Antigen-mediated phospholipase D activation in rat basophilic leukemia (RBL-2H3) cells: possible involvement of calcium/calmodulin.
The differential implication of protein kinase C (PKC) isozymes in antigen- or PMA-induced phospholipase D (PLD) activation was investigated in rat basophilic leukemia (RBL-2H3) cells. In [3H]oleic acid-labeled cells, both antigen (100 ng/ml) and phorbol 12-myristate 13-acetate (PMA) (100 nM) produced a specific product of PLD activation, [3H]phosphatidylbutanol (PBut) in the presence of butanol. Pretreatment of cells with a selective PKC inhibitor, Ro31-8425 (1-5 microM) inhibited PMA-stimulated PLD activity by 85%. In contrast, the antigen-stimulated PLD activity was much less sensitive to the inhibitor. RBL-2H3 cells express PKC alpha, beta, delta, epsilon and zeta isozymes and down-regulation of PKC by exposure to PMA (20 nM) for 1-2 h caused rapid decrease in PKC alpha and beta isozymes, leaving PKC delta, epsilon and zeta isozymes intact. Apparent decreases in the levels of PKC alpha and beta to about 50% were observed after adding 20 nM PMA for 1 h, when PMA-stimulated PLD activity was inhibited by up to 70%. Decrease in antigen-stimulated PLD activity was evident after 2 h PMA-treatment, when PKC alpha and beta decreased by nearly 70%. These results suggest that in the antigen-mediated PLD pathway PKC may be implicated but not play such a great role as PMA-stimulated pathway which is mediated through PKC alpha or beta. Then, we have examined the involvement of calcium/calmodulin (CaM) in PLD activation by antigen, since the antigen-stimulated PLD activation showed the absolute requirement for extracellular calcium. Preincubation of RBL-2H3 cells with a CaM antagonist W-7 (20 microM) inhibited the antigen-stimulated PLD activity by 90%, but W-5, a chlorine-deficient analogue of W-7 that only weakly interact with CaM, caused little inhibitory effect. Another non-specific CaM antagonist, trifluoperazine (TFP) also inhibited PLD activation. These results suggest that calcium/CaM may be involved in the antigen-stimulated PLD activation.
Kumada
T
T
Department of Otolaryngology, Gifu University School of Medicine, Japan.
Nakashima
S
S
Nakamura
Y
Y
Miyata
H
H
Nozawa
Y
Y
D000818:Animals; D000941:Antigens / Q000276:immunology*; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015536:Down-Regulation; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D015471:Leukemia, Basophilic, Acute; D008301:Maleimides / Q000494:pharmacology; D008407:Mast Cells / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D014407:Tumor Cells, Cultured
D000941:Antigens; D002147:Calmodulin; D004791:Enzyme Inhibitors; D007211:Indoles; D007527:Isoenzymes; D008301:Maleimides; C071924:Ro 31-8425; D011493:Protein Kinase C; D010739:Phospholipase D; D002118:Calcium