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C Inagaki

Publications and source records attributed to C Inagaki.

At least 19 recordsLinked to original sources
PubMed2006

Anti-prolactin (PRL) autoantibody-binding sites (epitopes) on PRL molecule in macroprolactinemia.

Macroprolactinemia, in which serum prolactin (PRL) mainly consists of PRL with a molecular mass greater than 100 kDa, has been demonstrated to be associated with hyperprolactinemia. We previously reported that anti-PRL autoantibody is the major cause of macroprolactinemia. In this study, the autoantibody-binding sites (epitopes) on the PRL molecule were examined using deletion mutant PRL. The sera from 159 patients with hyperprolactinemia were screened for macroprolactinemia using the polyethylene glycol method and 18 patients (11%) were diagnosed with macroprolactinemia. The sera from these patients were incubated with glutathione S-transferase-human prolactin (hPRL) fragment fusion proteins immobilized on glutathione sepharose and the amounts of bound immunoglobulin G (IgG) were measured using ELISA. IgG was bound to full-length hPRL1-199 in significantly greater amounts in sera from 14 of 18 patients with macroprolactinemia than in controls. hPRL, but not PRL of other species such as bovine, porcine, rat, or human GH, dose-dependently displaced the binding, suggesting that these patients had hPRL-specific autoantibodies. Deletion of 34 amino acid residues from N-and/or C-terminals significantly reduced the binding and N- or C-terminal fragment alone showed partial but significant binding, suggesting that the major epitopes recognized by anti-PRL autoantibodies are located in both N- and C-terminal residues of the PRL molecule.

Adolescent↗
PubMed2002

Protective effects of estradiol against amyloid beta protein-induced inhibition of neuronal Cl(-)-ATPase activity.

Low concentrations of amyloid beta proteins (Abetas, 1-10 nM) were recently demonstrated to reduce Cl(-)-ATPase activity in parallel with an increase in the intracellular Cl(-) concentration ([Cl(-)]i) and decreases in plasma membrane phosphorylated phosphatidylinositol (PIP and PIP2) levels in cultured rat hippocampal neurons. In this study, 17 beta-estradiol (estradiol) at a therapeutic concentration (1.8 nM) for Alzheimer's disease was found to block these Abeta (Abeta25-35)-induced changes. This protective effect of estradiol on Cl(-)-ATPase activity was antagonized by a pure estrogen receptor antagonist, ICI182780 and inhibitors for cyclic GMP-dependent protein kinase (PKG) (KT5823), Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) (KN62) and phosphatidylinositol (PI) 4-kinase (wortmannin and quercetin). Estradiol recovered Abeta-induced decreases in plasma membrane phosphoinositide (PIP and PIP2) levels, this effect being inhibited by KT5823 and KN62. Glutamate toxicity was augmented in neurons with elevated [Cl(-)]i either by Abeta-treatment or carbachol+KCl+LiCl-treatment. The increased glutamate toxicity in the Abeta-treated neurons was attenuated by estradiol. Thus, a therapeutic concentration of estradiol protected Abeta-treated neurons against inhibition of Cl(-)-ATPase activity and an increase in [Cl(-)]i through its receptor, probably via PKG- and CaMKII(-)mediated recovery of PI4P formation. Elevated [Cl(-)]i may be related to enhancement of glutamate toxicity.

Adenosine Triphosphatases↗
PubMed2001

Molecular cloning and characterization of the Cl(-) pump-associated 55-kDa protein in rat brain.

The Cl(-)-ATPase/pump in the plasma membrane of the rat brain is a candidate for active outwardly directed Cl(-) translocating systems. We recently isolated a Cl(-) pump, 520- or 580-kDa protein complex, which consisted of 51-, 55-, 60-, and 62-kDa proteins. In this study, we cloned a cDNA encoding a 55-kDa glycoprotein, designated as ClP55, which contained an open reading frame of 1512 base pairs encoding a protein of 504 amino acids including a signal peptide of 28 amino acids. Northern and Western blot analyses demonstrated expression of ClP55 mainly in the cerebrum. Application of antisense phosphorothioate oligonucleotides to cultured neurons resulted in a marked increase in the intracellular Cl(-) concentration ([Cl(-)](i)). Immunohistochemical analysis indicated that ClP55 was localized to the plasma membranes of neurons such as hippocampal pyramidal neurons and cerebellar Purkinje cells. Taken together, these results suggest that ClP55 is one of the Cl(-) pump subunits responsible for Cl(-) pump activity.

Adenosine Triphosphatases↗
PubMed2001

An anxiolytic agent, dihydrohonokiol-B, inhibits ammonia-induced increases in the intracellular Cl(-) of cultured rat hippocampal neurons via GABA(c) receptors.

The effects of an anxiolytic honokiol derivative, dihydrohonokiol-B (DHH-B) [3'-(2-propenyl)-5-propyl-(1,1'-biphenyl)-2,4'-diaol], on ammonia-induced increases in the intracellular Cl(-) concentration ([Cl(-)](i)) were examined using primary cultured rat hippocampal neurons. DHH-B (1-100 ng/ml), but not an inactive isomer of honokiol, magnolol (100 ng/ml), dose-dependently inhibited the ammonia-induced increases in [Cl(-)](i) without any changes in the control [Cl(-)](i). Such an effect of DHH-B was blocked by a gamma-aminobutylic acid A (GABA(A)) and GABA(C) Cl(-) channel blocker, 100 microM picrotoxin, and a GABA(C) receptor blocker, 10 microM (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid, but not by a GABA(A) receptor blocker, 10 microM bicuculline. Further, a GABA(C) receptor agonist, 200 microM cis-4-aminocrotonic acid, but not a GABA(A) receptor agonist, 10 microM muscimol, mimicked the effect of DHH-B. Thus, DHH-B appears to protect neurons from the ammonia-induced increases in [Cl(-)](i) through GABA(C) receptor stimulation.

Ammonia↗
PubMed2001

Pertussis toxin-sensitive and -insensitive mechanisms of alpha1-adrenoceptor-mediated inotropic responses in rat heart.

In rat left ventricular papillary muscle, phenylephrine, an alpha1-adrenoceptor agonist, induced a triphasic inotropic response; an initial transient, small, positive inotropic effect followed by a transient chloroethylclonidine-sensitive negative inotropic effect and a sustained 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB4101)-sensitive positive inotropic effect. Treatment with pertussis toxin for 2 days significantly inhibited only the transient negative inotropic effect without changing the sustained positive inotropic effect. This treatment also prevented the acetylcholine (1 microM)-induced negative inotropic effect. Further, phenylephrine-induced transient negative inotropic effect was attenuated in the presence of ouabain. These results suggest that pertussis toxin-sensitive or -insensitive G-protein may be responsible for alpha1-adrenoceptor subtype-mediated negative inotropic effect or positive inotropic effect, respectively, in which the transient negative inotropic effect was produced via the stimulation of Na+, K+ pump, presumably through pertussis toxin-sensitive G-protein-dependent pathway.

Adrenergic alpha-Antagonists↗
PubMed2001

Developmental changes in Cl(-)-ATPase activity in rat brains.

Developmental changes in brain Cl(-)-ATPase activity were examined using fetal, neonatal and adult rats. The Cl(-)-ATPase activity rapidly increased over 20 postnatal days to a level four-fold higher than that in an 18-day-old fetus. On Western blot analysis using an anti-Cl(-)-ATPase/pump 51 kDa subunit (ClP51) antibody, the amount of ClP51 protein increased in parallel with Cl(-)-ATPase activity. Immunohistochemistry using the same antibody showed Cl(-)-ATPase-like immunoreactivity on the cell membranes of neurons such as cerebral and hippocampal pyramidal cells and cerebellar Purkinje cells, where the immunoreactivity increased with developmental changes in the size and shape of the neurons. These findings suggest that neuronal Cl(-)-ATPase activity markedly increases during early postnatal development with an increase in the amount of Cl(-)-ATPase protein, which may support the formation of inwardly directed neuronal Cl(-) gradients.

Adenosine Triphosphatases↗
PubMed2001

Cl(-)-ATPase in rat brain and kidney.

Cl(-)-stimulated ATPase/ATP-dependent Cl(-) pump (Cl(-)-ATPase/pump) has been found as a candidate for an active outwardly directed Cl(-) transporter in brain neurons. (1) A 520-kDa protein complex with Cl(-)-ATPase/pump activity was isolated from rat brain. It consisted of four protein subunits (51, 55, 60, and 62 kDa proteins), the 51-kDa protein being a covalent phosphorylenzyme subunit. (2) An antiserum against the 51-kDa protein inhibited Cl(-)-ATPase/pump activity. Western blot analysis showed an immunoreactive 51-kDa protein in the brain, spinal cord, and kidney. By enzyme histochemistry and immunohistochemistry, Cl(-)-ATPase-like activity or immunoreactivity was observed on the plasma membranes of brain neurons, and on the baso-lateral membranes of type A intercalated cells of renal collecting ducts. (3) Reconstituted Cl(-)-ATPase/pump activity was highest in liposomes with phosphatidylinositol-4-monophosphate. LiCl, an inhibitor of inositolphosphatase, reduced Cl(-)-ATPase activity and increased intracellular Cl(-) concentrations in cultured rat hippocampal neurons with increased phosphatidylinositol turnover. (4) In the brains of patients with Alzheimer's disease (AD), where phosphatidylinositol 4-kinase activity is reduced, Cl(-)-ATPase activity was also reduced. Thus, Cl(-)-ATPase is likely an outwardly directed ATP-dependent Cl(-) transporter that consists of four subunits and is regulated by phosphatidylinositol-4-monophosphate. Changes in Cl(-)-ATPase activity may be related to the pathophysiology of human neurodegenerative diseases. J. Exp. Zool. 289:224-231, 2001.

Adenosine Triphosphatases↗
PubMed2001

Involvement of anion exchange in the hypoxia/reoxygenation-induced changes in pH(i) and.

The involvement of Cl(-)/HCO(3)(-) exchange in hypoxia/reoxygenation-induced changes in pH(i) and Ca(2+) concentration ([Ca(2+)](i)) was examined in rat ventricular myocytes. During 10-min hypoxia, the initial pH(i) (7.21+/-0.04) fell to below 6.8. Subsequent reperfusion with reoxygenated buffer returned this acidic pH(i) to the neutral range with increases in [Ca(2+)](i). These responses were reduced by the removal of Cl(-) or HCO(3)(-) and by the addition of anion exchange inhibitors, SITS (4-acetamido-4'isothiocyanato-stilbene-2,2'disulfonic acid) and DIDS (4,4'-diisothiocyano-stilbene-2,2'-disulfonic acid), while inhibitors for the Cl(-) channel and Na(+)/K(+)/2Cl(-) cotransport were without effects. The hypoxia-induced acidification was attenuated by protein kinase C inhibitors, calphostin C and chelerythrine, but not by a protein kinase A inhibitor, KT5720. Under normoxic condition, protein kinase C activation induced a SITS-sensitive acidification. Furthermore, in electrically driven rat papillary muscle, SITS and DIDS improved the recovery of developed tension during the reoxygenation. These results suggest that the hypoxia-induced decrease in pH(i) is mediated at least in part by anion exchange stimulation through protein kinase C activation, and this exchange takes part in the reoxygenation-induced Ca(2+) overload as well as contractile dysfunction.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2001

Amyloid beta proteins inhibit Cl(-)-ATPase activity in cultured rat hippocampal neurons.

Cl(-)-ATPase in the CNS is a candidate for an outwardly directed neuronal Cl(-) transporter requiring phosphatidylinositol-4-phosphate (PI4P) for its optimal activity. To test its pathophysiological changes in a phosphatidylinositol (PI) metabolism disorder, the effects of neurotoxic factors in Alzheimer's disease (AD), amyloid beta proteins (Abetas), on the Cl(-)-ATPase activity were examined using primary cultured rat hippocampal neurons. Amyloid beta proteins (1-40, 1-42 and 25-35) concentration-dependently (1-100 nM) and time-dependently (from 1 h to 6 day) decreased Cl(-)-ATPase activity and elevated intracellular Cl(-) concentrations ([Cl(-)]i), Abeta25-35 being the most potent. Addition of inositol or 8-Br-cyclic GMP completely reversed these Abeta-induced changes. The recoveries in enzyme activity were attenuated by an inhibitor of PI 4-kinase, 10 microM wortmannin or 20 microM quercetin, but not by a PI 3-kinase inhibitor, 50 nM wortmannin or 10 microM LY294002. The PI, PIP and PIP2 levels of the plasma membrane-rich fraction were lower in the Abeta-treated cells as compared with each control. In the Abeta-exposed culture, but not in control, stimulation by 10 microM glutamate for 10 min significantly increased fragmentation of DNA and decreased cell viability. Addition of inositol or 8-Br-cyclic GMP prevented the effect of Abeta-treatment on the neurotoxicity of glutamate. Thus, Abetas reduce neuronal Cl(-)-ATPase activity, resulting in an increase in [Cl(-)]i probably by lowering PI4P levels, and this may reflect a pre-apoptotic condition in early pathophysiological profiles of AD.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2001

GH, GH receptor, GH secretagogue receptor, and ghrelin expression in human T cells, B cells, and neutrophils.

We examined GH and GH receptor expression in human leukemic cell lines and leukocytes of normal subjects to elucidate the cell types expressing GH and GH receptor, the individual variations of their expressions, their correlation and the relationships with serum IgG and IGF-I concentrations. In addition, the expression of GH secretagogue receptor, which enhances GH secretion from the anterior pituitary by synthetic GH secretagogues and that of its endogenous ligand, ghrelin, were also examined in these immune cells. GH expression in human leukemic cell lines was observed mainly in B cell lines at both the mRNA and protein level [3.8 +/- 0.2 pg/10(6) cells in Raji and 19.9 +/- 3.3 pg/10(6) cells in Daudi vs. negligible in T cell lines (Jurkat and Hut-78) and in myeloid cell lines (K-562 and HL-60)]. B cells in normal subjects were also found to be the major immune cells expressing GH mRNA, with significant individual variation. GH receptor mRNA expression was detectable in all human leukemic cell lines, although the expression level varied widely among the cell lines and was weaker than that in the liver. On the other hand, GH receptor mRNA expression was mainly found in B cells, with marked individual variation in normal subjects. There was a positive correlation between the mRNA expressions of GH and GH receptor in B cells of normal subjects (r = 0.89; P < 0.001). Single cell RT-PCR revealed that some B cells expressed both GH and GH receptor transcripts, and others expressed only GH. GH/GH receptor expression levels in B cells did not show any correlation with serum IgG and IGF-I levels in normal subjects. Expression of GH secretagogue receptor and ghrelin was detectable in all immune cells regardless of the maturity and cell types with great individual variations. In summary, GH secreted from B cells may act locally on their own receptors, and their variable expressions may be related to individual immune functions. Widespread distribution of ghrelin and GH secretagogue receptor in human immune cells may indicate unknown biological functions other than enhancing GH secretion in the immune system.

B-Lymphocytes↗
PubMed2001

Molecular mechanisms for alpha2-adrenoceptor-mediated regulation of synoviocyte populations.

The sympathetic nervous system has been indicated to influence the severity of inflammatory disease including rheumatoid arthritis. In this study, we elucidated the effects of catecholamine on the synovial cell populations. Stimulation with epinephrine or norepinephrine for 1-2 weeks dose- and time-dependently increased the number of synovial A (macrophage-like) cells but decreased that of B (fibroblast-like) cells. These responses in A and B cells were inhibited by the alpha2-antagonist yohimbine, the G-protein inactivator pertussis toxin and the phospholipase C (PLC) inhibitor U-73122. Furthermore, the protein kinase C (PKC) inhibitor calphostin C and mitogen-activated protein (MAP) kinase inhibitors PD98059 and wortmannin also abolished the norepinephrine effects on A and B cell numbers. In A cells cloned from an A and B cell mixture, norepinephrine also increased the cell number. In immunoblotting and immunocytostaining analyses, among the PKC isozymes, only PKC betaII immunoreactivity was observed in the cytoplasm of unstimulated A and B cells. After alpha2-adrenoceptor stimulation, PKC betaII immunoreactivity increased in the plasma membranes of both A and B cells with decreases in the cytoplasm. These findings indicated that alpha2-adrenoceptor stimulation of type A and B synoviocytes produced an increase and a decrease in the respective cell number, probably through Gi-coupled PLC activation and the resulting stimulation of the PKC betaII/MAP kinase.

Adrenergic alpha-Agonists↗
PubMed2000

mRNA expression of kidney-specific ClC-K1 chloride channel in single-cell reverse transcription-polymerase chain reaction analysis of outer hair cells of rat cochlea.

Outer hair cells (OHCs) of cochlea have been suggested to have Cl(-) channels sensitive to an ototoxic diuretic, furosemide. We therefore examined whether kidney-specific chloride channels (ClC-K1 and ClC-K2) and ClC-5 are also expressed in OHCs of rat cochlea, assuming that these channels might be the targets of oto-nephrotoxic drugs, by single-cell reverse transcription-polymerase chain reaction (RT-PCR) technique. Single-cell RT-PCR revealed the presence of transcripts of ClC-K1 in OHCs which was verified by DNA sequencing, while ClC-K2 and ClC-5 were not detected. The possible roles of ClC-K1 in OHCs are discussed.

Animals↗
PubMed2000

Immunohistochemical demonstration of Cl- pump in type A intercalated cells of rat kidney.

In order to demonstrate the localization of an ethacrynic acid-sensitive Cl- pump in the rat kidney, immunohistochemical analysis was performed using an anti-Cl- pump antibody raised against rat brain Cl- pump protein with confocal laser scanning microscopy. The antibodies against Na+,K+-ATPase, aquaporin 2 and a type B intercalated cell marker, 43-kDa protein, were also used for comparison. Anti-Cl- pump antibody recognized a 51-kDa renal protein of the same size as that in the brain on Western blots. Cl- -pump-like immunoreactivity was observed on the basolateral membranes of 42+/-3% of cortical collecting duct (CCD) cells and of 38+/-1% of outer medullar collecting duct (OMCD) cells. Such immunoreactivity in CCD was sometimes co-localized with Na+,K+-ATPase, but in OMCD, the Cl- pump-like immunoreactivity co-existed with neither Na+,K+-ATPase, aquaporin 2 nor the type B intercalated cell marker 43-kDa protein. Thus, the Cl- pump was demonstrated to be localized on the basolateral membranes of type A intercalated cells of cortical and medullary collecting ducts in the rat kidney.

Animals↗
PubMed1999

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.

Animals↗
PubMed1999

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.

Animals↗
PubMed1999

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.

Adenosine Triphosphatases↗
PubMed1999

Long-term treatment with antidepressants increases glucocorticoid receptor binding and gene expression in cultured rat hippocampal neurones.

Since the glucocorticoid receptor (GR) and/or mineralocorticoid receptor (MR) in the hippocampus have been implicated in cortisol feedback of the hypothalamus-pituitary-adrenal (HPA) axis, abnormalities in those receptors might underlie the hyperactivity of the HPA axis described in patients with major depression. Animal studies have shown that long-term in-vivo treatment with antidepressants up-regulates hippocampal GR and/or MR, but it is not clear whether this up-regulation is evoked through a direct action of antidepressants on these receptors. We therefore examined the direct effects of long-term antidepressant treatment on GR binding and the levels of GR messenger RNA (mRNA) in primary cultures of rat hippocampal neurones. The time course of the effects of the tricyclic antidepressants desipramine and amitriptyline on GR binding, as assessed by [3H]dexamethasone binding using RU 28362, a specific agonist for GR, showed a biphasic mode of stimulation: desipramine significantly increased the GR binding with 2-day exposure by 36% over that in controls and by 99% and 60% with 10- and 14-day exposures, respectively. Amitriptyline also led to a significant increase in GR binding, with peaks at 2 (by 60%) and 14 days of exposure (by 60%). The effects of 14-day treatment with desipramine required at least the first 4-day exposure, and the first 10-day exposure was required for the full effect. Northern blot analysis demonstrated that the GR mRNA level was significantly increased by 14-day treatment with desipramine (+142% over control), amitriptyline (+108%), mianserin (+124%), paroxetine (+42%) and sulpiride (+92%), but not with haloperidol. Immunocytochemistry for GR revealed that 2- or 14-day treatment with desipramine significantly increased the number of GR-positive cells with dominant immunoreactivity in the nuclei of granule cell-like neurones or in perikarya of pyramidal cell- and granule cell-like neurones. These findings suggest that tricyclic antidepressants directly increase hippocampal GR by short-term (2-day) and long-term (14-day) exposure, and that the increase by long-term exposure is evoked commonly with different classes of antidepressants through transcriptional up-regulation of GR expression.

Amitriptyline↗