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H B Peng

Publications and source records attributed to H B Peng.

At least 55 records · Page 3Linked to original sources
PubMed1992

The influence of basic fibroblast growth factor on acetylcholine receptors in cultured muscle cells.

Acetylcholine receptors (AChRs) in Xenopus muscle cells undergo changes in channel kinetics during development in culture and these changes are somehow related to innervation. Recently we showed that basic fibroblast growth factor (bFGF), when locally applied, can mimic the effect of nerve in inducing a postsynaptic-type development. In this study, we examined whether bFGF can influence the developmental changes of AChRs. Patch clamp method was employed to record single AChR channel currents from cultured Xenopus myotomal muscle cells and the kinetics of low-conductance AChR channels were analyzed. In cultures treated with 1 microgram/ml bFGF at an early stage (stage 23), the burst duration of low-conductance AChR channels showed a 1.5-fold decrease between the first and second day in culture, while it underwent a remarkable 3-fold decrease during the same period in the control. Histogram analyses showed that the low-conductance channels were composed of a fast and a slow component and that the decrease in burst duration was due to a shift in the population from the slow to the fast. bFGF treatment appeared to slow down this shift by retaining the slow channels for a longer period of time. This effect is probably not due to channel modulation as the burst duration of short channel in older cells (stage 40) was not affected by bFGF. These data suggest that bFGF may enhance the metabolic stability of intrinsically short-lived AChRs. This effect seems to parallel the stabilization of junctional AChRs at the innervated endplate. Thus, bFGF, or a related polypeptide growth factor, may mediate this and other innervation-induced changes in the postsynaptic membrane.

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PubMed1992

Induction of acetylcholine receptor clustering by native polystyrene beads. Implication of an endogenous muscle-derived signalling system.

Aneural muscle cells in culture often form acetylcholine receptor (AChR) clusters, termed hot spots, which are similar to those found at the postsynaptic membrane both in structure and in molecular composition. Although hot spots form on both dorsal and ventral surfaces of the cell, the ventral ones are better characterized because of their association with sites of cell-substratum contact. To understand the stimuli and mechanisms involved in ventral hot spot formation, native, uncoated polystyrene beads were applied to cultured Xenopus myotomal muscle cells to create local membrane-substratum contacts. These beads were able to induce a postsynaptic-type development as evidenced by the clustering of AChRs and the development of a set of ultrastructural specializations, including membrane infoldings and a basement membrane. Whereas these native beads were effective in inducing clustering, beads coated with bovine serum albumin or treated with serum-containing medium were ineffective. Native beads were also capable of inducing clusters in serum-free medium, indicating that their effect was mediated by endogenous molecules that were locally presented by the beads, rather than by bead adsorption of components in the medium. Heparan sulfate proteoglycan (HSPG) is a major component of the muscle extracellular matrix and our previous study has shown that basic fibroblast growth factor (bFGF), a member of the heparin-binding growth factor (HBGF) family, and its receptor are present in Xenopus myotomal muscle during the period of synaptogenesis. Therefore, we tested the involvement of HBGF in bead induction. The results of this study show the following: (1) preincubation of cultures in heparin, which solubilizes matrix-bound HBGFs, suppressed the bead-induced AChR clustering. (2) Suramin, which interferes with the interaction between several growth factors and their receptors, also inhibited bead-induced clustering. (3) Tyrphostin, which blocks tyrosine kinase activity associated with a number of growth factor receptors, was also inhibitory to the bead effect. (4) The percentage of bead-induced AChR clusters was significantly enhanced by pretreating the cultures with bFGF prior to bead application. This exogenously applied bFGF could be largely removed by treatment of cultures with heparin, suggesting its association with HSPG at the cell surface. (5) An anti-bFGF neutralizing antiserum significantly reduced the efficacy of the bead stimulation. These data suggest that uncoated beads, which adhere to the cell surface and can mimic the cell-substratum interaction, effect a local presentation of HBGFs, such as bFGF, residing with the HSPG to their membrane receptors, thereby locally activating receptor-associated tyrosine kinases.(ABSTRACT TRUNCATED AT 400 WORDS)

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PubMed1992

Induction of dystrophin localization in cultured Xenopus muscle cells by latex beads.

The distribution of dystrophin in Xenopus myotomal muscle cells was examined in conventional and confocal immunofluorescence microscopy. By labeling dissociated single muscle fibers with a monoclonal or a polyclonal antibody against dystrophin, we found that dystrophin is ten times more concentrated at the myotendinous junction (MTJ) than at the extrajunctional sarcolemma. At the MTJ, dystrophin lines the membrane invaginations where myofibrils attach to the membrane. It is colocalized with talin, but is not related to the distribution of acetylcholine receptors (AChRs) which are clustered at the postsynaptic membrane in the vicinity of the MTJ in these fibers. We found that the localization of dystrophin can be induced in cultured Xenopus myotomal muscle cells by treating them with polystyrene latex beads. Dystrophin is discretely localized at the bead-muscle contacts. With electron microscopy, a sarcolemma specialization with all the salient features of the MTJ, including basal lamina-lined membrane invaginations along which myofibrils make attachment. Although these beads also induce clustering of AChRs, the patterns of dystrophin and AChR localization are distinct. The appearance of dystrophin at the bead-contacted sarcolemma is coincident with the development of the membrane invaginations. This, together with its concentration along membrane invaginations at the MTJ in vivo, suggests a role for dystrophin in the formation of this junctional specialization. Since the signal for MTJ development can be presented to cultured muscle cells in a temporally and spatially controlled manner by beads, this system offers a simple model for analyzing the mechanism of this sarcolemma specialization.

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PubMed1991

Induction of synaptic development in cultured muscle cells by basic fibroblast growth factor.

The role of basic fibroblast growth factor (bFGF) in signaling the development of the neuromuscular junction was examined. Beads coated with bFGF induced the formation of acetylcholine receptor (AChR) clusters in cultured Xenopus myotomal muscle cells. Tyrphostin, a tyrosine kinase inhibitor, abolished AChR clustering induced by bFGF beads, suggesting a role of tyrosine kinase activation in AChR clustering. Using specific antibodies, we demonstrated the presence of both bFGF and its receptor in the myotomal muscle in vivo during the period of neuromuscular connection. However, similar tissue from older animals with mature neuromuscular junctions showed an apparently truncated form of the bFGF receptor. These data suggest that bFGF may play a role in signaling synaptogenesis in skeletal muscle.

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PubMed1990

The influence of AChR clustering stimuli on the formation and maintenance of AChR clusters induced by polycation-coated beads in Xenopus muscle cells.

Nerve, polycation-coated beads, and electric fields not only induce acetylcholine receptors (AChRs) to cluster, but they also reduce the number of spontaneous AChR patches (hotspots) away from the induced cluster sites on embryonic Xenopus myotomal muscle cells grown in tissue culture (the global effect). In vivo, the ability of an AChR clustering stimulus to depress cluster formation elsewhere on the muscle cell may influence both the site at which the neuromuscular junction develops as well as which axons survive during synapse elimination. Since the causes of hotspot formation may be variable and cannot be controlled, we have further characterized the global effect by using AChR-clustering stimuli that can be controlled by the experimenter. We report that innervation inhibits the formation and maintenance of bead-associated AChR patches (BARPs) by a percentage of polycation-coated beads. We next investigated competition between beads and between beads and electric fields. In competition between beads added to the muscle cells at different times, however, the first set of beads had a competitive advantage over the second set of beads. This advantage was strengthened when the latency between bead applications was extended, or when a relatively large number of BARPs were formed by the first set of beads. Likewise, long-term electric fields were able to prevent the formation of BARPs, but were unable to disperse mature BARPs. Longer electric fields, or electric fields of greater magnitude competed better with the beads than brief or weak field treatments. None of the "winning" stimuli, including nerve, were able to totally block AChR patch formation or maintenance by competing stimuli. Thus, the global effect, at least in the case of competition between nonneuronal stimuli, favors the initial stimulus and appears to be graded.

Animals↗
PubMed1990

A protein homologous to the Torpedo postsynaptic 58K protein is present at the myotendinous junction.

The 58K protein is a peripheral membrane protein enriched in the acetylcholine receptor (AChR)-rich postsynaptic membrane of Torpedo electric organ. Because of its coexistence with AChRs in the postsynaptic membrane in both electrocytes and skeletal muscle, it is thought to be involved in the formation and maintenance of AChR clusters. Using an mAb against the 58K protein of Torpedo electric organ, we have identified a single protein band in SDS-PAGE analysis of Xenopus myotomal muscle with an apparent molecular mass of 48 kD. With this antibody, the distribution of this protein was examined in the myotomal muscle fibers with immunofluorescence techniques. We found that the 48K protein is concentrated at the myotendinous junctions (MTJs) of these muscle fibers. The MTJ is also enriched in talin and vinculin. By double labeling muscle fibers with antibodies against talin and the 48K protein, these two proteins were found to colocalize at the membrane invaginations of the MTJ. In cultured myotomal muscle cells, the 48K protein and talin are also colocalized at sites of membrane-myofibril interaction. The 48K protein is, however, not found at focal adhesion sites in nonmuscle cells, which are enriched in talin. These data suggest that the 48K protein is specifically involved in the interaction of myofibrillar actin filaments with the plasma membrane at the MTJ. In addition to the MTJ localization, 48K protein is also present at AChR clusters both in vivo and in vitro. Thus, this protein is shared by both the MTJ and the neuromuscular junction.

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PubMed1989

Development of calcitonin gene-related peptide (CGRP) immunoreactivity in relationship to the formation of neuromuscular junctions in Xenopus myotomal muscle.

Immunoreactivity (IR) to calcitonin gene-related peptide (CGRP) has recently been found in chick motoneurons. Bath application of this peptide causes an increase in surface acetylcholine receptor (AChR) density and cAMP level in cultured chick muscle cells. These results suggest a role for this peptide in the formation of synaptic specializations. In this study, we examined the development of CGRP IR in larval Xenopus myotomal muscle in relation to synaptogenesis. Using an antiserum against CGRP, a monoclonal antibody against the p65 synaptic vesicle antigen and a fluorescence conjugate of alpha-bungarotoxin, we followed the development of synaptic specializations with fluorescence microscopy in whole-mount specimens. We found that the postsynaptic specialization in the form of AChR clusters was first detected in stage 22 (24 hour) embryos. The presynaptic specializations, including the synaptic vesicle clusters as evidenced by p65 antibody staining and CGRP IR, were first detected at stage 32 (40 hours). The appearance of these two presynaptic specializations followed the same time course. Subsequently, all three structures, the AChR clusters, CGRP IR, and synaptic vesicle clusters, were colocalized at the neuromuscular junction (NMJ). This shows that CGRP is unlikely to be involved in signaling the development of the postsynaptic apparatus. This premise is further examined in cultures of Xenopus myotomal muscle cells. CGRP at concentrations up to 1 microM did not affect the number of AChR clusters, nor did it interfere with the formation of clusters induced by polycation-coated beads. In contrast, an extract from the basement membrane of Torpedo electric organ promoted the formation of AChR clusters and interfered with the clustering activity of the beads. These results suggest that CGRP, an integral component of the presynaptic specialization, is not involved in signaling synaptogenesis at the NMJ.

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PubMed1989

The role of lateral migration in the formation of acetylcholine receptor clusters induced by basic polypeptide-coated latex beads.

During the formation of the neuromuscular junction, the nerve induces the clustering of acetylcholine receptors (AChR) in the postsynaptic membrane. This process can be mimicked by treating cultured Xenopus myotomal muscle cells with basic polypeptide-coated latex beads. Using this bead-muscle coculture system, we examined the role of lateral migration of AChRs in the formation of the clusters. First, we studied the contributions of the preexisting and newly inserted AChRs. After the cluster formation was triggered by the addition of the beads, preexisting receptors were immediately recruited to the bead-muscle contacts and they remained to be the dominant contributor during the first 24 hr. New AChRs, which were inserted after the addition of the beads, appeared at the clusters after a 4-hr delay and, thereafter, there was a steady increase in their contribution. After 24-48 hr, newly inserted AChRs could be detected at the bead-induced clusters to the same extent as the preexisting AChRs. During this period, new receptors were continuously inserted into the plasma membrane, but there was no evidence of a local insertion at sites of new cluster formation. Concanavalin A (Con A) at a concentration of 100 micrograms/ml caused a fivefold decrease in the fraction of mobile AChRs and a large decrease in their diffusion coefficient. Pretreatment of cells with Con A suppressed clustering of preexisting AChRs, but left intact the contribution of the mobile newly inserted AChRs. Succinyl Con A, the divalent derivative of Con A which affected the mobility to a much less extent than Con A, had little effect on the clustering process. These results show that the formation of AChR clusters in Xenopus is mediated by lateral migration of AChRs within the plasma membrane and are consistent with the diffusion-trap hypothesis, which depicts freely diffusing AChR aggregating at the bead-muscle contacts where they bind to other localized molecular specializations induced by the beads.

Animals↗
PubMed1989

The relationship between talin and acetylcholine receptor clusters in Xenopus muscle cells.

Talin is involved in mediating the cytoskeleton-extracellular matrix interaction at focal contacts in cultured fibroblasts. Recently this protein has been localized at both the myotendinous junction (MTJ) and the neuromuscular junction (NMJ) in skeletal muscle. At the MTJ, talin may mediate the insertion of myofibrils into the plasma membrane, thus serving a function similar to that seen at focal contacts. However, the function of talin at the NMJ is unknown. In this study, we examined its distribution at both mature and developing acetylcholine receptor (AChR) clusters in Xenopus muscle cells both in vivo and in vitro with immunofluorescence. At the NMJs of both myotomal and submaxillaris muscles, talin was absent from the AChR clusters. In cultured myotomal muscle cells, it was absent from 40% of both the nerve-associated AChR patches and the spontaneously formed AChR clusters located on the top surface of the cells. We therefore conclude that this protein is not essential for maintenance of AChR clusters at the NMJ. In addition to MTJs, talin was invariably associated with AChR clusters induced by polyornithine-coated beads, and, to a large extent, with spontaneously formed clusters on the ventral side of cultured cells. A common feature of these talin-positive domains is the deep membrane infoldings, where bundles of actin filaments are inserted into the membrane. Thus, talin may be involved in the formation and maintenance of these structures. The deep membrane infoldings, though prominent at most NMJs, are absent from the two muscles under study in vivo. Our work thus suggests that the postjunctional membrane at the NMJ is heterogeneous, consisting of an AChR cluster domain and, often but not always, a domain for proteins involved in cytoskeleton-membrane linkage as exemplified by talin.

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PubMed1989

Localization of intracellular proteins at acetylcholine receptor clusters induced by electric fields in Xenopus muscle cells.

Electric fields cause acetylcholine receptor (AChR) patches to form on the cathodal sides of cultured muscle cells. These patches are stable for several hours following cessation of an electric field treatment, indicating that the receptors are anchored to the cluster sites. Furthermore, at the ultrastructural level, AChR patches induced by electric fields are marked by an accumulation of extracellular matrix material and a sarcolemmal density. Thus, these AChR patches are similar to those induced by other stimuli, including nerve, polycation-coated beads, and the tissue culture substratum. Proteins that may be involved in anchoring AChRs have been colocalized with AChR patches induced by the latter three stimuli, but not at AChR patches induced by electric fields. In this study, we demonstrate that three putative anchoring proteins, 43K (K = 10(3) Mr) protein, 58K protein and talin, are associated with field-induced AChR patches. We also show that these proteins persist at field-induced AChR patches following removal of the field, indicating that they are stabilized at the AChR patch. Our data are consistent with the possibility that these proteins contribute to the stabilization of AChRs at patches induced by the electric field. Since 43K, 58K and talin are intracellular proteins, and therefore could not undergo field-induced lateral electrophoresis, our observations support the notion that the electric field triggers the formation of an AChR-stabilizing specialization.

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PubMed1988

Localization of calcitonin gene-related peptide (CGRP) at a neuronal nicotinic synapse.

The localization of calcitonin gene-related peptide (CGRP) in the parasympathetic cardiac ganglion of the frog was investigated with an immunofluorescence method. By doubly staining the whole ganglion with antibodies against a synaptic-vesicle antigen and anti-CGRP antibodies, we found that all of the boutons at the nicotinic synapses on the postganglionic neurons contained immunoreactivity against this peptide. In contrast, neither the muscarinic nor the adrenergic terminals of the postganglionic fibers on the cardiac muscle contained CGRP immunoreactivity. This suggests that CGRP is involved in the functions of the neuronal nicotinic synapses.

Animals↗
PubMed1988

Increase in intracellular calcium induced by the polycation-coated latex bead, a stimulus that causes postsynaptic-type differentiation in cultured Xenopus muscle cells.

The polycation-coated latex bead is a potent stimulus for the induction of postsynaptic-type differentiation in cultured Xenopus myotomal muscle cells. Specializations characteristic of the neuromuscular junction, such as clusters of acetylcholine receptors and other postsynaptic-specific proteins, develop at the bead-muscle contact. Previous studies have shown that a deprivation of extracellular calcium inhibits the effect of the beads in causing the development of these specializations. This suggests that an increase in intracellular Ca2+ is a necessary condition for the development of this specialization. In this study, we tested whether an increase in intracellular calcium is observable upon the bead-muscle contact. The measurement was carried out on cells loaded with the fluorescent calcium indicator fura-2 AM by digitized video microscopy. When polycation-coated beads were added to cells, an increase in intracellular calcium concentration in the range of 5 to 57% of the resting level was observed within 10 sec after bead-muscle contact. Afterward, the calcium level gradually returned to the resting level with a time course of about 3 min. Uncoated beads, which do not induce the formation of acetylcholine receptor clustering, failed to elicit this calcium transient. Removal of extracellular calcium as well as blocking calcium channels with 50 microM verapamil also suppressed this transient induced by the polycation-coated beads. Both treatments are known to suppress the formation of receptor clusters by these beads. These results suggest that the polycation-coated beads cause an influx of calcium by increasing the membrane conductance to this ion. This process may underlie the signaling of the postsynaptic differentiation.

Animals↗
PubMed1988

Development of acetylcholinesterase induced by basic polypeptide-coated latex beads in cultured Xenopus muscle cells.

The formation of acetylcholine receptor (AChR) clusters can be induced by basic polypeptide-coated latex beads in cultured Xenopus muscle cells. Here we investigated the development of acetylcholinesterase (AChE) at the bead-induced AChR clusters. AChE activity began to appear at the clusters after 1 day of bead-muscle coculture and was present at all of the bead-induced clusters within 4-7 days. Electron microscopy revealed that AChE reaction products were discretely localized within the cleft and the membrane invaginations at the bead-muscle contacts. Thus, the beads can mimic the nerve in inducing a local accumulation of both the AChRs and AChE, suggesting that the development of both specializations can be effected by a common stimulus.

Acetylcholinesterase↗
PubMed1988

Patterns of N-CAM expression during myogenesis in Xenopus laevis.

The neural cell adhesion molecule (N-CAM) is seen in the membrane of nerves and muscles from several vertebrate species. Using indirect immunofluorescence, we have examined the expression of this protein during embryonic and postembryonic myogenesis in the African clawed frog, Xenopus laevis. While good staining for N-CAM was seen in neuronal tissues at all stages examined, no staining of embryonic muscle was observed, including both mononucleated and polynucleated myoblasts. In contrast, limb muscles formed at metamorphosis showed strong expression of N-CAM. The developing limb muscles eventually lose their N-CAM, but will reexpress it dramatically when denervated. These observations suggest that myogenesis programs executed at different stages of development can display distinct patterns of N-CAM expression.

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PubMed1987

Development of presynaptic specializations induced by basic polypeptide-coated latex beads in spinal cord cultures.

The development of the neuromuscular synapse is initiated by an interaction between the motoneuron processes and the skeletal muscle. Previously we showed that basic polypeptide-coated latex beads can mimic the nerve in effecting a postsynaptic differentiation (Peng, H.B. and Cheng, P.-C.: J. Neurosci., 2:1760-1774, 1982). In this study, we examined whether these beads can also induce a presynaptic differentiation along the nerve processes. Explant cultures were prepared from the spinal cords of Xenopus larvae and polyornithine-coated latex beads (0.45-4.5 micron in diameter) were applied. After 1-2 days, the cultures were processed for light and electron microscopy. For light microscopy, the cells were permeabilized and labeled with a monoclonal antibody against a 65-KD antigen on synaptic vesicles. Indirect immunofluorescence revealed that this antigen was concentrated at 60% of the bead-neurite contacts, therefore suggesting the clustering of synaptic versicles at these sites. This phenomenon was not observed at the contacts between neurites and negatively charged (polycarboxylate) beads. However, a concentration of this antigen was also observed at the varicosities along the neurites cultured on polylysine substrate. Thin-section electron microscopy showed the following features: (1) The neurites formed terminal-like varicosities on the bead. (2) Within these varicosities, clusters of 50-60-nm clear vesicles were prominent at the bead-neurite contact. (3) Large (80-100 nm) dense-cored vesicles were also present in the varicosity, but they did not form clusters. (4) Basement membrane did not form at the bead-neurite interface, in contrast to its appearance at the bead-muscle contact from our previous study.(ABSTRACT TRUNCATED AT 250 WORDS)

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