Stalmans P, Himpens B
Laboratory of Physiology, Leuven, Belgium.
Invest Ophthalmol Vis Sci. 1997 Jul;38(8):1598-609.
The intercellular communication between cultured rat retinal pigment epithelial (RPE) cells grown in increasing glucose concentrations or after modulation of the protein kinase C-induced protein phosphorylation was investigated by studying the conduction of the [Ca2+]i wave elicited by mechanical stimulation and by analyzing the fluorescence recovery after photobleaching (FRAP).
Subconfluent monolayers of RPE cells isolated from neonatal Long Evans rats were cultured in growth medium with various glucose levels and analyzed using the fluorescent dye fluo-3 for measurements of intracellular Ca2+ after mechanical stimulation and using 6-carboxyfluorescein diacetate to investigate the intercellular communication with FRAP.
Mechanical stimulation in 5 or 12 mM glucose resulted in a Ca2+ wave that spread centrifugally through the neighboring cells. An inhibition of the propagation of this wave, similar to that induced by halothane, could be observed in cells grown for 72 hours in 14-mM or higher concentrations of glucose. This inhibitory effect was not caused by a hyperosmotic effect, in that results of experiments on cells cultured in growth medium supplemented with mannitol instead of glucose did not differ from those of experiments in the control medium. Activation of protein kinase C by incubation of the cells for 30 minutes with phorbol 12-myristate 13-acetate (PMA) resulted in a strong inhibition of [Ca2+]i-wave propagation. This inhibition did not depend on the oxidizing effects of PMA because the addition of glaucine, a known antioxidant, did not prevent the inhibition. Cells grown for 72 hours in glucose-rich medium (25 or 50 mM) and in which all protein kinase C activity was downregulated by a previous 72-hour exposure to 1 microM PMA, did not display the inhibitory effect on the propagation of the Ca2+ wave that is normally induced by this elevated glucose level. Stimulation or inhibition of protein kinase A activity by incubating RPE cells with Sp-cyclic adenosine monophosphate or Rp-cyclic adenosine monophosphate respectively, or inhibition of tyrosine kinase activity with herbimycin A did not alter the intercellular communication after mechanical stimulation. To determine whether the observed changes were caused by alterations in gap junction conductance (GJC), FRAP experiments were performed in control conditions, after a 30-minute incubation with PMA, and in cells cultured in 50 mM glucose in the presence and in the absence of 1 microM PMA. The measured GJC was consistent with the inhibitory effect on propagation of an intercellular Ca2+ wave in all tested conditions.
In RPE cells, a glucose concentration of 14 mM (224 mg/dl) or higher inhibits Ca(2+)-wave propagation and intercellular GJC. This effect may be mediated by protein kinase C activity.
通过研究机械刺激引发的[Ca2+]i波传导以及分析光漂白后荧光恢复（FRAP），探讨在葡萄糖浓度升高或蛋白激酶C诱导的蛋白质磷酸化被调节后，培养的大鼠视网膜色素上皮（RPE）细胞之间的细胞间通讯。
从新生Long Evans大鼠分离的RPE细胞亚汇合单层在含有不同葡萄糖水平的生长培养基中培养，并使用荧光染料fluo-3分析机械刺激后细胞内Ca2+的测量，使用6-羧基荧光素二乙酸酯通过FRAP研究细胞间通讯。
在5或12 mM葡萄糖中进行机械刺激会导致Ca2+波离心扩散到相邻细胞。在14 mM或更高浓度葡萄糖中培养72小时的细胞中，可以观察到该波传播的抑制，类似于氟烷诱导的抑制。这种抑制作用不是由高渗效应引起的，因为在补充甘露醇而非葡萄糖的生长培养基中培养细胞的实验结果与对照培养基中的实验结果没有差异。用佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）孵育细胞30分钟激活蛋白激酶C会导致[Ca2+]i波传播受到强烈抑制。这种抑制不依赖于PMA的氧化作用，因为添加已知的抗氧化剂青藤碱并不能阻止这种抑制。在富含葡萄糖的培养基（25或50 mM）中培养72小时且所有蛋白激酶C活性通过先前72小时暴露于1 microM PMA而下调的细胞，对通常由这种升高的葡萄糖水平诱导的Ca2+波传播没有显示出抑制作用。分别用Sp-环磷酸腺苷或Rp-环磷酸腺苷孵育RPE细胞刺激或抑制蛋白激酶A活性，或用赫曲霉素A抑制酪氨酸激酶活性，均未改变机械刺激后的细胞间通讯。为了确定观察到的变化是否由间隙连接电导（GJC）的改变引起，在对照条件下、用PMA孵育30分钟后以及在存在和不存在1 microM PMA的情况下在50 mM葡萄糖中培养的细胞中进行了FRAP实验。在所有测试条件下，测得的GJC与对细胞间Ca2+波传播的抑制作用一致。
在RPE细胞中，14 mM（224 mg/dl）或更高的葡萄糖浓度会抑制Ca(2+)-波传播和细胞间GJC。这种作用可能由蛋白激酶C活性介导。
