Cellular mechanism underlying leptin-induced anion secretion of rat epididymal epithelial cells.

BACKGROUND: A large number of studies have shown that leptin plays an important role in the regulation of fertility via the hypothalamus-pituitary-gonad axis. However, its peripheral function in epididymis was still elusive. OBJECTIVE: The purpose of this study was to determine the pro-secretion effect of leptin on the rat epididymal epithelium. MATERIALS AND METHODS: In the present study, real-time quantitative polymerase chain reaction, western blot, and immunohistochemical analysis were employed to detect the expression pattern of leptin receptors in rat epididymis. The pro-secretion effect of leptin on epididymal epithelial cells was measured by short-circuit current, and the prostaglandin E2 and cyclic adenosine monophosphate level was evaluated by enzyme-linked immunosorbent assay. RESULTS: We verified that the leptin receptor was located on the epididymal epithelium, with a relatively high expression level in corpus and cauda epididymis. Ussing chamber experiments showed that leptin stimulated a significant rise of the short-circuit current in rat epididymal epithelial cells, which could be abolished by the specific leptin receptor antagonist peptide Allo-aca, or by removing the ambient Cl- and HCO3 -. Furthermore, the leptin-stimulated short-circuit current response could be abrogated by blocking the apical cystic fibrosis transmembrane regulator or the basolateral Na+-K+-2Cl- cotransporter. Our pharmacological experiments manifested that interfering with the prostaglandin H synthase-2-prostaglandin E2-EP2/EP4-adenylate cyclase pathways could significantly blunt the cystic fibrosis transmembrane regulator-mediated anion secretion induced by leptin. The enzyme-linked immunosorbent assay demonstrated that leptin could induce a substantial increase in prostaglandin E2 release and cyclic adenosine monophosphate synthesis of primary cultured rat cauda epididymal epithelial cells. Our data also suggested that JAK2, ERK, and PI3K-dependent phosphorylation may be involved in the activation of prostaglandin H synthase-2 and the subsequent prostaglandin E2 production. CONCLUSIONS: The present study demonstrated the pro-secretion function of leptin in rat epididymal epithelium via the activation of cystic fibrosis transmembrane regulator and Na+-K+-2Cl- cotransporter, which was dependent on the paracrine/autocrine prostaglandin E2 stimulated EP2/EP4-adenylate cyclase pathways, and thus contributed to the formation of an appropriate microenvironment essential for sperm maturation.

Aerobic exercises regulate the epididymal anion homeostasis of high-fat diet-induced obese rats through TRPA1-mediated Cl- and HCO3- secretion†.

Aerobic exercises could improve the sperm motility of obese individuals. However, the underlying mechanism has not been fully elucidated, especially the possible involvement of the epididymis in which sperm acquire their fertilizing capacity. This study aims to investigate the benefit effect of aerobic exercises on the epididymal luminal milieu of obese rats. Sprague-Dawley male rats were fed on a normal or high-fat diet (HFD) for 10 weeks and then subjected to aerobic exercises for 12 weeks. We verified that TRPA1 was located in the epididymal epithelium. Notably, aerobic exercises reversed the downregulated TRPA1 in the epididymis of HFD-induced obese rats, thus improving sperm fertilizing capacity and Cl- concentration in epididymal milieu. Ussing chamber experiments showed that cinnamaldehyd (CIN), agonist of TRPA1, stimulated an increase of the short-circuit current (ISC) in rat cauda epididymal epithelium, which was subsequently abolished by removing the ambient Cl- and HCO3-. In vivo data revealed that aerobic exercises increased the CIN-stimulated Cl- secretion rate of epididymal epithelium in obese rats. Pharmacological experiments revealed that blocking cystic fibrosis transmembrane regulator (CFTR) and Ca2+-activated Cl- channel (CaCC) suppressed the CIN-stimulated anion secretion. Moreover, CIN application in rat cauda epididymal epithelial cells elevated intracellular Ca2+ level, and thus activate CACC. Interfering with the PGHS2-PGE2-EP2/EP4-cAMP pathway suppressed CFTR-mediated anion secretion. This study demonstrates that TRPA1 activation can stimulate anion secretion via CFTR and CaCC, which potentially forming an appropriate microenvironment essential for sperm maturation, and aerobic exercises can reverse the downregulation of TRPA1 in the epididymal epithelium of obese rats.

Exercise Reduces Airway Smooth Muscle Contraction in Asthmatic Rats via Inhibition of IL-4 Secretion and Store-Operated Ca2+ Entry Pathway.

PURPOSE: Increased evidence has shown that aerobic exercise reduces airway hyperresponsiveness in asthmatic individuals. However, the underlying mechanisms of action remain elusive. This study aimed to investigate the effect of exercise on airway smooth muscle (ASM) contractile function in asthmatic rats, and uncover the possible involvement of interleukin 4 (IL-4) and the store-operated Ca2+ entry (SOCE) pathway. METHODS: In this study, chicken ovalbumin was used to induce asthma in male Sprague-Dawley rats. The exercise group received moderate-intensity aerobic exercise training for 4 weeks. IL-4 concentrations in bronchoalveolar lavage fluid (BALF) samples were evaluated by enzyme linked immunosorbent assay. The contractile function of the ASM was investigated using tracheal ring tension experiments and intracellular Ca2+ imaging techniques. Western blot analysis was used to evaluate expression levels of calcium-release activated calcium (CRAC) channel protein (Orai) and stromal interaction molecule 1 (STIM1) in ASM. RESULTS: Our data showed that the carbachol-stimulated, SOCE-mediated contraction of rat ASM was significantly increased in asthmatic rats, which could be abolished by exercise. Pharmacological studies revealed that GSK5498A and BTP-2, selective blockers of CRAC channels significantly inhibited SOCE-induced ASM contraction. In addition, exercise inhibited the up-regulation of IL-4 in BALF as well as STIM1 and Orai expression in the ASM of asthmatic rats. In line with these observations, we demonstrated that pretreatment of the ASM with IL-4 up-regulated the expression level of STIM1, Orai1 and Orai2, thereby promoting SOCE-mediated ASM contraction. CONCLUSIONS: The data in this study reveal that aerobic exercise may improve the ASM contractile function in asthmatic rats by inhibiting IL-4 secretion and by down-regulating the expression of STIM1, Orai1 and Orai2, thus decreasing excessive SOCE-mediated ASM contraction in asthmatic rats.

Toxoplasma gondii infection triggers ongoing inflammation mediated by increased intracellular Cl- concentration in airway epithelium.

Toxoplasma gondii is a widespread parasitic protozoan causing toxoplasmosis including pulmonary toxoplasmosis. As the first line of host defense, airway epithelial cells play critical roles in orchestrating pulmonary innate immunity. However, the mechanism underlying the airway inflammation induced by the T. gondii infection remains largely unclear. This study demonstrated that after infection with T. gondii, the major anion channel located in the apical membranes of airway epithelial cells, cystic fibrosis transmembrane conductance regulator (CFTR), was degraded by the parasite-secreted cysteine proteases. The intracellular Cl- concentration ([Cl-]i) was consequently elevated, leading to activation of nuclear factor-κB (NF-κB) signaling via serum/glucocorticoid regulated kinase 1. Furthermore, the heightened [Cl-]i and activated NF-κB signaling could be sustained in a positive feedback regulatory manner resulting from decreased intracellular cAMP level through NF-κB-mediated up-regulation of phosphodiesterase 4. Conversely, the sulfur-containing compound allicin conferred anti-inflammatory effects on pulmonary toxoplasmosis by decreasing [Cl-]i via activation of CFTR. These results suggest that the intracellular Cl- dynamically modulated by T. gondii mediates sustained airway inflammation, which provides a potential therapeutic target against pulmonary toxoplasmosis.

G protein-coupled estrogen receptor promotes acrosome reaction via regulation of Ca2+ signaling in mouse sperm†.

G protein-coupled estrogen receptor (GPER), a seven-transmembrane G protein-coupled receptor, mediates the rapid pre-genomic signaling actions of estrogen and derivatives thereof. The expression of GPER is extensive in mammal male reproductive system. However, the functional role of GPER in mouse sperm has not yet been well recognized. This study revealed that GPER was expressed at the acrosome and the mid-flagellum of the mouse sperm. The endogenous GPER ligand 17ß-estradiol and the selective GPER agonist G1 increased intracellular Ca2+ concentration ([Ca2+]i) in mouse sperm, which could be abolished by G15, an antagonist of GPER. In addition, the G1-stimulated Ca2+ response was attenuated by interference with the phospholipase C (PLC) signaling pathways or by blocking the cation channel of sperm (CatSper). Chlortetracycline staining assay showed that the activation of GPER increased the incidence of acrosome-reacted sperm. Conclusively, GPER was located at the acrosome and mid-flagellum of the mouse sperm. Activation of GPER triggered the elevation of [Ca2+]i through PLC-dependent Ca2+ mobilization and CatSper-mediated Ca2+ influx, which promoted the acrosome reaction of mouse sperm.

Mechanosensitive Piezo1 channel in rat epididymal epithelial cells promotes transepithelial K+ secretion.

The Piezo1 channel, a mechanosensitive channel that exhibit a preference for Ca2+, play multifarious physiological and pathological roles in the endothelium and epithelium of various tissues. However, the functional expression of Piezo1 channel in the epithelium of the male reproductive tract remains unknown. In the present study, the expression of Piezo1 channel in the rat epididymis was determined by real-time quantitative PCR, western blot and immunohistochemical analysis. Our data revealed that Piezo1 channel was located in the epithelial layer of the rat epididymis, with higher expression levels in the corpus and cauda regions. The pro-secretion function of Piezo1 channel was then investigated using short circuit current (ISC) and intracellular Ca2+ imaging techniques. Application of Yoda1, a selective Piezo1 channel activator, stimulated a remarkable decrease in the ISC of the epididymal epithelium. Pharmacological experiments revealed that the ISC response induced by Piezo1 channel activation was abolished by pretreating epithelial cells with the Yoda1 analogue, Dooku1, the selective mechanosensitive cation channel blocker, GsMTx4, or removal of basolateral K+. Meanwhile, we demonstrated that activation of Piezo1 channel triggered a robust Ca2+ influx in epididymal epithelial cells. The possible involvement of Ca2+- activated K+channels (KCa) in transepithelial K+ secretion was then evaluated. And that big conductance KCa (BK), but not small conductance or intermediate conductance KCa, mediated Piezo1-elicited transepithelial K+ secretion. Moreover, we demonstrated that NKCC and NKA were responsible for supplying substrate K+ during transepithelial K+ secretion. These data demonstrate that the activation of Piezo1 channel promotes BK-mediated transepithelial K+ secretion, and thus may plays an important role in the formation of a high K+ concentration in epididymal intraluminal fluid.

Activation of TRPV4 stimulates transepithelial K+ secretion in rat epididymal epithelium.

The maturation of sperms is dependent on the coordinated interactions between sperm and the unique epididymal luminal milieu, which is characterized by high K+ content. This study investigated the involvement of transient receptor potential vanilloid 4 (TRPV4) in the K+ secretion of epididymal epithelium. The expression level and cellular localization of TRPV4 and Ca2+-activated K+ channels (KCa) were analyzed via RT-PCR, real-time quantitative PCR, western blot and immunofluorescence. The functional role of TRPV4 was investigated using short-circuit current (ISC) and intracellular Ca2+ imaging techniques. We found a predominant expression of TRPV4 in the corpus and cauda epididymal epithelium. Activation of TRPV4 with a selective agonist, GSK1016790A, stimulated a transient decrease in the ISC of the epididymal epithelium. The ISC response was abolished by either the TRPV4 antagonists, HC067047 and RN-1734, or the removal of basolateral K+. Simultaneously, the application of GSK1016790A triggered Ca2+ influx in epididymal epithelial cells. Our data also indicated that the big conductance KCa (BK), small conductance KCa (SK) and intermediate conductance KCa (IK) were all expressed in rat epididymis. Pharmacological studies revealed that BK, but not SK and IK, mediated TRPV4-elicited transepithelial K+ secretion. Finally, we demonstrated that TRPV4 and BK were localized in the epididymal epithelium, which showed an increased expression level from caput to cauda regions of rat epididymis. This study implicates that TRPV4 plays an important role in the formation of high K+ concentration in epididymal intraluminal fluid via promoting transepithelial K+ secretion mediated by BK.

Cellular mechanism underlying oxytocin-stimulated Cl- secretion in rat cauda epididymal epithelium.

The neurohypophyseal hormone oxytocin (OT) plays critical roles in lactation and parturition, while its function in male reproduction system is largely unknown. This study aims to investigate the effect of OT on regulating transepithelial ion transport in rat cauda epididymal epithelium. With the use of RT-PCR, Western blot, and immunohistochemical analysis, we found that OT receptor (OTR) was expressed and localized at the basal membrane of rat cauda epididymal epithelium. The short-circuit current (Isc) measurement showed that basolateral application of OT to the primary cultured rat cauda epididymal epithelial cells elicited an increase in Isc, which was abrogated by pretreating the epithelial cells with CFTRinh-172, a blocker of cystic fibrosis transmembrane conductance regulator (CFTR). Pretreatment with the prostaglandin H synthase inhibitors indomethacin and piroxicam, or the nonselective antagonists of prostaglandin E2 (PGE2) receptor EP2 or EP4, AH-6809, and AH-23848, significantly attenuated OT-stimulated Isc response. Furthermore, the generation of PGE2 was measured using enzyme-linked immunosorbent assay, demonstrating that OT induced a substantial increase in PGE2 release from primary cultured rat cauda epididymal epithelial cells. In conclusion, activation of OTR by OT triggered PGE2 release, resulting in CFTR-dependent Cl- secretion through paracrine/autocrine pathways in rat cauda epididymal epithelium.

Cellular mechanisms underlying carbon monoxide stimulated anion secretion in rat epididymal epithelium.

Epididymal epithelium possesses active ion transport properties conducive to the maintenance of appropriate epididymal intraluminal microenvironment. The endogenous gasotransmitter carbon monoxide (CO) regulates numerous cellular processes including water and electrolyte transport in various epithelia. However, the functional role of CO in epididymal epithelium is still elusive. This study aims to explore the potential regulatory effect of CO on transepithelial ion transport in rat epididymis. Using qPCR technique, we verified that endogenous CO synthase heme oxygenase 1 was expressed in rat caput, corpus, and cauda epididymis. In addition, endogenous CO was detected in rat cauda epididymis. Ussing chamber experiments showed that CORM-2, a CO donor, induced an increase of the short-circuit current (ISC) in a concentration-dependent manner in rat cauda epididymal epithelium. The ISC response could be abrogated by removing the ambient Cl- or HCO3-. Interfering with the cAMP signaling pathway or blocking cystic fibrosis transmembrane regulator (CFTR) partially suppressed the CO-stimulated ISC response. Moreover, the CO-evoked ISC response was significantly attenuated by blocking Ca2+-activated Cl- channel (CaCC) or chelating intracellular Ca2+. Elevation of intracellular Ca2+ level was also observed after CO stimulation in rat cauda epididymal epithelial cells. Collectively, this study demonstrated that CO stimulated anion secretion via activation of CFTR and CaCC in rat cauda epididymal epithelium, which might contribute to the formation of the appropriate microenvironment essential for sperm storage.

The gasotransmitter hydrogen sulfide inhibits transepithelial anion secretion of pregnant mouse endometrial epithelium.

Endometrial epithelium exhibits a robust ion transport activity required for dynamical regulation of uterine fluid environment and thus embryo implantation. However, there still lacks a thorough understanding of the ion transport processes and regulatory mechanism in peri-implantation endometrial epithelium. As a gaseous signaling molecule or gasotransmitter, hydrogen sulfide (H2S) regulates a myriad of cellular and physiological processes in various tissues, including the modulation of ion transport proteins in epithelium. This study aimed to investigate the effects of H2S on ion transport across mouse endometrial epithelium and its possible role in embryo implantation. The existence of endogenous H2S in pregnant mouse uterus was tested by the detection of two key H2S-generating enzymes and measurement of H2S production rate in tissue homogenates. Transepithelial ion transport processes were electrophysiologically assessed in Ussing chambers on early pregnant mouse endometrial epithelial layers, demonstrating that H2S suppressed the anion secretion by blocking cystic fibrosis transmembrane conductance regulator (CFTR). H2S increased intracellular Cl- concentration ([Cl-]i) in mouse endometrial epithelial cells, which was abolished by pretreatment with the CFTR selective inhibitor CFTRinh-172. The cAMP level in mouse endometrial epithelial cells was not affected by H2S, indicating that H2S blocked CFTR in a cAMP-independent way. In vivo study showed that interference with H2S synthesis impaired embryo implantation. In conclusion, our study demonstrated that H2S inhibits the transepithelial anion secretion of early pregnant mouse endometrial epithelium via blockade of CFTR, contributing to the preparation for embryo implantation.

Cellular Mechanism Underlying Hydrogen Sulfide Mediated Epithelial K+ Secretion in Rat Epididymis.

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 ß-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 (I SC), 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.

In silico analysis of the effect of mutation on epidermal growth factor receptor in non-small-cell lung carcinoma: from mutational analysis to drug designing.

Tyrosine kinase inhibitors (TKI)-resistant mutation in epidermal growth factor receptor's (EGFR) kinase domain is an important anomaly to look into. Studying the mutations at atomic level using molecular dynamics simulations gave us an insight into the architectural changes happening at the microscopic level. The knowledge was used to design new TKI whose function is devoid of the affect of the mutations in kinase domain. Traditional Chinese medicinal library was used for structure-based drug designing, where virtual screening was followed by ADME/Tox analysis and the shortlisted compounds were docked into the kinase domain of EGFR and simulated there using atomic-level selection of the grid. The shortlisted compounds from molecular docking analysis were subjected to MM-PBSA calculations. The in silico data generated is giving a strong lead compound for further in vitro and in vivo analysis.

Relaxant Effect of Sodium Tanshinone IIA Sulphonate on Mouse Tracheal Smooth Muscle.

Sodium tanshinone IIA sulphonate, a water-soluble derivative of tanshinone IIA, has been proven to possess versatile biological properties, but its pharmacological effect on tracheal smooth muscle remains elusive. This paper presents a study on the relaxant effect and underlying mechanisms of sodium tanshinone IIA sulphonate on mouse tracheal smooth muscle. The relaxant effect of sodium tanshinone IIA sulphonate was evaluated in mouse tracheal rings using a mechanical recording system. Intracellular Ca2+ concentration was measured in primary cultured tracheal smooth muscle cells using confocal imaging system. The results showed that sodium tanshinone IIA sulphonate induced dose-dependent relaxation of mouse tracheal rings in a ß-adrenoceptor- and epithelium-independent manner. Pretreatment with the ATP-sensitive K+ channel blocker glibenclamide partly attenuated the relaxation response. Administration of sodium tanshinone IIA sulphonate notably inhibited the extracellular Ca2+-induced contraction. High KCl or carbachol-evoked elevation in the intracellular Ca2+ concentration was also abrogated by sodium tanshinone IIA sulphonate in tracheal smooth muscle cells. In conclusion, the tracheal relaxant effect of sodium tanshinone IIA sulphonate was independent of ß-adrenoceptor and airway epithelium, mediated primarily by inhibition of extracellular Ca2+ influx via L-type voltage-dependent Ca2+ channels and partially by activation of the ATP-sensitive K+ channel. These results indicate the potential therapeutic value of sodium tanshinone IIA sulphonate for asthma treatment.

Spin-Orbit Effects in Closed-Shell Heavy and Superheavy Element Monohydrides and Monofluorides with Coupled-Cluster Theory.

Bond lengths and force constants of a set of closed-shell sixth-row and superheavy element monohydrides and monofluorides are calculated in this work. Kramers restricted coupled-cluster approaches (KR-CC) with spin-orbit coupling (SOC) included at the self-consistent field (SCF) level as well as CC approaches with SOC included in post-SCF treatment (SOC-CC) are employed in calculations. Recently published relativistic effective core potentials are employed, and highly accurate results for superheavy element molecules are achieved with KR-CCSD(T). SOC effects on bond lengths and force constants of these molecules are investigated. Effects of electron correlation are shown to be affected by SOC to a large extent for some superheavy element molecules. Bond lengths and force constants with SOC-CC agree very well with those of KR-CC for most of the sixth-row element molecules. As for superheavy element molecules, SOC-CCSD is able to afford results that are in good agreement with those of KR-CCSD except for 111F, while the error of SOC-CCSD(T) is more pronounced. Large error would be encountered with SOC-CC approaches for molecules when both SOC and electron correlation effects are sizable.