TY - JOUR T1 - Binding of Iodinated <em>Beta</em> Adrenergic Antagonists to Proteins Derived from Rat Heart JF - Molecular Pharmacology JO - Mol Pharmacol SP - 1 LP - 15 VL - 12 IS - 1 AU - T. KENDALL HARDEN AU - BARRY B. WOLFE AU - PERRY B. MOLINOFF Y1 - 1976/01/01 UR - http://molpharm.aspetjournals.org/content/12/1/1.abstract N2 - Hydroxybenzylpindolol [Aurbach, G. D., Fedak, S. A., Woodard, C. J., Palmer, J. S., Hauser, D. &amp; Troxler, F. (1974) Science, 186, 1223-1224] and hydroxyphenyl derivatives of alprenolol and KL-255 have been prepared. These compounds were iodinated and studied as potential ligands for the investigation of beta adrenergic receptors in vitro. The iodinated derivatives were potent inhibitors of isoproterenol-stimulated adenylate cyclase (EC 4.6.1.1.) activity. Iodohydroxybenzylpindolol (IHYP) proved to be the most useful ligand, and the properties of the binding of IHYP to proteins derived from rat ventricular muscle have been characterized. The l stereoisomers of propranolol, isoproterenol, epinephrine and norepinephrine were more potent by an average of two orders of magnitude than the corresponding d isomers in the inhibition of IHYP binding. The Kd values of 10 antagonists were determined by direct binding assay and by inhibition of isoproterenol-stimulated adenylate cyclase. The Kd values of eight of these compounds, as determined by the two methods, were in good agreement, while in two cases they differed by approximately one order of magnitude. The EC50 values of isoproterenol, epinephrine, and norepinephrine for adenylate cyclase stimulation were determined and compared with their Kd values as determined by competition with IHYP for binding sites on rat ventricular muscle protein. The order of potency was the same in both cases, but the absolute potency of all three agonists was substantially lower in studies of adenylate cyclase than in binding studies. High concentrations of a variety of biologically active amines which would not be expected to interact with beta receptors did not affect the binding of IHYP. Specific binding was defined as the binding of IHYP in the presence of 1 µM d-isoproterenol minus its binding in the presence of 1 µM l-isoproterenol. When IHYP (0.02 nM) was incubated with 200-400 µg of protein, equilibrium was achieved in approximately 40-60 min. Dissociation of specific binding took place with a half-time of about 15 min. The Kd of IHYP was approximately 1.4 nM as determined both by Scatchard analysis and by direct measurements of rates of association and dissociation. The binding of IHYP was saturable, and there was approximately 0.16 pmole of IHYP binding sites per milligram of membrane protein. The binding sites have many of the properties expected of beta adrenergic receptors in vitro, and it is likely that these sites represent the physiologically significant receptors. ACKNOWLEDGMENTS The authors are grateful to Drs. Alfred Gilman and Gerald Aurbach for providing us with HYP, and to Dr. Robert Murphy for help in providing mass spectral analysis of HYP. Ms. Jane Andrews and Mr. Arnold Chacon provided excellent technical assistance. ER -