| EM1 and EM2 are newly discovered endogenousμOR ligands.However,the functional role of these EMs in colonic mobility remains unknown.The present investigation was undertaken to evaluate the effects of the EMs on the colonic mobility in vitro and to determine the mechanisms responsible for the muscle contraction induced by EMs.EMs(10-9-10-6M)caused significantly contractions in longitudinal and circular muscle of isolated colon.EMs-induced longitudinal or circular muscle contractions were not significantly affected by atropine,Nω-nitro-L-arginine methyl ester(L-NAME),indomethacin,phentolamine,propranolol and methysergide. Tetrodotoxin and naloxone completely abolished EMs-induced longitudinal or circular muscle contractions without affecting contractions in response to carbachol. Surprisingly,EMs(10-7M)-induced longitudinal muscle contractions were significantly attenuated by nor-binaltorphimine(nor-BNI)(3×10-6M).By contrast,pretreatment with naltrindole(NTI)(10-6M)did not significantly affect EMs-induced longitudinal or circular muscle contractions.Interestingly,the circular muscle contractions in response to EM2(10-7M)were not fully blocked byβ-funaltrexamine(β-FNA)(6×10-6M). Naloxonazine(10-6M)almost fully abolished the EMs-induced longitudinal or circular muscle contractions,and these effects could be only partially reversed by extensive washing.All the results indicated that activation of multiple subtypes of opioid receptors,possiblyμ1(naloxonazine-sensitive),μ2 and even other forms ofμORs(β-FNA-insensitive),was required for EMs-induced mouse colonic mobility.And these results also indicated that the machanisms responsible for the contractile effects induced by EMs in the longitudinal muscle of distal colon and in the circular muscle of proximal colon may be different.At 4 weeks and 8 weeks after the onset of diabetes,EMs and carbachol-induced contractions in the longitudinal muscle of distal colon were significantly reduced compared to those of non-diabetic mice.But mechanisms in both diabetes and non-diabetes were similar.All the results indicated that type 1 diabetes significantly attenuated the modulatory effects of EMs on the mouse colonic motility,but the mechanisms responsible for these effects were not significantly altered.We also characterized the effects of endomorphin-2(Tyr1-Pro2-Phe3-Phe4-NH2, EM2)and morphiceptin(Tyr1-Pro2-Phe3-Pro4-NH2)analogs,in which Phe3 or Phe4/Pro4 were substituted by naphthyl group-containing amino acids,on the colonic motility.[D-1-Nal4]EM2 fail to significantly induce but can dose-dependently antagonize endomorphins(EMs)(10-7M)-induced longitudinal muscle contractions.[1-Nal3]EM2 and[D-1-Nal3]morphiceptin displayed strong stimulatory effects(their EC50values were 34.84±6.06 nM and 55.21±11.49 nM,respectively)on the colonic motility,and these were in agreement with the results obtained from binding assays and GPI/MVD assays.Tetrodotoxin(TTX,10-7M)and naloxone(10-5M)but not NG-nitro-L-arginine methyl ester(L-NAME)(10-4M)completely abolished the[1-Nal3]EM2-(10-6M)and [D-1-Nal3]morphiceptin(10-6M)-induced colonic contractions.β-funaltrexamine(β-FNA)(2.5×10-6M)and naloxonazine(10-6M)significantly reduced but not fully blocked longitudinal and circular muscle contractions in response to[1-Nal3]EM2(10-6 M)and[D-1-Nal3]-morphiceptin(10-6M).Interestingly,nor-binaltorphimine(nor-BNI) (10-6M)significantly attenuated[D-1-Nal3]morphiceptin(10-6M)-but not[1-Nal3]EM2(10-6M)-induced colonic contractions.However,naltrindole(NTI)(10-6M) was able to antagonize[1-Nal3]EM2(10-6M)-but not[D-1-Nal3]morphiceptin(10-6 M)-induced colonic contractions.These results suggest that opoiod receptors especiallyμ-opioid receptor play critical role in the modulation of analogs[1-Nal3]EM2-and[D-1-Nal3]morphiceptin-induced mouse colonic motility and the heterocyclic modifications on the position 3 and 4 significantly change the effects and mechanisms of EM2 and morphiceptin on the colonic motility.
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