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Reproductive failure caused by porcine reproductive and respiratory syndrome virus (PRRSV) is characterized by embryonic death and weak-born piglets and is associated with placental cell apoptosis and impairment of endometrial integrity. Here, we aimed to determine whether endometrial epithelial barrier function and viability were altered following PRRSV type 1 or type 2 infection. PRRSV inoculation was examined at the apical or basolateral side of porcine glandular endometrial epithelial cell cultures isolated from 4- to 6-month-old PRRSV-free herd gilts (n = 7 pigs). On the apical side, four days postinfection (4 dpi) with type 2 PRRSV, transepithelial electrical resistance decreased by 31% ± 5%, and paracellular permeability to fluorescein isothiocyanate-dextran (4 kDa) increased by 10-fold as compared with the mock and type 1 infection. Real-time polymerase chain reaction results revealed that both PRRSV types upregulated the mRNA expression of the barrier builder tight junction protein (TJ) Cldn5, but downregulated pore-forming TJ Cldn7. Additionally, the expression of other TJ genes, i.e., Cldn3 and Cldn8, was differentially increased by PRRSV type 1 and that of zonula occludens-1 was increased by PRRSV type 2. MTT assays indicated an increase in porcine glandular endometrial epithelial cell culture at 2-6 dpi following type 2 infection. Analysis of apoptosis using Annexin/propidium iodide staining combined with flow cytometry showed that the percentage of viable cells decreased, accompanied by a significantly higher dead cell population following PRRSV type 2 infection at 2-4 dpi. PRRSV type 1 infection also induced dead cells (4%) at 2 dpi; however, the cell population recovered at 4 dpi. In conclusion, PRRSV type 2 infection caused more severe TJ barrier dysfunction and reduced cell viability compared with PRRSV type 1 infection in the porcine endometrium. Impairment in the membrane integrity of the maternal glandular endometrium may be the underlying mechanism of PRRSV-induced reproductive failure in pregnant sows.

Hydroxyxanthones, the synthetic form of xanthones that predominantly found in mangosteen (Garcinia mangostana), have been indicated as a potent anti-inflammatory action. This study aimed to examine the action of various HDX forms in preventing a tight junction (TJ) barrier disruption induced by TNF-α or IL-1β through the inhibition of p-MLC expression in Caco-2 cell model. TJ barrier function was determined by transepithelial electrical resistance (TER) and flux measurement of fluorescein isothiocyanate-dextran (FD-4). 1-monoHDX or 1,3-diHDX (10 μM) promoted TER but not FD-4 permeability in the absence of cytokines. Pretreatment with 1-monoHDX at 10 μM for 24 h prevented TNF-α or IL-1β effect on TJ permeability. p-MLC protein expression by Western blot analysis, which was induced by IL-1β was inhibited with all HDXs. Tricyclic scaffold as the basic skeleton of all HDXs may be a potential candidate therapeutic for treatment of diseases-related leaky gut.

This study aimed to investigate whether endometrial tissues and endometrial epithelial cells were capable of secreting beta-defensin (BD)-1 and -2, and soy isoflavones genistein or daidzein could promote these BD secretions. The effect of genistein on Cl- secretion in correlation with the BD secretion was also examined.Endometrial tissues or glandular epithelial cell monolayer were mounted in Ussing chamber for measurement of electrical parameters. The sample solutions from apical and basolateral compartments were collected before and after genistein or daidzein addition for the measurement of BD-1 and-2 levels by using ELISA technique.Endometrial tissues and epithelial cells constitutively secreted both BD-1 and -2 mostly at the apical compartment. Both genistein and daidzein induced BDs secretion which reached a peak at 5-15 min. The apical secretion of BDs was coincidence with increased Cl- secretion induced by genistein.Endometrial tissues and epithelial cells contributes to basal host defense against infection by secretion of BD-1 and -2, which could be enhanced by genistein or daidzein. This finding implies that these potent constituents of soy isoflavones may be useful to promote the innate immune function of human endometrium.

The present study aimed to investigate the anti-inflammatory effect of phytoestrogen genistein (Ge) on the secretion of interleukin 6 (IL6) under lipopolysaccharide (LPS) stimulated conditions in human endometrial epithelial cell line RL95-2. The effects of Ge on expression of TLRs 2, 3, 4 and 9 proteins in response to the inflammatory development induced by LPS were also examined and compared with those of 17β-estradiol (E2).The RL95-2 cells were cultured in the estrogen-deprived media with or without bacterial endotoxin LPS 30 min prior to incubation with Ge (10-7, 10-6 or 10-5 M) or E2 (10-9 M) for 48 h. The culture media were collected at 1 and 24 h for IL6 measurement by the enzyme linked immunosorbent assay and the cell lysate for TLR protein expression analyzed by semi-quantitative Western blot.Ge or E2 did not alter the IL6 level in the absence of LPS; however, treatment with either Ge or E2 for 1 h up to 24 h decreased the IL6 level stimulated by LPS. The cells challenged with LPS significantly upregulated the TLRs 2 and 9 but suppressed the TLRs 3 and 4 protein expression. Forty eight h-treatment with Ge had no effect on the TLRs expression, whereas E2 down-regulated the increased TLR9 induced by LPS.Ge suppressed the inflammatory response by decreasing the IL6 level, but not associated with the alteration of TLRs-mediated pathway inducedby bacterial infection. In contrast, E2 decreased the IL6 secretion possibly due to the opposition on LPS-induced TLR9 expression. This provides the potential evidence of soy isoflavone genistein in alleviating the inflammation of endometrium following bacterial invasion.

The objective of this study was to determine the molecular identity of ion channels involved in K+ secretion by the mammary epithelium and to examine their regulation by purinoceptor agonists. Apical membrane voltage-clamp experiments were performed on human mammary epithelial cells where the basolateral membrane was exposed to the pore-forming antibiotic amphotericin B dissolved in a solution with intracellular-like ionic composition. Addition of the Na+ channel inhibitor benzamil reduced the basal current, consistent with inhibition of Na+ uptake across the apical membrane, whereas the KCa3.1 channel blocker TRAM-34 produced an increase in current resulting from inhibition of basal K+ efflux. Treatment with two-pore potassium (K2P) channel blockers quinidine, bupivacaine and a selective TASK1/TASK3 inhibitor (PK-THPP) all produced concentration-dependent inhibition of apical K+ efflux. qRT-PCR experiments detected mRNA expression for nine K2P channel subtypes. Western blot analysis of biotinylated apical membranes and confocal immunocytochemistry revealed that at least five K2P subtypes (TWIK1, TREK1, TREK2, TASK1, and TASK3) are expressed in the apical membrane. Apical UTP also increased the current, but pretreatment with the PKC inhibitor GF109203X blocked the response. Similarly, direct activation of PKC with phorbol 12-myristate 13-acetate produced a similar increase in current as observed with UTP. These results support the conclusion that the basal level of K+ secretion involves constitutive activity of apical KCa3.1 channels and multiple K2P channel subtypes. Apical UTP evoked a transient increase in KCa3.1 channel activity, but over time caused persistent inhibition of K2P channel function leading to an overall decrease in K+ secretion.

Problem β-defensins are important innate chemical barriers that protect the endometrium from pathogen invasion. The effects of soy isoflavones, genistein and daidzein, on the expression and secretion of porcine β-defensins (PBD) in endometrial epithelial cells were investigated under normal or poly I:C-stimulated conditions. Method of study Primary cultured porcine endometrial epithelial (PE) cells were pretreated with genistein or daidzein followed by poly I:C inoculation. During treatment, the culture media were analyzed for PBD 1-4 secretion by ELISA and the total RNA for PBD gene expression by quantitative RT-PCR. Results Porcine endometrial epithelial cells constitutively expressed PBD 1-4 and secreted PBD-1, PBD-2, and PBD-4. Genistein and daidzein enhanced PBD-2 expression and PBD-2 and PBD-3 secretion. These compounds also potentiated PBD-2 and PBD-3 expression and secretion which were upregulated by poly I:C. Conclusion Soy isoflavones, genistein and daidzein, could be potentially used for promoting the innate host defense of endometrium against infection.

The present study aimed to investigate whether genistein, a potent phytoestrogen mainly found in soybean, modulated the expression of TLRs 2, 3, 4 and 9 proteins in human endometrial epithelial cell line RL95-2 under basal and polyinosinic-polycytidylic acid (poly I: C) stimulated conditions to mimic viral infection. The genistein effects were also compared with 17β-estradiol.The RL95-2 cells were cultured in the estrogen-deprived media with or without poly I: C 30 min prior to incubation with genistein (10(-7), 10(-6) or 10(-5) M) or 17β-estradiol (10(-9) M) for 48 h. The TLRs protein expression was analyzed by semi-quantitative Western blot.The cells expressed TLRs 3, 4 and 9 but a very low level of TLR2 proteins. Poly I: C significantly increased the TLRs 2 and 9 protein expressions whereas the TLRs 3 and 4 were reduced. Under basal condition, genistein at 10(-7) M increased the TLR2 while 17β-estradiol decreased the TLR4. All concentrations of genistein and 17β-estradiol attenuated the poly I: C induced increase in the TLR2. By contrast, both genistein at 10(-5) M and 17 β-estradiol further potentiated the TLR4 suppressed by poly I: C. Only 17β-estradiol was found to antagonize the poly I: C-induced changes in TLRs 3 and 9.Taken together, the present results that genistein increased the basal TLR2 and attenuated the viral component-induced TLR2 protein expression in human endometrial epithelial cells may indicate the potential role of this soy isoflavone in promoting the uterine immune function and probably alleviating the inflammation of endometrium following pathogen

The effect of prolactin (PRL) on ion transport across the porcine glandular endometrial epithelial cells was studied in primary cell culture using the short-circuit current technique. Addition of 1 μg/ml PRL either to the apical solution or to the basolateral solution produced a peak followed by a sustained increase in Isc, but with a lesser response when PRL was added apically. Basolateral addition of PRL increased the Isc in a concentration-dependent manner with a maximum effect at 1 μg/ml and an effective concentration value of 120 ng/ml. The PRL-stimulated Isc was significantly reduced by pretreatment with an apical addition of 5-nitro-2-(3-phenylpropylamino) benzoic acid (200 μM), diphenylamine-2-carboxylic acid (1 mM) or 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (200 μM), Cl− channel blockers, but not by amiloride (10 μM), a Na+ channel blocker. In addition, pretreatment with bumetanide (200 μM), a Na+–K+–2Cl− cotransporter inhibitor, in the basolateral solution significantly reduced the PRL-stimulated Isc. Replacement of Cl− or in the bathing solutions also decreased the Isc response to PRL. Pretreatment of the monolayer with AG490 (50 μM), an inhibitor of JAK2 activity significantly inhibited the PRL-induced increase in Isc. Western blot analysis of the porcine endometrial epithelial cells revealed the presence of short isoform of PRL receptor (PRLR-S) that could be regulated by 17β-estradiol. The results of this investigation showed that PRL acutely stimulated anion secretion across the porcine endometrial epithelial cells possibly through PRLR-S present in both apical and basolateral membranes. The PRL response appeared to be mediated by the JAK2-dependent pathway.

Contamination with bacterial endotoxin causes the disruption of the tight junction (TJ) barrier. We investigated the ameliorative effect of dietary flavonoids genistein (Ge) and daidzein (Di) in normal or lipopolysaccharide (LPS)-induced disruption of epithelial barrier function of the endometrium. Using the immortalized porcine glandular endometrial epithelial cells (PEG), transepithelial electrical resistance (TER) and FITC-dextran flux (FD-4) across the monolayer were measured. The mRNA expression of TJ proteins, zona occludens-1 (ZO1), and claudin-1, -3, -4, -7 and -8 was evaluated by real-time RT-PCR for coinciding effect of Ge or Di occurred at the gene transcription level. The results revealed that Ge and Di altered the TER, depending on times and concentrations. Low concentration (10−10 M) of both compounds decreased the TER, whereas higher concentrations (10−8and 10−6 M) increased the TER which was not related to the FD-4 flux. The increased TER by Ge or Di was parallel to the induction ofclaudin-3and-4or-8mRNA expression respectively. With LPS inoculation, all isoflavone treatments inhibited the decreased TER induced by LPS, but only Ge (10−8or 10−6 M) or Di (10−10or 10−6 M) was coincidence with the decreased FD-4 flux. Under this LPS-stimulated condition, some or all examined TJ gene expressions appeared to be promoted by specific concentration of Ge or Di respectively. Our findings suggest that the soy isoflavones treatment could promote and restore the impaired endometrial barrier function caused by LPS contamination.

The present study aimed to investigate the purgative effects of barakol, the purified extract of Cassia siamea Lam., on the longitudinal smooth muscle contractions of the rat ileum. The extract increased the force of spontaneous muscle contractions in a concentration-dependent manner (EC50=0.3 mM). Saxitoxin (0.3 microM) abolished the stimulatory effects of barakol, a result indicating a neural mechanism of action. In addition, atropine (10 microM) but not propanolol (10 microM) or phentolamine (10 microM), partially inhibited barakol-induced smooth muscle contractions suggesting that cholinergic nerves were involved. The motor effects of barakol were further examined in muscle strips treated with catecholamines to suppress spontaneous contractile activity and decrease muscle tone. Norepinephrine or dopamine (10 microM) decreased the amplitude of spontaneous contractions by 72% and 18%, respectively. Pretreatment of the tissues with barakol (1 mM) significantly decreased the inhibitory effect of norepinephrine by 60%, but not that of dopamine. Its ability to potentiate atropine- and saxitoxin-sensitive contractions and inhibit the antimotility actions of norepinephrine suggests that barakol may increase longitudinal smooth muscle contractions by decreasing the inhibitory effect of norepinephrine on excitatory cholinergic motor neurons. Barakol may produce a purgative action in small intestine which may be clinically important in patients with intestinal hypomotility disorders.

Genistein (Ge) and daidzein (Di), the active isoflavones primarily found in soybean, have various beneficial health effects. The present study aims to investigate the effects of these isoflavones on tight junction (TJ) barrier integrity in immortalized endometrial epithelial cells (IMM). Expressions of the TJ integrity markers, zona occludens-1 (ZO-1) and claudins (cldn)-1, -3, -4, -7 and -8 were analyzed by quantitative reverse transcription and real-time polymerase chain reaction (RT-PCR) and transepithelial electrical resistance (TER) by voltohmmeter. Treatment with Ge (10 nM or 1 μM) for 48 h increased the TER, and this was correlated with the mRNA expression of cldn-3 and/or ZO-1. However, low dose of Ge (0.1 nM) decreased both the TER and the expression of cldn-3 and/or ZO-1. Di did not alter the expression of the TJ proteins. Our results suggest that genistein increases barrier function possibly via the expression of cldn-3 and ZO-1. This represents an important protective role of genistein against...

β-defensins (BD) are antimicrobial peptides that contribute the mucosal innate immunity. Its expression is regulated by sex steroid hormones. Phytoestrogens genistein (Ge) and daidzein (Di) mostly ...

The objective of this study was to investigate acute and long-term effects of epidermal growth factor (EGF) and transforming growth factor alpha (TGFalpha) on basal ion transport activity of glandular endometrial epithelial cells in primary culture. The effects of EGF on insulin-dependent regulation of Na+ transport across this epithelium was also investigated. Addition of 1.6 nM EGF or 2 nM TGFalpha to the basolateral, but not the apical, solution inhibited both basal and insulin-stimulated Na+ transport with a maximum response within 45-60 min. This effect was mimicked by the calcium ionophore ionomycin. Incubation with EGF for 4 days inhibited insulin-stimulated Na absorption in a concentration-dependent fashion with an IC(50) value of 0.3 nM. Experiments using amphotericin B-permeabilized monolayers demonstrated that EGF inhibited Na transport by decreasing apical membrane Na conductance without affecting insulin-dependent stimulation of the Na+-K+ ATPase. Addition of EGF or TGFalpha for 24 h resulted in increased basal Cl- secretion in addition to inhibition of Na absorption. The EGF-induced increase in Cl- secretion was inhibited in part by indomethacin, suggesting that long-term regulation by EGF involves stimulation of arachidonic acid synthesis and prostaglandin release. The EGF-induced increase in indomethacin-insensitive Cl- secretion was prevented by the protein synthesis inhibitor cyclohexamide, and by the DNA transcription inhibitor actinomycin D indicating that EGF-stimulated anion secretion required DNA transcription and protein synthesis. The results of these studies demonstrated that the basal transport properties of endometrial epithelial cells are differentially regulated by EGF, TGFalpha, and insulin.

The objective of this study was to investigate the effects of insulin and insulin-like growth factor I on transepithelial Na(+) transport across porcine glandular endometrial epithelial cells grown in primary culture. Insulin and insulin-like growth factor I acutely stimulated Na(+) transport two- to threefold by increasing Na(+)-K(+) ATPase transport activity and basolateral membrane K(+) conductance without increasing the apical membrane amiloride-sensitive Na(+) conductance. Long-term exposure to insulin for 4 d resulted in enhanced Na(+) absorption with a further increase in Na(+)-K(+) ATPase transport activity and an increase in apical membrane amiloride-sensitive Na(+) conductance. The effect of insulin on the Na(+)-K(+) ATPase was the result of an increase in V(max) for extracellular K(+) and intracellular Na(+), and an increase in affinity of the pump for Na(+). Immunohistochemical localization along with Western blot analysis of cultured porcine endometrial epithelial cells revealed the presence of alpha-1 and alpha-2 isoforms, but not the alpha-3 isoform of Na(+)-K(+) ATPase, which did not change in the presence of insulin. Insulin-stimulated Na(+) transport was inhibited by hydroxy-2-naphthalenylmethylphosphonic acid tris-acetoxymethyl ester [HNMPA-(AM)(3)], a specific inhibitor of insulin receptor tyrosine kinase activity, suggesting that the regulation of Na(+) transport by insulin involves receptor autophosphorylation. Pretreatment with wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase as well as okadaic acid and calyculin A, inhibitors of protein phosphatase activity, also blocked the insulin-stimulated increase in short circuit and pump currents, suggesting that activation of phosphatidylinositol 3-kinase and subsequent stimulation of a protein phosphatase mediates the action of insulin on Na(+)-K(+) ATPase activation.

Background /Aim: Genistein, the most active isoflavone found primarily in soybeans, alters ion transport functions in intestinal and airway epithelia. The present study aims to investigate the acute effects and mechanisms of action of genistein in immortalized porcine endometrial epithelial cells. Methods: Ussing chamber technique was used for transepithelial electrical measurements. Results: Genistein increased short-circuit currents (Isc) which were inhibited by glibenclamide, NPPB, CFTRinh-172, DIDS or bumetanide, but not amiloride. In experiments with amphotericin B-permeabilized monolayers, genistein activated the apical Cl- current and barium-sensitive basolateral K+ current while inhibiting the apical K+ current. Genistein failed to increase the Isc in the presence of forskolin or IBMX, but did increase the Isc in UTP. Pretreatment with genistein also abolished the increase in the Isc when induced by forskolin, IBMX or UTP. However, Ca2+-chelating BAPTA-AM did not affect the genistein-induced increase in the Isc. The genistein-stimulated Isc was reduced by tyrosine kinase inhibitors, tyrphostin A23 or AG490. However, vanadate, a tyrosine phosphatase inhibitor, failed to inhibit the genistein response. Estrogen receptor antagonist ICI182,780 did not alter the genistein's action. Conclusion: The soy isoflavone, genistein, stimulates Cl- secretion in endometrial epithelial cells possibly via a direct activation of CFTR which appears to be modulated through a tyrosine kinase-dependent pathway. The present findings may be of benefit for the therapeutic application of genistein in the treatment of electrolyte transport disorders in the epithelia.

Cytochrome c (CytC) released from mitochondria induces apoptosis in both normal and tumor cells. Expression of Fas ligand (FasL) helps maintain tumor cell survival by inducing apoptosis of Fas-bearing anti-tumor immune cells. A risk of endometrial cancer has been reported to associate with phytoestrogen consumption. Therefore the effects of phytoestrogens, genistein and daidzein, on FasL and CytC protein expression were examined in primary cultured porcine endometrial cells (PE) and human cancerous endometrial cells (RL95-2) by Western blot analysis. Both cells were cultured in standard medium (SM) and switched to estrogen-deprived medium (SF) with or without 17beta-estradiol (E, 1 nM), genistein (10 microM) or daidzein (10 microM) for 48 h. FasL (25 kDa) which was found only in RL95-2 cells was upregulated in SF compared to SM. Treatment of RL95-2 cells with E, daidzein or genistein significantly increased the FasL expression by 7-10 folds. In the present study, low level of CytC was detected in both cells cultured in SM but markedly increased in SF by 1.5-2 folds. The SF-induced increase in CytC level was reversed by genistein or daidzein while E suppressed CytC in PE cells, but not in RL95-2 cells. The findings suggest that genistein and daidzein appear to act as a survival factor by inhibiting intracellular apoptogenic initiator in both normal and cancer endometrial cells. In addition, estrogen and phytoestrogens inducing the death signal FasL expressed by cancerous endometrial cells may cause the tumor progression. Thus, consuming phytoestrogen as a supplement should be awareness in patient with endometrial cancer.

Barakol, an active constituent extracted from Cassia siamea, has been shown to have anxiolytic effects similar to diazepam when treated intraperitoneally.Acute and chronic oral administrations of barakol on anxiolytic, locomotor and exploratory behaviors were examined in rats using an elevated plus maze followed by a holeboard apparatus in comparison with the anxiolytic diazepam.Male Wistar rats were divided into the acute and chronic treatment groups. The acutely-treated rats were given orally with vehicle control (distilled water, p.o.), diazepam (5 mg/kg, p.o.) and barakol (10, 30 and 100 mg/kg, p.o.) while the chronically-treated rats received the same treatment for 30 consecutive days. The anxiolytic behavior was tested on the elevated plus maze for 5 min and immediately followed by the holeboard to test for the directed exploratory behavior for 10 min.Acute and chronic oral administration of barakol (10, 30 and 100 mg/kg, p.o.) produced no significant changes in anxiolytic parameters tested on the elevated plus maze compared to diazepam which significantly increased the percentage of the open/total time and the time spent on the open arms. On the other hand, all parameters tested using the holeboard were not affected by barakol or diazepam when given acutely. When given chronically, all doses of barakol significantly decreased the number of head-dips and the total time spent head-dipping with no changes in the number of grooms or rears per minute.Acute and chronic oral administration of barakol had no anxiolytic and locomotor effects. However, it exerted a sedative effect as shown by a reduction in the directed exploratory behaviors.

The regulation of prolactin (PRL) on electrolyte transport was investigated in primary culture of porcine glandular endometrial epithelial cells using Ussing chamber technique. PRL produced a peak ...

Barakol is a purified extract of Cassia siamea, a plant that has been used as a laxative in traditional medicine. In this study, the effect of barakol on anion transport across the rat colon epithelium was investigated. Colonic epithelium was mounted in Ussing chambers and bathed with Ringer's solution. Addition of 1 mM barakol to the basolateral solution produced a slow increase in short-circuit current (Isc) in proximal colon and distal colon by 24.5 +/- 2.2 and 24.2 +/- 1.4 microA/cm(2), respectively. Barakol increased Isc in a concentration-dependent manner with an EC(50) value of 0.4 mM. The barakol-stimulated increase in Isc was inhibited by subsequent treatment with 500 microM diphenylamine-2-carboxylic acid or 400 microM glibenclamide added to the apical solution and 200 microM bumetanide added to the basolateral solution. Pretreatment of the tissues with 200 microM bumetanide, but not 10 microM amiloride, completely abolished the barakol-increased Isc. Ion substitution experiments showed an inhibition of barakol-stimulated Isc in chloride-free solution but not in bicarbonate-free solution. In addition, pretreatment of tissues with 10 microM tetrodotoxin or 10 microM indomethacin, but not 1 microM atropine or 10 microM hexamethonium, partially inhibited the Isc response by barakol. The present results demonstrated the stimulatory effect of barakol on the bumetanide-sensitive chloride secretion in rat colon. The effect of barakol was partly mediated by the stimulation of submucosal nerves and through the release of cyclooxygenase metabolites. These findings thus provide an explanation for the underlying mechanism of barakol as a secretagogue in mammalian colon.

The objective of this study was to investigate the mechanism of uridine 5'-triphosphate (UTP)-dependent inhibition of Na(+) absorption in porcine endometrial epithelial cells. Acute stimulation with UTP (5 microM) produced inhibition of sodium absorption and stimulation of chloride secretion. Experiments using basolateral membrane-permeabilized cell monolayers demonstrated a reduction in benzamil-sensitive Na(+) conductance in the apical membrane after UTP stimulation. The UTP-dependent inhibition of sodium transport could be mimicked by PMA (1 microM). Several PKC inhibitors, including GF109203X and Gö6983 (both nonselective PKC inhibitors) and rottlerin (a PKCdelta selective inhibitor), were shown to prevent the UTP-dependent decrease in benzamil-sensitive current. The PKCalpha-selective inhibitors, Gö6976 and PKC inhibitor 20-28, produced a partial inhibition of the UTP effect on benzamil-sensitive Isc. Inhibition of the benzamil-sensitive Isc by UTP was observed in the presence of BAPTA-AM (50 microM), confirming that activation of PKCs, and not increases in [Ca(2+)](i), were directly responsible for the inhibition of apical Na(+) channels and transepithelial Na(+) absorption.

The endometrial epithelium consists of a surface epithelium which faces the uterine lumen and a glandular epithelium which invaginates into the endometrial stroma (Davis & Blair, 1993). The endometrium is responsible for the transfer of nutrients, fluid and electrolytes between the circulatory system and the uterine lumen. The transport-related activity of the surface and glandular epithelium provides an optimal intrauterine environment for implantation and embryo development. Previous studies of uterine fluid ionic composition from pigs, humans and rats have revealed that the K+ concentration is 4- to 5-fold higher than that in plasma, and the Na+ concentration is significantly lower than its concentration in plasma (Clemetson, Kim, Mallikarjuneswara & Wilds, 1972; Iritani, Sato & Nishikawa, 1974; Casslen & Nilsson, 1984). Regulation of Na+ and K+ concentrations within uterine and oviduct fluids appears to be important for reproductive events including sperm capacitation, acrosomal reaction, sperm-egg fusion and implantation of the developing embryo (Clemetson et al. 1972; Boldt, Casas, Whaley, Creazzo & Lewis, 1991; Fraser, 1992, 1995). Other important functions of endometrial epithelial cells include regulation of luminal pH and uterine fluid volume. Active secretion of Cl− and HCO3− ions has been shown to establish a driving force for fluid secretion and to regulate luminal fluid pH in the intestine (Wenzl, Sjaastad, Weintraud & Machen, 1989; MacLeod, Redican, Lembessis, Hamilton & Field, 1996) and airways (Smith & Welsh, 1992). In rabbit, fluid secretion into the oviduct lumen is driven by active secretion of Cl− across the oviduct epithelium (Leese, 1988). Previous studies using primary cultures of human endometrial epithelial cells grown on permeable supports demonstrated that amiloride-sensitive Na+ absorption was accompanied by K+ secretion across the cell monolayers (Matthews, Thomas, Redfern & Hirst, 1993). Other studies with primary cultures of rodent endometrial epithelial cells grown on permeable supports showed that the short circuit current was stimulated by forskolin, an activator of adenylyl cyclase (Rochwerger, Dho, Parker, Foskett & Buchwald, 1994; Leung, Wong, Gabriel, Yankaskas & Boucher, 1995). In cultured mouse endometrial cells, adrenergic agonists were shown to produce an increase in Isc that was consistent with anion secretion. This response was mediated by β-adrenergic receptors and was blocked by apical addition of either glibenclamide or diphenylamine 2,2′-dicarboxylic acid (DPC) (Chan, Fong, Chung & Wong, 1997). These experiments indicate that endometrial epithelial cells in culture are capable of both Na+ absorption and anion secretion. Prostaglandins are produced by epithelial and stromal endometrial cells in human and other species (Alecozay, Harper, Scheken & Hanahan, 1991; Zhang, Paria & Davis, 1991; Chen, Yang, Hilsenrath, Le & Harper, 1995). They act in both an endocrine and paracrine manner to increase uterine blood flow, vascular permeability and to regulate menstruation and implantation (Ford, Reynolds & Magness, 1982; Keys, King & Kennedy, 1986; Chaudhuri, 1991; Yee & Kennedy, 1993). Prostaglandins, specifically PGE2, have been shown to regulate electrolyte transport function in rabbit cortical collecting duct (Sakairi & Jacobson, 1995), prairie dog gallbladder (Saunders-Kirkwood, Cates & Roslyn, 1993) and porcine distal colon epithelium (Traynor, Brown & O'Grady, 1993). However, little is known about the transport-related actions of prostaglandins on endometrial epithelial cells. Recent studies with porcine surface endometrium using amphotericin B-permeabilized tissues showed that PGF2α increased Na+ absorption by activation of K+ channels in the basolateral membrane (Vetter & O'Grady, 1996). The aim of this study was to characterize the transport properties of porcine glandular epithelial cells and to investigate the effect of PGE2 on regulation of anion transport.