Rat Cortex Membranes Research Articles

Changes in the cannabinoid (CB1) receptor expression level and G-protein activation in kainic acid induced seizures

It has been known for centuries that exogenous cannabinoids, such as tetrahydrocannabinol have anticonvulsant activity. Recent studies have advanced our understanding of the endogenous cannabinoid system and renewed the interest in cannabinoids as a potential treatment for epilepsy. The endogenous cannabinoid system is rapidly activated after seizure activity but still little is known about the molecular mechanisms underlying the role of the cannabinoid system in epilepsy. In this study epileptiform activity was induced by kainic acid (KA) and effects of the CB1 receptor agonists N-(2-Chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (ACEA) on G-protein signaling using the agonist-stimulated [(35)S]GTPγS binding assay were evaluated. Control and KA treated rat hippocampus and cortex membranes were used. Our results showed that the ACEA displayed a high potency and efficacy in stimulating the G-proteins and when compared to the control animals, significant enhancements were observed in tissues from the KA treated animals. Potency and efficacy values were in particular increased in the hippocampus tissues. Furthermore, gene expression levels of the cannabinoid receptor 1 (CB1) receptor and cannabinoid receptor interacting protein 1 (CRIP1) were measured by RT-PCR, where both CB1 and CRIP1 expressions were found to be elevated in the KA treated animals.

Effect of pulsed electromagnetic field exposure on adenosine receptors in rat brain

Different effects of pulsed electromagnetic field (PEMF) exposure on brain tissue have been described in pre-clinical models and in clinical settings. Nevertheless, the mechanism of action and the possible interaction with membrane receptors such as adenosine receptors (ARs) has not been investigated. The present study focused on the effect of PEMFs on A1 and A2A ARs in the rat cerebral cortex and cortical neurons. Affinity and density of ARs were evaluated by means of saturation binding experiments while mRNA expression was investigated through retro-transcription polymerase chain reaction (RT-PCR). PEMF treatment of the intact rat cerebral cortex or cortical neurons at 1.5 mT mediated a transient and significant increase in A2A ARs after 4 h (2.0-fold increase) and 6 h (1.4- and 1.8-fold increase, respectively) of exposure. In addition, PEMF treatment of the rat cerebral cortex and rat cortical neurons at 3 mT upregulated A2A ARs after 2 h (2.0- and 2.2-fold increase, respectively) and 4 h (1.6- and 1.9-fold increase, respectively). The treatment of rat cortex membranes with PEMFs at 1.5 and 3 mT induced an increase in A2A AR density after 2 h (1.9- and 2.2-fold increase, respectively) and was constant at all incubation times investigated. In rat cortical neurons, mRNA levels of A1 and A2A ARs were not affected by PEMF exposure for the times and intensities used. These results suggest that PEMF treatment has different biological effects in whole organs or cells in comparison with isolated membranes.

Pharmacological characterisation of the GlyT-1 glycine transporter using two novel radioligands

Inhibitors of the glycine transporter GlyT-1 are being developed as potential treatments for schizophrenia. Here we report on the use of two novel radioligands, [ 3H]-SB-733993 and [ 3H]-GSK931145, for the characterisation of GlyT-1 in both cells and native tissue. Binding was evaluated in membranes either from HEK293 cells expressing recombinant human GlyT-1 (hGlyT-1) or from rat cerebral cortex. Specific binding of both [ 3H]-SB-733993 and [ 3H]-GSK931145 to hGlyT-1 HEK293 cell membranes and rat cerebral cortex membranes was saturable and comprised >90% of total binding. K d and B max values for the two radioligands were fairly similar, with K d values of 1–2 nM and B max values of around 7000 fmol/mg protein in hGlyT-1 membranes and 3000 fmol/mg protein in rat cortex membranes. Association of [ 3H]-SB-733993 was faster, with binding reaching equilibrium within 30 min compared with 90 min for [ 3H]-GSK931145. Dissociation was also much slower for [ 3H]-GSK931145 than for [ 3H]-SB-733993, with 50% of specific binding being dissociated by approximately 40 min and 5 min, respectively. Autoradiography studies with [ 3H]-GSK931145 showed widespread distribution of binding in rat brain, with generally higher binding in caudal compared with rostral areas. Initial studies in human frontal cortex membranes showed clear specific binding of [ 3H]-GSK931145, though with much lower density ( B max 570 fmol/mg protein) and slightly lower affinity (K d 4.5 nM) compared with rat cortex. A human brain autoradiography study showed higher specific binding in cerebellum compared with frontal cortex. All GlyT-1 inhibitors tested, as well as glycine itself, competed fully for the binding of both [ 3H]-SB-733993 and [ 3H]-GSK931145 in both hGlyT-1 and rat cortex membranes. Studies on the effect of varying NaCl concentration showed that [ 3H]-SB-733993 binding was reduced by >90% in the absence of added Na + ions, whilst [ 3H]-GSK931145 binding was unaffected. Glycine produced concentration-dependent decreases in binding affinity of both radioligands without major changes in B max values, suggesting that both [ 3H]-SB-733993 and [ 3H]-GSK931145 bind to sites on GlyT-1 that are orthosteric to the site at which glycine itself binds. Overall, these results show that both [ 3H]-SB-733993 and [ 3H]-GSK931145 are useful radioligands for studies on GlyT-1 in both cell lines and native tissues, with [ 3H]-GSK931145 being the radioligand of choice for further studies on GlyT-1 expression and pharmacology.

Membrane alterations and fluidity changes in cerebral cortex during acute ammonia intoxication

Acute ammonia intoxication is known to cause alterations in activities of several membrane bound enzymes like Na + K + ATPase, acetylcholine esterase and glutamate uptake in brain. The alteration in these membrane associated activities could be a consequence of altered membrane architecture. To probe this, the effect of pathophysiological concentrations of ammonia on lipid composition and fluidity of membranes isolated from cerebral cortex of rats, were investigated in the present study. Administration of acute doses of ammonium acetate caused depletion of membrane sphingomyelin and cholesterol levels thereby reducing cholesterol: phospholipid (C: PL) ratio. Levels of phosphatidylserine increased while those of phosphatidylcholine and phosphatidylethanolamine remain unaltered. Membrane fluidity estimations using 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) indicated no changes in core and surface membrane fluidity following ammonium acetate administration. Acute ammonia toxicity induced no alteration in bulk fluidity but a decrease in annular fluidity of membranes, as determined using pyrene fluorescence. Elevated levels of malondialdehyde and declined level of total thiols in cerebral cortex membranes of rats under acute ammonia intoxication indicated the existence of oxidative stress.

Pharmacological properties of hydrophilic and lipophilic derivatives of octreotate

Derivatives of somatostatin (SST) represent the most important peptides for receptor targeting in oncological applications. Whereas the pharmacophor in somatostatin receptor-affine substances has been thoroughly investigated, the influence of modifications at the N-terminal has not yet been systematically studied. In order to investigate the influence of hydrophilic versus lipophilic modifications at the N-terminal end, a series of homologous derivatives of Tyr 3-octreotate modified with oligomers of ethylene glycol or fatty acids were synthesized. For this purpose, Tyr 3-octreotate was assembled using solid phase peptide synthesis and the fatty acids or oligomers of ethylene glycol were conjugated to the N-terminal end. The oligomers of ethylene glycol were activated by 4-nitrophenylchloroformate to obtain carbamate-linked hydrophilic compounds. The receptor affinities of these compounds were determined by competition experiments with [ 125I]Tyr 3-octreotide on rat cortex membranes. The hydrophilic derivatives and the short chain lipophilic derivatives revealed IC 50 values between 0.66 ± 0.02 nM and 2.16 ± 0.31 nM respectively. After labeling with 125I the organ distribution of selected derivatives was investigated in Lewis rats bearing the rat pancreatic tumor CA20948. All of the compounds showed high tumor uptake. The peptides conjugated to oligomers of ethylene glycol showed low uptake into the liver and kidneys. Increasing the length of the fatty acids resulted in a remarkable decrease in kidney uptake. In conclusion, the systematic modifications at the N-terminal result in a low effect on the receptor affinity but allow the modulation of the pharmacokinetic properties of octreotide derivatives.

Synthesis and Three-Dimensional Quantitative Structure-Activity Relationship Analysis of H3 Receptor Antagonists Containing a Neutral Heterocyclic Polar Group

Three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis was applied to a series of H(3) receptor antagonists characterized by an imidazole ring, an alkyl spacer, and a heterocyclic polar moiety containing an imidazole or a thiazole ring, with a view to investigate the requirements for H(3) receptor affinity on rat cortex membranes. The compounds were aligned based on the hypothesis that the presence of a H-bond donor group in the polar portion of the molecule can increase H(3) receptor affinity. The 3D-QSAR analysis, which was performed using both the CoMFA and CoMSIA protocols, revealed that the presence of a H-bond donor group is not statistically relevant for H(3) receptor affinity. Based on this result, another alignment was adopted that took into consideration the structural features common to all compounds, namely the imidazole ring and the N atom with a free lone pair in the polar portion. The 3D-QSAR models thus obtained showed that H(3) receptor affinity is modulated by the position and direction of the intermolecular interaction elicited by the polar group in the ligands.

Synthesis and Biological Effects of Novel 2-Amino-3-naphthoylthiophenes as Allosteric Enhancers of the A1Adenosine Receptor

The current study describes the synthesis and biological evaluation of a novel series of 2-amino-3-naphthoylthiophenes, with variable modifications at the 4- and 5-position of the thiophene as well as the naphthoyl ring. Allosteric enhancer activity was measured in several ways: (1) evaluating the effect on forskolin-stimulated cAMP accumulation in the presence of an A(1)-adenosine agonist (CPA) in Chinese hamster ovary (CHO) cells expressing the cloned human A(1)-adenosine receptor (hA(1)AR); (2) ability of these compounds to displace the binding of [(3)H]DPCPX, [(3)H]ZM 241385, and [(3)H]MRE 3008F20 to the ligand binding site of CHO cells expressing the hA(1), hA(2A), and hA(3) adenosine receptors, respectively; (3) effect on the binding of [(3)H]CCPA to membranes from CHO cells expressing hA(1)AR, to rat brain and human cortex membrane preparations containing native adenosine A(1) receptors; (4) kinetics of the dissociation of [(3)H]CCPA from CHO-hA1 membranes. The pharmacological assays compared the various activities to that of the reference compound PD 81,723 (compound 1). Several compounds appeared to be better than PD 81,723 to enhance the effect of CPA (and thus reduce cAMP content) in the CHO:hA(1) assay. The effect of these compounds at a concentration of 10 microM was slightly greater than that of the same concentration of the PD 81,723 and substantially greater than that of PD 81,723 when responses to 1 microM of each compound were compared. These include compounds 23, 25-29, 31-34, 38, 39, 43, and 58. Cycloalkylthiophenes tended to be more potent then their 4,5-dimethyl analogues, and in the series of cycloalkylthiophenes, tetrahydrobenzo[b]thiophene derivatives appeared to be more potent than the dihydrocyclopentadien[b]thiophene counterparts. Some of the most potent compounds were tested at a concentration of 10 microM for their affinity as competitors to the antagonist binding site of CHO cells expressing hA(1), hA(2A), and hA(3) adenosine receptors. None inhibited binding at the hA(2A)AR, but most of them inhibited binding to the hA(1)AR to varying extents (0-19%) as well as to the hA(3)AR to a substantial degree (0-57%). At a concentration of 10 microM, the compounds 31, 34, 37, 38, and 39 were more active than PD 81,723 to increase the binding of [(3)H]CCPA to CHO:hA(1), human brain and rat cortex membranes. Compound 37 was the most active compound increasing the binding to CHO:hA(1), human brain, and rat cortex membranes by 149, 43, and 27%, respectively (51, 15, and 22%, respectively, for PD 81,723). A good correlation was found between the increments [(3)H]CCPA binding to A(1) receptors expressed in different systems. Unlike the effect on agonist binding, the tested compounds did not increase the binding of the antagonist [(3)H]DPCPX on hCHO-A(1) membranes. Ligand dissociation studies revealed that two compounds (22 and 39) were more potent than 1 to slow the dissociation of [(3)H]CCPA from CHO:hA(1)AR membranes. No clear-cut structure-activity relationship can be observed based on data from the functional assay, but we have identified several compounds, in particular 37 and 39, which appeared to be more potent than 1 and that may be selected for further development.

Synthesis and Three-Dimensional Quantitative Structure-Activity Relationship Analysis of H3 Receptor Antagonists Containing a Neutral Heterocyclic Polar Group

Three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis was applied to a series of H3receptor antagonists characterized by an imidazole ring, an alkyl spacer, and a heterocyclic polar moiety containing an imidazole or a thiazole ring, with a view to investigate the requirements for H3receptor affinity on rat cortex membranes. The compounds were aligned based on the hypothesis that the presence of a H-bond donor group in the polar portion of the molecule can increase H3receptor affinity. The 3D-QSAR analysis, which was performed using both the CoMFA and CoMSIA protocols, revealed that the presence of a H-bond donor group is not statistically relevant for H3receptor affinity. Based on this result, another alignment was adopted that took into consideration the structural features common to all compounds, namely the imidazole ring and the N atom with a free lone pair in the polar portion. The 3D-QSAR models thus obtained showed that H3receptor affinity is modulated by the position and direction of the intermolecular interaction elicited by the polar group in the ligands.

Evaluation of the cholinomimetic actions of trimethylsulfonium, a compound present in the midgut gland of the sea hare Aplysia brasiliana (Gastropoda, Opisthobranchia).

Trimethylsulfonium, a compound present in the midgut gland of the sea hare Aplysia brasiliana, negatively modulates vagal response, indicating a probable ability to inhibit cholinergic responses. In the present study, the pharmacological profile of trimethylsulfonium was characterized on muscarinic and nicotinic acetylcholine receptors. In rat jejunum the contractile response induced by trimethylsulfonium (pD2 = 2.46 +/- 0.12 and maximal response = 2.14 +/- 0.32 g) was not antagonized competitively by atropine. The maximal response (Emax) to trimethylsulfonium was diminished in the presence of increasing doses of atropine (P<0.05), suggesting that trimethylsulfonium-induced contraction was not related to muscarinic stimulation, but might be caused by acetylcholine release due to presynaptic stimulation. Trimethylsulfonium displaced [3H]-quinuclidinyl benzilate from rat cortex membranes with a low affinity (Ki = 0.5 mM). Furthermore, it caused contraction of frog rectus abdominis muscles (pD2 = 2.70 +/- 0.06 and Emax = 4.16 +/- 0.9 g), which was competitively antagonized by d-tubocurarine (1, 3 or 10 microM) with a pA2 of 5.79, suggesting a positive interaction with nicotinic receptors. In fact, trimethylsulfonium displaced [3H]-nicotine from rat diaphragm muscle membranes with a Ki of 27.1 microM. These results suggest that trimethylsulfonium acts as an agonist on nicotinic receptors, and thus contracts frog skeletal rectus abdominis muscle and rat jejunum smooth muscle via stimulation of postjunctional and neuronal prejunctional nicotinic cholinoreceptors, respectively.

Pharmacological effects of naltriben as a ligand for opioid μ and κ receptors in rat cerebral cortex

Naltriben (NTB) has been used to differentiate the subtypes of δ opioid receptors, δ 1 and δ 2. However, there is considerable evidence suggesting that NTB may act on other types of opioid receptors too. We examined the effects of NTB on the specific binding of radiolabeled ligands for opioid μ and κ 2 receptors, and the effects on the release of [ 3H]norepinephrine ([ 3H]NE) in rat cerebral cortex slices. NTB displaced the specific binding of [ 3H]DAMGO with Ki value of 19.79 ± 1.12 nM in rat cortex membranes. Specific binding of [ 3H]diprenorphine ([ 3H]DIP) was inhibited by NTB with Ki value of 82.75 ± 6.32 nM in the presence of DAMGO and DPDPE. High K + (15 mM)–stimulated release of [ 3H]NE was attenuated by DAMGO in rat cerebral cortex slices. NTB (30 nM) shifted the dose-response curve of DAMGO to the right and attenuated the maximal effect. In the meantime, NTB inhibited high K +-stimulated [ 3H]NE release at concentrations above 100 nM. The inhibitory effect of NTB was not attenuated by CTAP (10 nM) and naloxone (3 nM) but by higher concentration of naloxone (30 nM), nor-BNI (300 nM) and bremazocine (3 nM). These results indicate that NTB, depending on the dosage, could acts not only as an antagonist at δ but also as a noncompetitive antagonist for μ receptors, and as an agonist for κ 2 receptors in rat cerebral cortex.

Structure-activity relationships in 1,4-benzodioxan-related compounds. 6. Role of the dioxane unit on selectivity for alpha(1)-adrenoreceptor subtypes.

WB 4101-related benzodioxans 3-9 were synthesized, and their biological profiles at alpha(1)-adrenoreceptor subtypes and 5-HT(1A) serotoninergic receptors were assessed by binding assays in CHO and HeLa cells membranes expressing the human cloned receptors. Furthermore, receptor selectivity of selected benzodioxan derivatives was further determined in functional experiments in isolated rat vas deferens (alpha(1A)) and aorta (alpha(1D)) and guinea pig spleen (alpha(1B)), in additional receptor binding assays in rat cortex membranes containing alpha(2)-adrenoreceptors and 5-HT(2) serotoninergic receptors, and in rat striatum membranes containing D(2) dopaminergic receptors. An analysis of the results of receptor binding experiments for benzodioxan-modified derivatives 3-9 showed high affinity and selectivity toward the alpha(1a)-adrenoreceptor subtype for compounds 3-5 and 7 and a reversed selectivity profile for 9, which was a selective alpha(1d) antagonist. Furthermore, the majority of structural modifications performed on the prototype 1 (WB 4101) led to a marked decrease in the affinity for 5-HT(1A) serotoninergic receptors, which may have relevance in the design of selective alpha(1A)-adrenoreceptor antagonists. The exception to these findings was the chromene derivative 8, which exhibited a 5-HT(1A) partial agonist profile.

Characterization of the binding of a novel radioligand to CCKB/gastrin receptors in membranes from rat cerebral cortex.

1. We have investigated the binding of a novel radiolabelled CCKB/gastrin receptor ligand, [3H]-JB93182 (5[[[(1S)-[[(3,5-dicarboxyphenyl)amino]carbonyl]-2-phenylethyla mino]-carbonyl]-6-[[(1-adamantylmethyl) amino]carbonyl]-indole), to sites in rat cortex membranes. 2. The [3H]-JB93182 was 97% radiochemically pure as assessed by reverse-phase HPLC (RP-HPLC) and was not degraded by incubation (150 min) with rat cortex membranes. 3. Saturation analysis indicated that [3H]-JB93182 labelled a homogeneous population of receptors in rat cortex membranes (pKD=9.48+/-0.08, Bmax=3.61+/-0.65 pmol g(-1) tissue, nH=0.97+/-0.02, n=5). The pKD was not significantly different when estimated by association-dissociation analysis (pKD=9.73+/-0.11; n=10). 4. In competition studies, the low affinity of the CCKA receptor antagonists, L-364,718; SR27897 and 2-NAP, suggest that, under the assay conditions employed, [3H]-JB93182 (0.3 nM) does not label CCKA receptors in the rat cortex. 5. The affinity estimates obtained for reference CCKB/gastrin receptor antagonists were indistinguishable from one of the affinity values obtained when a two site model was used to interpret [125I]-BH-CCK8S competition curves obtained in the same tissue (Harper et al., 1999). 6. This study provides further evidence for the existence of two CCKB/gastrin sites in rat cortex. [3H]-JB93182 appears to label selectively sites previously designated as gastrin-G1 and therefore it may be a useful compound for the further discrimination and characterization of these putative receptor subtypes.

Divalent cations influencing neuropeptide Y receptor subtype binding in rat hippocampus and cortex membranes as well as in recombinant cells

At least six types of neuropeptide Y (NPY) receptors (Y1–Y6) have been pharmacologically distinguished of which only the Y1, Y2, Y4 and Y5 subtypes have been thoroughly characterized. In order to further classify receptor subtypes in the brain, we performed receptor binding studies using rat cortical and hippocampal membranes and, in particular, studied the effects of different ion compositions of the buffer on the binding behaviour of several NPY agonists and the Y1 receptor antagonist BIBO3304. Ca 2+ was necessary for reliable Y1 receptor subtype classification in rat cortical membranes (with Hill coefficients close to unity) for the peptide agonists. This was further substantiated by the Y1 selective antagonist BIBO3304 displaying an IC 50 value of 0.9±0.5 nM for 80% of the total receptors, the remaining sites being BIBO3304 insensitive (IC 50>10 000 nM). Replacing Ca 2+ by Mn 2+ resulted in a complete loss of BIBO3304 sensitive sites. On the other hand, using hippocampal membrane preparations, displacement curves with Hill coefficients close to unity were only obtained in the presence of Mn 2+ ions, yielding a binding profile of receptors with low affinity for [Leu 31, Pro 34]NPY (IC 50=50 nM) and for BIBO3304 (IC 50>10 000 nM). Addition of Mn 2+ ions to cortical or of Ca 2+ ions to hippocampal membrane preparations resulted in binding profiles differing from typical receptor classification. Therefore, the influence of divalent cations on Y1 receptors expressed on recombinant cells was studied. In this monoreceptor system, Ca 2+ was necessary to detect high amounts of specific binding and Mn 2+ ions induced a change in the affinity state. These findings indicate that apparent NPY receptor heterogeneity does not only depend on the brain region examined and that divalent ions modulate ligand binding properties.

Synthesis and Characterisation of [ 90Y]-Bz-DTPA-oct: A Yttrium-90-Labelled Octreotide Analogue for Radiotherapy of Somatostatin Receptor-Positive Tumours

An investigation into the in vitro behaviour of two yttrium-90-labelled somatostatin analogues was performed. Further in vivo characterisation was performed with the most promising agent. A new DTPA-octreotide analogue (Bz-DTPA-oct) was synthesised by coupling a bifunctional DTPA chelator to the N-terminal amine of the D-Phe 1 of Tyr 3-octreotide. This new SRIF analogue and DTPA-octreotide (OctreoScan) were radiolabelled with 90Y prior to serum stability being evaluated. Receptor binding assays were also performed on the two radioligands using rat cortex membranes. The [ 90Y]-Bz-DTPA-oct was further evaluated in vivo using tumour-bearing rats. The first conjugate (DTPA-octreotide) bound with a high affinity to SRIF receptors and the 90Y complex was relatively stable in human serum ( t 1/2 3.8 d for 90Y lost to serum proteins). The second conjugate (Bz-DTPA-oct) also exhibited a high binding affinity to SRIF receptors, but it demonstrated an even slower loss of 90Y to serum proteins ( t 1/2 12.1 d). The in vivo evaluation of the more stable [ 90Y]-Bz-DTPA-oct showed a very rapid and high accumulation in somatostatin receptor-positive tumours, which after 1 h resulted in tumour/nontumour ratios of 3.8, 21, and 4.9 (for blood, muscle, and liver, respectively). These tumour/nontumour ratios increased, and were by 24 h postinjection 138, 285, and 6.1 (for blood, muscle, and liver). Yttrium-90-labelled Bz-DTPa-oct is rapidly and selectively accumulated in somatostatin receptor-positive tissue. Octadentate Bz-DTPA-oct could be ligand for 90Y radiotherapy of somatostatin receptor-positive tumours and their metastases.

Blockage of 5HT2C serotonin receptors by fluoxetine (Prozac).

Fluoxetine (Prozac) inhibited the membrane currents elicited by serotonin (5-hydroxytryptamine; 5HT) in Xenopus oocytes expressing either cloned 5HT2C receptors or 5HT receptors encoded by rat cortex mRNA. Responses of 5HT2C receptors, elicited by nM concentrations of 5HT, were rapidly and reversibly blocked by micromolar concentrations of fluoxetine. For responses elicited by 1 microM 5HT, the IC50 of fluoxetine inhibition was approximately 20 microM. In accord with the electrophysiological results, fluoxetine inhibited the binding of [3H]5HT to 5HT2C receptors expressed in HeLa cells (Ki approximately 65-97 nM), and the binding to 5HT receptors in rat cortex membranes was also inhibited but less efficiently (Ki approximately 56 microM). Our results show that fluoxetine is a competitive and reversible antagonist of 5HT2C receptors and suggest that some therapeutic effects of fluoxetine may involve blockage of 5HT receptors, in addition to its known blockage of 5HT transporters. Similar work may help to design more selective compounds for use in the treatment of brain disorders.

Effect of gamma-decanolactone on glutamate binding in the rat cerebral cortex.

Epilepsy is one of the most common neurological disorders. Even though antiepileptic drugs can afford a reasonably satisfactory treatment for 80% of diagnosed patients, chronic intractable epilepsy still affects a significant number of people and more effective and less harmful antiepileptic drugs are needed. Previous studies have shown that gamma-decanolactone has dose-dependent sedative effects, including hypnotic, anticonvulsant and hypothermic properties in mice. The present study reports an inhibitory effect of gamma-decanolactone on glutamate binding (96.8% with 5 mM) in rat cortex membranes. The non competitive nature of glutamate binding inhibition as a neurochemical correlate of the anticonvulsant activity of gamma-decanolactone may be a relevant mode of action for further drug development.

Binding thermodynamics of 5-HT 1A receptor ligands

The thermodynamic parameters ΔG°, ΔH° and ΔS° of the binding equilibrium of 15 ligands (eight agonists and seven antagonists) to the 5-HT 1A receptor subtype have been determined by affinity measurements carried out on rat cortex membranes (minus striatum) at six different temperatures (0, 10, 20, 25, 30, 35°C), and by van't Hoff plots. Most of the compounds studied are tryptamine, phenylpiperazine and tetralin derivatives. Affinity constants were measured by saturation experiments for the selective 5-HT 1A receptor agonist [ 3H]8-hydroxy- N,N-dipropyl-2-aminotetralin ([ 3H]8-OH-DPAT) and by inhibition assays of [ 3H]8-OH-DPAT binding for the other compounds. Scatchard plots were monophasic in the full range of temperatures, indicating a single class of high affinity binding sites. Van't Hoff plots of all ligands were linear in the temperature range investigated (0–30°C or 0–35°C). 5-Hydroxytryptamine (serotonin) and 5-methoxy-tryptamine (mexamine) displayed a positive slope. Experimental data indicate that for 5-HT 1A receptor subtype agonists and antagonists are not thermodynamically discriminated. The results are discussed from a quantitative point of view with the aim of obtaining new details on the nature of the forces driving the 5-HT 1A binding at a molecular level.

In vitro pharmacological properties of 4-bromodexetimide for muscarinic receptors

The decrease of m-AChR density observed hi neurodegenerative disorders has generated considerable interest in non-invasive mapping of muscarinic acetylcholine receptors (m-AChR) in the central nervous system. The ami of our study was to evaluate the selectivity of 4-bromodexetimide for the M1, M2, M3 and M4 m-AChR subtypes using in vitro binding analysis to determine the potential use of the bromine-76 labelled 4-bromodexetimide in the investigation of m-AChR subtypes in human brain with Positron Emission Tomography. Subtype selectivity of 4-bromodexetimide was determined in competition studies against tritiated subtype selective ligands using various rat or rabbit structure homogenates reflecting a single binding site and in optimal saturation and low non specific binding conditions. These conditions were reached for every subtype studied by analyzing the data from the saturation experiments of the tritiated ligands. 4-bromodexetimide displayed nanomolar affinities for the four m-AChR subtypes and a preferential selectivity for the M1 and M4 subtypes. The saturation analysis of [ 76Br]4-bromodexetimide, performed with rat cortex membranes showed high affinity for m-AChR receptors (K d = 1.8 nM). As in vivo studies of [ 76Br]4-bromodexetimide showed preferential localization in the cortex and the striatum which are M1 and M4 rich structures and since it binds preferentially to the M 1 and M 4 subtypes, this radiotracer can still allow a combined subtype specific measurement of these muscarinic receptors.

Pharmacological characterization and positron emission tomography evaluation of 4-[ 76Br]bromodexetimide and 4-[ 76Br]bromolevetimide for investigations of central muscarinic cholinergic receptors

4-[ 76Br]bromodexetimide and its inactive enantiomer 4-[ 76Br]bromolevetimide were prepared via electrophilic bromodesilylation using chloramine-T and no-carrier-added (NCA) [ 76Br]NH 4. In vitro, B max measured on rat cortex membranes were 3.7 ± 0.2 and <0.07 pmol/mg protein for 4-[ 76Br]bromodexetimide and 4-[ 76Br]bromolevetimide, respectively. The k D of 4-[ 76Br]bromodexetimide was 1.9 ± 0.3 nM. In vivo studies in rats showed specific uptake of 4-[ 76Br]bromodexetimide in cortex, striatum, thalamus and hippocampus. No specific uptake was observed with 4-[ 76Br]bromolevetimide. With [ 76Br]bromodexetimide, positron emission tomography (PET) studies in primates demonstrated a preferential accumulation of the radioactivity in the cortex and striatum which was displaced to the level of cerebellum by dexetimide. With 4-[ 76Br]bromolevetimide, the radioactivity concentrations in the cortex and striatum were similar to that of cerebellum.

Thermodynamics of 5-HT 3 receptor binding discriminates agonistic from antagonistic behaviour

Thermodynamic parameters ΔG°, ΔH° and ΔS° of the binding equilibrium of eleven ligands (seven agonists and four antagonists) to the serotonin 5-HT 3 receptor subtype have been determined by affinity measurements carried out on rat cortex membranes at six different temperatures (0, 10, 20, 25, 30, 35°C) and van't Hoff plots. Affinity constants were obtained from saturation experiments of [ 3H] endo- N-(9-methyl-9-azabicyclo[3.3.1]non-3-yl)-1-methyl-1- H-indazole-3-carboxamide ([ 3H]BRL 43694, a selective 5-HT 3 ligand) or by its displacement in inhibition assays for the other compounds. Van't Hoff plots were essentially linear in the temperature range investigated, showing that the ΔC p° of the binding equilibrium is nearly zero. Thermodynamic parameters are in the range 18 ≤ ΔH° ≤ 53 kJ mol −1 and 202 ≤ ΔS° ≤ 320 J K −1 mol −1 for agonists and − 16 ≤ ΔH° ≤ 0 kJ mol −1 and 70 ≤ ΔS° ≤ 179 J K −1 mol −1 for antagonists indicating that agonistic binding is totally entropy-driven while antagonistic binding is relatively less entropy- and more enthalpy-driven in the − TΔS° versus ΔH° plot the thermodynamic data are clearly arranged in separate clusters for agonists and antagonists, which, therefore, turn out to be thermodynamically discriminated. Experimental results are discussed according to the following main points: (i) the approximate linearity of the ΔH° versus ΔS° plot in terms of enthalpy-entropy compensation and (ii) the fact that ΔC p ° ⋍ 0 for practically all membrane receptors at variance with most reactions involving biomacromolecules in solution. Finally, the phenomenon of thermodynamical discrimination is reviewed and found to occur in five distinct membrane receptorial systems.