Induction of acetylcholine receptors on cultured skeletal muscle by a factor extracted from brain and spinal cord.

Abstract

Extracts of chicken brain and spinal cord increase the total number of acetylcholine receptors and the number of receptor clusters on uninnervated skeletal myotubes in culture. The active component in these extracts may be unique to neural tissue. Spinal cord cells grown in culture contain the active factor and they secrete it into the medium. Most of the activity is associated with a small molecule, possibly a peptide. Such a factor may be responsible for the clustering of receptors at newly formed nerve-muscle synapses.Acetylcholine receptors (AcChoRs) are distributed over the entire surface of embryonic skeletal myotubes (1-3). Soon after embryonic spinal cord neurites contact myotubes in vitro, synapses form and clusters of AcChoRs can be identified at sites of transmitter release (4, 5). AcChoR clusters are present on uninnervated myotubes in vitro, but it is now clear that newly arrived nerve processes induce new AcChoR clusters rather than seek out preexisting ones (4-7). In adult muscle, the density of extrajunctional receptors is 3-4 orders of magnitude lower than in the synaptic membrane (8, 9), so extrasynaptic receptors must disappear soon after innervation. Both the initial clustering of AcChoRs and the subsequent dramatic decrease in the number of extrajunctional receptors have been extensively studied.Muscle activity is an important determinant of extrajunctional acetylcholine (AcCho) sensitivity in adult and embryonic skeletal muscle (10-12). In-contrast, activity does not influence the clustering of AChoRs at synapses. New clusters appear at nerve-muscle contacts in vitro when cultures are grown in the presence of a-bungarotoxin (aBTX), curare, or tetrodotoxin (7, 13). This finding has renewed interest in the possibility that a chemical factor released from motor nerves may influence the number of AcChoRs.Some evidence for such a factor has already been obtained. In chicken spinal cord explant/muscle cocultures, AcCho sensitivity and the density of 125I-labeled aBTX binding sites on myotubes close to the explant are greater than on myotubes located farther away in the same culture (14). This may reflect the diffusion of a soluble factor from the spinal cord tissue. Rat brain and spinal cord extracts increase AcChoR numbers on L6 myotubes to levels found on primary rat myotubes and also promote the appearance of AcChoR clusters (15). Media conditioned by a hybrid neuroblastoma cell line increase the number of AcChoR clusters but not the total number of receptors on primary mouse myotubes (16).In this paper we document physiological effects of chicken brain and spinal cord extracts on primary chicken myotubes and describe the partial purification of a factor that increases the number and induces the clustering of AcChoRs.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 5397MATERIALS AND METHODS Cell Culture. Muscle cultures were ...

Neuroblastoma × spinal cord (NSC) hybrid cell lines resemble developing motor neurons

“…NSC-19 and NSC-34 cells express many of the morphological and physiological properties of primary motor neurons (Table 4; Fischbach and Dichter, 1974;Kato et al, 1985;Ransom et al, 1977;Peacock et al, 1973;O'Brien and Fischbach, 1986): they extend processes, establish contacts with cultured myotubes, synthesize and store acetylcholine, support action potentials, induce myotube twitching, and express neurofilament proteins. Upon coculture with myotubules, NSC-34 cells also induce AChR clustering, suggesting that these cells may model aspects of early neuromuscular synapse formation (Anderson and Cohen, 1977;MagillSolc and McMahan, 1988;Reist et al, 1987;Jessell et al, 1979;Falls et al, 1991). In a seprate report (Hunter et al, 1991), it has been shown that NSC-34 cells, similar to primary motor neurons and chick ciliary ganglion cells, adhere specifically to the leucine-arginineglutamate (LRE) motif of S-laminin, a neuromuscular synapse-specific basal lamina glycoprotein (Hunter et al, 1989a,b;.…”

“…Some myotubes displayed little or no stain at points of NSC contact, suggesting some NSC clones within a culture may have lost this activity. Whether AChR clusters represent redistribution of previously expressed AChR (Magill-Solc and McMahan, 1988;Reist et al, 1987), or AChR upregulation and membrane insertion (Jessell et al, 1979;Falls et al, 1991) is currently unknown. No distinct AChR aggregates were observed on myotubes cultured alone or with N18TG2 cells (confirming Busis et al, 1984) or NSC-19 cells.…”

Tyrosine Phosphorylation of Nicotinic Acetylcholine Receptor Mediates Grb2 Binding

“…An intriguing possibility is that the ARIA /erbB receptor tyrosine kinase pathway may be involved. ARIA (acetylcholine receptor-inducing activity) originally was isolated from chick brain extracts for its ability to promote the synthesis of AChR subunits in aneural myotubes (Jessell et al, 1979). Induction of AChR gene expression by ARIA recently was shown to require activation of the MAP kinase and PI3 kinase pathways via the erbB receptors (Si et al, 1996;Tansey et al, 1996).…”

The distribution of acetylcholine receptor clusters and sites of transmitter release along chick ciliary ganglion neurite-myotube contacts in culture.

“…Therefore, the ability to cluster ACh receptors cannot be used as a criterion for distinguishing axons from dendrites in culture. If a neuronal factor is responsible for inducing NARPs (Jessell et al, 1979;Buc-Caron et al, 1983) it must be transported throughout the neuritic arbor. The same conclusion was drawn in an earlier study of spinal cord motoneurons co-cultured with muscle (Role et al, 1985).…”