Abad Elena, Lorente Gisela, Gavara Núria, Morales Miguel, Gual Arcadi, Gasull Xavier
Laboratory of Neurophysiology, Faculty of Medicine, School of Medicine, University of Barcelona, Barcelona, Spain.
Invest Ophthalmol Vis Sci. 2008 Feb;49(2):677-86. doi: 10.1167/iovs.07-1080.
In nonexcitable cells, G(q)-coupled membrane receptor activation induces a biphasic increase in intracellular calcium (Ca(2+)) expressed as an initial IP(3)-dependent release from intracellular stores followed by a sustained Ca(2+) influx from the extracellular space that involves store-operated Ca(2+) channels (SOCs). In trabecular meshwork (TM) cells, contractile agonists such as bradykinin (BK) and endothelin-1 (ET-1) induce this type of Ca(2+) signaling. Given that trabecular outflow is modified by tissue contractility, the authors characterized SOCs and studied their participation in TM cell contractility.
Ca(2+) was measured in cultured bovine TM cells loaded with Fura-2. Ca(2+) currents were recorded using the patch clamp technique. Cell contractility measurements were assessed by traction microscopy.
BK and ET-1 activate a store-operated Ca(2+) entry that was greatly reduced in the absence of extracellular Ca(2+) or by preincubation with SOC blocker 2-APB or SKF96365. Store-operated Ca(2+) currents were also activated by intracellular dialysis with IP(3) + EGTA or after stimulation with thapsigargin. Electrophysiological characterization supports the presence of Ca(2+) release-activated Ca(2+) channels (CRACs) and nonselective cation channels, of which TRPC1 and TRPC4 channels may be candidate TRPs detected in TM cells. Extracellular Ca(2+) entry through SOCs is not required for TM cell contraction in response to BK or ET-1, but it modulates this process.
Extracellular Ca(2+) entry in TM cells in response to agonist stimulation and store-depletion is mediated by the activation of SOCs, which do not contribute to cell contraction but which may activate regulatory mechanisms to prevent excessive contraction. CRAC and TRPC channels involved represent interesting modulators of TM function to improve aqueous humor outflow.
在非兴奋性细胞中，G(q)偶联膜受体激活可诱导细胞内钙（[Ca(2+)]i）出现双相增加，表现为最初依赖肌醇三磷酸（IP(3)）从细胞内储存库释放钙，随后是持续的钙从细胞外空间内流，这涉及储存-操作性钙通道（SOCs）。在小梁网（TM）细胞中，收缩性激动剂如缓激肽（BK）和内皮素-1（ET-1）可诱导这种类型的钙信号传导。鉴于小梁网流出受组织收缩性影响，作者对SOCs进行了表征并研究了它们在TM细胞收缩性中的作用。
使用负载Fura-2的培养牛TM细胞测量[Ca(2+)]i。采用膜片钳技术记录钙电流。通过牵引显微镜评估细胞收缩性测量。
BK和ET-1激活一种储存-操作性钙内流，在无细胞外钙或用SOC阻滞剂2-APB或SKF96365预孵育时，该内流显著减少。用IP(3)+乙二醇双四乙酸（EGTA）进行细胞内透析或用毒胡萝卜素刺激后，也可激活储存-操作性钙电流。电生理特征支持存在钙释放激活钙通道（CRACs）和非选择性阳离子通道，其中瞬时受体电位通道蛋白1（TRPC1）和瞬时受体电位通道蛋白4（TRPC4）通道可能是在TM细胞中检测到的候选瞬时受体电位通道（TRPs）。响应BK或ET-1时，通过SOCs的细胞外钙内流并非TM细胞收缩所必需，但它可调节这一过程。
激动剂刺激和储存耗竭时，TM细胞中的细胞外钙内流由SOCs激活介导，SOCs对细胞收缩无作用，但可能激活调节机制以防止过度收缩。所涉及的CRAC和TRPC通道是改善房水流出的TM功能的有趣调节因子。
