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 signficant metabolism of [125I]E-2078 occurred during incubation with brain capillaries for 30 min at 37°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 μl/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 B(max) = 147 ± pmol/mg of protein, and a half-saturation constant (K(D)) = 4.62 ± 0.59 μM. 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), β-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 [125I]E-2078. These results demonstrate the E-2078 is internalized by brain capillaries via absorptive-mediated endocytosis, which is a polycation-sensitive pathway.
|Number of pages||7|
|Journal||Journal of Pharmacology and Experimental Therapeutics|
|Publication status||Published - 1989 Jan 1|
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