title: AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H+-ATPase accumulation in epididymal clear cells.
abstract: Acidic luminal pH and low [HCO(3)(-)] maintain sperm quiescent during maturation in the epididymis. The vacuolar H(+)-ATPase (V-ATPase) in clear cells is a major contributor to epididymal luminal acidification. We have shown previously that protein kinase A (PKA), acting downstream of soluble adenylyl cyclase stimulation by alkaline luminal pH or HCO(3)(-), induces V-ATPase apical membrane accumulation in clear cells. Here we examined whether the metabolic sensor AMP-activated protein kinase (AMPK) regulates this PKA-induced V-ATPase apical membrane accumulation. Immunofluorescence labeling of rat and non-human primate epididymides revealed specific AMPK expression in epithelial cells. Immunofluorescence labeling of rat epididymis showed that perfusion in vivo with the AMPK activators 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) or A-769662 induced a redistribution of the V-ATPase into subapical vesicles, even in the presence of a luminal alkaline (pH 7.8) buffer compared with that of controls perfused without drug. Moreover, preperfusion with AICAR blocked the PKA-mediated V-ATPase translocation to clear cell apical membranes induced by N(6)-monobutyryl-cAMP (6-MB-cAMP). Purified PKA and AMPK both phosphorylated V-ATPase A subunit in vitro. In HEK-293 cells [(32)P]orthophosphate in vivo labeling of the A subunit increased following PKA stimulation and decreased following RNA interference-mediated knockdown of AMPK. Finally, the extent of PKA-dependent in vivo phosphorylation of the A subunit increased with AMPK knockdown. In summary, our findings suggest that AMPK inhibits PKA-mediated V-ATPase apical accumulation in epididymal clear cells, that both kinases directly phosphorylate the V-ATPase A subunit in vitro and in vivo, and that AMPK inhibits PKA-dependent phosphorylation of this subunit. V-ATPase activity may be coupled to the sensing of acid-base status via PKA and to metabolic status via AMPK.
authors.affiliation: Renal-Electrolyte Division, Dept. of Medicine, Scaife A915, 3550 Terrace St., Pittsburgh, PA 15263, USA.
authors.forename: Kenneth R
authors.initials: KR
authors.lastname: Hallows
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Alzamora
authors.forename: Hui
authors.initials: H
authors.lastname: Li
authors.forename: Fan
authors.initials: F
authors.lastname: Gong
authors.forename: Christy
authors.initials: C
authors.lastname: Smolak
authors.forename: Dietbert
authors.initials: D
authors.lastname: Neumann
authors.forename: Núria M
authors.initials: NM
authors.lastname: Pastor-Soler
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001713:Biphenyl Compounds; D003994:Bucladesine / Q000031:analogs & derivatives / Q000378:metabolism; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D020536:Enzyme Activators / Q000494:pharmacology; D004822:Epididymis / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D008253:Macaca mulatta; D008297:Male; D051379:Mice; D010766:Phosphorylation; D021381:Protein Transport; D011753:Pyrones / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012265:Ribonucleotides / Q000494:pharmacology; D013876:Thiophenes / Q000494:pharmacology; D014162:Transfection; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*
chemical_list: D001713:Biphenyl Compounds; D020536:Enzyme Activators; D011753:Pyrones; D034741:RNA, Small Interfering; D012265:Ribonucleotides; D013876:Thiophenes; C002373:monobutyryl cyclic AMP; D000620:Aminoimidazole Carboxamide; D003994:Bucladesine; C526182:PRKAB1 protein, human; C526222:PRKAG1 protein, human; D017868:Cyclic AMP-Dependent Protein Kinases; D055372:AMP-Activated Protein Kinases; C526180:PRKAA1 protein, human; D025262:Vacuolar Proton-Translocating ATPases; C031143:AICA ribonucleotide; C512408:4-hydroxy-3-(4-(2-hydroxyphenyl)phenyl)-6-oxo-7H-thieno(2,3-b)pyridine-5-carbonitrile