Shoichi Shimada

Localization and rapid regulation of Na+/myo-inositol cotransporter in rat kidney.

myo-inositol, a major compatible osmolyte in renal medulla, is accumulated in several kinds of cells under hypertonic conditions via Na+/myo-inositol cotransporter (SMIT). To investigate the physiological role of the SMIT, we sought to determine its localization by in situ hybridization and its acute regulation by NaCl and furosemide administration. Northern analysis demonstrated that SMIT is strongly expressed in the medulla and at low levels in the cortex of kidney. Intraperitoneal injection of NaCl rapidly induced SMIT mRNA in both the cortex and medulla, and furosemide completely abolished this induction. In situ hybridization revealed that SMIT it predominantly present in the medullary and cortical thick ascending limbs of Henles loop (TALH) and macula densa cells. Less intense signals were seen in the inner medullary collecting ducts (IMCD). NaCl loading increased the signals throughout the TALH, and furosemide reduced the signals. SMIT in the IMCD is less sensitive to these kinds of acute regulation. Thus, the distribution pattern of SMIT does not correspond to the corticomedullary osmotic gradient, and SMIT in the TALH and macula densa cells is regulated very rapidly. These results suggest that SMIT expression in TALH may be regulated by intracellular and/or peritubular tonicity close to the basolateral membrane, which is supposed to be proportional to the magnitude of NaCl reabsorption.

Rapid and transient up-regulation of Na+/myo-inositol cotransporter transcription in the brain of acute hypernatremic rats

The osmoregulatory system is well developed in the brain. Osmolytes contribute to maintenance of cell volume and cellular functions without changing intracellular ionic composition. Myo-inositol is regarded as one of the major osmolytes in the brain. In the present study, we investigated the changes in expressions of sodium myo-inositol cotransporter (SMIT) mRNA in the brain of acute hypernatremic rats by in-situ hybridization and Northern blot methods. Under moderate acute hypernatremic conditions, SMIT mRNA level increased markedly at 1 h and returned to almost control levels at 3 h, in accordance with plasma Na+ concentrations. Especially, distinct increases in SMIT mRNA expression were observed in the granule cells and glial cells in the cerebellum. These findings indicate that SMIT plays an important role in osmoregulation, especially in the early stages of acute hypernatremia in the brain.

Developmental regulation of Na+/myo-inositol cotransporter gene expression

Abstract myo -Inositol plays a role in many important aspects of cellular regulation including membrane structure, signal transduction and osmoregulation. It is taken up into the cells by the Na + / myo -inositol cotransporter (SMIT). We investigated developmental changes in the expression of SMIT mRNA and protein in the rat. In the fetal rat brain, SMIT mRNA was abundantly and diffusely expressed throughout the whole brain and the spinal cord. Positive signals were expressed in neuronal and non-neuronal cells in these regions. SMIT is gradually down-regulated nearer birth, but intense signals were still detected in the brain at postnatal day one. In the adult rat brain, very weak hybridization signals were detected throughout whole brain except for the choroid plexus where SMIT mRNA expression remained high. In contrast, the pattern of developmental regulation of SMIT gene expression in the kidney was opposite to that seen in the brain. Signals in the kidney were very weak during embryonic stages, whereas SMIT expression increased significantly after birth. These results suggest that myo -inositol and its transporter play an important role in the CNS developmental stage.

Expression of dopamine transporter mRNA and its binding site in fetal nigral cells transplanted into the striatum of 6-OHDA lesioned rat

Neurological disorders in rat model of hemi-Parkinsons disease can be compensated by the transplantation of fetal nigral cells. However, the role of the dopamine transporter (DAT) in this recovery has not been clarified. To clarify this mechanism, we examined the expression of DAT in the caudate putamen (CPu) by in situ hybridization histochemistry (mRNA) and autoradiography (using the ligand [125I] beta-CIT, which labels DAT) and compared them with the recovery of motor disturbance revealed with methamphetamine-induced rotation. Models were made with the stereotaxic injection of 6-hydroxydopamine into the left side of the substantia nigra pars compacta. Cell suspensions from rat fetus (embryonic day 14-15) were transplanted into the lesioned side of CPu. Methamphetamine-induced rotation, expression of DAT mRNA, and [125I] beta-CIT binding were evaluated 2, 4 and 12 weeks after the transplantation. Methamphetamine-induced rotation recovered partly in the 2nd week and significantly in the 4th week. [125I] beta-CIT binding increased with time and the dense binding was detected 4 and 12 weeks after the transplantation. In all transplanted rats, cells expressing DAT mRNA were found in CPu. These results indicated that transplanted fetal dopaminergic cells maturated in CPu of host animals and extended nerve terminals where high density of DAT binding sites were found.

Induction of Na^+/myo-inositol cotransporter mRNA after focal cerebral ischemia : Evidence for extensive osmotic stress in remote areas

Myo-inositol is one of the major organic osmolytes in the brain. It is accumulated into cells through an Na+/myo-inositol cotransporter (SMIT) that is regulated by extracellular tonicity. To investigate the role of SMIT in the brain after cerebral ischemia, we examined expression of SMIT mRNA in the rat brain after middle cerebral artery occlusion, which would reflect alteration of extracellular tonicity. The expression of SMIT mRNA was markedly increased 12 h after surgery in the cortex of the affected side and lasted until the second day. Increased expression was also found in the contralateral cingulate cortex. Up-regulated expression was found predominantly in the neurons in remote areas, although nonneuronal cells adjacent to the ischemic core also expressed this mRNA. These results suggest that cerebral ischemia causes extensive osmotic stress in brain and that the neuronal cells respond to this stress by increasing SMIT expression.

Cloning and expression of a bovine glutamate transporter.

We have isolated a 3845 base-pair cDNA (BNGLUAS) encoding a bovine glutamate transporter (bovine GLAST) by screening a bovine retina cDNA library with an oligonucleotide probe corresponding to conserved regions of known glutamate transporters. The cDNA sequence predicted a protein of 542 amino acids and displayed 96% and 97% amino acid identity with the rat GLAST/GluT-1 and human GLAST, respectively. Expression of the bovine GLAST in Xenopus oocytes revealed Na(+)-dependent [14C]L-glutamate uptake and electrogenic glutamate uptake.

The differential osmoregulation and localization of taurine transporter mRNA and Na+/myo-inositol cotransporter mRNA in rat eyes

We studied the cellular localization and osmotic regulation of taurine transporter (TauT) mRNA in the rat eyes using in situ hybridization. TauT mRNA signals were expressed in the ciliary body, and the outer part of the inner nuclear layer (INL), the outer nuclear layer (ONL) and the inner segment (IS) of the adult rat retina. Chronic hypernatrema, induced by gavaging with 1 ml/100 g body weight of 5% NaCl every other day for 7 days, markedly increased in TauT mRNA in the retina compared with control rats. However, there was little change in TauT mRNA in the eyes in acute hypernatremic state that is induced by single injection of high concentration of NaCl. On the contrary, acute hypernatremic rats displayed markedly elevated Na+/myo-inositol cotransporter (SMIT) mRNA in the retina and the iris-ciliary body and the lens epithelium. Under chronic hypernatremic conditions, there was no significant increase in SMIT mRNA in rat eyes. These findings suggest that TauT mRNA is osmotically regulated in vivo to protect retinal neuronal function, especially against chronic hypernatremic conditions, in contrast to rapid up-regulation of SMIT mRNA in acute hypernatremic rats.

Changes in glutamate/aspartate transporter (GLAST/GluT-1) mRNA expression following facial nerve transection

Expression of glutamate/aspartate transporter (GLAST/GluT-1) was investigated in the axotomized facial nucleus by in-situ hybridization. Hybridization signals for GLAST mRNA were almost undetectable in the facial nucleus of sham-operated animals. However, the hybridization signals were seen from 3 days after facial nerve transection onward in the nucleus of the affected side. These signals lasted at least 5 weeks. Microautoradiograms showed that small non-neuronal cells in the ipsilateral facial nucleus expressed signals of GLAST mRNA after axotomy. These findings suggest that non-neuronal cells, presumably astrocytes, may protect axotomized motor neurons against glutamate toxicity via up-regulation of GLAST in the facial nucleus.

Expression of Na + /myo-inositol cotransporter mRNA in normal and hypertonic stress rat eyes

We studied the localization of Na+/myo-inositol cotransporter (SMIT) mRNA in normal and hypertonic stress rat eyes by in situ hybridization histochemistry using cRNA probes. SMIT mRNA signals were observed in the iris-ciliary body, the lens epithelial cells, and the ganglion cell layer and the inner nuclear layer of the retina. There was a rapid increase on SMIT mRNA in the retina of hypertonic stress rats compared with control rats. These findings suggest that Na+/myo-inositol cotransporter gene expression is osmotically regulated in vivo to protect retinal neuronal function against hypertonic stress.

GLAST mRNA expression in the periventricular area of experimental hydrocephalus

Glutamate transporters play an important role in maintaining the extracellular glutamate concentration below the neurotoxic level. We investigated the expression of glutamate/aspartate transporter (GLAST) mRNA in the periventricular region of rats with kaolin-induced hydrocephalus by in situ hybridization (ISH). The density of GLAST mRNA-positive cells and the level of hybridization signals per positive cell significantly increased in the acute stage of hydrocephalus. We also demonstrated co-localization of GLAST mRNA and GFAP immunoreactivity in a single cell using the combined methods of ISH and immunohistochemistry. These findings suggest that GLAST is expressed in the reactive astrocytes of the periventricular area and regulates extracellular glutamate concentration after hydrocephalic brain injury.