title: 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 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.
authors.affiliation: Research Service, North Florida/South Georgia Veterans Health System, and Department of Medicine, University of Florida College of Medicine, Gainesville, Florida 32610-0224, USA. xiasl@medicine.ufl.edu
authors.forename: Shen-Ling
authors.initials: SL
authors.lastname: Xia
authors.forename: Lanjun
authors.initials: L
authors.lastname: Wang
authors.forename: Melanie N
authors.initials: MN
authors.lastname: Cash
authors.forename: Xueling
authors.initials: X
authors.lastname: Teng
authors.forename: Ruth A
authors.initials: RA
authors.lastname: Schwalbe
authors.forename: Charles S
authors.initials: CS
authors.lastname: Wingo
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D005110:Extracellular Space / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D007679:Kidney Medulla; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D012333:RNA, Messenger / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; D000255:Adenosine Triphosphate; D002118:Calcium