Antigny Fabrice, Norez Caroline, Cantereau Anne, Becq Frédéric, Vandebrouck Clarisse
Institut de Physiologie et Biologie Cellulaires, Université de Poitiers, CNRS, 86022 Poitiers, France.
Respir Res. 2008 Oct 30;9(1):70. doi: 10.1186/1465-9921-9-70.
In airway epithelial cells, calcium mobilization can be elicited by selective autocrine and/or paracrine activation of apical or basolateral membrane heterotrimeric G protein-coupled receptors linked to phospholipase C (PLC) stimulation, which generates inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG) and induces Ca2+ release from endoplasmic reticulum (ER) stores.
In the present study, we monitored the cytosolic Ca2+ transients using the UV light photolysis technique to uncage caged Ca2+ or caged IP3 into the cytosol of loaded airway epithelial cells of cystic fibrosis (CF) and non-CF origin. We compared in these cells the types of Ca2+ receptors present in the ER, and measured their Ca2+ dependent activity before and after correction of F508del-CFTR abnormal trafficking either by low temperature or by the pharmacological corrector miglustat (N-butyldeoxynojirimycin).
We showed reduction of the inositol 1,4,5-trisphosphate receptors (IP3R) dependent-Ca2+ response following both correcting treatments compared to uncorrected cells in such a way that Ca2+ responses (CF+treatment vs wild-type cells) were normalized. This normalization of the Ca2+ rate does not affect the activity of Ca2+-dependent chloride channel in miglustat-treated CF cells. Using two inhibitors of IP3R1, we observed a decrease of the implication of IP3R1 in the Ca2+ response in CF corrected cells. We observed a similar Ca2+ mobilization between CF-KM4 cells and CFTR-cDNA transfected CF cells (CF-KM4-reverted). When we restored the F508del-CFTR trafficking in CFTR-reverted cells, the specific IP3R activity was also reduced to a similar level as in non CF cells. At the structural level, the ER morphology of CF cells was highly condensed around the nucleus while in non CF cells or corrected CF cells the ER was extended at the totality of cell.
These results suggest reversal of the IP3R dysfunction in F508del-CFTR epithelial cells by correction of the abnormal trafficking of F508del-CFTR in cystic fibrosis cells. Moreover, using CFTR cDNA-transfected CF cells, we demonstrated that abnormal increase of IP3R Ca2+ release in CF human epithelial cells could be the consequence of F508del-CFTR retention in ER compartment.
在气道上皮细胞中，通过顶端或基底外侧膜异源三聚体G蛋白偶联受体的选择性自分泌和/或旁分泌激活可引发钙动员，这些受体与磷脂酶C（PLC）刺激相关联，后者生成肌醇1,4,5 -三磷酸（IP3）和1,2 -二酰基甘油（DAG），并诱导内质网（ER）储存中的Ca2+释放。
在本研究中，我们使用紫外光光解技术监测胞质Ca2+瞬变，以将笼化Ca2+或笼化IP3释放到囊性纤维化（CF）和非CF来源的负载气道上皮细胞的胞质溶胶中。我们比较了这些细胞中内质网中存在的Ca2+受体类型，并在通过低温或药物校正剂米格列醇（N -丁基脱氧野尻霉素）校正F508del - CFTR异常转运之前和之后测量了它们的Ca2+依赖性活性。
我们发现，与未校正的细胞相比，两种校正处理后，肌醇1,4,5 -三磷酸受体（IP3R）依赖性Ca2+反应均降低，使得Ca2+反应（CF +处理组与野生型细胞相比）正常化。Ca2+速率的这种正常化并不影响米格列醇处理的CF细胞中Ca2+依赖性氯通道的活性。使用两种IP3R1抑制剂，我们观察到CF校正细胞中IP3R1在Ca2+反应中的作用降低。我们在CF - KM4细胞和CFTR - cDNA转染的CF细胞（CF - KM4 - 回复细胞）之间观察到类似的Ca2+动员。当我们在CFTR - 回复细胞中恢复F508del - CFTR转运时，特定的IP3R活性也降低到与非CF细胞相似的水平。在结构水平上，CF细胞的内质网形态在细胞核周围高度浓缩，而非CF细胞或校正后的CF细胞中内质网在整个细胞中延伸。
这些结果表明，通过校正囊性纤维化细胞中F508del - CFTR的异常转运，可逆转F508del - CFTR上皮细胞中IP3R功能障碍。此外，使用CFTR cDNA转染的CF细胞，我们证明CF人上皮细胞中IP3R Ca +释放的异常增加可能是F508del - CFTR保留在内质网区室的结果。
