Department of Physiology, University of Birmingham, Birmingham, UK.
J Cell Biochem. 2010 Jan 1;109(1):132-9. doi: 10.1002/jcb.22390.
HKC-8 cells are a human-derived renal proximal tubular cell line and provide a useful model system for the study of human renal cell function. In this study, we aimed to determine Ca(2+) signalling mediated by P2 receptor in HKC-8. Fura-2 and a ratio imaging method were employed to measure Ca(2+) in HKC-8 cells. Our results showed that activation of P2Y receptors by ATP induced a rise in Ca(2+) that was dependent on an intracellular source of Ca(2+), while prolonged activation of P2Y receptors induced a rise in Ca(2+) that was dependent on intra- and extracellular sources of Ca(2+). Pharmacological and molecular data in this study suggests that TRPC4 channels mediate Ca(2+) entry in coupling to activation of P2Y in HKC-8 cells. U73221, an inhibitor of PI-PLC, did not inhibit the initial ATP-induced response; whereas D609, an inhibitor of PC-PLC, caused a significant decrease in the initial ATP-induced response, suggesting that P2Y receptors are coupled to PC-PLC. Although P2X were present in HKC-8, The P2X agonist, alpha,beta me-ATP, failed to cause a rise in Ca(2+). However, PPADS at a concentration of 100 microM inhibits the ATP-induced rise in Ca(2+). Our results indicate the presence of functional P2Y receptors in HKC-8 cells. ATP-induced Ca(2+) elevation via P2Y is tightly associated with PC-PLC and TRP channel.
HKC-8 细胞是人肾近端小管细胞系，是研究人肾细胞功能的有用模型系统。在这项研究中，我们旨在确定 HKC-8 中 P2 受体介导的 Ca(2+)信号转导。我们使用 Fura-2 和比率成像方法测量 HKC-8 细胞中的 Ca(2+)。我们的结果表明，ATP 激活 P2Y 受体诱导 Ca(2+)的升高依赖于细胞内 Ca(2+)的来源，而 P2Y 受体的长期激活诱导 Ca(2+)的升高依赖于细胞内外 Ca(2+)的来源。本研究的药理学和分子数据表明，TRPC4 通道介导与 P2Y 激活偶联的 Ca(2+)内流在 HKC-8 细胞中。PI-PLC 的抑制剂 U73221 不能抑制初始 ATP 诱导的反应；而 PC-PLC 的抑制剂 D609 则导致初始 ATP 诱导的反应显著减少，表明 P2Y 受体与 PC-PLC 偶联。尽管 HKC-8 中存在 P2X，但 P2X 激动剂 α,β me-ATP 未能引起 Ca(2+)的升高。然而，浓度为 100 μM 的 PPADS 可抑制 ATP 诱导的 Ca(2+)升高。我们的结果表明，HKC-8 细胞中存在功能性 P2Y 受体。ATP 通过 P2Y 诱导的 Ca(2+)升高与 PC-PLC 和 TRP 通道密切相关。
