Hoffman S, Gopalakrishna R, Gundimeda U, Murata T, Spee C, Ryan S J, Hinton D R
Doheny Eye Institute, Los Angeles, CA, USA.
Exp Eye Res. 1998 Jul;67(1):45-52. doi: 10.1006/exer.1998.0491.
The effects of three calcium channel blockers, verapamil, diltiazem and nifedipine, were examined on in vitro proliferation and migration of human retinal pigment epithelial cells. Human retinal pigment epithelial cells were seeded in Dulbecco's modified essential medium with 10% fetal bovine serum and different concentrations of the three calcium channel blockers. After 3 days of treatment, cell proliferation was determined by cell counting and by [3H]-thymidine uptake. Cell viability was determined with trypan blue exclusion. For determination of cell migration, retinal pigment epithelial cells were grown to confluence and then growth-inhibited with mitomycin C. After a 3 mm zone was denuded, the cells were treated with different concentrations of the calcium channel antagonists. After 24 hr, the cells that had migrated over the wound edge were counted. To determine the involvement of protein kinase C in the verapamil effect, its activity was measured in both verapamil-treated and untreated cells. Verapamil dose dependently inhibited serum-induced proliferation of retinal pigment epithelial cells, when measured by cell number (IC50 14.6 microM) or [3H]-thymidine incorporation (IC50 11.3 microM). At concentrations of 15 microM and below, there was no effect on cell viability, as determined by morphology and trypan blue exclusion. Diltiazem inhibited cell proliferation at a concentration of 100 microM; however, 100 microM nifedipine had no effect. Verapamil showed a significant inhibition of serum-induced migration in the range of 10 microM to 0.1 microM. The IC50 of the inhibition of retinal pigment epithelial cell proliferation and migration by verapamil is significantly higher than that seen for effects on calcium channel blockage. Eight micromolar verapamil reversibly inhibited total protein kinase-C activity in retinal pigment epithelial cells suggesting the possibility that the drug may act by inhibiting the protein kinase-C pathway. These data suggest the potential of the calcium channel blocker verapamil as a pharmacological modulator of disorders such as proliferative vitreoretinopathy in which there is increased retinal pigment epithelial cell proliferation and migration.
研究了三种钙通道阻滞剂维拉帕米、地尔硫䓬和硝苯地平对人视网膜色素上皮细胞体外增殖和迁移的影响。将人视网膜色素上皮细胞接种于含10%胎牛血清及不同浓度这三种钙通道阻滞剂的杜尔贝科改良伊格尔培养基中。处理3天后，通过细胞计数和[3H] - 胸腺嘧啶核苷摄取来测定细胞增殖。用台盼蓝排斥法测定细胞活力。为了测定细胞迁移，将视网膜色素上皮细胞培养至汇合，然后用丝裂霉素C抑制生长。在刮除3mm区域后，用不同浓度的钙通道拮抗剂处理细胞。24小时后，对迁移至伤口边缘的细胞进行计数。为了确定蛋白激酶C是否参与维拉帕米的作用，在维拉帕米处理和未处理的细胞中均测量其活性。当通过细胞数量（IC50为14.6μM）或[3H] - 胸腺嘧啶核苷掺入（IC50为11.3μM）测量时，维拉帕米剂量依赖性地抑制血清诱导的视网膜色素上皮细胞增殖。在15μM及以下浓度时，通过形态学和台盼蓝排斥法测定，对细胞活力无影响。地尔硫䓬在100μM浓度时抑制细胞增殖；然而，100μM硝苯地平无作用。维拉帕米在10μM至0.1μM范围内对血清诱导的迁移有显著抑制作用。维拉帕米抑制视网膜色素上皮细胞增殖和迁移的IC50显著高于其对钙通道阻滞作用的IC50。8μM维拉帕米可逆性抑制视网膜色素上皮细胞中的总蛋白激酶C活性，提示该药物可能通过抑制蛋白激酶C途径发挥作用。这些数据表明钙通道阻滞剂维拉帕米作为增殖性玻璃体视网膜病变等疾病的药理学调节剂的潜力，在这些疾病中视网膜色素上皮细胞增殖和迁移增加。
