Characterization of the cyclic nucleotide phosphodiesterase isoenzymes present in rat epididymal fat cells.
Soluble and particulate fractions from extracts of rat epididymal fat cells were shown to exhibit a number of different phosphodiesterase activities, as determined by substrate specificity and sensitivity to activators and inhibitors. These activities were then further characterized following separation by MonoQ fast protein liquid chromatography (FPLC). A cyclic AMP-specific activity, unaffected by the presence of calcium and calmodulin and inhibited by rolipram, was the major soluble phosphodiesterase. This fraction also contained distinct calcium and calmodulin- and cyclic-GMP-stimulated activities. Over 80% of the phosphodiesterase activity in the particulate fraction could be accounted for by an insulin-activated cyclic AMP and cyclic GMP-hydrolysing enzyme, which was sensitive to inhibition by cyclic GMP, SKF 94120, SKF 95654 and cilostamide, and eluted as a single peak during MonoQ chromatography. At 1 microM cyclic AMP, the phosphodiesterase activity in the soluble fraction was about eight times greater than in the particulate fraction. Specific inhibitors to the particulate phosphodiesterase (cilostamide and SKF 95654) were added to incubations of isolated fat cells, and were able to potentiate sub-maximal concentrations of isoproterenol in the stimulation of lipolysis. These inhibitors were also able to reverse the antilipolytic effect of insulin, demonstrating the importance of the particulate phosphodiesterase in insulin action, despite the fact that its activity represents only a small proportion of the total phosphodiesterase activity in fat cells. Inhibitors of the major soluble phosphodiesterase had no effect on lipolysis.
Schmitz-Peiffer
C
C
Department of Biochemistry, School of Medical Sciences, University of Bristol, U.K.
Reeves
M L
ML
Denton
R M
RM
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000273:Adipose Tissue / Q000166:cytology / Q000201:enzymology*; D000818:Animals; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004822:Epididymis / Q000201:enzymology*; D007328:Insulin / Q000502:physiology; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008066:Lipolysis; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains
D007328:Insulin; D007527:Isoenzymes; D015105:3',5'-Cyclic-AMP Phosphodiesterases