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

Abstract

The involvement of extracellular matrix components in induction of the aggregation of acetylcholine (AcCho) receptors by factor(s) present in embryonic brain extract was investigated. Embryonic brain extract induced a three-fold increase in the number of AcCho receptor aggregates on the surface of cultured myotubes and a 5-to 10-fold increase in the synthesis of procollagen, which was secreted into the medium and converted to collagen. Adult brain extract, embryonic serum, and embryonic liver extract were less active in stimulating both collagen synthesis and AcCho receptor aggregation. A physiological connection between the two processes is suggested, since the number of AcCho receptor aggregates could be reduced to control levels by treating brain extract-stimulated myotubes with purified bacterial collagenase. In addition, stimulation of collagen secretion by ascorbic acid (50 ,Lg/ml) promoted a 1.6-fold increase in AcCho receptor aggregation. When ascorbic acid was added together with the brain extract, further increases in both collagen synthesis and AcCho receptor aggregation were observed.Functional synapses are formed when embryonic nerve cells are cocultured with embryonic myotubes (1,2). 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 (1-4). 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). These findings suggest that nerve termini release neurotrophic substances that may be responsible for the redistribution of AcCho receptors.Studies of the formation of neuromuscular synapses showed the development ofa specialized extracellular matrix in the synaptic cleft (8,9). Antiserum to basement membrane collagen from muscle and antiserum to lens capsule have been shown to interact with molecules present in the neuromuscular junctional area (10). In the regenerating muscle of adult frog, extracellular matrix components were able to induce localization of AcCho receptors to the original end plates (11). Moreover, particulate basal lamina isolated from the electric organ of Torpedo stimulated AcCho receptor aggregation activity when incubated with cultured myotubes (12).In this study, we have shown that embryonic brain extract contains factor(s) that stimulate the aggregation of AcCho receptors, as well as the synthesis, secretion, and conversion of collagenous macromolecules by cultured muscle cells. Treatments that affect collagen accumulation also affect receptor aggregation, indicating a possible physiological connection between the two processes. MATERIALS AND METHODSPurified bacterial collagenase, essentially free of nonspecifi...

Development of ultrastructural specializations during the formation of acetylcholine receptor aggregates on cultured myotubes

“…It is likely that brain extracts stimulate the deposition of extracellular matrix components onto the myotube surface. Consistent with this hypothesis, a brain extract treatment for 18 hr has been shown to increase collagen synthesis in myotube cultures (Kalcheim et al, 1982). As the effect of EBX on basal lamina in the current study occurs rapidly (2-4 hr), EBX-stimulated synthesis is not likely to be initially required.…”

Collagen synthesis inhibition reduces clustering of heparan sulfate proteoglycan and acetylcholine receptors but not agrin or p65, at neuromuscular contacts in vitro

“…Several lines of experimental evidence suggest that collagen synthesis and deposition are involved in the aggregation of AChRs in the muscle cell membrane, at least in the somewhat artificial situation in which clustering is evoked by ascorbate or brain extract (Kalcheim et al, 1982;Vogel et al, 1987 ). In our experiments, we used an inhibitor of collagen synthesis, cis-hydroxyproline ( cis-HP) , to address three questions about presynaptic and postsynaptic differentiation.…”

Molecular Components of the Synaptic Basal Lamina That Direct Differentiation of Regenerating Neuromuscular Junctions

“…AChR-aggregating molecules have also been extracted from other sources, including rat brain (Podleski et al 1978;Kalcheim et al 1982), chick brain and spinal cord (Jessell et al 1979), conditioned medium from neuronal cell cultures (Christian et al 1978;Schaffner and Daniels 1982), and the electromotor system of Torpedo (Connolly et al 1982). The active components from rat brain and neuronal cellconditioned medium have been reported to be proteins over 50 kD (Podleski et al 1978;Bauer et al 1981), whereas the components from chick brain and Torpedo electromotor system appear to be peptides of less than 1 kD (Connolly et al 1982;Buc-Caron et al 1983).…”