INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

16,16-DIMETHYL-PGE₂ (PGE₂) stimulates recovery of barrier function in porcine ischemia-injured ileal mucosa, but the PGE₂ receptor interactions involved in this model are unknown (3-5). PGE₂ may interact with at least four cell surface prostaglandin type E receptors (EP₁, EP₂, EP₃, and EP₄), which trigger a variety of intracellular responses depending on which G protein they are coupled to (12, 17). For example, PGE₂ stimulates production of cAMP via EP₂ and EP₄ receptors linked to G_s protein (6, 21) but inhibits production of cAMP via EP₃ receptors coupled to G_i protein (8, 19, 24). Furthermore, PGE₂ may increase intracellular Ca²⁺ via EP₁ (29) and EP₃ (16) receptors through G_q protein-phospholipase C interactions. From previous studies (5), we know that porcine mucosal tissue cAMP is elevated in response to PGE₂ and that the phosphodiesterase inhibitor theophylline heightens the effect of PGE₂ on recovery of transmucosal resistance (R). Although this data implicates a role for EP₂ or EP₄ receptors coupled to G_s protein, we do not know whether EP receptors that increase intracellular Ca²⁺ are also involved. Studies indicate a critical role for the EP₃ receptor in duodenal bicarbonate secretion in mice (26, 27) and for PGE₂-stimulated cytoprotection of gastric parietal cells (22). Work from other laboratories indicates that EP₁, EP₃, and EP₄ mRNA have all been detected in the mucosa or submucosa of the mouse (15) and rat (9), whereas EP₂ receptor mRNA was not detected in the gastrointestinal tract of rodents. However, EP₂ receptors have been detected in normal and inflamed human colonic mucosal epithelium (25).

In the present study, we sought to determine the nature of PGE₂-EP receptor interactions involved in PGE₂-stimulated recovery of R through a series of experiments using receptor-specific agonists and antagonists. Our data indicate that the response of ischemia-injured porcine ileal mucosa to PGE₂ involves an intriguing cross talk mechanism between EP receptors linked to the generation of cAMP and intracellular Ca²⁺, respectively.

	MATERIALS AND METHODS

TOP 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-wk-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 sodium (15 mg/kg iv) and maintained with intermittent infusion of pentobarbital sodium (6-8 mg · kg¹ · h¹). Pigs were placed on a heating pad and ventilated with 100% O₂ via a tracheotomy by 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 · kg¹ · h¹. 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 were 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 previously (1). 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 PD was short-circuited through Ag-AgCl electrodes using a voltage clamp that corrected for fluid resistance. R (in  · cm²) was calculated from the spontaneous PD and short-circuit current (I_sc). If the spontaneous PD was between 1 and 1 mV, tissues were current clamped at ±100 µA for 5 s and the PD was recorded. I_sc and PD were recorded every 15 min for 4 h.

Experimental treatments. Tissues were bathed in Ringer containing 5 µM indomethacin to prevent PG production while 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. Other treatments that were added before baseline electrical measurements were SC-19220 (Cayman Chemical), AH-6809 (Biomol), pertussis toxin, or TTX (Sigma Chemical, St. Louis, MO). 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 PGE₂, A-23187, thapsigargin (Sigma Chemical), sulprostone, 11-deoxy-PGE₁, misoprostol, or 11-deoxy-16,16-dimethyl-PGE₂ (Cayman Chemical).

cAMP RIA. Tissues were removed from Ussing chambers once I_sc peaked in response to receptor agonists and 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 vol 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 are reported as means ± SE. All data were analyzed using an ANOVA for repeated measures except when the peak response was analyzed using a standard one-way ANOVA or paired t-test (Sigmastat, Jandel Scientific, San Rafael, CA). Tukey's test was used to determine differences between treatments after 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 µM) and treated with the EP₁, EP₂, EP₃, and EP₄ agonist PGE₂ (1 µM) after a 30-min equilibration period showed marked elevations in R compared with tissues treated with indomethacin alone (Fig. 1A). However, neither the EP₂ and EP₄ agonist deoxy-PGE₁ (1 µM) nor the EP₁ and EP₃ agonist sulprostone (1 µM) had any effect on R, whereas a combination of deoxy-PGE₁ and sulprostone stimulated synergistic elevations in R similar in magnitude to that of PGE₂ (Fig. 1A). Because we have previously shown that recovery of R is preceded by increases in I_sc (3, 5), we also evaluated I_sc data for the presence of similar trends (Fig. 1B). Accordingly, there was no effect of either deoxy-PGE₁ or sulprostone on I_sc, but a combination of the two agents triggered elevations in I_sc similar in magnitude to that of PGE₂.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Electrical responses of ischemia-injured tissues treated with various prostanoid type E (EP) receptor agonists. A: serosal addition of the EP1, EP2, EP3, and EP4 receptor agonist 16,16-dimethyl-PGE2 (PGE2; 1 µM) to ischemia-injured tissues after an initial 30-min equilibration period resulted in ~2-fold elevations in transmucosal resistance (R), whereas treatment of tissues with the EP2 and EP4 receptor agonist deoxy-PGE1 (1 µM) or the EP1 and EP3 receptor agonist sulprostone (1 µM) had no apparent effect. However, addition of deoxy-PGE1 and sulprostone stimulated synergistic elevations in R. B: elevations in R after treatment with PGE2 or deoxy-PGE1 + sulprostone were preceded by elevations in short-circuit current (Isc). Values are means ± SE; n = 6. The significance of the synergism between deoxy-PGE1 and sulprostone was determined using 2-way ANOVA on repeated measures (P < 0.05).

Because we have previously demonstrated that elevations in R in response to PGE₂ were correlated with elevations in tissue cAMP (5), we next measured cAMP levels in response to various prostanoids. Tissues were taken for measurement of cAMP immediately after peak I_sc response. In ischemia-injured tissues treated with PGE₂, cAMP was elevated approximately twofold compared with tissues treated with indomethacin alone (Fig. 2). Although neither sulprostone nor deoxy-PGE₁ triggered elevations in cAMP above that of indomethacin-treated tissues, a combination of sulprostone and deoxy-PGE₁ elevated cAMP approximately twofold, similar to the effect of PGE₂. These data suggested cross talk between EP receptors wherein stimulation of EP₂ or EP₄ G_s-linked receptors with deoxy-PGE₁ was insufficient to trigger a rise in cAMP, but concurrent stimulation of EP₁ or EP₃ receptors resulted in elevations in cAMP.

View larger version (35K): [in this window] [in a new window]   Fig. 2.   Tissue homogenate cAMP content in tissues treated with various EP receptor agonists. Tissues were removed from Ussing chambers after peak Isc. Tissues treated with PGE2 (1 µM) or deoxy-PGE1 + sulprostone (1 µM) had significant elevations in cAMP compared with tissues treated with indomethacin alone or tissues additionally treated with sulprostone or deoxy-PGE1. cAMP elevations correlated with electrical data in that only treatments that triggered significant elevations in cAMP had elevated Isc and R. Values are means ± SE; n = 6. * P < 0.05 vs. tissues treated with indomethacin, indomethacin + deoxy-PGE1, or indomethacin + sulprostone (1-way ANOVA).

EP₁ receptors are coupled to G_q protein that results in increases in intracellular Ca²⁺ via activation of phospholipase C (11), whereas EP₃-G protein interactions depend on COOH terminal splice variation of EP₃ transcripts. There are at least four EP₃ splice variants: EP_3A coupled to G_o/G_i protein, EP_3B and EP_3C coupled to G_s protein, and EP_3D coupled to G_q protein (16). To exclude a role for EP₁ receptors in the response of tissues to PGE₂ or deoxy-PGE₁ plus sulprostone, tissues were pretreated with the EP₁ receptor antagonists SC-19220 or AH-6809. However, these antagonists had no effect on either PGE₂ (Table 1) or sulprostone plus deoxy-PGE₁-triggered elevations (data not shown) in R or I_sc at doses of 1-100 µM. This agreed with other studies (18) indicating that sulprostone has a preferential action on EP₃ receptors.

We next focused on which of the EP₃ receptor splice variants was involved in the response of tissues to PGE₂. Because G_o and G_i proteins have an inhibitory action on neuronal Ca²⁺ channels and adenylyl cyclase, respectively (10), it seemed unlikely that EP_3A would be responsible for the stimulatory action of prostanoids on R and I_sc in ischemia-injured ileum. To further exclude a role for EP_3A receptors, ischemia-injured tissues were pretreated for up to 60 min with 125 mg/ml pertussis toxin (to which G_o and G_i proteins are sensitive) and subsequently treated with PGE₂ or sulprostone plus deoxy-PGE₁. However, pertussis toxin had no effect on measurements of R and I_sc (Table 1). Because G_o protein is largely expressed in neuronal tissue (10), we also pretreated tissues with TTX (0.1 µM), but this neuronal inhibitor had no effect on subsequent treatment of ischemia-injured tissues with PGE₂ (Table 1) or sulprostone plus deoxy-PGE₁ (data not shown).

Of the remaining splice variants of EP₃, EP_3D linked to G_q protein, with associated elevations in intracellular Ca²⁺, was considered the most likely candidate for a cross talk response with EP₂- or EP₄-linked G_s protein. Therefore, tissues were treated with deoxy-PGE₁ and the calcium ionophore A-23187 (0.1 µM) to stimulate increases in Ca²⁺ independent of receptor interactions. Treatment of tissues with A-23187 stimulated increases in R compared with tissues treated with indomethacin alone (Fig. 3A). However, treatment of tissues with deoxy-PGE₁ and A-23187 triggered synergistic elevations in R, and evaluation of I_sc revealed similar trends (Fig. 3B). In addition, tissue cAMP measurements indicated that A-23187 in combination with deoxy-PGE₁ stimulated significant elevations in cAMP, whereas neither A-23187 nor deoxy-PGE₁ had any effect when given alone (Fig. 4). Although these results were suggestive of a role for intracellular Ca²⁺ in the cross talk response between EP receptors, A-23187 can also elevate phospholipid second messengers via stimulation of phospholipase C (13). Therefore, we next used thapsigargin, which has been shown to elevate intracellular Ca²⁺ from intracellular and extracellular Ca²⁺ sources without stimulating other second messenger signaling mechanisms (13). Tissues were treated with combinations of thapsigargin (0.1 µM) and deoxy-PGE₁ (1 µM) that, similar to experiments with A-23187 and deoxy-PGE₁, induced synergistic elevations in R and I_sc (Fig. 5). Thus elevations in intracellular Ca²⁺ appeared critical to the cross talk response with G_s-linked EP receptors.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Electrical responses of tissues treated with the calcium ionophore A-23187 and deoxy-PGE1. A: tissues treated with A-23187 (0.1 µM) had transient elevations in R, whereas tissues treated with deoxy-PGE1 (1 µM) did not differ in R from tissues treated with indomethacin alone. However, tissues treated with both A-23187 and deoxy-PGE1 had synergistic elevations in R, suggesting a requirement for elevated Ca2+ in the response of tissues to prostanoid agonists. B: similar trends were noted in the Isc responses of tissues to A-23187 and deoxy-PGE1. Values are means ± SE; n = 6. The significance of the synergistic response between A-23187 and deoxy-PGE1 was determined using 2-way ANOVA on repeated measures (P < 0.05).

View larger version (35K): [in this window] [in a new window]   Fig. 4.   Tissue homogenate cAMP content in tissues treated with A-232187 and/or deoxy-PGE1. Tissues were removed from Ussing chambers after peak Isc. Tissues treated with A-23187 (1 µM) and deoxy-PGE1 (1 µM) had significant elevations in cAMP compared with tissues treated with indomethacin + A-23187 or indomethacin + deoxy-PGE1. These data suggest that the EP2 and EP4 receptor agonist deoxy-PGE1 only triggers elevations in cAMP in the presence of elevated intracellular Ca2+. Values are means ± SE; n = 6. * P < 0.05 vs. tissues treated with indomethacin + A-23187 or indomethacin + deoxy-PGE1 (paired t-test).

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Electrical responses of tissues treated with thapsigargin, which elevates intracellular Ca2+, and deoxy-PGE1. A: synergistic elevations in R in tissues treated with thapsigargin (0.1 µM) and deoxy-PGE1 (1 µM) were noted that were qualitatively similar to those of tissues treated with A-23187 and deoxy-PGE1, indicating that an increase in intracellular Ca2+ is required for the full tissue response to E-type prostanoids. B: synergistic elevations in Isc were also noted in tissues treated with thapsigargin and deoxy-PGE1. Values are means ± SE; n = 6. The significance of the synergistic response between thapsigargin and deoxy-PGE1 was determined using 2-way ANOVA on repeated measures (P < 0.05).

In additional experiments, we sought to determine whether the activity of PGE₂ could be accounted for by solely by activation of EP₂ and EP₃ receptors. The tissue R and I_sc response to PGE₂ (1 µm) was compared with that of the EP₂ and EP₃ receptor agonists misoprostol (1 µM) or 11-deoxy-16,16-dimethyl-PGE₂ (1 µM) (27). The tissue R and I_sc responses to these agonists were similar in magnitude to those of PGE₂ (Fig. 6). Together, our data indicate that EP₂ and EP₃ receptors were the most likely receptors involved in the response of ischemia-injured porcine ileum to PGE₂ (Table 2).

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Electrical responses of tissues treated with the EP2 and EP3 agonists misoprostil and 11-deoxy-16,16-dimethyl PGE2. A: elevations in R similar to those of PGE2 (1 µM) were noted in tissues treated with misoprostil (1 µM) or 11-deoxy-16,16-dimethyl PGE2 (1 µM), suggesting that the action of PGE2 could be reproduced by agonists that activate only EP2 and EP3 receptors. B: similar elevations in Isc were also noted in tissues treated with misoprostil and 11-deoxy-16,16-dimethyl PGE2. Values are means ± SE; n = 6. The significance of the elevations in R and Isc noted in tissues treated with PGE2, misoprostil, or 11-deoxy-16,16-dimethyl PGE2 was determined using 2-way ANOVA on repeated measures (P < 0.05).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In previous studies (3, 4), we postulated that PGE₂ triggers recovery of R of ischemia-injured porcine ileum by stimulating closure of interepithelial spaces via cAMP. For example, PGE₂ stimulated increases in tissue cAMP, PGE₂-induced recovery of R was heightened by the phosphodiesterase inhibitor theophylline, and addition of cAMP to ischemic-injured tissues simulated the action of PGE₂ (4, 5). The present studies confirm an important role for cAMP in PGE₂-stimulated increases in R but indicate that EP receptor cross talk mechanisms are required to initiate generation of cAMP at prostanoid doses of 1 µm. This premise is based on the fact that deoxy-PGE₁, an agonist that interacts with the G_s protein-linked EP₂ and EP₄ receptors, was without effect on R or tissue cAMP levels unless it was applied to ischemia-injured tissue together with an agent that induces increases in intracellular Ca²⁺. Such agents included the Ca²⁺ ionophore A-23187 and thapsigargin. Candidate EP receptors that also trigger increases in intracellular Ca²⁺ were EP₁ and EP_3D receptors (16, 29), of which the EP_3D receptor appeared to be the most likely candidate, because EP₁ receptor antagonists failed to inhibit the action of PGE₂. In addition, two agonists that do not purportedly act on EP₁ receptors (the EP₂/EP₃ receptor agonists misoprostol and 11-deoxy-16,16-dimethyl PGE₂; Ref. 20) simulated the action of PGE₂ (27). Furthermore, the fact that an EP₂ and EP₃ agonist simulated the action of PGE₂ suggested that, of the G_s protein-linked EP receptors, EP₂ was most likely involved in the response of ischemia-injured tissue to PGE₂.

Although specific EP receptors have been implicated in various physiological responses of the gut to PGE₂ (2, 26), we are not aware of reports implicating cross talk between EP receptors. However, there are reports of cross talk between G_q and G_s protein-linked receptors in other tissues that we believe are relevant to the present study (23). For example, in cardiac fibroblasts, muscarinic agonists that signal increases in intracellular Ca²⁺ via G_q protein potentiated elevations in cAMP stimulated by ₂-agonists, which signal via G_s protein (14). Mechanisms proposed to account for G_q and G_s protein cross talk included stimulation of Ca²⁺/calmodulin sensitive adenylyl cyclase and release of -subunits, which act on adenylyl cyclase, by activated G_q protein (14). Although measurements of tissue cAMP in the present study allowed us to define prostanoid interactions required to activate adenylyl cyclase, the precise nature of intracellular Ca²⁺ signals could not be determined because of the complexity of native mucosa. However, based on experiments with A-23187 and thapsigargin, it appears that increased intracellular Ca²⁺ (and not activation of G_q protein per se) is required to activate adenylyl cyclase. Therefore, there may be other mediators triggered by intracellular Ca²⁺ that subsequently activate adenylyl cyclase. For example, increased intracellular Ca²⁺ may activate protein kinase C, which may in turn act on adenylyl cyclase (14).

One other mechanism that should be considered in the development of cross talk between intracellular Ca²⁺ and G_s protein-mediated agonists is the synergistic effect of Ca²⁺ and cAMP on intestinal epithelial Cl secretion. Such synergism has been attributed to Ca²⁺-induced opening of basolateral K⁺ channels, which increases the electromotive driving force for secretion of Cl, and cAMP-induced opening of apical Cl channels (7, 28). This may be relevant to the current study, because elevations in R were consistently preceded by elevations in I_sc, which we (3) have previously attributed to Cl secretion. However, the synergism documented in the present study appears to relate to the necessity for increases in intracellular Ca²⁺ to induce elevations in cAMP, rather than an interaction between Ca²⁺ and cAMP. The transient nature of elevations in R in the presence of deoxy-PGE₁ and A-23187 or thapsigargin was somewhat puzzling considering that PGE₂, which we presume also elevates intracellular Ca²⁺ via EP₃ receptors, had a more prolonged effect. One possible explanation is that A-23187 and thapsigargin induce only transient elevations in intracellular Ca²⁺. Such a transient response to A-23187 has been documented (7) in other intestinal tissues.

Having defined some of the complexities of PGE₂ receptor signaling in ischemia-injured porcine ileum, the clinical relevance of these findings may be considered. In general, we have previously shown (3) that PGE₂-induced elevations in R result in enhanced recovery of intestinal barrier function, based on mucosal-to-serosal fluxes of macromolecules such as mannitol and morphological evidence of "tightening" of interepithelial spaces. Although the current study confirms that a range of prostanoid agonists are capable of triggering increases in R, it appears that agents that selectively interact with EP₂ and EP₃ receptors are capable of reproducing the action of PGE₂, thereby lessening potential side effects of universal activation of all EP receptors.