Riochemical Pharmauolagy. Vol. 28. pp. 12% 1288. $3 Pergamon Prcsa I&.. 19 iY. Print& in Grektt zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFED Brita in. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 0006 352 :Y 0415~. 12X3 iOZ.OO/O zyxwvut SPECIFIC AND UNSPECIFIC BINDING OF I 3H I(-)DIHYDROALPRENOLOL TO CARDIAC TISSUE * WCUGANG KRAWIETZ and ERLANW ERDMANS Medizinische Klinik I der Universitiit Miinchen. Klinikum Grobhadern. D-8000 Miinchen 70. Germany (Receised 20 Ju& 19%: accepted 17 November 1978) Abort-Identifi~tion of ~-adrenergic receptors has been successfully performed by I “H I(-~ihydro~prenoloi binding to several tissues of a variety of species. Specific binding sites (receptors) have usually been defined by criteria as: velocity. reversibility. saturability and stereospecificity of binding. However. I ‘H I(-)dihydroalprenolol does not only bind to specific sites but also to unspecific ones. The given definitions of unspecific binding sites are somewhat confusing. l.‘H If-)DihydroalprenoloI bound (2670 c.p.m./mg prot.) to cardiac membranes is inhibited in the presence of unlabeled alprenolol in a concentration-dependent manner up to IO ’ M. From 10 ’ to IO” M this inhibition caused by (-) and (+) alprenolol was found to be stereospecific. In higher concentralions the measured inhibition of I >H I(-)dihydroalprenoloI binding to its receptor was no longer stereospecific. In heat denatured membranes 1 ZHl(-)dihydroalprenolol bound (1335 c.p.m./mg prot.) was identical with that of native membranes in the presence of 10.’ M (-) or (+) alprenolol. There was no inhibition of binding. however. in the presence of unlabeled (-) or (+) alprenolol in a concentration range below IO ’ M. At higher concentrations there was inhibition of binding. but it was not stereospecific. When an equilibrium of I ‘H I(-khhydroalprenolol binding to all cardiac membranes (native or heat denatured) had been established. addition of the unlabeled ligand led to a concentration-dependent and rapid displacement of the labeled l&and. Again. stereospecificity of the displacement was only observed in the native membranes. inhibition of adenylate cyclase activity was measured at the same (-) and (+) alprenolol concentrations and showed the same stereospecific effects. Thus. the results indicate that the loss of stereospecilicity in heat denatured membranes and in native membranes correlates with unspecific binding of l’HJ(-)dihydroalprenolol to cardiac membranes. It is quantitatively identical. It is concluded. that out of the criteria for specific binding mentioned. only stereospecificity seems to be valid for I ‘H I(-)dihydroalprenoloI binding to the /%adrenergic receptor. Receptors have formerly been defined rather indirectly by observations and calculations of certain physiologi- cal and ph~cologic~ effects after addition of hor- mones or drugs I 11. It should be kept in mind, however. that the term ‘%eceptor” is only a general one and its use implies a lack of knowledge of chemical structures involved in the interactions. Thus, a hormone receptor is supposed to be a bifunctional element that, in re- sponse to its interactions with the hormone. leads to the generation of a stimulus which in turn triggers some kind of a measurable response i 21. In the last few years there has been a great interest in characterizing recep- tors with labeled hormones and drugs by binding stud- ies 13-91. This way, it has become possible to note details of drug-(hormone)-receptor interactions, the initial step of an effect of a pharmacon. Labeled drugs and hormones, however, do not bind to their respective receptor only. Several inv~tigators have reported experiments demonstrating binding of radioactively labeled insulin to. for instance. talc, silicon, or glass tubes 1101 or of labeled nerve growth factor to glass beads 1111. Although this binding showed properties very similar to that of specific hor- mone-receptor binding (i.e. high affinity, time and - * Supported by the Deutsche Forschungsgemeinschaft (ER 65/l). concentration-dependent binding, displacement by un- labeled hormones etc.) it must lx termed as unspecific binding, because there is no biological activity as an answer to this “ho~on~~ptor binding” [ 121. Recently a great number of reports have demon- strated bindiig of 1 3HI(-~ihydro~pr~olol to a vari- ety of cell membranes I 13- 19 I. The specific binding of this jl-adrenergic receptor blocking agent to the mem- brane bound /&receptor causes an alteration of the activity of the receptor-coupled adenylate cyclase 15 1. There is, however, no generally accepted definition of “specific” and %nspecific” binding of this drug to its receptor. There are reports terming that binding of 13H1(-)dihydroalprenolol to membranes as “unspe- cific” which cannot be inhibited in the presence of unlabeled @-blocking agents in concentrations greater than 1 0w6 M (-) alprenolol [ 201, IO-’ M (-) proprano- 101 or (--) ~~enololl211, 10-M (t) proprano- 101122-231, 10m4 M (&) propranolol i 241, lo-’ M (-) propranolol or (-) alprenolol[251, or IO-” M (-) iso- prenaline 1261. The reason for this definition is not quite clear either, and “sometimes rather confus- ing” I 27 I as “unspecific” binding of this labeled drug to cell membranes may amount to 3-50 per cent of total binding [ 24-26 1. Thus, there is a need for an exact determination of specific and unspecific [ JH I(-klihydroalprenolol bind- ing to biological material. 1283