Photolabile opioid derivatives of D-Ala2-Leu5-enkephalin and their interactions with the opiate receptor

Photolabile derivatives of D-Ala2-Leu5-enkephalin were prepared by synthetic procedures in which a 2-nitro-4-azidophenyl group is linked to the terminal carboxyl group of the enkephalin by means of an ethylenediamine or ethylenediamine beta-alanine spacer. These peptides bind to opiate receptors with nanomolar affinities and inhibit electrically stimulated contractions of the mouse vas deferens and adenylate cyclase activity of NG108-15 neuroblastoma x glioma hybrid cell membranes. Both inhibitions are reversed by the opiate antagonist naloxone. Photolysis of the ligands bound to rat brain membranes results in the loss of approximately 50% of the receptor sites. This decrease in receptor number is blocked by naloxone and requires light. A photolabile [3H]enkephalin derivative labels an equivalent number of sites under similar irradiation conditions.     

Stability of opiate receptors of wet and lyophilized preparations of rat brain membranes

The effects of incubation of rat brain membranes at 0 degrees C on the specific binding of mu-ligands (naloxone, morphine) and the delta-ligand (D-Ala2, D-Leu5-enkephalin) to opiate receptors were studied. The effects of lyophilization of rat brain membranes on the properties of the opiate receptors were determined. The lyophilized brain membrane preparations revealed an extraordinarily high stability as compared to "wet" membranes. The experimental results suggest that morphine and D-Ala2, D-Leu5-enkephalin binding both to the high affinity and low affinity sites has different nature and point to the utility of stable and standard preparations of lyophilized membranes for the use in the receptor analysis of opiate and opioid peptides.     

Microsomal Opiate Receptors: Characterization of Smooth Microsomal and Synaptic Membrane Opiate Receptors

In continuing studies on smooth microsomal and synaptic membranes from rat forebrain, we compared the binding properties of opiate receptors in these two discrete subcellular populations. Receptors in both preparations were saturable and stereospecific. Scatchard and Hill plots of [3H]naloxone binding to microsomes and synaptic membranes were similar to plots for crude membranes. Both synaptic membranes and smooth microsomes contained similar enrichments of low- and high-affinity [3H]naloxone binding sites. No change in the affinity of the receptors was observed. When [3H]D-ala2-D-leu5-enkephalin was used as ligand, microsomes possessed 60% fewer high-affinity sites than did synaptic membranes, and a large number of low-affinity sites. In competition binding experiments microsomal opiate receptors lacked the sensitivity to (guanyl-5'-yl)imidodiphosphate [Gpp(NH)p] shown by synaptic and crude membrane preparations. In this respect microsomal opiate receptors resembled membranes that were experimentally guanosine triphosphate (GTP)-uncoupled with N-ethylmaleimide (NEM). Agonist binding to microsomal and synaptic membrane opiate receptors was decreased by 100 mM NaCl. Like NEM-treated crude membranes, microsomal receptors were capable of differentiating agonist and antagonists in the presence of 100 mM NaCl. MnCl2 (50-100 microM) reversed the effects of 100 mM NaCl and 50 microM GTP on binding of the mu-specific agonist [3H]dihydromorphine in both membrane populations. Since microsomal receptors are unable to distinguish agonists from antagonists in the presence of Gpp(NH)p, they are a convenient source of guanine nucleotide-uncoupled opiate receptors.     

Opiate-Inhibited Adenylate Cyclase in Rat Brain Membranes Depleted of Gs-Stimulated Adenylate Cyclase

Opiate agonists inhibit adenylate cyclase in brain membranes, but under normal conditions the maximal inhibition is small (10-15%). When rat brain membranes were preincubated at pH 4.5, washed, and then assayed for adenylate cyclase at pH 7.4, stimulation of activity by agents (fluoride, guanylyl-5'-imidodiphosphate, cholera toxin) that act through the stimulatory GTP-binding coupling protein (Gs) protein was lost. At the same time, inhibition of basal adenylate cyclase by opiate agonists was increased to a maximum of 30-40%. Opiate inhibition was maximal at low magnesium concentrations (less than 5 mM), required guanine nucleotides, and decreased the Vmax, not Km, of the enzyme. Incubation of membranes with pertussis toxin lowered the apparent affinity for agonists in inhibiting activity. The delta opioid agonists were more potent than mu agonists, and the Ke values for naloxone in blocking agonist inhibition were similar for both mu and delta agonists (50-90 nM). These results suggest that inhibition of adenylate cyclase in brain is not mediated by mu opiate receptors, but whether classic high-affinity delta and kappa receptors are involved with this enzyme cannot be confirmed by these experiments.     

Adrenocorticotropin- and opiate-like hormones from pituitaries of the sockeye salmon Oncorhynchus nerka

The pituitaries of vitellogenic sockeye salmon (Oncorhynchus nerka) were extracted with a mixture of acetone, water, and hydrochloric acid. The precipitate which formed upon the addition of a copious volume of acetone to the extract, designated acid acetone powder, was subjected to salt fractionation and desalting, followed by ion-exchange chromatography on CM-cellulose. An unadsorbed fraction (S-1) and four adsorbed fractions (S-2, S-3, S-4 and S-5) were obtained. Adrenocorticotropic activity was detected in the fractions by their ability to stimulate isolated rat adrenal decapsular cells to produce corticosterone and by their immunoreactivities in an adrenocorticotropin-specific radioimmunoassay. The steroidogenic activities of all fractions, except S-4, were blocked by corticotropin inhibiting peptide. Opiate activity was detected in the fractions by their ability to inhibit the binding of either [3H]naloxone or (D-ala2, D-leu5)-[3H]enkephalin to rat brain membranes. There was a discrepancy in the potencies of the five fractions in the two opiate radioreceptor assays, indicating the presence of opiate peptides with different affinities of binding to the micron- and delta-opiate receptors of the rat brain. There was a separation between adrenocorticotropic and opiate receptor binding activities, suggesting that the activities were due to separate molecular entities.     

5-[4-(3,3-Dimethylbutoxycarbonyl)phenyl]-4-pentynoic acid and its derivatives inhibit ionotropic gamma-aminobutyric acid receptors by binding to the 4'-ethynyl-4-n-propylbicycloorthobenzoate site

Acyclic noncompetitive antagonists of ionotropic gamma-aminobutyric acid (GABA) receptors, bearing an ester or ether linkage, were designed, synthesized, and assayed for their inhibition of the specific binding of [3H]4'-ethynyl-4-n-propylbicycloorthobenzoate (EBOB), a radiolabeled noncompetitive antagonist, to rat brain and housefly head membranes. 5-[4-(3,3-Dimethylbutoxycarbonyl)phenyl]-4-pentynoic acid (DBCPP), a butyl benzoate analogue, was found to competitively inhibit the binding of [3H]EBOB in rat brain membranes, with an IC50 of 88 nM. The potency conferred by the p-substituent decreased in the order C(triple bond)C(CH2)2COOH > C(triple bond)C(CH2)2COOCH3 > C(triple bond) CH > Br. Pentyl phenyl ethers were equally potent compared with butyl benzoates, while phenyl pentanoates and benzyl butyl ethers were less pont. These compounds were generally less active in housefly head membranes than in rat brain membranes. The introduction of an isopropyl group into the 1-position of the 3,3-dimethylbutyl group of a butyl benzoate and two benzyl butyl ethers caused an increase in potency in housefly GABA receptors, whereas this modification at the corresponding position of other compounds led to an unchanged or decreased potency. In the case of rat receptors, this modification resulted in a decrease in potency except for a phenyl pentanoate. To confirm that DBCPP interferes with GABA receptor function, we performed whole-cell patch clamp experiments with rat dorsal root ganglion neurons in the primary culture. Repeated co-applications of GABA and DBCPP suppressed GABA-induced whole-cell currents with an IC50 of 0.54 microM and a Hill coefficient of 0.7. These findings indicate that DBCPP and its derivatives inhibit ionotropic GABA receptors by binding to the EBOB site and that there might be structural difference in the noncompetitive antagonist-binding site between rat and housefly GABA receptors.     

pH Selectivity of N-Ethylmaleimide Reactions with Opiate Receptor Complexes in Rat Brain Membranes

N-Ethylmaleimide (NEM) decreases opiate agonist binding presumably by blocking crucial sulfhydryl (SH) groups at receptor binding sites. At physiological pH, NEM decreased GTP and manganese regulation but increased sodium effects on [3H]D-Ala2-Met5-enkephalinamide (D-Ala enk) binding to rat brain membranes. To determine the apparent pK values of putative SH groups in opiate receptors that react with NEM, rat brain membranes were incubated with 100-250 microM NEM in buffers ranging from pH 4.5 to 8.0. Results showed that lowering pH below 6.5 reduced the NEM effect on opiate receptor functions and that the apparent pK values of NEM-reacting SH groups in binding and regulatory sites ranged between 5.4 to 6.0. Most of the total SH groups in brain membranes continued to react with NEM at low pH, so that when nonspecific SH groups were blocked by incubating membranes at pH 4.5 with NEM, opiate receptors became sensitive to very low concentrations (1 microM) of NEM.     

Binding sites of opiates and endogenous opioids in the oocytes of the toad Bufo viridis

The binding of labelled naloxone, morphine and (D-Ala2,D-Leu5)enkephalin (DADL) to oocyte membranes of the toad Bufo viridis was investigated. The opiate antagonist naloxone binds to the membranes much more effectively than morphine or DADL. The binding of [3H]naloxone is reversible and saturating. The bound [3H]naloxone is readily replaced by unlabelled naloxone or bremazocine (kappa-agonist), far less effectively by morphine (mu-agonist) and SKF 10.047 (sigma-agonist) and is not practically replaced by DADL (delta-agonist), beta-endorphin (epsilon-agonist) and other neuropeptides. Analysis of experimental results in Scatchard plots revealed two types of binding sites with a high (Kd = 15 nM) and low (Kd = 10(3) nM) affinity for naloxone. The number of sites responsible for the binding of naloxone possessing a high affinity is 16 pmol-/mg of oocyte homogenate protein, i.e., 20-50 times as great as in the toad or rat brain. Trypsin and p-chloromercurybenzoate decrease the binding of [3H]naloxone. The oocyte extract is capable of replacing the membrane-bound [3H]naloxone, on the one hand, and of inhibiting the smooth muscle contracture of the rabbit vas deferens, on the other. This inhibition is reversed by naloxone and can also be induced by bremazocine, but not by morphine, DADL and SKF 10.047. In all probability oocytes contain compounds that are similar to opiate kappa-agonists. It may also be possible that these compounds mediate their effects via specific receptors and are involved in the control over maturation of oocytes and early development of toad eggs.     

3-[18F]Acetylcyclofoxy: a useful probe for the visualization of opiate receptors in living animals

A fluoro-analogue of the potent narcotic antagonist, naltrexone, was synthesized and shown to bind with high affinity to opiate receptors in vitro. 3-[18F]acetylcyclofoxy was prepared via a one-step triflate displacement reaction with the positron emitting 18F ion from tetraethylammonium [18F] fluoride. 3-[18F]acetylcyclofoxy accumulation in opiate receptor rich brain regions of both rat and baboon is shown to be completely displaced by the active enantiomer of naloxone [-)-naloxone) while the identical dose of the pharmacologically inert (+)-naloxone has no detectable effect. Moreover, both rat and baboon brain showed the well documented, typical opiate receptor distribution so that basal ganglia and thalamus are clearly visible in the living baboon brain up to 95 min after intravenous injection of 3-[18F] acetylcyclofoxy. We expect that 3-[18F )acetylcyclofoxy will be a useful probe for visualizing opiate receptors in living humans.     

Opiate binding sites and endogenous opioids in Bufo viridis oocytes

Binding sites with high affinity for [3H]naloxone, but not for [3H]morphine and [3H] (D-Ala2, D-Leu5) enkephalin, have been found in membranes of Bufo viridis oocytes. The binding is reversible and saturable. Bound [3H]naloxone is easily displaced both by unlabeled naloxone and bremazocine, much worse by morphine and SKF 10,047; (D-Ala2, D-Leu5) enkephalin and beta-endorphin practically fail to displace [3H]naloxone. Scatchard analysis is consistent with the existence of two classes of binding sites with Kd 15 nM and 10(3) nM. The number of binding sites with high affinity for naloxone is 16 pmol/mg protein of homogenized oocytes which is 20-50-fold higher than in, toad or rat brain. Oocyte extract displaces [3H]naloxone bound with oocytes' membranes and inhibits electrically evoked contractions of the rabbit vas deferens. This inhibition is reversed by naloxone. It is suggested that compounds similar to opiate kappa-agonists exist in oocytes. It cannot be ruled out that they participate via specific receptors in the regulation of oocyte maturation and egg development.     

Characterization of [3H]Met-enkephalin-Arg6-Phe7 binding to multiple sites in rat and guinea pig cerebellum

[3H]Met-enkephalin-Arg6-Phe7 (MERF) has been shown to label opioid (kappa2 and delta) and sigma2 sites in rat and frog brain membrane preparations, and no specific binding to kappa1 opioid receptors could be established (refs. 6 and 8). In this study the binding was examined in rat cerebellar membranes which are relatively rich in kappa2-sites, and in guinea pig cerebellar preparations where kappa1 opioid receptors are almost exclusively present. In accordance with our previous results, [3H]MERF binding could not be displaced in guinea pig cerebellar membranes neither with U-69,593 nor with naloxone or levorphanol suggesting no interaction with opioid sites, nevertheless a Kd of 2.8 nM was calculated in cold saturation experiments. In rat cerebellar membrane fractions about the half of the specific [3H]MERF binding sites was inhibited by opiate alkaloids such as naloxone, ethylketocyclazocine, or bremazocine. This portion of the heptapeptide binding sites was stereoselective as demonstrated by the difference in the affinities of the enantiomeric compounds levorphanol and dextrorphan, therefore it would represent an opioid site. In both tissues (-)N-allyl-normetazocine (SKF-10,047), which is also considered as sigma2 ligand, displayed the highest affinities. Among opioid peptides beta-endorphin and dynorphin(1-13) showed the highest potencies, displacing [3H]MERF also from its non-opioid sites. It was concluded therefore that [3H]MERF does not bind to kappa1 sites, and besides kappa2-opioid sites substantial binding to peptide preferring non-opioid sites, and/or sigma2 receptors also occurs.     

Effect of apomorphine on rat brain opiate receptors

Stereospecific binding of apomorphine to rat brain opiate receptors was shown by assaying the competition of 7,8(n)--3H--naloxone and D-ala2-tyrosyl-3,5-3H--enkephalin (5-D-leucine) for opiate receptor binding. EC-NaCl50, the concentration of apomorphine which inhibited 50% binding of the radioactive naloxone and D-ala2, D-leu5-enkephalin in the absence of NaCl were 20 and 42 microM, respectively. EC+NaCl 50, the concentration of apomorphine which inhibited 50% binding of the radioactive naloxone in the presence of 100 mM NaCl was 17 microM. From the ratio of EC+NaCl 50 to EC-NaCl the value of "sodium shift" of effective concentration can be calculated as 0.85. From the data obtained it is concluded that apomorphine, like naloxone, is a "pure" antagonist but it has much less affinity for enkephalin and opiate binding sites. The probable mechanisms of the pharmacological action of apomorphine are discussed.     

Receptor-mediated stimulation of brain GTPase by opiates in normal and dependent rats

In membranes from rat brain striatum, opiate agonists stimulated low-K GTPase. Half-maximal enhancement of enzyme activity was obtained with 0. 09m microM morphine and 3.8 microM levorphanol. This order of potency corresponded to that of the affinities of these compounds in binding to opiate receptor. The effect was inhibited by the antagonist naloxone. As shown by the use of the enantiomers levorphanol and dextrorphan, only the pharmacologically active stereoisomer stimulated GTPase. In membranes isolated from morphine-dependent rats, the activity of GTPase was reduced 20-40% relative to that in control rats. After the precipitation of morphine abstinence by naloxone, brain GTPase activity was intermediate between the respective values for naive and dependent animals.     

Evidence for coupling of the kappa opioid receptor to brain GTPase

In membranes from guinea pig cerebellum, a tissue which predominantly contains kappa opioid receptors, exogenous and endogenous kappa-selective opioid agonists stimulated low-km GTPase activity by 11-20% with concentrations for half-maximal stimulation of 3-23 microM. Opioid ligands of the mu and delta type had no effect on GTPase in these membranes. Similar stimulation of GTPase by kappa opiates was obtained in rat and monkey brain membranes pretreated with beta-funaltrexamine (beta-FNA) and cis-(+/-)-3-methylfentanyl isothiocyanate (superfit) to alkylate the mu and delta receptors, respectively. The stimulation of brain GTPase by kappa opiates in both types of membranes was inhibited by naloxone with IC50's of 0.35 microM and 0.40 microM. The results demonstrate the coupling of the kappa opioid receptor to high affinity GTPase, the Ni regulatory protein of the adenylate cyclase complex.     

Binding characteristics of a series of dimeric tripeptide enkephalins for delta opiate receptors in rat brain and NG108-15 cells

The N-terminal tripeptide enkephalin analogue, Tyr-D-Ala-Gly, was dimerized at the C-terminus systematically with a series of alpha,omega-diaminoalkanes, NH2-(CH2)n-NH2 (n = 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22). The binding affinities of dimers for delta opiate receptors in rat brain were evaluated and compared with those for delta receptors in NG108-15 cells. Although the monomeric tripeptide amide was almost inactive, dimers showed a dramatic increase in binding affinity (8-900 times). The enhancement of affinity was apparently related to the number of methylene chains in the crosslinking spacer moiety, and it was maximal at n = 14-18 in the rat brain. In NG cells the activity increased progressively from n = 2 to n = 22 without reaching any apparent peak. These results suggest that delta receptors in rat brain and NG cells may have slight structural differences.     

A novel analogue of clonidine with opiate-receptor agonist activity

A new analogue of the α₂-adrenergic receptor ligand clonidine, N-(4-hydroxphenacetyl)-4-aminoclonidine, was synthesized. The analogue possesses opiate-receptor agonist activity in addition to α-adrenergic partial agonist activity. The analogue elicits inhibition of adenylate cyclase of NG108-15 neuroblastoma × glioma hybrid cells; most of the inhibition is reversed by the opiate-receptor antagonist naloxone. The analogue also inhibits the binding of [³H]D-Ala²-Met⁵-enkephalinamide and [³H]dihydromorphine to rat brain opiate receptors. The structure of the analogue suggests common elements in the ligand binding sites of α- and opiate receptors and may lead to a new class of opiate analgesics.     

Dimeric pentapeptide enkephalin: a novel probe of delta opiate receptors

A dimeric pentapeptide enkephalin (DPE2) consisting of two molecules of [D-Ala 2, Leu 5] enkephalin linked at C-terminal leucine with ethylenediamine, (H-Tyr-D-Ala-Gly-Phe-Leu-NH-Ch2)2 is a bivalent ligand for the delta enkephalin receptors of rat brain and neuroblastoma-glioma hybrid (NG108-15) cells. This new enkephalin analog shows dramatically increased affinity in radioligand assays using whole brain membranes when delta but not mu specific radioligands are employed. When membranes from NG108-15 cells are used, the dimer shows greatly increased activity irrespective of the mu or delta specificity of the tracer. The dimer DPE2 shows a four-fold, "sodium shift" in its IC50 for competition with [3H]naloxone, suggestive of agonist behavior. Agonist activity was confirmed by demonstrating that DPE2 inhibits cyclic AMP production in prostaglandin E1 stimulated NG108-15 cells, and by demonstrating very high potency in the mouse vas deferens bioassay. DPE2 binds to the same delta sites as the delta-selective monomer [D-Ala2, D-Leu5] enkephalin, since the two ligands show complete crossdisplacement. Radiolabeled 3H-DPE2 shows a five-fold higher affinity constant, a 2.5-fold higher association rate constant, and a two-fold lower dissociation rate than the monomer. These results are consistent with the hypothesis that the dimeric pentapeptide enkephalin can bridge two delta receptors. This enkephalin dimer provides a valuable new probe of opiate receptors and their organization in cell membranes.     

Receptor-related interactions of opiates with PGE-induced adenylate cyclase in brain

The inhibition by opiates of the PGE2-induced formation of cAMP in slices from rat brain striatum was investigated. A maximal, 3.5-fold increase over the basal level of cAMP was obtained with an EC50 for PGE2 of 3 microM. Opiate agonists of both mu and kappa type were inhibitory. The IC50 values for morphine, levorphanol and ethylketocyclazocine (EKC) were 110 nM, 80 nM and 25 nM, respectively. These values were similar to the potencies of the compounds in displacing stereospecifically bound 3H-etorphine in rat brain membranes. As evidenced by the inactivity of dextrorphan, the inhibition of PGE2-dependent cAMP formation was stereospecific. Also ineffective were the opiate antagonists naloxone, naltrexone and MR 2266. These compounds did, however, reverse the inhibition by agonists, displaying thereby selectivity toward the putative mu and kappa opiates. Thus, the inhibition by morphine was antagonized to a greater degree by naloxone than by MR 2266, and the action of EKC was blocked more effectively by MR 2266 relative to naloxone.     

Interaction of [3H](–)-SKF-10,047 with Brain σ Receptors: Characterization and Autoradiographic Visualization

The sigma opiates differ from other opiates in their stimulatory and psychotomimetic actions. The sigma opiate [3H](-)-SKF-10,047 has been used to characterize sigma receptors in rat nervous tissue. Binding of [3H](-)-SKF-10,047 to rat brain membranes was of high affinity, saturable, and reversible. Scatchard analysis revealed the apparent interaction of this drug with two distinct binding sites characterized by affinities of 0.03 and 75 nM (5 mM Tris-HCl buffer, pH 7.4, at 4 degrees C). Competition analyses involving rank order determinations for a series of opiates and other drugs indicate that the high-affinity binding site is the mu opiate receptor. The lower-affinity site (revealed after suppression of mu and delta receptor binding) has been identified as the sigma opiate/phencyclidine receptor. In vitro autoradiography has been used to visualize neuroanatomical patterns of receptors labeled using [3H](-)-SKF-10,047 in the presence of normorphine and [D-Ala2,D-Leu5]enkephalin to block mu and delta interactions, respectively. Labeling patterns differ markedly from those for mu, delta, or kappa receptors. The highest densities (determined by quantitative autoradiography) are found in the medial portion of the nucleus accumbens, amygdaloid nucleus, hippocampal formation, central gray, locus coeruleus, and the parabrachial nuclei. Receptors in these structures could account for the stimulatory, mood-altering, and analgesic properties of the sigma opiates. Although not the most selective sigma opiate ligand, [3H](-)-SKF-10,047 binds to sigma opiate receptors in brain, and this interaction can be readily distinguished from its interactions with other classes of brain opiate receptors.     

Solubilization in good yield of active opiate binding sites from mammalian brain

Active opiate binding sites have been solubilized from mammalian brain cell membranes. The presence of 0.5-0.1 M NaCl during treatment of membranes from rat brain, human frontal cortex, and bovine corpus striatum with glycodeoxycholate or digitonin resulted in the extraction of active opiate binding sites in yields ranging up to 43%. The criteria for solubility of the sites were their inability to sediment at 10(5) x g after 2 hr and their apparent molecular weight of 3- 4 x 10(5) as determined by gel filtration. The receptors in solution resemble the membrane-bound sites with respect to saturability, stereo-specificity, sensitivity to heat and reagents, and high affinity for opioid ligands. The interaction of solubilized sites with immobilized lectins was used to demonstrate the glycoprotein nature of the opiate receptor. Soluble receptors from all species studied were retained by wheat germ agglutinin(WGA)-agarose and could be specifically eluted with N-acetylglucosamine. No retention of solubilized material was observed with eight other lectins examined, including horseshoe crab lectin, a sialic acid specific agglutinin. The receptor protein eluted from WGA columns was enriched 25-50-fold over the crude soluble fraction.