Enkephalin and Blood-Brain Barrier: Studies of Binding and Degradation in Isolated Brain Micro vessels*

Abstract

The literature regarding enkephalin transport through the brain endothelial wall,i.e. the blood-brain barrier (BBB), is conflicting. For example, some studies indicate that enkephalins traverse the BBB at low rates similar to other putative neurotransmitters, while other investigations have indicated that enkephalins readily cross the BBB. In addition, it is possible that enkephalins, like other peptides, such as insulin, do not cross the BBB but actively bind to specific peptide receptors on the luminal side of the BBB. Therefore, the present studies were undertaken with freshly isolated bovine brain cor- tical capillaries to determine if [tyrosyl-³H]leucine enkephalin is either specifically bound or taken up by the cell which comprises the BBB in vivo. Brain capillaries were incubated with radiochemically pure [tyrosyl-³H]leucine enkephalin and [^l4C]sucrose, an extracellular space marker, at 22–23 C. The amount of ³H radioactivity associatd with brain endothelia increased with time and reached equilibrium by 30 min. However, the uptake or binding of ³H radioactivity by brain endothelia was not saturated by unlabeled leucine enkephalin at concentrations as high as 1 μM, but was completely abolished by 5 mM tyrosine. The latter amino acid is the labeled N-terminal residue of enkephalin and is a neutral amino acid which is transported into brain endothelia by a specific carrier-mediated mechanism that is fully saturated by a 5-mM concentration of amino acid. Chromatographic analysis of the incubation medium indicated that extensive degradation of [tyrosyl-³H]leucine enkephalin to free [³H]tyrosine occurred, and this aminopeptidase activity was inhibited by 2 mM bacitracin. These results suggest that 1) a high affinity receptor or transport mechanism for leucine enkephalin does not exist in the BBB, 2) brain capillaries contain an active enkephalinase activity, which includes an aminopeptidase component, and 3) smilar to other circulating neurotransmitters, the interactions of enkephalins with the BBB are characterized by both a very low permeability and a high enzymic degradative activity.