Parr C E, Sullivan D M, Paradiso A M, Lazarowski E R, Burch L H, Olsen J C, Erb L, Weisman G A, Boucher R C, Turner J T
Division of Pulmonary Diseases, University of North Carolina, Chapel Hill 27599-7020.
Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3275-9. doi: 10.1073/pnas.91.8.3275.
The Cl- secretory pathway that is defective in cystic fibrosis (CF) can be bypassed by an alternative pathway for Cl- transport that is activated by extracellular nucleotides. Accordingly, the P2 receptor that mediates this effect is a therapeutic target for improving Cl- secretion in CF patients. In this paper, we report the sequence and functional expression of a cDNA cloned from human airway epithelial (CF/T43) cells that encodes a protein with properties of a P2U nucleotide receptor. With a retrovirus system, the human airway clone was stably expressed in 1321N1 astrocytoma cells, a human cell line unresponsive to extracellular nucleotides. Studies of inositol phosphate accumulation and intracellular Ca2+ mobilization induced by extracellular nucleotides in 1321N1 cells expressing the receptor identified this clone as the target receptor in human airway epithelia. In addition, we independently isolated an identical cDNA from human colonic epithelial (HT-29) cells, indicating that this is the same P2U receptor that has been functionally identified in other human tissues. Expression of the human P2U receptor (HP2U) in 1321N1 cells revealed evidence for autocrine ATP release and stimulation of transduced receptors. Thus, HP2U expression in the 1321N1 cell line will be useful for studying autocrine regulatory mechanisms and in screening of potential therapeutic drugs.
囊性纤维化（CF）中存在缺陷的氯离子分泌途径可被细胞外核苷酸激活的另一种氯离子转运途径所绕过。因此，介导这种效应的P2受体是改善CF患者氯离子分泌的治疗靶点。在本文中，我们报告了从人气道上皮（CF/T43）细胞克隆的一个cDNA的序列和功能表达，该cDNA编码一种具有P2U核苷酸受体特性的蛋白质。利用逆转录病毒系统，将人气道克隆稳定表达于1321N1星形细胞瘤细胞中，该细胞系对细胞外核苷酸无反应。对表达该受体的1321N1细胞中细胞外核苷酸诱导的肌醇磷酸积累和细胞内钙离子动员的研究确定该克隆为人气道上皮中的靶受体。此外，我们从人结肠上皮（HT-29）细胞中独立分离出一个相同的cDNA，表明这与在其他人体组织中已被功能鉴定的P2U受体相同。人P2U受体（HP2U）在1321N1细胞中的表达揭示了自分泌ATP释放和转导受体刺激的证据。因此，HP2U在1321N1细胞系中的表达将有助于研究自分泌调节机制和筛选潜在的治疗药物。
