Association of soluble adenylyl cyclase with the V-ATPase in renal epithelial cells.
Activation of soluble adenylyl cyclase (sAC) by bicarbonate causes local cAMP generation, indicating that sAC might act as a pH and/or bicarbonate sensor in kidney cells involved in acid-base homeostasis. Therefore, we examined the expression of sAC in renal acid-base transporting intercalated cells (IC) and compared its distribution to that of the vacuolar proton pumping ATPase (V-ATPase) under different conditions. In all IC, sAC and V-ATPase showed considerable overlap under basal conditions, but sAC staining was also found in other cellular locations in the absence of V-ATPase. In type A-IC, both sAC and V-ATPase were apically and subapically located, whereas in type B-IC, significant basolateral colocalization of sAC and the V-ATPase was seen. When apical membrane insertion of the V-ATPase was stimulated by treatment of rats with acetazolamide, sAC was also concentrated in the apical membrane of A-IC. In mice that lack a functional B1 subunit of the V-ATPase, sAC was colocalized apically in A-IC along with V-ATPase containing the alternative B2 subunit isoform. The close association between these two enzymes was confirmed by coimmunoprecipitation of sAC from kidney homogenates using anti-V-ATPase antibodies. Our data show that sAC and the V-ATPase colocalize in IC, that they are concentrated in the IC plasma membrane under conditions that "activate" these proton secretory cells, and that they are both present in an immunoprecipitated complex. This suggests that these enzymes have a close association and could be part of a protein complex that is involved in regulating renal distal proton secretion.
Paunescu
Teodor G
TG
Center for Systems Biology, Program in Membrane Biology and Division of Nephrology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.
Da Silva
Nicolas
N
Russo
Leileata M
LM
McKee
Mary
M
Lu
Hua A J
HA
Breton
Sylvie
S
Brown
Dennis
D
D000086:Acetazolamide / Q000494:pharmacology; D000136:Acid-Base Equilibrium; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology / Q000473:pathology; D004232:Diuretics / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D007527:Isoenzymes / Q000378:metabolism; D007668:Kidney / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D051381:Rats; D017207:Rats, Sprague-Dawley; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*
D004232:Diuretics; D007527:Isoenzymes; C480628:Atp6v1b1 protein, mouse; D025262:Vacuolar Proton-Translocating ATPases; D000262:Adenylyl Cyclases; D000086:Acetazolamide