The effects of extracellular nucleotides on [Ca 2+ ] i signalling in a human‐derived renal proximal tubular cell line (HKC‐8)

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+ ] i signalling mediated by P2 receptor in HKC‐8. Fura‐2 and a ratio imaging method were employed to measure [Ca 2+ ] i in HKC‐8 cells. Our results showed that activation of P2Y receptors by ATP induced a rise in [Ca 2+ ] i that was dependent on an intracellular source of Ca 2+ , while prolonged activation of P2Y receptors induced a rise in [Ca 2+ ] i 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, α,β me‐ATP, failed to cause a rise in [Ca 2+ ] i . However, PPADS at a concentration of 100 µM inhibits the ATP‐induced rise in [Ca 2+ ] i . Our results indicate the presence of functional P2Y receptors in HKC‐8 cells. ATP‐induced [Ca 2+ ] i elevation via P2Y is tightly associated with PC‐PLC and TRP channel. J. Cell. Biochem. 109: 132–139, 2010. © 2009 Wiley‐Liss, Inc.