CD3-induced preferential hydrolysis of polyphosphoinositides and calcium regulation of inositol phosphate metabolism in a permeabilized murine T cell clone.
Murine T helper cloned cells permeabilized with the bacterial lysin, tetanolysin, were used to investigate the role of intracellular Ca2+ in regulating myo-[2-3H] inositol phospholipid (InsPL) hydrolysis triggered upon perturbation of the T cell receptor-CD3 complex. [Ca2+] was controlled by a calcium/magnesium/EGTA buffer. Antibody (mAb) aggregation of CD3 induced InsPL hydrolysis in the absence of added Ca2+. However, stimulated InsPL hydrolysis increased with the free [Ca2+], reaching a maximum at 100-300 nM [Ca2+]. Ca2+ increased the overall efficiency of hydrolysis without changes in EC50 of the anti-CD3 mAb. The response diminished at > 300 nM [Ca2+] due to a mixed type inhibition. Ca2+ alone had no effect on inositol phosphate levels. Polyphosphoinositides were preferentially cleaved, since no accumulation of Ins(1)P/Ins(3)P was detected, indicating that direct hydrolysis of phosphatidylinositol did not occur, irrespective of the Ca2+ concentration. [Ca2+] above 300 nM shifted the relative amounts of CD3-induced inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) in favor of the latter. Unlabeled permeabilized cells exposed to > or = 100 nM [Ca2+] showed enhanced conversion of [3H]Ins(1,4,5)P3 to [3H]Ins(1,3,4,5)P4. In conclusion, InsPL hydrolysis is optimally triggered by CD3 perturbation at intracellular Ca2+ levels approximating those observed in intact resting lymphocytes (100 nM). Ca2+ concentrations similar to those triggered by InsPL-derived metabolites may inhibit InsPL hydrolysis and promote Ins(1,3,4,5)P4 production, thus controlling the amounts of Ins(1,4,5)P3.
Laboratory of Cell Biology, United States Food and Drug Administration, Bethesda, Maryland 20892.
A
A
Conti
C
C
Brando
K E
KE
DeBell
M A
MA
Alava
T
T
Hoffman
E
E
Bonvini
D000465:Algorithms; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D017252:CD3 Complex / Q000276:immunology / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002851:Chromatography, High Pressure Liquid; D002999:Clone Cells; D006868:Hydrolysis; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007295:Inositol Phosphates / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008954:Models, Biological; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols / Q000378:metabolism*; D006377:T-Lymphocytes, Helper-Inducer / Q000276:immunology* / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D017252:CD3 Complex; D007295:Inositol Phosphates; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; C081655:inositol-1,3,4,5-tetrakisphosphate; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium