Opioid pathways in an avian retina. I. The content, biosynthesis, and release of Met5-enkephalin

By means of an enzyme-linked immunosorbent assay, the concentration of enkephalin-immunoreactive substances was estimated to be about 25 nM in the chicken retina. The biosynthesis of 3H-Met5-enkephalin in this retina was studied by a pulse-chase incubation technique. Isolated retinas were incubated with 0.2 ml of oxygenated Ringer's solution containing 40 microCi of [3H]methionine and trasylol, a peptidase inhibitor, for 30 min at room temperature. The tissue was then rinsed three times in large volumes of Ringer's solution and incubated in the same solution containing unlabeled methionine (100 micrograms/ml) and trasylol for at least another hour. The products synthesized were extracted in acetic acid and assayed by high performance liquid chromatography (HPLC) and immunoassay. A peak of radioactivity that comigrated with Met5-enkephalin on HPLC and cross-reacted with antibodies against enkephalins was detected. The level of 3H-Met5-enkephalin radioactivity increased approximately 10-fold as the chase-incubation period increased from 0 to 120 min, suggesting that, as in other tissues, Met5-enkephalin may be synthesized as part of a larger precursor. The newly synthesized Met5-enkephalin could be released by depolarization of the retina with high extracellular K+ concentration. Furthermore, this K+-stimulated release was greatly suppressed by 5 mM Co2+ in the medium, suggesting that this release is Ca2+-dependent and may be synaptically mediated.