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Am J Physiol Cell Physiol 294: C1158-C1168, 2008. First published March 19, 2008; doi:10.1152/ajpcell.00020.2008
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Prolactin stimulates transepithelial calcium transport and modulates paracellular permselectivity in Caco-2 monolayer: mediation by PKC and ROCK pathways

Narongrit Thongon,1 La-iad Nakkrasae,2 Jirawan Thongbunchoo,2 Nateetip Krishnamra,1,2 and Narattaphol Charoenphandhu1,2

1Department of Physiology and 2Consortium for Calcium and Bone Research, Faculty of Science, Mahidol University, Bangkok, Thailand

Submitted 15 January 2008 ; accepted in final form 18 March 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Prolactin (PRL) was previously demonstrated to rapidly enhance calcium absorption in rat duodenum and the intestine-like Caco-2 monolayer. However, its mechanism was not completely understood. Here, we investigated nongenomic effects of PRL on the transepithelial calcium transport and paracellular permselectivity in the Caco-2 monolayer by Ussing chamber technique. PRL increased the transcellular and paracellular calcium fluxes and paracellular calcium permeability within 60 min after exposure but decreased the transepithelial resistance of the monolayer. The effects of PRL could not be inhibited by RNA polymerase II inhibitor (5,6-dichloro-1-β-D-ribobenzimidazole), confirming that PRL actions were nongenomic. Exposure to protein kinase C (PKC) or RhoA-associated coiled-coil forming kinase (ROCK) inhibitors (GF-109203X and Y-27632, respectively) abolished the stimulatory effect of PRL on transcellular calcium transport, whereas ROCK inhibitor, but not PKC inhibitor, diminished the PRL effect on paracellular calcium transport. Knockdown of the long isoform of PRL receptor (PRLR-L) also prevented the enhancement of calcium transport by PRL. In addition, PRL markedly increased paracellular sodium permeability and the permeability ratio of sodium to chloride, which are indicators of the paracellular charge-selective property and are known to be associated with the enhanced paracellular calcium transport. The permeability of other cations in the alkali metal series was also increased by PRL, and such increases were abolished by ROCK inhibitor. It could be concluded that PRL stimulated transepithelial calcium transport through PRLR-L and increased paracellular permeability to cations in the Caco-2 monolayer. These nongenomic actions of PRL were mediated by the PKC and ROCK signaling pathways.

charge selectivity; dilution potential; paracellular transport; phosphoinositide 3-kinase; protein kinase C; long form of prolactin receptor; RhoA; short interference RNA; transcellular transport

DURING PREGNANCY and lactation, prolactin (PRL) is a calcium-regulating hormone that mitigates negative calcium balance by increasing intestinal calcium absorption (10). In vivo studies in normal rats suggested that PRL stimulated both transcellular active and paracellular passive calcium transport in the duodenum and proximal jejunum (10, 33). Exposure to high physiological levels of PRL of 400–600 ng/ml, which are comparable to the levels attained during lactation and suckling (2), rapidly enhanced transepithelial calcium transport in isolated duodenal epithelium and intestine-like Caco-2 monolayer in a dose-dependent manner (25). However, the signaling pathways of PRL in the intestinal absorptive cells and the detailed mechanisms of the PRL-enhanced calcium transport were not completely understood.

Calcium traverses the intestinal epithelium via both transcellular and paracellular pathways (22). Transcellular active calcium transport is a metabolically energized process, consisting of apical calcium entry via the transient receptor potential vanilloid family Ca2+ channel 6 (TRPV6), cytoplasmic calcium translocation in a calbindin-D9K-bound form, and basolateral calcium extrusion via the plasma membrane Ca2+-ATPase (PMCA) (22). On the other hand, paracellular passive calcium transport is dependent on the transepithelial calcium gradient and transepithelial resistance (TER) and is absent when both sides of the epithelium contain equal calcium concentration (12, 29). In normal intestinal epithelia, movement of ions and nutrients across the paracellular pathway is generally regulated by the size- and charge-selective properties of the tight junction, which contains several charge-selective claudin proteins arranged in arrays of channellike barriers (32, 49, 54). Several mediators, e.g., phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), and RhoA-associated coiled-coil forming kinase (ROCK), can modulate permselectivity of the paracellular pathway (5, 15, 25, 47).

Our recent investigation (25) demonstrated that the Caco-2 monolayer, which strongly expressed short and long isoforms of the PRL receptor (PRLR-S and -L, respectively), responded to PRL by increasing both transcellular and paracellular calcium transport. However, little was known regarding the effect of PRL on the paracellular permselectivity and electrical properties of the epithelium. Moreover, it was not known whether PRLR-S or PRLR-L was responsible for PRL signaling in the intestinal absorptive cells, but there was a report that in mammary epithelia PRL augmented galactopoiesis and electrolyte transport through PRLR-L, whereas PRLR-S silenced those actions (3, 6, 24, 42). The signaling pathway of PRL in Caco-2 cells, in contrast to mammary epithelial cells, may be nongenomic since the actions were observed very rapidly within 60 min after PRL exposure (25). Interestingly, in Caco-2 cells and duodenal epithelium, the nongenomic actions of PRL were mediated by the PI3K pathway, but not the putative Janus kinase (JAK)2 or mitogen-activated protein kinase (MAPK) pathways (25). PI3K is the ultimate upstream kinase to several downstream targets, including PKC and ROCK, which are known to modulate transepithelial calcium transport and increase paracellular permeability, respectively (4, 21, 28, 36, 47). Thus PKC and ROCK pathways may mediate the actions of PRL.

In the present study, we used Caco-2 cells to demonstrate PRL-enhanced calcium absorption. Despite being human colorectal adenocarcinoma cells, confluent Caco-2 monolayers have been used widely in the studies of calcium and drug absorption because they have functional similarity to the small intestine, including the presence of brush border, expression of sucrase-isomaltase enzymes, and expression of the transcellular calcium transporters and charge-selective paracellular proteins, e.g., TRPV6, calbindin-D9K, PMCA, and claudin-1, -2, -3, and -5 (37–39, 57, 58). The Caco-2 monolayer also responds to PRL in a dose-dependent manner, with a maximal effective concentration of 600 ng/ml, similar to that seen in duodenal epithelium (25). However, the normal paracellular passive calcium transport and cationic permselectivity of the Caco-2 monolayer as well as its response to PRL by altering paracellular calcium transport have not been fully characterized.

Therefore, the objectives of this study were 1) to demonstrate that the rapid stimulatory effects of PRL on transepithelial calcium transport in the Caco-2 monolayer are nongenomic, 2) to show that PRL signaling involves the PKC and ROCK pathways, 3) to elucidate the normal characteristics of the paracellular calcium transport and cationic permselectivity of the Caco-2 monolayer, as well as the effects of PRL on these parameters, and 4) to demonstrate that PRLR-L is required for the PRL-enhanced calcium absorption.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. Caco-2 cells [American Type Culture Collection (ATCC) no. HTB-37] were grown in Dulbecco's modified Eagle's medium (DMEM; Sigma, St. Louis, MO) supplemented with 15% fetal bovine serum (FBS; GIBCO, Grand Island, NY), 1% L-glutamine (GIBCO), 1% nonessential amino acid (Sigma), and 100 U/ml penicillin-streptomycin. Cells were propagated in 75-cm2 T flasks (Corning) under a humidified atmosphere containing 5% CO2 at 37°C and subcultured as described in the ATCC protocol. Confluent Caco-2 monolayers were prepared by seeding cells on polyester Snapwell inserts with 12-mm diameter and 0.4-µm pore size (catalog no. 3801; Corning) at 5 x 105 cells/well. Culture medium was changed daily after 48 h of seeding. Monolayers were incubated at 37°C for 14 days in a humidified atmosphere containing 5% CO2.

On the experimental day, the Snapwell was mounted in a modified Ussing chamber with an exposed surface area of 1.13 cm2 to measure electrical parameters, calcium fluxes, and/or ion permeability, as previously described (25). The monolayer was incubated for 20 min in the chamber before the 60-min experiment was carried out.

Bathing solution. The bathing solution for the Ussing chamber experiments contained (in mmol/l) 118 NaCl, 4.7 KCl, 1.1 MgCl2, 1.25 CaCl2, 23 NaHCO3, 12 D-glucose, and 2 mannitol (all purchased from Sigma). The solution, continuously gassed with humidified 5% CO2 in 95% O2, was maintained at 37°C and pH 7.4 and had an osmolality of 290–293 mosmol/kgH2O as measured by a freezing point-based osmometer (model 3320; Advanced Instruments, Norwood, MA). Water used in the present work had a resistance >18.3 M{Omega}·cm and a free ionized calcium concentration <2.5 nmol/l.

Short interference RNA transfection. Two short interference RNA (siRNA) sequences targeted for human PRLR-L, i.e., 5'-GGGCUAUAGCAUGGUGACCTT-3' and 5'-GGUCACCAUGCUAUAGCCCTT-3', scrambled siRNAs, and the transfection reagent kit were supplied by Ambion (Austin, TX). Caco-2 cells were seeded in Snapwells at 5 x 105 cells/well for 12 days. Thereafter, in vitro transfection was performed with the 1:400 siPORT amine transfection reagent (Ambion) according to the manufacturer's instruction. The siRNAs were added in the culture medium to obtain a final concentration of 1 nmol/l. Control cells were treated with siPORT without siRNA. After a 48-h transfection, the PRL-stimulated calcium transport across the PRLR-L knockdown monolayer was measured. The siRNA efficiency was determined by quantitative real-time PCR (qRT-PCR). This PRLR-L knockdown study was approved by the Institutional Biosafety Committee (IBC) of the Faculty of Science, Mahidol University.

mRNA isolation, qRT-PCR, and sequencing. With the use of TRIzol reagent (Invitrogen, Carlsbad, CA), total RNA was prepared from the PRLR-L knockdown Caco-2 cells as previously described (13). One microgram of the total RNA was reverse-transcribed with the iScript kit (Bio-Rad, Hercules, CA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a housekeeping gene, served as a control gene to check the consistency of reverse transcription (% coefficient of variation <1%; n = 20). Sense and antisense primers of PRLRs and GAPDH were designed by OLIGO 6 (Molecular Biology Insights, Cascade, CO) and Primer Validator 1.4 (Naratt Software, Bangkok, Thailand), as shown in Table 1. The amplification reaction using real-time PCR (model MiniOpticon; Bio-Rad) was performed with the iQ SYBR Green SuperMix (Bio-Rad). Relative expression of PRLR over GAPDH was calculated from the threshold cycle (Ct) values by the 2{Delta}Ct method. After qRT-PCR, the PCR products were also visualized on a 1.5% agarose gel stained with 1.0 µg/ml ethidium bromide. Thereafter, all PCR products were extracted with the HiYield Gel/PCR DNA Extraction kit (Real Biotech, Taipei, Taiwan) and were sequenced with the ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA).


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  		Table 1. Homo sapiens oligonucleotide sequences used in qRT-PCR experiments

 
Measurement of electrical parameters. Three electrical parameters, i.e., potential difference (PD), short-circuit current (Isc), and TER, were determined as previously described (12). In brief, a pair of Ag/AgCl electrodes connected to agar bridges (3.0 mol/l KCl per 4 g% agar) was located near each surface of the mounted Snapwell for measurement of PD. The other ends of the PD-sensing electrodes were connected to a preamplifier (model EVC-4000; World Precision Instruments, Sarasota, FL). Another pair of Ag/AgCl electrodes connected in series to the EVC-4000 current-generating unit was placed at the end of each hemichamber to supply Isc. TER and conductance (G; G = 1/TER) were calculated from Ohm's equation.

Measurement of calcium, mannitol, and polyethylene glycol fluxes. Calcium transport across Caco-2 monolayer was determined by the method of Charoenphandhu et al. (12). After 20-min incubation, the Ussing chamber was filled with fresh bathing solution containing 45CaCl2 (final specific activity of 500 mCi/mol; Amersham, Little Chalfont, UK). Radioactivity of 45Ca was analyzed with a liquid scintillation spectrophotometer (model Tri-Carb 3100 TR; Perkin-Elmer, Shelton, CT). Total calcium concentration was analyzed by atomic absorption spectroscopy (model SpectrAA-300; Varian Techtron, Victoria, Australia). Unidirectional flux (JH->C, nmol·h–1·cm–2) of calcium from the hot side (H) to the cold side (C) was calculated with Eqs. 1 and 2.

Formula 1(1)

Formula 2(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 Snapwell (cm2); CH is the mean radioactivity in the hot side (cpm); and CT is the total calcium in the hot side (nmol).

Calcium fluxes in the absence of calcium concentration gradient (i.e., bathing solution in both hemichambers contained equal calcium concentration) represented the transcellular active calcium transport (12). The calcium gradient-dependent paracellular passive transport was studied by determining calcium fluxes in the presence of varying apical calcium concentrations (12), i.e., 1.25, 2.5, 5, 10, 20, 40, and 80 mmol/l.

In some experiments, the Caco-2 monolayer was bathed on both sides with solution containing 1 mmol/l mannitol and 1 mmol/l polyethylene glycol (PEG). Paracellular markers [3H]mannitol (Amersham; molecular weight 180; molecular radius ~350 pm) and [14C]PEG (Amersham; molecular weight 4,000; molecular radius ~2.5 nm) were added in the bathing solution to obtain final specific activities of 750 and 500 mCi/mol, respectively. Transepithelial mannitol flux and PEG flux were then measured.

In an Ussing chamber, the Caco-2 monolayer was directly exposed on the basolateral side for 60 min to 600 ng/ml recombinant human PRL (rhPRL) (purity >97%; catalog no. 682-PL; R&D Systems, Minneapolis, MN), which is the maximal effective concentration reported by Jantarajit et al. (25), or rhPRL plus inhibitors, which were an RNA polymerase II inhibitor [50 µmol/l 5,6-dichloro-1-β-D-ribobenzimidazole (DRB); Calbiochem, La Jolla, CA], a panspecific PKC inhibitor (0.8 or 1 µmol/l GF-109203X; A. G. Scientific, San Diego, CA), and a selective ROCK inhibitor (1 µmol/l Y-27632; Calbiochem).

Permeability measurement. Permeability of sodium (PNa) and chloride (PCl) were determined by the dilution potential technique, modified from the methods of Kahle et al. (26) and Hou et al. (23). In brief, Caco-2 monolayer was equilibrated for 20 min in normal bathing solution containing 145 mmol/l NaCl before the apical solution was replaced with 72.5 mmol/l NaCl-containing solution. Osmolality was maintained by an equivalent amount of mannitol. Changes in the electrical parameters before and after solution replacement were recorded until stable. The ion permeability ratio (PNa/PCl) was calculated from the dilution potential (V{delta}) with Eq. 3 (45)

Formula 3(3)
where PNa is the absolute permeability of sodium; PCl is the absolute permeability of chloride; Ca is the apical NaCl concentration; Cb is the basolateral NaCl concentration; and R, T, and F are the gas constant, temperature, and the Faraday constant.

When given {rho} = PNa/PCl, {phi} = Cb/Ca, and {nu} = FV{delta}/RT, Eq. 3 is rewritten as

Formula 4(4)

According to the Kimizuka-Koketsu equation (31),

Formula 5(5)
where G is the transepithelial conductance and PNa and PCl are calculated from G and {rho} by using Eqs. 6 and 7, respectively.

Formula 6(6)

Formula 7(7)

To study the permeability ratios of metal ions in the Group 1 series (alkali metals), i.e., PX/PCl (X+ is Li+, Na+, K+, Rb+, or Cs+ in the form of chloride salt; all purchased from Sigma), Na+ as the primary conductor in the bathing solution was substituted with X+, while pH was maintained with 10 mmol/l HEPES (Sigma). The solution used was titrated with 1 mol/l mannitol until the osmolality of 290 mosmol/kgH2O was obtained. During the experimental period, the Caco-2 monolayer was equilibrated for 20 min in normal bathing solution containing 145 mmol/l NaCl before the apical solution was replaced with 100 mmol/l X+-containing solution. Thereafter, the concentration of X+ was diluted to 50 mmol/l to create diffusion (dilution) potential. PX/PCl was calculated as described previously (45).

Calcium permeability (PCa) of the Caco-2 monolayer via the paracellular pathway was calculated from Eq. 8 (12)

Formula 8(8)
where JCa is the paracellular passive calcium flux and {Delta}C is the difference between the apical and basolateral calcium concentrations.

In some experiments, the monolayer was exposed to 1 µmol/l GF-109203X, 1 µmol/l Y-27632, or 75 µmol/l LY-294002 (PI3K inhibitor; Tocris Bioscience, Bristol, UK).

Statistical analyses. Results are expressed as means ± SE. Two sets of data were compared with the unpaired Student's t-test. Multiple comparisons were performed by one-way analysis of variance (ANOVA) with Dunnett's posttest. Linear regression with slope analysis was performed to obtain the apical calcium concentration-calcium flux and TER relationships. Nonlinear regression was performed with the one-phase exponential decay equation to demonstrate the {Delta}calcium-calcium permeability relationship as previously described (12). The level of significance for all statistical tests was P < 0.05. Data were analyzed by GraphPad Prism 4.0 for Mac OS X (GraphPad Software, San Diego, CA).


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Nongenomic action of PRL on transcellular active calcium transport involves PKC and ROCK pathways. As shown in Fig. 1, 600 ng/ml rhPRL significantly stimulated the transcellular active calcium transport in Caco-2 monolayer from the control value of 9.22 ± 0.48 (n = 10) to 18.04 ± 0.51 nmol·h–1·cm–2 (n = 12, P < 0.01). PRL concurrently decreased the TER of the monolayer by ~30% (Table 2). Here, 50 µmol/l DRB, a classic RNA polymerase II inhibitor, was used to inhibit gene transcription. DRB has been used to demonstrate nongenomic effects of several hormones, including vitamin D (43). Since the effect of PRL occurred within 60 min and was not inhibited by DRB, PRL exerted its rapid action via nongenomic signaling pathway(s).


Figure 1
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  		Fig. 1. Transcellular active calcium fluxes in Caco-2 monolayer exposed to RNA polymerase II inhibitor (50 µmol/l 5,6-dichloro-1-β-D-ribobenzimidazole; DRB), protein kinase C (PKC) inhibitor (0.8 and 1 µmol/l GF-109203X; GFX), or RhoA-associated coiled-coil forming kinase (ROCK) inhibitor (1 µmol/l Y-27632) in the presence (+rhPRL) or absence (–rhPRL) of 600 ng/ml recombinant human prolactin (rhPRL). DMSO 0.3% (vol/vol) was used as vehicle for preparation of inhibitors. **P < 0.01 compared with control group. Numbers in parentheses represent the number of independent Snapwells.

 

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  		Table 2. Epithelial electrical parameters for Caco-2 monolayer

 
In the presence of 0.8 µmol/l GF-109203X (PKC inhibitor) and 1 µmol/l Y-27632 (ROCK inhibitor), the effect of PRL on the transcellular active calcium transport was diminished (Fig. 1), while only Y-27632 inhibited the action of PRL on the TER (Table 2), suggesting that both PKC and ROCK pathways were involved in the signal transduction of PRL. A higher concentration of GF-109203X, i.e., 1 µmol/l, was used to confirm that inhibition of PKC completely abolished the effect of PRL on the transcellular calcium transport. Neither inhibitor alone nor 0.3% (vol/vol) DMSO, a vehicle for the preparation of inhibitors, had an effect on the transcellular active calcium flux or TER (Fig. 1).

PRL stimulates paracellular passive calcium transport via ROCK pathway. The relationship between apical calcium concentrations (CCa), which created the transepithelial calcium gradient ({Delta}calcium), and calcium flux (JCa) in Caco-2 monolayer shows linearity (r2 = 0.964, Fig. 2, A and B). JCa under this condition represents the {Delta}calcium gradient-dependent paracellular passive transport. After exposure to rhPRL, JCa at all CCa (1.25–80 mmol/l) were significantly increased compared with their respective controls (r2 = 0.956, P < 0.01). The slope, but not the y-intercept, of the PRL-exposed graph is higher than that of the control, i.e., 3.76 ± 0.13 vs. 6.41 ± 0.24 x 10–3 cm/h (P < 0.001). Interestingly, incubation of Caco-2 monolayer with rhPRL plus 1 µmol/l GF-109203X did not affect the paracellular passive calcium transport (Fig. 2A), whereas Y-27632 completely abolished the effect of PRL on this mode of calcium transport (Fig. 2B). In addition, rhPRL increased the paracellular PCa, and this effect was abolished by Y-27632 but not 1 µmol/l GF-109203X (Fig. 2, C and D).


Figure 2
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  		Fig. 2. Gradient-dependent paracellular passive calcium fluxes (A and B) and calcium permeability (PCa; C and D) in Caco-2 monolayer directly exposed to 600 ng/ml rhPRL, PKC inhibitor (1 µmol/l GFX), or ROCK inhibitor (1 µmol/l Y-27632). The apical bathing solution contained a calcium concentration of 1.25, 2.5, 5, 10, 20, 40, or 80 mmol/l. Inhibitors alone or vehicle (DMSO) had no effect on either calcium flux or permeability (data not shown). This experiment was performed on 210 independent Snapwell setups (n = 5 per value of apical calcium concentration). Control and rhPRL data in A and C were reused in panels B and D, respectively, for better presentation. *P < 0.01, control vs. rhPRL.

 
Since tight junctions of several epithelia, as an array of channellike structures, show a conductance block after exposure to divalent cations, including calcium (49), we measured the TER of the monolayer in the presence of transepithelial calcium gradient. As depicted in Fig. 3, increases in apical CCa led to an increase in TER with linear correlation (r2 = 0.882). Exposure to rhPRL, on the other hand, decreased the TER of the monolayer (P < 0.01) compared with the corresponding control values at the respective CCa. However, the TER of the PRL-exposed monolayer was still increased with the apical CCa (r2 = 0.842). The slopes of both regression lines are significantly different, i.e., 6.71 ± 0.43 vs. 3.67 ± 0.28 {Omega}·cm2·l·mmol–1 (P < 0.001). A reduction in TER after PRL exposure explained the PRL-stimulated paracellular passive calcium transport.


Figure 3
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  		Fig. 3. Effect of 600 ng/ml rhPRL on the relationship between apical calcium concentration and transepithelial resistance (TER) in Caco-2 monolayer (n = 5 per value of apical calcium concentration) incubated with PKC inhibitor (1 µmol/l GFX; A), ROCK inhibitor (1 µmol/l Y-27632; B), or phosphoinositide 3-kinase (PI3K) inhibitor (75 µmol/l LY-294002; C). Inhibitors alone or vehicle (DMSO) had no effect on TER (data not shown). Control and rhPRL data in A were reused in B and C for better comparison. *P < 0.01, control vs. rhPRL.

 
The ROCK inhibitor (Fig. 3B), but not the PKC inhibitor (Fig. 3A), abolished the effect of PRL on the apical calcium-induced alteration in TER. Since we recently reported (25) that PI3K inhibitor (75 µmol/l LY-294002) also compromised the paracellular passive calcium transport in Caco-2 monolayer, the effect of this inhibitor on the apical calcium-induced alteration in TER was also examined. Figure 3C shows that PI3K inhibitor, similar to ROCK inhibitor, totally abolished the effect of PRL on TER. Inhibitor alone or 0.3% (vol/vol) DMSO had no effect on the paracellular passive calcium transport and did not alter TER (data not shown).

PRL alters charge-selective property of paracellular pathway via ROCK pathway. Increased paracellular transport of calcium is usually associated with alterations in the size- and charge-selective properties of the tight junction, which is the principal barrier for paracellular ion transport (53, 54). However, in the present study, transepithelial fluxes of mannitol and PEG-4000, which are indicators of the widening of the tight junction or change in size selectivity (50), were not affected by PRL (Table 3), suggesting that charge selectivity rather than size selectivity was the determinant of the paracellular transport in the presence of PRL.


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  		Table 3. Transepithelial mannitol and PEG-4000 fluxes

 
The paracellular charge selectivity is determined by PNa, PCl, and PNa/PCl. By using the dilution potential technique, we found that rhPRL increased PNa (Fig. 4A), but not PCl (Fig. 4B), of Caco-2 monolayer from the control value of 7.81 ± 0.32 (n = 8) to 10.96 ± 0.42 x 10–6 cm/s (n = 8, P < 0.01), thereby increasing PNa/PCl from 3.73 ± 0.23 (n = 8) to 8.07 ± 0.82 (n = 8, P < 0.01) (Fig. 4C). The effects of PRL on PNa and PNa/PCl were completely abolished by Y-27632, but not by 1 µmol/l GF-109203X. Inhibitor alone or vehicle had no effect on the charge selectivity.


Figure 4
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  		Fig. 4. Effects of rhPRL on sodium permeability (PNa; A), chloride permeability (PCl; B), and PNa/PCl (C), which indicate charge-selective property of Caco-2 monolayer. Cells were incubated with PKC inhibitor (1 µmol/l GFX) or ROCK inhibitor (1 µmol/l Y-27632) in the presence (+rhPRL) or absence (–rhPRL) of 600 ng/ml rhPRL. DMSO 0.3% (vol/vol) used as vehicle had no effect on these parameters (data not shown). **P < 0.01 compared with control group. Numbers in parentheses represent the number of independent Snapwells.

 
Furthermore, we investigated permselectivity of the paracellular pathway to other cations in the alkali metal series with ionic radii ranging from 90 to 181 pm (ionic radii of Na+ and Ca2+ are 114 and 116 pm, respectively). Under normal conditions, the Caco-2 monolayer showed relative permeability to alkali metal ions as follows: PNa > PK > PRb > PCs > PLi (Fig. 5A). rhPRL also increased PX/PCl (where X+ is Li+, K+, Rb+, or Cs+) of all other alkali metal ions (Fig. 5, B, D–F), i.e., PLi/PCl from 1.09 ± 0.02 to 1.86 ± 0.09 (n = 6, P < 0.01), PK/PCl from 2.14 ± 0.07 to 3.41 ± 0.17 (n = 6, P < 0.01), PRb/PCl from 1.75 ± 0.07 to 2.18 ± 0.15 (n = 6, P < 0.01), and PCs/PCl from 1.70 ± 0.03 to 1.91 ± 0.08 (n = 6, P < 0.05). The effect of PRL on PX/PCl was diminished by Y-27632 and LY-294002, but not by 1 µmol/l GF-109203X. Therefore, the PRL-induced alteration in the cationic permselectivity in Caco-2 monolayer involved the ROCK pathway as well as the PI3K pathway.


Figure 5
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  		Fig. 5. A: effect of 600 ng/ml rhPRL on the relative paracellular permeability of the alkali metal ions over chloride (PX/PCl; X = Li+, Na+, K+, Rb+, or Cs+) plotted against their ionic radii. B–F: PX/PCl of Caco-2 monolayer incubated with PKC inhibitor (1 µmol/l GFX), ROCK inhibitor (1 µmol/l Y-27632), or PI3K inhibitor (75 µmol/l LY-294002) in the presence (+rhPRL) or absence (–rhPRL) of 600 ng/ml rhPRL. DMSO 0.3% (vol/vol) used as vehicle had no effect on PX/PCl (data not shown). *P < 0.05, **P < 0.01 compared with control group. Numbers in parentheses represent the number of independent Snapwells.

 
In another series of experiments, we investigated whether the TER-reducing action of PRL could be applied to other cations of variable sizes. The monolayer was bathed on both sides with solution containing the alkali metal ion of interest, while TER was measured simultaneously. Under control conditions, the TER-ionic radius graph showed a V-shaped relationship, with the lowest TER (or the highest conductance) when Na+ was used as the primary conductor (Fig. 6). Exposure to PRL significantly decreased TER (or increased ion conductance) no matter which alkali ion was used as the primary conductor. Similar to the permeability studies, this effect of PRL was abolished by Y-27632 (Fig. 6B) and LY-294002 (Fig. 6C), but not by 1 µmol/l GF-109203X (Fig. 6A).


Figure 6
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  		Fig. 6. Alterations of TER after apical Na+ being substituted with other alkali metal ions. Caco-2 monolayers (n = 6 per studied ion) were directly incubated with 600 ng/ml rhPRL with or without PKC inhibitor (1 µmol/l GFX; A), ROCK inhibitor (1 µmol/l Y-27632; B), or PI3K inhibitor (75 µmol/l LY-294002; C). TER values of the monolayer exposed to inhibitors alone or vehicle (DMSO) are comparable to those of control (data not shown). Control and rhPRL data in A were reused in B and C. *P < 0.01, control vs. rhPRL.

 
PRL exerts its stimulatory action on transepithelial calcium transport via PRLR-L. Since PRL normally uses PRLR-L to exert its actions, especially on the mammary gland epithelia (3), we investigated whether PRLR-L is required for the action of PRL in Caco-2 cells with a PRLR-L siRNA knockdown technique. After 48-h incubation with PRLR-L siRNA, expression of PRLR-L mRNA in Caco-2 cells was abolished (n = 5, P < 0.01) (Fig. 7, A and C), while the expression of PRLR-S was unaltered (Fig. 7, A and B). The siPORT used as a transfection reagent had no effect on the expression of PRLR-L or PRLR-S. Amplicon sequencing confirmed the results of qRT-PCR.


Figure 7
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  		Fig. 7. Representative electrophoretic image (A) and quantitative real-time PCR (B and C) demonstrate expression of short (PRLR-S) and long (PRLR-L) forms of PRL receptor in Caco-2 monolayer incubated for 48 h with siPORT (transfection agent) or siPORT + 1 nmol/l PRLR-L short interference RNA (siRNA). The absence of PRLR-L expression confirms the success of PRLR-L knockdown. **P < 0.01 compared with control group (–siPORT/–siRNA). Numbers in parentheses represent the number of independent Snapwells.

 
In the Ussing chamber experiments (Fig. 8), the PRLR-L knockdown monolayer (siPORT+, siRNA+) exhibited transcellular active calcium flux and electrical properties comparable to those of the control (siPORT–, siRNA–) or siPORT-treated (siPORT+, siRNA–) monolayers (Fig. 8 and Table 4). Similar to the effect of PRL on the normal Caco-2 monolayer in Fig. 1, rhPRL enhanced the transcellular active calcium flux of the siPORT-treated monolayer (siPORT+, siRNA–, rhPRL–) from 9.25 ± 1.08 (n = 5) to 17.05 ± 0.65 nmol·h–1·cm–2 (siPORT+, siRNA–, rhPRL+) (n = 5, P < 0.01). The TER of the siPORT-treated monolayer was also decreased by PRL (Table 4). After incubation with PRLR-L siRNA, the PRL-enhanced calcium transport and the PRL-altered TER in Caco-2 monolayer were completely reverted to basal levels. These findings indicated that PRL exerted its stimulatory action on calcium transport in the Caco-2 monolayer via PRLR-L.


Figure 8
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  		Fig. 8. Transcellular active calcium fluxes in Caco-2 monolayer exposed to 600 ng/ml rhPRL, siPORT (transfection agent), and/or 1 nmol/l siRNA targeting PRLR-L. **P < 0.01 compared with control group (–siPORT, –siRNA, –rhPRL). Numbers in parentheses represent the number of independent Snapwells.

 

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  		Table 4. Epithelial electrical parameters for PRLR-L knockdown experiment

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
PRL has been shown to exert both genomic and nongenomic actions on duodenum and proximal jejunum (25, 50, 51). In the present study, the effect of PRL on transepithelial calcium transport in Caco-2 monolayer was nongenomic, since the responses to PRL could be observed within 60 min and were not diminished by 50 µmol/l DRB, which is an inhibitor of gene transcription (43). We also demonstrated that the nongenomic action of PRL was mediated by the PKC and ROCK pathways.

The Caco-2 monolayer is a suitable model for investigating PRL action on transcellular active calcium transport because Caco-2 cells possess functional PRLR and transcellular calcium transporters (19, 25). In the absence of transepithelial calcium gradient, electrogenic transcellular active calcium transport involves the functions of TRPV6, calbindin-D9K, and PMCA (22). We previously showed (11) that PRL stimulated both apical calcium uptake and activity of the basolateral PMCA of purified duodenal membrane vesicles. The present results confirmed that transcellular calcium transport was increased by twofold after exposure to rhPRL, consistent with findings in the duodenal epithelium (25).

Interestingly, the TER of Caco-2 monolayer was decreased by PRL, similar to that observed after exposure to vitamin D, which is an important regulator of calcium absorption (14). Since the PD of the PRL-treated monolayer was not changed, it was conceivable that PRL did not interfere with the electrogenic transcellular sodium transport via Na+-K+-ATPases, which, in the absence of chemical gradient and diffusion potential, contributed up to 95% of the PD (46). Although PRL increased the electrogenic transcellular calcium transport, changes in the PD should be very small and not detectable by the Ussing technique. The PD change (V{Delta},Ca) due to the PRL-enhanced transcellular calcium transport, if present, could be estimated from the TER and the difference (J{Delta},Ca) between the control (9.22 nmol·h–1·cm–2) and PRL-exposed (18.04 nmol·h–1·cm–2) fluxes, i.e., V{Delta},Ca = J{Delta},CazF x TER (z = +2, F = 96,485.34 C/mol, TER = 255.67 {Omega}·cm2). Hence V{Delta},Ca was 120.88 µV, which could be considered negligible. When the monolayer was short-circuited, the external current (Isc) moved across the monolayer via the low-resistance pathway, i.e., the paracellular pathway (20). Therefore, a decrease in TER that was calculated from PD and Isc in the presence of PRL must have resulted from the PRL-induced increase in paracellular permeability, and thus enhanced paracellular calcium transport was anticipated.

Little is currently known regarding the paracellular calcium transport in Caco-2 monolayers. Generally, the paracellular passive calcium transport is more physiologically important since it is achieved at a small cost of energy and is nonsaturable (i.e., a graph of apical calcium vs. calcium flux shows linearity) (29). This transport mechanism, which occurs along the entire length of the small intestine, becomes significant when luminal calcium concentration exceeds 5 mmol/l, which is an average luminal calcium concentration in the rat duodenum (12, 55). When the apical calcium concentration was 20 mmol/l, the transcellular calcium flux contributed <10% of the total flux (Fig. 2). Nevertheless, transcellular calcium transport is still important during increased calcium demand, e.g., during pregnancy, lactation, and low calcium intake (8, 10). Here we found that PRL enhanced both transcellular and paracellular calcium transport as well as calcium permeability. The PRL-stimulated paracellular calcium flux at 5 mmol/l apical calcium was ~2.1-fold higher than that at 1.25 mmol/l calcium in the transcellular study, implicating the physiological significance of the paracellular transport. Moreover, as seen in Fig. 3, an increase in the apical calcium concentration also led to a divalent ion-induced conductance block, i.e., a linear increase in TER, as seen with many conventional ion channels (30, 40). Conductance block is probably caused by binding of calcium to the fixed negative charges of the paracellular proteins (49). A similar effect of high apical calcium has been reported in other epithelia, e.g., MDCK-II monolayer (49). After exposure to PRL, the increase in TER was attenuated, i.e., the paracellular conductance was increased, thus explaining increases in the paracellular calcium flux, calcium permeability, and slope of the apical calcium vs. paracellular calcium flux relationship.

Paracellular conductance and transport of ions can be regulated by changing the size- and/or charge-selective property of the paracellular pathway (32, 54). These properties are determined by different mechanisms (32). Paracellular size restriction, which is between 350 and 540 pm in the Caco-2 monolayer (9), is compromised by the myosin light chain kinase-mediated contraction of the perijunctional actomyosin ring complex and cytoskeletal arrangement (52). However, previous investigation using cytochalasin E, which widens the tight junction by disrupting actin polymerization, suggested that PRL-induced increase in duodenal calcium flux did not involve size selectivity of the paracellular pathway (50). This was confirmed by the present finding of no change in mannitol and PEG fluxes after exposure to PRL. Therefore, it appeared that the epithelial charge selectivity, but not the size selectivity, regulated the intestinal paracellular calcium transport. Generally, charge selectivity is defined by fixed negative or positive charges on the extracellular loops of tight junction proteins of the claudin family, which provide a hindrance for the permeation of ions with the opposite charge (32). Expressions of some claudins are associated with increased calcium permeability. For example, claudin-16 is widely recognized to be important for paracellular calcium and magnesium reabsorption in the thick ascending limb of the loop of Henle (17). Expression of claudin-3 in the duodenum is under the regulation of vitamin D (34), a hormone that also stimulates paracellular calcium transport (14). However, it is not known whether PRL alters the functions or localization of claudins in the Caco-2 monolayer and intestinal epithelia.

To access the charge-selective property of the Caco-2 monolayer, we used PNa/PCl as an indicator. Under control conditions, a PNa/PCl of 3.73 is comparable to those previously reported in Caco-2 monolayer (9) and small intestine (12, 41), indicating that these epithelia are more permeable to cations than anions. In contrast, normal colonic epithelium is more permeable to anions (16). It is noteworthy that the paracellular selectivity causes sodium and chloride movement to deviate from the mobility ratio of ~0.6 in free solution (20, 41). In addition, permeabilities of other cations in the alkali metal series, i.e., Li+, K+, Rb+, and Cs+, are also greater than that of Cl–, and comply with Eisenman sequence VII (i.e., PNa > PK > PRb > PCs > PLi) (for review, see Ref. 18). Similar to that reported in other epithelia (49), the conductance (reciprocal of TER) of the paracellular pathway was greatest when NaCl was used as the primary conductor, as seen in the ionic radius vs. TER curve, which showed a V-shaped relationship (Fig. 6). Such patterns were not altered by PRL. Although the paracellular pathway of the Caco-2 monolayer manifests a selective property similar to that of typical ion channels, whereby an ion current is not simply determined by the ionic or hydrated size of the ion, PRL enhanced the paracellular permeability of both sodium and calcium, which are of comparable ionic sizes. Our findings that PRL increased PNa and permeability to other alkali metal ions, but not PCl, suggested that PRL could increase the activity of the paracellular proteins with fixed negative charges by an unknown mechanism, thereby enhancing paracellular conductance, calcium permeability, and paracellular calcium transport.

PRL exerts its functions by binding to the membrane-bound PRLRs with a ratio of PRL to PRLRs of 1:2 (3). Two isoforms of PRLRs, i.e., PRLR-S and -L, have been identified in intestinal absorptive cells and Caco-2 cells (25). Investigations in mammary epithelia demonstrated that signal transduction of PRL was transferred by PRLR-L, whereas PRLR-S silenced PRL actions because it lacks the cytoplasmic tails required to activate downstream pathways (3, 6, 24, 42). On the other hand, PRLR-S may transmit PRL signals in the liver and endothelial cells (24, 44). In the PRLR-L knockdown Caco-2 monolayer, which expressed a normal level of PRLR-S, the effects of PRL on transcellular calcium transport and TER were totally abolished, indicating that actions of PRL are primarily mediated by PRLR-L.

Despite strong evidence pertaining to PRL-stimulated intestinal calcium absorption (10), little is known regarding PRL signal transduction in the intestinal epithelia and the Caco-2 monolayer. In mammary epithelia, PRL binding to PRLRs triggers dimerization of PRLRs, leading to activation of the putative JAK2 signaling (3). However, our recent investigation (25) elucidated that the PI3K pathway, but not the JAK2 pathway, is involved in the PRL-enhanced transcellular and paracellular calcium transport. The present data clearly showed that the PI3K pathway was involved in the rapid effects of PRL on the paracellular permeability to alkali metal ions and TER. Signal transduction of PRL via PI3K has also been reported in liver, pancreas, and T-lymphoma Nb2 cells (1, 7, 56). The PI3K pathway covers a large number of downstream targets, including PKC and ROCK (21). The Rho-related pathway was found to be associated with PRL signaling in endothelial cells (35), while the PKC pathway mediated PRL actions in cholangiocytes (48), adrenocortical cells (27), and the intestine, in which PKC enhanced intestinal calcium transport (4). Activation of the ROCK pathway increased paracellular permeability in several epithelia, such as brain endothelium and renal proximal tubule (28, 47). PKC-ROCK cross talk could also affect paracellular permeability in endothelial cells (47). Therefore, in the present study we focused on the PKC- and ROCK-mediated PRL actions in Caco-2 cells and were able to demonstrate that the ROCK pathway mediated PRL actions on transcellular and paracellular calcium transport, calcium permeability, charge selectivity, and TER. On the other hand, inhibition of the PKC pathway could only diminish transcellular calcium transport. Nevertheless, further investigations are required to demonstrate the detailed molecular mechanisms of PKC- and ROCK-mediated PRL actions in Caco-2 monolayer.

In conclusion, we provide evidence, for the first time, that PRL exerts its nongenomic stimulatory action on transcellular active calcium transport in the Caco-2 monolayer via the PKC and ROCK pathways. PRL also increased paracellular permeability and decreased TER via the ROCK pathway, thereby enhancing paracellular passive calcium transport. Moreover, PRL-induced increase in paracellular transport was by altering epithelial charge selectivity rather than size selectivity. The actions of PRL were mediated by the long isoform of PRLR. Our findings elaborate possible mechanisms of the PRL-stimulated intestinal calcium transport, which has long been reported in the rat (10).


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by grants from the Royal Golden Jubilee Program (PHD48K0063 to N. Thongon), the Mahidol University Postdoctoral Fellowship Program (to L-I. Nakkrasae), the Commission on Higher Education, and the Thailand Research Fund (RSA5180001 to N. Charoenphandhu and RTA5080008 to N. Krishnamra).


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. Charoenphandhu, Dept. of Physiology, Faculty of Science, Mahidol Univ., Rama VI Road, Bangkok 10400, Thailand (e-mail: naratt{at}narattsys.com)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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N. Thongon, L.-i. Nakkrasae, J. Thongbunchoo, N. Krishnamra, and N. Charoenphandhu
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