Little is known about the effect of alpha-MSH and other melanogenic stimulators on avian melanocytes. Tissue cultures of Barred Plymouth Rock regenerating feather melanocytes were established and the culture medium contained selected concentrations of alpha-MSH and other melanogenic 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 to the addition of 10(-5) M Forskolin, 10(-4) M IBMX, 10(-3) M c-GMP, and 10(-3) M db-c-AMP was observed in the cultures on days 5 and 7. However, 2.5 (optimum), 5, and 10 micrograms/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 significantly increased number of melanocytes containing numerous stage IV melanosomes. No increase in melanocyte cell number was observed in any of the experimental cultures. The addition of 1, 2 (optimum), or 3 mM calcium did enhance the increased pigmentation effect of 2.5 micrograms/ml alpha-MSH. Two very convincing experiments showed that c-AMP was the second messenger for alpha-MSH in these birds. First, the c-AMP inhibitor, 10(-3) M Rp-c-AMPS, completely inhibited the stimulatory effect of alpha-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-MSH. The results showed that these avian melanocytes have alpha-MSH receptors and were able to respond to the hormone. C-AMP was the second messenger in this system. Apparently db-c-AMP was not able to enter these mature, highly-differentiated cells and c-AMP agonists, Forskolin and IBMX, were also either unable to enter these older cells or, if they did enter the cells, were unable to stimulate c-AMP production. Evidently the more lipophilic 8-bromo-c-AMP was able to enter these cells and stimulate melanogenesis.
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