Glucose-induced synthesis of diacylglycerol de novo is associated with translocation (activation) of protein kinase C in rat adipocytes

Addition of glucose (5-20 mM) to rat adipocytes provoked dose-related increases in diacylglycerol, without increasing production of [3H]inositol phosphates. Cytosolic protein kinase C enzyme activity and immunoreactivity decreased within 1-5 min of 5 mM glucose addition, and further over 20 min. Membrane protein kinase C increased stoichiometrically during the first 5 min and then decreased. Higher concentrations (10 and 20 mM) of glucose provoked greater and more rapid decreases of cytosolic and membrane protein kinase C. Our findings suggest that glucose stimulates diacylglycerol production by providing substrate for phosphatidic acid synthesis de novo, and this is associated with translocative activation of protein kinase C.     

Evidence for the calcium-dependent activation of phospholipase D in thrombin-stimulated human erythroleukaemia cells.

Human erythroleukaemia (HEL) cells were exposed to thrombin and other platelet-activating stimuli, and changes in radiolabelled phospholipid metabolism were measured. Thrombin caused a transient fall in PtdInsP and PtdInsP2 levels, accompanied by a rise in diacylglycerol and phosphatidic acid, indicative of a classical phospholipase C/diacylglycerol kinase pathway. However, the rise in phosphatidic acid preceded that of diacylglycerol, which is inconsistent with phospholipase C/diacylglycerol kinase being the sole source of phosphatidic acid. In the presence of ethanol, thrombin and other agonists (platelet-activating factor, adrenaline and ADP, as well as fetal-calf serum) stimulated the appearance of phosphatidylethanol, an indicator of phospholipase D activity. The Ca2+ ionophore A23187 and the protein kinase C activator phorbol myristate acetate (PMA) also elicited phosphatidylethanol formation, although A23187 was at least 5-fold more effective than PMA. Phosphatidylethanol production stimulated by agonists or A23187 was Ca2(+)-dependent, whereas that with PMA was not. These result suggest that phosphatidic acid is generated in agonist-stimulated HEL cells by two routes: phospholipase C/diacylglycerol kinase and phospholipase D. Activation of the HEL-cell phospholipase D in response to agonists may be mediated by a rise in intracellular Ca2+.     

Effects of phorbol ester and cholecystokinin on the intracellular distribution of protein kinase C in rabbit pancreatic acini.

Treatment of rabbit pancreatic acini with the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), resulted in a time- and dose-dependent decrease of soluble protein kinase C activity coinciding with an increase of protein kinase C activity in the particulate fraction. After 5 min, soluble protein kinase C activity had decreased to almost 10% of the corresponding control. Total extractable protein kinase C activity, however, remained unchanged, indicating that the decrease of soluble protein kinase C activity was not due to TPA-induced inactivation of the enzyme. The biologically inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate, did not induce such a translocation of protein kinase C. The half-maximal concentration for TPA-induced translocation of protein kinase C was 40 nM, and was equal to that for TPA-induced amylase secretion from isolated acini. This suggests that translocation of protein kinase C to the particulate fraction is an important step in TPA-induced activation of protein kinase C and enzyme secretion. On the other hand, cholecystokinin, a secretagogue of the calcium-mobilizing type, whose secretory action is thought to be mediated, at least in part, by protein kinase C, did not change the subcellular distribution of protein kinase C. In the presence of R59022 6-(2-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl ) ethyl-7-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one, an inhibitor of diacylglycerol kinase activity, cholecystokinin produced a small but significant translocation of protein kinase C, suggesting that the inability of the hormone to induce translocation is not due to a rapid conversion of the diacylglycerol formed into phosphatidic acid.     

Insulin increases membrane and cytosolic protein kinase C activity in BC3H-1 myocytes

Insulin treatment stimulated the activity of the Ca2+- and phospholipid-dependent protein kinase (protein kinase C) in both cytosolic and membrane fractions of BC3H-1 myocytes. Within 60 s of insulin treatment, membrane protein kinase C activity increased 2-fold, diminished toward control levels transiently, and then increased 2-fold again after 15 min. Cytosolic protein kinase C activity increased more gradually and steadily up to 80% over a 20-min period. Increases in protein kinase C activity were dose-dependent and were not simply a result of translocation of cytosolic enzyme (although this may have occurred), as total activity was also increased. The increase in protein kinase C activity was not inhibited by cycloheximide (which also increased protein kinase C activity and 2-deoxyglucose transport) and was still evident following anion exchange chromatography. The insulin effect was decidedly different from those of 12-O-tetradecanoylphorbol-13-acetate and phenylephrine using histone III-S as substrate. Phenylephrine decreased cytosolic protein kinase C activity while increasing membrane activity; 12-O-tetradecanoylphorbol-13-acetate only decreased cytosolic protein kinase C activity. The early insulin-induced increases in membrane protein kinase C activity may be related to increased diacylglycerol generation from de novo phosphatidic acid synthesis, as there were rapid increases in [3H]glycerol incorporation into diacylglycerol, and transient increases in phospholipid hydrolysis, as there were transient rapid increases in [3H]diacylglycerol in cells prelabeled with [3H]arachidonate. Later, sustained increases in membrane and cytosolic protein kinase C activity may reflect the continuous activation of de novo phospholipid synthesis, as there were associated increases in [3H]glycerol incorporation into diacylglycerol at later, as well as very early time points.     

Messenger molecules of the phospholipase signaling system have dual effects on vascular smooth muscle contraction

Background and methods. In order to investigate the role of phospholipases and their immediately derived messengers in agonist-induced contraction of portal vein smooth muscle, we used the addition in the organ bath of exogenous molecules such as: phospholipases C, A(2), and D, diacylglycerol, arachidonic acid, phosphatidic acid, choline. We also used substances modulating activity of downstream molecules like protein kinase C, phosphatidic acid phosphohydrolase, or cyclooxygenase. Results. a) Exogenous phospholipases C or A(2), respectively, induced small agonist-like contractions, while exogenous phospholipase D did not. Moreover, phospholipase D inhibited spontaneous contractions. However, when added during noradrenaline-induced plateau, phospholipase D shortly potentiated it. b) The protein kinase C activator, phorbol dibutyrate potentiated both the exogenous phospholipase C-induced contraction and the noradrenaline-induced plateau, while the protein kinase C inhibitor 1-(-5-isoquinolinesulfonyl)-2-methyl-piperazine relaxed the plateau. c) When added before noradrenaline, indomethacin inhibited both phasic and tonic contractions, but when added during the tonic contraction shortly potentiated it. Arachidonic acid strongly potentiated both spontaneous and noradrenaline-induced contractions, irrespective of the moment of its addition. d) In contrast, phosphatidic acid inhibited spontaneous contractile activity, nevertheless it was occasionally capable of inducing small contractions, and when repetitively added during the agonist-induced tonic contraction, produced short potentiations of the plateau. Pretreatment with propranolol inhibited noradrenaline-induced contractions and further addition of phosphatidic acid augmented this inhibition. Choline augmented the duration and amplitude of noradrenaline-induced tonic contraction and final contractile oscillations. Conclusions. These data suggest that messengers produced by phospholipase C and phospholipase A(2) contribute to achieve the onset and maintenance of contraction, while phospholipase D-yielded messengers appear to provide a delayed "on/off switch" that ultimately brings relaxation.     

Protein kinase C: subcellular redistribution by increased Ca2+ influx. Evidence that Ca2+-dependent subcellular redistribution of protein kinase C is involved in potentiation of beta-adrenergic stimulation of pineal cAMP and cGMP by K+ and A23187

Phenylephrine is known to stimulate translocation of protein kinase C in rat pinealocytes (Sugden, D., Vanecek, J., Klein, D.C., Thomas, T.P., and Anderson, W. B. (1985) Nature 314, 359-361). In the present study, the receptor mediating this effect was found to belong to the alpha 1-adrenoceptor subclass. Activation of this receptor is also known to produce a sustained increase in [Ca2+]i by increasing net influx (Sugden, A. L., Sugden, D., and Klein, D. C. (1985) J. Biol. Chem. 261, 11608-11612), which points to the possible importance of Ca2+ influx in the subcellular redistribution (activation) of protein kinase C in intact cells. This possibility was investigated by reducing extracellular Ca2+ ((Ca2+]o) with EGTA or by inhibiting Ca2+ influx with inorganic Ca2+ blockers. These treatments reduced alpha 1-adrenoceptor-mediated translocation of protein kinase C. This suggested that elevation of Ca2+ influx alone triggers activation of protein kinase C. In support of this, it was found that treatments which elevate Ca2+ influx, including increased extracellular K+ and addition of the Ca2+ ionophore A23187, cause redistribution of protein kinase C. The effect of K+ was blocked by nifedipine and that of A23187 by EGTA, indicating that effects of these agents are Ca2+-dependent. The possible role of phospholipase C activation in these effects was examined by measuring the formation of [3H]diacylglycerol by cells labeled with [3H]arachidonic acid. Although [3H]diacylglycerol formation was easily detected in the presence or absence of an effective concentration of an inhibitor of diacylglycerol kinase, none of the agents which cause rapid translocation of protein kinase C were found to cause a rapid increase in the generation of [3H]diacylglycerol. These findings establish that an increase in Ca2+ influx is sufficient to trigger translocation of protein kinase C. In addition, we found that a very close correlation exists between translocation of protein kinase C by phenylephrine, K+, and A23187 and their ability to potentiate beta-adrenergic stimulation of cAMP and cGMP accumulation. This provides strong support to the proposal that translocation of protein kinase C is required for potentiation of beta-adrenergic stimulation of pinealocyte cAMP and cGMP accumulation.     

Cholinephosphotransferase in rat lung. In vitro formation of dipalmitoylphosphatidylcholine and general lack of selectivity using endogenously generated diacylglycerol

Diacylglycerol was generated in vitro in rat lung microsomes by forming phosphatidic acid via sn-glycerol-3-phosphate acyltransferase followed by the hydrolysis of the phosphatidic acid by phosphatidate phosphohydrolase. Diacylglycerol concentrations of 35 to 50 nmol/mg of microsomal protein were obtained. Cholinephosphotransferase activity was determined in microsomes by measuring the conversion of endogenously generated [14C]diacylglycerol to phosphatidylcholine. Reaction rates of 14 to 16 nmol/min/mg of protein were obtained with a 30-s reaction. Diacylglycerol which was primarily dipalmitoylglycerol was produced when palmitic acid was used in the sn-glycerol-3-phosphate acyltransferase reactions. Dipalmitoylphosphatidylcholine was formed via cholinephosphotransferase from the dipalmitoylglycerol with an apparent maximal velocity of 20 nmol/min/mg of protein. When oleic acid was used instead of palmitic acid, the apparent maximal velocity for cholinephosphotransferase was 26 nmol/min/mg of protein. The apparent Km values for the two different diacylglycerol substrates were the same (28.5 nmol/mg of protein). Diacylglycerols, with different molecular species composition, were generated using a variety of fatty acids and fatty acid mixtures. The phosphatidylcholine formed from these diacylglycerols had the same molecular species profiles as the diacylglycerol used as the substrate. The relative reaction rates with the different diacylglycerols were essentially the same except when 20:4 and 22:6 fatty acids were used individually, in which case the rates were lower. We conclude that cholinephosphotransferase readily forms dipalmitoylphosphatidylcholine from endogenously generated dipalmitoylglycerol and that the cholinephosphotransferase reaction is generally nonselective for the diacylglycerol substrate.     

Diacylglycerol mass measurements in stimulated HL-60 phagocytes

The mass of sn-1,2-diacylglycerol in crude lipid extracts from differentiated HL-60 phagocytes was measured by quantitative conversion of the diacylglycerol to [32P]-labeled phosphatidic acid catalyzed by E. coli diacylglycerol kinase. The chemotactic peptide N-formyl-Met-Leu-Phe caused a time- and concentration-dependent increase in diacylglycerol that was maximal at 4 min. Diacylglycerol returned toward basal levels by 15 min. The basal level of diacylglycerol was 290 +/- 25 pmol/10(7) cells (n = 36). Maximally effective concentrations of N-formyl-Met-Leu-Phe and N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys increased diacylglycerol to 176% +/- 16 of basal (n = 8) and 198% +/- 15 of basal (n = 4), respectively. t-Boc-Phe-Leu-Phe-Leu-Phe, a competitive antagonist of formyl peptide receptor function, competitively inhibited the N-formyl-Met-Leu-Phe-induced diacylglycerol increase. Pretreatment of the cells with pertussis toxin abolished the stimulated rise in diacylglycerol, whereas depletion of extracellular Ca2+ markedly inhibited the increase. The Ca2+ ionophore A23187 stimulated a large (450% of basal) and persistent (greater than 30 min) increase in diacylglycerol. These data suggest that agents which raise intracellular Ca2+ levels in differentiated HL-60 cells produce a prolonged increase in cellular diacylglycerol which may activate protein kinase C.     

Evidence for the presence of diacylglycerol kinase in rat brain myelin

The previous demonstration that incubation of brain slices with [³²P]phosphate brings about rapid tabeling of phosphatidic acid in myelin suggests that the enzyme involved should be present in this specialized membrane. DAG kinase (ATP:1,2-diacyglycerol 3-phosphotransferase, E.C. 2.7.1.107) is present in rat brain homogenate at a specific activity of 2.5 nmol phosphatidic acid formed/min/mg protein, while highly purified myelin had a much lower specific activity (0.29 nmol/min/mg protein). Nevertheless, the enzyme appears to be intrinsic to this membrane since it can not be removed by washing with a variety of detergents or chelating agents, and it could not be accounted for as contamination by another subcellular fraction. Production of endogenous, membrane-associated, diacylglycerol (DAG) by PLC (phospholipase C) treatment brought about translocation from soluble to particulate fractions, including myelin. Another level of control of activity involves inactivation by phosphorylation; a 10 min incubation of brain homogenate with ATP resulted in a large decrease in DAG kinase activity in soluble, particulate and myelin fractions.     

Ceramide is a competitive inhibitor of diacylglycerol kinase in vitro and in intact human leukemia (HL-60) cells.

Prior studies demonstrated that ceramide was phosphorylated by a novel Ca(2+)-dependent kinase distinct from diacylglycerol (DG) kinase in human myelogenous leukemia (HL-60) cells (Kolesnick, R. N., and Hemer, M. R. (1990) J. Biol. Chem. 265, 10900-10904). The present studies were initiated to determine whether mammalian DG kinase purified to homogeneity possessed phosphotransferase activity toward ceramide. A high molecular weight rat brain DG kinase demonstrated Mg(2+)-(but not Ca(2+)-) dependent DG kinase activity and did not phosphorylate ceramide in the presence of either cation. In contrast, ceramide served as a competitive inhibitor with an inhibition constant (Ki) 2-6-fold greater than the Km for DG. Inhibition was noncompetitive with respect to ATP and Mg2+. A cell-permeable ceramide, N-octanoyl sphingosine (C8-cer), was used to study effects of ceramide on DG kinase in intact HL-60 cells. C8-cer induced dose- and time-dependent increases in cellular DG levels. As little as 1 microM C8-cer increased DG from a basal level of 103 to 177 pmol.10(6) cells-1, and a maximal 2.9-fold elevation to 292 pmol.10(6) cells-1 occurred with 10 microM C8-cer. DG elevation was detected after 1 min, maximal by 7.5 min, and sustained for 30 min. The DG elevation was accompanied by a reduction in 32P incorporation in phosphatidic acid in cells short term-labeled with [32P]orthophosphoric acid, consistent with inhibition of DG kinase. In contrast, a similar elevation in the DG level induced by exogenous phospholipase C increased 32P incorporation into phosphatidic acid. C8-cer was not metabolized to sphingomyelin, indicating that DG was not generated through the phosphatidylcholine:ceramide cholinephosphotransferase reaction. DG elevation after C8-cer or phospholipase C treatment was sufficient to redistribute protein kinase C from cytosol to membrane. These findings provide evidence that ceramide may serve as a competitive inhibitor of DG kinase.     

Role of phospholipase D in the activation of protein kinase D by lysophosphatidic acid

Protein kinase D was auto-phosphorylated at Ser916 and trans-phosphorylated at Ser744/Ser748 in Rat-2 fibroblasts treated with lysophosphatidic acid. Both phosphorylations were inhibited by 1-butanol, which blocks phosphatidic acid formation by phospholipase D. The phosphorylations were also reduced in Rat-2 clones with decreased phospholipase D activity. Platelet-derived growth factor-induced protein kinase D phosphorylation showed a similar requirement for phospholipase D, but that induced by 4beta-phorbol 12 myristate 13-acetate did not. Propranolol an inhibitor of diacylglycerol formation from phosphatidic acid blocked the phosphorylation of protein kinase D, whereas dioctanoylglycerol induced it. The temporal pattern of auto-phosphorylation of protein kinase D closely resembled that of phospholipase D activation and preceded the trans-phosphorylation by protein kinase C. These results suggest that protein kinase D is activated by lysophosphatidic acid through sequential phosphorylation and that diacylglycerol produced by PLD via phosphatidic acid is required for the autophosphorylation that occurs prior to protein kinase C-mediated phosphorylation.     

Acth increases de novo synthesis of diacylglycerol and translocates protein kinase C in primary cultures of calf adrenal glomerulosa cells

Effects of ACTH on the production of diacylglycerol (DAG) and translocation of protein kinase C were studied in primary cultures of calf adrenal glomerulosa cells. To study DAG production two different labeling protocols were used: (a) cells were prelabeled for 3 days with [2-³H]glycerol before ACTH addition; (b) ACTH and [2-³ H]glycerol were added simultaneously to cells. In both cases, ACTH provoked rapid increases in the labeling of DAG which were maximal in 2 min, dose-dependent, and paralleled by increases in DAG mass. ACTH also increased the labeling of total glycerolipids including phosphatidic acid (PA), phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine and triacylglycerol. In both labeling protocols, the rates of increase in the labeling of DAG and PA were greater than those of other glycerolipids. Our results indicate that ACTH rapidly increases DAG, at least partly by stimulating the de novo synthesis of PA. In addition, we found that ACTH, like phorbol esters, stimulated the apparent translocation of immunoreactive protein kinase C from the cytosol to the membrane fraction.     

Stimulation of mitogenesis and glucose transport by 1-monooleoylglycerol in Swiss 3T3 fibroblasts

1-Monooleoylglycerol (MOG), a recently reported diacylglycerol kinase inhibitor (Bishop, W. R., Ganong, B. R., and Bell, R. M. (1986) J. Biol. Chem. 261, 6993-7000), exerts potent stimulatory effects on [3H]thymidine incorporation into DNA and glucose transport in Swiss 3T3 fibroblasts. MOG induces a rapid and sustained 2.5-fold increase in the cellular 1,2-diacylglycerol (1,2-DG) content, and phosphorylation of an acidic 80-kDa protein, a putative substrate for the protein kinase C (Ca2+/phospholipid-dependent protein kinase). The effect of MOG is additive to that of bombesin in terms of both an increase in tissue diacylglycerol content and phosphorylation of the 80-kDa proteins. In addition to these effects, MOG potently stimulates release of arachidonic acid from phospholipids. Inhibitors of cyclooxygenase and lipoxygenase have little effect, if any, on MOG-induced stimulation of glucose transport and DNA synthesis, while exogenously applied arachidonic readily stimulates both of these cellular responses. Furthermore, arachidonic acid, at its biologically active concentrations, is found to induce a rapid and sustained increase in cellular 1,2-DG content and stimulate the phosphorylation of the 80-kDa protein, although to a lesser extent than MOG. Prolonged pretreatment of the cells with phorbol 12,13-dibutyrate, which reduces the cellular protein kinase C content, markedly attenuates the effects of both MOG and arachidonic acid on glucose transport and DNA synthesis. These data indicate that MOG increases endogenous 1,2-DG content and thereby acts as a potent activator of protein kinase C, and that activation of protein kinase C is a crucial step in MOG-induced stimulation of mitogenesis and glucose transport.     

Cholinephosphotransferase in rat lung. The in vitro synthesis of dipalmitoylphosphatidylcholine from dipalmitoylglycerol

Active substrate preparations of dipalmitoylglycerol were obtained by sonicating dipalmitoylglycerol, phosphatidylglycerol, and Tween 20 together at 65 degrees C. The apparent Vmax for cholinephosphotransferase in lung microsomes was 30 nmol/min/mg of protein for dipalmitoylglycerol-phosphatidylglycerol-Tween 20 substrate preparations and 43 nmol/min/mg of protein for dioleoylglycerol-phosphatidylglycerol-Tween 20 preparations. Sonication at 65 degrees C was required for maximal activity with dipalmitoylglycerol but not for dioleoylglycerol. Highest activity was obtained when diacylglycerol was sonicated with phosphatidylglycerol and Tween 20 although phosphatidylglycerol also increased the activity when added separately. The presence of phosphatidylglycerol was critical particularly with dipalmitoylglycerol as substrate. Phosphatidylinositol, phosphatidylserine, and phosphatidic acid were slightly active but phosphatidylcholine and phosphatidylethanolamine were inactive.     

A diacylglycerol kinase inhibitor, R59022, potentiates secretion by and aggregation of thrombin-stimulated human platelets.

The diacylglycerol kinase inhibitor R59022 (10 microM) potentiates secretion and aggregation responses in human platelets challenged with sub-maximal concentrations of thrombin. Potentiation correlates closely with increased formation of diacylglycerol, increased phosphorylation of a 40 kDa protein, a known substrate for protein kinase C, and with decreased formation of phosphatidic acid, the product of diacylglycerol kinase. Phosphorylation of myosin light chains, formation of inositol phosphates and the mobilization of Ca2+ by thrombin are not affected by R59022 (10 microM). These data support a role for protein kinase C in platelet aggregation and secretion, and provide further evidence that endogenous diacylglycerols bring about the activation of this enzyme. These data also add further argument against a role for phosphatidic acid in platelet activation.     

Synthesis of saturated phosphatidylcholine and phosphatidylglycerol by freshly isolated rat alveolar type II cells

Saturated phosphatidylcholine and phosphatidylglycerol are important components of pulmonary surface active material, but the relative contributions of different pathways for the synthesis of these two classes of phospholipids by alveolar type II cells are not established. We purified freshly isolated rat type II cells by centrifugal elutriation and incubated them with [1-14C]palmitate as the sole exogenous fatty acid in one series of experiments or with [9,10-3H]palmitate, mixed fatty acids (16:0, 18:1 and 18:2), and [U-14C]glucose in another series of experiments. Type II cells readily incorporated [1-14C]palmitate into saturated phosphatidic acid (55-59% of total phosphatidic acid), saturated diacylglycerol (82-87% of total diacylglycerol), saturated phosphatidylcholine (69-76% of total phosphatidylcholine), and saturated phosphatidylglycerol (55-59% of total phosphatidylglycerol). Saturated phosphatidic acid, diacylglycerol and phosphatidylglycerol were nearly equally labeled in the sn-1 and sn-2 positions, whereas saturated phosphatidylcholine was preferentially labeled in the sn-2 position. With [9,10-3H]palmitate and [U-14C]glucose, the labeling patterns of phosphatidic acid, diacylglycerol and phosphatidylglycerol were similar to each other but different from that of phosphatidylcholine. The glucose label was found predominantly in the unsaturated phosphatidylcholines at early times (3-10 min) and in the saturated phosphatidylcholines at later times (30-90 min). Similarly, the 3H/14C ratio was very high in saturated phosphatidylcholine and always above that in saturated diacylglycerol. We conclude that freshly isolated type II cells synthesize saturated phosphatidic acid, diacylglycerol, phosphatidylcholine and phosphatidylglycerol and that under our in vitro conditions the deacylation-reacylation pathway is important for the synthesis of saturated phosphatidylcholine but is less important for the synthesis of saturated phosphatidylglycerol. By the assumptions stated in the text during the pulse chase experiment de novo synthesis of saturated phosphatidylcholine from saturated diacylglycerol accounted for 25% of the total synthesis of saturated phosphatidylcholine.     

Endothelin stimulates phosphatidic acid formation in cultured rat mesangial cells: role of a protein kinase C-regulated phospholipase D.

We have previously reported that endothelin-1 stimulates phospholipase C-induced hydrolysis of phosphatidylinositol-4,5-bisphosphate. Other signal transduction pathways that hydrolyze alternative phospholipids through phospholipase D may also mediate endothelin-stimulated cellular responses. We initially evaluated endothelin-dependent generation of 32P-phosphatidic acid as an indirect indication of phospholipase D activity in rat mesangial cells. Endothelin (10(-7) M) induced an elevation of phosphatidic acid that was maximal at 15 min and persisted upward of 60 min. Pretreatment with the diacylglycerol-kinase inhibitor, R59022, did not reduce formation of endothelin-stimulated 32P-phosphatidic acid, demonstrating that the sequential actions of phospholipase C/diacylglycerol kinase do not contribute to endothelin-stimulated phosphatidic acid formation. We next conclusively identified a role for phospholipase D in the generation of phosphatidic acid by assessing the formation of 3H-phosphatidylethanol from 3H-alkyl lyso glycerophosphocholine and exogenous ethanol. Endothelin stimulated 3H-alkyl phosphatidylethanol formation in the presence but not the absence of 0.5% ethanol. Also, endothelin induced a concomitant elevation of 3H-alkyl-phosphatidic acid that was significantly reduced when the cells were exposed to exogenous ethanol, reflecting the formation of phosphatidylethanol. In addition, endothelin stimulated the release of 3H-choline and 3H-ethanolamine, demonstrating that additional phospholipids may serve as substrates for phospholipase D. Phorbol esters and synthetic diglycerides mimicked the effects of endothelin to stimulate phospholipase D and inhibitors of protein kinase C significantly reduced endothelin-stimulated phospholipase D. In addition, endothelin did not stimulate phosphatidylethanol formation in protein kinase C down-regulated cells. The calcium ionophore, ionomycin, did not stimulate phospholipase D and mesangial cells pretreated with BAPTA to chelate cytosolic calcium did not show a diminished endothelin-stimulated phospholipase D. Thus these data demonstrate that mesangial cells possess a protein kinase C-regulated phospholipase D activity that can be stimulated with endothelin.     

Stereospecificity of diacylglycerol for stimulus-response coupling in platelets

In intact platelets, a permeable diacylglycerol having a 1,2-sn- but not 2,3-sn- configuration activated protein kinase C directly. In the presence of Ca2+-ionophore this diacylglycerol caused full activation of platelet release reaction. 1,3-Isomer was inactive. Among these isomers only 1,2-sn-diacylglycerol was converted rapidly to the corresponding phosphatidic acid in both intact and broken cell preparations. Thus, the diacylglycerol which functions in stimulus-response coupling possesses a 1,2-sn-glycerol backbone, and other isomers are not involved in the signal transduction through the protein kinase C pathway.     

Phosphorylation, transbilayer movement, and facilitated intracellular transport of diacylglycerol are involved in the uptake of a fluorescent analog of phosphatidic acid by cultured fibroblasts

We have previously shown that a fluorescent derivative of phosphatidic acid, 1-acyl-2-[N-(4-nitrobenzo-2-oxa-1,3-diazole) aminocaproyl]phosphatidic acid (C6-NBD-PA) is rapidly transferred from liposomes to Chinese hamster fibroblasts at 2 degrees C, resulting in intense labeling of the mitochondria, endoplasmic reticulum, and nuclear envelope, but not the plasma membrane. During this labeling, C6-NBD-PA is metabolized predominantly to fluorescent diacylglycerol (Pagano, R. E., Longmuir, K. J., Martin, O. C., and Struck, D. K. (1981) J. Cell Biol. 91, 872-877). In the present study we investigated the mechanism by which C6-NBD-PA enters cells and is translocated to intracellular membranes at low temperature. (i) When hydrolysis of C6-NBD-PA to diacylglycerol was prevented by using a nonhydrolyzable fluorescent phosphonate analog, intense labeling of the plasma membrane occurred but fluorescent lipid did not enter the cytoplasm of cells. (ii) Experiments using C6-NBD-PA and cells prelabeled with 32Pi demonstrated that some of the fluorescent diacylglycerol was rephosphorylated at 2 degrees C. (iii) When cells were treated with 1,3-[palmitoyl, N-(4-nitrobenzo-2-oxa-1,3-diazole)aminocaproyl]-glycerophosphate, the lipid was dephosphorylated to 1,3-diacylglycerol but its rephosphorylation could not be detected. Nevertheless, rapid labeling of cytoplasmic membranes occurred. (iv) Formation of fluorescent diacylglycerol at the plasma membrane by treatment of cells with fluorescent phosphatidylcholine followed by phospholipase C at 2 degrees C resulted in strong labeling of intracellular membranes. Based on these results, a working model is presented for the uptake and intracellular translocation of phosphatidic acid involving formation of diacylglycerol at the plasma membrane followed by its transbilayer movement, facilitated translocation to intracellular membranes, and rephosphorylation.     

Generation of phosphatidic acid and diacylglycerols following ligation of surface immunoglobulin in human B lymphocytes: potential role in PKC activation.

We have examined signal transduction via membrane IgM (mIgM) in resting and cycling human B cells. Crosslinking mIgM on all of the cell types studied transduced a signal through the phosphatidylinositol pathway, producing inositol 1,4,5-trisphosphate and release of intracellular free calcium. These second messengers were formed regardless of quantitative or qualitative differences in the surface expression of mIgM: cells that had low levels of surface IgM (T-51) or had no light chain associated with surface heavy chain (DB) signaled phosphatidylinositol pathway activation after mIgM crosslinking. Production of specific lipid products in nonquiescent B cells differed from that in normal resting cells. Ligation of surface immunoglobulin on resting B cells resulted in sustained increases of both diacylglycerol and phosphatidic acid, two lipids that can influence PKC activation. Whereas PKC was strongly activated in normal tonsillar B cells, several cell lines had reduced PKC activation following crosslinking of mIgM. The reduction in protein kinase C activation correlated with the absence or reduced levels of phosphatidic acid or diacylglycerol following stimulation: protein kinase C translocated and was activated only in cells that had elevated levels of both diacylglycerides and phosphatidic acid. Anti-IgM-induced phosphorylation of a protein kinase C substrate protein CD20, also increased in those cells having PKC activation and not in cells in which kinase activity was reduced. CD20 phosphorylation also increased following the direct addition of exogenous phosphatidic acid to resting B cells. Together, these observations show that the generation of lipid products following mIgM crosslinking in resting cells can vary from that in cycling cells and may relate to the different levels of PKC activation. In a companion study we report that ligation of surface IgM activates both an acyltransferase and phospholipase D to form phosphatidic acid.