Metabolic inhibition induces opening of unapposed connexin 43 gap junction hemichannels and reduces gap junctional communication in cortical astrocytes in culture
Metabolic inhibition induces opening of unapposed connexin 43 gap junction hemichannels and reduces gap junctional communication in cortical astrocytes in culture
复制标题
DOI：
10.1073/pnas.012589799
复制
发表时间：
2002-01-08
影响因子：
11.1
通讯作者：
Sáez, JC
中科院分区：
文献类型：
--
作者：
Contreras, JE;Sánchez, HA;Sáez, JC
大鼠皮质星形胶质细胞在正常条件下通过连接蛋白（Cx）43间隙连接与相邻细胞功能性偶联。小的荧光分子如荧光黄（LY）通过间隙连接在细胞内部之间通过，但在外部应用时不会进入细胞。通过抑制糖酵解和氧化代谢，使大鼠和小鼠皮质星形胶质细胞遭受“化学缺血”，诱导细胞对荧光黄和溴化乙锭透化，然后通过葡聚糖吸收和乳酸脱氢酶释放确定膜完整性丧失。差距连接阻断剂辛醇和18 α-大黄酸显著降低染料摄取，表明摄取是通过打开未对置的半通道介导的。细胞外La（3+）也降低了染料摄取和延迟细胞死亡。嘌呤能阻断剂氧化ATP无效。从具有Cx43编码DNA的靶向缺失的小鼠中分离的星形胶质细胞表现出大大降低的染料偶联和缺血诱导的染料摄取，这证明染料摄取是由Cx43半通道介导的。染料偶联减少，但不阻断代谢抑制。阻断脂氧合酶或用自由基清除剂处理减少大鼠星形胶质细胞的染料摄取，表明花生四烯酸副产物在半通道开放中的作用。此外，透化伴随着ATP水平的降低和Cx43的去磷酸化。虽然半通道开放将倾向于破坏垂死细胞质膜上的电化学和代谢梯度，但健康细胞可能通过Cx43间隙连接转移离子和必需代谢物来拯救垂死细胞。或者，垂死的星形胶质细胞可能通过Cx43间隙连接损害邻近细胞的健康，从而促进细胞死亡的传播。
Rat cortical astrocytes in pure culture are functionally coupled to neighboring cells via connexin (Cx) 43 gap junctions under ordinary conditions. Small fluorescent molecules such as Lucifer yellow (LY) pass between cell interiors via gap junctions, but do not enter the cells when externally applied. Subjecting rat and mouse cortical astrocytes to "chemical ischemia" by inhibition of glycolytic and oxidative metabolism induced permeabilization of cells to Lucifer yellow and ethidium bromide before loss of membrane integrity determined by dextran uptake and lactate dehydrogenase release. The gap junction blockers octanol and 18alpha-glycyrrhetinic acid markedly reduced dye uptake, suggesting that uptake was mediated by opening of unapposed hemichannels. Extracellular La(3+) also reduced dye uptake and delayed cell death. The purinergic blocker, oxidized ATP, was ineffective. Astrocytes isolated from mice with targeted deletion of the Cx43 coding DNA exhibited greatly reduced dye coupling and ischemia-induced dye uptake, evidence that dye uptake is mediated by Cx43 hemichannels. Dye coupling was reduced but not blocked by metabolic inhibition. Blockade of lipoxygenases or treatment with free radical scavengers reduced dye uptake by rat astrocytes, suggesting a role for arachidonic acid byproducts in hemichannel opening. Furthermore, permeabilization was accompanied by reduction in ATP levels and dephosphorylation of Cx43. Although hemichannel opening would tend to collapse electrochemical and metabolic gradients across the plasma membrane of dying cells, healthy cells might rescue dying cells by transfer of ions and essential metabolites via Cx43 gap junctions. Alternatively, dying astrocytes might compromise the health of neighboring cells via Cx43 gap junctions, thereby promoting the propagation of cell death.