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R Shingai

Publications and source records attributed to R Shingai.

At least 19 recordsLinked to original sources
PubMed2001

Adrenocorticotropic hormone activates an outward current in cultured mouse peritoneal macrophages.

To define the effects of adrenocorticotropic hormone (ACTH) in immunocompetent cells, ion channel activities in cultured mouse peritoneal macrophages were analyzed by the perforated patch-clamp method. ACTH induced outward currents at smaller holding potentials than K+ equilibrium potentials. Reversal potentials of ACTH-induced currents were dependent on external K+ concentrations, but not on external Cl- concentration. Quinine potently blocked the outward current and tetraethylammonium (TEA) partially suppressed that current. ACTH did not induce the response in Ca2+ free solution containing EGTA. These results suggest that ACTH can modulate macrophage functions through the activation of Ca2+ dependent K+ channels.

Adrenocorticotropic Hormone↗
PubMed2001

Calcium inhibits willardiine-induced responses in kainate receptor GluR6(Q)/KA-2.

A number of studies have demonstrated that willardiine [(S)-1-(2-amino-2-carboxyethyl) pyrimidine-2,4-dione] is a useful agonist for the activation of AMPA/kainate receptors. Here we examine the effect of extracellular calcium on currents evoked by willardiine in HEK 293 cells expressing the GluR6(Q)/KA-2 kainate receptor subunits. At a concentration of 1.8 mM, Ca2+ inhibited the currents induced by 100 microM willardiine by approximately 50%. When extracellular Na+ ions were replaced with Ca2+ ions there were no measurable inward currents. We conclude that Ca2+ inhibition of the willardiine-induced response is concentration dependent.

Alanine↗
PubMed2001

Kinetic analyses of alcohol-induced potentiation of the response of GABA(A) receptors composed of alpha(1) and beta(1) subunits.

To investigate the kinetics of both the potentiation and desensitization of the response of ionotropic GABA receptors (GABA(A) receptors) in the presence of various compounds, we expressed receptors composed of alpha(1) and beta(1) subunits by injecting cells with the cRNAs synthesized from cloned bovine GABA(A) receptor cDNAs and measured the electrical responses of the cells electrophysiologically with or without the compounds. The potentiation of the GABA(A) receptor-mediated response was quantitatively analyzed using a simple model with the assumption that the receptors have two identical binding sites for GABA molecules with a dissociation constant of K(1), and one potentiation site for the compound with a dissociation constant of K(p), and that the binding of the compound to the potentiation site only increases the affinity of the GABA binding sites, changing K(1) to K(1p). The estimated K(p) and K(1p) were dependent on the functional groups and the chain length of the compounds. These results could be satisfactorily analyzed using this simple model. The potentiation of the GABA(A) receptor-mediated response by the components of essential oils used for aromatherapy was also examined. These compounds accelerated the decay of the response, possibly due to desensitization of the receptors, which was also analyzed on the basis of the model.

Alcohols↗
PubMed2001

Effects of bisphenol A and its derivatives on the response of GABA(A) receptors expressed in Xenopus oocytes.

To study the effects of bisphenol-A (BPA) known to have estrogenic actions, and its derivatives, 3,5-dimethylphenol (DMP) and p-t-butylphenol (TBP), on ionotropic gamma-aminobutyric acid (GABA) receptors, GABA(A) receptors were expressed in Xenopus oocytes by injecting both poly(A)+ RNA prepared from rat whole brain and cRNAs synthesized from cloned cDNAs of alpha1 and beta1 subunit of the bovine receptors, and their electrical responses were measured by the voltage clamping method. BPA caused the potentiation and inhibition of the former receptor-responses, while it caused only inhibition of the latter ones. In the presence of low concentrations of GABA, DMP and TBP potentiated the responses of both receptors. DMP and TBP also increased the rate of decay of the response, possibly by desensitization of the receptors when GABA solution was continuously bath-applied. Diethyl terephthalate (DTP), which is also known to have estrogenic actions, had little effect on both the responses and the decay of both receptors.

Animals↗
PubMed2000

Durations and frequencies of free locomotion in wild type and GABAergic mutants of Caenorhabditis elegans.

We investigated how much time wild-type Caenorhabditis elegans (Bristol N2) nematodes and the GABA-deficient unc25 mutant and the vesicular GABA transporter-deficient unc47 mutant spent moving. The worms were allowed to move freely on the surface of agarose plates either with or without the food bacterium OP50. We identified forward movement, backward movement, resting and turns by watching images on video and computer displays. Forward movement lasted longer and rests were briefer without, than with, bacteria. Frequency distributions except for backward movement fitted a sum of two exponential functions. The duration of backward movement was not strongly influenced by exposure to bacteria, whereas the frequency of backward movements increased in their presence. The duration of forward movement of unc25 nematodes had no long component, thus differing from that of N2 and unc47 strain nematodes in treatments with and without bacteria. The durations of resting in these mutants were much longer than in the N2 strain, especially in the absence of bacteria. The turn frequency of unc47 nematodes had a higher short component than that of the wild type N2 and unc25 nematodes, in the absence of bacteria. A neural network model is discussed in conjunction with the features of mutants and current knowledge of GABAergic neural transmission.

Animals↗
PubMed1999

Modulation by L- and D-isoforms of amino acids of the L-glutamate response of N-methyl-D-aspartate receptors.

N-Methyl-D-aspartate (NMDA) receptor subtypes epsilon 1 and zeta 1 were coexpressed in Xenopus oocytes for the investigation of the magnitude of augmentation of the L-glutamate response by 20 common L-amino acids and their 19 D-isoforms. Simultaneous application of L- and D-alanine, -cysteine, and -serine, or glycine and L-glutamate potentiated the glutamate-induced current. Other amino acids produced only marginal effects. Analysis of the relationship between the response and amino acid size revealed that the critical threshold size is between those of cysteine and aspartate. No amino acid alone induced a current. The effects of L- and D-alanine, -cysteine, and -serine applied with L-glutamate were concentration-dependent. Molecular modeling of these three amino acids revealed a positive relationship between the charge at an atom of the side chain and the receptor sensitivity, which may explain the efficacies of these amino acids.

Alanine↗
PubMed1999

Localization of GABA receptor rho 2 and rho 3 subunits in rat brain and functional expression of homooligomeric rho 3 receptors and heterooligomeric rho 2 rho 3 receptors.

Ionotropic GABA receptors that are composed of rho subunits act to gate bicuculline-insensitive Cl- currents. Reverse transcription-polymerase chain reaction analysis revealed that the expression of rho 2 mRNA in adult rat brain was approximately eight times higher than mRNA in the rat brain at embryonic day 16, while that of rho 3 in the embryonic brain was approximately six times higher than in the adult brain. In the adult rat brain the rho 3 mRNA was present in the mesencephalon, hippocampus, cerebellum, thalamus and basal ganglia. In situ hybridization has been used to demonstrate the presence of rho 2 mRNA in the hippocampal CA1 region of the 8-day-old rat, and in the CAl region of the hippocampus, lateral geniculate nucleus, superficial gray layer of the superior colliculus and the pars compacta of the substantia nigra of the adult rat. When the homooligomeric rho 3 receptors were expressed in Xenopus oocytes, applications of agonists induced ionic currents. The order of potency of the agonists was muscimol > GABA = trans-4-amino-crotonic acid > cis-4-aminocrotonic acid. The ionic currents induced by GABA were blocked by picrotoxinin and Zn2+ in dose-dependent manner. In heterooligomeric rho 2 rho 3 receptors, picrotoxinin sensitivity was significantly reduced.

Amino Acid Sequence↗
PubMed1996

Expression of taste reception response of fleshfly in Xenopus oocytes.

Functional expression of the gustatory sensitivity to amino acids and sugars was investigated in Xenopus oocytes injected with poly(A+) RNA extracted from fleshfly labellar taste organs. The current induced by the application of amino acids and sugars that stimulate fleshfly sugar receptor cells was recorded under voltage clamp conditions. L-Phenylalanine and L-valine induced a large transient inward current in poly(A+) RNA-injected oocytes but only small responses in uninjected control oocytes. Glucose and fructose also produced inward currents in the RNA-injected oocytes. N-methylated compounds or D-isomers of these amino acids induced either no response or a much smaller response than L-amino acids as they did in the fleshfly sugar receptor cells. The oocyte expression system is a useful tool for characterizing the taste transduction mechanism.

Animals↗
PubMed1996

Cloning of a putative gamma-aminobutyric acid (GABA) receptor subunit rho 3 cDNA.

Cloned cDNA encoding a putative member of GABA receptor rho-subunit class was isolated from rat-retina-mRNA-derived libraries. The cDNA encodes a signal peptide of 21 amino acids followed by the mature rho 3 subunit sequence of 443 amino acids. The proposed amino acid sequence exhibits 63 and 61% homology to the previously-reported human rho 1 and rat rho 2 sequences, respectively. Northern blot analysis demonstrated the expression of mRNA for rho 3 subunit in retina.

Amino Acid Sequence↗
PubMed1996

Functional expression of GABA rho 3 receptors in Xenopus oocytes.

Homomeric rat GABA rho 3 receptors were expressed in Xenopus oocytes, and their pharmacological profile was investigated electrophysiologically. GABA activated the rho 3 receptors with an EC50 value of 7.5 microM and a Hill coefficient of 1.6. The GABA-induced current was not antagonized by bicuculline (100 microM), but was blocked by picrotoxin (IC50: 0.68 microM for 100 microM GABA). The current was almost insensitive to pentobarbital, diazepam and a neurosteroid, 3 alpha-OH-DHP. Many of the pharmacological properties of the rho 3 subunit were similar to those of the previously reported rat rho 1 and rho 2 subunits and GABAC receptors.

Animals↗
PubMed1996

A phenomenological model describing pharyngeal pulsing in the nematode Caenorhabditis elegans anesthetized by alcohol.

The recovery process of the suppressed pharyngeal pulsation in the nematode has been investigated for several concentrations of a homologous primary alcohol series (CnH2n-1OH, n = 1,2,3). A mathematical model describing the time course of the recovery process is phenomenologically constructed by using two time constants of delay time tD and recovery time tau. The values of tD and tau are obtained by fitting the equation to experimental data. The obtained values increase with increasing alcohol concentration. To observe the characteristics of tD and tau against the alcohol of order n, the inverse of these time constants are computed at 25 v/v% concentration and plotted on a semi-logarithmic scale. The plot curves decrease non-linearly and are dissimilar to the well-known curves illustrating the importance of lipid solubility in the cell membrane in anesthetic phenomena.

Alcohols↗
PubMed1995

Autoantibody against 70 kD heat shock protein in patients with autoimmune liver diseases.

BACKGROUND/AIMS: It has recently been suggested that heat shock proteins are implicated in the pathogenesis and teh pathophysiology of various immunological disorders, and the presence of antibodies against heat shock proteins has been reported in several autoimmune diseases. METHODS: We investigated autoantibodies against the two major human heat shock proteins (hsp70 and hsp90) in sera from patients with primary biliary cirrhosis and autoimmune hepatitis, the two major autoimmune liver disease. Reactivity with human heat shock proteins obtained from phytohemagglutinin stimulated cells was investigated by immunoblots with sera at 1:20 dilution. RESULTS: Reactivity with human hsp90 was not found in any sera from patients or normal controls. In contrast, reactivity with human hsp70 was found in 16 of 35 (45.7%) primary biliary cirrhosis patients and in 9 of 17 (52.9%) autoimmune hepatitis patients, but similar reactivity was found in only 2 of 15 patients with chronic hepatitis B and 1 of 13 patients with chronic hepatitis C. All the normal controls showed a negative reaction. Two-dimensional immunoblots and immunoabsorption experiments established that the autoantibody recognized only human hsc70 (73 kD/pI 5.5), a constitutive form of the hsp70 family. CONCLUSIONS: Although the pathological significance of the autoantibody against hsc70 in these autoimmune liver diseases remains unknown, the serum autoantibody detected in primary biliary cirrhosis patients is closely related to clinical variables including serum total bilirubin, alanine aminotransferase, IgG, IgM, titers of antimitochondrial antibodies, and major symptoms (pruritus and/or icterus). These observations may suggest that the anti-hsc70 antibody is an indicator for the disease activity of primary biliary cirrhosis.

Aged↗
PubMed1995

Identification of GABAA receptor subunits in rat retina: cloning of the rat GABAA receptor rho 2-subunit cDNA.

We identified GABAA receptor subunits in rat retina using PCR. The high degree of conservation among previously described members of ligand-gated anion channels in transmembrane domains was used to design degenerate sense and antisense oligonucleotides. These oligonucleotides were used as primers for PCR, which was applied to the rat retina cDNA. Analysis of clones derived from the PCR amplification identified the GABAA alpha 1, beta 1, beta 3, and gamma 2 subunits and the glycine alpha 1 subunit. In addition, two clones closely related to the human GABAA rho-subunit class were obtained. Molecular cloning revealed one of them as the rat counterpart of the human rho 2 subunit. Northern blot analysis demonstrated the expression of mRNAs for rho subunits in retina. These results further support the hypothesis that bicuculline-insensitive GABA channels in rat retina are comprised of rho subunits.

Amino Acid Sequence↗
PubMed1994

Modulation of ionic currents through GABAA receptor subtypes by endogenous steroids.

GABAA receptors were expressed in Xenopus oocytes by injecting messenger RNAs from chick retina and chick cortex, and the potency of 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-OH-DHP) and 5 alpha-pregnane-3 alpha, 21-diol-20-one (THDOC) was investigated by electrophysiology under voltage clamp conditions. For the receptors of chick retina, 3 alpha-OH-DHP (100 nM) and THDOC (100 nM) augmented GABA actions (peak current) 1.6 times (n = 6) and 1.5 times (n = 7), respectively. The currents induced by 0.3-10 microM GABA for the receptors of the chick retina were sensitive to bicuculline, pentobarbital, and diazepam. GABAA receptors in the chick retina in vivo as well as the receptors in the brain may be functionally modulated by endogenous steroids.

Animals↗
PubMed1993

Relationship between resting cytosolic Ca2+ and responses induced by N-methyl-D-aspartate in hippocampal neurons.

Cytosolic calcium concentrations ([Ca2+]i) in cultured hippocampal neurons from rat embryos were measured using fura-2. Neurons with higher resting [Ca2+]i showed greater [Ca2+]i responses to N-methyl-D-aspartate (NMDA) and K+ depolarization. There was a strong relationship between resting [Ca2+]i and the maximal changes in [Ca2+]i (delta[Ca2+]i), which fit the our proposed equation to describe this relationship.

Animals↗
PubMed1992

Effects of subunit types of the recombinant GABAA receptor on the response to a neurosteroid.

When vertebrate brain poly(A)+ RNA is expressed in Xenopus oocytes the response of the GABA receptors formed is found to be inhibited allosterically by a neurosteroid, pregnenolone sulphate (PS). This negative modulation was reproduced after expressing RNAs encoding bovine GABAA receptor subunits in the combinations alpha i + beta 1, or alpha i + beta 1 + gamma 2 (where i = 1, 2 or 3). The characteristics of this inhibition vary significantly with the type of the alpha subunit (alpha 1, alpha 2, or alpha 3) used. When the bovine gamma 2L alternate form of the gamma 2 subunit was replaced by the human gamma 2S subunit, the behaviour was unchanged: the human gamma 2S subunit used is a newly-cloned form, which encodes a polypeptide with two amino acid differences from the human gamma 2 subunit previously described. The results of co-application of PS and 3 alpha-hydroxy-5 alpha-pregnan-ol-20-one, a neurosteroid which is a positive modulator of the GABAA receptor, indicate that these act at different sites on the receptor. PS also increases the desensitisation of the receptor by GABA. This effect, also, is alpha-subunit-type dependent and occurs by an acceleration of the fast phase of desensitisation.

Animals↗
PubMed1992

N-methyl-D-aspartate increases cytosolic Ca2+ via G proteins in cultured hippocampal neurons.

The changes of the cytosolic Ca2+ concentrations ([Ca2+]i) induced by N-methyl-D-aspartate (NMDA) in fura-2-loaded cultured hippocampal neurons from rat embryos were investigated by the fast application method, using a fine pipe under extracellular Mg(2+)-free conditions. In the presence of Ca2+, NMDA, at concentrations in excess of 3 microM, induced a biphasic increase of [Ca2+]i, which consisted of an initial increase with a second rise that occurred after cessation of drug application. Under Ca(2+)-free conditions, NMDA (greater than 100 microM) in the absence of glycine or NMDA (greater than 50 microM) in the presence of glycine (greater than 10 microM) induced intracellular Ca2+ mobilization, which was blocked by 30 microM 2-amino-5-phosphonovaleric acid (APV) and reduced by islet-activating protein. When the neurons were superfused with Ca(2+)-free solution, the application of 3-10 microM NMDA, which had been dissolved in Ca(2+)-containing solution, induced the second phase [Ca2+]i increase, whereas application of kainate, quisqualate, or stimulation by 50 mM K+ did not. Islet-activating protein, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), and D-sphingosine reduced the second phase [Ca2+]i increase. These results suggest that NMDA-induced intracellular Ca2+ mobilization is potentiated by the initial entry of Ca2+ into the cells and is regulated in an islet-activating protein-sensitive manner.

Animals↗