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.     

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.     

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.     

Inhibition of IgE-mediated histamine release from rat basophilic leukemia cells and rat mast cells by inhibitors of transmethylation

This study evaluated the effect of inhibitors of transmethylation on histamine release from rat mast cells and rat basophilic leukemia cells. IgE-mediated histamine release from rat basophilic leukemia cells (RBL-2H3 cells) was inhibited by 3-deazaadenosine (DZA) in the presence of L-homocysteine thiolactone (Hcy) or the combination of adenosine, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), and Hcy in a dose-dependent fashion. There were no significant changes in the cellular cAMP levels by these inhibitors. Histamine release induced by anti-IgE or dextran from normal rat mast cells was also blocked by DZA plus Hcy in a dose-dependent manner. DZA at 10(-3) M in the presence of 10(-4) M Hcy or the combination of 10(-3) M adenosine, 10(-4) M EHNA, and 10(-3) M Hcy inhibited lipid (perhaps phospholipid) methylation into RBL-2H3 cells without affecting choline incorporation. In the presence of 10(-3) M DZA plus 10(-4) M Hcy there was a 170-fold increase in [35S]AdoHcy with the concomitant appearance of 3-deaza-AdoHcy when the cells were incubated with [35S]methionine, thus indicating that these drugs inhibited methylation reaction(s) through the intracellular accumulation of AdoHcy and 3-deaza-AdoHcy. In contrast, histamine release from rat mast cells induced by the calcium ionophore A23187, compound 48/80, polymyxin B, or ATP was not inhibited by these compounds. These results suggest that IgE- or dextran-mediated histamine release involves methylation reactions(s), whereas the other secretagogues bypass this early step.     

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.     

Antigen-triggered membrane potential changes in IgE-sensitized rat basophilic leukemia cells: evidence for a repolarizing response that is important in the stimulation of cellular degranulation

We have studied Ag-induced membrane potential changes of rat basophilic leukemia cells by using the potential-sensitive dye, bis-(1,3-diethylthiobarbiturate)trimethineoxonol. A rapid membrane depolarization is triggered by a multivalent Ag, and it has a bell-shaped dose dependence that parallels the degranulation response but not the extent of cross-linking of the IgE-receptor complexes. As the temperature is reduced from 37 degrees C, this depolarization response slows and decreases in magnitude until complete inhibition is observed at 15 degrees C, similar to the temperature dependence previously observed for the Ag-stimulated rise in cytoplasmic Ca2+ and for degranulation. The results imply that a highly temperature-dependent step subsequent to Ag binding and cross-linking is necessary for the depolarization response. A partial return to the resting potential is seen to follow the depolarization response to Ag. This repolarization process is inhibited by quinidine.HCl and Ba2+ in parallel with an inhibition of the degranulation response. Repolarization is not affected by 4-aminopyridine or by the absence of K+ in the external buffer. These data suggest that the repolarization is caused by a previously uncharacterized K+ channel.     

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.     

Activation of rat basophilic leukemia cells. Temporal identification of the signal calcium influx mediated by the receptor-operated channel pathway

Antigen-induced 45calcium influx into rat basophilic leukemia (RBL) cells was examined with emphasis on the early time domain under conditions that exclude loss of the cation during the subsequent washing step. Such preparations demonstrate a distinct, temporally separate influx peaking at 3 min, followed by a substantial efflux. This internalized 45Ca2+ approaches millimolar total intracellular concentration and is therefore either sequestered or becomes bound to intracellular components (proteins). The amplitude of this influx is linearly proportional to IgE-receptor occupancy at low receptor occupancies, and is sensitive to the anti-allergic drug cromolyn. Furthermore, the timing of both the maximal uptake and the maximal susceptibility to cromolyn correlates with the Quin-2 signal in these cells, and the initial degranulation pattern bears some resemblance to trends in the 45Ca2+ uptake curve. These qualities suggest that the early peak at 2-3 min, rather than any later 45Ca2+ uptake, comprises the initial signalling Ca2+ pool. Maximal apparent inhibition by cromolyn for 45Ca2+ uptake was about 65% and required a 10-15-min preincubation with the cells. The inhibitory effect was limited to the peak at 3 min, suggesting that tracer incorporation beyond 5-6 min largely involves other pools or pathways, triggered by receptor aggregation, yet only indirectly related to channel activity or to the signal proper. A quantitative similarity was found between the early peak measured on intact cells and the single channel conductance measured on reconstituted planar bilayers containing the purified receptor for IgE and the purified cromolyn-binding protein [Corcia, A. et al. (1986) EM BO J. 5, 849-854]. This, as well as the effects of cromolyn, support the assumption that cromolyn-binding protein is a major constituent involved in this early influx, or that influx is a principal pathway for the signaling calcium mass.     

Characterization of the chirality of the monohydroxy-eicosatetraenoic acids produced by rat basophilic leukemia cells

Incubation of rat basophilic leukemia cells with exogenous arachidonic acid and permeabilizing concentrations of ethanol resulted in the production of 5-, 12-, and 15-hydroxyeicosatetraenoic acids. With chiral phase high performance liquid chromatography, it was demonstrated that the 5-hydroxyeicosatetraenoic acid had strict (S) stereospecificity while contrary to expectation, the 12- and the 15-hydroxyeicosatetraenoic acids were non-racemic mixtures of the stereoisomers with the S/R ratios averaging 8.6 and 2.2, respectively. If the strict (S) stereospecificity of mammalian lipoxygenases holds true, these results suggest that the 15- and 12-hydroxyeicosatetraenoic acids may be derived from non-lipoxygenase sources. Examination of the chirality of the oxygenase products of unsaturated fatty acids may be of value in defining the enzymes which are activated in vivo in pathological states.     

Characterization of glucocorticoid inhibition of antigen-induced inositolphosphate formation by rat basophilic leukemia cells: possible involvement of phosphatases.

The suppressive effect of glucocorticoids (GC) upon antigen-induced phosphatidylinositol phospholipase C (PI-PLC) activity and inositol phosphate formation by rat basophilic leukemia cells (RBL-2H3) has been characterized. Addition of antigen for a period of 1-30 min enhanced production of [3H]inositol monophosphate (IP1), inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3) by about 5-10-fold. Pretreatment with hydrocortisone (HC) reduced formation of the various inositol phosphates (IPs) and degradation of phosphatidylinositol 4,5-bisphosphate (PIP2) by an average of 50%. Maximal inhibition of hydrolysis of PIP2 and reduction in stimulation of IP3 formation was reached after 4 h of preincubation with 2.10(-6) M of HC. Cycloheximide and RU486, a GC receptor antagonist, completely prevented the inhibitory effect of HC on IP formation. Other GC, dexamethasone (DEX) and triamcinolone (each at 2.10(-7) M) markedly suppressed antigen induced IP3 production, while aldosterone and sex steroids such as estradiol and progesterone (each at 2.10(-6) M) were virtually inactive. Antigen-stimulated phosphorylation of a 18 kDa and other proteins was inhibited by about 60% following pretreatment with the GC. This inhibition was in turn prevented by cycloheximide. DEX also doubled the activity of cellular acid phosphatase activity. The results suggest that the inhibitory effect of GC is specific, receptor-mediated, dependent on protein synthesis and possibly mediated by protein phosphatase activity.     

Regulation of the antigen-induced F-actin response in rat basophilic leukemia cells by protein kinase C

Multivalent antigen that is capable of binding to and crosslinking the IgE receptors on rat basophilic leukemia (RBL) cells, induces a rapid and sustained rise in the content of filamentous actin. This reorganization of the actin may be responsible for changes in cellular morphology during the degranulation process. The antigen-stimulated polymerization of actin can be blocked in a dose-dependent manner by protein kinase inhibitors which also block degranulation. Conversely, reagents such as PMA, 1,2-dioctanoyl-sn-glycerol (diC8), and 1-oleoyl-2-acetyl-glycerol (OAG) which stimulate protein kinase C (PKC) also activate the rise in F-actin, although they have no effect on degranulation by themselves. The actin response which can be stimulated by the PKC activators can also be blocked by protein kinase inhibitors indicating that the PMA- and OAG-induced response is probably through activation of a protein kinase. Depletion of PKC activity through long term (20 h) exposure of RBL cells to PMA, also inhibited the F-actin response when the cells were stimulated with either multivalent antigen or OAG. External Ca++, which is an absolute requirement for degranulation, is not necessary for the rise in F-actin, but may modulate the response. Furthermore, ionomycin, which induces a large Ca++ influx, does not stimulate the F-actin increase even at doses that cause degranulation. These results suggest that activation of a protein kinase, such as PKC, may be responsible for signaling the polymerization of actin in RBL cells and that a rise in intracellular Ca++ is neither necessary nor sufficient for this response.     

Self-renewal of acute lymphocytic leukemia cells is limited by the Hedgehog pathway inhibitors cyclopamine and IPI-926

Conserved embryonic signaling pathways such as Hedgehog (Hh), Wingless and Notch have been implicated in the pathogenesis of several malignancies. Recent data suggests that Hh signaling plays a role in normal B-cell development, and we hypothesized that Hh signaling may be important in precursor B-cell acute lymphocytic leukemia (B-ALL). We found that the expression of Hh pathway components was common in human B-ALL cell lines and clinical samples. Moreover, pathway activity could be modulated by Hh ligand or several pathway inhibitors including cyclopamine and the novel SMOOTHENED (SMO) inhibitor IPI-926. The inhibition of pathway activity primarily impacted highly clonogenic B-ALL cells expressing aldehyde dehydrogenase (ALDH) by limiting their self-renewal potential both in vitro and in vivo. These data demonstrate that Hh pathway activation is common in B-ALL and represents a novel therapeutic target regulating self-renewal and persistence of the malignant clone.     

Hydroperoxy fatty acid formation in selenium deficient rat platelets: coupling of glutathione peroxidase to the lipoxygenase pathway

After incubation with [1-14C]-arachidonic acid, washed platelets from selenium deficient rats produced a sevenfold greater amount of 12-hydroperoxytetraenoic acid than platelets from control animals. When stimulated with either arachidonic acid or t-butyl-hydroperoxide, antimycin-A1 treated platelets from the deficient rats also converted markedly lower amounts of [1-14C]-glucose to [14C]-CO2 than platelets from control rats. These results indicate a significant role for platelet selenium-dependent glutathione peroxidase in the enzymatic reduction of platelet-produced hydroperoxides.     

Induction of histidine decarboxylase in rat basophilic leukemia cells by interferon and prevention of its effect in coincubation with ADP-ribosyltransferase inhibitors

Treatment of rat basophilic leukemia cell line (2H3) with interferon-alpha significantly increased intracellular histamine levels. On the other hand, the histidine content was decreased reciprocally by interferon in a dose-dependent manner. Concomitantly, the activity of histidine decarboxylase, the enzyme responsible for histamine synthesis, was augmented. The increase in histidine decarboxylase activity was partially abolished in co-incubation with inhibitors of ADP-ribosyltransferase, such as 3-aminobenzamide or nicotinamide. These results suggest the pivotal role of activation of histidine decarboxylase, presumably through ADP-ribosylation of the enzyme, in interferon-induced histamine synthesis.     

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.     

IgE receptor-activated calcium permeability pathway in rat basophilic leukemia cells: measurement of the unidirectional influx of calcium using quin2-buffered cells

The intracellular calcium indicator and buffer quin2 has been used to generate a large calcium buffering capacity in the cytoplasm of rat basophilic leukemia cells. Above 3 mM intracellular quin2, there is no further increase in the initial rate of antigen-induced 45Ca uptake, suggesting that 45Ca buffering by quin2 is now sufficient to prevent the immediate efflux of 45Ca from the cells. Thus, the initial rate of 45Ca uptake should reflect the true unidirectional influx of calcium that occurs when immunoglobulin E (IgE) receptors are aggregated by antigen. The antigen-induced calcium permeability pathway appears to be saturable, with a Km of about 0.7 mM and a Vmax of 0.9 nmol of calcium (10(6) cells)-1 min-1. Although net 45Ca uptake reaches a plateau a few minutes after antigen stimulation, the increase in plasma membrane permeability is maintained for at least an hour, provided that receptors for IgE remain aggregated. The initial rate of 45Ca influx correlates well with the subsequent secretion of [3H]serotonin in response to different concentrations of antigen. Both 45Ca uptake and [3H]serotonin secretion are maximal when only 10% of the receptors are occupied with antigen-specific IgE. Thus, 45Ca influx correlates more closely with secretion than with the number of IgE receptors aggregated by antigen.