Zsembery Akos, Boyce Amanda T, Liang Lihua, Peti-Peterdi János, Bell P Darwin, Schwiebert Erik M
Department of Physiology and Biophysics, and the Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama, Birmingham, Alabama 35294-0005, USA.
J Biol Chem. 2003 Apr 11;278(15):13398-408. doi: 10.1074/jbc.M212277200. Epub 2003 Feb 3.
Purinergic receptor stimulation has potential therapeutic effects for cystic fibrosis (CF). Thus, we explored roles for P2Y and P2X receptors in stably increasing Ca(2+) in human CF (IB3-1) and non-CF (16HBE14o(-)) airway epithelial cells. Cytosolic Ca(2+) was measured by fluorospectrometry using the fluorescent dye Fura-2/AM. Expression of P2X receptor (P2XR) subtypes was assessed by immunoblotting and biotinylation. In IB3-1 cells, ATP and other P2Y agonists caused only a transient increase in Ca(2+) derived from intracellular stores in a Na(+)-rich environment. In contrast, ATP induced an increase in Ca(2+) that had transient and sustained components in a Na(+)-free medium; the sustained plateau was potentiated by zinc or increasing extracellular pH. Benzoyl-benzoyl-ATP, a P2XR-selective agonist, increased Ca(2+) only in Na(+)-free medium, suggesting competition between Na(+) and Ca(2+) through P2XRs. Biochemical evidence showed that the P2X(4) receptor is the major subtype shared by these airway epithelial cells. A role for store-operated Ca(2+) channels, voltage-dependent Ca(2+) channels, or Na(+)/Ca(2+) exchanger in the ATP-induced sustained Ca(2+) signal was ruled out. In conclusion, these data show that epithelial P2X(4) receptors serve as ATP-gated calcium entry channels that induce a sustained increase in Ca(2+). In airway epithelia, a P2XR-mediated Ca(2+) signal may have therapeutic benefit for CF.
嘌呤能受体刺激对囊性纤维化（CF）具有潜在的治疗作用。因此，我们探讨了P2Y和P2X受体在稳定增加人CF（IB3-1）和非CF（16HBE14o(-)）气道上皮细胞内钙离子浓度（Ca(2+)）方面的作用。使用荧光染料Fura-2/AM通过荧光光谱法测量胞质钙离子浓度。通过免疫印迹和生物素化评估P2X受体（P2XR）亚型的表达。在IB3-1细胞中，ATP和其他P2Y激动剂在富含Na(+)的环境中仅引起源自细胞内储存的Ca(2+)短暂增加。相比之下，ATP在无Na(+)培养基中诱导Ca(2+)增加且具有短暂和持续成分；锌或增加细胞外pH可增强持续平台期。P2XR选择性激动剂苯甲酰-苯甲酰-ATP仅在无Na(+)培养基中增加Ca(2+)，表明Na(+)和Ca(2+)通过P2XR存在竞争。生化证据表明P2X(4)受体是这些气道上皮细胞共有的主要亚型。排除了储存操纵性钙离子通道、电压依赖性钙离子通道或Na(+)/Ca(2+)交换体在ATP诱导的持续钙离子信号中的作用。总之，这些数据表明上皮P2X(4)受体作为ATP门控的钙内流通道，可诱导Ca(2+)持续增加。在气道上皮中，P2XR介导的钙离子信号可能对CF具有治疗益处。
