SLC26A3 (DRA) is stimulated in a synergistic, intracellular Ca21-dependent manner by cAMP and ATP in intestinal epithelial cells

In polarized intestinal epithelial cells, downregulated in adenoma (DRA) is an apical Cl-/HCO- 3 exchanger that is part of neutral NaCl absorption under baseline conditions, but in cyclic adenosine monophosphate (cAMP)-driven diarrheas, it is stimulated and contributes to increased anion secretion. To further understand the regulation of DRA in conditions mimicking some diarrheal diseases, Caco-2/BBE cells were exposed to forskolin (FSK) and adenosine 50-triphosphate (ATP). FSK and ATP stimulated DRA in a concentration-dependent manner, with ATP acting via P2Y1 receptors. FSK at 1 lM and ATP at 0.25 lM had minimal to no effect on DRA given individually; however, together, they stimulated DRA to levels seen with maximum concentrations of FSK and ATP alone. In Caco-2/BBE cells expressing the Ca2+ indicator GCaMP6s, ATP increased intracellular Ca2+ (Ca2+ i) in a concentration- dependent manner, whereas FSK (1 lM), which by itself did not significantly alter Ca2+ i, followed by 0.25 lM ATP produced a large increase in Ca2+ that was approximately equal to the elevation caused by 1 lM ATP. 1,2-Bis(2-aminophenoxy) ethane-N,N,N0,N0-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) pretreatment prevented the ATP and FSK/ATP synergistically increased the DRA activity and the increase in Ca2+ i caused by FSK/ATP. FSK/ATP synergistic stimulation of DRA was similarly observed in human colonoids. In Caco-2/BBE cells, subthreshold concentrations of FSK (cAMP) and ATP (Ca2+) synergistically increased Ca2+ i 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, whereas separation of DRA from Na+/H+ exchanger isoform-3 (NHE3) contributes to reduced NaCl absorption.