[3H]5,7-Dichlorokynurenic Acid Recognizes two Binding Sites in Rat Cerebral Cortex Membranes

Abstract

Binding of [³H]5,7-dichlorokynurenic acid ([³H]DCKA), a competitive antagonist of the strychnine-insensitive glycine site of the N-methyl-D-aspartate (NMDA) receptor channel complex, was characterized in synaptic plasma membranes from rat cerebral cortex. Non linear curve fitting of [³H]DCKA saturation and homologous displacement isotherms indicated the existence of two binding sites: a specific, saturable, high affinity site, with a pK_D value of 7.24 (K_D = 57.5 nmol/1) and a maximum binding value (B_max) of 6.9 pmol/mg of protein and a second site, with micromolar affinity. The pharmacological profile of both binding components was determined by studying the effect on [³H]DCKA and [³H]glycine binding of a series of compounds known to interact with different excitatory and inhibitory amino acid receptors. These studies confirmed the identity of the high affinity site of [³H]DCKA binding with the strychnine-insensitive glycine site of the NMDA receptor channel complex. 3-[2-(Phenylaminocarbonyl)ethenyl]-4,6-dichloroindole-2-carboxylic acid sodium salt (GV 150526A), a new, high affinity, selective glycine site antagonist (1), was the most potent inhibitor of this component of binding (pK_i = 8.24, K_i = 5.6 nmol/1). The low affinity component of [³H]DCKA binding was insensitive to the agonists glycine and D-serine and the partial agonist (±)-3-amino-1-hydroxy-2-pyrrolidone (HA 966), though recognised by glycine site antagonists. The precise nature of this second, low affinity [³H]DCKA binding site remains to be elucidated.     

Characterization of binding of [3H]PD 128907, A selective Dopamine D3 receptor agonist ligand,to CHO-K1 cells

PD 128907 [4a R, 10 b R-(+)-trans- 3, 4, 4a, 10 b - tetrahydro - 4- n-propy12 H,5H-[1] benzopyrano[4,3-b]1,4-oxazin-9-ol.], a selective dopamine (DA) D3 receptor agonist ligand exhibits about a 1000-fold selectivity for human D3 receptors (K_i, 1 nM) versus human D₂ receptors (K_i, 1183 nM) and a 10000-fold selectivity versus human D₄ receptors (K_i, 7000 nM) using [³H]spiperone as the radioligand in CHO-K1-cells. Studies with [³H]PD 128907, showed saturable, high affinity binding to human D₃ receptors expressed in CHO-K1 cells (CHO-K1-D₃) with an equilibrium dissociation constant (K_d) of 0.99 nM and a binding density (B_max) of 475 fmol/mg protein. Under the same conditions, there was no significant specific binding in CHO-K1-cells expressing human D₂ receptors (CHO-K1-D₂). The rank order of potency for inhibition of [³H]PD 128907 binding with reference DA agents was consistent with reported values for D₃ receptors. These results indicate that [³H]PD 128907 is a new, highly selective D₃ receptor ligand with high specific activity, high specific binding and low non-specific binding and therefore should be useful for further characterizing the DA D₃ receptors.     

Labelling of 5-Hydroxytryptamine3 Receptors with a Novel 5-HT3 Receptor Ligand, [3H]RS-42358–197

Abstract: RS-42358–197{(S)-N-(1-azabicyclo[2.2.2]oct-3-yl)-2,4,5,6-tetrahydro-1H-benzo[de]isoquinolin-1-one hydrochloride} displaced the prototypic 5-hydroxytryptamine₃ (5-HT₃) receptor ligand [³H]quipazine in rat cerebral cortical membranes with an affinity (pK_i) of 9.8 ± 0.1, while having weak affinity (pK_i < 6.0) in 23 other receptor binding assays. [³H]RS-42358–197 was then utilized to label 5-HT₃ receptors in a variety of tissues. [³H]RS-42358–197 labelled high-affinity and saturable binding sites in membranes from rat cortex, NG108–15 cells, and rabbit ileal myenteric plexus with affinities (K_D) of 0.12 ± 0.01, 0.20 ± 0.01, and 0.10 ± 0.01 nM and densities (B_max) of 16.0 ± 2.0, 660 ± 74, and 88 ± 12 fmol/mg of protein, respectively. The density of sites labelled in each of these tissues with [³H]RS-42358–197 was similar to that labelled with [³H]GR 65630, but was significantly less than that found with [³H]-quipazine. The binding of [³H]RS-42358–197 had a pharmacological profile similar to that of [³H]quipazine, as indicated by the rank order of displacement potencies: RS-42358–197 > (S)-zacopride > tropisetron > (R)-zacopride > ondansetron > MDL72222 > 5-HT. However, differences in 5-HT₃ receptors of different tissues and species were detected on the basis of statistically significant differences in the affinities of phenylbiguanide, and 1-(m-chlorophenyl)biguanide when displacing [³H]RS-42358-197 binding. [³H]RS-42358–197 also labelled a population (B_max= 91 ± 17 fmol/mg of protein) of binding sites in guinea pig myenteric plexus membranes, with lower affinity (K_D= 1.6 ± 0.3 nM) than those in the other preparations. Moreover, the rank order of displacement potencies of 15 5-HT₃ receptor ligands in guinea pig ileum was found not to be identical to that in other tissues. Binding studies carried out with [³H]RS-42358–197 have detected differences in 5-HT₃ receptor binding sites in tissues of different species and further underscore the unique nature of the guinea pig 5-HT₃ receptor.     

Identification of α2 adrenergic receptors in human frontal cortex membranes by binding of [H]RX 821002, the 2-methoxy analog of [H]idazoxan

The antagonist [³H]idazoxan binds with comparable affinity to α₂ adrenergic receptors and to phentolamine-displaceable non-stereoselective sites in human frontal cortex membranes. In contrast, idazoxan analogs possessing alkyl and alkoxy substituents at the 2-position of the benzodioxan moiety (i.e. RX 821002: 2-methoxy-1,4-[6,7-³H]benzodioxan-2-yl-2-imidazolin HCl, 43.8 Ci/mmol) possess 300–1200 times lower affinity for the non-stereoselective sites. Their affinity for the α₂ receptors is increased as well, resulting in more than a 1000-fold selectivity towards the receptors as compared to the non-stereoselective sites. [³H]RX 821002, the 2-methoxy analog of idazoxan possesses an approx. 10-fold higher affinity for the α₂ receptors (K_D = 2.8 nM than [³H]idazoxan (K_D = 24 nM) and about equal affinity as [³H]rauwolscine (K_D = 3.6 nM).[³H]Rauwolscine binds with comparable affinity to α₂ receptors and to 5-HT_1A receptors, and competition studies indicate that the K_i value of unlabelled RX 821002 for the 5-HT_1A receptors (30 nM) is about one order in magnitude above its K_i value for the α₂ receptors (4.1 nM). Labelling of the 5-HT_1A receptors by [³H]RX 821002 and by [³H]rauwolscine can be prevented by selective masking with 8-OH-DPAT (30 nM) or 5-HT (0.3 μM). Under these conditions, specific binding of [³H]RX 821002 to the α₂ receptors represents 84% of total binding (at its K_D), as compared to 77% for [³H]rauwolscine and 20% for [³H]idazoxan.[³H]RX 821002 labels the α₂ receptors as a single class of non-cooperative sites. Association and dissociation kinetics are very fast at 37°C. Antagonist competition curves are steep with Hill coefficients close to one and the agonist curves can be analysed in terms of two affinity sites, confirming the antagonistic properties of [³H]RX821002. About 60% of the α₂ receptors possess high agonist affinity.     

Differential Effects of Cocaine-Induced Seizures and Lethality on M1-Like Muscarinic and Dopaminergic D1- and D2-Like Binding Receptors in Mice Brain

Summary This work was designed to study the changes produced by cocaine-induced seizures and lethality on dopaminergic D₁- and D₂-like receptors, muscarinic M₁-like binding sites, as well as acetylcholinesterase activity in mice prefrontal cortex (PFC) and striatum (ST). Binding assays were performed in brain homogenates from the PFC and ST and ligands used were [³H]-N-methylscopolamine, [³H]-NMS (in the presence of carbachol), [³H]-SCH 23390 and [³H]-spiroperidol (in presence of mianserin), for muscarinic (M₁-like), D₁- and D₂-like receptors, respectively. Brain acetylcholinesterase (AChE) activity was also determined in these brain areas. Cocaine-induced SE decreased [³H]-SCH 23390 binding in both ST and PFC areas. A decrease in [³H]-NMS binding and an increase in [³H]-spiroperidol binding in PFC was also observed. Cocaine-induced lethality increased [³H]-spiroperidol binding in both areas and decreased [³H]-NMS binding only in PFC, while no difference was seen in [³H]-SCH 23390 binding. Neither SE, nor lethality altered [³H]-NMS binding in ST. AChE activity increased after SE in ST while after death the increase occurred in both PFC and ST. In conclusion, cocaine-induced SE and lethality produces differential changes in brain cholinergic and dopaminergic receptors, depending on the brain area studied suggesting an extensive and complex involvement of these with cocaine toxicity in central nervous system.     

Characterization of Muscarinic Receptor Subtypes in Canine Left Ventricular Membranes

Abstract

The pharmacological characteristics of muscarinic receptor (mAChR) subtypes in canine left ventricular membranes (LVM) were determined using [³H]quinuclidinyl benzilate ([³H]QNB) and [³H] N-methyl scopolamine ([³H]NMS) as ligands. Binding of [³H]QNB and [³H]NMS was saturable with respect to the radioligand concentrations. Analysis of binding isotherms by Scatchard plot showed that [³H]QNB and [³H] NMS bound to an apparently homogeneous population of mAChRs in LVM, with K_D values of 390 ± 100 and 285 ± 34 pM and B_max values of 240 ± 20 and 133 ± 9 fmol/mg protein, (n=6), respectively. The Hill coefficients for [³H]QNB and [³H]NMS binding were 0.95 ± 0.02 and 0.99 ± 0.01, respectively. Based on the competitive inhibition of [³H] ligand binding, atropine and NMS as well as the selective M₁ antagonist PZ revealed no selectivity for these mAChRs. PZ competed with [³H]QNB or [³H]NMS for a single binding site with a K_i value of 0.23 ± 0.03 μM and 0.62 ± 0.10 μM, (n = 6), respectively, which is close to the values of M² or M³ receptors. The data indicate that the M¹ receptor subtype did not exist in canine LVM. Competition of [³H] ligand binding with selective M₂ antagonists, AF-DX 116 and methoctramine and the selective M₃ antagonists, 4-DAMP and hexahydrosiladifenidol, gave a best fit for a two-binding site model. The inhibition of carbachol-mediated phosphoinositide hydrolysis by PZ, AF-DX 116 and 4-DAMP, generated an affinity profile for this response also dissimilar to that described for the classical cardiac M₂ response. Although no other muscarinic receptor mRNA has been detected in this tissue, these data suggest the presence of a second population of muscarinic sites, which may signify an M₂ receptor diversity.     

Properties of [3H]diazepam binding sites on rat blood platelets

Intact rat blood platelets are shown to possess benzodiazepine binding sites of the peripheral type, binding of [³H]diazepam being strongly inhibited by Ro5-4864 (K_i = 3.6 ± 0.5 nM) but only weakly inhibited by clonazepam (K_i = 35.1 ± 18.2 μM). Binding of [³H]diazepam is specific and saturable. Scatchard analysis reveals a single class of binding sites with K_D = 14.7 ± 1.0 nM and B_max = 564 ± 75 fmoles/10⁸ platelets. The Hill coefficient is 0.94, indicating a lack of binding site heterogeneity or negative cooperativity. Binding reaches equiliibrium at 6 min, with k₊₁ = 2.9 × 10⁷ M^?1 min^?1, and is rapidly reversible ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext><mtext>\; =\; 2.2\; </mtext><mtext>min</mtext>}}$ with K_?1 = 0.315 min^?1. K_D derived from the rate constants agrees with that estimated by Scatchard analysis. K_D of the crude membrane fraction of platelets is also close to that of intact platelets. Binding of [³H]diazepam is linear with platelet number (between 0.25–2 × 10⁸ platelets), is temperature sensitive with maximum binding at 0°C, and has a broad optimal pH range between pH 5–9.     

Solubilization of High-Affinity,Guanine Nucleotide-Sensitive μ-Opioid Receptors from Rat Brain Membranes

Abstract: High-affinity μ-opioid receptors have been solubilized from rat brain membranes. In most experiments, rats were treated for 14 days with naltrexone to increase the density of opioid receptors in brain membranes. Occupancy of the membrane-associated receptors with morphine during solubilization in the detergent 3-[(3-cholamidopropyl)dimethyl]-1-propane sulfonate appeared to stabilize the μ-opioid receptor. After removal of free morphine by Sephadex G50 chromatography and adjustment of the 3-[(3-cholamidopropyl)dimethyl]-1-propane sulfonate concentration to 3 mM, the solubilized opioid receptor bound [³H][d -Ala²,N-Me-Phe⁴,Gly-ol⁵]-enkephalin ([³H]DAMGO), a μ-selective opioid agonist, with high affinity (K_D = 1.90 ± 0.93 nM; B_max = 629 ± 162 fmol/mg of protein). Of the membrane-associated [³H]-DAMGO binding sites, 29 ± 7% were recovered in the solubilized fraction. Specific [³H]DAMGO binding was completely abolished in the presence of 10 µM guanosine 5′-O-(3-thiotriphosphate). The solubilized receptor also bound [³H]diprenorphine, a nonselective opioid antagonist, with high affinity (K_D = 1.4 ± 0.39 nM, B_max = 920 ± 154 fmol/mg of protein). Guanosine 5′-O-(3-thiotriphosphate) did not diminish [³H]diprenorphine binding. DAMGO at concentrations between 1 nM and 1 µM competed with [³H]diprenorphine for the solubilized binding sites; in contrast, [d -Pen²,d -Pen⁵]-enkephalin, a δ-selective opioid agonist, and U50488H, a κ-selective opioid agonist, failed to compete with [³H]diprenorphine for the solubilized binding sites at concentrations of <1 µM. In the absence of guanine nucleotides, the DAMGO displacement curve for [³H]diprenorphine binding sites better fit a two-site than a one-site model with K_Dhigh = 2.17 ± 1.5 nM, B_max = 648 ± 110 fmol/mg of protein and K_Dlow = 468 ± 63 nM, B_max = 253 ± 84 fmol/mg of protein. In the presence of 10 µM guanosine 5′-O-(3-thiotriphosphate), the DAMGO displacement curve better fit a one- than a two-site model with K_D = 815 ± 33 nM, B_max = 965 ± 124 fmol/mg of protein.     

Identification of stereospecific [3H]spiroperidol binding sites in mammalian lymphocytes

Direct binding to intact rat lymphocytes has been shown for the potent dopaminergic antagonist [³H]spiroperidol. The specific binding is saturable with two components (K_D1 = 1.9 nM, K_D2 = 36.2 nM). Determination of the K_D by kinetic studies measuring rate constants for association and dissociation provided K_D values similar to those obtained in equilibrium experiments. The specific binding is proportional to cell concentration and temperature dependent with a maximum at 37°C. [³H]spiroperidol binding is stereospecific since (+)butaclamol was more effective than (?)butaclamol. The relative potencies of different antidopaminergic agents in competing for [³H]spiroperidol binding sites parallel their activity in the striatum. Dopaminergic receptors have also been demonstrated in other mammalian lymphocytes (rabbit, dog, human). Lymphocyte dopaminergic receptors could be implicated in lymphocytes mediated immune response.     

Affinities of muscarinic drugs for [3H]N-methylscopolamine (NMS) and [3H]oxotremorine (OXO) binding to a mixture of M1−M4 muscarinic receptors: Use of NMS/OXO-M ratios to group compounds into potential agonist,partial agonist,and antagonist classes

The relative affinities of various muscarinic drugs in the antagonist ([³H]N-methyl scopolamine ([³H]NMS)) and agonist ([³H]Oxotremorine-m ([³H]OXO-M)) binding assays using a mixture of tissues containing M₁–M₄ receptor subtypes have been determined. [³H]NMS bound with high affinity (K_d=25±5.9 pM; n=3) and to a high density (B_max=11.8±0.025 nmol/g wet weight) of muscarinic receptors. [³H]OXO-M appeared to bind to two binding sites with differing affinities (K_d1=2.5±0.1 nM; K_d2=9.0±4.9 M; n=4) and to a different population of binding sites (B_max1=5.0±0.26 nmol/g wet weight; B_max2=130±60 nmol/g wet weight). Well known antagonists exhibited high affinity for [³H]NMS binding but a lower affinity for [³H]OXO-M binding. The opposite was true for acetylcholine and other known agonists. However, pilocarpine and McN-A-343 had similar affinities for sites labeled by both radioligands. Using the ratios of antagonist-to-agonist binding affinities, it was possible to group compounds into apparently distinct full agonist (ratios of 180–665; e.g. carbachol, muscarine, OXO-M, OXO-S and arecoline), partial agonist (ratios of 14–132; e.g. McN-A-343, pilocarpine, aceclidine, bethanechol, OXA-22 and acetylcholine) and antagonist (ratios of 0.22–1.9; e.g. atropine, NMS, pirenzepine, methoctramine, 4-DAMP and p-fluorohexahydrosialo-difenidol) classes. These data suggest that the NMS/OXO-M affinity ratios using a mixture of M₁–M₄ muscarinic receptors may be a useful way to screen and group a large number of compounds into apparent agonist, partial agonist, and antagonist classes of cholinergic agents.     

Behavior of rat hypothalamic and extrahypothalamic immuno-reactive TRF in thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC)

[³H] quinuclidinyl benzilate (QNB), a specific muscarinic antagonist, was utilized to identify muscarinic cholinergic receptors on dispersed anterior pituitary cells. Scatchard analysis of [³H] QNB binding to receptors departs from linearity with upward concavity. A high affinity binding site having a dissociation constant (K_d) of 1.5 nM was observed when the [³H] QNB concentration was varied from 0.15 to 20 nM. A low affinity binding site (K_d 20 nM) was observed when [³H] QNB concentration was above 20 nM. Using 10 nM [³H] QNB for binding, the second order association rate constant (k₁) of 0.064 nM^?1 min^?1 and first order dissociation rate constant (k₂) of 0.078 min^?1$\text{(}\text{T}\text{1}\text{2}\text{8}\text{min}\text{)}$ were observed. k₂/k₁ = K_d of 1.22 nM is in good agreement with K_d = 1.5 nM from equilibrium data. Muscarinic cholinergic receptor antagonists, atropine and scopolamine, and agonist oxtoremorine potently competed with [³H] QNB binding. A nicotinic cholinergic receptor agonist was 50 times less potent as a competitor of [³H] QNB binding than the muscarinic agonist.     

Recombinant Human NMDA Homomeric NR1 Receptors Expressed in Mammalian Cells Form a High-Affinity Glycine Antagonist Binding Site

Abstract: The cDNA NMDAR1 (NR1) encodes a single polypeptide that forms a receptor-channel complex with electrophysiological and pharmacological properties characteristic of the N-methyl-d -aspartate receptor. Homomeric NR1 recombinant receptors expressed in Xenopus oocytes show functional responses with low levels of conductance. In this study we have characterized, by radioligand binding techniques, the pharmacological properties of homomeric receptors of two human NR1 isoforms (NR1a and NR1e, which differ in their C-terminal region), transiently expressed in human embryonic kidney 293 cells. The glycine site antagonist (±)-4-(trans)-2-carboxy-5,7-dichloro-4-[³H]phenylaminocarbonylamino-1,2,3,4-tetrahydroquinoline ([³H]L-689,560) bound to NR1a- and NR1e-transfected cells with high affinity (K_D = 3.29 and 1.61 nM, respectively). B_max values for NR1a- and NR1e-transfected cells were 3.82 and 1.69 pmol/mg of protein, respectively, and Hill coefficients were close to unity. K_i values for glycine site antagonists inhibiting [³H]L-689,560 binding to NR1e-transfected cells were similar to those observed with rat brain membranes. Affinity values for agonists and partial agonists were four- to 16-fold weaker, indicating that the glycine site of homomeric NR1 receptors is in an antagonist-preferring state. K_i values obtained with NR1a-transfected cells were approximately twofold lower than those obtained with NR1e-transfected cells. High-affinity binding to NR1-transfected cells was not observed with the transmitter recognition site radioligands l -[³H]glutamate and d,l -(ε)-2-[³H]amino-4-propyl-5-phosphono-3-pentanoic acid ([³H]CGP-39653) or the ion-channel radioligand [³H]dizocilpine ([³H]MK-801). These results indicate that although transfection of mammalian cells with homomeric NR1 recombinant receptors does not appear to result in functional receptors, a glycine binding site is formed that may have a physiological role if present in vivo.     

Characterization of Muscarinic Acetylcholine Receptors in Cultured Bovine Aortic Endothelial Cells

Abstract

The binding characteristics of [³H]quinuclidinyl benzilate ([³H]QNB) to isolated crude membranes of cultured bovine aortic endothelial cells were investigated. [³H]QNB bound to endothelial cell membranes with high affinity (k_D = 0.056 nM) and limited capacity (132 fmol/mg DNA). The binding specificity, order of affinity and inhibition constants (K_i) were determined by displacement of bound [³H]QNB with unlabeled ligands. The order of affinity was QNB > atropine > 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP) > p-fluoro-hexahydro-sila-difenidol (p-F-HHSiD) (M₃ antagonist) > pirenzepine (M₁ antagonist) > AFDX-116 (M₂ antagonist) > (4-hydroxy-2-butynyl) trimethylammonium chloride m-chlorocarbanilate (McN-A-343, M₁ agonist). These observations suggest that muscarinic receptors of endothelial cells in culture are likely to be of M₃ and M₁ subtype. Northern blot analysis of receptor subtypes using cDNA probes did not provide conclusive results due to the low level expression of these receptors in cultured cells. Solubilization of protein bound [³H]QNB with 1% digitonin and 0.02% cholate followed by analysis on sucrose density gradients demonstrated the presence of a specifically bound [³H]QNB-protein complex sedimenting at the 6.2S region of the gradient. These data demonstrate the presence of muscarinic acetylcholine receptor protein in cultured bovine aortic endothelial cells.     

An [3H]oxotremorine binding method reveals regulatory changes by guanine nucleotides in cholinergic muscarinic receptors of cerebral cortex

A rapid, reliable filtration method for [³H]oxotremorine binding to membranes of the cerebral cortex that allows the direct study of regulation by guanine nucleotides of muscarinic receptors was developed. [³H]Oxotremorine binds to cerebral cortex membranes with high affinity (K _D, 1.9 nM) and low capacity (B _max, 187 pmol/g protein). These sites, which represent only about 18% of those labeled with [³H]quinuclidinyl benzilate, constitute a population of GTP-sensitive binding sites. Association and dissociation binding experiments revealed a similar value ofK _D (2.3 nM). Displacement studies with 1–4000 nM oxotremorine showed the existence of a second binding site of low affinity (K _D, 1.2 M) and large capacity (B _max, 1904 pmol/g protein). Gpp(NH)p, added in vitro, produced a striking inhibition of [³H]oxotremorine binding with an IC 50 of 0.3 M. Saturation assays, in the presence of 0.5 M Gpp(NH)p, revealed a non-competitive inhibition of the binding with little change in affinity. These results are discussed from the viewpoint of conflicting reports in the literature about guanine nucleotide regulation of muscarinic receptors in reconstituted systems and membranes from different tissues.     

Binding characteristics of [3H]flunitrazepam in pentobarbital-withdrawal rats

The effects of chronic pentobarbital (PB) treatment on the binding characteristics of [³H]flunitrazepam (FLU) in rat brain were examined. Saline or sodium PB (500 g/10l/hr) was infused into the lateral cerebral ventricles of rats for 6 days using osmotic pumps. Immediately before withdrawal, there were no significant differences in [³H]FLU binding constants (K_D and B_max) between saline and PB groups. However, 24 hr withdrawal caused an increase in B_max with no changes in K_D. The enhancement of [³H]FLU binding by in vitro addition of chloride ions and PB was not affected after the PB infusion. The PB enhancement of [³H]FLU binding was inhibited by the convulsant, picrotoxicin. PB withdrawal did not cause significant differences in the binding constants of [³H]Ro 15-1788, a benzodiazepine (BZ) antagonist, between the saline and PB groups. Pretreatment of membranes with 0.02 mM of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), a zwitterionic detergent, caused decreases in both K_D and B_max in FLU binding in PB-withdrawal membrane, but not in the saline-treated membrane. The enhancement of [³H]FLU binding by chloride ions and PB was not affected by the CHAPS treatment. These results suggest that the change in BZ receptors induced by PB withdrawal is functionally linked to the GABA-BZ-barbiturate receptor complex and that PB withdrawal induces some conformational changes in BZ receptors.     

[3H] verapamil binding sites in skeletal muscle tranverse tubule membranes

[³H]verapamil binding to muscle tubule membrane has the following properties. K_D = 27 ± 5 nM and maximum binding capacity B_max = 50 ± 5 pmol/mg of protein. A 1 = 1 stoichiometry of binding was found for the ratio of [³H]verapamil versus [³H] nitrendipine binding sites. The dissociation constant found at equilibrium is near that determined from the ratio of the rate constants for association (k₁) and dissociation (k_?1). Antiarrhythmic drugs like D600, diltiazem and bepridil are competitive inhibitors of [³H]verapamil binding with K_D values between 40 and 200 nM. Dihydropyridine analogs are apparent non competitive inhibitors of [³H]verapamil binding with half-maximum inhibition values (K_0.5) between 1 and 5 nM.     

Solubilization of muscarinic acetylcholine receptor by zwitterionic detergent from rat brain cortex

The muscarinic acetylcholine receptor was solubilized from rat brain cortex by zwitterionic detergent 3-[(3-chloramidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS). About 15% of the binding activity was solubilized and 40% of the activity was destroyed by the detergent. Binding of the muscarinic antagonist [³H]-N-methyl-4-piperidyl benzilate (4NMPB) was saturable. Scatchard analysis revealed a single population of binding sites with K_D value of 0.7 nM and a B_max value of 340 fmoles/mg protein. The homogenate and the CHAPS treated pellet and soluble receptors showed similar affinity for the agonists oxotremorine and carbamylcholine and for the antagonists QNB and atropine. The dissociation of 4NMPB from the soluble receptors appears slightly slower than from the membrane bound receptors.     

Characterization and Subcellular Localization of l-[3H]Carnitine Binding Sites in Rat Brain

Abstract: In the present study, we investigated the existence of a binding site for l -carnitine in the rat brain. In crude synaptic membranes, l -[³H]carnitine bound with relatively high affinity (K_D = 281 nM) and in a saturable manner to a finite number (apparent B_max value = 7.3 pmol/mg of protein) of binding sites. Binding was reversible and dependent on protein concentration, pH, ionic strength, and temperature. Kinetic studies revealed a K_off of 0.018 min^?1 and a K_on of 0.187 × 10^?3 min^?1 nM^?1. Binding was highest in spinal cord, followed by medulla oblongata-pons ≥ corpus striatum ≥ cerebellum = cerebral cortex = hippocampus = hypothalamus = olfactory bulb. l -[³H]Carnitine binding was stereoselective for the l -isomers of carnitine, propionylcarnitine, and acetylcarnitine. The most potent inhibitor of l -[³H]carnitine binding was l -carnitine followed by propionyl-l -carnitine. Acetyl-l -carnitine and isobutyryl-l -carnitine showed an affinity ～500-fold lower than that obtained for l -carnitine. The precursor γ-butyrobetaine had negligible activity at 0.1 mM. l -Carnitine binding to rat crude synaptic membrane preparation was not inhibited by neurotransmitters (GABA, glycine, glutamate, aspartate, acetylcholine, dopamine, norepinephrine, epinephrine, 5-hydroxytryptamine, histamine) at a final concentration of 0.1 mM. In addition, the binding of these neuroactive compounds to their receptors was not influenced by the presence of 0.1 mMl -carnitine. Finally, a subcellular fractionation study showed that synaptic vesicles contained the highest density of l -carnitine membrane binding sites whereas l -carnitine palmitoyltransferase activity was undetectable, thus excluding the possibility of the presence of an active site for carnitine palmitoyltransferase. This finding indicated that the localization of the l -[³H]carnitine binding site should be essentially presynaptic.     

[3H]N-Methylscopolamine binding to heart atrium and four brain regions from the mallard (Anas platyrhynchos)

1. Binding characteristics of membrane preparations from four brain regions and heart (HT) right artria from mallard ducks were documented using the muscarinic ligand [³H]N-methylscopolamine([³H]NMS).2. Brain regions used were: cerebellum (CL), medulla/pons (MP), corpus striatum (CS), and hippocampus (HI). Cholinesterase (ChE) activity from these same tissues was also measured.3. High affinity binding sites were identified in all tissue preparations, and were shown to be saturable and linear. Maximal number of [³H]NMS binding sites (Bmax) (fmoles [³H]NMS/mg protein) were: CL = 90.6, MP = 104.1, HI = 276.0, CS = 229.3, HT = 65.52. Dissociation constants (K_D) (nM [³H]NMS) for the same tissues were: CL = 0.204, MP = 0.293, HI = 0.347, CS = 0.163, HT = 0.447.     

Decrease or increase in cardiac muscarinic cholinergic receptor number in rats treated with methacholine or atropine

The effects of chronic treatment of the rat with methacholine and atropine on the cardiac muscarinic cholinergic receptors were investigated. [³H]Quinuclidinyl benzilate ([³H]QNB) was used to directly estimate the number and affinity of the receptors in the heart ventricular membrane. Methacholine treatment decreased, in a dose-related and time-dependent manner, the specific binding of [³H]QNB by 34% as compared to the control. Atropine treatment, on the other hand, resulted in a dose-related increase (28 to 66%) in the number of the receptors. The equilibrium dissociation constant (K_D) of the receptors for the ligand was the same (about 200 pM) for the control and the methacholine treated groups of rats, whereas a dose-related increase (39 to 105%) in the K_D was noted for the atropine treated rats. Similarly, the concentration of acetylcholine causing a 50 percent inhibition (IC₅₀) of the [³H]QNB binding was unaltered for the methacholine treated rats (4 μM), but it was increased 43% for the atropine treated rats.