Okada Seiko F, Nicholas Robert A, Kreda Silvia M, Lazarowski Eduardo R, Boucher Richard C
Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, NC, USA.
J Biol Chem. 2006 Aug 11;281(32):22992-3002. doi: 10.1074/jbc.M603019200. Epub 2006 Jun 5.
Extracellular ATP and its metabolite adenosine regulate mucociliary clearance in airway epithelia. Little has been known, however, regarding the actual ATP and adenosine concentrations in the thin ( approximately 7 microm) liquid layer lining native airway surfaces and the link between ATP release/metabolism and autocrine/paracrine regulation of epithelial function. In this study, chimeric Staphylococcus aureus protein A-luciferase (SPA-luc) was bound to endogenous antigens on primary human bronchial epithelial (HBE) cell surface and ATP concentrations assessed in real-time in the thin airway surface liquid (ASL). ATP concentrations on resting cells were 1-10 nm. Inhibition of ecto-nucleotidases resulted in ATP accumulation at a rate of approximately 250 fmol/min/cm2, reflecting the basal ATP release rate. Following hypotonic challenge to promote cell swelling, cell-surface ATP concentration measured by SPA-luc transiently reached approximately 1 microm independent of ASL volume, reflecting a transient 3-log increase in ATP release rates. In contrast, peak ATP concentrations measured in bulk ASL by soluble luciferase inversely correlated with volume. ATP release rates were intracellular calcium-independent, suggesting that non-exocytotic ATP release from ciliated cells, which dominate our cultures, mediated hypotonicity-induced nucleotide release. However, the cystic fibrosis transmembrane conductance regulator (CFTR) did not participate in this function. Following the acute swelling phase, HBE cells exhibited regulatory volume decrease which was impaired by apyrase and facilitated by ATP or UTP. Our data provide the first evidence that ATP concentrations at the airway epithelial surface reach the range for P2Y2 receptor activation by physiological stimuli and identify a role for mucosal ATP release in airway epithelial cell volume regulation.
细胞外ATP及其代谢产物腺苷调节气道上皮的黏液纤毛清除功能。然而，关于天然气道表面内衬的薄（约7微米）液体层中实际的ATP和腺苷浓度，以及ATP释放/代谢与上皮功能的自分泌/旁分泌调节之间的联系，人们所知甚少。在本研究中，嵌合型金黄色葡萄球菌蛋白A-荧光素酶（SPA- luc）与原代人支气管上皮（HBE）细胞表面的内源性抗原结合，并实时评估薄气道表面液体（ASL）中的ATP浓度。静息细胞的ATP浓度为1 - 10纳米。抑制外核苷酸酶导致ATP以约250飞摩尔/分钟/平方厘米的速率积累，反映了基础ATP释放速率。在进行低渗刺激以促进细胞肿胀后，通过SPA- luc测量的细胞表面ATP浓度短暂达到约1微米，与ASL体积无关，反映出ATP释放速率瞬间增加了3个对数。相比之下，通过可溶性荧光素酶在大量ASL中测量的ATP峰值浓度与体积呈负相关。ATP释放速率与细胞内钙无关，这表明在我们的培养物中占主导的纤毛细胞以非胞吐方式释放ATP介导了低渗诱导的核苷酸释放。然而，囊性纤维化跨膜传导调节因子（CFTR）不参与此功能。在急性肿胀期之后，HBE细胞表现出调节性体积减小，这一过程被腺苷三磷酸双磷酸酶抑制，并被ATP或UTP促进。我们的数据首次证明气道上皮表面的ATP浓度达到了通过生理刺激激活P2Y2受体的范围，并确定了黏膜ATP释放在气道上皮细胞体积调节中的作用。
