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Y Oyama

Publications and source records attributed to Y Oyama.

At least 163 records · Page 9Linked to original sources
PubMed1989

Electrophysiological estimation of the actions of acetylcholinesterase inhibitors on acetylcholine receptor and cholinesterase in physically isolated Aplysia neurones.

1. The actions of representative cholinesterase inhibitors on the acetylcholine responses of physically isolated single neurones from the pedal ganglion of Aplysia californica were studied, using electrophysiological techniques and rapid agonist application to analyse both the inhibitory actions on the acetylcholine receptor-channel complex and the degree of inhibition of acetylcholinesterase activity on the same neurone. The inhibitors used were physostigmine, edrophonium and diisopropylfluorophosphate (DFP). 2. When selected neurones were suddenly exposed to 50 microM acetylcholine by a 'concentration clamp' technique a large Na-dependent inward current was initiated, and decayed in the continued presence of acetylcholine without external perfusion. However, if perfusion of the acetylcholine solution was reinitiated the current increased somewhat, indicating that the decay of current was due to some combination of receptor desensitization and local depletion of acetylcholine at the membrane by acetylcholinesterase. 3. With simultaneous application of acetylcholine (50 microM) and physostigmine (0.1 to 100 microM) there was a dose-dependent reduction of peak amplitude of the acetylcholine response. However, physostigmine at low concentrations (0.1 to 10 microM) caused a time-dependent increase in the current amplitude alone with a time- and dose-dependent inhibition of acetylcholinesterase activity. At the highest concentration of physostigmine (100 microM) acetylcholinesterase activity was abolished but the current peak was very depressed. After removal of physostigmine from the bathing solution, the current amplitude decreased toward the control at the two lower concentrations as the inhibitory actions on acetylcholinesterase activity were almost reversible, while at the two higher concentrations (10 and 100 microM) the current increased and the inhibition of acethylcholinesterase remained. 4. When acetylcholine (50 microM) and edrophonium (0.1 to 10 microM) were applied simultaneously, edrophonium caused a dose-dependent increase in the peak amplitude that was correlated with a dose-dependent inhibition of acetylcholinesterase activity. Prolonged exposure to edrophonium did not change the peak amplitude and there was no time-dependent change in the inhibition of acetylcholinesterase activity. At the highest concentration of edrophonium used (100 microM), simultaneous application with acetylcholine augmented the peak amplitude relative to control, but to a lesser extent than 10 microM. Prolonged exposure to the highest concentration of edrophonium caused a time-dependent reduction in the peak amplitude. The effects of edrophonium were quickly reversible after the removal of the drug from the bathing solution. 5. DFP (1 and 10mM), similar to 1OO microM physostigmine, caused a dramatic reduction of the peak current on simultaneous application with ftetylcholine. During exposure to DFP the current amplitude and acetylcholinesterase activity were very depressed. After removing DFP from the bathing solution the current amplitude increased to more than the control level after 1 mm DFP, while it did not recover to the control level after 10mM DFP. The inhibition of acetylcholinesterase activity remained at both concentrations. 6. These results indicate that all three cholinesterase inhibitors have dose-dependent actions both at the acetylcholine receptor-channel complex and at acetylcholinesterase. The methods we have developed may be useful in the evaluation of various cholinesterase inhibitors.

Animals↗
PubMed1989

Effects of benzodiazepines and non-benzodiazepine compounds on the GABA-induced response in frog isolated sensory neurones.

1. The effects of benzodiazepines and non-benzodiazepine compounds on the gamma-aminobutyric acid (GABA)-induced chloride current (ICl) were studied in frog isolated sensory neurones by use of a concentration-jump (termed 'concentration-clamp') technique, under single-electrode voltage-clamp conditions. The drugs used were classified into four categories as follows: full benzodiazepine receptor agonists (diazepam, clonazepam, nitrazepam, midazolam, clotiazepam and etizolam), partial agonists (CL 218,872, Ro 16-6028, Ro 17-1812 and Ro 23-0364), inverse agonists (Ro 15-3505, FG 7142 and beta-CCE) and a benzodiazepine receptor antagonist, Ro 15-1788 (flumazenil). 2. All full agonists at concentrations of 3 x 10(-6) M or less increased dose-dependently the peak amplitude of ICl elicited by 3 x 10(-6) M GABA to twice to three times larger than the control. However, no further augmentation of the GABA response was observed at concentrations of 1 x 10(-5) M or higher. Partial agonists also showed a dose-dependent augmentation of the GABA response at concentrations ranging from 3 x 10(-8) M to 3 x 10(-5) M, but their efficacies of augmentation of the GABA response were only about half or less of those of full agonists. Of the inverse agonists, beta-CCE had a unique dose-dependent effect on the GABA response. Beta-CCE reduced dose-dependently the GABA response at concentrations of less than 3 x 10(-6) M, but augmented it at concentrations of 3 x 10(-5) M and 6 x 10(-5) M. The inverse agonists reduced dose-dependently the GABA response. The benzodiazepine antagonist, flumazenil, slightly augmented the GABA response at concentrations between 3 x 10 7M and 3 x 10 5 M. 3. These results show clear differences in the effects on the GABA response between these four categories of compounds known to affect the benzodiazepine recognition site of the GABA/ benzodiazepine receptor-chloride channel complex. Our experimental system of frog isolated sensory neurones and a 'concentration-clamp' technique appears to be useful for evaluating efficacy of compounds on responses mediated by the GABA/benzodiazepine receptor-chloride channel complex.

Animals↗
PubMed1988

Characterization of an inward current elicited by edrophonium in physically isolated and internally perfused Aplysia neurons.

We have studied the ionic and pharmacological properties of an inward current elicited by edrophonium, a cholinesterase inhibitor, on physically isolated and internally perfused Aplysia neurons using the voltage clamp, internal perfusion and rapid external perfusion techniques. The current amplitude was dependent on the external Na concentration [(Na)o] in an almost linear manner. However, complete replacement of (Na)o with Tris or sucrose failed to abolish the current. Internal application of Na [increased (Na)i] reduced the current amplitude. In normal (Na)o, changing (Ca)o (both increases and decreases in (Ca)o) reduced the current amplitude. In the sucrose-substituted (Na)o-free condition, edrophonium still could cause a small current (less than 5% of the control). However, an increased (Ca)o did not augment this residual current. Cs and Li carried the edrophonium-activated current when substituted for (Na)o. With sucrose-substituted Na-free sea water outside, edrophonium elicited an outward current when the neuron was internally perfused with Cs, but not when the neuron was internally perfused with K. Therefore, it is unlikely that K is permeant. External application of tetrodotoxin, a blocker of voltage-dependent Na channels, external application of Cd and internal application of F did not affect the current. The edrophonium response was most sensitive to strychnine, which was about 10 times more potent than D-tubocurarine. Hexamethonium, however, had no effect. The local anesthetics, lidocaine and procaine, inhibited the response over the same concentration range as D-tubocurarine. We conclude that edrophonium opens a monocationic channel (presumably a type of Na channel) which is sensitive to (Ca)o.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
PubMed1988

A single amino acid substitution converts cytochrome P450(14DM) to an inactive form, cytochrome P450SG1: complete primary structures deduced from cloned DNAS.

Genes for lanosterol 14-demethylase, cytochrome P450(14DM), and a mutated inactive cytochrome P450SG1 were cloned from S. cerevisiae strains D587 and SG1, respectively. A single nucleotide change resulting in substitution of Asp for Gly-310 of cytochrome P450(14DM) was found to have occurred in cytochrome P450SG1. In this protein the 6th ligand to heme iron is a histidine residue instead of a water molecule, which may be the ligand for the active cytochrome P450(14DM). Molecular models of the active sites of the cytochrome P450(14DM) and cytochrome P450SG1 were built by computer modeling on the basis of the known structure of that of cytochrome P450CAM whose crystallographic data are available. The mechanisms which may cause a histidine residue to gain access to the heme iron are discussed.

Amino Acid Sequence↗
PubMed1988

Some properties of inhibitory action of lidocaine on the Ca2+ current of single isolated frog sensory neurons.

The action of lidocaine on the Ca2+ current (ICa) was studied on single isolated neurons of frog dorsal root ganglia using a 'concentration-clamp' technique which combines intracellular perfusion with a rapid external solution change. Lidocaine decreased the peak amplitude of ICa at a threshold concentration of 10 microM. Higher concentrations gave a dose-dependent increase in inhibition of ICa. Lidocaine also depressed the Na+ current (INa) at a threshold concentration 10 times lower than that for decreasing the amplitude of ICa of neurons isolated from same ganglia. The rate of inhibitory action on ICa was slowed at more negative holding potentials (VH). Lidocaine appears to block both closed and open Ca2+ channels, but acts more profoundly on open channels.

Animals↗
PubMed1988

Edrophonium-induced inward membrane current in single neurons physically isolated from Aplysia californica.

The action of edrophonium on Aplysia neurons was studied using a concentration clamp technique which combines internal perfusion and a rapid drug application. Edrophonium elicited a dose-dependent inward current in the concentration range 10(-6) to 10(-4) M. At higher concentrations (10(-3) and 10(-2) M), the amplitude of the current often decreased and there was a rapid decay of the current. At these high concentrations, the current increased immediately after washing the neuron with normal solution. These results suggest that edrophonium blocks the ion channel which it opens. Removal of Na+ from the external solution greatly reduced the current amplitude by more than 90%. Removal of Ca2+ also reduced the amplitude of the response; however an increase of Ca2+ did not augment the response. These results suggest that Ca2+ does not carry the current, but is necessary for generation of an Na+-dependent inward current. Edrophonium, 10(-2) M, which completely blocked the current it induced within 20 s, did not significantly affect the voltage-dependent Na+ current. Tetrodotoxin, 1 x 10(-6) M, did not affect the edrophonium response. Hexamethonium, 1 x 10(-4) M, did not change the response elicited by edrophonium, while it significantly reduced the ACh response mediated by Na+. In some neurons edrophonium elicited an inward current, but ACh induced an outward current. Therefore the Na+ channels opened by edrophonium appear to be distinct from both the voltage-gated and ACh receptor-operated Na+ channels.

Acetylcholine↗
PubMed1988

Separation of current- and voltage-dependent inactivation of calcium current in frog sensory neuron.

The inactivation process of the calcium current was studied in frog dorsal root ganglion cells using a newly developed 'concentration clamp' technique, and an inactivation component independent of Ca influx was separated from the Ca-dependent component. The neuron was first placed in Ca-free solution, and pre-pulses of various amplitude were applied, thereby inducing depolarization without the Ca influx. Successive test pulses were applied immediately after a rapid exchange to a solution containing normal Ca. The amount of inactivation under this condition was much smaller than that measured when both pre- and test pulses were applied in the normal solution, and reached a steady level on pre-pulses over +50 mV. We suggest that the inactivation process of the Ca current consists of two components, i.e. a dominant component which is dependent on Ca influx, and a smaller one which is presumably voltage-dependent.

Animals↗
PubMed1988

Strychnine decreases the voltage-dependent Ca2+ current of both Aplysia and frog ganglion neurons.

1. The effects of strychnine on the voltage-dependent Ca2+ current (ICa) were studied in physically isolated Aplysia neurons and enzymatically dissociated frog sensory neurons of the dorsal root ganglion. Neurons were studied under the internal perfusion and the voltage clamp condition. 2. Strychnine decreased the ICa with threshold concentrations for effect at 1 to 10 microM. The depression of ICa increased with strychnine dose without effects on the current-voltage relation of ICa. The effects of low concentrations of strychnine were reversible, but recovery was incomplete at higher concentrations. The potency of strychnine was about 10 times less than that of diltiazem, an organic Ca2+ antagonist. At 100 microM the ICa of Aplysia neurons was reduced to about half of the control. This concentration of strychnine also reduced the peak amplitude of ICa of frog sensory neurons. 3. These results indicate that, in addition to its actions on transmitter responses and on Na+ and K+ currents, strychnine has effects on ICa that have not previously been appreciated.

Animals↗
PubMed1988

Intracellular picrotoxin blocks pentobarbital-gated Cl- conductance.

Using the 'inside-out' configuration of frog sensory neurons, we studied the effect of intracellular picrotoxin on the pentobarbital-gated single channel response of Cl- -current (iCl). The pentobarbital-induced iCl showed no voltage-dependency and the single channel conductance (gamma Cl) was 16 +/- 3.1 pS (n = 6). Picrotoxin caused the pentobarbital-gated Cl- channels to react in a flickering pattern and then finally caused them to cease their opening altogether. This inhibitory action of picrotoxin was reversible.

Animals↗
PubMed1988

Electrophysiological detection of acetylcholinesterase activity using concentration clamp on physically isolated Aplysia neurons.

In this study, we have used electrophysiological techniques to evaluate the acetylcholinesterase (AChE) activity on single neurons physically isolated from the pedal ganglia of Aplysia californica and kurodai. Acetylcholine (ACh) was applied to cells having Na-dependent response using a concentration clamp technique which allows a step-like complete change of external medium around the cell (diameter more than 120 micron) within 30 msec. When the neuron was exposed to ACh (50 microM) initially by rapid and brief (400-800 msec) flow and continuously after stopping the perfusion, the induced current rose to a peak and then decayed in the presence of ACh. However, the current increased again when the rapid flow of the same ACh solution around the cell was resumed. The increase in the current by the resumption of the perfusion was not seen when carbachol was substituted for ACh or when extraordinarily high concentration (10 mM) of ACh was used. Furthermore, this increase in the current was blocked by the antiAChE agent, edrophonium, in a dose-dependent manner. These results suggest that acetylcholinesterase (AChE) causes a local depletion of ACh at the membrane surface where the rate of hydrolysis of ACh exceeds the rate of ACh diffusion from bulk solution and the increase in the current by the resumption of the perfusion resulted from the restoration of ACh concentration at the membrane surface. This electrophysiological indication of AChE activity may be a useful tool for the study of AChE by other than biochemical means.

Acetylcholine↗
PubMed1988

Inhibition of drug-gated chloride currents by calcium influx in frog sensory neurons.

In isolated and internally perfused frog sensory neurons, muscimol-, pentobarbital- and alpha-chloralose-induced Cl- currents were suppressed by voltage-dependent Ca2+ currents. The amount of chloride currents inhibited by Ca2+ influx developed in a hyperbolic manner as a function of Ca2+ influx. The increase in intracellular Ca2+ concentration also suppressed the currents carried by monovalent anions, such as Br-, I-, NO3- and HCOO-.

Animals↗
PubMed1988

Effects of Ca2+ on the transient outward current of single isolated Helix central neurones.

1. Single Helix neurones were studied under voltage-clamp conditions with internal perfusion in order to examine the contribution of internal and external Ca2+ and the effects of 4-aminopyridine (4-AP) on the transient outward current (IA). 2. In Na+ -free snail Ringer, replacement of external Ca2+ [( Ca2+]o) with equimolar Co2+ reduced the maximum amplitude of IA and induced a shift of the steady-state part of the IA inactivation curve (I-V curve), in a positive direction along the voltage axis when the neurone was internally perfused with K-aspartate. 3. In Ca2+ -free solutions, precipitation or chelation of internal Ca2+ [( Ca2+]i) by internal perfusion with KF or EGTA shifted the curve in a more negative direction without affecting the maximum amplitude of IA. Thus, the kinetics of IA are dependent not only upon [Ca2+]o, as previously suggested, but also upon [Ca2+]i. 4. In the presence of 4-AP the I-V curve for IA shifted in a hyperpolarizing direction and the maximal amplitude was reduced when the neurone was internally perfused with K-aspartate. However, 4-AP had little or no effect on the I-V relationship of IA when the neurone was internally perfused with the Ca2+ precipitating or chelating agent, KF or EGTA. These results suggest that the actions of 4-AP on IA are at least partly dependent upon [Ca2+]i.

4-Aminopyridine↗