ATP release from human airway epithelial cells studied using a capillary cell culture system
ATP release from human airway epithelial cells studied using a capillary cell culture system
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DOI：
10.1113/jphysiol.2002.030148
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发表时间：
2002-11-15
影响因子：
5.5
通讯作者：
Hanrahan, JW
中科院分区：
文献类型：
--
作者：
Guyot, A;Hanrahan, JW
三磷酸腺苷（ATP）是一种重要的自分泌和旁分泌信号分子，其上皮释放具有急性机械敏感性，因此难以研究。我们在这里描述了一种新的制剂，最大限度地减少机械和代谢的扰动，并用它来检查上皮细胞的ATP分泌。将来自人气道粘膜下腺体的Calu-3细胞系在中空纤维生物反应器中在通过再循环培养基泵灌注的多孔毛细管上培养。细胞开始极化，培养物稳定> 5个月，这可以通过显微镜和乳酸盐产生（类似于250 μ g（10（8）个细胞）（-1）天（-1））来证明。心尖流速升高5倍使ATP分泌从200增加到6618 fmol min（-1）。将心尖渗透压降低25- 43%也会增加ATP分泌，然后ATP分泌自发下降至平台率，只要维持低渗灌注，该平台率就持续存在。释放失活后迅速剪切和渗透应力被终止，并没有与可检测的细胞裂解。降低顶端[Ca 2 +]，以增加连接蛋白半通道的通透性也刺激ATP的释放和分泌增加在低渗和剪切应力;然而，连接蛋白43阻滞剂氟芬那酸抑制剪切诱导的ATP释放仅在低钙溶液中，因此另一个分泌途径可能与生理（即毫米）钙。无论机制，目前的结果量化ATP对机械和渗透刺激的反应，并证明毛细血管培养研究上皮分泌的有用性。
Epithelial release of adenosine triphosphate (ATP), an important autocrine and paracrine signalling molecule, is acutely mechanosensitive and therefore difficult to study. We describe here a novel preparation that minimizes mechanical and metabolic perturbations, and use it to examine ATP secretion by epithelial cells. The Calu-3 cell line derived from human airway sub-mucosal glands was cultured in a hollow fibre bioreactor on porous capillaries that were perfused by a recirculating medium pump. Cells became polarized and cultures were stable for > 5 months, as evidenced by microscopy and lactate production (similar to250 mug (10(8) cells)(-1) day(-1)). Elevating apical flow rate 5-fold increased ATP secretion from similar to200 to 6618 fmol min(-1). Reducing apical osmolarity by 25-43 % also increased ATP secretion, which then declined spontaneously to a plateau rate that persisted as long as hypotonic perfusion was maintained. Release deactivated rapidly after shear and osmotic stresses were terminated, and was not associated with detectable cell lysis. Lowering apical [Ca2+] to increase connexin hemichannel permeability also stimulated ATP release and increased secretion during both hyposmotic and shear stress; however, the connexin 43 blocker flufenamic acid inhibited shear-induced ATP release only in low-Ca2+ solution, and therefore another secretory pathway may operate with physiological (i.e. mm) calcium. Regardless of the mechanism, the present results quantify ATP responses to mechanical and osmotic stimuli and demonstrate the usefulness of capillary cultures for studying epithelial secretion.