THE ROLE OF ALPHA-MSH, ITS AGONISTS, AND CAMP IN IN-VITRO AVIAN MELANOCYTES

Little is known about the effect of alpha-MSH and other melanogenic st imulators on avian melanocytes. Tissue cultures of Barred Plymouth Roc k regenerating feather melanocytes were established and the culture me dium contained selected concentrations of alpha-MSH and other melanoge nic stimulators in Ham's F-10 medium supplemented with antibiotics and 10% new born calf serum. Cultures were maintained at 37 degrees C in 95% air/5% CO2. No increase in melanogenesis over control levels due t o the addition of 10(-5) M Forskolin, 10(4) M IBMX, 10(-3) M c-GMP, an d 10(3) M db-c-AMP was observed in the cultures on days 5 and 7. Howev er, 2.5 (optimum), 5, and 10 mu g/ml alpha-MSH and 10(-3) M 8-bromo-c- AMP significantly increased melanogenesis over control levels on days 5 and 7. The stimulation of melanogenesis was detectable by a signific antly increased number of melanocytes containing numerous stage IV mel anosomes. No increase in melanocyte cell number was observed in any of the experimental cultures. The addition of 1, 2 (optimum), or 3 mM ca lcium did enhance the increased pigmentation effect of 2.5 mu g/ml alp ha-MSH. Two very convincing experiments showed that c-AMP was the seco nd messenger for alpha-MSH in these birds. First, the c-AMP inhibitor, 10(-3) M Rp-c-AMPS, completely inhibited the stimulatory effect of al pha-MSH in these in vitro melanocytes. Second, direct measurements of c-AMP levels in feather tissue showed a significant increase in c-AMP levels 10 min after alpha-MSH treatment. Controls received no alpha-MS H. The results showed that these avian melanocytes have alpha-MSH rece ptors and were able to respond to the hormone. C-AMP was the second me ssenger in this system. Apparently db-c-AMP was not able to enter thes e mature, highly-differentiated cells and c-AMP agonists, Forskolin an d IBMX, were also either unable to enter these older cells or if they did enter the cells, were unable to stimulate c-AMP production. Eviden tly the more lipophilic 8-bromo-c-AMP was able to enter these cells an d stimulate melanogenesis.