Jean Costentin

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

Abstract The pattern of hydrolysis of [ 3 H]Met 5 -enkephalin by slices from rat striatum has been determined by Chromatographic isolation of [ 3 H]peptide fragments and by assessment of the effects of various selective peptidase inhibitors. It consists in the formation of [ 3 H]Tyr, [ 3 H]Tyr-Gly and [ 3 H]Tyr-Gly-Gly which represent 80%, 2% and 18%, respectively, of total 3 H-metabolite formation. The formation of [ 3 H]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 [ 3 H]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, [ 3 H]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 [ 3 H]Tyr-Gly level is slightly increased. These data indicate that the hydrolysis of exogenous Met 5 -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 Met 5 -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.

Potent inhibition of cerebral aminopeptidases by carbaphethiol, a parenterally active compound

We designed phethiol (1-amino-1-benzyl-2-mercaptoethane) as a potent and selective inhibitor of Zn-containing aminopeptidases. This compound inhibited purified aminopeptidase M (EC.3.4.11.2) with a Ki of 5 nM but was at least 1000 times less potent against other metallopeptidases comprising angiotensin-converting enzyme EC 3.4.15.1), enkephalinase (EC 3.4.24.11), thermolysin (EC 3.4.24.4), or dipeptidylaminopeptidases. Phethiol alone significantly but partially protected endogenous (Met5) enkephalin released from depolarized brain slices, total protection being achieved when it was associated with an enkephalinase inhibitor. In order to obtain a parenterally-active inhibitor of cerebral aminopeptidases, the prodrug carbaphetiol, a readily hydrolyzable S-phenylcarbamoyl derivative of phethiol, was designed. Carbaphethiol (i.v.) elicited a rapid rise in mouse striatal level of Tyr-Gly-Gly, a characteristic extracellular metabolite of enkephalins. Carbapethiol alone and, even more, when associated with an enkephalinase inhibitor, exerted a potent naloxone-reversible antinociceptive activity. Carbaphethiol appears as the first parenterally-active inhibitor of cerebral aminopeptidases, potentially useful in neuropeptides degradation studies and as a pain-suppressing agent.