[I]2-(2-Chloro-4-iodo-phenylamino)-5-methyl-pyrroline (LNP 911), a High-Affinity Radioligand Selective for I1 Imidazoline Receptors
نویسندگان
چکیده
The I1 subtype of imidazoline receptors (I1R) is a plasma membrane protein that is involved in diverse physiological functions. Available radioligands used so far to characterize the I1R were able to bind with similar affinities to 2-adrenergic receptors ( 2-ARs) and to I1R. This feature was a major drawback for an adequate characterization of this receptor subtype. New imidazoline analogs were therefore synthesized and the present study describes one of these compounds, 2-(2-chloro-4-iodophenylamino)-5-methyl-pyrroline (LNP 911), which was of high affinity and selectivity for the I1R. LNP 911 was radioiodinated and its binding properties characterized in different membrane preparations. Saturation experiments with [I]LNP 911 revealed a single high affinity binding site in PC-12 cell membranes (KD 1.4 nM; Bmax 398 fmol/mg protein) with low nonspecific binding. [I]LNP 911 specific binding was inhibited by various imidazolines and analogs but was insensitive to guanosine-5 -O-(3-thio)triphosphate. The rank order of potency of some competing ligands [LNP 911, PIC, rilmenidine, 4-chloro-2-(imidazolin-2ylamino)-isoindoline (BDF 6143), lofexidine, and clonidine] was consistent with the definition of [I]LNP 911 binding sites as I1R. However, other high-affinity I1R ligands (moxonidine, efaroxan, and benazoline) exhibited low affinities for these binding sites in standard binding assays. In contrast, when [I]LNP 911 was preincubated at 4°C, competition curves of moxonidine became biphasic. In this case, moxonidine exhibited similar high affinities on [I]LNP 911 binding sites as on I1R defined with [ I]PIC. Moxonidine proved also able to accelerate the dissociation of [I]LNP 911 from its binding sites. These results suggest the existence of an allosteric modulation at the level of the I1R, which seems to be corroborated by the dose-dependent enhancement by LNP 911 of the agonist effects on the adenylate cyclase pathway associated to I1R. Because [ I]LNP 911 was unable to bind to the I2 binding site and 2AR, our data indicate that [ I]LNP 911 is the first highly selective radioiodinated probe for I1R with a nanomolar affinity. This new tool should facilitate the molecular characterization of the I1 imidazoline receptor. Most of the imidazoline ligands, such as clonidine, idazoxan, and related compounds, are known to bind to 2adrenergic receptors as well as to imidazoline receptors (Ruffolo et al., 1995). Extensive biochemical and physiological studies led to the subclassification of imidazoline receptors in three main classes: I1, I2, and I3 (Regunathan and Reis, 1996; Ernsberger, 1999; Bousquet et al., 2000). Cloning strategies and biochemical studies have assigned I2 binding sites (I2BS) to a modulatory site on monoamine oxidases A and B (Tesson et al., 1995). The existence of I3 receptors (non-I1/non-I2) has been suggested according to insulin release properties of some imidazolines in pancreatic cells (Chan et al., 1991; Zaitsev et al., 1996; Rustenbeck et al., 1997). Although KATP channel closure has been implicated in these effects, recent results (Efanov et al., 2001a,b; Chan et al., 2001) have characterized a KATP independent pathway activation leading to enhanced insulin release by imidazolines. However, attempts to characterize the associated imidazoline binding sites have been unsuccessful because of the lack of specific radioligands. Conversely, the I1 subtype of imidazoline receptors has been amply characterized by binding assays using radiolabeled clonidine or analogs (Molderings et al., 1993; Piletz et al., 1996; Ernsberger et al., 1997) and its pharmacological selectivity assessed. I1 receptor (I1R) is a plasma membrane receptor protein (Heemskerk et al., 1998) that has been shown to be coupled to a G protein in human platelets, bovine chromaffin cells, and PC-12 cells (Molderings et al., 1993; Piletz et al., 1996; Greney et al., 2000). Transduction pathways have already been associated with this receptor in the G.H. and U.D. contributed equally to this work. ABBREVIATIONS: I2BS, I2 binding site(s); I1R, I1 receptor(s); PIC, para-iodoclonidine; LNP 911, 2-(2-chloro-4-iodo-phenylamino)-5-methylpyrroline; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; HME, HEPES/MgCl2/EGTA; 2AR, 2-adrenoceptor(s); PBS, phosphate-buffered saline; CHO, Chinese hamster ovary; BDF6143, 4-chloro-2-(imidazolin-2-ylamino)-isoindoline; GTP S, guanosine-5 -O-(3thio)triphosphate. 0026-895X/02/6201-181–191$7.00 MOLECULAR PHARMACOLOGY Vol. 62, No. 1 Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics 1282/993859 Mol Pharmacol 62:181–191, 2002 Printed in U.S.A. 181 at A PE T Jornals on O cber 3, 2017 m oharm .aspeurnals.org D ow nladed from PC-12 cells: activation of a phosphocholine-specific phospholipase C (Separovic et al., 1996) and inhibition of an adenylate cyclase (Greney et al., 2000). Moreover, pharmacological studies have shown that this receptor is involved in several functions such as regulation of the cardiovascular function (Bousquet et al., 1984; Ernsberger et al., 1990), modulation of the ocular pressure (Ogidigben et al., 2001), control of the catecholamine release from chromaffin cells (Nguyen and De Lean, 1987), and renal sodium excretion (Smyth and Penner, 1999). So far, all the radioligands used to characterize the I1 receptors were “hybrid” molecules able to bind with similar affinities both to I1 receptors and to 2-adrenoceptors (Molderings et al., 1993; Piletz et al., 1996; Ernsberger et al., 1997). The lack of selective ligands hindered the use of these radioligands in binding assays on membranes bearing the two types of receptors. In such membrane preparations, 2-adrenoceptor blocking conditions were essential to reveal and characterize the I1 receptors. In an effort to fully characterize these receptors, we developed a series of new imidazolines or analogs to obtain selective and high-affinity I1 receptor ligands. We recently synthesized a series of pyrroline analogs with no detectable affinities for I2BS as well as for 2-adrenoceptors (Schann et al., 2001). Among the available pyrroline compounds, LNP 911 was found to be highly selective for I1R and interestingly exhibited nanomolar affinity for these receptors. As unlabeled LNP 911 exhibited high-affinity for I1 receptors detected by [I]para-iodoclonidine (PIC) in PC-12 cell membranes and high selectivity for I1 receptors compared with 2-adrenoceptors and I2 binding sites (S. Schann, H. Greney, M. Dontenwill, D. Urosevic, C. Rascente, G. Lacroix, L. Monassier, V. Bruban, J. Feldman, B. Pfeiffer, et al. Methylation of imidazoline related compounds leads to loss of 2-adrenoceptor affinity. Synthesis and biological evaluation of new selective I1 imidazoline receptor tools, manuscript in preparation), we decided to radioiodinate this drug and to investigate the resulting radioligand as a selective probe for the I1 receptors. This report describes the properties of [I]LNP 911 as the first selective I1 receptor radioligand and, hence, for the first time, an allosteric modulation of the I1 receptor binding site has been suggested by the use of this ligand. In addition, LNP 911 behaves as an allosteric enhancer of I1 receptor transduction pathway. Experimental Procedures Materials. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Invitrogen (Cergy-Pontoise, France). Benazoline was synthesized by Prof. Pigini (Camerino, Italy), BDF6143 was kindly provided by Beiersdorf-Lilly (Hamburg, Germany). Moxonidine was kindly provided by Solvay Pharmaceuticals GmbH (Hannover, Germany). Cirazoline was a gift from Synthelabo (Bagneux, France). Rilmenidine was a gift from Laboratoires Servier (Courbevoie, France). All other compounds were purchased from Sigma (L’Isle d’Abeau Chesnes, Saint-Quentin Fallavier, France). [H]Idazoxan and [H]RX 821002 was obtained from Amersham Biosciences (Orsay, France). [I]PIC was purchased from PerkinElmer Life Sciences (Paris, France). The chemical structures of the imidazoline and pyrroline compounds are shown in Fig. 1. LNP 911 Synthesis. The LNP 911 [2-(2-chloro-4-iodo-phenylamino)-5-methyl-pyrroline] synthesis will be described in detail elsewhere. Briefly, 2-(2-chloro-4-bromo-phenylamino)-5-methyl-pyrroline was obtained by reaction of 4-bromo-2-chloro-aniline with 5-methyl-pyrrolidinone in the presence of POCl3. This intermediate was then transformed into the stannous compound 2-(2-chloro-4tributylstannyl-phenylamino)-5-methyl-pyrroline. The radioiodination step was made as follow: 2-(2-chloro-4-tributylstannyl-phenylamino)-5-methyl-pyrroline (50 g) in MeOH (50 l) was mixed with HCl (0.5 M; 42 l) and [I]NaI (IMS30; 185 MBq; 5 mCi; 50 l; Amersham Biosciences, Little Chalfont, Buckinghamshire, England). Reaction was initiated by addition of chloramine-T (50 l; 1 mg/ml) and was allowed to react for 10 min. The reaction mixture was loaded onto a Jupiter C-18 reversed phase–high-performance liquid chromatography column (250 4.6 mm; Phenomenex, Macclesfield, Cheshire, England) and purified to obtain 70% yield of [I]LNP 911 (74 TBq/mmol; 2000 Ci/mmol) using a linear gradient with water, methanol, and trifluoroacetic acid. This product was diluted with ethanol to 100 Ci/ml and stored at 4°C. The cold marker [I]LNP 911 was demonstrated to coelute with the same retention time as [I]LNP 911. The radiochemical purity of [I]LNP 911 was shown to be 95% with less than 1% free iodide at initial analyses on a C-18 column using a linear gradient with water, propanol, and trifluoroacetic acid. The synthesis of intermediates was made in our laboratory by S.S. and J.D.E. and the custom radioiodination step was carried out by Iodine Ligand Development, Amersham Biosciences. Cell Cultures. PC-12 cells were obtained from Dr. G. Rebel (IRCAD, Strasbourg, France). They were cultured in 75-cm flasks in DMEM (1000 mg/l glucose) supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 g/ml streptomycin. When the cells reached confluence (3 to 4 days after plating), they were harvested by 2-min exposure to 0.25% trypsin at 37°C. For binding assays, after removing the medium, cells at confluence were frozen in the flasks at 20°C until use to prepare membranes. HT29 cells were obtained from Dr. H. Paris (Institut National de la Santé et de la Recherche Médicale U338, Toulouse, France) and cultured in 75-cm flasks in DMEM (4500 mg/l glucose) supplemented with 10% heatinactivated FBS, 100 U/ml penicillin, and 100 g/ml streptomycin. Cells were harvested at confluence after 48-h incubation in fresh DMEM without FBS, and membranes were prepared immediately. CHO2A, CHO2B, and CHO2c cell lines expressing the human Fig. 1. Chemical structures of imidazoline and pyroline compounds. 182 Greney et al. at A PE T Jornals on O cber 3, 2017 m oharm .aspeurnals.org D ow nladed from recombinant adrenoceptors (provided by Prof. A. D. Strosberg, Paris, France) were grown in Ham’s F12 medium supplemented with 2 mM glutamine, 10% fetal bovine serum, 100 IU/ml penicillin, 100 g/ml streptomycin, and 400 g/ml G418, in 5% CO2 at 37°C. Cells were passaged every 3 to 4 days. These CHO2A, CHO2B, and CHO2c cell lines expressed 1.8, 10, and 1.3 pmol of receptors/mg of protein, respectively, as determined by saturation experiments with [H]RX
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