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.

Developmental maturation of primate placental trophoblast: placental cytosolic and secretory phospholipase A2 expression after estrogen suppression of baboons.

We have demonstrated that the baboon placenta expressed the mRNAs and proteins for secretory and cytosolic phospholipase A2 (PLA2) enzymes and that cPLA2 expression increased with advancing gestation in association with the increase in placental estrogen production. To determine whether estrogen regulates placental PLA2 expression, as it does other aspects of syncytiotrophoblast functional differentiation, we compared sPLA2 and cPLA2 mRNA levels in placentas obtained on day 165 of gestation (term = day 184) from baboons that were untreated or treated during the second half of gestation with the aromatase inhibitor CGS 20267 or CGS 20267 and estradiol. Maternal saphenous and uterine vein estradiol levels were reduced (P < 0.05) by approximately 95% by treatment with CGS 20267 and restored by concomitant administration of CGS 20267 and estrogen. However, sPLA2 and cPLA2 mRNA levels expressed as a ratio of beta-actin were similar in whole villous placenta from baboons that were untreated or treated with CGS 20267 or CGS 20267 plus estrogen. PLA2 expression in an enriched fraction of nontrophoblast cells of the baboon placenta was also not altered by CGS 20267 treatment. Collectively these findings indicate that placental cPLA2 and sPLA2 expression is not estrogen-dependent. Because estrogen has been shown to regulate other aspects of placental steroidogenesis, we suggest that the regulatory role of estrogen on syncytiotrophoblast functional maturation is specific.

Developmental maturation of baboon placental trophoblast: expression of messenger ribonucleic acid and protein levels of cytosolic and secretory phospholipases A2.

Although the human placenta at term exhibits high levels of phospholipase A2 (PLA2) enzyme activity, our understanding of the ontogenesis, regulation, and specific roles of placental phospholipases A2 remains relatively incomplete. Using the baboon as the experimental model, the present study determined whether the levels of the messenger ribonucleic acid (mRNA) for the cytosolic (cPLA2) and/or type IIa, nonpancreatic secretory (sPLA2) enzymes are developmentally regulated and modulated by glucocorticoid treatment. Total RNA was extracted from whole villous placenta obtained on days 60 (early; n = 3), 100 (mid; n = 3), and 165 (late; n = 4) of gestation (term = day 184) from untreated baboons and on day 100 (n = 4) after maternal administration on days 60-99 of betamethasone (3 mg/day). The complementary DNA to cPLA2 recognized a single 2.9-kb mRNA transcript in both baboon and human placenta. Mean (+/-SE) levels of cPLA2 mRNA, expressed, in arbitrary units as a ratio to that of beta-actin, were similar at early (0.19 +/- 0.05) and midgestation (0.34 +/- 0.17) and increased (P < 0.005) 10-fold (2.53 +/- 0.53) by late gestation. Levels of cPLA2 protein were also greater (P < 0.05) on day 165 (2.6 +/- 0.3 arbitrary units) than on day 60 (0.6 +/- 0.2). Like that in the human, the baboon placenta contained very high levels of a single 0.9-kb mRNA transcript for sPLA2. In contrast to that of cPLA2, normalized levels of sPLA2 mRNA were similar at all three time points and were associated with high levels of sPLA2 protein throughout gestation. Treatment with betamethasone increased (P < 0.02) cPLA2 mRNA levels on day 100 by over 4-fold, but had no effect on sPLA2 mRNA levels. Additional studies indicated that the mRNAs for sPLA2 and cPLA2 were detected in an enriched fraction of nontrophoblast cells isolated by collagenase dispersion and Percoll density centrifugation. The mRNA for cPLA2 was also expressed in cytotrophoblast and syncytiotrophoblast cell fractions. Collectively, these findings indicate that the baboon placenta expresses mRNA and protein for both the cytosolic and secretory PLA2 enzymes, and that expression of cPLA2 is developmentally regulated and modulated by glucocorticoids. We previously demonstrated an estrogen-dependent developmental increase in placental expression of specific components of the progesterone steroidogenic pathway during the second half of baboon pregnancy. The current findings indicate that the developmental increase in placental function also includes expression of at least one specific PLA2 enzyme controlling arachidonic acid mobilization and eicosanoid synthesis.

PX-52, A novel inhibitor of 14 kDa secretory and 85 kDa cytosolic phospholipases A2.

Previously we reported that PGBx, a prostaglandin oligomer with anti-inflammatory activity, inhibited 14 kDa phospholipase A2 (PLA2) activity and blocked arachidonic acid mobilization in prelabeled human neutrophils (Biochim. Biophys. Acta 1006:272-277, 278-286, 1989) This study describes a new inhibitor of phospholipase A2, PX-52, that also blocks agonist induced arachidonic acid mobilization in prelabeled cells. PX-52, a fatty acid polymer, inhibited hydrolysis of 14C-oleate labeled E.coli by a variety of 14 kDa PLA2s including human PMN, sperm, synovial fluid and disc, as well as porcine pancreas, N. naja, and bee venom in a dose-dependent manner with IC50s ranging from 1.0-3.7 uM. Inhibition of activity was comparable at different Ca2+ concentrations, but was relieved by increasing substrate concentration or by methylation of PX-52. Hydrolysis of [14C]-arachidonyl phosphatidylcholine by 85 kDa, cytosolic PLA2 from U937 cells was similarly inhibited by PX-52, the IC50 = 5 uM. Arachidonic acid mobilization induced by A23187 in prelabeled human PMNs was blocked by PX-52; IC50 = 10-15 uM while concentrations of up to 80 uM oleate had no effect. These results demonstrate that PX-52 inhibits the in vitro activity of secretory and cytosolic PLA2s and agonist-induced arachidonic acid release from human cells. Given its ability to block the arachidonic acid cascade, PX-52 may be useful in the control of inflammation.

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.

Human neutrophils store type II 14-kDa phospholipase A2 in granules and secrete active enzyme in response to soluble stimuli.

Although "secretory" type II 14-kDa phospholipase A2 (sPLA2) activity has been described in neutrophils, direct evidence of enzyme secretion has been elusive. We have used immunogold electron microscopy with polyclonal and monoclonal antibodies to sPLA2 to demonstrate localization of the enzyme to granules of resting human neutrophils and translocation to phagolysosomes. Soluble stimuli such as calcium ionophore A23187 stimulate loss of cell-associated enzymatic activity. Supernatant fluids from stimulated neutrophils lack measurable PLA2 but contain proteases which inactivate exogenous sPLA2. The use of alpha-1-antitrypsin as a protease inhibitor permitted this first demonstration of secretion of PLA2 activity from stimulated human neutrophils.

Cellular regulation of arachidonate mobilization and metabolism.

Synthesis of eicosanoids is initiated by signal transduction cascades which result in the hydrolysis of free arachidonic acid from membrane phospholipids. Both a cytosolic 85 kDa and a nonpancreatic 14 kDa PLA2 may contribute to cellular arachidonate mobilization. In many cells, agonist-stimulated fatty acid release is dependent upon increases in intracellular free calcium and is enhanced by pretreatment with agents such as phorbol esters and soluble diglycerides. The response is specific for arachidonate and structurally similar polyunsaturated fatty acids containing a cis 5, 6 double bond. DMSO-differentiation of U937 cells markedly enhances the A23187-stimulated release of [3H]arachidonate, which appears to be coupled to differentiation-induced enhancement of capacitance calcium entry. Although both phorbol esters and soluble diglycerides enhance subsequent fMLP or A23187-stimulated arachidonate release in human neutrophils, several lines of evidence indicate that the effects of oleoylacetylglycerol and 1,2-dioctanoylglycerol are protein kinase C-independent. Soluble diglycerides, but not phorbol esters, enhance the coupling of arachidonate mobilization to subsequent leukotriene B4 synthesis. Further studies will be required to elucidate the mechanisms which regulate activation of cellular phospholipases and subsequent synthesis.

Separation of agonist-stimulated arachidonate mobilization from subsequent leukotriene B4 synthesis in human neutrophils: different effects of oleoylacetylglycerol and phorbol myristate acetate as priming agents.

Preincubation of human neutrophils with phorbol esters or soluble diglycerides enhances subsequent f-Met-Leu-Phe (fMLP)-stimulated arachidonate mobilization and leukotriene B4 (LTB4) synthesis. We have recently reported that 1,3-dioctanoylglycerol (1,3-diC8) is equipotent with 1,2-sn-dioctanoylglycerol (1,2-diC8) as priming agent, thus suggesting that the priming effects of diacylglycerols are protein kinase C (PKC) independent (Rosenthal et al., 1993, Biochim. Biophys. Acta 1177:79-86). In order to further investigate this question, the present study has directly compared the effects of oleoylacetylglycerol (OAG) and the PKC activator, phorbol 12-myristate 13-acetate (PMA), on agonist-stimulated lipid metabolism. The results indicate that both OAG and PMA dose dependently enhance f-Met-Leu-Phe (fMLP)-stimulated release of [3H]arachidonate. Optimal concentrations of OAG (5 microns) and PMA (10 nM) are equipotent in increasing fMLP-stimulated arachidonate mobilization as quantitated either with total radioactivity or by mass measurements of free arachidonate. By contrast OAG is sixfold more effective than PMA in enhancing synthesis of 5-lipoxygenase (5-LO) metabolites by mass and two to threefold more effective than PMA in enhancing synthesis of [3H]eicosanoids. Furthermore, OAG, but not PMA, enhances fMLP-stimulated synthesis of platelet-activating factor. By contrast, PMA directly stimulates [3H]arachidonate mobilization, while OAG (20 microM) does not; despite these differences, the combined effects of PMA + OAG on subsequent agonist-stimulated arachidonate release are not greater than those of PMA alone. In cells challenged with subthreshold concentrations (< 0.1 microM) of the calcium ionophore A23187, both OAG and PMA stimulate [3H]arachidonate release but not [3H]LTB4 synthesis. These findings suggest that OAG does not directly activate 5-LO, but instead couples arachidonate mobilization to leukotriene synthesis in a PKC-independent manner.

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.

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.

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.

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.

Mechanism(s) of cytoprotective and anti-inflammatory activity of PGB1 oligomers: PGBx has potent anti-phospholipase A2 and anti-oxidant activity.

We postulate that the anti-PLA2 and anti-oxidant activities may account for the broad spectrum protective effects of PGBx that were previously described. These dual properties are demonstrable in vitro, in situ and in vivo, and would have profound effects on stabilization of membrane structure and function, which in turn, would protect organelles, cells, tissues, and organs from inflammation and injury, and possibly alter patterns of aging involving senescence and cell death.

Retroconversion and delta 4 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.

C20 polyunsaturated fatty acids and phorbol myristate acetate enhance agonist-stimulated synthesis of 1-radyl-2-acetyl-sn-glycero-3-phosphocholine in vascular endothelial cells.

This study has investigated the effect of supplementation of vascular endothelial cells with arachidonate and other polyunsaturated fatty acids on the agonist-stimulated synthesis of platelet activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; 1-alkyl-2-acetyl-GPC). Incubation of calf pulmonary artery endothelial cells for 48 h in medium containing 40 microM arachidonate resulted in a 2-3-fold enhancement of [3H]acetate incorporation into 1-radyl-2[3H]acetyl-GPC in response to either bradykinin or calcium ionophore A23187. The effects of arachidonate supplementation were both dose- and time-dependent, requiring a minimum exogenous arachidonate concentration of 2.5 microM and an incubation time of 4-6 h. Eicosapentaenoate and docosahexaenoate also enhanced the synthesis of 1-radyl-2-[3H]acetyl-GPC, but were less potent than arachidonate; alpha-linolenate, linoleate and oleate were without effect. Although not effective as an agonist, phorbol myristate acetate potentiated A23187- and bradykinin-stimulated synthesis of 1-radyl-2-[3H]acetyl-GPC. The effects of arachidonate supplementation were synergistic with potentiation by phorbol myristate acetate. Sphingosine inhibited agonist-stimulated incorporation of [3H]acetate into 1-radyl-2-[3H]acetyl-GPC both in the presence and absence of PMA. Characterization of the radiolabeled material indicated that the primary product was the acyl analogue of PAF (1-acyl-2-acetyl-GPC) rather than PAF. The results from this study suggest that agonist-stimulated synthesis of 1-radyl-2-acetyl-GPC in vascular endothelial cells is modulated both by cellular fatty acyl composition and activation of protein kinase C. Enrichment of vascular endothelial cells with fatty acids, which are mobilized by agonist-stimulated phospholipase A2, may enhance subsequent deacylation of choline phospholipids and, thus, increase synthesis of both 1-acyl-2-acetyl-GPC and PAF.

Chain elongation of polyunsaturated fatty acids by vascular endothelial cells: studies with arachidonate analogues.

This study has utilized radiolabeled analogues of arachidonic acid to study the substrate specificity of elongation of long-chain polyunsaturated fatty acids. Human umbilical vein endothelial cells were incubated for 2-72 hr in medium supplemented with 0.9-2.6 microM [14C]fatty acid, and cellular glycerolipids were analyzed by gas-liquid chromatography with radioactivity detection. Elongation of naturally occurring C20 polyunsaturated fatty acids occurred with eicosapentaenoate (20:5(n-3] greater than Mead acid (20:3(n-9] greater than arachidonate (20:4(n-6]. Chain length markedly influenced the extent of elongation of 5,8,11,14-tetraenoates (18:4 greater than 19:4 greater than 20:4 greater than 21:4); effects of initial double bond position were also observed (6,9,12,15-20:4 greater than 4,7,10,13-20:4. Neither 5,8,14- nor 5,11,14-20:3 was elongated to the extent of 5,8,11-20:3. Differences between polyunsaturated fatty acids were observed both in the initial rates and in the maximal percentages of elongation, suggesting that the content of cellular C20 and C22 fatty acids may represent a balance between chain elongation and retroconversion. Umbilical vein endothelial cells do not exhibit significant desaturation of either 22:4(n-6) or 22:5(n-3). By contrast, incubation with 5,8,11,14-[14C]18:4(n-4) resulted in formation of both [14C]20:5(n-4) and [14C]22:5(n-4). The respective time courses for the appearances of [14C]22:5(n-4) and [14C]20:5(n-5) suggests delta 6 desaturation of [14C]22:4(n-4) rather than delta 4 desaturation of [14C]20:4(n-4).

Inhibition of human phospholipases A2 by cis-unsaturated fatty acids and oligomers of prostaglandin B1.

Inhibition of human phospholipases A2 by cis-unsaturated fatty acids and their oxidative metabolites and/or polymers was studied using partially purified human phospholipases A2 and [1-14C]oleate labelled, autoclaved E. coli as substrate. As previously reported for other phospholipases A2, oleic and arachidonic acids inhibited human synovial fluid phospholipase A2 with IC50s of 15 and 30 microM respectively. Air oxidation of arachidonic acid or hydroxylation of oleic acid (12-hydroxy-oleate) substantially relieved that inhibition. Similarly, the enzymatically oxidatized metabolite of arachidonate, prostaglandin B1 (PGB1), did not inhibit enzymatic activity. However, prostaglandin Bx (PGBx), an oligomer (n = 6) of PGB1, was a potent inhibitor of Ca(++)-dependent, neutral-active phospholipase A2 activities. Enzymatic activity in acid extracts from human neutrophils, platelets, sperm, plasma, synovial fluid, endometrium, degenerative disc, and snake venom was inhibited by PGBx with IC50s ranging from 0.5-7.0 microM. Inhibition was independent of substrate phospholipid concentration over a 24-fold range (5-120 microM) and PGBx quenched the tryptophan fluorescence of snake venom phospholipase A2 in a dose-dependent manner. Agonist-induced (A23187) release of arachidonic acid from prelabelled human neutrophils and cultured human endothelial cells was also inhibited by PGBx with IC50s of 3 and 20 microM, respectively. These results illustrate that oxidative reactions of cis-unsaturated fatty acids relieve their natural inhibitory activity, and polymerization of an inactive fatty acid metabolite yields a potent inhibitor of in vitro and in situ phospholipase A2 activity.

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.

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.

Substrate specificity of the agonist-stimulated release of polyunsaturated fatty acids from vascular endothelial cells.

Stimulation of vascular endothelial cells with agonists such as histamine and thrombin results in release of arachidonic acid from membrane lipids and subsequent eicosanoid synthesis. As shown previously, the agonist-stimulated deacylation is specific for arachidonate, eicosapentaenoate, and 5,8,11-eicosatrienoate. This study has utilized radiolabeled fatty acids differing in chain length and position of double bonds to further elucidate the fatty acyl specificity of agonist-stimulated deacylation. Replicate wells of confluent human umbilical vein endothelial cells were incubated with 14C-labeled fatty acids and then challenged with histamine, thrombin, or the calcium ionophore A23187. Comparison of the results obtained with isomeric eicosatetraenoic fatty acids with initial double bonds at carbons 4, 5, or 6 indicated that the deacylation induced by all three agonists exhibited marked specificity for the cis-5 double bond. Lack of stringent chain length specificity was indicated by agonist-stimulated release of 5,8,11,14- tetraenoic fatty acids with 18, 19, 20, and 21 carbons. Release of 5,8,14-[14C]eicosatrienoate was two-to threefold that of 5,11,14-[14C]eicosatrienoate, thus indicating that the cis-8 double bond may also contribute to the stringent recognition by the agonist-sensitive phospholipase. The present study has also demonstrated that histamine, thrombin, and A23187 do not stimulate release of docosahexaenoate from endothelial cells.