A new in vitro melanophore bioassay for MSH using tail-fins of Xenopus tadpoles.

A new in vitro melanophore system is described, which employs pieces from the ventral tail-fin of Xenopus laevis tadpoles. Tail-fin melanophores in vitro retain the ability to disperse their pigment in darkness and to reaggregate it upon illumination. In the light, alpha-MSH, cAMP, dibutyryl-cAMP and theophylline induce a concentration-dependent pigment dispersion. The log dose-response curve obtained with alpha-MSH is sigmoidal with a linear portion between 0.5 and 2.0 ng alpha-MSH/ml. In this range, the log dose-response curve can be used as the standard curve in a bioassay for melanotropic activity, applying either the melanophore index (assay I) or a photometric transmittance measurement (assay II) for the quantification of the melanophore response. To prevent interference from the light/darkness response, light of 400-500 nm (to which the melanophores are most sensitive) was used during the assay. Both assays show high precision (lambda I = 0.13, lambda II = 0.11). Several peptides derived from alpha-MSH were tested for their melanotropic activity. The in vitro Xenopus melanophore system offers unique properties for the study of alpha-MSH action: (1) the melanophore system is uncontaminated with other chromatophores; (2) to date it is the only system suitable for photoaffinity labelling of alpha-MSH receptors; and (3) the melanophore receptor requirements differ from those of Rana.