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.

scholarly journals Relative contributions of Na+-dependent phosphate co-transporters to phosphate transport in mouse kidney: RNase H-mediated hybrid depletion analysis

1997 ◽  
Vol 327 (3) ◽  
pp. 735-739 ◽  
Author(s):  
Ken-ichi MIYAMOTO ◽  
Hiroko SEGAWA ◽  
Kyoko MORITA ◽  
Tomoko NII ◽  
Sawako TATSUMI ◽  
...  
Keyword(s):  
Antisense Oligonucleotides ◽  
Xenopus Oocytes ◽  
Mouse Kidney ◽  
Rnase H ◽  
Transport Systems ◽  
Kidney Cortex ◽  
Type I ◽  
Type Ii ◽  
High Affinity ◽  
Pi Transport

Reabsorption of Pi in the proximal tubule of the kidney is an important determinant of Pi homoeostasis. At least three types (types I-III) of high-affinity Na+-dependent Pi co-transporters have been identified in mammalian kidneys. The relative roles of these three types of Na+/Pi co-transporters in Pi transport in mouse kidney cortex have now been investigated by RNase H-mediated hybrid depletion. Whereas isolated brush-border membrane vesicles showed the presence of two kinetically distinct Na+/Pi co-transport systems (high Km-low Vmax and low Km-high Vmax), Xenopus oocytes, microinjected with polyadenylated [poly(A)+] RNA from mouse kidney cortex, showed only the high-affinity Pi uptake system. Kidney poly(A)+ RNA was incubated in vitro with antisense oligonucleotides corresponding to Npt-1 (type I), NaPi -7 (type II) or Glvr-1 (type III) Na+/Pi co-transporter mRNAs, and then with RNase H. Injection of such treated RNA preparations into Xenopus oocytes revealed that an NaPi-7 antisense oligonucleotide that resulted in complete degradation of NaPi-7 mRNA (as revealed by Northern blot analysis), also induced complete inhibition of Pi uptake. Degradation of Npt-1 or Glvr-1 mRNAs induced by corresponding antisense oligonucleotides had no effect on Pi transport, which was subsequently measured in oocytes. These results indicate that the type II Na+/Pi co-transporter NaPi-7 mediated most Na+-dependent Pi transport in mouse kidney cortex.

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 Cloning, functional expression and dietary regulation of the mouse neutral and basic amino acid transporter (NBAT)

1997 ◽  
Vol 328 (2) ◽  
pp. 657-664 ◽  
Author(s):  
Hiroko SEGAWA ◽  
Ken-ichi MIYAMOTO ◽  
Yoshio OGURA ◽  
Hiromi HAGA ◽  
Kyoko MORITA ◽  
...  
Keyword(s):  
Amino Acid ◽  
Xenopus Oocytes ◽  
Physiological Role ◽  
Basic Amino Acid ◽  
Functional Expression ◽  
Amino Acid Transporter ◽  
Amino Acid Transporters ◽  
Transport Activity ◽  
Acid Transporter ◽  
Mouse Ileum

The Na+-independent dibasic and neutral amino acid transporter NBAT is among the least hydrophobic of mammalian amino acid transporters. The transporter contains one to four transmembrane domains and induces amino acid transport activity via a b0,+-like system when expressed in Xenopus oocytes. However, the physiological role of NBAT remains unclear. Complementary DNA clones encoding mouse NBAT have now been isolated. The expression of mouse NBAT in Xenopus oocytes also induced an obligatory amino acid exchange activity similar to that of the b0,+-like system. The amount of NBAT mRNA in mouse kidney increased during postnatal development, consistent with the increase in renal cystine and dibasic transport activity. Dietary aspartate induced a marked increase in cystine transport via the b0,+ system in mouse ileum. A high-aspartate diet also increased the amount of NBAT mRNA in mouse ileum. In the ileum of mice fed on the aspartate diet, the extent of cystine transport was further increased by preloading brush border membrane vesicles with lysine. Hybrid depletion of NBAT mRNA from ileal polyadenylated RNA revealed that the increase in cystine transport activity induced by the high-aspartate diet, as measured in Xenopus oocytes, was attributable to NBAT. These results demonstrate that mouse NBAT has an important role in cystine transport.

Metauothionein and Cadmium in Human Kidney Cortex: Influence of Smoking

1986 ◽  
Vol 5 (1) ◽  
pp. 27-33 ◽  
Author(s):  
K.H. Summer ◽  
G.A. Drasch ◽  
H.E. Heilmaier
Keyword(s):  
Renal Cortex ◽  
Human Kidney ◽  
Major Portion ◽  
Kidney Cortex ◽  
Cadmium Content ◽  
Post Mortem ◽  
The Mean ◽  
Protein B

1 Post-mortem specimens of human kidney cortex of 47 individuals classified according to their imoking habits were analysed for tissue cadmium and cadmium bound to metallothionein. 2 The cadmium content in the kidney cortex of all individuals was 5-99 μg/g wet wt. In smokers consuming more than 20 cigarettes/day the mean content of renal cortex cadmium was twice that of ion-smokers and amounted to 33.3 ± 12.5 μg/g wet wt. 3 The amount of cadmium bound to metallothionein of all individuals was 0.3-66 μg/g wet wt. iirectly correlating with the cadmium content of the kidney cortex (r = 0.932). 4 More than 50% of renal cortex cadmium was associated with the metallothionein fractions. Due to constant values of zinc and copper in metallothionein the relative amount of zinc and copper to cadmium in metallothionein decreased with increasing tissue cadmium. 5 Together with the elevated binding of cadmium to renal cortex metallothionein, cadmium ncreasingly was bound to non-metallothionein ligands. 6 These results suggest that, in the renal cortex of smokers with elevated cadmium, a major portion of cadmium is bound to metallothionein. However, it is unclear yet whether (a) the binding of cadmium to metallothionein with subsequent liberation of the metal during degradation of the protein, (b) the impairment of metallothionein functions by the binding of cadmium or (c) the increased binding to non-metallothionein ligands contribute to the toxicity of cadmium in highly exposed individuals.

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 Two oligopeptide transporters from Caenorhabditis elegans:molecular cloning and functional expression

1998 ◽  
Vol 332 (2) ◽  
pp. 565-572 ◽  
Author(s):  
You-Jun FEI ◽  
Takuya FUJITA ◽  
David F. LAPP ◽  
Vadivel GANAPATHY ◽  
Frederick H. LEIBACH
Keyword(s):  
Amino Acid ◽  
Functional Expression ◽  
Amino Acid Sequences ◽  
Xenopus Laevis Oocytes ◽  
Cdna Clones ◽  
Transport Activity ◽  
Animal Kingdom ◽  
Peptide Substrates ◽  
Electrogenic Transport ◽  
High Affinity

Two novel oligopeptide transporter cDNA clones, CPTA and CPTB, were identified by screening a Caenorhabditis elegans cDNA library using homology hybridization. The transporter proteins deduced from the cDNAs possess multiple transmembrane domains and reveal a moderate similarity to their mammalian counterparts in amino acid sequences. CPTA and CPTB, when expressed in Xenopus laevis oocytes and studied by both radiotracer flux and microelectrode voltage-clamp protocol, displayed a saturable electrogenic transport activity driven by a proton gradient with an overlapping broad spectrum of substrate specificity. Both transporters recognize di-, tri- and tetra-peptides including phenylalanylmethionylarginylphenylalaninamide (FMRFamide) and N-acetylaspartylglutamate, members of a large neuropeptide family commonly found throughout the animal kingdom. Kinetic analysis, however, revealed that CPTA and CPTB differed in their affinity for the peptide substrates, the former being a high-affinity type and the latter a low-affinity type. CPTA and CPTB are encoded by two distinct genes localized on separate chromosomes and are expressed during the whole life span of the organism.

Metal accumulation in human kidney cortex: mutual interrelations and effect of human factors

1995 ◽  
Vol 14 (4) ◽  
pp. 335-340 ◽  
Author(s):  
M. López-Artíguez ◽  
A. Cameán ◽  
G. González ◽  
M. Repetto
Keyword(s):  
Drug Abuse ◽  
Human Factors ◽  
Renal Cortex ◽  
Metal Accumulation ◽  
Human Kidney ◽  
Kidney Cortex ◽  
Individual Factors ◽  
Post Mortem ◽  
Metallic Ions ◽  
Accumulation Of Metals

1 Different metals were analysed in 77 post mortem samples of human renal cortex. 2 The concentrations of the metallic ions conformed to known distribution frequencies. In abnormal cases calcu lations were based on non parametric techniques. 3 There was no appreciable difference between the val ues found for each element and those described by other authors in other populations. 4 Statistically established correlations indicated that Zn was the element which was most strongly related to the others. 5 The influence of individual factors on metal concentra tions was considered. Significant differences occurred only in Pb between sexes, and in Cd with age. There was no sign that drug abuse influenced the accumulation of metals in renal cortex.

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.

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