Characterization of the analgesic and anti-inflammatory activities of ketorolac and its enantiomers in the rat.

The marked analgesic efficacy of ketorolac in humans, relative to other nonsteroidal anti-inflammatory drugs (NSAIDs), has lead to speculation as to whether additional non-NSAID mechanism(s) contribute to its analgesic actions. To evaluate this possibility, we characterized (R,S)-ketorolac's pharmacological properties in vivo and in vitro using the nonselective cyclooxygenase (COX) inhibitors [indomethacin (INDO) and diclofenac sodium (DS)] as well as the selective COX-2 inhibitor, celecoxib, as references. The potency of racemic (R,S)-ketorolac was similar in tests of acetic acid-induced writhing, carrageenan-induced paw hyperalgesia, and carrageenan-induced edema formation in rats; ID50 values = 0.24, 0. 29, and 0.08 mg/kg, respectively. (R,S)-ketorolac's actions were stereospecific, with (S)-ketorolac possessing the biological activity of the racemate in the above tests. The analgesic potencies for (R,S)-, (S)-, and (R)-ketorolac, INDO, and DS were highly correlated with their anti-inflammatory potencies, suggesting a common mechanism. (R,S)-ketorolac was significantly more potent than INDO or DS in vivo. Neither difference in relative potency of COX inhibition for (R,S)-ketorolac over INDO and DS nor activity of (S)-ketorolac at a number of other enzymes, channels, or receptors could account for the differences in observed potency. The distribution coefficient for (R,S)-ketorolac was approximately 30-fold less than for DS or INDO, indicating that (R,S)-ketorolac is much less lipophilic than these NSAIDs. Therefore, the physicochemical and pharmacokinetics properties of (R,S)-ketorolac may optimize the concentrations of (S)-ketorolac at its biological target(s), resulting in greater efficacy and potency in vivo.

Synthesis of prostaglandin E2 ethanolamide from anandamide by cyclooxygenase-2.

Because of its structural similarity to polyunsaturated fatty acids, anandamide could serve as substrate for enzymes such as lipoxygenases and cyclooxygenases, which metabolize polyunsaturated fatty acids to potent bioactive metabolites. Here the ability of recombinant human cyclooxygenase-1 (hCOX-1) and cyclooxygenase-2 (hCOX-2) to metabolize anandamide was studied. Baculovirus-expressed and -purified hCOX-2, but not hCOX-1, effectively oxygenated anandamide. Reverse phase high pressure liquid chromatography analysis of the products derived from 1-14C-labeled anandamide showed that the products formed are similar to those formed with arachidonic acid as substrate. The major prostanoid product derived from anandamide was determined by mass spectrometry to be prostaglandin E2 ethanolamide. Incubation of anandamide with lysates and the intact cell line expressing COX-2 but not that of COX-1 produced prostaglandin E2 ethanolamide. These results demonstrate the existence of a COX-2-mediated pathway for anandamide metabolism, and the metabolites formed represent a novel class of prostaglandins.

Differential allosteric regulation of prostaglandin H synthase 1 and 2 by arachidonic acid.

Prostaglandins are synthesized by prostaglandin H synthase (PGHS) 1 and 2. PGHS2 is regulated through inducible expression. We report here the regulation of PGHS1 activity by substrate-dependent cooperative activation. The cooperativity is characterized by a Hill coefficient of 1.29 +/- 0.06, a curved Eadie-Scatchard plot, and activation by low concentrations of competitive inhibitors. The activation also appears to induce a conformational change in the cyclooxygenase site. The cooperativity produces a 2-4-fold greater rate of PGHS2-dependent prostaglandin formation compared with PGHS1-dependent prostaglandin formation at arachidonic acid concentrations below 0.5 microM. A consequence of the PGHS1 cooperativity is that the affinity of many cyclooxygenase inhibitors for PGHS1 decreases in parallel to the activation by arachidonic acid. In contrast, the affinity of these inhibitors for PGHS2 is unaffected by the changes in arachidonic acid concentration. This results in a dramatic difference in PGHS2/PGHS1 selectivity at different arachidonic acid concentrations.

Cytoplasmic phospholipase A2 activity and gene expression are stimulated by tumor necrosis factor: dexamethasone blocks the induced synthesis.

The interaction of tumor necrosis factor alpha (TNF) with its two membrane-bound receptors initiates intracellular events in which arachidonic acid and its derivatives are involved. In HeLa cells, TNF treatment induces an arachidonic acid-selective, Ca(2+)-dependent cellular phospholipase A2 (cPLA2). By itself, TNF causes a modest increase in cPLA2 activity, but with the Ca2+ ionophore A23187 it provides a strong synergistic action. Within minutes in response to TNF, cPLA2 becomes phosphorylated and in the presence of Ca2+ produces a 3- to 4-fold increase in activity. TNF also increases cPLA2 mRNA and protein expression, an estimated 5-fold increase in an 8-hr period. This increase in cPLA2 activity occurs, therefore, in a biphasic time-dependent manner. Dexamethasone, known to antagonize the action of TNF, is here shown to inhibit TNF-induced gene expression and to prevent the second phase of increase in cPLA2 activation. Our results suggest that the cPLA2 activation may provide a regulatory function and may explain the proinflammatory action of TNF.

Detection of arachidonoyl-selective phospholipase A2 in human neutrophil cytosol.

The present report describes the presence of an arachidonic-acid-selective, dithiothreitol-insensitive phospholipase A2 enzyme activity in human neutrophil cytosol. The enzyme activity is eluted from Mono Q FPLC column between 350 to 450 mM salt and translocates from cytosol to membrane in a calcium dependent fashion. Furthermore, the PLA2 activity in the cytosol is quantitatively precipitated by the antibodies against U937 cPLA2. The neutrophil enzyme also migrates as approximately 110 kDa protein on SDS polyacrylamide gels. These studies indicate that the PLA2 enzyme present in human neutrophil cytosol is identical to the previously reported U937 cPLA2 (Clark et al. (1990) Proc. Natl. Acad. Sci. USA 87, 7708 and Clark et al. (1991) Cell 65, 1043).

Resistance to killing by tumor necrosis factor in an adipocyte cell line caused by a defect in arachidonic acid biosynthesis.

We have found that TA1-R6, which are resistant to the cytotoxic effects of tumor necrosis factor (TNF) in the presence of cycloheximide (Reid, T. R., Torti, F., and Ringold, G. M. (1989) J. Biol. Chem. 264, 4583-4589), have reduced ability to release arachidonic acid (20:4) from membrane phospholipids in response to either TNF or the calcium ionophore A23187 treatment. However, no defect in the activity of phospholipase A2, the principal enzyme responsible for the release of 20:4 from phospholipids, was observed in these cells. Detailed biochemical characterization of these TNF-resistant cells has revealed that these cells are unable to synthesize 20:4 endogenously because of a defect in delta 6-desaturase, the rate-limiting enzyme of 20:4 biosynthesis. This deficiency leads to a marked decrease in the steady-state levels of 20:4 present in choline-containing phospholipid (PC) and ethanolamine-containing phospholipid (PE). The TA1-R6 cells, however, are capable of incorporating exogenous 20:4 into PC and PE, and when loaded in such manner they become significantly more sensitive to the cytotoxic effects of TNF in the presence of cycloheximide. Therefore, the release of arachidonic acid from phospholipids appears to be a critical element in the signaling pathway utilized by TNF and is essential to the rapid cytotoxic response elicited by TNF in the absence of protein synthesis in wild-type TA1 cells.

A novel arachidonic acid-selective cytosolic PLA2 contains a Ca(2+)-dependent translocation domain with homology to PKC and GAP.

We report the cloning and expression of a cDNA encoding a high molecular weight (85.2 kd) cytosolic phospholipase A2 (cPLA2) that has no detectable sequence homology with the secreted forms of PLA2. We show that cPLA2 selectively cleaves arachidonic acid from natural membrane vesicles and demonstrate that cPLA2 translocates to membrane vesicles in response to physiologically relevant changes in free calcium. Moreover, we demonstrate that an amino-terminal 140 amino acid fragment of cPLA2 translocates to natural membrane vesicles in a Ca(2+)-dependent fashion. Interestingly, we note that this 140 amino acid domain of cPLA2 contains a 45 amino acid region with homology to PKC, p65, GAP, and PLC. We suggest that this homology delineates a Ca(2+)-dependent phospholipid-binding motif, providing a mechanism for the second messenger Ca2+ to translocate and activate cytosolic proteins.

Measurement of arachidonic acid release from human polymorphonuclear neutrophils and platelets: comparison between gas chromatographic and radiometric assays.

a simple gas chromatographic method for the assay of phospholipase A2 (PLA2) has been described in which arachidonic acid released from endogenous phospholipid pools is measured following its extraction and derivatization to pentafluorobenzyl esters. Using this assay, PLA2 activities in control and calcium ionophore-stimulated human neutrophils, as well as in control, thrombin, and calcium ionophore stimulated human platelets, have been measured. These values are compared with those obtained by monitoring the release of radioactivity from [3H]- or [14C]arachidonic acid prelabeled cells. While the radiometric assay measures only the release of exogenously incorporated radioactive arachidonic acid, the gas chromatographic assay measures arachidonic acid released from all the endogenous pools. Thus, the apparent increase in PLA2 activity in stimulated cells measured by the gas chromatographic assay is four- to fivefold higher than that by the radiometric assay. Inclusion of fatty acid free bovine serum albumin in the reaction buffer significantly increases the amount of arachidonic acid that is measured by gas chromatography. The gas chromatographic method has also been successfully utilized for measuring PLA2 activity in cell-free preparations derived from physically disrupted human neutrophils.

TNF induces c-fos via a novel pathway requiring conversion of arachidonic acid to a lipoxygenase metabolite.

Tumour necrosis factor (TNF), a lymphokine released by activated macrophages, has diverse effects on a wide variety of cell types. TNF exerts these effects via specific cell surface receptors; however little is known of the biochemical events that ensue. We have shown that TNF rapidly induces the proto-oncogenes c-fos and c-jun in the adipogenic TA1 cell line and have used these responses to characterize the intracellular mediators of TNF action. We find that arachidonic acid, which is released in response to TNF, induces c-fos, but not c-jun mRNA in quiescent TA1 cells. Pretreatment of the cells with lipoxygenase inhibitors abolishes the induction of c-fos by TNF, while the induction of c-jun is unaffected; in contrast, a cyclooxygenase inhibitor has no effect on either response. Finally, we have demonstrated that TNF stimulates production of lipoxygenase metabolites in TA1 cells and that one of these, 5-HPETE, induces c-fos, but not c-jun. These data suggest that TNF activates two second messenger pathways, one of which is dependent on release of arachidonic acid and its subsequent conversion to a lipoxygenase metabolite.

Sensitive method for the analysis of phospholipid subclasses and molecular species as 1-anthroyl derivatives of their diglycerides.

A sensitive high-performance liquid chromatographic (HPLC) method for the separation and quantitation of phospholipid subclasses and molecular species has been developed. Phospholipids for analysis are hydrolyzed to the diradyl glycerols (DGs) with phospholipase C and the resulting DGs reacted with a molar excess of 1-anthroyl nitrile in the presence of quinuclidine or 4-dimethylaminopyridine to form a stable adduct. The anthroyl-DGs were separated into alkenylacyl, alkylacyl, and diacyl subclasses either by using normal-phase HPLC or by thin-layer chromatography on silica gel G plates. Molecular species within alkenylacyl, alkylacyl, and diacyl subclasses were separated using reversed-phase HPLC. Separation of the individual subclasses was achieved for ethanolamine phosphoglycerides from bovine brain, as well as choline and ethanolamine phosphoglycerides from human neutrophils. Separation and quantitation of individual molecular species were carried out for alkenylacyl, alkylacyl, and diacyl subclasses of bovine brain ethanolamine phosphoglycerides by their absorbance at 254 nm with correction for recoveries as normalized to the internal standard 1,2-dipentadecanoyl-3-phosphatidylcholine added before the hydrolysis of phospholipids with phospholipase C or 1,2-dipentadecanoyl-3-anthroyl glycerol added after complete derivatization. The extinction coefficient of the 1-anthroyl derivatives were greater than 68,000 permitting the generation of concentration-dependent determinations which were linear to less than 1 pmol when monitored at 254 nm. Thus, this procedure provides a new and very sensitive method for the quantitation of picomole quantities of phospholipids or DGs by HPLC techniques.

Human neutrophil platelet-activating factor: molecular heterogeneity in unstimulated and ionophore-stimulated cells.

The molecular heterogeneity of platelet-activating factor (PAF) in resting and ionophore (A23187) -stimulated human neutrophils was measured by a very sensitive gas chromatography-negative ion chemical ionization mass spectrometric method. The molecular species compositions of PAF, which are due to variations in the 1-O-alkyl chain length, were significantly different between resting and ionophore-stimulated polymorphonuclear leukocytes. The major species of PAF produced by unstimulated polymorphonuclear leukocytes were 16:0, 17:0, 18:1 and 18:0, representing 55, 14, 8 and 10%, respectively, of the total PAF; 16:0 was the predominant PAF (74%) in A23187-stimulated polymorphonuclear leukocytes. The PAF molecular species from unstimulated polymorphonuclear leukocytes was similar to compositions from those of the precursor 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine, whereas those from the ionophore-stimulated polymorphonuclear leukocytes differed from the precursor 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine, thus indicating a very high degree of substrate selectivity for PAF synthesis. Although the physiological implications of the variations in PAF composition are not known, these studies indicate that the PAF produced by resting polymorphonuclear leukocytes are significantly different from those produced in response to ionophore.

Identification and quantitation of PAF from psoriatic scales.

Platelet activating factor was isolated from scales of psoriatic patients by the procedure of Bligh and Dyer and purified by silica gel thin layer chromatography. The purified PAF was digested with phospholipase C and the resulting diglyceride was derivatized into PFB ethers. The PAF-PFB ethers were analyzed using fused silica capillary chromatography-negative ion chemical ionization mass spectrometry. Different molecular species of PAF were identified by their negative ion mass spectra and by their elution time from the capillary column. All the molecular species had high abdundance (greater than 90%) of the molecular anion. 1-0-Hexadecyl-2-acetyl-GPC (16:0) was the major PAF species representing 51% of the total PAF. 17:0 and 18:1 were the next abundant species representing 15 and 16%, respectively. Several minor PAF molecular species were also present. The amount of each PAF molecular species was quantitated from 1-0-hexadecyl-2-2H3 acetyl-GPC used as the internal standard. Nanogram quantities of PAF were recovered from 100 mg of psoriatic scales. Significant amounts of lysoPAF were also present in these scales. The alkyl chain of the lysoPAF was compared with that of PAF.

Ether phospholipids in control and 20:4-depleted rat PMN: additional evidence for a 1-O-alkyl-2-20:4-sn-glycerol-3-phosphocholine specific phospholipase A2.

Earlier studies from out laboratory have shown that PAF and LTB4 biosynthesis are inhibited in rat PMN depleted of its 20:4 (JBC. '86, 261, 7592). To test whether these cells contain sufficient 1-O-alkyl-2-acyl-GPC to support PAF synthesis, phosphotidyl choline was isolated from these 20:4-depleted cells, fractionated into different subclasses and their fatty acid composition determined. These results were compared with those obtained with control PMN. Both control and 20:4-depleted PMN contained significantly large amounts of alkylacyl-GPC and diacyl-GPC. Small amounts (4%) of alkenyl-acyl-GPC were also present. The amount of 20:3 in 20:4-depleted cells was more or less equal to the amount of 20:4 in control cells. About 62% of PC-bond 20:4 in control PMN and about 56% of PC-bound 20:3 in the 20:4-depleted PMN was found associated with the alkylacyl species. These results show that both control and 20:4 depleted PMN have ample precursor substrates to support PAF biosynthesis and these substrates are enriched with 20:4 in control cells and with 20:3 in 20:4-depleted cells. These findings are consistant with the existance of a highly specific phospholipase A2 capable of distinguising 20:4 from 20:3 containing phospholipids.

Species-specific variations in the molecular heterogeneity of the platelet-activating factor.

The molecular heterogeneity of platelet-activating factor (PAF) synthesized by unstimulated and Ca2+ ionophore (A23187)-stimulated PMN from rat, mouse, and guinea pig and by rat basophilic leukemia (RBL) cells was investigated by gas chromatography-negative ion chemical ionization mass spectrometry. Several molecular species of PAF ranging from C14:0 to C19:0 were detected in all of the cells studied. PAF produced by each cell type exhibited a unique pattern of molecular species distribution. Although C16:0 was the major PAF molecular species of rat PMN and RBL cells representing 96% and 85% of the total PAF, respectively, PAF from mice PMN contained 81% of C16:0, 10% of C18:1, and 6% of C18:0. Alternatively, A23187-stimulated guinea pig PMN yielded PAF molecular species 35% in C16:0, 35% in C17:0, 8% in C18:1, and 3% in C18:0. Small but significant differences in the PAF molecular species distribution of resting and ionophore stimulated cells were also observed. In contrast to the PAF molecular species composition, the precursor 1-O-alkyl-2-acyl-glycero-3-phosphocholine of all the cell types was predominantly hexadecyl (C16:0) alkyl chain in the sn-1 position, representing 60 to 80% of the total 1-O-alkyl-2-acyl-glycero-3-phosphocholine. Thus, these results not only indicate a high degree of selectivity for utilization of precursor substrates for PAF biosynthesis, but also demonstrate that the selectivity is species specific.

Fatty acid composition of diacyl, alkylacyl, and alkenylacyl phospholipids of control and arachidonate-depleted rat polymorphonuclear leukocytes.

Phospholipid fatty acid composition and phospholipid subclass distribution of control and arachidonate-depleted rat polymorphonuclear leukocytes (PMN) were compared. The 20:4-depleted PMN contained significantly higher amounts of 16:1, 18:1 and 20:3 (delta 5,8,11) and lower amounts of 18:2 and 20:4 than the phospholipids from control cells. Choline-containing glycerophospholipids (CGP) were the major phospholipids of both control and 20:4-depleted cells representing 34% and 37% of the total phospholipids, respectively. Significant amounts of ethanolamine-containing glycerophospholipids (EGP) (29% and 30%) and sphingolipids (20% and 18%) were also present in both cell types. Serine-containing glycerophospholipids (SGP) together with inositol-containing glycerophospholipids (IGP) constituted 16% and 13% of the phospholipids in control and 20:4-depleted cells, respectively. CGP from control cells had significantly higher amounts of 16:0 and 18:2 and lower amounts of 18:0 and 20:4 than EGP, whereas CGP from 20:4-depleted cells has higher amounts of 16:0 and 16:1 and lower amounts of 20:3 than EGP. Analysis of the subclass composition of CGP and EGP revealed that both control and 20:4-depleted cells contained significantly large amounts of alkylacyl-GPC and alkenylacyl-GPE. Small amounts of alkylacyl-GPE and alkenylacyl-GPC were also observed. The predominant fatty acyl residues found in the 1,2-diacyl-GPC, alkylacyl-GPC of control cells were 16:0, 18:0, 18:1, 18:2, and 20:4, while those of 20:4-depleted cells were 16:0, 16:1, 18:0, 18:1, and 20:3. More than 60% of CGP-bound 20:4 of control cells and about 70% of the CGP-bound 20:3 of 20:4-depleted cells were found in their alkylacyl species.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of platelet-activating factor by arachidonic acid-depleted rat polymorphonuclear leukocytes.

1-O-[3H]Alkyl-2-acetyl-sn-glycero-3-phosphocholine ([3H]PAF) and 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphocholine ([3H]lyso-PAF) when incubated with rat polymorphonuclear leukocytes (PMN) were rapidly metabolized to 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine ([3H]alkyl-acyl-GPC) containing long chain acyl groups in the sn-2 position. The specificity and the absolute requirements of arachidonate (20:4) for acylation into PAF and lyso-PAF were investigated by comparing the rate of [3H]PAF and [3H]lyso-PAF metabolism by control rat PMN with that by rat PMN depleted of 20:4. Comparable rates of metabolism of [3H]PAF and [3H]lyso-PAF by both control and 20:4-depleted PMN were observed at all the concentrations of PAF and lyso-PAF studied. The nature of the fatty acyl group incorporated into the sn-2 position of the [3H]alkyl-acyl-GPC formed was analyzed by argentation chromatography. Dienoic fatty acids were the major fatty acid incorporated into the alkyl-acyl-GPC by both control and 20:4-depleted PMN at all the incubation times studied. At 3 min of incubation with [3H]PAF and [3H]lyso-PAF, control PMN had small but significant amounts of [3H]alkyl-acyl-GPC containing tetraenoic fatty acids, the concentration of which gradually increased as the incubation time progressed. On the other hand, under similar conditions, 20:4-depleted PMN had only trace amounts of the [3H]alkyl-acyl-GPC with tetraenoic fatty acid and the concentration of which remained at the low level throughout the incubation time. At 3 min of incubation, the 20:4-depleted PMN had small but significant amounts of [3H]alkyl-acyl-GPC with saturated fatty acids, the amount of which declined by 10 min and remained at that level as the incubation time progressed. While the concentration of [3H]alkyl-acyl-GPC with dienoic fatty acids in the 20:4-depleted cells gradually increased with the progress of incubation time, these molecular species of GPC in the control PMN remained more or less constant. In spite of a very high concentration (equivalent to that of 20:4 in control PMN) of eicosatrienoic acid (20:3 delta 5,8,11) in the 20:4-depleted PMN, no significant amounts of [3H]alkyl-acyl-GPC with trienoic fatty acid were formed by these cells. The rate of metabolism of [3H]PAF and [3H]lyso-PAF by the resident macrophages isolated from control and 20:4-depleted rats was similar.(ABSTRACT TRUNCATED AT 400 WORDS)

Platelet-activating factor and leukotriene biosynthesis is inhibited in polymorphonuclear leukocytes depleted of arachidonic acid.

Rat peripheral or elicited polymorphonuclear leukocytes 90% deficient in arachidonic acid incorporate, after stimulation with the calcium ionophore A23187, 86% less acetate into platelet-activating factor than control. The total amount of platelet-activating factor in the ionophore stimulated elicited polymorphonuclear leukocytes deficient in arachidonate, measured by gas chromatography-negative ion chemical ionization mass spectrometry, was 84% less than that of control. The mass spectrometry also revealed the presence of various molecular species of platelet-activating factor ranging from 1-O-tetradecyl to 1-O-nonadecyl forms in both the deficient and control cells. However, the 1-O-hexadecyl was the predominate molecular species representing 79 and 96% of the total platelet-activating factor in the respective deficient and control cells. Other molecular species were less than 1.5 and 8.5% of the total for control and deficient polymorphonuclear leukocytes, respectively. Leukotriene B4 formation was also inhibited by 90% in the deficient cells. Both platelet-activating factor and leukotriene B4 biosynthesis could be partially restored in arachidonic acid-deficient cells by prelabeling the cells with arachidonate. This represents the first dietary link with platelet-activating factor biosynthesis.

Measurement of sub-picogram quantities of platelet activating factor (AGEPC) by gas chromatography/negative ion chemical ionization mass spectrometry.

A very sensitive method of 1-O-alkyl-2-acetyl-sn-glycerophosphocholine (platelet activating factor) quantitation is described. 1-O-Alkyl-2-acetyl-sn-glycerophosphocholine is converted to 1-O-alkyl-2-acetylglycerol by treating with phospholipase-C which is in turn acylated with pentaflurobenzoyl chloride. The resulting diglyceryl ester of pentafluorobenzene is analysed by gas chromatography/negative ion chemical ionization mass spectrometry. With methane as the reagent gas the pentafluorobenzoyl acyl derivative yields mainly the molecular anion ([M]-.) representing more than 92% of the total ion current. Deuterium-labeled 1-O-hexadecyl-2-(2H3)acetyl-sn-glycerophosphocholine is used as the internal standard for quantitation. The standard curves are linear and typically have a correlation coefficient of greater than 0.999. Using 1 pg of the deuterium-labeled internal standard, quantities as low as 100 fg are measured.