A.D. Corbett

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.

Inhibitory effects of certain enantiomeric cannabinoids in the mouse vas deferens and the myenteric plexus preparation of guinea‐pig small intestine

1 The psychoactive cannabinoids (−)‐Δ9‐tetrahydrocannabinol ((−)‐Δ9‐THC) and the 1,1‐dimethylheptyl homologue of (−)‐11‐hydroxy‐Δ8‐tetrahydrocannabinol ((−)‐DMH) both inhibited electrically‐evoked contractions of the mouse isolated vas deferens and the myenteric plexus‐longitudinal muscle preparation of the guinea‐pig small intestine. 2 Concentrations of (−)‐Δ9‐THC and (−)‐DMH that decreased twitch heights by 50% were 6.3 and 0.15 nm respectively in the mouse vas deferens and 60 nm and 1.4 nm respectively in the myenteric plexus preparation. (−)‐DMH was about 40 times more potent than (−)‐Δ9‐THC in both preparations, supporting the notion that their mode of action in each tissue is the same. 3 The psychically inactive cannabinoid, (+)‐DMH, had no inhibitory effect in the mouse vas deferens at a concentration of 30 nm, showing it to be at least 1000 times less potent than (−)‐DMH. In the myenteric plexus preparation, (+)‐DMH was about 500 times less potent than its (−)‐enantiomer. 4 The inhibitory effects of sub‐maximal concentrations of (−)‐Δ9‐THC were not attenuated by 300 nm naloxone. 5 The findings that (−)‐Δ9‐THC and (−)‐DMH are highly potent as inhibitors of the twitch response of the mouse vas deferens and guinea‐pig myenteric plexus preparation and that DMH shows considerable stereoselectivity suggest that the inhibitory effects of cannabinoids in these preparations are mediated by cannabinoid receptors.

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.

Bremazocine is an agonist at k-opioid receptors and an antagonist at μ-opioid receptors in the guinea-pig myenteric plexus

1 The agonist and antagonist activity of bremazocine at opioid receptors in the guinea‐pig myenteric plexus preparation was determined in untreated tissues and in tissues in which either μ‐ or k‐opioid receptors were blocked preferentially. 2 After pretreatment of the tissue with β‐funaltrexamine for 90 min followed by washing out, the IC50 value of the selective μ‐ligand [D‐Ala2,MePhe4,Gly‐ol5]enkephalin was increased 67 fold whereas the IC50 values of the selective k‐ligand U‐69,593 and of the non‐selective k‐ligand bremazocine were not significantly changed. In this experimental design bremazocine acted only on k‐receptors. 3 After pretreatment of the tissue with β‐chlornaltrexamine and 10 μM of the μ‐ligand for 30 min followed by washout, the IC50 value of the μ‐ligand was increased 2 fold whereas the IC50 value of the selective k‐ligand was increased 32 fold and that of bremazocine 62 fold. Under these experimental conditions, it was shown that bremazocine is an antagonist against [D‐Ala2,MePhe4,Gly‐ol5]enkephalin at the μ‐receptor (Ke. = 1.6 nM). The residual agonist activity of bremazocine is at the k‐receptor. 4 In naive myenteric plexus preparations the μ‐antagonist activity of bremazocine cannot be demonstrated because its potency at the k‐receptor is very high. This dual action may be of importance for the responses of bremazocine in other peripheral and central tissues.

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.

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.

Depressant effects of hypoxia and hypoglycaemia on neuro-effector transmission of guinea-pig intestine studied in vitro with a pharmacological model.

Since intermittent ischaemia may play an important role in the ætiology of Inflammatory Bowel Disease, particularly Crohns Disease, a pharmacological model of neuronal ischaemia was applied to guinea‐pig isolated intestinal preparations to mimic the acute effects of reduced blood flow on intestinal motility. Neuro‐effector transmission and smooth muscle performance were examined in myenteric plexus‐longitudinal muscle preparations of guinea‐pig ileum exposed to sodium cyanide (NaCN), in order to inhibit oxidative phosphorylation, or to iodoacetic acid (IAA), to block glycolysis. Comparisons were made with the effects due to simple deprivation of oxygen or glucose. Depressions of cholinergic neuro‐effector transmission induced by hypoxia or NaCN (effective concentration range 0.1–3 mm), given as separate treatments, singly or repetitively over 60–90 min, were apparent within 30 s and were reversible. The maximum inhibition was 90% and the IC50 for NaCN was 0.3 mm. A conspicuous component of these inhibitions was prejunctional. Non‐cholinergic neuro‐effector contractions were inhibited by up to 90% by anoxia or NaCN but recovery was incomplete and slower than with cholinergic contractions. Glucose‐free solutions also caused a reversible failure of cholinergic neuro‐effector transmission but of slower onset. In contrast, IAA (0.06–1 mm) abolished contractions irreversibly, apparently by a direct depressant effect on smooth muscle contraction. Unlike NaCN, IAA caused an initial potentiation of electrically‐induced contractions, partly by a prejunctional potentiation of cholinergic neuro‐effector transmission. It is concluded that a disruption of intestinal activity in pathological conditions associated with intestinal ischaemia may result from disturbances in the function of enteric neurones.

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.