Regulation of V-ATPase recycling via a RhoA- and ROCKII-dependent pathway in epididymal clear cells

Abstract

ϩ -ATPase (V-ATPase) in their apical membrane and are major contributors to proton secretion. We showed that this process is regulated via recycling of V-ATPase-containing vesicles. We now report that RhoA and its effector ROCKII are enriched in rat epididymal clear cells. In addition, cortical F-actin was detected beneath the apical membrane and along the lateral membrane of "resting" clear cells using a pan-actin antibody or phalloidin-TRITC. In vivo luminal perfusion of the cauda epididymal tubule with the ROCK inhibitors Y27632 (10 -30 M) and HA1077 (30 M) or with the cell-permeable Rho inhibitor Clostridium botulinum C3 transferase (3.75 g/ml) induced the apical membrane accumulation of V-ATPase and extension of V-ATPase-labeled microvilli in clear cells. However, these newly formed microvilli were devoid of ROCKII. In addition, Y27632 (30 M) or HA1077 (30 M) decreased the ratio of F-actin to G-actin detected by Western blot analysis in epididymal epithelial cells, and Y27632 also decreased the ratio of F-actin to G-actin in clear cells isolated by fluorescence activated cell sorting from B1-enhanced green fluorescence protein (EGFP) transgenic mice. These results provide evidence that depolymerization of the cortical actin cytoskeleton via inhibition of RhoA or its effector ROCKII favors the recruitment of V-ATPase from the cytosolic compartment into the apical membrane in clear cells. In addition, our data suggest that the RhoA-ROCKII pathway is not locally involved in the elongation of apical microvilli. We propose that inhibition of RhoA-ROCKII might be part of the intracellular signaling cascade that is triggered upon agonist-induced apical membrane V-ATPase accumulation.actin; proton pump; luminal acidification; proton ATPase; epididymis SEVERAL SPECIALIZED biological tissues, such as kidney tubules (8 -10, 66), the male reproductive tract (1,6,32,33,43,57), the inner ear (27, 59) salivary glands (51, 65), and osteoclasts (30, 52) establish an acidic extracellular local environment. These tissues all have in common a high expression of the proton pumping V-ATPase, which is localized in the plasma membrane of specialized acidifying cells. In the male reproductive tract, luminal acidification is a critical process for sperm maturation and for their storage in a quiescent state. We have shown that the V-ATPase localizes in the apical membrane of clear cells in the epididymis (2,3,6,42,43,57) and that these cells are important players in luminal acidification, especially in the distal region of the epididymis, where they are present in high numbers. Impairment of the acidification process in the epididymis, either by genetic mutations or environmental factors, such as the heavy metal cadmium, reduces male fertility (4,5,11,22,64).We have shown previously that luminal bicarbonate via bicarbonate-regulated adenylyl cyclase (sAC)-dependent intracellular cAMP production (42), or luminal angiotensin II via the nitric oxide (NO)/soluble guanylate cyclase (sGC)-dependent pathway (57), induce the apical membrane ...

Seasonal variation in estrogen receptor ERα, but not ERβ, androgen receptor and aromatase, in the efferent ductules and epididymis of the big fruit-eating bat Artibeus lituratus

“…Conversely, the basal cells of the A. lituratus epithelium were weakly reactive for AR, which corroborated a previous study that described little dependency on androgens for these cells [43]. The basal cells have multifunctional roles in the epididymis, such as endocytosis, detoxification, and immune protection [45,54]. An additional function of the basal cells is paracrine regulation of the principal and clear cells for electrolyte and water transport [31,45].…”

Relative contribution of clear cells and principal cells to luminal pH in the mouse epididymis†

“…The Cd was perfused in vivo through the lumen, as we have described previously [24,42,43]. Briefly, the distal epididymis was exposed by an abdominal incision under a dissecting microscope and the vas deferens was cannulated using a micro cannula (0.61 mm O. D., 0.28 mm I. D., Warner Instruments, Hamden, CT, USA).…”

“…Double-immunofluorescence labeling for the B1 subunit of the VATPase (ATP6V1B1), specific for CCs, and clathrin, a subapical marker of CCs and PCs, was performed on cryosections, as previously described [12,24,42]. Sections were hydrated in PBS for 15 min and incubated in PBS containing 1% SDS (v/v) for 4 min.…”

“…The level of accumulation of V-ATPase in microplicae (previously known as microvilli) was quantified in CCs double labeled for the V-ATPase and clathrin, as we have previously described [24,42,46]. The area occupied by V-ATPase-positive microplicae was quantified and normalized for the width of the cell measured along the apical border [25].…”

Circulating aldosterone induces the apical accumulation of the proton pumping V-ATPase and increases proton secretion in clear cells in the caput epididymis

“…This is accompanied by an increase in apical membrane surface area resulting in the formation and elongation of apical microvillar projections. Thus microvilli elongation directly correlates with V-ATPase activity and has been used as a measure of clear cell activation (6,7,19,39,49). The importance of clear cells to male fertility has been demonstrated, as impairment of V-ATPase activity by genetic manipulation or by environmental factors causes male infertility (11,27,61).…”