dextromethorphan and ropizine

Dextromethorphan (DM) binds to high- and low-affinity sites in the rat brain. The high-affinity DM binding is inhibited by nonnarcotic antitussives, opipramol and sigma ligands with nanomolar affinities. Computer-assisted modeling of homologous and heterologous competition studies between DM and (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [(+)-3-PPP] were performed at pH 8.4. These experiments confirmed the existence of the common high-affinity DM1/sigma 1 site (R1) for which DM and (+)-3-PPP have Kd values of 20 and 10 nM, respectively. DM also binds to a second high-affinity site (R2, Kd, 20 nM) for which (+)-3-PPP has only micromolar affinity. Similarly, (+)-3-PPP binds to another high-affinity site (R3, Kd, 60 nM) for which DM has micromolar affinity. The common high-affinity DM1/sigma 1 site is allosterically modulated by the anticonvulsant ropizine, and is (+)-pentazocine sensitive, as is the homologous site in the guinea pig. However, in the rat the common DM1/sigma 1 site is 10 times smaller than in the guinea pig. This explains the apparently different effects of the allosteric modifiers in both species. The multiplicity of binding sites for DM and (+)-3-PPP resolved in this investigation will help to establish the physiological role and the pharmacological potential of the different sites. Meanwhile, the pharmacological effects of DM and sigma ligands cannot be summarily attributed to any particular binding site or receptor. This investigation also demonstrates that the use of multiple labeled and unlabeled ligands, combined with computer-assisted modeling, is essential to resolve multiple binding sites with similar affinities and to characterize the complex effects of allosteric modifiers.

Topics: Allosteric Regulation; Animals; Antitussive Agents; Binding Sites; Brain; Computer Simulation; Dextromethorphan; Guinea Pigs; Hydrogen-Ion Concentration; Male; Pentazocine; Piperazines; Piperidines; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, sigma

Computer-assisted analysis of self- and cross-displacement studies between dextromethorphan (DM) and (+)-3-(3-hydroxyphenyl)-N-(1-propyl) piperidine ((+)-3-PPP) demonstrated in the rat brain the existence of two high-affinity and one low-affinity binding site for each ligand. One high-affinity site is the common DM1/sigma 1 site, the affinity of which is allosterically increased 4 to 5-fold by 10 microM ropizine. The Kd values of the DM1/sigma 1 for DM and (+)-3-PPP are 17 and 11 nM respectively. DM binds to the second high-affinity site (R2) with a Kd of 15 nM; this site has low affinity for (+)-3-PPP. Conversely, (+)-3-PPP binds with high affinity (Kd 53 nM) to another site (R3), that has low-affinity for DM. The Bmax of the common DM1/sigma 1 site in the rat is about ten times smaller than that in the guinea pig. Thus, extreme caution should be exercised in extrapolating from one species to another. Since DM and most sigma ligands bind to more than one site, not all of which are shared, it is important not to attribute the complex pharmacological effects of these ligands to a single hypothetical receptor.

Topics: Allosteric Site; Animals; Binding, Competitive; Brain; Dextromethorphan; Electronic Data Processing; In Vitro Techniques; Kinetics; Models, Biological; Pentazocine; Piperazines; Piperidines; Rats; Rats, Inbred Strains; Tritium

Topics: Allosteric Regulation; Animals; Autoradiography; Binding Sites; Brain; Dextromethorphan; Guinea Pigs; In Vitro Techniques; Levorphanol; Piperazines; Piperidines; Tissue Distribution

Ropizine (10 microM) produces a simultaneous enhancement and inhibition of [3H]dextromethorphan (DM) high-affinity binding to different areas of the guinea pig brain. These results imply that there are two distinct types of high-affinity [3H]DM binding sites, which are present in variable proportions in different brain structures. The ropizine-enhanced [3H]DM binding type was preferentially inhibited by (+)-pentazocine. This is consistent with the presumption that the (+)-pentazocine-sensitive site is identical with the common site for DM and 3-(-3-Hydroxyphenyl)-N-(1-propyl)piperidine ((+)-3-PPP). The second binding type, which is inhibited by ropizine and is not so sensitive to (+)-pentazocine, has not been fully characterized. This study demonstrates that the biphasic effects of ropizine are due, at least in part, to the effects of ropizine on two different types of [3H]DM binding sites. However, this study does not rule out that common DM/(+)-3-PPP site also might be inhibited by higher concentrations of ropizine.

Topics: Animals; Anticonvulsants; Autoradiography; Binding Sites; Brain; Dextromethorphan; Guinea Pigs; Levorphanol; Pentazocine; Piperazines

Equilibrium binding analysis demonstrated that (+)-[3H]3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [(+)-[3H]3-PPP] binds in guinea pig brain homogenates to high and low affinity sites with Kd values of 25 nM and 0.9 microM, respectively. Competition studies with dextromethorphan (DM) site ligands and other drugs against (+)-[3H]3-PPP demonstrated that their Ki values and rank order of potency are identical to those found previously against [3H] DM. Most significant, ropizine produced a concentration-dependent increase in the binding of (+)-[3H]3-PPP, with an inhibitory component at high concentrations, as described previously for [3H]DM. Similarly, phenytoin increased the binding of (+)-[3H]3-PPP in the same fashion as that of [3H]DM. Computer-assisted analysis of equilibrium binding of (+)-[3H]3-PPP in the presence of 10 microM ropizine demonstrated that the binding increase produced is due to a 3-fold increase in the affinity for (+)-[3H]3-PPP. These results, and our previous finding that sigma ligands inhibit [3H] DM binding with a rank order of potency similar to that for sites labeled with (+)-[3H]3-PPP or (+)-[3H]SKF10,047 strongly suggest that sigma ligands bind to the high affinity DM site. These findings, and the inability of DM and other antitussives to produce psychotomimetic side effects, suggest that the high affinity DM sites can mediate only the nonpsychotomimetic effects of sigma ligands. However, further studies are necessary to determine the physiological role and therapeutic potential of the DM high affinity sites.

Topics: Allosteric Regulation; Animals; Binding Sites; Binding, Competitive; Dextromethorphan; Guinea Pigs; In Vitro Techniques; Levorphanol; Phenytoin; Piperazines; Piperidines; Receptors, Opioid; Receptors, sigma

Dextromethorphan (DM) is an antitussive with anticonvulsant activity that binds to high- and low-affinity sites in guinea pig brain homogenates. We examined the autoradiographic localization of [3H]DM using the anticonvulsant ropizine, an allosteric modifier that decreases the dissociation rate of [3H]DM. Competition studies demonstrated that the binding to brain sections was identical to that of brain homogenates [Craviso and Musacchio: Mol Pharmacol 23:629-640, 1983b]. Computer-assisted quantitative analysis of the autoradiographic images demonstrated that [3H]DM binds to discrete structures throughout the brain, but with higher density in the midbrain, pons, and medulla oblongata. The most intense labeling was observed in the rhabdoid, dorsal raphe, median raphe, caudal linear raphe nuclei, and cranial motor nerve nuclei. The central gray showed moderate to high-density labeling throughout its entire rostro-caudal extent, with very high binding in the dorsal tegmental nucleus and the locus coeruleus. Moderate and high binding was also seen in several hypothalamic structures. Distinct bands of moderate binding were seen in the pyramidal cell layer of the piriform cortex, the retrosplenial cortex, the granular cell layer of the dentate gyrus, the pyramidal cell layer of the hippocampus, and the Purkinje cell layer of the cerebellum. The striking similarity between the binding distribution of [3H]DM and sigma ligands, plus competition studies in brain homogenate, support the hypothesis that DM and sigma ligands share a common high-affinity binding site [Musacchio et al: Mol Pharmacol 35:1-5, 1989]. The distribution of [3H]DM binding provides possible anatomical substrates for both the antitussive and anticonvulsant actions of DM.

Topics: Allosteric Regulation; Animals; Anticonvulsants; Antitussive Agents; Autoradiography; Binding, Competitive; Dextromethorphan; Guinea Pigs; Image Processing, Computer-Assisted; Levorphanol; Male; Piperazines

Dextromethorphan (DM), a non-narcotic antitussive with anticonvulsant activity, binds to high (Kd, 57 nM)- and low-affinity sites (Kd, 24 microM) in the guinea pig brain. This work, done at physiological pH, expands previous results obtained at pH 8.3. Phenytoin (PHT) produces an allosteric increase in the binding of [3H]DM, which is more marked than that reported previously: PHT (100 microM,) at pH 7.4, increased the binding affinity of [3H]DM to brain homogenates 4-fold, without changing the concentration of DM sites. Moreover, ropizine (SC-13504) an anticonvulsant benzhydryl piperazine, also produced a marked concentration-dependent increase in the binding of [3H]DM, which is mediated by a decrease in the dissociation rate of [3H]DM. Importantly, the effects of ropizine are fully apparent at 10 microM, 10-fold lower than those of PHT. The effects of PHT and ropizine show that the affinity of the DM sites can be increased by other ligands, suggesting that these sites are located on macromolecules that can exist in at least two conformational states. [3H]DM also binds to peripheral tissues, but the brain displays the highest affinity. Besides, the central and peripheral sites are different as determined by competition studies with caramiphen and carbetapentane, which are DM site ligands with antitussive and anticonvulsant activity. The results reported in this communication are consistent with the hypothesis that the high-affinity DM binding sites mediate antitussive and anticonvulsant activity when occupied by the appropriate ligand.

Topics: Animals; Binding Sites; Brain; Dextromethorphan; Ethanol; Guinea Pigs; Hydrogen-Ion Concentration; Kinetics; Levorphanol; Male; Phenytoin; Piperazines; Protein Conformation; Stereoisomerism; Temperature