Alkaline pH- and cAMP-induced V-ATPase membrane accumulation is mediated by protein kinase A in epididymal clear cells.
In the epididymis, low luminal bicarbonate and acidic pH maintain sperm quiescent during maturation and storage. The vacuolar H(+)-ATPase (V-ATPase) in epididymal clear cells plays a major role in luminal acidification. We have shown previously that cAMP, luminal alkaline pH, and activation of the bicarbonate-regulated soluble adenylyl cyclase (sAC) induce V-ATPase apical accumulation in these cells, thereby stimulating proton secretion into the epididymal lumen. Here we examined whether protein kinase A (PKA) is involved in this response. Confocal immunofluorescence labeling on rat epididymis perfused in vivo showed that at luminal acidic pH (6.5), V-ATPase was distributed between short apical microvilli and subapical endosomes. The specific PKA activator N(6)-monobutyryl-3'-5'-cyclic monophosphate (6-MB-cAMP, 1 mM) induced elongation of apical microvilli and accumulation of V-ATPase in these structures. The PKA inhibitor myristoylated-PKI (mPKI, 10 microM) inhibited the apical accumulation of V-ATPase induced by 6-MB-cAMP. Perfusion at pH 6.5 with 8-(4-chlorophenylthio)-2-O-methyl-cAMP (8CPT-2-O-Me-cAMP; 10 microM), an activator of the exchange protein activated by cAMP (Epac), did not induce V-ATPase apical accumulation. When applied at a higher concentration (100 microM), 8CPT-2-O-Me-cAMP induced V-ATPase apical accumulation, but this effect was completely inhibited by mPKI, suggesting crossover effects on the PKA pathway with this compound at high concentrations. Importantly, the physiologically relevant alkaline pH-induced apical V-ATPase accumulation was completely inhibited by pretreatment with mPKI. We conclude that direct stimulation of PKA activity by cAMP is necessary and sufficient for the alkaline pH-induced accumulation of V-ATPase in clear cell apical microvilli.
Pastor-Soler
Núria M
NM
Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, A915 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15263, USA. pastorn@dom.pitt.edu
Hallows
Kenneth R
KR
Smolak
Christy
C
Gong
Fan
F
Brown
Dennis
D
Breton
Sylvie
S
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology*; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004822:Epididymis / Q000201:enzymology* / Q000648:ultrastructure; D004847:Epithelial Cells / Q000201:enzymology* / Q000648:ultrastructure; D006863:Hydrogen-Ion Concentration; D008297:Male; D018613:Microscopy, Confocal; D008871:Microvilli / Q000201:enzymology / Q000648:ultrastructure; D051381:Rats; D017207:Rats, Sprague-Dawley; D025262:Vacuolar Proton-Translocating ATPases / Q000187:drug effects / Q000378:metabolism*
D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D025262:Vacuolar Proton-Translocating ATPases