CFTR-mediated anion secretion in parathyroid hormone-treated Caco-2 cells is associated with PKA and PI3K phosphorylation but not intracellular pH changes or Na+/K+-ATPase abundance

Abstract

Parathyroid hormone (PTH) has previously been shown to enhance the transepithelial secretion of Cl − and HCO 3 − across the intestinal epithelia including Caco-2 monolayer, but the underlying cellular mechanisms are not completely understood. Herein, we identified the major signaling pathways that possibly mediated the PTH action to its known target anion channel, i.e., cystic fibrosis transmembrane conductance regulator anion channel (CFTR). Specifically, PTH was able to induce phosphorylation of protein kinase A and phosphoinositide 3-kinase. Since the apical HCO 3 − efflux through CFTR often required the intracellular H + /HCO 3 − production and/or the Na + -dependent basolateral HCO 3 − uptake, the intracellular pH (pH i ) balance might be disturbed, especially as a consequence of increased endogenous H + and HCO 3 − production. However, measurement of pH i by a pH-sensitive dye suggested that the PTH-exposed Caco-2 cells were able to maintain normal pH despite robust HCO 3 − transport. In addition, although the plasma membrane Na + /K + -ATPase (NKA) is normally essential for basolateral HCO 3 − uptake and other transporters (e.g., NHE1), PTH did not induce insertion of new NKA molecules into the basolateral membrane as determined by membrane protein biotinylation technique. Thus, together with our previous data, we concluded that the PTH action on Caco-2 cells is dependent on PKA and PI3K with no detectable change in pH i or NKA abundance on cell membrane.

Potentiometric pH Nanosensor for Intracellular Measurements: Real-Time and Continuous Assessment of Local Gradients

“…The results were validated via the commercially available fluorometric assay based on the cell-permeable fluorescent indicator BCFL-AM. 58 , 59 In all the cases using the pH nanosensor, six different cells were analyzed to obtain average pH values with their corresponding deviations ( n = 6).…”

“…As a control experiment, the pH i of a single PC12 cell was traced during the same time frame and without the addition of cariporide. The results were validated via the commercially available fluorometric assay based on the cell-permeable fluorescent indicator BCFL-AM. , In all the cases using the pH nanosensor, six different cells were analyzed to obtain average pH values with their corresponding deviations ( n = 6).…”

Vasoactive intestinal peptide and cystic fibrosis transmembrane conductance regulator contribute to the transepithelial calcium transport across intestinal epithelium-like Caco-2 monolayer

“…Indeed, the exact mechanism of CFTR action as a crucial player in calcium transport remains elusive. We previously reported that the salient calcium-regulating hormone PTH could directly stimulate CFTR opening, thereby enhancing HCO 3 transport across Caco-2 monolayer [7,37], while incubating Caco-2 monolayer in HCO 3 --free solution markedly diminished PTH-induced anion transport [38]. Thus, Caco-2 cells used CFTR to transport HCO 3 -, which might, in turn, affect calcium transport.…”