SYNTHESIS AND CHARACTERIZATION OF A HIGH-AFFINITY RADIOIODINATED PROBE FOR THE ALPHA-2-ADRENERGIC RECEPTOR

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  • 1 March 1986
    • journal article
    • research article
    • Vol. 29  (3) , 219-227
Abstract
The availability of radioiodinated probes has facilitated the localization and molecular characterization of cell membrane receptors for hormones and neurotransmitters. However, such probes are not available for the study of the .alpha.2-adrenergic receptor. This report describes the synthesis and characterization of functionalized derivatives of the selective .alpha.2-adrenergic antagonists, rauwolscine and yohimbine, which can be radiolabeled to high specific activity with 125I. Following demethylation of rauwolscine or yohimbine, the resultant carboxylic acid derivatives were reacted with 4-aminophenethylamine to yield the respective 4-aminophenethyl carboxamides, 17.alpha.-hydroxy-20.alpha.-yohimban-16.beta.-[N-4-amino-phenethyl]carboxamide (rau-pAPC) and 17.alpha.-hydroxy-20.beta.-yohimban-16.alpha.-[N-4-aminophenethyl]carboxamide. In competitive inhibition studies using rat renal membranes and the radioligand [3H]rauwolscine, rau-pAPC (Ki = 11 .+-. 1 nM) exhibited a 14-fold greater affinity than the corresponding yohimbine derivative (Ki = 136 .+-. 45 nM). The higher affinity compound, rau-pAPC, was radioiodinated by the chloramine T method, and the product, 125I-rau-pAPC [17 .alpha.-hydroxy-20-.alpha.-yohimban-16.beta.-(N-4-amino-3-[125I]-iodophenethyl)carboxamide], was purified by reverse phase HPLC to high specific activity (2175 Ci/mmol) and its binding characteristics were investigated in rat kidney membranes. Specific binding of 125I-rau-pAPC was saturable and of high affinity as determined by Scatchard analysis (KD = 1.8 .+-. 0.3 nM) or from kinetic studies (KD = k2/k1 = 0.056 .+-. 0.013 min-1/ 4.3 .+-. 0.2 .times. 107 M-1 min-1 = 1.3 .+-. 0.3 nM). In competition studies, .alpha.-adrenergic antagonists and agonists inhibited the binding of 125I-rau-pAPC with a potency order consistent with an interaction at .alpha.2-adrenergic receptors (rauwolscine > phentolamine > prazosin; clonidine > (-)-epinephrine > (-)-norepinephrine > dopamine > (+)-epinephrine). In rat liver and human platelet membranes, high affinity binding of 125I-rau-pAPC was also observed (liver, KD = 1.2 .+-. 0.4 nM; platelet, KD = 3.2 .+-. 1.5 nM). In addition, the density of .alpha.2-adrenergic receptors identified from binding studies with 125I-rau-pAPC in kidney, liver, and platelet membranes was similar to that observed in parallel studies with [3H]rauwolscine. These findings indicate that 125I-rau-pAPC is a high affinity probe that selectively identifies .alpha.2-adrenergic binding sites. Availability of this radioligand should facilitate the localization and biochemical characterization of this .alpha.-adrenergic.

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