Skip to main content

Advertisement

The inhibitory role of purinergic P2Y receptor on Mg2+ transport across intestinal epithelium-like Caco-2 monolayer

Abstract

The mechanism of proton pump inhibitors (PPIs) suppressing intestinal Mg2+ uptake is unknown. The present study aimed to investigate the role of purinergic P2Y receptors in the regulation of Mg2+ absorption in normal and omeprazole-treated intestinal epithelium-like Caco-2 monolayers. Omeprazole suppressed Mg2+ transport across Caco-2 monolayers. An agonist of the P2Y2 receptor, but not the P2Y4 or P2Y6 receptor, suppressed Mg2+ transport across control and omeprazole-treated monolayers. Omeprazole enhanced P2Y2 receptor expression in Caco-2 cells. Forskolin and P2Y2 receptor agonist markedly enhanced apical HCO3 secretion by control and omeprazole-treated monolayers. The P2Y2 receptor agonist suppressed Mg2+ transport and stimulated apical HCO3 secretion through the Gq-protein coupled-phospholipase C (PLC) dependent pathway. Antagonists of cystic fibrosis transmembrane conductance regulator (CFTR) and Na+-HCO3 cotransporter-1 (NBCe1) could nullify the inhibitory effect of P2Y2 receptor agonist on Mg2+ transport across control and omeprazole-treated Caco-2 monolayers. Our results propose an inhibitory role of P2Y2 on intestinal Mg2+ absorption.

Introduction

Although there is an abundant amount of magnesium (Mg2+) within human cells, and it has vital roles in numerous biological functions [1], knowledge regarding regulatory mechanisms of Mg2+ homeostasis is still minimal. Theoretically, plasma Mg2+ level is regulated within a narrow range by the synergistic actions of intestinal absorption, bone and soft tissue storage, and renal excretion [1]. Since dietary intake is the only source of Mg2+, intestinal absorption is vital for normal Mg2+ homeostasis. However, the understanding of the regulatory mechanism of intestinal Mg2+ absorption is still elusive. Principally, enterocyte epithelium absorbs Mg2+ via both saturable transcellular and non-saturable paracellular transport [1]. Approximately 90% of total intestinal Mg2+ uptake is processed through a Mg2+ channel-independent paracellular passive mechanism which exclusively occurs in the small intestine [1,2,3]. The Mg2+ channel-dependent transcellular active Mg2+ uptake plays an important role during low dietary Mg2+ intake [3].

In the small intestine, enterocyte epithelial cells are equipped with acid sensors, e.g., acid-sensing ion channel (ASIC), ovarian cancer G protein-coupled receptor 1 (OGR1), and transient receptor potential vanilloid (TRPV) that are implicated in mucosal defense by detecting mucosal protons and stimulating mucosal HCO3 secretion [4,5,6,7,8,9]. In addition to this mucosal defense, intestinal acid sensors also regulate ion transport across the enterocyte epithelium. Reiter et al. [10] reported that TRPV4 enhanced transcellular K+ transport and paracellular permeability through Ca2+ signaling in HC11 epithelial monolayers. OGR1 enhanced Mg2+ absorption in intestinal epithelium-like Caco-2 monolayers through protein kinase C (PKC) signaling [11, 12]. On the other hand, an activation of ASIC1a led to a suppression of Mg2+ absorption across Caco-2 monolayers through a Ca2+-signaling dependent pathway [12]. In addition to acid sensors, the purinergic P2Y2 receptor is also involved in mucosal acid sensing and defense of duodenocytes [8]. Activation of Gq-associated P2Y2 stimulated duodenal mucosal HCO3 secretion in a Ca2+ signaling-dependent mechanism [4]. Besides HCO3 secretion, the P2Y2 receptor also regulates Na+, K+, and Cl transport in various epithelial tissues [13]. However, the role of P2Y on intestinal Mg2+ absorption is still unknown.

PPI-induced hypomagnesemia (PPIH) has been reported since 2006 [14,15,16,17,18,19]. Suppression of intestinal Mg2+ absorption is a major underlying mechanism of PPIH in chronic users and animal models [14, 15, 17, 19,20,21]. We previously reported that a higher apical HCO3 secretion contributed to a suppression of intestinal Mg2+ absorption in PPIH [21]. Apical acidity in the small intestine [22] required for stabilizing mineral solubility [23] and stimulates intestinal Mg2+ absorption [11, 12, 24]. An increase in luminal pH from ~ 5 to 7.8 led to a decrease in soluble Mg2+ from 79.61 to 8.71% [25]; thus, secreted HCO3 increases luminal pH and subsequently suppresses Mg2+ absorption. Omeprazole, the most common PPI, significantly enhanced HCO3 secretion in human duodenum and intestinal epithelium-like Caco-2 monolayers [12, 26]. Antagonists of mucosal HCO3 secretion markedly increased duodenal Mg2+ absorption in PPIH rats [21]. P2Y2 regulated mucosal HCO3 secretion [4], but the involvement of P2Y2 on apical HCO3 secretion and Mg2+ absorption in PPI-treated intestinal epithelium is unknown.

Therefore, the present study aimed to investigate the role of purinergic P2Y receptors in the regulation of Mg2+ absorption in normal and omeprazole-treated intestinal-like Caco-2 monolayers. Caco-2 cells express P2Y receptors, i.e., P2Y2, P2Y4, and P2Y6 [27, 28]. They are also equipped with apical HCO3 secretion transporting machineries, e.g., cystic fibrosis transmembrane conductance regulator (CFTR) and Na+-HCO3 cotransporter-1 (NBCe1), which are modulated by parathyroid hormone (PTH) and HCl [12, 29]. In addition, Caco-2 monolayers have been used as a model for studying the regulation of intestinal Mg2+ absorption [11, 12, 30, 31].

Methods

Cell culture

Caco-2 cells (ATCC No. HTB-37, passage 25–40th) were grown in Dulbecco’s modified Eagle medium (DMEM) (Sigma, St. Louis, MO, USA) supplemented with 12.5% fetal bovine serum (FBS-Gold) (PAA Laboratories GmbH, Pasching, Austria), 1% l-glutamine (Gibco, Grand Island, NY, USA), 1% non-essential amino acid (Sigma), and 1% antibiotic–antimycotic solution (Gibco) and maintained in a humidified atmosphere containing 5% CO2 at 37 °C. Culture medium was changed 3 times a week. For epithelial electrical parameter measurement and Mg2+ flux studies, the monolayers were developed by seeding cells (5.0 × 105 cells/cm2) onto permeable polyester Snapwell inserts (12 mm diameter and 0.4 µm pore size filter) (Corning, Corning, NY, USA). After being maintained for 14 days, the Snapwell was inserted into a Ussing chamber (World Precision Instrument, Sarasota, FL, USA). For HCO3 secretion studies, the cells were plated (5.0 × 105 cells/cm2) onto permeable polyester Transwell-clear inserts (Corning) and maintained for 14 days. For western blot analysis, cells were plated (5.0 × 105 cells/well) on 6-well plates (Corning) and maintained for 14 days.

In the omeprazole-treated group, Caco-2 monolayers were grown in a culture medium containing omeprazole (Calbiochem, San Diego, CA, USA), from day 7 to day 14 of culture [31], at concentrations of 200 and 400 ng/ml that resembled those found in human plasma [32].

Bathing solutions

The physiological bathing solution contained (in mM) 118 NaCl, 4.7 KCl, 1.1 MgCl2, 1.25 CaCl2, 23 NaHCO3, 12 d-glucose, 2.5 l-glutamine, and 2 mannitol.

For the apical to basolateral total Mg2+ transport studies, the apical solution contained (in mM) 40 MgCl2, 1.25 CaCl2, 4.5 KCl, 12 d-glucose, 2.5 l-glutamine, 115 d-mannitol, and 10 HEPES, whereas the basolateral solution contained (in mM) 1.25 CaCl2, 4.5 KCl, 12 d-glucose, 2.5 l-glutamine, 250 d-mannitol, and 10 HEPES.

For apical HCO3 secretion experiments, the composition of the NaHCO3-free apical solution was as follows (in mM): 1.25 CaCl2, 4.5 KCl, 1 MgCl2, 12 d-glucose, 2.5 l-glutamine, 230 d-mannitol, and 10 HEPES; and the NaHCO3-containing basolateral solution contained (in mM) 25 NaHCO3, 1.25 CaCl2, 4.5 KCl, 1 MgCl2, 12 d-glucose, 2.5 l-glutamine, 200 d-mannitol, and 10 HEPES.

All solutions were continuously gassed with humidified 5% CO2 in 95% O2, maintained at 37 °C, pH 7.4, and had an osmolality of 290–295 mmol kg−1 water as measured by a freezing-point depression-based Fiske® micro-osmometer (model 210; Fiske® Associates, Norwood, MA, USA). All chemicals were purchased from Sigma.

Transepithelial electrical resistance

Snapwell inserts containing Caco-2 monolayers were rinsed gently, mounted in a Ussing chamber, and bathed on both sides with physiological bathing solution. Transepithelial potential difference (PD) and short-circuit current (Isc) were determined by Ag/AgCl electrodes and an epithelial voltage/current clamp apparatus (model ECV-4000; World Precision Instrument) as previously described [33]. Transepithelial electrical resistance (TEER) was calculated from PD and Isc by Ohm’s law.

Mg2+ transport study

Caco-2 monolayers were rinsed and mounted in a Ussing chamber as described above. After being equilibrated in physiological bathing solution for 15 min, total Mg2+ transport studies were performed by substituting the physiological bathing solution with apical and basolateral bathing solutions for Mg2+ transport. To investigate the Mg2+ channel-independent Mg2+ transport, apical sites of Caco-2 monolayers from the same passage and culture plate were pre-incubated for 10 min with Mg2+-channel inhibitor Cobalt(III)hexaammine [Co(III)hex, Table 1], which suppressed Mg2+ influx in Caco-2 epithelium and blocked Mg2+ channel-dependent Mg2+ transport. After that the apical and basolateral solutions were substituted with bathing solution for Mg2+ transport. At 30, 60, and 120 min after solution replacements, 50 μl of solution was collected from the basolateral side, as well as from the apical side. The Mg2+ concentration and the rate of Mg2+ flux were determined by the method of Thongon and Krishmanra [11]. The rate of Mg2+ channel-dependent Mg2+ transport was calculated by subtracting the rate of Mg2+ channel-independent Mg2+ transport from the rate of total Mg2+ transport. However, Co(III)hex might somehow interfered Mg2+ channel-independent paracellular Mg2+ transport, which was not demonstrated in the present study.

Table 1 Agonist, antagonist, or chelator used in the study of Mg2+ transport and HCO3 secretion across Caco-2 monolayers

In some experiments the monolayers were pre-incubated with agonists or antagonist, as demonstrated in Table 1, for 40 min prior to performing experiments.

Measurements of HCO3 secretion

Apical HCO3 secretion was studied by the modified method of Thongon et al. [12]. The Caco-2 monolayer was gently rinsed 3 times and incubated for 15 min in the physiological bathing solution. Then, apical and basolateral solutions were substituted with bathing solutions for HCO3 secretion. The apical membrane-bound carbonic anhydrase (CA) activity was suppressed by the selective CA IX inhibitor U-104 (Table 1). After 20 min, HCO3 secretion was stimulated by adding MRS2768 or forskolin (Table 1) and incubation proceeded for 5 min. After removal of the MRS2768- or forskolin-containing solutions, the monolayers were gently rinsed 3 times and further incubated for 25 min. Aliquots of apical solution at various time points (Fig. 5) were individually sampled. The concentration of HCO3 was immediately determined as previously described [12].

In some experiments the monolayers were pre-incubated with agonists or antagonist, as demonstrated in Table 1, for 40 min prior to performing experiments.

Western blot analysis

Western blot analysis was performed as previously described [11]. In brief, protein samples of Caco-2 cells were prepared by using Piece® Ripa Buffer (Thermo Fisher Scientific Inc., Rockford, IL, USA). Protein samples (35 μg each) were separated on 12.5% SDS-PAGE gels, and then transferred onto nitrocellulose membranes (Amersham, Buckinghamshire, UK) by electroblotting. Membranes were blocked and probed overnight at 4 °C with 1:1000 rabbit polyclonal antibodies raised against human P2Y2 receptor and CFTR (Santa Cruz Biotechnology, Santa Cruz, CA). Membranes were also re-probed with actin monoclonal antibodies (Santa Cruz Biotechnology) diluted at 1:5000. After 2 h incubation at 25 °C with goat anti-rabbit IgG-HRP-conjugated secondary antibodies (Santa Cruz Biotechnology) diluted at 1:10,000, blots were visualized by Thermo Scientific SuperSignal® West Pico Substrate (Thermo Fisher Scientific Inc.) and captured on CL-XPosure Film (Thermo Fisher Scientific Inc.). Densitometric analysis was performed using ImageJ for Mac Os X.

Statistical analysis

Results were expressed as mean ± SE. Two sets of data were compared using unpaired Student’s t-test. One-way analysis of variance (ANOVA) with Dunnett’s post test was used for comparison of multiple sets of data. The level of significance was P < 0.05. All data were analyzed by GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA).

Results

Omeprazole modulated paracellular Mg2+ transport

Previous Mg2+ transport kinetic analysis demonstrated that omeprazole selectively impeded non-saturable passive Mg2+ transport but not saturable active Mg2+ transport across Caco-2 monolayers [31]. By using competitive Mg2+-channel inhibitor Co(III)hex (Fig. 1), we observed total (white bars), Mg2+ channel-independent (gray bars), and Mg2+ channel-dependent Mg2+ transport (black bars). The results showed that 200 and 400 ng/ml omeprazole significantly suppressed total (Fig. 1d) and Mg2+ channel-independent Mg2+ transport (Fig. 1e) compared to the control group. Furthermore, we had studied the effect of omeprazole on Mg2+ channel-independent Mg2+ transport. Previously, it was reported that protein kinase C activator carbachol (CCh) could increase paracellular permeability and decrease TEER in Caco-2 monolayers [34, 35]. In control (Fig. 1a), 200-ng/ml omeprazole-treated group (Fig. 1b), and 400-ng/ml omeprazole-treated group (Fig. 1c), CCh significantly increased total and Mg2+ channel-independent Mg2+ transport compared to the corresponding vehicle-treated group. The rates of Mg+ channel-dependent Mg2+ transport of all experiments were not changed (Fig. 1a–c, f).

Fig. 1
figure1

Effect of omeprazole on Mg2+ transport across Caco-2 monolayers. The rate of Mg2+ transport across control (a), 200-ng/ml omeprazole-treated (b), and 400-ng/ml omeprazole-treated Caco-2 monolayers (c). White bar; total Mg2+ transport, gray bar; Mg2+ channel-independent Mg2+ transport, black bar; Mg2+ channel-dependent Mg2+ transport. *P < 0.05, **P < 0.01, ***P < 0.001 compared with its corresponding CCh-untreated group. (n = 6). The rate of total (d), Mg2+-channel independent (e), and Mg2+-channel dependent (f) Mg2+ transport of control and omeprazole-treated Caco-2 monolayers. *P < 0.05, **P < 0.01, ***P < 0.001 compared with its corresponding control group (n = 6)

We also studied TEER to confirm the permeability of Caco-2 monolayers. As demonstrated in Fig. 2c, Co(III)hex, which impeded Mg2+ channel-dependent Mg2+ transport, had no effect on TEER of control and omeprazole-treated monolayers when compared to its corresponding vehicle-treated monolayers. On the other hand CCh, which increased Mg2+ channel-independent Mg2+ transport, significantly decreased TEER of control and omeprazole-treated (Fig. 2c) Caco-2 monolayers. We further observed the involvement of Ca2+-activated K+ and Ca2+-activated Cl channels on CCh-suppressed TEER by using charybdotoxin (ChTX) and benzbromarone. ChTX and benzbromarone had no effect on CCh-suppressed TEER of control and omeprazole-treated Caco-2 monolayers. However, CCh might somehow modulate some ion channels or transports, which could affect TEER of Caco-2 monolayers. TEER of 400-ng/ml omeprazole-treated monolayers (458.56 ± 14.97 Ω cm2) was significantly higher than that of vehicle-treated control monolayers (362.31 ± 17.69 Ω cm2, P = 0.0003) (Fig. 2c). This series of experiments suggested that alteration of total Mg2+ transport across Caco-2 monolayers was the result of the modulation of Mg2+ channel-independent Mg2+ transport. These results also agreed with a previous study [31] that omeprazole exclusively suppressed nonsaturable passive Mg2+ transport in intestinal epithelium-like Caco-2 monolayers. In addition, TEER could determine the change of Mg2+ channel-independent Mg2+ transport, but not Mg2+ channel-dependent Mg2+ transport. Thus, Mg2+ channel-dependent Mg2+ transport was ignored in the rest of the experiments.

Fig. 2
figure2

Transepithelial electrical resistance (TEER). TEER of control and 400-ng/ml omeprazole-treated Caco-2 monolayers in various experiments. *P < 0.05, **P < 0.01 compared with its corresponding vehicle-treated group (n = 6). Benzb benzbromarone

P2Y2 receptor modulated Mg2+ transport

To observe the roles of P2Y2, P2Y4, and P2Y6 activities on Mg2+ transport across Caco-2 monolayers we incubated the monolayers with selective agonists of P2Y2, P2Y4, or P2Y6 receptors (Table 1). In control monolayers (Fig. 3a) the rate of Mg2+ transport (in nmol/h/cm2) of the P2Y2 agonist-treated group (81.14 ± 3.45), but not P2Y4 or P2Y6 agonist-treated groups, was significantly lower than that of the vehicle-treated group (138.89 ± 4.85). As demonstrated in Fig. 3b, the rate of Mg2+ transport (in nmol/h/cm2) of the P2Y2 agonist-treated group (41.94 ± 5.91) was significantly lower than that of vehicle-treated group (106.74 ± 5.12) in the 200-ng/ml omeprazole-treated condition. In 400-ng/ml omeprazole-treated monolayers (Fig. 3c), Mg2+ transport (in nmol/h/cm2) of the P2Y2 agonist-treated group (35.28 ± 2.72) was also significantly suppressed compared to the vehicle-treated group (95.24 ± 4.96). When compared to the corresponding vehicle-treated group, P2Y2 agonist suppressed the rate of Mg2+ transport by about 41.58, 60.71, and 62.96% of control, in the 200-ng/ml omeprazole-treated, and 400-ng/ml omeprazole-treated monolayers, respectively. In addition, TEER of MRS2768 treated monolayers was significantly higher than that of the corresponding vehicle-treated control (Fig. 2a) or omeprazole-treated monolayers (Fig. 2b). We further performed a western blotting study to confirm the enhancing effect of omeprazole on P2Y2 receptor activation in suppressing intestinal Mg2+ absorption. As demonstrated in Fig. 3d, 200 and 400 ng/ml omeprazole significantly enhanced P2Y2 expression when compared to the control cells.

Fig. 3
figure3

The effect of P2Y receptor agonists on Mg2+ transport across Caco-2 monolayers. The rate of Mg2+ transport across control (a), 200-ng/ml omeprazole-treated (b), and 400-ng/ml omeprazole-treated Caco-2 monolayers (c) with agonist of P2Y2 receptor MRS2768, P2Y4 receptor MRS4062, and P2Y6 receptor MRS2693 pre-incubations. Representative immunoblotting and densitometric analysis of P2Y2 expression in control and omeprazole-treated Caco-2 cells (d). ***P < 0.001 compared with its vehicle-treated group (n = 6)

Signaling pathway of P2Y2 receptor activation suppressed Mg2+ transport

This series of experiments aimed to observe the underlying mechanism by which P2Y2 receptor activation mediated the suppression of intestinal Mg2+ absorption. In control (Fig. 4a) and omeprazole-treated Caco-2 monolayers (Fig. 4b), MRS2768 significantly suppressed the rate of total Mg2+ transport. P2Y2 receptor antagonist, PLC antagonist, IP3 receptor antagonist, and intracellular Ca2+ chelator markedly normalized the effect of MRS2768 on Mg2+ transport. However, the antagonist of PKC, MEK1/2, PI3K, PKA, or voltage-gated Ca2+ channel had no effect on MRS2768-suppressed Mg2+ transport in either control or omeprazole-treated Caco-2 monolayers (Fig. 4a–b). In the TEER study, P2Y2 receptor antagonist, PLC antagonist, IP3 receptor antagonist, and intracellular Ca2+ chelator also normalized the effect of MRS2768-increased TEER of control (Fig. 2a) and omeprazole-treated (Fig. 2b) monolayers. These results suggested that P2Y2 receptor activation mediated the suppression of intestinal Mg2+ absorption through PLC, IP3 receptor, and intracellular Ca2+ signaling pathway.

Fig. 4
figure4

The signaling pathway of P2Y receptor activation suppressed Mg2+ transport across Caco-2 monolayers. The rate of Mg2+ transport across control (a) and 400-ng/ml omeprazole-treated Caco-2 monolayers (b) with agonist or antagonist pre-incubations. ***P < 0.001 compared with its vehicle-treated group (n = 6)

Contribution of HCO3 secretion on P2Y2 receptor activation suppressed Mg2+ transport

Previously, we reported the contribution of mucosal HCO3 secretion on omeprazole-suppressed duodenal Mg2+ absorption in PPIH rats [21]. Then, we further studied the contribution of HCO3 secretion on P2Y2 receptor activation-suppressed Mg2+ transport in Caco-2 monolayers. As demonstrated in Fig. 5a and b, P2Y2 agonist significantly suppressed Mg2+ transport in control and omeprazole-treated monolayers. The antagonist of NBCe1, CFTR, and CA could relieve the inhibitory effect of P2Y2 activation on Mg2+ transport across control and omeprazole-treated monolayers (Fig. 5a–b). By using HCO3-free bathing solution in both apical and basolateral sites, the P2Y2 agonist had no effect on Mg2+ transport in normal Caco-2 monolayers (Fig. 5a). In omeprazole-treated monolayers (Fig. 5b), the HCO3-free condition significantly increased Mg2+ transport when compared to its corresponding vehicle-treated group. Under the HCO3-free condition, the P2Y2 agonist also had no effect on Mg2+ transport in omeprazole-treated Caco-2 monolayers (Fig. 5b). Moreover, the antagonist of CFTR, CA, and NBCe1 also normalized the effect of P2Y2 activation-increased TEER of control (Fig. 2a) and omeprazole-treated (Fig. 2b) Caco-2 monolayers. Therefore, apical HCO3 secretion was involved in P2Y2 receptor activation-suppressed Mg2+ transport.

Fig. 5
figure5

Contribution of HCO3 secretion on P2Y receptor activation suppressed Mg2+ transport across Caco-2 monolayers. The rate of Mg2+ transport across control (a), and 400-ng/ml omeprazole-treated Caco-2 monolayers (b) with agonist or antagonist pre-incubations. *P < 0.05, ***P < 0.001 compared with its vehicle-treated group (n = 6)

P2Y2 receptor activation stimulated HCO3 secretion

Previously, it was reported that P2Y2 stimulated mucosal HCO3 secretion in rat duodenum [4]. The present study observed the effect of P2Y2 receptor activation on apical HCO3 secretion in Caco-2 monolayers. Since forskolin stimulated duodenum HCO3 secretion in mice [36] and apical HCO3 secretion in Caco-2 monolayers [29], we used forskolin as positive control for the stimulation of apical HCO3 secretion. Our results showed that P2Y2 agonist MRS2768 and forskolin significantly increased the rate of apical HCO3 secretion by control (Fig. 6a, b), 200-ng/ml omeprazole-treated (Fig. 6c, d), and 400-ng/ml omeprazole-treated (Fig. 6e, f) monolayers. Under the HCO3-free condition, the rate of basal, forskolin-stimulated, and MRS2768-stimulated HCO3 secretions were suppressed in control and omeprazole-treated Caco-2 monolayers (Fig. 6b, d, f). In the pre-stimulating condition (Fig. 6g), the basal HCO3 secretions (in µmol/h/cm2) of 200-ng/ml omeprazole-treated (4.45 ± 0.46) and 400-ng/ml omeprazole-treated (5.19 ± 0.49) monolayers were significantly higher than that of the control monolayers (2.49 ± 0.41). The rate of peak HCO3 secretions (in µmol/h/cm2) in forskolin-stimulated and MRS2768-stimulated conditions (Fig. 6h) of 200-ng/ml omeprazole-treated (12.48 ± 0.46 and 13.01 ± 0.57, respectively) and 400-ng/ml omeprazole-treated (14.12 ± 0.44 and 14.59 ± 0.48, respectively) monolayers were significantly higher than those of the control monolayers (9.16 ± 0.68 and 8.66 ± 0.57, respectively). We further observed the expression of CFTR protein in omeprazole-treated monolayers. As demonstrated in Fig. 7a, omeprazole had no effect on CFTR protein expression in Caco-2 monolayers.

Fig. 6
figure6

P2Y2 receptor agonist stimulated HCO3 secretion. Time course and rate of apical HCO3 secretion by control (a, b), 200-ng/ml omeprazole-treated (c, d), and 400-ng/ml omeprazole-treated Caco-2 monolayers (e, f) that were induced by forskolin or MRS2768. Basal (at 20 min; g) and peak forskolin- or MRS2768-stimulated HCO3 secretion (at 33 min; h) by control or omeprazole-exposed Caco-2 monolayers. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the corresponding control group. P < 0.05, ††P < 0.01, †††P < 0.001 compared with the pre-treated control group (n = 6)

Fig. 7
figure7

The signaling pathway of P2Y2 receptor agonist stimulated HCO3 secretion. Representative immunoblotting and densitometric analysis of CFTR expression in control and omeprazole-treated Caco-2 cells (a). The rate of peak MRS2768-stimulated HCO3 secretion by control (b), and 400-ng/ml omeprazole-treated Caco-2 monolayers (c) that were induced by agonist or antagonist. The pH of apical culture media of Caco-2 monolayers at 24 h after culture media change (d). *P < 0.05, **P < 0.01, ***P < 0.001 compared with the corresponding vehicle group (n = 6)

Signaling pathway of P2Y2 receptor activation stimulated HCO3 secretion

Previously, it was found that P2Y2 activation enhanced mucosal HCO3 secretion through PLC, IP3 receptor, and intracellular Ca2+ signaling pathway [4]. This series of experiments, therefore, showed the underlying signaling transduction pathway of P2Y2 receptor activation that mediated the stimulation of apical HCO3 secretion in Caco-2 monolayers. In control (Fig. 7b) and omeprazole-treated monolayers (Fig. 7c), the rate of peak MRS2768-stimulated HCO3 secretion was significantly suppressed in the monolayers treated with the antagonist of P2Y2 receptor, PLC, PI3K receptor, CFTR, NBCe1, and CA, as well as intracellular Ca2+ chelator. These results showed that P2Y2 receptor activation mediated the stimulation of HCO3 secretion through PLC, IP3 receptor, and intracellular Ca2+ signaling pathway. We further observed the pH of apical culture media of Caco-2 monolayers at 24 h after culture media change. MRS2768 and 400 ng/ml omeprazole significantly increased apical pH compared to vehicle treated group (Fig. 7d). CFTR and NBCe1 antagonists significantly abolished MRS2768 and omeprazole effects on apical pH.

Discussion

Intestinal Mg2+ absorption can be processed through saturable transcellular and non-saturable paracellular mechanisms. Transcellular Mg2+ transport is an active process that requires the activity of transient receptor potential melastatin 6 (TRPM6), TRPM7, and basolateral Na+/Mg2+ exchanger [1, 37,38,39] or other transport pathways. Paracellular Mg2+ transport is a passive mechanism modulated by the tight junction associated Claudin (Cldn) [11, 40]. However, the regulatory mechanism of intestinal Mg2+ absorption is largely unknown.

Our previous study demonstrated that intestinal-associated proton sensors ASIC1a and OGR1 could modulate intestinal-like Mg2+ transport across Caco-2 monolayers [12]. OGR1 activation increased Mg2+ transport across Caco-2 monolayers while ASIC1a activation decreased it. In the present study we focused on the role of the purinergic P2Y1 receptor family, which was regulating trans-epithelial Na+, K+, and Cl transport [13], on Mg2+ transport across Caco-2 monolayers. The Gq-coupled P2Y1 receptor family is composed of P2Y1, P2Y2, P2Y4, P2Y6 and P2Y11 receptors. However, Caco-2 monolayers expressed P2Y2, P2Y4, and P2Y6 [27], which are exclusively localized in the apical membrane [28]. Our results suggested that only P2Y2 was involved in the modulation of Mg2+ transport across Caco-2 monolayers. Generally, P2Y2 receptors regulate epithelial ion transport through Gq-dependent pathways which activate PLC and stimulate intracellular Ca2+ mobilization [4, 13]. In agreement with our results, P2Y2 receptor activation suppressed Mg2+ transport through PLC, IP3 receptor, and the intracellular Ca2+ signaling pathway. Li et al. [41] reported the inhibitory role of P2Y2 receptor on Cldn-1 expression. Purinergic P2Y receptor agonist adenosine triphosphate (ATP) rapidly suppressed epithelial paracellular permeability and increased epithelial TEER [42]. In addition, purinergic P2 receptor agonist also suppressed the activity of TRPM6 and TRPM7 [43, 44]. However, the role of P2Y2 on Cldn, TRPM6, and TRPM7 expressions and functions required further studies.

Duodenal mucosal bicarbonate secretion (DMBS) is the critical process of duodenal defense against intermittent duodenal epithelial exposure to a luminal acidic environment (pH < 2). Luminal H+ is the potent activator of DMBS by stimulating a duodenal associated acid sensor, e.g., ASIC1a [5]. In addition, luminal uridine triphosphate, a P2Y2 agonist, also stimulates DMBS [4]. Previously, apical HCO3 secretion had been observed in Caco-2 monolayers [12, 29]. Laohapitakworn et al. [29] reported that PTH rapidly stimulated CFTR-, CA-, and NBCe1-mediated apical HCO3 secretion in Caco-2 monolayers. Our group demonstrated that activation of ASIC1a stimulated an apical HCO3 secretion CFTR-dependent mechanism [12]. In the present study we reported the activation of P2Y2 receptor stimulating CFTR-, CA-, and NBCe1-mediated apical HCO3 secretion in Caco-2 monolayers. Our results agreed with the finding of a previous report [4] that showed P2Y2 receptor activation mediated the stimulation of intestinal HCO3 secretion through PLC, IP3 receptor, and the intracellular Ca2+ signaling pathway.

The underlying mechanism of PPI-suppressed intestinal Mg2+ uptake is still unclear. A previous mathematically simulated study suggested that only a 1% reduction of intestinal Mg2+ absorption could induce 80% Mg2+ depletion within 1 year of PPIs used [45]. Previous studies proposed that PPIs mainly affected colonic Mg2+ absorption in PPIH mice [2, 20]. Our group reported that PPIs impeded duodenal Mg2+ absorption in PPIH rats [21]. We hypothesized that a higher luminal HCO3 secretion could lead to a suppression of small intestinal Mg2+ absorption in PPIH [12, 21]. Omeprazole markedly enhanced HCO3 secretion in human duodenum and intestinal epithelium-like Caco-2 monolayers [12, 26]. Secreted HCO3 increased luminal pH and probably decreased Mg2+ solubility, since luminal soluble Mg2+ decreased from 79.61 to 8.71% when luminal pH increased from ~ 5 to 7.8 [25]. Therefore, antagonists of CFTR and NBCe1 significantly increased duodenal Mg2+ absorption in PPIH rats [21]. These findings agreed with the present study that omeprazole induced basal and peak-stimulated HCO3 secretion. Interestingly, in the HCO3-free condition the rate of Mg2+ transport markedly increased in omeprazole-treated Caco-2 monolayers, suggesting the inhibitory role of secreted HCO3 on intestinal Mg2+ absorption. However, luminal Mg2+ solubility and precipitation in the PPI-treated animal model requires further study.

The present study showed higher P2Y2 expression in omeprazole-treated Caco-2 monolayers. Thus, P2Y2-activated HCO3 secretion was also significantly higher in omeprazole-treated monolayers. These results explained the higher degree of suppression of Mg2+ absorption in P2Y2-activated 200-ng/ml omeprazole-treated (60.71%) and 400-ng/ml omeprazole-treated monolayers (62.96%) in comparison to control monolayers (41.58%). Moreover, P2Y2 activation enhanced HCO3 secretion and suppressed Mg2+ absorption were also mediated by PLC, IP3 receptor, intracellular Ca2+ mobilization, CFTR, CA, and NBCe1. In addition to higher HCO3 secretion, another possible mechanism of omeprazole-suppressed Mg2+ absorption is phosphatidylinositol 4,5-bisphosphate (PIP2)-mediated TRPM6 function. Since TRPM6 function required an interaction with membrane-associated PIP2, hydrolysis of PIP2 through activation of the Gq-protein coupled PLC-dependent pathway fully inactivated TRPM6 channels [46]. The higher Gq-associated P2Y2 expression and function might have induced PIP2 degradation which then inactivated TRPM6 channels in omeprazole-treated Caco-2 monolayers. However, our results suggested that Mg2+ channel-dependent Mg2+ absorption could not be involved in omeprazole-suppressed Mg2+ transport across Caco-2 monolayers.

Our results in the present study agreed with the previous study [8] that omeprazole suppressed Mg2+ channel-independent, but not Mg2+ channel-dependent, Mg2+ transport across Caco-2 monolayers. There are two possible answers to explain why omeprazole had no effect on Mg2+ channel-dependent Mg2+ transport across Caco-2 monolayers. Since omeprazole has no effect on TRPM6 expression in Caco-2 cells [21], Mg2+ channel-dependent Mg2+ transport was maintained in the same fraction. Regarding our recent results, from 100% of total Mg2+ transport, the percentages of Mg2+ channel-dependent Mg2+ transport were 18.55, 19.02, and 18.58% in control, 200-ng/ml omeprazole-treated, and 400-ng/ml omeprazole-treated monolayers. On the other hand, our recent method may not be sensitive enough to detect a very small change of Mg2+ channel-dependent Mg2+ transport of omeprazole-treated Caco-2 monolayers.

In conclusion, the present study reported the role of P2Y2 function on the modulation of intestinal Mg2+ absorption. P2Y2 agonist enhanced HCO3 secretion and suppressed Mg2+ transport through the activation of PLC, IP3 receptor, intracellular Ca2+ mobilization, CFTR, CA, and NBCe1. Inhibition of HCO3 secretion could restore Mg2+ transport in P2Y2 agonist-treated monolayers. The higher P2Y2 expression was found in omeprazole-treated Caco-2 monolayers. Therefore, the higher degree of HCO3 secretion and Mg2+ transport suppression was demonstrated in P2Y2-activated omeprazole-treated Caco-2 monolayers. Our results propose an inhibitory role of P2Y2 on intestinal Mg2+ absorption.

References

  1. 1.

    de Baaij JHF, Hoenderop JG, Bindels RJM (2015) Magnesium in man: implications for health and disease. Physiol Rev 95(1):1–46

  2. 2.

    Lameris ALL, Hess MW, van Kruijsbergen I, Hoenderop JGJ, Bindels RJM (2013) Omeprazole enhances the colonic expression of the Mg2+ transporter TRPM6. Pflugers Arch Eur J Physiol 465(11):1613–1620

  3. 3.

    Quamme GA (2008) Recent developments in intestinal magnesium absorption. Curr Opin Gastroenterol 24(2):230–235

  4. 4.

    Dong X, Smoll EJ, Ko KH, Lee J, Chow JY, Kim HD, Insel PA, Dong H (2009) P2Y receptors mediate Ca2+ signaling in duodenocytes and contribute to duodenal mucosal bicarbonate secretion. Am J Physiol Gastrointest Liver Physiol 296(2):G424–G432

  5. 5.

    Dong X, Ko KH, Chow J, Tuo B, Barrett KE, Dong H (2011) Expression of acid-sensing ion channels in intestinal epithelial cells and their role in the regulation of duodenal mucosal bicarbonate secretion. Acta Physiol 201:97–107

  6. 6.

    Holzer P (2007) Taste receptors in the gastrointestinal tract. V. Acid-sensing in the gastrointestinal tract. Am J Physiol Gastrointest Liver Physiol 292:G699–G705

  7. 7.

    Holzer P (2009) Acid-sensitive ion channels and receptors. Handb Exp Pharmacol 194:283–332

  8. 8.

    Kaunitz JD, Akiba Y (2011) Purinergic regulation of duodenal surface pH and ATP concentration: implications for mucosal defence, lipid uptake and cystic fibrosis. Acta Physiol 201(1):109–116

  9. 9.

    Xu Y, Casey G (1996) Identification of human OGR1, a novel G protein-coupled receptor that maps to chromosome 14. Genomics 35:397–402

  10. 10.

    Reiter B, Kraft R, Günzel D, Zeissig S, Schulzke J-D, Fromm M, Harteneck C (2006) TRPV4-mediated regulation of epithelial permeability. FASEB J 20:1802–1812

  11. 11.

    Thongon N, Krishnamra N (2012) Apical acidity decreases inhibitory effect of omeprazole on Mg2+ absorption and claudin-7 and -12 expression in Caco-2 monolayers. Exp Mol Med 44(11):684–693

  12. 12.

    Thongon N, Ketkeaw P, Nuekchob C (2014) The roles of acid-sensing ion channel 1a and ovarian cancer G protein-coupled receptor 1 on passive Mg2+ transport across intestinal epithelium-like Caco-2 monolayers. J Physiol Sci 64(2):129–139

  13. 13.

    Leipziger J (2003) Control of epithelial transport via luminal P2 receptors. Am J Physiol Ren Physiol 284(3):F419–F432

  14. 14.

    Cundy T, Dissanayake A (2008) Severe hypomagnesemia in long-term users of proton-pump inhibitors. Clin Endocrinol 69:338–341

  15. 15.

    Cundy T, Mackay J (2011) Proton pump inhibitors and severe hypomagnesemia. Curr Opin Gastroenterol 27(2):180–185

  16. 16.

    Danziger J, William JH, Scott DJ, Lee J, Lehman LW, Mark RG, Howell MD, Celi LA, Mukamal KJ (2013) Proton-pump inhibitor use is associated with low serum magnesium concentrations. Kidney Int 83(4):692–699

  17. 17.

    Epstein M, McGrath S, Law F (2006) Proton-pump inhibitors and hypomagnesemic hypoparathyroidism. N Engl J Med 355:1834–1836

  18. 18.

    Luk CP, Parsons R, Lee YP, Hughes JD (2013) Proton pump inhibitor-associated hypomagnesemia: what do FDA data tell us? Ann Pharmacother 47(6):773–780

  19. 19.

    Shabajee N, Lamb EJ, Sturgess I, Sumathipala RW (2008) Omeprazole and refractory hypomagnesemia. BMJ 337:a425

  20. 20.

    Hess MW, de Baaij JHF, Gommers LMM, Hoenderop JGJ, Bindels RJM (2015) Dietary inulin fibers prevent proton-pump inhibitor (PPI)-induced hypocalcemia in mice. PLoS One 10(9):e0138881

  21. 21.

    Thongon N, Penguy J, Kulwong S, Khongmueang K, Thongma M (2016) Omeprazole suppressed plasma magnesium level and duodenal magnesium absorption in male Sprague-Dawley rats. Pflugers Arch Eur J Physiol 468(11–12):1809–1821

  22. 22.

    Nugent SG, Kumar D, Rampton DS, Evans DF (2001) Intestinal luminal pH in inflammatory bowel disease: possible determinants and implications for therapy with aminosalicylates and other drugs. Gut 48:571–577

  23. 23.

    Evenepoel P (2001) Alteration in digestion and absorption of nutrients during profound acid suppression. Best Pract Res Clin Gastroenterol 15:539–551

  24. 24.

    Heijnen AM, Brink EJ, Lemmens AG, Beynen AC (1993) Ileal pH and apparent absorption of magnesium in rats fed on diets containing either lactose or lactulose. Br J Nutr 70:747–756

  25. 25.

    Ben-Ghedalia D, Tagari H, Zamwel S, Bondi A (1975) Solubility and net exchange of calcium, magnesium and phosphorus in digesta flowing along the gut of the sheep. Br J Nutr 33(1):87–94

  26. 26.

    Mertz-Nielsen A, Hillingsø J, Bukhave K, Rask-Madsen J (1996) Omeprazole promotes proximal duodenal mucosal bicarbonate secretion in humans. Gut 38:6–10

  27. 27.

    McAlroy HL, Ahmed S, Day SM, Baines DL, Wong HY, Yip CY, Ko WH, Wilson SM, Collett A (2000) Multiple P2Y receptor subtypes in the apical membranes of polarized epithelial cells. Br J Pharmacol 131(8):1651–1658

  28. 28.

    Wolff SC, Qi AD, Harden TK, Nicholas RA (2005) Polarized expression of human P2Y receptors in epithelial cells from kidney, lung, and colon. Am J Physiol Cell Physiol 288(3):C624–C632

  29. 29.

    Laohapitakworn S, Thongbunchoo J, Nakkrasae LI, Krishnamra N, Charoenphandhu N (2011) Parathyroid hormone (PTH) rapidly enhances CFTR-mediated HCO3 secretion in intestinal epithelium-like Caco-2 monolayer: a novel ion regulatory action of PTH. Am J Physiol Cell Physiol 301(1):C137–C149

  30. 30.

    Ekmekcioglu C, Ekmekcioglu A, Marktl W (2000) Magnesium transport from aqueous solutions across Caco-2 cells—an experimental model for intestinal bioavailability studies. Physiological considerations and recommendations. Magnes Res 13:93–102

  31. 31.

    Thongon N, Krishnamra N (2011) Omeprazole decreases magnesium transport across Caco-2 monolayers. World J Gastroenterol 17(12):1574–1583

  32. 32.

    Macek J, Klíma J, Ptácek P (2007) Rapid determination of omeprazole in human plasma by protein precipitation and liquid chromatography-tandem mass spectrometry. J Chromatogr B Anal Technol Biomed Life Sci 852:282–287

  33. 33.

    Thongon N, Nakkrasae LI, Thongbunchoo J, Krishnamra N, Charoenphandhu N (2008) Prolactin stimulates transepithelial calcium transport and modulates paracellular permselectivity in Caco-2 monolayer: mediation by PKC and ROCK pathways. Am J Physiol Cell Physiol 294:C1158–C1168

  34. 34.

    Blais A, Aymard P, Lacour B (1997) Paracellular calcium transport across Caco-2 and HT29 cell monolayers. Pflugers Arch 434(3):300–305

  35. 35.

    Stenson WF, Easom RA, Riehl TE, Turk J (1993) Regulation of paracellular permeability in Caco-2 cell monolayers by protein kinase C. Am J Physiol 265(5 Pt 1):G955–G962

  36. 36.

    Singh AK, Liu Y, Riederer B, Engelhardt R, Thakur BK, Soleimani M, Seidler U (2013) Molecular transport machinery involved in orchestrating luminal acid-induced duodenal bicarbonate secretion in vivo. J Physiol 591(21):5377–5391

  37. 37.

    Ryazanova LV, Rondon LJ, Zierler S, Hu Z, Galli J, Yamaguchi TP, Mazur A, Fleig A, Ryazanov AG (2010) TRPM7 is essential for Mg2+ homeostasis in mammals. Nat Commun 1:109

  38. 38.

    Li M, Jiang J, Yue L (2006) Functional characterization of homo- and heteromeric channel kinases TRPM6 and TRPM7. J Gen Physiol 127(5):525–537

  39. 39.

    Yamazaki D, Funato Y, Miura J, Sato S, Toyosawa S, Furutani K, Kurachi Y, Omori Y, Furukawa T, Tsuda T, Kuwabata S, Mizukami S, Kikuchi K, Miki H (2013) Basolateral Mg2+ extrusion via CNNM4 mediates transcellular Mg2+ transport across epithelia: a mouse model. PLoS Genet 9(12):e1003983

  40. 40.

    Hou J, Renigunta A, Konrad M, Gomes AS, Schneeberger EE, Pual DL, Waldegger S, Goodenough DA (2008) Clauin-16 and claudin-19 interact and form a cation-selective tight junction complex. J Clin Investig 118:619–628

  41. 41.

    Li WH, Qiu Y, Zhang HQ, Liu Y, You JF, Tian XX, Fang WG (2013) P2Y2 receptor promotes cell invasion and metastasis in prostate cancer cells. Br J Cancer 109(6):1666–1675

  42. 42.

    Gorodeski GI, Hopfer U (1995) Regulation of the paracellular permeability of cultured human cervical epithelium by a nucleotide receptor. J Soc Gynecol Investig 2(5):716–720

  43. 43.

    de Baaij JH, Blanchard MG, Lavrijsen M, Leipziger J, Bindels RJ, Hoenderop JG (2014) P2X4 receptor regulation of transient receptor potential melastatin type 6 (TRPM6) Mg2+ channels. Pflugers Arch 466(10):1941–1952

  44. 44.

    Demeuse P, Penner R, Fleig A (2006) TRPM7 channel is regulated by magnesium nucleotides via its kinase domain. J Gen Physiol 127(4):421–434

  45. 45.

    Bai JP, Hausman E, Lionberger R, Zhang X (2012) Modeling and simulation of the effect of proton pump inhibitors on magnesium homeostasis. 1. Oral absorption of magnesium. Mol Pharm 9(12):3495–3505

  46. 46.

    Xie Jia, Sun Baonan, Jianyang Du, Yang Wenzhong, Chen Hsiang-Chin, Overton Jeffrey D, Runnels Loren W, Yue Lixia (2011) Phosphatidylinositol 4,5-bisphosphate (PIP2) controls magnesium gatekeeper TRPM6 activity. Sci Rep 1:146

Download references

Acknowledgements

This study was supported by research grants from Burapha University through the National Research Council of Thailand (138/2560), and the Faculty of Allied Health Sciences, Burapha University (AHS06/2560) to N. Thongon. We express our gratitude to Dr. Prasert Sobhon of the Faculty of Allied Health Sciences, Burapha University for his helpful suggestions and proofreading. We also thank Dr. Petcharat Trongtorsak of Allied Health Sciences, Burapha University for a very kind gift of forskolin, CCh, and nifedipine. We also thank Ms. Pattamaporn Ketkeaw and Mr. Chanin Nuekchob of the Faculty of Allied Health Sciences, Burapha University and Mr. Phongthon Kanjanasirirat of the Excellent Center for Drug Discovery (ECDD), Faculty of Science, Mahidol University for their excellent technical assistance.

Funding

This study was funded by Burapha University through the National Research Council of Thailand (138/2560), and the Faculty of Allied Health Sciences, Burapha University (AHS06/2560) to N. Thongon.

Author information

TN designed and performed the experiments, analyzed and interpreted the results, and wrote and edited the manuscript. CS performed the experiments, analyzed the results, and wrote and edited the manuscript.

Correspondence to Narongrit Thongon.

Ethics declarations

Conflict of interest

All authors declare that they have no competing interests.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent

Not applicable.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Thongon, N., Chamniansawat, S. The inhibitory role of purinergic P2Y receptor on Mg2+ transport across intestinal epithelium-like Caco-2 monolayer. J Physiol Sci 69, 129–141 (2019). https://doi.org/10.1007/s12576-018-0628-2

Download citation

Keywords

  • Caco-2 monolayers
  • Intestinal HCO3 secretion
  • Mg2+ absorption
  • Proton pump inhibitor
  • P2Y2 receptor