Phospholipase A2 stimulation during cell secretion in rat basophilic leukemia cells.
The bridging of IgE receptors on rat basophilic leukemia cells (RBL-2H3) results in a number of biochemical events that accompany histamine secretion. Prominent among these is the release of arachidonic acid from cellular phospholipids, which could be due to the activation of phospholipase enzymes. In the present experiments we studied the intracellular activation of phospholipase A2 (PLA2) during histamine release. RBL-2H3 cells were stimulated through the IgE receptor, and the homogenates were prepared and tested for phospholipase A2 activity on 1-stearoyl-2-[14C]arachidonyl-sn-3-phosphatidylcholine. The amount of activity in the homogenates was dependent on the concentration of secretagogue used to activate the cells. Under optimal conditions there was a 1.86 +/- 0.12-fold (mean +/- SEM, N = 44) increase in the activity found in homogenates of stimulated cells. Activity was present in homogenates prepared 30 sec after cell activation, was optimal between 5 and 10 min, and decreased later. In time course experiments the PLA2 activation preceded histamine release. The activation of the enzyme in the cell occurred in the presence of 10 microM EGTA in the extracellular medium, which completely inhibited release of arachidonic acid and histamine. However, the activity of the enzyme required Ca2+. The PLA2 activity in the homogenates and the extent of cell stimulation for histamine release were maximal at the same concentration of antigen, and both were blocked by the addition of a monovalent hapten. The enzyme in the homogenates was capable of cleaving arachidonic acid from different phospholipids. The production of lysophospholipids could play a critical role in histamine release from cells. These results demonstrate the activation of PLA2 enzyme in cellular homogenates during the secretory process.
M
M
Garcia-Gil
R P
RP
Siraganian
D000614:Aminocaproates / Q000276:immunology; D000818:Animals; D001491:Basophils / Q000201:enzymology* / Q000276:immunology; D002118:Calcium / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006241:Haptens / Q000276:immunology; D006636:Histamine Release / Q000187:drug effects; D007942:Leukemia, Experimental / Q000201:enzymology* / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D051381:Rats; D013379:Substrate Specificity
D000614:Aminocaproates; D006241:Haptens; C031746:6-N-(2,4-dinitrophenyl)aminohexanoic acid; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium