Chiyoko Inagaki

Cl--ATPase and Na+/K+-ATPase activities in Alzheimer's disease brains

Abstract The enzyme activities and the protein levels of Cl − -ATPase and Na + /K + -ATPase were examined in Alzheimers disease (AD) brains. Cl − -ATPase and Na + /K + -ATPase activities in AD brains ( n =13) were significantly lower than those in age-matched control brains ( n =12). In contrast, there was no significant difference in anion-insensitive Mg 2+ -ATPase activity between the two groups. Western blot analysis revealed that the protein levels of Cl − -ATPase, Na + /K + -ATPase and neuron specific Na + /K + -ATPase α 3 isoform were also significantly reduced in AD brains, while the amount of protein disulfide isomerase, one of the house keeping membrane proteins, was not different between the two groups. The data first demonstrated that Cl − -ATPase and Na + /K + -ATPase are selectively impaired in AD brains, which may reduce the gradients of Na + , K + and Cl − across the cell membranes to cause excitotoxic cellular response and the resulting neuronal death.

Single-cell RT-PCR demonstrates expression of voltage-dependent chloride channels (ClC-1, ClC-2 and ClC-3) in outer hair cells of rat cochlea

We investigated whether voltage-dependent chloride channels (ClC-1, ClC-2 and ClC-3) are expressed in outer hair cells (OHCs) of rat cochlea using a single-cell reverse transcription-polymerase chain reaction (RT-PCR) technique. The OHCs were isolated from rat cochlea and the cytoplasm of each OHC was suctioned into a glass pipette containing RT-PCR reaction buffer with RNase inhibitor. RT-PCR revealed the presence of transcripts of ClC-1, ClC-2 and ClC-3, which were verified by DNA sequencing. The possible roles of these chloride channels in OHCs are discussed.

Chloride concentration in cultured hippocampal neurons increases during long-term exposure to ammonia through enhanced expression of an anion exchanger

The effects of long-term exposure to ammonia on [Cl-]i in cultured hippocampal neurons were examined. Ammonia increased the [Cl-]i time- (>/=24 h) and concentration- (>/=2 mM) dependently, resulting in a depolarizing shift of the equilibrium potential of the GABAA receptor-Cl- channel opening (EGABA). Such an effect of ammonia was diminished by the inhibitors of Cl-/HCO3- exchangers, 0.1 mM 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS) and 0.1 mM 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), and a carbonic anhydrase inhibitor, 2 mM acetazolamide, but not by a Na+/K+/2Cl-cotransport inhibitor, 50 microM bumetanide, suggesting an enhanced Cl-/HCO3- exchange activity by ammonia. The ammonia-induced increase in [Cl-]i was also abolished by the inhibitors of protein kinase C (PKC), 0.1 microM calphostin C and 10 microM 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine dihydrochloride (H-7), and of transcription and de novo protein synthesis, 1 microM actinomycin D and 0.5 microg/ml cycloheximide, while a PKC activator, 0.1 h microM phorbor 12-myristate 13-acetate (PMA), increased the [Cl-]i. The mRNA level of the AE3 Cl-/HCO3- exchanger was increased by ammonia in a calphostin C- and H-7-sensitive manner. The AE3-like immunoreactivity was also increased by ammonia. These findings suggest that long-term exposure to ammonia increases the expression of AE3 through the activation of PKC, resulting in an increase in [Cl-]i in neurons and a reduction of inhibitory postsynaptic potentials.

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.

Immunological aspects of human growth hormone and prolactin

We demonstrated that human lymphocytes secrete growth hormone and that the secretion is regulated in a different manner from that in the endocrine system. We could also show that some patients with hyperprolactinemia, especially asymptomatic type, have anti-prolactin autoantibody, which may be a novel case of hyperprolactinemia.