Identification and characterization of multiple corticotropin-releasing factor type 2 receptor isoforms in the rat esophagus

The rat esophagus shares some cellular features with skin squamous epithelium and striated muscle that express high levels of corticotropin- releasing factor type 2 (CRF₂) receptors or their cognate ligand urocortin (Ucn) 1, 2, and 3. We investigated the expression and cell signaling of CRF₂ receptors and ligands in the rat esophagus and lower esophageal sphincter (LES) by RT-PCR and quantitative PCR in normal and corticosterone-treated whole esophageal tissue, laser capture microdissected layers, and isolated esophageal cells. The expression of CRF₂ receptor protein and intracellular cAMP and ERK1/2 responses to CRF agonists and CRF₂ antagonist were determined in cultured esophageal cells and HEK-293 cells transfected with CRF_2b receptors. CRF₂ was abundantly expressed in the mucosa and longitudinal muscle layers of the esophagus and LES, whereas CRF₁ expression was scarce. CRF _2b wild-type transcript was predominantly expressed in the esophagus, and in addition, several new CRF₂ splice variants including six CRF_2a isoforms were identified. Expression of Ucn 1, Ucn 2, and to a smaller extent Ucn 3, but not CRF mRNA, was detected in the esophagus and LES. Ucn 1 and Ucn 2 stimulated dose-dependent cAMP production and ERK1/2 phosphorylation in the esophageal cells, whereas CRF and CRF₁ agonist, cortagine, had less potent effects. In addition, Ucn 2-stimulated cAMP and ERK responses were blocked by the CRF₂ antagonist, astressin ₂-B. These data established the presence of a prominent CRF ₂ signaling system in the esophagus and LES-encompassing multiple CRF₂ receptor variants and Ucn, suggesting a functional role in secretomotor activity and epithelial and muscle cell proliferation.