ABSTRACT
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.
Subject(s)
Epididymis , Epithelial Cells , Animals , Epithelial Cells/metabolism , Epithelium , Male , RatsABSTRACT
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.
