Nerve extract induces increase and redistribution of acetylcholine receptors on cloned muscle cells

Abstract

The effect of rat spinal cord explants and cell-free nerve extract on acetyicholine receptor site density and distribution was studied using 125I-and rhodamine-labeled abungarotoxin on Le, a cloned rat muscle cell line. Control Lt myotubes have a low and uniform distribution of acetylcholine receptors (20 i 3 sites per pm3 in the present study) The addition of spinal cord explants caused an increase in average receptor site density of abut 6 times on myotubes within 2 mm of the explant, while a smaller increase of 3 times was observed at distances greater than 5 mm. The formation of high-density patches of receptors was also stimulated. These observations suggested that a diffusible substance originating from the explant was responsible for these changes. Cell-free homogenates of the central nervous system were prepared and found to produce the same effects. The effect of the homogenate was not strongly dependent on the age of the fetus from which the tissue was isolated, and fetal liver had little or no effect. The active component~s) appears to be a protein(s) with a molecular weight of abut 100,000. Because the nerve homogenates make the L6 cells resemble primary muscle cultures, we suggest that a common factor is responsible for regulating the acetylcholine receptor in the two types of muscle culture. The normally acetylcholine receptor-poor L4 cells may provide a more sensitive assay for these factors than do primary muscle cultures.Many studies on neurotrophic phenomena have dealt with the control of synthesis and distribution of the acetylcholine receptors (AcChR) in embryonic and adult innervated and denervated muscle. One favored system for these studies has been muscle cells grown in culture [see recent reviews by Nelson (1) and Gutman (2)].In primary muscle cultures the AcChR tends to form in high density patches on myotubes. Several authors have suggested that these patches play a role in the subsequent development of the cells (3,4).The L6 line of cloned muscle cells (5) resembles primary muscle cultures in its developmental sequence and ability to form synaptic connections (6). Furthermore, the conductance change per AcChR in response to AcCh is the same in L6 as in primary rat myotubes (7). However, these cells have a uniform distribution and considerably lower site density of AcChR than the myotubes in primary cultures (7-9). These characteristics make L6 a desirable system for studying the possible control of AcChR distribution in skeletal muscle prior to innervation.The present study deals with the effects of isolated explants of nervous system tissue and of cell-free nerve extracts on AcChR in L6 cells. We have found that both are capable of inducing an increase in average AcChR site density and the formation of high-density patches.The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 2035 METHODSThe acetylcholine rec...

Early events in neuromuscular junction formation in vitro: induction of acetylcholine receptor clusters in the postsynaptic membrane and morphology of newly formed synapses.

“…Alternatively, a chemical factor that influences receptor number and/or distribution may be released in relatively high concentration at synapses. Recent studies have shown that extracts of spinal cord or brain and media conditioned by cultured neurons can increase the total number of receptors (29,44) and the number of receptor clusters (11, 29, 44) on uninnervated myotubes .…”

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

“…Acetylcholine (AcCho) receptors, which are distributed over the entire surface of noninnervated embryonic skeletal myotubes, aggregate at the sites of nerve-muscle contact on synaptogenesis (1)(2)(3)(4). 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).…”

Dependence of in vitro myogenesis on a trophic protein present in chicken embryo extract.

“…After 2-3 days in culture, the myoblast population increased as a result of active cell division, and at the same time myoblasts began fusing into multinucleated myotubes. [7][8][9][10] days, muscle cultures showed matured myotubes (Fig. 3A).…”

“…The best evidence for the trophic influence of a neurally derived substance has been obtained from in vitro experiments (4)(5)(6)(7)(8)(9)(10)(11). By using embryonic muscle culture, Oh (12,13) demonstrated that extracts of central and peripheral nervous tissue enhanced the morphological development of aneural muscle cells, increased protein synthesis and acetylcholinesterase activity, and maintained matured, cross-striated muscle fibers for several months in the absence of innervation.…”