The effects of extracellular nucleotides on [Ca2+]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 [Ca2+]i signalling mediated by P2 receptor in HKC-8. Fura-2 and a ratio imaging method were employed to measure [Ca2+]i in HKC-8 cells. Our results showed that activation of P2Y receptors by ATP induced a rise in [Ca2+]i that was dependent on an intracellular source of Ca2+, while prolonged activation of P2Y receptors induced a rise in [Ca2+]i that was dependent on intra- and extracellular sources of Ca2+. Pharmacological and molecular data in this study suggests that TRPC4 channels mediate Ca2+ 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 [Ca2+]i. However, PPADS at a concentration of 100 µM inhibits the ATP-induced rise in [Ca2+]i. Our results indicate the presence of functional P2Y receptors in HKC-8 cells. ATP-induced [Ca2+]i elevation via P2Y is tightly associated with PC-PLC and TRP channel. J. Cell. Biochem. 109: 132–139, 2010. © 2009 Wiley-Liss, Inc.