Sándor Benyhe

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

Morphine: New aspects in the study of an ancient compound

Morphine is the most widely used compound among narcotic analgesics and remains the gold standard when the effects of other analgetic drugs are compared. Apart from its presence in the poppy plant Papaver somniferum, morphine has been shown to be present in milk, cerebrospinal fluid and also in nervous tissue extracts. Recent evidence suggests that biosynthetic pathways for morphine exist in animal and even human tissues such as liver, blood and brain. The most characteristic effect of morphine is the modulation of pain perception resulting in an increase in the threshold of noxious stimuli. Antinociception induced by morphine is mediated via opioid receptors and therefore can be inhibited by opioid antagonists, e.g., naloxone. Nevertheless, consideration of morphine as endogenous ligand for opioid receptors seems to be speculative. Recently, the primary receptor for morphine-type drugs called the mu-opioid receptor has been cloned from rat brain. There is accumulating evidence that morphine actions are, at least partly, due to one of its major metabolite morphine-6-glucuronide in man. It is concluded that further investigations are necessary to elucidate the mechanisms, whereby multiple actions of morphine are expressed in the nervous system.

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.

Noladin ether, a putative endocannabinoid, inhibits μ-opioid receptor activation via CB2 cannabinoid receptors

We examined the occurrence of possible changes in mRNA expression and the functional activity of opioid receptors after acute in vivo and in vitro treatment with the putative endogenous cannabinoid noladin ether. While noladin ether (NE) demonstrates agonist activity at CB1 cannabinoid receptors, recent data indicate that NE acts as a full agonist at CB2 cannabinoid receptors too. Considering the functional interactions between opioids and cannabinoids, it is of interest to examine whether NE affects the opioid system. To that end, we studied the influence of NE on mu-opioid receptor (MOR) mRNA expression and MOR mediated G-protein signaling. We used real-time PCR and [35S]GTPgammaS binding assays to examine the changes of MOR mRNA levels and the capability of the mu-opioid agonist peptide ([D-Ala2,(NMe)Phe4,Gly5-ol]enkephalin (DAMGO) in activating regulatory G-proteins via MORs in forebrain membrane fractions of wild-type (w.t., CB1+/+) and CB1 receptor deficient transgenic mice (knockout, CB1-/-). We found, that the expression of MOR mRNAs significantly decreased both in CB1+/+ and CB1-/- forebrain after a single injection of NE at 1 mg/kg when compared to control. Consequently, MOR-mediated signaling is attenuated after acute in vivo treatment with NE in both CB1+/+ and CB1-/- mice. Inhibition on MOR mediated activation is observed after in vitro NE administration as well. Radioligand binding competition studies showed that the noticed effect of NE on MOR signaling is not mediated through MORs. Both in vivo and in vitro attenuations of NE can be antagonized by the CB2 selective antagonist SR144528. Taken together, our data suggest that the NE caused pronounced decrease in the activity of MOR is mediated via CB2 cannabinoid receptors.

CB2 cannabinoid receptor antagonist SR144528 decreases mu-opioid receptor expression and activation in mouse brainstem: Role of CB2 receptor in pain

Formerly considered as an exclusively peripheral receptor, it is now accepted that CB(2) cannabinoid receptor is also present in limited amounts and distinct locations in the brain of several animal species, including mice. However, the possible roles of CB(2) receptors in the brain need to be clarified. The aim of our work was to study the mu-opioid receptor (MOR) mRNA expression level and functional activity after acute in vivo and in vitro treatments with the endocannabinoid noladin ether (NE) and with the CB(2) receptor antagonist SR144528 in brainstem of mice deficient in either CB(1) or CB(2) receptors. This study is based on our previous observations that noladin ether (NE) produces decrease in the activity of MOR in forebrain and this attenuation can be antagonized by the CB(2) cannabinoid antagonist SR144528, suggesting a CB(2) receptor mediated effect. We used quantitative real-time PCR to examine the changes of MOR mRNA levels, [(35)S]GTPgammaS binding assay to analyze the capability of mu-opioid agonist DAMGO to activate G-proteins and competition binding assays to directly measure the ligand binding to MOR in mice brainstem. After acute NE administration no significant changes were observed on MOR signaling. Nevertheless pretreatment of mice with SR144528 prior to the administration of NE significantly decreased MOR signaling suggesting the involvement of SR144528 in mediating the effect of MOR. mRNA expression of MORs significantly decreased both in CB(1) wild-type and CB(1) knockout mice after a single injection of SR144528 at 0.1mg/kg when compared to the vehicle treated controls. Consequently, MOR-mediated signaling was attenuated after acute in vivo treatment with SR144528 in both CB(1) wild-type and CB(1) knockout mice. In vitro addition of 1microM SR144528 caused a decrease in the maximal stimulation of DAMGO in [(35)S]GTPgammaS binding assays in CB(2) wild-type brainstem membranes whereas no significant changes were observed in CB(2) receptor knockouts. Radioligand binding competition studies showed that the noticed effect of SR144528 on MOR signaling is not mediated through MORs. Our data demonstrate that the SR144528 caused pronounced decrease in the activity of MOR is mediated via CB(2) cannabinoid receptors.

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.