Membrane Recognition Sites Research Articles

Tailor-Made Specific Recognition of Cyromazine Pesticide Integrated in a Potentiometric Strip Cell for Environmental and Food Analysis.

Screen-printed ion-selective electrodes were designed and characterized for the assessment of cyromazine (CYR) pesticide. A novel approach is to design tailor-made specific recognition sites in polymeric membranes using molecularly imprinted polymers for cyromazine (CR) determination (sensor I). Another sensor (sensor II) is the plasticized PVC membrane incorporating cyromazine/tetraphenyl borate ion association complex. The charge-transfer resistance and water layer reached its minimal by incorporating Polyaniline (PANI) solid-contact ISE. The designed electrodes demonstrated Nernstain response over a linear range 1.0 × 10−2–5.2 × 10−6 and 1.0 × 10−2–5.7 × 10−5 M with a detection limit 2.2 × 10−6 and 8.1 × 10−6 M for sensors I and II, respectively. The obtained slopes were 28.1 ± 2.1 (r2 = 0.9999) and 36.4 ± 1.6 (r2 = 0.9991) mV/decade, respectively. The results showed that the proposed electrodes have a fast and stable response, good reproducibility, and applicability for direct measurement of CYR content in commercial pesticide preparations and soil samples sprayed with CYR pesticide. The results obtained from the proposed method are fairly in accordance with those using the standard official method.

Solution NMR analysis of the interaction between the actinoporin Sticholysin I and DHPC micelles—Correlation with backbone dynamics

Sticholysin I (StI), an actinoporin expressed as a water-soluble protein by the sea anemone Stichodactyla helianthus, binds to natural and model membranes, forming oligomeric pores. It is proposed that the first event of a multistep pore formation mechanism consists of the monomeric protein attachment to the lipid bilayer. To date there is no high-resolution structure of the actinoporin pore or other membrane-bound form available. Here we evaluated StI:micelle complexes of variable lipid composition to look for a suitable model for NMR studies. Micelles of pure or mixed lysophospholipids and of dihexanoyl phosphatidylcholine (DHPC) were examined. The StI:DHPC micelle was found to be the best system, yielding a stable sample and good quality spectra. A comprehensive chemical shift perturbation analysis was performed to map the StI membrane recognition site in the presence of DHPC micelles. The region mapped (residues F(51), R(52), S(53) in loop 3; F(107), D(108), Y(109), W(111), Y(112), W(115) in loop 7; Q(129), Y(132), D(134), M(135), Y(136), Y(137), G(138) in helix-α2) is in agreement with previously reported data, but additional residues were found to interact, especially residues V(81), A(82), T(83), G(84) in loop 5, and A(85), A(87) in strand-β5. Backbone dynamics measurements of StI free in solution and bound to micelles highlighted the relevance of protein flexibility for membrane binding and suggested that a conformer selection process may take place during protein-membrane interaction. We conclude that the StI:DHPC micelles system is a suitable model for further characterization of an actinoporin membrane-bound form by solution NMR.

Anti-Alcohol and Anxiolytic Properties of a New Chemical Entity, GET73.

N-[(4-trifluoromethyl)benzyl]4-methoxybutyramide (GET73) is a newly synthesized compound structurally related to the clinically used, alcohol-substituting agent, gamma-hydroxybutyric acid (GHB). The present study was designed to assess whether GET73 may share with GHB the capacity to reduce alcohol intake in rats. Additionally, the effect of treatment with GET73 on anxiety-related behaviors and cognitive tasks in rats was investigated. A series of in vitro binding assays investigated the capacity of GET73 to bind to the GHB binding site and multiple other receptors. GET73 (10−9–10−3 M) failed to inhibit [3H]GHB binding at both high- and low-affinity GHB recognition sites in rat cortical membranes. GET73 displayed minimal, if any, binding at dopamine, serotonin, GABA, and glutamate receptors in membranes from different rat brain areas. Acute treatment with low-to-moderate, non-sedative doses of GET73 (5–50 mg/kg, i.g. or i.p.) (a) reduced alcohol intake and suppressed “alcohol deprivation effect” (a model of alcohol relapse) in selectively bred, Sardinian alcohol-preferring (sP) rats, (b) exerted anxiolytic effects in Sprague-Dawley (SD) and sP rats exposed to the Elevated Plus Maze test, and (c) tended to induce promnestic effects in SD rats exposed to a modified water version of the Hebb–Williams maze test. Although the mechanism of GET73 action is currently unknown, the results of the present study suggest that GET73 has a multifaceted pharmacological profile, including the capacity to reduce alcohol drinking and anxiety-related behaviors in rats.

Phosphatidylinositol metabolism and membrane fusion

Membrane fusion underlies many cellular events, including secretion, exocytosis, endocytosis, organelle reconstitution, transport from endoplasmic reticulum to Golgi and nuclear envelope formation. A large number of investigations into membrane fusion indicate various roles for individual members of the phosphoinositide class of membrane lipids. We first review the phosphoinositides as membrane recognition sites and their regulatory functions in membrane fusion. We then consider how modulation of phosphoinositides and their products may affect the structure and dynamics of natural membranes facilitating fusion. These diverse roles underscore the importance of these phospholipids in the fusion of biological membranes.

Estradiol and its membrane-impermeable conjugate estradiol–BSA inhibit tamoxifen-stimulated prolactin secretion in incubated rat pituitaries

In the absence of estrogen (E), the selective E receptor modulator tamoxifen (TX) has two agonist effects in the rat pituitary: induction of progesterone receptor (PR)-dependent GnRH self-priming in the gonadotrope, and stimulation of prolactin (PRL) secretion in the lactotrope. TX-induced gonadotropin (GnRH) self-priming is absent when 10(-8) M estradiol-17beta (E2) is added to the incubation medium of pituitaries from TX-treated rats. The present experiments investigated whether PR-independent PRL release into the incubation medium of pituitaries from TX-treated ovariectomized (OVX) rats was affected by E2, and the effect of different ER ligands (ICI182780, TX, estradiol-17alpha, E2 -BSA) on TX-stimulated PRL secretion. Moreover, the effect of E2 on TRH-stimulated PRL secretion in pituitaries collected from estradiol benzoate- and TX-treated OVX rats was studied. It was found that: i) incubation with E2 supressed the PRL releasing effect of injected TX; ii) whereas coincubation with the pure anti-E type II ICI182780 antagonized the inhibitory effect of E2, coincubation with the anti-E type I TX did not; iii) estradiol-17alpha lacked inhibitory action, whereas a dose-dependent inhibitory effect of both E2 and E2 -BSA was noticed; and iv) TRH stimulatory effect on PRL release in pituitaries from TX-treated rats was blocked by addition of E2 to the medium. Taken together, these data argue in favor of the presence of specific membrane recognition sites for E in the lactotrope involved in steroid-specific E2 inhibition of TX-stimulated PRL secretion.

ChemInform Abstract: Imprinting of Specific Molecular Recognition Sites in Inorganic and Organic Thin Layer Membranes Associated with Ion‐Sensitive Field‐Effect Transistors.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Imprinting of specific molecular recognition sites in inorganic and organic thin layer membranes associated with ion-sensitive field-effect transistors

Molecular recognition sites were imprinted in inorganic TiO 2 films, and acrylamide–acrylamidephenylboronic acid copolymer membranes, associated with ion-sensitive field-effect transistors, ISFETs, that act as transduction devices for the association of the substrates to the imprinted membranes. Molecular structures of carboxylic acids, e.g. 4-chlorophenoxyacetic acid ( 1 ), 2,4-dichlorophenoxyacetic acid ( 2 ), fumaric acid ( 3 ), and maleic acid ( 4 ), are imprinted in TiO 2 films. The imprinted sites reveal high specificity, and substrates, structurally-related to the imprinted compounds are fully differentiated by the imprinted membranes. The specificity of the imprinted sites originates from the complementary structural fitting and ligation of the guest carboxylic acid residues to the Ti(IV)–OH sites in the host carboxylic acids to the imprinted cavities. An acrylamide–acrylamidephenylboronic acid copolymer acts as a functional polymer for the imprinting of nucleotides, e.g. adenosine 5′-monophosphate, AMP, ( 7 ), guanosine 5′-monophosphate, GMP, ( 8 ), or cytosine 5′-monophosphate, CMP, ( 9 ). The specificity of the imprinted nucleotide sites originates from the cooperative binding interactions between the nucleotides and the boronic acid ligand and acrylamide H-bonds. The detection regions and sensitivities for sensing of the different substrates by the functional polymers are determined.

Euphorbium: Modern research on its active principle, resiniferatoxin, revives an ancient medicine

Resiniferatoxin, an ultrapotent capsaicin analog present in the latex of Euphorbia resinifera, interacts at a specific membrane recognition site (referred to as the vanilloid receptor), expressed by primary sensory neurons mediating pain perception as well as neurogenic inflammation. Desensitization to resiniferatoxin is a promising approach to mitigate neuropathic pain and other pathological conditions in which sensory neuropeptides released from capsaicin-sensitive neurons play a crucial role. Clinical trials to evaluate the potential of topical resiniferatoxin treatment to relieve pain associated with diabetic polyneuropathy and postherpetic neuralgia are in progress. Though resiniferatoxin was isolated only two decades ago, the dried latex of Euphorbia resinifera, called Euphorbium, has been in medicinal use since the time of recorded history. This review highlights the most important events in the history of this ancient medicine, from the first written record of the therapeutic potential of Euphorbium (at the time of the reign of the Roman Emperor Augustus) to the identification of its active principle as resiniferatoxin in 1975. A brief overview of the enormous contribution of resiniferatoxin to our current understanding of the anatomical localization, function, and pharmacology of vanilloid receptors is provided. Lastly, the mechanisms are summarized by which capsaicin and resiniferatoxin, despite sharing receptors, may have dissimilar biological actions.

Steroid Hormones and Excitability in the Mammalian Brain

Adrenocortical and gonadal steroid hormones can pass the blood–brain barrier and bind to intracellular receptors in the brain. In addition to steroid hormones binding to intracellular steroid receptors, metabolites of these steroids and steroid hormones produced in the brain (neurosteroids) are thought to bind to membrane recognition sites. Actions mediated by the intracellular receptors are generally delayed in onset and are of prolonged duration, whereas the hormones binding to membrane recognition sites induce fast effects. Both fast and delayed actions by steroid hormones potentially alter the electrical properties of neuronal membranes and thus the firing patterns of neurons carrying receptors for the hormones. We here review the fast and delayed actions by steroid hormones on single cell electrical properties in the mammalian nervous system. In general, fast effects by corticosteroids—presumably mediated by membrane receptors—induce inhibitory effects on cellular firing, although regional differences seem to exist. Delayed effects by corticosteroid hormones via mineralocorticoid receptors serve to maintain or enhance fast transmission in the brain, while modulatory inputs are suppressed. By contrast, corticosteroids acting through glucocorticoid receptors suppress transmission carried by amino acids, particularly when the activity is elevated in comparison to resting level; modulatory inputs are enhanced. Prolonged activation of glucocorticoid receptors can implicate the integrity of neuronal circuits by allowing considerable influx of calcium ions during depolarization. Of the gonadal hormones, estradiol mainly exerts excitatory actions, in both a rapid and a delayed mode. Progesterone on the other hand is predominantly inhibitory, usually with a short delay in onset. The effect of androgens on neuronal excitability has not yet been studied in great detail. Finally, neurosteroids and A-ring reduced steroids in general induce rapid effects on firing patterns, probably by acting on ligand gated ion channels. The diverse actions of steroid hormones on single cell activity have consequences for the excitability in local circuits in which these cells participate. This is illustrated in this review for two processes that depend on circuit rather than single cell function, i.e., long term potentiation and epilepsy. The diverging character of steroid hormones with regard to the time frame, space, and nature of their effects is also reflected in the functional processes that are linked to the activity of the networks responding to steroids. In this way steroid hormones add an essentially new aspect to the regulation of functional processes in the brain, during physiological conditions but also when networks are implicated during diseases and disorders. Future research on steroid modulation of cellular excitability will gain considerably from attempts to either link the changed excitability to the underlying molecular events or study the effects on cellular activity in close connection with behavioral functions.

The future

Within the next 20 years, nuclear medicine could become a major focus of medical practice and biomedical research as medicine enters a new age of certainty, in which surgery, radiation, and chemotherapy will only be used when a benefit is certain to result from the treatment. Not only in neuropsychiatric disease, but also in other diseases, such as cancer, disease will be viewed as molecular dissonance, or, a communication disorder. Cells become cancerous and people become anxious, neurotic, or even psychotic because specific tissue cells or neurons either do not get the right messages because of a deficiency in DNA transcription and wrong messages are produced, or because of a failure in reception of the correct messages carried to cells. Nuclear medicine makes it possible to characterize these transmitters and receptors as they are distributed throughout various parts of the living human body. No field of clinical medicine is better equipped to transfer the advances in molecular biology and genetics to the care of the sick. Nuclear medicine takes a physiological and biochemical, rather than an anatomical or ontological, view of disease. Disease is characterized as a statistically defined deviation of one or more functions or regional biochemical processes from those of healthy people under circumstances as close as possible to those of a person of the same sex and age as the patient. The emission of photons from radioactive tracers that can penetrate from the inside of the body to external radiation detectors is what makes it possible to examine molecular interactions involving picomolar concentrations of molecules involved in biochemical interactions between macromolecular recognition sites, enzymes, and substrates within different parts of the living body. Perturbations (stress tests) will be increasingly used to detect abnormal responsiveness to external forces early in the course of a disease. Movement of the diagnostic process from an orientation toward anatomy to a more fundamental level, the molecular level, will be the hallmark of medicine in the next millennium. Gross or histopathologic changes will no longer suffice. Radiotracers will be able to characterize plasma membrane and intracellular recognition sites and will also provide in vivo probes, making it possible to identify changes in specific genes, as well as their molecular expression, even before the disease or disorder has manifest itself by symptoms and physical signs. Such molecular characterization of disease in situ will make it possible to treat the patient as an individual and not just as a representative member of a group of patients who have similar problems and have been labeled as having a particular disease. Diagnosis will become increasingly specific despite the heterogeneity of the present diagnostic labels.

3H]Aniracetam Binds to Specific Recognition Sites in Brain Membranes

[3H]Aniracetam bound to specific and saturable recognition sites in membranes prepared from discrete regions of rat brain. In crude membrane preparation from rat cerebral cortex, specific binding was Na+ independent, was still largely detectable at low temperature (4 degrees C), and underwent rapid dissociation. Scatchard analysis of [3H]aniracetam binding revealed a single population of sites with an apparent KD value of approximately 70 nM and a maximal density of 3.5 pmol/mg of protein. Specifically bound [3H]aniracetam was not displaced by various metabolites of aniracetam, nor by other pyrrolidinone-containing nootropic drugs such as piracetam or oxiracetam. Subcellular distribution studies showed that a high percentage of specific [3H]aniracetam binding was present in purified synaptosomes or mitochondria, whereas specific binding was low in the myelin fraction. The possibility that at least some [3H]aniracetam binding sites are associated with glutamate receptors is supported by the evidence that specific binding was abolished when membranes were preincubated at 37 degrees C under fast shaking (a procedure that substantially reduced the amount of glutamate trapped in the membranes) and could be restored after addition of either glutamate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) but not kainate. The action of AMPA was antagonized by DNQX, which also reduced specific [3H]aniracetam binding in unwashed membranes. High levels of [3H]aniracetam binding were detected in hippocampal, cortical, or cerebellar membranes, which contain a high density of excitatory amino acid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Characteristics of [3H]inositol(1,3,4,5)tetrakisphosphate recognition sites in human cerebellar membranes.

The characteristics of specific [3H]Ins(1,3,4,5)P4 binding sites in human cerebellar membranes were determined in this study. Binding rapidly reached steady state, possessed a pH optimum of 4.5-5.1 and was greater in the absence of BSA than in its presence. Heparin inhibited both specific and pseudospecific binding of the ligand, whereas only the specific binding was inhibited by non-radioactive Ins(1,3,4,5)P4. Calcium at a concentration of 1 mM, reduced binding by 27%. Competition studies with other inositol phosphates showed specificity for Ins(1,3,4,5)P4 with a pI50 value of 6.87 and a Hill coefficient of 0.27, indicating two sites. Ins(1,2,5,6)P4, Ins(1,3,4,6)P5, Ins(3,4,5,6)P4 displaced binding with IC50 values ranging from 0.1-1 microM, Ins(1,2,5,6)P4 and Ins(1,3,4,5,6)P5 being the most potent. Ins(1,4)P2 and Ins(1,5,6)P3 had lesser effects on binding. Rosenthal analysis of [3H]Ins(1,3,4,5)P4 saturation binding data at low ligand concentrations gave a KD of 27 nM and a Bmax of 33 pmol/mg protein. It is concluded that [3H]Ins(1,3,4,5)P4 binding sites in human cerebellar membranes have similar characteristics to these sites reported in the literature in animal cerebellar tissue, but are in greater abundance.

The interaction of antidepressant drugs with central and peripheral (enteric) 5‐HT3 and 5‐HT4 receptors

1. A combined study of receptor binding in central neuronal cell membranes and functional responses in isolated segments of guinea-pig small intestine allowed characterization of the interaction of four antidepressant drugs with central and peripheral 5-HT3 and 5-HT4 receptors. 2. Clomipramine, paroxetine and fluoxetine inhibited [3H]-DAU 6215 binding to 5-HT3 recognition sites in NG 108-15 cells with IC50 values in the range 1.3-4 microM. Litoxetine had an IC50 of 0.3 microM. The specific binding of [3H]-GR 113808 to 5-HT4 recognition sites in pig striatal membranes was inhibited by all four antidepressants with negligible potency (IC50 values > or = 20 microM). 3. In whole ileal segments, concentration-response curves to 5-HT were biphasic, with the high- and low-potency phases involving 5-HT4 and 5-HT3 receptors, respectively. Curves to 2-methyl-5-hydroxytryptamine (2-methyl-5-HT: a 5-HT3 receptor agonist) and 5-methoxytryptamine (5-MeOT: a 5-HT4 receptor agonist) were monophasic. All antidepressants were used at concentrations lacking anticholinoceptor properties, as demonstrated in both electrically stimulated longitudinal muscle-myenteric plexus preparations (LMMPs) and in unstimulated LMMPs following addition of acetylcholine (100 nM). 4. Fluoxetine (0.1-1 microM) and litoxetine (0.3-3 microM) antagonized both the high- and low-potency phases of the 5-HT curve. Schild analysis for the low-potency phase yielded pA2 estimates of 6.6 +/- 0.3 (Schild slope of 1.1) and of 6.6 +/- 0.1 (Schild slope of 1.1), respectively. At higher concentrations (3 microM), fluoxetine markedly inhibited the 5-HT response maximum. Clomipramine (10-300 nM) inhibited, by a mechanism independent of concentration, both phases of the 5-HT curve with a reduction of the maximum response. Paroxetine (1 microM) was ineffective on the high-potency phase, but caused a rightward shift of the low-potency phase (pKB: 6.1 +/- 0.01). 5. Responses to 2-methyl-5-HT were inhibited by 1 microM fluoxetine (pKB: 5.4 +/- 0.02). Like clomipramine(30 and 100 nM), litoxetine (1 and 3 microM) produced rightward displacements of 2-methyl-5-HT-induced contractions, which were virtually independent of antidepressant concentration (pKB values: 6.0 +/- 0.02 and 5.5 +/- 0.01, respectively). At higher concentrations, fluoxetine (3 microM) and clomipramine (300 nM)markedly reduced the 2-methyl-5-HT response maximum. Paroxetine (1 micro M) was ineffective.6. Responses to 5-MeOT were shifted to the right by fluoxetine (0.1-1 micro M) and litoxetine (1 and 3 microM)in a concentration-dependent manner. At higher concentrations, fluoxetine (3 microM) markedly reduced the 5-MeOT response maximum, an effect also observed with 100 and 300 nM clomipramine. Paroxetine(1 microM) was ineffective.7. In unstimulated LMMPs, the excitatory effects evoked by 5-HT, 2-methyl-5-HT and 5-MeOT and the antagonism produced by 300 nM clomipramine were comparable to those obtained in whole ileal segments. This suggests that 5-HT contained in the mucosa of whole preparations does not interfere with agonist-induced contractile responses and with the inhibitory effect of antidepressant drugs.8. In conclusion, our results show that clomipramine, fluoxetine, paroxetine and litoxetine possess low to moderate potency/affinity at both central and peripheral (enteric) 5-HT3 receptors. In contrast, all four antidepressants are virtually ineffective at central 5-HT4 receptors. Inhibition of 5-HT4 receptor mediated ileal contractions by fluoxetine, litoxetine and clomipramine may result from allostericant agonism or, more likely, from post-receptor blockade of second messenger generation. The interaction of antidepressants with central and peripheral 5-HT3 and 5-HT4 receptors may be relevant for both potential therapeutic action and adverse effects at gastrointestinal level.

Oligosaccharide behavior of complex natural glycosphingolipids in multicomponent model membranes.

Wideline 2H NMR of model membranes was used to consider the molecular consequences of factors often suggested as modulators of complex glycosphingolipid oligosaccharide arrangement and motional characteristics at cell surfaces. GM1, asialo-GM1, and globoside were studied as examples of plasma membrane recognition sites. The experimental approach involved substitution of deuterons (D) for protons at specific locations within the carbohydrate chains. Deuterated glycolipids were then dispersed at 7-10 mol% in unsonicated bilayers of 1-palmitoyl-2-oleoylphosphatidylcholine. Factors tested for their significance to carbohydrate chain conformation and dynamics included glycolipid natural alkyl and acyl chain variability, membrane fluidity, and the presence of cholesterol and a charged sugar residue (neuraminic acid). Effects of Ca2+ and membrane-associated protein were briefly considered. Two distinct strategies were employed in substituting deuterons for selected protons of carbohydrate residues. Neither approach necessitated alteration of the glycolipid natural fatty acid composition. (i) Protons of the exocyclic hydroxymethyl group on the terminal Gal residue of GM1 and asialo-GM1, and on the terminal N-acetylgalactosamine (GalNAc) residue of globoside, were replaced with deuterium (producing -CDHOH) by an enzymatic oxidation/reduction cycle. This represents the first application of such an approach to deuteration of complex neutral glycolipids. Spectral results were compared to those obtained for the similarly-deuterated monoglycosyl lipid, galactosylceramide (GalCer), with natural fatty acid composition. Efficacy of this labeling method may in principle be influenced by structural variations within a given glycolipid family. Also, asymmetric rotation of the deuterated group made it less attractive than the second method for relating spectral features to receptor geometry. (ii) A general synthetic, nonenzymatic method was investigated for replacing amino sugar N-acetyl groups with deuterated acetate (-COCD3). The acetate group of the GalNAc residue of globoside, GM1, and asialo-GM1, as well as that on neuraminic acid in GM1, was replaced with -COCD3. This second method afforded better signal-to-noise--an important consideration for 2H NMR. The NMR technique employed had the potential for detecting changes of as little as 10% in oligosaccharide orientation or motional order. Each glycolipid demonstrated clear evidence of preferred average oligosaccharide conformations in all (fluid) membrane environments examined. The most striking observation was that, in fluid matrices, conformation and motional order of the complex oligosaccharide chains were only modestly influenced by factors tested, including natural variation in the glycolipid hydrocarbon chains, membrane fluidity, temperature, and the presence of cholesterol or the N-acetylneuraminic acid (NeuAc) residue on GM1.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential effects of divalent cations on specific 3H-GR 65630 binding to 5-HT 3 receptors in rat cortical membranes

We have examined the ability of divalent cations to inhibit 3H-GR 65630 binding to 5-HT 3 receptor recognition sites in rat brain cortical membranes. Among the divalent metal cations tested, Cd 2+, Zn 2+ and Cu 2+ inhibited the specific 3H-GR 65630 binding activity to 5-HT 3 receptors at a concentration of 0.1–1 mM. The other divalent metal cations tested (i.e. Co 2+, Ni 2+, Ba 2+, Ca 2+, Mg 2+ and Mn 2+) produced no inhibition of the specific 3H-GR 65630 binding. Cd 2+, Zn 2+ and Cu 2+ did not change the B max value of the binding activity, but significantly increased the K d value. It was suggested that these cations inhibited the binding activity by reducing affinity of the 5-HT 3 receptor for the antagonist, resulting in apparent inhibition of the binding activity. As to the binding association rate, Cd 2+, Cu 2+ and Zn 2+ were found to have an inhibitory effect. The binding dissociation rate, however, was shown to be decreased by Cu 2+ but not by Cd 2+ and Zn 2+. Furthermore, the Zn 2+-induced inhibition of 3H-GR 65630 binding was suggested to be antagonized by both concanavalin A and wheatgerm agglutinin. The Cu 2+-induced inhibition, however, was not influenced by these lectins, indicating that Cu 2+ has a different lectin sensitivity for its inhibitory effect. The different mechanism of action between Cu 2+ and Zn 2+ was suggested in their inhibitory effect on the specific 3H-GR 65630 binding activity.

Binding of [ 3H]FH-510 to σ ligand recognition sites in guinea-pig brain membranes

We examined the characteristics of the binding of radiolabeled 5,8-dimethyl-4-(2-di-n-propylaminoethyl)carbazol monohydrochloride ([ 3H]FH-510), a highly potent and selective σ ligand, to guinea-pig brain membranes. [ 3H]FH-510 showed saturable and reversible binding to σ binding sites. The association rate constant (k +1) and dissociation rate constant (k −1) and dissociation (k −1) of [ 3H]FH-510 were 0.23 min −1·nM −1 and 0.081 min −1, respectively. Scatchard plot analysis showed a dissociation constant (K d and maximal number of binding sites (B max) of 6.0 ± 0.63 nM and 1763.3 ± 177.4 fmol/mg protein (n = 7), respectively. The rank order of potency (K i) of several structurally dissimilar σ ligands obtained for the displacement of [ 3H]FH-510 binding was highly correlated with that determined for [ 3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ([ 3H](+)-3-PPP) binding. The binding of [ 3H]FH-510 was not influenced by histaminergic, dopaminergic, adrenergic, serotonergic or cholinergic agents at 10 −7 M. Higher [ 3H]FH-510 binding to brain regions was observed in the cerebellum and pons-plus-medulla. Except for the nuclear fraction, the highest level of [ 3H]FH-510 and [ 3H](+)-3-PPP binding to subcellular fractions was observed in the microsomal fraction. From these results, it is suggested that FH-510 selectivity binds with high affinity to σ binding sites in guinea-pig membranes.

Pancopride, a potent and long-acting 5-HT 3 receptor antagonist, is orally effective against anticancer drug-evoked emesis

Pancopride ((±)N-(1-azabicyclo-[2,2,2]-oct-3-yl)-2-cyclopropylmethoxy-4-amino-5-chlorobenzamide) is a new potent and selective 5-HT 3 receptor antagonist, orally and parenterally effective against cytotoxic drug-induced emesis. In vitro, pancopride displayed high affinity (K i = 0.40 nM) for [ 3H]GR65630-labelled 5-HT 3 recognition sites in membranes from the cortex of rat brains. In vivo, pancopride antagonized 5-HT-induced bradycardia in anaesthetized rats when administered i.v. 5 min (ID 50 = 0.56 μg/kg) or p.o. 60 min (ID 50 = 8.7 μg/kg) before 5-HT challenge. A single oral dose (10 μg/kg) of pancopride produced a significant inhibition of the bradycardic reflex over an 8-h period. Pancopride dose dependcntly inhibited the number of vomiting episodes and delayed the onset of vomiting induced by cisplatin in dogs (ID 50 = 3.6 μg/kg i.v. and 7.1 μg/kg p.o.). Pancopride was also effective in blocking mechlorcthamine- and dacarbazine-induced emesis. Unlike metoclopramide, pancopride was shown to lack any measurable antidopamincrgic activity both in vitro and in vivo. These results support clinical data, indicating that pancopride will be a useful drug for treating cytostatic-induced emesis in humans.

A corticosteroid Receptor in Neuronal Membranes

Steroids may rapidly alter neuronal function and behavior through poorly characterized, direct actions on neuronal membranes. The membrane-bound receptors mediating these behavioral responses have not been identified. [3H]Corticosterone labels a population of specific, high-affinity recognition sites (dissociation constant = 0.51 nanomolar) in synaptic membranes from an amphibian brain. These binding sites were localized by receptor autoradiography in the neuropil, outside the regions of perikarya. The affinities of corticoids for this [3H]corticosterone binding site were linearly related to their potencies in rapidly suppressing male reproductive behavior. Thus, it appears that brain membranes contain a corticosteroid receptor that could participate in the regulation of behavior.

Synthesis and biochemical evaluation of tritium-labeled 1-methyl-N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-1H-indazole-3-carboxamide, a useful radioligand for 5HT3 receptors

The advent of potent, highly selective 5HT3 receptor antagonists has stimulated considerable interest in 5HT3 receptor mediated physiology and pharmacology. To permit detailed biochemical studies regarding interaction of the indazole class of serotonin (5HT) antagonists with 5HT3 receptors in multiple tissues, we synthesized 1-methyl-N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-1H-indazole- 3-carboxamide (LY278584, compound 9) in high specific activity, tritium-labeled form. This radioligand was selected as a synthetic target because of its potency as a 5HT3-receptor antagonist, its selectivity for this receptor viz a viz other 5HT-receptor subtypes, and the ability to readily incorporate three tritia via the indazole N-CH3 substituent. Alkylation of N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-1H-indazole-3-carboxamide (8) with sodium hydride and tritium-labeled iodomethane, followed by HPLC purification, resulted in [3H]-9 with a radiochemical purity of 99% and a specific activity of 80.5 Ci/mmol. This radioligand bound with high affinity to a single class of saturable recognition sites in membranes isolated from cerebral cortex of rat brain. The Kd was 0.69 nM and the Bmax was 16.9 fmol/mg of protein. The specific binding was excellent, and accounted for 83-93% of total binding at concentrations of 2 nM or less. The potencies of known 5HT3-receptor antagonists as inhibitors of [3H]-9 binding correlated well with their pharmacological receptor affinities as antagonists of 5HT-induced decreases in heart rate and contraction of guinea pig ileum, suggesting the central recognition site for this radioligand may be extremely similar to or identical with peripheral 5HT3 receptors.

Effect of aspartame on N-methyl- d-aspartate-sensitive l-[ 3H]glutamate binding sites in rat brain synaptic membranes

Aspartame ( l-aspartyl- l-phenylalanine methyl ester), an artificial low-calorie sweetener, was shown to dose-dependently inhibit l-[ 3H]glutamate binding to its N-methyl- d-aspartate-specific receptors. l-Aspartic acid, a major endogenous metabolite of aspartame, inhibited the binding more stronger than aspartame, while the other metabolites, l-phenylalanine and methanol, had no effect at the same concentration. Aspartame caused a significant change in the affinities of l-[ 3H]glutamate binding without altering the V max values of the binding, suggesting the inhibition is competitive. These in vitro findings suggested that aspartame may act directly on the N-methyl- d-aspartate-sensitive glutamate recognition sites in the brain synaptic membranes.