Na /H exchanger 3 inhibitor diminishes hepcidin-enhanced duodenal calcium transport in hemizygous b-globin knockout thalassemic mice

Narattaphol Charoenphandhu1,2,4 • Kamonshanok Kraidith1,2 • Kornkamon Lertsuwan1,3 • Chanakarn Sripong1,2 • Panan Suntornsaratoon1,2 • Saovaros Svasti6 • Nateetip Krishnamra1,2 • Kannikar Wongdee1,5

Abstract

Recent investigation has shown that the liverderived iron-regulating hormone, hepcidin, can potentiate intestinal calcium absorption in hemizygous b-globin knockout thalassemic (BKO) mice. Since the upregulation of Fe2?andH?cotransporter,divalentmetaltransporter(DMT)1, has been shown to correlate with thalassemia-induced intestinalcalciumabsorptionimpairment,theinhibitionofthe apical Na?/H? exchanger (NHE)-3 that is essential for cytoplasmic pH regulation and transepithelial sodium absorption washypothesizedtonegativelyaffecthepcidinaction.Herein, the positive effect of hepcidin on the duodenal calcium transportwasevaluated using Ussingchambertechnique.The results showed that BKO mice had lower absorptive surface area and duodenal calcium transport than wild-type mice. Besides, paracellular transport of zinc in BKO mice was compromised. Hepcidin administration completely restored calcium transport. Since this hepcidin action was totally abolishedbyinhibitorsofthebasolateralcalciumtransporters, Na?/Ca2? exchanger (NCX1) and plasma membrane Ca2?ATPase (PMCA1b), the enhanced calcium flux potentially occurred through the transcellular pathway rather than paracellular pathway. Interestingly, the selective NHE3 inhibitor, 100 nM tenapanor, markedly inhibited hepcidin-enhanced calcium transport. Accordingly, hepcidin is one of the promising therapeutic agents for calcium malabsorption in bthalassemia. It mainly stimulates the transcellular calcium transport across the duodenal epithelium in an NHE3-dependent manner.

Keywords Beta-thalassemia Calcium absorption Hepcidin Tenapanor Ussing chamber

Introduction

b-Thalassemia is caused by loss-of-function mutation of bglobin gene, leading to inadequate or absence of b-globin protein for red blood cell production. This genetic disease profoundly affects various organs, such as heart, liver, pancreas, bone, and intestine in both direct (i.e., anemia) and indirect manners (e.g., intestinal iron hyperabsorption and iron overload). We recently demonstrated in hemizygous bglobin knockout (BKO) mice that b-thalassemia is associated with osteopenia throughout the body—both trabecular and cortical sites—which mainly results from the suppression of bone formation and the acceleration of bone resorption [1, 2]. Impaired intestinal calcium absorption in thalassemia would highly worsen this pathological process; however, the underlying mechanism remains elusive.
How to restore intestinal calcium absorption in b-thalassemic individuals is not straightforward. Our recent study in BKO mice suggested that iron hyperabsorption may be a salient cause of impaired intestinal calcium absorption, and therefore calcium transport could be rescued by a liver-derived, iron absorption-inhibiting hormone, hepcidin [3, 4]. In other words, the way to stimulate calcium absorption in thalassemia is to prevent excessive iron absorption. Under normal conditions, divalent metal transporter (DMT)-1 mediates the apical Fe2? and H? uptake into the duodenal enterocytes using the H? gradient that could be maintained by Na?/H? exchanger (NHE)-3 in the apical membrane [4, 5]. Meanwhile, NHE3 serves to eliminate excessive H? accumulation in the cytoplasm during Fe2? uptake. DMT1 overexpression was recently observed in BKO mice [3], leading to a significantly higher H? influx and intracellular H? accumulation. Since direct exposure to acidic pH diminishes calcium absorption by interfering calcium binding to calbindin-D9k [6], the blockade of NHE3 would nullify cytoplasmic calcium translocation. Although hepcidin is believed to enhance calcium absorption by inducing DMT1 degradation and suppressing iron transport [3, 7], we hypothesized that NHE3 inhibitor is able to abolish the hepcidin-enhanced calcium absorption owing to its requirement for cytoplasmic pH regulation.
In general, the duodenal calcium absorption occurs through both paracellular and transcellular pathways, the latter of which is a three-step active transport process including apical calcium uptake through calcium channels (e.g., TRPV6), pH-dependent cytoplasmic calcium translocation, such as in calbindin-D9k-bound form, and basolateral calcium egress through NCX and PMCA1b [8, 9]. Whether hepcidin enhances calcium transport through the transcellular pathway is not known, but the calbindin-D9k-mediated pathway is highly sensitive to cytoplasmic pH [10]. Specifically, it has been known that cytoplasmic acidification is capable of diminishing calcium binding to calbindin-D9k [10].Ontheotherhand,theparacellularpathwaymayalsobe impaired by acid exposure, which deteriorates the paracellular mineral permeability in general [6].
Therefore, the principal objectives of the present study were to determine whether hepcidin stimulated calcium absorption across the transcellular pathway, and whether NHE3 played a role in the hepcidin-enhanced calcium transport in BKO mice.

Materials and methods

Animals

Female hemizygous b-globin knockout (b?/th3; BKO) thalassemic mice and wild-type C57BL/6 littermates (WT; 8 weeks old, weighing 15–30 g) were obtained from the Thalassemia Research Center, Institute of Molecular Biosciences, Mahidol University, Bangkok, Thailand. All BKO mice exhibited a phenotype of b-thalassemia intermedia [11]. The genotype was verified from tail DNA as described previously [12]. Mice were housed in the husbandry unit (polystyrene shoebox cages) with room temperature of 25 ± 2 C and humidity of 50–60% under a 12:12-h dark–light cycle with average illuminance of 150–200 lux in the day time (lights on at 0600). They were fed regular chow containing 0.9% wt/wt phosphorus, 1.0% wt/wt calcium, and 4000 IU/kg vitamin D (CP, Bangkok, Thailand) and reverse-osmosis water ad libitum. Body weights were recorded before sample collections. Our study has been approved by the Institutional Animal Care and Use Committee of the Faculty of Science, Mahidol University.

Inhibitors

Trifluoperazine (100 lM TFP), a PMCA inhibitor, was purchased from Sigma, St. Louis, MO, USA. The selective NHE3 inhibitor (1–100 nM tenapanor; catalog no. A14011) was purchased from AdooQ Bioscience, CA, USA. Tenapanor (100 nM) has been found to have similar potency to 100 lM amiloride (high dose for NHE inhibition) and 10 lM ethylisopropyl amiloride in inhibiting the parathyroid hormone-induced ion transport across the intestinal epithelial-like Caco-2 monolayer, a transporting process known to be dependent on NHE3 (Charoenphandhu et al. 2016; unpublished observation). The specific NCX1 inhibitor used in the present study was 100 lM KBR7943 (catalog no. 420336; Calbiochem, San Diego, CA, USA), which has been reported to block both forward and reverse modes of NCX1 but with different concentrations [i.e., different half maximal inhibitory concentrations (IC50)] [13]. In general, the IC50 of the reverse mode is \2.4 lM, whereas that of the forward mode is [30 lM. Since Iwamoto et al. [13] reported that high-dose KBR7943 ([30 lM) could inhibit the sodium-dependent calcium efflux, we used 100 lM KB-R7943 to block the NCX1-mediated basolateral calcium efflux in this study.

Experimental design

Before sample collection, mice were anesthetized by intraperitoneal injection of 70 mg/kg sodium pentobarbitone (Ceva Sante´ Animale, Libourne, France). Venous blood for hematological analysis was collected from tail vein to verify characteristics of thalassemic anemia using an automated analyzer (model ADVIA 120; Bayer, Tarrytown, NY, USA). Liver weight and spleen weight were recorded. Duodenal tissues were collected for histomorphometric analysis and ion transport studies. To identify the transporters responsible for the transcellular calcium transport in BKO mice, duodenal tissues were preincubated for 30 min in Ussing chamber with 200 nM hepcidin plus various inhibitors, i.e., 1–100 nM tenapanor (apical side), 100 lM TFP (basolateral side), or 100 lM KB-R7943 (high-dose; basolateral side).
To determine changes in serum hepcidin level in BKO mice, some BKO mice were randomly divided into three groups, i.e., (i) BKO mice daily injected with 0.5 lg/kg 1,25(OH)2D3 for 3 days, (ii) BKO mice injected with a single dose of 100 lg/kg hepcidin, and (iii) those injected with distilled water subcutaneously 24 h before sacrifice. Arterial blood samples were also collected from the left ventricle using a commercial sterile heparinized syringe (model REF364314; BD Diagnostics, Plymouth, UK) for hepcidin level measurement according to the manufacturer’s instruction (catalog no. MBS2600497; MyBioSource, San Diego, CA, USA).

Histomorphometric analysis of duodenal mucosa

Duodenal tissues were removed, cleaned off adhering connective tissues in ice-cold sterile normal saline, fixed at 4 C in 4% paraformaldehyde for 12 h. Then the tissues were dehydrated and cleared by graded ethanol and xylene, respectively, before embedded in paraffin. The paraffin block was longitudinally cut into 5-lm thickness and stained with hematoxylin and eosin. Histomorphometric measurements of villous height, villous width, crypt depth, crypt width, and mucosa/serosa amplification ratio were complied with Kisielinski et al. method [14]. The tissue slides were examined under a light microscope (Olympus BX51TRF, Tokyo, Japan) with NIS-Elements BR Analysis 4.00 (Nikon Instruments). Duodenal sections were first verified for the presence of Brunner’s glands, which is a histological marker for duodenum [15]. For accurate measurement of villous and crypt parameters, each examined villus was cut through its center perpendicular to the longitudinal axis of the intestine. Crypt depths were obtained by drawing a line from the top of the crypt down to the junction between crypt and smooth muscle layer (3–6 animals per group; 10 sections per animal; 1009 magnification).

Measurement of transepithelial calcium flux

Transepithelial calcium fluxes were determined as previously described [3, 6]. The tissue was equilibrated in Ussing chamber for 10 min with physiological bathing solution (118 mM NaCl, 4.7 mM KCl, 1.25 mM CaCl2, 1.1 mM MgCl2, 23 mM NaHCO3, 12 mM D-glucose, and 2 mM mannitol). In some experiments, duodenal tissues were preincubated with transport inhibitors for 10 min during equilibration, or preincubated with 200 nM hepcidin and/or inhibitors for 30 min. Thereafter, the basolateral chamber was refilled with fresh bathing solution containing drug or hormone, while the apical chamber was replaced by solution with radioactive 45Ca (0.45 lCi/ mL; final specific activity of 360 mCi/mol; catalog no. NEZ013; PerkinElmer, Boston, USA). Unidirectional calcium flux (JH?C; nmol/h/cm2) from the hot side (H; apical side) to the cold side (C; basolateral side) was calculated using Eqs. 1 and 2.
where RH?C is the rate of tracer appearance in the cold side (cpm/h); SH is the specific activity in the hot side (cpm/ nmol); A is the surface area of the tissue (cm2); CH is the mean radioactivity in the hot side (cpm); and CTo is the total calcium content in the hot side (nmol). Radioactivity of 45Ca was analyzed by liquid scintillation spectrophotometer (model Tri-Carb 3100; Packard, Meriden, CT, USA). Total calcium concentration in the bathing solution was analyzed by an atomic absorption spectrophotometer (model SpectrAA-300; Varian Techtron, Canada). Calcium concentration in both apical and basolateral hemichambers was 1.25 mM; thus, the measured calcium flux represented active calcium transport.

Measurement of paracellular zinc flux

The duodenal tissue of WT and BKO were equilibrated in the chamber filled with physiological bathing solution for 10 min, then the mucosal solution was replaced with bathing solution containing radioactive 65Zn (0.7 lCi/mL; final specific activity of 5 9 1012 mCi/mol; catalog no. NEZ111001; PerkinElmer). To determine the paracellular passive zinc transport, the apical and basolateral hemichamber contained 40 and 20 lM zinc, respectively.

Statistical analyses

Results are expressed as mean ± SE. Two sets of data were compared by unpaired Student’s t test. Multiple sets of data were compared by one-way analysis of variance (ANOVA) with Dunnett’s post-test. The level of significance was P\0.05. All data were analyzed using GraphPad Prism 6.0.

Results

Complete blood count analysis confirmed that BKO mice were anemic with the lower hemoglobin, hematocrit, and mean corpuscular volume (MCV) as compared to WT littermates (Fig. 1a–c). Blood smears from BKO mice also exhibited anisocytosis and poikilocytosis consistent with an increase in red cell distribution width (RDW) (Fig. 1d). BKO mice also showed a sign of hepatosplenomegaly from an increased liver and spleen weights (Fig. 1e–f). Photomicrographs of the duodenal tissues from BKO and WT mice are shown in Fig. 2a. Histomorphometric analysis of the duodenal mucosa revealed decreases in the villous height and mucosa/serosa amplification ratio (Fig. 2b, f), indicating that thalassemic mice had significantly less absorptive surface area than the WT mice. No difference in the duodenal villous width, crypt depth, or crypt width was observed (Fig. 2c–e). A decrease in the absorptive surface area generally compromised the paracellular passive transport of minerals as indicated by Fick’s law of diffusion. According to this notion, transepithelial zinc transport was tested. We confirmed that in the presence of transepithelial zinc gradient (mucosal zinc concentration of 40 lM vs. serosal concentration of 20 lM), the transepithelial zinc flux across the duodenum of BKO mice was *65% lower than that of WT mice (Fig. 3a).
Previously, we have shown that hepcidin potently increased the duodenal calcium flux in BKO mice comparable to prolonged administration of 0.5 lg/kg 1,25(OH)2D3 [3], and some investigators reported relationship between hepcidin and vitamin D [16]. It was, therefore, possible that 1,25(OH)2D3 mayalsoalterthecirculatinglevelsofhepcidin. To test this hypothesis, we injected the BKO mice with P[0.05; Fig. 4)—thereby alleviating the thalassemia-associated calcium malabsorption.
The hepcidin-enhanced calcium transport in BKO mice was completely abolished by 100 lM KB-R7943 (highdose) and TFP, inhibitors of NCX1 and PMCA1b, respectively (Fig. 4a, b). This suggests that hepcidin-enhanced calcium flux predominantly occurred through the transcellular pathway rather than the paracellular pathway. Interestingly, 100 nM tenapanor—an inhibitor of NHE3— also abolished the hepcidin-enhanced calcium transport (Fig. 4), confirming our hypothesis that NHE3 was essential for optimization of calcium absorption.

Discussion

Previously, our group has demonstrated a reciprocal correlation between calcium and iron absorption across the duodenal epithelia of thalassemic mice, which naturally exhibited anemia-associated iron hyperabsorption [3]. However, the underlying mechanism of the thalassemiaassociated calcium malabsorption is not completely understood. Interestingly, the thalassemia-associated impairment of calcium transport was rescued by hepcidin, which is a liver-derived negative regulator of duodenal iron absorption [4]. This hepcidin-enhanced calcium transport was completely abolished by inhibitors of NCX1 and PMCA1b, which extrude calcium across the basolateral membrane, and NHE3 inhibitor, indicating that hepcidin enhanced calcium transport via the transcellular rather than paracellular pathway. Meanwhile, mineral absorption across the paracellular pathway was also compromised under thalassemic condition as shown by a decrease in the paracellular transport of zinc (Fig. 3a), despite having no effect on the transcellular zinc transport [3].
Our results also showed a substantial reduction in the villous height and mucosa/serosa amplification ratio in thalassemic mice compared with the WT mice. This indicated the association of calcium absorption impairment and a decrease in absorptive surface area. The villi, especially the surface of the upper 2/3 of villous height, are the main site for calcium and iron transport in the duodenum [4, 17–19]. Thus, a decrease in villous height and consequently a decrease in mucosa/serosa amplification ratio, which represents surface area, are directly responsible for decreased absorption surface for both calcium and zinc. Furthermore, this morphological change could possibly impair the absorption of other nutrients that normally stimulate calcium absorption, such as vitamin D [2].
Nevertheless, several lines of evidence suggested that mucosal change was not the sole factor that impaired calcium absorption. Specifically, high iron uptake could be one of the major factors that impair calcium transport. Although the exact mechanism remained elusive, it was suggested that iron might compete with calcium during the cytoplasmic translocation [3, 20, 21], thereby diminishing the transcellular calcium flux in BKO mice. These explanations correspond to our previous results showing that hepcidin-suppressed duodenal iron transport was able to restore or normalize calcium transport in thalassemic mice [3]. The pathway responsible for the hepcidin-enhanced calcium transport through the transcellular pathway was proposed as it is sensitive to NCX1 and PMCA1b inhibitors. These two transporters are essential for the basolateral calcium extrusion and probably function in an interdependent manner. As suggested by the present data as well as a previous investigation [22], blockade of one transporter (either PMCA or NCX) appeared to interfere with the function of another transporter, thereby completely abolishing the entire transcellular calcium transport.
It was apparent that a potent stimulator of transcellular calcium transport, i.e., 0.5 lg/kg 1,25(OH)2D3, markedly increased serum hepcidin levels comparable to 100 lg/kg hepcidin injection (Fig. 3). The underlying mechanism of 1,25(OH)2D3-induced hepcidin production requires further investigation, but this finding suggests that the response of iron-hepcidin axis to vitamin D in b-thalassemia is different to that in chronic kidney disease, in which there is an inverse correlation between hepcidin and 1,25(OH)2D3 levels [16]. Nevertheless, long-term 1,25(OH)2D3 administration might benefit thalassemic patients by preventing iron toxicity through hepcidin induction as well as direct stimulation of intestinal calcium absorption.
Interestingly, our data showed that NHE3 was also another salient transporter responsible for the hepcidinenhanced calcium transport in BKO mice. The rationale behind this could be explained from NHE3 activities. Generally, NHE3 induces apical sodium influx into the cell as well as regulating the intracellular pH by inducing apical H? efflux [23]. Both of NHE3 activities can affect the intestinal calcium transport though different mechanisms. In details, sodium that enters the cell via NHE3 is extruded from cell through Na?/K?-ATPase lining the lateral plasma membrane into the paracellular space, thus creating paracellular osmotic gradient for solvent drag mechanism [6, 8, 24, 25]. As the result, luminal calcium moves across the paracellular space to the serosal side along with the water stream of solvent drag mechanism (for review, please see [6, 8, 25]). However, since hepcidin-enhanced calcium transport was completely abolished by NCX1 and PMCA1b inhibitors (Fig. 4), contribution of the solvent drag-induced paracellular calcium transport in hepcidin-stimulated transepithelial calcium absorption was likely to be negligible.
On the other hand, the pH-regulating activity of NHE3 turned out to be an important component of calcium transport mechanism in thalassemia-induced iron hyperabsorption. The upregulation of DMT1 in thalassemia [3] could lead to a surplus amount of H? accumulation in the cytoplasm, which, in turn, diminished calcium absorption [6]. It has been shown that direct exposure to acidic environment also inhibited calcium absorption in the rat duodenum [6]. The possible explanation is due to the protonation of calcium-binding sites on calcium-transporting proteins, including calbindin-D9k, which can hinder cytoplasmic calcium translocation [10]. Consequently, NHE3 becomes an essential player that maintains low intracellular H? concentration and allows calcium to be transported via the transcellular pathway in the thalassemic iron hyperabsorption. Recently, hepcidin has been reported to downregulate the duodenal DMT1 expression and iron transport, and eventually enhanced the calcium transport [3]. However, we previously showed that hepcidin even in a high dose did not completely abolish transport of DMT1 substrate (Fe2?) in the duodenum of thalassemic mice [3]. Therefore, the risk of calcium absorption impairment from H? accumulation remained, and NHE3 was still required for intracellular pH regulation. This explained why inhibition of NHE3 dramatically diminished the hepcidin-enhanced calcium transport as observed in the present study.
In conclusion, we elaborated possible mechanisms by which hepcidin enhanced duodenal calcium transport in BKO thalassemic mice. In the present study, BKO mice were found to have reduced mucosal surface area, which partially explained why the malabsorption of vitamin D, calcium, and other trace minerals (such as zinc) was found in b-thalassemia. Since iron hyperabsorption negatively affected calcium absorption [3], it was not surprising that iron absorption inhibitors, such as hepcidin, had a stimulating effect on calcium transport. We showed that this hepcidin-enhanced calcium transport occurred through the transcellular pathway rather than the paracellular pathway. Finally, the hepcidin-enhanced duodenal calcium transport in BKO mice required NHE3, presumably to prevent Tenapanor development of acidic cellular pH which would interfere with transcellular calcium translocation [10]. Although further experiments are required to demonstrate the detailed molecular mechanism of hepcidin on calcium absorption, our finding provides evidence that hepcidin is a promising therapeutic agent for restoring both iron and calcium balance in b-thalassemic patients.

References

1. Thongchote K, Svasti S, Teerapornpuntakit J, Krishnamra N,Charoenphandhu N (2014) Running exercise alleviates trabecular bone loss and osteopenia in hemizygous b-globin knockout thalassemic mice. Am J Physiol Endocrinol Metab 306:E1406– E1417
2. Charoenphandhu N, Kraidith K, Teerapornpuntakit J, ThongchoteK, Khuituan P, Svasti S, Krishnamra N (2013) 1,25-Dihydroxyvitamin D3 -induced intestinal calcium transport is impaired in b-globin knockout thalassemic mice. Cell Biochem Funct 31:685–691
3. Kraidith K, Svasti S, Teerapornpuntakit J, Vadolas J, Chaimana R, Lapmanee S, Suntornsaratoon P, Krishnamra N, Fucharoen S, Charoenphandhu N (2016) Hepcidin and 1,25(OH)2D3 effectively restore Ca2? transport in b-thalassemic mice: reciprocal phenomenon of Fe2? and Ca2? absorption. Am J Physiol Endocrinol Metab 311:E214–E223
4. Gulec S, Anderson GJ, Collins JF (2014) Mechanistic and regulatory aspects of intestinal iron absorption. Am J Physiol Gastrointest Liver Physiol 307:G397–G409
5. Orlowski J, Grinstein S (1997) Na?/H? exchangers of mammalian cells. J Biol Chem 272:22373–22376
6. Charoenphandhu N, Tudpor K, Pulsook N, Krishnamra N (2006) Chronic metabolic acidosis stimulated transcellular and solvent drag-induced calcium transport in the duodenum of female rats. Am J Physiol Gastrointest Liver Physiol 291:G446–G455
7. Brasse-Lagnel C, Karim Z, Letteron P, Bekri S, Bado A, Beaumont C (2011) Intestinal DMT1 cotransporter is down-regulated by hepcidin via proteasome internalization and degradation. Gastroenterology 140(1261–1271):e1261
8. Wongdee K, Charoenphandhu N (2015) Vitamin D-enhancedduodenal calcium transport. Vitam Horm 98:407–440
9. Christakos S (2012) Recent advances in our understanding of1,25-dihydroxyvitamin D3 regulation of intestinal calcium absorption. Arch Biochem Biophys 523:73–76
10. Kesvatera T, Jo¨nsson B, Telling A, To˜ugu V, Vija H, Thulin E, Linse S (2001) Calbindin D9k: a protein optimized for calcium binding at neutral pH. Biochemistry 40:15334–15340
11. Lewis J, Yang B, Kim R, Sierakowska H, Kole R, Smithies O,Maeda N (1998) A common human b globin splicing mutation modeled in mice. Blood 91:2152–2156
12. Jamsai D, Zaibak F, Khongnium W, Vadolas J, Voullaire L,Fowler KJ, Gazeas S, Fucharoen S, Williamson R, Ioannou PA (2005) A humanized mouse model for a common b0-thalassemia mutation. Genomics 85:453–461
13. Iwamoto T, Watano T, Shigekawa M (1996) A novel isothioureaderivative selectively inhibits the reverse mode of Na?/Ca2? exchange in cells expressing NCX1. J Biol Chem 271: 22391–22397
14. Kisielinski K, Willis S, Prescher A, Klosterhalfen B, Schumpelick V (2002) A simple new method to calculate small intestine absorptive surface in the rat. Clin Exp Med 2:131–135
15. Paulsen DF (2010) Digestive tract. In: Paulsen DF (ed) Histologyand cell biology: examination and board review. McGraw-Hill companies, Singapore, pp 207–228
16. Zughaier SM, Alvarez JA, Sloan JH, Konrad RJ, Tangpricha V(2014) The role of vitamin D in regulating the iron-hepcidinferroportin axis in monocytes. J Clin Transl Endocrinol 1:19–25
17. Smith MW, Debnam ES, Dashwood MR, Srai SK (2000) Structural and cellular adaptation of duodenal iron uptake in rats maintained on an iron-deficient diet. Pflugers Arch 439:449–454
18. Walters JR, Weiser MM (1987) Calcium transport by rat duodenal villus and crypt basolateral membranes. Am J Physiol 252:G170–G177
19. Bikle DD, Zolock DT, Munson S (1984) Differential response ofduodenal epithelial cells to 1,25-dihydroxyvitamin D3 according to position on the villus: a comparison of calcium uptake, calcium-binding protein, and alkaline phosphatase activity. Endocrinology 115:2077–2084
20. Moriya M, Linder MC (2006) Vesicular transport and apotransferrin in intestinal iron absorption, as shown in the Caco-2 cell model. Am J Physiol Gastrointest Liver Physiol 290:G301–G309
21. Nemere I, Norman AW (1990) Transcaltachia, vesicular calciumtransport, and microtubule-associated calbindin-D28K: emerging views of 1,25-dihydroxyvitamin D3-mediated intestinal calcium absorption. Miner Electrolyte Metab 16:109–114
22. Dorkkam N, Wongdee K, Suntornsaratoon P, Krishnamra N,Charoenphandhu N (2013) Prolactin stimulates the L-type calcium channel-mediated transepithelial calcium transport in the duodenum of male rats. Biochem Biophys Res Commun 430:711–716
23. Donowitz M, Mohan S, Zhu CX, Chen TE, Lin R, Cha B, ZachosNC, Murtazina R, Sarker R, Li X (2009) NHE3 regulatory complexes. J Exp Biol 212:1638–1646
24. Pan W, Borovac J, Spicer Z, Hoenderop JG, Bindels RJ, ShullGE, Doschak MR, Cordat E, Alexander RT (2012) The epithelial sodium/proton exchanger, NHE3, is necessary for renal and intestinal calcium (re)absorption. Am J Physiol Renal Physiol 302:F943–F956
25. Tanrattana C, Charoenphandhu N, Limlomwongse L, KrishnamraN (2004) Prolactin directly stimulated the solvent drag-induced calcium transport in the duodenum of female rats. Biochim Biophys Acta 1665:81–91