Priscilla J. Piper

The effect of leukotrienes C4 and D4 on the microvasculature of guinea-pig skin

The effects of chemically-synthesised leukotrienes C4 and D4 (5(S) hydroxy-6(R)-gamma-glutamylcysteinylglycinyl-7,9,11,14-eicosa-4 tetraenoic acid, LTC4; 5(S) hydroxy-6(R)-cysteinylglycinyl-7,9,11,14-eicosatetraenoic acid, LTD4 on the microvasculature have been measured in guinea-pig skin using [125I]-albumin accumulation to measure plasma exudation and 133Xe clearance to measure blood flow changes. As previously shown using biosynthetic material, LTD4 caused vasoconstriction resulting in reduced blood flow. Similarly, LTC4 was found to have vasoconstrictor activity but was more potent and had a steeper dose-response curve than LTD4. There was no evidence of conversion of exogenous arachidonic acid to vasoconstrictor activity in the skin in vivo (in the absence of another stimulus): intradermally injected arachidonic acid produced vasodilatation, but induced little change in blood flow in animals pretreated with indomethacin. The vasodilator effect of arachidonic acid is presumed to be due to conversion to either PGE2 or PGI2. These results suggest that cyclo-oxygenase is normally active in the skin, whilst lipoxygenase requires activation in some way. As reported in a previous study, LTD4 induced plasma exudation when injected into the skin, but pronounced responses could only be induced by LTD4 mixed with a vasodilator prostaglandin such as PGE2. In contrast, LTC4 induced no exudation when tested alone and little when PGE2 was added. However, evidence was obtained that LTC4 has some permeability-increasing activity which is masked by its potent vasoconstrictor activity.

Release of rabbit aorta contracting substance (RCS) and prostaglandins induced by chemical or mechanical stimulation of guinea‐pig lungs

1 Rabbit aorta contracting substance (RCS) and prostaglandins were released from guinea‐pig isolated perfused lungs by gentle massage and also by infusion of Prosparol. 2 RCS and prostaglandins were also released by infusion into the pulmonary artery of bradykinin, arachidonic and dihomo‐γ‐linolenic acids or shock perfusate (containing RCS‐releasing factor). 3 Arachidonic and dihomo‐γ‐linolenic acids caused a prolonged release of RCS and prostaglandins whereas bradykinin and shock perfusate gave a short‐lasting output. 4 RCS and prostaglandins, together with histamine were released when super‐fused chopped lung tissue was agitated. 5 Challenge of sensitized guinea‐pigs in vivo led to the release of an RCS‐like substance into the carotid arterial blood. 6 Intravenous injection of bradykinin into guinea‐pigs also released an RCS‐like substance. 7 The release of RCS and prostaglandins was inhibited by aspirin or indomethacin in all experiments. 8 RCS contracted all vascular tissues investigated and also rat stomach strip. 9 The half‐life of RCS was estimated as 1–2 minutes.

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.

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 μg/ml), sodium aspirin (1–10 μg/ml), sodium meclofenamate (0.1–1 μg/ml) or ketoprofen (0.5–5 μg/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 μg/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.

Inhibitors of nitric oxide synthase selectively reduce flow in tumour-associated neovasculature

1 The effects of l‐arginine analogues, NG‐nitro‐l‐arginine methyl ester (l‐NAME) and NG‐monomethyl‐l‐arginine (l‐NMMA) and methylene blue on blood flow in a murine adenocarcinoma and melanoma have been investigated. 2 Sponge implants in Balb/c and C57/BL mice were used to host proliferating tumour cells while the washout of 133Xe was employed to assess local blood flow in the implanted sponges. 3 Pharmacological inhibition of nitric oxide (NO) reduced blood flow in both tumours but this effect was reversed by administration of l‐arginine. 4 In marked contrast, the effect of these same NO inhibitors on the blood flow in sponge‐induced non‐neoplastic granulation tissue was negligible. 5 These results strongly suggest that: (a) flow in tumour vessels is modulated by nitric oxide which maintains a dilator tone in neoplastic tissue; (b) the constrictor activity (as monitored by an increase in t½ of 133Xe) of NO inhibitors may be attributed to the removal of such dilator tone; (c) many of the abnormalities described in tumour vasculature, such as hyporeactivity or unresponsiveness to vasoactive mediators and maximum vasodilatation, may be due to an increase in NO synthesis in cancers.

STIMULATION OF ARACHIDONIC ACID METABOLISM AND GENERATION OF THROMBOXANE A2 BY LEUKOTRIENES B4, C4 AND D4 IN GUINEA‐PIG LUNG in vitro

1 Leukotriene C4 (LTC4), LTD4, slow‐reacting substance of anaphylaxis (SRS‐A) (from guinea‐pig lung), bradykinin (Bk) and arachidonic acid (AA) release thromboxane A2 (TxA2) and prostaglandin‐like materials from guinea‐pig isolated perfused lungs. 2 Release of TxA2 induced by LTC4 and LTD4 is inhibited by a thromboxane synthetase inhibitor, imidazole (2.9 mm). 3 Mepacrine (200 μm), a phospholipase inhibitor, inhibits release of TxA2 and prostaglandin‐like materials caused by SRS‐A and Bk but not that due to exogenous AA 4 Leukotrienes B4, C4 and D4 are approximately equipotent in inducing dose‐related contractions of guinea‐pig parenchymal strips (GPPs). 5 Leukotriene‐induced contractions of GPPs are greatly inhibited by imidazole (2.9 mm), carbox‐yheptylimidazole (24 μm) and mepacrine (400 μm). 6 FPL 55712 (1.9 μm), the SRS‐A antagonist, blocks contractions of GPPs induced by LTC4 and LTD4 but not those due to LTB4 or Bk. 7 Tachyphylaxis to LTB4 occurs in GPPs but not to LTC4 or LTD4. 8 These results suggest that in guinea‐pig lung in vitro, LTB4, LTC4 and LTD4 activate a phospholipase with subsequent generation of cyclo‐oxygenase products of which TxA2 plays an important role.

THE ACTIONS OF LEUKOTRIENES C4 AND D4 ON GUINEA‐PIG ISOLATED HEARTS

1 Infusions of leukotrienes C4 (LTC4) and LTD4 (5 min; 4 × 10−10‐2 × 10−8 m) produced dose‐dependent decreases in coronary flow in Langendorff preparations of guinea‐pig isolated hearts. 2 LTC4 was more active than LTD4 and produced a greater reduction in coronary flow. 3 LTC4 and LTD4 also caused reduction in contractility in perfused hearts but not in isolated atria or driven ventricular strips. 4 There was a greater reduction in contractility to LTD4 compared with LTC4 at doses which produced approximately 50% reduction in coronary flow. 5 Indomethacin (1.4 × 10−5 m) inhibited the effects of LTC4 but only reduced the initial LTD4‐induced effects. 6 The effects of FPL 55712 (3.8 × 10−6 m) were similar to those of indomethacin. 7 LTC4 and LTD4 may therefore contribute to the abnormalities of cardiac function that occur in immediate hypersensitivity reactions, particularly reduction in coronary flow.

Generation of a leukotriene-like substance from porcine vascular and other tissues.

When chopped porcine vascular tissue was incubated with A23187 in the presence of a cyclo-oxygenase inhibitor, a substance with the biological properties of a leukotriene was released into the supernatant. The highest concentrations of leukotriene-like material were released by coronary and pulmonary arteries and the adventitia removed from these vessels, the greatest amount being released by the adventitia. Smaller quantities of leukotriene-like substance were released from other blood vessels but not from aorta, atrial or ventricular smooth muscle. Release of the leukotriene-like material was time-dependent. The activity of the leukotriene-like substance was antagonised by FPL 55712 and its release was inhibited by BW 755c. Leukotriene-like material was also generated from guinea-pig, rat, rabbit and dog blood vessels incubated with A23187 and from guinea-pig vessels during antigen challenge.

The release of spasmogenic substances from human chopped lung tissue and its inhibition

1 Human lung tissue, passively sensitized with reaginic antibodies, released prostaglandins E1, E2 and F2α in addition to histamine and slow reacting substance (SRS‐A), when exposed to the appropriate antigen. No rabbit aorta contracting substance (RCS) was detected. 2 Experiments with rats and guinea‐pigs showed that the release of RCS is not confined to anaphylactic reactions mediated by non‐reaginic antibodies but may be a feature of anaphylaxis in guinea‐pigs alone. 3 Human lung tissue gently agitated with a blunt nylon rod liberated an E‐type prostaglandin and RCS in addition to histamine and SRS‐A. 4 Human isolated bronchial muscle was contracted by RCS. 5 Disodium cromoglycate antagonized the release of prostaglandins during anaphylaxis but not during agitation of human lung tissue, whereas indomethacin blocked the release of prostaglandins during agitation and anaphylaxis. 6 The release of an E‐type prostaglandin during anaphylaxis in human lung tissue, which inhibits the further release of histamine could be another example of the regulatory role of prostaglandins in body functions.

The action of chemically pure SRS-A on the microcirculation in vivo

The purification of SRS-A for the purpose of structure determination has enabled us to investigate whether pure SRS-A has activity on the microvasculature. SRS-A from challenged sensitised lung in vitro was purified using five stages of purification. At each stage SRS-A activity was assayed against an in-house standard using the guinea-pig ileum blocked with mepyramine and hyoscine. The material obtained at each stage was then tested for its ability to induce plasma exudation (measured using the accumulation of intravenously-injected [131I]-albumin) in guinea-pig skin. It was found that vascular permeability-increasing activity corresponded with guinea-pig ileum contracting activity throughout the purification procedure. The final product, homogeneous SRS-A, at doses of 4 - 6 ng, produced a clear increase in vascular permeability. Two other lipoxygenase products which have been proposed to be derived from the same hydroperoxide intermediate as SRS-A, 5-hydroxyeicosatetraenoic acid and 5,12-dihydroxyeicosatetraenoic acid (leukotriene B), showed little effect on vascular permeability. PGE1 was found to potentiate plasma exudation induced by SRS-A to a greater extent than that induced by histamine. SRS-A, as a permeability-increasing agent in the presence of PGE1, was approximately 400 times more potent (on a molar basis) than histamine. When 133Xe was used to measure blood flow changes, chemically pure SRS-A was found to reduce flow in skin; 4 - 6 ng of SRS-A producing a 40-50% reduction. It is suggested that these actions of SRS-A may be important in pathophysiological conditions.