T1675 EGF Chronically Upregulates Colonic Epithelial Cl − Secretion By Mechanisms Involving PI3-K, ERK and p38 MAPK and Increases in Transport Protein Expression

Abstract

Background: Cholinergic activation of epithelial muscarinic M3 receptors (M3R) rapidly promotes colonic Cland fluid secretion. However, whether M3R activation has long-term consequences for epithelial secretory capacity is unknown. Aim: To investigate long-term effects of acute M3R activation on colonic epithelial secretory function.Methods: Clsecretion was measured as changes in short-circuit-current (Isc) across voltage-clamped T84 cells grown on permeable supports. M3R expression was assessed by immunoblotting. Results: As expected, the cholinergic M3R agonist, carbachol (CCh, 100 μM), rapidly stimulated Clsecretion across T84 cells (120 ± 8 μA/cm2; n = 39) with maximal responses occuring within 2 min. To determine long-term effects of M3R activation, cells were pretreated with CCh (100 μM for 15 min) and subsequent Isc responses to the agonist were measured 6 and 24 hrs later. Responses to CCh 6hrs after initial challenge with the agonist were unchanged (109 ± 15%; n = 4), while those measured 24 hrs later were reduced to 47 ± 4% (p < 0.001, n = 39) of those in control cells. CCh pretreatment did not chronically alter Isc responses to other secretagogues, including forskolin and thapsigargin. Treatment of T84 cells with conditionedmedium fromCCh-pretreated cells for 24 hrs also reduced Isc responses to the agonist to 51 ± 9% of those in control cells (n = 6, p < 0.001), implying a soluble factor is involved. As we have previously shown an important role for Src-dependent EGFr transactivation in acutely regulating cholinergic-induced Clsecretion, we examined if this pathway also mediates chronic downregulation of secretion upon agonist exposure. However, an EGFR inhibitor, AG1478 (100 nM), a metalloprotease inhibitor, GM6001 (10 μM), and a Src inhibitor, PP2 (20 μM), were all without effect in restoring responses to CCh 24 hrs after initial agonist challenge. Finally, CCh-pretreatment did not alter total cellular or basolateral surface expression of theM3R after 24 hrs.Conclusion: AcuteM3R activation chronically, and specifically, inhibits the capacity of colonic epithelial cells to elicit secretory responses upon repeated agonist exposure. This effect is slow in onset but is not due to receptor degradation or downregulation of cell surface expression implying a specific uncoupling of M3Rs to downstream effectors. The mechanism involved is independent of the EGFr-dependent antisecretory pathway we have previously described but involves release of an epithelialderived factor. We propose this novel antisecretory mechanism may function to prevent excessive colonic fluid loss upon repeated cholinergic stimulation of epithelial cells.