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

In polarized intestinal epithelial cells, downregulated in adenoma (DRA) is an apical Cl^-/[Formula: see text] 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 5'-triphosphate (ATP). FSK and ATP stimulated DRA in a concentration-dependent manner, with ATP acting via P2Y1 receptors. FSK at 1 µM and ATP at 0.25 µM 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 µM), which by itself did not significantly alter Ca²⁺_i, followed by 0.25 µM ATP produced a large increase in Ca²⁺ that was approximately equal to the elevation caused by 1 µM ATP. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-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.NEW & NOTEWORTHY The BB Cl^-/[Formula: see text] exchanger DRA takes part in both neutral NaCl absorption and stimulated anion secretion. Using intestinal cell line, Caco-2/BBE high concentrations of cAMP and Ca²⁺ individually stimulated DRA activity, whereas low concentrations, which had no/minimal effect, synergistically stimulated DRA activity that required a synergistic increase in intracellular Ca²⁺. This study increases understanding of diarrheal diseases, such as bile salt diarrhea, in which both cAMP and elevated Ca²⁺ are involved.