Linda E. Robson

CHARACTERIZATION OF THE k‐SUBTYPE OF THE OPIATE RECEPTOR IN THE GUINEA‐PIG BRAIN

1 In homogenates of guinea‐pig brain, the characteristics of the binding of [3H]‐ethylketazocine, an agonist for the putative k‐receptor, were determined by estimation of the affinity and capacity of binding, by competitive inhibition for the binding site by unlabelled ligands and by selective protection of the binding site from alkylation by phenoxybenzamine. 2 At 25°C the maximum number of binding sites for [3H]‐ethylketazocine was about 14pmol/g fresh brain, of which about 50% were high affinity sites. 3 In competition experiments, the high affinity binding of [3H]‐ethylketazocine to the k‐binding site was readily displaced by several k‐agonists but not by the selective μ‐ligand, D‐Ala2, MePhe4, Glyol5‐enkephalin or the selective δ‐ligand, D‐Ala2, D‐Leu5‐enkephalin. In contrast, the k‐agonists tested so far exhibit a high degree of cross‐reactivity with the μ‐binding site but somewhat less with the δ‐binding site. Similar specificities were observed in protection experiments. 4 The approximate proportions of the three subtypes of opiate receptor in the guinea‐pig brain are 25% μ‐binding sites, 45% δ‐binding sites and 30% k‐binding sites. 5 The endogenous opioids, Met‐enkephalin, Leu‐enkephalin and porcine β‐endorphin have only a low affinity for the k‐binding site.

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.

Opioid Peptides and their Receptors

The three agonists, methionine-enkephalin, leucine-enkephalin and beta-endorphin have different pharmacological patterns. It may be of particular importance that they vary in their relative affinities to the enkephalin and naltrexone binding sites in the brain; the former are probably related to delta-receptors prevalent in the mouse vas deferens and the later to mu-receptors prevalent in the guinea-pig. It is possible that mu-receptors are more important for the mediation of analgesic effects than delta-receptors. An understanding of the pharmacokinetics of the opioid peptides will be of basic importance for the design of enkephalin analogues suitable for use as analgesics in man.

Specific Protection of the Binding Sites of D-Ala

Phenoxybenzamine causes a long-lasting inactivation of the opiate receptors of the μ- and δ-type in homogenates of guinea-pig brain. This effect is selectively prevented when, before exposure to phenoxybenzamine, the homogenate is pre-incubated with ligands of high affinity for either of the two binding sites, i. e. dihydromorphine for the μ-receptor and Tyr-D-Ala-Gly-Phe-D-Leu for the δ-receptor. In contrast, Tyr-D-Ala- Gly-Phe-L-Leu amide, which has high affinities for both binding sites, protects both receptor sites.

^{2}

Binding at the mu, delta- and kappa-types of opioid binding sites was compared in homogenates from the brains of guinea-pig, rabbit, rat and two mouse strains, under conditions of selective labelling. Species differences were shown by two observations. Firstly, analysis of saturation curves in homogenates of brain from which the cerebellum had been removed showed that in guinea-pig brain the opioid binding sites consist of 24% mu-sites, 32% delta-sites and 44% kappa-sites. In contrast, in rabbit brain the corresponding values are 43% mu-sites, 19% delta-sites and 37% kappa-sites and in rat brain, 46% mu-sites, 42% delta-sites and 12% kappa-sites. In the brains of DBA/2 mice the opioid binding sites are comprised of 51% mu-sites, 29% delta-sites and 20% kappa-sites and in C57BL/10 mice, of 44% mu-sites, 35% delta-sites and 21% kappa-sites; these strain differences are due to significant differences in the concentrations of the mu-sites. Secondly, species differences were found when the binding of single concentrations of tritiated ligands (1 X KD value in whole brain) was determined at mu-, delta- and kappa-sites in six brain regions from guinea-pig, rat or rabbit.

-D-Leu

: By selective labelling techniques together with analysis of saturation curves it is shown that the concentrations of the mu-, delta- and chi-binding sites increase during postnatal development particularly in the first few weeks after birth. There are little or no changes in affinity. The rate of development is different for each type of site. The most striking finding is that the development of mu- and chi-sites precedes that of delta-sites.

^{5}

1 Isolated ilea from guinea‐pigs implanted with morphine pellets were stimulated coaxially, either with or without morphine present in the bath fluid, and the longitudinal contractions recorded. 2 In the absence of morphine the inhibitory effects of the presynaptic α‐adrenoceptor agonists, clonidine and oxymetazoline were much reduced and the dose‐response curve was flat. This state of ‘withdrawal’ was readily reversed by morphine and levorphanol but not its inactive (+)‐isomer, dextrorphan. 3 The κ‐agonists, ketazocine and ethylketazocine, also restored the effects of clonidine as did the opioid peptides Tyr‐d‐Ala‐Gly‐Phe‐d‐Leu, acting preferentially on δ‐receptors, and Tyr‐d‐Ala‐Gly‐MePhe‐Met(O)‐ol, acting mainly on μ‐receptors. 4 The inhibitory effects of adrenaline and adenosine 3′,5′‐diphosphate were reduced at low but not at high concentrations. 5 In contrast, the inhibitory effect of clonidine on the electrically evoked contractions of vasa deferentia from mice implanted with morphine pellets was not abolished by the lack of morphine in the bath fluid or by addition of naloxone. 6 A possible explanation is suggested for the loss of the inhibitory effects of presynaptic α‐adrenoceptor agonists in the withdrawn state of the dependent ileum.

-enkephalin (

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.

\delta

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.

-receptors) and Dihydromorphine (

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.