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

Mayuree Rodrat; Kannikar Wongdee; Jarinthorn Teerapornpuntakit; Jirawan Thongbunchoo; Duangrudee Tanramluk; Ratchaneevan Aeimlapa; Nithipak Thammayon; Natchayaporn Thonapan; Pathnaree Wattano; Narattaphol Charoenphandhu

doi:10.1371/journal.pone.0277096

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

PLoS One. 2022 Nov 18;17(11):e0277096. doi: 10.1371/journal.pone.0277096. eCollection 2022.

Authors

Mayuree Rodrat^{1

2

3

4}, Kannikar Wongdee^{1

5}, Jarinthorn Teerapornpuntakit^{1

6}, Jirawan Thongbunchoo^{1

2}, Duangrudee Tanramluk^{3

7}, Ratchaneevan Aeimlapa^{1

2}, Nithipak Thammayon^{1

8}, Natchayaporn Thonapan^{1

8}, Pathnaree Wattano¹, Narattaphol Charoenphandhu^{1

2

3

9}

Affiliations

¹ Faculty of Science, Center of Calcium and Bone Research (COCAB), Mahidol University, Bangkok, Thailand.
² Faculty of Science, Department of Physiology, Mahidol University, Bangkok, Thailand.
³ Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, Thailand.
⁴ Center of Research and Development for Biomedical Instrumentation, Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, Thailand.
⁵ Faculty of Allied Health Sciences, Burapha University, Chonburi, Thailand.
⁶ Department of Physiology, Faculty of Medical Science, Naresuan University, Phitsanulok, Thailand.
⁷ Integrative Computational BioScience (ICBS) Center, Mahidol University, Nakhon Pathom, Thailand.
⁸ Faculty of Science, Graduate Program in Molecular Medicine, Mahidol University, Bangkok, Thailand.
⁹ The Academy of Science, The Royal Society of Thailand, Bangkok, Thailand.

Abstract

Vasoactive intestinal peptide (VIP) as a neurocrine factor released by enteric neurons has been postulated to participate in the regulation of transcellular active calcium transport across intestinal epithelium, but the preceding evidence is scant and inconclusive. Herein, transepithelial calcium flux and epithelial electrical parameters were determined by Ussing chamber technique with radioactive tracer in the intestinal epithelium-like Caco-2 monolayer grown on Snapwell. After 3-day culture, Caco-2 cells expressed mRNA of calcium transporters, i.e., TRPV6, calbindin-D9k, PMCA1b and NCX1, and exhibited transepithelial resistance of ~200 Ω cm2, a characteristic of leaky epithelium similar to the small intestine. VIP receptor agonist was able to enhance transcellular calcium flux, whereas VIP receptor antagonist totally abolished calcium fluxes induced by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Since the intestinal cystic fibrosis transmembrane conductance regulator (CFTR) could be activated by VIP and calciotropic hormones, particularly parathyroid hormone, we sought to determine whether CFTR also contributed to the 1,25(OH)2D3-induced calcium transport. A selective CFTR inhibitor (20-200 μM CFTRinh-172) appeared to diminish calcium fluxes as well as transepithelial potential difference and short-circuit current, both of which indicated a decrease in electrogenic ion transport. On the other hand, 50 μM genistein-a molecule that could rapidly activate CFTR-was found to increase calcium transport. Our in silico molecular docking analysis confirmed direct binding of CFTRinh-172 and genistein to CFTR channels. In conclusion, VIP and CFTR apparently contributed to the intestinal calcium transport, especially in the presence of 1,25(OH)2D3, thereby supporting the existence of the neurocrine control of intestinal calcium absorption.

Copyright: © 2022 Rodrat et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Caco-2 Cells
Calcium* / metabolism
Calcium, Dietary / metabolism
Cystic Fibrosis Transmembrane Conductance Regulator* / genetics
Genistein / metabolism
Humans
Intestinal Mucosa / metabolism
Ion Transport
Molecular Docking Simulation
Receptors, Vasoactive Intestinal Peptide / metabolism
Vasoactive Intestinal Peptide / metabolism
Vasoactive Intestinal Peptide / pharmacology

Substances

Cystic Fibrosis Transmembrane Conductance Regulator
Calcium
Vasoactive Intestinal Peptide
Receptors, Vasoactive Intestinal Peptide
Genistein
Calcium, Dietary

Grants and funding

Our study was supported by grants from the National Research Council of Thailand-Mahidol University (to NC), National Science and Technology Development Agency (to NC), Thailand Science Research and Innovation (TSRI)-Mahidol University (Fundamental Fund/Basic Research Fund: fiscal year 2022; to NC), TSRI-Burapha University (Fundamental Fund: fiscal year 2022; to KW), TRF-Royal Golden Jubilee Ph.D. Program (PHD/0105/2557 to MR), Faculty of Science, Mahidol University (CIF/CNI Grant; to NC), and MU-MiniRC Grant from Mahidol University (to DT). Nithipak Thammayon was supported by TRF/TSRI-International Research Network Program (IRN60W0001).