dihydromorphine and bremazocine

Topics: Animals; Benzomorphans; Brain; Dihydromorphine; Ethylmaleimide; In Vitro Techniques; Naloxone; Rana esculenta; Receptors, Opioid; Receptors, Opioid, kappa

Opioid binding sites were localized in cryostat sections of guinea-pig brain by in vitro autoradiography using (-)-[3H]bremazocine. Quantification of binding sites in gray matter was accomplished using standard samples of tritium mixed in brain gray matter. The binding sites in fiber tracts were quantified using standards made from white matter to compensate for the quenching caused by myelin. Binding in gray matter corroborated previous findings that the moderately high densities of opioid sites in the cerebellum are of the kappa type, and that the V and VI laminae of the cerebral cortex, the substantia nigra, and olfactory bulb contain high levels of opioid sites in the guinea-pig. Several fiber tracts such as cerebellar white matter and the corpus callosum contained densities of (-)-[3H]bremazocine binding sites equal to, or higher than, the most densely labeled gray matter areas. The dorsal hippocampal commissure and the splenium of the corpus callosum contained 2200 fmol sites/mg protein, two and one half times more than the most densely labeled gray matter areas, the external plexiform layer of the olfactory bulb. These sites may be receptors in transport, but their density constitutes a massive volume of receptors for which a physiological role still needs to be more clearly defined.

Topics: Animals; Autoradiography; Benzomorphans; Brain; Dihydromorphine; Guinea Pigs; Neural Pathways; Rats; Receptors, Opioid; Species Specificity

Receptor binding experiments with masking of mu- and delta-receptors in the presence of an excess of unlabelled dihydromorphine and [D-Ala2, D-Leu5]enkephalin have been carried out. They indicate that 12 to 17% of original high affinity binding of [3H]Mr 2034 [-)(1R,5R,9R,2"S)-5,9-dimethyl-2-tetrahydrofurfuryl-2'-hydroxy-6, 7-benzomorphan hydrochloride), an opioid kappa-agonist, could then be detected as kappa-receptor sites in brain membranes both from untreated rats and from rats pretreated with naloxazone, too. Because of the irreversible blockade of the high affinity binding sites by naloxazone (naloxone hydrazone) treatment, the masking effects of mu- and delta-selective ligands seem to be mediated by the low affinity binding sites of these opioid receptor types. As could be shown before with [3H]Mr 2034, another kappa-agonist, [3H]bremazocine does not seem to be affected in its binding properties by naloxazone treatment of the rat in vivo. Displacement studies with several opioid agonists and antagonists, [3H]Mr 2034 and [3H]ethylketocyclazocine as radioligands in brain membranes from naloxazone treated rats and untreated controls provided further support for the evidence of two different kappa-receptors.

Topics: Animals; Benzomorphans; Binding, Competitive; Cyclazocine; Dihydromorphine; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Ethylketocyclazocine; In Vitro Techniques; Kinetics; Male; Morphinans; Naloxone; Rats; Receptors, Opioid; Receptors, Opioid, kappa

A new potent analgesic drug, 1-(1-phenylcyclohexyl)-4-phenyl-4-piperidinol (PCP-4-Ph-4-OH), derived from phencyclidine was tested for its interactions with different types of opioid receptors. The antinociceptive effect of PCP-4-Ph-4-OH in the mouse writhing test (ED50 = 0.3 mg/kg) is reversed by low doses of naloxone (pA2 = 6.98). The potency of PCP-4-Ph-OH in the inhibition of the electrically induced contractions of the guinea-pig ileum (IC50 = 17 nM) is 8-fold higher than that in the mouse vas deferens preparation (IC50 = 130 nM). The concentration of naloxone required to double the IC50 (Ke) of PCP-4-Ph-4-OH is 1.5 to 1.9 nM in both preparations. In opioid radioreceptor assays, PCP-4-Ph-4-OH displays 60- to-300 fold higher affinity for the [3H] dihydromorphine (mu) and D-[3H]Ala2-MePhe-Gly-ol5-enkephalin (mu) binding sites than for D-[3H]Ala2-D-Leu5-enkephalin (delta) sites in rat brain and [3H]bremazocine (kappa) sites in guinea-pig cerebellar membrane preparations. These results suggest that PCP-4-Ph-4-OH interacts with high affinity and selectivity with mu opioid receptors.

Topics: Analgesia; Animals; Benzomorphans; Binding, Competitive; Cerebellum; Dihydromorphine; Electric Stimulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Guinea Pigs; Ileum; Male; Mice; Muscle Contraction; Naloxone; Phencyclidine; Rats; Receptors, Opioid; Receptors, Opioid, mu; Vas Deferens

3H-Tifluadom labels specifically recognition sites of opioid kappa receptors. Membranes of guinea-pig whole brain bind 3H-tifluadom with two affinities, in contrast to the cerebellum where almost all opioid sites are kappa.

Topics: Animals; Benzodiazepines; Benzomorphans; Binding Sites; Brain; Cyclazocine; Dihydromorphine; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Ethylketocyclazocine; Guinea Pigs; In Vitro Techniques; Male; Morphine; Receptors, Opioid

The opiate agonists [3H]dihydromorphine (DHM, mu-selective ligand), [3H]bremazocine (potent kappa ligand), and [3H]etorphine bound stereospecifically, with high affinity, and reversibly to partially purified 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate (CHAPS)-solubilized extract from rat brain membranes. Recoveries of the three binding activities were as follows: [3H]DHM, 47%; [3H]bremazocine, 55%; and [3H]etorphine, 17%. Each ligand exhibited (by Scatchard analysis) binding to a class of high-affinity sites (Kd = 0.8-2 nM). Hill analyses revealed Hill coefficients of n = 1.1-1.3. Many of the properties of solubilized brain opiate receptors are similar to those of membrane-associated opiate receptors. Opiate binding in soluble fractions was inhibited by a variety of protein-modifying agents, including trypsin, proteinase K, and N-ethylmaleimide as well as by heat treatment (60 degrees, 15 min). The relative potencies of a series of opiate narcotic agonists and antagonists in displacing 2 nM [3H]etorphine binding to the CHAPS-solubilized extract was similar to that determined for rat brain homogenates. In contrast, D-Ala2, D-Leu5-enkephalin (DADLE, putative delta-selective ligand) exhibited a much lower affinity for solubilized brain opiate receptors than for the membrane-bound receptors unless assayed in the presence of manganese chloride, sodium chloride, and GTP. Mu agonist binding to solubilized receptors was inhibited relatively selectively by sodium and guanyl nucleotides. These findings lend support to the pharmacological relevance of the solubilized opiate-binding component(s). The pI of the solubilized brain opiate receptor(s) was estimated by liquid isoelectrofocusing to be pH 4. The sizes of the solubilized, prelabeled [3H]etorphine-receptor complex of the solubilized mu and kappa receptor subtypes, as assayed by stereospecific binding of [3H]DHM and [3H]bremazocine binding, respectively, were estimated by molecular exclusion chromatography. The [3H]etorphine-receptor complex migrated as a broad radioactive peak at a position corresponding to a protein of Stoke radius 63 A. A secondary peak of radioactivity was observed at the salt peak. Mu receptor activity chromatographed as two major peaks. The first of these eluted just behind, but significantly separated from, the protein void peak and corresponded to a Stokes radius of 70 A; the second eluted just ahead of the salt peak and corresponded to a radius of less than 20 A. Kappa rece

Topics: Animals; Benzomorphans; Brain Chemistry; Dihydromorphine; Etorphine; Isoelectric Focusing; Male; Molecular Weight; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, kappa; Receptors, Opioid, mu; Solubility