Extracellular ATP-induced calcium signaling in mIMCD-3 cells requires both P2X and P2Y purinoceptors

Abstract

Kidney tubules are targets for the activation of locally released nucleotides through multiple P2 receptor types. Activation of these P2 receptors modulates cellular Ca2+signaling and downstream cellular function. The purpose of this study was to determine whether P2 receptors were present in mIMCD-3 cells, a mouse inner medullary collecting duct cell line, and if so, to examine their link with intracellular Ca2+homeostasis. To monitor intracellular Ca2+concentration ([Ca2+]i), experiments were conducted using the fluorescent dye fura 2. ATP (0.1–100 μM) produced a dose-dependent increase in [Ca2+]iin a physiological Ca2+-containing solution, with an EC50of 2.5 μM. The P2-receptor antagonist PPADS reduced the effect of ATP on [Ca2+]i, and the P1-receptor agonist adenosine caused only a small increase in [Ca2+]i. Preincubation of cells with the phospholipase C antagonist U-73122 blocked the ATP-induced increase in [Ca2+]i, indicating P2Y receptors were involved in this process. In a Ca2+-free bath solution, thapsigargin and ATP induced intracellular Ca2+release from an identical pool. Nucleotides caused an increase in [Ca2+]iin the potency order of UTP = ATP > ATPγS > ADP > UDP that is best fitted with the P2Y2subtype profile. Although the P2Y agonist UTP induced a similar large transient increase in [Ca2+]ias did ATP, a small but sustained increase in [Ca2+]ioccurred only in ATP-stimulated cells, suggesting the role of P2X receptors in Ca2+influx. The sustained increase in [Ca2+]icould be blocked by either nonselective cation channel blockers Gd3+or P2X antagonists PPADS and PPNDS. Furthermore, when either Gd3+or PPNDS was applied to the bath solution before ATP application, the ATP-induced increase in [Ca2+]iwas significantly reduced. Both RT-PCR and Western blotting corroborated the presence of P2X1and P2Y2receptors. These studies demonstrate that mIMCD-3 cells have both P2X and P2Y subtype receptors and that the activation of both P2X and P2Y receptors by extracellular ATP appears to be required to regulate intracellular Ca2+signaling.