Hitomi Otani

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.

Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells

BackgroundProteinase-activated receptors (PARs; PAR1–4) that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR4, a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR4 stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transition (EMT) which contributes to the increase in myofibroblast population.MethodsEMT was assessed by measuring the changes in each specific cell markers, E-cadherin for epithelial cell, α-smooth muscle actin (α-SMA) for myofibroblast, using primary cultured mouse alveolar epithelial cells and human lung carcinoma-derived alveolar epithelial cell line (A549 cells).ResultsStimulation of PAR with thrombin (1 U/ml) or a synthetic PAR4 agonist peptide (AYPGKF-NH2, 100 μM) for 72 h induced morphological changes from cobblestone-like structure to elongated shape in primary cultured alveolar epithelial cells and A549 cells. In immunocytochemical analyses of these cells, such PAR4 stimulation decreased E-cadherin-like immunoreactivity and increased α-SMA-like immunoreactivity, as observed with a typical EMT-inducer, tumor growth factor-β (TGF-β). Western blot analyses of PAR4-stimulated A549 cells also showed similar changes in expression of these EMT-related marker proteins. Such PAR4-mediated changes were attenuated by inhibitors of epidermal growth factor receptor (EGFR) kinase and Src. PAR4-mediated morphological changes in primary cultured alveolar epithelial cells were reduced in the presence of these inhibitors. PAR4 stimulation increased tyrosine phosphorylated EGFR or tyrosine phosphorylated Src level in A549 cells, and the former response being inhibited by Src inhibitor.ConclusionPAR4 stimulation of alveolar epithelial cells induced epithelial-mesenchymal transition (EMT) as monitored by cell shapes, and epithelial or myofibroblast marker at least partly through EGFR transactivation via receptor-linked Src activation.

Mechanisms of oxyradical production in substance P stimulated rheumatoid synovial cells

We examined the intracellular mechanisms of substance P induced oxyradical production in rheumatoid synovial cells by the luminol-dependent chemiluminescence method. After stimulation with substance P (30 μM), single synovial A (macrophage-like) or B (fibroblast-like) cells released oxyradicals such as superoxide anions (OZ) and/or hypochlorous anions (OCl−) under a microscope equipped with an ultrasensitive photonic image intensifier. The substance P induced oxyradical production was blocked by a tachykinin NK1 (NK1) receptor antagonist, GR82334, GTP-binding protein (G-protein) inactivators, GDPβS and islet-activating protein (IAP), and a phospholipase C (PLC) inhibitor, U-73122. Substance P (30 μM) also induced a transient increase in the intracellular Ca2+ concentration ([Ca2+]i) in both synovial A and B cells as measured by a Ca2+ indicator, fura 2. BAPTA-AM and an inositol-1,4-5-triphosphate (IP3) receptor antagonist, heparin, inhibited the substance P induced increase in [Ca2+]i, but they had no effects on oxyradical production. In contrast to the effects of BAPTA-AM and heparin, protein kinase C (PKC) inhibitors, H-7 and calphostin C, completely inhibited substance P induced oxyradical production without any significant effects on [Ca2+]i increase. These findings suggest that the NK1 receptor/PLC-linked diacylglycerol (DAG) formation with the resulting activation of PKC is the main signal transduction pathway for substance P stimulated oxyradical production in synovial cells.

Involvement of anion exchange in the hypoxia/reoxygenation-induced changes in pHi and [Ca2+]i in cardiac myocyte

Abstract 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.

Inhibitory effects of calcitonin gene-related peptide on substance-P-induced superoxide production in human neutrophils

We examined the mechanisms of the inhibitory effects of calcitonin gene-related peptide (CGRP) on substance-P-induced superoxide anion (O2-) production in human neutrophils. Substance P (30 microM) caused O2- production associated with an inositol-1,4,5-trisphosphate (IP3)-induced transient increase in intracellular Ca2+ concentrations ([Ca2+]i). CGRP (10 microM) significantly inhibited substance-P-induced O2- production and transient increase in [Ca2+]i, but it only slightly suppressed IP3 formation. In addition, CGRP inhibited IP3-induced O2- production and transient increase in [Ca2+]i, caused by exogenous addition of IP3 in saponin-permeabilized neutrophils. These findings suggest that CGRP inhibits the response of neutrophils to substance P through the inhibition of IP3-induced Ca2+ release from intracellular Ca2+ stores. The inhibitory effects of CGRP on substance P- or IP3-induced O2- production and increases in [Ca2+]i were abolished by pretreating the neutrophils with a CGRP receptor antagonist, CGRP-(8 - 37), or cyclic AMP (cAMP)-dependent protein kinase inhibitors, N-[2-(methylamino) ethyl]-5-isoquinoline-sulfonamide dihydrochloride (H-8) and 9-n-hexyl ester derivative of K-572a (8R, 9S, 11 S)-(--)-9-hydroxy-9-methoxycarbonyl-8-methyl-8-methyl-2,3,9,10- tetrahydro-8,11-epoxy-1H,8H, 11 H-2,7b,11a-triazadibenzo (a,g)cycloocta(cde)trinden-1-one (KT5720). We concluded that CGRP receptor stimulation reduces substance-P-induced O2- production by the inhibition of IP3-induced transient increase in [Ca2+]i, probably via the phosphorylation of IP3 receptor by cAMP-dependent protein kinase.

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.

Involvement of Protein Kinase C and RhoA in Protease-Activated Receptor 1–Mediated F-Actin Reorganization and Cell Growth in Rat Cardiomyocytes

Protease-activated receptor 1 (PAR1) that can be activated by serine proteinases such as thrombin has been demonstrated to contribute to the development of cardiac remodeling and hypertrophy after myocardial injury. Here, we investigated the mechanisms by which PAR1 leads to hypertrophic cardiomyocyte growth using cultured rat neonatal ventricular myocytes. PAR1 stimulation with thrombin (1 U/ml) or a synthetic agonist peptide (TFLLR-NH2, 50 μM) for 48 h induced an increase in cell size and myofibril formation associated with BNP (brain natriuretic peptide) production. This actin reorganization assessed by fluorescein isothiocyanate (FITC)-conjugated phalloidin staining appeared at 1 h after PAR1 stimulation, and this response was reduced by a protein kinase C (PKC) inhibitor, chelerythrine, inhibitors of Rho (simvastatin) and Rho-associated kinase (ROCK) (Y-27632), but not by pertussis toxin (PTX). By Western blot analysis, translocation of PKCα or PKCε from the cytosol to membrane fractions was observed in cells stimulated with thrombin or TFLLR-NH2 for 2 - 5 min. In addition, PAR1 stimulation for 3 - 5 min increased the level of active RhoA. Furthermore, inhibitors of PKC and ROCK and Rho abrogated PAR1-mediated increase in cell size. Depletion of PKCα or PKCε by specific small interfering RNA also suppressed both actin reorganization and cell growth. These results suggest that PAR1 stimulation of cardiomyocytes induces cell hypertrophy with actin cytoskeletal reorganization through activation of PKCα and PKCε isoforms and RhoA via PTX-insensitive G proteins.

Protease-activated receptor-2-mediated Ca2+ signaling in guinea pig tracheal epithelial cells

The protease-activated receptor-2 (PAR-2), a G protein-coupled receptor activated by trypsin, contributes to the pathogenesis of inflammatory disease including asthma. Here, we examined the mechanisms by which stimulation of PAR-2 induces an increase in intracellular Ca2+ concentration ([Ca2+]i) in guinea pig tracheal epithelial cells. Trypsin (0.01-3 units/ml) dose-dependently induced a transient increase in [Ca2+]i, the increase being blocked by soybean trypsin inhibitor (SBTI 1 microM). An increase in [Ca2+]i was also induced by an agonist peptide for PAR-2 (SLIGRL-NH2, 0.001-10 microM) but not by thrombin (3 units/ml, an activator for PAR-1, PAR-3 or PAR-4). Repeated or cross stimulation of trypsin or SLIGRL-NH2 caused marked desensitization of the [Ca2+]i response. These responses of [Ca2+]i to trypsin and SLIGRL-NH2 were attenuated by a phospholipase C inhibitor, U-73122, and a Ca2+-ATPase inhibitor, thapsigargin (100 nM), while removal of Ca2+ and a L-type Ca2+-channel blocker, verapamil, were without significant effects. Further, trypsin was without effect on the rate of fura 2 quenching by Mn2+ entry as an indicator of Ca2+ influx. Thus, stimulation of PAR-2 appears to increase [Ca2+]i through the mobilization of Ca2+ from intracellular stores probably via phospholipase Cbeta-linked generation of a second messenger.

Different patterns of protein kinase C redistribution mediated by alpha 1-adrenoceptor stimulation and phorbol ester in rat isolated left ventricular papillary muscle.

1 In rat left ventricular papillary muscle, phenylephrine, an α1‐adrenoceptor agonist, had a staurosporine‐sensitive positive inotropic effect and increased the particulate‐associated protein kinase C (PKC) activity without significant changes in total PKC activity or in cytosolic Ca2+/phospholipid‐independent kinase (PKI) activity. 2 A PKC stimulant, phorbol 12,13‐dibutyrate (PDBu), decreased contractility and slightly increased PKC activity in the particulate fractions, with a marked decrease and increase in total PKC and PKI activities, respectively. 3 The PDBu‐induced negative inotropic response was attenuated by two protease inhibitors, leupeptine and a microbial peptide isolated from Aspergillus japonicus (E‐64), which are known to inhibit the conversion of particulate‐associated PKC to PKI. 4 Such differences in the patterns of PKC redistribution, i.e. marked increases in particulate PKC and cytosolic PKI activities caused by phenylephrine and PDBu, respectively, may account for the opposite inotropic effects of PKC stimulation by an α1‐agonist and a phorbol ester.

Chloride-dependency of amyloid β protein-induced enhancement of glutamate neurotoxicity in cultured rat hippocampal neurons

In our previous studies, pathophysiological concentrations of amyloid-beta (Abeta) proteins increased intracellular Cl(-) concentration ([Cl(-)]i) and enhanced glutamate neurotoxicity in primary cultured neurons, suggesting Cl(-)-dependent changes in glutamate signaling. To test this possibility, we examined the effects of isethionate-replaced low Cl(-) medium on the Abeta-induced enhancement of glutamate neurotoxicity in the primary cultured rat hippocampal neurons. In a normal Cl(-) (135 mM) medium, treatment with 10 nM Abeta25-35 for 2 days increased neuronal [Cl(-)]i to a level three times higher than that of control as assayed using a Cl(-)-sensitive fluorescent dye, while in a low Cl(-) (16 mM) medium such an Abeta25-35-induced increase in [Cl(-)]i was not observed. The Abeta treatment aggravated glutamate neurotoxicity in a normal Cl(-) medium as measured by mitochondrial reducing activity and lactate dehydrogenase (LDH) release, while in a low Cl(-) medium the Abeta treatment did not enhance glutamate toxicity. Upon such Abeta plus glutamate treatment under a normal Cl(-) condition, activated anti-apoptotic molecule Akt (Akt-pS473) level monitored by Western blot significantly decreased to 74% of control. Under a low Cl(-) condition, a resting Akt-pS473 level was higher than that under a normal Cl(-) condition and did not significantly change upon Abeta plus glutamate treatment. Tyrosine phosphorylation levels of 110 and 60 kDa proteins (pp110 and pp60) increased upon Abeta plus glutamate treatment under a normal Cl(-), but not low Cl(-), condition. These findings indicated that Abeta-induced enhancement of glutamate neurotoxicity is Cl(-)-dependent. Chloride-sensitive Akt pathway and tyrosine phosphorylation of proteins (pp110 and pp60) may be involved in this process.