The effect of blue light on Ca<sup>2+</sup>-protein kinase C signaling pathway in human retinal pigment epithelial cells in vitro_Chinese Journal of Ocular Fundus Diseases

Authors：

WuZhipeng ,  CaiShanjun , LyuJianping , 李海辉 , 宫鑫 , 宿罡 , 汪利敏 , 王丽丽

Department of Ophthalmology, The Affiliated Hospital of Zunyi Medical University, Zunyi 563003, China;

Corresponding author：

CaiShanjun, Email: caishanjun@163.com

Keywords：

Eye injuries/physiopathology; Retinal pigment epithelium; Apoptosis; Photic stimulation

DOI：

10.3760/cma.j.issn.1005-1015.2014.03.014

Video：

Export PDF Favorites Scan Get Citation

Abstract Full text Figures/Tables Video References Cited by

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.

Citation： WuZhipeng, CaiShanjun, LyuJianping. The effect of blue light on Ca²⁺-protein kinase C signaling pathway in human retinal pigment epithelial cells in vitro. Chinese Journal of Ocular Fundus Diseases, 2014, 30(3): 284-288. doi: 10.3760/cma.j.issn.1005-1015.2014.03.014 Copy

1.	Organisciak DT, Darrow RM, Barsalou L, et al. Light history and age-related changes in retinal light damage[J]. Invest Ophthalmol Vis Sci,1998,39:1107-1116.
2.	蔡善君,严密,张军军.蓝光诱导体外培养的人视网膜色素上皮细胞凋亡[J].中华眼底病志,2005,21:384-387.
3.	蔡善君,严密,毛咏秋.蓝光致人视网膜色素上皮细胞凋亡与线立体膜电位和细胞色素C的关系[J].中华眼科杂志,2006,42:1095-1102.
4.	Parekh AB, Putney JW Jr. Store-operated calcium channels[J]. Physiol Rev,2005,85:757-810.
5.	宿罡,宫鑫,蔡善君,等.蓝光所致人视网膜色素上皮细胞损伤与细胞内钙离子含量变化的关系[J].中华实验眼科杂志,2013,31:734-738.
6.	毛俊峰,刘双珍,秦文娟,等.蛋白激酶C活性调节剂对豚鼠近视眼视网膜Müller细胞的作用及意义[J].眼科研究,2009,27:5-9.
7.	王中群,李丽华,赵卫星,等.蛋白激酶C活性变化影响内皮-单核细胞黏附[J].中华老年心脑血管病杂志,2008,10:529-532.
8.	宫鑫,蔡善君,李海辉,等.蓝光照射对人视网膜色素上皮细胞L-型钙通道mRNA表达的影响[J].中华眼底病杂志,2013,29:411-415.
9.	Lucas M, Sánchez-Margalet V. Protein kinase C involvement in apoptosis[J]. Gen Pharmacol,1995,26:881-887.
10.	Westlund BS, Cai BL, Zhou JL, et al. Involvement of c-Abl, p53 and the MAP kinase JNK in the cell death program initiated in A2E-laden ARPE-19 cells by exposure to blue light[J]. Apoptosis,2009,14:31-41.
11.	Kumar S, Kain V, Sitasawad SL. High glucose-induced Ca²⁺ overload and oxidative stress contribute to apoptosis of cardiac cells through mitochondrial dependent and independent pathways[J]. Biochim Biophys Acta,2012,1820:907-920.
12.	Pang DB, Hong J.[Ca²⁺]i homeostasis and caspase-3 gene expression in verapamil-induced retinal pigment epithelium cells apoptosis in vitro[J]. Int J Ophthalmol,2010,3:288-290.

1. Organisciak DT, Darrow RM, Barsalou L, et al. Light history and age-related changes in retinal light damage[J]. Invest Ophthalmol Vis Sci,1998,39:1107-1116.
2. 蔡善君,严密,张军军.蓝光诱导体外培养的人视网膜色素上皮细胞凋亡[J].中华眼底病志,2005,21:384-387.
3. 蔡善君,严密,毛咏秋.蓝光致人视网膜色素上皮细胞凋亡与线立体膜电位和细胞色素C的关系[J].中华眼科杂志,2006,42:1095-1102.
4. Parekh AB, Putney JW Jr. Store-operated calcium channels[J]. Physiol Rev,2005,85:757-810.
5. 宿罡,宫鑫,蔡善君,等.蓝光所致人视网膜色素上皮细胞损伤与细胞内钙离子含量变化的关系[J].中华实验眼科杂志,2013,31:734-738.
6. 毛俊峰,刘双珍,秦文娟,等.蛋白激酶C活性调节剂对豚鼠近视眼视网膜Müller细胞的作用及意义[J].眼科研究,2009,27:5-9.
7. 王中群,李丽华,赵卫星,等.蛋白激酶C活性变化影响内皮-单核细胞黏附[J].中华老年心脑血管病杂志,2008,10:529-532.
8. 宫鑫,蔡善君,李海辉,等.蓝光照射对人视网膜色素上皮细胞L-型钙通道mRNA表达的影响[J].中华眼底病杂志,2013,29:411-415.
9. Lucas M, Sánchez-Margalet V. Protein kinase C involvement in apoptosis[J]. Gen Pharmacol,1995,26:881-887.
10. Westlund BS, Cai BL, Zhou JL, et al. Involvement of c-Abl, p53 and the MAP kinase JNK in the cell death program initiated in A2E-laden ARPE-19 cells by exposure to blue light[J]. Apoptosis,2009,14:31-41.
11. Kumar S, Kain V, Sitasawad SL. High glucose-induced Ca²⁺ overload and oxidative stress contribute to apoptosis of cardiac cells through mitochondrial dependent and independent pathways[J]. Biochim Biophys Acta,2012,1820:907-920.
12. Pang DB, Hong J.[Ca²⁺]i homeostasis and caspase-3 gene expression in verapamil-induced retinal pigment epithelium cells apoptosis in vitro[J]. Int J Ophthalmol,2010,3:288-290.

Chinese Journal of Ocular Fundus Diseases

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

Abstract Full text Figures/Tables Video References Cited by

Previous Article

Next Article

Format

Content

Chinese Journal of Ocular Fundus Diseases

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

Abstract Full text Figures/Tables Video References Cited by

Previous Article

Next Article

Format

Content

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