Pheochromocytoma PC12 cells influence the differentiation of cocultured skeletal myotubes

Rat skeletal myotubes were cultured in the presence or absence of pheochromocytoma PC12 cells under experimental conditions permitting the maintenance of the muscle cells for up to 5 weeks. The number of acetylcholine receptors (AChRs), estimated using radioiodinated alpha-bungarotoxin (alpha-BTX), was comparable in both types of culture for the first 2 weeks. After prolonged incubation (4 weeks), however, alpha-BTX binding sites were two times more abundant in the PC12-myotube cultures than in the myotube cultures. This is likely to reflect a higher number of AChRs at the surface of the myotubes, for control experiments showed that the contribution of PC12 cells was negligible. Presence of PC12 cells also increased the number of AChR aggregates. Use of fluorescent alpha-BTX demonstrated that mixed cultures contained two times more receptor patches than did myotubes alone during the first 2 weeks of culture, and up to five times more after 4 weeks. Essentially all the tight contacts established between PC12 cells and myotubes were characterized by a receptor cluster and, in some cases, by an aggregate of an acetylcholine esterase (AChE), as visualized by immunohistochemistry. Furthermore an antiserum specific for the adult (fast) isoform of the myosin heavy chain stained some myotubes in long-term, mixed cultures. The antigen was never observed in sister cultures of myotubes alone. These data show that PC12 cells, which are thought to derive from the medullary part of the adrenal gland, can influence the differentiation of skeletal muscle cells in several ways.