- Original Paper
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
The Journal of Physiological Sciences volume 64, pages 129–139 (2014)
Intestinal passive Mg2+ absorption, which is vital for normal Mg2+ homeostasis, has been shown to be regulated by luminal proton. We aimed to study the regulatory role of intestinal acid sensors in paracellular passive Mg2+ transport. Omeprazole enhanced the expressions of acid-sensing ion channel 1a (ASIC1a), ovarian cancer G protein-coupled receptor 1 (OGR1), and transient receptor potential vanilloid 4 in Caco-2 cells. It also inhibited passive Mg2+ transport across Caco-2 monolayers. The expression and activation of OGR1 resulted in the stimulation of passive Mg2+ transport via phospholipase C- and protein kinase C-dependent pathways. ASIC1a activation, on the other hand, enhanced apical HCO3 − secretion that led, at least in part, by a Ca2+-dependent pathway to an inhibition of paracellular Mg2+ absorption. Our results provided supporting evidence for the roles of OGR1 and ASIC1a in the regulation of intestinal passive Mg2+ absorption.
Magnesium (Mg2+) is essential for many physiological processes such as muscle contraction and relaxation, energy metabolism, neuronal function, and bone formation. Dietary intake is the sole source of Mg2+ in human, therefore adequate intestinal absorption of Mg2+ is vital for normal Mg2+ balance. Intestinal Mg2+ uptake comprises saturable transcellular active and non-saturable paracellular passive mechanisms [1–3], with the latter contributing about 90 % of the total intestinal Mg2+ absorption . This transport mechanism is driven by the electrochemical gradient set up by a higher luminal Mg2+ concentration and lumen positive voltage with respect to the basolateral side [1, 3, 4]. The tight junction-associated claudins (Cldn) have been reported to act as a paracellular Mg2+ channel within the tight junction [5, 6]. However, the regulation of the paracellular passive intestinal absorption of Mg2+ is unknown.
Apical proton has been shown to modulate paracellular Mg2+ transport [6–8]. Suppression of apical proton accumulation by a proton pump inhibitor (PPI) omeprazole altered paracellular permselectivity, suppressed Cldn-7 and -12 expressions, and inhibited paracellular passive Mg2+ transport across the intestinal-like Caco-2 monolayers [6, 9]. On the other hand, apical acidity (pH 7.0–5.5) was reported to increase paracellular passive Mg2+ uptake, affinity of paracellular channel for Mg2+, and expression of Cldn-7 and -12 in both control and omeprazole-exposed epithelia . However, the underlying mechanism of apical proton regulation of the passive Mg2+ absorption remains elusive.
Epithelial cells in the small intestine are regularly exposed to strong gastric acid. When luminal pH decreases, the intestinal epithelium cells can directly detect and modulate their cellular response through the proton sensors, e.g., ASIC1a, OGR1, and transient receptor potential vanilloid 4 (TRPV4) [10–14]. The pH of half (pH0.5) and full activation of OGR1 are 7.4–7.2 and 6.8, respectively [15, 16]. OGR1 is associated with Gq proteins and acts through phospholipase C (PLC)–protein kinase C (PKC) signaling pathway to activate the epithelial Na+/H+ exchanger (NHE) and H+-ATPase activity [15, 17]. ASIC1a is activated by extracellular pH below 6.9 with pH0.5 of 6.2–6.8 , whereas TRPV4 requires a more acidic pH for activation (pH > 6.0) and is fully activated at pH 4.0 . Both ASIC1a and TRPV4 are Ca2+ channels that trigger Ca2+ signaling to regulate epithelial HCO3 − secretion [11, 19]. Activation of TRPV4 also modulates paracellular permeability and Cldn expressions by a Ca2+-dependent mechanism . It is not known whether intestinal acid sensing ASIC1a, OGR1, and TRPV4 have any role in apical acidity-induced stimulation of paracellular passive Mg2+ absorption. The present study investigated the role of intestinal acid sensors in the regulation of paracellular passive Mg2+ absorption. The results showed that OGR1 enhanced whereas ASIC1a decreased the passive intestinal Mg2+ absorption.
The human intestinal Caco-2 cells (ATCC No. HTB-37) were grown and maintained for 14 days as previously described . For the experiments, the cells were plated on the permeable polyester Transwell-clear inserts (1.0 × 106 cells cm−2; Corning, Corning, NY, USA), 6-well plates (5.0 × 105 cells per well; Corning), or 96-well plates (5.0 × 104 cells per well; Corning) and maintained for 7 days . From days 8 to 14 of culture, cells were grown in media with or without 5 mM HCL-activated omeprazole (200 or 400 ng/mL; Calbiochem, San Diego, CA, USA). In some experiments, the apical side of Caco-2 monolayers was intermittently exposed over a 2-h period, 3 times a day, to acidic culture media (pH 6.5 or 5.5) with or without 5 mM HCL-activated omeprazole (Calbiochem) from days 8 to 14 (Fig. 1d). The cell monolayers grown on Transwell-clear inserts, 6-well plates, or 96-well plates were used for ion flux studies, western blot analysis, or MTT reduction assay, respectively. Apical pH of Caco-2 monolayers grown for 14 days in media with or without 5 mM HCL-activated omeprazole was also determined as previously described .
For the apical to basolateral 40 mM concentration gradient-driven passive 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 pH 7.4, 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 pH 7.4. In the apical acid-activated Mg2+ transport studies, the apical solution of pH 7.4 was substituted with apical solution of pH 7.0, 6.5, 6.0, or 5.5 . During Mg2+ transport studies, pH of all apical bathing solutions was simultaneously measured and not changed throughout the experiments.
For apical HCO3 − secretion experiments, the composition of 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 pH 7.4; 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 pH 7.4.
All solutions were maintained at 37 °C, pre-gassed with 100 % O2 for 30 min, and had an osmolality of 290–295 mosM as measured by a freezing-point depression-based Fiske® micro-osmometer (model 210; Fiske® Associates, Norwood, MA, USA). All chemicals were purchased from Sigma (St. Louis, MO, USA). Bioresearch grade deionized water used in the present work had a resistance of >18.3 MΩ cm, total organic compound of <10 part per billion, and pyrogen contamination of <0.005 endotoxin units/mL.
Measurements of paracellular Mg2+ flux
Apical to basolateral 40 mM MgCl2 gradient-driven paracellular Mg2+ flux study and determination of Mg2+ concentration were performed as previously described . Apical acidity-dependent passive Mg2+ transport was observed under apical bathing solution pH 7.0, 6.5, 6.0, or 5.5 with basolateral bathing solution remaining at pH 7.4. In some experiments, Caco-2 monolayers were pre-incubated with various compounds, namely; ASIC1a activator (1 or 10 μM amitriptyline; AMT; Sigma), ASIC1a inhibitor [30 nM psalmotoxin 1 (PcTx1); Phoenix Pharmaceuticals, Burlingame, CA, USA], TRPV4 inhibitor [10 μM ruthenium red (RR); Sigma], OGR1 inhibitor [10 μM CuCl2 (Cu2+) or 10 μM ZnCl2 (Zn2+); Sigma], polyclonal antibody raised against an extracellular domain of human OGR1 (1:10 or 1:100 OGR1 Ab; Santa Cruz Biotechnology, Santa Cruz, CA, USA), PLC inhibitor [10 μM 1-[6-((17β-3-Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione; U-73122; Calbiochem], PKC inhibitor (1 μM Bisindolylmaleimide I; Gö 6850; Calbiochem), phosphoinositide 3-kinase (PI3K) inhibitor (200 nM Wortmannin, Wort.; Calbiochem], and intracellular Ca2+ chelator [50 μM 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl ester); BAPTA-AM; Calbiochem] prior to the performed experiments.
Measurements of HCO3 − secretion
Bicarbonate secretion was measured in humidified atmosphere with 5 % CO2 at 37 °C. After removal of the culture media, 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 apical and basolateral solutions for the HCO3 − secretion experiment. To inhibit apical membrane-bound carbonic anhydrase (CA) activity and prevent apical HCO3 − degradation, the selective CA IX/XII inhibitor (45 nM 4-[[[(4-fluorophenyl)amino]carbonyl]amino]-benzenesulfonamide; U-104; Sigma) was added to the apical solution. After 20 min, HCL was added to the apical solution (the final concentration of 10 mM) and incubation proceeded for 5 min. After removal of the HCL-containing apical solution, the apical side of the monolayer was gently rinsed and further incubated for 50 min in the apical solution. Aliquots of apical solution at various time points (Fig. 7a) were individually sampled. The concentration of HCO3 − was immediately determined by using a clinical chemistry analyzer (ILab Taurus; Instrumentation Laboratory, Bedford, MA, USA). In some experiments, Caco-2 monolayers were pre-incubated with 30 nM PcTx1 (Phoenix Pharmaceuticals), 50 μM BAPTA-AM (Calbiochem) or cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor [50 μM N-(2-naphthalenyl)-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycine hydrazide; GlyH-101; Calbiochem].
MTT reduction assay
Percent viability of control Caco-2 cells and cells exposed to 200 or 400 ng/mL omeprazole (Calbiochem), and intermittently exposed to acidic apical medium with or without omeprazole was evaluated as previously described .
Western blot analysis
Western blot analysis was performed as previously described . In brief, protein samples were prepared by using Piece® Ripa Buffer (Thermo Fisher Scientific, Rockford, IL, USA). 35 μg of protein sample or 5 μL Cruz Marker™ Molecular Weight Standards (Santa Cruz Biotechnology) were separated on 12.5 % SDS-PAGE gel, and then transferred onto a nitrocellulose membrane (Amersham, Buckinghamshire, UK) by electroblotting. Membranes were blocked and probed overnight at 4 °C with 1:1,000 rabbit polyclonal antibodies (Santa Cruz Biotechnology) raised against human ASIC1a, OGR1, or TRPV4. Membranes were also reprobed with rabbit polyclonal antibodies (Santa Cruz Biotechnology) raised against actin (1:5,000), ASIC1a, OGR1, or TRPV4 antibodies. After 2 h incubation at 25 °C with 1:10,000 goat anti-rabbit IgG-HRP-conjugated secondary antibodies (Santa Cruz Biotechnology), blots were visualized by Thermo Scientific SuperSignal® West Pico Substrate (Thermo Fisher Scientific) and captured on CL-XPosure Film (Thermo Fisher Scientific). Densitometric analysis was performed using ImageJ for Mac Os X .
Results were expressed as mean ± SE. Two sets of data were compared using unpaired Student’s t test. One-way analysis of variance 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, San Diego, CA, USA).
Apical acidity nullified omeprazole effect on passive Mg2+ transport
These series of experiments were performed to demonstrate the effect of direct exposure to intermittent acidic apical culture medium, apical acidity, and omeprazole on paracellular passive Mg2+ transport. Since an acidic extracellular pH and omeprazole have previously been reported to affect cell viability [23, 24], MTT cell proliferation assay was performed. As shown in Fig. 1a, neither exposure to intermittent acidic apical culture medium (6.5 and 5.5) nor 200 or 400 ng/mL omeprazole, concentrations resembling those found in human plasma , had cytotoxic effect on cell viability when compared to control. Similar to our previous report , 200 and 400 ng/mL omeprazole significantly increased apical pH compared to control condition (data not shown), therefore omeprazole suppressed apical acidification.
Intermittent exposure to acidic apical culture medium at pH 6.5 and 5.5 had no effect on passive Mg2+ transport when compared to control pH 7.4 (Fig. 1b). Both 200 and 400 ng/mL omeprazole (pH 7.4) significantly decreased the passive Mg2+ transport from 134.82 ± 3.02 to 110.45 ± 4.32 and 96.42 ± 4.86 nmol h−1 cm−2, respectively (Fig. 1b). In the 200 ng/mL omeprazole-exposed groups, intermittent exposure to acidic apical culture medium of pH 6.5 and 5.5 abolished the inhibitory effect of omeprazole on passive Mg2+ transport (120.71 ± 6.36 and 130.70 ± 4.44 nmol h−1 cm−2, respectively; Fig. 1b). Acidic apical culture medium of pH 6.5 and 5.5 also normalized passive Mg2+ transport (115.16 ± 3.98 and 122.45 ± 2.93 nmol h−1 cm−2, respectively) in the 400 ng/mL omeprazole exposed groups (Fig. 1b).
To study the effect of apical acidity on passive Mg2+ transport, Caco-2 monolayers grown in culture medium pH 7.4 with or without omeprazole were used. As shown in Fig. 1c, apical acidity significantly increased passive Mg2+ transport from 133.60 ± 3.81 nmol h−1 cm−2 at pH 7.4 to 163.71 ± 6.94, 214.78 ± 7.43, 241.56 ± 9.07, and 270.42 ± 10.54 nmol h−1 cm−2 when apical bathing solution was changed to pH 7.0, 6.5, 6.0, and 5.5, respectively. Similar to Fig. 1b, 200 and 400 ng/mL omeprazole (apical pH 7.4) significantly decreased passive Mg2+ transport to 105.85 ± 5.19 and 98.95 ± 4.05 nmol h−1 cm−2, respectively (Fig. 1c). Apical acidity abolished the inhibitory effect of 200 and 400 ng/mL omeprazole on passive intestinal Mg2+ transport (Fig. 1c). These results demonstrated that apical proton directly stimulated passive intestinal Mg2+ absorption.
Intermittent acidic apical culture medium and omeprazole altered intestinal proton sensor expression
Since previous results demonstrated the stimulatory effect of apical acidity on the intestinal passive Mg2+ transport (Fig. 1) , these experiments aimed to examine the expression of proton sensitive ASIC1a, OGR1, and TRPV4 in Caco-2 cells. Omeprazole (200 and 400 ng/mL) significantly increased the expressions of ASIC1a, OGR1, and TRPV4 (Fig. 2a–d) in Caco-2 cells. On the other hand, intermittent exposure to acidic apical culture medium (pH 6.5 and 5.5) markedly suppressed ASIC1a, OGR1, and TRPV4 expressions (Fig. 2e–h). Intermittent exposure to acidic apical culture medium also abrogated the up-regulatory effect of 400 ng/mL omeprazole on ASIC1a, OGR1, and TRPV4 expressions (Fig. 3). These findings demonstrated the regulatory role of extracellular protons on the expression of intestinal acid sensors.
Intestinal acid sensors modulated passive Mg2+ transport
This experiment aimed to investigate the effect of activation or inhibition of ASIC1a, OGR1, and TRPV4 on passive Mg2+ transport across Caco-2 monolayers. Unexpectedly, at apical pH 7.4, OGR1 inhibitors (10 μM Cu2+ and 10 μM Zn2+ [15, 17]) and 1:10 OGR1 Ab significantly decreased passive Mg2+ transport from 133.60 ± 3.82 to 96.93 ± 5.09, 99.20 ± 5.66, and 102.65 ± 6.18 nmol h−1 cm−2, respectively (Fig. 4a). Neither ASIC1a inhibitor 30 nM PcTx1 , TRPV4 inhibitor 10 μM RR , nor 100 °C-heated OGR1 Ab had modulatory effect on passive Mg2+ transport in apical pH at 7.4 (Fig. 4a). In the presence of mild acidic apical pH 7.0, the rate of passive Mg2+ transport was significantly decreased from 161.18 ± 10.34 to 92.80 ± 7.58 and 96.67 ± 8.29 nmol h−1 cm−2 in the presence of Cu2+ and Zn2+, respectively, and to 126.91 ± 7.44 and 107.71 ± 8.65 nmol h−1 cm−2 by 1:100 and 1:10 OGR1 Ab, respectively (Fig. 4b). In the 400 ng/mL omeprazole exposed groups, the rate of passive Mg2+ transport (nmol h−1 cm−2) of 98.95 ± 4.05 was decreased by Cu2+ to 63.79 ± 4.36 at apical pH 7.4 (Fig. 5a) and from 122.36 ± 4.72 to 78.78 ± 3.52 at apical pH 7.0 (Fig. 5b), and by Zn2+ to 66.23 ± 4.63 at apical pH 7.4 and 81.39 ± 3.92 at apical pH 7.0, and by 1:10 OGR1 Ab to 67.33 ± 3.82 at apical pH 7.4 and 83.53 ± 3.58 at apical pH 7.0. These results indicated that OGR1 activation could have stimulatory effect on passive intestinal Mg2+ absorption.
As expected, PcTx1 significantly increased passive Mg2+ transport by about 14 % at acidic apical pH of 6.5 (241.56 ± 5.15 vs. 211.32 ± 5.58 nmol h−1 cm−2 of control group; Fig. 4c), by 17 % at acidic apical pH of 6.0 (280.96 ± 8.14 vs. 239.32 ± 7.58 nmol h−1 cm−2 of control group; Fig. 4d), and by 14 % at acidic apical pH of 5.5 (308.51 ± 8.74 vs. 270.42 ± 10.54 nmol h−1 cm−2 of control group; Fig. 4e). On the other hand, ASIC1a activator AMT  at 1 and 10 μM significantly decreased passive Mg2+ transport at apical pH 7.4 from 133.60 ± 3.82 to 99.41 ± 6.00 and 105.39 ± 4.92 nmol h−1 cm−2, and at apical pH 7.0 from 165.18 ± 6.60 to 111.54 ± 9.04 and 117.26 ± 6.76 nmol h−1 cm−2, respectively (Fig. 4f). In the 400 ng/mL omeprazole exposed groups, PcTx1 also increased passive Mg2+ transport by about 42 % at acidic apical pH of 6.5 (Fig. 5c), by about 49 % at acidic apical pH of 6.0 (Fig. 5d), and by about 67 % at acidic apical pH of 5.5 (Fig. 5e). On the other hand, ASIC1a activator AMT at 1 and 10 μM significantly reduced passive Mg2+ transport at apical pH 7.4 from 98.95 ± 4.05 to 73.31 ± 4.14 and 75.87 ± 6.47 nmol h−1 cm−2, and at apical pH 7.0 from 122.36 ± 4.72 to 80.89 ± 6.24 and 83.62 ± 8.05 nmol h−1 cm−2, respectively (Fig. 5f). These results demonstrated the inhibitory effect of ASIC1A on the passive intestinal Mg2+ absorption.
Signaling pathway of passive Mg2+ transport modulation
These series of experiments were performed to elucidate the signaling pathways of OGR1 and ASIC1a activation in the regulation of passive Mg2+ transport. Previous reports showed that active OGR1 acted through PLC and PKC to stimulate epithelial ion transport [15, 17]. In the present study, OGR1 inhibitor 10 μM Cu2+, PLC inhibitor 10 μM U-73122, and PKC inhibitor 1 μM Gö 6850 significantly decreased the rate of passive Mg2+ transport (nmol h−1 cm−2) in the absence (92.80 ± 7.58, 84.55 ± 6.51, and 94.95 ± 7.20, respectively, vs. 161.18 ± 10.34 of control monolayers) and presence of 400 ng/mL omeprazole (78.78 ± 3.52, 82.44 ± 5.52, and 78.11 ± 6.53, respectively, vs. 122.36 ± 4.72 of control monolayers) (Fig. 6a). Neither PI3K inhibitor 200 nM Wort. nor intracellular Ca2+ chelator 50 μM BAPTA-AM had a modulatory effect on passive Mg2+ transport in mild acidic apical pH at 7.0 (Fig. 6a). Therefore, active OGR1 probably stimulated intestinal passive Mg2+ absorption by PLC- and PKC-dependent mechanisms.
At acidic apical pH 5.5, 30 nM PcTx1 and 50 μM BAPTA-AM were found to increase the rate of passive Mg2+ transport (nmol h−1 cm−2) in the absence (328.46 ± 10.42 and 324.95 ± 6.31, respectively, vs. 266.68 ± 10.81 of control) and presence of 400 ng/mL omeprazole (299.97 ± 13.67 and 252.97 ± 16.35, respectively, vs. 178.61 ± 6.89 of control) (Fig. 6b). Inhibitors of Ca2+-sensitive signaling mediator (200 nM Wort. and 1 μM Gö 6850) had no effect on passive Mg2+ transport at acidic apical pH of 5.5 (Fig. 6b). Therefore, inhibition of ASIC1a and prevention of intracellular Ca2+ elevation could stimulate the intestinal passive Mg2+ absorption.
ASIC1a-stimulated HCO3 − secretion
After having demonstrated the expression and function of ASIC1a in Caco-2 epithelium, we sought to discover the possible underlying mechanism of ASIC1a-induced decrease in the passive Mg2+ transport. It was previously reported that activation of ASICs stimulated duodenal HCO3 − secretion in vivo . As shown in Fig. 7a, apical acid was found to stimulate HCO3 − secretion in both control and omeprazole-exposed monolayers at various time points. HCO3 − secretion in response to acid peaked at 50 min in control, 200 ng/mL omeprazole-exposed, and 400 ng/mL omeprazole-exposed groups (Fig. 7a). Omeprazole at 200 and 400 ng/mL significantly increased both the basal (4.33 ± 0.31 and 5.09 ± 0.5, respectively, vs. 2.43 ± 0.48 μmol h−1 cm−2 of control group) and peak acid-stimulated HCO3 − secretion (12.15 ± 0.55 and 13.85 ± 0.45, respectively, vs. 9.07 ± 0.54 μmol h−1 cm−2 of control group) (Fig. 7b, c). CFTR inhibitor 50 μM GlyH-101 on the other hand markedly decreased the basal HCO3 − secretion in both control and 400 ng/mL omeprazole-exposed monolayers (Fig. 7d). As expected, PcTx1, GlyH-101, and BAPTA-AM significantly reduced peak acid stimulated HCO3 − secretion in both control (3.01 ± 0.37, 2.53 ± 0.83, and 2.90 ± 0.67 μmol h−1 cm−2, respectively) and 400 ng/mL omeprazole exposed monolayers (5.92 ± 1.19, 5.03 ± 0.53, and 5.85 ± 1.01 μmol h−1 cm−2, respectively) (Fig. 7e). These results suggested that omeprazole induced HCO3 − secretion by ASIC1a-, Ca2+-, and CFTR-dependent mechanisms.
Adequate intestinal absorption of Mg2+ is vital for maintaining normal Mg2+ balance. We have previously reported a direct effect of apical acidity on passive Mg2+ transport across human intestinal Caco-2 epithelium. Suppression of apical proton secretion and elevation of apical pH by omeprazole decreased the paracellular cation selectivity, negative electrical field strength of paracellular pore, Mg2+ affinity of the paracellular channel, Cldn-7 and -12 expression, and paracellular Mg2+ absorption [6, 9]. On the other hand, increase in apical acidity markedly enhanced Mg2+ affinity of the paracellular channel, Cldn-7 and -12 expression, and paracellular Mg2+ absorption . In the present study, we proposed that intestinal passive Mg2+ absorption was regulated by the proton-sensitive OGR1 and ASIC1a.
In human tissues, OGR1 has previously been detected in the small intestine, spleen, testis, brain, lung, placenta, heart, and kidney, but not in the colon, liver, or skeletal muscle . OGR1 is known as a proton-sensitive G-protein-coupled receptor [15, 17] that requires extracellular histidine residues 17, 20, 84, 169, and 269 for its proton detection . It is coupled to Gq proteins and PLC that triggers an increase in the intracellular Ca2+ transient and PKC, which in turn activates the epithelial NHE and H+-ATPase in OGR1-transfected HEK293 cells [15, 17]. In the present study, activation of OGR1 by mild acidic apical pH of 7.0 was found to stimulate passive Mg2+ transport. This effect was inhibited by OGR1 inhibitors and OGR1 Ab. However, because OGR1 activity declined to an inactivation state when the extracellular pH declined to 6.5 , the stimulation of passive Mg2+ transport seen at more acidic apical pH of 6.5, 6.0, and 5.5 must be OGR1-independent. Our results also showed that the OGR1 induced stimulation of passive Mg2+ transport was mediated by PLC and PKC signaling pathways, but not by the intracellular Ca2+-dependent mechanism. The controversial reports regarding the role of intracellular Ca2+ as a downstream mediator of OGR1 activation in the present and previous studies [15, 17] was probably due to the different cell types used in the studies. Moreover, the observed up-regulation of OGR1 expression was probably a compensatory response of omeprazole-exposed monolayers to oppose the inhibitory effect of omeprazole on passive intestinal Mg2+ absorption.
Previously, the expression and function of ASIC1a has been extensively studied in the nervous system [12, 13, 28]. Although ASICs were cloned from the human small intestine , expression and function of ASICs were mainly observed in neurons in the gastrointestinal tract [12, 13]. Dong et al.  demonstrated the expression and function of ASIC1a in rat duodenal epithelium and human HT29 cells. In the present study, we proposed that ASIC1a activation had an inhibitory effect on intestinal passive Mg2+ absorption. Since ASIC1a is active at extracellular pH below 6.9 , it might directly regulate the passive Mg2+ transport under acidic apical pH of 6.5, 6.0, and 5.5. By using specific inhibitor or activator of ASIC1a in the monolayer incubation, we were able to show the inhibitory role of activated ASIC1a on passive intestinal Mg2+ absorption. Since ASIC1a can act as a Ca2+ channel , activation of ASIC1a by apical acidity probably involves an intracellular Ca2+ elevation leading to inhibition of intestinal passive Mg2+ absorption that in turn was relieved by PcTx1 and BAPTA-AM. However, it was possible that other acid sensors may also mediate the stimulatory effect of apical pH below 7.0 on the intestinal passive Mg2+ absorption.
Although TRPV4 has been reported to regulate paracellular permeability in mammary HC11 epithelium  and act as a proton sensor that is fully activated at pH 4.0 in Chinese hamster ovary cells , it was probably not involved in the apical acidity-induced paracellular passive Mg2+ transport in human intestinal Caco-2 epithelium. There are four possible reasons to explain this assumption: (1) being located in the basolateral membrane of human Caco-2 epithelium , TRPV4 cannot be directly activated by apical acidity, (2) the acidic apical pH used in the present study is not low enough to effectively activate TRPV4, (3) TRPV4 activation has not been known to affect passive Mg2+ transport, and (4) in the present study used different cell type from those used in the previous studies [18, 20].
It is widely accepted that duodenal mucosal HCO3 − secretion is an important mechanism of enterocyte epithelium to defend itself against exposure to strong gastric acid [30, 31]. The duodenal epithelial cells can directly detect and regulate mucosal HCO3 − secretion via ASIC1a and purinoceptors P2Y [11, 32]. Our results agreed with the previous study  that apical HCL stimulated HCO3 − secretion in ASIC1a by an intracellular Ca2+-dependent pathway. Apical HCO3 − secretion in the small intestine occurs via: (1) anion conductive CFTR-dependent, (2) anion Cl−/HCO3 − exchanger-dependent, and (3) paracellular hydrostatic pressure-dependent mechanisms . Because the specific CFTR inhibitor GlyH-101 could not totally suppress HCO3 − secretion in the present study, therefore the Cl−/HCO3 − exchanger-dependent and paracellular pathways may also play a role in the acid-stimulated HCO3 − secretion. Furthermore, from the finding that ASIC1a activation suppressed paracellular Mg2+ absorption, as well as enhanced apical HCO3 − secretion, it was possible that apical HCO3 − secretion could contribute to the suppression of paracellular Mg2+ absorption. In the small intestine, luminal proton from gastric acid was believed to play an important role in providing appropriate environment for mineral absorption by stabilizing their ionized forms . Not only reducing luminal proton [30, 31], secreted HCO3 − may also trigger the precipitation of luminal Mg2+ as MgCO3 , thus reducing the intestinal Mg2+ absorption. In addition, secreted HCO3 − may reduce the luminal positive voltage that is required as a driving force for passive Mg2+ absorption [1, 3, 4]. Therefore, apical secreted HCO3 − may, at least in part, suppress the intestinal passive Mg2+ absorption by decreasing Mg2+ bioavailability and luminal positive voltage.
Hypomagnesemia that occurred with a long-term use of PPI probably resulted from a decrease in the intestinal absorption [35–38]. Previously, we reported an inhibitory effect of the PPI omeprazole on the paracellular passive Mg2+ absorption in human enterocyte-like Caco-2 monolayers [6, 9]. In the present study, we showed an enhancing effect of omeprazole on the expression of ASIC1a, an activation of which increased the apical HCO3 − secretion and suppressed paracellular passive Mg2+ absorption in CFTR- and intracellular Ca2+-dependent manners. Since omeprazole increased paracellular anion selectivity , it was likely that paracellular HCO3 − secretion was also involved in the omeprazole-induced HCO3 − secretion in the present study. Our results agreed with a previous report of omeprazole stimulating human duodenal mucosal HCO3 − secretion in vivo . Therefore, the higher rate of HCO3 − secretion by omeprazole-exposed epithelium in the present study could, at least in part, decrease the intestinal passive Mg2+ absorption by increasing MgCO3 precipitation  and reducing the luminal-positive voltage that acted as a driving force for passive Mg2+ absorption.
In conclusion, we showed that the expression and activation of OGR1 resulted in the stimulation of paracellular intestinal Mg2+ absorption via PLC- and PKC-dependent pathways. ASIC1a activation in the presence of acidic apical condition, on the other hand, enhanced apical HCO3 − secretion that led, at least in part, by a Ca2+-dependent pathway to an inhibition of paracellular Mg2+ absorption.
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This study was supported by the research grants from the Thailand Research Fund (RSA5680005) and the Faculty of Allied Health Sciences, Burapha University (AHS 2-1/2556) to N. Thongon. We express our gratitude to Prof. Dr. Nateetip Krishnamra of the Faculty of Science, Mahidol University and Dr. Kukiat Tudpor of Faculty of Health Science, Srinakharinwirot University for their helpful suggestions and proofreading. We also thank Asst. Prof. Dr. Siriporn Chamniansawat, Ms. Penpisut Boonthongthalerng, and Ms. Pimsupa Madato of the Faculty of Allied Health Sciences, Burapha University and Mr. Phongthon Kanjanasirirat of the Faculty of Science, Mahidol University for their excellent technical assistance. Gö 6850, Wort., and BAPTA-AM were kind gifts from Dr. Siriporn Chamniansawat. Caco-2 cells were kindly provided by Prof. Dr. Nateetip Krishnamra.
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The authors declare no conflicts of interest.
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Thongon, N., Ketkeaw, P. & Nuekchob, C. 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, 129–139 (2014). https://doi.org/10.1007/s12576-013-0301-8
- Acid sensor
- Intestinal HCO3 − secretion
- Paracellular Mg2+ absorption
- Proton pump inhibitor