cAMP Stimulates SLC26A3 Activity in Human Colon by a CFTR-Dependent Mechanism That Does Not Require CFTR Activity

Abstract

Background & Aims SLC26A3 (DRA) is an electroneutral Cl - /HCO 3 - exchanger that is present in the apical domain of multiple intestinal segments. An area that has continued to be poorly understood is related to DRA regulation in acute adenosine 3′,5′-cyclic monophosphate (cAMP)-related diarrheas, in which DRA appears to be both inhibited as part of NaCl absorption and stimulated to contribute to increased HCO 3 - secretion. Different cell models expressing DRA have shown that cAMP inhibits, stimulates, or does not affect its activity. Methods This study re-evaluated cAMP regulation of DRA using new tools, including a successful knockout cell model, a specific DRA inhibitor (DRA inh -A250), specific antibodies, and a transport assay that did not rely on nonspecific inhibitors. The studies compared DRA regulation in colonoids made from normal human colon with regulation in the colon cancer cell line, Caco-2. Results DRA is an apical protein in human proximal colon, differentiated colonoid monolayers, and Caco-2 cells. It is glycosylated and appears as 2 bands. cAMP (forskolin) acutely stimulated DRA activity in human colonoids and Caco-2 cells. In these cells, DRA is the predominant apical Cl - /HCO 3 - exchanger and is inhibited by DRA inh -A250 with a median inhibitory concentration of 0.5 and 0.2 μmol/L, respectively. However, there was no effect of cAMP in HEK293/DRA cells that lacked a cystic fibrosis transmembrane conductance regulator (CFTR). When CFTR was expressed in HEK293/DRA cells, cAMP also stimulated DRA activity. In all cases, cAMP stimulation of DRA was not inhibited by CFTR inh -172. Conclusions DRA is acutely stimulated by cAMP by a process that is CFTR-dependent, but appears to be one of multiple regulatory effects of CFTR that does not require CFTR activity.

Chronic exposure of ErbB TKIs inhibits DRA expression and activity through an ERK/Elk-1/CREB/AP-1 dependent pathway

“…DRA activity was quantified as the extent of intracellular alkalinization initiated by extracellular Clremoval, and then exit of intracellular Clin exchange for HCO 3 entry. This process is entirely inhibited in these cells by the specific DRA inhibitor, DRA inh -A250, as reported in our previous publication (Tse et al, 2019). As shown in figure 4A, a rapid intracellular alkalinization occurred immediately after removal of Clfrom the apical surface of Caco-2/bbe cells in both vehicle (DMSO) and non-ErbB TKIs (dasatinib and suntinib) treated cells.…”

“…This intriguing and contradictory phenomenon might be explained by the interrupted interaction between CFTR and DRA. CFTR and DRA proteins are co-expressed in the luminal membrane of a large population of ileal lower villus and upper crypt cells and in some surface epithelial cells and upper crypt cells of human proximal colon (Tse et al, 2019). It has been reported that the FSK-stimulated activity (open probability) of CFTR is dramatically increased by six fold when DRA is present, which uses its STAS domain to bind to the phosphorylated R domain of CFTR (Ko et al, 2004).…”

“…Measurement of Cl -/HCO 3 exchange activity. A detailed procedure for measuring Cl -/HCO 3 exchange activity in Caco-2/bbe monolayers has been described previously (Tse et al, 2019). In brief, the activity was fluorometrically measured by using the pHi-sensitive dye BCECF-AM in a customized chamber allowing simultaneous but separate apical and basolateral superfusion.…”

Adaptation to inflammatory acidity through neutrophil-derived adenosine regulation of SLC26A3

“…It has been reported previously that forskolin is able to stimulate SLC26A3 expression and activity in a cAMP dependent fashion, we extended these studies to determine the signaling pathway(s) involved in SLC26A3 regulation by Ado 32 . Utilizing pharmacological inhibition, we targeted three significant points within the Ado signaling pathway: namely adenylyl cyclase, CREB, and Erk.…”

Cyclic nucleotides, gut physiology and inflammation

“…In addition to its role in CFTR activation, cAMP could contribute to cholera pathogenesis through additional mechanisms. For instance, attenuation of Na + absorption due to reduced expression of Na + /H + exchangers (e.g., NHE3) and enhancement of HCO 3− secretion due to stimulation of Cl − /HCO3 − exchangers (e.g., DRA/downregulated in adenoma) could act in parallel to cause diarrhea . Furthermore, CTX‐driven cAMP increase was reported to inhibit autophagy by suppressing autophagosome maturation, which might help bacteria escape host defense .…”