{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T09:24:48Z","timestamp":1790241888131,"version":"4.1.0"},"reference-count":47,"publisher":"American Physiological Society","issue":"2","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["American Journal of Physiology-Cell Physiology"],"published-print":{"date-parts":[[2008,2]]},"abstract":"<jats:p>\n                    In the epididymis, low luminal bicarbonate and acidic pH maintain sperm quiescent during maturation and storage. The vacuolar H\n                    <jats:sup>+<\/jats:sup>\n                    -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\n                    <jats:sup>6<\/jats:sup>\n                    -monobutyryl-3\u2032-5\u2032-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 \u03bcM) 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 \u03bcM), an activator of the exchange protein activated by cAMP (Epac), did not induce V-ATPase apical accumulation. When applied at a higher concentration (100 \u03bcM), 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.\n                  <\/jats:p>","DOI":"10.1152\/ajpcell.00537.2007","type":"journal-article","created":{"date-parts":[[2007,12,26]],"date-time":"2007-12-26T20:25:10Z","timestamp":1198700710000},"page":"C488-C494","source":"Crossref","is-referenced-by-count":80,"title":["Alkaline pH- and cAMP-induced V-ATPase membrane accumulation is mediated by protein kinase A in epididymal clear cells"],"prefix":"10.1152","volume":"294","author":[{"given":"N\u00faria M.","family":"Pastor-Soler","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kenneth R.","family":"Hallows","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christy","family":"Smolak","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fan","family":"Gong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dennis","family":"Brown","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sylvie","family":"Breton","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"24","reference":[{"key":"R1","doi-asserted-by":"crossref","unstructured":"Beaulieu V\n                      , Da Silva N, Pastor-Soler N, Brown CR, Smith PJ, Brown D, Breton S.\n                      Modulation of the actin cytoskeleton via gelsolin regulates vacuolar H\n                      +\n                      -ATPase recycling.\n                      J Biol Chem\n                      280\n                      : 8452\u20138463, 2005.","DOI":"10.1074\/jbc.M412750200"},{"key":"R2","doi-asserted-by":"crossref","unstructured":"Bhalla V\n                      , Oyster NM, Fitch AC, Wijngaarden MA, Neumann D, Schlattner U, Pearce D, Hallows KR.\n                      AMP-activated kinase inhibits the epithelial Na\n                      +\n                      channel through functional regulation of the ubiquitin ligase Nedd4-2.\n                      J Biol Chem\n                      281\n                      : 26159\u201326169, 2006.","DOI":"10.1074\/jbc.M606045200"},{"key":"R3","doi-asserted-by":"crossref","unstructured":"Blomqvist SR\n                      , Vidarsson H, Soder O, Enerback S.\n                      Epididymal expression of the forkhead transcription factor Foxi1 is required for male fertility.\n                      EMBO J\n                      25\n                      : 4131\u20134141, 2006.","DOI":"10.1038\/sj.emboj.7601272"},{"key":"R4","doi-asserted-by":"crossref","unstructured":"Bos JL\n                      .\n                      Epac proteins: multi-purpose cAMP targets.\n                      Trends Biochem Sci\n                      31\n                      : 680\u2013686, 2006.","DOI":"10.1016\/j.tibs.2006.10.002"},{"key":"R5","doi-asserted-by":"crossref","unstructured":"Breton S\n                      , Brown D.\n                      New insights into the regulation of V-ATPase-dependent proton secretion.\n                      Am J Physiol Renal Physiol\n                      292\n                      : F1\u2013F10, 2007.","DOI":"10.1152\/ajprenal.00340.2006"},{"key":"R6","doi-asserted-by":"crossref","unstructured":"Breton S\n                      , Hammar K, Smith PJ, Brown D.\n                      Proton secretion in the male reproductive tract: involvement of Cl\n                      \u2212\n                      -independent HCO\n                      3\n                      \u2212\n                      transport.\n                      Am J Physiol Cell Physiol\n                      275\n                      : C1134\u2013C1142, 1998.","DOI":"10.1152\/ajpcell.1998.275.4.C1134"},{"key":"R7","doi-asserted-by":"crossref","unstructured":"Breton S\n                      , Nsumu NN, Galli T, Sabolic I, Smith PJ, Brown D.\n                      Tetanus toxin-mediated cleavage of cellubrevin inhibits proton secretion in the male reproductive tract.\n                      Am J Physiol Renal Physiol\n                      278\n                      : F717\u2013F725, 2000.","DOI":"10.1152\/ajprenal.2000.278.5.F717"},{"key":"R8","doi-asserted-by":"crossref","unstructured":"Breton S\n                      , Smith PJ, Lui B, Brown D.\n                      Acidification of the male reproductive tract by a proton pumping (H\n                      +\n                      )-ATPase.\n                      Nat Med\n                      2\n                      : 470\u2013472, 1996.","DOI":"10.1038\/nm0496-470"},{"key":"R9","doi-asserted-by":"crossref","unstructured":"Brown D\n                      .\n                      The ins and outs of aquaporin-2 trafficking.\n                      Am J Physiol Renal Physiol\n                      284\n                      : F893\u2013F901, 2003.","DOI":"10.1152\/ajprenal.00387.2002"},{"key":"R10","doi-asserted-by":"crossref","unstructured":"Brown D\n                      , Breton S.\n                      Mitochondria-rich, proton-secreting epithelial cells.\n                      J Exp Biol\n                      199\n                      : 2345\u20132358, 1996.","DOI":"10.1242\/jeb.199.11.2345"},{"key":"R11","doi-asserted-by":"crossref","unstructured":"Brown D\n                      , Lydon J, McLaughlin M, Stuart-Tilley A, Tyszkowski R, Alper S.\n                      Antigen retrieval in cryostat tissue sections and cultured cells by treatment with sodium dodecyl sulfate (SDS).\n                      Histochem Cell Biol\n                      105\n                      : 261\u2013267, 1996.","DOI":"10.1007\/BF01463929"},{"key":"R12","doi-asserted-by":"crossref","unstructured":"Christensen AE\n                      , Selheim F, de Rooij J, Dremier S, Schwede F, Dao KK, Martinez A, Maenhaut C, Bos JL, Genieser HG, Doskeland SO.\n                      cAMP analog mapping of Epac1 and cAMP kinase. Discriminating analogs demonstrate that Epac and cAMP kinase act synergistically to promote PC-12 cell neurite extension.\n                      J Biol Chem\n                      278\n                      : 35394\u201335402, 2003.","DOI":"10.1074\/jbc.M302179200"},{"key":"R13","doi-asserted-by":"crossref","unstructured":"Dames P\n                      , Zimmermann B, Schmidt R, Rein J, Voss M, Schewe B, Walz B, Baumann O.\n                      cAMP regulates plasma membrane vacuolar-type H\n                      +\n                      -ATPase assembly and activity in blowfly salivary glands.\n                      Proc Natl Acad Sci USA\n                      103\n                      : 3926\u20133931, 2006.","DOI":"10.1073\/pnas.0600011103"},{"key":"R14","doi-asserted-by":"crossref","unstructured":"de Rooij J\n                      , Zwartkruis FJ, Verheijen MH, Cool RH, Nijman SM, Wittinghofer A, Bos JL.\n                      Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP.\n                      Nature\n                      396\n                      : 474\u2013477, 1998.","DOI":"10.1038\/24884"},{"key":"R15","doi-asserted-by":"crossref","unstructured":"Enserink JM\n                      , Christensen AE, de Rooij J, van Triest M, Schwede F, Genieser HG, Doskeland SO, Blank JL, Bos JL.\n                      A novel Epac-specific cAMP analogue demonstrates independent regulation of Rap1 and ERK.\n                      Nat Cell Biol\n                      4\n                      : 901\u2013906, 2002.","DOI":"10.1038\/ncb874"},{"key":"R16","doi-asserted-by":"crossref","unstructured":"Frokiaer J\n                      , Nielsen S, Knepper MA.\n                      Molecular physiology of renal aquaporins and sodium transporters: exciting approaches to understand regulation of renal water handling.\n                      J Am Soc Nephrol\n                      16\n                      : 2827\u20132829, 2005.","DOI":"10.1681\/ASN.2005080850"},{"key":"R17","doi-asserted-by":"crossref","unstructured":"Garcia F\n                      , Kierbel A, Larocca MC, Gradilone SA, Splinter P, LaRusso NF, Marinelli RA.\n                      The water channel aquaporin-8 is mainly intracellular in rat hepatocytes, and its plasma membrane insertion is stimulated by cyclic AMP.\n                      J Biol Chem\n                      276\n                      : 12147\u201312152, 2001.","DOI":"10.1074\/jbc.M009403200"},{"key":"R18","doi-asserted-by":"crossref","unstructured":"Gekle M\n                      , Wunsch S, Oberleithner H, Silbernagl S.\n                      Characterization of two MDCK-cell subtypes as a model system to study principal cell and intercalated cell properties.\n                      Pfl\u00fcgers Arch\n                      428\n                      : 157\u2013162, 1994.","DOI":"10.1007\/BF00374853"},{"key":"R19","doi-asserted-by":"crossref","unstructured":"Glass DB\n                      , Lundquist LJ, Katz BM, Walsh DA.\n                      Protein kinase inhibitor-(6-22)-amide peptide analogs with standard and nonstandard amino acid substitutions for phenylalanine 10. Inhibition of cAMP-dependent protein kinase.\n                      J Biol Chem\n                      264\n                      : 14579\u201314584, 1989.","DOI":"10.1016\/S0021-9258(18)71718-3"},{"key":"R20","doi-asserted-by":"crossref","unstructured":"Glunde K\n                      , Guggino SE, Solaiyappan M, Pathak AP, Ichikawa Y, Bhujwalla ZM.\n                      Extracellular acidification alters lysosomal trafficking in human breast cancer cells.\n                      Neoplasia\n                      5\n                      : 533\u2013545, 2003.","DOI":"10.1016\/S1476-5586(03)80037-4"},{"key":"R21","doi-asserted-by":"crossref","unstructured":"Hays S\n                      , Kokko JP, Jacobson HR.\n                      Hormonal regulation of proton secretion in rabbit medullary collecting duct.\n                      J Clin Invest\n                      78\n                      : 1279\u20131286, 1986.","DOI":"10.1172\/JCI112712"},{"key":"R22","doi-asserted-by":"crossref","unstructured":"Hudson RL\n                      .\n                      Bafilomycin-sensitive acid secretion by mantle epithelium of the freshwater clam,\n                      Unio complanatus\n                      .\n                      Am J Physiol Regul Integr Comp Physiol\n                      264\n                      : R946\u2013R951, 1993.","DOI":"10.1152\/ajpregu.1993.264.5.R946"},{"key":"R23","doi-asserted-by":"crossref","unstructured":"Isnard-Bagnis C\n                      , Da Silva N, Beaulieu V, Yu AS, Brown D, Breton S.\n                      Detection of ClC-3 and ClC-5 in epididymal epithelium: immunofluorescence and RT-PCR after LCM.\n                      Am J Physiol Cell Physiol\n                      284\n                      : C220\u2013C232, 2003.","DOI":"10.1152\/ajpcell.00374.2001"},{"key":"R24","doi-asserted-by":"crossref","unstructured":"Kelada AS\n                      , Macaulay SL, Proietto J.\n                      Cyclic AMP acutely stimulates translocation of the major insulin-regulatable glucose transporter GLUT4.\n                      J Biol Chem\n                      267\n                      : 7021\u20137025, 1992.","DOI":"10.1016\/S0021-9258(19)50530-0"},{"key":"R25","doi-asserted-by":"crossref","unstructured":"Keyes SR\n                      , Rudnick G.\n                      Coupling of transmembrane proton gradients to platelet serotonin transport.\n                      J Biol Chem\n                      257\n                      : 1172\u20131176, 1982.","DOI":"10.1016\/S0021-9258(19)68170-6"},{"key":"R26","doi-asserted-by":"crossref","unstructured":"Kleizen B\n                      , Braakman I, de Jonge HR.\n                      Regulated trafficking of the CFTR chloride channel.\n                      Eur J Cell Biol\n                      79\n                      : 544\u2013556, 2000.","DOI":"10.1078\/0171-9335-00078"},{"key":"R27","doi-asserted-by":"crossref","unstructured":"Lambers TT\n                      , Oancea E, de Groot T, Topala CN, Hoenderop JG, Bindels RJ.\n                      Extracellular pH dynamically controls cell surface delivery of functional TRPV5 channels.\n                      Mol Cell Biol\n                      27\n                      : 1486\u20131494, 2007.","DOI":"10.1128\/MCB.01468-06"},{"key":"R28","doi-asserted-by":"crossref","unstructured":"Levine N\n                      , Kelly H.\n                      Measurement of pH in the rat epididymis in vivo.\n                      J Reprod Fertil\n                      52\n                      : 333\u2013335, 1978.","DOI":"10.1530\/jrf.0.0520333"},{"key":"R29","doi-asserted-by":"crossref","unstructured":"Levine N\n                      , Marsh DJ.\n                      Micropuncture studies of the electrochemical aspects of fluid and electrolyte transport in individual seminiferous tubules, the epididymis and the vas deferens in rats.\n                      J Physiol\n                      213\n                      : 557\u2013570, 1971.","DOI":"10.1113\/jphysiol.1971.sp009400"},{"key":"R30","doi-asserted-by":"crossref","unstructured":"Lu H\n                      , Sun TX, Bouley R, Blackburn K, McLaughlin M, Brown D.\n                      Inhibition of endocytosis causes phosphorylation (S256)-independent plasma membrane accumulation of AQP2.\n                      Am J Physiol Renal Physiol\n                      286\n                      : F233\u2013F243, 2004.","DOI":"10.1152\/ajprenal.00179.2003"},{"key":"R31","doi-asserted-by":"crossref","unstructured":"Myers M\n                      , Forgac M.\n                      The coated vesicle vacuolar (H\n                      +\n                      )-ATPase associates with and is phosphorylated by the 50-kDa polypeptide of the clathrin assembly protein AP-2.\n                      J Biol Chem\n                      268\n                      : 9184\u20139186, 1993.","DOI":"10.1016\/S0021-9258(18)98332-8"},{"key":"R32","doi-asserted-by":"crossref","unstructured":"Noda Y\n                      , Sasaki S.\n                      Regulation of aquaporin-2 trafficking and its binding protein complex.\n                      Biochim Biophys Acta\n                      1758\n                      : 1117\u20131125, 2006.","DOI":"10.1016\/j.bbamem.2006.03.004"},{"key":"R33","doi-asserted-by":"crossref","unstructured":"Pastor-Soler N\n                      , Bagnis C, Sabolic I, Tyszkowski R, McKee M, Van Hoek A, Breton S, Brown D.\n                      Aquaporin 9 expression along the male reproductive tract.\n                      Biol Reprod\n                      65\n                      : 384\u2013393, 2001.","DOI":"10.1095\/biolreprod65.2.384"},{"key":"R34","doi-asserted-by":"crossref","unstructured":"Pastor-Soler N\n                      , Beaulieu V, Litvin TN, Da Silva N, Chen Y, Brown D, Buck J, Levin LR, Breton S.\n                      Bicarbonate-regulated adenylyl cyclase (sAC) is a sensor that regulates pH-dependent V-ATPase recycling.\n                      J Biol Chem\n                      278\n                      : 49523\u201349529, 2003.","DOI":"10.1074\/jbc.M309543200"},{"key":"R35","doi-asserted-by":"crossref","unstructured":"Pastor-Soler N\n                      , Pietrement C, Breton S.\n                      Role of acid\/base transporters in the male reproductive tract and potential consequences of their malfunction.\n                      Physiology Bethesda\n                      20\n                      : 417\u2013428, 2005.","DOI":"10.1152\/physiol.00036.2005"},{"key":"R36","doi-asserted-by":"crossref","unstructured":"Pfister MF\n                      , Forgo J, Ziegler U, Biber J, Murer H.\n                      cAMP-dependent and -independent downregulation of type II Na-Pi cotransporters by PTH.\n                      Am J Physiol Renal Physiol\n                      276\n                      : F720\u2013F725, 1999.","DOI":"10.1152\/ajprenal.1999.276.5.F720"},{"key":"R37","doi-asserted-by":"crossref","unstructured":"Prat AG\n                      , Cunningham CC, Jackson GR Jr, Borkan SC, Wang Y, Ausiello DA, Cantiello HF.\n                      Actin filament organization is required for proper cAMP-dependent activation of CFTR.\n                      Am J Physiol Cell Physiol\n                      277\n                      : C1160\u2013C1169, 1999.","DOI":"10.1152\/ajpcell.1999.277.6.C1160"},{"key":"R38","doi-asserted-by":"crossref","unstructured":"Seino S\n                      , Shibasaki T.\n                      PKA-dependent and PKA-independent pathways for cAMP-regulated exocytosis.\n                      Physiol Rev\n                      85\n                      : 1303\u20131342, 2005.","DOI":"10.1152\/physrev.00001.2005"},{"key":"R39","doi-asserted-by":"crossref","unstructured":"Shapiro M\n                      , Matthews J, Hecht G, Delp C, Madara JL.\n                      Stabilization of F-actin prevents cAMP-elicited Cl- secretion in T84 cells.\n                      J Clin Invest\n                      87\n                      : 1903\u20131909, 1991.","DOI":"10.1172\/JCI115215"},{"key":"R40","doi-asserted-by":"crossref","unstructured":"Steinmetz PR\n                      .\n                      Cellular organization of urinary acidification.\n                      Am J Physiol Renal Fluid Electrolyte Physiol\n                      251\n                      : F173\u2013F187, 1986.","DOI":"10.1152\/ajprenal.1986.251.2.F173"},{"key":"R41","doi-asserted-by":"crossref","unstructured":"Stevens AL\n                      , Breton S, Gustafson CE, Bouley R, Nelson RD, Kohan DE, Brown D.\n                      Aquaporin 2 is a vasopressin-independent, constitutive apical membrane protein in rat vas deferens.\n                      Am J Physiol Cell Physiol\n                      278\n                      : C791\u2013C802, 2000.","DOI":"10.1152\/ajpcell.2000.278.4.C791"},{"key":"R42","doi-asserted-by":"crossref","unstructured":"Sun-Wada GH\n                      , Wada Y, Futai M.\n                      Diverse and essential roles of mammalian vacuolar-type proton pump ATPase: toward the physiological understanding of inside acidic compartments.\n                      Biochim Biophys Acta\n                      1658\n                      : 106\u2013114, 2004.","DOI":"10.1016\/j.bbabio.2004.04.013"},{"key":"R43","doi-asserted-by":"crossref","unstructured":"Szaszi K\n                      , Kurashima K, Kaibuchi K, Grinstein S, Orlowski J.\n                      Role of the cytoskeleton in mediating cAMP-dependent protein kinase inhibition of the epithelial Na+\/H+ exchanger NHE3.\n                      J Biol Chem\n                      276\n                      : 40761\u201340768, 2001.","DOI":"10.1074\/jbc.M106724200"},{"key":"R44","doi-asserted-by":"crossref","unstructured":"Voss M\n                      , Vitavska O, Walz B, Wieczorek H, Baumann O.\n                      Stimulus-induced phosphorylation of V-ATPase by protein kinase A.\n                      J Biol Chem\n                      282\n                      : 33735\u201333742, 2007.","DOI":"10.1074\/jbc.M703368200"},{"key":"R45","doi-asserted-by":"crossref","unstructured":"Wagner CA\n                      , Finberg KE, Breton S, Marshansky V, Brown D, Geibel JP.\n                      Renal vacuolar-ATPase.\n                      Physiol Rev\n                      84\n                      : 1263\u20131314, 2004.","DOI":"10.1152\/physrev.00045.2003"},{"key":"R46","doi-asserted-by":"crossref","unstructured":"Wu MS\n                      , Bens M, Cluzeaud F, Vandewalle A.\n                      Role of F-actin in the activation of Na(+)-K(+)-Cl- cotransport by forskolin and vasopressin in mouse kidney cultured thick ascending limb cells.\n                      J Membr Biol\n                      142\n                      : 323\u2013336, 1994.","DOI":"10.1007\/BF00233439"},{"key":"R47","doi-asserted-by":"crossref","unstructured":"Yip KP\n                      .\n                      Epac-mediated Ca\n                      2+\n                      mobilization and exocytosis in inner medullary collecting duct.\n                      Am J Physiol Renal Physiol\n                      291\n                      : F882\u2013F890, 2006.","DOI":"10.1152\/ajprenal.00411.2005"}],"container-title":["American Journal of Physiology-Cell 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