HEPATIC INACTIVATION OF LEU-ENKEPHALIN

Leu-enkephalin radiolabelled at the N-terminal tyrosine by two differe nt methods was presented to isolated perfused rat livers. Approximatel y 10% of a pulse of tritiated Leu-enkephalin was taken up first-pass; this was increased to 62% when the peptide was iodinated with Bolton a nd Hunter reagent. Uptake of both forms of radiolabelled Leu-enkephali n was inhibited by taurocholate in a concentration-dependent manner. T he proportion of internalised radioactivity secreted into bile also di ffered but in both cases showed a very rapid time-course similar to th at of [24-C-14]taurocholate and suggestive of non-endocytic transfer v ia membrane transport proteins. Pre-perfusion with the aminopeptidase inhibitor bestatin increased uptake of H-3-labelled Leu-enkephalin fro m 10% to 23%; no further increase occurred when the endopeptidase 24.1 1 inhibitor thiorphan was also present. On infusion of the native pept ide into rat livers, 80% of Leu-enkephalin immunoreactivity was lost b etween the pre- and post-hepatic perfusate; this was reduced to 65% in the presence of 10(-5) M bestatin. The almost total release of the N- terminal tyrosine from 3H-labelled Leu-enkephalin which escaped first- pass uptake confirmed that substantial sinusoidal metabolism had occur red. Low levels of aminopeptidase N were visualised in the sinusoidal membrane using a specific monoclonal antibody coupled to peroxidase st aining. Thus, hepatic inactivation of Leu-enkephalin is primarily via hydrolysis mediated by cell surface peptidases (including aminopeptida ses) whilst uptake of the intact peptide, probably by a bile salt tran sport protein, is quantitatively minor unless the N-terminus is blocke d by Bolton and Hunter reagent or peptidase inhibitors are present.