A.T. McKnight

Selectivities of opioid peptide analogues as agonists and antagonists at the δ-receptor

1 The endogenous opioid ligands interact with more than one of the μ‐, δ‐ and κ‐binding sites. By the use of binding assays and bioassays, enkephalin analogues have been assessed for their selectivity for binding at the (5‐binding site and for their agonist and antagonist activities at the δ‐receptor. The electrically‐induced contractions of myenteric plexus‐longitudinal muscle preparations of the guinea‐pig ileum were inhibited by μ‐ and κ‐receptor ligands. Inhibitions were seen with μ‐, δ‐ and κ‐receptor ligands in the mouse vas deferens, mainly with μ‐receptor ligands in the rat vas deferens and only with κ‐receptor ligands in the rabbit vas deferens. 2 From observations on a considerable number of [Leu5] enkephalin analogues, it has been concluded that [d‐Pen2, d‐Pen5] enkephalin and [d‐Pen2, l‐Pen5] enkephalin are the most selective δ‐agonists and that N,N‐diallyl‐Tyr‐Aib‐Aib‐Phe‐Leu‐OH is the most selective antagonist (Aib = α‐aminoisobutyric acid). The binding of these peptides at the δ‐site is 99% of the total binding. As to potency, the agonists are superior to the antagonists.

Increase in potencies of opioid peptides after peptidase inhibition

Various agents that have been reported to reduce the enzymatic degradation of the enkephalins have been tested for their ability to potentiate the activity of [Met5]enkephalin in three in vitro assay tissues. The greatest effect was obtained with the combination of bestatin (10 microM or 30 microM), captopril (10 microM), thiorphan (0.3 microM) and L-Leucyl-L-leucine (2 mM) which increased the potency of [Met5]enkephalin 18-fold in the guinea-pig myenteric plexus, 13-fold in the mouse vas deferens and 200-fold in the rat vas deferens. The increased potency is attributed to inhibition of the peptidases since the mixture of inhibitors did not change the activity of either normorphine or the metabolically stable synthetic opioid peptides. The potencies of the hexa-, hepta- and octapeptide C-terminus extensions of [Met5]enkephalin and [Leu5]enkephalin were increased by the peptidase inhibitors in all three preparations; the greatest effects were found in the rat vas deferens. No significant changes in the potencies of fragments of beta-endorphin longer than beta-endorphin-(1-19) were obtained. It may now be possible to inhibit enzymatic degradation of opioid peptides sufficiently to measure their release from neurones activated by electrical field stimulation.

Proenkephalin- and prodynorphin- derived opioid peptides in guinea-pig heart

Consecutive high performance liquid chromatography (HPLC) fractionation and the mouse vas deferens assay were used to characterize opioid peptides in the guinea-pig heart. Atria were found to contain at least nine different opioid peptides derived from proenkephalin and prodynorphin. In ventricles at least seven different molecular species which may be derived only from prodynorphin were present. The total opioid activity in atria averaged 22 pmol and in ventricles 11 pmol [Met]enkephalin equivalents per g wet wt. The high content of [Leu]enkephalin in relation to [Met]enkephalin indicates the possibility of a dynorphinergic pathway of cardiac [Leu]enkephalin. Multiple cardiac opioid ligands and receptors, including kappa, may be functionally important in the peripheral control of cardiac performance and coronary circulation.

Characterization of opioid peptides in guinea-pig heart and skin.

Using reverse phase HPLC separations, and assay of eluate fractions in the mouse vas deferens, extracts of heart and skin from guinea-pigs were shown to contain several species of opioid-active material in low amounts (3-20 pmol/g as [Met5]enkephalin). Immunohistochemical studies revealed in the heart a small number of enkephalin-immunoreactive nerve fibres, particularly in cardiac ganglia and some small cells (paraganglionic cells, APUD cells). In the skin, enkephalin-immunoreactivity was confined to Merkel cells. Cardiac and cutaneous opioid peptides may modulate peripheral cardiovascular and sensory functions.

The opioid receptors in the hamster vas deferens are of the δ-type

The motor responses of the isolated vas deferens of the hamster were unaffected by opioid-receptor agonists which are selective for the mu- or kappa-receptor, while agonists which show degrees of selectivity for the delta-opioid receptor caused dose-related inhibition of the stimulation-evoked contractions. The agonist action of the enkephalins and their congeners was only apparent when various inhibitors of tissue peptidases were present. Responses to opioid agonists were antagonised in a competitive manner by naloxone and by the selective delta-receptor antagonist, ICI 174864. It is noteworthy that the benzomorphans bremazocine, ethylketocyclazocine and Mr 2034, which are agonists at kappa-receptors in other tissues, are antagonists at the delta-receptor of the hamster vas deferens. Thus, the vas deferens of the hamster contains opioid receptors only of the delta-type and may therefore provide a simple and specific test for the assay of activity at the delta-opioid receptor.

k-Binding and Degradation of [3H]Dynorphin A (1–8) and [3H]Dynorphin A (1–9) in Suspensions of Guinea Pig Brain Membranes

Abstract: Following incubation of [3H]dynorphin A (1–8) and [3H]dynorphin A (1–9) with suspensions of guinea pig brain membranes, analysis of the supernatants by HPLC has shown that both peptides are degraded at 25°C and at 0°C. Bestatin and captopril reduce degradation at 0°C but for a similar degree of protection at 25°C argininecontaining dipeptides are also required. The effects of these peptidase inhibitors on the degradation profiles indicate that [3H]dynorphin A (1–8) has three main sites of cleavage: the Tyr1‐Gly2, Arg6‐Arg7, and Leu5‐Arg6 bonds. With [3H]dynorphin A (1–9) as substrate the Arg7‐Ile8 and Ile8‐Arg9 bonds are also liable to cleavage. In binding assays, in contrast to the effects of peptidase inhibitors on the degradation of unbound [3H]dynorphin A (1–8) and [3H]dynorphin A (1–9), bestatin and captopril have little effect on the binding characteristics of the tritiated dynorphin A fragments at the k‐site at 0°C. However, at 25°C binding is low in the absence of peptidase inhibitors. When binding at μ‐ and δ‐sites is prevented, the maximal binding capacities of [3H]dynorphin A (1–8), [3H]dynorphin A (1–9), and [3H](–)‐bremazocine at the k‐site are similar; [3H]dynorphin A (1–9) has 5–10 times higher affinity for the k‐site than [3H]dynorphin A (1–8). Comparison of the effects of peptidase inhibitors on unbound dynorphin A fragments with their effects in binding assays suggests that the bound peptides are protected from the action of peptidases.

Pre-incubation of guinea-pig myenteric plexus with β-funaltrexamine: discrepancy between binding assays and bioassays

1 The acute effects of β‐funaltrexamine and the effects of pre‐incubation with this compound were examined in five in vitro assay tissues and in selective binding assays in homogenates of guinea‐pig brain and myenteric plexus. 2 In competitive displacement assays with selective ligands, β‐funaltrexamine had highest affinity for the μ‐binding site in the myenteric plexus and brain of guinea‐pig. Its affinity for the k‐site was about 15% of that for the μ‐site. 3 Pre‐incubation of the assay tissues with β‐funaltrexamine caused an increase in the IC50 values of μ‐and δ‐receptor agonists but not of k‐agonists. Although in bioassays on the myenteric plexus‐longitudinal muscle preparation of the guinea‐pig, the IC50 value of the μ‐receptor ligand [D‐Ala2, MePhe4, Gly‐ol5] enkephalin was increased up to 124 fold, its binding at the μ‐site in homogenates of the preparation was not affected by this treatment. 4 These findings indicate that the effects of pre‐incubation with β‐funaltrexamine on agonist potency of the μ‐receptor ligand are due to an interference with the coupling mechanism between the μ‐binding site and the effector system.

Radioligands for Probing Opioid Receptors

The three endogenous opioid precursors of almost 30000 Da are pro-opiocortin, proenkephalin and prodynorphin. Pro-opiocortin contains beta-endorphin, melanotropins and ACTH. Proenkephalin yields one [Leu5]enkephalin, three [Met5]enkephalins, one [Met5] enkephalyl-Arg-Arg-Val-NH2 (metorphamide or adrenorphin), one [Met5]enkephalyl-Arg-Gly-Leu and one [Met5]enkephalyl-Arg-Phe. [Leu5]enkephalin is common to all fragments of prodynorphin; its carboxyl extension by Arg-Lys leads to alpha- and beta-neo-endorphin and its carboxyl extension by Arg-Arg gives two dynorphins A and B of 17 and 13 amino acids, respectively. Another endogenous peptide is dynorphin A (1-8). The three main opioid binding sites are mu, delta and kappa. Their analysis has been facilitated by the synthesis of analogues of peptides and non-peptide compounds, which have selective agonist or antagonist action at only one site. The various physiological roles of the three types of the opiate receptor have so far not been sufficiently investigated.

Effects of electrical stimulation on teh enkephalinsin guinea-pig small intestine

Abstract When myenteric plexus-longitudnal muscle preparations of the guinea-pig small intestine are simulated electrically in the presence of cycloheximide there is a decrease in teh content of enkephalins which is taken to be due to their release. With supramaximal stimuli, the release per pulse is similar at 1 and 10 Hz but it is lower with submaximal than with maximal stimuli. Met-enkephalin is more readily released than Leu-enkephalin.

Hamster vas deferens contains δ-opioid receptors

Abstract Electrical field stimulation of the hamster, isolated vas deferens produces regular contractions that are unaffected by opioid-receptor agonists which are selective for the μ- or κ-receptor types. However, agonists which show a selectivity for the δ-opioid receptor produce dose-related inhibitions of the stimulation-evoked contractions. Responses of δ-opioid receptor agonists are antagonized in a competitive fashion by the selective δ-receptor antagonist ICI 174864, and also by naloxone. The “κ-opioid-agonist” benzomorphans, bremazocine and ethylketocyclazocine are antagonists in the hamster vas deferens; bremazocine is particularly potent in this regard. In conclusion, the hamster, isolated vas deferens may contain only δ-opioid receptors and provides a simple and specific test for the assay of activity at the δ-opioid receptor.