DEVELOPMENT OF A HIGH-AFFINITY RADIOIODINATED LIGAND FOR IDENTIFICATION OF IMIDAZOLINE-GUANIDINIUM RECEPTIVE SITES (IGRS) - INTRATISSUE DISTRIBUTION OF IGRS IN LIVER, FOREBRAIN, AND KIDNEY

Imidazoline/guanidinium receptive sites (IGRS) are membrane proteins t hat exhibit high affinity for various compounds with an imidazoline or guanidinium moiety. The structure of these binding sites and their si gnificance in the broad pharmacological action of such ligands are unc lear. To address this issue, we developed selective high affinity comp ounds that could be radioiodinated and used as molecular probes for st ructural characterization of these proteins. This report describes the synthesis and characterization of such a molecule, 2-(3-amino-4-[I-12 5]iodophenoxy)methylimidazoline ([I-125]AMIPI). [I-125]AMIPI is struct urally related to cirazoline, an imidazoline exhibiting high affinity for IGRS and the family of related imidazoline binding sites. The phen yl-substituted analogue of cirazoline, 2-(3-aminophenoxy)methylimidazo line, was generated by alkylation of acetamidophenol with 2-chlorometh ylimidazoline. 2-(3-Aminophenoxy)methylimidazoline exhibited high affi nity for IGRS in rabbit kidney membranes, as determined in competition binding studies with [H-3]idazoxan (K-i = 12.5 +/- 7.5 nM), and was r adioiodinated by chloramine-T oxidation to yield [I-125]AMIPI. The bin ding properties of [I-125]AMIPI were determined in membranes prepared from two representative tissues, rabbit kidney cortex and rat liver. S pecific binding of [I-125]AMIPI was saturable and of high affinity, as determined by Scatchard analysis of saturation binding isotherms (rab bit kidney, K-d = 2.0 +/- 0.9 nM, B-max = 554 +/- 201 fmol/mg, five ex periments; rat liver, K-d = 2.6 +/- 1.3 nM, B-max = 73 +/- 10 fmol/mg, three experiments). [I-125]AMIPI binding in rabbit kidney membranes w as inhibited by various imidazolines and guanidinium compounds, with t he following rank order of potency: cirazoline K-i = 3.6 +/- 0.96 nM)> idazoxan (high-affinity site K-i= 0.2 +/- 0.1 nM and low-affinity sit e K-i= 76 +/- 30 nM)> guanabenz (high-affinity site K-i= 1.7 +/- 0.84 nM and low-affinity site K-i 201 +/- 72.7 nM)> amiloride (K-i= 625 +/- 130 nM)> clonidine (K-i = 2200 +/- 200 nM) > p-aminoclonidine (K-i = 3422 +/- 172 nM). [I-125]AMIPI binding was not inhibited by the alpha( 2)-adrenergic receptor antagonist rauwolscine or endogenous agonists f or neurotransmitter receptors (epinephrine, histamine, serotonin, and dopamine). The rank order of competing ligands is consistent with the definition of the [I-125]AMIPI binding site as an IGRS. Receptor autor adiography was used to determine the intratissue distribution of IGRS in rat liver, rat forebrain, and canine kidney. Autoradiograms indicat ed homogeneous specific binding of [I-125] AMIPI in liver. Renal [I-12 5]AMIPI binding was observed as discrete cortical rays. Autoradiograms of rat forebrain tissue sections indicated high densities of specific binding in the hypothalamic arcuate nucleus and the subfornical organ . [I-125]AMIPI binding was inhibited by both imidazoline and guanidini um ligands but was not competed for by the selective alpha(2)-adrenerg ic receptor antagonist rauwolscine. These data indicate that [I-125]AM IPI is a high affinity probe that is specifically recognized by the IG RS and thus should facilitate pharmacological and molecular characteri zation of this entity.