Characterization of the two major species of slow reacting substance from rat basophilic leukemia cells as glutathionyl thioethers of eicosatetraenoic acids oxygenated at the 5 position. Evidence that peroxy groups are present and important for spasmogenic activity.

The most prominent slow reacting substance from rat basophilic leukemia cells (type I) was characterized by radiochemical, chemical and physical methods and shown to contain a C20 unsaturated fatty acid oxygenated at the 5 position and a sulfur containing side chain in thioether linkage at the 6 position. Its spasmogenic action on guinea pig ileal muscle was largely inactivated under reducing conditions which suggested that a peroxy group was present and important for contractile activity. This was supported by ferrous thiocyanate analysis. The peroxy group is almost certainly at the 5 position, probably in the form of a peroxy ester or hydroperoxide. Based on amino acid hydrolysis (0.85 moles of glycine and 0.30 moles of glutamic acid per mole SRS), the sulfur containing side chain is apparently a mixture of glutathione and cysteinyl-glycine, but by chromatography the side chain is predominantly glutathione and the low yield of glutamic acid may be due to complexing of its alpha COOH group in a peroxy ester linkage. The fatty acid moiety has 3 conjugated double bonds, probably at the 7,8, 9,10 and 11,12 positions. Type II SRS, the second major species, differs in that the sulfur containing side chain is linked at the 12 or 13 position and is almost certainly glutathione and in the failure of alkaline borohydride to produce inactivation. These observations strongly implicate the lipoxygenase pathway in slow reacting substance biosynthesis.     

Arylsulfatase inactivation of slow reacting substance. Evidence for proteolysis as a major mechanism when ordinary commercial preparations of the enzyme are used

Type II B arylsulfatases are known to inactivate slow reacting substance (SRS), but the mechanism is unclear. In the present study, ordinary commercial preparations of Sigma limpet arylsulfatase largely inactivated the glutathionyl and cysteinyl-glycyl forms of SRS, but the cysteinyl form of SRS was largely resistant to the enzyme. Evidence is presented which established that a major mechanism for the inactivation of the glutathionyl and cysteinyl-glycyl SRS types, at least by the particular enzyme preparations we have studied, involves cleavage of the glycine moiety from the sulfur containing side chain. This was confirmed by digestion studies with glutathione itself. In addition, there is ome evidence to indicate that the enzyme may destabilize the double bond structure of the SRS molecule, contributing to the overall inactivation.     

Aryl sulfatase inactivation of slow reacting substance. Evidence for proteolysis as a major mechanism when ordinary commercial preparations of the enzyme are used

Type II B arylsulfatases are known to inactivate slow reacting substance (SRS), but the mechanism is unclear. In the present study, ordinary commercial preparations of Sigma limpet arylsulfatase largely inactivated the glutathionyl and cysteinyl-glycyl forms of SRS, but the cysteinyl form of SRS was largely resistant to the enzyme. Evidence is presented which established that a major mechanism for the inactivation of the glutathionyl and cysteinyl-glycyl SRS types, at least by the particular enzyme preparations we have studied, involves cleavage of the glycine moiety from the sulfur containing side chain. This was confirmed by digestion studies with glutathione itself. In addition, there is some evidence to indicate that the enzyme may destabilize the double bond structure of the SRS molecule, contributing to the overall inactivation.     

Sequential conversion of the glutathionyl side chain of slow reacting substance (SRS) to cysteinyl-glycine and cysteine in rat basophilic leukemia cells stimulated with A-23187

In rat basophilic leukemia (RBL-1) cells stimulated with A-23187, the major slow reacting substance (SRS) species contain glutathione, cysteinyl-glycine, or cysteine in their side chains, corresponding or closely related to leukotrienes LTC4, LTD4, and LTE4, respectively. Evidence is presented that most of the SRS produced during the first few minutes of stimulation by the ionophore has a glutathionyl side chain which is sequentially converted to cysteinyl-glycine and cysteine.     

Sequential conversion of the glutathionyl side chain of slow reacting substance (SRS) to cysteinyl-glycine and cysteine in rat basophilic leukemia cells stimulated with A-23187

In rat basophilic leukemia (RBL-1) cells stimulated with A-23187, the major slow reacting substance (SRS) species contain glutathione, cysteinyl-glycine, or cysteine in their side chains, corresponding or closely related to leukotrienes LTC₄, LTD₄, and LTE₄, respectively.³ Evidence is presented that most of the SRS produced during the first few minutes of stimulation by the ionophore has a glutathionyl side chain which is sequentially converted to cysteinyl-glycine and cysteine.     

Formation of the cysteinyl form of slow reacting substance (leukotriene E4) in human plasma

The two major species of slow reacting substance (SRS) contain either a glutathionyl or cysteinyl-glycyl side chain. Incubation of these SRS's with undiluted or diluted (usually 1:10 or 1:50) human plasma at 37°C resulted in marked losses of smooth muscle contracting activity due primarily to conversion of their oligopeptide side chains to cysteine.     

Slow reacting substance (SRS) from ionophore A23187-stimulated peritoneal mast cells of the normal rat. II. Evidence for a precursor role of arachidonic acid and further purification.

The generation of slow reacting substance (SRS) from ionophore A23187-stimulated rat peritoneal mast cells was enhanced by arachidonic acid (AA). This SRS generation was inhibited by 5,8,11,14-eicosatetraynoic acid (ETYA), an acetylenic analogue of AA and an inhibitor of both fatty acid cyclooxygenase and lipoxygenase. Indomethacin, a fatty acid cyclooxgenase inhibitor, had an enhancing effect upon SRS generation. This suggests SRS generation occurred through an ETYA sensitive step--perhaps a lipoxygenase. Radiolabel from [14C]-AA was incorporated into SRS with comigration of radioactivity and bioreactivity in silicic acid and thin layer chromatographies. Upon silicic acid chromatography, the active principle was eluted in the methanol fraction. Two-dimensional thin layer chromatography revealed chromatographic separation from other known spasmogenic substances and phospholipids. Mast cell SRS was found to display physiochemical properties similar to those of rat basophilic leukemia cell SRS, namely: that mast cell SRS generation was 1) enhanced by arachidonic acid; 2) inhibited by ETYA but not by indomethacin; 3) incorporation of [14C]-AA into the active principle; and 4) similar behavior during purification in silicic acid and thin layer chromatographies.     

Slow reacting substance of anaphylaxis, SRS-A: Assignment of the stereochemistry

We have recently described the structure elucidation of slow reacting substance of anaphylaxis (SRS-A) from lung and of a slow reacting substance (SRS) from basophilic leukaemia cells as 5-hydroxy-6-cysteinylglycinyl-7,9,11,14-eicosatetraenoic acid. The stereochemistry of this molecule has now been shown to be 5(S)-hydroxy-6(R)-cysteinylglycinyl-7,9-trans-11,14-cis-eicosatetraenoic acid by comparison of the synthetic and natural products and their derivatives using mass spectrometric and HPLC chromatographic techniques. The synthetic and natural compounds are also indistinguishable by their pharmacological properties, their conversion by soybean lipoxygenase, and their UV spectra.     

Stimulated production of slow reacting substances by mercaptans from ionophore A 23187-induced mononuclear cells: mercaptan structure-activity studies.

When isolated mononuclear cells from the peritoneal cavity of the rat are challenged with the ionophore, A 23187, slow reacting substance (SRS) is produced. SRS production is markedly enhanced by the addition of mercaptans to the incubations. In confirmation of previously published reports, this enhancement is dependent on the duration of a preincubation of the cells with the mercaptans before addition of the ionophore, a two minute preincubation being optimal. Quantitative structure-activity studies revealed that a variety of mercapto carboxylic acids, where the mercapto group was one or two carbon atoms removed from the carboxyl group, were all active in enhancing SRS production and the enhancement followed parallel dose response curves. A 4-mercapto acid, while weakly active, had a distinctly different dose response curve and cysteamine, which lacks a carboxyl function, was inactive. Preliminary characterization of the products of the incubations produced in the presence of different mercaptans has revealed that, while they all qualitatively meet the criteria of stability to alkali, lability in acid and destruction by arylsulfatase which are associated with slow reacting substance of anaphylaxis, there are quantitative differences in stability which suggest that the products may not be identical.     

Characterization of slow reacting substance as a family of thiolipids derived from arachidonic acid.

Using radiolabeled cysteine and arachidonic acid as biosynthetic precursors, the slow reacting substance (SRS) produced by the rat basophilic cell line, RBL-1, has been characterized as a family of thiolipids derived from arachidonic acid.     

Fatty acid structural requirements for leukotriene biosynthesis

Utilizing a variety of fatty acids, differing in chain length, degree and position of unsaturation, we investigated the substrate specificity for the enzymatic production of biologically active slow reacting substances (SRS) and of the other leukotrienes. A cell-free enzyme system obtained from RBL-1 cells was used in this study. The primary structural requirement observed for the conversion by this lipoxygenase enzyme system was a delta 5,8,11 unsaturation in a polyenoic fatty acid. Such fatty acids as 20:4 (5,8,11,14) 20:5 (5,8,11,14,17), 20:3 (5,8,11), 19:4 (5,8,11,14) and 18:4 (5,8,11,14) were readily converted to compounds that comigrated with 5-HETE and 5,12-DiHETE and to biologically active SRS. Chain length did not have an influence on the formatin of these hydroxyacids. Fatty acids with the initial unsaturation at delta 4, delta 6, delta 7, or delta 8 were a poor substrate for the leukotriene enzyme system. Therefore, this lipoxygenase pathway in leukocytes is quite different from the lipoxygenase in platelets which does not exhibit this specificity.     

Incorporation of radiolabel from [1-14C]5-hydroperoxy-eicosatetraenoic acid into slow reacting substance

When synthetic [1-¹⁴C]5-hydroperoxy-eicosatetraenoic acid was incubated with rat basophilic cells, incorporation of the radiolabel into slow reacting substance (SRS) could be demonstrated as evidenced by comigration of spasmogenic activity and radioactivity after purification by high pressure liquid chromatography. This provides direct evidence that SRS is a product of the lipoxygenase pathway.     

Effect of the 5-hydroperoxide of eicosatetraenoic acid and inhibitors of the lipoxygenase pathway on the formation of slow reacting substance by rat basophilic leukemia cells; direct evidence that slow reacting substance is a product of the lipoxygenase pathway

Previous studies in a line of rat basophilic leukemia (RBL 1) cells have indicated that the slow reacting substance (SRS) made during stimulation with the divalent cation ionophore, A23187, is derived from arachidonic acid (AA). In the present report, various inhibitors of AA metabolism were compared with regard to their effects on SRS formation and incorporation of radioactivity from [1-14C]-AA into known metabolites of the lipoxygenase and cyclooxygenase pathways. An apparently close parallel between lipoxygenase product formation and SRS synthesis is demonstrated. In addition, exogenous 5-hydroperoxy-eicosatetraenoic acid (5-HPETE) has been shown to markedly enhance SRS synthesis, even when A23187 is absent. The data provide very strong evidence that SRS is produced through the lipoxygenase pathway.     

On the structure of slow reacting substance of anaphylaxis: evidence of biosynthesis from arachidonic acid.

The mononuclear cells in peritoneal washings from normal rats can be induced to produce large amounts of slow reacting substance of anaphylaxis by incubation with 10 mM cysteine in the presence of the calcium ionophore A-23187. This production of slow reacting substance could be inhibited by the addition of non-steroidal anti-inflammatory drugs, e.g., indomethacin, ibuprofen and flurbiprofen, Furthermore, mediator production was inhibited by eicosatetraynoic acid, the substrate analog of arachidonic acid, and by 9,11-azoprosta-5, 13-dienoic acid (AZO analog 1), a structural analog of the prostaglandin endoperoxide, PGH2, which known to inhibit thromboxane synthesis. Relatively high concentrations of hydrocortisone acetate inhibited mediator production; this inhibition could be partly reversed by the addition of arachidonic acid or to a lesser extent by eicosatrienoic acid. Preliminary results suggest that a small fraction of the 3H-labled arachidonic acid which was taken up by these cells in vitro was associated with slow reacting substance. We postulate that slow reacting substance of anaphylaxis may be derived from a prostaglandin endoperoxide which is formed during the oxidation of arachidonic acid by the prostaglandin fatty acid cyclooxygenase.     

Fatty acid structural requirements for leukotriene biosynthesis

Utilizing a variety of fatty acids, differing in chain length, degree and position of unsaturation, we investigated the substrate specificity for the enzymatic production of biologically active slow reacting substances (SRS) and of the other leukotrienes. A cellfree enzyme system obtained from RBL-1 cells was used in this study. The primary structural requirement observed for the conversion by this lipoxygenase enzyme system was a Δ^5,8,11 unsaturation in a polyenoic fatty acid. Such fatty acids as 20:4 (5,8,11,14), 20:5 (5,8,11,14,17), 20:3 (5,8,11), 19:4 (5,8,11,14) and 18:4 (5,8,11,14) were readily converted to compounds that comigrated with 5-HETE and 5,12-DiHETE and to biologically active SRS. Chain length did not have an influence on the formation of these hydroxyacids. Fatty acids with the initial unsaturation at Δ⁴, Δ⁶, Δ⁷ or Δ⁸ were a poor substrate for the leukotriene enzyme system. Therefore, this lipoxygenase pathway in leukocytes is quite different from the lipoxygenase in platelets which does not exhibit this specificity.     

Site-specific mutagenesis of two histidine residues in fatty acid ethyl ester synthase-III.

Human myocardial fatty acid ethyl ester synthase-III is a newly described acidic glutathione S-transferase that metabolizes both ethanol and carcinogens. Structure-function studies have not been performed relating these two distinct enzymatic activities. Since there are only two histidine residues in fatty acid ethyl ester synthase-III (His 72 and His 163), the role of each was examined by site-specific mutagenesis. Fatty acid ethyl ester synthase-III mutagenized at position 72 to contain either Gln, Pro or Ala had less than 5% of control glutathione S-transferase activity but retained fatty acid ethyl ester synthase activity under standard assay conditions. In contrast, substitution of histidine 163 with proline had no effect on glutathione S-transferase activity, but it slightly increased synthase activity. Thus, this study indicates that histidine plays a differential role in fatty acid ethyl ester synthase III depending on the nucleophilic substrate.     

On the structure of slow reacting substance of anaphylaxis: Evidence of biosynthesis from arachidonic acid

The mononuclear cells in peritoneal washings from normal rats can be induced to produce large amounts of slow reacting substance of anaphylaxis by incubation with 10 mM cysteine in the presence of the calcium ionophore A-23187. This production of slow reacting substance could be inhibited by the addition of non-steroidal anti-inflammatory drugs, e.g., indomethacin, ibuprofen and flurbiprofen. Furthermore, mediator production was inhibited by eicosatetraynoic acid, the substrate analog of arachidonic acid, and by 9,11-azoprosta-5,13-dienoic acid (AzO analog 1), a structural analog of the prostaglandin endoperoxide, PGH₂, which is known to inhibit thromboxane synthesis. Relatively high concentrations of hydrocortisone acetate inhibited mediator production; this inhibition could be partly reversed by the addition of arachidonic acid or to a lesser extent by eicosatrienoic acid. Preliminary results suggest that a small fraction of the ³H-labeled arachidonic acid which was taken up by these cells in vitro was associated with slow reacting substance. We postulate that slow reacting substance of anaphylaxis may be derived from a prostaglandin endoperoxide which is formed during the oxidation of arachidonic acid by the prostaglandin fatty acid cyclooxygenase.     

Introduction of a nomenclature: Leukotrienes

The structure of a slow reacting substance (SRS) from mouse mastocytoma cells was recently reported (Murphy, R.C., Hammarström, S. and Samuelsson, B. (1979) Proc. Natl. Acad. Sci. USA, in press). We proposed that SRS is formed from a previously described unstable epoxide intermediate in the formation of dihydroxylated arachidonic acid metabolites in leukocytes. The term is introduced for compounds which like SRS are non-cyclized C₂₀ carboxylic acids with one or two oxygen substituents and three conjugated double bonds.     

Inhibition of fatty acid amidohydrolase, the enzyme responsible for the metabolism of the endocannabinoid anandamide, by analogues of arachidonoyl-serotonin

Arachidonoyl-serotonin inhibits in a mixed-type manner the metabolism of the endocannabinoid anandamide by the enzyme fatty acid amidohydrolase. In the present study, compounds related to arachidonoyl-serotonin have been synthesised and investigated for their ability to inhibit anandamide hydrolysis by this enzyme in rat brain homogenates. Removal of the 5-hydroxy from the serotonin head group of arachidonoyl-serotonin produced a compound (N-arachidonoyltryptamine) that was a 2.3-fold weaker inhibitor of anandamide hydrolysis, but which also produced its inhibition by a mixed-type manner (Ki(slope) 1.3 microM; Ki(intercept) 44 microM). Replacement of the amide linkage in this compound by an ester group further reduced the potency. In contrast, replacement of the arachidonoyl side chain by a linolenoyl side chain did not affect the observed potency. N-(Fur-3-ylmethyl) arachidonamide (UCM707), N-(fur-3-ylmethyl)linolenamide and N-(fur-3-ylmethyl)oleamide inhibited anandamide hydrolysis with pI50 values of 4.53, 5.36 and 5.25, respectively. The linolenamide derivative was also found to be a mixed-type inhibitor. It is concluded that the 5-hydroxy group of arachidonoyl-serotonin contributes to, but is not essential for, inhibitory potency at fatty acid amidohydrolase.     

The nature of fatty acid modifies the equilibrium position in the esterification catalyzed by lipase

The equilibrium position in lipase mediated esterification of various fatty acids and butanol was studied. The influence of the chain length and the presence of unsaturations in the fatty acids on the equilibrium position was measured and predicted. To predict equilibrium position the program TREP extended (TREPEX) based on the UNIFAC group contribution method was used. Using an equilibrium constant of 35, calculated on the basis of thermodynamic activities, the equilibrium position between butanol and saturated and/or unsaturated fatty acids with different chain lengths can be predicted. The ester mole fraction at equilibrium increases with the fatty acid chain length, and for fatty acids with the same carbon number, the highest values are found for unsaturated fatty acids. For reaction systems containing two saturated fatty acids, a slightly higher mole fraction is obtained for the fatty acid with the higher chain length, while for mixtures consisting of saturated and unsaturated fatty acids, the mole fractions of the unsaturated esters are lower than those of the saturated ones, regardless the chain length of the fatty acid. These experimental results are in good agreement with the calculations with TREPEX.