Kidney tubules are targets for the activation of locally released nucleotides through multiple P2 receptor types. Activation of these P2 receptors modulates cellular Ca(2+) 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 Ca(2+) homeostasis. To monitor intracellular Ca(2+) concentration ([Ca(2+)](i)), experiments were conducted using the fluorescent dye fura 2. ATP (0.1-100 microM) produced a dose-dependent increase in [Ca(2+)](i) in a physiological Ca(2+)-containing solution, with an EC(50) of 2.5 microM. The P2-receptor antagonist PPADS reduced the effect of ATP on [Ca(2+)](i), and the P1-receptor agonist adenosine caused only a small increase in [Ca(2+)](i). Preincubation of cells with the phospholipase C antagonist U-73122 blocked the ATP-induced increase in [Ca(2+)](i), indicating P2Y receptors were involved in this process. In a Ca(2+)-free bath solution, thapsigargin and ATP induced intracellular Ca(2+) release from an identical pool. Nucleotides caused an increase in [Ca(2+)](i) in the potency order of UTP = ATP > ATP gamma S > ADP > UDP that is best fitted with the P2Y(2) subtype profile. Although the P2Y agonist UTP induced a similar large transient increase in [Ca(2+)](i) as did ATP, a small but sustained increase in [Ca(2+)](i) occurred only in ATP-stimulated cells, suggesting the role of P2X receptors in Ca(2+) influx. The sustained increase in [Ca(2+)](i) could be blocked by either nonselective cation channel blockers Gd(3+) or P2X antagonists PPADS and PPNDS. Furthermore, when either Gd(3+) or PPNDS was applied to the bath solution before ATP application, the ATP-induced increase in [Ca(2+)](i) was significantly reduced. Both RT-PCR and Western blotting corroborated the presence of P2X(1) and P2Y(2) receptors. 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 Ca(2+) signaling.