The effect of blue light on Ca2+-protein kinase C signaling pathway in human retinal pigment epithelial cells in vitro

Abstract

Objective To investigate the effect of blue light on Ca2+-protein kinase C (PKC)signaling pathway in human retinal pigment epithelial (RPE) cells in vitro.Methods Primary human RPE cells were cultured in vitro and characterized.The experiments were carried out using the 4th generation of human RPE cells.The PKC protein level was measured by Western blot to determine the most appropriate concentration of phorbol ester (PMA) and calcium phosphate binding protein (calphostin C) on PKC expression.Non-radioactive isotope method was used to determine the effect of blue light on PKC expression of cultured cells.Blue-light damage model of human RPE cells was established by 6 hour irradiation of medical blue-light lamp [20 W,450-500 nm wavelength,(2000± 500) Lux],and 24 hours prolongation of post-exposure culture.The human RPE cells were randomly divided into 5 groups.Group A did not receive light irradiation,group B only received blue light irradiation,group C was blue light irradiation and 0.1 mmol/L nifedipine treatment,group D was blue light irradiation and 100.0 nmol/L calphostin C treatment,group E was blue light irradiation and 100.0 nmol/L PMA treatment.Intracellular Ca2-concentration was measured by acetoxymethyl ester (Fluo 3-AM) labelling and confocal microscope imaging.Results The PKC protein expression in 100.0 nmol/L or 200.0 nmol/L PMA-treated groups was higher than 0.1,1.0,10.0,and 50.0 nmol/L PMA-treated groups,the difference was statistically significant (F=217.537,P＜0.05),but there was no statistically difference between 100.0 nmol/L and 200.0 nmol/L PMA-treated groups (P =0.072).The PKC protein expression in 100.0 nmol/L or 200.0 nmol/L calphostin C-treated groups was lower than 5.0,25.0,50.0,and 75.0 nmol/L calphostin C-treated groups,the difference was statistically significant (F=164.543,P＜0.05),but there was no statistically difference between 100.0 nmol/L and 200.0 nmol/L calphostin C treated groups (P=0.385).PKC level in blue light group was higher than non-light group,the difference was statistically significant (t =-9.869,P＜0.05).The Ca2+ fluorescence intensity values in group B,C,D and E was higher than group A,the difference was statistically significant (F=26 764.92,P＜0.05).The Ca2+ fluorescence intensity values in group E was higher than group B,C and D (P＜0.05),and that in group B was higher than group C and D (P＜0.05).Conclusions The PKC activity and intracellular Ca2+ concentration in human RPE cells increase after blue light irradiation.Both calcium channel inhibitor nifedipine and PKC inhibitor calphostin C can reduce intracellular Ca2+ concentration in human RPE cells.PMA can induce intracellular Ca2+ concentration in human RPE cells after blue light irradiation. Key words: Eye injuries/physiopathology; Retinal pigment epithelium; Apoptosis; Photic stimulation