Association of soluble adenylyl cyclase with the V-ATPase in renal epithelial cells

摘要

First published October 24, 2007; doi: 10.1152/ajprenaI.00406.2007.-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 (1C) and compared its distribution to that of the vacuolar proton pumping ATPase (V-ATPase) under different conditions. In all 1C, 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 Bl 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 coimmuno-precipitation of sAC from kidney homogenates using anti-V -ATPase antibodies. Our data show that sAC and the V-ATPase colocalize in 1C, that they are concentrated in the 1C 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.proton pump; kidney; intercalated cells; acid-base homeostasisa major unresolved issue in renal physiology is how extracellular acid-base status is sensed by renal epithelial cells to initiate their homeostatic response to such stimuli. Among the factors that have been suggested are, not surprisingly, pH, CO2, and bicarbonate as well as a number of potential hormonal stimuli (48). Early elegant studies showed that basolateral C02 elevation, together with an initial increase in calcium, is an important stimulus for inducing proton secretion by the proximal tubule and collecting ducts (42, 47). This occurs at least in part by inducing the apical accumulation of the vacuolar proton pumping ATPase (V-ATPase) in these cell types. Our previous work using epididymal proton-secreting cells as a model system provided strong evidence that bicarbonate plays a central role in this process via its stimulatory effecton the generation of cAMP by the soluble adenylyl cyclase (sAC) (35).