Examination of relative rates of cAMP synthesis and degradation in crude membranes of adipocytes treated with hormones.
The impact of changes in the activation state of the low Km cAMP phosphodiesterase (PDE) on cAMP output in adipocyte membranes was assessed by measuring the product of cAMP synthesis and degradation in the membrane preparation simultaneously. Crude membranes were prepared from adipocytes treated with the cAMP analog, 8-pCl phi S-cAMP and from adipocytes treated with 2 nM insulin. Using membranes from control and treated cells, adenylate cyclase was activated with various concentrations of forskolin and cAMP production (synthesis minus degradation) was measured with and without complete PDE inhibition using the specific inhibitor CI-914. Half maximal inhibition of the low Km cAMP PDEs in control membranes was produced by 1.16 +/- 0.07 microM CI-914 and greater than 98% of the activity was inhibited by 100 microM CI-914. The I50 and the concentration of CI-914 producing complete PDE inhibition in membranes from 8-pCl phi S-cAMP or insulin-treated cells were identical to those seen in membranes from control cells. Treatment of adipocytes with 8-pCl phi S-cAMP or with insulin did not modify basal rates of cAMP synthesis or alter the ability of adenylate cyclase to be activated by forskolin. The impact of PDE activity on cAMP accumulation was relatively small in membranes from control cells, but treatment of adipocytes with 8-pCl phi S-cAMP or with insulin activated the low Km cAMP PDE and caused a marked decrease in cAMP accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)
Gettys
T W
TW
Department of Medicine, Duke Medical Center, Durham, NC.
Okonogi
K
K
Tarry
W C
WC
Johnston
J
J
Horton
C
C
Taylor
I L
IL
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism; D000273:Adipose Tissue / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D004789:Enzyme Activation; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008297:Male; D011724:Pyridazines / Q000494:pharmacology; D051381:Rats
D007328:Insulin; D011724:Pyridazines; D005576:Colforsin; C042527:imazodan; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D000262:Adenylyl Cyclases