Wulin Zuo

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.

Relaxant effect of flavonoid naringenin on contractile activity of rat colonic smooth muscle.

ETHNOPHARMACOLOGICAL RELEVANCE Disturbed gastrointestinal (GI) motility can be associated with smooth muscle abnormalities and dysfunction. Exploring innovative approaches that can modulate the disturbed colonic motility are of great importance for clinical therapeutics. Naringenin, a flavonoid presented in many traditional Chinese herbal medicines, has been shown to have a relaxant effect on different smooth muscles. The aim of the present study was to investigate the effect of naringenin on regulation of GI motility. MATERIAL AND METHODS Mechanical recording was used to investigate the effect of naringenin on isolated rat colonic smooth muscle spontaneous contractions. Whole cell patch clamp, intracellular [Ca(2+)] concentration ([Ca(2+)]i) and membrane potential measurements were examined on primary cultures of colonic smooth muscle cells (SMCs). A neostigmine-stimulated rat model was utilized to investigate the effect of naringenin in vivo. RESULTS Naringenin induced a concentration-dependent inhibition (1-1000 μM) on rat colonic spontaneous contraction, which was reversible after wash out. The external Ca(2+) influx induced contraction and [Ca(2+)]i increase were inhibited by naringenin (100 μM). In rat colonic SMCs, naringenin-induced membrane potential hyperpolarization was sensitive to TEA and selective large-conductance calcium-activated K(+) (BKCa) channel inhibitor iberiotoxin. Under whole cell patch-clamp condition, naringenin stimulated an iberiotoxin-sensitive BKCa current, which was insensitive to changes in the [Ca(2+)]i concentration. Furthermore, naringenin significantly suppressed neostigmine-enhanced rat colon transit in vivo. CONCLUSION Our results for the first time demonstrated the relaxant effect of flavonoid naringenin on colon smooth muscle both in vitro and in vivo. The relaxant effect of naringenin was attributed to direct activation of BKCa channels, which subsequently hyperpolarized the colonic SMCs and decreased Ca(2+) influx through VDCC. Naringenin might be of therapeutic value in the treatment of GI motility disorders.

Cellular Mechanisms of Carbachol-Stimulated Cl− Secretion in Rat Epididymal Epithelium

Abstract Neurotransmitter-controlled Cl− secretions play an important role in maintenance of the epididymal microenvironment for sperm maturation. This study was carried out to investigate the effect of carbachol (CCH) on the cultured rat epididymal epithelium and the signal transduction mechanisms of this response. In normal K-H solution, CCH added basolaterally elicited a biphasic Isc response consisting of a transient spike followed by a second sustained response. Ca2+ activated Cl− channel blocker disulfonic acid stilbene (DIDS, 300 μM) only inhibited part of the CCH-induced Isc response, while nonselective Cl− channel blocker diphenylamine-dicarboxylic acid (DPC, 1 mM) reduced all, indicating the involvement of different conductance pathways. Both peaks of the CCH-induced Isc response could be significantly inhibited by pretreatment with an adenylate cyclase inhibitor, MDL12330A (50 μM). An increase in intracellular cAMP content upon stimulation of CCH was measured. All of the initial peak and part of the second peak could be inhibited by pretreatment with Ca2+-chelating agent BAPTA/AM (50 μM) and an endoplasmic reticulum Ca2+ pump inhibitor, Thapsigagin (Tg, 1 μM). In a whole-cell patch clamp experiment, CCH induced an inward current in the single cell. Two different profiles of currents were found; the first component current exhibited an outward rectifying I-V relationship in a time and voltage-dependent manner, and the current followed showed a linear I-V relationship. The carbachol-induced current was found to be partially blockable by DIDS and could be completely blocked by DPC. The above results indicate that the CCH-induced Cl− secretion could be mediated by Ca2+ and cAMP-dependent regulatory pathways.

Stimulating Effects of Dopamine on Chloride Transport Across the Rat Caudal Epididymal Epithelium in Culture

Abstract The present study investigated the effects of dopamine on chloride transport across cultured rat caudal epididymal epithelium. The results showed that dopamine induced a biphasic short-circuit current (Isc) in a concentration-dependent manner. The dopamine-induced response consisted of an initial rapid spike followed by a sustained phase. The alpha and beta adrenoreceptor inhibitors, phentolamine and propranolol, inhibited the initial spike and the sustained phase, respectively, suggesting a contribution of adrenergic receptors. The response was almost abolished by removing the extracellular Cl−, suggesting that the dopamine-induced short-circuit current is primarily a Cl− current. The response was inhibited by the apical Cl− channel blocker, diphenylamine-dicarboxylic acid, and the Ca2+-activated Cl− channel blocker, disulfonic acid stilbene, indicating that Cl− may pass through two types of Cl− channels on the apical side. Preloading monolayers with the intracellular Ca2+ chelator BAPTA/AM abolished the initial spike and greatly reduced the second phase in the Isc response to dopamine. Pretreating the monolayers with an adenylate cyclase inhibitor, MDL12330A, inhibited all of the second Isc response and part of the initial spike. Also, characteristics of the Cl− currents induced by dopamine were observed in whole-cell patch-clamp recording. The increases of intracellular cAMP and Ca2+ induced by dopamine were also measured. The results suggest that extracellular dopamine activates Ca2+-dependent and cAMP-dependent regulatory pathways, leading to activation of both Ca2+-dependent and cAMP-dependent Cl− conductances in epididymal epithelial cells.

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.

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.