Airway epithelial cells harbor the capacity of active Cl transepithelial transport and play critical roles in modulating innate immunity. However, whether intracellular Cl accumulation contributes to relentless airway inflammation remains largely unclear. This study showed that, in airway epithelial cells, intracellular Cl concentration ([Cl]) was increased after Pseudomonas aeruginosa lipopolysaccharide (LPS) stimulation via nuclear factor-κB (NF-κB)-phosphodiesterase 4D (PDE4D)-cAMP signaling pathways. Clamping [Cl] at high levels or prolonged treatment with LPS augmented serum- and glucocorticoid-inducible protein kinase 1 (SGK1) phosphorylation and subsequently triggered NF-κB activation in airway epithelial cells, whereas inhibition of SGK1 abrogated airway inflammation in vitro and in vivo. Furthermore, Cl-SGK1 signaling pathway was pronouncedly activated in patients with bronchiectasis, a chronic airway inflammatory disease. Conversely, hydrogen sulfide (HS), a sulfhydryl-containing gasotransmitter, confers anti-inflammatory effects through decreasing [Cl] via activation of cystic fibrosis transmembrane conductance regulator (CFTR). Our study confirms that intracellular Cl is a crucial mediator of sustained airway inflammation. Medications that abrogate excessively increased intracellular Cl may offer novel targets for the management of airway inflammatory diseases.
SARS-CoV-2, the culprit pathogen of COVID-19, elicits prominent immune responses and cytokine storms. Intracellular Cl− is a crucial regulator of host defense, whereas the role of Cl− signaling pathway in modulating pulmonary inflammation associated with SARS-CoV-2 infection remains unclear. By using human respiratory epithelial cell lines, primary cultured human airway epithelial cells, and murine models of viral structural protein stimulation and SARS-CoV-2 direct challenge, we demonstrated that SARS-CoV-2 nucleocapsid (N) protein could interact with Smad3, which downregulated cystic fibrosis transmembrane conductance regulator (CFTR) expression via microRNA-145. The intracellular Cl− concentration ([Cl−]i) was raised, resulting in phosphorylation of serum glucocorticoid regulated kinase 1 (SGK1) and robust inflammatory responses. Inhibition or knockout of SGK1 abrogated the N protein-elicited airway inflammation. Moreover, N protein promoted a sustained elevation of [Cl−]i by depleting intracellular cAMP via upregulation of phosphodiesterase 4 (PDE4). Rolipram, a selective PDE4 inhibitor, countered airway inflammation by reducing [Cl−]i. Our findings suggested that Cl− acted as the crucial pathological second messenger mediating the inflammatory responses after SARS-CoV-2 infection. Targeting the Cl− signaling pathway might be a novel therapeutic strategy for COVID-19.
Introduction Hydrogen sulfide (H2S) plays a large role in female and male sexual responses characterized by a smooth muscle relaxant effect. Moreover, H2S is a novel pro-secretory neuromodulator that modulates epithelial ion transport. However, whether H2S has a role in regulating vaginal epithelial ion transport and fluid secretion has not been extensively studied. Aim To identify the effects of H2S on vaginal epithelial ion transport and lubrication in an exploratory investigation. Methods The mRNA, protein expression, and localization of cystathionine γ-lyase (CSE) and H2S production in vaginal epithelium were examined by reverse transcriptase polymerase chain reaction, Western blot, H2S synthesizing activity assay, and immunohistochemistry, respectively. The effect of H2S on vaginal epithelial ion transport, vaginal fluid secretion, and ionic concentration was investigated using a short-circuit current (ISC), a measurement of vaginal lubrication, and ion chromatography, respectively. Main Outcome Measures The mRNA, protein expression, and localization of CSE, H2S formation, changes of ISC responses, vaginal lubrication, and K+ and Cl− concentrations were studied. Results CSE mRNA and protein were predominantly expressed in vaginal epithelium. Sodium hydrosulfide hydrate (NaHS) caused concentration-dependent changes in ISC across isolated rat vaginal epithelium, which consisted of an initial decrease phase and then an increase phase. The increase phase in ISC was mainly Cl− dependent and abolished by cystic fibrosis transmembrane conductance regulator inhibitor, whereas the decrease phase was sensitive to the adenosine triphosphate–sensitive K+ (KATP) channel blocker. Furthermore, intravaginal treatment of NaHS significantly enhanced vaginal lubrication in vivo, and this effect was prevented by cystic fibrosis transmembrane conductance regulator and KATP channel inhibitors. In addition, the ionic concentrations of K+ and Cl− in rat vaginal fluid were significantly increased by NaHS treatment. Conclusion The CSE-H2S pathway participates in the regulation of vaginal epithelial K+ and Cl− ion transport to modulate lumen fluid secretion.
As a novel gasotransmitter, hydrogen sulfide (H2S) elicits various physiological actions including smooth muscle relaxation and promotion of transepithelial ion transport. However, the pro-secretory function of H2S in the male reproductive system remains largely unclear. The aim of this study is to elucidate the possible roles of H2S in modulating rat epididymal intraluminal ionic microenvironment essential for sperm storage. The results revealed that endogenous H2S-generating enzymes cystathionine β-synthetase (CBS) and cystathionine γ-lyase (CSE) were both expressed in rat epididymis. CBS located predominantly in epithelial cells whilst CSE expressed primarily in smooth muscle cells. The relative expression level of CBS and CSE escalated from caput to cauda regions of epididymis, which was paralleled to the progressively increasing production of endogenous H2S. The effect of H2S on epididymal epithelial ion transportation was investigated using short-circuit current (ISC), measurement of intracellular ion concentration and in vivo rat epididymal microperfusion. Our data showed that H2S induced transepithelial K+ secretion via adenosine triphosphate-sensitive K+ (KATP) channel and large conductance Ca2+-activated K+ (BKCa) channel. Transient receptor potential vanilloid 4 (TRPV4) channel-mediated Ca2+ influx was implicated in the activation of BKCa channel. In vivo studies further demonstrated that H2S promoted K+ secretion in rat epididymal epithelium. Inhibition of endogenous H2S synthesis caused a significant decrease in K+ concentration of cauda epididymal intraluminal fluid. Moreover, our data demonstrated that high extracellular K+ concentration actively depressed the motility of cauda epididymal sperm in a pH-independent manner. Collectively, the present study demonstrated that H2S was vital to the formation of high K+ concentration in epididymal intraluminal fluid by promoting the transepithelial K+ secretion, which might contribute to the maintenance of the cauda epididymal sperm in quiescent dormant state before ejaculation.
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