Xun-Ting Zeng

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.

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.

Amyloid β 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 Alzheimers disease (AD), amyloid β proteins (Aβs), on the Cl−‐ATPase activity were examined using primary cultured rat hippocampal neurons. Amyloid β 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), Aβ25–35 being the most potent. Addition of inositol or 8‐Br‐cyclic GMP completely reversed these Aβ‐induced changes. The recoveries in enzyme activity were attenuated by an inhibitor of PI 4‐kinase, 10 µm wortmannin or 20 µm quercetin, but not by a PI 3‐kinase inhibitor, 50 nm wortmannin or 10 µm LY294002. The PI, PIP and PIP2 levels of the plasma membrane‐rich fraction were lower in the Aβ‐treated cells as compared with each control. In the Aβ‐exposed culture, but not in control, stimulation by 10 µm 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 Aβ‐treatment on the neurotoxicity of glutamate. Thus, Aβs 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.

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.

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.

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.

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.

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 Alzheimers 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.

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.

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.