Differential innate immune response of endometrial cells to porcine reproductive and respiratory syndrome virus type 1 versus type 2.

Modification of cellular and immunological events due to porcine reproductive and respiratory syndrome virus (PRRSV) infection is associated with pathogenesis in lungs. PRRSV also causes female reproductive dysfunction and persistent infection which can spread to fetus, stillbirth, and offspring. In this study, changes in cellular and innate immune responses to PRRSV type 1 or type 2 infection, including expression of PRRSV mediators, mRNA expression of Toll-like receptors (TLRs) and cytokine, and cytokine secretion, were examined in primary porcine glandular endometrial cells (PGE). Cell infectivity as observed by cytopathic effect (CPE), PRRSV nucleocapsid proteins, and viral nucleic acids was detected as early as two days post-infection (2 dpi) and persisted until 6 dpi. A higher percentage of CPE and PRRSV-positive cells were observed in type 2 infections. PRRSV mediator proteins, CD151, CD163, sialoadhesin (Sn), integrin and vimentin, were upregulated following type 1 and type 2 infection. CD151, CD163 and Sn were upregulated by type 2. In both PRRSV types, mRNA expression of TLR1 and TLR6 was upregulated. However, TLR3 was upregulated by type 1, but TLR4 and TLR8 mRNA and protein were downregulated by type 2 only. Interleukin (IL)-1ß, IL-6 and tumor necrotic factor (TNF)-α were upregulated by type 2, but IL-8 was upregulated by type 1. Both PRRSV type 1 and 2 stimulated IL-6 but suppressed TNF-α secretion. In addition, IL-1ß secretion was suppressed only by type 2. These findings reveal an important mechanism underlying the strategy of PRRSV infection in the endometrium and associated with the viral persistence.

Porcine reproductive and respiratory syndrome virus induces tight junction barrier dysfunction and cell death in porcine glandular endometrial epithelial cells.

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.

P2Y receptor regulation of K2P channels that facilitate K+ secretion by human mammary epithelial cells.

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.

Soy isoflavones enhance ß-defensin synthesis and secretion in endometrial epithelial cells with exposure to TLR3 agonist polyinosinic-polycytidylic acid.

PROBLEM: ß-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 ß-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.

Anti-inflammatory Effect of Genistein in Human Endometrial Cell Line Treatment with Endotoxin Lipopolysaccharide.

Objective: 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 17ß-estradiol (E2). Material and Method: 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. Results: 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. Conclusion: 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.

Enhanced Secretion of Beta-Defensins in Endometrial Tissues and Epithelial Cells by Soy Isoflavones.

Objective: 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. Material and Method: 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. Results: 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. Conclusion: 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.

Soy isoflavones improves endometrial barrier through tight junction gene expression.

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(-8) and 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 of claudin-3 and -4 or -8 mRNA expression respectively. With LPS inoculation, all isoflavone treatments inhibited the decreased TER induced by LPS, but only Ge (10(-8) or 10(-6) M) or Di (10(-10) or 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.

Isoflavone Genistein Modulates the Protein Expression of Toll-like Receptors in Cancerous Human Endometrial Cells.

OBJECTIVE: 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 17ß-estradiol. MATERIAL AND METHOD: 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 17ß-estradiol (10(-9) M) for 48 h. The TLRs protein expression was analyzed by semi-quantitative Western blot. RESULTS: 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 17ß-estradiol decreased the TLR4. All concentrations of genistein and 17ß-estradiol attenuated the poly I: C induced increase in the TLR2. By contrast, both genistein at 10(-5) M and 17 ß-estradiol further potentiated the TLR4 suppressed by poly I: C. Only 17ß-estradiol was found to antagonize the poly I: C-induced changes in TLRs 3 and 9. CONCLUSION: 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

Activation of chloride secretion by isoflavone genistein in endometrial epithelial cells.

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.

Site-specific regulation of ion transport by prolactin in rat colon epithelium.

The effect of prolactin (PRL) on ion transport across the rat colon epithelium was investigated using Ussing chamber technique. PRL (1 µg/ml) induced a sustained decrease in short-circuit current (I(sc)) in the distal colon with an EC(50) value of 100 ng/ml and increased I(sc) in the proximal colon with an EC(50) value of 49 ng/ml. In the distal colon, the PRL-induced decrease in I(sc) was not affected by Na(+) channel blocker amiloride or Cl(-) channel blockers, NPPB, DPC, or DIDS, added mucosally. However, the response was inhibited by mucosal application of K(+) channel blockers glibenclamide, quinidine, and chromanol 293B, whereas other K(+) channel blockers, Ba(2+), tetraethylammonium, clotrimazole, and apamin, failed to have effects. The PRL-induced decrease in I(sc) was also inhibited by Na(+)-K(+)-2Cl(-) transporter inhibitor bumetanide, Ba(2+), and chromanol 293B applied serosally. In the transverse and proximal colon, the PRL-induced increase in I(sc) was suppressed by DPC, glibenclamide, and bumetanide, but not by NPPB, DIDS, or amiloride. The PRL-induced changes in I(sc) in both distal and proximal colon were abolished by JAK2 inhibitor AG490, but not BAPTA-AM, the Ca(2+) chelating agent, or phosphatidylinositol 3-kinase inhibitor wortmannin. These results suggest a segment-specific effect of PRL in rat colon, by activation of K(+) secretion in the distal colon and activation of Cl(-) secretion in the transverse and proximal colon. Both PRL actions are mediated by JAK-STAT-dependent pathway, but not phosphatidylinositol 3-kinase pathway or Ca(2+) mobilization. These findings suggest a role of PRL in the regulation of electrolyte transport in mammalian colon.

Modulatory effects of phytoestrogens on the expression of Fas ligand and the release of cytochrome C in normal and cancerous endometrial cells.

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.

Behavioral effects of acute and chronic oral administration of barakol in rats.

INTRODUCTION: Barakol, an active constituent extracted from Cassia siamea, has been shown to have anxiolytic effects similar to diazepam when treated intraperitoneally. OBJECTIVE: 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. MATERIAL AND METHOD: 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. RESULTS: 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. CONCLUSION: 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.

Regulation of electrolyte transport across cultured endometrial epithelial cells by prolactin.

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 microg/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 microg/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 microM), diphenylamine-2-carboxylic acid (1 mM) or 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (200 microM), Cl(-) channel blockers, but not by amiloride (10 microM), a Na(+) channel blocker. In addition, pretreatment with bumetanide (200 microM), 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 microM), 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 17beta-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.

Barakol suppresses norepinephrine-induced inhibition of spontaneous longitudinal smooth muscle contractions in isolated rat small intestine.

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.

Barakol extracted from Cassia siamea stimulates chloride secretion in rat colon.

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.

UTP-dependent inhibition of Na+ absorption requires activation of PKC in endometrial epithelial cells.

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.