[HCO(3)(-)]-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. HCO(3)(- )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 HCO(3)(- )may also regulate the expression of sAC and thereby affect the production of cAMP upon activation by HCO(3)(- )and the stimulation of CFTR in BCECs. RESULTS: RT-competitive PCR indicated that sAC mRNA expression in BCECs is dependent on [HCO(3)(-)] and incubation time in HCO(3)(-). Immunoblots showed that 10 and 40 mM HCO(3)(- )increased sAC protein expression by 45% and 87%, respectively, relative to cells cultured in the absence of HCO(3)(-). Furthermore, 40 mM HCO(3)(- )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 μM adenosine or 10 μM forskolin, which increase cAMP levels, showed decreased sAC mRNA expression by 20% and 30%, respectively. Intracellular cAMP production by sAC paralleled the time and [HCO(3)(-)]-dependent expression of sAC. Bicarbonate-induced apical Cl(- )permeability increased by 78% (P < 0.01) in BCECs cultured in HCO(3)(-). However for cells cultured in the absence of HCO(3)(-), apical Cl(- )permeability increased by only 10.3% (P > 0.05). CONCLUSION: HCO(3)(- )not only directly activates sAC, but also up-regulates the expression of sAC. These results suggest that active cellular uptake of HCO(3)(- )can contribute to the basal level of cellular cAMP in tissues that express sAC.