Xia Shen-Ling, Wang Lanjun, Cash Melanie N, Teng Xueling, Schwalbe Ruth A, Wingo Charles S
Research Service, North Florida/South Georgia Veterans Health System, and Department of Medicine, University of Florida College of Medicine, Gainesville, Florida 32610-0224, USA.
Am J Physiol Renal Physiol. 2004 Aug;287(2):F204-14. doi: 10.1152/ajprenal.00281.2003. Epub 2004 Apr 6.
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+)), experiments were conducted using the fluorescent dye fura 2. ATP (0.1-100 microM) produced a dose-dependent increase in Ca(2+) 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+), and the P1-receptor agonist adenosine caused only a small increase in Ca(2+). Preincubation of cells with the phospholipase C antagonist U-73122 blocked the ATP-induced increase in Ca(2+), 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+) 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+) as did ATP, a small but sustained increase in Ca(2+) occurred only in ATP-stimulated cells, suggesting the role of P2X receptors in Ca(2+) influx. The sustained increase in Ca(2+) 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+) 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.
肾小管是局部释放的核苷酸通过多种P2受体类型激活的靶标。这些P2受体的激活调节细胞内Ca(2+)信号传导和下游细胞功能。本研究的目的是确定P2受体是否存在于mIMCD-3细胞（一种小鼠髓质内集合管细胞系）中，如果存在，则研究它们与细胞内Ca(2+)稳态的联系。为了监测细胞内Ca(2+)浓度(Ca(2+))，使用荧光染料fura 2进行实验。在含有生理Ca(2+)的溶液中，ATP(0.1 - 100 microM)使Ca(2+)呈剂量依赖性增加，EC(50)为2.5 microM。P2受体拮抗剂PPADS降低了ATP对Ca(2+)的作用，而P1受体激动剂腺苷仅使Ca(2+)有小幅增加。用磷脂酶C拮抗剂U - 73122预孵育细胞可阻断ATP诱导的Ca(2+)增加，表明P2Y受体参与了这一过程。在无Ca(2+)的浴液中，毒胡萝卜素和ATP诱导细胞内Ca(2+)从同一池中释放。核苷酸使Ca(2+)增加的效力顺序为UTP = ATP > ATPγS > ADP > UDP，这与P2Y(2)亚型特征最相符。尽管P2Y激动剂UTP诱导的Ca(2+)瞬时大幅增加与ATP相似，但仅在ATP刺激的细胞中出现了Ca(2+)的小幅持续增加，提示P2X受体在Ca(2+)内流中的作用。Ca(2+)的持续增加可被非选择性阳离子通道阻滞剂Gd(3+)或P2X拮抗剂PPADS和PPNDS阻断。此外，在加入ATP之前将Gd(3+)或PPNDS应用于浴液中，ATP诱导的Ca(2+)增加会显著降低。RT-PCR和蛋白质印迹法均证实了P
