Increased intracellular cyclic AMP inhibits inositol phospholipid hydrolysis induced by perturbation of the T cell receptor/CD3 complex but not by G-protein stimulation. Association with protein kinase A-mediated phosphorylation of phospholipase C-gamma 1.
Modulation of inositol phospholipid (InsPL) hydrolysis in response to increasing intracellular concentrations of cyclic AMP (cAMP) was studied in a murine T helper type II (Th2) lymphocyte clone, 8-5-5. Intact 8-5-5 cells produced maximal amounts of cAMP in response to prostaglandin E2 (PGE2), cholera toxin (CTx) or 7 beta-deacetyl-7 beta-(gamma-N-methylpiperazino)butyryl forskolin (dmpb-forskolin). cAMP generation reached a plateau after 5 min of treatment with dmpb-forskolin (300 microM) or PGE2 (1 microM), but required 60 min of treatment with CTx (1 microgram/ml). Preincubation of 8-5-5 cells with 1 microM-PGE2 or 300 microM-dmpb-forskolin (10 min at 37 degrees C) or with 1 microgram of CTx/ml (60 min at 37 degrees C) completely inhibited InsPL hydrolysis induced by perturbation of the T cell receptor (TCR)/CD3 complex with the monoclonal antibody 145.2C11. Preincubation with the cAMP analogue 8-bromo-cyclic AMP (8-Br-cAMP) also inhibited InsPL hydrolysis. Tetanolysin-permeabilized 8-5-5 cells produced cAMP in response to PGE2, dmpb-forskolin and guanosine 5'-[gamma-thio]triphosphate (GTP[S]), a non-cell-permeating, non-hydrolysable analogue of GTP that directly activates G-proteins. No inhibition of TCR/CD3-induced InsPL hydrolysis was observed under these conditions. InsPL hydrolysis was also unaffected when permeabilized cells were incubated with up to 10 mM-8-Br-cAMP, suggesting that permeabilized cells lost (a) soluble effector molecule(s) involved in mediating the inhibitory effect observed in intact cells. Treatment of 8-5-5 cells with dmpb-forskolin or CTx prior to permeabilization resulted in inhibition of TCR/CD3-induced InsPL hydrolysis, but did not affect InsPL hydrolysis induced via G-protein stimulation with GTP[S]. Treatment of permeabilized 8-5-5 cells with purified cAMP-dependent protein kinase (PKA) resulted in inhibition of TCR/CD3- but not GTP[S]-induced InsPL hydrolysis. This effect was associated with phosphorylation of phospholipase (PLC)-gamma 1 in the absence of phosphorylation of components of the TCR/CD3 complex. These results suggest that PKA-mediated phosphorylation of PLC may regulate TCR/CD3-induced InsPL hydrolysis.
Alava
M A
MA
Division of Hematology, Center for Biologics Evaluation and Research, US Food and Drug Administration, Bethesda, MD 20892.
DeBell
K E
KE
Conti
A
A
Hoffman
T
T
Bonvini
E
E
D000818:Animals; D000945:Antigens, Differentiation, T-Lymphocyte / Q000187:drug effects* / Q000502:physiology; D017252:CD3 Complex; D002417:Cattle; D002463:Cell Membrane Permeability / Q000187:drug effects; D002772:Cholera Toxin / Q000494:pharmacology; D005576:Colforsin / Q000031:analogs & derivatives / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000096:biosynthesis*; D015232:Dinoprostone / Q000494:pharmacology; D004224:Diterpenes; D015536:Down-Regulation / Q000502:physiology; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000187:drug effects; D006868:Hydrolysis; D007295:Inositol Phosphates / Q000378:metabolism*; D007424:Intracellular Fluid / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism*; D051379:Mice; D008819:Mice, Nude; D010743:Phospholipids / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism* / Q000494:pharmacology / Q000502:physiology; D011948:Receptors, Antigen, T-Cell / Q000037:antagonists & inhibitors / Q000187:drug effects* / Q000502:physiology; D013268:Stimulation, Chemical; D013601:T-Lymphocytes / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D006377:T-Lymphocytes, Helper-Inducer / Q000187:drug effects / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism*
D000945:Antigens, Differentiation, T-Lymphocyte; D017252:CD3 Complex; D004224:Diterpenes; D007295:Inositol Phosphates; D007527:Isoenzymes; D010743:Phospholipids; D011948:Receptors, Antigen, T-Cell; C055627:L 858051; D005576:Colforsin; D002772:Cholera Toxin; D000242:Cyclic AMP; D011494:Protein Kinases; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D015232:Dinoprostone