1. Exogenously applied acetylcholine (ACh) is a modulator of human myoblast fusion. Using a chemiluminescent method, we examined whether an endogenous ACh‐like compound (ACh‐lc) was present in, and released by, pure human myogenic cells. 2. Single, freshly isolated satellite cells and proliferating myoblasts contained 15 and 0.5 fmol ACh‐lc, respectively. Cultured myotubes contained ACh‐lc as well. Also, ACh‐like immunoreactivity was detected in all myogenic cells. 3. Part of the ACh‐lc was synthesized by choline acetyltransferase (ChAT), as indicated by the reduction of ACh‐lc content when bromoACh was present in the culture medium, and by direct measurements of ChAT activity. Also, ChAT‐like immunoreactivity was observed in all myogenic cells. 4. Myoblasts and myotubes released ACh‐lc spontaneously by a partially Ca(2+)‐dependent mechanism. 5. The application by microperfusion of medium conditioned beforehand by myoblasts (thus presumably containing ACh‐lc) onto a voltage‐clamped myotube induced inward currents resembling ACh‐induced currents in their kinetics, reversal potential, and sensitivity to nicotinic antagonists. 6. In vitro, the spontaneously released ACh‐lc promoted myoblast fusion but only in the presence of an anticholinesterase. 7. Our observations indicate that human myogenic cells synthesize and release an ACh‐lc and thereby promote the fusion process that occurs in muscle during growth or regeneration.

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Acetylcholine and choline chemiluminescent assays have limitations when these compounds are detected in small areas of mammalian nervous tissue. Use of 7-dimethyl-aminonaphthalene-1,2-dicarbonic acid hydrazide (7-DMAN), instead of luminol, gives a threefold increase in emitted light in the chemiluminescent assay for acetylcholine based on the coupled choline oxidase-peroxidase reaction. Addition of light enhancers, such as para-iodophenol or D-luciferin, to luminol or 7-DMAN further increased the light emission. Under these conditions the detection limit for acetylcholine was 650 femtomoles. This enhanced chemiluminescent assay should be convenient for the detection of in vivo and in vitro acetylcholine release from mammalian neurons.

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