title: [125I]2-(2-chloro-4-iodo-phenylamino)-5-methyl-pyrroline (LNP 911), a high-affinity radioligand selective for I1 imidazoline receptors.
abstract: 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 I(1)R were able to bind with similar affinities to alpha2-adrenergic receptors (alpha2-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-iodo-phenylamino)-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 [125I]LNP 911 revealed a single high affinity binding site in PC-12 cell membranes (K(D) = 1.4 nM; B(max) = 398 fmol/mg protein) with low nonspecific binding. [125I]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-2-ylamino)-isoindoline (BDF 6143), lofexidine, and clonidine] was consistent with the definition of [125I]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 [125I]LNP 911 was preincubated at 4 degrees C, competition curves of moxonidine became biphasic. In this case, moxonidine exhibited similar high affinities on [125I]LNP 911 binding sites as on I1R defined with [125I]PIC. Moxonidine proved also able to accelerate the dissociation of [125I]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 [125I]LNP 911 was unable to bind to the I2 binding site and alpha2AR, our data indicate that [125I]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.
authors.affiliation: Laboratoire de Neurobiologie et Pharmacologie Cardiovasculaire, Faculté de Medecine, Strasbourg, France.
authors.forename: Hugues
authors.initials: H
authors.lastname: Greney
authors.forename: Dragan
authors.initials: D
authors.lastname: Urosevic
authors.forename: Stephan
authors.initials: S
authors.lastname: Schann
authors.forename: Laurence
authors.initials: L
authors.lastname: Dupuy
authors.forename: Véronique
authors.initials: V
authors.lastname: Bruban
authors.forename: Jean-Daniel
authors.initials: JD
authors.lastname: Ehrhardt
authors.forename: Pascal
authors.initials: P
authors.lastname: Bousquet
authors.forename: Monique
authors.initials: M
authors.lastname: Dontenwill
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004347:Drug Interactions; D007093:Imidazoles / Q000494:pharmacology; D054755:Imidazoline Receptors; D007457:Iodine Radioisotopes; D016716:PC12 Cells; D011759:Pyrrolidines / Q000494:pharmacology*; D011869:Radioligand Assay; D051381:Rats; D018341:Receptors, Adrenergic, alpha-2 / Q000378:metabolism; D011955:Receptors, Drug / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: C459181:2-(2-chloro-4-iodophenylamino)-5-methylpyrroline; D007093:Imidazoles; D054755:Imidazoline Receptors; D007457:Iodine Radioisotopes; D011759:Pyrrolidines; D018341:Receptors, Adrenergic, alpha-2; D011955:Receptors, Drug; C410887:imidazoline I1 receptors; C043482:moxonidine