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.

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.

Localization of the high-affinity glutamate transporter EAAC1 in rat kidney

1997 ◽  
Vol 273 (6) ◽  
pp. F1023-F1029 ◽  
Author(s):  
Chairat Shayakul ◽  
Yoshikatsu Kanai ◽  
Wen-Sen Lee ◽  
Dennis Brown ◽  
Jeffrey D. Rothstein ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Rat Kidney ◽  
Cell Volume Regulation ◽  
Glutamate Transporter ◽  
Transport Systems ◽  
Kidney Cortex ◽  
Acid Base Balance ◽  
Western Blots ◽  
High Affinity

Most amino acids filtered by the glomerulus are reabsorbed in the kidney via specialized transport systems. Recently, the cDNA encoding a high-affinity glutamate transporter, EAAC1, has been isolated and shown to be expressed at high levels in the kidney. To determine the potential role of EAAC1 in renal acidic amino acid reabsorption, the distribution of EAAC1 mRNA and protein in rat kidney was examined. In situ hybridization revealed that EAAC1 mRNA is expressed predominantly in S2 and S3 segments of the proximal tubules and at low levels in the inner stripe of outer medulla and inner medulla. Polyclonal antibodies raised against the carboxy terminus of EAAC1 recognized a single band of ∼70 kDa on Western blots of membrane protein from kidney cortex and medulla. Immunofluorescence microscopy revealed intense signals in the luminal membrane of S2 and S3 segments and weaker signals in S1 segments, descending thin limbs of long-loop nephrons, medullary thick ascending limbs, and distal convoluted tubules. These results are consistent with EAAC1 encoding the previously described apical high-affinity glutamate transporter in the kidney that mediates reabsorption of acidic amino acids in tubules beyond early proximal tubule S1 segments. Potential additional roles of EAAC1 in acid/base balance, cell volume regulation, and amino acid metabolism are discussed.

Assessment of High-Affinity Hybridization, RNase H Cleavage, and Covalent Linkage in Translation Arrest by Antisense Oligonucleotides

1998 ◽  
Vol 8 (2) ◽  
pp. 103-111 ◽  
Author(s):  
JAY E. GEE ◽  
IAN ROBBINS ◽  
ALEXANDER C. VAN DER LAAN ◽  
JACQUES H. VAN BOOM ◽  
CAROLINE COLOMBIER ◽  
...  
Keyword(s):  
Antisense Oligonucleotides ◽  
Rnase H ◽  
Translation Arrest ◽  
Covalent Linkage ◽  
High Affinity

Pyruvate shuttle in muscle cells: high-affinity pyruvate transport sites insensitive to trans-lactate efflux

2003 ◽  
Vol 285 (6) ◽  
pp. E1196-E1204
Author(s):  
Raymond Mengual ◽  
Kaoukib el Abida ◽  
Nassima Mouaffak ◽  
Michel Rieu ◽  
Michele Beaudry
Keyword(s):  
High Capacity ◽  
Muscle Cells ◽  
Type I ◽  
Maximal Velocity ◽  
Type Ii ◽  
Lactate Shuttle ◽  
Transport Efficiency ◽  
High Affinity ◽  
High K ◽  
Transport Kinetic

The specificity of the transport mechanisms for pyruvate and lactate and their sensitivity to inhibitors were studied in L6 skeletal muscle cells. Trans- and cis-lactate effects on pyruvate transport kinetic parameters were examined. Pyruvate and lactate were transported by a multisite carrier system, i.e., by two families of sites, one with low affinity and high capacity (type I sites) and the other with high affinity and low capacity (type II). The multisite character of transport kinetics was not modified by either hydroxycinnamic acid (CIN) or p-chloromercuribenzylsulfonic acid (PCMBS), which exert different types of inhibition. The transport efficiency (TE) ratios of maximal velocity to the trans-activation dissociation constant ( Kt) showed that lactate and pyruvate were preferentially transported by types I and II sites, respectively. The cis-lactate effect was observed with high Ki values for both sites. The trans-lactate effect on pyruvate transport occurred only on type I sites and exhibited an asymmetric interaction pattern ( Kt of inward lactate > Kt of outward lactate). The inability of lactate to trans-stimulate type II sites suggests that intracellular lactate cannot recruit these sites. The high-affinity type II sites act as a specific pyruvate shuttle and constitute an essential relay for the intracellular lactate shuttle.

Reconstitution of the Functional Mouse Oncostatin M (OSM) Receptor: Molecular Cloning of the Mouse OSM Receptor β Subunit

Blood ◽  
1999 ◽  
Vol 93 (3) ◽  
pp. 804-815 ◽  
Author(s):  
Minoru Tanaka ◽  
Takahiko Hara ◽  
Neal G. Copeland ◽  
Debra J. Gilbert ◽  
Nancy A. Jenkins ◽  
...  
Keyword(s):  
Amino Acid Level ◽  
Embryonic Stem ◽  
Oncostatin M ◽  
Chromosome 15 ◽  
Type I ◽  
Type Ii ◽  
Β Subunit ◽  
High Affinity ◽  
High Affinity Receptor ◽  
Species Specific

Oncostatin M (OSM) is a member of the interleukin-6 (IL-6) family of cytokines that share the gp130 receptor subunit. Of these family members, leukemia inhibitory factor (LIF) is most closely related to OSM, and various overlapping biologic activities have been described between human LIF and OSM (hLIF and hOSM). Two types of functional hOSM receptors are known: the type I OSM receptor is identical to the LIF receptor that consists of gp130 and the LIF receptor β subunit (LIFRβ), and the type II OSM receptor consists of gp130 and the OSM receptor β subunit (OSMRβ). It is thus conceivable that common biologic activities between hLIF and hOSM are mediated by the shared type I receptor and OSM-specific activities are mediated by the type II receptor. However, in contrast to the human receptors, recent studies have demonstrated that mouse OSM (mOSM) does not activate the type I receptor and exhibits unique biologic activity. To elucidate the molecular structure of the functional mOSM receptor, we cloned a cDNA encoding mOSMRβ, which is 55.5% identical to the hOSMRβ at the amino acid level. mOSM-responsive cell lines express high-affinity mOSM receptors, as well as mOSMRβ, whereas embryonic stem cells, which are responsive to LIF but not to mOSM, do not express mOSMRβ. mOSMRβ alone binds mOSM with low affinity (kd = 13.0 nmol/L) and forms a high-affinity receptor (kd = 606 pmol/L) with gp130. Ba/F3 transfectants expressing both mOSMRβ and gp130 proliferated in response to mOSM, but failed to respond to LIF and human OSM. Thus, the cloned mOSMRβ constitutes an essential and species-specific receptor component of the functional mOSM receptor. Reminiscent of the colocalization of the mOSM and mLIF genes, the mOSMRβ gene was found to be located in the vicinity of the LIFRβ locus in the proximal end of chromosome 15.

Reconstitution of the Functional Mouse Oncostatin M (OSM) Receptor: Molecular Cloning of the Mouse OSM Receptor β Subunit

Blood ◽  
1999 ◽  
Vol 93 (3) ◽  
pp. 804-815 ◽  
Author(s):  
Minoru Tanaka ◽  
Takahiko Hara ◽  
Neal G. Copeland ◽  
Debra J. Gilbert ◽  
Nancy A. Jenkins ◽  
...  
Keyword(s):  
Amino Acid Level ◽  
Embryonic Stem ◽  
Oncostatin M ◽  
Chromosome 15 ◽  
Type I ◽  
Type Ii ◽  
Β Subunit ◽  
High Affinity ◽  
High Affinity Receptor ◽  
Species Specific

Abstract Oncostatin M (OSM) is a member of the interleukin-6 (IL-6) family of cytokines that share the gp130 receptor subunit. Of these family members, leukemia inhibitory factor (LIF) is most closely related to OSM, and various overlapping biologic activities have been described between human LIF and OSM (hLIF and hOSM). Two types of functional hOSM receptors are known: the type I OSM receptor is identical to the LIF receptor that consists of gp130 and the LIF receptor β subunit (LIFRβ), and the type II OSM receptor consists of gp130 and the OSM receptor β subunit (OSMRβ). It is thus conceivable that common biologic activities between hLIF and hOSM are mediated by the shared type I receptor and OSM-specific activities are mediated by the type II receptor. However, in contrast to the human receptors, recent studies have demonstrated that mouse OSM (mOSM) does not activate the type I receptor and exhibits unique biologic activity. To elucidate the molecular structure of the functional mOSM receptor, we cloned a cDNA encoding mOSMRβ, which is 55.5% identical to the hOSMRβ at the amino acid level. mOSM-responsive cell lines express high-affinity mOSM receptors, as well as mOSMRβ, whereas embryonic stem cells, which are responsive to LIF but not to mOSM, do not express mOSMRβ. mOSMRβ alone binds mOSM with low affinity (kd = 13.0 nmol/L) and forms a high-affinity receptor (kd = 606 pmol/L) with gp130. Ba/F3 transfectants expressing both mOSMRβ and gp130 proliferated in response to mOSM, but failed to respond to LIF and human OSM. Thus, the cloned mOSMRβ constitutes an essential and species-specific receptor component of the functional mOSM receptor. Reminiscent of the colocalization of the mOSM and mLIF genes, the mOSMRβ gene was found to be located in the vicinity of the LIFRβ locus in the proximal end of chromosome 15.

Characterization of L-glutamine transport by type II alveolar cells

1992 ◽  
Vol 262 (4) ◽  
pp. L459-L465 ◽  
Author(s):  
R. D. Hautamaki ◽  
B. Greene ◽  
W. W. Souba
Keyword(s):  
Maximum Velocity ◽  
Kinetic Studies ◽  
Transport Systems ◽  
Type Ii ◽  
Alveolar Cells ◽  
High Affinity ◽  
System A ◽  
Sodium Dependent ◽  
Type Ii Alveolar Cells ◽  
Dependent Transport

This study characterized the sodium-dependent transport of L-glutamine (L-Gln) by rat type II alveolar cells. Uptake of 50 microM glutamine (Gln) was determined and found to be linear for at least 15 min. The sodium-dependent velocity represented greater than 80% of the total uptake at all time points. Kinetic studies of sodium-dependent Gln uptake at concentrations between 0.005 and 8 mM showed uptake to occur via two saturable transport systems, a high-affinity carrier [Michaelis constant (Km) = 259 +/- 19 microM, maximum velocity (Vmax) = 0.942 +/- 0.08 nmol.mg protein-1.30 s-1] and a low-affinity transporter (Km = 4.96 +/- 1.2 mM, Vmax = 2.98 +/- 0.19 nmol.mg protein-1.30 s-1). Uptake of Gln via the low-affinity system was nearly completely blocked by 10 mM 2-methylaminoisobutyric acid (MeAIB), and increasing concentrations of Gln almost totally inhibited transport of MeAIB, indicating the presence of system A. Further inhibition studies of the high-affinity transporter showed marked inhibition by serine, cysteine, and nonradioactive Gln. Lithium did not substitute for sodium, strongly suggesting that L-Gln was not transported by system N. Furthermore, transport of Gln by the high-affinity carrier was not affected by hormones or by changes in external pH. We conclude that sodium-dependent L-Gln transport by rat type II alveolar cells occurs predominantly via system A and system ASC.

Evidence that the transport-related proteins BAT and 4F2hc are not specific for amino acids: induction of Na+-dependent uridine and pyruvate transport activity by recombinant BAT and 4F2hc expressed in Xenopus oocytes

1998 ◽  
Vol 76 (5) ◽  
pp. 859-865 ◽  
Author(s):  
Sylvia YM Yao ◽  
William R Muzyka ◽  
Carol E Cass ◽  
Christopher I Cheeseman ◽  
James D Young
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Transport ◽  
Xenopus Oocytes ◽  
Transport Systems ◽  
Choline Uptake ◽  
Type I ◽  
Organic Substrates ◽  
Acid Transport ◽  
Cationic Amino Acids

Members of the BAT and 4F2hc gene family have one or, in the case of BAT, up to four transmembane domains and induce amino acid transport systems bo,+ (BAT) and y+L (4F2hc) when expressed in Xenopus oocytes. System bo,+ is a Na+-independent process with a broad tolerance for cationic and zwitterionic amino acids, whereas y+L exhibits Na+-independent transport of cationic amino acids (e.g., lysine) and Na+-dependent transport of zwitterionic amino acids (e.g., leucine). Mutations in the human BAT gene are associated with type I cystinuria, a genetic disease affecting the ability of intestinal and renal brush border membranes to transport cationic amino acids and cystine. An unresolved question is whether BAT and 4F2hc themselves have catalytic (i.e., transporting) activity or whether they operate as activators of other, as yet unidentified, transporter proteins. In this report, we have investigated the transport of representatives of four different classes of organic substrates in Xenopus oocytes following injection with rat BAT or 4F2hc RNA transcripts: leucine (a control amino acid substrate), uridine (a nucleoside), pyruvate (a monocarboxylate), and choline (an amine). Both recombinant proteins induced small, statistically significant Na+-dependent fluxes of uridine and pyruvate but had no effect on choline uptake. In contrast, control oocytes injected with transcripts for conventional nucleoside and cationic amino acid transporters (rat CNT1 and murine CAT1, respectively) showed no induction of transport of either leucine or pyruvate (CNT1) or uridine or pyruvate (CAT1). These findings support the idea that BAT and 4F2hc are transport activators and minimize the possibility that they have intrinsic transport capability. The transport-regulating functions of these proteins may extend to permeants other than amino acids.Key words: amino acid transport, uridine, pyruvate, BAT, 4F2hc, Xenopus oocytes.

scholarly journals The TGFβ type I receptor TGFβRI functions as an inhibitor of BMP signaling in cartilage

2019 ◽  
Vol 116 (31) ◽  
pp. 15570-15579 ◽  
Author(s):  
Weiguang Wang ◽  
Hyelim Chun ◽  
Jongseung Baek ◽  
Joshua Elyahu Sadik ◽  
Anna Shirazyan ◽  
...  
Keyword(s):  
Growth Plate ◽  
Bmp Signaling ◽  
Type I ◽  
Type Ii ◽  
Tgfβ Signaling ◽  
Major Function ◽  
High Affinity ◽  
Bmp Receptor ◽  
Tgfβ Receptor ◽  
Intracellular Mediators

The type I TGFβ receptor TGFβRI (encoded by Tgfbr1) was ablated in cartilage. The resulting Tgfbr1Col2 mice exhibited lethal chondrodysplasia. Similar defects were not seen in mice lacking the type II TGFβ receptor or SMADs 2 and 3, the intracellular mediators of canonical TGFβ signaling. However, we detected elevated BMP activity in Tgfbr1Col2 mice. As previous studies showed that TGFβRI can physically interact with ACVRL1, a type I BMP receptor, we generated cartilage-specific Acvrl1 (Acvrl1Col2) and Acvrl1/Tgfbr1 (Acvrl1/Tgfbr1Col2) knockouts. Loss of ACVRL1 alone had no effect, but Acvrl1/Tgfbr1Col2 mice exhibited a striking reversal of the chondrodysplasia seen in Tgfbr1Col2 mice. Loss of TGFβRI led to a redistribution of the type II receptor ACTRIIB into ACVRL1/ACTRIIB complexes, which have high affinity for BMP9. Although BMP9 is not produced in cartilage, we detected BMP9 in the growth plate, most likely derived from the circulation. These findings demonstrate that the major function of TGFβRI in cartilage is not to transduce TGFβ signaling, but rather to antagonize BMP signaling mediated by ACVRL1.

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