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.     

Release of slow reacting substance (SRS) from rat basophilic leukemia (RBL-1) cells.

When rat basophilic leukemia (RBL-1) cells were exposed to the ionophore A23187, a substance was released that produced a prolonged contraction of guinea pig ileum resembling that seen with slow reacting substances (SRSs) from various sources. The response was temperature, dose, and the time dependent with no activity being demonstrated in unstimulated cells. Several lines of evidence indicated that the RBL-1 product was markedly similar or identical to SRSs obtained from non-neoplastic tissues: 1) appropriate behavior in seven different chromatographic systems, 2) an appropriate profile of activity on various smooth muscle preparations, 3) an ability of low concentrations of the selective SRS inhibitor FPL 55712 to block the guinea pig ileal response, 4) failure of chymotrypsin to destroy activity, 5) loss of the activity after incubation with arylsulfatase, and 6) an ability to release activity from cells preincubated with indomethacin. Since RBL-1 cells can be grown in considerable guantity and under optimal conditions an average of 1500 SRS units/10(7) cells can be obtained, these cells should be useful as a biosynthetic source in further attempts to purify and characterize the SRS molecule.     

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.     

Modulation by phorbol 12-myristate 13-acetate of arachidonic acid release from rat basophilic leukemia cells stimulated with A23187

Rat basophilic leukemia (RBL-2H3) cells were cultured in medium containing [3H]arachidonic acid and labelling of the different lipid fractions was followed with time. After up to 4 h of culture, the label was found mostly in phosphatidylcholine. After 8 h, labelling of phosphatidylethanolamine gradually exceeded that of phosphatidylcholine, until at 24 h, approximate equilibrium labelling of the lipid fractions was attained and 45% of the label was found in phosphatidylethanolamine, 35% in phosphatidylcholine, 18% in the phosphatidylserine/inositide fraction and the remainder in the neutral lipid fraction. Stimulation of cells with A23187 after 30 min of labelling caused release of [3H]arachidonic acid which was accountable by a decrease in radioactivity of phosphatidylcholine, whereas stimulation of cells after 24 h of labelling caused the release of radioactive arachidonic acid, which was accompanied by a decrease of label in both phosphatidylcholine and phosphatidylethanolamine. Incubation of the labelled cells with phorbol 12-myristate 13-acetate prior to ionophore addition enhanced both the release of [3H]arachidonic acid and its metabolites and the decrease in label of the same phospholipids as those affected by ionophore alone. Under our conditions, the enhancement effects of phorbol ester were greatest after 2-5 min of preincubation, prior to ionophore addition. The results suggest that in basophilic leukemia cells, arachidonic acid release proceeds from several pools of phospholipids and that the activity of the phospholipase(s) involved is modulated by protein kinase C.     

Slow reacting substance (SRS) from ionophore A23187-stimulated peritoneal mast cells of the normal rat. I. Conditions of generation and initial characterization.

When rat peritoneal mast cells were exposed to the ionophore A23187, a principle was released that possessed the biologic properties of slow reacting substance (SRS) from various sources. The response was dose, time, and temperature dependent with no activity being demonstrated in unstimulated cells. Supporting evidence that the mast cell product was similar or identical to SRS obtained from other sources include: 1) appropriate differential bioassay profile, 2) resistance to lipolysis and proteolysis, 3) acid lability and base stability, 4) inactivation by limpet arylsulfatase, and 5) inhibition by low concentrations FPL 55712. These data demonstrate that the isolated rat peritoneal mast cell contains the biosynthetic capacity to produce a bioreactive substance with the properties of SRS.     

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 α 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.     

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.     

Ionophore A-23187 induced histamine release from rat mast cells and rat basophil leukemia (RBL-1) cells.

Ionophore A-23187 releases histamine from normal mast cells apparently by promoting Ca++ influx (Foreman et al, Nature 245: 249, 1973). In our hands at concentrations of greater than 0.2 mug/ml release occurs in 1 to 2 min, is blocked by metabolic inhibitors, and is unaccompanied by cytotoxicity (trypan-blue uptake, lactic dehydrogenase (LDH) release). At higher doses (0.5 mug/ml) histamine release is followed by significant cytotoxicity, but again Ca++ is required. In parallel studies, we examined cultured rat basophilic leukemia (RBL-1) cells. These cells, which apparently have normal surface receptors for IgE, contained approximately 700 ng histamine/10(6) cells but did not release histamine when IgE-mediated release was looked for. They do not respond to doses of ionophore which would be expected to give non-cytotoxic histamine release. At higher doses histamine release is preceded by progressive LDH release: LDH release is 75% complete at 5 min whereas 10 min are required for 75% maximal histamine release. This reaction requires Ca++ and is temperature dependent but is not inhibited by metabolic poisons (2-deoxyglucose, dinitrophenol, CN-). These studies suggest that either Ca++ does not enter into these cells normally or that one or more mechanisms which are ordinarily triggered by the changes in Ca++ flow are unresponsive in the RBL-1 cells. These studies also underline the importance of ruling out cytotoxicity in ionophore-induced phenomena.     

Morphological changes induced by the calcium ionophore A23187 in rat basophilic leukemia (2H3) cells.

RBL-2H3 cells have been widely used to study histamine release in vitro. It was previously shown that these cells undergo striking morphological changes after IgE-mediated secretion. The present study was undertaken to examine if the morphological changes were dependent on activation of the Fc epsilon receptor. Therefore, the cells were stimulated to release histamine by two different mechanisms: activation of the Fc epsilon receptor by antigen and treatment with the calcium ionophore A23187. Cell surface and cytoskeletal changes were examined by fluorescence microscopy and scanning electron microscopy after either IgE- or ionophore-mediated histamine release. After exposure of the cells to either secretagogue, the cells spread over the surface of the culture dish and underwent rearrangement of the cytoskeleton. In addition, scanning electron microscopy revealed that deep ruffles developed on the surface of the cells undergoing IgE-mediated release. The surface changes were not as pronounced with the ionophore. The distribution of the cytoskeletal elements was examined by immunofluorescence using FITC-phalloidin and antibodies against vimentin and tubulin. In unstimulated cells actin was localized at the cell periphery, just under the plasma membrane. In the stimulated cells it was associated with the cell periphery and concentrated in the surface ruffles. As the stimulated cells spread, intermediate filaments and microtubules became distributed throughout the cell body, but there was no obvious association with the membrane ruffles. These morphological changes were dependent on the presence of extracellular calcium and on the concentration of ionophore or antigen, and were also correlated with the amount of histamine released. Additionally, IgE-mediated stimulation led to increased uptake of the soluble-phase tracer Lucifer yellow, whereas stimulation with the ionophore A23187 showed no increase in Lucifer yellow internalization. Ionophore A23187 produced changes similar but not identical to those seen in the RBL-2H3 cells after IgE-mediated histamine release. The differences may be owing to the involvement of the Fc epsilon receptor in IgE-mediated secretion.     

Release of slow reacting substance of anaphylaxis (SRS-A) from human leukocytes by the calcium ionophore A23187.

The ability of the calcium ionophore A23187 to release slow reacting substance of anaphylaxis (SRA-A) from human leukocytes was studied. About 25 times more SRS-A activity was released from aliquots of leukocytes by ionophore stimulation than by antigen stimulation, although comparable amounts of histamine were released. Cell separation studies revealed that granulocytes other than basophils were also capable of releasing SRS-A. The contractile activity released after challenge with ionophore appeared physicochemically identical to the SRS-A of rat or human origin released by antigen challenge in terms of its stability to base hydrolysis, inactivation by arylsulfatase, and chromatographic behavior on silicic acid and Sephadex LH-20 columns. We suggest that some mediators of allergic reactions previously associated, in man, only with antigen-IgE antibody interaction on mast cells or basophils may be released by other stimuli and from other cell types.     

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.     

Arachidonic acid metabolism in rat basophilic leukemia (RBL-1) cells

Rat basophilic leukemia (RBL-1) cells metabolized arachidonic acid through more than one enzymatic pathway. The major cyclooxygenase product was prostaglandin (PG) D₂ as established by chromatographic and chemical behavior and the effect on platelet aggregation. PGD₂ formation from exogenous arachidonic acid was inhibited by indomethacin, 1 μg/ml. RBL-1 incubated with exogenous arachidonic acid also formed SRS-A the synthesis of which was not inhibited by indomethacin. However, the SRS-A activity was blocked by the specific receptor antagonist FPL 55712. [¹⁴C]arachidonic acid was effectively incorporated into the phospholipids of RBL-1 cells. Challenge of such prelabelled cells or unlabelled cells with A 23187 caused release of PGD₂, SRS-A and another presently unidentified product. However, with A 23187 as a stimulus, the RBL-1 cyclo-oxygenase could not be blocked by low concentrations of indomethacin. This work further substantiates our earlier findings that SRS-A formed from arachidontic acid is not a cyclooxegenase product.     

Phospholipase activation in the IgE-mediated and Ca2+ ionophore A23187-induced release of histamine from rat basophilic leukemia cells

The importance of phospholipase(s) activation in the IgE-mediated and ionophoreinduced histamine release from the rat basophilic leukemia cell line has been examined. The activation of phospholipase(s) as measured by [¹⁴C]arachidonic acid release and the release of histamine both required Ca²⁺ and were temporally parallel. Inhibition of phospholipase(s) activity by the inhibitors mepacrine and α-parabromoacetophenone also correlated with the inhibition of histamine release. [¹⁴C]Arachidonic acid released by the phospholipase(s) was mainly metabolized to prostaglandin D₂. The inhibition of the cyclooxygenase pathway by indomethacin did not affect histamine release. 5,8,11,14-Eicosatetraynoic acid inhibited both histamine and [¹⁴C]arachidonic acid release suggesting an effect not only on the cyclooxygenase and lipoxygenase pathways but also on the phospholipase(s). These results suggest that activation of phospholipase appears to be necessary for histamine release in the rat bosophilic leukemia cells.     

Induction of histidine decarboxylase of rat basophilic leukemia (2H3) cells stimulated by higher oligomeric IgE or phorbol myristate acetate

When rat basophilic leukemia (2H3) cells were stimulated by higher oligomer, the chemically cross-linked oligomers of IgE, in the presence of calcium the activity of histidine decarboxylase (HDC, L-histidine carboxylase, E.C.4.1.1.22), a histamine-forming enzyme, was increased by 1 hr, reaching maximum activity by 2 hr, and returning to the original level by 8 hr. A similar increase in enzyme activity was observed in cells treated with phorbol myristate acetate (PMA) or oleoyl-acetylglycerol (OAG), which are known activators of protein kinase C. Removal of calcium from medium abolished the increase in HDC activity in response to higher oligomer but not that induced by PMA or OAG, suggesting that the increase in HDC activity may be mediated by protein kinase C. The increase in the HDC activity probably required induction of enzyme synthesis, because it was prevented by cycloheximide.     

Microfilaments regulate the rate of exocytosis in rat basophilic leukemia cells

Disruption of microfilaments in rat basophilic leukemia (RBL) cells by exposure to cytochalasin B is observed to potentiate the rate of antigen-stimulated secretion from these cells. Under these conditions, cytochalasin B is without effect on the antigen-stimulated production of inositol phosphates or 45Ca2(+)-influx. In streptolysin-O-permeabilized RBL cells, cytochalasin B is observed to potentiate the rate of secretion in response both to guanosine 5'-(2-thio)-O-triphosphate (GTP gamma S) and to Ca2+ (buffered between 0.1 and 10 microM). However, under these conditions, cytochalasin B does not affect to antigen-stimulated production of inositol phosphates. Consistent with these data, microfilaments are proposed to regulate a terminal step in exocytosis, in a physiologically relevant manner.     

Calcium specificity of the antigen-induced channels in rat basophilic leukemia cells

Ion channels, activated upon IgE-Fc epsilon receptor aggregation by specific antigen, were studied in micropipet-supported lipid bilayers. These bilayers were reconstituted with purified IgE-Fc epsilon receptor complex and the intact 110-kDa channel-forming protein, both isolated from plasma membranes of rat basophilic leukemia cells (line RBL-2H3). In order to identify the current carrier through these ion channels and to determine their ion selectivity, we investigated the currents flowing through the IgE-Fc epsilon receptor gated channels in the presence of a gradient of Ca2+ ions. Thus, the solution in which the micropipet-supported bilayer was immersed contained 1.8 mM CaCl2, while the interior of the micropipet contained 0.1 microM Ca2+ (buffered with EGTA). Both solutions also contained 150 mM of a monovalent cation chloride salt (either K+ or Na+). The currents induced upon specific aggregation of the IgE (by either antigen or anti-IgE antibodies) were examined over a range of potentials imposed on the bilayer. The type of conductance event most frequently observed under the employed experimental conditions was a channel that has a slope conductance of 3 pS and a reversal potential practically identical with the calculated value for the reversal potential of calcium (134 +/- 11 mV in the presence of sodium, 125 +/- 13 mV in the presence of potassium). These results indicate that this channel is highly selective for calcium against the monovalent cations sodium and potassium. This same channel has a conductance of 4-5 pS in the presence of symmetrical solutions containing only 100 mM CaCl2 and 8 pS in the presence of 0.5 M NaCl with no calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhancement of the release of inflammatory mediators by substance P in rat basophilic leukemia RBL-2H3 cells

Summary Substance P (SP), a neurotransmitter, may play an important role in neurogenic inflammation. Ginseng has been used extensively in traditional medicine; however, few studies were focused on their anti-allergic effect. Therefore, the effect and mechanism of ginsenoside Rb1 on the SP enhancement of allergic mediators were explored. In this study, SP and dinitrophenyl-bovine serum albumin (DNP-BSA) were used to activate rat basophilic leukemia (RBL)-2H3 cells. The cultured supernatants were assayed for histamine, leukotriene C₄(LTC₄) and interleulin-4 (IL-4) production. The mitogen-activated protein kinases (MAPKs) signaling pathway was determined by Western blotting analysis. We found that IgE/DNP-BSA, SP, ginsenoside Rb1, or MAPK specific inhibitors had no effect on cell viability and cytotoxicity. SP (30 μM) alone, did not induce histamine and LTC₄ release, but it enhanced allergen-induced histamine and LTC₄ release. In␣addition, SP significantly induced and enhanced allergen-activated IL-4. Ginsenoside Rb1 dose-dependently inhibited these effects. SP enhanced the allergen-activated ERK pathway in RBL-2H3 cells, and Rb1 effectively inhibited the ERK pathway activation. Although MAPK specific inhibitors suppressed LTC₄ and IL-4, only U0126 inhibited the SP enhanced histamine release. These results demonstrate that Rb1 dose-dependently inhibited SP enhanced allergen-induced mediator release and its mechanism was through the inhibition of the ERK pathway.     

Bifidobacterium suppresses IgE-mediated degranulation of rat basophilic leukemia (RBL-2H3) cells

Sixteen heat-killed bifidobacteria isolated from human intestine and a probiotic strain Lactobacillus GG were tested for their ability to influence IgE-mediated degranulation of rat basophilic leukemia (RBL-2H3) cells in vitro . The bifidobacteria suppressed IgE-mediated degranulation of RBL-2H3 cells by 1.6–56.4% in a strain-dependent manner. Bifidobacteria from healthy infants expressed high inhibitory effects on IgE-mediated degranulation (41–55%), while those from allergic infants varied greatly in their effects against degranulation. Bifidobacteria taxonomically identified as Bifidobacterium bifidum exhibited much stronger inhibitory effects against IgE-mediated degranulation than those taxonomically identified as B. adolescentis ( P < 0.05).These results indicate that the intestinal bifidobacteria might be one of factors influencing IgE-mediated allergic responses.     

Slow reacting substances from ionophore A23187-stimulated basophilic leukemia cells and peritoneal mast cells in the rat. I. Purification and comparison during sequential sephadex LH-20 and thin layer chromatography.

Radiolabeled slow reacting substance (SRS) from rat basophilic leukemia cells (RBL-1) or rat peritoneal mast cells was generated by stimulation with the divalent cation ionophore A23187 in the presence of [1^?14C]-arachidonic acid (AA). These radiolabeled SRSs were purified by sequential adsorption, gel filtration and partition chromatography on Sephadex LH-20 with correspondence of bio- and radioactivities. Two-dimensional high performance thin layer chromatography of the active principles continued to show comigration of bio- and radioactivities. RBL-1 and mast cells incorporated [¹⁴C]-AA into bioactive SRS which are analogous based upon similar behavior during purification.