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 G(Sα) and G(iα) are not associated with changes in their mRNAs

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-di-p ropyl xanthine. Saturation binding studies demonstrated a 47% decrease in total A₁AR density without a change in K(D). Agonist competition studies revealed a decreased percentage of receptors, from 55% to 35%, in the high affinity state following desensitization. An increase in G(Sα) of 49% was found by Western blotting using specific G(sα) antibodies. Further, an antibody that recognizes G(iα1) and G(iα2) was used to quantitate these subtypes of G(iα) and both were decreased by 59% following desensitization. However, when an antibody that recognizes G(iα3) was used, no change in G(iα3) was found, demonstrating, in this case, differential regulation of G(iα3)subtypes. The mechanisms responsible for changes in G(Sα) and G(iα) were studied by measuring the levels of their mRNAs from normal and desensitized adipocytes. Using either labeled cDNAs (G(iα2), G(iα3)) or oligonucleotides (G(Sα), G(iα1)), isoforms of G(iα) 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 G(Sα), a decrease in G(iα1) and G(iα2), and no change in G(iα3) were found. The regulation of G(sα) and the subtypes of G(iα) in this system does not occur by altering the levels of their respective transcripts.