Biology subjects

Nie, H.-G.

Publications and source records attributed to Nie, H.-G..

2 recordsLinked to original sources

bioRxiv2020

Ion transport mechanisms for smoke inhalation injured airway epithelial barrier

Smoke inhalation injury is the leading cause of death in firefighters and victims. Inhaled hot air and toxic smoke are the predominant hazards to the respiratory epithelium. We aimed to analyze the effects of thermal stress and smoke aldehyde on the permeability of the airway epithelial barrier. Transepithelial resistance (RTE) and short-circuit current (ISC) of mouse tracheal epithelial monolayers were digitized by an Ussing chamber setup. Zonula occludens-1 tight junctions were visualized under confocal microscopy. A cell viability test and fluorescein isothiocyanate-dextran assay were performed. Thermal stress (40°C) decreased RTE in a two-phase manner. Meanwhile, thermal stress increased ISC followed by its decline. Na+ depletion, amiloride (an inhibitor for epithelial Na+ channels [ENaCs]), ouabain (a blocker for Na+/K+-ATPase) and CFTRinh-172 (a blocker of cystic fibrosis transmembrane regulator [CFTR]) altered the responses of RTE and ISC to thermal stress. Steady-state 40°C increased activity of ENaCs, Na+/K+-ATPase, and CFTR. Acrolein, one of the main oxidative unsaturated aldehydes in fire smoke, eliminated RTE and ISC. Na+ depletion, amiloride, ouabain, and CFTRinh-172 suppressed acrolein-sensitive ISC, but showed activating effects on acrolein-sensitive RTE. Thermal stress or acrolein disrupted zonula occludens-1 tight junctions, increased fluorescein isothiocyanate-dextran permeability but did not cause cell death or detachment. The synergistic effects of thermal stress and acrolein exacerbated the damage to monolayers. In conclusion, the paracellular pathway mediated by the tight junctions and the transcellular pathway mediated by active and passive ion transport pathways contribute to impairment of the airway epithelial barrier caused by thermal stress and acrolein.Graphical HeadlightsThermal stress and acrolein are two essential determinants for smoke-inhalation injury, impairing airway epithelial barrier.Transcellular ion transport pathways via the ENaC, CFTR, and Na/K-ATPase are interrupted by both thermal stress and acrolein, one of the most potent smoke toxins.Heat and acrolein damage the integrity of the airway epithelium through suppressing and relocating the tight junctions.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsENaCsepithelial Na+ channelsCFTRcystic fibrosis transmembrane regulatorRTEtransepithelial resistanceISCshort-circuit currentMTEmouse tracheal epithelialFITCfluorescein isothiocyanateHBEhuman bronchial epithelialDMSOdimethyl sulfoxideZO-1zonula occludens-1P1phase 1P2phase 2ASIamiloride-sensitive ISCKCsK+ channelsROSreactive oxygen speciesCaCCsCa2+-activated Cl− channelsNKCCNa+/K+/2Cl−MAPKmitogen-activated protein kinaseView Full Text

pharmacology and toxicology↗

bioRxiv2020

Plasmin improves oedematous blood-gas barrier by cleaving epithelial sodium channels

Background and PurposeLung oedema in association with suppressed fibrinolysis is a hallmark of lung injury. We aimed to test whether plasmin cleaves epithelial sodium channels (ENaC) to resolve lung oedema fluid. Experimental ApproachesHuman lungs and airway acid-instilled mice were used for analysing fluid resolution. In silico prediction, mutagenesis, Xenopus oocytes, immunoblotting, voltage clamp, mass spectrometry, protein docking, and alveolar fluid clearance were combined for identifying plasmin specific cleavage sites and benefits. Key ResultsPlasmin led to a marked increment in lung fluid resolution in both human lungs ex vivo and injured mice. Plasmin specifically activated {beta}{gamma}ENaC channels in oocytes in a time-dependent manner. Deletion of four consensus proteolysis tracts ({Delta}432-444, {gamma}{Delta}131-138, {gamma}{Delta}178-193, and {gamma}{Delta}410-422) eliminated plasmin-induced activation significantly. Further, immunoblotting assays identified 7 cleavage sites (K126, R135, K136, R153, K168, R178, K179) for plasmin to trim both furin-cleaved C-terminal fragments and full-length human {gamma}ENaC proteins. In addition to confirming the 7 cleavage sites, 9 new sites (R122, R137, R138, K150, K170, R172, R180, K181, K189) in synthesized peptides were found to be cleaved by plasmin with mass spectrometry. These cleavage sites were located in the finger and the thumb, particularly the GRIP domain of human ENaC 3D model composed of two proteolytic centres for plasmin. Novel uncleaved sites beyond the GRIP domain in both and {gamma} subunits were identified to interrupt the plasmin cleavage-induced conformational change in ENaC channel complexes. Additionally, plasmin could regulate ENaC activity via the G protein signal. Conclusion and ImplicationsWe demonstrate that plasmin could cleave ENaC to benefit the blood-gas exchange by resolving oedema fluid as a potent fibrinolytic therapy for oedematous pulmonary diseases. Bullet point summaryO_ST_ABSWhat is already knowC_ST_ABSO_LISerine proteases proteolytically cleave epithelial sodium channels, including plasmin and uPA acutely. C_LIO_LIActivity of epithelial sodium channels is increased post proteolysis. C_LI What this study addsO_LIPlasmin cleaves up to 16 sites composed of two proteolytic centres in both full-length and furin-cleaved human {gamma} subunit of epithelial sodium channels in hours. C_LIO_LINon-proteolytic sites in both and {gamma} subunits interrupt the plasmin cleavage-induced channel gating. C_LIO_LIIntratracheally instilled plasmin facilitates alveolar fluid clearance in normal human and injured mouse lungs. C_LI Clinical significanceO_LIActivation of human lung epithelial sodium channels by plasmin may benefit lung oedema resolution as a novel therapy for ARDS. C_LI

pharmacology and toxicology↗