Kyoko Omori

An ATP-driven Cl− pump regulates Cl− concentrations in rat hippocampal neurons

To investigate the role of Cl(-)-stimulated Mg(2+)-ATPase (Cl(-)-ATPase) in neurons, we examined the effects of ethacrynic acid (0.3 mM), which completely inhibits Cl(-)-ATPase on the intracellular Cl- concentrations of cultured rat hippocampal neurons, using Cl(-)-sensitive fluorescent probes. Ethacrynic acid and ATP consuming treatment increased the intracellular Cl- concentration, but elevation of the extracellular K+ concentration up to 10 mM, inhibition of Na+/K(+)-ATPase, or dissolution of H+ gradients had no effect. Furosemide (0.1 mM), an inhibitor of Na+/K+/Cl- co-transport, decreased the intracellular Cl- concentrations. These results indicate that an ethacrynic acid-sensitive and ATP-driven Cl- pump functions to reduce intraneural Cl- concentrations.

Calcineurin-mediated BAD Ser155 dephosphorylation in ammonia-induced apoptosis of cultured rat hippocampal neurons

We previously reported that ammonia induced apoptosis in cultured rat hippocampal neurons with moderate increases in the intracellular calcium concentration and decreases in phospho-BAD levels. Since this suggested the involvement of calcineurin in the apoptosis, the effects of calcineurin inhibitors, 1 microM cyclosporin A and 1 microM FK506, on the ammonia-induced neuronal apoptosis were tested. Both of the inhibitors abolished the neuronal apoptosis assessed by double staining with Hoechst 33258 and anti-neurofilament antibody, and the ammonia-induced decrease in phospho-BAD Ser(155) level. Thus, calcineurin appeared to be involved in the dephosphorylation of BAD at the sites including Ser(155) in ammonia-induced apoptosis.

GABAC receptor agonist suppressed ammonia‐induced apoptosis in cultured rat hippocampal neurons by restoring phosphorylated BAD level

Ammonia‐induced apoptosis and its prevention by GABAC receptor stimulation were examined using primary cultured rat hippocampal neurons. Ammonia (0.5–5 mm NH4Cl) dose‐dependently induced apoptosis in pyramidal cell‐like neurons as assayed by double staining with Hoechst 33258 and anti‐neurofilament antibody. A GABAC receptor agonist, cis‐4‐aminocrotonic acid (CACA, 200 µm), but not GABAA and GABAB receptor agonists, muscimol (10 µm) and baclofen (50 µm), respectively, inhibited the ammonia (2 mm)‐induced apoptosis, and this inhibition was abolished by a GABAC receptor antagonist (1,2,5,6‐tetrahydropyridin‐4‐yl)methylphosphinic acid (TPMPA, 15 µm). Expression of all three GABAC receptor subunits was demonstrated in the cultured neurons by RT‐PCR. The ammonia‐treatment also activated caspases‐3 and ‐9 as observed in immunocytochemistry for PARP p85 and western blot. Such activation of the caspases was again inhibited by CACA in a TPMPA‐sensitive manner. The anti‐apoptotic effect of CACA was blocked by inhibitors for MAP kinase kinase and cAMP‐dependent protein kinase, PD98059 (20 µm) and KT5720 (1 µm), suggesting possible involvement of an upstream pro‐apoptotic protein, BAD. Levels of phospho‐BAD (Ser112 and Ser155) were decreased by the ammonia‐treatment and restored by coadministration of CACA. These findings suggest that GABAC receptor stimulation protects hippocampal pyramidal neurons from ammonia‐induced apoptosis by restoring Ser112‐ and Ser155‐phospho‐BAD levels.

Regulation of the expression of lysyl oxidase mRNA in cultured rabbit retinal pigment epithelium cells.

Lysyl oxidase, an extracellular amine oxidase, controls the maturation of collagen and elastin. We examined the regulation of lysyl oxidase mRNA in cultured rabbit retinal pigment epithelium (RPE) cells in relation to the changes in subretinal fluid transport and phenotype of RPE cells. The level of the mRNA in cells grown on microporous membranes was markedly increased by application of hyperosmotic mannitol solution on the apical side (191% of control), implying that RPE cells express more lysyl oxidase in the condition which may cause the accumulation of subretinal fluid. Platelet-derived growth factor increased the mRNA level in subconfluent cells in culture (137% of control) and basic fibroblast growth factor decreased it (79% of control). In addition, exposure of cells to retinoic acid alone or in combination with dibutyryl cAMP for 22 days markedly decreased the level of lysyl oxidase mRNA (52 or 35% of control) while increasing the level of mRNA of N-acetylglucosaminidase (NAG), a marker enzyme for lysosomes (162 or 142% of control). Moreover, the level of lysyl oxidase mRNA in cells grown on microporous membranes was lower than that in cells grown on plastic dishes, while the level of NAG mRNA in the former cells was higher than that in the latter. Taken together, the expression of lysyl oxidase seemed to increase during proliferation of RPE cells and decrease toward differentiation. beta-Aminopropionitrile, an inhibitor of lysyl oxidase, significantly inhibited the contraction of collagen gels by fetal calf serum, suggesting that lysyl oxidase may be involved in pathogenesis caused by RPE cells.

Intracerebroventricular injection of ethacrynic acid induces status epilepticus

The intracerebroventricular (i.c.v.) injection of ethacrynic acid to mice at a dose of more than 25 micrograms induced repeated tonic-clonic convulsions with subsequent death. Ethacrynic acid was more potent than other loop diuretics such as furosemide and bumetanide. Diazepam and 2-amino-5-phosphonovaleric acid notably reduced both the incidence of convulsion and the lethality seen after ethacrynic acid administration. Both phenobarbital and ketamine suppressed the incidence of convulsions but not the lethality. Without effects on the incidence of convulsions or lethality, dextromethorphan prolonged, while phenytoin or atropine shortened, the time to the onset of convulsion. Neither ethosuximide, carbamazepine, nor muscimol had a significant effect on the responses to ethacrynic acid. The present findings indicate that i.c.v. injected ethacrynic acid shows strong convulsive activity, probably due to impairment of Cl- transport processes, concomitant with enhancement of excitatory amino acid activity in the brain.

Long-lasting c-fos and NGF mRNA expressions and loss of perikaryal parvalbumin immunoreactivity in the development of epileptogenesis after ethacrynic acid-induced seizure.

A single cerebroventricular injection of ethacrynic acid (EA), a Cl(-)-ATPase inhibitor, induces generalized tonic-clonic convulsions in mice. To clarify whether such convulsive stimulus triggers a long-lasting rearrangement of the neural circuitry culminating in seizure susceptibility, we examined molecular, cellular and behavioral changes following the EA-induced seizure. The expression of immediate early gene c-fos mRNA as an index for cellular activation increased biphasically, with an early transient increase at 60 min and a late prolonged increase on the 10th to 14th day post-EA administration, most remarkably in the hippocampus and pyriform cortex. On the 14th day post-EA seizure, subconvulsive dose of kainic acid (5-17.5 mg/kg) caused severe (stage 5) seizure in 77% of the mice, with 70% mortality. In addition, the expression of nerve growth factor (NGF) also showed biphasic increases with close spatiotemporal correlation with c-fos expression. Moreover, the number of cell somata and the density of axon fibers of parvalbumin (PARV)-positive cells, a subpopulation of GABAergic interneurons, decreased in area dentata, CA1 and CA3 on the 7th and 14th day post-EA seizure. In area dentata and CA1, the density of glutamic acid decarboxylase (GAD)-positive cells also decreased on the 14th day. Thus, the transient EA-induced seizures appear to develop seizure susceptibility by causing damage of a subpopulation of inhibitory interneurons along with increases in the expression of c-fos and NGF in limbic structures.

Antiserum against Cl− pump complex recognizes 51 kDa protein, a possible catalytic unit in the rat brain

We purified Cl- pump in the rat brain and obtained 520 or 580 kDa protein complexes which consisted of 62, 60, 55 and 51 kDa proteins. An antiserum against 520 kDa protein complex recognized 51 kDa protein in both 520 and 580 kDa complexes, and reduced both Cl(-)-ATPase and Cl(-) pump activities. Such an immunoreactive 51 kDa protein was found in the brain, spinal cord and kidney. When incubated with [gamma-(32)P]ATP, the protein complex yielded phosphorylated 51 kDa protein, the label being hydroxylamine-sensitive and increased in the presence of Cl- and/or an inhibitor of Cl- pump, ethacrynic acid. Thus, the antibody appears to recognize a possible catalytic subunit of Cl- pump, 51 kDa protein, in the rat.

Lithium decreases Cl^--ATPase activity and increases intracellular Cl^- concentration in cultured rat hippocampal neurons

Under the conditions of stimulated phosphatidylinositol turnover (0. 1 mM carbachol plus 20 mM KCl), LiCl (0.1-10 mM) reduced the activity of Cl--ATPase in cultured rat hippocampal neurons without affecting Na+/K+- or anion-insensitive Mg2+-ATPase. This inhibition of Cl--ATPase was attenuated by the addition of 0.5 mM inositol to culture media. The intracellular Cl- concentrations of the LiCl-treated neurons increased in an inositol-sensitive manner.

Two Types of Clathrin-Coated Vesicles Isolated from Rat Brain: Analysis of Biochemical Properties and Cellular Origin

Abstract: Two major fractions rich in clathrin‐coated vesicles (CVs) (fraction I, ρ= 1.140 g/cm3; fraction II, ρ= 1.113 g/ cm3) were separated from rat brain using a sucrose gradient and compared for their cellular origins and Cl‐ translocation systems. Electron micrographs showed that both fractions contained CVs of different size distributions (fraction I, 85±9.5 nm in diameter; fraction II, 72±6.8 nm in diameter). Fraction II contained potent ouabain‐sensitive ATPase activity, whereas fraction I contained only a little activity. Im‐munoblot analysis for the Na+,K+‐ATPase catalytic subunit, a and α(+), demonstrated that fraction II exhibited predominantly α(+), whose proportion to α was analogous to that observed in the extracts of primary cultured neuronal cells. Furthermore, on a sucrose density gradient, cultured neuronal cells yielded fraction II but not fraction I, whereas primary cultured glial cells yielded fraction I but not fraction II. Labeling‐chase experiments using 125I‐transferrin in cultured neuronal cells showed the internalized ligand in fraction II and the surface‐bound ligand in the fraction with lower density (ρ= 1.090 g/cm3), a result suggesting that the involvement of Na+,K+‐ATPase in fraction II is attributable to endocytic vesicles. Cl‐ uptake in fraction II was approximately threefold higher than that in fraction I. ALEthylmaleimide (100 μM) completely inhibited the CI‐ uptake in fraction I but partially (∼50%) inhibited that in fraction II. These findings suggest that the two CV fractions isolated from rat brain originate from different cell types—glial and neuronal cells—and differ in size distribution of CVs, content of Na+,K+‐ATPase, and mechanism for Cl‐ uptake.

Continuous administration of antisense oligonucleotides to c-fos reduced the development of seizure susceptibility after ethacrynic acid-induced seizure in mice

We previously demonstrated that seizure susceptibility developed by the 14th day post-ethacrynic acid (EA)-induced seizure in mice, with a prolonged increase in the expression of c-fos mRNA in the brain during days 10-14. To examine whether such c-fos increase contributes to the development of seizure susceptibility, we administered antisense oligodeoxynucleotide to c-fos by continuous infusion into the lateral ventricle of mice that had shown a moderate stage of EA seizure, and evaluated the seizure susceptibility to kainic acid (10 mg/kg) on the 14th day. Antisense-infused mice displayed significant reduction of the c-Fos level in the hippocampus and cerebral cortex on the 7th and 14th days, and a significant decrease in seizure severity. These findings suggest that the prolonged increase in c-fos expression after EA seizure may lead to the development of seizure susceptibility.