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

¹Research Service, North Florida/South Georgia Veterans Health System, and ²Department of Medicine, University of Florida College of Medicine, Gainesville, Florida 32610-0224; and ³Department of Biochemistry, Brody School of Medicine, East Carolina University, Greenville, North Carolina 27858

Submitted 11 August 2003 ; accepted in final form 29 March 2004

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⁺]_i in a physiological Ca²⁺-containing solution, with an EC₅₀ of 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⁺]_i in the potency order of UTP = ATP > ATPS > ADP > UDP that is best fitted with the P2Y₂ subtype profile. Although the P2Y agonist UTP induced a similar large transient increase in [Ca²⁺]_i as did ATP, a small but sustained increase in [Ca²⁺]_i occurred only in ATP-stimulated cells, suggesting the role of P2X receptors in Ca²⁺ influx. The sustained increase in [Ca²⁺]_i could 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⁺]_i was significantly reduced. Both RT-PCR and Western blotting corroborated the presence of P2X₁ and P2Y₂ 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 Ca²⁺ signaling.

epithelia; purinergic receptors; collecting duct; calcium channel; kidney

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