Coupling of TRPV6 and TMEM16A in epithelial principal cells of the rat epididymis.

The epididymis establishes a congenial environment for sperm maturation and protection. Its fluid is acidic, and the calcium concentration is low and declines along the length of the epididymal tubule. However, our knowledge of ionic currents and mechanisms of calcium homeostasis in rat epididymal epithelial cells remains enigmatic. In this study, to better understand calcium regulation in the epididymis, we use the patch-clamp method to record from single rat cauda epididymal principal cells. We detect a constitutively active Ca(2+) current with characteristics that match the epithelial calcium channel TRPV6. Electrophysiological and pharmacological data also reveal a constitutively active calcium-activated chloride conductance (CaCC). Removal of extracellular calcium attenuates not only the TRPV6-like conductance, but also the CaCC. Lanthanide block is time dependent such that the TRPV6-like component is inhibited first, followed by the CaCC. The putative CaCC blocker niflumic acid partially inhibits whole-cell currents, whereas La(3+) almost abolishes whole-cell currents in principal cells. Membrane potential measurements reveal an interplay between La(3+)-sensitive ion channels and those that are sensitive to the specific TMEM16A inhibitor tannic acid. In vivo perfusion of the cauda epididymal tubule shows a substantial rate of Ca(2+) reabsorption from the luminal side, which is dose-dependently suppressed by ruthenium red, a putative blocker of epithelial Ca(2+) channels and CaCC. Finally, we discover messenger RNA for both TRPV6 and TMEM16A in the rat epididymis and show that their proteins colocalize in the apical membrane of principal cells. Collectively, these data provide evidence for a coupling mechanism between TRPV6 and TMEM16A in principal cells that may play an important role in the regulation of calcium homeostasis in the epididymis.

Cell-cell interaction underlies formation of fluid in the male reproductive tract of the rat.

The epithelia lining the epididymides of many species consists of several cell types. We have provided evidence that the basal cells are essential to the integrated functions of the epithelium. Basal cells, but not principal cells, and other cells in the epididymis express TRPC3 and COX-1. We have isolated basal cells from intact rat epididymis using antibody-coated Dynabeads and subjected them to whole-cell patch-clamp measurement of nonselective cation channel activity, a feature of TRPC3 protein, and Fluo-3 fluorescence measurement of intracellular Ca2+ concentration. The results show that a nonselective cation current blockable by La3+ (0.1 mM), Gd3+ (0.1 mM), or SKF96365 (20 microM) could be activated by lysylbradykinin (200 nM). In cells loaded with Fluo-3, addition of lysylbradykinin (100 nM) caused a sustained increase of intracellular Ca2+. This effect was blocked by Gd3+ (0.1 mM) or SKF96365 (20 microM) and was not observed in Fluo-3-loaded principal cells. Stimulation of basal cell/principal cell cocultures with lysylbradykinin (200 nM) evoked in principal cells a current with CFTR-Cl- channel characteristics. Isolated principal cells in the absence of basal cells did not respond to lysylbradykinin but responded to PGE2 (100 nM) with activation of a CFTR-like current. Basal cells, but not principal cells, released prostaglandin E2 when stimulated with lysylbradykinin (100 nM). The release was blocked by SKF96365 (20 microM) and BAPTA-AM (0.05 or 0.1 mM). Confluent cell monolayers harvested from a mixture of disaggregated principal cells and basal cells responded to lysylbradykinin (100 nM) and PGE2 (500 nM) with an increase in electrogenic anion secretion. The former response was dependent on prostaglandin synthesis as piroxicam blocked the response. However, cell cultures obtained from principal cells alone responded to PGE2 but not to bradykinin. These results support the notion that basal cells regulate principal cells through a Ca2+ and COX signaling pathway.