Binding of [H]MSX-2 (3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine) to rat striatal membranes — a new, selective antagonist radioligand for A2A adenosine receptors

The present study describes the preparation and binding properties of a new, potent, and selective A_2A adenosine receptor (AR) antagonist radioligand, [³H]3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine ([³H]MSX-2). [³H]MSX-2 binding to rat striatal membranes was saturable and reversible. Saturation experiments showed that [³H]MSX-2 labeled a single class of binding sites with high affinity (K_d=8.0 nM) and limited capacity (B_max=1.16 fmol·mg⁻¹ of protein). The presence of 100 μM GTP, or 10 mM magnesium chloride, respectively, had no effect on [³H]MSX-2 binding. AR agonists competed with the binding of 1 nM [³H]MSX-2 with the following order of potency: 5′-N-ethylcarboxamidoadenosine (NECA)>2-[4-(carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine (CGS-21680)>2-chloroadenosine (2-CADO)>N⁶-cyclopentyladenosine (CPA). AR antagonists showed the following order of potency: 8-(m-bromostyryl)-3,7-dimethyl-1-propargylxanthine (BS-DMPX)>1,3-dipropyl-8-cyclopentylxanthine (DPCPX)>(R)-5,6-dimethyl-7-(1-phenylethyl)-2-(4-pyridyl)-7H-pyrrolo[2,3-d]pyrimidine-4-amine (SH-128)>3,7-dimethyl-1-propargylxanthine (DMPX)>caffeine. The K_i values for antagonists were in accordance with data from binding studies with the agonist radioligand [³H]CGS21680, while agonist affinities were 3–7-fold lower. [³H]MSX-2 is a highly selective A_2A AR antagonist radioligand exhibiting a selectivity of at least two orders of magnitude versus all other AR subtypes. The new radioligand shows high specific radioactivity (85 Ci/mmol, 3150 GBq/mmol) and acceptable nonspecific binding at rat striatal membranes of 20–30%, at 1 nM.     

Labelling of I2B-imidazoline receptors by [3H]2-(2-benzofuranyl)-2-imidazoline (2-BFI) in rat brain and liver: characterization, regulation and relation to monoamine oxidase enzymes

The novel selective imidazoline radioligand [3H]2-(2-benzofuranyl)-2-imidazoline (2-BFI) was used to characterize and assess further the nature of I₂-imidazoline receptors in rat brain and liver. In the cerebral cortex, 2-BFI displayed high affinity (K _i= 9.8 nM) for a single class of [³H]2-BFI binding sites. Other imidazoline/guanidine compounds (e.g. aganodine, cirazoline and idazoxan) displayed biphasic competition curves, indicating the existence of high (K _iH= 2.9-78 nM; R _H= 61-83%) and low (K _iL= 4.7-158 μM) affinity sites. The pharmacological profile for [³H]2-BFI binding (aganodine > cirazoline > 2-BFI >> clonidine > amiloride >> efaroxan) was typical of that for I₂-sites. This profile was almost identical to that obtained against [³H]idazoxan (correlation between pK _i values, r = 0.97) which indicated that the sites characterized with [³H]2-BFI in brain corresponded to I₂-imidazoline receptors. The low affinity of amiloride against [³H]2-BFI (K _i= 900 nM) further indicated that these brain I₂-sites belong to the I_2B-subtype. [³H]2-BFI binding sites (B _max= 72 fmol/mg protein) in brain were differentially modulated by treatment (7 days) with cirazoline (up-regulation: 25%) and the MAO inhibitor phenelzine (down-regulation: 31%), indicating that these I₂-sites are regulated in vivo, as is the case for those labelled by [³H]idazoxan. Chronic treatment with 2-phenylethylamine, a phenelzine metabolite and endogenous amine, did not alter the density of brain of I₂-imidazoline receptors labelled by [³H]idazoxan. Preincubation of liver membranes with the MAO inhibitor clorgyline (10^-7 M) abolished the binding of [³H]Ro 41-1049 (N-(2-aminoethyl)-5-(m-fluorophenyl)-4-thiazole carboxamide) to MAO-A, but it did not alter the binding of [³H]Ro 19-6327 (N-(2-aminoethyl)-5-chloro-2-pyridine carboxamide) to MAO-B or that of [³H]2-BFI to I₂-sites. At 10^-4 M it also abolished MAO-B sites, but a substantial proportion of I₂-sites (40%) remained intact. Preincubation of liver membranes at 60 °C also abolished MAO-A/B sites, whereas still 22% of I₂-sites remained. The results indicate that [³H]2-BFI is a good tool for the identification of I₂-imidazoline receptors and suggest further that certain I₂-sites and MAO are different proteins. Received: 10 January 1997 / Accepted: 6 March 1997     

Correlation between the sites labelled by [3H]-8 hydroxy-2-(di-N-propylamino)tetralin and [3H]-imipramine in human platelets

The authors investigated the possible correlations between the binding parameters, maximum binding capacity (Bmax) and dissociation constant (Kd), of [³H]-8 hydroxy-2-(di-N-propylamino) tetralin and [³H]-imipramine in platelets of 13 healthy subjects. The in vitro pharmacological characterization was also carried out for a further comparison. The results, showing that the Bmax values were significantly and positively correlated, together with the findings of the displacement studies, suggest that in human platelets, [³H]-8 hydroxy-2-(di-N-propylamino)tetralin and [³H]-imipramine bind to the same protein.     

Synthesis of high specific activity (3,4-[3H]cyclohexyl)-N-[1 (2-benzo[b]thienyl)cyclohexyl]piperidine {[3H]BTCP}: A selective probe for the dopamine-reuptake complex

High specific activity [³H]BTCP, a radioligand for the dopaminereuptake complex was synthesized in 7-steps starting with the readily available starting materials, cyclohexane-1,4-dione monoethylene ketal and benzo[b]thiophene; the tritium label was introduced in the final step on the 3- and 4- positions of the cyclohexyl ring by catalytic tritiation of N-[4-(2-benzo[b]thienyl)cyclohexenyl]piperidine to give [³H]BTCP in 7.3% yield with a specific activity of 29.8 Ci/mmol (51.4% isotopic incorporation).     

Autoradiographic localisation of D3-dopamine receptors in the human brain using the selective D3-dopamine receptor agonist (+)-[3H]PD 128907

The selective D₃-dopamine receptor agonist 4aR,10bR-(+)-trans-3,4,4a,10b-tetrahydro-4-[N-propyl-2,3-³H]-2H,5H-[1]benzopyrano[4,3-b]-1,4-oxazin-9-ol ([³H]PD 128907) was used to visualise D₃-dopamine receptors in whole hemisphere cryosections from post-mortem human brain. [³H]PD 128907 has an 18- to 40-fold selectivity for D₃- over D₂-dopamine receptors as compared to a 7- to 24-fold selectivity of the more commonly used ligand [³H]7-OH-DPAT. [³H]PD 128907 accumulated markedly in the nucleus accumbens and in the ventral parts of caudate nucleus and putamen, with a slightly heterogeneous (patch-matrix like) distribution. The binding in the lateral parts of caudate nucleus and putamen was much less dense. No binding was obtained in any other regions. A very high proportion of [³H]PD 128907 was specifically bound, as judged from the low binding remaining in the presence of the D₂/D₃-dopamine receptor antagonist raclopride. This gives the ligand a potential for the detection of low density D₃-dopamine receptors in the human brain. The binding obtained with [³H]PD 128907 was qualitatively similar to that using [³H]7-OH-DPAT in the presence of GTP. However, [³H]7-OH-DPAT labelled, in contrast to [³H]PD 128907, also D₃-dopamine receptors in neocortex. The new compound [³H]PD 128907 appears to be a suitable radioligand for autoradiographic examination of the D₃-dopamine receptor localisation in the human brain, and should also be useful for pharmacological studies of this receptor subtype. Received: 20 November 1995/Final version: 2 May 1996     

Pharmacokinetics of inhibition of adenosine deaminase by erythro-9-(2-hydroxy-3-nonyl)adenine in CBA mice

The pharrnacokinetics oferythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) inhibition of adenosine deaminase (ADA) was measured in vivo in CBA mice. The in vivo assay utilized injection of 10–100 nmoles [2-³H]adenosine and measurement of blood ³H₂O 20 min later. A single oral dose of EHNA (50 mg/kg) totally inhibited ADA for 4 hr and caused a large increase in conversion of [2-³H]adenosine to [2-³H]ATP. EHNA (3 mg/kg) decreased deamination by 50% for 2–6 hr, depending on the dose of adenosine used. Mice dosed with EHNA (100 mg/kg) once daily for 7 days showed the same ADA recovery rate as mice dosed only once. High single oral doses of EHNA had no effect on blood ATP and GTP pools.     

Synthesis characterization and biological evaluation of some alkoxyphthalimide derivatives of 3-(4-substituted phenyl)-6,6-diphenyl-3,3a-dihydro-2H-imidazo[2,1-b]pyrazolo[3,4-d][1,3]thiazol-7(6H)-one

A series of 2-N-ethoxyphthalimido 3-(4-substitutedphenyl)-6,6-diphenyl-3,3a-dihydro-2H-imidazo[2,1-b]pyrazolo[3,4-d][1,3]thiazol-7(6H)-one(7a–e) and 4-(4-substituted phenyl)-2-(N-ethoxyphthalimido amino)-7,7-diphenylimidazo[2′,1′:2,3][1,3]thiazolo[4,5-d] pyrimidin-8(7H)-one (9a–e) have been designed and synthesized starting from thiohydentoin (1). The structure of all synthesized compounds has been established by IR, ¹H NMR, ¹³C NMR and mass studies. These compounds have been screened for antimicrobial activities in order to evaluate the possibility of the derivatives to be used as potential chemotherapeutic agents.     

Inhibition of the neuronal uptake of 5-hydroxytryptamine and noradrenaline in rat brain by (Z)- and (E)-3-(4-Bromophenyl)-N,N-dimethyl-3-(3-pyridyl) allylamines and their secondary analogues

(Z)-3-(4-Bromophenyl)-N,N-dimethyl-3-(3-pyridyl)allylamine (H $\text{102}\text{09}$) and the secondary amine analogue (A 24356) were about 10 times more potent in inhibiting the uptake of [¹⁴C]-hydroxytryptamine than the uptake of [³H]-(?)noradrenaline in homogenate and slices of the rat hypothalamus in vitro, whereas the corresponding tertiary (E)-isomer (A 23140) had the same activity on both uptake mechanisms and the secondary (E)-derivative (A 23889) was much more potent in inhibiting the noradrenaline uptake. All four amines had rather low activity on the dopamine uptake in homogenate of rat striatum. After oral administration in vivo, H $\text{102}\text{09}$ and A 24356 were about equally potent in inhibiting the 5-hydroxytryptamine uptake in slices of rat hypothalamus and were much less active on the noradrenaline uptake. On the other hand, A 23889 and A 23140 were very active inhibitors of the noradrenaline uptake but poor inhibitors of the 5-hydroxytryptamine uptake after administration in vivo. It was found that H $\text{102}\text{09}$ was rapidly transformed to A 24356 in vivo and that the main effect in vivo was caused by the secondary amine derivative.     

3,4-二氯-N-甲基-N-[反式-2-(1-△3-吡咯啉基)环己基]-苯乙酰胺盐酸盐的合成及其镇痛活性的研究

3, 4-Dichloro-N-methyl-N-[trans-2- (1-△³-pyrrolinyl)-cyclohexyl]-benzenacetamide hydrochloride (K-Ⅱ) was synthesized from N-methyl-7-azabicyclo [4.1.0] heptane by treatment with 2,5-dihydropyrrole to give N-[trans-2(1-△³-pyrrolinyl)-cyclohexyl]-N-methylamine which was amidated with 3,4-dichlorophenyl-acetic acid. K-Ⅱ is an analogue of U-50488 H(K-Ⅰ), a known kappa receptor agonist.The results of animal tests showed that K-Ⅱ is 3 times as potent as K-Ⅰ as an analgesic in the mouse hot plate test and 5 times in the mouse writhing test and that the affinity of K-Ⅱ for kappa receptor may be higher than that of K-Ⅰ. The general behavioural effects of these two agents are similar in mice.     

Mixed Dopaminergic/Serotonergic Properties of Several 2-Substituted 4-[2-(5-Benzimidazole)ethyl]-1-arylpiperazines

A series of substituted 4-[2-(5-benzimidazole)ethyl]-arylpiperazines was synthesized by introducing different substituents into position 2 of benzimidazole ring of 4-[2-(N,N-di-n-propyl-amino)ethyl]-1,2-diaminobenzenes. They were evaluated for in vitro binding affinity at the D₁ and D₂ dopamine and 5-HT_1A serotonin receptors using synaptosomal membranes of the bovine caudate nuclei and hippocampi, respectively. Tritiated SCH 23390 (D₁ receptor-selective), spiperone (D₂ receptor selective) and 8-OH-DPAT (5-HAT_1A receptor selective) were employed as the radioligands. Only compound 6 expressed a moderate binding affinity at the dopamine D₁ receptor, while the remaining ligands were inefficient or weak competitors of [³H]SCH 23390. Compound 12 was an absolutely inactive competitor of all three radioligands. Also, compound 7 was an inefficient displacer of [³H]-8-OH-DPAT. Compound 19 with a K_i value of 3.5 nM was the most potent competitor of [³H]spiperone and compound 13 (K_i = 3.3 nM) was the most efficient in displacing [³H]-8-OH-DPAT from the 5-HAT_1A serotonin receptor. Ligands 5, 6, 8–11 , and 13–20 expressed mixed dopaminergic/serotonergic activity in nanomolar range of concentrations with varying affinity ratios which strongly depended on the properties of the substituents introduced into position 2 of benzimidazole ring of the parent compounds.     

氚标记强效镇痛剂N-[1-(β-羟基-β-苯乙基)-3-甲基-4-哌啶基]-N-丙酰苯胺的制备以及与阿片受体的结合试验

本文报道以N-[1-(对-溴苯甲酰甲基)-3-甲基-4-哌啶基]-N-丙酰苯胺(Ⅲ)为前体,以PdO/BaSO₄作催化剂,用氚气进行卤—氚置换,氚化还原羰基的反应。反应产物经硅胶纸层析纯化后,用甲基橙比色法定量测定,得到N-{1-[β-羟基-β-氚-β-(对-氚苯基)乙基]-3-甲基-4-哌啶基}-N-丙酰苯胺(Ⅳ,[³H]F-7302),其比放射性为59 Ci/mM,放化纯度为98%。[³H]F-7302与小鼠脑内阿片受体的特异性结合在浓度为4.5×10^-9M时达到饱和,解离常数Kd=1.25×10^-9M,最大结合量B_max=93.08×10¹²M/g蛋白,其特异性结合与非特异性结合比值达10～15。     

Synthesis of 2-(3-Substituted-1,2,4-oxadiazol-5-yl)-8-methyl-8-azabicyclo[3.2.1]octanes and 2α-(3-Substituted-1,2,4-oxadiazol-5-yl)-8-methyl-8-azabicyclo[3.2.1]oct-2-enes as Potential Muscarinic Agonists

Radioligand binding affinities of seven muscarinic receptor ligands which possess an oxadiazole ring side chain have been determined in rat heart, rat brain, and m₁- or m₃-transfected CHO cell membrane preparations to determine the selectivity for subtypes of muscarinic receptor. The ratios of binding constants in brain membranes were measured as an indicator of potential agonist activity against [³H]QNB and [³H]Oxo-M. These muscarinic ligands did not discriminate the subtypes of muscarinic receptors. Six muscarinic ligands which have a 3-amino- or 3-methyl-1,2,4-oxadiazol-5-yl groups attached to the 8-methyl-8-azabicyclo[3.2.1]oct-2-ene or 8-methyl-8-azabicyclo[3.2.1]octane head group show binding constants between 2.04 x 10^–6 and 1.79 x 10^–5 M in rat heart, rat brain, and m₁- or m₃-transfected CHO cell membrane preparations. 1-Methyl-2-[3-amino-1,2,4-oxadiazol-5-yl]piperidine shows low binding constants of approximately 10^–4 M in rat heart and rat brain. (1R,5S)-2-[3-Amino-1,2,4-oxadiazol-5-yl]-8-methyl-8-azabicyclo-[3.2.1]oct-2-ene [(1R,5S)-17] was the most active compound.     

Metabolism of a glucuronide conjugate of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in rats

 Besides 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), [4-(methylnitrosamino)-1-(3-pyridyl)but-1-yl]-β-O-d-glucosiduronic acid (NNAL-Glu) is another important metabolite of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) which has been detected in the urine of tobacco users and non-smokers heavily exposed to sidestream cigarette smoke. In order to evaluate the toxicological significance of NNAL-Glu formation and excretion, the metabolism of [5-³H]-NNAL-Glu was studied in rats. Five male F344 rats were administered 3.7 mg/kg [5-³H]-NNAL-Glu by i.v. injection and the metabolites in urine analysed by HPLC. More than 90% of the radioactivity was excreted in urine within the first 24 h. Unchanged NNAL-Glu accounted for 81.2±3.1% of the total radioactivity; the remaining part of the dose appears to be deconjugated resulting in the urinary excretion of NNAL (3.6±1.7%) and its α-hydroxylation (11.5±2.2%) and N-oxidation (3.6±1.6%) products. The presence of α-hydroxylation products of NNAL-Glu in urine suggests that this NNK metabolite may be activated in vivo to carcinogenic intermediates. Received: 25 April 1994 / Accepted: 29 June 1994     

Resolution of multiple affinity states of 5-hydroxytryptamine-1a receptors labelled by [3H]-8-hydroxy-2-(di-n-propilamino) tetralin in rat hippocampal membranes

• 1.1. We examined the binding of [³H]-8-hydroxy-2.(DI-n-propilamino)tetralin ([³H]-8OH-DPAT) to 5-hydroxytriptamine-1A (5-HT1A) receptors in rat hippocampal membranes.
• 2.2. Computer analysis of [³H]-8OH-DPAT displacement curves in the absence and in the presence of 100 μM guanosine-5'-O-(3-thiotriphosphate) (GTPγS) were best fitted with a three-site model with apparent dissociation constants (K_d) of 0.45, 2.8 and 30 nM; the corresponding binding capacity (B_max) existing in the three affinity states were 4, 2 and 12 pmol/g of tissue (wet weight), respectively.
• 3.3. These results suggest that [³H]-8OH-DPAT binding can be resolved as complex isotherms and we provided evidence that [³H]-8OH-DPAT labels 2 high-affinity GTPγS-sensitive and one low-affinity GTPγS-insensitive state of 5-HT1A receptors.

     

Interactions of 7-[3-(4-[2,3-Dimethylphenyl]piperazinyl)-propoxy]-2(lH)-quinolinone (OPC-4392) with 3H-Spiperone and 3H-SCH 23390 Binding in Rat Striatum: Effects of Lesions

7-[3-(4-[2,3-Dimethylphenyl]piperazinyl)propoxy]-2(lH)-quinolinone (OPC-4392), a presynaptic dopamine autoreceptor agonist and postsynaptic D-2 receptor antagonist (Yasuda et al, Life Sci. 42:1941–1954,1988), was studied for its binding characteristics at ³H-SCH 23390-labeled dopamine D-l receptors and ³H-spiperone-labeled dopamine D-2 receptors in rat striatum. The binding affinity of OPC-4392 for ³H-spiperone-labeled D-2 receptors was 500 times higher than for ³H-SCH 23390-labeled D-l receptors. 6-Hydroxydopamine lesions of striatum and high-frequency current lesions of medial forebrain bundle did not affect the competition of OPC-4392 for ³H-spiperone binding. Kainic acid lesions of striatum significantly changed the one-site model fit to a two-site model fit of the competition curve of OPC-4392 for ³H-spiperone binding, suggesting that OPC-4392 competed with ³H-spiperone binding differently for postsynaptic dopamine D-2 receptors and for presynaptic dopamine autoreceptors.     

[3H]2-(2-Benzofuranyl)-2-imidazoline,a highly selective radioligand for I2-imidazoline receptor binding sites Studies in rabbit kidney membranes

2-(2-Benzofuranyl)-2-imidazoline (2-BFI) has recently been characterised as a selective ligand for the I₂-type of imidazoline-receptor binding site(s) (I₂-RBS). The present studies determined the relative levels of specific [³H]2-BFI binding to membrane homogenates of brain and kidney from rat, guinea pig and rabbit and identified the pharmacological characteristics of [³H]2-BFI binding sites in rabbit kidney membranes. Rabbit kidney membranes had the highest relative density of specific [³H]2-BFI binding of all tissues studied (2000?fmol/mg protein). Rabbit brain and guinea pig kidney had moderate levels of specific [³H]2-BFI binding (350–500?fmol/mg protein), while rat kidney and guinea pig and rat brain displayed much lower densities of binding (40–65?fmol/mg protein). Studies of [³H]2-BFI binding kinetics in rabbit kidney homogenates revealed binding to two distinct sites with K _d values of 0.10?±?0.01?nmol/l and 1.00?±?0.36?nmol/l respectively. Equilibrium saturation studies were also consistent with the presence of two binding sites – [³H]2-BFI (0.01–20?nmol/l) bound to sites with affinities of 0.10?± 0.01?nmol/l and 0.92?±?0.13?nmol/l and binding densities of 470?±?80 and 840?±?60?fmol/mg protein (n=3), representing 36 and 64% respectively. Drug inhibition studies revealed that l-adrenaline; α₁-adrenoceptor drugs (prazosin, l-phenylephrine) and α₂-adrenoceptor drugs (rauwolscine, methoxyidazoxan, 2-(2,4-(O-methoxyphenyl)-piperazin-1-yl)-ethyl-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione (ARC-239) had extremely low affinities for [³H]2-BFI binding sites (IC₅₀?≥?10?μmol/l). Putative I₁-RBS compounds, p-aminoclonidine, moxonidine, imidazole-4-acetic acid and cimetidine, inhibited [³H]2-BFI binding to rabbit renal membranes with low to very low affinities (K _i values 3 to ≥100?μmol/l), suggesting [³H]2-BFI does not label I₁-RBS in rabbit kidney membranes. I₂-RBS compounds – 2-(4,5-dihydroimidaz-2-yl)-quinoline (BU224), 2-(4,5-dihydroimidaz-2-yl)-quinoxaline (BU239), idazoxan and cirazoline – potently inhibited [³H]2-BFI binding (K _i values 0.37–1.6?nmol/l), confirming the labelling of I₂-RBS. Inhibition of [³H]2-BFI binding by certain compounds was consistent with their interaction with two binding site populations – for example (drug, K _i values) guanabenz, 0.65?nmol/l and 0.17?μmol/l; naphazoline, 0.94?nmol/l and 2.8?μmol/l; amiloride, 76?nmol/l and 26?μmol/l rilmenidine, 150?nmol/l and 50?μmol/l; and clonidine, 230?nmol/l and 70?μmol/l. The high affinity of amiloride for a high proportion (85%) of the binding is consistent with the presence of the I_2A-subtype of I-RBS in rabbit kidney. These results demonstrate that [³H]2-BFI is a highly selective and high affinity radioligand for I₂-RBS which should be useful for the further characterisation of these sites in mammalian tissues.     

[3H]R75231 — a new radioligand for the nitrobenzylthioinosine sensitive nucleoside transport proteins Characterization of (±)-[3H]R75231 binding to calf lung membranes,stereospecificity of its two stereoisomers,and comparison with [3H]nitrobenzylthioinosine binding

Summary The tritiated analogue of R75231 ((±)-2-(aminocarbonyl)-N-(4-amino-2,6-dichlorophenyl)-4-[5,5-bis(4-fluorophenyl)pentyl]-1-piperazineacetamide) has been examined as a new radioligand for (nitrobenzylthioinosine sensitive) nucleoside transport proteins. [³H]R75231 was prepared in two steps from R69064 (((±)-4-[5,5-bis[4-fluorophenyl)-4-pentenyl]-2-piperazinecarboxamide dihydrochloride) with a specific activity of 0.23 TBq/mmol (6.3 Ci/mmol). [³H]R75231 bound in a pseudo-irreversible and saturable manner to a membrane preparation of calf lung tissue. The new radioligand displayed high affinity (K_D = 0.32 ± 0.06 nmol/l at 25°C) and capacity (B_max = 6.1 ± 0.3 pmol/mg protein). Specific [³H]R75231 binding could be fully displaced by both structural analogues and reference inhibitors such as dipyridamole, NBI, dilazep and hexobendine, as well as by various nucleosides. The two stereoisomers of R75231, R88016 ((+)-R75231) and R88021 ((–)-R75231), potently displaced specific [³H]R75231 and [³H]NBI binding, R88021 being 30-fold more active than R88016. Pseudo-Hill coefficients derived from the shape of all the [³H]R75231 displacement curves were approximately unity. In contrast, R75231 and most of its analogues displaced specific [³H]NBI binding with pseudo-Hill coefficients consistently larger than unity under identical experimental conditions. This latter finding is suggestive for the existence of two distinct binding sites for the two radioligands, which may or may not overlap to some extent. Send offprint requests to A. P. IJzerman at the above address     

Mechanisms for the increase in electrically stimulated [3H]norepinephrine release from rat cortical slices by N-(n-propyl)-N-(4-pyridinyl)-1H-indol-1-amine

N-(n-propyl)-N-(4-pyridinyl)-1H-indol-1-amine (HP 749) is currently in clinical trials for the treatment of Alzheimer's disease (AD). While HP 749 has many pharmacological properties, the biochemical basis for its efficacy in animal models for AD remains unexplained. To this end, we have investigated some biochemical properties of HP 749 as they relate to its effect on electrically stimulated [³H]norepinephrine (NE) release. HP 749 was found to inhibit both [³H]NE uptake and [³H]yohimbine binding to cortical μ₂-adrenergic receptors. Consistent with this profile, HP 749 (1 and 10 μM) enhanced electrically stimulated release of [³H]NE from rat cortical slices. Both clonidine (1 μM) and nomifensine (10 μM) inhibited the effect of HP 749 (1 μM). The enhancement of [³H]NE release produced by the μ₂ adrenergic antagonist, idazoxan (0.1 μM), was completely reversed by the μ₂ agonist, clonidine (1 μM), but was not affected by the NE uptake inhibitor, nomifensine (10 μM). These results indicate that the HP 749 enhancement of electrically stimulated [³H]NE release is due, at least in part, to a combination of presynaptic μ₂-adrenergic receptor antagonism and NE reuptake blockade. These mechanisms may contribute to some of the adrenergic effects of HP 749.     

Alteration of DNA by 5-(3-methyl-1- triazeno)imidazole-4-carboxamide (NSC-407347)

5-(3-Methyl-1-triazeno)imidazole-4-carboxamide (NSC-407347, MTIC), an active intermediate in the metabolism of 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (NSC-45388, DTIC), was investigated for its effect on DNA. When [³H]MTIC was added to an aqueous solution of calf thymus DNA, it was rapidly hydrolyzed, and less than 0.5 per cent of the added ³H was recovered in DNA as methyl groups attached to its base and phosphate constituents. It was estimated that the hydrolysis of phosphotriesters in methylated DNA released 0.5 per cent of total bound ³H of DNA in the experiment in vitro, and 5.7 per cent in vivo. When MTIC was added to tissue culture cells which had been prelabeled with [³H]thymidine, there was a large and immediate increase in acid-soluble radioactivity of both the cell media and the cells. At the same time, there was an accelerated drop in the specific activity of DNA, indicating that degeneration of DNA had occurred; MTIC at 10^?3 M had a greater effect than at 10^?5 M. Paper chromatographic studies showed that the acid-soluble radioactivity was predominantly in the form of thymidine. MTIC at 10^?4 M induced repair synthesis of DNA but inhibited semi-conservative replication. The extent of repair synthesis was greater at 30 min than after 5 min. Treatment of cells with MTIC at 10^?3 M irreversibly reduced the sedimentation of DNA in alkaline sucrose gradient. At 10^?5 M, the reduction was reversible, and a normal pattern was restored in 30 min. Comparison of these results with those of sedimentation analysis of DNA in formamide gradient at neutral pH indicated that single strand breaks which were apparent in alkaline sucrose were probably the result of exposure of alkali-labile lesions in DNA to high pH.     

SR 33557, a novel calcium-antagonist: interaction with [3H]-(±)-nitrendipine and [3H]-(−)-desmethoxy-verapamil binding sites in cerebral membranes

Summary We investigated the pharmacological properties of SR 33557, a novel compound with calcium-antagonist properties, in both functional tests in vitro and radioligand binding studies. SR 33557 potently antagonized calcium-induced contraction of potassium-depolarized rat aorta in vitro with an IC₅₀ value of 5.6 ± 0.9 nM, but was a much weaker inhibitor of noradrenaline-induced contraction of the same tissue (IC₅₀ = 96 ± 22 nM). SR 33557 totally inhibited [³H]-(±)-nitrendipine binding to rat brain membranes with a K _i value of 0.19 ± 0.03 nM. Diltiazem, which used alone increases [³H]-(±)-nitrendipine binding, reversed this inhibition indicating that SR 33557 allosterically regulates [³H]-(±)-nitrendipine binding. SR 33557 also fully inhibited [³H]-(–)-desmethoxyverapamil binding to cerebral membranes, but inhibition curves were biphasic. IC₅₀ value calculated for that part of the curve which reflects the high affinity binding site of SR 33557 (IC₅₀ = 0.20 ± 0.02 nM) was very similar to the K _i value determined for inhibition of [³H]-(±)-nitrendipine binding. Kinetic evidences indicate that SR 33557 binds to a site which is distinct from the 1,4-dihydropyridine or the phenylalkylamine binding sites associated with the calcium channel. To test the pharmacological specificity of these interactions, the ability of SR 33557 to interact with eight other receptors in cerebral or heart membranes was assessed by binding assays. No high-affinity interaction was observed between SR 33557 and any of the receptors investigated. We conclude that SR 33557 binds specifically and with a high affinity to a site closely associated with the voltage-operated calcium channel in cerebral membranes. Send offprint requests to: P. Chatelain at the above address