Abstract
In epithelial cells, the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated Cl- channel, plays a key role in water and electrolytes secretion. A dysfunctional CFTR leads to the dehydration of the external environment of the cells and to the production of viscous mucus in the airways of cystic fibrosis patients. Here, we applied the quadriwave lateral shearing interferometry (QWLSI), a quantitative phase imaging technique based on the measurement of the light wave shift when passing through a living sample, to study water transport regulation in human airway epithelial CFBE and CHO cells expressing wild-type, G551D- and F508del-CFTR. We were able to detect phase variations during osmotic challenges and confirmed that cellular volume changes reflecting water fluxes can be detected with QWLSI. Forskolin stimulation activated a phase increase in all CFBE and CHO cell types. This phase variation was due to cellular volume decrease and intracellular refractive index increase and was completely blocked by mercury, suggesting an activation of a cAMP-dependent water efflux mediated by an endogenous aquaporin (AQP). AQP3 mRNAs, not AQP1, AQP4 and AQP5 mRNAs, were detected by RT-PCR in CFBE cells. Readdressing the F508del-CFTR protein to the cell surface with VX-809 increased the detected water efflux in CHO but not in CFBE cells. However, VX-770, a potentiator of CFTR function, failed to further increase the water flux in either G551D-CFTR or VX-809-corrected F508del-CFTR expressing cells. Our results show that QWLSI could be a suitable technique to study water transport in living cells. We identified a CFTR and cAMP-dependent, mercury-sensitive water transport in airway epithelial and CHO cells that might be due to AQP3. This water transport appears to be affected when CFTR is mutated and independent of the chloride channel function of CFTR.
Highlights
According to the studies realized with the Digital Holographic Microscopy (DHM) technique, optical path difference (OPD) values are expected to decrease during water influx mainly due to the dilution of intracellular content leading to intracellular refractive index (n2) decrease [20,23]
A hypotonic shock of 50% (-165 mOsmol) induced a transient OPD decrease in both CFBE WT-CFTR (Max OPD shift: -3.24 ± 0.70 nm, n = 7; Fig 2A left) and CHO WT-CFTR cells (Max OPD shift: -5.51 ± 0.73 nm, n = 8; Fig 2B left), suggesting an activation of water influx followed by a regulatory-volume decrease (RVD) mechanism
Our observations suggest that the OPD increase recorded after the forskolin stimulation of CHO WT-CFTR cells is both related to a cellular volume decrease and an intracellular refractive index increase
Summary
At the apical plasma membrane, the epithelial sodium channel (ENaC) is responsible for sodium absorption whereas the chloride secretion occurs by CFTR and TMEM16A, a Ca2+ activated Cl- channel These two ion channels are involved in Airway Surface Liquid (ASL) regulation which is necessary to mucus clearance and to the maintenance of lung sterility (reviewed in [2]). During the past two decades, a novel method to investigate transmembrane water transport regulation in living cells has been developed It is based on the Digital Holographic Microscopy (DHM), a non-invasive and label-free quantitative phase imaging technique detecting light retardation induced by transparent specimen called phase shift [19,20,21,22,23,24]. Our results highlighted the role of CFTR in a cAMP-dependent and mercury-sensitive water transport in airway epithelial and CHO cells
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