Focal Stimulation of the Mossy Fibers Releases Endogenous Dynorphins That Bind K1-Opioid Receptors in Guinea Pig Hippocampus

Physiological release of endogenous opioids in guinea pig hippocampal slices was detected in an in vitro competition binding assay using [3H]U69,593, a kappa 1-selective radioligand. Veratridine-induced opioid release caused a decrease in [3H]U69,593 binding that was blocked by either tetrodotoxin addition or the removal of calcium from the incubation buffer. Focal electrical stimulation of opioid peptide-containing afferent pathways resulted in a decrease in [3H]U69,593 binding, whereas stimulation of a major afferent lacking endogenous opioid immunoreactivity had no effect. The addition of 6-cyano-7-nitroquinoxaline-2,3-dione blocked the reduction in [3H]U69,593 binding caused by perforant path stimulation, but not the reduction caused by mossy fiber stimulation, suggesting that the primary source of endogenous kappa ligands was likely to be the dentate granule cells. Antisera against dynorphin A(1-8) or dynorphin B peptides inhibited the effects of mossy fiber stimulation in the [3H]U69,593 displacement assay. Antisera against other prodynorphin- and proenkephalin-derived opioid peptides had no effect. As shown by receptor autoradiography, the distribution of kappa 1 binding sites was limited to the molecular layer of the dentate gyrus and the presubiculum region of temporal hippocampal slices. These results indicate that prodynorphin-derived opioids released under physiological conditions from the mossy fibers act at kappa 1 receptors in the guinea pig dentate gyrus.     

Specificity of the dynorphin-processing endoprotease: comparison with prohormone convertases

The cleavage specificity of a monobasic processing dynorphin converting endoprotease is examined with a series of quench fluorescent peptide substrates and compared with the cleavage specificity of prohormone convertases. A dynorphin B-29-derived peptide, Abz-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr-Arg-Ser-Glneddnp (where Abz is o-aminobenzoyl and eddnp is ethylenediamine 2,4-dinitrophenyl), that contains both dibasic and monobasic cleavage sites is efficiently cleaved by the dynorphin converting enzyme and not cleaved by two propeptide processing enzymes, furin and prohormone convertase 1. A shorter prorenin-related peptide, Dnp-Arg-Met-Ala-Arg-Leu-Thr-Leu-eddnp, that contains a monobasic cleavage site is cleaved by the dynorphin converting enzyme and prohormone convertase 1 and not by furin. Substitution of the P1' position by Ala moderately affects cleavage by the dynorphin-processing enzyme and prohormone convertase 1. It is interesting that this substitution results in efficient cleavage by furin. The site of cleavage, as determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry, is N-terminal to the Arg at the P1 position for the dynorphin converting enzyme and C-terminal to the Arg at the P1 position for furin and prohormone convertase 1. Peptides with additional basic residues at the P2 and at P4 positions also serve as substrates for the dynorphin converting enzyme. This enzyme cleaves shorter peptide substrates with significantly lower efficiency as compared with the longer peptide substrates, suggesting that the dynorphin converting enzyme prefers longer peptides that contain monobasic processing sites as substrates. Taken together, these results suggest that the cleavage specificity of the dynorphin converting enzyme is distinct but related to the cleavage specificity of the prohormone convertases and that multiple enzymes could be involved in the processing of peptide hormones and neuropeptides at monobasic and dibasic sites.     

Dermorphin tetrapeptide analogs as potent and long-lasting analgesics with pharmacological profiles distinct from morphine

Dermorphin (Tyr-d-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂) is a heptapeptide isolated from amphibian skin. With a very high affinity and selectivity for μ-opioid receptors, dermorphin shows an extremely potent antinociceptive effect. The structure-activity relationship studies of dermorphin analogs clearly suggest that the N-terminal tetrapeptide is the minimal sequence for agonistic activity at μ-opioid receptors, and that the replacement of the d-Ala² residue with d-Arg² makes the tetrapeptides resistant to enzymatic metabolism. At present, only a handful of dermorphin N-terminal tetrapeptide analogs containing d-Arg² have been developed. The analogs show potent antinociceptive activity that is greater than that of morphine with various injection routes, and retain high affinity and selectivity for μ-opioid receptors. Interestingly, some analogs show pharmacological profiles that are distinct from the traditional μ-opioid receptor agonists morphine and [d-Ala²,NMePhe⁴,Gly-ol⁵]enkephalin (DAMGO). These analogs stimulate the release of dynorphins through the activation of μ-opioid receptors. The activation of κ-opioid receptors by dynorphins is suggested to reduce the side effects of μ-opioid receptor agonists, e.g., dependence or antinociceptive tolerance. The dermorphin N-terminal tetrapeptide analogs containing d-Arg² may provide a new target molecule for developing novel analgesics that have fewer side effects.     

Dynorphins Modulate DNA Synthesis in Fetal Brain Cell Aggregates

Abstract: Previously, opioid peptide analogues, β-endorphin, and synthetic opiates were found to inhibit DNA synthesis in 7-day fetal rat brain cell aggregates via κ-and μ-opioid receptors. Here dynorphins and other endogenous opioid peptides were investigated for their effect on DNA synthesis in rat and guinea pig brain cell aggregates. At 1 µ M , all dynorphins tested and β-endorphin inhibited [³H]thymidine incorporation into DNA by 20–38% in 7-day rat brain cell aggregates. The putative ε-antagonist β-endorphin (1–27) did not prevent the effect of β-endorphin, suggesting that the ε-receptor is not involved in opioid inhibition of DNA synthesis. The κ-selective antagonist norbinaltorphimine blocked dynorphin A or B inhibition of DNA synthesis, implicating a κ-opioid receptor. In dose-dependency studies, dynorphin B was three orders of magnitude more potent than dynorphin A in the attenuation of thymidine incorporation, indicative of the mediation of its action by a discrete κ-receptor subtype. The IC₅₀ value of 0.1 n M estimated for dynorphin B is in the physiological range for dynorphins in developing brain. In guinea pig brain cell aggregates, the κ-receptor agonists U50488, U69593, and dynorphin B reduced thymidine incorporation by 40%. When 21-day aggregates were treated with dynorphins, a 33–86% enhancement of thymidine incorporation was observed. Because both 7- and 21-day aggregates correspond to stages in development when glial cell proliferation is prevalent and glia preferentially express κ-receptors in rat brain, these findings support the hypothesis that dynorphins modulate glial DNA synthesis during brain ontogeny.