The biological effects of catecholamines in mammalian pigment cells are poorly understood. Our previous results showed the presence of alpha(1)-adrenoceptors in SK-Mel 23 human melanoma cells. The aims of this work were to (1) characterize catecholamine effects on proliferation, tyrosinase activity and expression, (2) identify the alpha(1)-adrenoceptor subtypes, and (3) verify whether chronic norepinephrine (NE) treatment modified the types and/or pharmacological characteristics of adrenoceptors present in SK-Mel 23 human melanoma cells. Cells treated with the alpha(1)-adrenergic agonist, phenylephrine (PHE, 10(-5) or 10(-4) M), for 24-72 h, exhibited decreased cell proliferation and enhanced tyrosinase activity, but unaltered tyrosinase expression as compared with the control. The proliferation and tyrosinase activity responses were inhibited by the alpha(1)-adrenergic antagonist prazosin, suggesting they were evoked by alpha(1)-adrenoceptors. The presence of actinomycin D, a transcription inhibitor, did not diminish PHE-induced effects. RT-PCR assays, followed by cloning and sequencing, demonstrated the presence of alpha(1A)- and alpha(1B)-adrenoceptor subtypes. NE-treated cells (24 or 72 h) were used in competition assays, and showed no significant change in the competition curves of alpha(1)-adrenoceptors as compared with control curves. Other adrenoceptor subtypes were not identified in these cells, and NE pretreatment did not induce their expression. In conclusion, the activation of SK-Mel 23 human melanoma alpha(1)-adrenoceptors elicit biological effects, such as proliferation decrease and tyrosinase activity increase. Desensitization or expression of other adrenoceptor subtypes after chronic NE treatment were not observed.

No abstract

No abstract

The biological effects of catecholamines in mammalian pigment cells are poorly understood, but in poikilothermic vertebrates they regulate the translocation of pigment granules. We have previously demonstrated in SK-Mel 23-human melanoma cells the presence of low affinity alpha(1)-adrenoceptors, which mediate a decrease in cell proliferation and increase in tyrosinase activity, with no change of tyrosinase expression. In this report, we investigated the signalling pathways involved in these responses. Calcium mobilization in response to phenylephrine (PHE), an alpha(1)-adrenergic agonist, was investigated by confocal microscopy, and no change of fluorescence during the treatment was observed, suggesting that calcium is not involved in the signalling pathway activated by alpha(1)-adrenoceptors in SK-Mel 23 cells. cAMP levels, determined by enzyme-immunoassay, were significantly increased by PHE (10(-5)-10(-4)M), that could be blocked by the alpha(1)-adrenergic antagonist benoxathian (10(-5)-10(-4)M). Several biological assays were then performed with PHE, for 72 h, in the absence or presence of various signalling pathway inhibitors, in an attempt to determine the intracellular messengers involved in the responses of proliferation and tyrosinase activity. Our results suggest the participation of p38 and ERKs in PHE-induced decrease of proliferation, and possibly also of cAMP and protein kinase A. Regarding PHE-induced increase of tyrosinase activity, it is suggested that the following signalling components are involved: cAMP/PKA, PKC, PI3K, p38 and ERKs.

No abstract