AGGREGATION OF PIGMENT GRANULES IN SINGLE CULTURED XENOPUS-LAEVIS MELANOPHORES BY MELATONIN ANALOGS

1 Isolated melanophores were differentiated from aggregates of neural crest obtained from neurula stage Xenopus laevis embryos after 2 days in culture. 2 Condensation of pigment granules in these cells by melatonin (5-methoxy N-acetyltryptamine, aMT) and various novel analogues was monitored with an image analysis system to quantitate the area occupied by pigment in individual cells. 3 Melanophores exposed to vehicle (a maximum of 0.1% MeOH) showed little (< 5%) change in pigment area. aMT produced a dramatic condensation of pigment granules (EC50 = the concentration producing a half maximal condensation, 9 pM). The response was rapid, reached a maximum (approximately 80% decrease in pigmented area) by 10 min, and was reversible after removal of aMT from the culture medium. 4 Aggregation to aMT was blocked by treating melanophores with pertussis toxin (1-mu-g ml-1, 7 h) indicating a role for a guanosine 5' triphosphate (GTP)-binding protein in transducing the aMT receptor signal. 5 Structure-activity studies indicated that analogues of aMT lacking a side-chain N-acyl substituent (5-methoxytryptamine, MT) or a group at the 5-position of the indole ring (N-acetyltryptamine, aT) were unable to induce pigment aggregation (EC50 > 10-mu-M). 6 Lengthening the side-chain N-acyl group (N-propionyl, N-butanoyl) was tolerated to some degree but eventually (N-valeroyl and larger) activity diminished. Of the 5-position analogues tested 5-methoxy (aMT) was by far the most potent. 7 Halogen substitution in the 6-position of the indole ring led to some loss of activity as did a 6-OH substitution. The 6-OCH3 compound was inactive. 8 These studies demonstrate the utility of this model in investigations of structure-activity relationships at the aMT receptor and suggest that it may be a valuable system for determining the transduction mechanisms coupled to the aMT receptor.