Synergistic signaling via cAMP and CA 2+1-coupled EP receptors is required for PGE 2-triggered recovery of intestinal barrier function

Abstract

We have recently shown that PGI2 and PG~ have a synergistic role in rescuing epithelial barrier function in porcine ischemic-injured ileum. While it appears that PGI2 signals via cholinergic nerve-induced increases in Ca2+ ;, and PGE2 signals via receptor-mediated release of cAMP, the precise nature of PGE2 receptor interactions that mediate recovery of mucosal barrier function is not known. PGE2 may induce increases in cAMP via EP2 or EP4 receptors and increases in Ca2+; via EP I receptors. However, PGErEP3 receptor signaling pathways are more complex, and may result in increases or decreases in cAMP or increases in Ca2+i depending upon the splice variant of the EP3 receptor present. The objective of the present study was to define the PGE2 signaling pathways involved in recovery of mucosal barrier function. Porcine ileum was subjected to 45-minutes of complete ischemia in vivo, after which the mucosa was stripped and mounted in Ussing chambers. Transepithelial resistance (R) was serially monitored over a 4-hour period as an indicator of mucosal recovery. All tissues were pre-treated with indomethacin (5 x 1O-6M). Ischemia resulted in 50% reductions in R (26::':2.70ecm2) compared to control tissues (51::':3.20ecm2). Treatment with the EP,IEP2IEPi EP4 agonist 16,16-dimethyl PG~ (PGE2, 1O6M) triggered 2-fold elevations in R (51::':4.20ecm2)within I hour, whereas the EP2IEP4 agonist deoxyPGE, (10-6M) or the EP,IEP3 agonist sulprostone (10-6M) had no effect. However, a combination of deoxyPGE, and sulprostone simulated the action of PGE2.cAMP was significantly elevated in tissues treated with PGE2 (23.2::':2.2 vs. 14.9::':2.2 pmol/ml for control) and deoxyPGErf sulprostone (49.1::':4.2 pmol/ml), but not with deoxyPGE, or sulprostone alone. Because of the potential for EP, and EP3 receptors to trig~er elevations in Ca2+;, we applied deoxyPGE, in combination with the Ca + ionophore A23187. Treatment with A23187 (1Oand deoxyl'Gfi, resulted in elevations in R similar in magnitude to those of PGE 2 (55::':6.40ecm2), whereas treatment with A23187 alone triggered significant but less marked elev~tions in R (45::':4.60ecm.j,. Pre-treatment with the EP I receptor antagonists SC-19220 (106 10 M) or AH-6809(l0sM)had no effect on PGE2 or sulprostone/deoxyPGE,-induced elevations in R. These studies suggest PGE2 signals recovery of R in ischemic-injured porcine ileum via complex si~a1ing pathways requiring both cAMP (via EP2 or EP4 receptors) and Ca +, (most likely via EP3 receptors).