Hiromi Haga

Identification of an Amino Acid Transporter Associated with the Cystinuria-related Type II Membrane Glycoprotein

We identified an amino acid transporter that is associated with the cystinuria-related type II membrane glycoprotein, rBAT (related to b0,+ amino acid transporter). The transporter designated BAT1 (b0,+-type amino acid transporter 1) from rat kidney was found to be structurally related to recently identified amino acid transporters for system L, system y+L, and system x−C, which are linked, via a disulfide bond, to the other type II membrane glycoprotein, 4F2hc (4F2 heavy chain). In the nonreducing condition, a 125-kDa band, which seems to correspond to the heterodimeric complex of BAT1 and rBAT, was detected in rat kidney with anti-BAT1 antibody. The band was shifted to 41 kDa in the reducing condition, confirming that BAT1 and rBAT are linked via a disulfide bond. The BAT1 and rBAT proteins were shown to be colocalized in the apical membrane of the renal proximal tubules where massive cystine transport had been proposed. When expressed in COS-7 cells with rBAT, but not with 4F2hc, BAT1 exhibited a Na+-independent transport of cystine as well as basic and neutral amino acids with the properties of system b0,+. The results from the present investigation were used to establish a family of amino acid transporters associated with type II membrane glycoproteins.

Sequence, tissue distribution and developmental changes in rat intestinal oligopeptide transporter

Complementary DNA clones encoding the rat PepT1 small-intestinal oligopeptide transporter were isolated from a jejunal library by cross-hybridization with a rabbit PepT1 cDNA probe. The cDNA sequence indicates that rat PepT1 is composed of 710 amino acids and shows 77% and 83% amino acid sequence identity with rabbit and human PepT1, respectively. Northern blot analysis detected rat PepT1 mRNA in the small intestine and kidney. Intestinal PepT1 mRNA levels were highest in 4-day old rats, and then decreased reaching the adult level by day 28 after birth. These results indicate that the expressions of PepT1 gene change markedly during development.

Regulation of the PepT1 peptide transporter in the rat small intestine in response to 5-fluorouracil–induced injury

BACKGROUND & AIMS The oligopeptide transport system of the small intestine is resistant to mucosal injury. The mechanism of this resistance was investigated by examining the activity level and expression of the peptide transporter PepT1 in the intestine of rats treated with 5-fluorouracil. METHODS The expression and localization of PepT1 were examined by immunoblot analysis of brush border membrane vesicles and immunohistochemical analysis of intestinal sections with PepT1-specific rabbit polyclonal antibodies. Also, Northern blot analysis was used for the expression of PepT1 messenger RNA (mRNA). RESULTS Although the amounts of sucrase and an Na+-dependent glucose transporter protein in intestinal vesicles decreased markedly after 5-fluorouracil treatment, the amount of PepT1 protein remained largely unaffected. Immunohistochemical analysis also showed that the PepT1 immunoreactivity level was preserved in the brush border membrane of the remaining villi of 5-fluorouracil-treated rats. Levels of amino acid, glucose, and phosphate transporter mRNAs were profoundly depressed in 5-fluorouracil-treated animals, whereas the level of PepT1 mRNA conversely increased. CONCLUSIONS The resistance of intestinal peptide transport to tissue injury may be attributable to increased synthesis of PepT1 rather than to a change in the kinetic properties of the residual absorbing cells.

The basic amino acid transporter (rBAT) -like immunoreactivity in paraventricular and supraoptic magnocellular neurons of the rat hypothalamus

In the rat hypothalamus, the basic amino acid transporter (rBAT)-like immunoreactivity was analyzed by immunohistochemistry using an antibody against the 15-amino acid sequence of the deduced rat rBAT protein. In the supraoptic and the paraventricular nuclei, magnocellular neurons exhibited the marked rBAT-like immunoreactivity in intracellular structures but not in the plasma membrane. The results suggest that the rBAT serves as an intracellular amino acid transport system in magnocellular neurons.

Abnormal expression of tyrosine hydroxylase immunoreactivity in cerebellar cortex of ataxic mutant mice

Expression of tyrosine hydroxylase (TH) was examined immunohistochemically in the cerebellum of two ataxic mutants, Rolling mouse Nagoya (RMN) and dilute-lethal mice (DL). In littermate controls of both mutants, a few TH-positive Purkinje cells were distributed sparsely and their number was smaller than in the mutants at any ages examined. In RMN, TH-positive Purkinje cells were distributed in lobule IX and X, and were arranged into parasagittal bands at 2 weeks of age. TH-positive Purkinje cells increased in number and were widely distributed throughout the vermis at 3 weeks of age. In adult RMN, TH-positive Purkinje cells were found in all lobules of the cerebellum. Their parasagittal bands also became evident in the hemisphere. In DL, TH-positive Purkinje cells were mainly distributed in vermal lobules IX and X, and the flocculus at 3 weeks of age. They were also found as bands in lobules IX and X. The results suggest that abnormal expression of TH in Purkinje cells may not be specific to the allelic group. Since TH promoter is activated by Ca2+, TH expression in the mutant Purkinje cells may predict neuronal dysfunction caused by alterations in cellular Ca2+ currents.

Immunohistochemical and RT-PCR detection of Na+-dependent inorganic phosphate cotransporter (NaPi-2) in rat brain

Expression of a renal Na+-dependent inorganic phosphate (Pi) cotransporter (NaPi-2) was studied in rat forebrain with reverse transcription and polymerase chain reaction (RT-PCR) and immunohistochemistry. RT-PCR analysis for total RNA from whole brain and sequencing of the PCR products showed expression of NaPi-2 mRNA in the brain. Immunohistochemical analysis revealed NaPi-2 staining in many nonpyramidal neurons of all six layers throughout neocortical areas and in neurons of proisocortical and periallocortical areas. NaPi-2-immunoreactive neurons were also detectable in the piriform cortex, hippocampal formation, caudate-putamen, amygdaloid nuclei and lateral geniculate nucleus. Furthermore, NaPi-2 staining was shown in ependymal cells and microvascular endothelial cells. The present results suggest that NaPi-2 is synthesized within the brain and involved in maintaining Pi homeostasis of certain neurons and/or the entire brain.

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 Δ511-685 mutant, stimulated the uptake of these amino acids. The Δ511-685 mutant significantly increased the uptake of arginine. In the presence of sodium, the Δ511-685 mutant also increased the uptake of leucine. The Δ511-685 mutant did not stimulate cystine uptake in the presence or absence of sodium. The stimulation of arginine uptake by the Δ511-685 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 Δ511-685 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 Δ511-685 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 (Δ511-685) 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.

Ataxic mutant mice with defects in Ca2+ channel α1A subunit gene: morphological and functional abnormalities in cerebellar cortical neurons

ABSTRACT This review summarizes recent studies in the morphological and functional abnormalities of cerebella in three ataxic mutant mice, i.e. tottering mouse, leaner mouse, and rolling mouse Nagoya (RMN). These mutants carry mutations in the Ca2+ channel α1A subunit gene, and become useful models for human neurological diseases such as episodic ataxia type‐2, familial hemiplegic migraine, and spinocerebellar ataxia type‐6. All three mutants exhibited altered morphology of the Purkinje cells, ectopic synaptic contacts between granule cell axons (parallel fibers) and Purkinje cell dendritic spines and abnormal expression of tyrosine hydroxylase in Purkinje cells. In leaner mice, Purkinje cell loss was observed in alternating sagittal compartments of the cerebellar cortex corresponding to the Zebrin II‐negative zones. The mutated Ca2+ channel α1A subunit was highly expressed in granule and Purkinje cells, and the P‐type Ca2+ currents in Purkinje cells were selectively reduced in the mutant mice. Therefore, we concluded that altered Ca2+ currents through the mutated Ca2+ channel α1A subunit might be involved in the functional and morphological abnormalities in granule and Purkinje cells, and might result in expressions of behavioral phenotypes including ataxia. Increased levels of corticotropin‐releasing factor and cholecystokinin in some climbing and mossy fibers were observed in RMN. These neuropeptides modulated the excitability of granule and Purkinje cells, indicating the possible expression of ataxic symptoms.

Abnormal expression of tyrosine hydroxylase immunoreactivity in Purkinje cells precedes the onset of ataxia in dilute–lethal mice

Expression of tyrosine hydroxylase (TH) immunostaining in the cerebellum was examined in dilute-lethal mice (DL) prior to and following the onset of ataxia. DL walked normally on postnatal days 7 and 8. Falling over when walking was exhibited by about 20% of DL on day 9 and by all DL by day 10. TH-positive Purkinje cells in lobules IX and X of the vermis of either ataxic or non-ataxic DL were clearly observed on day 9 when compared to control mice, and had drastically increased by day 10. These results revealed that abnormal TH expression occurred in some Purkinje cells of DL cerebella, preceding the onset of ataxia.

Dietary regulation of renal phosphate transporters in hypophosphatemic mice

Abstract: X-linked hypophosphatemic rickets (XLH) is known to impair renal adaptive response to Pi restriction. We investigated the effects of dietary Pi on the synthesis of renal sodium-dependent inorganic phosphate (Na/Pi) cotransporters (Npt1 and NaPi-7) in X-linked hypophosphatemic mice (Hyp). The NaPi-7 mRNA level in Hyp mice was reduced to 50% of that of normal mice while the Npt1 mRNA level was unchanged. After feeding a low-Pi diet, the amounts of NaPi-7 protein and mRNA were markedly increased in both normal and Hyp mice. In contrast, after feeding a high-Pi diet, the levels of protein and mRNA were largely decreased in both mice. Immunohistochemical analysis indicated that NaPi-7 staining was largely enhanced in the apical membrane of renal proximal tubular cells in the normal and Hyp mice fed the low-Pi diet. In contrast, NaPi-7 staining was decreased in both groups of rats fed the high-Pi diet. Npt1 immunoreactivity was detected in the apical membrane of proximal convoluted and straight tubular cells in Hyp and normal mice, and was unchanged regardless of dietary Pi manipulation in both mice. Thus, dietary regulation for the synthesis of the two cotransporters is not impaired in Hyp mice.