RESUMEN
Morphine differs from most opiates its poor ability to internalize µ opioid receptors (µORs). However, chronic treatment with morphine produces adaptational changes at the dynamin level, which enhance the efficiency of acute morphine stimulation to promote µOR internalization in enteric neurons. This study tested the effect of chronic treatment with fentanyl, a µOR-internalizing agonist, on ligand-induced endocytosis and the expression of the intracellular trafficking proteins, dynamin and ß-arrestin, in enteric neurons using organotypic cultures of the guinea pig ileum. In enteric neurons from guinea pigs chronically treated with fentanyl, µOR immunoreactivity was predominantly at the cell surface after acute exposure to morphine with a low level of µOR translocation, slightly higher than in neurons from naïve animals. This internalization was not due to morphine's direct effect, because it was also observed in neurons exposed to medium alone. By contrast, D-Ala2-N-Me-Phe4-Gly-ol5-enkephalin (DAMGO), a potent µOR-internalizing agonist, induced pronounced and rapid µOR endocytosis in enteric neurons from animals chronically treated with fentanyl or from naïve animals. Chronic fentanyl treatment did not alter dynamin or ß-arrestin expression. These findings indicate that prolonged activation of µORs with an internalizing agonist such as fentanyl does not enhance the ability of acute morphine to trigger µOR endocytosis or induce changes in intracellular trafficking proteins, as observed with prolonged activation of µORs with a poorly internalizing agonist such as morphine. Cellular adaptations induced by chronic opiate treatment might be ligand dependent and vary with the agonist efficiency to induce receptor internalization.
Asunto(s)
Analgésicos Opioides/farmacología , Sistema Nervioso Entérico/efectos de los fármacos , Fentanilo/farmacología , Neuronas/efectos de los fármacos , Receptores Opioides mu/metabolismo , Animales , Endocitosis/efectos de los fármacos , Sistema Nervioso Entérico/metabolismo , Cobayas , Íleon/efectos de los fármacos , Íleon/metabolismo , Inmunohistoquímica , Ligandos , Masculino , Morfina/farmacología , Neuronas/metabolismo , Técnicas de Cultivo de Órganos , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiologíaRESUMEN
Intestinal ischemia is a clinical emergency with high morbidity and mortality. We investigated whether activation of µ opioid receptor (µOR) protects from the inflammation induced by intestinal ischemia and reperfusion (I/R) in mice. Ischemia was induced by occlusion of the superior mesenteric artery (45 min), followed by reperfusion (5 hr). Sham-operated (SO) and normal (N) mice served as controls. Each group received subcutaneously 1) saline solution, 2) the µOR selective agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO; 0.01 mg kg(-1) ), 3) DAMGO and the selective µOR antagonist [H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2] (CTAP; 0.1 mg kg(-1) ), or 4) CTAP alone. I/R induced intestinal inflammation as indicated by histological damage and the significant increase in myeloperoxidase (MPO) activity, an index of tissue neutrophil accumulation. Tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) mRNA levels were also increased in I/R mice compared with SO. DAMGO significantly reduced tissue damage, MPO activity, and TNF-α mRNA levels in I/R, and these effects were reversed by CTAP. By contrast, DAMGO did not modify IL-10 mRNA levels or gastrointestinal transit. DAMGO's effects are receptor mediated and likely are due to activation of peripheral µORs, because it does not readily cross the blood-brain barrier. These findings suggest that activation of peripheral µOR protects from the inflammatory response induced by I/R through a pathway involving the proinflammatory cytokine TNF-α. Reduction of acute inflammation might prevent I/R complications, including motility impairment, which develop at a later stage of reperfusion and likely are due to inflammatory cell infiltrates.
Asunto(s)
Inflamación/metabolismo , Mucosa Intestinal/metabolismo , Receptores Opioides mu/metabolismo , Daño por Reperfusión/metabolismo , Animales , Citocinas/biosíntesis , Femenino , Inflamación/etiología , Inflamación/patología , Intestinos/irrigación sanguínea , Intestinos/patología , Mesenterio/irrigación sanguínea , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa , Daño por Reperfusión/complicaciones , Daño por Reperfusión/patología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND & AIMS: The µ opioid receptor (µOR) undergoes rapid endocytosis after acute stimulation with opioids and most opiates, but not with morphine. We investigated whether prolonged activation of µOR affects morphine's ability to induce receptor endocytosis in enteric neurons. METHODS: We compared the effects of morphine, a poor µOR-internalizing opiate, and (D-Ala2,MePhe4,Gly-ol5) enkephalin (DAMGO), a potent µOR-internalizing agonist, on µOR trafficking in enteric neurons and on the expression of dynamin and ß-arrestin immunoreactivity in the ileum of guinea pigs rendered tolerant by chronic administration of morphine. RESULTS: Morphine (100 µmol/L) strongly induced endocytosis of µOR in tolerant but not naive neurons (55.7% ± 9.3% vs 24.2% ± 7.3%; P < .001) whereas DAMGO (10 µmol/L) strongly induced internalization of µOR in neurons from tolerant and naive animals (63.6% ± 8.4% and 66.5% ± 3.6%). Morphine- or DAMGO-induced µOR endocytosis resulted from direct interactions between the ligand and the µOR because endocytosis was not affected by tetrodotoxin, a blocker of endogenous neurotransmitter release. Ligand-induced µOR internalization was inhibited by pretreatment with the dynamin inhibitor, dynasore. Chronic morphine administration resulted in a significant increase and translocation of dynamin immunoreactivity from the intracellular pool to the plasma membrane, but did not affect ß-arrestin immunoreactivity. CONCLUSIONS: Chronic activation of µORs increases the ability of morphine to induce µOR endocytosis in enteric neurons, which depends on the level and cellular localization of dynamin, a regulatory protein that has an important role in receptor-mediated signal transduction in cells.