Rat Brain Membranes Research Articles

Studies of peculiarities of binding of the Semax neuropeptide, with a labeled terminal proline residue to plasma membranes of rat brain

The peculiarities and characteristics of the binding of the Semax neuropeptide (Met-Glu-His-Phe-Pro-Gly-Pro) with a 3H-labeled terminal Pro to plasma membranes of the rat forebrain were studied. Specific binding sites were found that were characterized by a dissociation constant of the ligand-receptor complex of Kd = 100.4 ± 19.5 nM at 0–4°C. Significant biodegradation of Semax in the course of its interaction with plasma membranes was shown by HPLC. The rate of this degradation was considerably decreased at 0–4°C.

The Novel α7 Nicotinic Acetylcholine Receptor AgonistN-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]-7-[2-(methoxy)phenyl]-1-benzofuran-2-carboxamide Improves Working and Recognition Memory in Rodents

The relative contribution of alpha4beta2, alpha7 and other nicotinic acetylcholine receptor (nAChR) subtypes to the memory enhancing versus the addictive effects of nicotine is the subject of ongoing debate. In the present study, we characterized the pharmacological and behavioral properties of the alpha7 nAChR agonist N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-7-[2-(methoxy)phenyl]-1-benzofuran-2-carboxamide (ABBF). ABBF bound to alpha7 nAChR in rat brain membranes (Ki=62 nM) and to recombinant human 5-hydroxytryptamine (5-HT)3 receptors (Ki=60 nM). ABBF was a potent agonist at the recombinant rat and human alpha7 nAChR expressed in Xenopus oocytes, but it did not show agonist activity at other nAChR subtypes. ABBF acted as an antagonist of the 5-HT3 receptor and alpha3beta4, alpha4beta2, and muscle nAChRs (at higher concentrations). ABBF improved social recognition memory in rats (0.3-1 mg/kg p.o.). This improvement was blocked by intracerebroventricular administration of the alpha7 nAChR antagonist methyllycaconitine at 10 microg, indicating that it is mediated by alpha7 nAChR agonism. In addition, ABBF improved working memory of aged rats in a water maze repeated acquisition paradigm (1 mg/kg p.o.) and object recognition memory in mice (0.3-1 mg/kg p.o.). Rats trained to discriminate nicotine (0.4 mg/kg s.c.) from vehicle did not generalize to ABBF (0.3-30 mg/kg p.o.), suggesting that the nicotine cue is not mediated by the alpha7 nAChR and that selective alpha7 nAChR agonists may not share the abuse liability of nicotine. Our results support the hypothesis that alpha7 nAChR agonists may provide a novel therapeutic strategy for the treatment of cognitive deficits with low abuse potential.

Identification of a novel non-AT1, non-AT2 angiotensin binding site in the rat brain

Efforts to protect radiolabeled angiotensins from metabolism during receptor binding assays date back more than 30 years. However, this continues to be a problem. This study focused on the effects of a protease inhibitor, p-chloromercuribenzoate (PCMB), on the binding of 125I-Ang II to rat brain membranes. Addition of PCMB to the incubation medium revealed a high affinity binding site for 125I-Ang II in brain membranes ( K d = 1–4 nM) with a greater amount of binding than revealed in previous studies of brain Ang II receptors. Further characterization of this binding, revealed it to be insensitive to inhibition by losartan (an AT 1 receptor antagonist) and PD123319 (an AT 2 receptor antagonist). This non-AT1, non-AT2 binding site was not present in liver or adrenal membranes. It was activated by a limited range of concentrations of PCMB, with maximal activation at 0.3–1 mM. This binding site was equally abundant in cerebral cortex (a brain region with few Ang II receptors) and the hypothalamus (a brain region with abundant Ang II receptors). The binding site was also present in mouse brain, but not mouse liver. The binding site shows high affinity for Ang I, Ang II and Ang III ( K i ∼ 40–100 nM), but lesser affinity for smaller angiotensin fragments and other neuropeptides. This binding site shares some characteristics with the liver cytosolic Ang II binding proteins, later identified as endopeptidases EC 3.4.24.15 and/or EC 3.4.24.16. However, some unique characteristics of this non-AT1, non-AT2 binding site suggest that it may be a novel angiotensin binding substance.

Detection of α7 nicotinic acetylcholine receptors with the aid of antibodies and toxins

Neuronal nicotinic acetylcholine receptors (nAChRs) containing α7 subunit are well represented in the brain and some non-neuronal tissues, and their malfunctioning is associated with diverse pathologies. Therefore, detection and quantification of α7 nAChR are important tasks. The affinity-purified antibodies were prepared against the 1–23 and 179–190 fragments of the human and rat α7 nAChR extracellular domain. The specificity and selectivity of these α7 (1–23) and α7 (179–190) antibodies was tested by ELISA in model systems: the E. coli-expressed α7 subunit extracellular domain and the pituitary cell line GH4C1 stably expressing human α7 nAChR. On the rat brain slices two antibodies and biotinylated α-cobratoxin specifically stained the hippocampus region known to be rich in α7 nAChR. Western blot analysis revealed that in the human thalamus membranes and in rat brain membranes, antibodies α7 (1–23) stained a single band of 62 kDa, while the α7 (179–190) antibodies stained a doublet of 53–54 kDa. The results obtained show that utilization of model systems and a combination of several antibodies with appropriately labeled toxins may provide better ways for detection of α7 nAChR.

Xendorphin B1, a novel opioid- like peptide determined from a Xenopus laevis brain cDNA library, produces opioid antinociception after spinal administration in amphibians

Prodynorphins (PDYNs) from the African clawed frog ( Xenopus leavis), originally described as ‘proxendorphins’, are novel members of the family of opioid-like precursor polypeptides and were recently discovered based on polymerase chain reaction (PCR) isolates from a Xenopus brain cDNA library. This amphibian prodynorphin was found in two isoforms, XenPDYN-A and XenPDYN-B, consisting of 247 and 279 amino acids, respectively. Each prepropeptide contains five potential opioid-like peptides, collectively named xendorphins. One of these, xendorphin B1 ( XenPDYN-B sequence 96–111: YGGFIRKPDKYKFLNA), is a hexadecapeptide that displaced [ 3H]naloxone and the radiolabelled kappa opioid, [ 3H]dynorphin A (1–17), with nanomolar affinity from rat brain membranes. Using the acetic acid pain test, the present study examined the antinociceptive effects of spinally administered xendorphin B1 in amphibians. Xendorphin B1 produced a long-lasting and dose-dependent antinociceptive effect in the Northern grass frog ( Rana pipiens) with an ED 50 value of 44.5 nmol/frog. The antinociceptive effects of xendorphin B1 were significantly blocked by pretreatment with the non-selective opioid antagonist, naltrexone. This is the first report of the in vivo characterization of a non-mammalian prodynorphin-derived peptide and suggests that xendorphin peptides may play a role in the modulation of noxious information in vertebrates.

In Vitro Screening of Psychoactive Drugs by [35S]GTP.GAMMA.S Binding in Rat Brain Membranes

We constructed a reproducible, simple, and small-scale determination method of the psychoactive drugs that acted directly on the monoamine receptor by measuring the activation of [(35)S]guanosine-5'-O-(3-thio)-triphosphate binding to guanine nucleotide-binding proteins (G proteins). This method can simultaneously measure the effects of three monoamines, namely dopamine (DA), serotonin (5-HT), and norepinephrine (NE), in rat brain membranes using a 96-well microplate. Activation of D(1) and D(2) receptors in striatal membranes by DA as well as 5-HT and NEalpha(2) receptors in cortical membranes could be measured. Of 12 tested phenethylamines, 2,5-dimethoxy-4-chlorophenethylamine (2C-C), 2,5-dimethoxy-4-ethylphenethylamine (2C-E), and 2,5-dimethoxy-4-iodophenethylamine (2C-I) stimulated G protein binding. The other phenethylamines did not affect G protein binding. All 7 tryptamines tested stimulated G protein binding with the following rank order of potency; 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT)>5-methoxy-N,N-diallyltryptamine (5-MeO-DALT)>5-methoxy-alpha-methyltryptamine (5-MeO-AMT)>or=5-methoxy-N,N-methylisopropyltryptamine (5-MeO-MIPT)>5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT)>N,N-dipropyltryptamine (DPT)>or=alpha-methyltryptamine (AMT). This assay system was able to designate psychoactive drugs as prohibited substances in accordance with criteria set forth by the Tokyo Metropolitan government.

Ouabain-induced isoform-specific localization change of the Na +, K +-ATPase α subunit in the synaptic plasma membrane of rat brain

Na +, K +-ATPase is one of major membrane proteins that has two subunits, α and β. The α subunit has the ATPase activity and the ouabain binding site. Among four isoforms of the α subunit, expression of α1, α2, and α3, but not α4, is observed in matured rat brain. Ouabain is one of cardiac glycosides, and endogenous ouabain-like compounds have been recognized as a new class of steroid hormone. The α subunit is considered as their endogenous receptor. Recent studies envisaged the importance of membrane microdomains (MDs) as signaling platforms, which are recovered as a detergent-resistant membrane microdomain fraction (DRM). Although this ATPase has been considered as a non-DRM protein, some amount of the α subunit was found to be a component of the DRM prepared from the synaptic plasma membrane fraction (SPM) of rat brain. Ouabain treatment increased the amount of α3 isoform, but not α1, in the DRM derived from synaptosome fraction and SPM. These results suggest that the localization of the α subunit of Na +, K +-ATPase is regulated with isoform-specific mechanisms and the physiological importance of DRM in the signal transduction of the endogenous ouabain-like steroid hormone in neurons.

Isolation and Structure-Activity of μ-Conotoxin TIIIA, A Potent Inhibitor of Tetrodotoxin-Sensitive Voltage-Gated Sodium Channels

Mu-conotoxins are three-loop peptides produced by cone snails to inhibit voltage-gated sodium channels during prey capture. Using polymerase chain reaction techniques, we identified a gene sequence from the venom duct of Conus tulipa encoding a new mu-conotoxin-TIIIA (TIIIA). A 125I-TIIIA binding assay was established to isolate native TIIIA from the crude venom of Conus striatus. The isolated peptide had three post-translational modifications, including two hydroxyproline residues and C-terminal amidation, and <35% homology to other mu-conotoxins. TIIIA potently displaced [3H]saxitoxin and 125I-TIIIA from rat brain (Nav1.2) and skeletal muscle (Nav1.4) membranes. Alanine and glutamine scans of TIIIA revealed several residues, including Arg14, that were critical for high-affinity binding to tetrodotoxin (TTX)-sensitive Na+ channels. We were surprised to find that [E15A]TIIIA had a 10-fold higher affinity than TIIIA for TTX-sensitive sodium channels (IC50, 15 vs. 148 pM at rat brain membrane). TIIIA was selective for Nav1.2 and -1.4 over Nav1.3, -1.5, -1.7, and -1.8 expressed in Xenopus laevis oocytes and had no effect on rat dorsal root ganglion neuron Na+ current. 1H NMR studies revealed that TIIIA adopted a single conformation in solution that was similar to the major conformation described previously for mu-conotoxin PIIIA. TIIIA and analogs provide new biochemical probes as well as insights into the structure-activity of mu-conotoxins.

Mitochondrial Creatine Kinase Activity Prevents Reactive Oxygen Species Generation: ANTIOXIDANT ROLE OF MITOCHONDRIAL KINASE-DEPENDENT ADP RE-CYCLING ACTIVITY

As recently demonstrated by our group (da-Silva, W. S., Gómez-Puyou, A., Gómez-Puyou, M. T., Moreno-Sanchez, R., De Felice, F. G., de Meis, L., Oliveira, M. F., and Galina, A. (2004) J. Biol. Chem. 279, 39846-39855) mitochondrial hexokinase activity (mt-HK) plays a preventive antioxidant role because of steady-state ADP re-cycling through the inner mitochondrial membrane in rat brain. In the present work we show that ADP re-cycling accomplished by the mitochondrial creatine kinase (mt-CK) regulates reactive oxygen species (ROS) generation, particularly in high glucose concentrations. Activation of mt-CK by creatine (Cr) and ATP or ADP, induced a state 3-like respiration in isolated brain mitochondria and prevention of H(2)O(2) production obeyed the steady-state kinetics of the enzyme to phosphorylate Cr. The extension of the preventive antioxidant role of mt-CK depended on the phosphocreatine (PCr)/Cr ratio. Rat liver mitochondria, which lack mt-CK activity, only reduced state 4-induced H(2)O(2) generation when 1 order of magnitude more exogenous CK activity was added to the medium. Simulation of hyperglycemic conditions, by the inclusion of glucose 6-phosphate in mitochondria performing 2-deoxyglucose phosphorylation via mt-HK, induced H(2)O(2) production in a Cr-sensitive manner. Simulation of hyperglycemia in embryonic rat brain cortical neurons increased both DeltaPsi(m) and ROS production and both parameters were decreased by the previous inclusion of Cr. Taken together, the results presented here indicate that mitochondrial kinase activity performed a key role as a preventive antioxidant against oxidative stress, reducing mitochondrial ROS generation through an ADP-recycling mechanism.

New polyamine-sensitive inhibitors of the NMDA receptor: Syntheses and pharmacological evaluation

Derivatives of 5-(4-aminobutyl)-2-thiophene-octylamine, a potent polyamine-sensitive inhibitor of the NMDA receptor, were synthesized and evaluated as inhibitors of [ 3H]MK-801 binding to rat brain membranes. Alkylations of the terminal amino groups reduced inhibitory potency; only incorporation of the amino group of the short 4-aminobutyl arm into a piperidine ring was tolerated. Substitution of the thiophene nucleus with methyl or ethyl, and its replacement by a benzene nucleus, was of minor influence. The corresponding diguanidines exhibited high potency independent of chain length, whereas their sensitivity to spermine was sharply dependent on chain length. Insertion of an amide bond into the long octylamine arm increased sensitivity to spermine and to Tris buffer. Our results indicate that spermine sensitivity of [ 3H]MK-801 binding inhibition is responsive to subtle changes in inhibitor structure and represents a promising target for pharmaceutical research.

Synthesis, radiolabeling and receptor binding of [ 3H][(1 S,2 R)ACPC 2]endomorphin-2

Previously, we have shown that substitution of Pro 2 for cis-2-aminocyclopentanecarboxylic acid, ACPC in endomorphin-2 results in an analogue with greatly augmented proteolytic stability, high μ-opioid receptor affinity and selectivity. We now report the synthesis and biochemical characterization of [ 3H][(1 S,2 R)ACPC 2]endomorphin-2 with a specific activity of 1.41 TBq/mmol (38.17 Ci/mmol). Specific binding of [ 3H][(1 S,2 R)ACPC 2]endomorphin-2 was saturable and of high affinity with an equilibrium dissociation constant, K d = 1.80 ± 0.21 nM and receptor density, B max = 345 ± 27 fmol × mg protein −1 at 25 °C in rat brain membranes. Similar affinity values were obtained in kinetic and displacement assays. Both Na + and Gpp(NH)p decreased the affinity proving the agonist character of the radioligand. [ 3H][(1 S,2 R)ACPC 2]endomorphin-2 retained the μ-specificity of the parent peptide. The new radioligand will be a useful tool to map the topographical requirements of μ-opioid peptide binding due to its high affinity, selectivity and enzymatic stability.

Naloxone-blocked depriming effect of anxiolytic selank on apomorphine-induced behavioral manifestations of hyperfunction of dopamine system

Peptide anxiolytic selank (Thr-Lys-Pro-Arg-Pro-Gly-Pro) applied intraperitoneally in doses of 0.01, 0.1, 1.0, and 10.0 mg/kg to mice reduces behavioral manifestations of dopaminergic system induced by apomorphine in the verticalization test. This effect was comparable to that of atypical antipsychotic olanzapine in near-therapeutic doses (0.1 and 1.0 mg/kg, intraperitoneally) and was blocked with nonselective opioid receptor antagonist naloxone (10 mg/kg, intraperitoneally). Radioreceptor assay showed that selank did not displace nonselective D2-dopamine receptor antagonist (3)H-spiperone (EC50>100 microM) and delta- and micro-opioid receptor ligand 3H-DADLE (EC50>40 microM) from specific binding sites on rat brain membranes. It is hypothesized that the revealed behavioral effect of selank is mediated by its modulating effect on the endogenous opioid system and specifically, by its effect on activity of enkephalin-degrading enzymes.

Alkylene tether-length dependent γ-aminobutyric acid type A receptor competitive antagonism by tacrine dimers

Bis(7)-tacrine was previously demonstrated as an antagonist of γ-aminobutyric acid type A (GABA A) receptors. In this study, the effects of a series of alkylene-linked tacrine dimers on GABA A receptors were examined. In radioligand binding assay, the analogues differed in binding affinity for GABA A receptors, and potency monotonically increased as the tether was shortened from nine to two methylenes. Bis(2)-tacrine, the shortest tacrine dimer, could displace [ 3H]muscimol from rat brain membranes with an IC 50 of 0.48 μM, which was 11, 13 and 525 times more potent than the GABA A receptor antagonist (+)-bicuculline, bis(7)-tacrine and tacrine, respectively. In whole-cell patch-clamp recordings, these dimeric tacrine analogues competitively antagonized GABA-induced inward current with a rank order of potency of bis(2)-tacrine > bicuculline > bis(7)-tacrine > bis(9)-tacrine > tacrine, and the potency of bis(2)-tacrine was 11, 18 and 487 times higher than that of (+)-bicuculline, bis(7)-tacrine and tacrine, respectively. Bis(2)-tacrine shifted the GABA concentration–response curve to the right in a parallel manner, and the inhibition was voltage-independent between −80 and +20 mV. It can be concluded that the shorter the alkylene linkage in tacrine dimers the stronger the binding affinity and higher the antagonistic effect on the GABA A receptor will be.

In vitro and in vivo effect of BU99006 (5-isothiocyanato-2-benzofuranyl-2-imidazoline) on I 2 binding in relation to MAO: Evidence for two distinct I 2 binding sites

BU99006 is an irreversible I 2 ligand which selectively inactivates I 2 binding sites, making it an ideal tool with which to study I 2 site mechanism. We sought to determine the effects of BU99006 on I 2 binding in relation to monoamine oxidase (MAO), and the time course of these effects. In vitro, rat brain membranes that were pre-treated with 10 μM BU99006 showed no change in MAO activity, despite suffering a significant reduction in [ 3H]2BFI binding (52.5 ± 19.6 to 8.5 ± 3.8 fmol mg −1, 84%). Furthermore, reversible I 2 ligands 2BFI and BU224 were able to inhibit MAO, whether treated with BU99006 or not. In vivo, a 5 mg kg −1 i.v. dose of BU99006 in rats rapidly reduced [ 3H]2BFI binding with similar magnitude (85%, maximal reduction after 20 min), without effect on either MAO activity or the specific binding of selective MAO-A and MAO-B radioligands. Moreover, following this irreversible treatment, recovery of central [ 3H]2BFI binding occurred with a rapid half-life of 4.3 h in rat brain (2.0 h in mouse), which is not consistent with a site on MAO. These data indicate that the high affinity site which is occupied by [ 3H]2BFI and irreversibly binds BU99006, is not the same as that which causes inhibition of MAO, and may point to the existence of another I 2 binding site.

Immunolocalization of Na V1.2 channel subtypes in rat and cat brain and spinal cord with high affinity antibodies

High titer polyclonal antibodies were produced in rabbit against a peptide unique to Na V1.2 sodium channels. Na V1.2 antibodies displayed 500,000-fold greater affinity for the Na V1.2 peptide compared with Na V1.1 or Na V1.3 peptides from the same region. These antibodies, when coupled to Sepharose beads, retained saxitoxin binding sites from solubilized rat brain membranes. Eluted protein from this antibody-affinity column was recognized by antibodies directed against neuronal voltage-gated sodium channels. Rabbit antibodies, which had been partially purified, were used in immunocytochemical localization of the Na V1.2 channel in 50 μm rat brain slices at dilutions of 1:1000 or 1:2000. Na V1.2 channels were predominately localized in unmyelinated fibers in the cortex, hippocampus, spinal cord and hypothalamus. Varicosities were seen in fiber staining which may reflect true varicosities in the fiber or simply varying densities of sodium channels along the fiber. Cell body staining with the Na V1.2 antibody was primarily observed in the hypothalamus. Antibody staining in the cerebellum was complex, with staining observed primarily in posterior lobes and considerably lower amounts of staining observed in anterior lobes. Specific staining was limited to fibers located in the granule and molecular layer, in an orientation consistent with granule cell unmyelinated axon labeling.

The β-Adrenergic Receptor Antagonist Metipranolol Blunts Zinc-Induced Photoreceptor and RPE Apoptosis

To determine the effect of zinc on retinal cells at concentrations at which it is known to cause oxidative stress. Furthermore, the effects of metipranolol, known to prevent retinal damage, and of other antiglaucoma drugs were determined on zinc-injured retinal cells. Lipid peroxidation assays were conducted on rat brain and bovine retina-retinal pigment epithelial (RPE) membrane preparations. Immunohistochemistry, immunoblot analysis and the terminal-deoxynucleotidyl transferase dUTP-linked nick-end labeling (TUNEL) procedure determined the effects of zinc with or without trolox or metipranolol on photoreceptor death in situ. The effect of treatments on cultured RPE cells was analyzed using cell viability assays, immunoblot analysis, and the TUNEL procedure. Zinc-induced lipid peroxidation of rat brain and bovine retina-RPE membranes, although the effect of the latter was of a (twofold) greater magnitude. Both effects, however, were similarly attenuated by metipranolol, desacetylmetipranolol, and trolox. Antiglaucoma drugs other than metipranolol had no effect. Intraocular injection of 150 microM zinc and treatment of cultured RPE cells with zinc led to mainly photoreceptor apoptosis and apoptotic death of RPE cells (50% death at 18 microM rising to 10% at 50 microM), respectively. Zinc-induced apoptosis of cultured RPE cells and photoreceptors were attenuated only by metipranolol and trolox. The combined data suggest that oxidative injury to RPE cells and photoreceptors may be caused by elevated levels of zinc in diseases such as age-related macular degeneration (AMD) and that metipranolol may act as an efficacious antioxidant to blunt this process.

The synthesis and nicotinic binding activity of (±)-epiquinamide and (±)- C(1)- epiepiquinamide

The synthesis of (±)-epiquinamide 1 and (±)- C(1)- epiepiquinamide 2 based on the use of a Curtius rearrangement to introduce the C(1) amino residue is reported. In a competition binding assay for [ 3H]epibatidine binding to rat brain membranes neither (±)- 1 nor (±)- 2 showed any significant level of nicotinic activity.

Lack of interaction between etifoxine and CRF 1 and CRF 2 receptors in rodents

Hyperactivity of the corticotropin-releasing factor (CRF) system occurs in some patients with anxiety disorders and depression. Blockade of CRF 1 and CRF 2 receptors can underlie the anxiolytic effects of drugs. In the present investigation, in vivo and in vitro studies were designed to determine whether the anxiolytic drug etifoxine, known to enhance GABAergic synaptic transmission, behaves also as a CRF 1 and CRF 2 receptor antagonist. A drug exerting multiple actions may be of clinical interest in the treatment of various different forms of mood disorders. Using two animal models, it was found that etifoxine reversed the excess CRF-induced grooming but not the hypo-locomotion of the rat placed in an open field. Etifoxine attenuated the CRF-induced gastric emptying delay in the mouse. On the other hand, in vitro, binding of etifoxine to CRF 1 and CRF 2 receptors on rat brain membranes was negligible and functionally, etifoxine did not block the CRF 1 and CRF 2 activation-induced cAMP production in presence of CRF in human neuroblastoma SH-SY5Y cells. The selective anxiolytic properties of etifoxine appear unrelated to an antagonist activity at the CRF 1 and CRF 2 receptors. The decrease in CRF activity produced by etifoxine may be related to its GABAergic properties.

Preclinical Profile of PF9404C, a Nitric Oxide Donor with β Receptor Blocking Properties

PF9404C ((2'S),(2S)-3-isopropylamine, 1-[4-(2,3-dinitroxy)propoxymethyl]-phenoxy-2'-propranol) is the S-S diesteroisomer of a novel blocker of beta-adrenergic receptors with vasorelaxing properties. It causes a concentration-dependent relaxation of rat aorta helical strips precontracted with 10(-6) M norepinephrine (NE; IC50 33 nM). It is equipotent to nitroglycerin (NTG; IC50 49 nM), but much more potent than isosorbide dinitrate (ISD; IC50 15,000 nM). In rat aorta smooth muscle cells, at 10 microM, PF9404C increased the formation of cGMP from 3 pmol/mg protein in basal conditions to 53 pmol/mg protein, suggesting that the mechanism of its vasorelaxing effects involves the slow generation of NO. This is supported by the facts that (i) ODQ (a blocker of guanylate cyclase) inhibited the vasodilatory effects of PF9404C; and (ii) PF9404C generates NO, as indirectly measured by the Griess reaction. In the electrically driven guinea pig left atrium, PF9404C blocks the inotropic effects of isoproterenol in a concentration-dependent manner. Its IC50 (30 nM) was similar to that of S-propranolol (22.4 nM) and lower than that of metoprolol (120 nM) or atenolol (192 nM). The beta adrenergic ligand (-)-[3H]-CGP12177 (4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one hydrochloride) (0.2 nM) is displaced from its binding sites in rat brain membranes with a K(i) of 7, 17, 170, and 1200 nM for PF9404C, S-(-)propranolol, metoprolol, and atenolol, respectively. PF9404C blocks 45Ca2+ entry into bovine adrenal chromaffin cells induced by direct depolarization with 70 mM K+ or by the nicotinic agonist dimethylphenylpiperazinium (DMPP). PF9404C exhibits about 3-fold higher potency than NTG to relax the majority of the vessels studied, especially when they were contracted with K+, and shows a certain selectivity of action for the renal artery. It produces auto-tolerance that is ca. 20-fold less pronounced than that observed with NTG. Cross-tolerance in preparations pre-exposed to PF9404C and later relaxed with NTG, was much greater than auto-tolerance. This makes PF9404C a useful pharmacological tool for the development of novel NO-donor compounds with a lesser degree of vascular tolerance than those currently available.

In vitro and antinociceptive profile of HON0001, an orally active NMDA receptor NR2B subunit antagonist

The analgesic activity and side effect liabilities of a novel NR2B antagonist, 7-hydroxy-6-methoxy-2-methyl-1-(2-(4-(trifluoromethyl)phenyl)ethyl)-1,2,3,4-tetrahydroisoquinoline hydrochloride (HON0001) were investigated. HON0001 inhibited [ 3H]MK-801 binding to rat brain membranes in a biphasic manner, with IC 50 values of 54.68 ± 4.96 nM and 46.48 ± 5.85 μM for high- and low-affinity sites, respectively. HON0001 inhibited [ 3H]ifenprodil binding to membranes of rat cerebral cortex with an IC 50 value of 57.01 ± 3.4 nM, consistent with the results obtained for high-affinity sites of [ 3H]MK-801 binding. HON0001 exhibited no or negligible affinity for other receptors, transporters and ion channels, while HON0001 had a moderate agonistic activity at μ-opioid receptors and affinity for dopamine D 1 receptors. HON0001 exhibited an analgesic effect in carrageenan-induced mechanical hyperalgesia and in the Seltzer model of partial sciatic nerve ligation following oral administration. In contrast, unlike MK-801, HON0001 did not affect spontaneous locomotor activity, rotarod performance and step-through latency in a passive avoidance task even at doses much higher than antinociceptive doses. HON0001 exhibited excellent brain penetration with a brain-to-plasma ratio of 34.5. These findings show that HON0001 is an orally active NR2B antagonist and that it may be useful for treating patients with neuropathic and other conditions without causing the side effects often observed with currently available non-subtype selective NMDA receptor antagonists.