A factor from neurons increases the number of acetylcholine receptor aggregates on cultured muscle cells.

Abstract

There is an increase in the number of acetylcholine (AcCho) receptor aggregates on striated embryonic mouse myotubes when they are cocultured with clonal neuroblastoma-glioma hybrid cells. Medium conditioned by hybrid cells contains a factor which increases the number of AcCho receptor aggregates on myotubes cultured from mouse, rat or chick muscle. AcCho receptor-aggregating activity was present in medium conditioned by the neuroblastoma parent clone but was not detected in medium conditioned by cells of the parent glioma clone, fibroblasts, or HeLa cells. The factor increased the aggregation of AcCho receptors within 24 hr without a significant increase in the total number of AcCho receptors, and its action did not depend on myotube protein synthesis. The factor appears to rearrange the distribution of myotube AcCho receptors either by aggregating mobile AcCho receptors or by stabilizing labile receptor aggregates. Acetylcholine (AcCho) receptors of the innervated skeletal muscle are localized at the neuromuscular junction (1) seemingly by two processes: (i) accumulation of AcCho receptors at the neuromuscular contact region, and (ii) elimination of AcCho receptors from noncontact regions (2). Activation of the muscle fiber by electrical stimulation or synaptic activity can decrease the number of extrajunctional AcCho receptors (3); however, there is only speculation concerning the mechanism of AcCho receptor accumulation at muscle regions aligned with presynaptic acetylcholine release sites (4, 5). In various cells, randomly dispersed surface receptors form patches or caps when the cells are treated with specific immunoglobulins or lectins (6, 7). We here report a similar phenomenon: a factor produced by the cholinergic neuroblastoma-glioma hybrid cell line NG108-15 increases the number of AcCho receptor aggregates on cultured myotubes. We propose that such a factor may be involved in the aggregation of AcCho receptors at the site of nerve contact during synapse formation. MATERIALS AND METHODSCell Culture. Mouse muscle cultures were prepared from the hindlimbs of 18-20-day-old C57B/6N mouse embryos, as described (8), except that cell proliferation was suppressed by the addition of 10-5 M fluorodeoxyuridine on the fifth and sixth days of culture. Cultures were established by adding a suspension of 5 X 105 single cells to 35-mm plastic plates, or 1.5 X 105 cells to the 16-mm wells of plastic multiwell plates (Costar). Conditioned medium produced as described below was added to muscle cultures 10-21 days after plating.Rat muscle cultures were prepared from Fischer strain rat embryos by using the same methods.Chick muscle cultures were prepared from the pectoral muscle of 11-day-old chick embryos (9). A suspension of mechanically dissociated single cells was prepared in a medium of 85% Eagle's minimal essential medium, 10% horse serum, and 5% chick embryo extract, and 2,0 X 104 cells in 40ul 'of medium were added to the 5-mm round wells in Teflon-covered glass slides (Roboz Surgical Instrument Co.). On ...

Embryonic brain extract induces collagen biosynthesis in cultured muscle cells: involvement in acetylcholine receptor aggregation.

“…It was found that addition of rat embryonic brain and spinal cord extracts to the culture increased the number ofAcCho receptors and AcCho receptor aggregates on the cell surface ofa rat muscle cell line and on primary chicken myotubes (5,6). Similarly, medium conditioned by a hybrid neuroblastoma cell line induced aggregation ofAcCho receptors on the surface ofcultured myotubes (7). These findings suggest that nerve termini release neurotrophic substances that may be responsible for the redistribution of AcCho receptors.…”

Neural Influence on Protein Kinase C Isoform Expression in Skeletal Muscle

“…NG108-15 cells form functional cholinergic synapses on myotubes (Christian et al, 1977), and a factor from these cells promotes nAChR clustering (Christian et al, 1978). Coculture of myotubes with NG108-15 cells increases nAChR ␤ subunit tyrosine phosphorylation (K. Miles, unpublished observations), a phosphorylation pattern shared by mature nAChRs in the neuromuscular junction (Qu et al, 1990).…”

Increases in muscle Ca2+ mediate changes in acetylcholinesterase and acetylcholine receptors caused by muscle contraction.

“…These studies have also demonstrated repeatedly that although each event is regulated by some aspect of motor nerve-muscle interaction, the types of regulation may vary among the individual events. For instance, clustering ofAcChoR is independent of synaptic transmission, appearing, instead, to be dictated at first by molecules of neuronal origin and ultimately by a component of the muscle's basal lamina (5)(6)(7)(8)(9)(10). On the other hand, the levels of extrajunctional AcChoR and synaptic AcChoE seem to be strongly influenced by synaptic activity (10)(11)(12)(13)(14).…”