LYSOPHOSPHATIDIC ACID INHIBITS GAP-JUNCTIONAL COMMUNICATION AND STIMULATES PHOSPHORYLATION OF CONNEXIN-43 IN WB CELLS - POSSIBLE INVOLVEMENT OF THE MITOGEN-ACTIVATED PROTEIN-KINASE CASCADE
LYSOPHOSPHATIDIC ACID INHIBITS GAP-JUNCTIONAL COMMUNICATION AND STIMULATES PHOSPHORYLATION OF CONNEXIN-43 IN WB CELLS - POSSIBLE INVOLVEMENT OF THE MITOGEN-ACTIVATED PROTEIN-KINASE CASCADE
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DOI：
10.1042/bj3030475
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发表时间：
1994-10-15
影响因子：
4.1
通讯作者：
MURRAY, AW
中科院分区：
文献类型：
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作者：
HII, CST;OH, SY;MURRAY, AW
溶血磷脂酸（LPA）被证明是一个强大的抑制剂的培养大鼠肝WB细胞之间的间隙连接通信，确定通过转移的荧光黄，在约0.3 μ M LPA获得50%的抑制。通信抑制是快速的（5分钟），并保持至少80分钟。孵育3小时后，与LPA，通信能力部分恢复和染料转移是难治性的进一步添加LPA。通讯中的LPA-难治性细胞保持敏感性抑制佛波酯和表皮生长因子（EGF）。LPA诱导的抑制与连接蛋白-43蛋白的磷酸化相关，如通过蛋白质印迹上检测到的蛋白质的较慢迁移所检测到的，这可以通过将样品与碱性磷酸酶孵育来消除。观察到LPA对通信的作用的时间和剂量依赖性与诱导丝裂原活化蛋白激酶（MAP激酶）之间的密切对应关系。通过使用抗（MAP激酶R1）（erk 1-III）抗体在蛋白质印迹上的迁移率变化和通过Mono Q柱上的分级分离证实了42 kDa和44 kDa亚种的活化。用佛波酯预处理24 h的细胞对佛波酯抑制通讯或激活MAP激酶不敏感，但保留其对LPA的敏感性。结果表明，LPA启动WB细胞中的蛋白激酶级联的激活，这可能是独立的蛋白激酶C，并确定连接蛋白-43作为激活的激酶的底物之一。
Lysophosphatidic acid (LPA) was shown to be a powerful inhibitor of gap-junctional communication between cultured rat liver WB cells, as determined by the transfer of Lucifer Yellow, with 50% inhibition obtained at about 0.3 mu M LPA. Inhibition of communication was rapid (5 min) and was maintained for at least 80 min. After incubation for 3 h with LPA, communication competence was partially restored and dye transfer was refractory to further addition of LPA. Communication in LPA-refractory cells retained sensitivity to inhibition by phorbol ester and by epidermal growth factor (EGF). LPA-induced inhibition was associated with phosphorylation of connexin-43 protein, as detected by slower migration of the protein detected on Western blots, which could be eliminated by incubation of samples with alkaline phosphatase. A close correspondence was observed between the time- and dose-dependency of LPA effects on communication and the induction of mitogen-activated protein kinase (MAP kinase). Activation of both the 42 kDa and 44 kDa subspecies were confirmed by mobility shifts on Western blots using an anti-(MAP kinase R1) (erk 1-III) antibody and by fractionation on Mono Q columns. Cells pretreated with phorbol ester for 24 h were insensitive to phorbol ester inhibition of communication or activation of MAP kinase, but retained their sensitivity to LPA. The results indicate that LPA initiates the activation of protein kinase cascades in WB cells that are probably independent of protein kinase C and identifies connexin-43 as one substrate for the activated kinases.