Rat Cerebral Membranes Research Articles

Influence of Subchronic Administration of the HLDF-6 Peptide on an Efficacy of the Exploratory Behavior of Inbred Mice of the Balb/c and C57Bl/6 Strains

A comparative analysis of the psychotropic activity of the HDLF-6 peptide in the acidic form (TGENHR) and the amide form (HLDF-6-amide, TGENHR-NH2) was carried out using the method for the behavioral assessing of inbred mice of the Balb/c strain (with initial deficiency of exploratory activity) and the С57Bl/6 strain (as reference) in the “closed cross-maze” test (CCM). The peptide forms were intraperitoneally (at the subchronic daily doses of 100 μg/kg,) and intranasally (at daily doses of 30, 100, and 300 μg/kg) administrated for five days. Both intraperitoneal and intranasal administration of HLDF-6-amide (but not the HLDF-6 peptide) were shown to result in a development of the selective nootropic effect in the Balb/c mouse population. On the contrary, this effect was not reproduced in the group of the C57Bl/6 mice. The locomotor activity and the anxiety level were not changed in these groups of mice after injections of both forms of the peptide. Based on the results of behavioral experiments in the CCM test, we concluded that the HLDF-6-amide exhibited a selective pharmacological effect, which manifested itself as an increase in the exploratory activity only for the animals with an initial cognitive deficit. This property was characteristic of nootropic drugs. We first established by the radioreceptor analysis that a common target for the specific high affinity binding on the rat cerebral membranes existed for the HLDF-6 peptide and its derivatives. Thus, further development of an original intranasal nootropic drug on the basis of the HLDF-6-amide was promising.

Vascular and molecular pharmacology of the metabolically stable CGRP analogue, SAX

The main purpose of this study was to compare in vitro pharmacological properties of human αCGRP (CGRP) and a recently discovered metabolically stable CGRP analogue, SAX, in isolated rat and human artery segments.In rat, CGRP and SAX induced similar vasodilatory responses in isolated mesenteric artery with the potency of SAX being lower than that of CGRP (vasodilatory pEC50 8.2 ± 0.12 and 9.0 ± 0.11, respectively). A corresponding difference in receptor binding affinity of SAX and CGRP was determined in rat cerebral membranes (pKi 8.3 ± 0.19 and 9.3 ± 0.14, respectively). CGRP and SAX-induced vasodilation was antagonised with similar potencies by the CGRP receptor antagonist BIBN4096BS supporting a uniform receptor population for the agonists.In human tissue, SAX and CGRP induced similar pharmacological responses with different potencies in subcutaneous artery (vasodilatory pEC50 8.8 ± 0.18 and 9.5 ± 0.13, respectively) and human recombinant receptors (cAMP signalling pEC50 9.1 ± 0.16 and 10.2 ± 0.19). Like in the rat mesenteric artery, both SAX and CGRP-responses were inhibited by the CGRP receptor antagonist BIBN4096BS with similar antagonistic potencies.In conclusion, all pharmacological characteristics of SAX and CGRP in human and rat sources points towards action via a uniform BIBN4096BS sensitive receptor population with the potency of SAX being 5–10 fold lower than that of CGRP.

Syntheses and In-vitro Evaluation of Tetrahydroaminoacridine (THA) Based Analogues as High Affinity Choline Transporter (HAChT) Imaging Probe

Introduction: In cholinergic neurons, high affinity choline uptake (HACU) by the high affinity choline transporter (HAChT) is a rate-limiting and regulatory step for the synthesis of Acetylcholine (Ach).Thus, HAChT appear to be a relatively specific presynaptic marker for cholinergic neurons in Alzheimer’s disease.Objectives: The principle objective of the study is to check the affinity of tetrahydroaminoacridine (THA) derivatives for HAChT. Another objective of the research work is to clarify whether the hemicholinium-3 (ChT inhibitor) and HACU enhancer molecules share the same binding sites or not.Materials and Methods: The inhibition activities of tacrine, the 2,3-dimethylfuran derivative of tacrine (DMTA) and their corresponding 2-oxo-1-pyrrolidineacetyl derivatives, namely PTAA and MKC-231 were measured by displacement of a typical HAChT antagonist [3H]HC-3 in rat cerebral membrane. The percentage of inhibition against the binding of [3H]HC-3 to HAChT were calculated using GraphPad Prism v4 software.Results: Hemicholinium-3 showed affinity for HAChT (IC50 = 20 nM) in the in vitro binding assay. A very insignificant inhibition activity (IC50 = 1000 nM) of Tacrine was revealed. The newly synthesized tacrine derivatives, DMTA and PTAA did not show any affinity for HAChT. Although MKC-231 was reported to enhance cholinergic activity at synaptic terminals, it did not show any affinity for the HAChT in [3H]HC-3 binding assay.Conclusion: In vitro [3H]HC-3 binding assay revealed no affinity of MKC-231, tacrine and its corresponding2-oxo-1-pyrrolidineacetate derivative towards HAChT. So, it is worthy to develop radiolabeled HC-3 derivatives with high affinity for HAChT, which can diffuse the BBB, to enable the in vivo investigation of HACU system.Bangladesh J. Nuclear Med. 17(2): 97-102, July 2014

Development of novel PET probe [11C](R,R)HAPT and its stereoisomer [11C](S,S)HAPT for vesicular acetylcholine transporter imaging: A PET study in conscious monkey

Carbon-11-labeled (R,R)trans-8-methyl-2-hydroxy-3-[4-[2-aminophenyl]piperizinyl]-tetralin ([(11)C](R,R)HAPT) and its stereoisomer [(11)C](S,S)HAPT were developed for imaging vesicular acetylcholine transporters (VAChTs), exclusively located in presynaptic cholinergic neurons. Both positron emission tomography (PET) probes were evaluated in the brain of conscious monkey (Macaca mulatta) using high-resolution PET. Time-activity curves (TACs) of [(11)C](R,R)HAPT peaked within 5 min after the injection in all regions except the caudate and putamen, both of which showed peaks around 20 min postinjection. The regional distribution patterns of [(11)C](R,R)HAPT determined as total distribution volume (V(t)) were highest in the putamen, high in the caudate, intermediate in the amygdala, hippocampus, and thalamus, lower in the cingulate gyrus and frontal, temporal, and occipital cortices, and lowest in the cerebellum. In contrast, the distribution and TACs of [(11)C](S,S)HAPT were homogeneous in all regions. The uptake of [(11)C](R,R)HAPT was reduced by 1 mg/kg (-)-vesamicol, a specific VAChT antagonist, in all regions except the cerebellum, but not by 0.1 mg/kg SA4503, a specific sigma-1 receptor agonist. These results well reflect the in vitro affinity assessments using rat cerebral membranes. They also demonstrate that [(11)C](R,R)HAPT is a potential PET probe for noninvasive and quantitative imaging of VAChT in the living brain.

Syntheses and in vitro evaluation of decalinvesamicol analogues as potential imaging probes for vesicular acetylcholine transporter (VAChT)

A series of vesamicol analogues, o-iodo-trans-decalinvesamicol (OIDV) or o-bromo-trans-decalinvesamicol (OBDV), were synthesized and their affinities to vesicular acetylcholine transporter (VAChT) and σ receptors (σ-1, σ-2) were evaluated by in vitro binding assays using rat cerebral or liver membranes. OIDV and OBDV showed greater binding affinity to VAChT (Ki=20.5±5.6 and 13.8±1.2nM, respectively) than did vesamicol (Ki=33.9±18.1nM) with low affinity to σ receptors. A saturation binding assay in rat cerebral membranes revealed that [125I]OIDV had a single high affinity binding site with a Kd value of 1.73nM and a Bmax value of 164.4fmol/mg protein. [125I]OIDV revealed little competition with inhibitors, which possessed specific affinity to each σ (σ-1 and σ-2), serotonin (5-HT1A and 5-HT2A), noradrenaline, and muscarinic acetylcholine receptors. In addition, BBB penetration of [125I]OIDV was verified in in vivo. The results of the binding studies indicated that OIDV and OBDV had great potential to be VAChT imaging probes with high affinity and selectivity.

Comparison of the binding affinity of CGP-12177A at recombinant rat α 1D-adrenoceptors expressed in BHK-21 cell membranes and α 1-adrenoceptors present in rat cerebral cortex membranes

Recent in vitro studies, performed in rat aorta, mesenteric and intrapulmonary arteries, and human pulmonary artery, demonstrated that the β-adrenoceptor ligand CGP-12177A (4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one) is also provided with antagonist or partial agonist properties at α 1-adrenoceptors. These observations were supported by estimates of CGP-12177A binding affinity at α 1-adrenoceptors, which have been always performed in rat cerebral cortex membranes, as a surrogate of vascular tissue. Since α 1D-adrenoceptors are predominant in both rat aorta and mesenteric artery, in the present study, we measured, for the first time, the binding affinity of CGP-12177A at recombinant rat α 1D-adrenoceptors expressed in BHK-21 cell membranes. CGP-12177A binding affinity was also determined in rat cerebral cortex membranes, where various α 1-adrenoceptor subtypes are present. By means of [ 3H]prazosin binding competition experiments, we found that CGP-12177A bound to α 1D-adrenoceptor-expressing BHK-21 cell membranes, with a binding affinity (p K i = 5.39 ± 0.27) almost identical to that measured in cerebral membranes (p K i = 5.44 ± 0.07), indicating that it is a non-subtype selective α 1-adrenoceptor ligand. Moreover, CGP-12177A binding affinity was very close to its functional affinity evaluated in rat aorta in terms of antagonist potency against phenylephrine-induced contraction (p K B = 5.65 ± 0.07). In conclusion, our results demonstrate that, in order to evaluate CGP-12177A binding affinity at aorta and mesenteric artery α 1-adrenoceptors, estimates in rat cerebral membranes are as reliable as those in recombinant rat α 1D-adrenoceptors, since both values are very close to CGP-12177A functional affinities in isolated vessels.

Asymmetrical nitrido tc-99m heterocomplexes as potential imaging agents for benzodiazepine receptors.

The design, synthesis, and biological evaluation of nitrido technetium-99m complexes for imaging benzodiazepine receptors are described. The design was performed by selecting the precursor biologically active substrate desmethyldiazepam, and the reactive metal-containing fragment [(99m)Tc(N)(PXP)](2+) (PXP = diphosphine ligand) as molecular building-blocks for assembling the structure of the final radiopharmaceuticals through the application of the so-called 'bifunctional' and 'integrated' approaches. This required the synthesis of the ligands H(2)BZ1, H(2)C1, and H(2)C2 (Figures 1 and 2) derived from desmethyldiazepam. In turn, these ligands were reacted with [(99m)Tc(N)(PXP)](2+) to afford the complexes [(99m)Tc(N)(PXP)(L)] (L = BZ1, C1, C2). The chemical nature of the resulting Tc-99m radiopharmaceuticals was investigated using chromatographic methods, and by comparison with the analogous complexes prepared with the long-lived isotope Tc-99g and characterized by spectroscopic and analytical methods. Results showed that the complexes [(99m)Tc(N)(PXP)(L)] are neutral and possess an asymmetrical five-coordinated structure in which two different bidentate ligands, PXP and L, are coordinated to the same Tc[triple bond]N core. With the ligand H(2)BZ1, two isomers were obtained depending on the syn or anti orientation of the pendant benzodiazepine group relative to the Tc[triple bond]N multiple bond. Biodistribution studies of Tc-99m complexes were carried out in rats, and affinity for benzodiazepine receptors was assessed through in vitro binding experiments on isolated rat's cerebral membranes using the corresponding Tc-99g complexes.

In vitro characterization of radioiodinated (−)-m-iodovesamicol in rat cerebral membranes

We investigated the binding characteristics of 125I-(−)-m-iodovesamicol ( 125I- (−)-mIV), radioiodinated at the meta position of the 4-phenylpiperidine moiety, in cerebral membranes of the rat brain. The receptor binding affinity of (−)-mIV ( Ki = 37 nM) was comparable to that of (−)-vesamicol ( Ki = 30 nM). The stereoselectivity of (−)-mIV and (−)-vesamicol for the vesamicol receptor was very high [ both (−)mIY and (−)-vesamicol binding more than 20-fold more active than their (+)isomers], and 125I-(−)-mIV had low affinity for the sigma, dopamine, serotonin, adrenaline and acetylcholine receptors. Furthermore, in a saturation binding study using cerebral membrane preparations, (−)-mIV exhibited a K d of 18.2 nM with maximum number of binding site B max of 660 fmol/mg of protein. These results showed that the characteristics of binding between (−)-mIV and (−)-vesamicol to cholinergic binding sites in the rat brain were similar.

Flavonoids as antioxidant agents: Importance of their interaction with biomembranes

Flavonoids, a group of phenolic compounds widely occurring in the plant kingdom, have been reported to possess strong antioxidant activity. In the present study, four flavonoids (quercetin, hesperetin, naringenin, rutin), chosen according to their structural characteristics, were tested in two different in vitro experimental models: (1) Fe 2+-induced linoleate peroxidation (Fe 2+-ILP), by detection of conjugated dienes; and (2) autooxidation of rat cerebral membranes (ARCM), by using thiobarbituric acid for assay of free malondialdehyde production. The results obtained were also interpreted in the light of flavonoid interactions, studied by differential scanning calorimetry, with dipalmitoylphosphatidylcholine (DPPC) vesicles as a biological membrane model. The antilipoperoxidant activity of the flavonoids tested can be classified as follows: rutin > hesperetin > quercetin ⪢ naringenin in the Fe 2+-ILP test; quercetin > rutin ⪢ hesperetin > naringenin in the ARCM test. Quercetin, hesperetin, and naringenin interacted with DPPC liposomes causing different shifts, toward lower values, of the main transition peak temperature ( T m ) typical for DPPC liposomes; however, no change in T m of DPPC dispersion was observed in the presence of rutin. The hypothesis will be discussed that flavonoid capacity to modify membrane-dependent processes, such as free-radical-induced membrane lipoperoxidation, is related not only to their structural characteristics but also to their ability to interact with and penetrate the lipid bilayers.

Hydrolysis of exogenous [3H]phosphatidylcholine by brain membranes and cytosol

Phosphatidylcholine, in addition to the widely studied inositol phospholipids is cleaved to produce second messengers in neuronal signal transduction processes. Because of the difficulty in labelling and measuring the metabolism of endogenous phosphatidylcholine in brain tissue, we investigated the utility of measuring the hydrolysis of exogenous labelled substrate incubated with rat cerebral cortical cytosol and membrane fractions as has been successful in studies of phosphoinositide hydrolysis. In the cytosol [3H]phosphatidylcholine was hydrolyzed at a linear rate for 60 min of incubation and GTP gamma S stimulated hydrolysis by 63%. The products of phospholipase C and phospholipase D, phosphorylcholine and choline, contributed only 44% of the [3H]phosphatidylcholine hydrolytic products in the cytosol, with phospholipase D activity slightly predominating. GTP gamma S stimulated cytosolic phospholipase C and reduced phospholipase D activity. [3H]Phosphatidylcholine was hydrolyzed much more slowly by membranes than by cytosol. In membranes the production of [3H]phosphorylcholine and [3H]choline were approximately equal, contributing 27% of the total [3H]phosphatidylcholine hydrolysis, and GTP gamma S only caused a slight stimulation of phospholipase C activity. Chronic lithium treatment (4 weeks) appeared to slightly reduce [3H]phosphatidylcholine metabolism in the cytosol and in membranes, but no statistically significant reductions were achieved. Cytosol and membrane fractions from postmortem human brain metabolized [3H]phosphatidylcholine slowly, and GTP gamma S had no effects. In summary, exogenous [3H]phosphatidylcholine was hydrolyzed by brain cytosol and membranes, and this was stimulated by GTP gamma S, but the complex contributions of multiple metabolic pathways complicates the application of this method for studying individual pathways, such as phospholipase D which contributes only a fraction of the total processes hydrolyzing exogenous [3H]phosphatidylcholine.

Differential binding activity of erythrocyte ankyrin to the alpha-subunits of Na+, K(+)-ATPases from rat cerebral and axonal membrane.

Ankyrin is an important key protein transferring the signal between the inside and outside of eukaryotic cells, because of its ability to bind both to ionic channels of the plasma membranes and to cytoskeletal proteins. In this study, we investigated new ankyrin binding proteins in rat cerebral membrane. Five main proteins in the extract of the demyelinated membranes were bound to erythrocyte ankyrin as examined by affinity chromatography. One of the proteins had a molecular weight of 97 kD that was almost identical with that of the alpha-subunit of Na+, K(+)-ATPase. 125I-labeled erythrocyte ankyrin was bound to the alpha-subunit of cerebral Na+, K(+)-ATPase, which contains both alpha and alpha(+) subunits. The binding experiment also showed that 70% of the total erythrocyte ankyrin bound to cerebral Na+, K(+)-ATPase. On the other hand, erythrocyte ankyrin binds less to Na+, K(+)-ATPase prepared from rat brain stem axolemma which contained only alpha(+) subunit. These results suggest that erythrocyte ankyrin may bind with high affinity to a cerebral Na+, K(+)-ATPase isoform with alpha subunit, but with much lower affinity to axonal Na+, K(+)-ATPase containing alpha(+) subunit.

Ethanol Fails to Modify [3H]GR65630 Binding to 5‐HT3 Receptors in NCB‐20 Cells and in Rat Cerebral Membranes

Low concentrations of ethanol have been found to enhance the electrophysiologic effect of serotonin (5-HT) acting at 5-HT3 receptors on NCB-20 cells. To determine whether this action of ethanol reflects a change in the agonist-receptor interaction, the effect of ethanol (100 mM) on agonist and antagonist binding to 5-HT3 receptor was studied in vitro in membrane from NCB-20 cells and from cortex plus hippocampus of rat. The antagonist [3H]GR65630 was used to label 5-HT3 recognition sites. Ethanol did not change the characteristics of saturable [3H]GR65630 binding in either membrane preparation. In competition studies, the agonists 5-HT and 2-methyl-5-HT completely inhibited the binding of [3H]GR65630 to NCB-20 cell membranes, while in brain membranes the maximum displacement of specific [3H]GR65630 binding by 5-HT was approximately 30%. Ethanol decreased the affinity of the receptor for 2-methyl-5-HT, but not to 5-HT in NCB-20 cells, and had no effect on agonist binding in brain membranes. The results indicate that enhancement of 5-HT responses at 5-HT3 receptors by ethanol is not a result of changes in the equilibrium binding characteristics of the agonist.

Autoradiographic changes in central benzodiazepine binding sites and their coupling to gamma-aminobutyric acid receptors after seizures in the developing rat.

Benzodiazepines are psychoactive substances classically used for their anticonvulsant properties in neonates as well as in adults. In a previous work, we have shown that seizures lead to an age-dependent upregulation of central benzodiazepine binding sites measured in isolated rat cerebral membranes. However, information concerning regional changes in the receptor density was lacking. In our present study, the effects of bicuculline-induced seizures were investigated by quantitative autoradiography of central benzodiazepine receptors in developing rats and in adults. Animals were killed 30 min after an intraperitoneal injection of either saline or a convulsive dose of bicuculline. Benzodiazepine binding sites in brain sections were labeled by [3H]flunitrazepam. Generalized seizures induced a widespread increase in benzodiazepine receptors, with a marked enhancement in structures that mediate seizure activity, such as substantia nigra, amygdala, septum, and hippocampus. The addition of exogenous gamma-aminobutyric acid to the incubation medium increased benzodiazepine binding by the same order of magnitude whether rats were given saline or bicuculline, suggesting that additional benzodiazepine sites are also functionally linked to gamma-aminobutyric acid receptors. The age-related postictal increase in benzodiazepine receptors might reflect a compensatory response for protection against recurrent seizures, especially in newborns.

Autoradiographic changes in brain adenosine A 1 receptors and their coupling to G proteins following seizures in the developing rat

In the central nervous system, adenosine has been shown to be a major regulator of neuronal activity in convulsive disorders, mainly via the A 1 receptor subtype. In a previous work, we have shown that seizures lead to an age-dependent upregulation of cerebral adenosine A 1 sites measured in isolated rat cerebral membranes. However, information concerning regional changes in the receptor density was so far lacking. In the present study, the effects of bicuculline-induced seizures were investigated by quantitative autoradiography of central adenosine A 1 receptors in developing rats and in adults. Animals were sacrificed 30 min after an intraperitoneal injection of either saline or a convulsive dose of bicuculline. Adenosine A 1 receptors in brain section, were labeled by [ 3H]N 6-cyclohexyladenosine (CHA), a potent receptor agonist. Generalized seizures induced a widespread increase in CHA-specific binding, with a marked enhancement in structures that mediate seizure activity, such as substantia nigra, amygdala, septum and hippocampus. Moreover, the addition of guanylyl-5′-imidodiphosphate, a GTP analogue, to the incubation medium reduced CHA binding by the same order of magnitude whether rats were given saline or bicuculline, suggesting that additional adenosine A 1 receptors are also functionally linked to G proteins. The age-related postictal increase in adenosine receptors might contribute to facilitate adenosine anticonvulsant effect, especially in newborns.

Phencyclidine blocks histamine H 3-receptors in rat brain

Phencyclidine (PCP) was examined for its ability to modulate histamine release from rat brain slices labeled with L-[ 3H]histidine. PCP failed to mimic but completely reversed the autonihibitory effect of histamine at H 3-receptors with an apparent K i value of 13 ± 3 μM. A direct interaction of PCP with H 3-autoreceptors rather than PCP or σ receptor sites was confirmed by binding studies. PCP inhibited the binding of [ 3H](R)α-methylhistamine to H 3-receptor sites in rat cerebral membranes with a K i value of 25 ± 2 μM. It is concluded that PCP is a H 3-receptor antagonist of moderate potency.

Sex Differences in [3H]Nitrendipine Binding and Effects of Sex Steroid Hormones in Rat Cardiac and Cerebral Membranes

The sex differences and regulation by sex steroid hormones in calcium channels were studied by using [3H]nitrendipine binding to cardiac and cerebral membranes in 1 5-week old spontaneously hypertensive rats (SHRs). The maximal number of binding sites (Bmax) in the hippocampus of female SHRs increased by 24.1% over that in male SHRs. In the females, the Bmax values in the cardiac, striatal, thalamic and hippocampal membranes from ovariectomized SHRs decreased by 34.7, 29.9, 29.3 and 26.9%, respectively, compared to normal SHRs. This phenomenon, except for the hippocampus, was inhibited by estradiol but not by testosterone. In the male, the Bmax values in cardiac and cerebral membranes showed almost no changes after orchidectomy or treatment with estradiol or testosterone. After gonadectomy, the Bmax values in the cardiac, striatal and thalamic membranes of females decreased by 30.2, 33.0 and 35.6%, respectively, compared to those in males. The changes in apparent dissociation constant (KD) values were less remarkable than those in the Bmax values. These findings suggest that sex differences exist in the calcium channels of the heart, striatum, thalamus and hippocampus, and they suggest that estradiol, but not testosterone, may play a part in the regulation of the calcium channels in female SHRs.

Sex differences in [3H]nitrendipine binding and effects of sex steroid hormones in rat cardiac and cerebral membranes.

The sex differences and regulation by sex steroid hormones in calcium channels were studied by using [3H]nitrendipine binding to cardiac and cerebral membranes in 15-week old spontaneously hypertensive rats (SHRs). The maximal number of binding sites (Bmax) in the hippocampus of female SHRs increased by 24.1% over that in male SHRs. In the females, the Bmax values in the cardiac, striatal, thalamic and hippocampal membranes from ovariectomized SHRs decreased by 34.7, 29.9, 29.3 and 26.9%, respectively, compared to normal SHRs. This phenomenon, except for the hippocampus, was inhibited by estradiol but not by testosterone. In the male, the Bmax values in cardiac and cerebral membranes showed almost no changes after orchidectomy or treatment with estradiol or testosterone. After gonadectomy, the Bmax values in the cardiac, striatal and thalamic membranes of females decreased by 30.2, 33.0 and 35.6%, respectively, compared to those in males. The changes in apparent dissociation constant (KD) values were less remarkable than those in the Bmax values. These findings suggest that sex differences exist in the calcium channels of the heart, striatum, thalamus and hippocampus, and they suggest that estradiol, but not testosterone, may play a part in the regulation of the calcium channels in female SHRs.

Characterization of dihydropyridine binding sites in the rat brain: hypertension and age-dependent modulation of [ 3H](+)-PN 200-110 binding

The properties of [ 3H]dihydropyridine (DHP), nitrendipine and (+)-PN 200-110, binding to rat cerebral membranes were investigated. In normotensive Wistar-Kyoto (WKY) adult rats, the highest densities of [ 3H]DHP binding sites were found in the hippocampus. Frontal cerebral cortex and hypothalamus had intermediate levels and no specific binding of [ 3H]DHP and [ 125I]iodipine could be detected in the brainstem membranes and more precisely in the nucleus tractus solitarius and in the locus coeruleus. Changes in the maximal number of DHP binding sites ( B max ) were observed in spontaneously hypertensive rats (SHR) and in old Sprague-Dawley rats. In adult SHR, there was a significant increase in the B max values of [ 3H](+)-PN 200-110 binding in the hippocampus when compared to the values obtained in WKY. There was no difference in the B max values between young (3 weeks) prehypertensive SHR and age-matched WKY. In senescent (26 months) Sprague-Dawley rats, the B max values of [ 3H](+)-PN 200-110 binding were significantly reduced (30%) in the frontal cerebral cortex and the hippocampus, as compared with the number of DHP binding sites found in mature Sprague-Dawley rats (15 weeks).

Characterisation of two probes for the localisation of enkephalinase in rat brain: [ 3H]thiorphan and a 125I-labeled monoclonal antibody

We studied the binding of two radioactive probes, i.e. [ 3H]thiorphan and a 125I-labeled monoclonal antibody raised against the rabbit kidney enzyme, to enkephalinase (EC 3.4.24.11, membrane metalloendopeptidase) from rat cerebral membranes. [ 3H]Thiorphan binding at equilibrium to striatal membranes was monophasic with a K D (0.7 nM) and a pharmacology consistent with a selective labeling of the enzyme. The ratio of V max/B max was in the same range as the K cat of the enzyme purified from peripheral tissues. The monoclonal antibody immunoprecipitated to a similar extent the solubilised enkephalinase activity and [ 3H]thiorphan binding sites from striatum. The regional distributions of binding sites for the two probes established either on isolated membranes or autoradiographic sections were highly heterogeneous and similar to that of enkephalinase activity. Hence the two probes appear to label membrane-bound enkephalinase in rat brain but, from a technical point of a view, the 125I-monoclonal antibody is a more sensitive and flexible tool.

Modification of ligand binding to membranes by a soluble acceptor: Alpha-1-acid glycoprotein attenuates 3H-Imipramine binding to cerebral membranes

The effect of individual plasma proteins on the binding of 3H-imipramine ( 3H-IMI) was investigated, using rat cerebral membranes as the binding site source. Addition of alpha-1-acid glycoprotein (aAGLP) to an incubation medium containing 4 nm 3H-IMI resulted in a concentration-dependent inhibition of 3H-IMI binding, with an IC 50 value of 0.53 μM. Albumin (0.7 mM) and gamma globulin (3 μM) had no apparent effect. Scatchard analyses of 3H-IMI binding in the presence and absence of 0.5 μm aAGLP revealed that the inhibition of 3H-IMI binding was associated with an increased K d, with no appreciable change in B max. The inhibitory action of platelet-free plasma (PFP) on 3H-IMI binding to cerebral membranes was eliminated after treatment of PFP with specific antibodies to aAGLP. It is suggested that the inhibition of 3H-IMI binding to cerebral membranes by PFP is due, at least partially, to the presence of aAGLP in the plasma. The observed inhibitory effect is consistent with a competition for the ligand between the membrane binding site and the soluble protein acceptor. These findings may explain the serum effect confounding radioreceptor assays of various drugs.