M. Wollemann

The kappa-opioid receptor: Evidence for the different subtypes

Classification of drugs acting on the kappa-opioid receptors seems to be difficult, since some of these ligands are also sigma agonists and/or display non-opioid actions as well. Furthermore, certain benzomorphans having kappa-agonistic character, are shown to be mu-antagonists too. Therefore the classification of the kappa-opioid receptor has to be presently restricted to two subclasses that also have physiological meaning. Dynorphin and Met-enkephalin-Arg6-Phe7 are proposed as endogenous peptide ligands for kappa-receptors. Nonpeptide agonists are benzeneacetamides interacting with the kappa1 receptor. Benzomorphans bind to both subtypes of kappa-receptors. No selective nonpeptide ligand for the kappa2 receptor exists as yet. Nor-binaltorphimine, a specific kappa-antagonist also inhibits both kappa-subtypes. Further research for kappa2 selective drugs is necessary for clear distinction between the two kappa-opioid binding sites. Molecular cloning of opioid receptors including their subtypes are expected to provide direct proof of their existence.

Met5-enkephalin-Arg6-Phe7, an endogenous neuropeptide, binds to multiple opioid and nonopioid sites in rat brain.

Receptor binding properties of the naturally occurring opioid heptapeptide MERF were studied in rat brain membrane preparations using tritium‐labeled derivative of the peptide with 40 Ci/mmol specific radioactivity. Binding assays were performed in the presence of broad‐spectrum peptidase inhibitors at 0°C. Under these conditions, the equilibrium binding was achieved in 30–40 min, and approximately 90% of the applied radioligand remained unchanged as determined by HPLC analysis. The apparent affinity (Kd value) of [3H]Met‐enkephalin‐Arg6‐Phe7, calculated from saturation binding data, was 10.2 ± 2.5 nM, and the maximal number (Bmax) of the heptapeptide binding sites was found to be 468 ± 43 fmol/mg protein. About half the sites represent nonopioid sites because the Bmax was only 255 ± 30 fmol/mg, when the nonspecific binding was measured with 1 μM naloxone. The rank order potencies of the examined compounds revealed that the opioid component of [3H]Metenkephalin‐Arg6‐Phe7 recognition sites are probably not μ and certainly not κ1 sites, whereas these sites are characterized by a κ2‐like binding profile. Considering the discrepancies between rat and frog brain found in the affinity of some compounds, including naltrindole and norbinaltorphimine, the presence of a novel, MERF‐selective “heptapeptide” binding site in rat brain membranes is also suggested. A number of the heterologous competition curves could be described by a high‐affinity stereospecific component and a substantially lower‐affinity binding element, which could completely be displaced with several peptide ligands such as Met5‐enkephalin, dynorphin(1–13), and unlabeled MERF but not by other compounds such as [D‐Ala2‐(Me)Phe4‐Gly5‐ol]enkephalin, morphine, or naloxone. [3H] Met‐enkephalin‐Arg6‐Phe7 binding can also be inhibited by FMRF‐amide analogs and sigma receptor ligands, such as (+)N‐allyl‐normetazocine and haloperidol, although with moderate affinity. It is concluded that the stereo‐specific high‐affinity binding is of opioid in character, whereas the residual sites characterized with their lower affinity are naloxone‐insensitive nonopioid sites. J. Neurosci. Res. 48:249–258, 1997.

Kinetics and Physical Parameters of Rat Brain Opioid Receptors Solubilized by Digitonin and CHAPS

Abstract: Rat brain opioid receptors were solubilized with digitonin and a zwitterionic detergent, 3‐[(3‐cholamido‐propyl)‐dimethylammonio]‐1‐propanesulfonate (CHAPS). The yield of solubilization was 70–75% with digitonin and 30–35% with CHAPS. Kinetic and equilibrium studies performed from digitonin extracts resulted in KD values comparable with those of the membrane fractions. Two [3H]naloxone binding sites were obtained in the extracts similarly to membrane fractions. The rank order potency of drugs used in the competition experiments did not change during solubilization. The distributions of μ, δ, and κ opioid receptor binding sites were similar in membrane and digitonin‐solubilized fractions (48–50%μ, 35–37%κ, and 13–17%δ subtypes). The hydrodynamic properties of digitonin‐ and CHAPS‐solubilized preparations were studied by sucrose density gradient centrifugation and Sepharose‐6B chromatography. In all cases, two receptor populations were identified with the following parameters: sedimentation coefficients for the digitonin extracts were 9.2S and 13.2S and for CHAPS extract 8S and 15.6S; the Stokes radii were 45Å and 65Å for the digitonin extract and 31Å and 76Å for the CHAPS‐solubilized preparation.

Solubilization and Characterization of Opioid Binding Sites from Frog (Rana esculenta) Brain

Abstract: Active opioid receptors were solubilized from frog (Rana esculenta) brain membrane fractions by the use of 1% digitonin. It was found by kinetic as well as by equilibrium measurements that both the membrane and the solubilized fractions contain two binding sites. For the membrane preparations, KD values were 0.9 and 3.6 nM, and Bmax values were 293 and 734 fmol/mg protein. For the solubilized preparations, KD values were 0.4 and 2.6 nM, and Bmax values were 35 and 266 fmol/mg protein. The stereospecificity of the binding did not change during solubilization. Both the membrane‐bound and the solubilized receptors showed weak binding of enkephalin and μ‐specific drugs, suggesting that they are predominantly of the k‐type. The membrane‐bound and the soluble receptors showed the same distribution of subtypes, i.e., 70%k, 13%μ, and 17%δ for the membrane‐bound and 71%k, 17%μ, and 12%δ for the soluble receptors

Characterization of kappa1 and kappa2 opioid binding sites in frog (rana esculenta) brain membrane preparation

The distribution and properties of frog brain kappa-opioid receptor subtypes differ not only from those of the guinea pig brain, but also from that of the rat brain. In guinea pig cerebellum the kappa1 is the dominat receptor subtype, frog brain contains mainly the kappa2 subtype, and the distribution of the rat brain subtypes is intermediate between the two others. In competition experiments it has been established that ethylketocyclazocine and N-cyclopropylmethyl-norazidomorphine, which are nonselective kappa-ligands, have relatively high affinities to frog brain membranes. The kappa2 ligands (Met5)enkephalin-Arg6-Phe7 and etorphine also show high affinities to the frog brain. Kappa1 binding sites measured in the presence of 5 μM /D-Ala2-Leu5/enkephalin represent 25–30% of [3H]ethylketocyclazocine binding in frog brain membranes. The kappa2 subtype in frog brain resembles more to the mu subtype than the delta subtype of opioid receptors, but it differs from the mu subtype in displaying low affinity toward beta-endorphin and /D-Ala2-(Me)Phe4-Gly5-ol/enkephalin (DAGO). From our data it is evident that the opioid receptor subtypes are already present in the amphibian brain but the differences among them are less pronounced than in mammalian brain.

The effect of capsaicin on the adenylate cyclase activity of rat brain

The effect of capsaicin on the adenylate cyclase activity in different regions of the rat brain (preoptic area of the hypothalamus, cerebral cortex and cerebellum) was investigated. Capsaicin added in vitro (10(-7)-10(-5) M) increased the adenylate cyclase activity of different brain regions. Following systemic capsaicin desensitization adenylate cyclase activity was significantly increased in the preoptic area. The enhanced adenylate cyclase activity in the preoptic area was inhibited by the vitro addition of capsaicin or 5-HT, whereas desensitization did not affect the in vitro activating effect of capsaicin in other brain regions (cerebral cortex, cerebellum). It is assumed that the pharmacological effect of capsaicin in the preoptic area is mediated through the activation of adenylate cyclase. Since capsaicin induces irreversible impairment of the function of warmsensitive hypothalamic neurons it is assumed that adenylate cyclase is involved in maintaining normal thermoregulatory functions.

Purification of a kappa-opioid receptor subtype from frog brain

A kappa-opioid receptor subtype was purified from a digitonin solubilized preparation of frog brain membranes using affinity chromatography. The affinity resin was prepared by coupling D-Ala2-Leu5-enkephalin to Sepharose-6B matrix. After elution of the receptor by 50 mumol naloxone, the kappa-subtype was separated from the mu- and delta-subtypes by gel permeation chromatography on Sepharose-6B. The purified receptor binds 3,900 pmol [3H]-ethylketocyclazocine per mg protein (a 4,300-fold purification over the membrane-bound receptor) with a KD of 8.3 nM. The purified receptor protein exhibits high affinity for kappa-selective ligands. The purified fraction shows two bands (Mr 65,000 and 58,000) in sodium dodecyl sulfate gel electrophoresis.

Tyr-D-Ala-Gly-(Me)Phe-chloromethyl ketone: A mu specific affinity label for the opioid receptor

An alkylating tetrapeptide enkephalin derivative, Tyr-D-Ala-Gly-(Me)Phe-chloromethyl ketone (DAMK) was synthesized, and its binding characteristics on rat brain membranes were evaluated. In competition experiments, the product shows high affinity for the mu opioid binding site of the rat brain membranes, whereas its binding to the delta and kappa subtypes is weak. Micromolar concentrations of this ligand produce a dose-dependent, apparently irreversible inhibition of /3H/-naloxone binding, with apparent IC50 value of 1-5 uM. Neither reversibly binding opioids nor tosyl-amino acid chloromethyl ketones show these effects. Saturation binding analysis with /3H/-naloxone of membranes preincubated with Tyr-D-Ala-Gly-(Me)Phe-CH2Cl reveal a selective and irreversible inhibition of the high affinity /3H/-naloxone binding site. Irreversible blockade of mu-selective /3H/-ligand binding by Tyr-D-Ala-Gly-(Me)Phe-CH2Cl is much more effective than that of the binding of /3H/-enkephalin or /3H/-ethylketocyclazocine. The mu-selective binding properties of this new irreversible enkephalin analogue suggest that it could serve as an affinity label for the mu opioid receptor subtype.

Binding characteristics and analgesic activity of D-Ala2-Leu5-enkephalin chloromethyl ketone

The chloromethyl ketone derivative of D-Ala2-Leu5-enkephalin (DALECK) was synthesized and its potency was tested in competing for 3H-naloxone binding sites and inducing analgesia. It was established that the compound is a potent affinity reagent at alkaline pH, blocking selectively and irreversibly the high-affinity (KD less than 1 nM) binding site. Intracisternally given DALECK showed a long-lasting, dose-dependent antinociceptive effect in the rat tail-withdrawal test. This could be completely antagonized by naloxone administration showing the reversible nature of DALECK in this in vivo assay. It is suggested that DALECK binds reversibly to the morphine receptor which mediates analgesia but irreversibly to the enkephalin receptor, the function of which remains to be elucidated.

Separation of κ-opioid receptor subtype from frog brain

Complete separation of the [3H]ethylketocyclazocine ([3H]EKC) specific binding (k subtype) from tritiated Tyr‐D‐Ala2‐Me‐Phe4‐Gly‐ol5 enkephalin (DAGO) and Tyr‐D‐Ala2‐L‐Leu5‐enkephalin (DALA) binding (μ‐and δ‐subtypes, respectively) was achieved by Sepharose‐6B chromatography and sucrose density gradient centrifugation of digitonin solubilized frog brain membranes. The apparent sedimentation coefficient (S 20,w) for the k receptor‐detergent complex was 13.1 S and the corresponding Stokes radius 64 Å. The isolated fractions exhibited high affinity for EKC and bremazocine, whereas μ‐ and δ‐specific ligands were unable to compete for the [3H]EKC binding sites, indicating that the κ subtype represents a separate molecular entity from the μ and δ receptor sites.