Biomedical subjects

X T Zeng

Publications and source records attributed to X T Zeng.

14 recordsLinked to original sources

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

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

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↗

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

Induction of cytokines in a human colon epithelial cell line by Shiga toxin 1 (Stx1) and Stx2 but not by non-toxic mutant Stx1 which lacks N-glycosidase activity.

Stx1 and Stx2 produced by Shiga toxin-producing Escherichia coli are cytotoxic due to their N-glycosidase activity on 28S rRNA. In this study, we have shown that proinflammatory cytokine mRNAs, especially IL-8, were induced by Stx1 and Stx2 in Caco-2 cells. A non-toxic mutant of Stxl which lacks N-glycosidase activity did not induce cytokine mRNAs. IL-8 production at the protein level was enhanced by Stx1 and Stx2, but not by the mutant Stx1. These results demonstrate that Shiga toxins induce expression and synthesis of cytokines in Caco-2 cells and their N-glycosidase activity is essential for the induction.

Amino Acid Substitution↗

PubMed1998

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.

Adenosine Triphosphatases↗

PubMed1996

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.

Calcitonin Gene-Related Peptide↗

PubMed1996

A Cl- pump in rat brain neurons.

Cl(-)-stimulated and ethacrynic acid-sensitive ATPase (Cl(-)-ATPase) of plasma membrane origin in the rat brain is a candidate for an active outwardly directed Cl- translocating system. Biochemistry of Cl(-)-ATPase and ATP-dependent Cl- transport (Km values for ATP and Cl-, nucleotide specificity, pH dependency, and sensitivity to ethacrynic acid) suggested that Cl(-)-ATPase is an ATP-driven Cl- pump. Activity of the reconstituted Cl(-)-ATPase/pump increased in the presence of phosphatidylinositol-4-monophosphate, and this pump activity further increased at an inside-positive membrane potential or in the presence of a protonophore, suggesting that the Cl(-)-ATPase/pump is an electrogenic Cl- transporter, probably regulated by phosphoinositide turnover in vivo. In cultured hippocampal pyramidal cell-like neurons from embryonic rat brain, ethacrynic acid and ATP-consuming treatment increased, but furosemide, an inhibitor of Na+/K+/Cl- cotransporter, decreased, [Cl-]i when monitored using Cl(-)-sensitive fluorescent probes. The stationary levels of [Cl-]i were lower and the effects of ethacrynic acid were more prominent in perikarya than in dendrites, while the effects of furosemide were more obvious in dendrites than in perikarya. The lower perikaryonic [Cl-]i and the marked effects of ethacrynic acid were observed in the later stage rather than in the early stage of culture. Thus, region-specific localization and developmental changes in the activities of Cl- transporters probably result in uneven and age-dependent distribution of Cl- in the neurons.

Adenosine Triphosphatases↗

PubMed1995

Adrenergic receptor-mediated Cl- transport in rabbit corneal endothelial cells.

Adrenoceptor-mediated Cl- transport in cultured rabbit corneal endothelium was examined using a Cl(-)-sensitive fluorescent dye. The intracellular Cl- concentration ([Cl-]i) in the endothelial cells was estimated to be about 30 mM. Noradrenaline (0.001-0.1 mM) transiently decreased the [Cl-]i in a dose-dependent manner. Such a decrease in [Cl-]i was completely antagonized by pretreatment with the alpha-adrenoceptor antagonist phentolamine (0.1 mM). The selective alpha 2-adrenoceptor agonist UK 14304-18 (5-bromo-6-[(4H,5H-imidazol-2-yl)amino]quinoxaline, 0.1 mM) persistently decreased the [Cl-]i, but neither the alpha 1-adrenoceptor agonist phenylephrine (0.1 mM) nor the beta-adrenoceptor agonist isoproterenol (0.1 mM) had any effect. The alpha 2-adrenoceptor agonist/antagonist yohimbine (0.1 mM) persistently and more strongly decreased the [Cl-]i than UK 14304-18 did. The yohimbine-induced decrease in the [Cl-]i was not further altered by UK 14304-18 or phenylephrine, but partly reversed by noradrenaline, isoproterenol and an adenylate cyclase activator, forskolin (0.1 mM). The yohimbine-induced decrease in [Cl-]i was inhibited by the carbonic anhydrase inhibitor acetazolamide (1 mM), and Cl-/HCO3- exchange inhibitors, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, but not by the H(+)-ATPase inhibitor N,N'-dicylohexylcarbodiimide. The forskolin-induced recovery in [Cl-]i was inhibited by the Na+/K+/Cl- cotransport inhibitor bumetanide (0.1 mM), but not by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

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

PubMed1994

Electrogenic and phosphatidylinositol-4-monophosphate-stimulated Cl- transport by Cl- pump in the rat brain.

Rat brain Cl(-)-ATPase was solubilized and reconstituted in asolectin liposomes. Phosphatidylinositol-4-monophosphate increased Cl(-)-ATPase and ATP-dependent Cl- uptake activities in proteoliposomes. The ATP-dependent Cl- uptake was inhibited by a Cl(-)-ATPase inhibitor, ethacrynic acid, and increased at an inside-positive membrane potential or in the presence of a protonophore. These findings suggest that Cl(-)-ATPase is an electrogenic Cl- transporter, or a primary Cl- pump, probably regulated by phosphoinositide turnover in vivo.

Adenosine Triphosphatases↗

PubMed1992

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 alpha 1-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 alpha 1-agonist and a phorbol ester.

Adrenergic alpha-Agonists↗

PubMed1991

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.

Adenosine Triphosphatases↗

PubMed1990

Ethacrynic acid-induced convulsions and brain noradrenaline in mice.

The intracerebroventricular injection of ethacrynic acid (a 50% convulsive dose; 50 micrograms/mouse) accelerated brain noradrenaline turnover and decreased noradrenaline contents. The decrease in noradrenaline contents was antagonized by 2-amino-5-phosphonovalerate but not by diazepam. Both 2-amino-5-phosphonovalerate and diazepam suppressed the incidence of ethacrynic acid-induced convulsions while reserpine, alpha-methyl-para-tyrosine or FLA-63 augmented it. The results suggest that stimulation by ethacrynic acid of excitatory amino acid neurons enhances-noradrenergic neuronal anticonvulsive activity.

Animals↗