High-density Binding Research Articles

Comparison of Endothelin-A and Endothelin-B Receptor Distribution Visualized by Radioligand Binding versus Immunocytochemical Localization using Subtype Selective Antisera

Molecular studies have predicted the existence in human tissue of splice variants and modifications to the amino acid sequence of endothelin receptors that may modulate function. Endothelin-A (ETA) receptors were visualized by ligand binding and autoradiography to the renal vasculature throughout the cortex and medulla, including the large arcuate arteries, adjacent veins and arterioles. Lower binding densities were visualized to the vasa recta and glomeruli. A similar pattern of staining was revealed by ETA selective antisera, with the higher resolution demonstrating ETA receptors confined to smooth muscle cells. Staining was also detected to the vasa recta and glomeruli. Ligand binding revealed a more heterogeneous endothelin-B (ETB) receptor distribution with high densities concentrated in the medulla. Three different site-directed ETB antisera demonstrated a similar pattern of staining to the endothelium lining all renal vessels but not to the smooth muscle. Staining was also detected to glomerular endothelial cells as well as epithelial cells lining the renal tubule, particularly the collecting ducts, consistent with high binding densities observed in the medulla by autoradiography. There was no evidence for a differential distribution in either ETA or ETB receptors visualized by the two techniques that might have indicated modified receptors or further subtypes in the human kidney.

Characterization of [ 3H]ZM 241385 binding in wild-type and adenosine A 2A receptor knockout mice

The binding of the adenosine A 2A receptor antagonist [ 3H] 4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol ([ 3H]ZM 241385) to mouse brain and spinal cord was investigated. In brain homogenates, single-site binding was observed with a B max of 299±28 fmol mg −1 protein and a K d of 0.75±0.08 nM. In autoradiographic studies, there was a high density of specific binding of [ 3H]ZM 241385 in the striatum, with a very low density in the cortex and no binding elsewhere in the brain or in the spinal cord. All specific binding of [ 3H]ZM 241385 was lost in genetically modified mice lacking the adenosine A 2A receptor, confirming the selectivity of this radioligand.

Controlled Surface Functionalization of Silica Nanospheres by Covalent Conjugation Reactions and Preparation of High Density Streptavidin Nanoparticles

Silica nanoparticles with a diameter of 100 nm were covalently modified at their surface by adjustable amounts of amine and carboxyl functional groups. Bioconjugation studies of two proteins, streptavidin and streptactin, with the functional nanoparticles resulted in optimum binding of the proteins to a long-chain carboxyl-terminated linker. The surface functionalization of the nanoparticles was monitored by a variety of independent methods, including zeta-potential measurements, dynamic light scattering (DLS), scanning electron microscopy (SEM), particle charge detection titrations (PCD) and elemental analysis. At the surface of the nanoparticles, a functional surface group density of 1.8 amino groups per nm2 was realized. The amine functions were quantitatively transferred to carboxyl groups coupled with a linker elongation. Streptavidin was immobilized by covalent binding to the carboxyl linkers and resulted in a protein density at the surface of the nanoparticles that was three times higher than the highest binding densities at nanoparticles published to date. The binding capacity of the streptavidin-covered nanoparticles for ligand biotin was quantified by titration with biotin-4-fluorescein to 2.5 biotin binding sites per 100 nm2.

Distribution of muscimol, QNB, and 5HT binding in the vertebrate diencephalon: a comparative study of eight mammals and three non-mammals.

The distribution of muscimol, quinuclidinyl benzilate (QNB), and serotonin (5HT)-bound receptors in the diencephalon was examined by conventional receptor-binding methods in 11 species of amniotes including 2 reptiles, 1 bird, and 8 mammals, selected mostly on the basis of their differing last common ancestor with Anthropoids. We found that receptor binding can help define major subdivisions of the forebrain. The results show that in each of these species, the distribution of muscimol and QNB binding across the four major subdivisions of the diencephalon was consistent; densest in the dorsal thalamus, with hypothalamus and then either ventral thalamus or epithalamus with successively lesser amounts. However, the binding of serotonin (5HT) was most prevalent in the hypothalamus with equivalent amounts in the other diencephalic subdivisions. Myelin- and cell-stained materials showed that the pattern of high-density binding probably is not the secondary result of non-neurochemical factors such as differences in cell or neuropil density or in total available membrane. Perhaps more importantly, the receptor distributions suggest functional roles for major subdivisions across taxa. Results show that GABA-A and muscaranic Ach receptors are common in the dorsal diencephalon across vertebrate species and, therefore, are probably responsible for the gating of information to the cortex. Results show that serotonin is predominant in the hypothalamus. The lack of it in the dorsal thalamus indicates that it is probably not responsible for gating of information to the cortex. Results also show that in nonmammals the amount of GABA-A and muscaranic Ach differs from that found in mammals. For muscaranic Ach, the labeling in marsupials differs from that in placentals. Primates differ from other species (nonmammals and mammals combined) in the amount of 5HT found in the ventral diencephalon and the hypothalamus.

Simultaneous Binding of Ruthenium(II) [(1,10-Phenanthroline)2dipyridophenazine]2+ and Minor Groove Binder 4‘,6-Diamidino-2-phenylindole to Poly[d(A−T)2] at High Binding Densities: Observation of Fluorescence Resonance Energy Trasfer Across the DNA Stem

Photophysical properties of Δ- and Λ-[Ru(phenanthroline)2dipyrido[3,2-a:2‘,3‘-c]phenazine]2+ ([Ru(phen)2DPPZ]2+) bound to poly[d(A−T)2] in the presence and absence of 4‘,6-diamidino-2-phenylindole (DAPI) has been studied. In the presence of DAPI, the minor groove of poly[d(A−T)2] is blocked, allowing comparison of the binding mode of the ruthenium complexes. Absorption and circular dichroism (CD) as well as flow linear dichroism (LD) of the Λ-enantiomer are essentially unaffected when the minor groove is blocked by DAPI or not. However, with the Δ-enantiomer LD suggests a small but distinct change in binding geometry for both the DAPI and the ruthenium complex when DAPI is simultaneously bound to poly[d(A−T)2]. In all cases, the DPPZ ligand of the metal complex is intercalated between the nucleobases. From these data, it is concluded that the ruthenium complex still binds to DNA with the Ru(phen)2 moiety of both the Λ- and Δ-enantiomer located in the major groove when the minor groove is blocked. When DAPI and the ruthenium complex ions are simultaneously bound to poly[d(A−T)2], the luminescence of DAPI is decreased and that of the ruthenium complex is increased. The intensity ratio of the excitation relative to absorption spectra of the ruthenium complex is similar to the absorption spectrum of DAPI, demonstrating that excitation energy of DAPI is transferred to the ruthenium complex across the stem of DNA.

In vitro characterization of [ 3H]MethoxyPyEP, an mGluR5 selective radioligand

We have characterized the in vitro properties of 3-[ 3H]methoxy-5-(pyridin-2-ylethynyl)pyridine ([ 3H]MethoxyPyEP), an analogue of the mGluR 5 receptor subtype antagonist MPEP [2-methyl-6-(phenylethynyl)-pyridine], in rat tissue preparations using tissue homogenates and autoradiography. Binding of [ 3H]MethoxyPyEP to rat cortex, hippocampus, thalamus and cerebellum membrane preparations revealed saturable, high affinity binding (3.4 ± 0.4 nM, n = 4 in rat cortex) to a single population of receptors in all regions studied except for cerebellum. Binding was found to be relatively insensitive to pH and insensitive to DTT. High concentrations of NEM both reduce receptor concentration and binding affinity for the radioligand. In time-course studies at room temperature k on and k off were determined as 2.9 × 10 7 M −1 min −1 and 0.11 min −1 respectively. The rank order of affinities, as assessed by equilibrium competition studies, of a variety of ligands suggested binding of the radioligand selectively to mGluR5 (MPEP > trans-azetidine-2,4-dicarboxylic acid ≅ (S)-4-carboxyphenylglycine ≅ (+)MK801 ≅ CP-101,606 ≅ clozapine ≅ atropine ≅ ketanserin ≅ yohimbine ≅ benoxathian). Autoradiographic studies with [ 3H]MethoxyPyEP showed that binding was regioselective, with high density of binding in caudate and hippocampus, intermediate binding in thalamus and very low density in the cerebellum. These data show that [ 3H]MethoxyPyEP is a high affinity radioligand useful for the in vitro study of mGluR5 receptor distribution and pharmacologic properties in brain.

Localization of AT 2 receptors in the nucleus of the solitary tract of spontaneously hypertensive and Wistar Kyoto rats using [ 125I] CGP42112: upregulation of a non-angiotensin II binding site following unilateral nodose ganglionectomy

We have examined the binding distribution of a selective AT 2 receptor ligand [ 125I] CGP42112 in the brain of adult Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). AT 2 receptor localization was also examined in the rat brainstem following unilateral nodose ganglionectomy. Specific [ 125I] CGP42112 binding was observed in discrete brain regions from both rat strains, including the nucleus of the solitary tract (NTS), and did not differ between WKY and SHR. [ 125I] CGP42112 binding in the NTS revealed an AT 2 receptor component that was displaceable by PD 123319 and Ang II (50–58%), as well as a non-angiotensin II receptor component (42–49%). Following unilateral nodose ganglionectomy, [ 125I] CGP42112 binding density on the denervated side of the NTS was increased approximately two-fold in both WKY and SHR. This increased [ 125I] CGP42112 binding density in the ipsilateral NTS was comprised of a greater non-angiotensin II component than that observed in the sham groups, since only ∼30% was displaced by PD123319 and angiotensin II. Furthermore, [ 125I] CGP42112 also revealed high binding density on the denervated side in the dorsal motor nucleus and the nucleus ambiguus in both WKY and SHR. AT 2 receptor immunoreactivity was also visualised in the NTS of sham operated rats, but was not observed in the dorsal motor nucleus or the nucleus ambiguus, nor was it up-regulated following nodose ganglionectomy. These results demonstrate, for the first time, an AT 2 receptor binding site in the NTS, as well as a non-angiotensin II [ 125I] CGP42112 binding site. These studies also demonstrate that nodose ganglionectomy represents a useful model in which to study a non-angiotensin II [ 125I] CGP42112 binding site that is up-regulated following degeneration of afferent vagal nerves.

Laminar distribution of nicotinic receptor subtypes in cortical regions in schizophrenia

The laminar cortical distribution of the [ 125I]α-bungarotoxin, [ 3H]cytisine and [ 3H]epibatidine nicotinic acetylcholine receptor ligands was investigated by quantitative autoradiography in autopsy tissue from the cingulate, orbitofrontal and temporal cortices of control and schizophrenia subjects matched for age and smoking history. Different laminar binding patterns were observed for the various nicotinic ligands both in schizophrenic and control brains. [ 125I]α-Bungarotoxin binding was distributed homogeneously across all cortical layers in all three brain regions, with highest binding densities in the cingulate cortex. [ 3H]Cytisine and [ 3H]epibatidine binding varied across the cortical ribbon, with high binding in layers I, III, V and VI, within the three cortical regions. A significantly reduced [ 125I] α-bungarotoxin binding (−54%) was observed in the cingulate cortex of schizophrenia subjects, in comparison with normal individuals who smoked tobacco. In the same brain region also a significantly higher [ 3H]cytisine binding (48–77%) was observed in nearly all layers, except for layer I of the schizophrenia subjects, when compared to normal individuals with a history of tobacco use. No significant changes in [ 3H]epibatidine binding was observed within the individual cortical layers between control subjects and patients with schizophrenia, but when calculated as a whole region (i.e. measurements performed across the whole cortical ribbon), the temporal cortex showed a significant increase in [ 3H]epibatidine binding in schizophrenia subjects compared to control subjects. The results suggest opposite changes of the α4β2 and α7 nicotinic receptor subtypes in the cingulate cortex of patients with schizophrenia which might reflect involvement of two different nicotinic receptor mechanisms in schizophrenia brain.

Quantification of D1B(D5) receptors in dopamine D1A receptor-deficient mice.

The unavailability of selective D1A(D1) or D1B(D5) dopamine receptor ligands has prevented the direct localization of binding sites for these receptors. Thus, receptor autoradiography with long exposure times was used to detect minor D1-like binding sites in the brains of D1A null mutants. Coronal brain sections were prepared from the caudal portion of the prefrontal cortex of homozygous or heterozygous D1A knockout mice or wildtype mice, and labeled with the D1 receptor antagonist [3H]-SCH23390. Slides were dried, and apposed to film with polymer-calibrated standards for 90 days to allow visualization of any low abundance binding sites. No binding was detected in most regions of homozygote (-/-) mouse brains that have high densities of D1 binding in wildtype mice (e.g., the striatum, nucleus accumbens, olfactory tubercles or amygdala). Conversely, small, but detectable amounts of D1-binding were measured in the hippocampus, albeit with a density less than the lowest standard (ca. 20 fmol/mg). Saturation binding of [3H]-SCH23390 in hippocampal homogenates from homozygous mice confirmed a B(max) of 12.3 fmol/mg protein with a K(D) of 0.57 nM. The current work demonstrates directly the presence of D1B(D5) receptors in hippocampus, and also shows that the loss of functional D1A gene products almost completely eliminates detectable D1-binding sites in striatum, as well as in some regions (e.g., the amygdala) where a non-adenylyl cyclase coupled D1 receptor has been reported. This indicates that these non-adenylyl cyclase coupled D1-like receptors represent alternate signaling pathways rather than novel gene products(s).

Distribution of angiotensin IV binding sites (AT 4 receptor) in the human forebrain, midbrain and pons as visualised by in vitro receptor autoradiography

Angiotensin IV and other AT 4 receptor agonists, improve memory retention and retrieval in the passive avoidance and swim maze learning paradigms. Angiotensin IV binding sites (also known as the AT 4 receptors) are widely distributed in guinea pig and monkey ( Macaca fascicularis) brains where high densities of the binding sites have been detected in the hippocampus, neocortex and motor nuclei. However, the distribution of the binding sites in the human brain is not known. We have recently localised the angiotensin IV binding sites (AT 4 receptors) in post-mortem human brain using iodinated Nle-angiotensin IV, a higher affinity and more stable analogue of angiotensin IV. This radioligand bound with relatively high affinity and specificity to angiotensin IV binding sites. In competition studies on consecutive sections through the prefrontal cortex and claustrum, angiotensin IV, Nle-angiotensin IV and LVV-hemorphin 7 competed for the binding of 125I[Nle]-angiotensin IV with nanomolar affinities. Angiotensin II and the AT 1 and AT 2 receptor antagonists were ineffective in competing for the binding at concentrations of up to 10 μM. We found high densities of 125I[Nle]-angiotensin IV binding sites throughout the cerebral cortex including the insular, entorhinal, prefrontal and cingulate cortices. Very high densities of the binding sites were observed in the claustrum, choroid plexus, hippocampus and pontine nucleus. Some thalamic nuclei displayed high densities of binding including the anteroprincipal, ventroanterior, anteromedial, medial dorsal and ventrolateral nuclei. The caudate nucleus, putamen, many amygdaloid nuclei and the red nucleus all displayed moderate densities of binding with a higher level detected in the substantia nigra pars compacta. In the hypothalamus, high densities binding sites were found in the ventromedial nucleus with lower levels in the dorsomedial and paraventricular nuclei. The distribution of 125I[Nle]-angiotensin IV binding sites in the human brain is similar to that found in other species and supports multiple roles for the binding sites in the central nervous system, including facilitation of memory retention and retrieval.

Dopamine receptor immunohistochemistry in the rat choroid plexus.

1. Earlier studies have demonstrated a high density of dopamine D1-like receptor binding in the choroid plexus by light microscope autoradiography, but the dopaminergic specificity of this binding was questioned. 2. In this study the localization of dopamine receptor subtypes was investigated in the rat choroid plexus by Western blot analysis and immunohistochemistry using antibodies raised against dopamine D1-D5 receptor protein. 3. Western blot analysis revealed reactivity with immune bands of approximately 50 and 51 KDa corresponding to dopamine D1 and D5 receptors, respectively. Dopamine D1-like (D1 and D5) receptor protein immunoreactivity insensitive to superior cervical ganglionectomy was located in smooth muscle of choroid arteries and to a larger extent within choroid plexus epithelium. 4. Western blot analysis revealed reactivity with immune bands of approximately 53 KDa and 40-42 KDa corresponding to dopamine D2 and D4 receptors, respectively, and no dopamine D3 receptor reactivity. Dopamine D2-like receptor protein immunoreactivity displayed a distribution similar to that of tyrosine-hydroxylase (TH)-immunoreactive sympathetic fibres and disappeared after superior cervical ganglionectomy. It consisted in the expression of dopamine D2 and to a lesser extent of D4 receptor protein immunoreactivity perivascularly and associated with choroid epithelium. No D3 receptor protein immunoreactivity was found in rat choroid plexus. 5. The above results indicate that rat choroid plexus expresses dopamine receptor protein, being dopamine D1-like receptors predominant in epithelium and arterial smooth muscle and D2-like receptors in sympathetic nerve fibres supplying choroid plexus epithelium and vasculature. 6. These findings suggests that dopamine receptors with a different anatomical localization may modulate production of cerebrospinal fluid.

Increased high-density lipoprotein-3 binding to leukocytes following weight loss and improved glycemic control in type 2 diabetic patients

This study evaluates the effect on high-density lipoprotein (HDL) binding activity in cultured granulocytes before and after metabolic control of non-insulin-dependent diabetes mellitus ([NIDDM] type 2 diabetes) patients. In 20 type 2 diabetic patients, diabetic control was accomplished by administration of oral antidiabetic agents and dietary restrictions. Adequate metabolic control was reflected by a decrease in the fasting glucose, glycosylated hemoglobin (HbA1c), mean insulin, and body mass index (BMI). After control of the diabetes, the mean HDL3 cholesterol was increased from 0.918 +/- 0.05 to 1.008 +/- 0.05 mmol/L (P < .05) and apolipoprotein AI (apo AI) was increased from 103 +/- 5.8 to 115 +/- 5.1 mg/dL (P < .01). The HDL3 maximum specific binding was higher after versus before diabetic control, 77 +/- 6 versus 122 +/- 8 ng/mg cell protein (P < .01). This increase was related to an increase in maximum binding ([Bmax] from 4.97 x 10(-10) to 8.3 x 10(-10) mol/L, P < .001), and no significant changes were observed in the Kd (from 1.47 x 10(-7) v 2.04 x 10(-7) mol/L). These results suggest that the metabolic control of type 2 diabetes increases HDL3 binding activity.

Quantitative autoradiographic distribution of γ-hydroxybutyric acid binding sites in human and monkey brain

γ-Hydroxybutyric acid (GHB), a naturally occurring metabolite of GABA, is present in micromolar concentrations in various areas of the mammalian brain. Specific GHB binding sites, uptake system, synthetic and metabolizing enzymes have been identified in CNS. The present study shows the anatomical distribution of GHB binding sites in sections of primate (squirrel monkey) and human brain by radioligand quantitative autoradiography. In both species the highest densities of binding sites were found in the hippocampus, high to moderate densities in cortical areas (frontal, temporal, insular, cingulate and entorhinal) and low densities in the striatum; no binding sites were detected in the cerebellum. High density of GHB binding was found in the monkey amygdala. In addition the binding characteristics of [ 3H]GHB to membrane preparations of human brain cortex were examined. Scatchard analysis and saturation curves revealed both a high ( K d1 92±4.4 nM; B max1 1027±110 fmol/mg protein) and a low-affinity binding site ( K d2 916±42 nM; B max2 8770±159 fmol/mg protein). The present study is the first report on the autoradiographic distribution of specific GHB binding sites in the primate and human brain: such distribution is in both species in good agreement with the distribution found in the rat brain.

Lactose-containing starburst dendrimers: influence of dendrimer generation and binding-site orientation of receptors (plant/animal lectins and immunoglobulins) on binding properties.

Starburst glycodendrimers offer the potential to serve as high-affinity ligands for clinically relevant sugar receptors. In order to define areas of application, their binding behavior towards sugar receptors with differential binding-site orientation but identical monosaccharide specificity must be evaluated. Using poly(amidoamine) starburst dendrimers of five generations, which contain the p-isothiocyanato derivative of p-aminophenyl-beta-D-lactoside as ligand group, four different types of galactoside-binding proteins were chosen for this purpose, i.e., the (AB)(2)-toxic agglutinin from mistletoe, a human immunoglobulin G fraction, the homodimeric galectin-1 with its two binding sites at opposite ends of the jelly-roll-motif-harboring protein and monomeric galectin-3. Direct solid-phase assays with surface-immobilized glycodendrimers resulted in obvious affinity enhancements by progressive core branching for the plant agglutinin and less pronounced for the antibody and galectin-1. High density of binding of galectin-3 with modest affinity increases only from the level of the 32-mer onwards points to favorable protein-protein interactions of the monomeric lectin and a spherical display of the end groups without a major share of backfolding. When the inhibitory potency of these probes was evaluated as competitor of receptor binding to an immobilized neoglycoprotein or to asialofetuin, a marked selectivity was detected. The 32- and 64-mers were second to none as inhibitors for the plant agglutinin against both ligand-exposing matrices and for galectin-1 on the matrix with a heterogeneous array of interglycoside distances even on the per-sugar basis. In contrast, a neoglycoprotein with the same end group was superior in the case of the antibody and, less pronounced, monomeric galectin-3. Intimate details of topological binding-site presentation and the ligand display on different generations of core assembly are major operative factors which determine the potential of dendrimers for applications as lectin-targeting device, as attested by these observations.

Regional difference in correlation of 5-HT 4 receptor distribution with cholinergic transmission in the guinea pig stomach

Localization and function of 5-HT 4 receptors in the stomach were examined in mucosa-free preparations of antrum, corpus and fundus from guinea pig stomach by determination of acetylcholine release and in vitro receptor autoradiography. Specific [ 125 I ]SB207710, (1- n-butyl-4-piperidinyl) methyl-8-amino-7-iodo-1,4-benzodioxane-5-carboxylate, binding sites were detected in 3 regions of the stomach. High densities of binding were observed in the myenteric plexus of antrum and corpus, but not fundus. In mucosa-free preparations treated with 5-HT 1, 5-HT 2 and 5-HT 3 receptor antagonists, 5-HT (10 −8–10 −6 M) potentiated the electrically stimulated (0.5 Hz, 1 ms) outflow of [ 3 H ]acetylcholine from antrum and corpus strips preloaded with [ 3 H ]choline, but not from fundus strips, and the potentiation was antagonized by SB204070, (1- n-butyl-4-piperidinyl) methyl-8-amino-7-chloro-1,4-benzodioxane-5-carboxylate. Thus, 5-HT 4 receptors are located on myenteric cholinergic neurons in the antrum and corpus of guinea pig stomach and their activation evokes the release of acetylcholine.

Characterization of (2S,2'R,3'R)-2-(2',3'-[3H]-dicarboxycyclopropyl)glycine binding in rat brain.

[(2S,2'R,3'R)-2-(2',3'-[3H]Dicarboxycyclopropyl)glycine ([3H]DCG IV) binding was characterized in vitro in rat brain cortex homogenates and rat brain sections. In cortex homogenates, the binding was saturable and the saturation isotherm indicated the presence of a single binding site with a K(D) value of 180 +/- 33 nM and a Bmax of 780 +/- 70 fmol/mg of protein. The nonspecific binding, measured using 100 microM LY354740, was <30%. NMDA, AMPA, kainate, L(-)-threo-3-hydroxyaspartic acid, and (S)-3,5-dihydroxyphenylglycine were all inactive in [3H]DCG IV binding up to 1 mM. However, several compounds inhibited [3H]DCG IV binding in a concentration-dependent manner with the following rank order of potency: LY341495 = LY354740 > DCG IV = (2S,1'S,2'S)-2-(2-carboxycyclopropyl)glycine > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid > (2S,1'S,2'S)-2-methyl-2-(2-carboxycyclopropyl)glycine > L-glutamate = ibotenate > quisqualate > (RS)-alpha-methyl-4-phosphonophenylglycine = L(+)-2-amino-3-phosphonopropionic acid > (S)-alpha-methyl-4-carboxyphenylglycine > (2S)-alpha-ethylglutamic acid > L(+)-2-amino-4-phosphonobutyric acid. N-Acetyl-L-aspartyl-L-glutamic acid inhibited the binding in a biphasic manner with an IC50 of 0.2 microM for the high-affinity component. The binding was also affected by GTPgammaS, reducing agents, and CdCl2. In parasagittal sections of rat brain, a high density of specific binding was observed in the accessory olfactory bulb, cortical regions (layers 1, 3, and 4 > 2, 5, and 6), caudate putamen, molecular layers of the hippocampus and dentate gyrus, subiculum, presubiculum, retrosplenial cortex, anteroventral thalamic nuclei, and cerebellar granular layer, reflecting its preferential (perhaps not exclusive) affinity for pre- and postsynaptic metabotropic glutamate mGlu2 receptors. Thus, the pharmacology, tissue distribution, and sensitivity to GTPgammaS show that [3H]DCG IV binding is probably to group II metabotropic glutamate receptors in rat brain.

[formula omitted]]Rilmenidine-labelled imidazoline-receptor binding sites co-localize with [ [formula omitted]]2-(benzofuranyl)-2-imidazoline-labelled imidazoline-receptor binding sites and monoamine oxidase-B in rabbit, but not rat, kidney

The distribution and relative densities of imidazoline-receptor binding sites (I-RBS) and monoamine oxidase (MAO)-A and -B enzyme(s) in rat and rabbit kidney were compared autoradiographically using fixed nanomolar concentrations of [ 3 H ]rilmenidine and [ 3 H ]2-(benzofuranyl)-2-imidazoline ([ 3 H ]2-BFI) to label I-RBS, and [ 3 H ]RO41-1049 and [ 3 H ]RO19-6327 to label MAO-A and -B isoenzymes, respectively. In rat kidney, high densities of I-RBS labelled by [ 3 H ]rilmenidine were observed in the cortex and outer stripe (120–280 fmol/mg tissue), in contrast to low I-RBS densities labelled by [ 3 H ]2-BFI (<4 fmol/mg). A relatively high density of [ 3 H ]RO41-1049 binding to MAO-A enzyme was present in all regions of the rat kidney (160–210 fmol/mg) compared with a low density of [ 3 H ]RO19-6327 binding to MAO-B (<25 fmol/mg). Comparison of MAO-A and -B distributions with that of [ 3 H ]rilmenidine-labelled I-RBS strongly suggests a lack of association in rat kidney. Similarly, the extremely low densities of [ 3 H ]2-BFI-labelled I 2-RBS in rat kidney contrasts with the density of MAO-A, but is consistent with the low density of MAO-B. Rabbit kidney cortex and outer stripe contained high relative densities of [ 3 H ]rilmenidine-labelled I-RBS (200–215 fmol/mg) and [ 3 H ]2-BFI-labelled I 2-RBS (45–60 fmol/mg) with lower densities in the inner stripe and inner medulla (⩽100 and 30 fmol/mg respectively). A high density of MAO-A binding was observed in the inner stripe (515 fmol/mg) with lower levels in the cortex and outer stripe (100–240 fmol/mg), while high densities of MAO-B binding were observed in the cortex and outer stripe (290–450 fmol/mg) with lower levels in the inner stripe (65 fmol/mg). The correlation between the localization of [ 3 H ]rilmenidine-labelled I-RBS and [ 3 H ]RO19-6327-labelled MAO-B in rabbit kidney ( r=0.87, P=0.057) suggest that [ 3 H ]rilmenidine may label a binding site co-existent with MAO-B, but not MAO-A (n.s.), in this tissue, but rilmenidine did not inhibit [ 3 H ]RO41-1049 or [ 3 H ]RO19-6327 binding. The distribution of [ 3 H ]2-BFI-labelled I 2-RBS overlapped the combined distributions of both MAO-A and -B isoenzymes, suggesting that [ 3 H ]2-BFI may label sites on both enzymes in the rabbit, but [ 3 H ]2-BFI binding only correlated with [ 3 H ]RO19-6327 ( r=0.84, P=0.07), not [ 3 H ]RO41-1049 binding (n.s.). Moreover, 2-BFI only inhibited [ 3 H ]RO19-6327, not [ 3 H ]RO41-1049 binding. These data are consistent with reports that I 2-RBS are located on MAO-B and allosterically influence the catalytic site. The relationship of [ 3 H ]rilmenidine- and [ 3 H ]2-BFI-labelled I-RBS and the identity of non-MAO-associated [ 3 H ]rilmenidine-labelled I-RBS requires further investigation.

Characterization of the 5-HT4 binding site in human brain.

In this study, drug inhibition and saturation experiments on the binding of the highly selective 5-HT4 antagonist [3H]GR 113808 were performed in human brain membranes so as to better characterize this binding site. Drug competition studies were carried out by incubating 0.2 nM [3H]GR 113808 in the presence of increasing concentrations of six different drugs, i.e. 5-HT, 5-CT, ondansetron, tropisetron, BIMU 1 and BIMU 8 (mixed 5-HT3 and 5-HT4 agonists). The binding displaced by 5-HT showed a drug inhibition constant (Ki) value of 197nM. The use of 5-CT or ondansetron also showed the existence of single-site models albeit with Ki values in the micromolar range (11,5 microM). Tropisetron, BIMU 1 and BIMU 8 displaced bound [3H]GR 113808 according to a two-site binding model, with the high affinity component in the nanomolar range and the low affinity site in the micro or milimolar range. Saturation experiments revealed high binding densities in basal ganglia (187 fmol/mg in putamen, and 149 fmol/mg in caudate nucleus), while lower densities were observed in cortical regions (49 fmol/mg in temporal cortex, 45 fmol/mg in parietal cortex and 71 fmol/mg in cingulate cortex). The apparent affinity (Kd) was similar in the brain regions studied, ranging from 0.13 to 0.34 nmol/l. Despite the enrichment of 5-HT receptors in human brain, their functional correlate in brain diseases remains to be clarified.

Myocardial fibrosis associated with aldosterone or angiotensin II administration: attenuation by calcium channel blockade.

Chronic administration of either angiotensin II (Ang II) or aldosterone (ALDO) leads to myocardial fibrosis. Myofibroblasts (myoFb) play a major role in collagen accumulation at sites of tissue repair. Pathophysiologic bases of cardiac fibrosis in such chronic primary or secondary hyperaldosteronism are under investigation. In vitro studies have shown that Ang II and ALDO each increase intracellular calcium and this second messenger is involved in altered fibroblast collagen turnover and growth. In the present study, we tested our hypothesis that calcium channel blockade would attenuate myocardial fibrosis that accompanies administration of either circulating Ang II or ALDO. Five animal groups were studied: (1) untreated age- and sex-matched control rats; (2) intact rats receiving Ang II (75 ng/min) for 2 weeks; (3) rats receiving Ang II plus mibefradil (30 mg/kg/day p.o.), a calcium channel blocker, for 2 weeks; (4) uninephrectomized rats receiving ALDO (0.75 microgram/h) together with a high salt diet for 6 weeks; and (5) uninephrectomized rats receiving ALDO and high salt diet plus mibefradil. Myocardial fibrosis was assessed by hydroxyproline concentration and interstitial and perivascular collagen volume fraction examined by videodensitometry on heart sections stained with collagen-specific picrosirius red. MyoFb were identified by immunohistochemical alpha-smooth muscle actin (SMA) labeling. ACE binding was determined by in vitro quantitative autoradiography. Compared to controls, in rats receiving either Ang II or ALDO we found: (1) myocardial fibrosis, expressed as microscopic scars, and perivascular fibrosis in both right and left ventricles with increased (P<0.05) hydroxyproline concentration and collagen volume fraction; (2) myoFb at sites of fibrosis, where high ACE binding density was also present; and (3) hydroxyproline concentration and collagen volume fraction were significantly (P<0.05) attenuated and the extent of alpha-SMA labeling and ACE binding density were each markedly (P<0.01) reduced in rats receiving either hormone plus mibefradil. This study therefore suggests calcium may modulate fibrous tissue formation in rat models of hyperaldosteronism by altering MyoFb collagen turnover and cell growth. It further is our contention that these findings implicate calcium as a signal used by effector hormones of the RAAS to promote tissue repair and that calcium channel blockade may offer advantage as a cardioprotective strategy in this setting.

In VitroBinding Characteristics of a New Selective Group II Metabotropic Glutamate Receptor Radioligand, [3H]LY354740, in Rat Brain

The in vitro binding of [3H]LY354740, the first high affinity group II-selective metabotropic glutamate (mGlu) receptor radioligand, was characterized in rat cortical, hippocampal, and thalamic membranes as well as in rat brain sections. [3H]LY354740 binding was saturable in all regions investigated. Nonspecific binding (in the presence of 10 microM DCG-IV) was approximately 8% of the total. Ionotropic glutamate receptor agonists, N-methyl-D-aspartate, (R,S)-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid/kainate, a Na+-dependent glutamate uptake blocker as well as a group I-selective mGlu receptor agonist (all up to 1 mM) did not inhibit [3H]LY354740 binding to cortical membranes. However, several known metabotropic receptor ligands inhibited the binding with the following rank order of potency: LY354740 = LY341495 > (2S,2'R, 3'R)-2-(2',3'-dicarboxycyclopropyl)glycine = (2S,1'S, 2'S)-2-(2-carboxycyclopropyl)glycine > glutamate = (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid > (2S,1'S, 2'S)-2-methyl-2-(2-carboxycyclopropyl)-glycine > quisqualate > ibotenate > L-2-amino-3-phosphonopropionic acid = (S)-alpha-methyl-4-carboxyphenylglycine > L-(+)-2-amino-4-phosphonobutyric acid. N-Acetyl-aspartyl-glutamate, (2S)-alpha-ethylglutamic acid, and (R, S)-alpha-methyl-4-phosphonophenylglycine inhibited [3H]LY354740 binding in a biphasic manner. Guanosine-5'-O-(3-thiotriphosphate concentration-dependently and almost completely inhibited the binding. Finally, in parasagittal sections of rat brain, a high density of specific binding was observed in the accessory olfactory bulb, cortical regions (layers 1-3 > 4-6), caudate putamen, molecular layers of the hippocampus and dentate gyrus, presubiculum, retrosplenial cortex, anteroventral thalamic nuclei, and cerebellar granular layer, reflecting its preferential (perhaps not exclusive) affinity for presynaptic and postsynaptic mGlu2 receptors. Thus, the pharmacology, tissue distribution, and sensitivity to guanosine-5'-O-(3-thiotriphosphate show that [3H]LY354740 binding probably occurs to group II mGlu receptors in rat brain.