[HCO3-]-regulated expression and activity of soluble adenylyl cyclase in corneal endothelial and Calu-3 cells.
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.
Sun
Xing Cai
XC
School of Optometry, Indiana University, 800 E, Atwater Ave, Bloomington, IN 47405, USA. sxingcai@indiana.edu
Cui
Miao
M
Bonanno
Joseph A
JA
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
D001639:Bicarbonates; D002712:Chlorides; D003470:Culture Media; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000242:Cyclic AMP; D000262:Adenylyl Cyclases