title: Absorptive-mediated endocytosis of a dynorphin-like analgesic peptide, E-2078 into the blood-brain barrier.
abstract: The binding and internalization of a novel analog of dynorphin-like analgesic basic peptide, [125I]E-2078 (CH3-[125I] Tyr-Gly-Gly-Phe-Leu-Arg-CH3Arg-D-Leu-NHC2H5), by isolated bovine brain capillaries were investigated. High-performance liquid chromatographic analysis showed that no significant metabolism of [125I] E-2078 occurred during incubation with brain capillaries for 30 min at 37 degrees C. The binding of [125I]E-2078 to brain capillaries increased with time and the steady-state cell-to-medium concentration ratio was 58.5 +/- 2.6 microliters/mg of protein. Approximately one-fourth of the [125I]E-2078 binding was resistant to acid wash, and showed significant dependence on temperature and medium osmolarity. The acid sensitive binding of [125I]E-2078, which presumably represents surface binding, was saturable and the Scatchard plot gave a maximal binding capacity Bmax = 147 +/- 29 pmol/mg of protein, and a half-saturation constant (KD) = 4.62 +/- 0.59 microM. Pretreatment of brain capillaries with phenylarsine oxide, an endocytosis inhibitor, completely suppressed the acid resistant binding of [125I]E-2078, but did not influence the surface binding of [125I]E-2078. The acid resistant binding of [125I] E-2078 was inhibited by poly-L-lysine and protamine, but not inhibited by insulin, transferrin, dynorphin (1-8), beta-neoendorphin, naloxone or poly-L-glutamate. Moreover, in vivo brain extraction of [125I]E-2078 in rats was 368 +/- 55% higher than that of [3H] sucrose and was significantly inhibited by 1 mM of unlabeled E-2078. These results demonstrate that E-2078 is internalized by brain capillaries via absorptive-mediated endocytosis, which is a polycation-sensitive pathway.
authors.affiliation: Department of Pharmaceutics, Faculty of Pharmacetical Sciences, Kanazawa University, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Terasaki
authors.forename: K
authors.initials: K
authors.lastname: Hirai
authors.forename: H
authors.initials: H
authors.lastname: Sato
authors.forename: Y S
authors.initials: YS
authors.lastname: Kang
authors.forename: A
authors.initials: A
authors.lastname: Tsuji
mesh_terms: D000818:Animals; D001152:Arsenicals / Q000494:pharmacology; D001812:Blood-Brain Barrier / Q000502:physiology*; D001921:Brain / Q000098:blood supply / Q000187:drug effects / Q000378:metabolism*; D002196:Capillaries / Q000378:metabolism; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004399:Dynorphins / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D004705:Endocytosis; D066298:In Vitro Techniques; D008297:Male; D010446:Peptide Fragments / Q000493:pharmacokinetics*; D010455:Peptides / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D001152:Arsenicals; D010446:Peptide Fragments; D010455:Peptides; C029341:oxophenylarsine; C061285:E 2078; D004399:Dynorphins