Jansen L A, de Vrije T, Jongen W M
Agrotechnological Research Institute, Department of Food Safety, Wageningen, The Netherlands.
Carcinogenesis. 1996 Nov;17(11):2311-9. doi: 10.1093/carcin/17.11.2311.
Differences in calcium-mediated regulation of gap junctional intercellular communication (GJIC) between a cell line consisting of mouse epidermal initiated cells (3PC) and a mouse epidermal carcinoma-derived cell line (CA3/7) were studied. Under low extracellular calcium ((Ca2+)e) conditions (0.05 mM) CA3/7 cells showed a low level of GJIC compared with 3PC cells. High (Ca2+)e (1.20 mM) raised GJIC between CA3/7 cells to the GJIC level of 3PC cells, which in turn remained unchanged under these conditions. Raising the free intracellular calcium concentration ((Ca2+)i), using a calcium ionophore (ionomycin) or the Ca2+-ATPase inhibitor thapsigargin under low (Ca2+)e conditions, did not affect the GJIC level between 3PC cells, and increased GJIC between CA3/7 cells. Intracellular calcium chelation in 3PC cells under low (Ca2+)e conditions by ethylene glycol-bis(beta-amino-ethyl ether) N,N,N',N'-tetra-acetic acid acetoxy-methyl ester (EGTA-AM) decreased GJIC in this cell line. High (Ca2+)e conditions protected both cell lines from a decreased GJIC by EGTA-AM exposure. Inhibition of calmodulin (CaM) by calmidazolium (CDZ) or N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) under low (Ca2+)e conditions, inhibited GJIC in 3PC cells and increased GJIC in CA3/7 cells. Inhibition of Ca2+/CaM-dependent protein kinase (Ca2+/CaM-PK) by 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-7) decreased GJIC in both cell lines. Western analysis showed that Cx43 was more phosphorylated in both cell lines in concurrence with different effects on the GJIC level. Under conditions in which GJIC was inhibited, a decreased immunostaining of Cx43 on the plasma membrane was found. The level of immunostaining of the cell adhesion molecule E-cadherin on the plasma membranes of both cell types remained unchanged under conditions in which GJIC was changed by modulaters of (Ca2+)i, CaM activity, or the Ca2+/CaM-PK activity. These results indicate that differences exist between 3PC cells and CA3/7 cells in the GJIC regulation by intracellular calcium and calmodulin.
研究了由小鼠表皮起始细胞组成的细胞系（3PC）和小鼠表皮癌衍生细胞系（CA3/7）之间钙介导的间隙连接细胞间通讯（GJIC）调节的差异。在低细胞外钙（（Ca2+）e）条件（0.05 mM）下，与3PC细胞相比，CA3/7细胞的GJIC水平较低。高（Ca2+）e（1.20 mM）使CA3/7细胞之间的GJIC升高至3PC细胞的GJIC水平，而在这些条件下3PC细胞的GJIC水平保持不变。在低（Ca2+）e条件下，使用钙离子载体（离子霉素）或Ca2+-ATP酶抑制剂毒胡萝卜素提高细胞内游离钙浓度（（Ca2+）i），不影响3PC细胞之间的GJIC水平，但增加了CA3/7细胞之间的GJIC。在低（Ca2+）e条件下，用乙二醇双（β-氨基乙醚）N，N，N'，N'-四乙酸乙酰氧基甲酯（EGTA-AM）对3PC细胞进行细胞内钙螯合，降低了该细胞系的GJIC。高（Ca2+）e条件可保护两种细胞系免受EGTA-AM暴露导致的GJIC降低。在低（Ca2+）e条件下，用氯丙咪嗪（CDZ）或N-（6-氨基己基）-5-氯-1-萘磺酰胺（W-7）抑制钙调蛋白（CaM），抑制了3PC细胞的GJIC并增加了CA3/7细胞的GJIC。用1-（5-碘萘-1-磺酰基）-1H-六氢-1,4-二氮杂卓（ML-7）抑制Ca2+/CaM依赖性蛋白激酶（Ca2+/CaM-PK），降低了两种细胞系的GJIC。蛋白质免疫印迹分析表明，两种细胞系中Cx43的磷酸化程度更高，这与对GJIC水平的不同影响一致。在GJIC受到抑制的条件下，发现质膜上Cx43的免疫染色减少。在通过（Ca2+）i、CaM活性或Ca2+/CaM-PK活性调节剂改变GJIC的条件下，两种细胞类型质膜上细胞粘附分子E-钙粘蛋白的免疫染色水平保持不变。这些结果表明，3PC细胞和CA3/7细胞在细胞内钙和钙调蛋白对GJIC的调节方面存在差异。
