Rat Cerebral Cortex Membranes Research Articles

Regulation of m 1 muscarinic receptors and nNOS mRNA levels by autoantibodies from schizophrenic patients

In this paper we demonstrate that, circulating antibodies from schizophrenic patients interacting with cerebral M 1 muscarinic acetylcholine receptors (M 1 mAChRs), can act as an inducer of m 1 mAChR-mRNA, and neuronal nitric oxide synthase (nNOS) mRNA gene expression of rat frontal cortex. The different signaling pathways involved in the autoantibody's actions, were characterized. As previously reported serum autoantibodies from schizophrenic patients reacted against neural cells surface inhibiting the binding of the specific mAChR radioligand to rat cerebral frontal cortex membrane. Moreover, by ELISA using M 1 synthetic peptide (with identical aminoacid sequence to human M 1 mAChR) as coating antigen we demonstrated the reactivity against the second extracellular loop of human cerebral M 1 mAChR. The corresponding affinity-purified anti M 1 peptide IgG (anti M 1 peptide IgG) from schizophrenic patients by stimulation of M 1 mAChR exerted an increase in m 1 mAChR-mRNA and nNOS-mRNA levels, that significantly correlated with the accumulation of phosphoinositides (IPs) and activation of NOS (α = 0.05). All these effects were blunted by pirenzepine and mimicked the action of the authentic agonist. Concurrent analysis of the effects of nNOS, phospholipase C (PLC) and calcium/calmodulin (CaM) inhibition on both, m 1 mAChR-mRNA and nNOS-mRNA levels, showing that antibody up-regulation mRNA level is under the control of endogenous nitric oxide (NO) signaling system. On the basis of our results, the activation of M 1 mAChR by schizophrenic autoantibody appears to induce nNOS-mRNA expression and reciprocally, the activation of NOS up-regulates m 1 mAChR gene expression. These results gave support to the participation of an autoimmune process in a particular group of chronic schizophrenic patients.

NEW 4-ARYL-1,4-DIHYDROPYRIDINES AND 4-ARYLPYRIDINES AS P-GLYCOPROTEIN INHIBITORS

Efflux of cytotoxic agents mediated by P-glycoprotein is believed to be an important mechanism of multidrug resistance, which remains a serious limitation to successful chemotherapy in cancers such as metastatic breast cancer. A series of 4-aryl-1,4-dihydropyridines and corresponding aromatized 4-arylpyridines have been synthesized based on structure modifications of niguldipine to enhance multidrug resistance reversal activity, while minimizing calcium channel binding. Thirty new compounds were characterized. [(3)H]Vinblastine accumulation studies indicated that at a concentration level of 3 muM, 15 of 18 4-aryl-1,4-dihydropyridines and all 4-arylpyridines can successfully restore intracellular accumulation of vinblastine in a resistant human breast adenocarcinoma cell line, MCF-7/adr, which overexpresses P-glycoprotein. The most potent compounds led to an approximately 15-fold increase of vinblastine accumulation. All of the test compounds that significantly increased vinblastine accumulation in MCF/adr cells were able to substantially reduce IC(50) values of daunomycin and increase its cytotoxicity in MCF-7/adr-resistant cells, confirming the results of the vinblastine accumulation studies. Calcium channel binding assays for these newly synthesized compounds were conducted using rat cerebral cortex membrane. All but eight compounds demonstrated negligible calcium channel binding over the concentration range from 15 to 2500 nM. The results demonstrate that the newly synthesized series of 1,4-dihydropyridines and pyridines represent P-glycoprotein modulators with negligible calcium channel blocking activity.

Characterization of the interaction of indiplon, a novel pyrazolopyrimidine sedative-hypnotic, with the GABAA receptor.

Clinically used benzodiazepine and nonbenzodiazepine sedative-hypnotic agents for the treatment of insomnia produce their therapeutic effects through allosteric enhancement of the effects of the inhibitory neurotransmitter GABA at the GABA(A) receptor. Indiplon is a novel pyrazolopyrimidine sedative-hypnotic agent, currently in development for insomnia. Using radioligand binding studies, indiplon inhibited the binding of [(3)H]Ro 15-1788 (flumazenil) to rat cerebellar and cerebral cortex membranes with high affinity (K(i) values of 0.55 and 0.45 nM, respectively). [(3)H]Indiplon binding to rat cerebellar and cerebral cortex membranes was reversible and of high affinity, with K(D) values of 1.01 and 0.45 nM, respectively, with a pharmacological specificity consistent with preferential labeling of GABA(A) receptors containing alpha1 subunits. In "GABA shift" experiments and in measurements of GABA-induced chloride conductance in rat cortical neurons in culture, indiplon behaved as an efficacious potentiator of GABA(A) receptor function. In both the radioligand binding and electrophysiological experiments, indiplon had a higher affinity than zolpidem or zaleplon. These in vitro properties are consistent with the in vivo properties of indiplon as an effective sedative-hypnotic acting through allosteric potentiation of the GABA(A) receptor.

Actions of amphetamine derivatives and cathinone at the noradrenaline transporter

We have recently shown that methylenedioxymethamphetamine (MDMA), methylenedioxyamphetamine (MDA), cathinone and methylenedioxyethylamphetamine (MDEA) have a cocaine-like action to potentiate the contractile actions of noradrenaline but not isoprenaline in the 1-Hz paced rat right ventricle. The purpose of this study was to directly test the actions of these compounds at the noradrenaline transporter. In rat left ventricular slices, potency (−log IC 50) values at inhibiting uptake of [ 3H]noradrenaline were: cocaine 6.16±0.15, cathinone 6.03±0.16, MDMA 6.05±0.07, MDA 5.68±0.06 and MDEA 5.56±0.08. MDEA and MDA were significantly less potent. In rat cerebral cortex membranes, MDMA was significantly less potent at displacing [ 3H]nisoxetine binding; −log EC 50 values: cocaine 5.04±0.08, cathinone 5.40±0.14, MDA 4.66±0.11, MDEA 4.99±0.15, MDMA 4.22±0.07. The noradrenaline uptake studies showed that MDEA was least potent: MDEA was also least potent functionally in the paced rat right ventricle. The [ 3H]nisoxetine displacement studies did not compare with the functional studies.

Methyl-laudanosine: a new pharmacological tool to investigate the function of small-conductance Ca(2+)-activated K(+) channels.

Small-conductance Ca(2+)-activated K(+) channels (SK channels) underlie the prolonged postspike afterhyperpolarization (AHP) observed in many central neurons and play an important role in modulating neuronal activity. However, a lack of specific and reversible blockers of these channels hampers their study in various experimental conditions. Because previous work has shown that bicuculline salts block these channels, we examined whether related alkaloids, namely laudanosine quaternary derivatives, would produce similar effects. Intracellular recordings were performed on rat midbrain dopaminergic neurons and hippocampus CA1 pyramidal cells. Binding experiments were performed on rat cerebral cortex membranes. Laudanosine, methyl-laudanosine, and ethyl-laudanosine blocked the apamin-sensitive AHP of dopaminergic neurons with mean IC(50) values of 152, 15, and 47 microM, respectively. The benzyl and butyl derivatives were less potent. Methyl-laudanosine had no effect on the I(h) current, action potential parameters, or membrane resistance of dopaminergic cells, or on the decrease in input resistance induced by muscimol, indicating a lack of antagonism at GABA(A) receptors. Interestingly, 100 microM methyl-laudanosine induced a significant increase in spiking frequency of dopaminergic neurons but not of CA1 pyramidal cells, suggesting the possibility of regional selectivity. Binding experiments on laudanosine derivatives were in good agreement with electrophysiological data. Moreover, methyl-laudanosine has no affinity for voltage-gated potassium channels, and its affinity for SK channels (IC(50) 4 microM) is superior to its affinity for muscarinic (IC(50) 114 microM) and neuronal nicotinic (IC(50) > or =367 microM) receptors. Methyl-laudanosine may be a valuable pharmacological tool to investigate the role of SK channels in various experimental models.

Antibodies against cerebral M1 cholinergic muscarinic receptor from schizophrenic patients: molecular interaction.

We demonstrated the presence of circulating Abs from schizophrenic patients able to interact with cerebral frontal cortex-activating muscarinic acetylcholine receptors (mAChR). Sera and purified IgG from 21 paranoid schizophrenic and 25 age-matched normal subjects were studied by indirect immunofluorescence, flow cytometry, immunoblotting, dot blot, ELISA, and radioligand competition assays. Rat cerebral frontal cortex membranes and/or a synthetic peptide, with an amino acid sequence identical with that of human M(1) mAChR, were used as Ags. By indirect immunofluorescence and flow cytometry procedures, we proved that serum-purified IgG fraction from schizophrenic patients reacted to neural cell surfaces from rat cerebral frontal cortex. The same Abs were able to inhibit the binding of the specific M(1) mAChR radioligand [(3)H]pirenzepine. Immunoblotting experiments showed that IgG from schizophrenic patients revealed a band with a molecular mass coincident to that labeled by an anti-M(1) mAChR Ab. Using synthetic peptide for dot blot and ELISA, we demonstrated that these Abs reacted against the second extracellular loop of human cerebral M(1) mAChR. Also, the corresponding affinity-purified antipeptide Ab displayed an agonistic-like activity associated to specific receptor activation, increasing cyclic GMP production and inositol phosphate accumulation, and protein kinase C translocation. This paper gave support to the participation of an autoimmune process in schizophrenia.

In vitro and in vivo characterization of [125I]iodomethyllycaconitine in the rat.

The in vitro and in vivo binding characteristics of [125I]iodomethyllycaconitine ([125I]iodoMLA) were determined in the rat. [125I]iodoMLA binding to rat cerebral cortex membranes was saturable and reversible and its specific binding represented approximately 70-80% of the total binding. [125I]iodoMLA labeled a single site with Kd = 1.8 +/- 0.4 nM and Bmax = 68 +/- 3 fmol/mg protein. Kinetic analysis revealed a t1/2 for association and dissociation of 10.5 +/- 3.1 and 10.3 +/- 1.6 min, respectively. Pharmacological characterization of [125I]iodoMLA binding indicated that it was specific for the alpha7 nAChR. In vitro brain region binding studies revealed greater binding in regions known to contain high numbers of alpha7 nAChRs. The analysis of the biodistribution of intravenously administered [125I]iodoMLA indicated that it was rapidly cleared and exhibited poor brain penetration; nevertheless, the levels of [125I]iodoMLA in alpha7 nAChR-rich target regions were significantly increased compared to the nontarget region (cerebellum) 60-120 min after administration. No metabolism of MLA by human liver S9 fraction was detected. Our results suggest that [125I]iodoMLA will be a useful radioligand to study the alpha7 nAChR in vitro and in vivo.

Spermidine antagonizes the inhibitory effect of huperzine A on [ 3H]dizocilpine (MK-801) binding in synaptic membrane of rat cerebral cortex

Huperzine A, a novel cholinesterase inhibitor, was found to inhibit the N-methyl- d-aspartate (NMDA) receptors in the brain. In this study, the mechanisms of the NMDA receptor inhibition were investigated using [ 3H]dizocilpine (MK-801) binding in synaptic membrane of rat cerebral cortex. Changing the concentrations of l-glutamate and l-glycine did not alter the potency of huperzine A. Spermidine caused rightward shift of the concentration–response curve of huperzine A, and considerably increased its IC 50 value. Huperzine A did not affect the potency of unlabeled (+)-MK-801 in [ 3H]MK-801 binding. Saturation binding studies reveal that huperzine A exerts a negative allosteric modulation on the MK-801 binding site within the NMDA receptor-channel. The results suggest that huperzine A is a non-competitive antagonist of the NMDA receptors, acting at one of the polyamine binding sites.

The effect of the serotonergic receptor antagonist mianserine on the activity of Na, K-ATPase from rat cerebral cortex membranes.

The effect of the serotonergic receptor antagonist mianserine on the activity of Na, K-ATPase from rat cerebral cortex membranes.

Pharmacological analysis of immepip and imetit homologues. Further evidence for histamine H 3 receptor heterogeneity?

Following a previous report by our research group on discriminative properties of a series of aliphatic histamine homologues, we now studied immepip , imetit and its lower and higher sidechain homologues as ligands for the histamine H 3 receptor in a [ 125I]-iodophenpropit binding assay using rat cerebral cortex membranes, and two functional H 3 receptor models (inhibition of the neurogenic contraction of the guinea pig jejunum and inhibition of [ 3H]-noradrenaline release in rat cerebral cortex slices). The immepip homologues behaved as competitive H 3-receptor antagonists in both functional systems. The potencies (p A 2 values) observed at the guinea pig jejunum were 8.4 and 6.2 for the immepip homologues VUF 4929 and VUF 4735, respectively, whereas on the electrically evoked release of [ 3H]-noradrenaline from cortical slices the p A 2 values were 7.1 and 5.5 for VUF 4929 and VUF 4735, respectively. Moreover, immepip, but not the (R)-α-methylhistamine, showed almost a tenfold higher agonistic potency in the rat cerebral cortex than in the guinea pig jejunum. For imetit and its homologues important discrepancies in the potencies in the two functional assays were noticed as well. VUF 8328 acts as a potent (p D 2=8.0) partial agonist in the brain, but as a very active (p A 2=9.4) competitive antagonist in the guinea pig jejunum. The partial agonistic activity of VUF 8328 in the brain was confirmed by GTPγS-sensitive, biphasic displacement of [ 125I]-iodophenpropit binding to rat cerebral cortex membranes. The differences in potencies shown by the various ligands are discussed in relation to H 3 receptor heterogeneity.

Autoradiographic localization of [formula omitted]thiocolchicoside binding sites in the rat brain and spinal cord

Thiocolchicoside is used in humans as a myorelaxant drug with anti-inflammatory and analgesic activity. Recently we established the experimental conditions that allowed the identification of [ 3 H] thiocolchicoside binding sites in synaptic membranes of rat spinal cord and cerebral cortex. The pharmacological characterization of these sites indicated that GABA and several of its agonists and antagonists, as well as strychnine, were able to interact with [ 3 H] thiocolchicoside binding in a dose-dependent manner and with different affinities. In order to gain more insight into the nature and the anatomical distribution of the binding sites labeled by [ 3 H] thiocolchicoside, in the present study we examined the localization of these sites on parasagittal and coronal sections of the rat brain and spinal cord, respectively, using receptor autoradiography. In the spinal cord an intense signal was observed in the gray matter, with the highest density occurring in the superficial layers of the dorsal horns. Strychnine completely displaced [ 3 H] thiocolchicoside binding, whereas GABA only partially removed the radioligand from its binding sites. In the brain, specific binding occurred in several areas and was displaced by both GABA and strychnine. The distribution of [ 3 H] thiocolchicoside binding sites in brain sections, however, did not match that found for [ 3 H] muscimol. Furthermore, cold thiocolchicoside was not able to completely displace [ 3 H] muscimol binding, and showed a different efficacy in the various areas labeled by the radioligand. We conclude that thiocolchicoside may interact with a subpopulation of GABA A receptors having low-affinity binding sites for GABA. Furthermore, the observed sensitivity to strychnine in the spinal cord indicates an interaction also with strychnine-sensitive glycine receptors, suggesting that the pharmacological effects of thiocolchicoside may be the result of its interaction with different receptor populations.

Functional modulation of gamma-aminobutyric acid A receptors by etifoxine and allopregnanolone in rodents

We looked for an interaction between etifoxine and the neurosteroid allopregnanolone at central gamma-aminobutyric acid (GABA A) receptors. Etifoxine (2 μM) did not affect the affinity of allopregnanolone (IC 50=108 nM) for its site in preparations of Sprague–Dawley rat cerebral cortex membranes, as determined by the inhibition of [ 35S] t-butylbicyclophosphorothionate binding, a specific ligand of the GABA A receptor chloride channel site. Etifoxine and allopregnanolone were anticonvulsants, blocking the clonic convulsions induced by bicuculline (an antagonist of the GABA A receptor) in CD1 mice. A combination of subactive doses of the two compounds showed additive anticonvulsant effects. These results suggest that etifoxine and allopregnanolone bind to distinct putative recognition sites at or near the chloride channel site. Functionally, their binding may have an additive effect by enhancing GABA A inhibitory transmission.

(125)I]3beta-(4-ethyl-3-iodophenyl)nortropane-2beta-carboxylic acid methyl ester ([(125)I]EINT): a potent and selective radioligand for the brain serotonin transporter.

The binding characteristics of [(125)I]3beta-(4-ethyl-3-iodophenyl)nortropane-2beta-carboxylic acid methyl ester ([(125)I]EINT), a high-affinity selective ligand for the serotonin transporter (5-HTT), and its binding characteristics to rat brain membranes were determined. [(125)I]EINT binding to rat cerebral cortex membranes was saturable and reversible, and its specific binding represented approximately 90% of the total binding. [(125)I]EINT labeled a single site with K(d) = 0.22 +/- 0.03 nM and B(max) = 583 +/- 38 fmol/mg protein. Kinetic analysis revealed a t(1/2) for association and dissociation of 20 and 24 min, respectively. Pharmacological characterization of [(125)I]EINT confirmed its high specificity for the 5-HTT. The pattern of brain region distribution in vivo of intravenously administered [(125)I]EINT indicated greater accumulation of the radioligand in 5-HTT-rich brain regions. However, the signal-to-background ratio was low. Thus, [(125)I]EINT appears to be a useful radioligand for studying the 5-HTT in vitro, but it may not be a good in vivo ligand.

Similar potency of the enantiomers of huperzine A in inhibition of [ 3H]dizocilpine (MK-801) binding in rat cerebral cortex

The inhibition of huperzine A, a potential therapeutic agent to treat Alzheimer's disease, on rat cortical acetylcholinesterase was found to be highly stereospecific. In the present study the effect of the enantiomers of huperzine A on [ 3H]dizocilpine (MK-801) binding to synaptic membrane of rat cerebral cortex was compared. The natural (−)-huperzine A and the synthetic (+)-huperzine A inhibited the specific binding of [ 3H]MK-801 with a similar potency. The IC 50 values were 65±7 and 82±12 μM ( n=5 for each enantiomer, P=0.248), respectively. The result indicates that huperzine A inhibits N-methyl- d-aspartate (NMDA) receptor in rat cerebral cortex without stereoselectivity.

Distribution and characterisation of somatostatin receptor mRNA and binding sites in the brain and periphery

The distribution and nature of (somatostatin) SRIF receptors and receptor mRNAs was studied in the brain and periphery of various laboratory animals using in situ hybridisation, autoradiography and radioligand binding. The messenger RNA (mRNA) expression of SRIF receptors msst 1, msst 2, msst 3, msst 4 and msst 5 was studied in the adult mouse brain by in situ hybridisation histochemistry using specific oligonucleotide probes and compared to that of adult rats. As observed in rat brain, sst 3 receptor mRNA is prominently expressed across the mouse brain, although equivalent binding has not yet been identified in situ. Sst 1 and sst 2 receptor mRNA expression, was prominent and again comparable to that observed in rat brain, whereas sst 4 and especially sst 5 receptor mRNA show comparatively low levels, although the former appears to be widely distributed while the latter could only be identified in a few nuclei. Altogether, the data are compatible with current knowledge, i.e. sst 1 and sst 2 receptor mRNA is prominent (both receptors have been functionally identified in the brain and for sst 2 in the periphery), sst 3 mRNA is highly expressed but in the absence of any functional correlate remains elusive. The expression of sst 4 mRNA is comparatively low (especially when compared to what is seen in the lung, where high densities of sst 4 receptors are present) and it remains to be seen whether sst 5 receptor mRNA, which is confined to a few nuclei, will play a role in the brain, keeping in mind that high levels are found in the pituitary. Radioligand binding studies were performed in CCL39 cells expressing the five human recombinant receptors and compared to binding in membranes of rat cerebral cortex with [ 125I]Tyr 11-SRIF 14 which in the presence of 120 mM labels primarily sst 1 receptor as suggested by the better correlation hsst 1 and similar rank order of potency. The profile of [ 125I]Tyr 3-octreotide labelled sites in rat cortex correlates better with recombinant sst 2 than sst 3 or sst 5 binding profiles. Finally, [ 125I]LTT-SRIF 28-labelled sites in rat lung express a sst 4 receptor profile in agreement with previous findings. SRIF receptor autoradiography was performed in the brain and peripheral tissue of rat and/or guinea-pig using a number of ligands known to label recombinant SRIF receptors: [ 125I]LTT-SRIF 28, [ 125I]CGP 23996, [ 125I]Tyr 10-CST, or [ 125I]Tyr 3-octreotide. Although, [ 125I]Tyr 10-CST has been shown to label all five recombinant SRIF receptors, it is apparent that this radioligand is not useful for autoradiographic studies. By contrast, the other three ligands show good signal to noise ratios in rat or guinea-pig brain, rat lung, rat pancreas, or guinea-pig ileum. In most tissues, [ 125I]Tyr 3-octreotide represents a prominent part of the binding (when compared to [ 125I]LTT-SRIF 28 and [ 125I]CGP 23996), suggesting that sst 2 receptors are strongly expressed in most tissues; it is only in rat lung that [ 125I]LTT-SRIF 28 and [ 125I]CGP 23996 show marked binding, whereas [ 125I]Tyr 3-octreotide does apparently label no sites, in agreement with the sole presence of sst 4 receptors in this tissue.

The identification of a potent imidazoline-based vascular K(ATP) channel antagonist.

In this study the activity of a number of novel imidazoline-based compounds (IMID series) was assessed by functional and binding studies to determine their actions at K(ATP) channels. The novel compounds, which we synthesised, were methoxy-, methyl-, butyl- and fluorophenyl derivatives of clonidine. In functional studies we determined the potency (by calculating a pK(B) value) of the IMID compounds to antagonise levcromakalim responses in segments of isolated pig coronary artery. The most potent compounds identified (laboratory codes: IMID-1M, IMID-26F and IMID-4F) had apparent pK(B) values of approximately 7 which is similar to that for the sulphonylurea, glibenclamide and the lipophilic quaternary ion, tetraphenylphosphonium. This inhibitory action was specific for levcromakalim since the imidazoline antagonist IMID-1M failed to effect vasorelaxation response-curves to the non-KATP channel opener, sodium nitroprusside. In the spontaneously beating rat right atrium preparation the majority of the compounds were able to cause slowing of heart rate, but with low EC50 values (approximately 10-30 microM). In binding studies, the compounds were unable to displace binding of [3H]P1075 to bovine aortic smooth muscle preparations nor [3H]glibenclamide binding to rat cerebral cortex membranes. These studies show that some imidazoline-based compounds are potent antagonists of levcromakalim-mediated vasorelaxation responses in the pig coronary artery. The compounds displayed only minimal bradycardic activity. The site of action of the imidazoline compounds does not appear to be the same as that used by K(ATP) channel openers or sulphonylurea-based antagonists. It is likely that these compounds interact with the K(ATP) channel pore itself.

6-Chloro-3′-nitroflavone is a Potent Ligand for the Benzodiazepine Binding Site of the GABA A Receptor Devoid of Intrinsic Activity

6-Chloro-3′-nitroflavone integrates a list of nearly 70 flavone derivatives synthesized in our laboratories. The effects of 6-chloro-3′-nitroflavone on the benzodiazepine binding sites (BDZ-BSs) of the GABA A receptor were examined in vitro and in vivo. 6-Chloro-3′-nitroflavone inhibited the [ 3H]flunitrazepam ([ 3H]FNZ) binding to rat cerebral cortex membranes with a K i of 6.68 nM and the addition of GABA to extensively washed membranes did not modify its affinity for the BDZ-BSs (GABA-shift = 1. 16 ± 0.12). The binding assays performed in rat striatal and cerebellar brain membranes showed that this compound has similar affinity to different populations of BDZ-BSs. Electrophysiological experiments revealed that 6-chloro-3′-nitroflavone did not affect GABA A-receptors (GABA A-Rs) responses recorded in Xenopus oocytes expressing α 1β 2γ 2s subunits, but blocked the potentiation exerted by diazepam (DZ) on GABA-activated chloride currents. In vivo experiments showed that 6-chloro-3′-nitroflavone did not possess anxiolytic, anticonvulsant, sedative, myorelaxant actions in mice or amnestic effects in rats; however, 6-chloro-3′-nitroflavone antagonized diazepam-induced antianxiety action, anticonvulsion, short-term, and long-term amnesia and motor incoordination. These biochemical, electrophysiological, and pharmacological results suggest that 6-chloro-3′-nitroflavone behaves as an antagonist of the BDZ-BSs.

Compounds extracted from Phyllantus and Jatropha elliptica inhibit the binding of [3H]glutamate and [3H]GMP-PNP in rat cerebral cortex membrane.

Glutamate is to be considered a nociceptive neurotransmitter and glutamatergic antagonists present antinoceptive activity. In this study we investigated the effects of the naturally occurring antinociceptive compounds rutin, geraniin and quercetine extracted from Phyllanthus, as well as the diterpene jatrophone, extracted from Jatropha elliptica on the binding of [3H]glutamate and [3H]GMP-PNP [a GTP analogue which binds to extracellular site(s), modulating the glutamatergic transmission] in rat brain membrane. Jatrophone inhibited [3H]glutamate binding and geraniin inhibited [3H]GMP-PNP binding. Quercetine inhibited the binding of both ligands. These results may indicate a neurochemical parameter possibly related to the antinoceptive activity of these natural compounds.

Bolus Intravenous Injection of Phosphorothioate Oligonucleotides Causes Hypotension by Acting as α1-Adrenergic Receptor Antagonists

Bolus intravenous injections of phosphorothioate oligonucleotides (PS-ODN) into primates cause profound hypotension, which has been attributed to complement activation, the biochemical pathway leading to acute inflammatory response. Because the hypotension was not accompanied by peripheral or pulmonary edema and epinephrine was not effective, but administration of 200 ml Ringer's lactate was effective, we examined the possibility that the 15-base PS-ODN interferes with sympathetic tone. We administered doses ranging from 3.3 to 10 mg/kg of a 15-base PS-ODN as a 30–60 s iv bolus into the right atrium of conscious Macaca mulatta. Blood pressure fell to 27 mm Hg following a 5.0 mg/kg dose, but no hypotension was observed after a 3.3 mg/kg dose; 10 mg/kg was lethal. Adrenergic receptor binding was evaluated in radioligand binding assays using rat cerebral cortex membranes with radiolabeled prazosin. The 15-base PS-ODN competes with prazosin for the α1-adrenergic receptor with an IC50 of 14 μM, which favors binding over serum albumin (Kd = 37 to 48 μM). Admixing serum albumin with 5.0 mg/kg 15-base PS-ODN prior to injection prevented hypotension, suggesting that unbound PS-ODN interferes with sympathetic tone before binding to plasma proteins. Interactions of the 15-base PS-ODN with the α1-adrenergic receptor in vivo were confirmed by a decreased response to phenylephrine. Reducing the length from 15 to 9 or 5 bases abolished α1-adrenergic receptor binding in vitro and bolus infusion of 5.0 mg/kg of 9-base PS-ODN no longer produced hypotension. In conclusion, the 15-base PS-ODN shows cooperative binding to the α1-adrenergic receptor, which produces cardiovascular effects that are oligomer length, dose, and formulation dependent.

Comparison of the effects of cholinesterase inhibitors on [ 3H]MK-801 binding in rat cerebral cortex

Huperzine A, a selective inhibitor of acetylcholinesterase, was recently demonstrated to exert an antagonist effect on N-methyl- d-aspartate (NMDA) receptor in rat cerebral cortex. In the present study, the effects of six cholinesterase inhibitors, e.g. huperzine A, huperzine B, tacrine, donepezil (E2020), physostigmine and galanthamine on [ 3H]dizocilpine (MK-801) binding to synaptic membrane of rat cerebral cortex were compared. Their IC 50 values (mean ± SD) were 36.9±12.1, 316.8±93.2, 33.2±3.7, 135.0±15.1, 50.4±7.4, and 3344±295 μM, respectively. The rank order of potency is tacrine ≈ huperzine A>physostigmine>donepezil>huperzine B≫galanthamine. There is no correlation between their activities to inhibit [ 3H]MK-801 binding and to inhibit acetylcholinesterase ( r=+0.563, P=0.245). The results suggest that most cholinesterase inhibitors available exhibit an antagonist effect on NMDA receptor in rat cerebral cortex in addition to their inhibitory effect on acetylcholinesterase.