The bioactive phospholipid, lysophosphatidylcholine, induces cellular effects via G-protein-dependent activation of adenylyl cyclase.

The naturally occurring phospholipid, lysophosphatidylcholine (lyso-PC), regulates a broad range of cell processes, including gene transcription, mitogenesis, monocyte chemotaxis, smooth muscle relaxation, and platelet activation. Despite the growing list of cellular effects attributable to lyso-PC, the mechanism(s) by which it alters cell function have not been elucidated. In this report, we have examined the effects of exogenous lyso-PC on signal transduction processes within a variety of lyso-PC-responsive cells, including human platelets, monocyte-like THP-1 cells, and the megakaryoblastic cell line, MEG-01. Pretreatment of each of these cells with increasing concentrations of lyso-PC (25-150 microg/ml) was associated with a progressive increase in the cytosolic concentration of cAMP. The accumulation of cAMP in platelets correlated closely with the ability of lyso-PC to inhibit multiple platelet processes, including platelet aggregation, agonist-induced protein kinase C activation, thromboxane A2 generation, and the tyrosine phosphorylation of platelet proteins. In each of the cell types examined, the ability of lyso-PC to increase the cellular levels of cAMP was synergistically enhanced by pretreating the cells with the cAMP phosphodiesterase inhibitor, theophylline (5 mM), and was specifically inhibited by the P-site inhibitor of adenylyl cyclase, 2,5-dideoxyadenosine. A role for the stimulatory G-protein, Gs, in the lyso-PC-induced activation of adenylyl cyclase was suggested by the ability of the GTPase inhibitor, guanylyl 5'-thiophosphate (0.2 mM), to inhibit the lyso-PC-stimulated increase in cAMP, and also by the ability of cholera toxin to inhibit increases in membrane GTPase activity in response to lyso-PC. The functional significance of lyso-PC-induced activation of adenylyl cyclase was investigated in MEG-01 cells. Treatment of these cells with either lyso-PC or dibutyryl cAMP for 36-40 h resulted in a 3-5-fold increase in the surface expression of the natural anticoagulant protein, thrombomodulin (TM). The ability of lyso-PC to increase TM expression was abolished by pretreating these cells with the adenylyl cyclase inhibitor, 2,5-dideoxyadenosine, whereas the dibutyryl cAMP-induced increase in TM remained insensitive to adenylyl cyclase inhibition. These studies define an important role for the adenylyl cyclase signaling system in mediating cellular effects induced by lyso-PC.

The hydrolysis of phospholipids by phospholipase A 2 (PLA 2 ) 1 generates free fatty acids and lysophospholipids. While free fatty acids act as potential second messengers (1,2) and as precursors for biologically active eicosanoids (3), the role of lysophospholipids in cellular physiology remains unclear. The natural phospholipid, lysophosphatidylcholine (lyso-PC), has recently emerged as an important mediator of the atherogenic effects of oxidized low density lipoprotein (4,5). This lysophospholipid constitutes as much as 40% of the total lipid content of oxidized low density lipoprotein (6) and has been detected in elevated levels in atherosclerotic lesions from animals fed an atherosclerotic diet (7). In vitro, lyso-PC has multiple atherogenic effects. It is mitogenic for murine macrophages and has been postulated to play a critical role in oxidized low density lipoprotein-induced foam cell formation (8). Lyso-PC also induces endothelial cell gene expression of multiple growth factors involved in atherogenesis and inflammation (9). Furthermore, it can up-regulate the surface expression of endothelial cell adhesion molecules involved in the recruitment of mononuclear leukocytes into the arterial intima (4).
In addition to its atherogenic effects, there is growing evidence that in certain clinical settings the extracellular generation of lyso-PC by secretory PLA 2 may promote tissue inflammation and induce hemostatic disturbances (10 -12). Lyso-PC's proinflammatory effects include its ability to alter the adhesive properties of endothelial cells, induce chemotaxis of monocytes (5) and T-lymphocytes (13), and stimulate T-cell (14) and macrophage activation, leading to enhanced Fc-mediated phagocytosis (15,16). Lyso-PC also has vasoactive properties, stimulating endothelium-dependent vascular smooth muscle relaxation (17) and impairing endothelial-regulated vasomotor control (18). Furthermore, this lipid has potent platelet inhibitory effects and is essential for the inhibition of platelet aggregation by a subset of secretory PLA 2 (12).
A major outstanding issue regarding lyso-PC pathophysiology is the mechanism(s) by which this polar phospholipid alters cell function. Other naturally occurring phospholipids, such as lysophosphatidic acid (lyso-PA), appear to evoke cellular responses (19), through specific G protein-coupled receptor(s) linked to the activation of phospholipase C and inhibition of adenylyl cyclase (20,21). Adenylyl cyclase is a ubiquitous integral membrane signaling enzyme responsible for the generation of the second messenger, cyclic 3Ј,5-adenosine monophosphate (cAMP). The enzyme activity of adenylyl cyclase is tightly controlled by specific G-proteins, which either stimulate (via Gs␣ or ␤␥) (22)(23)(24) or inhibit (via G␣ i ) its catalytic activity (25). In human platelets, activation of adenylyl cyclase by prostacyclin or forskolin, results in a global down-regulation of all signaling events required for platelet activation (26). The stimulation of this signal transduction system in other cell types, by hormones, neurotransmitters, or other bioactive molecules, elicits diverse cellular effects related to cell cycle progression (27,28), energy metabolism, and gene transcription (29,30).
Lyso-PC has been demonstrated to regulate the activity of a number of intracellular signaling enzymes, including protein kinase C, as a necessary step for T-cell activation and possibly monocyte chemotaxis (14,31). Lyso-PC directly stimulates guanylate cyclase enzyme activity and inhibits adenylyl cyclase in 3T3 mouse fibroblast membrane preparations (32), indicating a potential role for this lysophospholipid in the co-ordinated regulation of cyclic nucleotide second messengers. In other studies, thrombin stimulation of the prostaglandin synthesis in mouse fibroblasts and seminal vesicle microsomes was attenuated by exogenous lyso-PC, suggesting a role for this lysophospholipid in the regulation of prostaglandin synthesis (33). Although these studies suggest that the generation of lyso-PC within the cell can directly regulate the activity of a variety of signaling enzymes, they provide limited information on the signal transduction processes stimulated by extracellular lyso-PC.
In this study, we have examined the signaling events induced by lyso-PC in a range of lyso-PC-responsive cells. Our results indicate that lyso-PC specifically stimulates the activation of adenylyl cyclase in human platelets, THP-1 and MEG-01 cell lines, leading to the production of cAMP. The increase in cellular cAMP correlated with the lyso-PC-mediated inhibition of platelet activation, and increased surface expression of thrombomodulin on MEG-01 cells. These studies suggest that the cellular effects induced by extracellular lyso-PC may be mediated, at least in part, by the adenylyl cyclase signaling pathway.
Platelet Aggregation Studies-Platelet aggregation was performed using a four channel automated platelet analyzer (Kyoto Daiichi, Japan) set to 950 rpm at 37°C. Each reaction mixture (400 l) contained fibrinogen (1 mg/ml), BSA (5 mg/ml), washed platelets (3 ϫ 10 8 /ml), and the indicated concentrations of platelet agonist. The rate of platelet aggregation was determined from the slope of aggregometer tracings. Inhibition of platelet aggregation was expressed as the percentage difference in the initial rate of platelet aggregation in the presence or absence of lyso-PC.
Phosphorylation of the Protein Kinase C Substrate, p47-Platelets (1.2 ϫ 10 9 /ml) were washed and resuspended in phosphate-free Tyrode's buffer supplemented with 10 mM theophylline. [ 32 P]H 3 PO 4 was added to the platelet suspension (0.5 mCi/ml platelets), and incubated for 2 h at 37°C. Free [ 32 P]H 3 PO 4 was removed by washing the platelets twice with the same buffer, before final resuspension in phosphate-free Tyrode's buffer. The 32 P-labeled platelets (3 ϫ 10 8 /ml, in 200 l of phosphate-free Tyrode's buffer) were stimulated with thrombin (0.05 unit/ml) in the presence of BSA (5 mg/ml) and a range of lyso-PC concentrations (10 -100 g/ml) for 10 min at 37°C. The reaction mixture was freeze-thawed three times using liquid nitrogen, boiled for 10 min in the presence of 3% sodium dodecyl sulfate (SDS) (w/v), and subjected to 12.5% SDS polyacrylamide gel electrophoresis (PAGE) under reducing conditions. The gel was fixed, dried, and autoradiographed.
Anti-phosphotyrosine Immunoblots-Washed platelets were stirred in the presence of 0.05 unit/ml thrombin or platelet resuspension buffer for 5 min. The cells were lysed with SDS-reducing buffer and immediately boiled for 10 min. After electrophoresis and transfer to polyvinylidene difluoride membranes, immunoblots of the platelet extracts were performed using the anti-phosphotyrosine antibody, PY20.
Preparation of Platelet Membranes-Platelet membranes were prepared using a modified method of Steer and Wood (35). Washed platelets (1.2 ϫ 10 9 ) were lysed with a hypotonic buffer (12 ml) containing 10 mM Tris, pH 7.6, 1 mM EGTA, 250 mM sucrose, 1 mM phenylmethylsulfonyl fluoride, 5 mM benzamidine, and 0.4 mM leupeptin. Platelet membranes were pelleted by centrifugation at 15,000 ϫ g for 10 min at 4°C. The pellet was washed four times with Tris buffer (150 mM), pH 8.0, 1 mM EGTA. The washed platelet membranes were finally resuspended in Tyrode's buffer (containing 10 mM theophylline) for the adenylyl cyclase assays, or in 20 mM MOPS buffer, pH 7.5, for the GTPase assays.
Measurement of cAMP-Washed platelets (1 ϫ 10 9 /ml), THP-1 (1 ϫ 10 7 /ml), or MEG-01 (5 ϫ 10 7 /ml) cells were resuspended in Tyrode's buffer (200 l) containing 5 mg/ml BSA, and 10 mM theophylline where indicated. In some experiments adenylyl cyclase was inhibited by preincubating cells with 1 mM 2,5-dideoxyadenosine for 30 min at 37°C. The cells were treated with the indicated concentration of lyso-PC or PGE 1 for 10 min at 37°C. The assays were terminated with the addition of 5 mM EDTA and the cells freeze-thawed three times. The samples were heated at 80°C for 20 min and centrifuged (15,000 ϫ g) for 10 min. The supernatant was removed and assayed for cAMP (picomole of cAMP/10 8 platelets) using a commercial kit (Amersham), according to the manufacturer's instructions.
Platelet membrane adenylyl cyclase assays were performed with 500 g/ml membrane protein, 1 mM ATP, in the presence or absence of GDP␤S (0.2-1.2 mM). Assays were initiated by the addition of PGE 1 (200 ng/ml) or the indicated concentration of lyso-PC, and incubated for 10 min at 37°C. In some experiments, the membranes were preincubated with 2,5-dideoxyadenosine for 5 min at room temperature, prior to the addition of lyso-PC or PGE 1 . At the completion of the assays, the membranes were heated to 80°C for 20 min, then centrifuged (15,000 ϫ g for 10 min), and the supernatant assayed for cAMP (picomole of cAMP/mg of membrane protein).
GTPase Assays-GTPase activity was measured by monitoring the liberation of 32 P from [␥-32 P]GTP using a modified method of Houslay et al. (36). Each reaction mixture (100 l) contained washed platelet membranes (50 g/ml), 20 mM MOPS buffer, pH 7.5, 5 mM MgCl 2 , 1 mM dithiothreitol, 0.5 mM EGTA, 0.5 mM ATP, 2 M [␥-32 P]GTP (2-3 ϫ 10 5 cpm/assay), in the presence of the indicated concentrations of lyso-PC, PGE 1 , or Triton X-100. Assays were performed for 30 min at 30°C and terminated by the addition of 400 l of ice-cold phosphate buffer (20 mM), pH 7.5, containing 5% activated Norit-A charcoal. The reaction mixtures were centrifuged at 15,000 ϫ g for 10 min, and the radioactivity measured from 200 l of supernatant. All assays were performed within a linear range with respect to time and protein concentration. GTPase activity was expressed as the % increase above the basal activity of unstimulated platelet membranes.
ADP-ribosylation of Gs was performed by incubating platelet membranes (500 g/ml) with 1 mM NAD, 0.5 mM GTP, 0.5 mM ATP, and 120 g/ml cholera toxin (preactivated with 50 mM dithiothreitol for 20 min at 37°C), in the presence of Tyrode's buffer, pH 7.4, for 30 min at 37°C. Control experiments were performed in the absence of cholera toxin. The membranes were pelleted by centrifugation at 15,000 ϫ g for 10 min, washed three times with 500 l of ice-cold Tyrode's buffer, and finally resuspended in MOPS buffer prior to the performance of GTPase assays.

FACS Analysis of Thrombomodulin (TM) Expression on MEG-01
Cells-MEG-01 cells were cultured using RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (Life Technologies, Inc.), 2 mM glutamine (ICN) at 37°C in the presence of 100 units/ml penicillin, 100 mg/ml streptomycin, and 5% CO 2 . For measurement of surface TM, MEG-01 cells (0.5-1 ϫ 10 6 /ml) were seeded on 24-well plates, with or without 2 mM theophylline, and cultured in the presence of control buffer, the indicated concentrations of lyso-PC or dibutyryl-cAMP (1 mM) for 36 -40 h. In some studies, the cells were preincubated with 1 mM 2,5-dideoxyadenosine for 40 min, prior to the addition of lyso-PC or dibutyryl-cAMP. The cells (0.5-1 ϫ 10 6 ) were then harvested and washed twice with phosphate-buffered saline (containing 5% fetal calf serum, 0.05% NaN 3 ), and incubated with an anti-TM monoclonal antibody (kindly donated by Dr. Phillip Bird, Melbourne, Australia) for 30 min at 4°C. After washing twice with phosphate-buffered saline, the cells were incubated with a fluorescence conjugated anti-mouse Ig (Sigma) for 30 min. These cells were washed a further two times with phosphate-buffered saline then resuspended to a final volume of 400 l, prior to FACS analysis.

RESULTS
To investigate the mechanism(s) by which lyso-PC regulates cell function, we examined signal transduction processes in a number of lyso-PC-responsive cells, including human platelets, monocyte-like THP-1 cells, and the megakaryoblastic cell line, MEG-01. Platelets are a particularly useful cell model for studying signal transduction processes, as these cells undergo rapid functional and morphological changes in response to stimulation, and the signaling processes regulating these cell processes have been investigated in considerable detail. Furthermore, these cells are responsive to several bioactive lipids, including platelet activating factor (37), lyso-PA (38), and lyso-PC (12). Both platelet activating factor and lyso-PA stimulate platelet aggregation via receptor-linked signaling pathways (19,20,37), while lyso-PC inhibits platelet function by an as yet undefined mechanism (12). In our initial studies, we investigated the effects of exogenous lyso-PC on signal transduction processes in human platelets.
Lysophosphatidylcholine Inhibits Agonist-induced Platelet Aggregation and Activation of Protein Kinase C, TXA 2 Generation, and the Tyrosine Phosphorylation of Multiple Platelet Proteins-Stimulation of platelets by a range of agonists is associated with the activation of cytosolic phospholipase A 2 , leading to the hydrolysis of membrane phospholipids and the subsequent generation of prostaglandin endoperoxides and thromboxane A 2 (TXA 2 ) (3). We examined the effect of lyso-PC on TXA 2 synthesis, by measuring the production of its stable metabolite thromboxane B 2 (TXB 2 ) in collagen-activated platelets. In these studies, we observed a close correlation between the inhibition of collagen-induced platelet aggregation by increasing concentrations of lyso-PC (12.5-100 g/ml), and the inhibition of TXB 2 generation (Fig. 1A, lanes 3-6). Increasing the concentration of collagen (5-20 g/ml), while maintaining a constant concentration of lyso-PC (100 g/ml), overcame the platelet inhibitory effects of lyso-PC and lead to increased production of TXB 2 (Fig. 1A, lanes 7-10). Qualitatively similar results were obtained in thrombin-stimulated platelets (data not shown).
Although lyso-PC has well described toxic effects on cells at FIG. 1. Lysophosphatidylcholine inhibits agonist-induced platelet aggregation, thromboxane A 2 production, protein kinase C activation, and the tyrosine phosphorylation of platelet proteins. A, correlation between the inhibition of platelet aggregation and thromboxane B 2 generation by lyso-PC. Washed platelets (3 ϫ 10 8 /ml) were activated with collagen (1 g/ml) for 5 min at room temperature, in the presence or absence of lyso-PC (12.5-100 g/ml). The rate of platelet aggregation (slashed bar) and TXB 2 concentration (black bar) were determined as described under "Experimental Procedures." P-labeled platelets (3 ϫ 10 8 /ml) were incubated for 10 min at 37°C with: 1) Tyrode's buffer; 2) 1 unit/ml thrombin alone; 3) 0.05 unit/ml thrombin alone, or in the presence of increasing concentrations of lyso-PC; 4) 10; 5) 20; 6) 40; 7) 60; or 8) 100 g/ml. Lane 9 represents platelets incubated with 100 g/ml lyso-PC in the absence of thrombin. At the end of the incubation period, platelets were freeze-thawed then boiled in SDS-reducing buffer. The platelet proteins were then separated using 12.5% SDS-polyacrylamide gel electrophoresis, and the 32 P-labeled proteins identified by autoradiography. These results are from one experiment, representative of three. It should be noted that in this particular experiment 40 g/ml lyso-PC did not abolish p47 phosphorylation nor platelet aggregation, which contrasts with collagenstimulated platelets (1 g/ml). This difference is a reflection of the high concentrations (39) due to its natural detergent properties, several lines of evidence suggest that its inhibitory effects on platelet function are unlikely to be due to membrane lysis. First, the extracellular concentration of lactose dehydrogenase did not increase following pretreatment of the cells with lyso-PC (12.5-150 g/ml). Second, the morphology of lyso-PCtreated platelets, as observed with phase-contrast microscopy, remained indistinguishable from untreated cells. Finally, the ability of high concentrations of collagen (20 g/ml) and thrombin (0.2 unit/ml) to overcome the platelet inhibitory effects of lyso-PC, confirms the functional integrity of these cells.
Previous studies have reported a role for free fatty acids and lyso-PC in the activation of protein kinase C (PKC) (1,2,14,31). We therefore examined the effect of lyso-PC on PKC activation in resting and thrombin-stimulated platelets, by measuring the phosphorylation of the PKC substrate, p47. Pretreatment of platelets with lyso-PC alone (100 g/ml) had minimal effect on the phosphorylation status of p47 (Fig. 1B, lane 9). In contrast, doses of thrombin (0.05 unit/ml) which can activate PKC independent of TXA 2 production (40, 41), induced prominent p47 phosphorylation (Fig. 1B, lane 3). Concentrations of lyso-PC which inhibited thrombin-induced aggregation (60 -100 g/ml) abolished p47 phosphorylation (Fig. 1B, lanes 7 and  8). In all experiments there was a close correlation between the inhibition of p47 phosphorylation and the abolition of thrombin-induced platelet aggregation. Furthermore, as with TXB 2 generation, the inhibitory effects of lyso-PC on PKC activation could be completely reversed by increasing the concentration of thrombin (data not shown). These studies indicate that the platelet inhibitory effects of lyso-PC are not confined to the inhibition of TXA 2 generation.
In addition to activating serine/threonine kinases, thrombin stimulation of platelets also leads to the activation of multiple non-receptor tyrosine kinases, leading to the tyrosine phosphorylation of multiple platelet proteins. These phosphorylation events are considered to play an important role in regulating platelet functional responses (42). We therefore examined the effects of lyso-PC on the level of protein tyrosine phosphorylation in human platelets. Immunoblot analysis of resting platelet lysates using anti-phosphotyrosine antibodies, revealed the presence of tyrosine-phosphorylated proteins of 109, 75, 67, and 60 kDa (Fig. 1C, lane 1). Pretreatment of platelets with lyso-PC (10 -100 g/ml) alone did not induce the tyrosine phosphorylation of platelet proteins (data not shown). In contrast, thrombin stimulation (0.05 unit/ml) of platelets led to an increase in the level of phosphorylation of the 109, 75, 67, and 60 kDa proteins, along with the appearance of additional minor bands at 124, 118, 112, 100, 87, 57, and 46 kDa (Fig. 1C, lane  2). Concentrations of lyso-PC (50 or 100 g/ml) (Fig. 1C, lanes  4 and 5) or PGE 1 (200 ng/ml) (Fig. 1c, lane 3), which negated platelet aggregation, inhibited the thrombin-stimulated tyrosine phosphorylation of these platelet proteins. These same concentrations of lyso-PC also abolished the thrombin-stimulated activation and cytoskeletal translocation of the major platelet non-receptor tyrosine kinase, pp60 c-src (data not shown). Collectively, these studies indicate that lyso-PC treatment of platelets leads to a global and reversible down-regulation of multiple signal transduction pathways required for platelet activation.
Effect of Lyso-PC on the Platelet Cytosolic Concentration of cAMP-Activation of adenylyl cyclase is associated with the enzymatic production of the soluble second messenger, cAMP, from cellular ATP. cAMP in turn regulates a myriad of cell processes by modulating the catalytic activity of protein kinase A (23, 43). This serine-threonine kinase phosphorylates a range of cellular proteins and is responsible for attenuating all sig-naling events required for platelet activation (26,44). We therefore investigated whether the inhibition of platelet function by lyso-PC may be due to the cellular accumulation of cAMP. Consistent with this possibility, treatment of platelets with lyso-PC caused a dose-dependent increase in cAMP ( Fig.  2A), which correlated closely with its ability to inhibit platelet aggregation (data not shown). The maximal increase in the cytosolic cAMP concentration was obtained using 150 g/ml lyso-PC, which resulted in a 5.5-fold increase in cAMP levels from 1.2 to 6.7 pmol/10 8 platelets. This level of increase in the intracellular concentration of cAMP was specific to lyso-PC, as similar concentrations of phosphatidylcholine, lysophosphatidic acid, lysophosphatidylglycerol, lysophosphatidylethanolamine, and lysophosphatidylserine had only a minor effect on cAMP levels ( Fig. 2A). Furthermore, this effect was not selec-
Lyso-PC Increases the Intracellular Concentration of cAMP by Activating Adenylyl Cyclase-The intracellular level of cAMP is dynamically regulated by adenylyl cyclase and cAMP phosphodiesterase (22,26,44). To determine whether the cellular accumulation of cAMP in response to lyso-PC was due to increased synthesis or reduced metabolism of the second messenger, we examined the effects of the phosphodiesterase inhibitor, theophylline, or the P-site inhibitor of adenylyl cyclase, 2,5-dideoxyadenosine, on the lyso-PC-induced increase in cAMP. Inhibition of cAMP phosphodiesterase by saturating concentrations of theophylline (10 mM) led to a modest increase in the cellular level of cAMP (Fig. 3A) (30). In contrast, when platelets were stimulated with PGE 1 (200 ng/ml), in the presence of theophylline (10 mM), there was a dramatic increase in the cellular concentration of cAMP. Similarily, the lyso-PC induced increase in cytosolic cAMP in human platelets, THP-1, and MEG-01 cells, was synergistically enhanced by theophylline, suggesting activation of adenylyl cyclase as the primary mechanism by which lyso-PC increases intracellular cAMP. Further evidence supporting this hypothesis stems from the observation that when near saturating concentrations of PGE 1 (200 ng/ml) and lyso-PC (100 g/ml) were combined, the cytosolic concentration of cAMP was not elevated above that achieved with PGE 1 alone.
To confirm that the cellular accumulation of cAMP was due to increased generation, rather than decreased metabolism of cAMP, we pretreated MEG-01 cells and human platelets with 2,5-dideoxyadenosine, prior to exposing the cells to lyso-PC. This inhibitor reduced the lyso-PC and PGE 1 -stimulated increase in cAMP by 60 -70% in both platelets and MEG-01 cells (data not shown). It is likely that the inability of 2,5-dideoxyadenosine to completely abolish the increase in cAMP in intact cells was due to its incomplete access to the intracellular pool of adenylyl cyclase, as the pretreatment of isolated platelet membranes with 2,5-dideoxyadenosine, completely inhibited the lyso-PC-stimulated increase in cAMP (Fig. 3B).
A Role for the Stimulatory G-protein, Gs, in the Lyso-PCinduced Activation of Adenylyl Cyclase-Adenylyl cyclase can be activated directly, by agents such as forskolin (45,46), or via the receptor-linked stimulatory G-protein, Gs␣ or ␤␥ (22,47). We investigated the possibility that Gs␣ may be required for the lyso-PC-induced activation of adenylyl cyclase. For these studies, we examined the ability of the specific GTPase inhibitor, GDP␤S, to block the lyso-PC-stimulated production of cAMP from isolated platelet membranes. Platelet membranes were exposed to either lyso-PC or PGE 1 , in the presence of theophylline (10 mM), and the level of cAMP determined, as described under "Experimental Procedures." As shown in Fig.  4A, in the presence of theophylline (10 mM), both PGE 1 (200 ng/ml) and lyso-PC (100 g/ml) increased the generation of cAMP from intact platelet membranes approximately 5-fold. GDP␤S (0.2 mM) alone had minimal effect on cAMP levels. However, the same concentration of GDP␤S dramatically reduced the PGE 1 and lyso-PC-stimulated production of cAMP. In control experiments, concentrations of GDP␤S up to 1.2 mM had no inhibitory effect on forskolin's ability to stimulate adenylyl cyclase.
To strengthen our hypothesis further that Gs␣ plays a critical role in inducing the activation of adenylyl cyclase by lyso-PC, we examined the ability of this lysolipid to stimulate platelet membrane GTPase activity. As demonstrated in Fig. 4B, lyso-PC (5-100 g/ml) stimulated membrane GTPase activity in a dose-dependent manner, up to a maximum of 250% above the level observed in non-stimulated platelet membranes. These assays were performed in the absence of added BSA, hence the apparent concentration of lyso-PC required to activate GTPase was significantly lower than that required to activate adenylyl cyclase (Fig. 2, A and B). This difference between the two assays is a reflection of albumin's ability to bind lysophospholipids and lower their effective "free concen-
tration" in solution. Previous studies have reported that lyso-PC can stimulate the GTPase activity of p21 ras (48), an effect which is mimicked in part by non-ionic detergents, such as Triton X-100. We therefore examined whether Triton X-100 could reproduce the lyso-PC-stimulated increase in platelet membrane GTPase activity, as these cells contain numerous members of the Ras GTPase superfamily. The results from these studies are presented in Fig. 4B and indicate that Triton X-100 increases membrane GTPase activity in a dose-dependent manner. The level of GTPase stimulation by Triton X-100 was approximately 40% of that observed with lyso-PC, suggesting that the stimulatory effect of this lipid on membrane GTPase activity is unlikely to be simply a nonspecific detergent effect.
Although an increase in membrane GTPase activity in response to lyso-PC supports the hypothesis that this lipid may activate G-proteins, they do not identify which GTPases are activated. To address this issue, we pretreated platelet membranes with cholera toxin to induce the specific ADP-ribosylation of Gs␣. This covalent modification is an effective method of specifically inhibiting the GTPase activity of Gs␣ (49). We confirmed ADP-ribosylation of Gs␣ by demonstrating complete inhibition of PGE 1 -stimulated increase in membrane GTPase activity (Fig. 4C) and by demonstrating ADP-ribosylation of Gs␣ with 32 P-labeled NAD (data not shown). Cholera toxin pretreatment of platelet membranes also reduced the lyso-PCstimulated increase in GTPase activity by approximately 65%, while having minimal effect on the Triton X-100-stimulated increase in GTPase activity (Fig. 4C). The residual increase in GTPase activity induced by lyso-PC after cholera toxin pretreatment was not investigated further but is presumably due to the activation of Ras proteins.
Activation of Adenylyl Cyclase Is Responsible for Cellular Effects of Lyso-PC-While the results to date clearly indicate that pathophysiologically relevant concentrations of exogenous lyso-PC can activate adenylyl cyclase, leading to the cellular accumulation of cAMP, they do not establish an essential role for this pathway in mediating cellular effects induced by lyso-PC. To address this issue, we initially focused our attention on the platelet inhibitory effects of lyso-PC by comparing platelet inhibition with that obtained with PGE 1 . PGE 1 is a well characterized inhibitor of platelet aggregation, which is thought to mediate its cellular effects by stimulating the production of cAMP (26). If the inhibition of platelet activation by lyso-PC is primarily mediated by cAMP, then the comparative doseresponse curves for cAMP generation and platelet inhibition by lyso-PC and PGE 1 would be expected to be similar. The dose- response studies outlined in Fig. 5 clearly indicate that increasing concentrations of both inhibitors elevate cAMP and inhibit platelet aggregation to a similar extent, supporting our hypothesis that an increase in the cellular level of cAMP is the primary mechanism by which lyso-PC inhibits platelet activation.
In other studies we attempted to reverse the lyso-PC-mediated inhibition of platelet aggregation by blocking the adenylyl cyclase signaling pathway with 2,5-dideoxyadenosine, however, as mentioned earlier, this inhibitor could only decrease adenylyl cyclase activity by approximately 60 -70% in intact cells. The residual increase in cAMP induced by lyso-PC or PGE 1 under these assay conditions was sufficient to prevent platelet activation, as these cells are exquisitely sensitive to small increases in cellular cAMP (26).
To further examine the role of adenylyl cyclase in mediating the cellular effects of lyso-PC, we studied the ability of 2,5dideoxyadenosine to inhibit the lyso-PC-stimulated surface expression of the natural anticoagulant protein, TM on MEG-01 cells. Incubation of these cells with lyso-PC (20 -40 g/ml) for 36 -40 h resulted in a 3-5-fold increase in the surface expression of TM. Pretreatment of the cells with 2,5-dideoxyadenosine inhibited the lyso-PC-stimulated increase in cAMP by approximately 75% and dramatically reduced the up-regulation of TM expression (Fig. 6). It is unlikely that the effects of 2,5-dideoxyadenosine on TM expression were due to the inhibition of cell growth or survival, as the total number of viable cells was no different in control or 2,5-dideoxyadenosine-pretreated cells (data not shown). Furthermore, in control studies, the addition of 2,5-dideoxyadenosine failed to inhibit the upregulation of TM by dibutyryl-cAMP, confirming that the cellular effects of 2,5-dideoxyadenosine are likely to be due to the inhibition of adenylyl cyclase. DISCUSSION While there is growing evidence that lyso-PC plays an important role in mediating the atherogenic effects of oxidized low density lipoprotein and the proinflammatory effects of secretory phospholipase A 2 , the mechanism by which this natural lysolipid alters cell function has remained elusive. The studies reported in this article indicate that non-toxic concentrations of lyso-PC stimulate the cytosolic accumulation of cAMP in a number of lyso-PC-responsive cells. Studies with specific inhibitors of adenylyl cyclase and cAMP phosphodiesterase clearly demonstrate that the lyso-PC-stimulated increase in intracellular cAMP involves the activation of adenylyl cyclase, rather than the inhibition of cAMP phosphodiesterase.
Several lines of evidence indicate that the synthesis of cAMP is important for the induction of specific cellular effects by lyso-PC. First, the ability of lyso-PC to reversibly inhibit all platelet responses, including shape change, serotonin release, and platelet aggregation, is consistent with the intracellular effects of elevated cAMP. Furthermore, cAMP accumulation causes a down-regulation of all signaling processes linked to platelet activation, including PKC activation, protein tyrosine phosphorylation, and thromboxane A 2 generation. Second, the similar dose-response curves for inhibition of platelet aggregation by lyso-PC and PGE 1 indicate that the stimulation of adenylyl cyclase is likely to be the predominant mechanism by which this lysolipid inhibits platelet function. Third, the increased surface expression of thrombomodulin on MEG-01 cells in response to lyso-PC was specifically inhibited by the adenylyl cyclase inhibitor, 2,5-dideoxyadenosine. While these studies do not exclude a role for other signaling pathways in mediating the cellular effects of lyso-PC, they demonstrate that activation of adenylyl cyclase is at least one means by which this polar phospholipid regulates cell function. In this regard, PKC activation has previously been suggested as a mechanism by which lyso-PC may mediate its cellular effects (14,31). Although PKC is a well characterized activator of adenylyl cyclase in certain cell types, it is unlikely that this mechanism operates in platelets, as lyso-PC appears to down-regulate PKC activity in these cells. In endothelial cells, signaling pathways uncoupled from PKC activation appear to be important for the lyso-PC-induced up-regulation of cell adhesion receptors (4) and for the enhanced gene expression of nitric oxide synthase (30). It is FIG. 5. Correlation between the inhibition of platelet aggregation and elevation in cAMP induced by lyso-PC and PGE 1 . Washed platelets (1 ϫ 10 9 /ml) were activated with collagen (1 g/ml) for 5 min in the presence of control buffer (Control); increasing concentrations of lyso-PC: 1) 12.5 g/ml; 2) 25 g/ml; 3) 37.5 g/ml; 4) 50 g/ml; 5) 100 g/ml; or increasing concentrations of PGE 1 , 25 (6), 50 (7), 100 (8), 200 (9), and 400 (10) ng/ml. The platelet cytosolic cAMP concentration (black bar) and the % inhibition of platelet aggregation (slashed bar) was quantitated as described under "Experimental Procedures." Results represent the mean Ϯ S.E. from four experiments. tempting to speculate that the adenylyl cyclase signaling system may be involved in modulating these cellular events, as cAMP is a well characterized regulator of gene transcription, and has previously been demonstrated to regulate the transcription of several lyso-PC-responsive genes, including thrombomodulin and cyclo-oxygenase II (29,50).
The mechanism by which non-toxic concentrations of lyso-PC activate adenylyl cyclase is clearly an important issue for future study. There are conflicting reports in the literature regarding the effects of lyso-PC on the enzyme activity of adenylyl cyclase. Lysophospholipids have recently been shown to directly activate yeast adenylyl cyclase in the absence of other membrane components (28). In contrast to our results, this study found that the stimulation of yeast adenylyl cyclase only occurred in the presence of very high concentrations of lysophospholipids. Using multiple cell types, we have demonstrated that low concentrations of lyso-PC (12.5 to 50 g/ml) can activate mammalian adenylyl cyclase leading to the cellular accumulation of cAMP. The actual concentration of free lyso-PC in our experiments is likely to be much lower than 25 g/ml, as each experiment was performed in the presence of 5 mg/ml albumin which has a high affinity for lysophospholipids (51). Direct comparison of yeast and mammalian adenylyl cyclase is complicated by the fact that there are several major differences between these two enzymes. The yeast enzyme is not an integral membrane protein and is structurally dissimilar from mammalian adenylyl cyclase (23,28). Furthermore, the yeast enzyme is associated with the membrane via its interaction with the GTPase-activating protein IRA1 and can be readily solubilized with high concentrations of salt, whereas the solublization of mammalian adenylyl cyclase requires detergent treatment of membranes. Extraction of adenylyl cyclase from its lipid environment under these conditions completely abolished its catalytic activity, 2 precluding any direct examination of the effects of lyso-PC on mammalian adenylyl cyclase. Despite this, two independent lines of evidence suggest that the effects of lyso-PC on mammalian adenylyl cyclase is unlikely to be due to a direct effect of this lipid on the enzyme but rather an indirect effect through the activation of Gs. First, the lyso-PC-stimulated increase in adenylyl cyclase activity could be abolished with the G-protein inhibitor, GDP␤S, and second, pretreatment of platelet membranes with lyso-PC was able to stimulate an increase in membrane GTPase activity. This increase is likely to involve activation of Gs, as cholera toxin was able to substantially reduce this increase in membrane GTPase activity. An additional study by Shier et al. (32) demonstrated that lyso-PC at concentrations greater than 1 mg/ml markedly inhibited sodium fluoride-stimulated adenylyl cyclase activity in 3T3 mouse fibroblast membranes, whereas concentrations less than 100 g/ml had a minimal effect on adenylyl cyclase. The inhibitory effects of high concentrations of lyso-PC on mammalian adenylyl cyclase are not surprising given the detergent properties of this lysolipid. The reason why lower concentrations of lyso-PC did not stimulate adenylyl cyclase in these studies is not immediately apparent, although it may reflect cell type specific differences.
A number of phospholipid-derived bioactive messengers have now been demonstrated to mediate cellular effects through the activation of G-proteins. The simplest natural phospholipid, lysophosphatidic acid (19 -21, 38), and the phospholipid, platelet-aggregating factor (37), induce cell changes through putative G-protein coupled receptors on the cell surface, while the products of arachidonic acid metabolism, prostaglandin I 2 and E 1 mediate their cellular effects through specific G-proteincoupled receptors linked to the activation of adenylyl cyclase (23,47,52). Our studies indicate that lyso-PC should be added to this list of lipid messengers which alter cell function through the activation of G-proteins. While the precise mechanism by which lyso-PC activates Gs is unclear from our studies, it is possible that lyso-PC may intercalate into the membrane lipid bilayer and directly activate Gs␣, in a receptor-independent manner, or alternatively, lyso-PC may bind to and activate a specific membrane surface receptor coupled to Gs␣. The exact mechanism by which lyso-PC stimulates intracellular cAMP synthesis remains to be defined and will ultimately require reconstitution of the response with purified components.