Development of a high-affinity radioiodinated ligand for identification of imidazoline/guanidinium receptive sites (IGRS) : intratissue distribution of IGRS in liver, forebrain, and kidney
Ivkovic, B.; Bakthavachalam, V.; Zhang, W.; Parini, A.; Diz, D.; Bosch, S.; Neumeyer, J.L.; Lanier, S.M.
Molecular Pharmacology 46(1): 15-23
1994
ISSN/ISBN: 0026-895X PMID: 8058049 Document Number: 423833
Imidazoline/guanidinium receptive sites (IGRS) are membrane proteins that exhibit high affinity for various compounds with an imidazoline or guanidinium moiety. The structure of these binding sites and their significance in the broad pharmacological action of such ligands are unclear. To address this issue, we developed selective high affinity compounds that could be radioiodinated and used as molecular probes for structural characterization of these proteins. This report describes the synthesis and characterization of such a molecule, 2-3-amino-4-(125I)iodophenoxy)methylimidazoline ((125I)AMIPI). (125I)AMIPI is structurally related to cirazoline, an imidazoline exhibiting high affinity for IGRS and the family of related imidazoline binding sites. The phenyl-substituted analogue of cirazoline, 2-(3-aminophenoxy)methylimidazoline, was generated by alkylation of acetamidophenol with 2-chloromethylimidazoline. 2-(3-Aminophenoxy)methylimidazoline exhibited high affinity for IGRS in rabbit kidney membranes, as determined in competition binding studies with (3H)idazoxan (K-i = 12.5 +- 7.5 nM), and was radioiodinated by chloramine-T oxidation to yield (125I)AMIPI. The binding properties of (125)AMIPI were determined in membranes prepared from two representative tissues, rabbit kidney cortex and rat liver. Specific binding of (125I)AMIPI was saturable and of high affinity, as determined by Scatchard analysis of saturation binding isotherms (rabbit kidney, K-d = 2.0 +- 0.9 nM, B-max = 554 +- 201 fmol/mg, five experiments; rat liver, K-d = 2.6 +- 1.3 nM, B-max = 73 +- 10 fmol/mg, three experiments). (125I)AMIPI binding in rabbit kidney membranes was inhibited by various imidazolines and guanidinium compounds, with the following rank order of potency: cirazoline (K-i = 3.6 +- 0.96 nM) gt idazoxan (high-affinity site K-i = 0.2 +- 0.1 nM and low-affinity site K-i = 76 +- 30 nM) gt guanabenz (high-affinity site K-i = 1.7 +- 0.84 nm and low-affinity site K-i = 201 +- 72.7 nM) gt amiloride (K-i = 625 +- 130 nM) gt clonidine (K-i = 2200 +- 200 nM) gt p-aminoclonidine (K-i = 3422 +- 172 nM). (125I)AMIPI binding was not inhibited by the alpha-2-adrenergic receptor antagonist rauwolscine or endogenous agonists for neurotransmitter receptors (epinephrine, histamine, serotonin, and dopamine). The rank order of competing ligands is consistent with the definition of the (125I)AMIPI binding site as an IGRS. Receptor autoradiography was used to determine the intratissue distribution of IGRS in rat liver, rat forebrain, and canine kidney. Autoradiograms indicated homogeneous specific binding of (125I) AMIPI in liver. Renal (125I)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. (125I)AMIPI binding was inhibited by both imidazoline and guanidinium ligands but was not competed for by the selective alpha-2-adrenergic receptor antagonist rauwolscine. These data indicate that (125I)AMIPI is a high affinity probe that is specifically recognized by the IGRS and thus should facilitate pharmacological and molecular characterization of this entity.