Studies on the release by somatic stimulation from rat and cat spinal cord of active materials which displace dihydromorphine in an opiate-binding assay.

Using the spinal superfusion procedure, in anesthetized rats and cats, the presence of active factors which displace dihydromorphine in brain opiate binding studies, has been observed. Separation of this activity on a Sephadex G-10 column reveals the presence of two fractions which occur before (Fraction I) and after (Fraction II) the salt peak which account for over 70% of the observed dihydromorphine-displacing activity. The ratio of activity in Fraction II/Fraction I is 33 and 21, in the resting spinal perfusates of the rat and cat, respectively. High intensity, bilateral stimulation of the sciatic nerve in cats, results in a 30- and 5.4-fold increase in the levels of Fraction I and Fraction II, respectively, over pre-stimulation levels. In rat, bilateral stimulation of the hind paws, resulted in a frequency-dependent increase in the levels of Fraction I (1.9- and 3.2-fold at 5 and 50 Hz, respectively). Dynorphin 1-13 fragment elutes at least partly in Fraction I. With regard to Fraction II, the peak co-chromatographs with hexapeptide derivatives of enkephalin. Met- and Leu-enkephalin (Fraction III), elute off the column at a point where opiate receptor displacing activity is relatively small. Electrophoretic separation of Fraction I radioreceptor activity of alkaline and acid pH on agarose columns revealed two principle peaks which co-migrated with alpha-neoendorphin and dynorphin 1-13. Fraction II activity appeared primarily in a single peak which was isographic with enkephalin hexapeptides. Using radioimmunoassays, detectable levels of dynorphin and Met-enkephalin were observed and sciatic nerve stimulation resulted in significant increases. Neither column-coupled radioreceptor assays nor radioimmunoassays revealed the presence of beta-endorphin. The present experiments demonstrate the releasability by high intensity somatic stimulation of a variety of opioid peptides present in spinal terminals. Significantly, however, the majority of this activity appears to be found in fractions different from those of the pentapeptide enkephalins.