The receptor-mediated activation of a phosphatidylcholine-hydrolysing phospholipase D (PLD) has recently been described. We investigated the effect of alpha-thrombin and epidermal growth factor (EGF) on cellular PLD activity in order to determine the role of this enzyme in mitogen-induced increases in phosphatidic acid and sn-1,2-diacylglycerol. In the presence of ethanol, stimulation of [3H]myristic acid-labelled quiescent IIC9 cells with alpha-thrombin or EGF resulted in a rapid increase in radiolabelled phosphatidyl-ethanol which reached a plateau at 1 min, indicating the rapid and transient activation of PLD. We observed a concomitant decrease in the mitogen-stimulated increase of radiolabelled phosphatidic acid. In contrast, ethanol did not significantly effect the elevation of sn-1,2-diacylglycerol levels stimulated by alpha-thrombin or EGF as determined by measurement of sn-1,2-diacylglycerol mass or the appearance of [3H]1,2-diacylglycerol. A novel lipid, detected by two-dimensional t.l.c. analysis, was generated in [3H]myristic acid-labelled cells stimulated with alpha-thrombin, but not EGF, in the presence of ethanol. Treatment in vitro of cellular lipids isolated from [3H]myristic acid-labelled cultures with PLD in the presence of ethanol also resulted in the generation of this novel lipid species, supporting the role of this enzyme in its production. These data indicate that in quiescent IIC9 cells: (a) alpha-thrombin or EGF rapidly and transiently activates a PLD; (b) although this activation is responsible for part of the mitogen-induced increases in phosphatidic acid, it does not contribute to induced increases in sn-1,2-diacylglycerol; and (c) activation of this enzyme appears to be involved in the formation of a novel lipid generated in response to alpha-thrombin, but not EGF, in IIC9 fibroblasts.

Diacylglycerols (DAGs) derived from phosphatidylcholine (PC) hydrolysis have been shown to activate protein kinase C (PKC) in vitro, but it is not known whether this event occurs in response to DAGs generated via agonist-induced PC hydrolysis in intact cells. In this report we have addressed this question directly, using alpha-thrombin stimulation of IIC9 fibroblasts. PKC activation in intact cells was assessed in two ways, by measuring: 1) PKC membrane association as determined by kinase activity and Western blot analysis and 2) the phosphorylation of an endogenous PKC substrate, an 80-kDa protein. Treatment with 500 ng/ml alpha-thrombin has been shown to stimulate both phosphoinositide and PC hydrolysis, whereas treatment with 100 pg/ml alpha-thrombin stimulates only PC breakdown. Using these two conditions, we show that DAG produced from phosphoinositide, but not PC hydrolysis, is associated with the activation of PKC.

alpha-Thrombin stimulates a biphasic increase in cellular 1,2-diacylglycerol mass in quiescent IIC9 fibroblasts. This report describes the use of hirudin, a high-affinity inhibitor of alpha-thrombin that renders it catalytically inactive, to investigate the dependence of elevated 1,2-diacylglycerol levels on the presence of catalytically active alpha-thrombin. When cultures were incubated in the presence of alpha-thrombin, 1,2-diacylglycerol levels remained elevated for greater than or equal to 4 h. Inactivation of alpha-thrombin after 15 s did not alter the kinetics of 1,2-diacylglycerol formation occurring over the next 1 h. However, sustained (1-4 h) increases in this lipid were eliminated. Inactivation of alpha-thrombin after 1 h of stimulation resulted in 1) an immediate and reversible decline in 1,2-diacylglycerol levels, 2) elimination of the sustained phase of 1,2-diacylglycerol production, 3) inhibition of the alpha-thrombin-stimulated generation of choline metabolites, and 4) a blunted mitogenic response to alpha-thrombin. These data indicate that early (0-1 h) and late (1-4 h) increases in 1,2-diacylglycerol are differentially dependent on the presence of catalytically active alpha-thrombin. Furthermore, sustained increases in 1,2-diacylglycerol in response to alpha-thrombin are regulated at least in part at the level of generation (via phosphatidylcholine hydrolysis). Our results also support a role for sustained 1,2-diacylglycerol levels in the mitogenic response.

A property common to many growth factors is that they must be present for several hours before the commitment to DNA synthesis and cell division occurs. The intracellular signals that are relevant during this period are poorly defined. We examined the formation of 1,2-diacylglycerol in IIC9 fibroblasts after stimulation with epidermal growth factor (EGF), and found that the mass of this lipid remained elevated for at least four hours. The concentration-dependence of EGF-stimulated 1,2-diacylglycerol production and [3H]thymidine incorporation were similar. Studies of phospholipid metabolism strongly suggested that phosphatidylcholine was the source of the 1,2-diacylglycerol generated in response to EGF. EGF did not stimulate the hydrolysis of other phospholipids, including the phosphoinositides, nor did it increase synthesis de novo of 1,2-diacylglycerol. This pattern of sustained 1,2-diacylglycerol formation from phosphatidylcholine may be important in the mitogenic signalling of EGF and potentially other growth factors.

Previous reports have demonstrated the hydrolysis of inositol phospholipids in polymorphonuclear leukocytes (PMN) in response to chemoattractants and in lymphocytes in response to the mitogen phytohemagglutinin. We investigated the role of 1,2-diacylglycerol, one of the products of receptor-linked phosphatidylinositol hydrolysis, in mediating the migratory response of leukocytes. In an under-agarose migration system, we found 1,2-dioctanoylglycerol to be a strong chemoattractant for human PMN, 6C3HED (a mouse thymic lymphoma), and Jurkat (a human T-cell leukemia). By using a modified Boyden chamber assay, the migratory response of PMN to 1,2-dioctanoylglycerol was determined to be primarily chemotactic. Analysis of structural analogs indicated that both the position and number of acyl chains are important in determining chemoattractant activity. These studies demonstrate that exogenous 1,2-diacylglycerol can stimulate the directed migration of leukocytes. They further suggest that the formation of 1,2-diacylglycerol following receptor-mediated stimulation may represent a common step in the migratory responses of myeloid and lymphoid cells.

We have examined the kinetics of 1,2-diacylglycerol production in quiescent IIC9 fibroblasts. alpha-Thrombin and epidermal growth factor (EGF) both stimulate an increase in the mass of cellular 1,2-diacylglycerol. The generation of 1,2-diacylglycerol is biphasic when stimulated by a high concentration of alpha-thrombin (500 ng/ml), with an early phase peaking at 15 s and a late phase peaking at 5 min. Production of 1,2-diacylglycerol is monophasic when stimulated by: (a) a low concentration of alpha-thrombin (100 pg/ml); (b) a high concentration of alpha-thrombin added to cultures which had been pretreated with chymotrypsin; or (c) EGF. In all cases the stimulation of 1,2-diacylglycerol was sustained for at least 30 min. In a previous report (Raben, D. M., Yasuda, K., and Cunningham, D. D. (1987) Biochemistry 26, 2759-2765), it was demonstrated that alpha-thrombin stimulates lipid metabolism in fibroblasts via two coupling mechanisms designated R1 and R2. We now present evidence that the early phase of alpha-thrombin-stimulated 1,2-diacylglycerol production is related to R1, which is characterized by: 1) increased release of arachidonic acid, 2) hydrolysis of polyphosphoinositides, and 3) inhibition by pretreating cultures with chymotrypsin. The late phase is related to R2 which is characterized by 1,2-diacylglycerol production in the absence of stimulated phosphoinositide hydrolysis and arachidonic acid release. In addition, EGF activates an R2-like mechanism in that it does not stimulate the release of arachidonic acid or hydrolysis of polyphosphoinositides but does stimulate a 2-fold increase in 1,2-diacylglycerol mass.

We have previously shown that antibody-sensitized mouse peritoneal macrophages release arachidonic acid (C20:4) and its oxygenated derivatives when treated with complement, and that the major part of the release depended on the terminal complement complexes (TCC). To further delineate the process(es) responsible for this release we have extended our studies to rat peritoneal poly-morphonuclear leukocytes (PMNs). Experiments were performed with antibody-sensitized rat PMNs labeled with [3H]C20:4 and carrying the TCC, C5b-7, C5b-8 or C5b-9. In contrast to the results of other studies, production of leukotriene B4 (LTB4), the major radiolabeled derivative, was strictly dependent on the presence of C9. However, low levels of C20:4 and prostaglandins (PGs) were produced prior to the C5b-9 stage. Kinetic studies demonstrated that release of LTB4 was rapid; the initial release occurred within 4–6 min and a second rise in release coincided with cell death. Virtually all the LTB4 produced was released as we found no evidence of retention of intracellular LTB4 at either the C5b-8 or C5b-9 stages. In the absence of extracellular calcium, the release of LTB4 was completely abolished and the release of C20:4 and PGs was drastically reduced. [3H]C20:4-labeled PMNs carrying C5b-9 did release substantial amounts of radiolabeled material in the presence of EGTA; however, the majority of this lipid was in the form of intact phospholipid and triglyceride. These results indicate that release of C20:4 and its oxygenated derivatives from rat PMNs is (1) dependent on the participation ofC9 in the preexisting C5b-8 complex in the cell membrane, and (2) largely dependent on the presence of calcium.