L-dopa binding sites in rodent melanoma cells.

Rapid, saturable, specific and stereoselective binding of L-dopa to crude membranes and purified nuclei from rodent amelanotic melanoma cells is reported. Cross-linking of [3H]dopa to melanoma cell surface emphasized proteins of approx. 55, 30, 25 and less than 20 kDa. It is suggested that these binding sites may regulate melanocyte activity.

Melanogenesis during the anagen-catagen-telogen transformation of the murine hair cycle.

Melanin synthesis of follicular melanocytes is strictly coupled to the growth stage of the hair cycle (anagen), ceases during follicle regression (catagen), and is absent throughout the resting stage (telogen). Having previously characterized the expression and activity of melanogenesis-related proteins during the telogen-anagen transition of the murine hair cycle (JID 96:172, 1991), we here report a biophysical and biochemical analysis of follicular melanogenesis during the anagen-catagen-telogen transformation of the C57 BL-6 mouse hair cycle. Tyrosinase activity and concentration as well as dopachrome tautomerase activity were compared with melanin synthesis, as measured by electron paramagnetic resonance spectroscopy (EPR). The visible changes in skin color and the histologically appreciable switch-off of melanin formation during the anagen-catagen transformation were accompanied by a steep decline in 1) the melanin-associated EPR signal of full-thickness mouse skin, 2) tyrosinase and dopachrome tautomerase activities, and 3) the skin concentration of 80-85-kD melanogenesis related protein and 66-68-kD tyrosinase protein. Telogen skin displayed a minimum of the EPR amplitude as well as of tyrosinase and dopachrome tautomerase activity detected. By EPR, only eumelanin was identified during all hair cycle stages. The gradual switch-off of melanogenesis during anagen VI started with an unexpectedly early decline of the EPR melanin signal, followed by dopachrome tautomerase activity and the concentration of 80-85-kD melanogenesis related protein. The initiation of catagen was characterized by a significant and rapid decrease in activity and concentration of tyrosinase, and was accompanied by a second drop in dopachrome tautomerase activity. Together, these biochemical and biophysical parameters of follicular melanogenesis serve as novel and differential markers for the imminent termination of anagen and the development of catagen. They also show that the switch-off of melanogenesis during the anagen-catagen-telogen transition is a stochastic process commencing already in mid anagen VI.

Identification and characterization of two isozymic forms of arylamine N-acetyltransferase in Syrian hamster skin.

Arylamine N-acetyltransferase (EC 2.3.1.5) activity was examined using skin from Syrian hamster. Two isozymes of arylamine N-acetyltransferase, designated NAT-1 and NAT-2, were detected on anion-exchange high-performance liquid chromatography analysis. Both enzyme activities had indistinguishable molecular masses (30 kDa), but differed significantly in their specificity toward the aromatic amines including serotonin, dopamine, methoxytryptamine, tryptamine, para-phenetidine, para-aminobenzoic acid, and sulphamethazine. Specifically, NAT-2 but not NAT-1 catalyzed acetylation of dopamine to N-acetyldopamine and acetylation of serotonin to form N-acetylserotonin, a direct precursor of melatonin. The two isozymes were also distinguishable based upon their sensitivity toward methotrexate inhibition (50% inhibiting dose for NAT-1 = 380 microM; NAT-2 > 2 mM). The presence of these two activities in the skin raises new questions about the physiologic role of this enzyme in general and in the skin-specific functions in particular.

Melatonin inhibits proliferation and melanogenesis in rodent melanoma cells.

The effects of melatonin on proliferation and on the induction of melanogenesis in rodent melanoma cells were investigated. It was found that melatonin at low concentrations (0.1-10 nM) inhibited cell growth but had no effect on melanogenesis, while at high concentrations (> or = 0.1 microM) it inhibited the induction of melanogenesis but not cell growth. These effects were specific since corresponding concentrations of the direct precursor and product of melatonin degradation N-acetylserotonin (N-Ac-5HT) and 5-methoxytryptamine (5MT), respectively, did not have any effect on cell proliferation or melanogenesis. At very high concentration (100 microM) both N-Ac-5HT and melatonin could stimulate melanoma proliferation while 5MT inhibited it. The demonstration of differential and unparalleled effects of melatonin on cell proliferation and melanogenesis suggests that melatonin can regulate or modify both processes via different mechanisms.