SLC26A3 (DRA) is stimulated in a synergistic, intracellular Ca2+ dependent manner by cAMP AND ATP in intestinal epithelial cells

In polarized intestinal epithelial cells, DRA is a brush border (BB) Cl-/HCO3- exchanger that is part of neutral NaCl absorption under baseline conditions but in cAMP driven diarrheas it is stimulated and contributes to increased anion secretion. To further understand regulation of DRA in conditions mimicking some diarrheal diseases, differentiated Caco-2/BBE cells were exposed to forskolin and ATP. Forskolin and ATP both acutely stimulated DRA in a concentration dependent manner. Forskolin at 1µM and ATP at 0.25 µM had minimal to no effect on DRA activity given individually; however, together, they stimulated DRA activity to levels seen with maximum concentrations of forskolin and ATP alone. In Caco-2/BBE cells expressing the Ca2+ indicator GCaMP6s, ATP alone increased Ca2+ in a concentration dependent manner, while forskolin (1 µM), that by itself did not significantly alter Ca2+, followed by 0.25 µM ATP produced a large increase in Ca2+ that was ∼equal to the elevation caused by 1 µM ATP. BAPTA-AM pretreatment prevented the ATP and forskolin/ATP synergistic increased DRA activity and the increase in intracellular Ca2+ caused by ATP/forskolin. Conclusion: In Caco-2/BBE cells subthreshold concentrations of forskolin (cAMP) and ATP (Ca2+) synergistically increased intracellular Ca2+ and stimulated DRA activity with both being blocked by BAPTA-AM pretreatment. Diarrheal diseases such as bile acid diarrhea, in which both cAMP and Ca2+ are elevated, are likely to be associated with stimulated DRA activity contributing to increased anion secretion, while separation of DRA from NHE3 contributes to reduced NaCl absorption.