Antagonism of NMDA receptors by sigma receptor ligands attenuates chemical ischemia-induced neuronal death in vitro

We investigated the effects of sigma receptor ligands on neuronal death induced by chemical ischemia using primary cultures of rat cerebral cortical neurons. The induction of chemical ischemia by sodium azide and 2-deoxy-D-glucose led to delayed neuronal death in a time- and concentration-dependent manner, as determined by trypan blue exclusion. The neurotoxicity was inhibited by N-methyl-D-aspartate (NMDA) receptor antagonists, indicating the involvement of glutamate. The sigma receptor ligands (+)-N-allylnormetazocine ((+)-SKF10,047) and haloperidol, but not carbetapentane and R(+)-3-(3-hydroxyphenyl)-N-propylpiperidine ((+)-3-PPP), prevented chemical ischemia-induced neurotoxicity in a concentration-dependent manner. The protective effects of (+)-SKF10,047 and haloperidol were not affected by the sigma receptor antagonists. (+)-SKF10,047 and haloperidol, but not carbetapentane and (+)-3PPP, inhibited the glutamate-induced increase in intracellular Ca(2+), and the inhibitory effects were not attenuated by sigma receptor antagonists. These results suggest that direct interaction with NMDA receptors but not sigma receptors is crucial to the neuroprotective effects of sigma receptor ligands with affinity for NMDA receptors.     

Involvement of sigma receptors in the modulation of the glutamatergic/NMDA neurotransmission in the dopaminergic systems

Extracellular single-unit recordings and iontophoresis were used to examine the effects of different selective sigma receptor ligands on dopaminergic and glutamatergic N-methyl-D-aspartate (NMDA) neurotransmissions both in origin (A10 and A9 areas) and terminal (nucleus accumbens and caudate nucleus) regions of the rat mesolimbic and nigrostriatal dopaminergic systems. The selective sigma1 receptor ligands 2-[4-(4-methoxy-benzyl)piperazin-1-yl-methyl]4-oxo[4H]-benzo-th iazolin-2-one (S-21377), systemically administered (1.2 mg/kg, i.v., cumulative dose), and 2[(4-benzyl piperazin-1-yl) mothyl] naphthalene, dichiorydrate (S-21378), iontophoretically applied, slightly increased the spontaneous firing rate and potentiated the NMDA-induced neuronal activation of dopaminergic neurons in the A9 and A10 regions. (+)N-cyclopropylmethyl-N-methyl-1,4-diphenyl-1-ethyl-butyl-2-N (JO-1784), another selective sigma1 receptor ligand produced no or little effect in these areas. The systemic administration of the selective sigma2 receptor ligand 1,4-bis-spiro[isobenzofuran-1(3H), 4'-piperidin-1'yl]butane (Lu 29-252) (2 mg/kg, i.v., cumulative dose) did not modify the firing activity of A9 and A10 dopaminergic neurons, but significantly potentiated the NMDA-induced increase in firing activity of A10 dopaminergic neurons. None of the sigma receptor ligands tested had any effects on the dopamine-induced suppression of firing. In the nucleus accumbens, the systemic administration of (JO-1784), (40 microg/kg, i.v.), (+)-pentazocine (30 microg/kg, i.v.), another selective sigma1 receptor ligand, and of the non selective sigma1 receptor ligand di-tolyl-guanidine (DTG) (20 microg/i.v.) produced a significant increase of NMDA-induced neuronal activation. Microiontophoretic applications of JO-1784 also potentiated the NMDA response. They also increased significantly the suppressant effect of dopamine on NMDA and kainate-induced activations of accumbens neurons. In the caudate nucleus, (+)-pentazocine, but not JO-1784, potentiated slightly the neuronal response to NMDA. None of the sigma receptor ligands tested did modify significantly the responses of caudate and accumbens neurons to kainate. These findings suggest that at least two subtypes of sigma1 receptors may affect differentially the glutamate NMDA neurotransmission in the terminal and origin regions of the mesolimbic and nigrostriatal dopaminergic systems. These results also demonstrate the existence of a functional interaction between sigma2 and NMDA receptors in the A10 region.     

Inhibition of hyperpolarization-activated cation currents by phencyclidine and some sigma ligands in rat hippocampal CA1 pyramidal neurons in vitro

Using whole-cell voltage-clamp recordings, hyperpolarization-activated cation currents (Ih) were elicited with hyperpolarizing voltage jumps in CA1 pyramidal neurons of rat hippocampal slices, and the effects of phencyclidine (PCP) and some sigma ligands on Ih were studied. PCP concentration-dependently (0.1-100 microM) suppressed Ih and shifted the activation curve of Ih to the negative direction. D-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP, 20 microM) and MK-801 (30 microM), competitive and non-competitive NMDA blockers, respectively, failed to mimic the inhibitory effect of PCP on Ih, and suppression of Ih by PCP was unaffected in the presence of these blockers. To explore the involvement of sigma1 receptors in the reduction of Ih, the effects of representative sigma1 ligands were studied. SKF10047 (100 microM), a sigma1 agonist, attenuated the maximal Ih and shifted the half-activation potential of Ih to the hyperpolarized direction. In the presence of the sigma1 antagonist NE-100 (1 microM), which alone did not affect Ih, the effect of SKF10047 on Ih was unaltered. By contrast, a higher concentration of NE-100 (10 microM) mimicked the effect of SKF10047. Again, no antagonism of Ih suppression by SKF10047 was obtained with rimcazole (100 microM), a sigma1 receptor antagonist that is structurally distinct from NE-100. This concentration of rimcazole alone resulted in a slight but significant reduction of Ih. Thus these major sigma1 ligands appear to suppress Ih independently of their agonistic or antagonistic properties. The results of this study suggest that PCP and some sigma ligands could modulate cell excitability partly through their action on Ih.     

Direct inhibition of the N-methyl-D-aspartate receptor channel by dopamine and (+)-SKF38393.

1. Dopamine is known to modulate glutamatergic synaptic transmission in the retina and in several brain regions by activating specific G-protein-coupled receptors. We have examined the possibility of a different type of mechanism for this modulation, one involving direct interaction of dopamine with ionotropic glutamate receptors. 2. Ionic currents induced by fast application of N-methyl-D-aspartate (NMDA) were recorded under whole-cell patch-clamp in cultured striatal, thalamic and hippocampal neurons of the rat and in retinal neurons of the chick. Dopamine at concentrations above 100 microM inhibited the NMDA response in all four neuron types, exhibiting an IC50 of 1.2 mM in hippocampal neurons. The time course of this inhibition was fast, developing in less than 100 ms. 3. The D1 receptor agonist (+)-SKF38393 mimicked the effect of dopamine, with an IC50 of 58.9 microM on the NMDA response, while the enantiomer (-)-SKF38393 was ineffective at 50 microM. However, the D1 antagonist R(+)-SCH23390 did not prevent the inhibitory effect of (+)-SKF38393. 4. The degree of inhibition by dopamine and (+)-SKF38393 depended on transmembrane voltage, increasing 2.7 times with a hyperpolarization of about 80 mV. The voltage-dependent block by dopamine was also observed in the presence of MgCl2 1 mM. 5. Single-channel recordings showed that the open times of NMDA-gated channels were shortened by (+)-SKF38393. 6. These data suggested that the site to which the drugs bound to produce the inhibitory effect was distinct from the classical D1-type dopamine receptor sites, possibly being located inside the NMDA channel pore. It is concluded that dopamine and (+)-SKF38393 are NMDA channel ligands.     

Factors regulating survival and death of midbrain dopamine neurons

We utilized organotypic midbrain slice cultures for the assessment of survival and degeneration of dopaminergic neurons in the substantia nigra. Application of N-methyl-D-aspartate (NMDA) to midbrain slice cultures for 24 h caused a concentration-dependent decrease in the number of surviving dopaminergic neurons visualized by tyrosine hydroxylase immunohistochemistry. Simultaneous application of (-)-deprenyl significantly attenuated the cytotoxic effect of NMDA. Because pretreatment with (-)-deprenyl failed to reduce NMDA toxicity, it is suggested that the neuroprotective effect of (-)-deprenyl is not mediated by its irreversible inhibitory action on monoamine oxidase B. We also prepared co-cultures of midbrain and striatal slices to investigate whether the presence of target tissue influences toxic actions of several drugs on dopaminergic neurons. Co-cultured dopaminergic neurons formed dense innervation to the striatal tissue. Dopaminergic neurons in midbrain--striatum co-cultures were more resistant to the cytotoxic actions of NMDA and a nitric oxide donor NOC-18, than the same neuronal population in single midbrain cultures. On the other hand, the toxicity of 1-methyl-4 phenylpyridinium ion or buthionine-[S,R]-sulfoximine was more prominent in midbrain--striatum co-cultures than that in single midbrain cultures. Organotypic slice cultures appeared to be a useful system for evaluation of dopaminergic neuronal death under experimental conditions relevant to physiological/pathophysiological situations.     

Multiple affinity binding states of the sigma receptor: effect of GTP-binding protein-modifying agents

The sigma receptor, which is labeled with (+)-[3H]3-(3-hydroxyphenyl)-N- 1-(propyl)piperidine [(+)-[3H]3-PPP], is a site that binds several psychotomimetic opiate benzomorphans and certain antipsychotics, such as haloperidol. In order to elucidate the mechanisms involved in sigma receptor ligand binding, equilibrium binding analysis and kinetics of association and dissociation of the relatively selective sigma receptor ligand (+)-[3H]3-PPP were determined in rat brain membranes in the absence and presence of 5'-guanylylimidodiphosphate [Gpp(NH)p]. In the absence of Gpp(NH)p, (+)-3-PPP, cyclazocine, pentazocine, and (+)-SKF 10047 bind to high and low affinity sites (KH = 1.3-7.5 nM; KL = 84-500 nM), as determined by computer assisted analysis of the inhibition of (+)-[3H]3-PPP binding by the sigma ligands. The antipsychotics haloperidol and chlorpromazine inhibit (+)-[3H]3-PPP binding in a manner indicating interaction with a single state of the receptor. Gpp(NH)p (0.1 mM) abolished the high affinity binding component of the sigma agonist-like compounds tested but had no effect on the affinities of the antipsychotics for the receptor. Gpp(NH)p decreased the association rate of (+)-[3H]3-PPP binding 5-fold and also converted the biexponential dissociation kinetics of the ligand, observed in the absence of Gpp(NH)p, to a rapid monophasic dissociation process. Pretreatment of membranes with N-ethylmaleimide and pertussis toxin inhibited (+)-[3H]3-PPP binding and abolished the effect of Gpp(NH)p on the sigma ligand binding. These findings indicate of the sigma receptor is capable of existing in two discrete states, having high and low affinity for sigma agonist-like drugs. The regulation of the high affinity binding state by GTP-binding protein-modifying agents suggests its coupling to GTP-binding protein(s).     

Deprenyl rescues dopaminergic neurons in organotypic slice cultures of neonatal rat mesencephalon from N-methyl-D-aspartate toxicity.

The potential neuroprotective effect of (-)-deprenyl (R-N,alpha-dimethyl-N-2-propynylbenzeneethanamine) against N-methyl-D-aspartate (NMDA) excitotoxicity was investigated on rat mesencephalic dopaminergic neurons in organotypic slice cultures. While 24 h application of NMDA (100 microM) caused a marked decrease in the number of surviving dopaminergic neurons, simultaneous application of (-)-deprenyl significantly attenuated the cytotoxic effect of NMDA. (+)-Deprenyl showed a less potent but still significant protective effect against NMDA insult. Pre-treatment of cultures with (-)-deprenyl conferred no protection against subsequent NMDA insult, suggesting that the protective effect of (-)-deprenyl may be independent of its irreversible inhibitory action on monoamine oxidase B. (-)-Deprenyl was also ineffective in preventing cell death induced by H2O2. These results indicated that (-)-deprenyl protects dopaminergic neurons from NMDA excitotoxicity through a mechanism distinct from monoamine oxidase inhibition or detoxification of reaction oxygen species.     

The sigma agonist 1,3-di-o-tolyl-guanidine directly blocks SK channels in dopaminergic neurons and in cell lines

Small conductance Ca²⁺-activated K⁺ (SK) channels are widely expressed in the brain and underlie medium-duration afterhyperpolarizations (mAHPs) in many types of neurons. It was recently reported that the activation of sigma-1 (σ₁) receptors inhibits SK currents in rat hippocampus. Because many interactions between σ receptors and brain dopaminergic systems have been reported, we set out to examine putative effects of σ receptor ligands on the SK mediated mAHP in midbrain dopaminergic neurons. We found that 1,3-di-o-tolyl-guanidine (DTG) inhibited the mAHP in a concentration-dependent manner (∼ 60% inhibition at 100 µM), while other σ receptor agonists (carbetapentane, (+)-SKF10047 and PRE-084) had little effect. Moreover, the effect of DTG was not affected by high concentrations of the σ₁ receptor antagonist BD 1047. A role for σ₂ receptors could also be excluded by the lack of effect of the σ₂ receptor ligand 5-bromo-tetrahydroisoquinolinylbenzamide. These results argue against a coupling of σ receptors to SK channels in dopaminergic neurons. We next hypothesized that DTG could directly block the channel. This hypothesis was tested in HEK-293 cells which were transiently transfected with rSK2 or hSK3 subunits. DTG inhibited the current flowing through both subtypes with mean IC₅₀s ∼ 200 µM. This action was also unaffected by BD 1047. Other σ receptor ligands had little or no effect. We conclude that DTG directly blocks SK channels. This pharmacological action may be important to consider in future experimental settings.     

Interaction of 1,3-di(2-[5-3H]tolyl) guanidine with sigma 2 binding sites in rat heart membrane preparations.

Membrane preparations of rat hearts displayed specific binding activity for the prototypic sigma (sigma) receptor ligand, 1,3-di(2-[5-3H]tolyl) guanidine [( 3H]DTG), but not for the phencyclidine (PCP) receptor ligand, [3H]MK-801. Scatchard plot analysis of [3H]DTG binding revealed the presence of one high affinity saturable binding site with a KD of 8.7 nM and a Bmax of 100 pmol/g protein. The drug specificity profile of the receptor correlated with that of the sigma receptor with the following order of potency: DTG greater than haloperidol greater than (-)-pentazocine greater than (-)-butaclamol greater than (+)-butaclamol greater than (-) SKF-10047 greater than (+)pentazocine greater than PCP greater than TCP greater than MK-801 greater than (+)SKF-10047. [3H]DTG binding was sensitive to the Ca2+ channel blocker, verapamil (Ki 202 nM) but not to the K+ channel blocker, 4-aminopyridine. The reverse stereoselectivity of [3H]DTG binding for (-)-SKF-10047 and (-)-pentazocine (Ki of 1289 and 140 nM as compared with 17,582 and 2190 nM for (+)-SKF-10047 and (+)-pentazocine, respectively) indicated that the heart contains sigma receptors with characteristics of the sigma 2 subtype.     

The lack of utility of the rat vas deferens as a functional bioassay for sigma ligands

The present study examined the utility of the rat vas deferens preparation as a bioassay for sigma site ligands. sigma Ligands such as (+/-)-pentazocine, phencyclidine (PCP) and (+)-SK&F 10047 potentiated neurogenic twitch contractions. However, neither the order of potency nor the absolute potency of (+/-)-pentazocine and (+)-SK&F 10047 correlated with their affinity at central sigma sites. Furthermore, another potent sigma ligand, ditolyl-ortho guanidine (DTG) neither affected neurogenic twitch contractions nor inhibited twitch potentiation by PCP or (+)-SK&F 10047 at concentrations up to 30 mumol/l. These data indicate that the rat vas deferens is not a useful bioassay for the evaluation of sigma ligands. PCP, (+)-SK&F 10047 and (+/-)-pentazocine probably enhance neurogenic contractions in rat vas deferens primarily by inhibition of the neuronal uptake of noradrenaline.     

Presence of sigma and phencyclidine (PCP)-like receptors in membrane preparations of bovine adrenal medulla

Receptor binding studies with [3H]-(+)SKF-10047 were carried out to characterize the putative sigma (sigma) and phencyclidine (PCP) receptors in membrane preparations of bovine adrenal medulla. Specific binding of the radiolabelled compound was observed after incubation with the membrane preparation at 37 degrees, the equilibrium being reached at 20 min and the maximal binding being observed with 0.6 mg/ml protein. Saturation binding studies were performed at equilibrium (30 min at 37 degrees with 0.5 mg/ml of membrane protein) in the presence of haloperidol (1 microM) or 1-[1-(2-thienyl) cyclohexyl] piperidine (TCP; 0.2 microM) to block sigma or PCP receptors, respectively. The binding of [3H]-(+)SKF-10047 was characterized by two distinct components. A high affinity binding site (haloperidol sensitive) had an apparent KD of 8.3 nM and a Bmax of 67 pmol/g protein. A lower affinity binding site (TCP sensitive) had an apparent KD of 32.7 nM and a Bmax of 83 pmol/g protein. The drug specificity of the high affinity binding site resembled that of the putative sigma receptor, being potently inhibited by haloperidol and pentazocine. The binding pharmacology of the low affinity site resembled that of the phencyclidine receptor, being potently displaced by TCP and PCP. The binding of [3H]-(+)SKF-10047 to both receptors showed marked stereoselectivity for the dextrorotatory (+) isomer of SKF-10047 and was insensitive to the receptor specific opioid ligands DAGO (mu), DSLET (delta) and U-69593 (kappa). These data indicate that bovine adrenal medulla contains sigma and PCP-like receptors.     

The function of sigma receptors--electrophysiological approach

The functions of sigma (sigma) receptors were reviewed based on electrophysiological studies. Systemic administration of sigma ligands reportedly produces a variety of effects on dopamine (DA) neurons. In the mesolimbic DA system, (+)SKF-10,047 suppressed activities of the ventral tegmental area. In the substantia nigra, DTG also suppressed these activities, while BMY-14802 increased the activity of neurons. In the cerebellum, however, activities of Purkinje cells were suppressed by locally applied DTG, with probable involvement of the catecholaminergic system. This effect on Purkinje cells may explain the action of sigma ligands on the motor system. In the hippocampus, neuronal activities were inhibited by SR31742A in vivo (CA3 region) and by (+)SKF10,047 in vitro (CA1 region). DTG at high concentration (1 mM) completely suppressed population spikes (PS) in the CA1 region of hippocampal slice preparations. In our experiments, a novel sigma ligand, OPC-24439, suppressed PS in CA1 at concentrations of 1-100 microM. However, NMDA-induced neuronal firings in CA3 in vivo were augmented by low doses of DTG in a haloperidol-sensitive manner, but high doses were ineffective. In contrast, non-NMDA responses were not affected by DTG. In addition, several sigma ligands having no effect on the NMDA response have been reported. In addition, endogenous ligands, neuropeptide Y and dehydroepiandrosterone, augmented the NMDA-induced firing. In whole-cell patch clamp recording, NMDA-induced currents were suppressed by a relatively higher concentration of DTG in a concentration-dependent manner, while non-NMDA responses were only slightly suppressed. These findings suggest that effects of sigma ligands on NMDA receptor responses are biphasic, and sigma ligands may modulate memory and learning and suppress neuronal death by anoxia. In addition, sigma ligands are also reported to suppress Ca2+ channels in hippocampal culture neurons and induce current by closing K+ channels in NCB-20 cells. Thus, sigma receptors may be involved in the modulation of a variety of neurons that relate to psychiatric function and plasticity.     

Role of the sigma receptor in the inhibition of [3H]-noradrenaline uptake in brain synaptosomes and adrenal chromaffin cells.

1. Rat brain synaptosomes and cultured bovine adrenal chromaffin cells were used to monitor the inhibitory effects of phencyclidine (PCP) and sigma (sigma)-receptor ligands on the uptake of [3H]-noradrenaline ([3H]-NA). 2. A Na(+)-dependent high affinity uptake was observed in synaptosomes (30 degrees C) and chromaffin cells (37 degrees C) with Km of 0.22 and 0.56 microM and Vmax of 2.5 pmol min-1 mg-1 protein and 0.7 pmol min-1 per 10(6) cells, respectively. 3. PCP and haloperidol inhibited the high affinity uptake with IC50 of 0.17 and 0.42 microM, respectively in synaptosomes and 0.24 and 0.47 microM, respectively in adrenal chromaffin cells. 4. A close correlation (r = 0.96) was established between the ability of various PCP and sigma-receptor ligands to inhibit [3H]-NA uptake in both systems: PCP greater than TCP greater than haloperidol greater than 3-(+)-PPP greater than MK-801 greater than or equal to (-)-butaclamol greater than (+)-SKF-10047 greater than DTG. Spiperone and opioid receptor ligands were ineffective at 20 microM. 5. These results indicate that the central and peripheral inhibitory effects of PCP and sigma-receptor ligands on [3H]-NA uptake involves a receptor (sigma 1-like) which is distinct from that (PCP2) recognized for the inhibition of [3H]-dopamine uptake by PCP.     

Quantitative autoradiography of [3H]-MK-801 binding sites in mammalian brain.

1. An in vitro receptor autoradiography procedure is described for visualizing binding sites for the excitatory amino acid antagonist radiolabelled MK-801, in rat and gerbil brain sections. 2. Ten micron sections were labelled by incubation at room temperature for 20 min in 30 nM [3H]-MK-801. This was followed by 2 rinses for 20 s in fresh buffer solution. Specifically bound ligand determined with 100 microM unlabelled MK-801 amounted to 55-60% of total. 3. Phencyclidine, (+/-)-SKF 10047, ketamine and 2-aminophosphonovaleric acid (APV) (all 100 microM) prevented the specific binding of [3H]-MK-801. L-Glutamate and N-methyl D-aspartate (NMDA) (100 microM) had no effect. However, L-glutamate prevented the inhibition by APV. 4. The highest concentrations of [3H]-MK-801 binding sites occurred in the hippocampal formation, cerebral cortex, olfactory bulb and thalamus. Very low levels were detected in the brain stem and cerebellum. 5. The distribution of [3H]-MK-801 binding sites was comparable to that of NMDA sites and phencyclidine sites (labelled with [3H]-TCP) but not with high-affinity sigma sites labelled with [3H]-3-PPP. 6. The density of [3H]-MK-801 binding sites in the gerbil hippocampus was examined 1, 2, 6 and 22 days after unilateral carotid artery occlusion for 10 min. Only at 6 and 22 days was the binding reduced (by 36% and 46% respectively) in the CA1 region whereas a significant neuronal loss was apparent at day 2. In CA2 a decrease in binding was only evident at day 22. 7. These results indicate that binding sites for [3H]-MK-801 can be detected in mammalian brain sections by receptor autoradiography. Their distribution supports an association with the NMDA receptor complex and the loss in the hippocampus after carotid artery occlusion indicates their presence on pyramidal cells is vulnerable to ischaemic insult.     

Heterogeneous binding of sigma radioligands in the rat brain and liver: possible relationship to subforms of cytochrome P-450.

The binding of four sigma receptor ligands, 3H-(+)-N-allyl-N-normetazocine (3H-(+)-SKF 10,047), 3H-(+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine (3H-(+)-3-PPP), 3H-haloperidol and 3H-N,N'-di(o-totyl)guanidine (3H-DTG), and the cytochrome P450IID6 ligand and dopamine uptake inhibitor 3H-1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine (3H-GBR 12935) to membranal preparations of rat liver or whole rat brain was examined regarding kinetical properties and inhibition by various compounds with affinity for sigma binding sites or cytochrome P-450. In rat brain the density of binding sites was increased in order (+)-SKF 10,047 less than (+)-3-PPP less than DTG much less than GBR 12935. In liver the corresponding order was (+)-SKF 10,047 less than DTG less than haloperidol less than (+)-3-PPP less than GBR 12935. The inhibition pattern of each ligand was similar in brain and liver, indicating that the binding sites were similar in the two tissues. With the exception of 3H-(+)-SKF 10,047 which appears to bind to a homogeneous haloperidol-sensitive site, there were quite marked differences between the ligands studied, suggesting heterogeneous binding sites. For instance, (+)-SKF 10,047 and progesterone were potent inhibitors of the binding of 3H-(+)-SKF 10,047, 3H-(+)-3-PPP and 3H-haloperidol but inhibited only a minor fraction of the binding of 3H-DTG to the brain and liver preparations. Multiple binding sites were also indicated by the low Hill coefficients found for most of the compounds studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

3H]ethylketocyclazocine binding to NCB-20 hybrid neurotumor cells

Ethylketocyclazocine (EKC) binds to two sites on NCB-20 neuroblastoma X Chinese hamster brain hybrid cells (KDH = 2 nM, Bmax = 21,000 sites/cell; KDL = 27 nM, Bmax = 140,000 sites/cell. The high-affinity site has been characterized as a delta opiate receptor. The low-affinity site is relatively benzomorphan-specific; opioid peptides, morphine, etorphine, and naloxone do not compete at it. Rank order of potency among benzomorphans is (+)-EKC greater than Mr 2267 greater than (+)-ketocyclazocine greater than (+)-SKF 10047 greater than bremazocine greater than cyclazocine. Among other drugs of interest that inhibit [3H]EKC binding are phencyclidine and its analogues, Ki values for which are 0.2-40 microM. Stereoselectivity is the reverse of other opioid receptors: (+)-EKC much much greater than (-)-EKC, Mr 2267 greater than Mr 2266, (+)-SKF 10047 greater than (-)-SKF 10047. The site is sensitive to trypsin, but not to N-ethylmaleimide. Binding is insensitive to nucleotides, slightly sensitive to physiological concentrations of sodium, magnesium, and manganese ions and to EDTA but not EGTA.     

L-arginine and arginine analogues: effects on isolated blood vessels and cultured endothelial cells.

1. The involvement of the haloperidol-sensitive, sigma recognition site and the N-methyl-D-aspartic acid (NMDA) receptor in the mediation of the discriminative stimulus properties of (+)-N-allylnormetazocine [+)-NANM, (+)-SKF 10,047), has been investigated in the rat by use of a two-lever, operant drug discrimination paradigm. 2. Six compounds with nanamolar affinity for the sigma recognition site [+/-)-pentazocine, (+)-3-(hydroxyphenyl)-N-propylpiperidine [+)-3-PPP), ditolylguanidine (DTG), haloperidol, (-)-butaclamol and BMY 14802) were investigated for their ability to generalise or antagonise the (+)-NANM discriminative stimulus. Each drug was tested at doses found in an ex vivo radioligand binding assay to displace [3H]-DTG from the central sigma recognition site by more than 40%. 3. While (+/-)-pentazocine (in the presence of naloxone) generalised and (+)-3-PPP partially antagonised the (+)-NANM cue, the other putative sigma ligands were ineffective either as agonists or antagonists at doses clearly occupying the sigma site in vivo. 4. Dose-dependent generalisation to the (+)-NANM cue was seen with the selective non-competitive NMDA receptor antagonist, MK-801, a compound devoid of significant affinity for the sigma recognition site. 5. (+/-)-Pentazocine was found to antagonise seizures induced in the mouse by NMDLA, a model reflecting antagonism of central NMDA receptors, and a strong correlation was found between the rank order of potency of compounds to generalise to the (+)-NANM discriminative stimulus and their potencies as anticonvulsants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophysiological effects of monoamine oxidase inhibition on rat midbrain dopaminergic neurones: an in vitro study.

1 The effects of the inhibition of monoamine oxidase (MAO) type A and B have been evaluated on the spontaneous firing activity of the dopaminergic (principal) neurones of the rat midbrain intracellularly recorded from a slice preparation. 2 The non-specific MAO inhibitor, pargyline, superfused at a concentration of 10-100 microM, decreased or abolished the spontaneous firing discharge of the principal neurons in the subtantia nigra pars compacta and ventral tegmental area. This effect had a slow onset and appeared to be sustained. 3 The administration of the dopamine D2/3 receptor antagonist, sulpiride (100-300 nM), antagonized the pargyline-induced effect, while the superfusion of the dopamine D1 receptor antagonist, SCH 23390 (1-3 microM) did not counteract the induced inhibition of the firing rate. 4 The inhibitor for the MAO A, clorgyline (30-100 microM), reduced the firing rate of the dopaminergic neurones. A similar depressant effect was also observed when a MAO B inhibitor, deprenyl (30-100 microM), was applied. Lower concentrations of both drugs (300 nM-10 microM) did not produce consistent effects on neuronal discharge. 5 Our data suggest that only the blockade of both types of MAO enzymes favours the inhibitory action of endogenous dopamine on somato-dendritic D2/3 autoreceptors.     

Enantiomeric N-substituted N-normetazocines: a comparative study of affinities at sigma, PCP, and mu opioid receptors.

The optical antipodes of N-allyl-N-normetazocine (2; SKF 10047, NANM) were the original compounds used for the classification of the sigma receptor as distinct from other receptors such as the PCP (NMDA), opioid, and dopamine receptors. Later studies showed that (+)-N-(dimethylallyl)-N-normetazocine [(+)-4, (+)-pentazocine] was more potent and selective for the sigma receptor. In order to gain additional structure-activity relationship information, several N-substituted N-normetazocine analogs were prepared and evaluated for their sigma-1 ([3H]-(+)-3-PPP or [3H]-(+)-pentazocine), PCP ([3H]TCP), and mu opioid ([3H]DAMGO) receptor binding affinities. (+)-N-Benzyl-N-normetazocine [(+)-10)] possessed subnanomolar affinities for the sigma site, Ki = 0.67. The analog (+)-10 showed greater than 14,000- and 2400-fold selectivity, respectively, for the sigma receptor relative to the PCP and mu opioid receptors. The N-substituted N-normetazocines were enantioselective for the sigma site. The (+)-N-benzyl analog, (+)-10, showed a 55-fold selectivity relative to (-)-10. Analysis of the data also revealed that (+)-normetazocine [(+)-1] [Ki = 30 nM] possessed the highest affinity for the PCP receptor. However, (+)-metazocine [(+)-5] (Ki = 41 nM) was the most selective compound for the PCP receptor relative to the sigma (51-fold) and mu opioid (greater than 200-fold) sites.