Crow J M, Atkinson M M, Johnson R G
Department of Genetics and Cell Biology, University of Minnesota, St. Paul.
Invest Ophthalmol Vis Sci. 1994 Jul;35(8):3332-41.
To investigate in bovine and embryonic chicken lens cultures the effects of elevated intracellular calcium on the permeability of gap junctions. To determine the changes in intracellular calcium using fura-2. To detect any changes in the phosphorylation of connexin43 after ionophore treatment.
Lucifer yellow was micro-injected into individual cells, and dye spread to neighboring cells was evaluated. Intracellular calcium levels were measured using the calcium indicator, fura-2. Cultures were also labeled with 32P-orthophosphate followed by immunoprecipitation with antibodies specific for the gap junction protein, connexin43.
Bovine lens cultures incubated in the presence of either A23187 or ionomycin showed a reduction in intercellular dye transfer. The intracellular calcium concentrations in bovine cells were increased from a mean value of 0.11 +/- .009 microM in the controls to a mean of 0.40 +/- .073 microM with ionomycin treatment. Subsequent addition of EGTA to the medium decreased the intracellular calcium concentrations to a mean of 0.26 +/- .113 microM and reversed the inhibition of dye spread found with ionomycin. With ionomycin in the medium, the phosphorylated form of connexin43 was not as prominent as in the controls. In contrast, these same treatments had no detectable effect on junctional permeability in the embryonic chicken lens cultures. Dye spread was equally extensive and rapid under control and ionophore conditions, even though fura studies showed an elevation in intracellular calcium levels.
In the bovine cultures, physiologically relevant changes in the levels of cytoplasmic calcium markedly reduced dye transfer. The increase in cytoplasmic calcium was correlated with a change in the phosphorylation level of connexin43. The regulation of junctional communication in the chick lens cultures appears to differ significantly from that in the bovine system.
在牛和胚胎鸡晶状体培养物中研究细胞内钙升高对缝隙连接通透性的影响。使用fura-2测定细胞内钙的变化。检测离子载体处理后连接蛋白43磷酸化的任何变化。
将荧光黄显微注射到单个细胞中，并评估染料向相邻细胞的扩散。使用钙指示剂fura-2测量细胞内钙水平。培养物还用32P-正磷酸盐标记，然后用缝隙连接蛋白43特异性抗体进行免疫沉淀。
在A23187或离子霉素存在下孵育的牛晶状体培养物显示细胞间染料转移减少。牛细胞内钙浓度从对照组的平均值0.11±0.009微摩尔增加到离子霉素处理后的平均值0.40±0.073微摩尔。随后向培养基中添加乙二醇双四乙酸（EGTA）将细胞内钙浓度降低到平均值0.26±0.113微摩尔，并逆转了离子霉素引起的染料扩散抑制。在培养基中加入离子霉素后，连接蛋白43的磷酸化形式不如对照组突出。相比之下，这些相同的处理对胚胎鸡晶状体培养物中的连接通透性没有可检测到的影响。即使fura研究显示细胞内钙水平升高，在对照和离子载体条件下染料扩散同样广泛且迅速。
在牛培养物中，细胞质钙水平的生理相关变化显著降低了染料转移。细胞质钙的增加与连接蛋白43磷酸化水平的变化相关。鸡晶状体培养物中连接通讯的调节似乎与牛系统中的调节有显著差异。
