Mitsuyoshi Hara

Uneven distribution of intracellular Cl− in rat hippocampal neurons

Electrophysiological observations of neurons suggest that perikarya and dendrites differ in local intracellular Cl- concentration ([Cl-]i), that has not been demonstrated yet. Regional [Cl-]i in cultured hippocampal neurons was estimated using a Cl(-)-sensitive fluorescent dye. Calibration showed that perikaryonic [Cl-]i was lower than dendritic [Cl-]i. Ethacrynic acid, an inhibitor of the outwardly directed Cl(-)-pump, increased the perikaryonic but not dendritic [Cl-]i. A decrease in [Cl-]i induced by furosemide or bumetanide, inhibitors of Na+/K+/2Cl- cotransporters, was more prominent in dendrites than in perikarya. These findings suggest that uneven distribution of Cl- is generated by the region-specific localization of these transporters.

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.

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.

Atrial natriuretic peptide stimulates Cl- transport in retinal pigment epithelial cells

To study the regulatory role of atrial natriuretic peptide (ANP) on the Cl- transport activity of retinal pigment epithelial (RPE) cells, RPE cells from rabbits were cultured and exposed to ANP and other reagents under perfusion. The changes in intracellular Cl- concentration ([Cl-]i) were continuously recorded using a Cl(-)-sensitive fluorescent dye. The cGMP content was estimated by radioimmunoassay. ANP increased the cGMP content and the [Cl-]i in RPE cells. A guanylate cyclase activator, nitric oxide, and a cell permeable cGMP precursor, 8-Br-cGMP, also increased the level of cGMP and the [Cl-]i. A guanylate cyclase inhibitor, LY83583, an inhibitor of cGMP-dependent protein kinase, KT5823, and an inhibitor of Na+/K+/2Cl- cotransporter, bumetanide, diminished or abolished the ANP-induced increase in [Cl-]i. ANP facilitates Cl- accumulation in RPE cells, which is mediated by guanylate cyclase, cGMP-dependent protein kinase, and the Na+/K+/2Cl- cotransporter.

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.

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.

Effects of protein kinase and phosphatase inhibitors on slow shortening of guinea pig cochlear outer hair cells.

The intracellular mechanisms of slow shortening in isolated guinea pig cochlear outer hair cells were investigated using inhibitors and/or an activator of protein kinases and protein phosphatases. The slow shortening was induced by tetanic electrical field stimulation, and changes in the cell length, volume and intracellular Cl- concentration were microscopically monitored using a chloride-sensitive fluorescent dye. The slow shortening was inhibited by a calmodulin inhibitor, W-7, and a calcium calmodulin-dependent protein kinase II (CaMKII) inhibitor, KN-62. The inhibition by W-7 or KN-62, was abolished by the supplemented conductance of K+ with valinomycin. Among the protein phosphatase inhibitors tested, a type 1 and 2A protein phosphatase inhibitor, calyculin A, inhibited the slow shortening. The inhibition by calyculin A was abolished by the increased Cl- permeability, but neither by the increased K+ conductance with valinomycin nor by the increased Ca2+ conductance with A23187. A protein serine/threonine phosphatase activator, N-acetylsphingosine, inhibited the shortening, which was abolished by either valinomycin or a type 2A protein phosphatase inhibitor, okadaic acid, but not by calyculin A. These findings suggest the following signaling mechanisms in the slow shortening of outer hair cells; the K+ channel opening is facilitated through protein phosphorylation by CaMKII and suppressed via okadaic acid-sensitive dephosphorylation, and the Cl- channel opening depends on calyculin A-sensitive protein phosphatase activity.

Transporting Cl−-ATPase in Rat Brain

Intracellular Cl− concentration ([Cl−]i) is an essential factor in the regulation of neuronal excitability through receptor-operated Cl− channels (Alger 1985; Barker 1985). Evidence for chloride activities higher or lower than those expected from a passive distribution (Alvarez-Leefmans 1990) has suggested the presence of inwardly or outwardly directed active Cl− transport mechanisms. In neurons, uphill accumulation of Cl− is known to be mediated by Na+/K+/Cl− co-transporters in both invertebrate and vertebrate neurons (Russell 1983; Ballanyi and Grafe 1985; Alvarez-Leefmans et al. 1988). However, the mechanisms underlying the outwardly directed Cl− transport have not been fully elucidated. Thompson et al. (1988a, b) and Thompson and Gahwiler (1989a, b) have suggested that active Cl− extrusion from mammalian cortical neurons is mediated by outwardly directed Cl−/cation co-transport mechanism. On the other hand, neuronal Cl−-stimulated ATPase acting as an ATP-driven Cl− pump has also been a candidate for such an outwardly directed Cl− transport system (Inagaki et al. 1985, 1987; Inagaki and Shiroya 1988; Shiroya et al. 1989b; Inoue et al. 1991a; Hara et al. 1992). We report in this chapter on the Cl− stimulated ATPase which outwardly transports Cl− across neuronal plasma membranes.