RESUMEN
Chronic visceral pain in patients with irritable bowel syndrome (IBS) has been difficult to treat effectively partially because its pathophysiology is not fully understood. Recent studies show that norepinephrine (NE) plays an important role in the development of visceral hypersensitivity. In this study, we designed to investigate the role of adrenergic signaling in visceral hypersensitivity induced by heterotypical intermittent stress (HIS). Abdominal withdrawal reflex scores (AWRs) used as visceral sensitivity were determined by measuring the visceromoter responses to colorectal distension. Colon-specific dorsal root ganglia neurons (DRGs) were labeled by injection of DiI into the colon wall and were acutely dissociated for whole-cell patch-clamp recordings. Blood plasma level of NE was measured using radioimmunoassay kits. The expression of ß2-adrenoceptors was measured by western blotting. We showed that HIS-induced visceral hypersensitivity was attenuated by systemic administration of a ß-adrenoceptor antagonist propranolol, in a dose-dependent manner, but not by a α-adrenoceptor antagonist phentolamine. Using specific ß-adrenoceptor antagonists, HIS-induced visceral hypersensitivity was alleviated by ß2 adrenoceptor antagonist but not by ß1- or ß3-adrenoceptor antagonist. Administration of a selective ß2-adrenoceptor antagonist also normalized hyperexcitability of colon-innervating DRG neurons of HIS rats. Furthermore, administration of ß-adrenoceptor antagonist suppressed sustained potassium current density (IK) without any alteration of fast-inactivating potassium current density (IA). Conversely, administration of NE enhanced the neuronal excitability and produced visceral hypersensitivity in healthy control rats, and blocked by ß2-adrenoceptor antagonists. In addition, HIS significantly enhanced the NE concentration in the blood plasma but did not change the expression of ß2-adrenoceptor in DRGs and the muscularis externa of the colon. The present study might provide a potential molecular target for therapy of visceral hypersensitivity in patents with IBS.
Asunto(s)
Síndrome del Colon Irritable/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Estrés Fisiológico , Dolor Visceral/metabolismo , Animales , Butoxamina/química , Colon/metabolismo , Relación Dosis-Respuesta a Droga , Ganglios Espinales/metabolismo , Síndrome del Colon Irritable/fisiopatología , Masculino , Neuronas/metabolismo , Norepinefrina/sangre , Técnicas de Placa-Clamp , Fentolamina/química , Propranolol/química , Ratas , Ratas Sprague-Dawley , Transducción de SeñalRESUMEN
Recently we predicted the 3D structure of the human beta2-adrenergic receptor (beta2AR) and of the binding site of several agonists and antagonists to beta2AR. These predictions (MembStruk and HierDock) included no explicit water and only a few lipid molecules. Here we include explicit H(2)O and an infinite lipid bilayer membrane in molecular dynamics (MD) simulations of three systems: apo-beta2AR, epinephrine-bound beta2AR, and butoxamine-bound beta2AR (epinephrine is an endogenous agonist, and butoxamine is a beta2AR selective antagonist). The predicted structures for apo-beta2AR and butoxamine-beta2AR are stable in MD, but in epinephrine-beta2AR, extracellular water trickles into the binding pocket to mediate hydrogen bonding between the catechol of epinephrine and Ser-204 on helix 5. The epinephrine-beta2AR structure shows dynamic flexibility with small, piston-like movements of helices 3 and 6 and transient interhelical hydrogen bonding between Ser-165 on transmembrane 4 and Ser-207 on transmembrane 5. These couplings and motions may play a role in protein activation. The apo-beta2AR shows less dynamic flexibility, whereas the antagonist-beta2AR structure is quite rigid. This MD validation of the structure predictions for G protein-coupled receptors in explicit lipid and water suggests that these methods can be trusted for studying the mechanism of activation and the design of subtype-specific agonists and antagonists.