Effects of DMSO-induced differentiation on arachidonate mobilization in the human histiocytic lymphoma cell line U937: Responsiveness to sub-micromolar calcium ionophore A23187 and phorbol esters

Abstract

The human histiocytic lymphoma cell line, U937, is a rich source for isolation and purification of the 85 kDa cytosolic phospholipase A 2 (cPLA 2). Recent studies suggest that this enzyme catalyzes the agonist-stimulated release of arachidonate from membrane phospholipids, thereby initiating eicosanoid synthesis. We therefore investigated in situ regulation of phospholipase A 2 activity in intact U937 cells. The results indicate that calcium ionophore A23187 stimulatable release in intact undifferentiated U937 is low and only weakly dose dependent. Dimethyl sulfoxide (DMSO) differentiation of U937 cells results in a dramatic increase of A23187-stimulated arachidonate mobilization. Consistent with the characteristics of cPLA 2 in vitro, A23187-stimulated arachidonate release in differentiated U937 cells is highly specific for arachidonate and is activated by submicromolar A23187 concentrations. Phorbol myristate acetate (PMA) further potentiates arachidonate release in differentiated U937 cells by 4–6-fold over A23187 alone. However, treatment of differentiated U937 cells with PMA alone is an ineffective stimulus for arachidonate release, suggesting that a calcium transient is necessary for in situ arachidonate mobilization. A23187-stimulated arachidonate release increases during distinct temporal phases of differentiation (0–36 h, 84–96 h). By contrast PMA enhancement of the response to A23187 develops early in differentiation, and is complete by 36 h. These results suggest that differentiation-induced alterations in cPLA 2 regulatory elements, such as intracellular free calcium and/or phosphorylation, may regulate mobilization of arachidonate in U937 cells.