, has been cloned from a human intestinal cDNA library by using a PCR-based strategy (GenBank accession no. AY571960 ). The encoded protein is comprised of 524 amino acid residues and shares 68% similarity and 53% identity with GLUT5, its most closely related isoform. When GLUT7 was expressed in Xenopus oocytes, it showed high-affinity transport for glucose ( Km = 0.3 mM) and fructose (IC50 = 0.060 mM). Galactose, 2-deoxy-d-glucose, and xylose were not transported. Uptake of 100 μM d-glucose was not inhibited by 200 μM phloretin or 100 μM cytochalasin B. Northern blotting indicated that the mRNA for GLUT7 is present in the human small intestine, colon, testis, and prostate. Western blotting and immunohistochemistry of rat tissues with an antibody raised against the predicted COOH-terminal sequence confirmed expression of the protein in the small intestine and indicated that the transporter is predominantly expressed in the enterocytes' brush-border membrane. The unusual substrate specificity and close sequence identity with GLUT5 suggest that GLUT7 represents an intermediate between class II GLUTs and the class I member GLUT2. Comparison between these proteins may provide key information as to the structural determinants for the recognition of fructose as a substrate.

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Age- and tissue-specific induction of NHE3 by glucocorticoids in the rat small intestine

AJP Cell Physiology ◽

10.1152/ajpcell.2000.278.4.c629 ◽

2000 ◽

Vol 278 (4) ◽

pp. C629-C637 ◽

Cited By ~ 33

Author(s):

Pawel R. Kiela ◽

Yigit S. Guner ◽

Hua Xu ◽

James F. Collins ◽

Fayez K. Ghishan

Keyword(s):

Small Intestine ◽

Glucocorticoid Receptor ◽

Western Blot ◽

Mrna Level ◽

Rat Small Intestine ◽

Adult Rats ◽

Tissue Specific ◽

Distal Small Intestine ◽

Proximal Small Intestine ◽

Specific Induction

Of the two known apical isoforms of the Na+/H+ exchanger (NHE) family, only the NHE3 gene is regulated by glucocorticoids. The aim of these studies was to investigate the mechanisms underlying the effects of methylprednisolone (MP) on expression of NHE3 in the proximal and distal small intestine of suckling and adult rats. Immunoblots showed that the glucocorticoid responsiveness in the proximal small intestine was greatest in suckling animals (NHE3/β-actin: 0.43 ± 0.09 control vs. 1.57 ± 0.15 MP; P < 0.001), and responsiveness decreased with age with no effect in adults (0.56 ± 0.14 vs. 0.64 ± 0.17). Distal small intestine was responsive only in adult rats (0.49 ± 0.13 vs. 1.65 ± 0.09; P < 0.001). This pattern was confirmed at the mRNA level and by 22Na+ uptake. Western blot and [3H]dexamethasone mesylate binding showed that the responsiveness of NHE3 to glucocorticoids is directly related to the expression of glucocorticoid receptor (GR) in the small intestine. These studies suggest that loss and gain of glucocorticoid responsiveness in the proximal and distal small intestine, respectively, are related to age- and segment-dependent expression of GR.

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Differential tissue expression of the glutathione transferase multigene family

Biochemical Journal ◽

10.1042/bj2380373 ◽

1986 ◽

Vol 238 (2) ◽

pp. 373-378 ◽

Cited By ~ 9

Author(s):

S E Pemble ◽

J B Taylor ◽

R K Craig ◽

B Ketterer

Keyword(s):

Glutathione Transferase ◽

Cdna Probe ◽

Tissue Expression ◽

Northern Blotting ◽

Cdna Clones ◽

Rat Tissues ◽

Specific Distribution ◽

Free Translation ◽

Multigenic Family

The content of GSH transferase mRNAs in poly(A)-containing RNA isolated from eight rat tissues was examined by immunoprecipitation of cell-free translation products and by Northern blotting. Considerable tissue-specific distribution and heterogeneity of immunoprecipitable GSH transferase subunits 1 and 2 synthesized in vitro was observed. These results were confirmed by Northern blotting using a 32P-labelled subunit 1 cDNA probe. The same probe, used in a Southern blot analysis of genomic DNA, provided confirmation that GSH transferase subunits 1 and 2 comprise a multigenic family in the rat. The results show that the selection of cDNA clones coding for chosen subunits can be made easier by making use of qualitative and quantitative tissue differences in GSH transferase mRNAs.

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Cloning and expression of rat lung acidic Ca2+-independent PLA2 and its organ distribution

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1998.274.5.l750 ◽

1998 ◽

Vol 274 (5) ◽

pp. L750-L761 ◽

Cited By ~ 21

Author(s):

Tae-Suk Kim ◽

Chandra Dodia ◽

Xi Chen ◽

Brian B. Hennigan ◽

Mahendra Jain ◽

...

Keyword(s):

Enzymatic Activity ◽

Translational Control ◽

Consensus Sequence ◽

Cdna Probe ◽

Cloning And Expression ◽

Organ Distribution ◽

Alveolar Type ◽

Cdna Clones ◽

Rat Tissues ◽

Ph Optimum

A clone for a rat acidic Ca2+-independent phospholipase A2(aiPLA2) was isolated from a cDNA library prepared from rat granular pneumocytes with a probe based on the human aiPLA2 sequence (T. S. Kim, C. S. Sundaresh, S. I. Feinstein, C. Dodia, W. R. Skach, M. K. Jain, T. Nagase, N. Seki, K. Ishikawa, N. Nomura, and A. B. Fisher. J. Biol. Chem. 272: 2542–2550, 1997). In addition, a consensus sequence for mouse aiPLA2 was constructed from several mouse cDNA clones in the GenBank and dbEST databases. Each sequence codes for a 224-amino acid protein with 88% identity of the amino acids among the three species and conservation of a putative lipase motif (GDSWG). Translation of mRNA produced from the rat clone in a wheat germ system resulted in expression of PLA2 activity with properties similar to those of the human enzyme, i.e., acidic pH optimum and Ca2+ independence. The localization of aiPLA2 in rat tissues was studied with the human cDNA probe, polyclonal and monoclonal antibodies, and aiPLA2activity. aiPLA2 is present in the lung as evidenced by high levels of mRNA and protein expression and by enzymatic activity that is inhibited by anti-PLA2 antibody and by the transition state analog 1-hexadecyl-3-trifluoroethylglycero- sn-2-phosphomethanol (MJ33). Immunocytochemistry showed the presence of aiPLA2 in alveolar type II cells, alveolar macrophages, and bronchiolar epithelium. In the brain, heart, liver, kidney, spleen, and intestine, aiPLA2 mRNA content was <50% of that in the lung, immunoreactive protein was not detectable, and enzymatic activity was not inhibited by MJ33 or aiPLA2 antibody. These results show marked enrichment of aiPLA2in the lung compared with the other organs and suggest translational control of aiPLA2 expression.

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Sulphonation of dehydroepiandrosterone and neurosteroids: molecular cloning, expression, and functional characterization of a novel zebrafish SULT2 cytosolic sulphotransferase

Biochemical Journal ◽

10.1042/bj20031050 ◽

2003 ◽

Vol 375 (3) ◽

pp. 785-791 ◽

Cited By ~ 24

Author(s):

Takuya SUGAHARA ◽

Yuh-Shyong YANG ◽

Chau-Ching LIU ◽

T. Govind PAI ◽

Ming-Cheh LIU

Keyword(s):

Molecular Cloning ◽

Expressed Sequence Tag ◽

Expression System ◽

Functional Characterization ◽

Amino Acid Identity ◽

Cdna Clones ◽

Reverse Transcription Pcr ◽

Prokaryotic Expression System ◽

Partial Cdna

By searching the zebrafish EST (expressed-sequence tag) database, we have identified two partial cDNA clones encoding the 5′ and 3′ regions of a putative zebrafish sulphotransferase (ST). Using the reverse transcription-PCR technique, a full-length cDNA encoding this zebrafish ST was successfully cloned. Sequence analysis revealed that this novel zebrafish ST displays 44%, 43% and 40% amino acid identity with mouse SULT2B1, human SULT2B1b and human SULT2A1 ST respectively. This zebrafish ST therefore appears to belong to the SULT2 cytosolic ST gene family. Recombinant zebrafish ST, expressed using the pGEX-2TK prokaryotic expression system and purified from transformed Escherichia coli cells, migrated as a 34 kDa protein upon SDS/PAGE. Purified zebrafish ST displayed a strong sulphonating activity toward DHEA (dehydroepiandrosterone), with a optimum pH of 9.5. The enzyme also exhibited activities toward several neurosteroids with differential Km and Vmax values. A thermostability experiment revealed the enzyme to be relatively stable over a temperature range between 20 °C and 43 °C. Among ten different divalent metal cations tested, Fe2+ and Cd2+ exhibited small, but significant, stimulatory effects, whereas Hg2+ and Cu2+ displayed considerably stronger inhibitory effects on the DHEA-sulphonating activity of the enzyme. These results constitute the first study on the molecular cloning, expression, and characterization of a zebrafish cytosolic SULT2 ST.

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