- Date de publication : 2011-06-28
Shum WW, Da Silva N, Belleannée C, McKee M, Brown D, Breton S. Regulation of V-ATPase recycling via a RhoA- and ROCKII-dependent pathway in epididymal clear cells. Am. J. Physiol., Cell Physiol. 2011;301:C31-43. doi: 10.1152/ajpcell.00198.2010. PubMed PMID: 21411727.
adp ribose transferases actins amides animals blotting, western botulinum toxins cytoskeleton enzyme inhibitors epididymis epithelial cells flow cytometry green fluorescent proteins male mice mice, transgenic microvilli proton pumps pyridines rats rats, sprague-dawley reverse transcriptase polymerase chain reaction signal transduction vacuolar proton-translocating atpases rho-associated kinases rhoa gtp-binding protein
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 μM) and HA1077 (30 μM) or with the cell-permeable Rho inhibitor Clostridium botulinum C3 transferase (3.75 μg/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 μM) or HA1077 (30 μM) 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.