Molecular cloning and functional expression of cDNAs encoding a human Na+-nucleoside cotransporter (hCNT1)

1997 ◽  
Vol 272 (2) ◽  
pp. C707-C714 ◽  
Author(s):  
M. W. Ritzel ◽  
S. Y. Yao ◽  
M. Y. Huang ◽  
J. F. Elliott ◽  
C. E. Cass ◽  
...  
Keyword(s):  
Molecular Cloning ◽  
Xenopus Oocytes ◽  
Polymerase Chain Reaction Amplification ◽  
Human Kidney ◽  
Functional Expression ◽  
Nucleoside Transporter ◽  
Renal Secretion ◽  
Transporter Protein ◽  
Reaction Amplification ◽  
Nucleoside Drugs

We report identification of a new human nucleoside transporter protein by molecular cloning and functional expression of its cDNA. Previously, we used expression selection in Xenopus oocytes to isolate a cDNA from rat jejunal epithelium encoding the pyrimidine-selective Na+-dependent nucleoside transporter rCNT1 (Q.-Q. Huang, S. Y. M. Yao, M. W. L. Ritzel, A. R. P. Paterson, C. E. Cass, and J. D. Young. J. Biol. Chem. 269: 17757-17760, 1994). cDNAs for a human homologue of rCNT1, designated hCNT1, have been isolated from human kidney by hybridization cloning and reverse transcriptase polymerase chain reaction amplification strategies. hCNT1 was 83% identical to rCNT1 in amino acid sequence and exhibited the transport characteristics of an Na+-dependent nucleoside transporter with selectivity for pyrimidine nucleosides and adenosine when expressed in Xenopus oocytes. Deoxyadenosine, which undergoes net renal secretion, and guanosine were poor permeants. hCNT1 did, however, transport 3'-azido-3'-deoxythymidine. This is the first demonstration that members of the CNT family exist in human cells and provides evidence of their involvement in the renal transport of physiological nucleosides and nucleoside drugs. The hCNT1 gene was mapped to chromosome 15q25-26.

scholarly journals Molecular cloning and characterization of a nitrobenzylthioinosine-insensitive (ei) equilibrative nucleoside transporter from human placenta

1997 ◽  
Vol 328 (3) ◽  
pp. 739-743 ◽  
Author(s):  
Mark GRIFFITHS ◽  
Y. M. Sylvia YAO ◽  
Fatima ABIDI ◽  
E. V. Simon PHILLIPS ◽  
E. Carol CASS ◽  
...  
Keyword(s):  
Molecular Cloning ◽  
Human Placenta ◽  
Ovarian Tissue ◽  
Functional Expression ◽  
Nucleoside Transporter ◽  
Intact Cells ◽  
Equilibrative Nucleoside Transporter ◽  
Equilibrative Nucleoside Transporters ◽  
Sequence Deletion

Mammalian equilibrative nucleoside transporters are typically divided into two classes, es and ei, based on their sensitivity or resistance respectively to inhibition by nitrobenzylthioinosine (NBMPR). Previously, we have reported the isolation of a cDNA clone encoding a prototypic es-type transporter, hENT1 (human equilibrative nucleoside transporter 1), from human placenta. We now report the molecular cloning and functional expression in Xenopus oocytes of a cDNA from the same tissue encoding a homologous ei-type transporter, which we designate hENT2. This 456-residue protein is 46% identical in amino acid sequence with hENT1 and corresponds to a full-length form of the delayed-early proliferative response gene product HNP36, a protein of unknown function previously cloned in a form bearing a sequence deletion. In addition to placenta, hENT2 is found in brain, heart and ovarian tissue. Like hENT1, hENT2 mediates saturable transport of the pyrimidine nucleoside uridine (Km 0.2±0.03 mM) and also transports the purine nucleoside adenosine. However, in contrast with hENT1, which is potently inhibited by NBMPR (Ki 2 nM), hENT2 is NBMPR-insensitive (IC50 < 1 μM). It is also much less sensitive to inhibition by the coronary vasoactive drugs dipyridamole and dilazep and to the lidoflazine analogue draflazine, properties that closely resemble those reported for classical ei-type transport in studies with intact cells.

Na+-dependent purine nucleoside transporter from human kidney: cloning and functional characterization

1997 ◽  
Vol 273 (6) ◽  
pp. F1058-F1065 ◽  
Author(s):  
Juan Wang ◽  
Sheng-Fang Su ◽  
Mark J. Dresser ◽  
Marci E. Schaner ◽  
Carla B. Washington ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Human Kidney ◽  
Functional Expression ◽  
Purine Nucleoside ◽  
Northern Analysis ◽  
Xenopus Laevis Oocytes ◽  
Chromosome 15 ◽  
Nucleoside Transporter ◽  
Chain Reactions ◽  
Multiple Transcripts

Many purine nucleosides and their analogs are actively transported in the kidney. Using homology cloning strategies and reverse transcriptase-polymerase chain reactions, we isolated a cDNA encoding a Na+-dependent nucleoside transporter, hSPNT1, from human kidney. Functional expression in Xenopus laevis oocytes identified hSPNT1 as a Na+-dependent nucleoside transporter that selectively transports purine nucleosides but also transports uridine. The Michaelis constant ( Km) of uridine (80 μM) in interacting with hSPNT1 was substantially higher than that of inosine (4.5 μM). hSPNT1 (658 amino acids) is 81% identical to the previously cloned rat Na+-nucleoside transporter, SPNT, but differs markedly from SPNT in terms of its primary structure in the NH2terminus. In addition, an Alu repetitive element (∼282 bp) is present in the 3′-untranslated region of the hSPNT1 cDNA. Northern analysis revealed that multiple transcripts of hSPNT1 are widely distributed in human tissues including human kidney. In contrast, rat SPNT transcripts are absent in kidney and highly localized to liver and intestine. The hSPNT1 gene was localized to chromosome 15. This is the first demonstration of a purine nucleoside transporter in human kidney.

Functional expression of cAMP-dependent and independent urea transporters in Xenopus oocytes

1993 ◽  
Vol 265 (2) ◽  
pp. C514-C520 ◽  
Author(s):  
H. Hasegawa ◽  
A. S. Verkman
Keyword(s):  
Internal Control ◽  
Xenopus Oocytes ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Human Kidney ◽  
Functional Expression ◽  
Kidney Cortex ◽  
Kidney Medulla ◽  
Urea Uptake ◽  
Medullary Collecting Duct

Facilitated transport of urea by the inner medullary collecting duct in kidney is important for the urinary concentrating mechanism. To examine the nature and tissue distribution of urea transporters, mRNA was isolated from different tissues and expressed in Xenopus oocytes. [14C]urea and [3H]methylglucose uptake were measured at 21 degrees C at 64 h after microinjection of mRNA. Relative urea uptake in oocytes injected with 50 ng of unfractionated mRNA was (n = 6-42): 1.0 (water-injected control), 1.0 +/- 0.3 (human kidney cortex), 2.9 +/- 0.5 (rat kidney papilla), 2.5 +/- 0.5 (human kidney papilla), 2.7 +/- 0.3 (rat liver), 1.1 +/- 0.3 (rat brain), 1.2 +/- 0.3 (rat muscle), and 2.6 +/- 0.3 (rabbit reticulocyte). Urea uptake was inhibited to near control values by 0.2 mM phloretin and 0.2 mM p-chloromercuribenzenesulfonate (pCMBS) in oocytes injected with mRNA from kidney medulla, liver, and reticulocyte; phloretin and pCMBS had no effect in control oocytes and oocytes injected with mRNA from kidney cortex, brain, and muscle. Urea uptake was strongly increased in oocytes injected with kidney medulla mRNA (4.4-fold over control) by a 5-min preincubation with the adenosine 3',5'-cyclic monophosphate (cAMP) agonist adenosine-3',5'-cyclic monophosphorothioate (Sp-cAMPS) or a mixture of CPT-cAMP, forskolin, and 3-isobutyl-1-methylxanthine; cAMP agonists did not affect urea uptake in oocytes expressing the reticulocyte and liver urea transporters. As an internal control, (phloretin inhibitable) glucose uptake was enhanced in all oocytes (up to 5-fold greater than control), and was not affected by pCMBS and the cAMP agonists.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Functional expression of the nitrobenzylthioinosine-sensitive nucleoside transporter of human choriocarcinoma (BeWo) cells in isolated oocytes of Xenopus laevis

1994 ◽  
Vol 299 (3) ◽  
pp. 769-773 ◽  
Author(s):  
C E Boumah ◽  
C M Harvey ◽  
A R P Paterson ◽  
S A Baldwin ◽  
J D Young ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Xenopus Oocytes ◽  
Functional Expression ◽  
Transport Processes ◽  
Nucleoside Transport ◽  
Xenopus Laevis Oocytes ◽  
Nucleoside Transporter ◽  
Transport Activity ◽  
Bewo Cells ◽  
Ic50 Value

Cultured human choriocarcinoma (BeWo) cells have previously been shown to exhibit, in comparison with other cultured cell types, elevated nitrobenzylthioinosine (NBMPR)-sensitive transport activity and large numbers (> 10(7)/cell) of high-affinity NBMPR-binding sites [Boumah, Hogue and Cass (1992) Biochem. J. 288, 987-996]. The present study investigates whether NBMPR-sensitive nucleoside transport activity could be induced in Xenopus laevis oocytes by microinjection of poly(A)+ RNA isolated from proliferating cultures of BeWo cells. Expression of uridine transport activity was assayed by comparing rates of uptake (22 degrees C) of 100 microM [3H]uridine by RNA-injected oocytes with uptake by water-injected or uninjected oocytes. A 4-fold stimulation of uridine uptake (2.0 versus 0.5 pmol/90 min per oocyte) was seen when oocytes were injected with 50 ng of BeWo poly(A)+ RNA, and this stimulation was abolished when the RNA-injected oocytes were assayed in the presence of 10 microM NBMPR. The expressed uridine transport activity in oocytes was highly sensitive to NBMPR, with a 50% reduction seen at 1.1 nM NBMPR (IC50 value). The IC50 value for NBMPR inhibition of uptake of 100 microM [3H]uridine by intact BeWo cells was 1.4 nM. Inward fluxes of [3H]uridine in the RNA-injected oocytes were greatly reduced in the presence of high concentrations (2 mM) of non-radioactive nucleosides (adenosine, thymidine, inosine) that are known permeants of NBMPR-sensitive nucleoside transport processes. These results establish that the abundance of NBMPR-sensitive nucleoside transporter mRNA in poly(A)+ RNA preparations from BeWo cells is sufficient to achieve production of functionally active transporter protein in Xenopus oocytes and that, when expressed in Xenopus oocytes, the transporters exhibit NBMPR sensitivity and permeant selectively similar to that of the native transporters.

scholarly journals Cloning and functional expression of a Na+-dependent phosphate co-transporter from human kidney: cDNA cloning and functional expression

1995 ◽  
Vol 305 (1) ◽  
pp. 81-85 ◽  
Author(s):  
K Miyamoto ◽  
S Tatsumi ◽  
T Sonoda ◽  
H Yamamoto ◽  
H Minami ◽  
...  
Keyword(s):  
Antisense Oligonucleotides ◽  
Xenopus Oocytes ◽  
Renal Cortex ◽  
Human Kidney ◽  
Functional Expression ◽  
Northern Blotting ◽  
Kidney Cortex ◽  
Kinetic Characterization ◽  
Transport Activity ◽  
High Affinity

A cDNA clone encoding a protein 69% identical in amino acid sequence with that of the Na/P(i) co-transporter NaP(i)-1 was isolated from a human kidney cDNA library. The DNA sequence was identical with that of NPT-1 cDNA published by Chong, Kristjansson, Zoghbi and Hughe (1993) (Genomics, 18, 355-359). In the present study, we have characterized the function of the encoded protein and the tissue distribution of its mRNA. Injection of RNA transcribed from NPT-1 into Xenopus oocytes resulted in expression of Na/P(i) co-transport activity showing a high affinity for P(i) transport (Km 0.29 mM). Kinetic characterization ([P(i)], [Na+]) demonstrated that the expressed transport activity has properties similar to those displayed by oocytes injected with human kidney poly(A)+ RNA. Northern blotting demonstrated that NPT-1 mRNA is expressed in renal cortex, liver and brain but not in other tissues. Hybrid depletion with antisense oligonucleotides to NaP(i)-3 and NPT-1 completely inhibited poly(A)+ RNA-induced Na(+)-dependent P(i) uptake in oocytes. These findings indicate that two high-affinity Na/P(i) cotransporters (NaP(i)-3 and NPT-1) are present in human kidney cortex.

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