Jiehong Huang

Sodium Coupled Bicarbonate Influx Regulates Intracellular and Apical pH in Cultured Rat Caput Epididymal Epithelium

Background The epithelium lining the epididymis provides an optimal acidic fluid microenvironment in the epididymal tract that enable spermatozoa to complete the maturation process. The present study aims to investigate the functional role of Na+/HCO3 − cotransporter in the pH regulation in rat epididymis. Method/Principal Findings Immunofluorescence staining of pan cytokeratin in the primary culture of rat caput epididymal epithelium showed that the system was a suitable model for investigating the function of epididymal epithelium. Intracellular and apical pH were measured using the fluorescent pH sensitive probe carboxy-seminaphthorhodafluor-4F acetoxymethyl ester (SNARF-4F) and sparklet pH electrode respectively to explore the functional role of rat epididymal epithelium. In the HEPES buffered Krebs-Henseleit(KH) solution, the intracellular pH (pHi) recovery from NH4Cl induced acidification in the cultured caput epididymal epithelium was completely inhibited by amiloride, the inhibitor of Na+/H+ exchanger (NHE). Immediately changing of the KH solution from HEPES buffered to HCO3 − buffered would cause another pHi recovery. The pHi recovery in HCO3 − buffered KH solution was inhibited by 4, 4diisothiocyanatostilbene-2, 2-disulfonic acid (DIDS), the inhibitor of HCO3 − transporter or by removal of extracellular Na+. The extracellular pH measurement showed that the apical pH would increase when adding DIDS to the apical side of epididymal epithelial monolayer, however adding DIDS to the basolateral side had no effect on apical pH. Conclusions The present study shows that sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured caput epididymal epithelium.

Tyrosine phosphorylation modulates store-operated calcium entry in cultured rat epididymal basal cells†

Store‐operated calcium entry (SOCE) is essential for many cellular processes. In this study, we investigated modulation of SOCE by tyrosine phosphorylation in rat epididymal basal cells. The intracellular Ca2+([Ca2+]i) measurement showed that SOCE occurred in rat epididymal basal cells by pretreating the cells with thapsigargin (Tg), the inhibitor of sarco‐endoplasmic reticulum Ca2+‐ATPase. To identify the role of Ca2+ channels in this response, we examined the effects of transient receptor potential canonical channel blockers 2‐aminoethoxydiphenyl borate (2‐APB), 1‐[β‐[3‐(4‐methoxyphenyl)pro‐poxy]‐4‐methoxyphenethyl]‐1H‐imidazole hydrochloride(SKF96365), Gd3+, and non‐selective cation channel blocker Ni2+ respectively on SOCE and found that these blockers could inhibit the Ca2+ influx to different extent. Furthermore, we studied the regulation of SOCE by tyrosine kinase pathway. The inhibitor of tyrosine kinase genistein remarkably suppressed the SOCE response, whereas sodium orthovanadate, the inhibitor of tyrosine phosphatase, greatly enhanced it. The results suggest that tyrosine kinase pathway plays a significant role in the initiation of SOCE and positively modulates SOCE in epididymal basal cells. J. Cell. Physiol. 226: 1069–1073, 2011.

Functional studies of acid transporter in cultured rat epididymal cell

OBJECTIVE To explore the functional role of vacuolar H(+)-ATPase in the pH regulation of epididymal fluid and its effect on sperm motility. DESIGN Experimental study. SETTING Physiology laboratory in a university. ANIMAL(S) Immature male Sprague-Dawley rats. INTERVENTION(S) The H(+)-ATPase inhibitor was applied to the primary culture of epididymal cells. MAIN OUTCOME MEASURE(S) The intracellular luminal fluid pH and sperm percent motility were recorded. RESULT(S) Double immunofluorescence of H(+)-ATPase and carbonic anhydrase II in primary culture of cauda epididymal epithelial cells showed that the system was a suitable model for investigation of acid secretion by clear cells. Clear cells were pharmacologically distinct from principal cells in acid/base transportation. The intracellular pH recovery from cellular acidification was suppressed by the H(+)-ATPase inhibitor bafilomycin A1(100 nM) and the Na(+)/H(+) exchanger inhibitor amiloride (1 mM) by 85% and 54%, respectively. These results suggest that, in addition to Na(+)/H(+) exchanger, clear cells actively pump proton from cytoplasm into extracellular space through H(+)-ATPase. In addition, inhibition of H(+)-ATPase by bafilomycin A1 blocked the acidification of luminal fluid with IC(50) values of 12 nM, which supports that H(+)-ATPase acidifies the luminal fluid. We also confirm that the acid fluid regulates rat cauda sperm motility. CONCLUSION(S) The present work shows that clear cells, the minority cell type of epididymal cell population, play an important role in the pH regulation of epididymal fluid by H(+)-ATPase.

Functional expression of G protein‐coupled receptor 30 in immature rat epididymal epithelium

The aim of this study is to investigate the functional role of G protein‐coupled receptor 30 (GPR30) in the epididymis. We found that GPR30 is expressed in the epithelium of the immature rat epididymis and is involved in chloride secretion into the caudal epididymis lumen. The short‐circuit current (Isc) experiments showed that in primary cultured caudal epididymis epithelium, activation of GPR30 by its specific agonist G1 induced a mono‐phasic current increase, and G15, the specific antagonist of GPR30, could completely inhibit the current induced by G1. The G1‐induced Isc was largely blocked by application of the non‐specific chloride channel inhibitor diphenylamine‐dicarboxylic acid (DPC), or by the cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTRinh‐172, suggesting that the current was mainly mediated through CFTR. In addition, after stimulating GPR30 by G1, the intracellular concentration of cAMP in the epithelium was significantly increased, indicating that the cAMP signal pathway is involved and could be responsible for the CFTR activation. Finally, to further investigate the function of GPR30 in vivo, G15 was administrated into rats subcutaneously. The osmotic pressure of the micro perfusion solution from epididymis was measured and the sperms were collected. Results showed that there was an osmotic pressure increase of the perfusion solution from G15 treated rats. When the GPR30 was inhibited by G15 endogenously, the motility of sperms decreased. Our data demonstrated that GPR30 is involved in the formation of caudal epididymis fluid micro‐environment thus affecting sperm motility.

Increased intracellular Cl − concentration promotes ongoing inflammation in airway epithelium

AbstractsAirway 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−]i) was increased after Pseudomonas aeruginosa lipopolysaccharide (LPS) stimulation via nuclear factor-κB (NF-κB)–phosphodiesterase 4D (PDE4D)–cAMP signaling pathways. Clamping [Cl−]i 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 (H2S), a sulfhydryl-containing gasotransmitter, confers anti-inflammatory effects through decreasing [Cl−]i 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.

Sodium tanshinone IIA sulfonate stimulated Cl− secretion in mouse trachea

Sodium tanshinone IIA sulfonate (STS) is a derivate of tanshinone IIA, a lipophilic compound in Salvia miltiorrhiza. This study aimed to investigate the effect of STS on ion transport in mouse tracheal epithelium and the mechanisms underlying it. Short-circuit current (Isc) was measured to evaluate the effect of STS on transepithelial ion transport. Intracellular Ca2+ imaging was performed to observe intracellular Ca2+ concentration ([Ca2+]i) changes induced by STS in primary cultured mouse tracheal epithelial cells. Results showed that the apical application of STS at mouse trachea elicited an increase of Isc, which was abrogated by atropine, an antagonist of muscarinic acetylcholine receptor (mAChR). By removing ambient Cl− or applying blockers of Ca2+-activated Cl− channel (CaCC), the response of STS-induced Isc was suppressed. Moreover, STS elevated the [Ca2+]i in mouse tracheal epithelial cells. As a result, STS stimulated Cl− secretion in mouse tracheal epithelium via CaCC in an mAChR-dependent way. Due to the critical role of Cl− secretion in airway hydration, our findings suggested that STS may be used to ameliorate the airway dehydration symptom in cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD).

Hydrogen sulfide protects against the development of experimental cerebral malaria in a C57BL/6 mouse model

Hydrogen sulfide (H2S) has anti-inflammatory and neuroprotective properties, particularly during pathological processes. Experimental cerebral malaria (ECM), which is caused by vascular leakage into the brain, is characterized by inflammation, neurological deficits and cerebral hemorrhage. The present study investigated the correlation between ECM genesis and the levels of H2S. The results indicated that the levels of H2S derived from the brain decreased over time following ECM infection, and that the low H2S bioavailability was partially caused by decreased expression of the H2S generating enzyme, cystathionine-β-synthase. Administration of NaHS (an exogenous donor of H2S) provided protection against ECM. NaHS inhibited the destruction of the blood brain barrier and the secretion of proinflammatory biomarkers, including interluekin-18, matrix metalloproteinase-9 and serum cluster of differentiation 40 into the brain during ECM. In conclusion, these results suggested that low levels of H2S in brain contributed to the progression of ECM, and that H2S donor administration may represent a potential protective therapy against ECM.

Role of GPR30 in estrogen-induced prostate epithelial apoptosis and benign prostatic hyperplasia

Several studies have implicated estrogen and the estrogen receptor (ER) in the pathogenesis of benign prostatic hyperplasia (BPH); however, the mechanism underlying this effect remains elusive. In the present study, we demonstrated that estrogen (17β-estradiol, or E2)-induced activation of the G protein-coupled receptor 30 (GPR30) triggered Ca2+ release from the endoplasmic reticulum, increased the mitochondrial Ca2+ concentration, and thus induced prostate epithelial cell (PEC) apoptosis. Both E2 and the GPR30-specific agonist G1 induced a transient intracellular Ca2+ release in PECs via the phospholipase C (PLC)-inositol 1, 4, 5-triphosphate (IP3) pathway, and this was abolished by treatment with the GPR30 antagonist G15. The release of cytochrome c and activation of caspase-3 in response to GPR30 activation were observed. Data generated from the analysis of animal models and human clinical samples indicate that treatment with the GPR30 agonist relieves testosterone propionate (TP)-induced prostatic epithelial hyperplasia, and that the abundance of GPR30 is negatively associated with prostate volume. On the basis of these results, we propose a novel regulatory mechanism whereby estrogen induces the apoptosis of PECs via GPR30 activation. Inhibition of this activation is predicted to lead to abnormal PEC accumulation, and to thereby contribute to BPH pathogenesis.