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Y Kidokoro

Publications and source records attributed to Y Kidokoro.

At least 55 records · Page 3Linked to original sources
PubMed1989

Heparin and heparan sulfate partially inhibit induction of acetylcholine receptor accumulation by nerve in Xenopus culture.

It has been demonstrated that ACh receptors in Xenopus nerve-muscle cultures migrate in the membrane to the nerve contact area during junction formation (Anderson et al., 1977) and that "diffusion trapping" is the major mechanism for nerve-induced receptor accumulation (Kidokoro and Brass, 1985; Kuromi et al., 1985; Kidokoro et al., 1986). A crucial remaining question is how the nerve induces the trap for randomly diffusing ACh receptors. In this study we examined the effect of various glycosaminoglycans in the culture medium on the nerve-induced receptor accumulation and found that heparin and heparan sulfate partially inhibited nerve-induced receptor accumulation, but similar molecules, chondroitin sulfate type A and type C, did not. By chemical modification of heparin we also showed that N-sulfate residues and a large-molecular-weight molecule are essential for this inhibitory effect. Heparin did not affect ACh receptor clustering (hot-spot formation) in myocytes cultured without nerve. By changing the time and duration of heparin application, we found that heparin was effective in inhibiting nerve-induced receptor accumulation only when it was present in the culture medium during the period that neurites are actively forming contact with muscle membrane.

Animals↗
PubMed1988

Early cross-striation formation in twitching Xenopus myocytes in culture.

Spontaneous release of neurotransmitter has been demonstrated in various types of synapses. Its physiological significance, however, is still unknown. In nerve-muscle cultures of embryonic Xenopus laevis, we observed that acetylcholine, which is released spontaneously at the synaptic terminal, caused frequent twitches of muscle cells. These muscle cells developed cross-striations earlier than neighboring non-twitching cells. This effect of innervation was unaffected by tetrodotoxin but was blocked by alpha-bungarotoxin. Repeated iontophoretic application of acetylcholine or KCl to muscle cells caused twitches and also accelerated the formation of cross-striations. Thus twitching apparently promotes lateral alignment of myofibrils. It is also known that myosin synthesis is higher in twitching muscle cells. Therefore, successfully innervated twitching muscle cells may have an advantage for faster differentiation over neighboring non-twitching muscle cells. We suggest that spontaneously released transmitter may serve as a mediator for trophic interaction at forming synapses.

Acetylcholine↗
PubMed1987

Acetylcholine dose-response relation and the effect of cesium ions in the rat adrenal chromaffin cell under voltage clamp.

Acetylcholine(ACh)-induced inward current was examined in the rat adrenal chromaffin cell in culture. Cells were voltage clamped with one electrode using the giga-seal technique. The ACh dose-response relationship was determined by applying various concentrations of ACh with the "puffer" method. Although there were several technical difficulties, we found tentative figures as 135 to 480 microM for the dissociation constant and 1.2 to 1.4 for the Hill coefficient. External Cs ions had a depressing effect on the ACh response. Cs ions may slowly block ACh receptor channels from outside at the resting state. The ACh response in normal saline recorded with the Cs-filled electrode reversed the polarity at -7.1 mV. When the amplitude of ACh-induced current was plotted against holding membrane potential the curve showed strong rectification at the region more negative than the reversal potential. Between +10 and +30 mV there was a region of a negative slope. We discussed possible mechanisms for this characteristic I-V curve.

Acetylcholine↗
PubMed1987

Two types of acetylcholine receptor channels in developing Xenopus muscle cells in culture: further kinetic analyses.

1. Further developmental changes in acetylcholine (ACh) receptor channel function were examined in Xenopus muscle cultures using the cell-attached patch-clamp technique. 2. Two types of acetylcholine (ACh) receptor channel events, low- and high-conductance channel events, were distinguished as reported earlier. Apparent open-time histograms for high-conductance channel events were well fitted by a single exponential but those for low-conductance channel events were sometimes fitted better by two exponentials. 3. In low-conductance channel events when the open-time histogram was well fitted by two exponentials, successive open times were correlated: an event with a long open time tended to be followed after a brief interval (less than 1 ms) by another long-duration event. A short-duration event was less frequently followed by an event within a short interval (1 ms) with a long-duration event. 4. Closed-time histograms for the interval between successive low-conductance channel events and between successive high-conductance channel events were both fitted by two exponentials. The fast time constant was 0.36 ms for the high-conductance channel event and 0.31 ms for the low-conductance channel event. There was an indication that a third and faster component was hidden in the first bin (0-200 microseconds) in the closed-time histogram of both types of channel events. 5. Defining a burst as successive openings separated by closures briefer than 1 ms, the number of gaps per burst was different for the two types of channel events. They were 0.16 for high- and 0.37 for low-conductance channel events. In both types of channels, neither the fast component in the closed-time histogram nor the number of gaps per burst changed with time in culture. 6. The apparent open time of both types of channels increased progressively as ACh concentration was increased, suggesting an increasing number of unresolved closures at higher concentrations. At 100 microM-ACh the apparent open time became shorter probably due to channel blockade by ACh molecules. Closed-time histograms were fitted by two exponentials. The time constant of the fast component remained similar to that at low concentration (0.2 microM) up to 20 microM, but the relative number of closures belonging to this component increased with ACh concentration. In contrast, the slow component shortened its time constant as ACh concentration increased and the relative frequency decreased. Again, there was an indication that another faster component existed in the closed-time histogram.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed1986

Concanavalin A prevents acetylcholine receptor redistribution in Xenopus nerve-muscle cultures.

During neuromuscular junction formation ACh receptors accumulate at the nerve-contact region. It has been shown that this is at least partly due to lateral migration of existing receptors in the membrane (Anderson et al., 1977). Randomly diffusing ACh receptor molecules in the membrane may be trapped at the nerve-contact region to form a high receptor density area. If this were the major mechanism, cross-linking ACh receptors by tetravalent concanavalin A (Con A) should immobilize receptors and prevent nerve-induced receptor accumulation. We examined the effect of Con A on nerve-induced receptor accumulation and on the mobility of ACh receptors in cultured Xenopus muscle cells. ACh receptors were stained with tetramethyl rhodamine conjugated alpha-bungarotoxin. The cells were then treated briefly with Con A, and neural tube cells were added to these cultures. The mobility of ACh receptors was measured by the fluorescence photobleaching recovery method. The Con A treatment prevented rapid diffusion of ACh receptors as well as nerve-induced receptor accumulation. Functional synapse formation was not inhibited by this treatment. In contrast, divalent succinyl Con A did not affect the mobility of ACh receptors nor prevent nerve-induced ACh receptor accumulation. When the Con A concentration was varied, the blocking effect on the nerve-induced receptor accumulation changed in parallel with the mobile fraction of receptors. Newly inserted ACh receptors after the Con A treatment were found to be mobile and to accumulate at the nerve-contact region. In these cultures, new receptors accumulated around old, immobilized receptors in some areas along the nerve contact. This observation suggests that new receptors were inserted elsewhere and migrated to the nerve-contact region surrounding immobilized old ones. In addition to the accumulation of receptors, the nerve disperses preexisting receptor clusters prior to induction of high-density regions along the contact area, and, at this early stage, denervation disperses nerve-induced receptor clusters in Xenopus cultures (Kuromi and Kidokoro, 1984a, b). When cultures were treated with Con A, neither of these events occurred, suggesting that these are also diffusion-mediated.

Animals↗
PubMed1985

Formation of acetylcholine receptor clusters at neuromuscular junction in Xenopus cultures.

The formation of acetylcholine receptor (AChR) clusters at the neuromuscular junction was investigated by observing the sequential changes in AChR cluster distribution on cultured Xenopus muscle cells. AChRs were labeled with tetramethylrhodamine-conjugated alpha-bungarotoxin (TMR-alpha BT). Before innervation AChRs were distributed over the entire surface of muscle cells with occasional spots of high density (hot spots). When the nerve contacted the muscle cell, the large existing hot spots disappeared and small AChR clusters (less than 1 micron in diameter) initially emerged from the background along the area of nerve contact. They grew in size, increased in number, and fused to form larger clusters over a period of 1 or 2 days. Receptor clusters did not migrate as a whole as observed during "cap" formation in B lymphocytes. The rate of recruitment of AChRs at the nerve-muscle junction varied from less than 50 binding sites to 1000 sites/hr for alpha BT. In this study the diffusion-trap mechanism was tested for the nerve-induced receptor accumulation. The diffusion coefficient of diffusely distributed AChRs was measured using the fluorescence photobleaching recovery method and found to be 2.45 X 10(-10) cm2/sec at 22 degrees C. There was no significant difference in these values among the muscle cells cultured without nerve, the non-nerve-contacted muscle cells in nerve-muscle cultures, and the nerve-contacted muscle cells. It was found that the diffusion of receptors in the membrane is not rate-limiting for AChR accumulation.

Animals↗
PubMed1985

Ontogeny of electrically excitable cells in cultured olfactory epithelium.

A primary system has been developed in which it is possible to study the production of electrically excitable neuron-like cells from a precursor population of olfactory epithelial cells. Rat nasal epithelium was dissociated and placed in culture. The initial surviving cells are flat and ciliated and contain glial fibrillary acidic protein (GFAP). After 3-5 days electrically excitable cells appear that contain neuron-specific enolase but not GFAP. These round cells originate by means of the differentiation of the GFAP-positive flat cell to a round cell, followed by the division of the round cell. Therefore, neuron-like cells can be derived from cells that synthesize GFAP.

Action Potentials↗
PubMed1985

Development and subsequent neural tube effects on the excitability of cultured Xenopus myocytes.

We examined both the development of electrical excitability in cultured Xenopus muscle over a period of 7 days, and the effects of neural tube on the muscle action potential. During muscle development, delayed and anomalous rectification were present in most cases within 24 hr. The action potential was dependent on Na at all times examined, and the rate of rise of the action potential (Vmax) increased substantially (seven-fold) from the first 2 days to 6 to 7 days in vitro, reflecting an increase in Na current density. In order to determine the mechanism for the increase in Vmax, we examined single-channel Na currents using the gigaseal technique. Single-channel conductance (gamma) did not increase substantially when measured using the patch clamp technique: gamma = 24 pS at 1 to 2 days, and gamma = 28 pS at 4 to 6 days. The channel open time at 14 degrees C was 0.6 msec for 1- to 2-day-old cells and 0.5 msec in 4- to 6-day-old cells at a step potential 40 mV from rest. The time constant for current decay as well as the time-to-peak current also did not change over time. Thus, channel kinetics appear unchanged. The maximum inward current from summed records was statistically greater for older cells, and the frequency of patches displaying single-channel events increased from 75 to 98%. Thus, we conclude that during development in vitro, Na current density increases as a result of an increase in channel density without detectable alterations in single-channel properties. Neural tube addition led to a further increase in Vmax (two-fold), even in muscle cells with no apparent nerve contact. Single channel analysis of cells in coculture revealed gamma to be 28 pS in three cells displaying a single amplitude peak for individual Na currents. In the majority of cases (9/12), however, there appeared to be two classes of Na channels present which were difficult to separate. The larger conductance channel likely corresponds to the 28 pS class. The smaller channels, when present, did not contribute substantially to the population of events comprising the amplitude histogram. Other single-channel kinetic parameters also did not change. We, therefore, conclude that neural tube addition does not effect activation or inactivation kinetics but likely causes a further increase in channel density and possibly the induction of a second type of Na channel.

Action Potentials↗
PubMed1985

Redistribution of acetylcholine receptors during neuromuscular junction formation in Xenopus cultures.

At the adult neuromuscular junction, acetylcholine (ACh) receptors are highly localized at the subsynaptic membrane, whereas, embryonic myotubes before innervation have receptors distributed over the entire surface. Thus sometime during development, ACh receptors accumulate to the nerve contact area. This nerve-induced receptor accumulation can be reproduced in Xenopus nerve-muscle cultures, which provides us with a unique opportunity to investigate the underlying molecular mechanism of this event. Anderson and Cohen (1977) have shown that nerve-induced receptor accumulation is, at least partly, due to migration of pre-existing receptors. It is, thus, plausible that freely diffusing receptors in the membrane are trapped at the nerve-contact region and form clusters. We tested this diffusion trap model. First, receptors in the background region are indeed predominantly mobile and those in the cluster are immobile. Second, the diffusion of receptors in the membrane is fast enough to account for the rate of receptor accumulation. Third, when receptors were immobilized by a lectin, Concanavalin A, the nerve no longer induced receptor accumulation. Thus the diffusion trap model seems adequate to accommodate these observations. Aside from this diffusion mediated mechanism, it is conceivable that newly formed receptors are preferentially inserted at the nerve contact site and these new receptors become immobilized at the site of insertion. To test this hypothesis we stained new receptors separately from old ones and quantitatively compared their distribution. For this purpose we developed a method to quantify fluorescence micrographs. We found that the ratio between old and new receptors was similar at all nerve-induced clusters examined and at the diffusely distributed region.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Nerve disperses preexisting acetylcholine receptor clusters prior to induction of receptor accumulation in Xenopus muscle cultures.

We have investigated the sequential changes of acetylcholine receptor (AChR) distribution on identified Xenopus laevis muscle cells in culture before and after innervation. AChRs on muscle cells were stained with tetramethylrhodamine-conjugated alpha-bungarotoxin and the distribution of AChR clusters was examined on a fluorescence microscope using an image intensifier. Large receptor clusters were identified on muscle cells and their fate was followed afterward. In muscle cells cultured without neural tube cells, about one-half of the identified AChR clusters survived for 2 days. In nerve-muscle cocultures, preexisting AChR clusters survived longer on non-nerve-contacted muscle cells than on muscle cells cultured without nerve. However, in nerve-contacted muscle cells the great majority of preexisting AChR clusters dispersed within 2 days. The dispersal of preexisting AChR clusters preceded receptor accumulation along the path of nerve contact by about 12-16 hr. Therefore, an accelerated dispersal of receptor clusters in innervated muscle cells is not a consequence of receptor accumulation along the nerve. The preexisting AChR clusters located near and far from the nerve contact sites dispersed along a similar time course. Protease inhibitors, trasylol and leupeptin, reduced the nerve-induced dispersal of the preexisting AChR clusters in the period before AChR accumulation at the nerve contact sites but did not do so during the period when AChRs began to accumulate at nerve-muscle contact. The significance of the dispersal of preexisting receptor clusters is discussed with regard to neuromuscular junction formation.

Animals↗
PubMed1984

Denervation disperses acetylcholine receptor clusters at the neuromuscular junction in Xenopus cultures.

The effect of denervation on acetylcholine receptor (AChR) cluster distribution on cultured Xenopus muscle cells has been examined in order to study the role of intact nerve in the maintenance of clusters at the nerve-muscle junction during development. AChRs on the muscle cell were labeled with tetramethyl rhodamine-conjugated alpha-bungarotoxin and sequential changes in AChR cluster distribution were examined with a fluorescence microscope using an image intensifier. Denervation was carried out by exposing the nerve cell body to a focused laser light of a high intensity. After this procedure the neurites originating from the cell quickly disintegrated and large AChR clusters associated with nerve divided into smaller clusters. Individual clusters subsequently decreased in size and finally disappeared. In about 30% of the cases new AChR clusters appeared at the extrajunctional region after denervation. These observations indicate that intact nerves are necessary for the maintenance of receptor localization at the nerve-muscle junction and that nerve-induced accumulation is seemingly reversible during the early period of synapse formation. We tested the idea that receptor clusters were lost due to diffusion of receptors in the muscle membrane after denervation. However, the rate of receptor cluster dispersal after denervation was much slower than that predicted by the diffusion model, suggesting that diffusion of receptors is not a rate-limiting step. Furthermore, we found that receptor clusters at the junction stabilize during days in culture. Thus, 80-90% of receptor clusters at the nerve-muscle junction disappeared at 7 hr after denervation in 1-day cocultures, while about 50% of receptor clusters remained after denervation in 3-day cocultures.

Animals↗
PubMed1984

Two types of miniature endplate potentials in Xenopus nerve-muscle cultures.

The frequency distribution of miniature endplate potential (mepp) amplitudes was examined in Xenopus nerve-muscle cultures. In the early days of nerve-muscle co-culture (1-2 days) the mepp amplitude histogram was skewed in the majority of muscle cells and had a peak approximately at 0.6 mV. Innervated muscle cells in older cultures (3-4 days), however, often had two peaks. The distribution at the smaller amplitude was skewed and the peak was approximately at 0.6 mV, whereas the peak at the larger amplitude was more-or-less symmetrical and about 18 times greater than the small one. The time-to-peak value of mepps in the peak at the smaller amplitude was slightly smaller than those in the peak at the larger amplitude. Small mepps were not due to electrotonic spread of mepps generated at distant sites nor due to focal development of acetylcholinesterase. Two peaks were also observed in the amplitude histogram of extracellularly recorded miniature endplate currents. Therefore, these two types of mepps must be generated practically at the same site. Mepps in the peak at the smaller amplitude had similar properties as sub-miniature endplate potentials (s-mepps) described in the adult animal. The second symmetrical peak in older cultures may correspond to classical mepps (c-mepps). The age-dependent shift from the amplitude distribution with one peak to that with two peaks has also been reported in developing and regenerating neuromuscular junctions. Therefore, I suggest that during development in culture, mepps appear at first as s-mepps and subsequently c-mepps develop, forming the second peak in the amplitude histogram.

Age Factors↗
PubMed1984

Acetylcholine receptor channel properties during development of Xenopus muscle cells in culture.

Developmental changes in acetylcholine (ACh) receptor channel function on aneural cultures of embryonic myotomal muscle cells were examined using the patch-clamp technique. At all stages of differentiation two different unitary-event amplitudes were observed, corresponding to high-gamma (single-channel conductance) (64 pS) and low-gamma (46 pS) channel types. No change in conductance occurred for either channel type during the 6-day in vitro period examined. At resting membrane potential (-85 mV) the low-gamma channel exhibited a mean open time of approximately 2 ms which, on the average, was 2-3-fold longer than that measured for the high-gamma channel. Neither the estimated mean channel open time nor the voltage dependence of the open state measured for either channel type changed during development. In recordings with low ACh concentration (0.1-0.25 microM) both high-gamma and low-gamma channel types exhibited non-stationary opening probabilities over the recording period. Usually the opening rate of both channel types decreased with time following seal formation, however, the 'drop-out' rate was faster for the low-gamma channel. A developmental increase in the proportion of high-gamma events occurred between day 1 (16%) and day 5 (56%) in culture, paralleling the time-dependent changes in the channel kinetics based on ACh-activated membrane noise. We conclude that the development of non-junctional muscle membrane is associated with increased expression of high-gamma channels and that this process is primarily responsible for the previously reported developmental alterations in macroscopic ACh receptor channel currents.

Animals↗
PubMed1984

Acetylcholine-induced potential fluctuations and underlying currents in rat adrenal chromaffin cells.

Adrenal chromaffin cells are innervated by splanchnic nerves. The neurotransmitter acetylcholine (ACh), released at the terminal, somehow triggers the catecholamine secretion. On ACh stimulation the chromaffin cell in culture depolarizes with prominent potential fluctuations and generates repetitive action potentials. Because these cells are small (10-16 micron in diameter) and have a large input resistance (about 6 G omega), the stochastic nature of channel opening and closing can generate considerable voltage noises that underlie the repetitive generation of action potentials. The action potential in the adrenal chromaffin cell is mediated by Na as well as Ca inward currents. Thus, Ca2+ entering the cell during the action potential may link the ACh stimulation and the hormone secretion. In addition, Ca2+ entering the cell through ACh receptor channels may also significantly contribute to secretion. It is most likely, however, that ACh receptors are highly localized at the subsynaptic area, which is only a small portion of the cell surface. Ca influx through ACh receptor channels is thus limited to the area close to the synapse, whereas voltage-dependent Ca channels may provide Ca2+ through the entire surface. Ca channels probably play an important role in stimulus-secretion coupling in the adrenal chromaffin cell.

Acetylcholine↗