Effect of tranilast on the release of slow reacting substance of anaphylaxis (SRS-A) and contraction of the smooth muscle

Slow reacting substance of anaphylaxis (SRS-A) and slow reacting substance (SRS) were released from actively sensitized guinea-pig lung with bovine serum albumin and from rat peritoneal exudate cells with ionophore A23187, respectively. FPL55712 markedly inhibited the contraction induced by SRS-A and SRS in guinea-pig ileum which was treated with atropine (10(-7) g/ml), mepyramine (10(-6) g/ml), and cyproheptadine (10(-7) g/ml). Tranilast and isoproterenol markedly suppressed the release of SRS-A in a dose-dependent manner; the concentrations of these drugs that gave 50% inhibition (IC50) were 1.1 X 10(-4)M and 8.3 X 10(-9)M, respectively. Although the inhibitory effect of tranilast (10(-3)M) was not affected in the presence of propranolol (3 X 10(-6)M), the inhibitory effect of isoproterenol was greatly diminished by propranolol. Also, tranilast markedly suppressed the release of SRS in a dose-dependent manner, its IC50 being 6.4 X 10(-5)M. However isoproterenol slightly inhibited the release of SRS. Disodium cromoglycate did not suppressed the release of SRS-A at all, and it suppressed SRS release a little. Tranilast inhibited the contraction induced by leukotriene C4 (0.5 ng/ml) and D4 (1 ng/ml) in guinea-pig trachea in a dose-dependent manner; the IC50 values were 2.2 X 10(-4)M and 2.0 X 10(-4)M, respectively, for these inhibitions. These results suggest that the inhibition of SRS-A release and SRS-A-induced contraction of smooth muscle by tranilast participates in the anti-asthmatic effect of tranilast, and its inhibitory mechanism is different from that of isoproterenol.     

Enzymatic study to characterize the slow reacting substance of anaphylaxis (SRS-A) and leukotrienes

Slow reacting substance of anaphylaxis (SRS-A) has been shown to be one of the major mediators in hypersensitive reactions and to be composed of leukotriene (LT) C4, LTD4 and LTE4. In the present study, we examined the properties of SRS-A released from sensitized guinea pig lungs by antigen and SRS released from rat peritoneal exudate cells and from human leucocytes by ionophore A23187 (0.5 and 0.2 microgram/ml, respectively). By the incubation with SRS-A, SRS and LTs with arylsulfatase (type V) in pH 5.7 buffered solution at 37 degrees C for 30 min, SRS-A and LTD4 were greatly inactivated and rat SRS was slightly inactivated, but human SRS and LTC4 were not inactivated at all. The same results were obtained when aminopeptidase was used in place of arylsulfatase. Moreover, when SRS-A, LTC4 and LTD4 were incubated with 0.02 mg/ml of gamma-glutamyltranspeptidase (gamma-GTP) pH 8.0 buffered solution at 37 degrees C for 30 min, the activities of SRS-A and LTD4 were slightly decreased, but those of SRS and LTC4 were obviously potentiated. On the other hand, incubation with a large amount of gamma-GTP (0.2 mg/ml) a dose at which this enzyme preparation showed clear aminopeptidase activity, SRS-A, SRS, LTC4 and LTD4 were obviously inactivated. In addition, we found a peak of LTD4 in guinea pig SRS-A, that of LTC4 in human SRS, and that of LTC4 in rat SRS on high performance liquid chromatograms. From these results, we demonstrated that guinea pig lung SRS-A is mainly composed of LTD4, human leukocyte SRS is mainly LTC4, and rat peritoneal SRS is composed of both LTC4 and LTD4. The inactivation of LTD4 and SRS-A by arylsulfatase may be due to aminopeptidase contamination in the enzyme preparation.     

Further evidence on the structure of slow reacting substance of anaphylaxis (SRS-A).

Arachidonic acid stimulates the release of SRS-A from the peritoneal cavity of sensitized rats or from rat peritoneal cells incubated in vitro. When rat peritoneal cells are incubated in the presence of tritiated arachidonic acid, significant amounts of radioactivity migrate in parallel to bioactivity on purification with Amberlite XAD-8, DE-52, Silicic acid and Sephadex LH-20. Lipoxidase (E.C. 1.13.1.13 and E.C. 1.13.11.12) inactivates mouse, rat and human SRS-A in a concentration-dependent pattern. Following extensive purification, rat SRS-A is also inactivated by the 2 x crystalline suspension of lipoxidase. These findings suggest (a) that SRS-A is a metabolite of arachidonic acid and (b) because of the strict specificity of lipoxidase, that the SRS-A molecule contains a cis, cis-1, 4-pentadiene and a structure very close either to arachidonic acid, to linoleic acid or to linolenic acid.     

On the preparation of highly purified slow reacting substance of anaphylaxis (SRS-A) from biological extracts.

1 Very highly purified (greater than 100,000 u/mg) slow reacting substance of anaphylaxis (SRS-A) has been prepared by reversed phase high pressure liquid chromatographic (HPLC) techniques. 2 High resolution liquid chromatography suggests that SRS-A may exist in at least three distinct forms which are possible tautomeric. 3 SRS-A collected by antigen challenge in vivo and by calcium ionophore-induced release in vitro are chromatographically indistinguishable. 4 Treatment of SRS-A with diazomethane but not sodium borohydride results in a loss of biological activity but treatment of the methyl ester with base results in a partial recovery of activity. 5 Highly purified SRS-A was examined by infrared and ultra-violet spectroscopy, and found to have a benzene-aromatic and probably an amino acid.     

The partial purification of slow reacting substance of anaphylaxis from rat peritoneal anaphylactic fluid and its separation from an arachidonic acid releasing substance

A method is described for the separation of Slow Reacting Substance of Anaphylaxis (SRS-A) from the other mediators of anaphylaxis. This method has the advantage of a high overall yield of biologically pure SRS-A, although chemical homogeneity is not obtained. Silicic acid chromatography of the material extracted by ethyl acetate revealed the presence of an arachidonic acid releasing substance (ARS). ARS is less polar than SRS-A and releases arachidonic acid and thromboxanes from perfused guinea-pig lungs. ARS can be distinguished from the peptide RCS-RF by its solubility in apolar solvents.     

The release of prostaglandins and thromboxanes from guinea-pig lung by slow reacting substance of anaphylaxis, and its inhibition.

1 Rabbit aorta contracting substance (RCS; consisting mainly of thromboxane A2) and prostaglandin-like material were released from guinea-pig isolated perfused lungs by injection of slow reacting substance of anaphylaxis (SRS-A). 2 SRS-A was resistant to boiling and proteolytic enzymes and was therefore distinguished from rabbit aorta contracting substance releasing factor (RCS-RF). 3 The release of RCS and prostaglandin-like material by SRS-A was anatagonized by indomethacin (1 microgram/ml), betamethasone and dexamethasone (4 to 50 microgram/ml). 4 Imidazole (200 microgram/ml) inhibited the formation of thromboxane A2 but not that of prostaglandins. 5 The activity of SRS-A on guinea-pig ileum and its ability to release RCS and prostaglandins were destroyed by incubation with arylsulphatase (0.83 microgram to 1 mg/ml) and with lipoxidase (16.5 to 50 microgram/ml): SRS-A lost activity on incubation with bovine serum albumin (9 microgram/ml) due to protein binding.     

Release of mediators of anaphylaxis: inhibition of prostaglandin synthesis and the modification of release of slow reacting substance of anaphylaxis and histamine.

1 When isolated perfused lungs from sensitized guinea-pigs were challenged with antigen, histamine, slow reacting substance of anaphylaxis (SRS-A) and prostaglandin-like substances were released into the effluent. 2 Treatment of the lungs before and during challenge with indomethacin (0.5--10 microgram/ml), sodium aspirin (1--10 microgram/ml), sodium meclofenamate (0.1--1 microgram/ml) or ketoprofen (0.5--5 microgram/ml) inhibited the release of prostaglandins while increasing the output of histamine and SRS-A between three- and five-fold. 3 Diethylcarbamazine (0.2--1 mg/ml) reduced the release of SRS-A and histamine but increased the amount of prostaglandin-like substances produced. 4 Eicosatetraynoic acid (10 microgram/ml) inhibited formation of prostaglandins but did not modify release of histamine and SRS-A. 5 The results with non-steroid anti-inflammatory drugs and diethylcarbamazine suggest that prostaglandins, or some other product of the cyclo-oxygenase system, depress the anaphylactic release of SRS-A and histamine.     

Effect of variation in endogenous levels of ascorbic acid on the in vitro immunological release of histamine and slow reacting substance of anaphylaxis from actively sensitized guinea-pig lung fragments

1. The in vitro immunological release of histamine and slow reacting substance of anaphylaxis (SRS-A) from actively sensitized guinea-pig lung fragments was greatly reduced when the animals were maintained on an ascorbic acid-deficient diet. Excessive dietary levels of ascorbic acid did not increase mediator release above normal levels. 2. Restoration of ascorbic acid in the diet of scorbutic guinea-pigs restored in vitro immunological histamine to normal levels. 3. Variation in dietary levels of ascorbic acid had no effect on lung histamine content. 4. The lung ascorbic acid content was proportional to the dietary intake. Approximately 60% of the total lung ascorbic acid was removed by the process of chopping and washing of the tissue. This relationship was independent of dietary intake. 5. The results indicate that the immunological release of mediators of inflammation from guinea-pig lung is dependent on adequate endogenous levels of ascorbic acid.     

Increase in antigen-induced release of slow reacting substance of anaphylaxis from guinea pig lung by sodium fluoride

Some of the mechanisms involved in the release of mediators of anaphylaxis from passively sensitized guinea pig lung were examined. NaF (10 mM) markedly enhanced antigen-induced release of slow reacting substance of anaphylaxis (SRS-A), whereas release of histamine was not changed. Similar results were obtained with equimolar amounts of KF but not with NaCl. When SRS-A, released from lung in the presence of NaF, was applied to guinea pig ileum, the tissue contracted. This response was selectively antagonized by FPL 55712, an SRS-A antagonist. A23187, a divalent cation ionophore, released histamine and SRS from guinea pig lung. NaF and KF, but not NaCl, reduced equally the amounts of mediators released by A23187. These results indicate that antigen-induced release of histamine and SRS-A can be dissociated by fluoride ions. Furthermore, they suggest that NaF may prove useful in dissecting and understanding some of the complex biochemical steps involved in the release of SRS-A.     

Effects of modulators of arachidonic acid metabolism on the synthesis and release of slow-reacting substance of anaphylaxis.

1 Slow-reacting substance of anaphylaxis (SRS-A) was released in the peritoneum of passively sensitized rats challenged with ovalbumin and from rat isolated peritoneal cells stimulated with calcium ionophore A23187. 2 Both monocytes (macrophages) and mast cells appear to be involved in the synthesis and release of SRS-A. 3 The immunological release of SRS-A in vivo is enhanced by indomethacin and inhibited by dexamethasone, mepacrine, 1-phenyl-3-pyrazolidone (1-P-3-P), and methylimidazole. 4 SRS-A release induced by A23187 in vitro is inhibited by dexamethasone, indomethacin 1-P-3-P, eicosatetraynoic acid (ETA) and 15-hydroperoxy arachidonic acid. The inhibition induced by dexamethasone, indomethacin and 1-P-3-P is reduced by an increase in the calcium concentration from 1 mM to 5 mM, whereas the inhibition induced by ETA is increased. 5 The results suggest that a lipoxygenase is important in the synthesis and release by SRS-A.