title: Nonpigmented ciliary epithelial cells respond to acetazolamide by a soluble adenylyl cyclase mechanism.
abstract: OBJECTIVE
  The nonpigmented ciliary epithelium (NPE) is rich in soluble adenylyl cyclase (sAC), a proposed cytoplasmic bicarbonate sensor. Here, we examine the contribution of sAC to an increase in cyclic AMP (cAMP) and changes in a key ion transporter, H(+)-ATPase, in NPE exposed to acetazolamide, a carbonic anhydrase inhibitor (CAI).
  
  
  METHODS
  Cyclic AMP was measured by radioimmunoassay in primary cultured porcine NPE. The pH-sensitive dye BCECF was used to examine cytoplasmic pH regulation. Subcellular protein translocation was examined by Western blot.
  
  
  RESULTS
  A transient cAMP increase, detectable within minutes of acetazolamide treatment, was prevented by KH7, a specific sAC inhibitor. Following 10-minute exposure to acetazolamide, the abundance of H(+)-ATPase B1 subunit and sAC was doubled in a plasma membrane-rich fraction, suggesting subcellular translocation. Similar evidence of H(+)-ATPase translocation was observed in NPE exposed to 8-Bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP). Consistent with increased capacity for proton export, acetazolamide increased the rate of pH recovery from acidification. KH7 and bafilomycin A1, an inhibitor of H(+)-ATPase, both prevented the stimulatory effect of acetazolamide on pH recovery. In a parallel study, H(+)-ATPase abundance was found to be higher in the plasma membrane of HEK293 cells that overexpress sAC compared to the normal HEK293 cells. HEK cells that overexpress sAC and had higher H(+)-ATPase abundance displayed a faster rate of pH recovery and greater sensitivity to KH7.
  
  
  CONCLUSIONS
  Acetazolamide increases cAMP in a response that involves activation of sAC. Subcellular translocation of H(+)-ATPase and an increase in the capacity for proton export by acetazolamide-treated NPE cells is a cAMP-dependent response.
authors.affiliation: Department of Physiology, University of Arizona, Tucson, Arizona.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Shahidullah
authors.forename: Amritlal
authors.initials: A
authors.lastname: Mandal
authors.forename: Guojun
authors.initials: G
authors.lastname: Wei
authors.forename: Lonny R
authors.initials: LR
authors.lastname: Levin
authors.forename: Jochen
authors.initials: J
authors.lastname: Buck
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Delamere
mesh_terms: D000086:Acetazolamide / Q000494:pharmacology*; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D002924:Ciliary Body / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D017136:Ion Transport; D013552:Swine
chemical_list: D002257:Carbonic Anhydrase Inhibitors; D000262:Adenylyl Cyclases; D000086:Acetazolamide
keywords: H+-ATPase; acetazolamide; cytoplasmic pH; nonpigmented ciliary epithelium; soluble adenylyl cyclase