Gi down-regulation as a mechanism for heterologous desensitization in adipocytes.
Prolonged incubation of rat adipocytes with (-)N6-phenylisopropyl adenosine (PIA) (an A1 adenosine receptor agonist) leads to down-regulation of each of the three subtypes of Gi (Green, A., Johnson, J. L., and Milligan, G. (1990) J. Biol. Chem. 265, 5206-5210). To determine whether other inhibitors of adenylylcyclase would have similar actions, we incubated adipocytes in primary culture with PIA, prostaglandin E1 (PGE1), or nicotinic acid. After various times cells were homogenized, and crude membrane fractions were analyzed on Western blots using antipeptide antisera to alpha- and beta-subunits of G-proteins (SG1 (which binds to alpha i1 and alpha i2), I3B (which binds to alpha i3), BN2 (binds to beta-subunits) and CS1 (recognizes forms of alpha s)). PIA and PGE1 caused approximately 90% down-regulation of alpha i1 and alpha i3, and about 50% loss of alpha i2 and beta-subunits. In contrast, nicotinic acid at concentrations up to 1 mM had no effect on levels of any of these Gi subtypes. None of the compounds altered levels of either a 43- or 47-kDa form of alpha s. PIA caused about a 50% decrease in binding of [3H]DPCPX (an A1 adenosine receptor antagonist), indicating adenosine receptor down-regulation; however, neither PGE1 nor nicotinic acid treatment altered [3H]DPCPX binding. None of the treatments affected the activity of adenylylcyclase when measured in the presence of 100 microM forskolin and 10 mM Mn2+, indicating that the catalytic subunit of adenylylcyclase is not altered. To determine whether Gi down-regulation results in heterologous desensitization, we incubated adipocytes with maximally effective concentrations of PIA (300 nM), PGE1 (3 microM), or nicotinic acid (1 mM) for 4 days. The cells were then washed and incubated for an additional 30 min with various concentrations of these compounds to determine their ability to inhibit lipolysis. PIA caused a (marked) decrease in the sensitivity of the cells to both PIA and PGE1, thus indicating heterologous desensitization. Similarly, PGE1 decreased the sensitivity of the cells to both PGE1 and PIA, again demonstrating heterologous desensitization. In contrast, prolonged incubation with nicotinic acid decreased the sensitivity of the cells to nicotinic acid but had no effect on the sensitivity of the cells to PIA. Adenylylcyclase in membranes from PGE1-treated cells showed decreased sensitivity to inhibition by PIA. In contrast, adenylylcyclase showed normal sensitivity to PIA in membranes from nicotinic acid-treated cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Green
A
A
Department of Internal Medicine, University of Texas Medical Branch, Galveston 77555.
Milligan
G
G
Dobias
S B
SB
D000262:Adenylyl Cyclases / Q000378:metabolism; D000273:Adipose Tissue / Q000187:drug effects / Q000378:metabolism*; D000527:Alprostadil / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D015536:Down-Regulation; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008066:Lipolysis / Q000187:drug effects; D046911:Macromolecular Substances; D008297:Male; D009525:Niacin / Q000494:pharmacology; D010660:Phenylisopropyladenosine / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D011983:Receptors, Purinergic / Q000378:metabolism
D046911:Macromolecular Substances; D011983:Receptors, Purinergic; D009525:Niacin; D010660:Phenylisopropyladenosine; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D000527:Alprostadil