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Y Kidokoro

Publications and source records attributed to Y Kidokoro.

At least 37 records · Page 2Linked to original sources
PubMed1997

Permeation properties of Na+ and Ca2+ ions through the mouse epsilon2/zeta1 NMDA receptor channel expressed in Xenopus oocytes.

Ion permeation properties of the mouse e2/zeta1 NMDA receptor channel expressed in Xenopus oocytes were studied using the outside-out patch-clamp technique. In symmetrical Na+ solutions, the single-channel I-V relations were almost linear at low electrolyte concentrations, but rectified inwardly for Na+ concentrations above 50 mm. In symmetrical Na+ solutions, the "zero-current conductance" increased with Na+ concentration and saturated according to a hyperbolic curve, the half-maximal saturating activity, KM(Na), being 14.2 mm and the maximal conductance, Gmax(Na), 53.9 pS. When Ca2+ was present with Na+ in the external solution, the single-channel current was lower than in pure Na+, although the reversal potential indicated a higher permeability for Ca2+ than for Na+. Using ion activities, PCa/PNa was found to be about 17. The I-V data were fitted with a model based on the Eyring's rate theory, assuming a one-ion pore with three energy barriers and two sites. The KM(Ca) and Gmax (Ca) were 76.5 microm and 21.2 pS, respectively. According to the estimated rate constants, KM for Ca2+ is mainly determined by the binding strength of a site located 80% away from the channel opening at the external membrane-solution interface, a position similar to that postulated previously for the Mg2+ blocking site.

Animals↗
PubMed1997

An inhibitory role of calcineurin in endocytosis of synaptic vesicles at nerve terminals of Drosophila larvae.

In this study, we tested a hypothesis that activation of calcineurin, Ca2+/calmodulin-dependent protein phosphatase 2B, is an initiating signal for synaptic vesicle endocytosis. We examined effects of calcineurin inhibitors, cyclosporin A or FK506 and calmodulin inhibitors on stimulus-induced FM1-43 uptake into nerve terminals of Drosophila larvae. Fluorescent FM1-43 labels recycling synaptic vesicles in nerve terminals. Pretreatment with cyclosporin A (5-40 microM) or with FK506 (5-10 microM) enhanced FM1-43 uptake induced by high (60 mM) K+ in a dose-dependent manner. The effect required some preincubation time of about 10 min. The nerve terminals loaded with FM1-43 were destained by electrical nerve stimulation in the cyclosporin A-pretreated preparations, confirming that FM1-43 was taken up into synaptic vesicles. Pretreatment with rapamycin (2 or 20 microM), a structural analog of FK506 which has no effect on calcineurin, or calyculin A (0.3-50 nM), an inhibitor of protein phosphatase 1 and 2A, had no detectable effect on FM1-43 uptake. On the other hand, pretreatment with trifluoperazine (1-50 microM) or with phenoxybenzamine (100 microM), inhibitors of calmodulin, enhanced FM1-43 uptake. Since endocytosis is coupled with exocytosis, it is possible that the enhancement of FM1-43 uptake results from facilitation of exocytosis. However, the frequency of spontaneous junctional potentials and the mean amplitude of evoked potentials did not change after the cyclosporin A treatment, suggesting that the exocytosis process was not significantly affected by the drug. Furthermore, we can temporally separate synaptic vesicle exocytosis and endocytosis in a Drosophila mutant, shibire (shi(ts1)). By taking advantage of this mutation, we showed that cyclosporin A and trifluoperazine enhanced synaptic vesicle recycling by directly acting on the endocytotic process. Present results are not compatible with the hypothesis, but suggest that calcineurin inhibits synaptic vesicle recycling.

Animals↗
PubMed1997

Permeation of organic cations and ammonium through the glutamate receptor channel in Drosophila larval muscle.

The inside-out configuration of the patch-clamp technique was used to study the effects of large organic cations on the single-channel current through the glutamate receptor channel in muscles of Drosophila larvae. Control experiments with symmetrical Na+ showed slightly supra-linear I-V curves. When external Na+ was equiosmolarly replaced with either arginine+ or N-methyl-D-glucamine+ (NMDG+), the reversal potential changed almost according to VNa, suggesting that both these ions are only slightly permeant through the channel. However, both ions strongly reduced the current amplitude in a voltage-dependent manner, the effect being most pronounced for the inward current. Tris+ had similar effects on the Na+ current, although the reversal potential indicated that this ion is somewhat permeant. All of these results could be fitted with a "one-ion" Eyring model for the channel with internal binding sites. The ion-channel dissociation constants for arginine+, NMDG+, and Tris+ were found to be 17, 27, and 23 mM, respectively. In order to acquire evidence for the "one-ion" hypothesis used in the model, I-V data were taken with different mixtures of Na+ and a comparably permeant ion, NH4+. All of the data could be accurately fitted with the results of the Eyring theory for singly occupied channels.

Animals↗
PubMed1997

Functional organization of chromaffin cells and cholinergic synaptic transmission in rat adrenal medulla.

Optical recordings of membrane depolarization and whole-cell patch-clamp recordings of membrane potentials and currents were obtained from chromaffin cells in slices of rat adrenal medulla. The stimulation of splanchnic nerve fibers caused a discontinuous spread of electrical activity across the slice. Cells in clusters with diameters of about 80 microns were excited simultaneously, suggesting that the adrenal medulla is organized into descrete cell complexes with common innervation. The electrical properties of chromaffin cells in situ were in agreement with previous reports on cultured cells. A fraction of the recorded cells displayed excitatory postsynaptic currents (EPSCs) of 0.2-1 nA upon the stimulation of presynaptic nerve fibers. The EPSC was blocked by hexamethonium, suggesting that nicotinic ACh receptors were involved. The decay phase of the EPSC was well fit by the sum of two exponentials with time constants of 6.3 and 57.3 ms. The relative amplitude of the fast component was 84.1%. These two exponentials may reflect activation of both fast and slow time-constant ACh receptor channels by presynaptic release of ACh. There were multiple peaks in the EPSC amplitude histograms in low-[Ca2+] saline, the first peak was at 37 pA. To resolve the quantal size, miniature EPSCs were recorded in a tetrodotoxin-containing high-[K+] solution. The miniature EPSC amplitude histograms were also multimodal with the first peak at 25 pA, which probably represents the quantal size of the synapse. The second and third peaks were at the integer multiples of the first one.

Action Potentials↗
PubMed1996

p53 gene alterations and p53 protein in oral epithelial dysplasia and squamous cell carcinoma.

To examine the expression of p53 protein and gene alterations in oral epithelial lesions including epithelial dysplasias and primary squamous cell carcinomas, immunohistochemical and temperature gradient gel electrophoresis (TGGE) methods were applied to formalin-fixed and paraffin-embedded tissues. Morphologically normal mucosal epithelium stained negatively for p53 protein. Three out of 11 (27.3 per cent) epithelial dysplasias and 19 out of 57 (33.3 per cent) primary squamous cell carcinomas stained positively for p53 protein. Although more than half of the cases were positive for p53 protein in stage I, the positive cancer cases were found at other stages with variable frequency. Immunoreactive products were localized in the nucleus, especially in the basal and suprabasal layers. The analysis by TGGE revealed gene alterations in exons 5-8 in 3 out of 3 epithelial dysplasias and 17 out of 19 (89.5 per cent) primary squamous cell carcinomas which were immunohistochemically positive for p53 protein. These results suggest that p53 gene mutation may be involved in carcinogenesis in the oral squamous epithelium even in the early stage of the dysplasia-carcinoma sequence.

Adult↗
PubMed1996

Longitudinal body wall muscles are electrically coupled across the segmental boundary in the third instar larva of Drosophila melanogaster.

Longitudinal body wall muscles in the third instar larva of the fruitfly, Drosophila melanogaster, were systematically examined for electrical and dye coupling. These muscle cells were found to be electrically coupled but rarely dye-coupled across the segmental boundary. The inter-segmental coupling coefficients between muscle #6s and muscle #7s across the segmental boundary were 0.33 +/- 0.09 (mean +/- S.D., n = 12) and 0.43 +/- 0.09 (n = 5), respectively, which are much larger than values previously reported in Drosophila but similar to those reported in the blowfly and hawkmoth. By contrast, the intra-segmental coupling coefficient between muscles #6 and #7 was smaller, 0.16 +/- 0.08 (n = 28). Other muscle cells which had apparent physical contacts with these longitudinal muscles were examined but were not electrically coupled to them. Nerve-evoked as well as miniature excitatory junctional potentials were found also electrotonically spread across the segmental boundary. The inter-segmental coupling between muscle #6s was not blocked by the gap junction inhibitors halothane or 1-octanol. Functional significance of this electrical coupling is apparently in coordination of larval body movements.

1-Octanol↗
PubMed1996

Kinetic properties of glutamate receptor channels in cultured embryonic Drosophila myotubes.

Glutamate receptor channels are ubiquitous agonist-gated channels. Pharmacologically they are classified into several subtypes but may have common fundamental channel properties. To build foundation for future molecular biological and genetic studies, we studied kinetics of the glutamate receptor channel in embryonic Drosophila myotubes in culture using the patch clamp technique. There were many brief lasting channel events together with prolonged ones. Brief events were frequently observed in low concentrations whereas the frequency of prolonged events increased with agonist concentrations. Long openings (> 5 ms) were often interrupted by brief closures, most of which lasted less than 100 mu s, thus showing a bursting behavior. At all agonist concentrations, the burst duration was fitted with three exponential components (brief, intermediate and long). The mean duration of the long component increased linearly with the glutamate concentration. The mean closed time and number of brief closures per ms within long bursts were independent of agonist concentration. The mean burst durations of the brief (30-250 mu s) and intermediate component (300-1050 mu s) did not change significantly with agonist concentration. The closing episodes within bursts were rare in the brief and intermediate burst components. The ratio of the fractional areas of the brief or intermediate and long burst components increased linearly with agonist concentration in the log-log plot with a slope of one. These findings suggest that the brief and intermediate components are due to singly-liganded openings and the long component is the result of doubly-liganded openings.

Animals↗
PubMed1995

Glomus tumor of the upper lip.

A case of glomus tumor of the upper lip in a 57-year-old Japanese male is described. The patient had a tender swelling of the upper lip, and this was completely excised. The lesion was found to be encapsulated by fibrous connective tissue and composed of blood capillaries surrounded by sheets of epitheloid cells. Immunohistochemical examination revealed that the neoplastic cells were positive for S-100 protein, vimentin, desmin and actin. Although the endothelial cells of blood capillaries in the tumor tissue were positive for factor VIII, no reaction product was found in the tumor cells. These results suggested that the tumor cells had characteristics of smooth muscle cells.

Actins↗
PubMed1995

Junctional and extrajunctional glutamate receptor channels in Drosophila embryos and larvae.

Glutamate receptor channels in Drosophila embryos and larvae were examined with the patch-clamp technique in various configurations. In the cell-attached mode, only one type of channel was observed in the extrajunctional region at any stages. The burst duration histogram was fit with three exponentials. The burst duration of long component lengthened with increasing glutamate concentration. In excised outside-out patches the unitary channel current was 7.1 pA at -60 mV and direction of current reversed at zero membrane potential. In contrast, junctional receptor channels had different properties. In the whole-cell configuration, spontaneous synaptic currents with steps on the falling phase were observed. The step amplitudes had two discrete values of 9.4 and 18.5 pA at -60 mV, due to openings of junctional glutamate receptor channels. Synaptic currents changed amplitudes linearly with the membrane potential in the negative potential range but nonlinearly above zero. With 1 mM glutamate in the bath, synaptic currents were no longer observed. Instead, there were single channel events with the current amplitude varying between 8 and 12 pA at -60 mV. Their long burst duration depended on glutamate concentration indicating that they are glutamate receptor channel events. The extrapolated reversal potential of these channel currents was around +12 mV. These junctional receptor channels were strictly localized at the junction. Our findings suggest that the channel conversion mechanism in Drosophila is different from that observed in vertebrates. Further close examination of other intermediate steps during neuromuscular junction formation is needed.

Animals↗
PubMed1994

Ion permeation properties of the glutamate receptor channel in cultured embryonic Drosophila myotubes.

Ion permeation properties of the glutamate receptor channel in cultured myotubes of Drosophila embryos were studied using the inside-out configuration of the patch-clamp technique. Lowering the NaCl concentration in the bath (intracellular solution), while maintaining that of the external solution constant, caused a shift of the reversal potential in the positive direction, thus indicating a higher permeability of the channel to Na+ than to Cl- (PCl/PNa < 0.04), and suggesting that the channel is cation selective. With 145 mM Na+ on both sides of the membrane, the single-channel current-voltage relation was almost linear in the voltage range between -80 and +80 mV, the conductance showing some variability in the range between 140 and 170 pS. All monovalent alkali cations tested, as well as NH4+, permeated the channel effectively. Using the Goldman-Hodgkin-Katz equation for the reversal potential, the permeability ratios with respect to Na+ were estimated to be: 1.32 for K+, 1.18 for NH4+, 1.15 for Rb+, 1.09 for Cs+, and 0.57 for Li+. Divalent cations, i.e. Mg2+ and Ca2+, in the external solution depressed not only the inward but also the outward Na+ currents, although reversal potential measurements indicated that both ions have considerably higher permeabilities than Na+ (PMg/PNa = 2.31; PCa/PNa = 9.55). The conductance-activity relation for Na+ was described by a hyperbolic curve. The maximal conductance was about 195 pS and the half-saturating activity 45 mM. This result suggests that Na+ ions bind to sites in the channel. All data were fitted by a model based on the Eyring's reaction rate theory, in which the receptor channel is a one-ion pore with three energy barriers and two internal sites.

Animals↗
PubMed1994

Miniature endplate currents at the newly formed neuromuscular junction in Drosophila embryos and larvae.

Miniature endplate currents (mepcs) were recorded in embryos and larvae of Drosophila melanogaster with the patch clamp technique in the whole cell configuration from abdominal ventral oblique muscles. Shortly after nerve-muscle contacts (16 h after egg laying) mepcs were small and infrequent. The mean amplitude of mepcs increased abruptly during late embryonic stages but did not change any further up to second instar larvae. The amplitude distribution of mepcs was skewed toward larger amplitudes and changed drastically to a more broader distribution at the time of hatching, but did not change any further. These muscle cells were found to be electrically coupled to those in the rostral neighbor segment. The synaptic current has a fast rising phase and decayed with a single exponential throughout the developmental period examined. The decay time constant increased with amplitude but the rise time did not. Neither the rise time nor the decay time constant changed with development. Many synaptic currents decayed in a stepwise manner revealing underlying single channel events. Two discrete steps were discerned, with amplitudes of about 7.6 and 16 pA at -60 mV. These two types of channels were observed throughout the stages examined.

Animals↗
PubMed1993

Inhibition of nerve- and agrin-induced acetylcholine receptor clustering on Xenopus muscle cells in culture.

During an early stage of neuromuscular junction formation the nerve induces acetylcholine (ACh) receptors to accumulate at the contact area. To elucidate the induction process we tested various glycosaminoglycans for their ability to inhibit nerve-induced receptor accumulation. The potency sequence was found as follows: fucyoidin > dextran sulfate > heparin = heparan sulfate > chondroitin sulfate type A and B. This sequence is similar to that for agrin-induced receptor clustering in chick myotubes [J. Neurosci., 10 (1990) 3576-3582], suggesting that agrin-like molecules are involved in nerve-induced receptor accumulation in the Xenopus system. We further tested whether agrin in the culture medium competes with the endogenous inducing substance and found that agrin partially inhibited nerve-induced receptor accumulation. We compared nerve- and agrin-induced receptor accumulation under various experimental conditions. Generally, they behaved similarly except in the presence of heparin. Heparin in the culture medium partially blocked nerve-induced receptor accumulation, whereas it totally inhibited agrin-induced receptor clustering. Our observations are consistent with the hypothesis that an agrin-like molecule released by the nerve is the induction signal for receptor accumulation in the Xenopus system.

Agrin↗
PubMed1992

Initial uncoordinated expression of three types of voltage-gated currents in cultured Xenopus myocytes.

The expression of three types of voltage-gated ionic currents, namely the Na+ inward current, K+ outward rectifier current and K+ inward rectifier current, was examined in cultured developing Xenopus myocytes. In the population of myocytes, the Na+ inward current and K+ outward rectifier current appeared at around 32 h after fertilization (stage 27) and gradually increased during the period of observation, up to more than 44 h after fertilization (stage 33/34). The developmental time course of these two types of currents was similar. However, during the transition period individual myocytes did not necessarily express these two types of currents in a coordinated fashion. Some myocytes had a large Na+ inward current and small K+ outward rectifier current or vice versa. The K+ inward rectifier current was observed in some cells earlier than stage 27 and also gradually increased during the observation period. The initial expression of this current was not correlated with the K+ outward rectifier current or with the Na+ inward current.

Animals↗
PubMed1992

Synaptic activation of rat adrenal medulla examined with a large photodiode array in combination with a voltage-sensitive dye.

The adrenal medulla is innervated by sympathetic preganglionic nerve fibers in the splanchnic nerve. Synaptic activation of the adrenal medulla causes catecholamine secretion which is known to be modified by various neuropeptides and other factors. To understand the neuronal control mechanism of catecholamine secretion, it is necessary to know the transfer function at the synapse and how it is affected by such factors. By using a large photodiode array in combination with a voltage-sensitive dye, membrane potential changes in a slice of the rat adrenal gland were recorded upon brief local electrical stimulation. Electrical signals were recorded only on the portion of the diode array corresponding to the medulla. In a typical record, a spike and an underlying slow potential were observed following a small deflection due to a presynaptic nerve action potential. Both the spike and slow potential were blocked in Ca(2+)-free solution or by hexamethonium, a nicotinic antagonist, but were not affected by atropine, a muscarinic antagonist. The slow potential was interpreted as a nicotinic synaptic potential in the chromaffin cells and the spike as a population action potential. A double pulse experiment revealed that the chromaffin cell action potential began to fail only when the stimulus interval was less than 50 ms (20 Hz). When the stimulus intensity was reduced, the minimal response was found to behave in an all-or-none fashion. This suggested that one nerve fiber is innervating a cluster of chromaffin cells, which may correspond to a previously histologically identified "complex" of cells [Hillarp (1946) Acta. anat. 4, Suppl. 1]. Each complex was innervated by approximately four nerve fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Medulla↗
PubMed1990

Acetylcholine receptor channels in Xenopus myocyte culture; brief openings, brief closures and slow desensitization.

1. Single acetylcholine (ACh) receptor channel currents were analysed in Xenopus myocyte culture. Channel events activated by ACh were recorded using the cell-attached patch clamp technique at low temperatures (11.2-15.8 degrees C) and at 100 mV hyperpolarization over the average resting membrane potential of -75 mV. There were two types of channels, low- and high-conductance channels, as reported previously, with unitary current amplitudes 4.7 +/- 0.4 pA (mean +/- S.D., n = 17) and 6.6 +/- 1.2 pA (n = 14), respectively. 2. At low concentrations of ACh many brief openings (less than 1 ms in duration) were observed. Almost all of them occurred in isolation. The mean open time, which was not dependent on ACh concentration, was 63.1 +/- 18.1 microseconds (n = 15) for the low-conductance channel and 80.3 +/- 18.7 microseconds (n = 2) for the high-conductance channel. 3. Brief openings were frequently observed at 20 nM-ACh, at which concentration the frequency of longer openings (longer than 1 ms) was rare. The frequency of brief openings reached maximum at 50 nM-ACh and declined at high concentrations. The occurrence ratio between the long and brief openings increased linearly with ACh concentration. This finding suggests strongly that the brief openings are due to singly liganded receptor channels, since the long openings are most likely due to doubly liganded receptor channels. 4. A burst of openings was defined as a group of openings separated by brief closures (gaps) lasting less than 1 ms. The main component in the closed time histogram had a time constant of 25.0 +/- 5.1 microseconds (n = 16) for the low-conductance channel and 32.0 +/- 10.2 microseconds (n = 10) for the high-conductance channel. These values were not dependent on ACh concentration. The number of gaps per burst was 2.22 +/- 0.69 (n = 16) for the low-conductance channel and 0.85 +/- 0.23 (n = 9) for the high-conductance channel. The former is significantly greater than the latter. 5. During the recording from a single patch, the frequency of low-conductance channel events declined with time at all ACh concentrations, and the rate of decline was slower in the high-conductance channel in the same patch. The rate of decline was dependent linearly on the ACh concentration. It is most likely that this decline is due to desensitization of the receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed1990

Changes in kinetics of acetylcholine receptor channels after initial expression in Xenopus myocyte culture.

1. Developmental changes in single acetylcholine (ACh) receptor channel properties were analysed in cultured Xenopus myocytes at low temperatures (11-16 degrees C) using the cell-attached patch clamp technique. Single-channel recordings were done at fourteen stages of development ranging from several hours (stage 21) to 5 days (stage 47) after ACh receptors first appear in the muscle membrane. 2. Two types of channels, low conductance and high conductance, which have been described previously, were observed at all stages. At low concentrations of ACh, channel events often occurred as bursts of openings separated by closures briefer than 1 ms. Such bursts were treated as one event. Many brief, isolated channel openings, which were described in the preceding paper, were also observed. Developmental changes in burst duration, brief openings and brief closures were assessed for the period studied in culture. 3. Throughout development, most of the burst duration histograms for the low-conductance channel were not well fitted by a single exponential, having an excess of brief openings. The brief component could be largely accounted for by singly liganded openings, as described in the preceding paper. The burst durations reported here represent the main component of the distribution. At 150 nM-ACh, high-conductance channel burst duration histograms were well fitted by single exponentials. 4. There was a developmental increase in the percentage of single-channel events belonging to the high-conductance class. The percentage of high-conductance events remained low (less than 10%) for the first day after ACh receptors appeared (stages 20-34), and increased to 60% at stage 47. 5. In addition to a shift in channel population, there was a decrease in both low- and high-conductance channel burst durations during early stages of development (mostly within the first day in culture). The effect was more dramatic for the low-conductance channel: overall, low-conductance channel burst duration at 50 mV hyperpolarization over the resting potential decreased from 24 ms at stage 24/25 to 6 ms at stage 47, a 4-fold decrease. During the same period, high-conductance burst duration decreased from 10.6 to 4 ms. a 2.5-fold decrease. In contrast to the burst duration, the mean open time of 60 microseconds for the brief isolated class of opening in the low-conductance channel did not change with development. 6. Neither the single-channel conductance nor the voltage dependence of burst duration changed with development.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed1989

Action potentials and sodium inward currents of developing neurons in Xenopus nerve-muscle cultures.

Action potentials and voltage-gated Na+ inward currents from cultured embryonic neurons of Xenopus laevis were recorded using the patch-clamp technique in the whole cell configuration. Neurons together with muscle cells were dissociated from embryos shortly after completion of gastrulation. Under the voltage-clamp condition the voltage-gated Na+ inward current was isolated from other currents by pharmacological means and by ion substitution. A small Na+ current was observed in round cells without neurites (presumptive neurons). The mean amplitude of the peak Na+ current was 2.5 times larger in neurons with short processes than in presumptive neurons. As they developed further by extending longer processes, the maximum amplitude of the Na+ inward current recorded at the soma decreased. In varicosities, the Na+ inward current density was greater than that at the soma of neurons with extended neurites but kinetic properties and voltage-dependency were similar.

Action Potentials↗