The Mechanism Of Glutamate Signaling Pathway In The Epidermis

Objective :To investigate the mechanism of glutamate signaling pathway in the epidermis.Methods:1.After culturing the human epidermal melanocyte and keratinocyte respectively, we observed the intracellular distribution of NMDAR1 and NMDAR2A in melanocyte and keratinocyte using immunofluorescence microscopy. Confocal microscopy was performed to measure intracellular Ca2+ concentration and observe the intracellular distribution ofβ-tubulin in melanocyte and keratinocyte .The apoptosis of keratinocyte was measured by flow cytometry.2.Melanocyte and keratinocyte were co-cultured, then the morphology changes were imaged by scanning microscopy. The microplate reader was performed to determine the absorbance of melanin particles in keratinocyte.Results:1.Immunofluorescence microscopy showed that NMDAR1 and NMDAR2A were distributed in the cytoplasm of melanocyte.Stimulation with 100μM NMDA or MK-801 induced an increase or decrease in intracellular Ca2+ concentration of melanocyte.The responses to 100μM NMDA were blocked afer 5min or 1h incubation of the melanocyte with the NMDAR inhibitor MK-801(100μM).After incubation of the melanocyte for 24h with 100μM MK-801,dendrites were retracted and there was more intense staining forβ-tubulin around the nuclei of the melanocyte.2.Immunofluorescence microscopy showed that NMDAR1 and NMDAR2A were distributed in the cytoplasm of keratinocyte.Stimulation with 100μM NMDA induced an increase in intracellular Ca2+ concentration of keratinocyte.After incubation of the keratinocyte for 24h with 100μM MK-801, there was more intense staining forβ-tubulin around the nuclei of thekeratinocyte. 3.Scanning electron microscope found that NMDAR inhibitor MK-801 reduced the number of the filopodias in the melanocyte.The absorbance of melanin particles was reduced in keratinocyte with 100μM MK-801.Conclutions:1.Melanocyte and keratinocyte have the expression of NMDAR1 and NMDAR2A.2.Glutamate signaling pathway can affect the intracellular calcium concentration in MC.3.Glutamate signaling pathway can affect morphology and the number of the filopodias by acting onβ-tubulin of the melanocyte .4.Glutamate signaling pathway can affect morphology,the intracellular calcium concentration and the intracellular distribution ofβ-tubulin in keratinocyte,but this had nothing to do with the apoptosis of the keratinocyte.