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The role of alpha-MSH, its agonists, and C-AMP in in vitro avian melanocytes
R R Bowers1, A Biboso, O Chavez
1Department of Biology and Microbiology, California State University, Los Angeles 90032, USA. rbowers@calstatela.edu
Abstract:
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.
Insights
Alpha-melanocyte-stimulating hormone (alpha-MSH) stimulates melanogenesis in avian feather melanocytes by activating cyclic AMP (c-AMP) as its second messenger. Other tested compounds did not increase melanogenesis, suggesting specific cellular responses to alpha-MSH.
Area of Science:
- Cell Biology
- Endocrinology
- Developmental Biology
Background:
- Limited understanding of melanogenic stimulators' effects on avian melanocytes.
- Feather regeneration involves melanocyte activity and pigment production.
Purpose of the Study:
- To investigate the effects of alpha-MSH and other compounds on melanogenesis in avian feather melanocytes.
- To elucidate the signaling pathway, specifically the role of cyclic AMP (c-AMP), in alpha-MSH-induced melanogenesis in birds.
Main Methods:
- Establishment of Barred Plymouth Rock regenerating feather melanocyte tissue cultures.
- Treatment with varying concentrations of alpha-MSH, Forskolin, IBMX, c-GMP, db-c-AMP, and 8-bromo-c-AMP.
- Assessment of melanogenesis via melanosome stage and melanocyte counts; calcium's role was also evaluated.
- Inhibition studies using Rp-c-AMPS and direct measurement of intracellular c-AMP levels post-alpha-MSH treatment.
Main Results:
- Alpha-MSH (2.5-10 µg/ml) significantly increased melanogenesis, indicated by more stage IV melanosomes.
- Forskolin, IBMX, c-GMP, and db-c-AMP did not enhance melanogenesis.
- Calcium (2 mM) potentiated alpha-MSH's pigment-enhancing effect.
- Rp-c-AMPS inhibited alpha-MSH's effect, and alpha-MSH treatment increased intracellular c-AMP levels, confirming c-AMP as the second messenger.
Conclusions:
- Avian feather melanocytes possess functional alpha-MSH receptors and respond to the hormone.
- Cyclic AMP (c-AMP) serves as the crucial second messenger mediating alpha-MSH action in these cells.
- The inability of db-c-AMP, Forskolin, and IBMX to stimulate melanogenesis suggests limitations in cell entry or signaling activation in mature avian melanocytes, unlike the effective 8-bromo-c-AMP.