The Role And Mechanism Of P2Y₁ Receptor Regulating Opioid Induced Constipation

Objective To observe the expression changes of P2Y₁ receptor in the distal colonic submucosa of rats with opioid induced constipation（OIC）its correlation with the occurrence of OIC.Methods OIC model was created by intraperitoneal injection of loperamide hydrochloride,a selective agonist ofμopioid receptor（MOR）,and the model was evaluated by observing the fecal and stool characteristics of rats.Seven days later,the model was evaluated by detecting the fecal traits.The distribution of P2Y₁-containing neurons in the distal colonic submucosal plexus of rat was demonstrated by the immunofluorescence histochemistry.RT-PCR and Western Blot were used to detect the mRNA and protein expressions of MOR and P2Y₁ receptor in the distal colonic submucosa tissues of OIC rats.At the same time,electrophysiological method was used to observe the effect of P2Y₁receptor on distal colon motor function of rats.Results 1.After five days,the feces of OIC rats appeared like sausage shaped pieces.2.Immunofluoresence histochemical staining showed the co-existence of MOR andβ-Adenosine triphosphate（ATPB）,P2Y₁ and calbindin（CB）,P2Y₁ and calcitonin generelated peptide（CGRP）in nerve cells of distal colonic submucosal plexus.3.RT-PCR experiments showed that the mRNA expression of MOR was significantly increased in the distal colonic submucosa of OIC rats,while the P2Y₁ was decreased.Western Blot results showed that MOR protein expression was increased and the P2Y₁ protein was significantly decreased in the distal colonic submucosa of OIC rats.4.In vitro tensile test results showed that endomorphine-2（EM2）enhanced the autonomic contraction of distal colon segment,while the MOR selective receptor antagonistβ-Funaltrexamine hydrochloride（β-FNA）reduced the autonomic contraction amplitude（P<0.05）.Adenosine5’-[β-thio]diphosphate trilithium salt（ADPβS）,was the agonist of P2Y₁ receptor,inhibited the autonomic contraction of distal colon fragments,continuing to add EM2 could inhibit the effect of ADPβS on the autonomy;then upside down to join the order of the drug,ADPβS inhibited the effect of EM2（P<0.05）.MRS2500 tetraammonium salt（MRS2500）could enhance the autonomic contraction of distal colon segment,and it also inhibited theβ-FNA inhibition effect;reverse the order in which drugs were added,β-FNA reduced MRS2500 effect on colon clip（P<0.05）.ADPβS further enhanced the inhibitory effect ofβ-FNA on colon fragments（P<0.05）;but in contrast,it was found thatβ-FNA had no effect on ADPβS（P>0.05）.5.In vitro electrical field stimulation experiments showed that Electride field stimulation（EFS）significantly inhibited the autonomic contraction of distal colonic fragments,EM2 could inhibit EFS on distal colon fragments,continued addition of inhibited the effect（P<0.05）;reversing the adding order of the two showed that EM2 had no effect on the effect ofβ-FNA on EFS（P>0.05）.The addition of ADPβS further enhanced the inhibitory effect of EFS on the autonomic contraction of distal colon segments（P<0.05）.Continued addition of EM2 could attenuate this effect,but the inhibitory effect of ADPβS on EFS was further enhanced by the addition ofβ-FNA（P<0.05）;Reversing the order of drug addition,it can be seen that ADPβS significantly inhibited the effect of EM2 on EFS（P<0.05）,but it had no significant effect onβ-FNA（P>0.05）.MRS2500 also had a significant inhibitory effect on EFS,continuous addition ofβ-FNA could significantly inhibit the action of MRS2500（P<0.05）,but EM2 had no effect on MRS2500（P>0.05）;reversing the order of drug addition,MRS2500 also reduced the action ofβ-FNA on EFS（P<0.05）,but had no change in the action of EM2 on EFS（P>0.05）.Conclusion 1.P2Y₁ receptor is distributed in the submucosal plexus nerve cells;2.P2Y₁receptor is closely related to the occurrence of OIC;3.The occurrence of OIC may be associated with MOR dysfunction leading to abnormal P2Y₁ receptor function;4.The regulation of MOR on colonic motility may be related to the regulation of release of purine transmitters such as ATP in the colonic nervous system,so as to regulate the function of P2Y₁receptor.