An assessment of phosphodiesterase activity in situ after treatment of hepatocytes with hormones.
The role of phosphodiesterase activation in controlling adenosine 3',5'-cyclic monophosphate (cAMP) levels within hepatocytes was investigated by preloading hepatocytes with the hydrolyzable cAMP analogue 8-para-chlorophenylthio-cAMP (8-pCl phi S-cAMP) and measuring disappearance of the analogue after treating the cells with various hormones. Incubation of hepatocytes with 15 nM 8-pCl phi S-cAMP increased the intracellular concentration of the analogue at 0.5 and 2 min, but by 5 min the concentration plateaued and remained constant or declined slightly at 7 and 10 min. Treatment of hepatocytes with 5 nM glucagon led to a rapid 50% decline in intracellular concentration of the analogue. However, 6 nM insulin produced no detectable change in analogue concentration, and a combination of 5 nM glucagon and 6 nM insulin produced no greater lowering of 8-pCl phi S-cAMP than did glucagon alone. Treatment of hepatocytes with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (50 microM) blocked approximately 30% of the glucagon-mediated decrease in 8-pCl phi S-cAMP concentration, and in separate cell incubations, it blocked 50% of the cAMP lowering produced by 125 nM 8-pCl phi S-cAMP. Treatment of analogue-preloaded hepatocytes with effective concentrations of phenylephrine, vasopressin, or angiotensin resulted in no change in intracellular analogue or cAMP concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)
Gettys
T W
TW
Howard Hughes Medical Institute, Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Blackmore
P F
PF
Corbin
J D
JD
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism; D005934:Glucagon / Q000494:pharmacology*; D066298:In Vitro Techniques; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D051381:Rats; D013873:Thionucleotides / Q000378:metabolism
D007328:Insulin; D013873:Thionucleotides; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D005934:Glucagon; D000242:Cyclic AMP; D010727:Phosphoric Diester Hydrolases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D002118:Calcium