title: [HCO3-]-regulated expression and activity of soluble adenylyl cyclase in corneal endothelial and Calu-3 cells.
abstract: BACKGROUND
  Bicarbonate activated Soluble Adenylyl Cyclase (sAC) is a unique cytoplasmic and nuclear signaling mechanism for the generation of cAMP. HCO3- activates sAC in bovine corneal endothelial cells (BCECs), increasing [cAMP] and stimulating PKA, leading to phosphorylation of the cystic fibrosis transmembrane-conductance regulator (CFTR) and increased apical Cl- permeability. Here, we examined whether HCO3- may also regulate the expression of sAC and thereby affect the production of cAMP upon activation by HCO3- and the stimulation of CFTR in BCECs.
  
  
  RESULTS
  RT-competitive PCR indicated that sAC mRNA expression in BCECs is dependent on [HCO3-] and incubation time in HCO3-. Immunoblots showed that 10 and 40 mM HCO3- increased sAC protein expression by 45% and 87%, respectively, relative to cells cultured in the absence of HCO3-. Furthermore, 40 mM HCO3- up-regulated sAC protein expression in Calu-3 cells by 93%. On the other hand, sAC expression in BCECs and Calu-3 cells was unaffected by changes in bath pH or osmolarity. Interestingly, BCECs pre-treated with10 microM adenosine or 10 microM forskolin, which increase cAMP levels, showed decreased sAC mRNA expression by 20% and 30%, respectively. Intracellular cAMP production by sAC paralleled the time and [HCO3-]-dependent expression of sAC. Bicarbonate-induced apical Cl- permeability increased by 78% (P < 0.01) in BCECs cultured in HCO3-. However for cells cultured in the absence of HCO3-, apical Cl- permeability increased by only 10.3% (P > 0.05).
  
  
  CONCLUSIONS
  HCO3- not only directly activates sAC, but also up-regulates the expression of sAC. These results suggest that active cellular uptake of HCO3- can contribute to the basal level of cellular cAMP in tissues that express sAC.
authors.affiliation: School of Optometry, Indiana University, 800 E, Atwater Ave, Bloomington, IN 47405, USA. sxingcai@indiana.edu
authors.forename: Xing Cai
authors.initials: XC
authors.lastname: Sun
authors.forename: Miao
authors.initials: M
authors.lastname: Cui
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Bonanno
mesh_terms: D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001639:Bicarbonates / Q000494:pharmacology*; D002417:Cattle; D016764:Cell Polarity; D002478:Cells, Cultured / Q000187:drug effects / Q000201:enzymology; D002712:Chlorides / Q000378:metabolism; D003470:Culture Media / Q000494:pharmacology; D000242:Cyclic AMP / Q000096:biosynthesis; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology; D004728:Endothelium, Corneal / Q000166:cytology / Q000201:enzymology*; D004790:Enzyme Induction / Q000187:drug effects; D005260:Female; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport / Q000187:drug effects; D009994:Osmolar Concentration; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D001639:Bicarbonates; D002712:Chlorides; D003470:Culture Media; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000242:Cyclic AMP; D000262:Adenylyl Cyclases