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

ABSTRACT: 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 CRF2b receptors. CRF2 was abundantly expressed in the mucosa and longitudinal muscle layers of the esophagus and LES, whereas CRF1 expression was scarce. CRF2b wild-type transcript was predominantly expressed in the esophagus, and in addition, several new CRF2 splice variants including six CRF2a 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, astressin2-B. These data established the presence of a prominent CRF2 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.

SUBMITTER: Wu SV

PROVIDER: S-EPMC8103778 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Corticotropin-releasing factor (CRF) orchestrates our body's response to stressful stimuli. Pain is often stressful and counterbalanced by activation of CRF receptors along the nociceptive pathway, although the involvement of the CRF receptor subtypes 1 and/or 2 (CRF-R1 and CRF-R2, respectively) in CRF-induced analgesia remains controversial. Thus, the aim of the present study was to examine CRF-R1 and CRF-R2 expression within the spinal cord of rats with Freund's complete adjuvant-induced unilateral inflammation of the hind paw using reverse transcriptase polymerase chain reaction, Western blot, radioligand binding, and immunofluorescence confocal analysis. Moreover, the antinociceptive effects of intrathecal (i.t.) CRF were measured by paw pressure algesiometer and their possible antagonism by selective antagonists for CRF-R1 and/or CRF-R2 as well as for opioid receptors. Our results demonstrated a preference for the expression of CRF-R2 over CRF-R1 mRNA, protein, binding sites and immunoreactivity in the dorsal horn of the rat spinal cord. Consistently, CRF as well as CRF-R2 agonists elicited potent dose-dependent antinociceptive effects which were antagonized by the i.t. CRF-R2 selective antagonist K41498, but not by the CRF-R1 selective antagonist NBI35965. In addition, i.t. applied opioid antagonist naloxone dose-dependently abolished the i.t. CRF- as well as CRF-R2 agonist-elicited inhibition of somatic pain. Importantly, double immunofluorescence confocal microscopy of the spinal dorsal horn showed CRF-R2 on enkephalin (ENK)-containing inhibitory interneurons in close opposition of incoming mu-opioid receptor-immunoreactive nociceptive neurons. CRF-R2 was, however, not seen on pre- or on postsynaptic sensory neurons of the spinal cord. Taken together, these findings suggest that i.t. CRF or CRF-R2 agonists inhibit somatic inflammatory pain predominantly through CRF-R2 receptors located on spinal enkephalinergic inhibitory interneurons which finally results in endogenous opioid-mediated pain inhibition.

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

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