Rabbit Aorta Contracting Substance Research Articles

Nutrition classics. Proceedings of the National Academy of Sciences of the United States of America, August 1975, Volume 72: Thromboxanes: a new group of biologically active compounds derived from prostaglandin endoperoxides. By Mats Hamberg, Jan Svensson, and Bengt Samuelsson.

An unstable [t1/2 at 37 degrees = 32 +/- 2 (SD) sec] intermediate, thromboxane A2, was detected in the conversion of prostaglandin G2 into 8-(1-hydroxy-3-oxopropyl)-9,12L-dihydroxy-5,10-heptadecadienoic acid (thromboxane B2) in platelets. The intermediate was trapped by addition of methanol, ethanol, or sodium azide to suspensions of washed human platelets incubated for 30 sec with arachidonic acid or prostaglandin G2. The structures of the resulting derivatives demonstrated that the intermediate possessed an oxane ring as in thromboxane B2 but lacked its hemiacetal hydroxyl group. Additional experiments using 18O2 or [2H8]arachidonic acid in the formation of thromboxane B2 and CH3O2H for the trapping of thromboxane A2, together with information on the t1/2 of the intermediate, indicated the presence of an oxetane structure in thromboxane A2. Incubation of arachidonic acid or prostaglandin G2 with washed platelets led to formation of an unstable factor that induced irreversible platelet aggregation and caused release of [14C]serotonin from platelets that had been incubated with [14C]serotonin. The properties and the mode of formation of this factor indicated that it was identical with thromboxane A2. Furthermore, evidence is presented that the more unstable and major component of rabbit aorta contracting substance (RCS) formed in platelets and guinea pig lung is also thromboxane A2.

Thromboxane A2 antagonist--discovery of seratrodast

We were interested in RCS (rabbit aorta contracting substance) and SRS-A (slow reacting substance of anaphylaxis) and their involvement in human bronchial asthma. When we started our anti-asthmatic drug research in the 1970's. We synthesized a lot of chemical compounds and eventually discovered that AA-861 inhibited the generation of SRS-A from the lung tissue of actively sensitized guinea pigs. AA-861 was found to be a potent 5-lipoxygenase inhibitor. This compound reduced experimental allergic asthma in guinea pigs, but it is easily metabolized in the body. More recently, we found a novel compound, AA-2414 (seratrodast), which is not metabolized in the body. AA-2414 proved to be not a 5-lipoxygenase inhibitor, but a thromboxane A2 (TXA2) receptor antagonist. Seratrodast is the first receptor antagonist that is being developed as an anti-asthmatic drug. Seratrodast inhibits both immediate-, late asthmatic responses in guinea pigs, and also reduces airway hyperresponsiveness in dogs. The anti-asthmatic action of seratrodast in animal models indicates that the drug should be of use in the treatment of human asthmatics. In clinical studies, seratrodast showed a marked effect to improve clinical parameters in bronchial asthma. It is also reported that seratrodast is free from harmful aftereffects. Clinical trials are under way in the US.

FPL 55712—An antagonist of slow reacting substance of anaphylaxis (SRS-A): A review

Compound FPL 55712 is a highly selective antagonist of SRS-A derived from guinea-pig, rat, man and dog. It equally antagonizes SRS (slow reacting substance) released by calcium ionophore from rat basophilic leukemia cells and peritoneal mono-nuclear cells and by compound 48/80 from cat paw. In comparatively high doses FPL 55712 significantly inhibits passive cutaneous anaphylaxis in rat and anaphylactic bronchoconstriction in guinea-pig and rat and antigen-induced rhythmic mechanical activity in chicken ileum and late phase of Schultz-Dale reaction in guinea-pig trachea and human bronchus. In addition, FPL 55712 inhibits the SRS-A-induced release of rabbit aorta contracting substance (RCS) from guinea-pig lung. FPL 55712 facilitates sputum clearance in allergic canine asthma. This compound also inhibits the guinea-pig ECF-A (eosinophil chemotactic factor of anaphylaxis)-induced eosinophil chemotactic response of guinea-pig eosinophils in vitro. Compound FPL 55712 is an invaluable pharmacological tool for the identification of SRS(A) and to help define the role of SRS-A in the pathophysiology of immediate hypersensitivity (e.g. anaphylaxis and allergic asthma). Clinical trials of FPL 55712 as an aerosol in allergic obstructive respiratory diseases in man and animals may help to further understand the mechanism(s) and chemical mediator(s) involved in these conditions.

RCS from human platelets: Is it only thromboxane?

To determine if inhibition of a rabbit aorta contracting substance (RCS) corresponds to inhibition of thromboxane synthelase human washed platelets were stimulated with thrombin. RCS formation was bioassayed on rabbit aorta strips in Tyrode solution containing selective blocking agents and thromboxane (TX)B 2 in the same probes by specific radioimmunoassay. Dazoxiben inhibited the formation of TXB 2 but not of RCS, whereas indomethacin inhibited both RCS and TX formation. The data indicate that the rabbit aorta cannot be used alone to predict inhibition of TX formation with specific TX synthetase inhibitors.

Lipid metabolism in cultured cells. Activators of endogenous thromboxane A2 synthesis in cultured lung fibroblasts.

Thromboxane A2 (rabbit aorta-contracting substance) is a proaggregatory vasoconstrictive, oxygenated metabolite of arachidonic acid which was originally discovered in guinea pig lung perfusates during antigen-induced anaphylaxis. The specific stimuli which activate synthesis and the cellular source in the lung remain undefined. In order to study pulmonary thromboxane A2 (TXA2) synthesis, a cultured lung cell model has been used. Monolayer cultures of human diploid embryonic lung fibroblast (WI-38) metabolized exogenously supplied [14C]arachidonic acid to TXA2 as well as prostaglandin E2. Both were unequivocally identified by gas chromatography/mass spectrometry. Cellular phospholipids were labeled by preincubating cultures overnight with [14C]arachidonic acid. Release of thromboxane A2 into the culture fluid from these prelabeled cultures was stimulated by two phospholipase activating agents, mellitin and the calcium ionophore A23187. The lung cells also released TXA2 and prostaglandin in a dose-dependent fashion when treated with thrombin but not when exposed to trypsin. Bradykinin, an anaphylactic mediator in vivo, was a potent TXA2 releasing agent in this in vitro system whereas histamine was inactive. In addition, anaphylactic shock perfusates from guinea pig lung were shown to contain a factor (other than bradykinin) which activates fibroblasts TXA2 synthesis in these cultured lung cells. These experiments indicate that the lung fibroblast is probably a source of pulmonary thromboxane in vivo and that the cultured lung cell system described here is a useful model for defining the complex interactions of mediators of anaphylaxis and asthma.

Vascular smooth muscle reactivity to rabbit aorta contracting substance (RCS) and production of prostacyclin-like substance in normotensive and hypertensive rats.

Male SHR of young prehypertensive (5-6 weeks) and mature hypertensive (24-28 weeks) ages and age and sex matched control normotensive Wistar rats (NWR) were studied for aortic smooth muscle contractility in response to rabbit aorta contracting substance (RCS), serotonin, norepinephrine and potassium. Production of prostacyclin (PGI2) in SHR and NWR aortae of these two age groups was also compared. The contractility of young SHR in response to the agonists was all depressed as compared with the matched NWR. With age advances to 24-28 weeks, aortic responsiveness of NWR to all agonists were reduced and the contractility became the same for SHR and NWR except for the response to RCS which was now greater in SHR than in NWR. The PGI2-like substance released from young SHR aortae was the same as that from the matched NWR. The PGI2-like substance in mature aged rat aortae was much higher than that in young rat aortae and the activity in mature SHR was 1.8 times higher than that of the age matched NWR. The increased production of PGI2-like substance in aorta walls of the mature aged SHR may be a compensatory mechanism to reduce the elevated blood pressure.

The action of leukotriene B 4 (LTB 4) on the lung

The actions of leukotriene B 4 (LTB 4), a member of a newly discovered pathway of metabolism of arachidonic acid, were investigated both on the guinea-pig perfused lung preparation and on the parenchymal strip and compared to histamine and Slow Reacting Substance of Anaphylaxis (SRS-A). LTB 4 was prepared from human polymorphonuclear leukocytes, extracted and purified by chromatography (Silicic acid and HPLC) and its purity was determined by gas chromatography and mass spectrometry. LTB 4 is three times more potent than histamine (molar concentration) to contract the parenchymal strips and the contraction to LTB 4 as well as to SRS-A lasted longer. The contraction to LTB 4 is blocked by indomethacin (20 μg/ml), reduced by polyphloretin phosphate (50 μg/ml) and unaffected by FPL-55712 (1 μg/ml). Following its injection in the pulmonary artery of a perfused lung, LTB 4 (1 μg) induced the release of RCS (Rabbit Aorta Contracting Substance:a mixture of prostaglandins and thromboxanes) which can be abolished by indomethacin (1 μg/ml). These findings suggest (a) that in the lung, LTB 4 is a myotropic agent three times more powerful than histamine (b) that LTB 4 stimulated a receptor which is different of histamine or SRS-A receptors, and (c) that its contractile action in the lung is mediated by prostaglandins and thromboxanes.

Angiotensin-II-stimulated release of thromboxane a 2 and prostacyclin (PGI 2 ..) in isolated, perfused kidneys of spontaneously hypertensive rats

Angiotensin-II(A-II)-stimulated release of rabbit aorta-contracting substance (RCS), prostaglandin-like substance (PLS) and dog coronary-relaxing substance (DRS) was studied in isolated, perfused kidneys of spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY). When the perfusion pressure was set at 60–70 mmHg, the dose-related pressor response of the kidney to A-II did not differ significantly between the strains, or between the ages of 6, 12 and 18 weeks. The A-II-induced release of RCS from the kidney was most remarkable in 6-week SHR, slight in 12- and 18-week SHR, but negligible for all ages of WKY. The DRS release was remarkable in 12- and 18-week SHR, but was hardly detectable in 6-week SHR and all ages of WKY. The PLS release was much more remarkable in 18-week SHR than in 6- and 12-week SHR and all ages of WKY. The RCS was confirmed to be thromboxane A 2 (TXA 2) by: (1) its half-life of 38 sec at 37°, (2) a concomitant release of radio-immunoassayable TXB 2: (a stable metabolite of TXA 2) into the perfusate and (3) specific inhibition of its release by L-8027 (a TXA 2 synthetase inhibitor). The DRS was considered to be prostacyclin (PGI 2) by: (1) its half-life of 5.2 min at 37° and (2) its anti-platelet aggregation activity. It is speculated that an enhanced TXA 2 synthesis in the 6-week SHR kidney may be involved in increasing the renal vascular resistance and in initiating the process of hypertension.

Rat aortic strip as a bioassay tissue for thromboxane A 2 and rabbit aorta contracting substance (RCS) released from guinea pig lung by bradykinin or anaphylaxis

Rat aortic strips and rabbit aortic strips were superfused in series with Krebs solution. Comparison of the sensitivity of the tissues to thromboxane A 2 (TXA 2), generated by mixing prostaglandin (PG)H 2 with human platelet microsomes (HPM), indicated that the rat aorta was just as sensitive an assay tissue as the rabbit aorta. Furthermore, it was equally selective in that it did not respond to low levels of the other metabolites of arachidonic acid. When the two tissues were used simultaneously to assay aortic contracting activity released from perfused guinea pig lung by bradykinin, both tissues detected activity that could be matched with similar known amounts of TXA 2. However, when ovalbumin was used to release aortic contracting activity from sensitized guinea pig lung, the amounts of TXA 2 needed to match the responses of the assay tissues often differed by 2–3 fold. This suggested that other substances, as well as TXA 2, released during anaphylaxis can affect the aortic strips and thus influence the bioassay of TXA 2. This discrepancy in assay of TXA 2 can be detected only when more than one assay tissue is used. In the series of experiments in which we used the assay tissues to detect TXA 2 released by bradykinin, we noted that bradykinin released more TXA 2 from unsensitized lungs than from sensitized ones. Although the significance of this observation remains unclear, it suggests that there are quantitative differences in the PG biosynthetic pathways induced by the sensitization process.

Vascular responsiveness to the products of platelet aggregation

Platelets have been known to produce a labile but potent vasoactive substance as well as to release ADP, ATP and serotonin (5-HT) on stimulation. This substance reported as rabbit aorta contracting substance (RCS) by Piper and Vane2 has been identified as mixture of prostaglandin (PG) endoperoxides (PGG2, PGH2) and thromboxane A2.3 We have reported that; 1) intravascular platelet aggregation induced by ADP produced profound constriction and resultant injury of the pulmonary artery in rabbits; 2) intravenous injection of ADP elicited significantly attenuated cardiorespiratory disturbance and thrombus formation in spontaneously hypertensive rats (SHR) as compared with normal Wistar rats. In this paper in vitro responsiveness of the pulmonary artery of rabbit and the aorta of SHR and vitamine E deficient rat (VEDR) to RCS and 5-HT was investigated.Materials and Methods: 1) The right or left pulmonary artery and thoracic aorta of 9 rabbits, 2) thoracic aorta of 7 pairs of 24-26 weeks old SHR and NWR and 6 pairs of 6 weeks old SHR and NWR, and 3) thoracic aorta of 3 pairs of 11 weeks old VEDR and NWR were excised promptly after death. Each vessels were cut spirally to produce 35-40×2.5-3.0mm strips and the strips were suspended in gassed Krebs medium at 37°C. RCS was generated by agitation of washed rabbit platelets with 5 units of thrombin or 6.25μg of sodium arachidonate for 40sec at 37°C.Results: 1) The contractile response of pulmonary artery to RCS was about 50% of the maximum tension developed by 40mM K+ and 1μM 5-HT. The pulmonary artery response to RCS was as twice greater as the aorta and one to 5-HT was 1.5 times greater (Fig. 1A, B). 2) The contraction of 24-week SHR aorta by RCS was not different from NWR but one by 5-HT was significantly increased. However, the contraction of 6-week SHR aorta was not different from NWR (Fig. 2A, B). 3) Aorta responsiveness of VEDR was not different from NWR (Fig. 3).Comment: Rabbit pulmonary artery contracted in great extent by RCS as well as by 5-HT. Vascular responsiveness of SHR was almost same to NWR or rather increased. So the mechanism of attenuated cardiorespiratory disturbance by intravascular platelet aggregation in SHR may not be on the vessels but on the platelet. Vascular responsiveness of VEDR will be further studied in connection with PGI2.

Stimulation of arachidonic acid metabolism by human slow-reacting substances

Human slow-reacting substance of anaphylaxis (SRS-A) and clacium ionophore-induced human SRS released prostaglandin-like substances and rabbit aorta contracting substance (RCS) from guinea-pig lungs. This effect was abolished by incubation of SRS-A and SRS with arysulphatase or pretreatment of the lungs with indometahcin. Human SRS-A and SRS therefor resembled guinea-pig SRS-A in stimulating arachidonic acid metabolism. These results provide further evidence for a similar (or identical) nature of human SRS-A and SRS and suggest a possible role for slow-reacting substances in the release of prostaglandins during anaphylaxis.

Enhancement of anaphylactic mediator release from guinea-pig perfused lungs by fatty acid hydroperoxides

Fragments of chopped lung from indomethacin treated guinea-pigs had an anti-aggregating effect when added to human platelet rich plasma (PRP), probably due to the production of prostacyclin (PGI 2) since the effect was inhibited by 15-hydroperoxy arachidonic acid (15-HPAA, 10 μg ml −1). Both 15-HPAA (1–20 μg ml −1 min −1) and 13-hydroperoxy linoleic acid (13-HPLA, 20 μg ml −1 min −1) caused a marked enhancement of the anaphylactic release of histamine, slow-reacting substance of anaphylaxis (SRS-A) and rabbit aorta contracting substance (RCS) from guinea-pig isolated perfused lungs. This enhancement was not reversed by the concomitant infusion of either PGI 2 (5 μg ml −1 min −1) or 6-oxo-prostaglandin F 1α (6-oxo-PGF 1α, 5 μg ml −1 min −1). Anaphylactic release of histamine and SRS-A from guinea-pig perfused lungs was not inhibited by PGI 2 (10 ng - 10 μg ml −1 min −1) but was inhibited by PGE 2 (5 and 10 μg ml −1 min −1). Antiserum raised to 5,6-dihydro prostacyclin (PGI 1) in rabbits, which also binds PGI 2, had no effect on the release of anaphylactic mediators. The fatty acid hydroperoxides may enhance mediator release either indirectly by augmenting thromboxane production or by a direct effect on sensitized cells. Further experiments to distinguish between these alternatives are described in the accompanying paper (27).

Transformation of arachidonic acid and prostaglandin endoperoxides by the guinea pig heart. Formation of RCS and prostacyclin

The metabolism of arachidonic acid (AA) was studied in perfused isolated hearts from guinea pigs. The coronary effluent was continuously bioassayed for prostaglandin-like substances (PLS) using the cascade technique of Vane. Injections of AA in doses between 1–50 μg into the perfusion fluid prior to the heart produced vasodilatation of the coronary vascular bed followed by a contraction of the rat stomach strip (RSS), chick rectum (CR) and rat colon (RC) as well as relaxation of the bovine coronary artery (BCA). At the higher doses of AA there was also contraction of the rabbit aorta (RbA). The same pattern of effects on the bioassay tissues was seen when prostaglandin endoperoxide (PGH 2) was perfused through the heart. The response of the bank of superfused tissues provided evidence for the formation of prostacyclin (PGX or PGI 2), PGE 2 and PGF 2α. Chromatographic studies showed that 6-oxo-PGF 1α together with other prostaglandins was present in the perfusate after acidification, which suggested that the bovine coronary relaxing substance consists mainly of PGI 2. Moreover, the rabbit aorta contracting substance (RCS) released in the perfusate was due to prostaglandin endoperoxides and not to thromboxane (TXA 2. The formation of PLS from AA was completely blocked after treatment of the heart with the cyclo-oxygenase inhibitors, indomethacin or meclofenamic acid. Pretreatment of the heart with 15-hydroperoxyarachidonic acid (15-HPAA), a selective inhibitor of prostacyclin synthetase, inhibited the effect of AA on the coronary vasculature and diverted the metabolic transformation of AA towards PGE 2 and PGF 2α.

Thrombin, collagen and A23187 stimulated endogenous platelet arachidonate metabolism: Differential inhibition by PGE 1, local anesthetics and a serine-protease inhibitor

The formation of malondialdehyde (MDA) and rabbit aorta contracting substance (RCS) induced by treatment of platelets with thrombin and collagen, but not that produced from exogenous arachidonic acid, is inhibited by prostaglandin E 1 (10 −8 − 10 −7M), the local anesthetics tetracaine, SKF 525-A and dibucaine (1 mM), and the serine-protease inhibitor phenylmethanesulfonyl fluoride (PMSF). The burst in oxygen consumption which accompanies platelet stimulation by thrombin and collagen in the presence of antimycin A, known to be due to the oxidation of endogenous arachidonate, is also markedly suppressed by PGE 1, tetracaine and PMSF. The inhibitory effect of PGE 1 is strongly potentiated by theophylline (1.0 mM). Addition of the Ca 2+ ionophore A23187 to platelet suspensions overcomes PGE 1 and PMSF inhibition of MDA and RCS formation, and induces a vigorous increase in O 2 consumption. Tetracaine and dibucaine, however, block the responses to A23187. Formation of MDA and RCS (a mixture of PG endoperoxides and TXA 2) due to stimulation by thrombin and collagen depends upon activation of Ca 2+-dependent phospholipase A 2 (PLA 2) to supply free arachidonate from specific membrane phospholipids. These experiments therefore indicate that increased cellular cAMP, induced by PGE 1, antagonizes the mobilization of the Ca 2+ which is normally required for PLA 2 activity. Thrombin-stimulated platelets exhibit enhanced 45Ca uptake which probably reflects exchange of extracellular Ca 2+ with an increased available pool of exchangeable intracellular Ca 2+. PGE 1 strongly suppresses this 45Ca uptake, providing more direct evidence supporting the view that cAMP prevents the rise in free cytoplasmic Ca 2+ induced by thrombin. Under conditions which make sufficient free cytoplasmic Ca 2+ available (i.e., A23187), despite high cellular cAMP, formation of RCS and MDA, and O 2 uptake are nearly normal indicating that activation of PLA 2 can occur. Local anesthetics on the other hand since they abolish the response to A23187 as well, appear to directly antagonize the ability of Ca 2+ to activate PLA 2. The effect of PMSF suggests that stimulus-specific proteases may be involved in the thrombin and collagen-induced activation of PLA 2 activity.

Cyclic adenosine 3',5'-monophosphate inhibits the availability of arachidonate to prostaglandin synthetase in human platelet suspensions.

When thrombin is added to washed human platelets, one of its actions results in activation of a phospholipase that hydrolyzes arachidonic acid from phospholipids. The arachidonate is converted to the cyclic endoperoxides (prostaglandin G2 and prostaglandin H2) by fatty acid cyclo-oxygenase. These compounds are then converted to thromboxane A2, also called rabbit aorta-contracting substance, by thromboxane synthetase. These labile, pharmacologically active compounds then break down to inactive products including thromboxane B2 and malonaldehyde. Incubation of platelets with either dibutyryl cyclic adenosine 3',5'-monophosphate (dBcAMP) or prostaglandin E1 (PGE1) before thrombin addition blocks the subsequent formation of oxygenated products of arachidonic acid including thromboxane A2, thromboxane B2, and malonaldehyde. In contrast, when arachidonic acid is added directly to platelets, prior incubation with dBcAMP or PGE1 does not inhibit production of the prostaglandins or their metabolites. Thrombin treatment of platelets also blocks the acetylation of cyclo-oxygenase by aspirin since the hydrolyzed arachidonic acid competes with aspirin for the active site on cyclo-oxygenase. Prior treatment of platelets with dBcAMP or PGE1 reverses the thrombin inhibition of the acetylation of cyclo-oxygenase. We conclude that agents which elevate platelet cAMP levels inhibit the hydrolysis of arachidonic acid from platelet phospholipids. We also find that prostaglandin synthesis can be dissociated, in part, from platelet aggregation and release, and that cAMP has separate actions on these processes. Higher thrombin concentrations are required to stimulate prostaglandin synthesis (0.05-2 U/ml) than are required to induce [14C]serotonin release (0.02-0.1 U/ml). Furthermore, dBcAMP and PGE1 both inhibit platelet aggregation induced by either arachidonic acid or prostaglandin H2 without affecting the production of prostaglandin metabolites from these compounds.

Mechanism of action of bradykinin-induced release of prostaglandin E.

1. The mode of action of bradykinin-induced release/biosynthesis of prostaglandin E (PGE) was investigated using the method of the isolated perfused rabbit ear. 2. Phentolamine (1 μg/ml) hardly reduced the total amount of PGE released by bradykinin. Only in the first fraction (3 min) following bradykinin injection was PGE release significantly inhibited. 3. Papaverine (5–10 μg/ml) did not affect the bradykinin-induced PGE release but totally abolished the vasoconstriction evoked by the peptide. 4. Mepacrine (10 μg/ml) greatly inhibited the bradykinin-stimulated PGE release but did not reduce the conversion of arachidonic acid by cyclo-oxygenase into PGs when used in this concentration. 5. Phospholipase A2 — injected intra-arterially into the perfused rabbit ear — strongly stimulated the release of PGE. As with bradykinin, the phospholipase A2-induced PGE release was also significantly inhibited by meparcrine (10 μg/ml). 6. Bradykinin or arachidonic acid, even in high doses (20 or 100 μg respectively), were unable to promote the release of rabbit aorta contracting substance (RCS) from the rabbit ear. 7. PGs were not metabolized when injected intraarterially through the rabbit ear. 8. The results suggest that PGE release induced by bradykinin is hardly mediated via liberating catecholamines or the constricting vessel wall. It is concluded that bradykinin stimulates PGE release by selectively activating a phospholipase A2 without affecting cyclo-oxygenase and subsequent enzymes. The phosholipase A2 catalyzes the splitting of PG precursors from membrane phospholipids, before they are converted by the active PG synthetase system into PGs.

Prostaglandin endoperoxides, serotonin and the superfused rabbit aorta: Possible pitfalls in the bio-assay of rabbit aorta contracting substance (RCS)

The specificity of the isolated rabbit aorta for identification and assay of prostaglandin endoperoxides, formed during aggregation of platelet rich plasma (PRP) from rats, was investigated in the superfused organ cascade. The use of cumulative dose-response curves in this design is quicker than conventional step-wise dosing while delivering identical results. On the rabbit aorta, in contrast to the rat fundus strip, methysergide is too weak an antagonist to rule out responses due to 5-HT. After pretreatment with phenoxybenzamine, in the presence of methysergide, the rabbit aorta retains its sensitivity towards oxygenated fatty acids, while being virtually unreactive towards 5-HT. The prostaglandin endoperoxide PGH2 is more potent than prostaglandin E2 when assayed on the rabbit aorta, while on the rat fundus the situation is reversed. Combining the rabbit aorta and rat fundus strip with pre-filtration on Amberlite XAD-2 columns showed that during collagen induced aggregation rat PRP generated both a labile RCS-most probably a prostaglandin endoperoxide- and a lipophylic prostaglandin-like material, along with 5-HT.

Platelets, acute inflammation and inflammatory mediators

The anti-inflammatory activity of aspirin-like drugs could derive, at least in part, by inhibiting synthesis and release of prostaglandins or rabbit aorta-contracting substance from platelets. Indeed, aggregation of platelets and the consequent release of inflammatory mediators has been frequently evoked as a factor in the development of the inflammatory reaction. The participation of platelets in acute inflammation was tested in three types of trauma in rats rendered thrombocytopenic with anti-platelet serum. Oedema in response to carrageenin, anti-platelet serum or passive cutaneous anaphylaxis was no different from the controls in thrombocytopenic rats.

Release od smooth muscle-contracting substances from isolated perfused lungs

Infusion of tryptamine (1–4 μg/ml) through the pulmonary of rat isolated lung perfused with Krebs solution caused release of a mixture of spasmogens contracting isolated smooth muscle preparations. One component of this mixture had biological activity comparable to E-type prostaglandins. Other components included a slow reacting substances comparable to SRS-A and a rabbit aorta-contracting substance comparable to RCS. Infusions of 5-hydroxytryptamine, acetylcholine and histamine (0.5–2 μg/ml) also caused released. Released induced by the tryptamines but not that by acetylcholine and histamine was prevented by methysergide whereas acetylcholine-induced release was prevented by hyoscine which did not affect tryptamine-induced release. The tryptamines and histamine released spasmogens from dog isolated lungs but only histamine was effective in guinea-pig lung. We concluded that amine-induced release from isolated lungs is a fairly general phenomenon and that it may represent an endocrine functions of lung.

Bioassay of histamine in the presence of prostaglandins.

1. An Amberlite XAD-2 column in a heated (37 degrees C) jacket was incorporated in between two banks of bioassay organs superfused with Krebs bicarbonate solution in cascade. The column removed prostaglandins, E1, E2 and F2alpha and also rabbit aorta contracting substance (RCS) and possibly slow reacting substance of anaphylaxis (SRS-A). 2. The column gave free passage to histamine in concentrations up to 3000 ng/ml. The method described improved the accuracy of histamine bioassay in the.presence of prostaglandins.