36 – Glucocorticoids and Sgk1- Potent Regulators of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in the Intestine

Background: ST inhibitS intestinal NHE3 activity as part of Enterotoxigenic E. coli diarrhea (Traveler's Diarrhea).Based largely on studies in murine intestine, the defined mechanism involved includes activation of guanylate cyclase C, increased cGMP to activate cGKII to phosphorylate and inhibit NHE3.For drug development and physiologic/disease studies it is often assumed that studies in animal intestine, colon cancer cell lines and human intestine are equivalent.However, drug development with this model have usually failed.We tested this assumption by comparing how ST inhibits NHE3 in mouse jejunum and polarized Caco-2 cells and demonstrate that this assumption is not always accurate.METHODS: Parallel studies were performed in mouse (C57Bl/6N) jejunum and Caco-2 cells with study of NHERF2 and NHERF3 KO mice and shRNA KD in Caco-2 cells; and include: In vivo loop studies, two photon microscopy/SNARF-4F intracellular pH determination, surface biotinylation, measurement of cGMP and cytosolic free Ca 2+ using cytosolic and membrane bound GcAMP.Results: 1) ST rapidly inhibited NHE3 in mouse jejunum and Caco-2 cells.This involved an acute reduction in BB NHE3 determined by cell surface biotinylation without altering the specific activity of BB NHE3.2) The ST effect on NHE3 required both NHERF2 and NHERF3; mutations in NHERF3 to prevent its heteromerization with NHERF2 also prevented the ST effect.3) ST activated guanylate cyclase C-cGMP in both models, which did not require either NHERF2 or NHERF3.4) cGKII determined by immunoblotting and qRT-PCR was present in mouse jejunum but not Caco-2.5) In mouse jejunum, ST inhibited NHE3 via activation of cGKII (based on studies with a specific cGKII inhibitor) but did not act by cAMP or elevated Ca 2 .6) In Caco-2 cells, ST effect on NHE3 was prevented by BAPTAM but not H-89 (PKA inhibitor) and there was no effect of 8-pCPT-cGMP.ST elevated intracellular Ca 2+ rapidly that did not depend on external Ca 2+.CONCLU-SIONS: 1) ST causes similar inhibition of NHE3 in mouse jejunum and Caco-2 cells by a process that entirely occurs by altering trafficking and involves similar signaling in both models up to the increase in cGMP.2) ST did not alter NHE3 in the absence of either NHERF2 or NHERF3 which acted as a heterodimer in this effect.3) ST activated GCC-cGMP in both models independently of NHERF2 or NHERF3.4) cGKII was present in mouse jejunum but not in Caco-2.5) ST acts by activation of cGKII in mouse intestine but not in Caco-2 cells in which ST inhibits NHE3 by elevating intracellular Ca 2+ .6) This demonstration of different intracellular signaling in two models of NHE3 inhibition serves as a warning that it can not be assumed that intestinal transport models all behave equivalently and documentation of mechanisms of action are required, including when developing drugs for GI disorders. 35