The Study On Physiological And Pharmacological Activity Of Dual Peptidase Inhibitor Opiorphin

Opiorphin,an endogenous human peptide mediator,was identified in human saliva by Wisher et al.They demonstrated that opiorphin is a dual inhibitor of human neutral ecto-endopeptidase NEP(EC 3.4.24.11) and human ecto-aminopeptidase APN(EC 3.4.11.2),in vitro.It has been reported that opiorphin displays potent analgesic activity by potentiating endogenous opioid-dependent enkephalinergic pathway.In the present study,we investigated the effects of human opiorphin in serial of in vitro and in vivo assays.In in vitro bioassay,opiorphin(10^-6 to 10^-4 M) caused colonic contraction in a concentration-dependent manner,which was completely blocked by naloxone and partially attenuated byβ-funaltrexamine(β-FNA) and naltrindole. Moreover,opiorphin(10^-4 M) significantly enhanced the contractile response induced by Met-enkephalin.The data suggested that the effect of opiorphin on colonic contraction may be due to the protection of enkephalins.In in vivo bioassay, intracerebroventricular(i.c.v.) administration of opiorphin(1.25-10μg/kg) dose- and time-dependently induced potent analgesic effect(ED₅₀=3.22μg/kg).This effect was fully blocked by naloxone and significantly inhibited by co-injection(i.c.v.) withβ-FNA or naltrindole,but not by nor-binaltorphimine,indicating the involvement of bothμ- andδ-opioid receptors in the analgesic response evoked by opiorphin.In addition,i.c.v,administration of 5μg/kg opiorphin produced the comparative effect as 10μg/kg morphine on the analgesia,suggesting that opiorphin displayed more potent analgesic effect than that induced by morphine.We also found that intravenous injection(i.v.) of opiorphin(50-300 nmol/kg) dose-dependently increased mean arterial pressure(MAP) and heart rate(HR) in urethane-anesthetized rats.The hypertensive and tachycardic responses induced by opiorphin(200,300 nmol/kg) were significantly antagonisted by angiotensin converting enzyme inhibitor captopril(2 mg/kg,i.v.) and angiotensinâ…¡AT₁ recepter antagonist valsartan(10 mg/kg,i.v.).Interestingly,the pressor response to opiorphin was also blocked by pretreatment withα-adrenoceptors antagonist phentolamine(2 mg/kg,i.v.) orβ-adranoceptors antagonist propranolol(2 mg/kg,i.v.).However, propranolol(2 mg/kg,i.v.) significantly inhibited the tachycardic response induced by opiorphin,but phentolamine(2 mg/kg,i.v.) did not.Nevertheless,opioid receptor antagonist naloxone did not significantly reduce the cardiovascular effects of opiorphin.These data suggest that the cardiovascular responses induced by opiorphin could be attributed to the up-regulation of endogenous angiotensin levels,and the sympathetic nervous system may also be involved but not including opioid-dependent way.In in vitro assay,opiorphin can not cause significant vasocontdction on the rat arota.Therefore,the cardiovascular effects induced by opiorphin are mainly due to the protection of the endogenous circulation angiotensin at peripheral levels through inhibition of both neutral endopeptidase and aminopeptidase N,but not vacular-formed angiotensin.In conclusion,all findings suggested that opiorphin play important physiological and pharmacological functions in both central and peripheral tissues by inhibiting endogenous enkephalins or angiotensin inactivation.