Tumor necrosis factor-alpha induces stress fiber formation through ceramide production: role of sphingosine kinase.

Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine that activates several signaling cascades. We determined the extent to which ceramide is a second messenger for TNF-alpha-induced signaling leading to cytoskeletal rearrangement in Rat2 fibroblasts. TNF-alpha, sphingomyelinase, or C(2)-ceramide induced tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, and stress fiber formation. Ly 294002, a phosphatidylinositol 3-kinase (PI 3-K) inhibitor, or expression of dominant/negative Ras (N17) completely blocked C(2)-ceramide- and sphingomyelinase-induced tyrosine phosphorylation of FAK and paxillin and severely decreased stress fiber formation. The TNF-alpha effects were only partially inhibited. Dimethylsphingosine, a sphingosine kinase (SK) inhibitor, blocked stress fiber formation by TNF-alpha and C(2)-ceramide. TNF-alpha, sphingomyelinase, and C(2)-ceramide translocated Cdc42, Rac, and RhoA to membranes, and stimulated p21-activated protein kinase downstream of Ras-GTP, PI 3-K, and SK. Transfection with inactive RhoA inhibited the TNF-alpha- and C(2)-ceramide-induced stress fiber formation. Our results demonstrate that stimulation by TNF-alpha, which increases sphingomyelinase activity and ceramide formation, activates sphingosine kinase, Rho family GTPases, focal adhesion kinase, and paxillin. This novel pathway of ceramide signaling can account for approximately 70% of TNF-alpha-induced stress fiber formation and cytoskeletal reorganization.

Increased concentrations of phosphatidate, diacylglycerol and ceramide in ras- and tyrosine kinase (fps)-transformed fibroblasts.

Concentrations of the bioactive lipids, phosphatidate and diacylglycerol, increased with time in culture in ras- and tyrosine kinase (fps)-transformed fibroblasts but not in control fibroblasts. On Day 3, diacylglycerol and phosphatidate concentrations were about 3.3- and 5.5-fold higher respectively in the ras-transformed compared to control fibroblasts. These concentrations in fps-transformed fibroblasts were increased about twofold. The changes in phosphatidate and diacylglycerol resulted from enhanced phospholipid turnover rather than from synthesis de novo. The increased ratio of phosphatidate to diacylglycerol is explained by decreased activities of two distinct phosphatidate phosphohydrolases and increased diacylglycerol kinase in ras-transformed fibroblasts. Ceramide concentrations were about 2.5- and threefold higher in the fps- and ras-transformed cells respectively on Day 3 compared to the controls. Incubating control fibroblasts from Days 1 to 3 with phosphatidylcholine-specific phospholipase C increased diacylglycerol, phosphatidate and ceramide concentrations, and decreased Mg2+-independent-phosphatidate phosphohydrolase activity. 8-(4-chlorophenylthio)-cAMP had a cytostatic effect in ras-transformed cells, it decreased the concentrations of phosphatidate and diacylglycerol, but increased that of ceramide. The consequences of increased ceramide and phosphatidate concentrations in ras-transformed cells are discussed in relation to signal transduction, cell division and the transformed phenotype.

Factors controlling the metabolism of phosphatidate by phosphohydrolase and phospholipase A-type activities. Effects of magnesium, calcium and amphiphilic cationic drugs.

1. The simultaneous deacylation and dephosphorylation of 1,2-diacyl-sn-[3H]glycerol 3-phosphate by the microsomal and soluble fraction of rat liver was studied. The substrate was either in the form of an emulsion or bound to microsomal membranes. 2. Mg2+ stimulated the deacylation and dephosphorylation of phosphatidate emulsions by both fractions, although the stimulation of both microsomal activities was less than that in the soluble fraction. The preparations of membrane-bound phosphatidate contained Mg2+. Further addition of Mg2+ inhibited dephosphorylation, whereas low concentrations of EDTA stimulated. Additional Mg2+ had little effect on the deacylation of membrane-bound phosphatidate and EDTA inhibited it. 3. Ca2+ inhibited the phosphohydrolase reactions in both fractions, but had little effect on the deacylation of phosphatidate emulsions or membrane-bound phosphatidate. 4. In the absence of Mg2+, lower concentrations of amphiphilic cations (chlorpromazine and benfluorex) stimulated the deacylation and dephosphorylation of phosphatidate emulsions by the soluble fraction. They also stimulated deacylation by the microsomal fraction, but inhibited dephosphorylation. In the present of 5 mM MgCl2, these drugs inhibited the dephosphorylation and deacylation of phosphatidate emulsions, the deacylation reaction being slightly less sensitive. Chlorpromazine (0.4 and 0.8 mM) also inhibited the dephosphorylation of membrane-bound Mg2+-phosphatidate by microsomal and microsomal plus soluble fractions. The deacylation was stimulated by 0.4 mM chlorpromazine and by 1 and 2 mM norfenfluramine. Chlorpromazine (0.8 mM) inhibited the deacylation by microsomal plus soluble fractions, but not by microsomal fractions alone. 5. The possible importance of the deacylation of phosphatidate in the physiological and pharmacological control of glycerolipid synthesis is discussed.

The metabolism of CDP-diacylglycerol and phosphatidylinositol in the microsomal fraction of rat liver. Effects of chlorpromazine, magnesium and manganese.

1. The metabolism of CDPdiacylglycerol and phosphatidylinositol was measured using substrates bound to the microsomal membranes of rat liver. 2. Chlorpromazine inhibited the degradation of [14C]CDPdiacylglycerol and the concomitant inositol-independent release of 14C in water-soluble products in the presence of various concentrations of Mg2+ and Mn2+. 3. The activity of CDPdiacylglycerol inositol phosphatidyltransferase was measured by determining the rate of incorporation of [3H]inositol into phosphatidylinositol, and by the inositol-dependent release of water-soluble 14C from [14C]CDPdiacylglycerol. Both of these parameters were inhibited by chlorpromazine in incubations that contained rate-limiting concentrations of Mg2+. However, chlorpromazine stimulated the reaction when 20 mM Mg2+, 0.5 mM Mn2+, 2 mM Mn2+ or 20 mM Mn2+ was added to the incubations. 4. Low concentrations of chlorpromazine increased an inositol-exchange reaction in the presence of 0.5 mM Mn2+ whereas higher concentrations of chlorpromazine inhibited. Chlorpromazine had relatively less effect on the inositol-exchange reaction at higher concentrations of Mn2+. 5. The action of chlorpromazine in decreasing the breakdown of CDPdiacylglycerol and in stimulating its conversion to phosphatidylinositol could explain part of the mechanism by which this compound and other amphiphilic cations increase the synthesis of acidic phospholipids.

Age-related changes in the translocation of phosphatidate phosphohydrolase from the cytosol to microsomal membranes in rat liver.

The effects of oleate, spermine and chlorpromazine were assayed in the presence or absence of 0.15 M KCl on the translocation of phosphatidate phosphohydrolase activity from cytosol to endoplasmic reticulum membranes in liver homogenates obtained from rats aged 1, 30, 60, 180 and 360 days. Marked age-associated decreases in phosphatidate phosphohydrolase distribution onto the membranes were demonstrated under nearly all conditions. In liver homogenates taken from 1-day-old rats and incubated with 0.15 M KCl, most of the enzyme was active (associated with the membranes). Physiological salt concentration (0.15 M KCl) produced a 2-fold increase of oleate-induced translocation of phosphatidate phosphohydrolase activity in liver homogenates from 1-day-old rats; it had no effect on those from 60-day-old rats, and produced a notable decline in liver homogenates obtained from 180- and 360-day-old rats. The promoting effect of spermine on oleate-induced translocation of this enzyme activity was higher in younger rats when incubated in the absence of 0.15 M KCl. Chlorpromazine did not show its usual antagonizing effect on oleate-induced translocation of phosphatidate phosphohydrolase when added to homogenates taken from 1-day-old rats. The antagonizing effect was slightly apparent in liver homogenates from 30-day-old rats and was more pronounced in those from 60-day-old rats in which the values diminished to one-half and to one-third either in the presence or absence of 0.15 M KCl.

Decreased serum lipids, serum insulin and triacylglycerol synthesis in adipose tissue of JCR:LA-corpulent rats treated with benfluorex.

Rats of the JCR:LA-corpulent strain were treated with benfluorex daily at a dose of 25 mg/kg body weight. This strain of rat, if homozygous for the cp gene (cp/cp), is hyperphagous, obese, hypertriglyceridemic, insulin resistant and in the case of male rats, atherosclerosis prone. The benfluorex treatment produced a sharp reduction in food intake which remained suppressed despite recovery toward normal after 2 weeks of treatment. This was accompanied by sustained decreases in body weight and adipose tissue mass. The ability of adipose tissue from female rats to take up glucose and convert it to lactate, glyceride-glycerol and fatty acids was decreased. This decrease was largely due to decreased adipose tissue mass. The serum concentrations of glucose, lactate, triacylglycerol, cholesterol, phospholipids and insulin were decreased in both sexes. The treatment also improved glucose tolerance and decreased corticosterone concentrations in male rats only. While reduction of food consumption contributes to the effects seen, benfluorex clearly had significant direct metabolic effects. The effects are consistent with an improved insulin sensitivity leading to a decrease in circulating triacylglycerol. The changes produced by benfluorex are all in directions that should inhibit atherogenesis in this animal model for the human obesity/hypertriglyceridemia/insulin resistant syndrome.

Sexual dimorphism in the preferential secretion of unsaturated lysophosphatidylcholine by rat hepatocytes but no secretion by sheep hepatocytes.

(1) Rat and ovine hepatocytes were incubated in monolayer culture with various fatty acids to determine their effects on the composition of the lysophosphatidylcholine that was secreted. (2) No lysophosphatidylcholine was detected in the medium from the ovine hepatocytes even though these cells were hormonally responsive and they secreted phosphatidylcholine and triacylglycerol in very-low-density lipoprotein. (3) Lysophosphatidylcholine was readily detected in the incubation medium of rat hepatocytes. The predominant fatty acids in this lipid were unsaturated. Stearate and arachidonate contributed 15 and 34%, and 24 and 26% of the total fatty acids when hepatocytes from male and female rats were used, respectively. The relative proportions of stearate and arachidonate in the phosphatidylcholine secreted from the hepatocytes were 20 and 14%, and 28 and 21% for the males and females, respectively. The equivalent values for stearate and arachidonate for phosphatidylcholine in the hepatocytes were 18 and 17% and 33 and 22% for male and female rats. These results provide further indications of sex differences in hepatic phospholipid metabolism and extend this to the secretion of phosphatidylcholine and lysophosphatidylcholine. (4) The addition of 1 mM stearate to the incubation medium did not significantly decrease the proportion of arachidonate in the lysophosphatidylcholine obtained from the hepatocytes of the male rats. However, the relative proportion of arachidonate was decreased in incubations that contained 1 mM oleate or linoleate. (5) The results provide evidence that the preferential secretion of unsaturated lysophosphatidylcholine by the liver may provide a system for transporting unsaturated fatty acids and choline to other organs in non-ruminant animals. However, this mechanism may not operate for ruminants.

Effects of sphingosine, albumin and unsaturated fatty acids on the activation and translocation of phosphatidate phosphohydrolases in rat hepatocytes.

The activities of two phosphatidate phosphohydrolases were measured in cultured rat hepatocytes incubated with 0.1 mM albumin. The activity, which is inhibited by N-ethylmaleimide (PAP-1) is located in the cytosolic and membrane fractions. PAP-1 activity is stimulated by Mg2+ and it can be translocated from the cytosol to the membranes by relatively low (0.5-1 mM) concentrations of fatty acids. In addition, higher concentrations (1-3 mM) of fatty acids cause an increase in the total PAP-1 activity. Translocation of PAP-1 activity in the hepatocytes is preferentially promoted by unsaturated fatty acids (C18:1, C18:2, C18:3, C20:4 and C20:5), rather than by saturated acids (C14:0, C16:0, C18:0). Increasing the extracellular concentration of albumin from 30 microM to 1 mM displaces PAP-1 activity from the membrane fraction. Sphingosine, but not staurosporine, can inhibit the redistribution of PAP-1 activity induced by oleate. The amphiphilic amines, sphingosine, chlorpromazine and propranolol, also decrease membrane-bound PAP-1 activity in the absence of fatty acids, but they do not alter, significantly, the activity of the cytosolic PAP-1. In the presence of 1 mM oleate, sphingosine, chlorpromazine and propranolol decrease the translocation of PAP-1 from the cytosol to the membranes. The phosphohydrolase activity, which is insensitive to N-ethylmaleimide (PAP-2), is specifically located in the plasma membrane (Jamal, Z., Martin, A., Gomez-Muñoz, A. and Brindley, D.N. (1991) J. Biol. Chem. 266, 2988-2996) and it is not stimulated by Mg2+. Saturated fatty acids, albumin, sphingosine and propranolol have no significant effects on PAP-2 activity. However, chlorpromazine decreases PAP-2 activity by about 14%. Linolenate, arachidonate and eicosapentaenoate at 1 mM also produced small (7-10%) decreases in PAP-2 activity. It is proposed that both PAP-1 and PAP-2 activities may be involved in signal transduction, although the main function of PAP-1 seems to be involved in the synthesis of glycerolipids.

Relationship between the displacement of phosphatidate phosphohydrolase from the membrane-associated compartment by chlorpromazine and the inhibition of the synthesis of triacylglycerol and phosphatidylcholine in rat hepatocytes.

Glycerolipid synthesis was studied in isolated hepatocytes by using 177 microM [14C]oleate and 1 mM [3H]glycerol. Chlorpromazine (25-400 microM) inhibited the synthesis of phosphatidylcholine and triacylglycerol. This was accompanied by an average increase of 12-fold in the accumulation of the labelled precursors in phosphatidate at 200 microM chlorpromazine and a decrease in the conversion of phosphatidate to diacylglycerol of 76%. These results indicate that part of the inhibition of the synthesis of phosphatidylcholine and triacylglycerol occurs at the level of phosphatidate phosphohydrolase. The relative rate of triacylglycerol synthesis at different concentrations of chlorpromazine was approximately proportional to the rate of conversion of phosphatidate to diacylglycerol. Phosphatidylcholine synthesis increased at higher rates of conversion of phosphatidate to diacylglycerol, but it was relatively independent of the latter rate when this was inhibited by more than about 30% with chlorpromazine. The addition of oleate to the hepatocytes caused a translocation of phosphatidate phosphohydrolase from the cytosol to the membrane-associated compartment. Chlorpromazine had the opposite effect and displaced the phosphohydrolase from the membranes in the presence or absence of oleate. There was a highly significant correlation between the activity of phosphatidate phosphohydrolase that was associated with the membranes of the hepatocytes and the calculated conversion of [3H]phosphatidate to diacylglycerol. Chlorpromazine also antagonized the association of the phosphohydrolase with microsomal membranes when cell-free preparations were incubated with combinations of oleate and spermine. Furthermore, it inhibited the transfer of the soluble phosphohydrolase to microsomal membranes that were labelled with [14C]phosphatidate and thereby decreased diacylglycerol production. It is concluded that part of the action of chlorpromazine in inhibiting the synthesis of triacylglycerol and phosphatidylcholine occurs because it prevents the interaction of the soluble phosphatidate phosphohydrolase with the membranes on which glycerolipid synthesis occurs. This in turn prevents the conversion of phosphatidate to diacylglycerol.

Structural organization of mammalian lipid phosphate phosphatases: implications for signal transduction.

This article describes the regulation of cell signaling by lipid phosphate phosphatases (LPPs) that control the conversion of bioactive lipid phosphates to their dephosphorylated counterparts. A structural model of the LPPs, that were previously called Type 2 phosphatidate phosphatases, is described. LPPs are characterized by having no Mg(2+) requirement and their insensitivity to inhibition by N-ethylmaleimide. The LPPs have six putative transmembrane domains and three highly conserved domains that define a phosphatase superfamily. The conserved domains are juxtaposed to the proposed membrane spanning domains such that they probably form the active sites of the phosphatases. It is predicted that the active sites of the LPPs are exposed at the cell surface or on the luminal surface of intracellular organelles, such as Golgi or the endoplasmic reticulum, depending where various LPPs are expressed. LPPs could attenuate cell activation by dephosphorylating bioactive lipid phosphate esters such as phosphatidate, lysophosphatidate, sphingosine 1-phosphate and ceramide 1-phosphate. In so doing, the LPPs could generate alternative signals from diacylglycerol, sphingosine and ceramide. The LPPs might help to modulate cell signaling by the phospholipase D pathway. For example, phosphatidate generated within the cell by phospholipase D could be converted by an LPP to diacylglycerol. This should change the relative balance of signaling by these two lipids. Another possible function of the LPPs relates to the secretion of lysophosphatidate and sphingosine 1-phosphate by activated platelets and other cells. These exogenous lipids activate phospholipid growth factor receptors on the surface of cells. LPP activities could attenuate cell activation by lysophosphatidate and sphingosine 1-phosphate through their respective receptors.

Cholesterol feeding induces hypertriglyceridaemia in hamsters and increases the activity of the Mg(2+)-dependent phosphatidate phosphohydrolase in the liver.

(1) Feeding increased cholesterol to hamsters resulted in a dose-dependent increase in cholesterol and triacylglycerol in very-low-density lipoprotein (VLDL), in serum non-esterified fatty acids and in the activity of the Mg(2+)-dependent phosphatidate phosphohydrolase in liver. (2) The effects of increasing dietary cholesterol by 0.12% (w/w) in addition to feeding fat (14%, w/w) were dependent upon the nature of the fat. Lard in the presence of 0.12% (w/w) cholesterol increased serum triacylglycerols as did olive oil. By contrast, sunflower oil did not cause a significant change in serum triacylglycerol concentrations. (3) There was a highly positive correlation between VLDL triacylglycerol and VLDL cholesterol concentrations suggesting that, at least in this model, there is a close relationship between hypertriglyceridaemia and hypercholesterolaemia.

Effect of alcohol on the activity and subcellular distribution of phosphatidate phosphohydrolase in rat liver.

The activity and subcellular distribution, before and after translocation with oleic acid, of hepatic phosphatidate phosphohydrolase (PAH: E.C.3.1.3.4) was measured following acute and chronic administration of ethanol with either 18% or 4.4% total calories as fat, to male Wistar rats. Acute injection produced a significant increase in PAH activity, without affecting the subcellular distribution or translocation of activity from the cytosol into the membraneous compartments induced by oleic acid. PAH activity, subcellular distribution and the translocation of activity by oleic acid was similar in all groups following chronic liquid feeding. Chronic ethanol feeding was associated with significantly increased plasma corticosterone in the ethanol-fed rats compared with controls, independent of the fat content of the diet. Plasma insulin was similar in chronic alcohol fed rats and controls. This suggests that enhanced PAH activity may contribute to triacylglycerol accumulation following acute alcohol injection, but activity normalises as hepatic lipid accumulation progresses and this effect is not due to attenuation of the hormonal effects of ethanol.

Effects of cell-permeable ceramides and tumor necrosis factor-alpha on insulin signaling and glucose uptake in 3T3-L1 adipocytes.

Incubation of 3T3-L1 adipocytes with C2- and C6-ceramides (N-acetyl- and N-hexanoylsphingosines) but not dihydro-C2-ceramide increased 2-deoxyglucose uptake in the absence of insulin. This effect was inhibited by PD 98059, LY 294002, and rapamycin, which block the activation of mitogen-activated protein kinase, phosphatidylinositol (PI) 3-kinase, and ribosomal S6 kinase, respectively. Long-term increases in PI 3-kinase activity associated with insulin receptor substrate 1 (IRS-1) increased GLUT1 and GLUT4 concentrations in plasma membranes. This together with increased GLUT1 (but not GLUT4) synthesis explains the increase in non-insulin-dependent glucose uptake. C2-ceramide inhibited insulin-stimulated glucose uptake after 2 h by decreasing insulin-induced translocation of GLUT1 and GLUT4 to plasma membranes. This occurred when there was no increase in basal glucose uptake or decrease in activation of IRS-1 or PI 3-kinase. Incubation for 24 h with tumor necrosis factor-alpha (TNF-alpha) but not C2-ceramide decreased the concentration and insulin-induced tyrosine phosphorylation of IRS-1 in this experimental system. Cell-permeable ceramides mimic some effects of TNF-alpha, especially in stimulating basal glucose uptake. We identified a site for inhibiting insulin-stimulated glucose uptake that is downstream of PI 3-kinase. Our work provides further mechanisms for the effects of TNF-alpha and ceramides in increasing non-insulin-dependent glucose uptake and decreasing insulin-stimulated uptake in vivo.

Platelet-released phospholipids link haemostasis and angiogenesis.

Considerable attention has focused on identifying mediators of neovascularization at sites of growth and abnormal tissue development. By contrast, mediators of angiogenesis at sites of injury and wound repair are not well defined but factors generated during blood coagulation (haemostasis) are attractive candidates. In addition to proteins generated, activated and released during the activation of clotting cascades, platelet-derived lipid mediators are now known to play a key role in many aspects of the angiogenic response. The first indication of lipid mediator involvement in angiogenesis was the discovery that lysophosphatidate (LPA), phosphatidic acid (PA) and sphingosine 1-phosphate (SPP) are high affinity agonists for G-protein coupled EDG (endothelial differentiation gene) receptors. The prototype for this family, EDG-1, was cloned from genes expressed when endothelial cells were activated to assume an angiogenic phenotype in vitro. The subsequent finding that SPP is a high affinity ligand for EDG-1 led Spiegel, Hla and associates (Lee et al., Science 1998;279:1552-1555) to hypothesize that platelet-released phospholipids play an important role in angiogenesis. These investigators and others demonstrated that SPP, LPA and phosphatidate (PA) induce many important endothelial cell responses associated with angiogenesis, including liberation of endothelial cells from established monolayers, chemotactic migration, proliferation, adherens junction assembly and morphogenesis into capillary-like structures. Although these studies indicated the potential involvement of platelet-derived phospholipids in angiogenesis, their physiological importance was not established. However, recent work demonstrates that >80% of the potent endothelial cell chemoattractive activity generated in human serum during clotting--an activity necessary for optimal angiogenesis--results from platelet-derived SPP. Other factors released from platelets during clotting, including LPA and PA, exert profound effects on endothelial cells that contribute unique aspects to the angiogenic response. These combined studies establish that SPP and other platelet-derived lipid mediators provide a novel link between haemostasis and angiogenesis.

Activation of phospholipase D in FRTL-5 thyroid cells by forskolin and dibutyryl-cyclic adenosine monophosphate.

We demonstrated previously that TSH activates phospholipase D (PLD) via stimulation of protein kinase C (PKC) in Fischer rat thyroid line (FRTL)-5 thyroid cells. To examine the role of the cAMP pathway in the regulation of PLD, we studied the effects of forskolin (0-100 microM; 30 min) and dibutyryl cAMP (dbcAMP; 0-1 mM; 30 min) on PLD activation. FRTL-5 thyroid cells were labeled mainly in phosphatidylcholine with [3H]myristate followed by incubation with 200 mM ethanol before the addition of agonist. PLD was assessed by the measurement of [3H]phosphatidylethanol. Forskolin (100 nM to 100 microM) and dbcAMP (100 pM to 100 microM) increased PLD activity significantly. Maximal responses to forskolin and dbcAMP exceed the PLD responses produced by 100 microU/ml of TSH. To determine whether the effects of forskolin and dbcAMP on PLD occurred as a consequence of PKC activation, FRTL-5 thyroid cells were preincubated for 10 min with the PKC inhibitors, chelerythrine (1 microM) or calphostin C (1 microM), or they were pretreated for 24 h with phorbol myristate acetate (100 nM) to down-regulate PKC. Unlike TSH-mediated PLD activation, these treatments had no effect on PLD activation by cAMP agonists. Forskolin (10 microM; 30 min) had no effect on the subcellular distribution of PKC alpha-, epsilon-, or zeta-isoforms, confirming the lack of involvement of PKC. The protein kinase A (PKA) inhibitors, H-89 (10 microM; 30 min) and dideoxyadenosine (5 nM; 10 min) significantly decreased the forskolin- and dbcAMP-mediated PLD activation without any effect on the phorbol ester-mediated PLD response. Following pretreatment with H-89 or dideoxyadenosine, the TSH-mediated PLD response was also significantly reduced. These studies indicate that forskolin and dbcAMP stimulate PLD in FRTL-5 thyroid cells directly via PKA without involvement of PKC. Studies of cells in the presence and absence of ethanol revealed approximately 60% of the phosphatidate plus diacylglycerol produced via TSH occurs via PLD activation. Although TSH-mediated inositol phosphate generation occurred with similar concentrations of TSH that led to PLD activation, 10-fold higher TSH concentrations were required to increase intracellular Ca2+. These results and the lack of a rapid Ca2+ transient following physiological TSH concentrations suggest that alternatives to conventional hydrolysis of phosphatidylinositol 4,5-bisphosphate may initiate PKC activation. Thus, the two major signal transduction systems in the FRTL-5 thyroid cell (PKA and PKC) appear to converge on PLD activation. Stimulation of both of these pathways by TSH may be required for optimal physiological activation of PLD.

Thyroid-stimulating hormone activates phospholipase D in FRTL-5 thyroid cells via stimulation of protein kinase C.

We studied whether TSH or phorbol myristate acetate (PMA) stimulates the hydrolysis of phospholipids, predominantly phosphatidylcholine, via phospholipase D (PLD) in FRTL-5 thyroid cells and whether this occurs as a consequence of protein kinase C (PKC) activation. FRTL-5 thyroid cells were labeled with [3H]myristate followed by incubation with 200 mM ethanol before the addition of agonist. PLD activity was assessed by the measurement of [3H]phosphatidylethanol from [3H]phospholipid (predominantly [3H]phosphatidylcholine). Compared to control values, bovine TSH (100 microU/ml) increased PLD activity by 480% and 600%, respectively, after 30 and 60 min of exposure. Studies with purified human and bovine TSH revealed similar results, indicating that this effect was due to TSH itself. PMA (100 nM) increased PLD activity at 10 min (630% of the control value), and this effect persisted up to 60 min (600% of the control value). To determine whether the effects of TSH on PLD occurred as a consequence of PKC activation, FRTL-5 thyroid cells were preincubated with the PKC inhibitor, chelerythrine (1 microM for 10 min), or were pretreated for 24 h with PMA (100 nM) to down-regulate PKC. PLD stimulation by TSH and PMA was largely abolished by such treatments. These studies indicate that in FRTL-5 thyroid cells, TSH and PMA are capable of stimulating PLD, and that PKC activation is responsible for this stimulation. The role of PLD activation could be to amplify and prolong the PKC signal by further production of diacylglycerol.

Spermine promotes the translocation of phosphatidate phosphohydrolase from the cytosol to the microsomal fraction of rat liver and it enhances the effects of oleate in this respect.

Spermine (0.5-2 mM) promoted the translocation of phosphatidate phosphohydrolase from the soluble to the microsomal fraction in a cell-free system derived from rat liver. By contrast, spermidine (1 mM) and putrescine (1 mM) had no significant effect on the translocation when added alone. Spermine, and to a lesser extent, spermidine, enhanced the translocating action of oleate and increased its effectiveness in transferring the phosphohydrolase from the soluble to the microsomal fraction. It is proposed that the phosphohydrolase becomes metabolically active when it combines with membranes and that polyamines might help to regulate this interaction. This could facilitate the action of fatty acids and enable cells to increase their capacity for triacylglycerol synthesis to match an increased availability of fatty acids.

Effects of the lipase inhibitors, Triton WR-1339 and tetrahydrolipstatin, on the synthesis and secretion of lipids by rat hepatocytes.

The lipase inhibitors, Triton WR-1339 and tetrahydrolipstatin, were incubated with rat hepatocytes. Triton WR-1339 increased the recovery of triacylglycerol in the hepatocytes and incubation medium by 31% and 38%, respectively. Tetrahydrolipstatin decreased the accumulation of newly synthesized, and of total triacylglycerol in the medium. This compound might be useful in determining mechanisms involved in intracellular triacylglycerol metabolism and the secretion of very low density lipoproteins.

Long-chain fatty acids and their acyl-CoA esters cause the translocation of phosphatidate phosphohydrolase from the cytosolic to the microsomal fraction of rat liver.

A translocation of phosphatidate phosphohydrolase from the cytosolic to the microsomal fraction was promoted in cell-free extracts of rat liver by oleate and palmitate and their CoA esters. Oleate was more potent in this respect than palmitate and the CoA esters were more effective than the unesterified acids. Octanoate, octanoyl-CoA and CoA did not cause the translocation. It is proposed that the interaction of phosphatidate phosphohydrolase with the membranes that synthesize glycerolipids causes it to become metabolically active. This enables the liver to increase its capacity for triacylglycerol synthesis in response to an increased supply of fatty acids.

Spermine antagonises the effects of dexamethasone, glucagon and cyclic AMP in increasing the activity of phosphatidate phosphohydrolase in isolated rat hepatocytes.

Rat hepatocytes were incubated in monolayer culture, under serum free conditions, for 8 h. Glucagon (10 nM), 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (100 microM) and dexamethasone (100 nM) increased the activity of phosphatidate phosphohydrolase by approx. 2-, 3.6- and 3.3-fold, respectively. Spermine alone had no significant effect. Spermine (2.5 mM) almost completely inhibited the glucagon induced increase in phosphohydrolase activity. It only partially inhibited the dexamethasone and cyclic AMP mediated inductions. Spermidine had no significant effect in this respect. The results are discussed in relation to the known effects of polyamines on glycerolipid synthesis, in particular, and on intermediary metabolism.