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.     

The mechanism of action of leukotrienes C4 and D4 in guinea-pig isolated perfused lung and parenchymal strips of guinea pig, rabbit and rat

The biological actions of pure slow-reacting substance of anaphylaxis (SRS-A) from guinea-pig lung, pure slow-reacting substance (SRS) from rat basophilic leukaemia cells (RBL-1) and synthetic leukotrienes C4 (LTC4) and D4 (LTD4) have been investigated on lung tissue from guinea pig, rabbit and rat. In the guinea pig, the leukotrienes released cyclo-oxygenase products from the perfused lung and contracted strips of parenchyma. The effects of SRS-A, SRS and LTD4 were indistinguishable. LTC4 and LTD4 had similar actions although LTD4 was more potent than LTC4. Indomethacin (1 microgram/ml) inhibited the release of cyclo-oxygenase products from perfused guinea-pig lung and caused a marked reduction in contractions of guinea-pig parenchymal strips (GPP) due to LTC4 and LTD4. The residual contraction of the GPP was abolished by FPL 55712 (0.5 - 1.0 microgram/ml). It appears, therefore, that a major part of the constrictor actions of LTC4 and LTD4 in guinea-pig lung are mediated by myotropic cyclo-oxygenase products, i.e. thromboxane A2 (TxA2) and prostaglandins (PGs). In rabbit and rat lung, however, SRS-A, SRS and the leukotrienes were much less potent in contracting parenchymal strips and there was little evidence of the release of cyclo-oxygenase products. FPL 55712 at a concentration of 1 microgram/ml failed to antagonise leukotriene-induced contractions.     

Identification and characterization of leukotriene D4 receptors and signal transduction processes in rat basophilic leukemia cells

The leukotriene D4 (LTD4) receptor on rat basophilic leukemia (RBL-1) cell membranes was characterized using a radioligand binding assay. [3H]LTD4 binding to RBL-1 membrane receptors was stereoselective, specific, and saturable. The binding affinity and maximum binding density of [3H]LTD4 to RBL-1 membrane receptors were 0.9 +/- 0.2 nM and 800 +/- 125 fmol/mg protein, respectively. Binding of [3H]LTD4 to the receptors was enhanced by divalent cations (Ca2+, Mg2+, and Mn2+) and inhibited by guanine nucleotides and sodium ions, specifically, indicating that a guanine nucleotide-binding protein may regulate the agonist-receptor interaction. LTD4, LTE4 agonist and antagonist analogs competed with the radioligand in binding to the RBL-1 LTD4 receptors. The binding affinities of these analogs correlated with (a) those determined from the guinea pig lung LTD4 receptors and (b) the pharmacological activities in smooth muscle contraction. LTD4 and related agonists also induced time- and concentration-dependent phosphatidylinositol hydrolysis in RBL-1 cells. The LTD4 induction of inositol 1-phosphate was potent, stereoselective, specific, and was blocked by LTD4 receptor antagonists. The rank order potency of agonist-induced inositol 1-phosphate formation in RBL-1 cells was equivalent to the receptor binding affinity determined using either RBL-1 cell or guinea pig lung membranes. These studies have demonstrated the G protein coupled LTD4 receptors on RBL-1 cell membranes. Binding of agonists to the receptor may activate the G protein-regulated phospholipase C to induce hydrolysis of phosphatidylinositol. The hydrolytic products of phosphatidylinositol, possibly inositol trisphosphate and diacylglycerol, may be the intracellular messengers for LTD4 receptors in RBL-1 cells.     

Purification and characterization of a phosphoinositide-specific phospholipase C from guinea pig uterus. Phosphorylation by protein kinase C in vivo

Two peaks of phosphoinositide-specific phospholipase C (PI-PLC) activity were resolved when guinea pig uterus cytosolic proteins were chromatographed on a DEAE-Sepharose column. The first peak of enzyme activity eluting from the DEAE-Sepharose column (PI-PLC I) was further purified to homogeneity, whereas the second peak of enzyme activity was enriched 300-fold. PI-PLC I migrated as a 62-kDa protein on sodium dodecyl sulfate-polyacrylamide gels. Antibodies prepared against PI-PLC I failed to react with PI-PLC II. PI-PLC I hydrolyzed all three phosphoinositides, exhibiting a greater Vmax for phosphatidylinositol 4,5-bisphosphate greater than phosphatidylinositol 4-phosphate greater than phosphatidylinositol. Hydrolysis of phosphatidylinositol was calcium-dependent, whereas significant hydrolysis of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate occurred in the presence of 2.5 mM EGTA. At physiological concentrations of calcium, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were the preferred substrates. Antibodies specific for PI-PLC I reacted with a 62-kDa protein in both the cytosol and membrane fractions from guinea pig uterus. Quantitation of the immunoblots revealed that 25% of the 62-kDa protein was membrane-associated, whereas only 5% of the total enzyme activity was membrane-associated. Approximately 20% of the membrane-bound phospholipase C activity and immunoreactive material were loosely bound, whereas the remainder required detergent extraction for complete solubilization. The 62-kDa protein associated with the membrane fractions did not bind lectin affinity columns, suggesting that it was not glycosylated. PI-PLC I was identified as a phosphoprotein in [32P]orthophosphate-labeled rat basophilic leukemia (RBL-1) cells by two-dimensional gel electrophoresis followed by immunoblotting. In untreated cells, 32P-labeled PI-PLC I was found in the cytosolic fraction. Treatment of RBL-1 cells with those phorbol esters which are known to activate the Ca2+/phospholipid-dependent enzyme protein kinase C, resulted in a time-dependent increase in the phosphorylation of both membrane-bound and cytosolic PI-PLC I. Thus, in RBL-1 cells, protein kinase C may play an important role in the regulation of phospholipase C through protein phosphorylation.     

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.     

Metabolism of cyclic GMP in peritoneal macrophages of rat and guinea pig

The aim of our studies was to establish which enzymes constitute the "cGMP pathway" in rat and guinea pig peritoneal macrophages (PM). We found that in guinea pig PM synthesis of the nucleotide was significantly enhanced in response to activators of soluble guanylyl cyclase (sGC) and it was only slightly stimulated by specific activators of particulate guanylyl cyclases (pGC). In contrast, rat PM responded strongly to atrial natriuretic peptide (ANP), the activator of pGC type A. The rat cells synthesized about three-fold more cGMP than an equal number of the guinea pig cells. The activity of phosphodiesterases (PDE) hydrolyzing cGMP was apparently regulated by cGMP itself in PM of both species and again it was higher in the rat cells than in those isolated from guinea pig. However, guinea pig PM revealed an activity of Ca(2+)/calmodulin-dependent PDE1, which was absent in the rat cells. Using Western blotting analysis we were unable to detect the presence of cGMP-dependent protein kinase 1 (PKG1) in PM isolated from either species. In summary, our findings indicate that particulate GC-A is the main active form of GC in the rat PM, while in guinea pig macrophages the sGC activity dominates. Since the profiles of the PDE activities in rat and guinea pig PM are also different, we conclude that the mechanisms regulating cGMP metabolism in PM are species-specific. Moreover, our results suggest that targets for cGMP other than PKG1 should be present in PM of both species.     

Comparative enzymology of 11 beta -hydroxysteroid dehydrogenase type 1 from glucocorticoid resistant (Guinea pig) versus sensitive (human) species

Type 1 11 beta-hydroxysteroid dehydrogenase constitutes a prereceptor control mechanism through its ability to reduce dehydroglucocorticoids to the receptor ligands cortisol and corticosterone in vivo. We compared kinetic characteristics of the human and guinea pig 11 beta-hydroxysteroid dehydrogenase isozymes derived from species differing in glucocorticoid sensitivity. Both orthologs were successfully expressed as full-length enzymes in yeast and COS7 cells and as soluble transmembrane-deleted constructs in Escherichia coli. Both isozymes display Michaelis-Menten kinetics in intact cells and homogenates and show low apparent micromolar K(m) values in homogenates, which are lowered by approximately one order of magnitude in intact cells, allowing corticosteroid activation at physiological glucocorticoid levels. Recombinant soluble proteins were expressed and purified with high specific dehydrogenase and reductase activities, revealing several hundred-fold higher specificity constants than those reported earlier for the purified native enzyme. Importantly, these purified soluble enzymes also display a hyperbolic dependence of reaction velocity versus substrate concentration in 11-oxoreduction with K(m) values of 0.8 microm (human) and 0.6 microm (guinea pig), close to the values obtained from intact cells. Active site titration was carried out with the human enzyme using a novel inhibitor compound and reveals a fraction of 40-50% active sites/mol total enzyme. The kinetic data obtained argue against the involvement of 11 beta-hydroxysteroid dehydrogenase as a modulating factor for the glucocorticoid resistance observed in guinea pigs. Instead, the expression of 11 beta-hydroxysteroid dehydrogenase type 1 in the Zona glomerulosa of the guinea pig adrenal gland suggests a role of this enzyme in mineralocorticoid synthesis in this hypercortisolic species.     

Stimulation of T-cell activation by UV-treated, antigen-pulsed macrophages: evidence for a requirement for antigen processing and interleukin 1 secretion

The nature of the defect(s) in the ability of UV-treated guinea pig macrophages to stimulate the proliferative response of guinea pig T cells to soluble protein antigens was investigated. T cells proliferated vigorously when cultured with peritoneal exudate cells (PEC) which had been pulsed with soluble protein antigens, but failed to proliferate when cultured with soluble antigen or with antigen-pulsed, UV-treated PEC. UV-treated macrophages were unable to secrete interleukin 1 (IL-1). Addition of IL-1 partially restored the T-cell proliferative response stimulated by antigen-pulsed, UV-treated PEC. However, IL-1 was able to restore such a response only when the PEC were pulsed with antigen before being exposed to UV. Similar results were obtained when antigen-pulsed PEC were used to stimulate T cells to secrete interleukin 2 (IL-2). These results demonstrate that UV-treated macrophages are defective both in their ability to properly process and present antigen for T-cell recognition and in their ability to secrete IL-1.     

The mechanism of action of leukotrienes C4 and D4 in guinea-pig isolated perfused lung and parenchymal strips of guinea pig, rabbit and rat

The biological actions of pure slow-reacting substance of anaphylaxis (SRS-A) from guinea-pig lung, pure slow-reacting substances (SRS) from rat basophilic leukaemia cells (RBL-1) and synthetic leukotrienes C₄ (LTC₄) and D₄ (LTD₄) have been investigated on lung tissue from guinea pig, rabbit and rat. In the guinea pig, the leukotrienes released cyclo-oxygenase products from the perfused lung and contracted strips of parenchyma. The effects of SRS-A, SRS and LTD₄ were indistinguishable. LTC₄ and LTD₄ had similar actions although LTD₄ was more potent than LTC₄. Indo-methacin (1 μg/ml) inhibited the release of cyclo-oxygenase products from perfused guinea-pig lung and caused a marked reduction in contractions of guinea-pig parenchymal strips (GPP) due to LTC₄ and LTD₄. The residual contraction on the GPP was abolished by FPL 55712 (0.5 – 1.0 μg/ml). It appears, therefore, that a major part of the constrictor actions of LTC₄ and LTD₄ in guinea-pig lung are mediated by myotropic cyclo-oxygenase products, i.e. thromboxane A₂ (TxA₂) and prostaglandins (PGs).In rabbit and rat lung, however, SRS-A, SRS and the leukotrienes were much less potent in contracting parenchymal strips and there was little evidence of the release of cyclo-oxygenase products. FPL 55712 at a concentration of 1 μg/ml failed to antagonise leukotriene-induced contractions.     

Production of migration inhibitory factor (MIF) by human leukocytes following exposure to Epstein-Barr virus.

The ability of Epstein-Barr virus (EBV) to induce a cell-mediated immune response (CMI) in cultures of human leukocytes was investigated. Partially purified EBV, obtained from culture fluids of AV-1 cells, was inactivated by uv-irradiation. Inactivated virus was mixed with peripheral leukocytes from Hodgkin's disease (HD), infectious mononucleosis (IM) and malignant lymphoma patients as well as from normal individuals in an in vitro culture system. Production of migration inhibitory factor (MIF), as measured by guinea pig macrophage migration inhibition (MMI), was utilized as an indicator of CMI response. Significant differences in MIF response were observed subsequent to exposure of the cells to EBV. Leukocytes from patients in each of the disease categories tested exhibited greater MIF production than did those from the normal controls. There were significant differences in MIF production by leukocytes from the malignant and non-malignant disease categories. Serum from each subject was examined for immunoglobulin specific for EBV capsid antigen (anti-VCA). Although the majority of individuals within the disease categories tested had elevated anti-EBV serum titers, no correlation could be made between elevated anti-VCA titer and levels of MIF production.     

Species comparison of adenosine A1 receptors in isolated mammalian atrial and ventricular myocardium

Various species have been used as models to study the effects of adenosine (ADO) on atrial and ventricular myocardium, but few direct tissue comparisons between species have been made. This study further characterizes adenosine A(1) receptor binding, adenylate cyclase activity and direct and indirect A(1) receptor-mediated functional activity in atrial and ventricular tissue from Sprague-Dawley rats and Hartley guinea pigs. Rat right atria (RA) were found to be significantly more sensitive to cyclopentyladenosine (CPA), while guinea pig left atria (LA) were more sensitive to CPA. After the addition of isoproterenol (ISO), the reduction of CPA response in rat RA was significantly greater than in guinea pig; however, after ISO treatment, the guinea pig LA was more sensitive to CPA than the rat. Adenylate cyclase inhibition by CPA was significantly greater in atria and ventricles obtained from guinea pig than rat. In competition binding experiments, guinea pig RA had significantly more high affinity sites than rat, but the K(i)s were not significantly different. There were no significant differences between guinea pig LA and rat LA. Guinea pig ventricular tissue had fewer high affinity sites than rat without any differences in their K(i) values. In antagonist saturation experiments, the density and affinity of A(1) receptors in guinea pig cardiac membranes were significantly greater than in rat. Our results indicate definite species differences as well as tissue differences between rat and guinea pig. These differences must be considered when interpreting studies using rat and guinea pig tissue as models for cardiac function.     

Possible role of macrophage glycolipids as receptors for migration inhibitory factor (MIF).

Guinea pig peritoneal exudate cells incubated with water soluble glycolipids obtained from macrophages show an enhanced response to migration inhibitory factor. Incorporation of these glycolipids into liposomes greatly facilitates their interaction with indicator cells. Enhancement of peritoneal exudate cell responsiveness to migration inhibitory factor was specific for glycolipids from guinea pig macrophages. Glycolipids extracted from guinea pig brain and polymorphonuclear leukocytes as well as several bovine and porcine glycolipids had no effect. Specificity of enhancement was not due merely to a preferential association of macrophage glycolipids with indicator cells. The possible role of macrophage glycolipids as receptors for MIF is discussed.     

Structure of the immunodominant surface antigen from the Toxoplasma gondii SRS superfamily

Toxoplasma gondii is a persistent protozoan parasite capable of infecting almost any warm-blooded vertebrate. The surface of Toxoplasma is coated with a family of developmentally regulated glycosylphosphatidylinositol (GPI)-linked proteins (SRSs), of which SAG1 is the prototypic member. SRS proteins mediate attachment to host cells and interface with the host immune response to regulate the virulence of the parasite. The 1.7 A structure of the immunodominant SAG1 antigen reveals a homodimeric configuration in which the dimeric interface is mediated by an extended beta-sheet that forms a deep groove lined with positively charged amino acids. This basic groove seems to be conserved among SRS proteins and potentially serves as a sulfated proteoglycan-binding site on target cell surfaces, thus rationalizing the promiscuous attachment properties of Toxoplasma to a broad range of host cell types.     

Leukotriene C4 production during hypoxic pulmonary vasoconstriction in isolated rat lungs

Leukotriene inhibitors preferentially inhibit hypoxic pulmonary vasoconstriction in isolated rat lungs. If lipoxygenase products are involved in the hypoxic pressor response they might be produced during acute alveolar hypoxia and a leukotriene inhibitor should block both the vasoconstriction and leukotriene production that occurs in response to hypoxia. We investigated in isolated blood perfused rat lungs whether leukotriene C4 (LTC4) could be recovered from whole lung lavage fluid during ongoing hypoxic vasoconstriction. Lung lavage from individual rats had slow reacting substance (SRS)-like myotropic activity by guinea pig ileum bioassay. Pooled lavage (10 lungs) as analyzed by reverse phase high performance liquid chromatography had an ultraviolet absorbing component at the retention time for LTC4. At radioimmunoassay, and SRS myotropic activity by bioassay. LTC4 was not found during normoxic ventilation, during normoxic ventilation after a hypoxic pressor response, or during vasoconstriction elicited by KCl. Diethylcarbamazine citrate, a leukotriene synthesis blocker, concomitantly inhibited the hypoxic vasoconstriction and LTC4 production. Thus 5-lipoxygenase products may play a role in the sequence of events leading to hypoxic pulmonary vasoconstriction.     

Tumor necrosis factor as an interleukin 1-dependent differentiation inducing factor (D-factor) for mouse myeloid leukemic cells

Experiments were conducted to purify the differentiation-inducing factor (D-factor), which induces differentiation of mouse myeloid leukemic cell line, Ml, into macrophage-like cells, in a conditioned medium of guinea pig peritoneal macrophages stimulated with lipopolysaccharide. On gel filtration under high performance liquid column chromatography (HPLC), D-factor eluted at the position of 45-15 KD. By the subsequent separation on DEAE HPLC the D-factor activity disappeared. However, in the presence of recombinant human IL 1 alpha the D-factor activity appeared at a position where tumor necrosis factor (TNF) eluted. Even after fractionation on hydroxyapatite HPLC the IL 1-dependent D-factor was co-chromatographed with TNF. Recombinant human TNF as well as the partially purified guinea pig TNF induced differentiation of Ml cells in conjunction with either the partially purified guinea pig IL 1 or recombinant human IL 1 alpha, although these factors by themselves did not induce differentiation. These findings suggest that a part of D-factor activity in the conditioned medium resulted from the cooperative effects between TNF and IL 1.     

Prokaryotic Expression and In Vitro Functional Analysis of IL-1β and MCP-1 from Guinea Pig

The Guinea pig (Cavia porcellus) is an excellent animal model for studying human tuberculosis (TB) and also for a number of other infectious and non-infectious diseases. One of the major roadblocks in effective utilization of this animal model is the lack of readily available immunological reagents. In order to address this issue, guinea pig interleukin 1 beta (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) were efficiently cloned and expressed in a prokaryotic expression vector, and the expressed proteins in soluble form from both the genes were confirmed by N-terminal sequencing. The biological activity of recombinant guinea pig IL-1β was demonstrated by its ability to drive proliferation in thymocytes, and the recombinant guinea pig MCP-1 exhibited chemotactic activity for guinea pig resident peritoneal macrophages. These biologically active recombinant guinea pig proteins will facilitate an in-depth understanding of the role they play in the immune responses of the guinea pig to TB and other diseases.     

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.     

Rat sperm 2B1 glycoprotein (PH20) contains a C-terminal sequence motif for attachment of a glycosyl phosphatidylinositol anchor. Effects of endoproteolytic cleavage on hyaluronidase activity

Rat sperm 2B1 antigen (the orthologue of guinea pig sperm PH20) is a plasma membrane-bound glycoprotein that is endoproteolytically cleaved during passage through the epididymis and subsequently migrates from the tail to the acrosomal domain during capacitation. Unlike guinea pig PH20, however, sperm surface 2B1 is insensitive to phosphatidylinositol phospholipase C, nor is it known how endoproteolytic cleavage affects its hyaluronidase activity. In this investigation we have expressed 2B1 cDNA in Chinese hamster ovary cells; we have shown that it contains an internal sequence motif for attachment of a glycosyl phosphatidylinositol (GPI) anchor and that cleavage from a single- into a two-chain molecule causes a significant shift in the optimum pH for hyaluronidase activity. Functionally, these results suggest that 1) 2B1 glycoprotein on rat spermatozoa is attached to the plasma membrane via a GPI anchor and that this is an important factor in its ability to migrate from the tail to the acrosomal domain during capacitation; and 2) endoproteolytic cleavage of 2B1 serves to optimize its hyaluronidase activity immediately before fertilization, thereby facilitating penetration of spermatozoa through the cumulus oophorus.