Participation of both 'enkephalinase' and aminopeptidase activities in the metabolism of endogenous enkephalins

The pattern of hydrolysis of ³H]Met⁵-enkephalin by slices from rat striatum has been determined by Chromatographic isolation of ³H]peptide fragments and by assessment of the effects of various selective peptidase inhibitors. It consists in the formation of ³H]Tyr, ³H]Tyr-Gly and ³H]Tyr-Gly-Gly which represent 80%, 2% and 18%, respectively, of total³H-metabolite formation.The formation of ³H]Tyr, presumably occurring under the action of membrane-bound aminopeptidases, is reduced in the presence of either 10^?4 M puromycin or 2.10^?5 M bestatin by about 40% and 75%, respectively. Both aminopeptidase inhibitors display significantly higher potency on a crude particulate fraction from rat striatum. The formation of ³H]Tyr-Gly-Gly in the slice preparation is strongly reduced by addition of 10^?7 M thiorphan, an enkephalin dipeptidylcar?ypeptidase (‘enkephalinase’) inhibitor, but only slightly in the presence of 10^?6 M captopril, an angiotensin-converting enzyme inhibitor.When the aminopeptidase pathway is inhibited by bestatin, ³H]Tyr-Gly-Gly represents more than 60% of total hydrolysis products, suggesting that the tripeptide is partly hydrolysed after being formed under the action of ‘enkephalinase’. In the presence of both bestatin and thiorphan, the total hydrolysing activity of the slice preparation is reduced by 75% while ³H]Tyr-Gly level is slightly increased. These data indicate that the hydrolysis of exogenous Met⁵-enkephalin in the extracellular space of the slice preparation primarily involves the ‘enkephalinase’ as well as the aminopeptidase pathways.The participation of both pathways in the inactivation of endogenous Met⁵-enkephalin has also been evidenced by evaluating the recovery of the opioid peptide released by K⁺-induced depolarisation of striatal slices in the presence of bestatin and thiorphan. While the presence of either inhibitor allows a partial protection of the released peptide, their co-presence results in total recovery of the enkephalin in intact form in the medium.Finally, administration of either the aminopeptidase or the ‘enkephalinase’ inhibitor in mice results in antinociceptive effects as evaluated in the hot-plate jump test. These effects are additive and prevented by naloxone, an opiate receptor antagonist.Thus, the various experimental approaches used indicate that both (but only) the ‘enkephalinase’ and the aminopeptidase pathways play a critical role in the inactivation of endogenous enkephalins.