Abstract:

Objective To investigate the roles of glutamate signaling pathway in melanin transfer. Methods Epidermal melanocytes and keratinocytes were isolated from human foreskin tissue followed by purification and primary culture. Immunofluorescence microscopy was conducted to observe the intracellular distribution of N-methy-D-aspartate receptor 1 （NMDAR1） and NMDAR2A in melanocytes. Some melanocytes were classified into 4 groups to be pretreated with MK801 （the NMDAR antagonist dizocilpine maleate） at 100 μmol/L for 5 minutes followed by treatment with NMDA （an NMDAR agonist） at 100 μmol/L （MK801-pretreated group 1）, pretreated with MK801 at 100 μmol/L for 1 hour followed by treatment with NMDA at 100 μmol/L （MK801-pretreated group 2）, treated with MK801 at 100 μmol/L for 5 minutes （MK801 group）, treated with NMDA at 100 μmol/L for 5 minutes （NMDA group）, respectively, then, confocal microscopy was performed to measure the intracellular calcium （Ca2+） concentration of the melanocytes. The distribution of β-tubulin was visualized by confocal microscopy in melanocytes treated with MK801 at 100 μmol/L for 24 hours. Some melanocytes and keratinocytes were cocultured with or without MK801 at 100 μmol/L for 24 or 48 hours, then, scaning microscopy was carried out to observe the junction structure between melanocytes and keratinocytes, and alkali method coupled with spectrophotometric analysis to determine melanin content in keratinocytes. Results The intracellular calcium concentration of melanocytes was decreased by MK-801, but increased by NMDA at 100 μmol/L, and the increase was blocked by the pretreatment with MK-801 for 5 minutes or 1 hour. After incubation with MK-801 at 100 μmol/L for 24 hours, a more intense staining for β-tubulin was observed around the nuclei of melanocytes. There was a significant reduction in the number of filopodia on the surface of and between melanocytes and keratinocytes after treatment with MK-801 at 100 μmol/L for 48 hours. Also, the content of melanin （represented as the absorbance value at 375 nm） transferred from melanocytes into keratinocytes was statistically reduced in coculture system treated with MK-801 at 100 μmol/L compared with that without treatment （0.158 ± 0.003 vs. 2.203 ± 0.006, t = 6.323, P < 0.01）. Conclusions The glutamate signaling pathway exerts a regulatory effect on intracellular calcium concentration of, distribution of β-tubulin in, filopodia formation of melanocytes and melanin transfer between melanocytes and keratinocytes.

Key words: filopodia