Genistein augments prostaglandin-induced recovery of barrier function in ischemia-injured porcine ileum

Anthony T. Blikslager1, Malcolm C. Roberts2, Karen M. Young1, J. Marc Rhoads4, and Robert A. Argenzio3

Departments of 1 Clinical Sciences, 2 Food Animal Health and Resource Management, and 3 Anatomy, Physiological Sciences, and Radiology, College of Veterinary Medicine, North Carolina State University, Raleigh, 27606; and 4 Department of Pediatrics, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina 27599.


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have previously shown that PGE2 enhances recovery of transmucosal resistance (R) in ischemia-injured porcine ileum via a mechanism involving chloride secretion. Because the tyrosine kinase inhibitor genistein amplifies cAMP-induced Cl- secretion, we postulated that genistein would augment PGE2-induced recovery of R. Porcine ileum subjected to 45 min of ischemia was mounted in Ussing chambers, and R and mucosal-to-serosal fluxes of [3H]N-formyl-methionyl-leucyl phenylalanine (FMLP) and [3H]mannitol were monitored as indicators of recovery of barrier function. Treatment with genistein (10-4 M) and PGE2 (10-6 M) resulted in synergistic elevations in R and additive reductions in mucosal-to-serosal fluxes of [3H]FMLP and [3H]mannitol, whereas treatment with genistein alone had no effect. Treatment of injured tissues with genistein and either 8-bromo-cAMP (10-4 M) or cGMP (10-4 M) resulted in synergistic increases in R. However, treatment of tissues with genistein and the protein kinase C (PKC) agonist phorbol myristate acetate (10-5-10-6 M) had no effect on R. Genistein augments recovery of R in the presence of cAMP or cGMP but not in the presence of PKC agonists.

mucosa; chloride secretion; transmucosal resistance; tyrosine kinase


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE INTESTINAL MUCOSAL BARRIER is composed of a single layer of epithelial cells anchored to one another by a series of interepithelial junctions (25, 26). In terms of barrier function, the most critical of the interepithelial junctions is the apically situated tight junction because it regulates paracellular flux of solutes and bacterial toxins (25, 26). For example, it has recently been shown that the bacterial toxin N-formyl-methionyl-leucyl phenylalanine (FMLP), a potent neutrophil chemoattractant, may gain access to subepithelial tissues across submature tight junctions (35). Breakdown of the intestinal barrier, such as occurs during intestinal ischemia-reperfusion injury, increases the ability of bacteria and their toxins to traverse the gut and may lead to onset of sepsis and multiple organ failure (39). We have recently shown that PGE2 and PGI2 trigger recovery of barrier function in ischemia-injured intestinal mucosa via a pathway that principally involves closure of interepithelial spaces (8, 9).

PGE2 stimulates recovery of barrier function via cAMP (8). This intracellular second messenger has diverse cellular effects, making it difficult to determine the exact role of this mediator in recovery of barrier function. For example, cAMP may trigger elevations in transepithelial resistance via direct effects on tight junctions (16) but cAMP also signals Cl- secretion (11, 41). Our recent studies indicate that inhibition of PGE2-triggered Cl- secretion in ischemia-injured porcine ileum largely abolishes the reparative action of PGE2 (8). Because of the apparent role of Cl- secretion in recovery of barrier function, we developed an interest in genistein, a novel Cl- secretagogue. There has been a great deal of recent interest in isoflavinoids such as genistein because these compounds are antioxidants (10) and anticarcinogenic in the gut (21, 42) and may be derived from dietary sources such as soybean meal (38). Genistein augments Cl- secretion in a cAMP-dependent fashion, possibly by interacting directly with phosphorylated cystic fibrosis transmembrane conductance regulator (CFTR) (17). However, genistein is a tyrosine kinase inhibitor that may have direct effects on recovery of barrier function via its effects on tight junction-associated tyrosine kinases (13, 29, 36). In the present studies, we postulated that genistein would augment PGE2-stimulated recovery of mucosal barrier function in acutely injured intestinal epithelium via a signaling pathway that involved Cl- secretion rather than an action involving inhibition of tyrosine kinases.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental animal surgeries. All studies were approved by the North Carolina State University Institutional Animal Care and Use Committee. Six- to eight-week-old Yorkshire crossbred pigs of either sex were housed singularly and maintained on a commercial pelleted feed. Pigs were held off feed for 24 h before experimental surgery. General anesthesia was induced with xylazine (1.5 mg/kg im), ketamine (11 mg/kg im), and pentobarbital (15 mg/kg iv) and was maintained with intermittent infusion of pentobarbital (6-8 mg · kg-1 · h-1). Pigs were placed on a heating pad and ventilated with 100% O2 via a tracheotomy using a time-cycled ventilator. The jugular vein and carotid artery were cannulated, and blood gas analysis was performed to confirm normal pH and partial pressures of CO2 and O2. Lactated Ringer solution was administered intravenously at a maintenance rate of 15 ml · kg-1 · h-1. Blood pressure was continuously monitored via a transducer connected to the carotid artery. The ileum was approached via a ventral midline incision. Ileal segments were delineated by ligating the intestinal lumen at 10-cm intervals and subjected to ischemia by clamping the local mesenteric blood supply for 45 min.

Ussing chamber studies. After the ischemic period, the mucosa was stripped from the seromuscular layer in oxygenated (95% O2/5% CO2) Ringer solution and mounted in 3.14 cm2 aperture Ussing chambers, as described in a previous study (5). Tissues were bathed on the serosal and mucosal sides with 10 ml Ringer solution. The serosal bathing solution contained 10 mM glucose and was osmotically balanced on the mucosal side with 10 mM mannitol. Bathing solutions were oxygenated (95% O2/5% CO2) and circulated in water-jacketed reservoirs. The spontaneous potential difference (PD) was measured using Ringer-agar bridges connected to calomel electrodes, and the PD was short-circuited through Ag-AgCl electrodes using a voltage clamp that corrected for fluid resistance. Resistance (Omega  · cm2) was calculated from the spontaneous PD and short-circuit current (Isc). If the spontaneous PD was between -1.0 and 1.0 mV, tissues were current clamped at ±100 µA for 5 s and the PD was recorded. Isc and PD were recorded every 15 min for 4 h.

Experimental treatments. Tissues were bathed in Ringer containing 5 × 10-6 M indomethacin to prevent PG production while the mucosa was stripped from the seromuscular tissues, and indomethacin was added to the serosal and mucosal bathing solutions in the same concentration before tissues were mounted on Ussing chambers. In addition, bumetanide (10-4 M) was added to the serosal side of tissues at the beginning of the experiment where appropriate. Baseline electrical readings were taken for 30 min, after which further treatments were added to the tissues depending on the study. Treatments added after the 30-min equilibration included genistein (10-4 M, serosal and mucosal), 16,16-dimethyl PGE2 (10-6 M, serosal), vasoactive intestinal peptide (10-7 M, serosal), 8-bromo-cAMP (10-4 M, serosal and mucosal), 8-bromo-cGMP (10-4 M, serosal and mucosal), phorbol myristate acetate (PMA; 10-5 M-10-6 M, serosal and mucosal), carbachol (10-5 M, serosal), and theophylline (10-2 M, serosal and mucosal).

Isotopic NaCl flux studies. All fluxes were conducted under short-circuit conditions (tissues clamped to 0 mV). To assess transmucosal Na+ and Cl- fluxes, 0.3 µCi/ml 22Na and 36Cl were added to the mucosal or serosal solutions of tissues paired according to their conductance (conductance within 25% of each other) (4, 5). After a 15-min equilibration period and before addition of treatments, standards were taken from the bathing reservoirs. A 30-min flux period was initiated at the same time as addition of treatments by taking samples from the bathing reservoirs opposite from the side of isotope addition. Samples were counted for 22Na and 36Cl in a liquid scintillation counter. The contribution of 22Na beta -counts to 36Cl beta -counts were determined and subtracted. Unidirectional Na+ and Cl- fluxes from mucosa to serosa (Jms) and serosa to mucosa (Jsm) and the net flux (Jnet) were determined using standard equations (4, 5).

[3H]FMLP and mannitol fluxes. These studies were performed in much the same way as NaCl fluxes, except that 0.2 µCi/ml [3H]FMLP or 0.2 µCi/ml [3H]mannitol (Sigma Chemical, St. Louis, MO) was placed on the mucosal surface of affected tissues 15 min after addition of treatments (PGE2 and/or genistein). Radiolabeled probes were diluted in 10-4 M FMLP and 10-2 M mannitol, respectively. Jms were calculated over a 1-h time period starting 30 min after the addition of treatments. Permeability was calculated from flux data using the following formula
Permeability (cm/s) 

= [(<IT>J</IT><SUB>ms</SUB>) × (1 h/3,600 s)] × [1/(M/1,000 cm<SUP>3</SUP>)]

Electron and light microscopy. Tissues were taken at 0, 15, 30, 60, 120, and 240 min for routine histological evaluation. Tissues were sectioned (5 µm) and stained with hematoxylin and eosin. For each tissue, three sections were evaluated. Four well-oriented villi were identified in each section. The length of the villus and the width at the midpoint of the villus were obtained using a light microscope with an ocular micrometer. In addition, the height of the epithelial-covered portion of each villus was measured. The surface area of the villus was calculated using the formula for the surface area of a cylinder. The formula was modified by subtracting the area of the base of the villus and multiplying by a factor accounting for the variable position at which each villus was cross-sectioned. In addition, the formula was modified by a factor that accounted for the hemispherical shape of the upper portion of the villus (4). The percentage of the villous surface area that remained denuded was calculated from the total surface area of the villus and the surface area of the villus covered by epithelium. The percent-denuded villous surface area was used as an index of epithelial restitution.

In experiments designed to assess epithelial ultrastructure under the influence of PGE2 and genistein, tissues were removed from Ussing chambers after 120 min (peak resistance) during three separate experiments (n = 3 for each treatment). Tissues were placed in Trump's 4F:1G fixative and prepared for transmission electron microscopy using standard techniques (10). For each tissue evaluated, five well-oriented interepithelial junctions were evaluated.

cAMP RIA. Tissues were removed from Ussing chambers once Isc had peaked in response to genistein and PGE2 treatment, and they were immediately frozen in liquid N2. Tissues were stored at -70°C before extraction and RIA. One part tissue (100 mg) was homogenized with nine parts 5% TCA. The homogenate was centrifuged at 2,500 g at 4°C for 15 min and extracted three times with 5 volumes of water-saturated ether. Excess ether was discarded after each extraction, and the samples were evaporated to dryness. RIA for cAMP was performed using a commercial kit according to the manufacturer's instructions (Biomedical Technologies, Stoughton, MA).

Data analysis. Data were reported as means ± SE. All data were analyzed using ANOVA for repeated measures, except where peak or single time-point responses were analyzed using a standard one-way ANOVA (Sigmastat, Jandel Scientific, San Rafael, CA). A Tukey's test was used to determine differences between treatments after the ANOVA, and P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Ischemia-injured tissues bathed in indomethacin (5 × 10-6 M) and treated with 16,16-dimethyl PGE2 (10-6 M) after a 30-min equilibration period showed marked elevations in transmucosal resistance (R) compared with tissues treated with indomethacin alone (Fig. 1A). Additional treatment after the 30-min equilibration period with the novel Cl- secretagogue genistein (10-4 M) augmented the R response to PGE2, whereas treatment with genistein and indomethacin in the absence of PGE2 was without effect (Fig. 1A). The response between PGE2 and genistein was significantly greater than the additive effect of each individually, indicating synergism between PGE2 and genistein. Evaluation of Isc, which is closely correlated with Cl- secretion in this tissue (5), also showed a synergistic response (Fig. 1B).



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Fig. 1.   Electrical responses of ischemia-injured tissues treated with 10-6 M 16,16-dimethyl PGE2 and 10-4 M genistein. A: serosal addition of PGE2 to ischemia-injured tissues after an initial 30-min equilibration period resulted in approximately twofold elevations in resistance (R), whereas treatment of tissues with mucosal and serosal genistein after the 30-min equilibration period had no apparent effect. However, addition of PGE2 and genistein triggered synergistic elevations in R. B: synergistic elevations in R were preceded by synergistic elevations in short-circuit current (Isc). Isc is associated with Cl- secretion in this tissue. C: recovery of R in presence of PGE2 was markedly reduced by basolateral Na+-K+-2Cl- inhibitor bumetanide (10-4 M, applied to serosal side of tissues at time 0), whereas recovery of R in presence of PGE2 and genistein was completely inhibited. D: bumetanide also inhibited elevations in Isc triggered by PGE2 and genistein. Values are means ± SE; n = 6. Significance of synergism between genistein and PGE2 was determined using two-way ANOVA on repeated measures (P < 0.05).

To determine whether the synergistic effects of PGE2 and genistein on Isc were attributable to Cl- secretion, unidirectional Na+ and Cl- fluxes were performed over a 30-min period commencing 30 min after the addition of PGE2 and genistein. These studies indicated that PGE2 and genistein had synergistic effects on net Cl- secretion (Table 1). To directly assess the role of elevated Isc in the recovery of R, ischemia-injured tissues were pretreated with the basolateral Na+-K+-2Cl- transport inhibitor bumetanide (10-4 M). Subsequent stimulation of tissues with PGE2 resulted in significantly reduced recovery of R (Fig. 1C) and complete inhibition of Isc (Fig. 1D) compared with tissues treated with PGE2 in the absence of bumetanide (Fig. 1, A and B). Furthermore, genistein/PGE2-stimulated recovery of R was completely abolished in the presence of bumetanide (Fig. 1C), although limited elevations in Isc were detected (Fig. 1D). These experiments suggested that Isc (as a measure of Cl- secretion) is correlated with recovery of R.

                              
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Table 1.   Unidirectional Na+ and Cl- fluxes across ischemia-injured porcine ileal mucosa treated with indomethacin genistein, PGE2, or genistein and PGE2

We have previously shown that PGE2-stimulated Cl- secretion is associated with closure of dilated interepithelial spaces rather than an effect on epithelial restitution (8). In the present studies, ~20% of the villous surface area was denuded by the ischemic episode, but the denuded area was reduced to ~10% within 30 min (Table 2). Tissues continued to restitute between 30 and 60 min in the presence indomethacin (resulting in ~4% denuded villous surface). The addition of genistein, PGE2, or both genistein and PGE2 to recovering tissues at 30 min had no significant effect on denuded villous surface area (Table 2). In addition, there were no apparent morphological differences on light microscopy between recovering tissues under the influence of the various treatments after 60 min of recovery time (Fig. 2). Epithelium at the tips of repairing villi appeared flattened regardless of treatment. However, evaluation of restituted epithelium with electron microscopy revealed dilatation of interepithelial spaces in tissues treated with indomethacin alone, whereas tissues treated with PGE2 or PGE2 and genistein had closely apposed interepithelial spaces (Fig. 3). As in previous studies (8), ~75% of the interepithelial spaces were dilated in indomethacin-treated tissues, whereas tissues additionally treated with PGE2 had ~10% of the interepithelial spaces dilated, and there was no apparent dilatation of interepithelial spaces in tissues treated with both PGE2 and genistein.

                              
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Table 2.   Morphometric assessment of epithelial restitution in ischemia-injured porcine ileal mucosa treated with indomethacin, genistein, PGE2, or genistein and PGE2



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Fig. 2.   Histological appearance of ischemia-injured porcine ileal mucosa. A: ischemia for 45 min resulted in lifting and sloughing of epithelium from tips of villi. B: after a 60-min recovery period in an Ussing chamber in presence of indomethacin (5 × 10-6 M), villi have contracted and epithelial restitution is nearly complete. C: treatment of tissues with PGE2 (10-6 M) had no observable effect on restitution in tissues recovered for 60 min. D: similarly, tissues treated with genistein (10-4 M) and PGE2 have restituted within 60 min. Bar = 100 µm.



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Fig. 3.   Representative electron micrographs of ischemia-injured tissues. Restituted epithelium treated with indomethacin (5 × 10-6 M) had evidence of dilated interepithelial spaces in 3 separate tissues (A1, A2, and A3), whereas repairing tissues treated with PGE2 (B) or PGE2 and genistein (C) had evidence of closely apposed interepithelial spaces. Bar in A1 = 8 µm (applies to A1, A2, and A3). Bar in B and C = 6 µm.

We have previously shown that PGE2 triggers increases in Isc and R via the intracellular second messenger cAMP (9). To explore the possibility that genistein's synergism with PGE2 was mediated via an interaction with cAMP, we assessed the effect of treating tissues with genistein and the cAMP agonist VIP (10-7 M) after a 30-min equilibration period. Similar to experiments using PGE2, tissues treated with genistein and VIP showed synergistic elevations in R and Isc, although these electrical responses were transient (Fig. 4). To expand on these results, we next determined the ability of genistein to synergize with a range of putative Cl- secretagogues that signal via distinct intracellular signaling pathways. Tissues were treated with genistein and one of the following: 10-4 M 8-bromo-cAMP, 10-4 M 8-bromo-cGMP, the protein kinase C stimulant PMA (10-6 M) (14), or the Ca2+-mediated agonist carbachol (10-5 M) (41). Tissues treated with 8-bromo-cGMP or 8-bromo-cAMP and genistein after a 30-min equilibration period showed similar synergistic elevations in R and Isc, whereas tissues treated with either carbachol or PMA alone or in the presence of genistein had little or no discernible effect on R or Isc (Fig. 4). To rule out a dose-dependent lack of response of tissues to PMA and carbachol, higher doses were used (10-5 M PMA and 10-4 M carbachol). However, these higher dosages produced no further effect on Isc either alone or in the presence of genistein. PMA at higher doses (10-5 M) tended to show reduced peaks in Isc (peak Isc in the presence of 10-5 M PMA and 10-4 M genistein, 2 ± 1.7 µA/cm2, n = 4) compared with lower doses of PMA (Fig. 5), possibly indicating near-toxic levels.


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Fig. 4.   Electrical responses of ischemia-injured tissues treated with vasoactive intestinal peptide (VIP) (10-7 M) and genistein (10-4 M). A: serosal addition of VIP to ischemia-injured tissues after an initial 30-min equilibration period stimulated approximately twofold elevations in R, whereas treatment of tissues with both VIP and genistein resulted in additional synergistic elevations in R. B: synergistic elevations in R in response to genistein and VIP were preceded by synergistic elevations in Isc. Values are means ± SE; n = 6. Significance of synergism between genistein and VIP was determined using two-way ANOVA on repeated measures (P < 0.05).



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Fig. 5.   Peak electrical responses of ischemia-injured tissues treated with genistein (10-4 M) and Cl- secretagogues cAMP (10-4 M), cGMP (10-4 M), phorbol myristate acetate (PMA; 10-6 M), and carbachol (10-5 M). A: serosal and mucosal addition of cAMP and cGMP after a 30-min equilibration period resulted in significant elevations in R. This response was significantly elevated by pretreatment of tissues with genistein. In contrast, PMA and carbachol addition after a 30-min equilibration period had no effect on R either in presence or absence of genistein. B: synergistic elevations in R in response to genistein and either cAMP or cGMP were preceded by synergistic elevations in Isc. In addition, PMA, but not carbachol, triggered significant elevations in Isc in presence of genistein. Values are means ± SE; n = 6. * P < 0.05 vs. indomethacin/genistein-treated tissues and dagger  P < 0.05 vs. indomethacin/Cl- secretagogue-treated tissues by one-way ANOVA.

Because genistein had the most dramatic effects in tissues treated with cAMP or cGMP, we questioned whether genistein was simply acting as a phosphodiesterase inhibitor as has been demonstrated in pituitary (30) and neural tissue (40). To assess the effect of phosphodiesterase inhibition on recovery of ischemia-injured porcine mucosa, tissues were treated after a 30-min equilibration period with theophylline (10-2 M) or theophylline and PGE2. In contrast to genistein, theophylline triggered marked elevations in Isc and R when administered alone. However, theophylline stimulated additive elevations in Isc and synergistic elevations in R when administered in combination with PGE2 (Fig. 6). These studies indicated that inhibition of phosphodiesterase amplified the effects of PGE2, presumably as a result of additive increases in intracellular cAMP levels. To explore the potential of genistein to mediate its action on mucosal recovery via this mechanism, we measured cAMP levels in ischemia-injured tissues during the peak Isc response (when the cAMP signal was presumed to be maximal). Treatment with PGE2 resulted in approximately twofold increases in cAMP levels compared with tissues treated with indomethacin alone (114.4 ± 7.7 vs. 47.8 ± 12.7 pmol/ml, respectively). Genistein tended to reduce rather than increase cAMP levels in PGE2-treated tissues (83.1 ± 13.6 pmol/ml), but there was no statistically significant effect of genistein on cAMP levels.


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Fig. 6.   Response of ischemia-injured porcine mucosa to inhibition of phosphodiesterase. A: mucosal and serosal addition of phosphodiesterase inhibitor theophylline (Theo) after a 30-min equilibration period resulted in elevations in R that were further heightened in presence of theophylline and PGE2. B: synergistic elevations in R in response to theophylline and PGE2 were preceded by additive elevations in Isc. Values are means ± SE; n = 6. Significance of synergism between theophylline and PGE2 was determined using two-way ANOVA on repeated measures (P < 0.05).

Although it appeared that the synergistic effect of PGE2 and genistein on recovery of R was attributable to increased Cl- secretion, it has recently been shown that genistein may stimulate recovery of barrier function via an action on tight junction regulatory proteins (13, 29, 36). To assess the potential role of genistein-mediated tyrosine kinase inhibition on recovery of R in our model, we pretreated tissues with varying doses (10-4 M-10-6 M) of the alternative tyrosine kinase inhibitor tyrphostin 47 (37). However, tyrphostin 47 did not augment the effects of PGE2 on recovery of R as genistein had done, and tyrphostin 47 had no effects of its own on ischemia-injured mucosa (data not shown). In further experiments, tissues were pretreated with the distinct tyrosine kinase inhibitor herbimycin A (10-6 M), but, similar to the results with tyrphostin 47, this agent had no effect on recovery of R in the presence of PGE2 (data not shown).

To assess the ability of PGE2 and genistein to enhance barrier function of ischemia-injured porcine ileum, we performed Jms of the small bacterial toxin [3H]-FMLP (5-6 Å, 180 Da). As predicted from measurements of R, application of PGE2 or genistein and PGE2 resulted in additive and significant reductions in Jms of FMLP compared with tissues treated with indomethacin alone. However, genistein had no effect in the absence of PGE2 (Fig. 7A). Although it was initially thought that FMLP traversed the epithelium solely by the paracellular route (35), more recent studies indicate that FMLP may be transported across the epithelium by the hPepT1 transporter (27). Therefore, we chose to further assess the effects of PGE2 and genistein on paracellular permeability by performing Jms of [3H]mannitol (Fig. 7B). Mannitol fluxes showed similar trends to those of FMLP fluxes, but significant reductions in mannitol flux were only noted in tissues treated with both genistein and PGE2 compared with tissues treated with indomethacin alone.


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Fig. 7.   Evaluation of effects of PGE2 and genistein on mucosal-to-serosal fluxes (Jms) of N-formyl-methionyl-leucyl phenylalanine (FMLP) and mannitol. Fluxes were commenced 30-min after addition of treatments and were conducted over a 1-h period. A: tissues in presence of indomethacin (5 × 10-6 M) had significantly greater Jms of FMLP over 1-h time period after treatment compared with control tissues. Addition of PGE2 and genistein resulted in incremental and significant decreases in transmucosal passage of FMLP. B: similar results were obtained for Jms of mannitol, except that reductions in Jms of mannitol were only observed after addition of both PGE2 and genistein. Values are means ± SE; n = 6 for Jms of FMLP and n = 8 for Jms of mannitol. * P < 0.05 vs. indomethacin and genistein/indomethacin-treated tissues.

To further assess the effect of PGE2 and genistein on paracellular permeability, we calculated permeability from flux data for both Na+ and mannitol using standard equations (see MATERIALS AND METHODS). We used Jsm of Na+ to assess permeability to passive movement of Na+ (rather than active transport). Ischemia-injured tissues treated with indomethacin were significantly more permeable to Na+ (PNa = 2.7 ± 0.07 × 10-5 cm/s) than mannitol (Pmannitol = 1.4 ± 0.1 × 10-5 cm/s, P < 0.001 vs. PNa), as might be expected on the basis of molecular size. Treatment with PGE2 and genistein resulted in significant reductions in PNa (2.0 ± 0.2 × 10-5 cm/s, P < 0.01 vs. PNa in tissues treated with indomethacin alone) and Pmannitol (1.0 ± 0.1 × 10-5 cm/s, P < 0.05 vs. Pmannitol in tissues treated with indomethacin alone). The degree of reduction in PNa (~26%) was similar to that of mannitol (~29%).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present studies indicate that PGE2, but not genistein alone, enhances recovery of barrier function in ischemia-injured porcine mucosa. However, the reparative action of PGE2 is heightened in the presence of genistein, as determined by synergistic elevations in transmucosal resistance and additive reductions in Jms of FMLP and mannitol. In previous studies we have shown that PGE2 stimulates recovery of barrier function by triggering closure of interepithelial spaces (8), a process that would be expected to increase paracellular but not transcellular resistance. We believe a similar process is responsible for amplified recovery of R in the present studies because PGE2 and genistein had no effect on epithelial restitution but resulted in closure of dilated interepithelial spaces evident in tissues treated with indomethacin alone. In addition, PGE2 and PGE2 in combination with genistein incrementally reduced Jms of FMLP, a molecule that at least in part traverses the epithelium via the paracellular pathway (35), and Jms of mannitol, a molecule that exclusively traverses the epithelium via the paracellular route (24). Pmannitol was reduced by 29% by treatment with PGE2 and genistein, which correlated well with a 26% reduction in PNa after the same treatment. In previous studies, significant correlations between Jsm of Na+ and Jms of mannitol (which were used in the present studies to calculate permeability) were indicative of selective remodeling of interepithelial tight junctions (24). Reductions in mannitol fluxes (Fig. 7) were not as dramatic as the corresponding elevations in transepithelial resistance (Fig. 1) in tissues treated with PGE2 and genistein. However, mannitol fluxes were begun at 60 min and continued until 120 min recovery time, whereas R peaked at 60 min but was declining at 120 min. Because peak R was transient, it was not possible to directly correlate mannitol fluxes with maximal changes in R in the presence of PGE2 and genistein.

The predominant cellular activity assigned to genistein is that of a tyrosine kinase inhibitor (37), and it has recently been determined that tight junction regulatory proteins are acted on by tyrosine kinases (1). Accordingly, one plausible theory regarding the mechanism by which genistein augmented recovery of R in the present study is inhibition of tight junction-associated tyrosine kinases. For example, treatment of Madin-Darby canine kidney cells with vanadate and H2O2 resulted in rapid increases in paracellular permeability associated with increased phosphotyrosine immunofluorescence in tight junction-associated proteins. The effect of vanadate/H2O2 was inhibited by genistein or tyrphostin 25, both of which inhibited tyrosine phosphorylation (13). Conversely, the tyrosine kinase inhibitors tyrphostin 47 and herbimycin A had no effect on recovery of R in ischemia-injured porcine tissues, suggesting that genistein's action on recovery of barrier function is not attributable to inhibition of tyrosine kinases. However, we did not directly assess the action of genistein on tyrosine kinases in this study.

Another action that has been attributed to genistein is that of a phosphodiesterase inhibitor (30, 40). However, the action of genistein differed from that of theophylline, a known phosphodiesterase inhibitor in porcine intestinal epithelium (3, 6, 7), in that theophylline triggered recovery of R by itself, whereas genistein had no noticeable effect on R by itself. Although both genistein and theophylline caused synergistic elevations in R, measurements of cAMP levels in tissues treated with PGE2 and genistein revealed no additive action of genistein on cAMP levels, indicating an effect of genistein distinct from inhibition of phosphodiesterase.

The synergistic response between PGE2 and genistein on recovery of barrier function was preceded by synergistic elevations in Isc and net Cl- secretion, consistent with our previous work that has shown that changes in epithelial Cl- transport trigger recovery of barrier function (8). However, the kinetic relationship between changes in Cl- secretion (as indicated by Isc) and changes in R in the present studies were not direct. For instance, Fig. 1 demonstrates marked increases in Isc immediately after addition of the treatment, whereas R is maximal ~1 h later. This might suggest that the physical structures signaled by changes in Isc and responsible for elevations in R are relatively slow to respond. Such a hypothesis is supported by studies in Necturus gallbladder, wherein addition of cAMP triggered maximal elevations in Isc within 5 min, whereas R was maximal by 30 min associated with changes in ultrastructural morphology of tight junctions (16). Additional disparities in Isc and R elevations in the present study included the fact that the magnitude of Isc did not correlate with the magnitude of elevations in R in tissues treated with PGE2 and theophylline, whereas elevations in Isc and R were closely correlated in tissues treated with PGE2 and genistein. This likely relates to differences in mechanisms of action between theophylline and genistein. For example, we have previously shown that a significant proportion of PGE2-stimulated elevations in R are associated with inhibition of electroneutral Na+-Cl- absorption, a process that would not reflect in elevations in Isc (8). Theophylline would be expected to amplify effects of PGE2 via the same signaling pathways because theopylline increases intracellular cAMP, the second messenger responsible for inhibition of Na+-Cl- absorption (4). Conversely, genistein had no demonstrable effects on cAMP levels in the present study but appeared to have direct effects on Isc and R in the presence of PGE2. The mechanism of genistein's action on Isc has not been conclusively determined (15, 18, 19, 22, 31, 37), although a recent study indicated that genistein augmented Isc via a direct interaction with activated CFTR (17).

The relative contribution of Cl- secretion and inhibition of Na+-Cl- absorption is highlighted by experiments using bumetanide, which only partially inhibited PGE2-stimulated elevations in R despite complete inhibition of Isc (Fig. 1C). The bumetanide-insensitive component of PGE2-stimulated R recovery has been shown in previous studies to be associated with inhibition of neutral Na+-Cl- absorption by the PGE2 second messenger cAMP (8). However, the effect of bumetanide on tissues treated with both PGE2 and genistein was puzzling, since the Na+-K+-2Cl- inhibitor fully inhibited elevations in R in these tissues, suggesting the lack of an important role for PGE2 inhibition of Na+-Cl- absorption in the presence of genistein. This may be explained by a recent study that indicates that neutral Na+-Cl- absorption is intimately associated with CFTR function (12), a function that may have been altered by genistein. We speculate that treatment with both PGE2 and genistein results in failure of PGE2 to inhibit Na+-Cl- absorption. Another puzzling aspect of the effects of bumetanide on tissues treated with PGE2 and genistein was the incomplete inhibition of Isc. This could not be explained by Cl- secretion, which is inhibited by bumetanide, or changes in neutral Na+-Cl- absorption, which contribute no net change in Isc. However, it is conceivable that tissues treated with PGE2 and genistein secreted HCO-3, which would not be inhibited by bumetanide. Electrogenic HCO-3 secretion has previously been demonstrated in porcine ileum (2).

In addition to net Cl- secretion in the presence of PGE2 and genistein, there was also evidence for net Na+ secretion (Table 1). Previous studies in porcine jejunum (32, 33) and rabbit ileum (28) have shown a role for the cAMP-signaling pathway in the stimulation of net Na+ secretion. Similarly in our studies, net Na+ secretion was noted primarily in response to the cAMP agonist PGE2 (addition of genistein resulted in no significant additive effect on net Na+ secretion). Na+ secretion in rabbit ileum and porcine jejunum is dependent on the presence of HCO-3 but not Cl-; therefore, a Na+-HCO-3 electrogenic transport mechanism has been postulated (28). Whether or not this transport mechanism exists in porcine ileum is not known (33, 34). However, net Na+ secretion is consistently present in porcine jejunum under a variety of conditions, including rotavirus infection (33) and porcine cryptosporidiosis (4).

In further studies, we showed that genistein increases Isc and enhances recovery of R in the presence of cGMP or cAMP-mediated agonists (including 8-bromo-cAMP and VIP), but there was no evidence of enhanced recovery of R in the presence of mediators that signal via protein kinase C (PMA) or intracellular Ca2+ (carbachol). Recent studies suggest that genistein has a direct action on CFTR by interacting with select nucleotide-binding sites. However, phosphorylation of CFTR by cyclic nucleotides is necessary for genistein to hyperactivate these Cl- channels (17). This may explain why genistein had little or no effect in the presence of carbachol, an intracellular Ca2+ agonist that mediates its action on Cl- secretion via basolateral K+ channels (41), or PMA, which may phosphorylate CFTR but at a distinct site from that of the cyclic nucleotides (14).

The mechanisms by which genistein-augmented Cl- secretion in the presence of either cAMP or cGMP agonists results in enhanced recovery of barrier function are unclear. Assuming that this reparative process is a paracellular phenomenon (based on the morphological appearance of the interepithelial spaces and a lack of an effect on epithelial restitution), there are two general possibilities: physical collapse of interepithelial spaces as ion-rich fluid is drawn out of the paracellular space or a transmucosal osmotic gradient developed during apical secretion of Cl- that signals closure of tight junctions (8). Madara (23) has previously concluded that a transmucosal osmotic gradient results in rapid alterations in tight junction structure, and we have also shown that placing an osmotic load on the mucosal surface of injured tissues simulates the action of Cl- secretagogues by inducing marked increases in R (8). However, other investigators have induced elevations in R in the presence of Cl- secretagogues and ascribed this action to collapse of lateral interepithelial spaces (20). Our previous finding that the cytoskeletal contractile agent cytochalasin D inhibits recovery of transmucosal resistance in the presence of PGE2 and PGI2 suggests that ischemia-injured tissues do not repair in the presence of open tight junctions (9). However, we have documented distinct morphological differences in the appearance of the interepithelial spaces in tissues treated with indomethacin, PGE2, and genistein compared with those treated with indomethacin alone. Therefore, we have concluded that closure of both tight junctions and interepithelial spaces likely contributes to recovery of mucosal barrier function.


    ACKNOWLEDGEMENTS

We thank Julia Vorobiov and the Immunotechnologies Core of the University of North Carolina Center for Gastrointestinal Biology and Disease (National Institutes of Health Center Grant DK-34987) for assistance with cAMP assays.


    FOOTNOTES

This work was supported by National Institutes of Health Grant DK-53284 and United States Department of Agriculture Grant 9802537 (both to A. T. Blikslager).

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: A. T. Blikslager, College of Veterinary Medicine, North Carolina State Univ., 4700 Hillsborough St., Raleigh, NC 27606 (E-mail: anthony_blikslager{at}ncsu.edu).

Received 2 April 1999; accepted in final form 7 October 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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