Modification of the rat adipocyte A1 adenosine receptor-adenylate cyclase system during chronic exposure to an A1 adenosine receptor agonist: alterations in the quantity of GS alpha and Gi alpha are not associated with changes in their mRNAs

JP Longabaugh, J Didsbury, A Spiegel and GL Stiles

Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.

The A1-adenosine receptor (A1AR) adenylate cyclase system in rat adipocytes undergoes heterologous desensitization following chronic in vivo exposure to an A1AR agonist (+)-N6-(R-phenylisopropyl)adenosine [J. Biol. Chem. 262:841-847 (1987)]. This desensitization involves an absolute increase in adenylate cyclase activity and a refractoriness to receptor ligands that are inhibitory to adenylate cyclase. In this study, receptor changes were characterized using an A1AR antagonist radioligand, [3H]8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl] oxy]phenyl]-1,3-dipropyl xanthine. Saturation binding studies demonstrated a 47% decrease in total A1AR density without a change in KD. Agonist competition studies revealed a decreased percentage of receptors, from 55% to 35%, in the high affinity state following desensitization. An increase in GS alpha of 49% was found by Western blotting using specific GS alpha antibodies. Further, an antibody that recognizes Gi alpha 1 adn Gi alpha 2 was used to quantitate these subtypes of Gi alpha and both were decreased by 59% following desensitization. However, when an antibody that recognizes Gi alpha 3 was used, no change in Gi alpha 3 was found, demonstrating, in this case, differential regulation of Gi alpha subtypes. The mechanisms responsible for changes in GS alpha and Gi alpha were studied by measuring the levels of their mRNAs from normal and desensitized adipocytes. Using either labeled cDNAs (Gi alpha 2, Gi alpha 3) or oligonucleotides (GS alpha, Gi alpha 1), Northern analysis demonstrated that mRNAs for GS alpha and all three isoforms of Gi alpha are present in adipocytes but that there are no changes in the levels of any of these transcripts following desensitization. These data suggest that desensitization of the A1AR-adenylate cyclase system involves a down- regulation of A1ARs and an additional loss of A1AR agonist high affinity sites. Further, an increase in GS alpha, a decrease in Gi alpha 1 and Gi alpha 2, and no change in Gi alpha 3 were found. The regulation of GS alpha and the subtypes of Gi alpha in this system does not occur by altering the levels of their respective transcripts.

Volume 36, Issue 5, pp. 681-688, 11/01/1989
Copyright © 1989 by American Society for Pharmacology and Experimental Therapeutics

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