Rat Cortical Membranes Research Articles

Evidence for binding of the ectodomain of amyloid precursor protein 695 and activated high molecular weight kininogen

To identify ligands that bind to the N-terminal portion of human amyloid precursor protein (APP), we sought binding partners for a fragment of the ectodomain of human APP695 (sAPP 695T). The probe bound to fragments of high molecular weight kininogen (HK) in rat cortical membrane preparations in vitro. Laser confocal microscopy indicated that APP and HK colocalize near cerebral blood vessels, in the neuropil, and in many neurons of rat brain. sAPP 695T bound to human activated kininogen (HKa) ( K d=0.3±0.1 nM), but not to inactivated or low molecular weight kininogen. Binding was specific for the light chain sequence of HKa. Biotinylated human HKa also bound to sAPP 695 ( K d=0.3±0.5 nM). sAPP 695 and HKa form tight complexes in solution that can be coimmunoprecipitated. These results support the hypothesis that forms of APP and kininogen can interact in brain tissue. Considering the implications of APP in neurite outgrowth, the APP–HKa interaction could modulate neurogenesis.

E-p-methoxycinnamic acid protects cultured neuronal cells against neurotoxicity induced by glutamate.

1. We previously reported that four new phenylpropanoid glycosides and six known cinnamate derivatives isolated from roots of Scrophularia buergeriana Miquel (Scrophulariaceae) protected cultured cortical neurons from neurotoxicity induced by glutamate. Here, we have investigated the structure-activity relationships in the phenylpropanoids using our primary culture system. 2. The alpha,beta-unsaturated ester moiety and the para-methoxy group in the phenylpropanoids appeared to play a vital role in neuroprotective activity. This suggested that E-p-methoxycinnamic acid (E-p-MCA) might be a crucial component for their neuroprotective activity within the phenylpropanoid compounds. E-p-MCA significantly attenuated glutamate-induced neurotoxicity when added prior to an excitotoxic glutamate challenge. 3. The neuroprotective activity of E-p-MCA appeared to be more effective in protecting neurons against neurotoxicity induced by NMDA than from that induced by kainic acid. E-p-MCA inhibited the binding of [propyl-2,3-(3)H]-CGP39653 and [2-(3)H]-glycine to their respective binding sites on rat cortical membranes. However, even high concentrations of E-p-MCA failed to inhibit completely [propyl-2,3-(3)H]-CGP39653 and [2-(3)H]-glycine binding. 4. Indeed, E-p-MCA diminished the calcium influx that routinely accompanies glutamate-induced neurotoxicity, and inhibited the subsequent overproduction of nitric oxide and cellular peroxide in glutamate-injured neurons. 5. Thus, our results suggest that E-p-MCA exerts significant protective effects against neurodegeneration induced by glutamate in primary cultures of cortical neurons by an action suggestive of partial glutamatergic antagonism.

Amphetamine inhibits the N-methyl-D-aspartate receptor-mediated responses by directly interacting with the receptor/channel complex.

Amphetamine (AMPH) induces behavioral sensitization and neurotoxicity primarily by enhancing the dopamine-mediated neurotransmission. However, the involvement of the N-methyl-D-aspartate (NMDA) receptor in AMPH-induced neuropathology is also known. Recent investigation has found that high concentration of dopamine could inhibit NMDA receptor-mediated responses by blocking the NMDA receptor channel. By virtue of the structure similarity between dopamine and AMPH, we determined whether d-AMPH and its analogs, l-AMPH and methamphetamine (MAMH), could affect the NMDA receptor-mediated [3H]N-[1-(2-thienyl)cyclohexyl] piperidine ([3H]TCP) binding in rat cortical membrane preparations and intracellular 45Ca2+ accumulation and cell death in the rat primary cortical cell cultures. AMPH concentration-dependently inhibited NMDA- and glycine-stimulated [3H]TCP binding and intracellular 45Ca2+ accumulation with two distinct potencies; a minor inhibition with high potency and a major inhibition with low potency. [3H]TCP binding suggested that the high-potency inhibition was produced by decreasing agonist-induced activation of the NMDA receptor channel. On the other hand, the low-potency inhibition was produced by competing with [3H]TCP binding in the NMDA receptor channel, like the action of noncompetitive antagonist of the NMDA receptor. However, AMPH analogs were less potent in inhibiting NMDA- and glycine-induced cultured cell death. Thus, this result indicates that AMPH could antagonize the NMDA receptor-mediated responses in vitro by two different mechanisms, probably, through directly interacting with two distinct sites on this receptor/channel complex.

Syntheses and binding affinities of 6-nitroquipazine analogues for serotonin transporter. part 2: 4-Substituted 6-nitroquipazines

Eleven 4-substituted derivatives of 6-nitroquipazine were synthesized and evaluated for their abilities to displace [ 3H]citalopram binding to the rat cortical synaptic membranes. Among them, 4-chloro-6-nitroquipazine was shown to possess the highest binding affinity ( K i=0.03 nM) which was approximately 6 times higher than that of 6-nitroquipazine ( K i=0.17 nM) itself. In this paper, we describe the syntheses of 4-substituted 6-nitroquipazine derivatives, the results of corresponding biological evaluation and the SAR study.

Synthesis and structure-activity relationships of 4-amino-5-chloro-N-(1,4-dialkylhexahydro-1,4-diazepin-6-yl)-2-methoxybenzamide derivatives, novel and potent serotonin 5-HT3 and dopamine D2 receptors dual antagonist.

In search of a dopamine D2 and serotonin 5-HT3 receptors dual antagonist as a potential broad antiemetic agent, a number of benzamides were prepared from 4-amino-5-chloro-2-methoxybenzoic acid derivatives and 6-amino-1,4-dialkylhexahydro-1,4-diazepines and evaluated for their binding affinity for the dopamine D2 and the serotonin 5-HT3 receptors using rat brain synaptic and rat cortical membranes, respectively. From the results of both in vitro receptor binding and in vivo biological assays for the dopamine D2 receptor, 1-ethyl-4-methylhexahydro-1,4-diazepine ring was selected as an optimum amine moiety. Introduction of one methyl group on the nitrogen atom at the 4-position and/or modification of the substituent at the 5-position of the 4-amino-5-chloro-2-methoxybenzoyl moiety caused a marked increase in the dopamine D2 receptor binding affinity along with a potent 5-HT3 receptor binding affinity. Among the compounds, 5-chloro-N-(1-ethyl-4-methylhexahydro-1,4-diazepin-6-yl)-2-methoxy-4-methylaminobenzamide (82), 5-bromo (110), and 5-iodo (112) analogues exhibited a much higher affinity for the dopamine D2 receptor than that of metoclopramide (IC50=17.5-61.0 nM vs. 483 nM). In particular, 82 showed a potent antagonistic activity for both receptors in vivo tests. Optical resolution of the racemate 82 brought about a dramatic change in the pharmacological profile with the (R)-enantiomer exhibiting a strong affinity for both the dopamine D2 and the 5-HT3 receptors, while the corresponding (S)-enantiomer had a potent and selective serotonin 5-HT3 receptor binding affinity.

Bis(7)-Tacrine, a promising anti-Alzheimer's agent, attenuates glutamate-induced cell injury in primary cultured cerebrocortical neurons of rats

The effects of bis(7)-tacrine, a novel dimeric acetylcholinesterase (AChE) inhibitor, on glutamate-induced cell injury were investigated in primary cerebral cortical neurons of rats. Exposure of cultured neurons (12 days after plating) to 0.5 mmol/L glutamate for 30 min resulted in significant cell damage. Pretreatment with bis(7)-tacrine (0.03–1.0 μmol/L) reduced the glutamate-induced neurotoxicity in a concentration dependent manner and the maximal response was seen at 1 μmol/L with approximately, 30% protection. A receptor binding assay showed that bis(7)-tacrine can completely displace MK-801 binding to rat cortical membrane with an IC50 of 0.57 μmol/L. These findings suggest that bis(7)-tacrine can directly interact with N-methyl-D-aspartate receptor channel complex, which may contribute to the inhibitor's protective effects against glutamate-induced excitotoxicity. Thus, it is possible that anti-glutamate/anti-AChE synergism is responsible for potentially better Alzheimer's therapy of bis(7)-tacrine relative to tacrine.

Novel Potent 5-HT 3 Receptor Ligands Based on the Pyrrolidone Structure: Synthesis, Biological Evaluation, and Computational Rationalization of the Ligand–Receptor Interaction Modalities

Novel conformationally constrained derivatives of classical 5-HT 3 receptor antagonists were designed and synthesized with the aim of probing the central 5-HT 3 receptor recognition site in a systematic way. The newly-synthesized compounds were tested for their potential ability to inhibit [ 3H]granisetron specific binding to 5-HT 3 receptor in rat cortical membranes. These studies revealed subnanomolar affinity in some of the compounds under study. The most potent ligand in this series was found to be quinuclidine derivative ( S)- 7i, which showed an affinity comparable with that of the reference ligand granisetron. The potential 5-HT 3 agonist/antagonist activity of some selected compounds was assessed in vitro on the 5-HT 3 receptor-dependent [ 14C]guanidinium uptake in NG 108-15 cells. Both of the tropane derivatives tested in this functional assay ( 7a and 9a) showed antagonist properties, while the quinuclidine derivatives studied [the enantiomers of compounds 7i, 8g, and 9g, and compound ( R)- 8h] showed a full range of intrinsic efficacies. Therefore, the functional behavior of these 5-HT 3 receptor ligands appears to be affected by the structural features of both the azabicyclo moiety and the heteroaromatic portion. In agreement with the data obtained on NG 108-15 cells, investigations on the 5-HT 3 receptor-dependent Bezold–Jarisch reflex in urethane-anaesthetized rats confirmed the 5-HT 3 receptor antagonist properties of compounds 7a and ( S)- 7i showing for these compounds ID 50 values of 2.8 and 181 μg/kg, respectively. Finally, compounds 7a, ( S)- 7i and 9a (at the doses of 0.01, 1.0, and 0.01 mg/kg ip, respectively) prevented scopolamine-induced amnesia in the mouse passive avoidance test suggestive of a potential usefulness in cognitive disorders for these compounds. Qualitative and quantitative structure–affinity relationship studies were carried out by means of theoretical descriptors derived on a single structure and ad-hoc defined size and shape descriptors (indirect approach). The results showed to be useful in capturing information relevant to ligand–receptor interaction. Additional information derived by the analysis of the energy minimized 3-D structures of the ligand–receptor complexes (direct approach) suggested interesting mechanistic and methodological considerations on the binding mode multiplicity at the 5-HT 3 receptors and on the degree of tolerance allowed in the alignment of molecules for the indirect approach, respectively.

Calmodulin effect on purified rat cortical plasma membrane Ca 2+-ATPase in different phosphorylation states

The plasma membrane Ca 2+-ATPase in neuronal tissue plays an important role in fine tuning of the intracellular Ca 2+ concentration. The enzyme exhibits a high degree of tissue specificity and is regulated by several mechanisms. Here we analysed the relationship between separate modes of Ca 2+-ATPase regulation, i.e., reversible phosphorylation processes mediated by protein kinases A and C, protein phosphatases PP1 and PP2A, and stimulation by calmodulin. The activity of PKA- or PKC-phosphorylated Ca 2+-ATPase was influenced by the further addition of calmodulin, and this effect was more pronounced for PKC-phosphorylated calcium pump. In both cases the fluorescence study revealed the increased calmodulin binding, and for PKA-mediated phosphorylation it was correlated with a higher affinity of calcium pump for calmodulin. The incubation of Ca 2+-ATPase with CaM prior to protein kinases action revealed that CaM presence counteracts the stimulatory effect of PKA and PKC. Under the in vitro assay cortical Ca 2+-ATPase was a substrate for PP1 and PP2A. Protein phosphatases decreased both the basal activity of Ca 2+-ATPase and its affinity for calmodulin. Fluorescence analysis confirmed the lowered ability of dephosphorylated Ca 2+-ATPase for calmodulin binding. These results may suggest that interaction of CaM with calcium pump and its stimulatory action could be a partly separate phenomenon that is dependent on the phosphorylation state of Ca 2+-ATPase.

Effect of Li+ upon the Mg2+-Dependent Activation of Recombinant Giα1

Although lithium salts have been used in the treatment and prophylaxis of manic-depressive or bipolar patients for 50 years, the mechanism of the pharmacologic action of Li+ is unknown. Based on activity studies of inhibitory and stimulatory guanine-binding (G) proteins in rat cortical membranes, it was proposed that Li+ inhibition of G-proteins may account for its pharmacologic action. We used the purified α subunit of the recombinant inhibitory G-protein, rGiα1, and found that Li+ at therapeutic levels significantly inhibited the formation of the GDP.AlF−4.rGiα1 complex. Because our studies were conducted with a purified, metal-reconstitutedG-protein rather than with cell membrane suspensions, our Li+ inhibition results lend additional support to the G-protein hypothesis for Li+ action.

Binding characteristics of GYKI-46 903, a non-competitive ligand at 5-HT3receptors

GYKI-46903 [(+)-(5 s,6r )-4-(4-fluorophenyl)-6-propionyloxy-1-aza-bicyclo[3.3.1]-non-3-ene-hy- drochloride], a cognition enhancer identified as a non-competitive antagonist of 5-HT3receptors in isolated guinea-pig ileum, was investigated for allosteric action at 5-HT3receptors in rat cortical membranes by using [3H]granisetron. Equilibrium and kinetic protocols were applied and the competitive antagonist granisetron was included as a negative control. In competition studies, both granisetron and GYKI-46 903 displaced the radioligand with Kivalues of 0.20 ± 0.02 and 79.84 ± 0.28 nM, respectively. The inhibition curve for GYKI-46 903 resulted in a Hill slope significantly greater than unity ( 1.37 ± 0.11), whereas the slope for granisetron was 0.88 ± 0.08, not different from unity. These results indicate non-competitive and competitive interactions, respectively. Scatchard analysis yielded a linear plot, suggesting a single population of binding sites with a Kdof 0.13 ± 0.01 nM and a Bmaxof 13.15 ± 0.34 fmol per mg of protein. Scatchard plots obtained in the absence and presence of granisetron (0.1–3 nM) or GYKI-46 903 (30–1000 nM) revealed a concentration-dependent increase inKd values by either of these compounds. Granisetron left the Bmaxunchanged, but there was a significant increase in the Bmaxby GYKI-46 903, which could point to an atypical allosteric interaction. The Schild plot derived from the Kdshifts induced by granisetron was linear with a slope of 1.02, not different from unity, as expected from a competitive interaction. The Schild regression for GYKI-46 903 was linear with a slope of 1.20, deviating significantly from unity, which may also indicate an allosteric interaction. Both the association and dissociation curves of [3H]granisetron were monoexponential. The dissociation rate constant (K−1) and the association rate constant (K+1) were 0.32 ± 0.01 min−1and 1.15 min−1nM−1, respectively. The dissociation driven by an excess concentration of ondansetron ( 1 μ M) in the absence and presence of granisetron (0.1–3 nM) or GYKI-46 903 (30–10 000 nM) was not influenced by the compounds under study, as compared with the control, indicating the lack of an allosteric effect on the dissociation. Summing up, the binding profile of GYKI-46 903 may reflect a mixed type of action, including a negative allosteric interaction.

MODULATION OF [3H]-8-OH-DPAT BINDING TO RAT BRAIN MEMBRANES BY METAL IONS

The binding of ( 3 H)-8-OH-DPAT to rat hippocampal and cortical membranes was specific and saturable with Kd = 0.87 ± 0.18 nM and Kd = 2.4 ± 0.9 nM, respectively. Guanine nucleotides decreased the ( 3 H)-8-OH-DPAT binding affinity without significant influence on the number of binding sites. The radioligand binding affinity was only slightly affected by the pH in the interval from 6 to 9. The 5-HT 1A receptors in cortical membranes were considerably more stable than in hippocampus, indicating that the lipid environment determines the stability of the receptor in these brain regions. All chlorides of m onovalent metals studied at concentrations above 30 mM decreased the ( 3 H)-8-OH-DPAT binding. A significant increase in 2 nM ( 3 H)-8-OH-DPAT binding to hippocampal membranes was found in the presence of millimolar concentrations of MgCl2, CaCl2, BaCl2, MnCl2, CoCl2, and NiCl2, while the radioligand binding to cortical membranes was inhibited. It is proposed that different G proteins are coupled to 5-HT 1A receptors in rat hippocampus and cerebral cortex.

CHANGES IN MEMBRANE FLUIDITY DURING THE MICELLE FORMATION DETERMINE THE EFFICIENCY OF THE SOLUBILIZATION OF MUSCARINIC RECEPTORS

The influence of various concentrations of 15 detergents at different temperatures on the fluidity of membranes was analysed by fluorescence anisotropy of inserted 1,6-diphenyl-1,3,5- hexatriene in rat cortical and Sf9 cell membranes. Comparison of the obtained data with the abilities of the detergents to solubilize muscarinic receptor subtypes indicated that not only the fluidity of the micelles formed but also the micelle formation process, the lipid/protein ratio, and presence of sterols have significant influence on the efficiency of the solubilization of mAChR. Digitonin seems to exploit efficiently all these factors by its biphasic modulatory effect on membrane fluidity during the receptor solubilization.

Differential abilities of phorbol esters in inducing protein kinase C (PKC) down-regulation in noradrenergic neurones.

1. The ability of several phorbol ester protein kinase C (PKC) activators (phorbol 12, 13-dibutyrate, PDB; phorbol 12, 13-diacetate, PDA; and 12-deoxyphorbol 13-acetate, dPA) to down-regulate PKC was studied by assessing their effects on electrical stimulation-induced (S-I) noradrenaline release from rat brain cortical slices and phosphorylation of the PKC neural substrate B-50 in rat cortical synaptosomal membranes. 2. In cortical slices which were incubated for 20 h with vehicle, acute application of PDB, PDA and dPA (0.1 - 3.0 microM) enhanced the S-I noradrenaline release in a concentration-dependent manner to between 200 - 250% of control in each case. In slices incubated with PDB (1 microM for 20 h), subsequent acute application of PDB (0.1 - 3.0 microM) failed to enhance S-I release, indicating PKC down-regulation. However, in tissues incubated with PDA or dPA (3 microM) for 20 h, there was no reduction in the facilitatory effect of their respective phorbol esters or PDB (0.1 - 3.0 microM) when acutely applied, indicating that PKC was not down-regulated. This was confirmed using Western blot analysis which showed that PDB (1 microM for 20 h) but not PDA (3 microM for 20 h) caused a significant reduction in PKCalpha. 3. Incubation with PDB for 20 h, followed by acute application of PDB (3 microM) failed to increase phosphorylation of B-50 in synaptosomal membranes, indicating down-regulation. In contrast, tissues incubated with PDA or dPA for 20 h, acute application of their respective phorbol ester (10 microM) or PDB (3 microM) induced a significant increase in B-50 phosphorylation. 4. Acutely all three phorbol esters elevate noradrenaline release to about the same extent, yet PDA and dPA have lower affinities for PKC compared to PDB, suggesting unique neural effects for these agents. This inability to cause functional down-regulation of PKC extends their unusual neural properties. Their neural potency and lack of down-regulation may be related to their decreased lipophilicity compared to other phorbol esters. 5. We suggest that PKC down-regulation appears to be related to binding affinity, where agents with high affinity, irreversibly insert PKC into artificial membrane lipid and generate Ca(2+)-independent kinase activity which degrades and deplete PKC. We suggest that this mechanism may also underlie the ability of PDB to down-regulate PKC in nerve terminals, in contrast to PDA and dPA.

Serotonin transporter inhibitors: synthesis and binding potency of 2′-methyl- and 3′-methyl-6-nitroquipazine

Racemic 2′-methyl- and 3′-methyl-6-nitroquipazine ligands were selected as targets, synthesized and evaluated at the serotonin transporter employing an in vitro competitive inhibition assay with [ 3H]paroxetine and rat cortical membrane. The 2′-methyl-6-nitroquipazine was found to be 50 times more potent than the 3′-methyl-substituted counterpart and of comparable potency to the known high affinity agent 5-iodo-6-nitroquipazine.

3H]ac-RYYRWK-NH2, a novel specific radioligand for the nociceptin/orphanin FQ receptor.

The hexapeptide ac-RYYRWK-NH2 has been described as a potent partial agonist at the nociceptin (NC)/orphanin FQ receptor which has no affinity for mu-, kappa- or delta-opioid receptors. However, it is not clear whether ac-RYYRWK-NH2 is truly selective for the NC receptor, and ac-RYYRWK-NH2 has therefore been radiolabelled and characterised in receptor-binding experiments. Saturation experiments with [3H]ac-RYYRWK-NH2 binding to rat cortical membranes revealed a single high affinity site for [3H]ac-RYYRWK-NH2 (Kd=0.071 +/- 0.018 nM; Bmax=22+/-2 fmol/mg protein). Uncoupling of the G-proteins resulted in a significant 45% increase in Kd and no change in Bmax. [3H]ac-RYYRWK-NH2 binding to rat cortical membranes or to membranes from baby hamster kidney cells expressing human orphan opioid receptor-like (ORL1) was displaced by NC and ac-RYYRWK-NH2 to the same extent. The following rank order of potency was observed: ac-RYYRWK-NH2 > [Tyr14]NC-OH = NC-OH = NC-NH2 > NC, H-(1-13)-NH2 > NC(1-12)-NH2 >> NC(1-11)-NH2 and, thus, displayed a typical NC receptor pharmacology. Novel cyclic analogues of ac-RYYRWK-NH2 were prepared but these structures were much less active when compared to ac-RYYRWK-NH2. In vitro receptor autoradiography with [3H]ac-RYYRWK-NH2 to rat brain sections revealed high levels of binding in the cerebral cortex, amygdala, hypothalamus and superior colliculus, but low levels in the cerebellum and striatum. Overall, the regional distribution was very similar to that of [3H]NC. Ac-RYYRWK-NH2 seems indeed to be selective for the NC receptor and [3H]ac-RYYRWK-NH2 is a novel radioligand which may be useful for further exploring the pharmacology and receptor-ligand interaction of the NC receptor.

ChemInform Abstract: Syntheses and Binding Affinities of 6-Nitroquipazine Analogues for Serotonin Transporter. Part 1.

6-Nitroquipazine has been known as one of the most potent and selective inhibitors of serotonin transporter in vitro and in vivo. Nine derivatives of 6-nitroquipazine were synthesized and tested for their potential abilities to displace [3H]citalopram binding to the rat cortical membranes.

Isolation and Characterization of a Novel ConusPeptide with Apparent Antinociceptive Activity

Isolation and Characterization of a Novel ConusPeptide with Apparent Antinociceptive Activity

Blockade of voltage-sensitive Na + channels by the 5-HT 1A receptor agonist 8-OH-DPAT: possible significance for neuroprotection

The present study was undertaken to determine whether 5-hydroxytryptamine 1A (5-HT 1A) receptor agonists interact with voltage-sensitive Na + or N- and P/Q-type Ca 2+ channels to reduce the influx of Na + and/or Ca 2+. The 5-HT 1A receptor agonist 8-hydroxy-2-(di- n-propylamino)tetralin (8-OH-DPAT) inhibited both [ 3H]batrachotoxinin binding to neurotoxin site 2 of the Na + channel in rat cortical membranes (IC 50=5.1 μM) and veratridine-stimulated Na + influx into rat synaptosomes (EC 50=20.8 μM). The 5-HT 1A receptor agonist flesinoxan and the 5-HT 1A receptor antagonist N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)- N-(2-pyridinyl) cyclohexanecarboxamide (WAY-100635) also displaced [ 3H]batrachotoxinin binding with similar affinities to 8-OH-DPAT, but were much less effective in reducing veratridine-stimulated Na + influx. All three serotonergic agents also increased [ 3H]saxitoxin binding to neurotoxin site 1 of the Na + channel. In contrast, none of these agents interacted with radioligand binding to N- or P/Q-type Ca 2+ channels. These data show that 8-OH-DPAT directly interacts with voltage-sensitive Na + channels to reduce Na + influx so providing an additional mechanism to explain how it functions as a neuroprotectant.

A novel series of 2-carboxytetrahydroquinolines provides new insights into the eastern region of glycine site NMDA antagonists.

A series of potent 4-substituted tetrahydroquinolines has been synthesized and biologically tested in order to refine the eastern region of the pharmacophore model for glycine site NMDA antagonists concerning the assessment of lipophilicity, flexibility, and hydrogen bonding. Displacement studies on rat cortical membranes using [3H]-5,7-dichlorokynurenic acid as a radioligand indicated that binding affinities are markedly enhanced when additional hydrogen-accepting groups are introduced into the eastern region of the 2-carboxytetrahydroquinolines. Among the most potent ligands were some urea, sulfonylurea, and crown ether compounds as interesting leads for new diagnostics, especially for the evaluation of PET tracers, which allow biodistribution studies and NMDA receptor studies in the living organism.

Pharmacological characterization of (10bS)-1,2,3,5,6,10b-hexahydropyrrolo[2,1-a]isoquinoline oxalate (YSL-3S) as a new alpha2-adrenoceptor antagonist.

Alpha2-adrenoceptor antagonists, which can enhance synaptic norepinephrine levels by blocking feedback inhibition processes, are potentially useful in the treatment of disease states such as depression, memory impairment, impotence and sexual dysfunction. (10bS)-1,2,3,5,6,10b-Hexahydropyrrolo[2,1-a]isoquinoline oxalate (YSL-3S) was evaluated in several in vitro biological tests to establish its pharmacological profile of activities as an alpha2-adrenoceptor antagonist. Saturation binding assay revealed that [3H]rauwolscine bound to the alpha2-adrenoceptors with a Kd value of 6.3+/-0.5 nM and a Bmax value of 251+/-39 fmol/mg protein in rat cortical synaptic membranes. Competitive binding assay showed that YSL-3S inhibited the binding of [3H]rauwolscine (1 nM) in a concentration-dependent manner with a Ki value of 98.2+/-12.1 nM while it did not inhibit the binding of [3H]cytisine (1.25 nM) to neuronal nicotinic cholinergic receptors. The Ki values of yohimbine, clonidine and norepinephrine for [3H]rauwolscine binding were 15.8+/-1.0, 40.1+/-5.9 and 40.0+/-11.5 nM, respectively. In addition, the binding affinity of YSL-3S for alpha2-adrenoceptors was higher than that of its antipode and the racemic mixture. The functional activity of YSL-3S at the presynaptic alpha2-adrenoceptors was assessed using the prostatic portion of the rat vas deferens. Clonidine inhibited field-stimulated contractions of the vas deference in a dose-dependent manner. The presence of YSL-3S or yohimbine caused a parallel, rightward the dose-response curve of clonidine in a dose-dependent manner, indicating an antagonistic action at the presynaptic alpha2-adrenoceptors. The pA2 values of yohimbine and YSL-3S were 7.66+/-0.13 and 6.64+/-0.18, respectively. The results indicate that YSL-3S acts as a competitive antagonist at presynaptic alpha2-adrenoceptors with a potency approximately ten times lower than yohimbine, but is devoid of binding affinity for neuronal nicotinic cholinergic receptors.