Protein Kinase A (PKA) Regulates Vacuolar H + ‐ATPase (V‐ATPase) Recycling in Epididymal Clear Cells

Abstract

In the epididymis, low luminal bicarbonate and acidic pH maintain sperm quiescent during maturation and storage. The V-ATPase, located in clear cells, plays a major role in luminal acidification. We have shown that cAMP and the bicarbonate-regulated soluble adenylyl cyclase induces V-ATPase apical accumulation in these cells. We examined whether or not PKA is involved in this response. Confocal immunofluorescence labeling on epididymis perfused in vivo showed that at luminal acidic pH (6.5), V-ATPase was distributed between short apical microvilli and sub-apical endosomes. The specific PKA activator, N6-monobutyryl-cAMP, induced elongation of apical microvilli and accumulation of V-ATPase in these structures. The PKA inhibitor H89 (10 μM) partially inhibited the apical accumulation of V-ATPase induced by the cAMP permeant analogue, 8-cpt-cAMP. Finally, perfusion at pH 6.5 with 8-pCPT-2′-O-methyl-cAMP, an activator of the exchange factors directly activated by cAMP (Epac), can partially mimic the cAMP-induced V-ATPase apical accumulation. These results indicate that direct stimulation of PKA activity by cAMP leads to the accumulation of V-ATPase in apical microvilli, although direct activation of Epac by cAMP may also participate in V-ATPase recycling in epididymal clear cells.