Changes in glucose oxidation during growth of embryonic heart cells in culture.

A rapid and convenient method has been utilized to investigate glucose oxidation during growth of chick embryo heart cells in tissue culture. Primary isolates of chick embryo heart cells showed exponential growth when plated at low densities and exhibited density-inhibited growth as cultures became confluent. The density-dependent growth inhibition of chick embryo heart cells is associated with a marked decrease in the specific activity of glucose oxidation to CO(2). This decrease in glucose oxidation was observed as density increased as either a function of time in culture or as related to initial plating density. The decrease in (14)CO(2) production associated with density-dependent inhibition of growth is due to a marked decrease in activity of the pentose phosphate pathway.

Interaction of fatty acid and glucose oxidation by cultured heart cells.

Chick embryo heart cells in tissue culture actively oxidize [1-(14)C]palmitate to (14)CO(2). Fatty acid oxidation by cell monolayers was linear with time and increasing protein concentration. The addition of carnitine to the assay medium resulted in a 30-70% increase in the rate of fatty acid oxidation. The specific activity of palmitic acid oxidation did not change significantly with time in culture and was also the same in rapidly proliferating and density-inhibited cell cultures. Addition of unlabeled glucose to the assay medium resulted in a 50% decrease in (14)CO(2) production from [1-(14)C]palmitate. Conversely, palmitate had a similar sparing effect on [(14)C]glucose oxidation to (14)CO(2). Lactate production accounted for most of the glucose depleted from the medium and was not inhibited by the presence of palmitate in the assay. Thus, the sparing action of the fatty acids on glucose oxidation appears to be at the mitochondrial level. The results indicate that although chick heart cells in culture are primarily anaerobic, they can oxidize fatty acid actively.

Neuromuscular excitability changes in the vastus medialis following anterior cruciate ligament reconstruction.

PURPOSE: Quadriceps weakness following anterior cruciate ligament reconstruction (ACLR) is prevalent despite intensive rehabilitation. Diminished neuromuscular excitability is one potential factor that may limit muscular recovery following injury or surgery. The H-reflex provides a measure of alpha motorneuron (neuromuscular) excitability in the sensory-motor pathway of the respective muscle and nerve. To date the vastus medialis (VM) and soleus (SOL) H-reflexes have been examined primarily in control subjects with induced knee joint effusion. This prospective, randomized clinical trial evaluated the affect of ACLR, utilizing hamsting (HS) or bone-patellar tendon-bone (BTB) autograft, on VM and SOL H-reflex latency and amplitude in twenty subjects. METHODS: Preoperatively bilateral VM and SOL H-reflex tests were conducted. VM and SOL H-reflexes were subsequently conducted on the involved lower extremity at 1 and 3 months post surgery. At each test session subjects completed visual analog scales and knee girth was measured. RESULTS: The VM H-reflex amplitude increased in the HS group at 3 months compared to 1-month post surgery (p<.05). Significant changes over time were also noted in the visual analog pain and functional scales and the mid-patella girth. CONCLUSIONS: The increased VM H-reflex amplitude at 3 months following HS autograft ACLR demonstrates an increase in VM neuromuscular excitability. Increased VM neuromuscular excitability was not evident in patients following BTB reconstruction. The increased neuromuscular excitability, observed only in the HS group, warrants consideration when selecting graft type for patients with extensive preoperative quadriceps dysfunction.

Fatty acid metabolism of isolated mammalian cells.

It is now clear that a wide variety of differentiated cells in culture exhibit essentially the full spectrum of mammalian fatty acid metabolism. These cells readily incorporate free fatty acids into membrane phosphoglycerides, modify exogenous fatty acids by desaturation and elongation, and store excess fatty acyl groups, primarily as triacylglycerols. Similarly, many different types of cells synthesize cyclooxygenase and lipoxygenase derivatives of long chain polyunsaturated fatty acids. Furthermore, although the fatty acid composition of cellular phospholipids can be modified by medium supplementation, cells in culture exhibit definite fatty acyl specificities for the various steps of fatty acid activation, transesterification and release. As the extensive repertoire of fatty acid metabolism in mammalian cells has been elucidated, and as the ability to grow differentiated cells in culture has increased, new questions have arisen. There is still much to be learned about the enzymes involved in synthesizing and maintaining the unique fatty acid composition of the different cellular phospholipids and the processes which regulate the desaturation, elongation and retroconversion of polyunsaturated fatty acids. Other areas of great current interest are the mechanisms by which certain long chain polyunsaturated fatty acids are made available for conversion to oxygenated, biologically-active derivatives, the metabolic interactions between different polyunsaturated fatty acids, particularly n-3 and n-6 fatty acids, the cellular roles of the C22 polyunsaturated fatty acids, and the functions of particular molecular species of phospholipids in membrane-mediated events. Further research in these areas will contribute to unravelling the role of fatty acids and fatty acid derivatives in the physiological processes of mammalian cells.

Apparent specificity of the thrombin-stimulated deacylation of endothelial glycerolipids for polyunsaturated fatty acids with a delta-5 desaturation.

Human umbilical vein endothelial cells readily incorporate exogenous polyunsaturated fatty acids. Subsequent stimulation with thrombin results in the release of both arachidonate and eicosapentaenoate from cellular phospholipids. The present study has investigated the utilization of 8,11,14-[14C]eicosatrienoate, the precursor of prostaglandin E1. Analysis of released 14C-fatty acids by radio-gas chromatography indicated that thrombin stimulated the release of 6-10% of the [14C]arachidonate synthesized by desaturation of the [14C]eicosatrienoate, but did not stimulate release of [14C]eicosatrienoate per se (less than 1%). As determined by digestion of cellular lipid extracts with pancreatic phospholipase A2, both 8,11,14-[14C]eicosatrienoate and [14C]arachidonate were esterified primarily in the 2-position. Similarly, separation of phospholipid classes by two-dimensional thin-layer chromatography did not indicate any major differences in the distribution of the incorporated 14C-fatty acids. Experiments with additional 14C-fatty acids indicated that 5,8,11-eicosatrienoate is released in response to thrombin but that 8,11,14,17-eicosatetraenoate is not. These results suggest that the delta-5 double bond is required for the thrombin-stimulated release of free fatty acids from endothelial phospholipids and their subsequent availability as substrates for eicosanoid synthesis.

Retention of phospholipid acyl groups is not characteristic of neoplastic cells in culture.

Phospholipid acyl group retention of normal and mouse sarcoma virus transformed cell lines has been examined. The cells were fed [1-14C]oleate for 24 h and then grown to non-radioactive medium for 4 days. Both the transformed rat and mouse cell lines continued to release 14C-labeled fatty acids and phospholipids into the culture medium similarly to the uninfected cells, and showed shifts in 14C between phospholipid subclasses. Thus, the phospholipid acyl group stability reported for L cells is not characteristic of oncogenic cells in culture.

Elongation of C20 polyunsaturated fatty acids by human skin fibroblasts.

Human skin fibroblasts actively elongate a portion of incorporated C20 polyunsaturated fatty acids to their respective C22 derivatives. As much as 40% of incorporated [14C]eicosapentaenoate is elongated within 8 h and 85% by 48 h. Elongation of [14C]arachidonate is initially less than half that of [14C]eicosapentaenoate and plateaus at 20-30% of incorporated 14C-labeled fatty acid. The elongation of 5,8,11-[14C]eicosatrienoate is intermediate between that of 20:4(n-6) and 20:5(n-3). Docosatetraenoate is not an effective inhibitor of the elongation of arachidonate, thus suggesting that the observed plateau is not due to product inhibition. When concentrations of exogenous fatty acids are increased, these cells elongate substantial quantities of C20 polyunsaturated fatty acids; elongation of eicosapentaenoate is consistently more extensive than that of arachidonate. Eicosapentaenoate is also an effective inhibitor of the elongation of [14C]arachidonate. Increases in exogenous arachidonate up to 10 microM result in an increase in elongation of [14C]arachidonate both in absolute quantities and as a percentage of that incorporated; the arachidonate thus acts as a positive modulator of its own elongation. Increased eicosapentaenoate also enhances the elongation of [14C]eicosapentaenoate, but only at lower concentrations (0.02-0.15 microM). The factors which regulate the elongation of C20 polyunsaturated fatty acids in human skin fibroblasts serve to permit extensive elongation of eicosapentaenoate while retaining incorporated arachidonate primarily in its C20 form.

Elongation of arachidonic and eicosapentaenoic acids limits their availability for thrombin-stimulated release from the glycerolipids of vascular endothelial cells.

This study has examined the thrombin-stimulated release of polyunsaturated fatty acids from endothelial glycerolipids. Human umbilical vein endothelial cells were incubated with 1.25 microM [14C]arachidonate or [14C]eicosapentaenoate and then exposed to thrombin in buffered saline plus albumin. After an incorporation period of 0.5 h, the thrombin-stimulated release of the two radiolabeled fatty acids was quite similar. By contrast, after 24 h of fatty acid incorporation, the thrombin-stimulated release of radiolabeled fatty acid from cells incubated with [14C]eicosapentaenoate was only 25-30% of that from cells with [14C]arachidonate. Analysis of cellular glycerolipids indicated that 23 and 72%, respectively, of the incorporated [14C]arachidonate and [14C]eicosapentaenoate had been elongated to 22-carbon fatty acids in 24 h. Both 20- and 22-carbon 14C-labeled fatty acids were released to albumin in the medium in control incubations. Addition of thrombin stimulated the release of [14C]arachidonate and [14C]eicosapentaenoate, but not of their respective elongation products. Furthermore, endothelial cells incorporated exogenous [14C]docosatetraenoate into cellular glycerolipids but did not release it in response to thrombin. Thus, the thrombin-stimulated release of polyunsaturated fatty acids from vascular endothelial cells is highly selective for arachidonate and eicosapentaenoate. These results suggest that the extensive elongation of eicosapentaenoate by these cells serves to remove n - 3 polyunsaturated fatty acids from the pool of cellular acyl groups which are released in response to thrombin and are thus made available for metabolism by cyclooxygenase and lipoxygenase enzymes.

Oligomers of prostaglandin B1 inhibit arachidonic acid mobilization in human neutrophils and endothelial cells.

The previous paper (Biochim. Biophys. Acta 1006 (1989) 272-277) has demonstrated that oligomers of prostaglandin B1 are effective in vitro inhibitors of a wide range of both cell-derived and extracellular phospholipases A2. The present study has investigated the effects of prostaglandin oligomers on agonist-stimulated phospholipase activity on intact human cells. PGBx, an oligomer (n = 6) or PGB1, and PGB-trimer inhibit as much as 95% of the A23187-stimulated release of arachidonic acid from human neutrophils. The effect is dose-dependent, with an IC50 of 4-5 microM; near maximal inhibition is obtained with as little as 1 min of preincubation with PGB-trimer. Consistent with its role as a phospholipase A2 inhibitor, PGB-trimer also inhibits the A23187-stimulated incorporation of [3H]acetate into platelet-activating factor. PGBx and PGB-trimer also inhibit the release of arachidonic acid from human umbilical vein endothelial cells stimulated with histamine, thrombin, or ionophore A23187; inhibition of the basal or unstimulated turnover of both arachidonic acid and oleic acid is also observed. Inhibition by PGB-trimer can be blocked by simultaneous addition of 50 microM albumin; cells preincubated with PGB-trimer are not affected by albumin. Furthermore, removal of exogenous PGB-trimer prior to challenge with A23187 does not reverse the inhibition of either endothelial cells and neutrophils. Thus, prostaglandin B1 oligomers are taken up by human neutrophils and vascular endothelial cells and serve as potent inhibitors of arachidonic acid mobilization. One mechanism for the pharmacological effects of PGBx may be inhibition of cell-associated and extracellular phospholipase A2.

Oligomers of prostaglandin B1 inhibit in vitro phospholipase A2 activity.

Oligomers of prostaglandin B1 inhibited phospholipase A2 extracted from human neutrophils in a dose-dependent manner (IC50 = 5 microM), while the monomer was not inhibitory at concentrations of 10 microM or less. The inhibitory activity of PGB1 oligomers increased with increasing polymer size; PGB dimer had approximately one-half the maximal inhibitory activity of PGBx, while a trimer was almost as inhibitory as a tetramer and PGBx (n = 6). PGBx as an oil or as a water-soluble sodium-salt-inhibited Ca2(+)-dependent phospholipase A2 from snake venom, bovine pancreas, human neutrophil and platelet, human synovial fluid, and human sperm with IC50 values ranging from 0.5-7.5 microM. Inhibition was independent of added Ca2+ and was independent of substrate phospholipid concentration. Interaction of purified snake venom phospholipase A2 (Naja mocambique) with PGBx resulted in dose-dependent quenching of the enzyme's tryptophan fluorescence; 50% quench was noted with a molar ratio of PGBx/enzyme of 1.5. Inhibition of phospholipase A2 activity by PGBx was relieved in a dose-dependent manner by either defatted or untreated bovine serum albumin. PGBx is a potent in vitro inhibitor of a wide spectrum of phospholipases A2, and as illustrated in the accompanying paper, has profound inhibitory effects on arachidonic acid mobilization in human neutrophils and vascular endothelial cells. Modulation of cellular and extracellular phospholipases A2, and the bioactive transmitters generated by this catalytic event, may be a basic mechanism by which oligomers of prostaglandin B1 exert their reported membrane-protective effects.

Effects of DMSO-induced differentiation on arachidonate mobilization in the human histiocytic lymphoma cell line U937: responsiveness to sub-micromolar calcium ionophore A23187 and phorbol esters.

The human histiocytic lymphoma cell line, U937, is a rich source for isolation and purification of the 85 kDa cytosolic phospholipase A2 (cPLA2). Recent studies suggest that this enzyme catalyzes the agonist-stimulated release of arachidonate from membrane phospholipids, thereby initiating eicosanoid synthesis. We therefore investigated in situ regulation of phospholipase A2 activity in intact U937 cells. The results indicate that calcium ionophore A23187 stimulatable release in intact undifferentiated U937 is low and only weakly dose dependent. Dimethyl sulfoxide (DMSO) differentiation of U937 cells results in a dramatic increase of A23187-stimulated arachidonate mobilization. Consistent with the characteristics of cPLA2 in vitro, A23187-stimulated arachidonate release in differentiated U937 cells is highly specific for arachidonate and is activated by submicromolar A23187 concentrations. Phorbol myristate acetate (PMA) further potentiates arachidonate release in differentiated U937 cells by 4--6-fold over A23187 alone. However, treatment of differentiated U937 cells with PMA alone is an ineffective stimulus for arachidonate release, suggesting that a calcium transient is necessary for in situ arachidonate mobilization. A23187-stimulated arachidonate release increases during distinct temporal phases of differentiation (0-36 h, 84-96 h). By contrast PMA enhancement of the response to A23187 develops early in differentiation, and is complete by 36 h. These results suggest that differentiation-induced alterations in cPLA2 regulatory elements, such as intracellular free calcium and/or phosphorylation, may regulate mobilization of arachidonate in U937 cells.

Phospholipid acyl group stability in cultured fibroblasts. Differences between human cell lines of fetal and adult origin.

Human fibroblasts of both fetal and adult origin incorporated [1-14C] acetate primarily into phospholipid acyl groups (70-80% of total radioactivity). When these labeled cells were replated in non-radioactive medium, there was continuous loss of 14C from steroids, triacylglycerols and non-lipid material. In contrast, after some initial loss, cell lines of fetal origin completely retained 14C in cellular phospholipids during continued cell division. Unlike cells of fetal origin, fibroblasts of adult origin continued to lose radioactivity from their phospholipid acyl groups during growth in unlabeled medium. Loss of radioactivity from [1-3H] acetate incorporated into phospholipids of adult cells cannot be attributed to cell death since it was not accompanied by any loss of previously incorporated [ME-14C] thymidine. If cellular phospholipids were labeled with [U-14C] glycerol, both fetal and adult fibroblasts continued to lose radioisotope from the cells during growth in nonradioactive medium. Thus, there is turnover of the phospholipid molecules themselves in fetal human fibroblasts grown in vitro, but their acyl groups are retained within cellular phospholipids. In this respect, fibroblasts of fetal origin resemble established cell lines such as the L fibroblast. Fibroblasts of adult origin do not exhibit this complete conservation of their phospholipid acyl groups.

High-affinity incorporation of 20-carbon polyunsaturated fatty acids by human skin fibroblasts.

This study has examined the acyl specificity of incorporation of polyunsaturated fatty acids into cellular glycerolipids of human skin fibroblasts. At low exogenous fatty acid concentrations (0.2-1.2 microM) the extent of incorporation of arachidonate, eicosapentaenoate, 8,11,14-eicosatrienoate and 5,8,11-eicosatrienoate is 60-150% greater than that of oleate or linoleate. As the concentration of exogenous free fatty acid is increased to 25 microM, there is little decrease in the percentage of exogenous oleate incorporated into cellular glycerolipids. Under these conditions, the percentage incorporation of arachidonate and eicosapentaenoate drops 2-3-fold and approaches that of oleate. In contrast, the percentage incorporation of 8,11,14-eicosatrienoate remains high as exogenous fatty acid concentrations are increased. Incorporation of arachidonate, eicosapentaenoate, 8,11,14-eicosatrienoate and 5,8,11-eicosatrienoate is inhibited by addition of any of the other C20 polyunsaturated fatty acids but not by palmitate or oleate. C20 polyunsaturated fatty acids other than the eicosanoid precursors are also not effective inhibitors of arachidonate incorporation. The high affinity incorporation of C20 polyunsaturated fatty acids does not appear to be due to their selective esterification in any one class of cellular phospholipids. These results are compatible with a model of two pathways of fatty acids incorporation into mammalian cells. One pathway utilizes all exogenous long-chain fatty acids and, at least in fibroblasts, is not readily saturable. The second is a high-affinity, low-capacity uptake mechanism specific for arachidonate and other precursors of eicosanoids. The acyl specificity of this latter pathway appears to be similar to that of platelet arachidonyl- CoA synthetase. Results obtained with 8,11,14-eicosatrienoate would indicate, however, that at high concentrations, additional mechanisms influence the acyl specificity of fatty acid incorporation in these cells.

Fatty acyl delta 6 desaturation activity of cultured human endothelial cells. Modulation by fetal bovine serum.

Human endothelial cells from umbilical vein actively desaturate [14C]linoleate and synthesize icosatrienoate, arachidonate and docosatetraenoate. Desaturation and chain elongation of 9,12,15-[14 C]linolenate (n - 3) by these cells is more extensive than that of [14 C]linoleate. Both confluent primary monolayers and subconfluent subcultures exhibit greater fatty acyl CoA delta 6-desaturase activity when growth and incubation media contain 2.5% fetal bovine serum instead of 10%. Prior growth with 20% serum diminishes the extent of subsequent linoleate desaturation. Use of medium supplemented with 20-100 microM oleate results in up to 67% inhibition of [14 C]arachidonate synthesis. These results indicate that, despite previously published reports to the contrary, human vascular endothelial cells are similar to other normal mammalian cells in having fatty acyl delta 6-desaturase activity. Suppression of endogenous arachidonate synthesis by elevated levels of serum lipids may impair endothelial cell function.

Selective effects of isomeric cis and trans fatty acids on fatty acyl delta 9 and delta 6 desaturation by human skin fibroblasts.

Human skin fibroblasts incorporate and actively desaturate long-chain fatty acids. Growth of these cells in lipid-free medium can be used to enhance delta 9 and delta 6 desaturation of [14C]stearate and [14C]linoleate, respectively. Medium supplementation with cis fatty acids inhibits delta 9 desaturation; effectiveness as inhibitors is linoleate (9c,12c-18:2) greater than oleate (9c-18:1) greater than vaccenate (11c-18:1). Linoelaidate (9t,12t-18:2), trans-vaccenate (11t-18:1) and saturated fatty acids are without effect; elaidate (9t-18:1) appears stimulatory. By contrast, the trans fatty acids elaidate and linoelaidate are potent inhibitors of delta 6 desaturation; inhibition by trans-vaccenate is 50% of that of elaidate. Desaturation of [14C]linoleate is only slightly inhibited by oleate, cis-vaccenate, or (6c,9c,12c)-linolenate. The relative effectiveness of isomeric cis- and trans-octadecenoic acids as inhibitors of delta 9 and delta 6 desaturation in intact human cells is different from that found in microsomal studies. The cell culture system can thus be important in evaluating physiological effects of isomeric fatty acids on cellular metabolic processes.

Human vascular endothelial cells synthesize and release 24- and 26-carbon polyunsaturated fatty acids.

Vascular endothelial cells from human umbilical vein readily incorporate [14C]eicosatrienoate (20:3 (n - 6) and desaturate it to synthesize [14C]arachidonate (20:4) and [14C]docosatetraenoate (22:4). Both substrate and metabolites are extensively esterified in cellular phospholipids and triacylglycerol. After these cells are incubated for 24-48 h with 4.5 microM [14C]20:3 in culture medium plus 10% fetal bovine serum, the medium contains a number of radiolabeled free fatty acids. In addition to arachidonate and docosatetraenoate, these include still longer-chain polyunsaturated fatty acids. We have identified these as 24:4, 24:5, 26:4 and 26:5 by both radio-gas chromatography and HPLC. Although the 24- and 26-carbon polyunsaturated fatty acids represent a negligible percentage of cellular 14C-labeled fatty acids, they are each present in the medium at a concentration of 10-40 nM, whereas [14C]arachidonate is 60-100 nM. In particular, products of delta 4 desaturation are a significant component of radiolabeled polyunsaturated fatty acids in medium but not in the cells. Since docosapolyenoic fatty acids have recently been shown to give rise to biologically active oxygenated derivatives, the selective release and possible subsequent metabolism of even longer polyunsaturated fatty acids warrants further investigation.

1,3-Dioctanoylglycerol modulates arachidonate mobilization in human neutrophils and its inhibition by PGBx: evidence of a protein-kinase-C-independent role for diacylglycerols in signal transduction.

Preincubation of human neutrophils with 1-oleoyl-2-acetylglycerol (OAG) enhances subsequent f-Met-Leu-Phe (fMLP)-stimulated arachidonate mobilization. We have recently demonstrated that preincubation of neutrophils with OAG also reverses inhibition of A23187 stimulated [3H]arachidonate mobilization by the phospholipase A2 inhibitors, PGBx and aristolochic acid. The present study has compared the effects of 1,2-sn-dioctanoylglycerol (1,2-diC8) and 1,3-dioctanoylglycerol (1,3-diC8) on these cellular events. Dose-dependent priming (ED50 < 2.5 microM) of fMLP-stimulated [3H]arachidonate mobilization is obtained with both 1,2-diC8 and 1,3-diC8. Both diC8s also enhance fMLP-stimulated synthesis of leukotriene B4, 5-hydroxyeicosatetraenoic acid and platelet-activating factor, and generation of superoxide. Furthermore, both 1,2-diC8 and 1,3-diC8 reverse the effects of PGBx on A23187-stimulated [3H]arachidonate mobilization and platelet-activating factor synthesis. By contrast, higher concentrations (5-10 microM) of 1,2-diC8, but not 1,3-diC8, directly stimulate both [3H] arachidonate mobilization and superoxide generation. Since 1,3-diC8 does not activate protein kinase C (PKC), these results suggest that PKC is involved in direct activation of neutrophils by diacylglycerols but not in priming. Furthermore, reversal of the inhibitory effects of PGBx by diacylglycerols also appears to involve a PKC-independent mechanism.

The effects of the phospholipase A2 inhibitors aristolochic acid and PGBx on A23187-stimulated mobilization of arachidonate in human neutrophils are overcome by diacylglycerol or phorbol ester.

Aristolochic acid and PGBx, two structurally unrelated, protein-targeted inhibitors of isolated phospholipases A2, are effective antagonists of calcium ionophore A23187-stimulated mobilization of [3H]arachidonate from human neutrophils. We now report that preincubation of neutrophils with oleoylacetylglycerol (OAG, 15 microM) substantially reverses the inhibitory effect of 200 microM aristolochic acid (from 70 to 24% inhibition). Similarly, OAG increases the IC50 for PGBx from 2.5 to greater than 20 microM. The effects of OAG on inhibition by either aristolochic acid or PGBx are dose-dependent, with an ED50 of 2.5 microM. Protection against inhibition by either aristolochic acid or PGBx is also observed with phorbol myristate acetate (PMA, ED50 3 nM), but not 4-alpha-phorbol didecanoate. Aristolochic acid and PGBx do not inhibit PMA-stimulated superoxide generation, and are thus not protein kinase C inhibitors. Furthermore, neither aristolochic acid nor PGBx inhibit diglyceride generation through the phospholipase D/phosphatidate phosphohydrolase pathway. A23187-stimulated [3H]arachidonate mobilization is increased by 20-50% when neutrophils are preincubated with OAG or PMA. The present results indicate that OAG and PMA also modulate the A23187-stimulated [3H]arachidonate mobilization so as to render it less sensitive to inhibitors of phospholipase A2.

Sphingolipid metabolism and signal transduction: inhibition of in vitro phospholipase activity by sphingosine.

Sphingosine inhibits protein kinase C activity in vitro and has been used to implicate this enzyme in signal transduction and cell function. We report that sphingosine directly inhibits phospholipases A2 and D. Sphingosine inhibits Ca(2+)-dependent phospholipases A2 from Naja naja, porcine pancreas, Crotalus adamanteus, human disc and neutrophil in a dose-dependent manner with IC50 values ranging from 5-40 microM using [1-14C]oleate-labelled autoclaved E. coli (20 microM) as substrate. Inhibition is comparable using the same concentrations (20 microM) of [1-14C]oleate-labelled C. albicans or E. coli, or aqueous dispersions of 1-acyl-2-[1-14C]linoleoylglycerophosphoethanolamine or -choline. Sphinganine and stearylamine are as inhibitory as sphingosine; monoolein is less inhibitory (IC50 = 70 microM), while octylamine, N-acetylsphingosine, sphingomyelin and ceramide have no effect. Inhibition is relieved by increasing concentrations of substrate phospholipid. The molar ratio of sphingosine to phospholipid required for 50% inhibition ranges from 0.5 to 1.0 with 2-100 microM E. coli phospholipid. In contrast, sphingosine has a biphasic effect on the hydrolysis of E. coli by S. chromofuscus phospholipase D; concentrations less than or equal to 25 microM stimulate activity while concentrations greater than 25 microM are inhibitory. Addition of Triton X-100 eliminates both the stimulatory and inhibitory effects of sphingosine on phospholipase D activity.

Effects of aristolochic acid on phospholipase A2 activity and arachidonate metabolism of human neutrophils.

Aristolochic acid is an alkaloid which has recently been shown to have anti-inflammatory activity against edema in mouse foot pads induced by phospholipases A2 from human synovial fluid. The present study has investigated the effects of aristolochic acid on phospholipase activity and arachidonic acid mobilization in human neutrophils. We find that aristolochic acid is a dose-dependent inhibitor of the calcium-dependent neutral active phospholipase A2 isolated from human neutrophils. As much as 90% of the A23187-stimulated release of previously incorporated [3H]arachidonate from intact neutrophils is inhibited by aristolochic acid; the effect is dose-dependent, with an IC50 of 40 microM, and quite rapid, with near maximal inhibition within 5 min. Aristolochic acid inhibits the A23187-stimulated loss of [3H]arachidonate from both choline- and inositol-phospholipids. Decreased release of free [3H]arachidonate is accompanied by a concomitant decrease in synthesis of [3H]leukotriene B4 and [3H]hydroxyeicosatetraenoic acids. Furthermore, aristolochic acid also inhibits the A23187-stimulated synthesis of [3H]alkylacetylglycerophosphocholine from cellular [3H]alkylacylglycerophosphocholine. These results indicate that aristolochic acid is an effective inhibitor of the A23187-stimulated phospholipase A2 activity in human neutrophils.