ABSORPTIVE-MEDIATED ENDOCYTOSIS OF A DYNORPHIN-LIKE ANALGESIC PEPTIDE, E-2078, INTO THE BLOOD-BRAIN-BARRIER

  • 1 October 1989
    • journal article
    • research article
    • Vol. 251  (1) , 351-357
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.degree.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 .mu.l/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 .mu.M. Pretreatment of brain capillaries with phenylarsine oxide, an endocytosis inhibitor, completely suppressed 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. Moveover, 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.