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Biomedical subjects

C Inagaki

Publications and source records attributed to C Inagaki.

At least 55 records · Page 3Linked to original sources
PubMed1995

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.

Aminoquinolines↗
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↗
PubMed1994

A cytosolic peptide potentiates the GTP effect on beta-adrenergic response of adenylate cyclase.

A cytosolic peptide-GTP complex that stimulates l-isoproterenol-responsive adenylate cyclase activity was identified in the rat liver. The peptide component was purified and characterized with regard to its interaction with GTP. The peptide was isolated as a complex form with GTP on a Sephadex G-25 column in 1 mM NaHCO3, and was purified as a dissociated form, with relative molecular weight (M(r)) approximately 3,000 and GTP-binding ability, in 200 mM ammonium acetate. The purified peptide alone displayed little stimulatory effect on adenylate cyclase activity, but its reassociated form with GTP clearly enhanced the effect of GTP on the enzyme activity. The isoproterenol competition curve using l-[3H]dihydroalprenolol as an antagonist ligand shifted to lower affinity by the addition of the peptide reassociated with GTP (16.5-fold shift), whereas the same concentration of GTP (1 microM) or the peptide alone had little or no effect (1.5- or 0.9-fold shift, respectively). Furthermore, the peptide enhanced the GTP effect in response to l-isoproterenol but not to glucagon, prostaglandin E1, or fluoride. These results suggest that the cytosolic peptide potentiates the effect of GTP on the agonist-beta-adrenergic receptor-stimulatory guanine nucleotide-binding regulatory component of the adenylate cyclase ternary complex.

Adenylyl Cyclases↗
PubMed1994

Purification and characterization of neurotrophic factor for retinal cholinergic neurons derived from cultured hippocampal neurons.

A neurotrophic factor that supports the development of cholinergic retinal neurons was purified from media conditioned by a primary culture of embryonic hippocampal neurons. Retinal choline acetyltransferase (ChAT), which is located exclusively in amacrine cells, served as a marker for the development of retinal cholinergic neurons. In a serum-free control culture, retinal neurons from 17-day-old rat fetus displayed little increase in the enzyme activity and a low proportion of neurite-bearing cells (15-20%) within 7 days. The conditioned media, when added to the retinal neuron culture, dose-dependently increased ChAT activity and the number of neurite-bearing cells (40-60%), the maximum ChAT activity being approximately sixfold higher than that in the control. The fraction with these stimulatory activities was purified by Sephadex G-15 column chromatography and two times reverse-phase HPLC. The final fraction showed approximately 3,000-fold higher purification as compared with that in the Sephadex G-15 fraction. Gas-phase protein sequencing analysis of the final fraction yielded a peptide sequence: Tyr-Leu-Leu-Pro-Ala-Gln-Val-Asn-Ile-Asp. A synthetic peptide with this sequence dose-dependently stimulated ChAT activity in the retinal cell culture and dissociated cell culture of the septal nucleus. These findings suggest that the developing hippocampal neurons produce a neurotrophic peptide that stimulates the development of cholinergic neurons.

Amino Acid Sequence↗
PubMed1993

Regulation of Cl- conductance in delayed shortening and shrinkage of outer hair cells.

Tetanic electrical field stimulation induces slow and delayed shortening of isolated outer hair cells (OHCs) from guinea pigs concomitant with a decrease in cell volume and intracellular Cl- concentration ([Cl-]i). These responses are dependent on the entry of Ca2+ and the activation of both Ca(2+)-activated K+ channels and furosemide-sensitive Cl- channels. In this study, the regulatory mechanisms of the Cl- channels of OHCs were investigated. Tetanic stimulation induced shrinkage and a decrease in [Cl-]i, the latter being unrelated to the activity of the Na+/K+/2Cl- cotransporter. These responses were inhibited by tetraethylammonium, and the inhibition was overcome by an increase in the K+ conductance by valinomycin, though valinomycin alone did not affect the resting cell volume and [Cl-]i. The deprivation of extracellular Ca2+ inhibited the stimulation-induced changes in volume and [Cl-]i even in the presence of valinomycin. These results suggest that electrical stimulation-induced delayed shortening and shrinkage of OHCs are induced by Cl- efflux, which is electrically coupled to K+ efflux and independently activated following an increase in intracellular Ca2+ concentration.

Animals↗
PubMed1993

[Chloride channels in cardiac muscle].

This section describes properties, signal transduction pathways for activation and the physiological roles of cardiac Cl- channels. Recent application of patch clamp technics have revealed that a wide variety of Cl- channels are present in cardiac cells, which are regulated by different cellular stimuli, such as alpha 1- or beta-adrenoceptor stimulation, ATP, adenosine and cell swelling. When these channels are activated, Cl- is transported ECl-dependently in both inward and outward directions, affecting the action potential durations and/or resting potentials. Analysis of cardiac Cl- channel mechanisms yields new aspects for understanding pathophysiological bases of heart diseases or arrests such as arrhythmia.

Chlorides↗
PubMed1992

Ethacrynic acid-induced convulsions and brain neurotransmitters in mice.

Intracerebroventricular injection of ethacrynic acid (50% convulsive dose; 50 micrograms/mouse) accelerated the synthesis/turnover of 5-hydroxytryptamine (5-HT) but suppressed the synthesis of gamma-aminobutyric acid and acetylcholine in mouse brain. These effects were completely antagonized by pretreatment with a glutamate/N-methyl-D-aspartate antagonist, aminophosphonovaleric acid. In ethacrynic acid-induced convulsions, these neurotransmitter systems may be differentially modulated, probably through activation of glutaminergic neurons in the brain.

2-Amino-5-phosphonovalerate↗
PubMed1992

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.

Animals↗
PubMed1992

Enflurane-induced release of an excitatory amino acid, glutamate, from mouse brain synaptosomes.

To clarify the mechanisms of enflurane-induced convulsions, we examined the effects of enflurane, halothane, and diethyl ether on the release of an excitatory neurotransmitter, glutamate, from isolated pinched-off nerve terminals (synaptosomes) of the mouse cerebral cortex. At concentrations corresponding to those used clinically (0.75 and 1.25 mM), enflurane released more glutamate than did halothane. Diethyl ether (10 and 58 mM) had no effect on glutamate release. Enflurane (0.75-15 mM) increased glutamate and aspartate release in a dose-dependent manner but had little effect on the release of the inhibitory neurotransmitters glycine and gamma-aminobutyric acid or on the release of glutamine. A glutamate uptake inhibitor, kainic acid (1 mM), did not affect enflurane-induced glutamate release. Replacement of the medium's Ca2+ by Co2+, or exposure to cold (about 2 degrees C), suppressed the enflurane-induced glutamate release. Depolarization caused by 40 mM K+ increased the basal level of glutamate released, and enflurane-induced glutamate release was lower after depolarization. Enflurane had no effect in synaptosomes prepared from the cerebellum, diencephalon and pons, or medulla oblongata. Thus, enflurane increased Ca(2+)- and temperature-dependent glutamate release, especially from synaptosomes of the cerebral cortex. These data provide a pathophysiologic explanation for enflurane-induced convulsions.

Animals↗
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↗
PubMed1992

Delayed shortening and shrinkage of cochlear outer hair cells.

Slow shortening of cochlear outer hair cells has been speculated to modify cochlear sensitivity. Tetanic electrical field stimulation of isolated outer hair cells from guinea pigs shortened the cells for 2-3 min. Electrical stimulation reduced cell length and volume (-13.5 +/- 1.5 and -37.3 +/- 3.0% of initial values, respectively, n = 16) and decreased the intracellular Cl- concentration. Cytochalasin B (100 microM) inhibited electrical stimulation-induced shortening but not volume reduction. The following chemicals or manipulations inhibited the responses: 10 microM furosemide, 0.1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), 1 mM anthracene-9-carboxylic acid (AC9), 25 mM tetraethylammonium, 2.3 microM charybdotoxin (ChTX), 250 nM omega-conotoxin, and Ca(2+)-free medium. These findings suggest that both electrical stimulation-induced shortening and shrinkage of outer hair cells result not only from an actin-mediated contractile force, but also from Cl- efflux through furosemide-, DIDS-, and AC9-sensitive Cl- channels, and K+ efflux through ChTX-sensitive K+ channels.

Actins↗
PubMed1992

[Neuronal intracellular chloride ion concentrations and their regulatory mechanisms].

Intracellular chloride ion concentration ([Cl-]i) plays an important role in cellular functions including the control of membrane potential and excitability. In neurons, Cl- equilibrium potential (ECl) is lower or higher than the membrane potential (Em), suggesting that [Cl-]i is lower or higher than that expected from passive distribution. As the mechanisms to control [Cl-]i, active outwardly or inwardly directed Cl- transport systems have been reported. The former includes Na(+)-dependent Cl-/HCO3- exchanger, K+/Cl- cotransporter and ATP-dependent Cl- pump; and the latter includes Na+/K+/2Cl- cotransporter and amino acid-dependent Na+/Cl- cotransporter. In hippocampal pyramidal cells, recent studies using a Cl(-)-sensitive fluorescent probe to monitor [Cl-]i revealed the presence of an ATP-dependent Cl- pump and a Na+/K+2Cl- cotransporter, and an uneven distribution of [Cl-]i (cell body less than dendrite) and these Cl- transport systems. Intracerebroventricular administration of an inhibitor of the ATP-dependent Cl- pump, ethacrynic acid, induces status epilepticus in mice. Thus, it appears to be necessary to elucidate cellular and molecular mechanisms of Cl- transporters and their control systems for a better understanding of Cl(-)-related functions in neurons.

Animals↗
PubMed1992

Comparison of converting enzyme inhibitor and calcium channel blocker in SHR with nephrotoxic serum nephritis.

In order to compare the protective effects of angiotensin converting enzyme inhibitors (ACEI) and calcium channel blockers (CCB) on the renal function in experimental nephritis, nephrotoxic serum nephritis was induced in male spontaneously hypertensive rats (SHR). The above drugs were then chronically administered to different groups, as follows: the ACEI-treated group (n = 7) received captopril (150 mg/kg/day), and the CCB-treated group (n = 6) was given both nifedipine (40 mg/kg/day) and nisoldipine (20 mg/kg/day). The control group (n = 8) received a placebo. Although the control group developed marked hypertension and proteinuria, the rats treated with either ACEI or CCB demonstrated a significant and equivalent decrease in mean arterial pressure and urinary protein excretion. At 15 weeks after the injection of nephrotoxic serum, all rats were anesthetized with Inactin, and the glomerular filtration rate (GFR) and renal plasma flow (RPF) were measured. In the control group, GFR and RPF were markedly attenuated. However, both were preserved at much higher levels in the ACEI-treated group, and GFR was also maintained to a similar degree in the CCB-treated group. Histological studies were carried out after the clearance studies. As a result, it was found that the ACEI treatment significantly limited the development of glomerulosclerosis, whereas CCB modestly ameliorated the glomerular structural lesions. Moreover, ACEI significantly reduced the serum cholesterol, while CCB did not exert such an effect. These results suggest that both ACEI and CCB have a therapeutic effect in experimental glomerulonephritis models which are accompanied by hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
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↗
PubMed1991

Ethacrynic acid-induced glutamate release from mouse brain synaptosomes.

A loop diuretic, ethacrynic acid (0.3 mM), released glutamate from mouse brain synaptosomes as potently as 40 mM K+, and was more potent than furosemide and bumetanide. Ethacrynic acid-induced glutamate release was suppressed by depletion of Ca2+ or Cl- from the incubation medium. The findings suggest that ethacrynic acid enhances glutamate release through Cl(-)-related depolarization of nerve endings.

4-Aminopyridine↗
PubMed1991

Two types of clathrin-coated vesicles isolated from rat brain: analysis of biochemical properties and cellular origin.

Two major fractions rich in clathrin-coated vesicles (CVs) (fraction I, rho = 1.140 g/cm3; fraction II, rho = 1.113 g/cm3) were separated from rat brain using a sucrose gradient and compared for their cellular origins and Cl- translocation systems. Electron micrographs showed that both fractions contained CVs of different size distributions (fraction I, 85 +/- 9.5 nm in diameter; fraction II, 72 +/- 6.8 nm in diameter). Fraction II contained potent ouabain-sensitive ATPase activity, whereas fraction I contained only a little activity. Immunoblot analysis for the Na+,K(+)-ATPase catalytic subunit, alpha and alpha(+), demonstrated that fraction II exhibited predominantly alpha(+), whose proportion to alpha was analogous to that observed in the extracts of primary cultured neuronal cells. Furthermore, on a sucrose density gradient, cultured neuronal cells yielded fraction II but not fraction I, whereas primary cultured glial cells yielded fraction I but not fraction II. Labeling-chase experiments using 125I-transferrin in cultured neuronal cells showed the internalized ligand in fraction II and the surface-bound ligand in the fraction with lower density (rho = 1.090 g/cm3), a result suggesting that the involvement of Na+,K(+)-ATPase in fraction II is attributable to endocytic vesicles. Cl- uptake in fraction II was approximately threefold higher than that in fraction I. N-Ethylmaleimide (100 microM) completely inhibited the Cl- uptake in fraction I but partially (approximately 50%) inhibited that in fraction II. These findings suggest that the two CV fractions isolated from rat brain originate from different cell types--glial and neuronal cells--and differ in size distribution of CVs, content of Na+,K(+)-ATPase, and mechanism for Cl- uptake.

Adenosine Triphosphate↗
PubMed1991

Ethacrynic acid-sensitive and ATP-dependent Cl- transport in the rat kidney.

Ouabain- and furosemide-insensitive and ATP-dependent Cl- uptake was demonstrated in rat renal membrane vesicles. Such a Cl- uptake activity was prominent in cortical plasma membrane fractions with high activities of Cl(-)-ATPase and Na+, K(+)-ATPase. The membrane vesicles accumulated Cl- in an osmotically reactive manner with the following sequence of nucleotide specificity: ATP greater than ITP greater than UTP greater than GTP greater than CTP., beta, gamma-Methylene ATP, ADP and AMP had no effect. ATP-dependent Cl- uptake was markedly inhibited by a Cl(-)-ATPase inhibitor, ethacrynic acid (0.3 mM), but not affected by an H(+)-ATPase inhibitor, N,N'-dicyclohexylcarbodiimide (0.1 mM). These findings suggest that an ethacrynic acid-sensitive and ATP-driven

Adenosine Triphosphatases↗