Rapid regulation of PDE-2 and PDE-4 cyclic AMP phosphodiesterase activity following ligation of the T cell antigen receptor on thymocytes: analysis using the selective inhibitors erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) and rolipram.
The PDE2, cyclic GMP-stimulated, and the PDE4, cyclic AMP-specific enzymes provide the major, detectable cyclic AMP phosphodiesterase activities in murine thymocytes. In the absence of the cyclic GMP, PDE4 activity predominated (approximately 80% total) but in the presence of low (10 microM) cyclic GMP concentrations, PDE2 activity constituted the major PDE activity in thymocytes (approximately 80% total). The PDE4 selective inhibitor rolipram dose-dependently inhibited thymocyte PDE4 activity (IC50 approximately 65 nM). PDE2 was dose-dependently activated (EC50 approximately 1 microM) by cyclic GMP and inhibited by erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) (IC50 approximately 4 microM). EHNA was shown to serve as a selective inhibitor of PDE-2 activity as assessed from studies using separated PDE1, PDE2, PDE3 and PDE4 species from hepatocytes as well as human PDE2 and PDE4 enzymes. EHNA completely ablated the ability of cyclic GMP to activate PDE2 activity, whilst having a much smaller inhibitory effect on the unstimulated PDE2 activity. EHNA exhibited normal Michaelian kinetics of inhibition for the cyclic GMP-stimulated PDE2 activity with Hill plots near unity. Apparent negative co-operative effect were seen in the absence of cyclic GMP with Hill coefficients of approximately 0.3 for inhibition of PDE2 activity. Within 5 min of challenge of thymocytes with the lectin phytohaemagglutinin (PHA) there was a transient decrease (approximately 83%) in PDE-4 activity and in PDE2 activity (approximately 40%). Both anti-TCR antibodies also caused an initial reduction in the PDE4 activity which was followed by a sustained and profound increase in activity. In contrast to that observed with PHA, anti-TCR/CD3 antisera had little effect on PDE2 activity. It is suggested that, dependent upon the intracellular concentrations of cyclic GMP, thymocyte cyclic AMP metabolism can be expected to switch from being under the predominant control of PDE4 activity to that determined predominantly by PDE2 activity. These activities may be rapidly and differentially regulated following ligation of different cell surface receptors.
Michie
A M
AM
Division of Biochemistry & Molecular Biology, University of Glasgow, Scotland, UK.
Lobban
M
M
Müller
T
T
Harnett
M M
MM
Houslay
M D
MD
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001667:Binding, Competitive / Q000502:physiology; D017252:CD3 Complex / Q000276:immunology; D002478:Cells, Cultured / Q000737:chemistry / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D006152:Cyclic GMP / Q000494:pharmacology; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006157:Guanosine Monophosphate / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000187:drug effects / Q000378:metabolism; D008213:Lymphocyte Activation / Q000502:physiology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D010835:Phytohemagglutinins; D011760:Pyrrolidinones / Q000494:pharmacology*; D011817:Rabbits; D011948:Receptors, Antigen, T-Cell / Q000276:immunology / Q000378:metabolism*; D020889:Rolipram; D012680:Sensitivity and Specificity; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013950:Thymus Gland / Q000166:cytology
D000911:Antibodies, Monoclonal; D017252:CD3 Complex; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D010726:Phosphodiesterase Inhibitors; D010835:Phytohemagglutinins; D011760:Pyrrolidinones; D011948:Receptors, Antigen, T-Cell; C010192:9-(2-hydroxy-3-nonyl)adenine; D006157:Guanosine Monophosphate; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D006152:Cyclic GMP; D000225:Adenine; D020889:Rolipram