PURIFICATION AND CHARACTERIZATION OF (-)[I-125]HYDROXYPHENYLISOPROPYLADENOSINE, AN ADENOSINE R-SITE AGONIST RADIOLIGAND AND THEORETICAL-ANALYSIS OF MIXED STEREOISOMER RADIOLIGAND BINDING

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  • 1 January 1984
    • journal article
    • research article
    • Vol. 26  (3) , 414-423
Abstract
(-)-N6-(R-4-Hydroxyphenylisopropyl)adenosine (HPIA) was iodinated with NaI and trace 125I. Mono- and diiodinated reaction products and the starting material were separated by high pressure liquid chromatography and the structures of the reaction products were verified by NMR. (-)-N6-(R-Phenylisopropyl)adenosine (PIA), IHPIA, and I2HPIA decreased rat atrial contractility with ED50 values of 24, 28 and 33 nM, respectively. The contractile effects of these compounds were competitively blocked by theophylline (Ki = 7.9 .mu.M), but were not affected by adenosine deaminase. IHPIA also inhibited (-)isoproterenol-stimulated cAMP accumulation in adipocytes with an ED50 (10 nM) and to an extent (83%) nearly identical to PIA. [125I]HPIA prepared using carrier-free 125I bound to adenosine receptors on membranes from rat cerebral cortex, adipocyte ghosts and heart ventricles. Binding was inhibited stereospecifically by PIA and by other adenosine analogs and alkylxanthines. The Kd of [125I]HPIA determined kinetically using brain membranes at 21.degree. was 0.94 nM (Ki = 2.55 .times. 107 M-1 min-1; K-1 = 0.024 min-1) in gooid agreement with the equilibrium determination of 1.94 nM. The density of adenosine receptors in brain membranes was 871 fmol/mg of protein. When normalized to protein, the density of receptors in heart membranes and adipocyte ghosts, respectively, was 39- and 2.3-fold less than in brain membranes. Evidently, [125I] HPIA can be rapidly synthesized and purified, binds to adenosine R-sites and is an agonist radioligand resistant to adenosine deaminase. Computer modeling of the equilbrium binding resulting from the use of mixed stereoisomers of a radioligand indicates that the combined use of (-)[125I]HPIA and (+)[125I]HPIA would result in the generation of nonlinear Scatchard plots.

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