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 I₁R were able to bind with similar affinities to α₂-adrenergic receptors (α₂-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°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 I₂binding site and α₂AR, 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.