Swiss 3T3 cells preferentially incorporate sn-2-arachidonoyl monoacylglycerol into sn-1-stearoyl-2-arachidonoyl phosphatidylinositol.

The sn-1-stearoyl-2-arachidonoyl phospholipids of animal cells appear to be formed by special mechanisms. To determine whether monoacylglycerol (MG) incorporation pathways are involved we incubated quiescent Swiss 3T3 cells with [3H]glycerol-labeled sn-2-arachidonoyl MG, then analyzed the radioactive cell lipids that accumulated. We also examined cell homogenates to identify enzyme activities that might promote the incorporation of sn-2-arachidonoyl MG into other cell lipids. The cell incubation experiments demonstrated rapid labeling of several lipids, including diacylglycerol, lysophosphatidic acid, phosphatidic acid, and phosphatidylinositol. They also demonstrated selective labeling of sn-1-stearoyl-2-arachidonoyl species of phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine. The cell homogenate experiments identified an sn-2-acyl MG acyltransferase activity, an MG kinase activity that phosphorylates sn-2-arachidonoyl MG in preference to sn-2-oleoyl MG, and a stearoyl-specific acyl transferase activity that converts sn-2-arachidonoyl lysophosphatidic acid into sn-1-stearoyl-2-arachidonoyl phosphatidic acid. The results also showed that this stearoyl transferase could act with other enzymes to convert sn-2-arachidonoyl lysophosphatidic acid into sn-1-stearoyl-2-arachidonoyl phosphatidylinositol. The combined results indicate that Swiss 3T3 cells incorporate sn-2-arachidonoyl MG into phospholipids by at least two different pathways, including one that specifically forms sn-1-stearoyl-2-arachidonoyl phosphatidylinositol.     

Arachidonic acid metabolism by rabbit synovial cells in culture. Studies of non-cyclooxygenase pathways

We have investigated arachidonic acid (20:4) metabolism by rabbit synovial cells in culture. The lipoxygenase products 5-HETE, 12-HETE and 15-HETE were not detected, despite the presence of a cyclooxygenase inhibitor sodium meclofenamate (20 microM), nor after incubation with ionophore A23187 (1 microM), 20:4 (10 microM), prostaglandin E2, (1 microM), N-formylmethionylleucylphenylalanine (0.01 microM), or murine spleen cell-conditioned medium. [3H]20:4 (10 microM) was incorporated into phospholipids, triacylglycerols and diacylglycerols. A majority of the 3H content of phosphatidylinositol/phosphatidylserine and of diacylglycerols was already present at 1 min, in contrast to the slower accumulation of 3H in triacylglycerols, phosphatidylcholine and phosphatidylethanolamine. The diacylglycerol fraction contained sn-glycerol-1-acyl-2-20:4. These observations are consistent with phospholipase C activity in synovial cells under those culture conditions. The products generated by these enzymes may play important roles in the physiological processes of synovium.     

The mitogenic effect of 15- and 12-hydroxyeicosatetraenoic acid on endothelial cells may be mediated via diacylglycerol kinase inhibition

15-Hydroxyeicosatetraenoic acid (15-HETE), a major lipoxygenase metabolite of arachidonic acid in fetal bovine aortic endothelial cells, was a mitogen for these cells, stimulating both cell proliferation and DNA synthesis in the presence of serum and serum-deprived cells. In [14C]arachidonic acid-labeled confluent endothelial cell monolayers, 15-HETE (30 microM) caused an elevation of [14C]diacylglycerol (DAG) with a concomitant decrease in cellular [14C]phosphatidylinositol (PI) in both unstimulated and stimulated cells. 1-Oleoyl-2-acetylglycerol, a synthetic DAG analog, stimulated endothelial cell DNA synthesis in a concentration-dependent manner. In [3H]inositol-labeled cells, 15-HETE also caused a decrease in cellular PI content under both basal and stimulated conditions. 15-HETE, however, had no effect on either isolated phospholipase C activity or phosphoinositide turnover in lithium chloride-treated cells. In intact cells, 15-HETE (30 microM) inhibited the synthesis of [3H]PI from [3H]inositol (80% inhibition, p less than 0.001). In human red cell membranes, the production of phosphatidic acid from endogenous DAG was inhibited by 15-HETE in a concentration-dependent manner with an IC50 of 41 microM. Although 12-HETE had effects similar to those of 15-HETE, the parent compound arachidonic acid did not affect DNA synthesis or DAG kinase activity. Our study thus demonstrates that the mitogenic activity of 15- and 12-HETE on endothelial cells may be mediated via DAG kinase inhibition with the concomitant accumulation of cellular DAG.     

Carbachol and Bradykinin Increase the Production of Diacylglycerol from Sources Other than Inositol-Containing Phospholipids in PC12 Cells

Both carbachol and bradykinin increased diacylglycerol formation in PC12 pheochromocytoma cells. The effect of carbachol was apparent only in cells that had been treated with nerve growth factor. Incubation of the cells in Ca2(+)-free medium attenuated carbachol-stimulated diacylglycerol formation but did not reduce the response to bradykinin. Pretreatment of the cells with pertussis toxin did not affect either carbachol- or bradykinin-stimulated diacylglycerol formation; therefore, the inhibitory guanine nucleotide Gi probably does not mediate this response. The time course of carbachol-stimulated diacylglycerol accumulation did not coincide with the time course of inositol 1,4,5-trisphosphate (IP3) production. IP3 was elevated at the earliest time measured, 15 s, and then slowly declined so that by 5 min IP3 levels were only 50% of maximal. Diacylglycerol levels, in contrast, were not elevated for the first 2 min and then peaked at 5 min. These data indicate that hydrolysis of phosphatidylinositol 4,5-bisphosphate was not the major source of the diacylglycerol peak at 5 min. To investigate the source of diacylglycerol, I examined the fatty acid composition of the diacylglycerol by prelabeling the cells with [3H]palmitic acid and [14C]stearic acid. The 14C/3H ratio in diacylglycerol should reflect the phospholipid(s) from which it is derived. The 14C/3H ratio of the increment in diacylglycerol produced by carbachol and bradykinin was intermediate between the 14C/3H ratios of phosphatidylcholine and phosphatidylinositol. The 14C/3H ratio in triacylglycerol was similar to that of phosphatidylcholine. These data indicate that carbachol and bradykinin stimulate the formation of diacylglycerol from sources other than inositol-containing phospholipids; phosphatidylcholine and triacylglycerol are two possible sources of this diacylglycerol.     

G Proteins Coupled to Phospholipase C: Molecular Targets of Long-Term Ethanol Exposure

Long-term ethanol exposure is known to inhibit bradykinin-stimulated phosphoinositide hydrolysis in cultures of neuroblastoma x glioma 108-15 cells. In the present study, [3H]bradykinin binding, GTP-binding protein function, and phospholipase C activity were assayed in cells grown for 4 days in 100 mM ethanol with the aim of elucidating the molecular target of ethanol on signal transduction coupled to inositol trisphosphate and diacylglycerol formation. Ethanol exposure reduced guanosine 5'-O-(3-thiotriphosphate) [GTP(S)]- and, to a lesser extent, NaF/AlCl3-stimulated phosphoinositide hydrolysis, whereas it had no effect on the enzymatic activity of a phosphatidylinositol 4,5-bisphosphate-specific phospholipase C. [3H]Bradykinin binding in the absence of GTP(S) was not influenced by ethanol exposure. However, the reduction in [3H]bradykinin binding seen in control cells after addition of GTP analogue was inhibited in cells grown in ethanol-containing medium. The results indicate that long-term ethanol exposure exerts its effects on receptor-stimulated phosphoinositide hydrolysis primarily at the level of the GTP-binding protein.     

Bradykinin stimulates phospholipase C in rat renal medullary slices

Rat renal medullary slices prelabeled with [14C]arachidonic acid generate [14C]diacylglycerol within 1 min of exposure to bradykinin action. Production of [14C]diacylglycerol is transient. 2 min after the addition of bradykinin, the levels of metabolite reach the maximum, but decrease thereafter. Simultaneously, bradykinin induces a parallel decrease of the radioactivity in phosphatidylinositol. No degradation of other phospholipids is observed, and triacylglycerol is not affected. The degradation of [14C]phosphatidylinositol to [14C]diacylglycerol indicated the presence of phospholipase C activity. Preincubation of prelabeled slices with 2 mM dibutyryl cyclic AMP prevents both the generation of diacylglycerol and the degradation of phosphatidylinositol. Neither mepacrine nor indomethacin block diacylglycerol production and phosphatidylinositol breakdown. We conclude that, when rat renal medullary slices are stimulated with bradykinin, phosphatidylinositol-specific phospholipase C is activated.     

Bradykinin Stimulates Arachidonic Acid Release Through the Sequential Actions of an sn-1 Diacylglycerol Lipase and a Monoacylglycerol Lipase

In cultured dorsal root ganglion (DRG) neurons prelabeled with [3H]arachidonic acid [( 3H]AA), bradykinin (BK) stimulation resulted in increased levels of radioactive diacylglycerol, monoacylglycerol, and free AA. The transient increases in content of radioactive diacylglycerol and monoacylglycerol preceded the increase in level of free AA, suggesting the contribution of a diacylglycerol lipase pathway to AA release. An analysis of the molecular species of diacylglycerols in unstimulated cultures revealed the presence of two primary [3H]AA-containing species, 1-palmitoyl-2-arachidonoyl and 1-stearoyl-2-arachidonoyl diacylglycerol. BK stimulation resulted in a preferential increase in content of 1-stearoyl-2-arachidonoyl diacylglycerol. When DRG cultures were labeled with [3H]stearic acid, treatment with BK increased the amount of label in diacylglycerol and free stearic acid, but not in monoacylglycerol. This result suggested that AA release occurred through the successive actions of an sn-1 diacylglycerol lipase and monoacylglycerol lipase. Other data supporting a diacylglycerol lipase pathway was the significant inhibition of [3H]AA release and consequent accumulation of diacylglycerol by RG 80267, which preferentially inhibits diacylglycerol lipase. Analysis of the molecular species profiles of individual phospholipids in DRG neurons indicated that phosphoinositide hydrolysis may account for a significant portion of the rapid increase in content of 1-stearoyl-2-arachidonoyl diacylglycerol. We were unable to obtain evidence that the phospholipase A2 pathway makes a significant contribution to BK-stimulated AA release in DRG cultures. Under our assay conditions there were no BK-stimulated increases in levels of radioactive lysophosphatidylinositol, lysophosphatidylcholine, or lysophosphatidylethanolamine in cultures prelabeled with [3H]inositol, [3H]choline, or [3H]-ethanolamine, respectively.     

Insulin Promotes the Hydrolysis of a Glycosyl Phosphatidylinositol in Cultured Rat Astroglial Cells

Abstract: Glycosyl phosphatidylinositols have been implicated in insulin signaling through their action as precursors of second messenger molecules in peripheral tissues. In the present study, cultured rat astrocytes were used to investigate whether glycosyl phosphatidylinositol might be involved in the mechanism of insulin signal transduction in neural cells. A glycosyl phosphatidylinositol sensitive to hydrolysis by both phosphatidylinositol-specific phospholipase C and glycosyl phosphatidylinositol-specific phospholipase D and to nitrous acid deamination was purified. When astrocytes were exposed to 10 n M insulin, a rapid and significant reduction in the content of glycosyl phosphatidylinositol was observed within 1–2 min. In addition, an inverse concentration-dependent relationship between glycosyl phosphatidylinositol and diacylglycerol levels was found, suggesting a phospholipase C-mediated hydrolysis of glycosyl phosphatidylinositol in response to insulin. The effects of insulin were mediated through its own receptors and not through insulin-like growth factor (IGF)-I and/or IGF-II receptors, as demonstrated by affinity cross-linking studies. Also, the effects of 5 n M IGF-I or 5 n M IGF-II on glycosyl phosphatidylinositol and diacylglycerol levels were different from those caused by insulin and were not essentially modified by pretreatment of the cells with either platelet-derived growth factor (PDGF) or epidermal growth factor (EGF). When cells were sequentially incubated with PDGF and EGF, a reduction in both glycosyl phosphatidylinositol and diacylglycerol contents was observed; the diacyl-glycerol but not the glycosyl phosphatidyl content was reversed after incubation with IGF-I, and especially with IGF-II, for 10 min. Despite the remarkable homology among insulin, IGF-I, and IGF-II, our results indicate that in astrocytes these compounds probably use different signal transduction pathways.     

Decreased myo-lnositol Uptake Is Associated with Reduced Bradykinin-Stimulated Phosphatidylinositol Synthesis and Diacylglycerol Content in Cultured Neuroblastoma Cells Exposed to L-Fucose

Abstract: L-Fucose is a potent, competitive inhibitor of myo-inositol transport by cultured mammalian cells. Chronic exposure of neuroblastoma cells to L-fucose causes a concentration-dependent decrease in myo-inositol content, accumulation, and incorporation into phosphoinositides. In these studies, L-fucose supplementation of culture medium was used to assess the effect of decreased myo-inositol metabolism and content on bradykinin-stimulated phosphatidylinositol synthesis and diacylglycerol production. Chronic exposure of cells to 30 mML-fucose caused a sustained decrease in bradykinin-stimulated, but not basal, ³H-inositol phosphate release and ³²P incorporation into phosphatidylinositol in cells incubated in serum-free, unsupplemented medium. In addition, ³²P incorporation into phosphatidylinositol 4-phosphate and phosphatidylinositol 4, 5-bisphosphate was not altered in L-fucose-conditioned cells. Acute exposure of cells to serum-free medium containing 30 mM L-fucose did not affect either basal or bradykinin-stimulated ³²P incorporation into phosphatidylinositol. Basal diacylglycerol content was decreased by 20% in cells chronically exposed to 30 mM L-fucose, although analysis of the molecular species profile revealed no compositional change. Bradykinin stimulated diacylglycerol production in neuroblastoma cells by increasing the hydrolysis of both phosphoinositides and phosphatidylcholine. Bradykinin-stimulated production of total diacylglycerol was similar for control and L-fucose-conditioned cells. However, there was a decrease in the bradykinin-induced generation of the 1 -stearoyl-2-arachidonoyl diacylglycerol molecular species in the cells chronically exposed to 30 mM L-fucose. This molecular species accounts for about 70% of the composition of phosphoinositides, but only 10% of phosphatidylcholine. The results suggest that a decrease in myo-inositol uptake results in diminished agonist-induced phosphatidylinositol synthesis and phosphoinositide hydrolysis in cultured neuroblastoma cells grown in L-fucose-containing medium.     

Interleukin 2 does not induce phosphatidylinositol hydrolysis in activated T cells

Hydrolysis of phosphatidylinositol-4,5-bisphosphate to diacylglycerol and myoinositol-1,4,5-trisphosphate is thought to be a primary event in the activation of cells by some growth factors, mitogenic lectins, and oncogenes. The mechanism whereby interleukin 2 (IL 2) binding to its receptor on activated T lymphocytes leads to cell proliferation has not been determined. Because the mitogenic has not been determined. Because the mitogenic action of IL 2 resembles that of some growth factors, the possible role of phosphatidylinositol breakdown in the activation of T cells by IL 2 was examined. In human or murine IL 2-sensitive cells, incubation with IL 2 did not alter the rate of turnover of phosphatidylinositol, phosphatidylinositol-5-phosphate, phosphatidylinositol-4,5-bisphosphate, or phosphatidylcholine in 32PO4-loaded cells. IL 2 also did not alter either the isotopic labeling of diacylglycerol or [3H]arachidonic acid release from cells. In addition, IL 2 did not alter the rate of formation of the phosphatidylinositol breakdown products myoinositol-1,4,5-trisphosphate, myoinositol-1,4-bisphosphate, or myoinositol-1-phosphate. In contrast, under similar conditions, IL 2 induced significant increases in [3H]thymidine incorporation and cell proliferation. Mitogenic lectins such as concanavalin A and phytohemagglutinin gave significant changes in isotopic labeling of phosphoinositols, diacylglycerols, and phosphatidylinositols, indicating that phosphatidylinositol hydrolysis induced by mitogenic lectins was detectable in the assay systems. IL 2, in contrast to other growth factors, does not appear to signal cells by increasing phosphatidylinositol breakdown.     

Physiological implications of the contrasting modulation of the activities of the epsilon- and zeta-isoforms of diacylglycerol kinase

We have shown that the requirement of the epsilon-isoform of diacylglycerol kinase for diacylglycerols containing arachidonic acid is specific for this substrate and cannot be replaced by the presence of an arachidonoyl group in other places in the membrane; rather, it has to be present on the substrate itself. In addition, we demonstrate that the increased activity shown toward 1-stearoyl-2-arachidonoylglycerol by the epsilon-isoform of diacylglycerol kinase is not a consequence of altered membrane physical properties but is rather a specific interaction with the arachidonoyl group. We have also compared the modulation of the activity of the epsilon-isoform of diacylglycerol kinase with that of the zeta-isoform with regard to some of the intermediates involved in phosphatidylinositol cycling. One of the products of the hydrolysis of phosphatidylinositol diphosphate is diacylglycerol enriched in arachidonic acid. The activity of the epsilon-isoform is known to be specific for this form of diacylglycerol. We show that in contrast, the activity of the zeta-isoform is lower against 1-stearoyl-2-arachidonoylglycerol compared with dioleoylglycerol. We demonstrate that addition of phosphatidylserine, as well as other anionic phospholipids including L-alpha-phosphatidylinositol 4,5-bisphosphate, strongly inhibits the epsilon-isoform, but these anionic lipids increase the activity of the zeta-isoform. Addition of Ca(2+), which is released from internal stores as a consequence of phosphatidylinositol cycling, promotes the activity of the epsilon-isoform of this enzyme but has little effect on the zeta-isoform. The contrasting conditions required for maximal activity of these two isoforms of diacylglycerol kinase, as well as their different substrate specificity, suggest that they have different physiological roles in signal transduction.     

Substrate specificity of diacylglycerol kinase-epsilon and the phosphatidylinositol cycle

We show that diacylglycerol kinase-ε (DGKε) has less preference for the acyl chain at the sn-1 position of diacylglycerol (DAG) than the one at the sn-2 position. Although DGKε discriminates between 1-stearoyl-2-arachidonoyl-DAG and 1-palmitoyl-2-arachidonoyl-DAG, it has similar substrate preference for 1-stearoyl-2-arachidonoyl-DAG and 1,2-diarachidonoyl-DAG. We suggest that in addition to binding to the enzyme, the acyl chain at the sn-1 position may contribute to the depth of insertion of the DAG into the membrane. Thus, the DAG intermediate of the PI-cycle, 1-stearoyl-2-arachidonoyl-DAG, is not the only DAG that is a good substrate for DGKε, the DGK isoform involved in PI-cycling.     

A novel role for activating transcription factor-2 in 15(S)-hydroxyeicosatetraenoic acid-induced angiogenesis

To investigate the mechanisms underlying 15(S)-HETE-induced angiogenesis, we have studied the role of the small GTPase, Rac1. We find that 15(S)-HETE activated Rac1 in human retinal microvascular endothelial cells (HRMVEC) in a time-dependent manner. Blockade of Rac1 by adenovirus-mediated expression of its dominant negative mutant suppressed HRMVEC migration as well as tube formation and Matrigel plug angiogenesis. 15(S)-HETE stimulated Src in HRMVEC in a time-dependent manner and blockade of its activation inhibited 15(S)-HETE-induced Rac1 stimulation in HRMVEC and the migration and tube formation of these cells as well as Matrigel plug angiogenesis. 15(S)-HETE stimulated JNK1 in Src-Rac1-dependent manner in HRMVEC and adenovirus-mediated expression of its dominant negative mutant suppressed the migration and tube formation of these cells and Matrigel plug angiogenesis. 15(S)-HETE activated ATF-2 in HRMVEC in Src-Rac1-JNK1-dependent manner and interference with its activation via adenovirus-mediated expression of its dominant negative mutant abrogated migration and tube formation of HRMVEC and Matrigel plug angiogenesis. In addition, 15(S)-HETE-induced MEK1 stimulation was found to be dependent on Src-Rac1 activation. Blockade of MEK1 activation inhibited 15(S)-HETE-induced JNK1 activity and ATF-2 phosphorylation. Together, these findings show that 15(S)-HETE activates ATF-2 via the Src-Rac1-MEK1-JNK1 signaling axis in HRMVEC leading to their angiogenic differentiation.     

Ca2+-dependent and Ca2+-independent pathways for release of arachidonic acid from phosphatidylinositol in endothelial cells

The pathways for degradation of phosphatidylinositol (PI) were investigated in sonicated suspensions prepared from confluent cultures of bovine pulmonary artery endothelial cells. The time courses of formation of 3H-labeled and 14C-labeled metabolites of phosphatidyl-[3H]inositol ([3H]Ins-PI) and 1-stearoyl-2-[14C] arachidonoyl-PI were determined at 37 degrees C and pH 7.5 in the presence of 2 mM EDTA with or without a 2 mM excess of Ca2+. The rates of formation of lysophosphatidyl-[3H]inositol ([3H]Ins-lyso-PI) and 1-lyso-2-[14C] arachidonoyl-PI were similar in the presence and absence of Ca2+, and the absolute amounts of the two radiolabeled lyso-PI products formed were nearly identical. This indicated that lyso-PI was formed by phospholipase A1, and phospholipase A2 was not measurable. In the presence of EDTA, [14C]arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI paralleled release of glycerophospho-[3H]inositol ([3H]GPI) from [3H]Ins-PI. Formation of [3H]GPI was inhibited by treatment with the specific sulfhydryl reagent, 2,2'-dithiodipyridine, and this was accompanied by an increase in [3H]Ins-lyso-PI. In the presence of Ca2+, [14C] arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI was increased 2-fold and was associated with Ca2+-dependent phospholipase C activity. Under these conditions, [3H]inositol monophosphate production exceeded formation of [14C]arachidonic acid-labeled phospholipase C products, diacylglycerol plus monoacylglycerol, by an amount that was equal to the amount of [14C]arachidonic acid formed in excess of [3H]GPI. Low concentrations of phenylmethanesulfonyl fluoride (15-125 microM) inhibited Ca2+-dependent [14C]arachidonic acid release, and the decrease in [14C] arachidonic acid formed was matched by an equivalent increase in 14C label in diacylglycerol plus monoacyclglycerol. These data supported the existence of two pathways for arachidonic acid release from PI in endothelial cells; a phospholipase A1-lysophospholipase pathway that was Ca2+-independent and a phospholipase C-diacylglycerol lipase pathway that was Ca2+-dependent. The mean percentage of arachidonic acid released from PI via the phospholipase C-diacylglycerol lipase pathway in the presence of Ca2+ was 65 +/- 8%. The mean percentage of nonpolar phospholipase C products of PI metabolized via the diacylglycerol lipase pathway to free arachidonic acid was 28 +/- 3%.     

Blockade of Receptor-Mediated Cyclic GMP Formation by Hydroxyeicosatetraenoic Acid

Receptor-mediated cyclic GMP formation in N1E-115 murine neuroblastoma cells appears to involve oxidative metabolism of arachidonic acid. Evidence in support of this includes the blockade of this response by lipoxygenase inhibitors, e.g., eicosatetraynoic acid (ETYA) or other metabolic perturbants, e.g., methylene blue. It was recently discovered that the lipoxygenase products 15-hydroxyeicosatetraenoic (15-HETE) acid and 12-HETE, like ETYA, were inhibitors of M1 muscarinic receptor-mediated cyclic GMP formation. In the present report, the effects of monoHETEs are explored in more detail, particularly with regard to the function of the muscarinic receptor. Like 12-HETE and 15-HETE (IC50 = 13 and 11 microM, respectively), 5-HETE inhibited the cyclic GMP response to the muscarinic receptor (IC50 = 10 microM). All three of these monoHETEs were shown also to be inhibitors of the cyclic GMP responses to receptors stimulated by carbachol, histamine, thrombin, neurotensin, and bradykinin. 15-HETE was shown to inhibit the muscarinic receptor-mediated response in a complex manner (apparent noncompetitive and uncompetitive components; IC50 = 18 and 2 microM, respectively). 15-HETE did not inhibit either the M1 muscarinic receptor-stimulated release of [3H]inositol phosphates from cellular phospholipids or the M2 muscarinic receptor-mediated inhibition of hormone (prostaglandin E1)-induced AMP formation. It seemed possible that the monoHETEs could enter into biochemical pathways for arachidonate in N1E-115 cells. [3H]Arachidonate and the three [3H]-monoHETEs all rapidly labeled the membrane lipids of intact N1E-115 cells, with each [3H]eicosanoid producing a unique labeling profile. [3H]15-HETE labeling was noteworthy in that 85% of the label found in the phospholipids was in phosphatidylinositol (PI;t1/2 to steady state = 3 min). Exogenous 15-HETE inhibited the labeling of PI by [3H]arachidonate (IC50 = 28 microM) and elevated unesterified [3H]arachidonate levels. Thus, the mechanism of blockade of receptor-mediated cyclic GMP responses by monoHETEs is likely to be more complex than the simple inhibition of cytosolic mechanisms, e.g., generation of a putative second messenger by lipoxygenase, and may involve also alterations of membrane function accompanying the redistributions of esterified arachidonate.     

Regulation of diacylglycerol kinase reaction in Swiss 3T3 cells. Increased phosphorylation of endogenous diacylglycerol and decreased phosphorylation of didecanoylglycerol in response to platelet-derived growth factor

We studied the influence of platelet-derived growth factor (PDGF) on diacylglycerol phosphorylation in Swiss 3T3 cells. Rates of incorporation of 32P into phosphatidic acid (PA) and phosphatidylinositol (PtdIns) were determined in prelabeled cells into which sn-1,2-didecanoylglycerol (diC10) had been introduced. PDGF stimulated the formation of [32P]PA and -PtdIns from endogenous substrates but decreased the formation of [32P]PA10 and -PtdIns10. Direct measurements of diacylglycerol phosphorylation in lysates of quiescent and stimulated cells showed that PDGF stimulated the phosphorylation of endogenous diacylglycerol 2-fold in parallel with diacylglycerol accumulation but decreased by 50% the phosphorylation of diC10. Total diacylglycerol kinase activity, measured in a mixed micellar assay, was not changed by PDGF treatment. The maximum activity of diacylglycerol kinase exceeded that needed to phosphorylate all of the endogenous diacylglycerol, suggesting that the PDGF-dependent increase in diacylglycerol mass would account for the increase in PA formation. The increased mass of diacylglycerol also could explain the inhibition of diC10 phosphorylation, via substrate competition. The predominant species of endogenous diacylglycerol was 1-stearoyl-2-arachidonoyl-glycerol (18:0/20:4 diacylglycerol). In mixed micelles, the rate of phosphorylation of 18:0/20:4 diacylglycerol was 8-fold higher than that of diC10, and the 18:0/20:4 species competed with diC10 for phosphorylation. Studies showed that a membrane-bound enzyme accounted for the PDGF effect on PA formation; there was no evidence for translocation of cytosolic enzyme to the membrane. The results support these conclusions: 1) PDGF stimulates the phosphorylation of cellular diacylglycerol by promoting a transient accumulation of this lipid. 2) The stimulated phosphorylation is catalyzed by a diacylglycerol kinase that preferentially phosphorylates 18:0/20:4 diacylglycerol over diC10. 3) The diacylglycerol kinase responsible for the PDGF effect is membrane-bound.     

Specific incorporation of 5-hydroxy-6,8,11,14-eicosatetraenoic acid into phosphatidylcholine in human endothelial cells

The incorporation of hydroxyeicosatetraenoic acids (HETEs) into cellular lipids was studied in cultures of human umbilical vein endothelial cells. 5-[3H]HETE was incorporated into the phospholipids (8%) and neutral lipids (15.5%). The uptake was at half maximum after 15 min and reached a plateau after 1 h. The incorporation occurred mainly into phosphatidylcholine (6.3%) with minimal uptake into phosphatidylserine and phosphatidylinositol (0.6%) or phosphatidylethanolamine (1.2%). There was no uptake of 12-[3H]HETE, 15-[3H]HETE or [3H]leukotriene B4 into phospholipids. Treatment of the phosphatidylcholine fraction with phospholipase A2 released 64% of the 5-[3H]HETE with 26% remaining in the lysophosphatidylcholine fraction. This indicates that the majority of the 5-HETE was in the sn-2 position. Unlabeled 5-HETE and arachidonic acid inhibited the uptake of 5-[3H]HETE into phosphatidylcholine with an ID50 of 2.5 and 1.25 microM, respectively. Stearic acid and 15-HETE were not effective inhibitors. Histamine, which activates phospholipases, increased the uptake of 5-[3H]HETE into phosphatidylcholine by 3-fold. Both 5-[3H]HETE and 12-[3H]HETE were incorporated into the neutral lipids of the cells. Analysis of the neutral lipid fraction revealed that 5-[3H]HETE was incorporated into mono-, di- and triacylglycerols but not cholesterol esters. Incorporation of 5-HETE into cellular lipids reduced histamine- and arachidonic acid-stimulated synthesis of 6-ketoprostaglandin F1 alpha and prostaglandin E2 in a concentration-related manner. Angiotensin I converting enzyme activity was not changed. Thus, 5-HETE is incorporated specifically into phosphatidylcholine and glycerol esters of human endothelial cells and this incorporation inhibits prostaglandin synthesis in these cells.     

Rapid dephosphorylation of protein kinase C substrates by protein kinase A activators results from inhibition of diacylglycerol release

The biochemical events encompassing the dephosphorylation of protein kinase C substrates by protein kinase A activators have been investigated in a neurotumor cell line, NCB-20. Treatment of [32P]orthophosphate-labeled cells with protein kinase A activators (e.g. forskolin, dibutyryl cAMP, prostaglandin E1) resulted in an inhibition of protein kinase C activity due to a failure of the protein kinase C complex to translocate into the membrane. Phospholipase C activity, as measured by the synchronous release of diacylglycerol and inositol phosphates (inositol 1,4,5-trisphosphate, inositol 1,4-bisphosphate, and inositol 1-phosphate) in response to bradykinin, was inhibited up to 50% following exposure to protein kinase A activators. At the same time, phospholipase C-specific inositol phospholipid substrates (phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate) were found to accumulate in NCB-20 cells following treatment with protein kinase A activators. This suggests that phospholipase C may be altered through protein kinase A-mediated protein phosphorylation. Second messenger generation (inositol phosphates, diacylglycerol, and Ca2+) is therefore inhibited through cyclic AMP-mediated shutdown of the inositol lipid cycle at the level of phospholipase C.