biostudies-literatureShum WWNICHD NIH HHSNIDDK NIH HHSC31-43https://www.ebi.ac.uk/biostudies/studies/S-EPMC3129830biostudies-literatureUnknown301(1)Luminal acidification in the epididymis is critical for sperm maturation and storage. Clear cells express the vacuolar H(+)-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.American journal of physiology. Cell physiologyRegulation of V-ATPase recycling via a RhoA- and ROCKII-dependent pathway in epididymal clear cells.PMC3129830DK-38452R37 DK042956R01 HD040793P01 DK038452P30 DK043351HD-40793Shum WWBelleannee CBreton SDa Silva NMcKee MBrown DfalseRegulation of V-ATPase recycling via a RhoA- and ROCKII-dependent pathway in epididymal clear cells.Luminal acidification in the epididymis is critical for sperm maturation and storage. Clear cells express the vacuolar H(+)-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.2011-01-01T00:00:00Z2011 Jul2021-02-20T09:16:53Z2019-03-27T03:06:42ZS-EPMC31298302141172710.1152/ajpcell.00198.2010