604 H 2S Induces Analgesia Is Mediated In Vivo Activation of μ Opioid Receptor and PI3K/AKT Pathway Activation

Abstract

Background and aim. Hydrogen sulfide (H2S) functions as a neuromodulator and reverses colorectal distension (CRD)-induced pain. Opioid system is widely involved in analgesia. Whether the opioid receptors are involved in the H2S -induced analgesia is unknown. Methods. Four, graded (0.4-1.6 ml water) CRD were produced in awake rats and colorectal pain were assessed by measuring the abdominal withdrawal response (AWR) after i.p. administration of saline or Na2S, a donor of H2S (100 mMol/kg). Spinal cFos expression was also detected. To determine whether the opioid system was involved in the H2S -induced antinociception, pre-treatment with non-selective opioid antagonist naloxone (0.5 mg/kg i.p.) or selective antagonists of μ(MOR) (CTAP, 0.09 mg/kg), k (KOR) (GNTI, 0.08 mg/kg) and δ (DOR) (NTI, 4 mg/kg) opioid receptors or antisense oligodeoxynucleotides directed against specific exons of the DOR, KOR and MOR (10 μg each probe/10 μl saline). i.c.v. administered were performed and the AWR score detected. The H2S (50 μM) In Vitro effects of H2S on MOR phosphorylation, β-arrestins recruitment and MOR internalization were determined in the neuronal cell line SKNMC and compared with those obtained with the MOR agonist DAMGO (1 μ). The effects of H2S and DAMGO on PI3K/AKT pathway was also detected. Results. H2S significantly inhibited the CRD-induced pain and spinal cFos overexpression. The H2S-induced analgesia was significantly reverted by naloxone, CTAP and antisense oligodeoxynucleotides against MOR, while both DOR and KOR inhibition appeared less effective. In the SKNMC cells, both H2S and DAMGO caused the timedependent MOR phosphorylation on serine 377 and the co-immunoprecipitation between MOR and β-arrestin, indicating that H2S induced the interaction of b-arrestins with MOR. Finally, plasma membrane fraction and immunoflurescence staining demonstrated that both DAMGO and H2S caused the translocation of MOR from the plasma membrane to the cytoplasma and that MOR internalization was inhibited by pre-treating cells with CTAP (1 μM). Both DAMGO and H2S induced AKT phosphorylation. CTAP inhibited the DAMGOinduced AKT phosphorylation but not those induced by H2S. Finally, pre-treating neuronal cells with the PI3K inhibitor LY294002 caused the blockade of H2S-induced MOR internalization. Conclusions. This study provides evidence that: 1) 2Sinhibits CRD-induced pain by interacting with peripheral and central MOR; 2) H2S causes MOR phosphorylation, the association between MOR and β-arrestins and MOR internalization; 3) H2S induces AKT phosphorylation; 4) inhibition of PI3K/AKT pathway results in the blockade of H2S-induced MOR internalization.