By J. Linden, C. Q. Earl, A. Patel, R. H. Craig, S. M. Daluge (auth.), Professor Dr. med. Eckehart Gerlach, Dr rer nat. Bernhard Friedrich Becker (eds.)

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Extra resources for Topics and Perspectives in Adenosine Research: Proceedings of the 3rd International Symposium on Adenosine, Munich, June 1986

Sample text

Although the solubilization of adenosine receptors was reported previously, the effect of adenosine deaminase treatment was not evaluated [7, 12, 16, 22]. Indeed, in these studies the adenosine Rj receptor appeared to be unusual in that a high-affinity form of the receptor that was regulated by guanine nucleotides was extracted without the addition of stabilizing ligands. However, the present study demonstrates that the adenosine Rj receptor is similar to other inhibitory receptors coupled to adenylate cyclase, such as aradrenergic [20] and Drdopaminergic receptors [9], in that occupancy of the receptor with agonists prior to solubilization promotes the formation of a high-affinity state of the receptor.

Hydrodynamic Studies The hydrodynamic properties of the adenosine R j receptor were determined with cholate extracts that were prebound with [3H]PIA. In these experiments, membranes were incubated with 2-4 nM[3H]PIA in buffer containing 3 mMMgCi 2 for 2 h at 24° C. The membranes were then centrifuged at 12 000 gfor 20 min at 4° C. The supernatant was discarded, and the membranes were washed once and sedimented as above. The pellet was resuspended in buffer and solubilized as described above. Treatment of these cholate extracts with GTP abolished all the specific [3H]PIA bound, indicating that the radioactivity was associated specifically with adenosine receptors that interacted with N j • Prior to centrifugation, these preparations were filtered over a lO-cm column of Sephadex G50 to separate the free from the bound radioligand [22].

T\ 5 4 +\ 3 -.... 1 2 Cuii a.. :E « :::'7 0 -""'-------- reversible JL " E -6 Y If-~ 5 __ ~ ""1 \ 4 t. ~ R-PIA ~ ..... R-AHPIA "'+"'-... 3 2 1 +- R-AHPIA covalent II o" 10-10 10-9 10-8 10-7 Concentration of R-AHPlAIR-PIA (M) Fig. 3. Reduction of cyclic AMP levels in isolated rat fat cells by R-AHPIA. The upper graph shows the direct reduction caused by the presence of the indicated concentrations of R-AHPIA. The lower graph shows the reduction caused by pretreatment with the indicated concentrations of either R-AHPIA or R-PIA and UV irradiation, followed by blockade with 1 mM theophylline of all Al receptors not covalently labelled.

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