Molecular cloning and hormonal regulation of PiT-1, a sodium-dependent phosphate cotransporter from rat parathyroid glands.

The extracellular concentration of inorganic phosphate (Pi) is an important determinant of parathyroid cell function. The effects of Pi may be mediated through specific molecules in the parathyroid cell membrane, one candidate molecule for which would be a Na+-dependent Pi cotransporter. A complementary DNA encoding a Na+-Pi cotransporter, termed rat PiT-1, has now been isolated from rat parathyroid. The 2890-bp complementary DNA encodes a protein of 681 amino acids that shows sequence identities of 97% and 93% with the type III Na+-Pi cotransporters mouse PiT-1 and human PiT-1, respectively. Expression of rat PiT-1 in Xenopus oocytes revealed that it possesses Na+-dependent Pi cotransport activity. PiT-1 messenger RNA (mRNA) is widely distributed in rat tissues and is most abundant in brain, bone, and small intestine. The amount of PiT-1 mRNA in the parathyroid of vitamin D-deficient rats was reduced compared with that in normal animals and increased markedly after administration of 1,25-dihydroxyvitamin D3. Furthermore, the abundance of PiT-1 mRNA in the parathyroid was much greater in rats fed a low-Pi diet than in those fed a high-Pi diet. Thus, rat PiT-1 may contribute to the effects of Pi and vitamin D on parathyroid function.

Gene structure and functional analysis of the human Na+/phosphate co-transporter.

Three lambda phage clones encompassing the Na+/phosphate co-transporter (NaPi-3) gene and its 5' flanking region were isolated from a human genomic DNA library. The gene comprises 13 exons and 12 introns and spans approx. 14 kb. All exon-intron junctions conform to the GT/AG rule. The major transcription-initiation site was determined by primer-extension analysis and is an adenosine residue 57 bp upstream of the 3' end of the first exon. There is a typical TATA box 28 bp upstream of the major transcription-initiation site and various cis-acting elements, including a cAMP-responsive element, AP-1, AP-2 and SP-1 sites in the 5' flanking region. This region also contains three direct-repeat-like sequences that resemble the consensus binding sequence for members of the steroid-thyroid hormone receptor superfamily, including vitamin D. Deletion analysis suggests that the region from nt-2409 to nt-1259 in the 5' flanking region may be involved in kidney-specific gene expression. Vitamin D responsiveness of the NaPi-3 promoter was also detected in COS-7 cells co-transfected with a human vitamin D receptor expression vector. The presence of the three vitamin D receptor- responsive elements in the NaPi-3 promoter may be important in mediating the enhanced expression of the gene by 1,25-dihydroxyvitamin D3.

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

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.

Effects of truncation of the COOH-terminal region of a Na+-independent neutral and basic amino acid transporter on amino acid transport in Xenopus oocytes.

To determine the role of a neutral and basic amino acid transporter (NBAT) in amino acid transport, we microinjected several COOH-terminal deletion mutants of NBAT cRNA into Xenopus oocytes and measured transport activity for arginine, leucine, and cystine in the presence and absence of sodium. Wild-type NBAT significantly stimulated the uptake of all three amino acids 10-20-fold compared with controls. On the other hand, no mutant, except a Delta511-685 mutant, stimulated the uptake of these amino acids. The Delta511-685 mutant significantly increased the uptake of arginine. In the presence of sodium, the Delta511-685 mutant also increased the uptake of leucine. The Delta511-685 mutant did not stimulate cystine uptake in the presence or absence of sodium. The stimulation of arginine uptake by the Delta511-685 mutant was inhibited by a 100-fold excess of unlabeled leucine in the presence of sodium. Inhibition of L-arginine uptake by L-homoserine was seen only in the presence of sodium, and an increase in the inhibition of L-arginine uptake by L-histidine was seen when the extracellular pH was decreased. Furthermore, an inward current in oocytes injected with the Delta511-685 mutant was recorded electrophysiologically when basic amino acids were applied. Homoserine was also taken up, but sodium was necessary for their transport. These properties of the Delta511-685 mutant correspond to those of the y+ amino acid transporter. If NBAT is a component of the b0,+-like amino acid transport system, it is unlikely that a mutant protein (Delta511-685) is able to stimulate an endogenous y+-like transport system. These results suggest that NBAT functions as a activator of the amino acid transport system in Xenopus oocytes.

Mutations of the basic amino acid transporter gene associated with cystinuria.

To investigate the function of a basic and neutral amino acid transporter-like protein (rBAT) which is a candidate gene for cystinuria, we analysed the rBAT gene in cystinuric patients. Patient 1 is a compound heterozygote with mutations in the rBAT gene causing a glutamine-to-lysine transition at amino acid 268, and a threonine-to-alanine transition at amino acid 341, who inherited these alleles from his mother (E268K) and father (T341A), respectively. Injection of T341A and E268K mutant cRNAs into oocytes decreased transport activity to 53.9% and 62.5% of control (L-cystine transport activity in oocytes injected with wild-type rBAT cRNA), respectively. Co-injection of E268K and T341A into oocytes strongly decreased amino acid transport activity to 28% of control. On the other hand, co-injection of wild-type and mutant rBAT did not decrease transport activity. Furthermore, immunological studies have demonstrated that the reduction of amino acid transport is not due to a decrease in the amount of rBAT protein expressed in oocyte membranes. These results indicate that mutations in the rBAT gene are crucial disease-causing lesions in cystinuria. In addition, co-injection experiments suggest that rBAT may function as a transport activator or regulatory subunit by homo- or hetero-multimer complex formation.

Cloning and functional expression of a Na(+)-dependent phosphate co-transporter from human kidney: cDNA cloning and functional expression.

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.

[Malignant lymphoma from left renal capsule: report of case].

A 59-year-old woman was admitted to our clinic with the complaint of left flank pain. Excretory urogram showed left hydronephrosis. Computed tomographic (CT) scan and renal angiography showed a left renal capsular tumor. Histological specimen obtained by a sure cut needle suggested malignant lymphoma. She was treated with a combined treatment of 8 MHz radiofrequency hyperthermia in a total of 10 sessions and 5,440 rads irradiation for 5 weeks. After the treatment, CT scan showed 92% tumor regression. After that, a recurrent tumor in left shoulder muscle became manifest. She received combination chemotherapy with 3 courses of ABEP regimen (aclacinomycin, cytosine arabinoside, etoposide, prednisolone) and 7 courses of ACOPE regimen (adriamycin, cyclophosphamide, vincristine, prednisolone, etoposide) and complete remission was obtained.

Pancreatography with contrast medium containing polyethylene hydrogenated castor oil.

A new contrast medium has been used for endoscopic pancreatography. Good visualization of the pancreas was obtained by this method. No side-effects of this method were observed by clinical and histologic examination, either in clinical or experimental studies. Clinically, the new method provided more detailed information regarding pancreatic changes in chronic pancreatitis. It is suggested that this method may make detection of a small tumor of the pancreas in the parenchyma and permit earlier diagnosis of carcinoma of the pancreas.