title: Regulation of V-ATPase recycling via a RhoA- and ROCKII-dependent pathway in epididymal clear cells.
abstract: 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.
authors.affiliation: Center for Systems Biology, Program in Membrane Biology/Nephrology Division, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA.
authors.forename: Winnie Waichi
authors.initials: WW
authors.lastname: Shum
authors.forename: Nicolas
authors.initials: N
authors.lastname: Da Silva
authors.forename: Clémence
authors.initials: C
authors.lastname: Belleannée
authors.forename: Mary
authors.initials: M
authors.lastname: McKee
authors.forename: Dennis
authors.initials: D
authors.lastname: Brown
authors.forename: Sylvie
authors.initials: S
authors.lastname: Breton
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D036002:ADP Ribose Transferases / Q000008:administration & dosage / Q000494:pharmacology; D000199:Actins / Q000276:immunology / Q000378:metabolism; D000577:Amides / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D001905:Botulinum Toxins / Q000008:administration & dosage / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism; D004791:Enzyme Inhibitors; D004822:Epididymis / Q000166:cytology / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008871:Microvilli / Q000378:metabolism; D017494:Proton Pumps; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*
chemical_list: D000199:Actins; D000577:Amides; D004791:Enzyme Inhibitors; D017494:Proton Pumps; D011725:Pyridines; C108830:Y 27632; D049452:Green Fluorescent Proteins; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D054460:rho-Associated Kinases; D001905:Botulinum Toxins; D025262:Vacuolar Proton-Translocating ATPases; D020742:rhoA GTP-Binding Protein; C049347:fasudil