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

J. P. Longabaugh, J. Didsbury, Allen M. Spiegel, G. L. Stiles

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)681-688
Number of pages8
JournalMolecular Pharmacology
Volume36
Issue number5
StatePublished - 1989
Externally publishedYes

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Adenosine A1 Receptor Agonists
Adenosine A1 Receptors
Adenylyl Cyclases
Adipocytes
Messenger RNA
Antibodies
Adenosine A1 Receptor Antagonists
Xanthine
Oligonucleotides
Adenosine
Protein Isoforms
Down-Regulation
Complementary DNA
Western Blotting
Ligands

ASJC Scopus subject areas

  • Pharmacology

Cite this

@article{b1571b5b9aa24a3e96d9a6e68522093d,
title = "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",
abstract = "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.",
author = "Longabaugh, {J. P.} and J. Didsbury and Spiegel, {Allen M.} and Stiles, {G. L.}",
year = "1989",
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TY - JOUR

T1 - Modification of the rat adipocyte A1 adenosine receptor-adenylate cyclase system during chronic exposure to an A1 adenosine receptor agonist

T2 - Alterations in the quantity of G(Sα) and G(iα) are not associated with changes in their mRNAs

AU - Longabaugh, J. P.

AU - Didsbury, J.

AU - Spiegel, Allen M.

AU - Stiles, G. L.

PY - 1989

Y1 - 1989

N2 - 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.

AB - 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.

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