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1.
M. Assumpció Boronat Gabriel Olmos Duane D. Miller Popat N. Patil Jesús A. García-Sevilla 《Naunyn-Schmiedeberg's archives of pharmacology》1998,357(3):351-355
Isothiocyanatobenzyl imidazoline (IBI), the 4′-NCS analogue of tolazoline, has been used to alkylate several receptor sites
in rabbit iris muscles. Because of the high affinity of tolazoline for the I2-imidazoline binding sites (Ki = 16–130 nM), this study was designed to assess whether IBI is also an alkylating agent for these sites. In competition studies,
IBI displayed moderate affinity (Ki∼ 2–3 μM) against I2A-imidazoline sites in the rabbit cerebral cortex and I2B-imidazoline sites in the rat cerebral cortex labelled by [3H]2-(2-benzofuranyl)-2-imidazoline ([3H]2-BFI). However, preincubation (30 min at 25° C) of rat cortical and liver membranes with IBI (10–7 M to 10–3 M), followed by extensive washing, markedly decreased (17% to 96%) the specific binding of [3H]2-BFI to I2B-imidazoline sites. IBI (10–5 M to 10–3 M) also bound irreversibly to I2A-imidazoline sites in rabbit cerebral cortex but with a lesser efficacy (27% to 83% reduction of [3H]2-BFI binding). Saturation curves of [3H]2-BFI binding in the rat cerebral cortex indicated that preincubation with 10–6 M IBI reduced the total density (Bmax) without affecting the affinity (Kd) of I2B-imidazoline sites for IBI. Acute treatments (6 h) with IBI (10 and 30 mg/kg, i.p.) also dose-dependently reduced (26% and
41%; respectively) the total density of I2B-imidazoline sites. These results demonstrate the ability of IBI to alkylate I2-imidazoline binding sites in vitro and in vivo and provide evidence for the use of IBI as a new tool for the study of the
functional implications of imidazoline binding sites.
Received: 15 October 1997 / Accepted: 2 December 1997 相似文献
2.
A. R. Hosseini P. R. King W. J. Louis A. L. Gundlach 《Naunyn-Schmiedeberg's archives of pharmacology》1996,355(1):131-138
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 I2A-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. 相似文献
3.
Gerhard J. Molderings Liane Kundt Manfred Göthert 《Naunyn-Schmiedeberg's archives of pharmacology》1994,350(3):252-257
Bovine adrenal chromaffin cells, which have been shown to lack 2-adrenoceptors, were used to investigate the pharmacological characteristics of [3H]idazoxan binding sites. The binding of [3H]idazoxan was very rapid, reversible, partly specific (as defined by cirazoline 0.1 mmol/l; 50% specific binding at [3H]idazoxan 10 nmol/l), saturable and of high affinity (KD 13 nmol/l) and, hence, was compatible with the criteria for the identification of an imidazoline binding site (IBS). Since in competition experiments rauwolscine and (–)-adrenaline showed only negligible affinity for these adrenal medullary binding sites, the lack of 2-adrenoceptors was confirmed. Histamine and amiloride also did not inhibit [3H]idazoxan binding or caused only negligible inhibitor. In contrast, the specific binding of [3H]idazoxan was concentration-dependently inhibited by several imidazolines and guanidines with the following rank order of potency which conforms to the characteristics of the previously defined I2-IBS (in parentheses: Ki, nmol/l): idazoxan (4) = cirazoline (4) clonidine (272) = BDF 6143 (299; 4-chloro-2-(2-imidazoline-2-ylamino) isoindoline) > BDF 6100 (563; 2-(2-imidazolin-2-ylamino)-isoindoline) BDF 7579 (868; (4-chloro-2-isoindolinyl)guanidine) > phentolamine (1424) = naphazoline (1451). Equilibrium [3H]idazoxan binding was reduced by K+ but not by Na+ or the non-hydrolysable GTP-analogue Gpp(NH)p (5-guanylylimidodiphos-phate; 100 mol/l). In conclusion, membranes of the bovine adrenal medulla are endowed with non-adrenergic high-affinity [3H]idazoxan sites which exhibit the pharmacological properties of the amiloride-insensitive subtype of I2-IBS and probably are not coupled to a G-protein.
Correspondence to: G. J. Molderings at the above address 相似文献
4.
L F Callado A I Maeztu J Ballesteros M Gutiérrez J J Meana 《European journal of pharmacology》2001,423(2-3):109-114
[(3)H]2-(2-benzofuranyl)-2-imidazoline (2-BFI) and [(3)H]idazoxan are the most used tools to characterise imidazoline I(2) receptors. We evaluated the binding of both radioligands to human postmortem frontal cortex membranes. Saturation binding analyses revealed that [(3)H]idazoxan (in the presence of 2 microM efaroxan to avoid radioligand binding to alpha(2)-adrenoceptors and imidazoline I(1) receptors) and [(3)H]2-BFI bound with high affinity to an apparent single population of sites. However, in competition studies whereas [(3)H]idazoxan (10 nM) binding was displaced monophasically by idazoxan and 2-BFI, both drugs displayed biphasic curves for [(3)H]2-BFI (1 nM). The proportion of the low-affinity binding site increased from 17% to 25% when 10 nM [(3)H]2-BFI was displaced by idazoxan. Amiloride inhibited [(3)H]2-BFI (10 nM) binding with low affinity and in a monophasic way. These data indicate that [(3)H]2-BFI recognises in human postmortem brain membranes a second binding site different from the imidazoline I(2) receptors labelled by [(3)H]idazoxan. 相似文献
5.
Christa E. Müller Juris Maurinsh Roland Sauer 《European journal of pharmaceutical sciences》2000,10(4)
The present study describes the preparation and binding properties of a new, potent, and selective A2A 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 (Kd=8.0 nM) and limited capacity (Bmax=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 Ki 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 A2A 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. 相似文献
6.
Juan F. López-Giménez G. Mengod J. M. Palacios M. Teresa Vilaró 《Naunyn-Schmiedeberg's archives of pharmacology》1997,356(4):446-454
The recently developed 5-HT2A receptor selective antagonist [3H]MDL100,907 ((+/–)2,3-dimethoxyphenyl-1-[2-(4-piperidine)-methanol]) has been characterized as a radioligand for the autoradiographic
visualization of these receptors. [3H]MDL100,907 binding to rat brain tissue sections was saturable, had sub-nanomolar affinity (Kd=0.2–0.3nM), and presented
a pharmacological profile consistent with its binding to 5-HT2A receptors (rank order of affinity for [3H]MDL100,907-labelled receptors: MDL100,907 > spiperone > ketanserin > mesulergine). The distribution of receptors labelled
by [3H]MDL100,907 was compared to the autoradiographical patterns obtained with [3H]Ketanserin, [3H]Mesulergine, and [3H]RP62203 (N-[3-[4-(4-fluorophenyl)-piperazin-1-y1]propyl]-1,8-naphtalenesultam) and to the distribution of 5-HT2A receptor mRNA as determined by in situ hybridization. As opposed to the other radioligands, [3H]MDL100,907 labelled a single population of sites (5-HT2A receptors) and presented extremely low levels of non-specific binding. The close similarity of the distributions of [3H]MDL100,907-labelled receptors and 5-HT2A mRNA further supports the selectivity of this radioligand for 5-HT2A receptors and suggests a predominant somatodendritic localization of these receptors. The present results point to [3H]MDL100,907 as the ligand of choice for the autoradiographic visualization of 5-HT2A receptors.
Received: 7 April / Accepted: 18 May 1997 相似文献
7.
G. Olmos R. Alemany J. A. García-Sevilla 《Naunyn-Schmiedeberg's archives of pharmacology》1996,354(6):709-716
I2-imidazoline receptors labelled with [3H]-idazoxan in the rabbit and rat brains displayed high and low affinity, respectively, for the guanidide amiloride; reinforcing the previous definition of I2A-imidazoline receptors expressed in the rabbit brain and Its-imidazoline receptors expressed in the rat brain. Other drugs tested displayed biphasic curves in competition experiments, indicating the existence of high and low affinity sites for both subtypes of I2-imidazoline receptors. Among the drugs studied, bromoxidine, moxonidine, (+)- and (-)-medetomidine and clorgyline were more potent on the high and/or low affinity sites of 12B- than on their corresponding of I2A-imida-zoline receptors (K
iH
ratios 20 to 65). No correlation was found for the potencies of the drugs tested at the low affinity sites of both I2-imidazoline receptor subtypes. Preincubation (30 min at 25°°C) with 10-6 M clorgyline reduced by 60% the B
max of [3H]-idazoxan binding to I2 B-imidazoline receptors in the rat brain, but it did not affect the binding parameters of the radioligand saturation curves to I2A-imidazoline receptors in the rabbit brain. These results indicated that I2A- and I2B-imidazoline receptor subtypes differ in the pharmacological profiles of their high and low affinity sites and in the ability to irreversibly bind clorgyline. In rat cortical membranes western blot detection of immunoreactive imidazoline receptor proteins revealed a double band of 29/30 kDa and two less intense bands of 45 and 66 kDa. In rabbit cortical membranes the antibody used detected proteins of 30, 57 and 66 kDa. It is suggested that different imidazoline receptor proteins (45 vs 57 kDa) may account for the different pharmacological profiles of I2-imidazoline receptor subtypes. 相似文献
8.
I. Rustenbeck Cornelia Herrmann Peter Ratzka Arnold Hasselblatt 《Naunyn-Schmiedeberg's archives of pharmacology》1997,356(3):410-417
To elucidate the β-cytotropic effect of imidazoline compounds their inhibitory effect on ATP-dependent K+ channels (KATP channels) in pancreatic B-cells was compared with their binding to membranes from insulin-secreting HIT T15 cells. KATP channels in inside-out patches from B-cells were closed with the following rank order of efficacy at 10 μM: guanabenz > phentolamine
= alinidine > clonidine > idazoxan > rilmenidine = amiloride. The last four compounds achieved an incomplete inhibition only.
In contrast to sulfonylureas, the inhibitory action of imidazolines was not enhanced by ADP. With intact cells the site which
mediates inhibition is less easily accessible for protonated compounds, suggesting a location at the inner face of the plasma
membrane. Competition binding experiments were performed by masking α-adrenoceptors and using [3H]clonidine as ligand. Homologous displacement of [3H]clonidine revealed two distinct binding sites in HIT cell membranes characterized by dissociation constants of 38 nM and
4,911 nM and maximal binding capacities of 118 fmol/mg protein and 18 pmol/ mg protein. Generally, ligands for I2 imidazoline receptors were more potent than ligands for I1 imidazoline receptors to displace [3H]clonidine from the high affinity site, which does not fit into the current classification of imidazoline receptors. Binding
to the second site had affinities in the micromolar range, similar to the concentrations necessary to inhibit KATP channels in B-cells. However, alinidine and phentolamine inhibited KATP channels already at concentrations at which they displaced [3H] clonidine only from the high affinity site, but not yet from the low affinity site. Since the proportion of the low and
high affinity site varied in dependence of the competitor, the imidazoline binding sites in HIT cells may not be independent,
but may rather represent two interacting or interconvertible sites both of which may be involved in KATP channel closure.
Received: 1 April 1997 / Accepted: 2 June 1997 相似文献
9.
Gerhard J. Molderings Daniel Moura Klaus Fink Heinz Bönisch Manfred Göthert 《Naunyn-Schmiedeberg's archives of pharmacology》1993,348(1):70-76
Summary Imidazolines bind with high affinity not only to -adrenoceptors but also to specific imidazoline binding sites (IBS) labelled by either [3H]clonidine or [3H]idazoxan and termed I1- and I2-IBS, respectively. Since bovine adrenal chromaffin cells lack 2-adrenoceptors, we investigated the pharmacological characteristics of [3H]clonidine binding sites in the bovine adrenal medulla. The binding of [3H]clonidine was rapid, reversible, partly specific (as defined by naphazoline 0.1 mmol/l; 55% specific binding at [3H]clonidine 10 nmol/l), saturable and of high affinity. The specific binding of [3H]clonidine to bovine adrenal medullary membranes was concentration-dependently inhibited by various imidazolines, guanidines and an oxazoline derivative but not, or with negligible affinity, by rauwolscine and (–)-adrenaline. In most cases, the competition curves were best fitted to a two-site model. The rank order of affinity for the high affinity site (in a few cases the single detectable site) was as follows: naphazoline >- BDF 7579 (4-chloro-2-isoindolinyl guanidine) >-clonidine>- cirazoline >_ BDF 6143 (4-chloro-2-(2-imidazolin-2-ylamino)isoindoline hydrochloride) > BDF 7572 (4,7-chloro-2-(2-imidazolin-2-ylamino)-isoindoline) > moxonidine = rilmenidine > BDF 6100 (2-(2-imidazolin-2-ylamino)-isoindoline) = idazoxan > phentolamine > aganodine = guanabenz > amiloride > histamine. This rank order is compatible with the pharmacological properties of the I1-IBS. The non-hydrolysable GTP-analogue Gpp(NH)p (5guanylylimidodiphosphate; 100 mol/l) inhibited specific [3H]clonidine binding by about 50%. Equilibrium [3H]clonidine binding was also significantly reduced by K+ and Mg2+ In conclusion, [3H]clonidine labels non-adrenergic high-affinity sites in plasma membranes of the bovine adrenal medulla; these sites exhibit the pharmacological properties of I1-IBS, but not of I2-IBS. Furthermore, the IBS in the adrenal medulla appear to be coupled to a G-protein.Correspondence to G. J. Molderings at the above address 相似文献
10.
H. B. Niznik D. E. Grigoriadis I. Pri-Bar O. Buchman P. Seeman 《Naunyn-Schmiedeberg's archives of pharmacology》1985,329(4):333-343
Summary In order to label dopamine D2 receptors selectively we tritiated the potent benzamide neuroleptic, YM-09151-2 (26.7 Ci/mmol). The binding of [3H]-YM-09151-2 to canine striatal membranes was saturable and specific with a K
D of 57 pmol/l and B
max of 36 pmol/g tissue as determined by Scatchard analysis. The K
D, but not the B
max, of [3H]-YM-09151-2 increased 6-fold in the absence of sodium chloride. [3H]-YM-09151-2 labeled 40% more sites than [3H]-spiperone in the same tissue homogenate. [3H]-YM-09151-2 binding was inhibited by dopaminergic drugs in a concentration and stereoselective manner with the appropriate dopamine D2 receptor profile. Thus, dopamine agonists inhibited [3H]-YM-09151-2 binding to canine striatal membranes with the following rank order of potency: (–)-N-n-propylnorapomorphine > apomorphine > (±)-6,7-dihydroxy-2-aminotetralin > (+)-N-n-propylnorapomorphine > dopamine > (–)-noradrenaline > serotonin > (–)-isoprenaline. Dopaminergic antagonists competed for [3H]-YM-09151-2 binding with the following order of potency: spiperone > (+)-butaclamol > haloperidol > clebopride > (–)-sulpiride > SCH-23390 > (–)-butaclamol. Furthermore, dopamine agonists recognized 2 states of the receptor labeled by [3H]-YM-09151-2, D
2
high
and D
2
low
. The D
2
high
state of the receptor could be converted to D
2
low
by guanine nucleotides and sodium ions as is the case for [3H]-spiperone binding to D2 receptors. [3H]-YM-09151-2 appears to be a more selective ligand for dopamine D2 receptors than [3H]-spiperone, since YM-09151-2 displays approximately 9-fold lower affinity than spiperone for cortical serotonergic (S2) receptors. [3H]-YM-09151-2 may become a useful tool for the selective characterization of dopamine D2 receptors.Abbreviations used (±)ADTN
(±)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene
- NPA
N-n-propylnorapomorphine
- Gpp(NH)p
5-guanylylimidodiphosphate 相似文献
11.
G. J. Molderings K. Donecker M. Göthert 《Naunyn-Schmiedeberg's archives of pharmacology》1995,351(5):561-564
We have identified and characterized non-adrenergic [3H]clonidine binding sites in rat stomach. The binding of [3H]clonidine was rapid, reversible, partly specific (as defined by cirazoline 0.1 mmol/l; 68% specific binding at [3H]clonidine 10 nmol/l), saturable and of high affinity. The specific binding of [3H]clonidine to rat stomach membranes was concentration-dependently inhibited by various imidazolines and guanidines including the sigma site ligand 1,2-di-(2-tolyl)guanidine (DTG), by the butyrophenone derivative haloperidol and by the piperidine derivative (+)-3-PPP[(R)-3-(3-hydroxyphenyl)-N-propylpiperidine]; the latter two compounds are also known to exhibit affinity for sigma sites. In contrast, rauwolscine, histamine, ranitidine and the non-hydrolysable GTP-analogue Gpp(NH)p (5 guanylylimidodiphosphate) did not, or with negligible affinity, inhibit [3H]clonidine binding. In most cases, the competition curves were best fitted to a two-site model. The rank order of affinity for the high affinity site (in a few cases for a single detectable site) was as follows: cirazoline>idazoxanDTG>(+)-3-PPP> clonidine>guanabenz>haloperidol. This rank order is not compatible with the pharmacological properties of either I1- or I2-imidazoline binding sites. However, the ability of haloperidol, (+)-3-PPP and DTG to displace [3H]clonidine (the latter two with high affinity) suggests that the [3H] clonidine binding sites in rat stomach may be related to sigma-like sites. 相似文献
12.
Donatella Marazziti Alessandra Rossi Gino Giannaccini Claudia Martini Silvia Castrogiovanni Maria Grazia Micali Antonio Lucacchini Giovanni Battista Cassano 《Human psychopharmacology》1995,10(5):417-421
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. 相似文献
13.
BU224 (2-(4,5-dihydroimidaz-2-yl)-quinoline) is a selective imidazoline I(2) binding site ligand characterised in both competition binding assays and functional studies. However, in some studies, BU224 has been reported to have a different functional effect from that seen with another selective imidazoline I(2) binding site ligand 2-BFI (2-(2-benzofuranyl)-2-imidazoline). This effect may reflect differing efficacies of the ligands or a difference in their brain distribution. The present study has investigated the distribution of the tritiated form of BU224 in rat brain and correlated this distribution with other imidazoline I(2) binding site ligands, [(3)H]idazoxan and [(3)H]2-BFI. Saturation studies revealed binding of [(3)H]BU224 was of high affinity and saturable. The central distribution of [(3)H]BU224 was similar to that previous reported for imidazoline I(2) binding site in rat brain. Autoradiography revealed that the highest levels of binding were in the arcuate nucleus, interpeduncular nucleus, area postrema, pineal gland and ependymal cell layer lining the ventricles. Correlation analysis of the binding distribution with our previous published studies revealed a highly significant correlation between [(3)H]BU224 and both [(3)H]idazoxan (r=0.94) and [(3)H]2-BFI (r=0.96). These data indicate [(3)H]BU224 labels the same population of imidazoline I(2) binding site in rat brain as seen with [(3)H]idazoxan and [(3)H]2-BFI. Therefore, the differences in functional effects observed with these compounds may reflect agonist and antagonist properties. 相似文献
14.
Roger G. Evans John M. Haynes 《Clinical and experimental pharmacology & physiology》1994,21(8):649-658
1. We characterized the binding of [3H]-rauwolscine, [3H]-p-aminoclonidine and [3H]-idazoxan in a dog kidney membrane preparation. Our aim was to determine the pharmacological nature of the α2-adrenoceptor- and imidazoline-preferring binding sites in this organ. 2. [3H]-Rauwolscine bound to an apparent single site with an affinity (KD) of 2.2 nmol/ L and a maximum density (Bmax) of 58.5 fmol/mg protein, when 10 μmol/L idazoxan defined non-specific binding. However displacement studies demonstrated that a number of compounds, including prazosin, inhibited [3H]-rauwolscine binding in a complex manner consistent with displacement from two distinct binding sites. The majority (69%) of the [3H]-rauwolscine binding sites had a relatively low affinity for prazosin (KI= 398 nmol/L), while the remainder had a relatively high affinity for prazosin (KI= 7.9 nmol/ L). 3. [3H]-p-Aminoclonidine bound to an apparent single site (KD= 5.2 nmol/L; Bmax= 72.4 fmol/mg protein), when 10 μmol/L phentolamine defined non-specific binding. When 1 μmol/L of the potent and selective α2-adrenoceptor antagonist 2-methoxyidazoxan was included in the incubate, no specific binding was detected. We therefore conclude that under the conditions of this experiment [3H]-p-aminoclonidine binds only to α2-adrenoceptors in the dog kidney. 4. [3H]-Idazoxan bound to two sites, with a higher (KD= 0.95 nmol/L; Bmax= 43.9 fmol/mg protein) and lower (KD= 9.1 nmol/L; Bmax= 93.8 fmol/mg protein) affinity, respectively, when 1 mmol/L phentolamine defined non-specific binding. When 10 μmol/ L GTPγS was included in the incubate, the low affinity site was unaffected but the maximum binding at the higher affinity site was reduced by 79%. 2-Methoxyidazoxan displaced [3H]-idazoxan in a monophasic manner and with low potency (IG50=11.5 μmol/L). Yohimbine, efaroxan, clonidine, rilmenidine, guanabenz and idazoxan itself displaced [3H]-idazoxan in a complex manner; the slope of the displacement curves being less than unity. 5. We conclude that the dog kidney contains a heterogeneous population of α2-adrenoceptors that can be labelled either with [3H]-rauwolscine or [3H]-p-aminoclonidine. The dog kidney also contains a heterogeneous population of non-adrenoceptor imidazoline-preferring binding sites of the I2-subtype, that can be labelled with [3H]-idazoxan. The binding site for which [3H]-idazoxan has the highest affinity appears to be coupled to a guanine nucleotide binding regulatory protein. 相似文献
15.
G. J. Molderings K. Schmidt H. Bönisch M. öthert 《Naunyn-Schmiedeberg's archives of pharmacology》1996,354(3):245-252
The influence of several imidazolines and -site ligands on cation influx through the 5-HT3 receptor channel in NIE-115 mouse neuroblastoma cells was studied by measuring the 2-min influx of the organic cation [14C] guanidinium induced by 1 M 5-HT (in the presence of 10 M substance P in all experiments). In addition, we determined specific binding of [3H]DTG (1,3-di(2-tolyl)-guanidine), a selective -site radioligand, and [3H] GR65630 (3-(5-methyl-1H-imidazol-4-yl)-1-(1-methyl-1H-indol-3-yl)-1-propanone), a selective 5-HT3 receptor antagonist, to membranes prepared from NIE-115 cells.The 5-HT-induced [14C]guanidinium influx was inhibited by the imidazolines, ondansetron, antazoline, idazoxan, BDF 6143 (4-chloro-2-(2-imidazolin-2-ylamino)-isoindoline), cirazoline, naphazoline, clonidine and by the guanidine agmatine, but not by the catecholamine adrenaline. The inhibitory effect of the imidazolines on cation influx through the 5-HT3 receptor channel was mimicked by the -site ligands, (±)-ifenprodil, (+)-3-PPP ((R)-3-(3-hydroxyphenyl)-N-propylpiperidine), DTG (1,3-di-tolyl-guanidine), haloperidol, dizocilpine, and ketamine as well as by the polyamines, arcane and spermidine. — Ondansetron inhibited [3H]GR65630 binding with high affinity, whereas inhibition of binding of this radioligand to the 5-HT3 receptor by antazoline, BDF 6143, idazoxan, cirazoline, (±)-ifenprodil, (+)-3-PPP, DTG and haloperidol occurred in the high micromolar range. In the competition experiments with [3H]DTG, (±)-ifenprodil, haloperidol, unlabelled DTG, BDF 6143 and (+)-3-PPP inhibited binding of the radioligand at moderate affinity (Ki values in the range of 1 M or lower), whereas ondansetron, amazoline, idazoxan, cirazoline, naphazoline, clonidine, tolazoline, efaroxan, RX821002 (2-[2-(2-methoxy-1,4-benzodioxanyl)]imidazoline), ketamine and spermidine exhibited affinity in the high micromolar or millimolar range only. Comparison of the potencies of the ligands (pIC50% values) in inhibiting 5-HT-induced [14C]guanidinium influx with their affinities (pKi values) at the 5-HT recognition sites of the 5-HT3 receptor and at the 2-sites of the N1E-115 cells by means of multiple regression analysis revealed a significant correlation with the affinities at both sites.In conclusion, our data suggest that imidazolines and -ligands, which as a rule possess low affinity for the 5-HT recognition site of the 5-HT3 receptor, may be assumed to exert their inhibitory effect on cation influx through the 5-HT3 receptor channels, at least in part, by interacting with 2-binding sites. 相似文献
16.
A. Newman-Tancredi L. Verrièle C. Chaput M. J. Millan 《Naunyn-Schmiedeberg's archives of pharmacology》1998,357(3):205-217
The novel benzodioxopiperazine, 5-HT1A receptor weak partial agonist, S 15535 (4-(benzodioxan-5-yl)1-(indan-2-yl)piperazine) bound with high affinity and selectivity
to membranes of Chinese Hamster Ovary cells stably expressing the human (h) 5-HT1A receptor (Ki = 0.6 nM versus [3H]-8-hydroxy-dipropylamino-tetralin, [3H]-8-OH-DPAT): its affinity at h5-HT1A receptors was more than 70-fold higher than its affinity at > 50 other binding sites. S 15535 was tritiated to high specific
activity (50 Ci/mmol) and its binding profile characterised. At 22° C, [3H]-S 15535 associated and dissociated from h5-HT1A receptors with half-times of 2.9 and 5.0 min, respectively, yielding a Kd estimate of 3.6 nM. In saturation binding experiments, [3H]-S 15535 displayed a Bmax value for h5-HT1A receptors (1630 fmol/mg), higher than that obtained with the agonist [3H]-8-OH-DPAT (1023 pmol/mg). Guanylyl imidodiphosphate (GppNHp, 100 μM) reduced the binding of [3H]-S 15535 by only 25% compared with 79% for [3H]-8-OH-DPAT at h5-HT1A receptors. [3H]-S 15535 also showed high affinity, saturable binding to rat hippocampal membranes (Bmax = 820 fmol/mg versus 647 fmol/mg for [3H]-8-OH-DPAT). For both h5-HT1A and rat 5-HT1A receptors, the Ki values for competition binding of 15 serotonergic ligands with [3H]-S 15535 was highly correlated with that of [3H]-8-OH-DPAT. However, important differences were also observed. The agonist, 5-hydroxytryptamine (5-HT), displayed biphasic
competition curves with [3H]-S 15535 but not with [3H]-8-OH-DPAT at h5-HT1A receptors. Similarly, the ‘antagonists’, spiperone, methiothepin and (+)butaclamol, showed biphasic competition isotherms
versus [3H]-S 15535 but not [3H]-8-OH-DPAT. When [3H]-S 15535 competition binding experiments were carried out in the presence of GppNHp (100 μM) the 5-HT and 8-OH-DPAT competition
curves shifted to the right, whereas the spiperone and methiothepin competition curves shifted to the left. In contrast, in
the presence of GppNHp, the competition isotherms for N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl)cyclohexanecarboxamide
(WAY 100,635) were not altered. Taken together, these data show that (i) [3H]-S 15535 is a highly selective 5-HT1A receptor ligand which labels both G-protein-coupled and uncoupled 5-HT1A receptors, (ii) antagonists, such as WAY 100,635, which yield monophasic isotherms in competition with both [3H]-agonists and [3H]-antagonists, are not sensitive to the G-protein coupling state of the receptor, but (iii) spiperone and methiothepin behaved
as inverse agonists, their competition isotherms with [3H]-S 15535 being modulated in an opposite manner to those of agonists.
Received: 12 September 1997 / Accepted: 1 December 1997 相似文献
17.
The muscarinic antagonist 1-[benzilic 4,4′-3H]quinuclidinyl benzilate ([3H] QNB) bound to a single class of muscarinic receptors with high affinity in rabbit ileal membranes. The K
D
and B
max
values for [3H]QNB calculated from analysis of saturation isotherms were 52.5 pM and 154 fmol/mg, respectively. Chlorpheniramine (CHP),
histamine H1 blocker, increased K
D
value for [3H]QNB without affecting the binding site concentrations and Hill coefficient. The K
i
value of CHP for inhibition of [3H]QNB binding in ileal membranes was 1.44μM and the pseudo-Hill coefficient for CHP was close to unit. In the functional assay
carbachol, muscarinic agonist, increased the contractile force of ileum with ED50 value of 0.11μM. CHP caused the rightward shift of the dose-response curve to carbachol. The pA2 value of CHP determined from Schild analysis of carbachol-induced contraction was 5.77 and the slope was unity indicating
competitive antagonism with carbachol. The dissociation constant (K
i
) of CHP obtained in competitive experiments with [3H]QNB was similar to the K
A
value (1.69μM) of CHP as inhibitor of carbachol-induced contraction in rabbit ileum. This result suggests that the binding
of H1 blocker, CHP, vs [3H]QNB to muscarinic receptors in ileal membranes represents an interaction with a receptor of physiological relevance. 相似文献
18.
Michael P. Johnson Barry W. Siegel Albert A. Carr 《Naunyn-Schmiedeberg's archives of pharmacology》1996,354(2):205-209
In studies using standard radioligands, unlabeled MDL 100,907 (R-(+)--(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-piperidinemethanol) has been shown to have a high degree of selectivity for the 5-HT2A receptor. The present study was undertaken to investigate the receptor binding characteristics of [3H]MDL 100,907 in rat cortical homogenates. [3H]MDL 100,907 was found to reach equilibrium at 37°C after 15 min. Saturation experiments indicated binding to a single site with a KD of 0.56 nM, Hill slope of 1.15, and a Bmax of 512 fmol/mg protein. In parallel experiments with the standard 5-HT2A receptor radioligand, [3H]ketanserin, with prazosin added to block 1 receptors, a similar Hill slope and Bmax was noted but a two-fold higher KD was found. In competition binding studies using 0.5 nM [3H]MDL 100,907, some 19 standard ligands to various receptors including the 5HT1A, D2, 1, and receptors resulted in estimated KI values that were consistent with [3H]MDL 100,907 selectively binding to the 5-HT2A receptor. A comparison of the KI values for 17 standard 5-HT2A receptor agonists and antagonists displacing [3H]MDL 100,907 versus [3H]ketanserin resulted in a highly significant linear correlation (R2 = 0.96, P<0.001). Taken together these results suggest that [3H]MDL 100,907 is binding to the 5-HT2A receptor with a sub-nanomolar affinity without the use of secondary blocking agents. 相似文献
19.
Luisa Ugedo Joseba Pineda José A Ruiz-Ortega Raúl Martín-Ruiz 《British journal of pharmacology》1998,125(8):1685-1694
- Imidazoline binding sites have been reported to be present in the locus coeruleus (LC). To investigate the role of these sites in the control of LC neuron activity, we studied the effect of imidazolines using in vivo and in vitro single-unit extracellular recording techniques.
- In anaesthetized rats, local (27 pmoles) and systemic (1 mg kg−1, i.v.) administrations of 2-(2-benzofuranyl)-2-imidazoline (2-BFI), a selective I-imidazoline receptor ligand, increased the firing rate of LC cells (maximal increase: 22±5%, P<0.001 and 16±7%, P<0.001 respectively). Chronic pretreatment with the irreversible monoamine oxidase inhibitor clorgyline (3 mg kg−1, i.p., every 12 h for 14 days) abolished this effect.
- In rat midpontine brain slices containing the LC, bath application (1 mM) of the imidazolines 2-BFI, 2-(4,5-dihydroimidaz-2-yl)-quinoline (BU224), idazoxan, efaroxan, phentolamine and (2-2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline (RX821002) reversibly stimulated LC cells. The maximal effect was ∼90% except for RX821002 and efaroxan which induced smaller maximal effects (∼58% and ∼35% respectively). Simultaneous application of idazoxan and 2BFI did not lead to additive effects.
- Bath application of the α2-adrenoceptor antagonists, yohimbine (1–10 μM) and N-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) (10 μM), failed to modify LC activity. The irreversible blockade of α2-adrenoceptors with EEDQ (10 μM) did not alter the effect of idazoxan or that of efaroxan. Previous application of clorgyline (10 μM) did not modify the excitatory effect of 2-BFI or efaroxan.
- Changes in the pH of the bathing solution (6.84–7.84) did not influence the effect caused by idazoxan. Bath application of 2-BFI (1 mM) reversed the inhibition induced by diazoxide (300 μM), an ATP-sensitive K+ channel opener, whereas application of glibenclamide (3 μM), an ATP-sensitive K+ channel blocker, partially blocked the effect of 2-BFI.
- This study shows that imidazoline compounds stimulate the firing rate of LC neurons. This effect is not mediated by α2-adrenoceptors nor by I1 or I2-imidazoline receptors but involves a different subtype of imidazoline receptor. Our results indicate that this receptor is located extracellularly and modulates ATP-sensitive K+ channels.
20.
Imidazoline I2 binding sites are heterogeneous in nature and have been observed in the brain of a number of species. Development of specific imidazoline I2 radioligands, such as [3H]2-BFI and [3H]BU224, that have a high affinity for the imidazoline I2 binding site, has enabled the central distribution of these sites to be mapped. Extensive studies have been conducted on the rat brain with a number of radioligands. However, to date a comprehensive analysis of imidazoline I2 ligand binding in mouse brain has not been completed. In the present work we describe levels of [3H]2-BFI specific binding found throughout the mouse brain. [3H]2-BFI (2 nM) showed discrete regional distribution which was readily displaced by saturating concentrations of the specific imidazoline I2 ligand BU224. The highest levels of [3H]2-BFI specific binding were found in the dorsal raphe, paraventricular thalamus and nucleus accumbens. Moderate levels were found throughout the lining of the aqueduct, lateral ventricle, lateral 4th ventricle, 4th ventricle, 3rd ventricle, but not the dorsal 3rd ventricle. Based on the loss of [3H]idazoxan binding in brain homogenates from monoamine oxidase-A and B (MAO-A and MAO-B) deficient mice it has been suggested that imidazoline I2 binding sites are predominantly on MAO. Consistent with this hypothesis the regional distribution of [3H]2-BFI shows some overlap with that previously reported for MAO. However, in the rat imidazoline I2 binding sites have been shown to be heterogeneous in nature and it is likely [3H]2-BFI is binding to multiple imidazoline I2 binding sites within mouse brain. 相似文献