The purpose of this study was to clarify the mechanism of the blood-brain barrier (BBB) transport of H-Tyr-D-Arg-Phe-β-Ala-OH (TAPA), which is a novel dermorphin analog with high affinity for the μ1-opioid receptor. The in vivo BBB permeation influx rate of [125I]TAPA after an i.v. bolus injection (7.3 pmol/g body weight) into mice was estimated to be 0.265 ± 0.025 μL/(min · g of brain). The influx rate of [125I]TAPA was reduced 70% by the coadministration of unlabeled TAPA (33 nmol/g of brain), suggesting the existence of a specific transport system for TAPA at the BBB. In order to elucidate the BBB transport mechanism of TAPA, a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4) was used as an in vitro model of the BBB. The acid-resistant binding of [125I]TAPA, which represents the internalization of the peptide into cells, was temperature- and concentration-dependent with a half saturation constant of 10.0 ± 1.7 μM. The acid-resistant binding of TAPA was significantly inhibited by 2,4-dinitrophenol, dansylcadaverine (an endocytosis inhibitor) and poly-L-lysine and protamine (polycations). These results suggest that TAPA is transported through the BBB by adsorptive-mediated endocytosis, which is triggered by binding of the peptide to negatively charged sites on the surface of brain capillary endothelial cells. Blood-brain barrier transport via adsorptive-mediated endocytosis plays a key role in the expression of the potent opioid activity of TAPA in the CNS.
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