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H Aoshima

Publications and source records attributed to H Aoshima.

At least 55 records · Page 3Linked to original sources
PubMed1990

Acetylcholine receptor-mediated membrane current in oocytes injected with Electrophorus electricus mRNA: analyses of nicotine, succinylcholine, and decamethonium responses on the basis of the minimal model.

Nicotinic acetylcholine receptor was synthesized in Xenopus oocytes after injection of the mRNA purified from Electrophorus electricus electroplax. Nicotine, succinylcholine, and decamethonium (agonist)-elicited membrane currents in the injected oocytes were measured electrophysiologically by the voltage-clamping method. The following four different measurements were made to establish the relationship between the agonist concentration and the membrane current: 1) the agonist-induced membrane current before desensitization, 2) the agonist-induced membrane current after desensitization equilibrium, 3) the fraction of the active form of the receptors after desensitization equilibrium, 4) the rate of recovery of desensitized receptors upon removal of the agonist. These results were analyzed on the basis of the minimal model proposed from receptor-mediated ion translocation measurements. The equilibrium and rate constants of the model were evaluated for nicotine, succinylcholine, and decamethonium, and could explain the observed electrical responses in the injected oocyte, i.e. the characteristics of the receptor response caused by these agonists.

Animals↗
PubMed1988

Expression of amino acid transport systems in Xenopus oocytes injected with mRNA of rat small intestine and kidney.

Xenopus and Cynops oocytes were injected with exogenous mRNA prepared from rat small intestine and kidney and their electrical responses to amino acids were measured by both the current clamped and the voltage clamped methods. Oocytes injected with mRNA of rat small intestine showed a depolarization response to several neutral and basic amino acids, and almost no response to acidic amino acids. The responses to amino acids increased with incubation time after injection of mRNA, and followed Michaelis-Menten type kinetics. The responses were dependent on both Na+ concentration and membrane potential, and were inactivated by a sulfhydryl reagent, 5,5-dithiobis(2-nitrobenzoate). These results are interpreted as due to the expression of Na+/amino acid cotransporter(s) in oocytes injected with rat small intestine mRNA. On the other hand, the oocyte injected with rat kidney mRNA showed a hyperpolarization response to neutral amino acids, a depolarization response to basic ones, and almost no response to acidic ones in frog Ringer solution. These responses were independent of Na+ concentration and followed Michaelis-Menten type kinetics. These amino acid response characteristics in oocytes injected with rat kidney mRNA are interpreted as due to the expression of facilitated diffusion carrier protein(s) (uniporter) of amino acids in the oocyte.

Amino Acids↗
PubMed1987

Effect of lipid hydroperoxide on Xenopus oocytes and on neurotransmitter receptors synthesized in Xenopus oocytes injected with exogenous mRNA.

The effect of 13-L-hydroperoxylinoleic acid (LOOH) on both Xenopus oocytes and neurotransmitter receptors synthesized in the oocytes was studied by electrophysiological and ion flux measurement. Addition of LOOH to the incubation mixture of the oocytes raised the membrane potential and decreased the membrane resistance of the oocytes. These effects of LOOH on the oocytes were reversed within a few hours by incubation with frog Ringer solution. Addition of LOOH also caused an increase of Li+ and 45Ca2+ uptake into the oocytes. However, production of alkoxy radicals by the addition of FeCl2 to the incubation mixture containing LOOH did not accelerate the damage to the oocytes by LOOH. So essential toxicity is caused possibly by an increase in the membrane permeability resulting from disturbance of the lipid bilayer arrangement, not from production of active alkoxy radicals during decomposition of LOOH. Nicotinic acetylcholine and gamma-aminobutyric acid receptors were synthesized in Xenopus oocytes by injecting mRNA prepared from Electrophorus electricus electroplax and rat brain. LOOH noncompetitively inhibited the function of these receptors and also increased the rate of desensitization of the receptors.

Animals↗
PubMed1987

Minimal model to account for the membrane conductance increase and desensitization of gamma-aminobutyric acid receptors synthesized in the Xenopus oocytes injected with rat brain mRNA.

gamma-Aminobutyric acid (GABA) receptors, which translocate chloride anion with binding GABA, were synthesized in Xenopus oocytes by injecting rat brain mRNA. GABA-elicited responses in the oocytes were measured electrophysiologically by the current-clamped method. Five different measurements were made to establish the relationship between GABA concentration and the electrical responses: (1) the GABA-elicited conductance increase before desensitization; (2) the rate of desensitization of GABA receptors; (3) the rate of recovery of desensitized receptors upon removal of GABA; (4) the GABA-elicited conductance increase after desensitization equilibrium; (5) the fraction of the active form of GABA receptors after desensitization equilibrium. These results were interpreted on the basis of the minimal model proposed for nicotinic acetylcholine receptor in Electrophorus electricus electroplax [Hess, G. P., Cash, D. J., & Aoshima, H. (1983) Annu. Rev. Biophys. Bioeng. 12, 443-473]. Estimated equilibrium and rate constants in the model for GABA receptors could successfully explain the results of the five above measurements.

Animals↗
PubMed1987

Induction of muscarinic acetylcholine, serotonin and substance P receptors in Xenopus oocytes injected with mRNA prepared from the small intestine of rats.

Serotonin and muscarinic acetylcholine (ACh) receptors were clearly induced in Xenopus oocyte injected with mRNA prepared from the small intestines of rats. Their response to ACh and serotonin was composed of 4 distinct components: fast and slow depolarization, slow hyperpolarization and large membrane potential fluctuation. About three-quarters of the injected oocytes responded to substance P. The response of the injected oocytes to substance P was transient and decayed even in the presence of substance P, indicating the presence of desensitization. However, the injected oocytes showed no response to 6 other drugs analyzed: adrenaline, noradrenaline, dopamine, gamma-aminobutyric acid, glycine and glutamate.

Animals↗
PubMed1987

Expression of the functional D-glucose transport system in Xenopus oocytes injected with mRNA of rat small intestine.

mRNA prepared from rat small intestine was injected into Xenopus oocytes. The injected oocytes showed a clear electrical response to D-glucose (Glu) in the form of membrane depolarization and conductance increase, while none was shown to D-fructose. The membrane electrical response of the injected oocytes evoked by Glu followed the Michaelis-Menten type kinetics and was dependent on the membrane potential of the oocyte. Replacing the Na+ of the bathing buffer with choline+ resulted in no response to Glu. Thus, a Glu transport system coupled to a Na+ gradient was expressed in Xenopus oocytes by injecting mRNA from rat small intestine.

Animals↗
PubMed1987

Acetylcholine receptor-controlled ion translocation caused by phenyltrimethylammonium and nereistoxin: simple estimation of equilibrium constants of the minimal model.

The rate of slow Li+ influx and the fraction of active form of acetylcholine receptor (AChR) of Electrophorus electricus membrane vesicles at equilibrium between the active and desensitized forms of the receptor were measured in the presence of various concentrations of phenyltrimethylammonium (PTA) and nereistoxin (NTX), by a simple filtration assay and flame emission spectroscopy. The equilibrium constants of these ligands in the minimal model, which accounts for the AChR-mediated ion flux, were estimated simply from these two measurements, since the equilibrium constants for acetylcholine (ACh) and carbamylcholine (Carb) estimated from two kinetic measurements agreed well with those estimated from five sophisticated kinetic measurements of AChR-mediated ion fluxes. PTA showed high potency but not high efficacy, and showed inhibition when large doses were applied. NTX showed both low potency and low efficacy and acted as an inhibitor when it was added with Carb. The apparent dissociation constants of these three agonists evaluated from the minimal model and the equilibrium constants agreed with those obtained by assay of inhibition of radiolabeled ligand binding.

Animals↗
PubMed1986

Li+ uptake into Xenopus and Cynops oocytes injected with exogenous mRNA, observed by flame emission spectroscopy.

Li+ uptake into Xenopus oocytes was measured by flame emission spectroscopy. Li+ uptake into the oocytes increased proportionally with incubation time and was dependent on either pH or temperature. Maximum uptake of Li+ was observed around pH 7. Li+ uptake into Xenopus oocytes increased by a factor of roughly 7 over the range 4-30 degrees C. When mRNA prepared from electroplax of Electrophorus electricus was injected into Xenopus or Cynops oocytes, Li+ uptake into the injected oocytes increased by the addition of carbamylcholine (Carb), an agonist of the acetylcholine receptor (AChR). This increase of Li+ uptake by Carb was inhibited by d-tubocurarine, an antagonist of nicotinic AChR. Thus, a new method was established for detection of the activity of nicotinic AChR synthesized in oocytes injected with exogenous mRNA.

Animals↗
PubMed1986

Time course of the induction of acetylcholine receptors in Xenopus oocytes injected with mRNA from Electrophorus electricus electroplax.

mRNA from the electroplax of adult Electrophorus electricus was injected into Xenopus oocytes. At various times after injection, the induction of the nicotinic acetylcholine (ACh) receptor in the oocyte membrane was studied electrophysiologically using a two-electrode current clamp. When the ACh sensitivity was induced, membrane potential and conductance rapidly rose from the resting value to their peaks and slowly fell (desensitization) in response to bath-applied 0.1 mM ACh. It took a latency period of 8 +/- 2.6 h (n = 10) from mRNA injection to the first appearance of the ACh sensitivity. During about 10 h incubation after the onset, the increasing speed in the peak of ACh-induced conductance change with incubation time was slow at first and accelerated later. The speed was then decelerated until the induction stages of 60 h. Apparent desensitization properties of the induced receptors changed with an increase of incubation time. During the early induction stages, the conductance decline after its peak followed two exponentials in a minute time region of ACh application: an early slow and a late fast one. The rate of decline in the late fast component slowed down markedly with incubation time. Finally the desensitization followed a single exponential.

Acetylcholine↗
PubMed1986

New translation system of mRNA coding for neurotransmitter receptors using oocytes of the newt, Cynops pyrrhogaster.

Eel electroplax mRNA was injected into oocytes of newts (Cynops pyrrhogaster) and the injection induced synthesis of a nicotinic acetylcholine receptor in the oocyte membrane. The time course of the induction and dose-response relationship of the receptor were recorded electrophysiologically. The receptor responses and their developmental changes were similar to those of Xenopus oocytes injected with eel mRNA. Newt oocytes lived much longer (7-10 days) than Xenopus oocytes (3-4 days) under our experimental conditions. In non-injected newt oocytes, native transmitter receptors were not observed. In addition, newts have large oocytes (1.6-1.9 mm in diameter), into which a large amount of mRNA could be easily injected. Thus, newt oocytes may be a more useful system to translate exogenous mRNAs coding for neurotransmitter receptors than Xenopus oocytes.

Animals↗
PubMed1984

A second, slower inactivation process in acetylcholine receptor-rich membrane vesicles prepared from Electrophorus electricus.

An agonist such as carbamylcholine or phenyltrimethylammonium induced a second, slower complete inactivation of acetylcholine receptor prepared from Electrophorus electricus. The rate of this inactivation of the receptor followed first-order kinetics. The rate constant of the inactivation increased with the agonist concentration until it reached a plateau, the value of which was 0.19 h-1 at 4.5 degrees C. The reaction was also temperature dependent, and the activation energy of the inactivation caused by 1 mM carbamylcholine was estimated to be 7.6 kcal/mol. The inactive receptor was reconverted to the active form with a rate constant of about 0.015 h-1 at 4.5 degrees C when the carbamylcholine concentration (0.1 mM) was reduced by 15-fold dilution in eel Ringer's solution. These results can be interpreted by adding, to the minimal reaction scheme proposed by the Hess group, a second, slower, reversible inactivation process either through the intact form or through the first desensitized form of the receptor binding two agonist molecules.

Animals↗
PubMed1983

Inhibition schemes for acetylcholine receptor-mediated ion translocation in the presence of various kinds of inhibitors.

Some basic inhibition schemes for acetylcholine receptor (AChR)-mediated ion translocation in the presence of agonist, cholinergic ligand, and inhibitors are proposed on the basis of the minimum reaction scheme (Hess, G.P., Cash, D.J., & Aoshima, H. (1983) Annu. Rev. Biophys. Bioeng. 12, 443-473). Equations for the rate coefficients of ion flux before and after desensitization, JA and JD, and for desensitization, a, were derived from each scheme, assuming that binding of inhibitors to AChR does not affect the values of rate and equilibrium constants of cholinergic ligand to the receptor. In the presence of inhibitors, AChR-mediated transmembrane Li+ influx caused by carbamylcholine (Carb) was measured by using Electrophorus electricus membrane vesicles, a simple filtration assay and flame emission spectroscopy. The dependence of the ratios of rate coefficients on the concentration of ligand and inhibitors was examined in detail. The inhibition constants of d-tubocurarine and caffeine were estimated to be about 31 nM and 0.84 mM, respectively, on the basis of a simple competitive inhibition scheme, and that of procaine was estimated to be 0.11 mM on the basis of a simple noncompetitive one.

Animals↗
PubMed1982

Cocaine and phencyclidine inhibition of the acetylcholine receptor: analysis of the mechanisms of action based on measurements of ion flux in the millisecond-to-minute time region.

The effects of cocaine and of phencyclidine and procaine on acetylcholine receptor-controlled ion flux were measured in the millisecond-to-minute time region. Chemical kinetic measurements of ion flux were made in membrane vesicles prepared from the electric organ of Electrophorus electricus and in PC-12 cells, a sympathetic neuronal cell line. A quench-flow technique was used to measure ion flux in the millisecond-to-second range in membrane vesicles. Cocaine and phencyclidine both inhibit acetylcholine receptor-controlled ion flux, but by different mechanisms. Both compounds decrease the initial rate of ion flux, an effect observed with the local anesthetic procaine. This inhibition cannot be prevented by saturating concentrations of acetylcholine (1 mM). These results from chemical kinetic experiments are consistent with electrophysiological measurements which indicate that local anesthetics act by interfering with the movement of ions through receptor-formed channels. The chemical kinetic experiments, however, give additional information about the action of phencyclidine. They indicate that phencyclidine also increases the rate of receptor inactivation (desensitization) and changes the equilibrium between active and inactive receptor conformations, effects not observed in the presence of cocaine or procaine.

Animals↗