Miriam D. Rosenthal

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.

Retroconversion and δ4 desaturation of docosatetraenoate (22:4(n−6)) and docosapentaenoate (22:5(n−3)) by human cells in culture

This study has investigated the metabolic modification of [3-14C]docosatetraenoate (22:4(n-6)) and [3-14C]docosapentaenoate (22:5(n-3)) by human cells in culture. Fetal skin fibroblasts converted as much as 20% of the incorporated [14C]22:4(n-6) to [14C]20:4(n-6) within 6 h and 41% within 48 h. Retroconversion of incorporated [14C]22:5(n-3) was less than 13% at all time points. Chain shortening of [14C]22:4(n-6) was also 2-6-fold greater than that of [14C]22:5(n-3) in retinoblastoma and vascular endothelial cells. Fibroblasts, vascular endothelial cells and retinoblastoma cells all elongated substantially more [14C]eicosapentaenoate than [14C]arachidonate to the respective C22 fatty acids. Within 3-4 days, fibroblasts incubated with either [14C]20:5(n-3) or [14C]22:5(n-3) had the same ratio of radiolabeled C22:C20 fatty acids in cellular glycerolipids. By contrast, the cells incubated with [14C]22:4(n-6) or [14C]20:4(n-6) did not reach a common C22/C20 equilibrium by 5 days. Although fibroblasts were found to desaturate [14C]22:5(n-3), a substantial lag time was observed; [14C]22:6(n-3) was 2% at 48 h and 20% at 96 h. By contrast, synthesis of [14C]22:6(n-3) by retinoblastoma cells was 51% within 6 h and greater than 90% at 96 h. Desaturation of [14C]22:4(n-6) was observed in retinoblastoma cells, but not in fibroblasts. These results thus suggest that the ratio of C22C20 polyunsaturated fatty acids in cells is regulated by the relative rates of retroconversion and chain elongation, with the net effect of the two processes favoring C20 for n-6 and C22 for the n-3 fatty acids. Furthermore, although fibroblasts desaturate [14C]22:5(n-3), the process appears to be qualitatively different from that of retinoblastoma cells.

The effects oftrans fatty acids on fatty acyl Δ5 desaturation by human skin fibroblasts

The effectiveness of different fatty acids as inhibitors of fatty acyl Δ5 desaturation activity in human skin fibroblasts has been investigated. When incubated with 2.25 μM [14C] eicosatrienoate (20∶3ω6) in otherwise lipid-free medium, these cells rapidly incorporate the radiolabeled fatty acid into cellular glycerolipids and desaturate it to produce both [14C] arachidonate and [14C] docosatetraenoate. The Δ5 desaturation activity can be enhanced by prior growth of the cells without serum lipids. Elaidate (9t–18∶1) is a potent inhibitor of Δ5 desaturation whiletrans-vaccenate (11t–18∶1) is virtually without effect. Oleate and linoleate are only mildly inhibitory. Linoelaidate (9t, 12t–18∶2) is more inhibitory than linoleate but significantly less effective than elaidate. The effects of elaidate can be readily overcome by increasing the concentration of exogenous eicosatrienoate. Studies with a variety oftrans monounsaturates of differing chain lengths indicate that the ω9trans fatty acids are potent inhibitors of Δ5 desaturation, while ω7trans fatty acids are relatively ineffective. Intact human fibroblasts could thus be important in characterizing novel fatty acids as selective inhibitors of arachidonate synthesis in vivo.

Fatty acyl Δ6 desaturation activity of cultured human endothelial cells modulation by fetal bovine serum

Human endothelial cells from umbilical vein actively desarurate [14C]linoleate and synthesize icosatrienoate, arachidonate and docosatetraenoate. Desaturation and chain elongation of 9,12,15-[14C]linolenate (n − 3) by these cells is more extensive than that of [14C]linoleate. Both confluent primary monolayers and subconfluent subcultures exhibit greater fatty acyl CoA Δ6-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 μM oleate results in up to 67% inhibition of [14C]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 Δ6-desaturase activity. Suppression of endogenous arachidonate synthesis by elevated levels of serum lipids may impair endothelial cell function.

Accumulation of neutral lipids by human skin fibroblasts: Differential effects of saturated and unsaturated fatty acids

The accumulation of neutral lipids by human skin fibroblasts grown in medium supplemented with fatty acids has been investigated. GM-10 cells incorporated exogenous fatty acids into both phospholipids and neutral lipids. More [14C] oleate, linoleate, or linolenate was incorporated into triacylglycerol than was [14C] palmitate or stearate. Supplementation of medium containing delipidized serum with unsaturated fatty acids resulted in far more stimulation of [14C] glycerol incorporation into triacylglycerol than did supplementation with saturated fatty acids. Palmitate- and stearate-fed cells incorporated sizable amounts of [14C] fatty acids and [14C] glycerol into diacylglycerol as well as triacylglycerol, especially at higher fatty acid concentrations. Increased oleate supplementation from 10–300 μM resulted in increased triacylglycerol synthesis and accumulation of discrete cytoplasmic lipid droplets; palmitate concentrations above 70 μm were toxic. Micrographs of the palmitate-fed cells showed electron translucent slits, suggesting solid depositions of saturated fat, rather than the discrete osmiophilic droplets found in oleate-fed cells. Although GM-10 cells can synthesize fully saturated triacylglycerols, these data suggest that in cells fed saturated fatty acids, solid depositions of neutral lipids may sequester diacylglycerols and thus limit triacylglycerol synthesis.

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.

Selective effects of isomeric cis and trans fatty acids on fatty acyl Δ9 and Δ6 desaturation by human skin fibroblasts

Abstract Human skin fibroblasts incorporate and actively desaturate long-chain fatty acids. Growth of these cells in lipid-free medium can be used to enhance Δ9 and Δ6 desaturation of [14C]stearate and [14C]linoleate, respectively. Medium supplementation with cis fatty acids inhibits Δ9 desaturation; effectiveness as inhibitors is linoleate (9c,12c–18:2) > oleate (9c-18:1) > vaccenate (11c–18:1). Linoelaidate (9t,12t-18:2), trans-vcccenate (11t-18:1) and saturated fatty acids are without effect; elaidate (9t–18:l) appears stimulatory. By contrast, the trans fatty acids elaidate and linoelaidate are potent inhibitors of Δ6 desaturation; inhibition by trans-vaccenate is 50% of that of elaidate. Desaturation of [14C]linoIeate 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 Δ9 and Δ6 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.

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.

Arachidonate mobilization is coupled to depletion of intracellular calcium stores and influx of extracellular calcium in differentiated U937 cells

We have previously reported that dimethylsulfoxide-differentiation of U937 cells induced significant A23187-stimulatable arachidonate mobilization, consistent with characteristics of cytosolic phospholipase A2 (Rzigalinski, B.A. and Rosenthal, M.D. (1994) Biochim. Biophys. Acta 1223, 219-225). The present report demonstrates that differentiated cells attained higher elevations of intracellular free calcium in response to A23187 and thapsigargin, consistent with enhancement of the capacitative calcium influx pathway. Differentiation induced as significant increase in the size of the intracellular calcium stores, as well as in the capacity for store-activated calcium influx. Alterations in the capacitative calcium influx pathway were coupled to differentiation-induced activation of cPLA2 and mobilization of arachidonate in response to thapsigargin and fMLP stimulation. Although cPLA2 activity is often associated with influx of extracellular calcium, arachidonate mobilization in response to thapsigargin or fMPL was not simply a consequence of calcium influx. Assessment of intracellular free calcium elevations during thapsigargin or fMPL-induced stimulation suggest that a low level of arachidonic acid release was initiated upon release of intracellular store calcium. This initial release of arachidonate was unaffected by inhibition of calcium influx with nickel, EGTA, or SKF96365. Arachidonate release was observed when extracellular calcium was replaced with extracellular strontium, suggesting activation of the cytosolic PLA2 rather than secretory PLA2. Inhibition of PLA2 with prostaglandin B oligomer prevented both thapsigargin and fMLP-stimulated influx of extracellular calcium. Furthermore, exogenous free arachidonate stimulated influx of extracellular calcium in differentiated U937 cells. These results suggest that cPLA2-mediated release of free arachidonate may participate in the formation of a calcium influx factor which controls influx of extracellular calcium through store-controlled channels in the plasma membrane.

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.