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Actin cytoskeleton remodeling by RhoA and ROCKII regulates vacuolar H+‐ATPase (V‐ATPase) recycling in epididymal clear cells

The V‐ATPase in clear cells is important for luminal acidification in the epididymis, a process that is critical for sperm maturation and storage. We demonstrated that proton secretion in these cells is regulated via V‐ATPase recycling. We show here that RhoA and ROCKII are enriched in clear cells, and that a narrow band of cortical F‐actin is located beneath the apical membrane of “resting” clear cells. In epididymis perfused in vivo, agents known to stimulate V‐ATPase membrane accumulation (cpt‐cAMP or cpt‐cGMP) significantly reduced the intensity of the cortical F‐actin staining. The Rho‐Kinase (ROCK) inhibitors Y27632 and HA1077, or the cell permeable Rho inhibitor, Clostridium C3 transferase, also decreased the subapical F‐actin staining and decreased the amount of F‐actin versus G‐actin detected by Western blotting. These agents induced apical membrane V‐ATPase accumulation and elongation of V‐ATPase‐labeled microvilli in clear cells. These results show that actin cytoskeleton depolymerization via inhibition of RhoA or ROCKII favors apical membrane V‐ATPase recruitment. Thus, cAMP‐ or cGMP‐dependent RhoA/ROCKII inhibition might be part of the intracellular signaling cascade that is triggered upon agonist‐induced apical membrane V‐ATPase accumulation and subsequent activation of V‐ATPase‐dependent proton secretion in clear cells. This research is supported by NIH grants DK38452 and HD40793.