NHE3 inhibition by cAMP and Ca²⁺ is abolished in PDZ‐domain protein PDZK1‐deficient murine enterocytes

Abstract

The PDZ-binding protein PDZK1 (NHERF3/CAP70/PDZ-dc-1) in vitro binds to NHE3, but its role in the regulation of NHE3 activity in native enterocytes is unknown. This study was undertaken to understand the physiological role of PDZK1 in regulating NHE3 activity in native murine colonic enterocytes. NHE3 transport rates were assessed fluorometrically in BCECF-loaded colonic crypts in the NHE3-expressing cryptal openings by measuring acid-activated, Na + -dependent, Hoe 642-insensitive proton efflux rates. NHE3 mRNA expression levels and NHE3 total enterocyte and brush border membrane (BBM) protein abundance were determined by quantitative PCR and Western analysis and immunohistochemistry. In pdzk1−/− colonic surface cells, acid-activated NHE3 transport rates were strongly reduced, and the inhibitory effect of forskolin and ionomcyin was virtually abolished. Hyperosmolarity, on the other hand, still had an inhibitory effect. In addition, the NHE3-selective inhibitor S1611 inhibited acid-activated NHE3 activity in pdzk1−/− and +/+ mice, suggesting that functional NHE3 is present in pdzk1-deficient colonocytes. NHE1 and NHE2 activity was not altered in pdzk1−/− colonic crypts. Immunohistochemistry revealed apical NHE3 staining in pdzk1−/− and +/+ proximal colon, and Western blot analysis revealed no difference in NHE3 abundance in colonic enterocyte homogenate as well as brush border membrane. Lack of the PDZ-adaptor protein PDZK1 in murine proximal colonic enterocytes does not influence NHE3 abundance or targeting to the apical membrane, but abolishes NHE3 regulation by cAMPergic and Ca 2+ -dependent pathways. It leaves NHE3 inhibition by hyperosmolarity intact, suggesting an important and selective role for PDZK1 in the agonist-mediated regulation of intestinal NHE3 activity.

Bile acids inhibit Na+/H+ exchanger and Cl−/HCO3 − exchanger activities via cellular energy breakdown and Ca2+ overload in human colonic crypts

“…It was shown previously that the Cl -/HCO 3 -exchanger SLC26A3 and NHE3 are inhibited by the pathological increase of intracellular Ca 2+ [6,26]. In our experiments, CDC induced a dose- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 the release from the ER via IP 3 R and/or RyR or Ca 2+ entry from the extracellular space [32,14,13] [26].…”

“…The activities of the different NHE isoforms are extracted by using the isoform selective NHE inhibitor HOE-642. The isoform selectivity of HOE-642 is dose-dependent, 1µM HOE642 inhibits NHE1 whereas 50µM HOE642 inhibits both NHE1 and 2 but not NHE3 [2,6]. The activities (A) of NHE isoforms can be calculated from the recoveries (R) as follows: …”

Intestinal Anion Exchanger Down-regulated in Adenoma (DRA) Is Inhibited by Intracellular Calcium

“…Furthermore, colonic surface cells from PDZK1 knock-out mice show loss of calcium inhibition of NHE3 (34). In these colonic surface cells NHE3 and DRA are coexpressed (32).…”

NHERF3 (PDZK1) Contributes to Basal and Calcium Inhibition of NHE3 Activity in Caco-2BBe Cells

“…Recent studies have begun evaluating the effect of NHERF3 on mouse intestinal Na ϩ and Cl Ϫ transport. Basal electroneutral sodium absorption was decreased by Ͼ40% in the NHERF3 null mouse jejunum (21) and by Ͼ80% in the colon (22). In addition, Cinar et al (22) demonstrated that cAMP and [Ca 2ϩ ] i inhibition of NHE3 activity was abolished in the NHERF3 null mouse colon.…”