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%.     

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.     

Secretogogue-stimulated phosphatidylinositol breakdown in the exocrine pancreas liberates arachidonic acid, stearic acid, and glycerol by sequential actions of phospholipase C and diglyceride lipase

When mouse pancreatic "minilobules" prelabeled with either [14C]arachidonic acid (AA), [14C]stearic acid (SA), or [3H]glycerol were stimulated with the secretogogue, caerulein, there was a 60-70% loss in radioactivity in phosphatidylinositol (PI) at 30 min. This loss was accompanied by the formation of [14C] phosphatidic acid (PA), [14C]diacylglycerol (DG), [14C] triacylglycerol (TG), and free [14C]AA, [14C]SA, and [3H]glycerol. The loss in radioactive PI was the same as the loss in chemically measured PI-phosphorus. Thirty to fifty per cent of the caerulein-induced loss of prelabeled PI could be accounted for as free [14C]AA, [14C]SA, or [3H]glycerol. Increased incorporation of fatty acid or glycerol residues into DG, PA, and TG accounted for the balance of the loss in PI. The specific DG-lipase inhibitor, RHC 80267, markedly inhibited the caerulein-stimulated release of [14C]AA, [14C]SA, and [3H]glycerol and roughly doubled the caerulein-induced increment in [14C]AA-, [14C]SA-, or [3H]glycerol-labeled DG, showing that the source of the caerulein-induced increment in fatty acids and glycerol was DG. When the PI was prelabeled with either [32P] orthophosphate, [3H]myoinositol, or [3H]glycerol, only 1% or less of the radioactivity in PI was in lysophosphatidylinositol (LPI), and there was no increase in radioactivity in LPI on stimulation with caerulein. These observations, taken together, argue strongly for a phospholipase C-catalyzed breakdown of PI followed by DG-lipase and argue against any significant involvement of phospholipase A2 in PI degradation in mouse pancreas. The formation of substantial amounts of free [14C]AA on stimulation supports the view that, among other things, the phosphoinositide effect in the exocrine pancreas serves to generate arachidonate (and its metabolites). The release of appreciable amounts of free fatty acids and glycerol shows that a significant portion of the DG formed as a result of caerulein-stimulated PI breakdown is not conserved in the phosphoinositide cycle.     

Role of phospholipase C and diacylglyceride lipase pathway in arachidonic acid release and acetylcholine-induced vascular relaxation in rabbit aorta

ACh stimulates arachidonic acid (AA) release from membrane phospholipids of vascular endothelial cells (ECs). In rabbit aorta, AA is metabolized through the 15-lipoxygenase pathway to form vasodilatory eicosanoids 15-hydroxy-11,12-epoxyeicosatrienoic acid (HEETA) and 11,12,15-trihydroxyeicosatrienoic acid (THETA). AA is released from phosphatidylcholine (PC) and phosphatidylethanolamine (PE) by phospholipase A2 (PLA2), or from phosphatidylinositol (PI) by phospholipase C (PLC) pathway. The diacylglycerol (DAG) lipase can convert DAG into 2-arachidonoylglycerol from which free AA can be released by monoacylglycerol (MAG) lipase or fatty acid amidohydrolase (FAAH). We used specific inhibitors to determine the involvement of the PLC pathway in ACh-induced AA release. In rabbit aortic rings precontracted by phenylephrine, ACh induced relaxation in the presence of indomethacin and N(omega)-nitro-L-arginine (L-NNA). These relaxations were blocked by the PLC inhibitor U-73122, DAG lipase inhibitor RHC-80267, and MAG lipase/FAAH inhibitor URB-532. Cultured rabbit aortic ECs were labeled with [14C]AA and stimulated with methacholine (10(-5) M). Free [14C]AA was released by methacholine. Methacholine decreased the [14C]AA content of PI, DAG, and MAG fractions but not PC or PE fractions. Methacholine-induced release of [14C]AA was blocked by U-73122, RHC-80267, and URB-532 but not by U-73343, an inactive analog of U-73122. The data suggested that ACh activates PLC, DAG lipase, and MAG lipase pathway to release AA from membrane lipids. This pathway is important in regulating vasodilatory eicosanoid synthesis and vascular relaxation in rabbit aorta.     

Attenuation of sn-1,2-diacylglycerol second messengers. Metabolism of exogenous diacylglycerols by human platelets

The metabolism of exogenous [3H]diacylglycerols by intact human platelets was studied in order to examine: the metabolic fate of these second messengers in an intact cell, the effect of diacylglycerol kinase and diacylglycerol lipase inhibitors on this metabolism, the effect of agonist stimulation on metabolism, and the dependence of metabolism on diacylglycerol chain length. When 2.5 microM [3H]dioctanoylglycerol (diC8) was added to 10(9) platelets it was rapidly metabolized; 80% was converted to various products in 2.5 min. Initially, 40% was recovered as 3H-labeled phospholipid (predominantly phosphatidic acid) reflecting the action of diacylglycerol kinase, 20% was recovered as [3H]glycerol due to the action of diacylglycerol and monoacylglycerol lipases, and small amounts were recovered as triacylglycerol and monoacylglycerol. Thrombin stimulation of platelets did not affect the rate or pathway of metabolism. Pretreatment of platelets with the diacylglycerol kinase inhibitors, diC8ethyleneglycol or 1-monooleoylglycerol, inhibited 3H-labeled phospholipid production 47% and 75%, respectively, and resulted in a longer lived diC8 signal. The diacylglycerol lipase inhibitor, RHC 80267, inhibited the production of water-soluble metabolites 75%. Despite inhibition of the lipase, the overall metabolism of exogenous [3H]diC8 occurred at a similar rate as in control platelets due to an increased flux towards phospholipid. The ability of exogenous diacylglycerols to be metabolized by diacylglycerol kinase correlated well with their ability to activate protein kinase C in platelets. [3H]Dibutyroylglycerol, didodecanoylglycerol, and ditetradecanoylglycerol, were not metabolized by this route. These diacylglycerols were still metabolized via the lipase pathway. The results indicate that platelets possess potent attenuation systems to defend against the accumulation of diacylglycerol second messengers, and that the primary metabolic fate of cell-permeable, exogenous diacylglycerols is conversion to phosphatidic acid.     

Phospholipid metabolism in bradykinin-stimulated human fibroblasts. II. Phosphatidylcholine breakdown by phospholipases C and D; involvement of protein kinase C

Bradykinin (BK) and phorbol 12-myristate 13-acetate (PMA) both stimulate the hydrolysis of phosphatidylcholine (PC) in human fibroblasts, resulting in the formation of phosphatidic acid (PA) and diacylglycerol (DG) (Van Blitterswijk, W.J., Hilkmann, H., de Widt, J., and Van der Bend, R.L. (1990) J. Biol. Chem. 266, 10337-10343). Stimulation with BK resulted in the rapid and synchronous formation of [3H]choline and [3H]myristoyl-PA from the correspondingly prelabeled PC, indicative of phospholipase D (PLD) activity. In the presence of ethanol or n-butanol, transphosphatidylation by PLD resulted in the formation of [3H]phosphatidylethanol or - butanol, respectively, at the cost of PA and DG formation. This suggests that PC-derived DG is generated via a PLD/PA phosphohydrolase pathway. A more pronounced but delayed formation of these products was observed by PMA stimulation. The Ca2+ ionophore ionomycin also activated PLD and accelerated (synergized) the response to PMA. Both [3H] choline and [3H]phosphocholine were released into the extracellular medium in a time- and stimulus-dependent fashion, without apparent changes in the high intracellular levels of [3H]phosphocholine. The protein kinase C (PKC) inhibitors staurosporin and 1-O-hexadecyl-2-O-methylglycerol inhibited BK- and PMA-induced activation of PLD. Down-regulation of PKC by long-term pretreatment of cells with phorbol ester caused a dramatic drop in background [3H]choline levels, while subsequent stimulation with BK, ionomycin, or PMA failed to increase these levels and failed to induce transphosphatidylation. From these results we conclude that PLD activation is entirely mediated by (downstream of) PKC. Unexpectedly, however, BK stimulation of these PKC-depleted cells caused a marked generation of DG from PC within 15 s, which was not seen in BK-stimulated control cells, suggesting PC breakdown by a phospholipase C (PLCc). We conclude that cells stimulated with BK generate DG via both the PLCc and the PLD/PA hydrolase pathway, whereas PMA stimulates mainly the latter pathway. BK stimulation of normal cells leads to activation of PKC and, by consequence, to attenuation of the level of PLCc-generated DG and to stimulation of the PLD pathway, whereas the reverse occurs in PKC-down-regulated cells.     

Nitric Oxide Synthase Inhibitors Do Not Attenuate Diacylglycerol or Monoacylglycerol Lipase Activities in Synaptoneurosomes

Neuron-enriched cultures and synaptoneurosomal fractions from 10 day-old rat brain contain diacylglycerol and monoacylglycerol lipase activities. Glutamate and its analogs stimulate the activities of diacylglycerol and monoacylglycerol lipases in a time-and dose-dependent manner. Stimulation of diacylglycerol and monoacylglycerol lipases by glutamate or NMDA can be blocked by MK-801 (non-competitive antagonist). Nitro L-arginine methyl ester and L-methylarginine have no effect on glutamate stimulated activities of diacylglycerol and monoacylglycerol lipases. Our studies suggest that synaptoneurosomal preparations from young rat brain are useful for obtaining important information on signal transduction.     

Potassium-evoked glutamate release liberates arachidonic acid from cortical neurons

Brain cells in situ contain low concentrations of free polyunsaturated fatty acids such as arachidonic acid (AA) that are released following pathological insults. As a large rise in extracellular [K(+)] accompanies cerebral ischemia, we explored whether this was a stimulus for cellular AA release employing a murine mixed cortical cell culture preparation radiolabeled with AA. Elevating the [K(+)](o) from 5 to 52 mm induced a time-dependent increase in [(3)H]AA release, which reached a plateau after 15 min. Removal of [Ca(2+)](o) or addition of CdCl(2) (100 microm) diminished the net high K(+)-induced AA release, as did treatment of the cultures with tetanus toxin (300 ng/ml) to block endogenous neurotransmitter release. Pharmacological antagonism of both ionotropic and metabotropic glutamate receptors completely prevented high K(+)-evoked AA release, indicating that glutamate was the neurotransmitter in question. Addition of exogenous glutamate mimicked precisely the characteristics of AA release that followed increases in [K(+)](o). Finally, glutamate and AA were released solely from neurons as tetanus toxin did not cleave astrocytic synaptobrevin-2, nor was AA released from pure astrocyte cultures using the same stimuli that were effective in mixed cultures. Taken in toto, our data are consistent with the following scenario: high [K(+)](o) depolarizes neurons, causing an influx of Ca(2+) via voltage-gated Ca(2+) channels. This Ca(2+) influx stimulates the release of glutamate into the synaptic cleft, where it activates postsynaptic glutamate receptors. Events likely converge on the activation of a phospholipase A(2) family member and possibly the enzymes diacylglycerol and monoacylglycerol lipases to yield free AA.     

Mobilization of diacylglycerol in intact HeLa cells by exogenous phospholipase C from Cl. perfringens is accompanied by release of fatty acids including arachidonic acid.

The second messenger diacylglycerol (DAG), chiefly derived from phosphatidylcholine (PC) or from phosphatidylinositol (PI), through the activation of specific phospholipases C (PLC), plays a key role in cellular stimulation. The activation of a particular PLC was simulated in intact HeLa cells by treatment with exogenous PC-PLC (Cl. perfringens) or with PI-PLC (B. cereus). Both enzymes rapidly mobilized DAG. However, only PC-PLC led, in Hela cells, to morphological changes (which were reversible on enzyme removal within the time frame of the experiments) and to an increase of intracellular calcium concentration with a lag of > 10 min. In cells prelabeled with [1-14C]arachidonic acid only PC-PLC but not PI-PLC induced the release of labeled fatty acid with a lag of > 10 min. Upon prelabeling of cells with [1-14C]oleic acid, PC-PLC led to a release of radioactive oleic acid. The release of arachidonic acid (AA) required a threshold dose of PC-PLC and a minimum time of treatment beyond which the AA release continued for a certain period, even in the absence of the exogenous enzyme. Under the conditions used, neither PLA2 nor DAG lipase activity were detectable in the PC-PLC preparation. Therefore, AA release was due to activation of a cellular enzyme, probably cellular PLA2 activity. The PC-PLC-induced AA release could be inhibited to a certain extent by EGTA and by quinacrine but not by the glucocorticoid fluocinolone acetonide. Only PC-PLC (but not PI-PLC) caused, in addition, an increase of the level of monoglycerol, which paralleled the appearance of AA. An increase of labeled monoglycerol was detectable in HeLa cells prelabeled with radioactive oleic acid or with 1-[1-14C]palmitoyl-lyso-PC but not in cells prelabeled with radioactive AA, thus indicating that the fatty acid originated from sn-2 position of the glycerol moiety. The 1-monoacylglycerol was probably generated from lysophospholipids by the bacterial PC-PLC. This enzyme preparation has been shown to catalyze such breakdown of lysophosphatidylcholine in vitro. PC-PLC-induced AA release occurred also after down-regulation of protein kinase C by an overnight pretreatment with phorbol ester TPA (TPA-pretreated cells, but not control cells, on treatment with PC-PLC, metabolized AA to prostaglandins).(ABSTRACT TRUNCATED AT 400 WORDS)     

Phosphatidic acid as the biosynthetic precursor of the endocannabinoid 2-arachidonoylglycerol in intact mouse neuroblastoma cells stimulated with ionomycin

In mouse neuroblastoma N18TG2 cells prelabeled with [3H]arachidonic acid ([3H]AA) the biosynthesis of 2-arachidonoylglycerol (2-AG) is induced by ionomycin in a fashion sensitive to an inhibitor of diacylglycerol (DAG) lipase, RHC 80267, but not to four different phospholipase C (PLC) blockers. Pulse experiments with [3H]AA showed that ionomycin stimulation leads to the sequential formation of [3H]phosphatidic acid ([3H]PA), [3H]DAG, and [3H]2-AG. [3H]2-AG biosynthesis in N18TG2 cells prelabeled with [3H]AA was counteracted by propranolol and N-ethylmaleimide, two inhibitors of the Mg2+/Ca2(+)-dependent brain PA phosphohydrolase. Pretreatment of cells with exogenous phospholipase D (PLD) led to a strong potentiation of ionomycin-induced [3H]2-AG formation. These data indicate that DAG precursors for 2-AG in intact N18TG2 cells are obtained from the hydrolysis of PA and not through the activation of PLC. The presence of 2% ethanol during ionomycin stimulation failed to elicit the synthesis of [3H]phosphatidylethanol and did not counteract the formation of [3H]PA, thus arguing against the activation of PLD by the Ca2+ ionophore. Selective inhibitors of secretory phospholipase A2 and the acyl-CoA acylase inhibitor thimerosal significantly reduced [3H]2-AG biosynthesis. The implications of these latter findings, and of the PA-dependent pathways of 2-AG formation described here, are discussed.     

Human cytomegalovirus stimulates arachidonic acid metabolism through pathways that are affected by inhibitors of phospholipase A2 and protein kinase C

Inhibitors of phospholipase A2, tetracaine and quinacrine, inhibitors of protein kinases, H-7 and H-8, and a diacylglycerol lipase inhibitor reduced the level of CMV-induced [3H]AA release. A combination of H-7 and quinacrine inhibited stimulation of [3H]AA by about 80%. LU cells chronically treated with TPA and infected with CMV, had a reduced level of CMV-induced [3H]AA release and in the presence of quinacrine it was completely inhibited. These results suggest that CMV-induced stimulation of AA metabolism is mediated by pathways which are associated with activation of PLA2 and protein kinase C.     

Carbachol stimulation of triacylglycerol lipase activity in pancreatic acinar cells.

Carbachol (CCh) reduced the levels of [3H]arachidonic acid in triacylglycerol (TG) of pancreatic acinar cells. In cells prelabeled with [14C]glycerol, CCh reduced [14C]TG and increased [14C]diacylglycerol levels. Using [3H]triolein as exogenous substrate, CCh enhanced TG lipase activity 3-fold in a particulate fraction derived from intact acinar cells. These results portray a mechanism for generating diacylglycerol and arachidonic acid in exocrine pancreas involving agonist stimulation of TG hydrolysis.     

A Possible Pathway of Phosphoinositide Metabolism Through EDTA-Insensitive Phospholipase A1 Followed by Lysophosphoinositide-Specific Phospholipase C in Rat Brain

Abstract— Incubation of [2-³H]glycerol-labeled phosphatidylinositol with a crude cytosol fraction of rat brain in the presence of EDTA yielded [³H]lysophosphatidylinositol predominantly without accumulation of labeled monoacylglycerol and diacylglycerol. The pH optimum of this Phospholipase A activity was 8.0. The activity for phosphatidylinositol was twofold higher than for phosphatidylethanolamine, whereas phosphatidylcholine, phosphatidylserine, and phosphatidic acid were not hydrolyzed significantly under the conditions used. The phospholipase A activity for phosphatidylethanolamine was resolved in part from that for phosphatidylinositol by ammonium sulfate fractionation of the cytosol, indicating the existence of at least two forms of EDTA-insensitive phospholipase A. The positional specificity of the phosphatidylinositol-hydrolyzing activity was found to be that of a phospholipase A₁, as radioactive lysophosphatidylinositol was produced from 1 -stearoyl-2-[1-¹⁴C]arachidonyl- sn -glycero-3-phosphoinositol without release of free arachidonate. A phospholipase C activity specific for lysophosphoinositides was found in a membrane fraction from rat brain, which was similar to that characterized in porcine platelets. The phospholipase C was demonstrated to hydrolyze the 2-acyl isomer as well as the 1-acyl isomer of lysophosphatidylinositol. Taken together, our results suggest a possible pathway through which phosphatidylinositol is selectively degraded to the 2-acyl isomer of lysophosphatidylinositol in a Ca²⁺-independeht manner, and subsequently converted to 2-monoacylglycerol in rat brain.     

Angiogenin activates phospholipase C and elicits a rapid incorporation of fatty acid into cholesterol esters in vascular smooth muscle cells

Angiogenin activates the phosphoinositide-specific phospholipase C (PLC) in cultured rat aortic smooth muscle cells to yield a transient (30 s) peak of 1,2-diacylglycerol (DG) and inositol trisphosphate. Within 1 min, the DG level falls below that of the control and remains so for at least 20 min. A transient increase in monoacylglycerol indicates that depletion of DG may be the consequence of hydrolysis by DG lipase. In addition to these changes in second messengers, a rapid increase in incorporation of radiolabeled tracer into cellular cholesterol esters is observed. Stimulated cholesterol ester labeling is inhibited by preincubation with either the DG lipase inhibitor RHC 80267 or the acyl coenzyme A:cholesterol acyltransferase inhibitor Sandoz 58035. Cells prelabeled with [3H]arachidonate show a sustained increase in labeling of cholesterol esters following exposure to angiogenin. In contrast, cells prelabeled with [3H]oleate show only a transient elevation that returns to the basal level by 5 min. This suggests initial cholesterol esterification by oleate followed by arachidonate that is released by stimulation of the PLC/DG lipase pathway.     

Protein kinase C-independent activation of a novel nonspecific phospholipase C pathway by phorbol myristate acetate releases arachidonic acid for prostaglandin synthesis in MC3T3-E1 osteoblasts

The effects of phorbol myristate acetate, an activator of protein kinase C, on the release of [³H]arachidonic acid and prostaglandin synthesis were studied in an osteoblast cell line (MC3T3-E1). Phorbol myristate acetate (20 uM) liberated 16 and 55% of the [³H]arachidonate in prelabeled phosphatidylinositol and phosphatidylethanolamine, respectively, and evoked a 19-fold stimulation in the synthesis of prostaglandin E₂. Phorbol myristate acetate doubled the cellular mass of 1,2-diacylglycerol and stimulated the liberation of [³H]arachidonate from the diacylglycerol pool in prelabeled cells. The diacylglycerol lipase inhibitor RHC 80267 blocked 75–80% of the phorbol ester-promoted (total) cellular liberation of [³H]arachidonic acid and production of prostaglandin E₂. In comparison, the release of [³H]arachidonate from phosphatidylethanolamine (but not phosphatidylinositol) was only partially antagonized (to the same degree) by the PLA₂ inhibitor p-bromophenacylbromide and the protein kinase C inhibitor Et-18-OMe. PMA-induced formation of diacylglycerol or synthesis of PGE₂ was not affected by the prior inhibition of protein kinase C. Therefore, we have shown a novel pathway for the liberation of arachidonic acid in osteoblasts involving the nonspecific hydrolysis of phosphatidylinositol and phosphatidylethanolamine by phospholipase C followed by the deesterification of diacylgycerol. This pathway can be activated by a phorbol ester through a protein kinase C-independent mechanism.     

Monoacylmonoalkylglycerol as a substrate for diacylglycerol hydrolase activity in adipose tissue

The synthesis and use of 1(3)-[3H]oleoyl-2-0-oleylglycerol as a substrate for the assay of diacylglycerol hydrolase activity in adipose tissue is described. Neither the compound nor its reaction product are hydrolyzed by purified adipose tissue monoacylglycerol lipase.     

Mechanisms of bombesin-induced arachidonate mobilization in Swiss 3T3 fibroblasts

A peptide mitogen bombesin, which activates the phospholipase C-protein kinase C signaling pathway, induces a mepacrine-sensitive, dose-dependent increase in the release of [3H]arachidonic acid and its metabolites ([3H]AA) from prelabeled Swiss 3T3 fibroblasts. The effect is temporally composed of two phases, i.e. an initial transient burst that is essentially independent of extracellular Ca2+, and a following sustained phase that is absolutely dependent on the extracellular Ca2+. The initial transient [3H]AA liberation occurs concomitantly with bombesin-induced 45Ca efflux from prelabeled cells: both responses being substantially attenuated by loading cells with a Ca2+ chelator quin2. However, bombesin-induced intracellular Ca2+ mobilization by itself is not sufficient as a signal for the initial transient [3H]AA liberation, since A23187 potently stimulates 45Ca efflux to an extent comparable to bombesin but fails to induce [3H]AA release in the absence of extracellular Ca2+. The second sustained phase of the bombesin-induced [3H]AA release is abolished by reducing extracellular Ca2+ to 0.03 mM, although bombesin effects on phospholipase C and protein kinase C activation are barely affected by the same procedure. A protein kinase C activator phorbol 12,13-dibutyrate induces an extracellular Ca(2+)-dependent, slowly developing sustained increase in [3H]AA release, and markedly potentiates both phases of bombesin-induced [3H]AA release. Down-regulation of cellular protein kinase C completely abolishes all of the effects of phorbol dibutyrate, and partially inhibits the second but not the first phase of bombesin-induced [3H]AA release. These results indicate that bombesin-induced receptor-mediated activation of phospholipase A2 involves multiple mechanisms, including intracellular Ca2+ mobilization for the first phase, protein kinase C activation plus Ca2+ influx for the second phase, and as yet unknown mechanism(s) independent of intracellular Ca2+ mobilization or protein kinase C for both of the phases.     

Tumor necrosis factor-alpha stimulates phosphatidylinositol breakdown by phospholipase C to coordinately increase the levels of diacylglycerol, free arachidonic acid and prostaglandins in an osteoblast (MC3T3-E1) cell line

The effects of (human recombinant) tumor necrosis factor-alpha on phosphatidylinositol breakdown, release of 1,2-diacylglycerols, mobilization of arachidonate from diacylglycerol and prostaglandin synthesis were examined in a model osteoblast cell line (MC3T3-E1). Tumor necrosis factor-alpha (10 nM) caused a specific (30%) decrease in the mass of phosphatidylinositol (and no other phospholipids) within 30 min of exposure. Tumor necrosis factor-alpha doubled the rate of incorporation of [32P]orthophosphoric acid into phosphatidylinositol, indicating that the turnover of inositol phosphate was enhanced, and increased the content of diacylglycerol in parallel with phosphatidylinositol breakdown. The cytokine (10-50 nM; 4 h) also promoted a specific release of 24-34% of the [3H]arachidonate from prelabeled phosphatidylinositol, a release of 80% of the 3H-fatty acid from the diacylglycerol pool, and a 30-fold increase in the synthesis of prostaglandin E2. The tumor necrosis factor-alpha induced liberation of [3H]arachidonate from diacylglycerol, cellular arachidonate release and the synthesis of prostaglandin E2 were each blocked by an inhibitor of diacylglycerol lipase, the compound RHC 80267 (30 microM). Therefore, we conclude that, in the MC3T3-E1 cell line, tumor necrosis factor-alpha activates a phosphatidylinositol-specific phospholipase C (phosphatidylinositol inositolphosphohydrolase; EC 3.1.4.3) to release diacylglycerol, and increases the metabolism of diacylglycerol to liberate arachidonate for prostaglandin synthesis.     

Lipases operative at the fat cell surface: attempt at an integrated approach

Extracellular acylglycerols are hydrolysed by lipases active at the surface of intact fat cells isolated from rat or human adipose tissue. During short-term incubation, rat fat cells hydrolyse di-[3H]oleyl-[14C]glycerol at a rate of 70 +/- 7.7 mU/10(6) cells (mean +/- S.E.) versus 440 +/- 62 mU/10(6) cells for the hydrolysis of mono-[3H]oleylglycerol; these relatively high lipolytic potencies may serve, among other functions, to counteract the cytolytic effect of both esters. Reaction rates with both substrates are unchanged by addition of various apolipoproteins C and by the nutritional state of the animals. Fat cells incorporate 15-20 per cent of the total [3H]-oleic chains liberated by hydrolysis, with no correlation between uptake and hydrolysis rates. [3H]-oleic chains in cell lipids are found mainly as diacylglycerol (15 per cent) and triacylglycerol (80 per cent). Both lipolytic processes differ from the hydrolysis of trioleylglycerol by cell-bound lipoprotein lipase, which occurs at lower rates (6.5 +/- 0.6 mU/10(6) cells) and depends on apolipoprotein C-II and nutritional state of the animals. The results support the accepted view that lipoprotein lipase and monoacylglycerol lipase are distinct enzymes. Differences between lipoprotein lipase and diacylglycerol lipase activities raise the possibility of different catalytic entities. In conclusion, isolated fat cells in suspension hydrolyse and incorporate lipids. This model should approximate physiological conditions more closely than the use of lipases in the free state.     

Bradykinin Activates a Phospholipase D that Hydrolyzes Phosphatidylcholine in PC12 Cells

In PC12 pheochromocytoma cells whose phospholipids had been prelabelled with [3H]palmitic acid, bradykinin increased the production of [3H]phosphatidic acid. The increase in [3H]phosphatidic acid occurred within 1-2 min. before the majority of the increase in [3H]diacylglycerol. When the phospholipids were prelabeled with [3H]choline, bradykinin increased the intracellular release of [3H]choline. The production of phosphatidic acid and choline suggests that bradykinin was increasing the activity of phospholipase D. Transphosphatidylation is a unique property of phospholipase D. In cells labeled with [3H]palmitic acid, bradykinin stimulated the transfer of phosphatidyl groups to both ethanol and propanol to form [3H]phosphatidylethanol and [3H]phosphatidylpropanol, respectively. The effect of bradykinin on [3H]phosphatidic acid and [3H]phosphatidylethanol formation was partially dependent on extracellular Ca2+. In cells treated with nerve growth factor, carbachol also increased [3H]phosphatidylethanol formation. To investigate the substrate specificity of phospholipase D, cells were labeled with [14C]stearic acid and [3H]palmitic acid, and then incubated with ethanol in the absence or presence of bradykinin. The 14C/3H ratio of the phosphatidylethanol that accumulated in response to bradykinin was almost identical to the 14C/3H ratio of phosphatidylcholine. The 14C/3H ratio in phosphatidic acid and diacylglycerol was higher than the ratio in phosphatidylcholine. These data provide additional support for the idea that bradykinin activates a phospholipase D that is active against phosphatidylcholine. The hydrolysis of phosphatidylcholine by phospholipase D accounts for only a portion of the phosphatidic acid and diacylglycerol that accumulates in bradykinin-stimulated cells: bradykinin evidently stimulates several pathways of phospholipid metabolism in PC12 cells.