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.