Hepatic inactivation of Leu-enkephalin.
Leu-enkephalin radiolabelled at the N-terminal tyrosine by two different methods was presented to isolated perfused rat livers. Approximately 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 and Hunter reagent. Uptake of both forms of radiolabelled Leu-enkephalin was inhibited by taurocholate in a concentration-dependent manner. The proportion of internalised radioactivity secreted into bile also differed but in both cases showed a very rapid time-course similar to that of [24-(14)C]taurocholate and suggestive of non-endocytic transfer via membrane transport proteins. Pre-perfusion with the aminopeptidase inhibitor bestatin increased uptake of 3H-labelled Leu-enkephalin from 10% to 23%; no further increase occurred when the endopeptidase 24.11 inhibitor thiorphan was also present. On infusion of the native peptide into rat livers, 80% of Leu-enkephalin immunoreactivity was lost between 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 occurred. Low levels of aminopeptidase N were visualised in the sinusoidal membrane using a specific monoclonal antibody coupled to peroxidase staining. Thus, hepatic inactivation of Leu-enkephalin is primarily via hydrolysis mediated by cell surface peptidase (including aminopeptidases) whilst uptake of the intact peptide, probably by a bile salt transport protein, is quantitatively minor unless the N-terminus is blocked by Bolton and Hunter reagent or peptidase inhibitors are present.
MacKinnon
K L
KL
School of Biomolecular Sciences, Liverpool John Moores University, UK.
Zuzel
K A
KA
Billington
D
D
D000818:Animals; D001659:Biliary Tract / Q000378:metabolism; D001711:Biotransformation; D018826:CD13 Antigens / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004743:Enkephalin, Leucine / Q000378:metabolism / Q000493:pharmacokinetics*; D006868:Hydrolysis; D007457:Iodine Radioisotopes; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D051379:Mice; D010477:Perfusion; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013388:Succinimides; D014316:Tritium; D014443:Tyrosine / Q000378:metabolism
D007457:Iodine Radioisotopes; D011480:Protease Inhibitors; D013388:Succinimides; D014316:Tritium; D014443:Tyrosine; D004743:Enkephalin, Leucine; C022581:Bolton-Hunter reagent; D018826:CD13 Antigens; D007930:Leucine; C012211:ubenimex