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Home , Leukotriene

Br. J. Pharmac. (1985), 86, 55-62

Differential release ofeicosanoids by bradykinin, and calcium ionophore A23 187 in guinea-pig isolated perfused Y.S. Bakhle*, S. Moncada, G. de Nucci' & J.A. Salmon Department ofProstaglandin Research, Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS and Department ofPharmacology*, Royal College of Surgeons ofEngland, 35-43 Lincoln's Inn Fields, London WC2A 3PN

I The effects of infusions of bradykinin (0.2 fLM), calcium ionophore A23 187 (0.5 LM) and arachidonic acid (13 LM) on the release ofeicosanoids from the guinea-pig isolated perfused lung were investigated using radioimmunoassay for B2 (TXB2), 6-oxo- Flm (6-oxo- PGF,.), PGE2, (LTB4) and LTC4 and bioassay using the superfusion cascade. 2 Bradykinin released more 6-oxo-PGF,, than TXB2, whereas arachidonic acid and ionophore released more TXB2 than 6-oxo-PGF,,. 3 The time course of release varied with the stimulus: bradykinin and arachidonic acid produced an immediate release, whereas the ionophore showed a slower onset of release. 4 Although the amounts of LTB4 and LTC4 released by the ionophore were very low according to radioimmunoassays, there was evidence from the bioassay of release of a leukotriene-like substance, thought to be LTD4. 5 The leukotriene antagonist FPL 55712 lacks specificity in the guinea-pig trachea; at the concentration used (2 tiM) it antagonized contractions ofthe tracheal strip to PGE2 as well as to LTC4. 6 Our results show that in the guinea-pig perfused lung the of exogenous arachidonic acid is both qualitatively and quantitatively different from the metabolism ofendogenous arachidonic acid; furthermore, the profile of eicosanoid production is stimulus-dependent.

Introduction Infusions or injections of arachidonic acid (AA) into metabolism in guinea-pig lung was similarly depen- the pulmonary circulation ofguinea-pig isolated dent on the stimulus utilized. lead to the formation of rabbit aorta contracting substance (RCS) (Piper & Vane, 1969), subsequently shown to be mainly composed of Methods (TXA2) (Hamberg et al., 1976). The metabolism of endogenous arachidonic acid leading to the synthesis Preparation of the lungs ofeicosanoids can also be triggered in guinea-pig lung by other stimuli, such as bradykinin and the calcium Male guinea-pigs (300-400 g body weight) were ionophore A23187 (Palmer et al., 1973; Al-Ubaidi & anaesthetized with pentobarbitone (60mg kg-1 i.p.). Bakhle, 1980). Following mid-thoracotomy the pulmonary artery There is evidence, however, from other tissues (e.g. was cannulated and perfused for 10 min with 50 ml of kidney, heart) that the formed from the heparinized Krebs bicarbonate solution (10uml-1). metabolism ofexogenous AA are different from those The trachea was cannulated and the lungs removed derived from endogenous AA released by bradykinin and suspended in a heated chamber. The lungs were (Isakson et al., 1976; Needleman et al., 1979). We perfused via the pulmonary artery with warmed (370C) undertook this study to determine whether or not AA and gassed (95% 02-5% CG2) Krebs bicarbonate solution at a constant rate of5 ml min-1 (Bakhle et al., 'To whom correspondence should be addressed. 1969). (D The Macmillan Press Ltd 1985 56 Y.S. BAKHLE et al.

Collection of lung effluent buffer but without prior extraction or purification. The specificity of the antisera used in these RIAs has Arachidonic acid, bradykinin and A23187 were given been previously established: TXB2, 6-oxo-PGF1. and either in 5 min infusions at 0.1 ml min-' or as a single PGE2 (Salmon, 1978), LTB4 (Salmon et al., 1982) and bolus injection. For radioimmunoassay (RIA) of LTC4 (Aehringhaus et al., 1982). eicosanoids, the lung effluent was collected in one minute fractions for the times shown (normally Materials 15 min). In some experiments, a single collection over 3 or 4 min was made. In the experiments involving The Krebs bicarbonate solution had the following RIA, the lungs were used for a single infusion of a composition (mM): NaCI 118, KCI 4.7, KH2PO4 1.2, single stimulus (AA, bradykinin or A23187). When MgSO4, 7H201.17, CaCI2, 6H200.25, NaHCO3 25 bioassay was used to identify eicosanoids, bolus and glucose 8.4. Calcium ionophore A23 187 (Calbio- injections of the stimuli were employed so that more chem, La Jolla U.S.A.) was initially dissolved in responses could be obtained within the experimental absolute ethanol and then diluted in Krebs soution. lifetime of the lung (about 60min). Bradykinin triacetate salt (Sigma Chemical Company, Poole) was dissolved in distilled water. Arachidonic Bioassayfor leukotriene-like substance acid (Sigma) was initially dissolved in hexane, evaporated under N2 and resuspended in 0.5 M meth- Lung effluent was used to superfuse a series of anolic NaOH to give a concentration of bioassay tissues in a cascade (Vane, 1964): rat stomach 10 mg AA ml- '. The methanol was removed under N2 strip (RSS), rabbit aorta (RbA), guinea-pig trachea and the residue resuspended in I M Tris buffer pH 9, (GPT) and guinea-pig ileum smooth muscle (GPISM). then further diluted in Krebs solution. Indomethacin The bioassay tissues were arranged in two banks (Merck, Sharp & Dohme Ltd, Hertfordshire) was separated by a delay coil, such that the superfusing dissolved in 5% NaHCO3. Other drugs used were: effluent after passing over the first bank of tissues was delayed by 2 min before passing over the second bank E 100 (Alabaster & Hawkeswood, 1978). The selectivity of the assay tissues was increased by an infusion into the E lung effluent of a mixture of antagonists to x 80 I- (mepyramine, 1 M), acetylcholine (atropine, 1.5 1M), E catecholamines (phenoxybenzamine, 0.3 tiM and o 60 1- propranolol, 7 tiM) and 5-hydroxytryptamine (methy- sergide bimaleate, 0.3 gM), (Gilmore et al., 1968) o 40 v together with indomethacin (5.6 tAM) to prevent en- dogenous synthesis of by the assay c 20 I- tissues (Eckenfels & Vane, 1972). U0 0 Responses of the tissues were detected with auxo- L) 0 L tonic levers (Paton, 1957) attached to Harvard heart smooth muscle transducers and displayed on a Watan- abe multichannel pen recorder. 12 3 4 5 6 7 10 15 The bioassay was used particularly to identify Time (min) leukotriene-like substance (LT-LS) in the eicosanoid Figure 1 Time course of (TXB2) mixture in lung effluent. This identification was based production from guinea-pig perfused lungs induced on the following criteria. (i) The LT-LS would con- by A23187 (O.5M, 0 0), bradykinin (0.2fLM, tract the GPT before and after the delay. (ii) No O- O) and arachidonic acid (AA; 13 1AM, inhibition of its production occurred when indometh- 0- -- -0). The points are the mean of data generated acin was infused through the lung. (iii) Inhibition of in 4 lungs and the vertical lines show the s.e.mean. The the GPT contraction to the LT-LS occurred after concentration of TXB2 in each 1 min fraction of lung infusion ofthe leukotriene antagonist FPL 55712 over effluent was measured by radioimmunoassay. Since there the assay tissues (Augstein et al., 1973). were marked variations in the actual concentrations of TXB2 released by different stimuli (see Figure 3), the data are presented as the percentage of the maximum concen- Radioimmunoassay of the lung effluent tration of TXB2 released by each stimulus. The hatched rectangle shows the duration of the infusion of the The concentrations of TXB2, 6-oxo-prostaglandin F1l agonist. The time course of TXB2 production for each (6-oxo-PGFlj), PGE2, LTB4 and LTC4 in the lung stimulus was very different, either in time of onset effluent were determined by specific radioimmunoas- (compare AA and bradykinin with ionophore) or in say (RIA) after suitable dilutions (1:2- 1: 100) in RIA duration (compare AA and ionophore with bradykinin). STIMULUS-DEPENDENT EICOSANOID RELEASE BY GUINEA-PIG LUNG 57 phenoxybenzamine hydrochloride (Dibenyline; SK & experiments. Student's unpaired t test was used to F Stevenage), methysergide bimaleate (Sandoz Prods. determine the significance of differences between Ltd, Leeds) mepyramine maleate (May & Baker, means and a P value of <0.05 was taken as sig- Dagenham), propranolol hydrochloride and atropine nificant. sulphate (Sigma). FPL 55712 (sodium 7-(3(4-acetyl-3- hydroxy-2-propylphenoxy)- 2-hydroxy-propoxy)-4- oxo-8-propyl- 4H- I-benzopyran- 2-carboxylate) was a gift from Fisons Ltd, Loughborough; U-46619 Results (1 la,9a-epoxymethano ) and PGE2 were gifts from the Upjohn Co., Kalamazoo, U.S.A. Time-course of eicosanoid release Antiserum against LTC4 was kindly provided by Dr B. Peskar (University of Bochum, West Germany). Infusion of arachidonic acid (13 fAM) into the guinea- 5,6,8,9,11,12,14,15 (n)-[3H]-TXB2, specific activity, pig lung led to the synthesis of TXA2 (measured as (s.a.) 140 Ci mmol-'; 5,6,8, 1 ,12,14,15(n)-[3HJ-PGE2, TXB2 by RIA). This synthesis reached a plateau in the s.a. 160 Ci mmol-'; 6-oxo-5,6,8,9, 11,14,1 5,(n)-[3H]- second minute and was maintained until the end ofthe PGFIZ, s.a. 150 Cimmol- and 5,6,8,9,11,12, infusion, falling immediately after the infusion was 14,15(n)-[3H]-LTB4, s.a. 221 Ci mmol ' were pur- finished (Figure 1). Bradykinin (0.2 p1M) induced syn- chased from Amersham International (Amersham, thesis ofTXB2 which peaked during the first 2 min and Bucks). 14,15(n)-[3H]-LTC4, s.a.34 Cimmol' was declined thereafter. The response to A23187 (0.51M) obtained from New England Nuclear (Boston, Mass., was, however, different from either of the first two U.S.A.). agonists. The release slowly increased during the infusion period and only reached a maximum after the Statistics end of the infusion. The fall in TXB2 production was also slow; by 10 min, TXB2 levels were about half the Results are shown as mean values ± s.e.mean for n maximal value and at 15min TXB2 levels were still higher than before stimulation. Measurements of 6- oxo-PGF1, in the same lung effluent samples showed that the production of this eicosanoid had the same 30- time course as that ofTXB2 for all three agonists. This is illustrated in Figure 2 in response to AA infusion. The time course for PGE2, LTB4 and LTC4 (the last two were detectable only when using the calcium ionophore A23 187) was similar to that of TXB2 (data not shown). C Ratios of eicosanoids formed U1 c 20 At the concentrations shown in Figure 3, all three 0 stimuli (AA, bradykinin and the ionophore) produced approximately equal concentrations of (PGI2) measured as immunoreactive 6-oxo-PGFI,. 10L The samples were taken at the time of maximum eicosanoid production. For infusion of AA, the effluent samples were taken from the second to the fifth minute; for bradykinin from the second to the 1 2 3 4 5 6 7 10 fourth minute and for A23187 from the sixth to the Time (min) ninth minute. Bradykinin released more 6-oxo-PGFI, Figure 2 Time course of thromboxane B2 (TXB2, than TXB2, whereas arachidonic acid and A23187 * *) and 6-oxo-prostaglandin Fl, (6-oxo-PGF,m, released more TXB2 than 6-oxo-PGF1.. The iono- -- - -0) production from guinea-pig perfused lungs phore was the most potent stimulus for releasing induced by arachidonic acid (1 3 pM). The points show the TXB2. Levels of PGE2 were low in comparison to mean concentrations of TXB2 and 6-oxo-PGFm from 4 those ofTXB2 and and the levels ofLTB4 lungs and the vertical lines show the s.e.mean. The 6-oxo-PGF1I hatched rectangle shows the period ofthe infusion. These and LTC4 were much lower still (less than 1 ng ml-') and are not shown. The ratio between experiments were carried out as in Figure 1. Note that TXB2 and 6- absolute concentrations ofeicosanoid are shown and that oxo-PGF I. did not change qualitatively by varying the about three times as much TXB2 as 6-oxo-PGFI. is dose ofany ofthe three stimuli but increased when the produced. dose of AA was augmented (Table 1). 58 Y.S. BAKHLE et al.

130 Bioassay of eicosanoids Using the same concentrations ofagonist (AA, 13 gM; bradykinin, 0.2 gM and A23 187, 0.5 gIM), we also 120 analysed the eicosanoid production by bioassay, using 7 the two banks ofassay tissues separated by a delay coil C (see Methods). Following the standard 5 min infusion ofeach ofthe agonists, contraction ofGPT before and c after the delay was observed, although the immun- -W0 oreactive LTC4 was always very low (see above). Co 20[ Because ofthe discrepancy between RIA and bioassay C._ for LT-LS, we examined the nature of the LT-LS 0(Dc further, but using injections rather than infusions of c) agonist. This enabled more responses to be obtained 10 within the experimental life time of the isolated lung preparation. Injection of AA into the lung caused the release of substances that contracted the RSS, GPT and RbA as illustrated by the experiment shown in Figure 4. Bradykinin Arachidonic A23187 Responses of the GPT and RSS in the second bank of acid tissues after the delay were still present but that of the Figure 3 Release of 6-oxo-prostaglandin F., (6-oxo- RbA had virtually disappeared. The next set of tissue PGFI.; hatched columns), thromboxane B2 (TXB2; stip- responses followed injection of the ionophore A23 187 pled columns) and PGE2 (open columns) from guinea-pig through the lung and showed a qualitatively similar perfused lungs induced by infusion of bradykinin pattern with contractions of the GPT and RSS before (0.2 pM), arachidonic acid (13 pM) and the ionophore and after the delay. The last two sets of responses A23187 (0.1 FM). Each column represents the mean of 5 demonstrate that even after the delay the RbA was experiments and the vertical lines show the s.e.mean. The values shown are the concentrations found in single capable of contracting to a stable agonist, the en- samples of lung effluent taken during the time of doperoxide analogue U46619, and that authentic maximum production of eicosanoid (see text). Whereas LTC4 contracted the GPT and RSS before and after the release of PGI2 (as 6-oxo-PGFI,) was approximately the delay but was without effect on the RbA. These the same for all three stimuli, release of TXA2 (measured suggestions ofthe presence of a LT-LS in lung effluent as immunoreactive TXB2) was markedly different for were further tested by using indomethacin. each stimulus. In the experiment illustrated in Figure 5, the initial responses to stimulation of the lung with AA were abolished by infusing indomethacin through the lung. However, during this infusion the ionophore was still Table 1 The ratio of thromboxane B2 (TXB2) to 6- able to release from lung a material contracting the oxo-prostaglandin Fl. (6-oxo-PGFj) produced GPT before and after the delay, although the RbA from guinea-pig isolated lung: effect of different contracting material was not apparent. Injections of concentrations of stimulant agonists bradykinin through the lung released biological activity comparable to that following AA, and as with Stimulus Concentration Ratio AA, all activity was absent after indomethacin treat- CuM) ment of the lung. Bradykinin 0.2 0.4+0.1 The third criterion of LT-LS, antagonism by Bradykinin 2 0.4+0.1 FPL 55712, was then applied. In Figure 6 the respon- A23187 0.1 8.5+0.6* ses of two GPTs, both after the delay, are shown. The A23187 0.5 10.9 + 1.0 first response on both tissues was to authentic LTC4 Arachidonic acid 13 3.1 +0.8 and then an infusion of FPL 55712 was started only Arachidonic acid 65 9.4 + 1.2* over the lower GPT. This prevented its response to a larger amount of LTC4 and to stimulation by AA The ratios shown are the mean values (± s.e.mean) through the lung, although the upper GPT contracted from 5 experiments (lungs) at each concentration to both injections. The last injection was of the for each stimulus. Only one stimulus was given to ionophore and here too the upper GPT contracted each lung. Eicosanoids in lung effluent were measured by radioimmunoassay as described for but the lower GPT was blocked by the FPL 55712 Figure 3. infusion. Results similar to those shown here for AA * Significant P < 0.05. stimulation were obtained with bradykinin stimula- STIMULUS-DEPENDENT EICOSANOID RELEASE BY GUINEA-PIG LUNG 59

Indomethacin 5.6 FM T.L. RSS5m RSS X > ~~~~~5min

GPT RbA 8 mn

RbA GPT

Dela- -elay------Delay Delay --- Delay - Delay GPT ,

GPT

RSS J O.T. **LTC4LC, ;AA A23187 15 ng 50 jig 3 jig _ T.L. RbA A AA A23187 50 Aig 50 g 3 Lg

O.T. U46619 LTC4 Figure 5 Effect of indomethacin on leukotriene-like 150ng 30ng substance (LT-LS) release by arachidonic acid (AA) and T.L. AA A23187 ionophore. In this experiment, the LT-LS generated by 35 tg 29g AA (50 gg) given through the lung was absent after indomethacin treatment of the lung (5.6 JM), but the Figure 4 Bioassay of eicosanoids produced from guinea-pig trachea (GPT) contracting activity stimulated guinea-pig lung following arachidonic acid (AA) or ionophore injections into the pulmonary circulation. by ionophore (3 ftg) was still produced. The first contrac- tion of the was an authentic Perfusate effluent from the isolated lung superfused two GPT induced by injection of LTC4 (15 ng) given directly to the tissues. The same banks of assay tissues separated by a 2min delay coil pattern of responses was observed in another four (marked Delay on the figure). The first set of responses experiments. T.L. = injection through the lung; was to AA (35 jig) injection; note the loss of rabbit aorta (RbA)-contracting activity after the delay and that the O.T. = injection over tissues; RbA = rabbit aorta. guinea-pig trachea (GPT) contracting activity survived although decreased relative to rabbit stomach strip (RSS) contracting activity. With the ionophore (2 gAg), all the FPL 55712. We therefore examined the specificity of contractions in both banks were larger than those to AA, FPL 55712 on the GPT. but the RbA-contracting activity was still lost while the Two isolated smooth muscle strips, GPT and GPT contracting activity still survived. The third set of GPISM, were superfused with Krebs solution contain- responses to U46619 (150 ng), a stable thromboxane A2 ing indomethacin (5.6 JM) and their responses to (TXA2)-mimetic, shows that both RbA tissues were capable of responding and that the lack ofcontraction of authentic LTC4 and PGE2 observed. One of four the second RbA (after the delay) in the previous sets of experiments is illustrated in Figure 7; the GPT and responses was due to the degradation to TXA2 in the GPISM contracted to both LTC4 and PGE2. After delay coil. Comparison of the last set of responses, to treatment with FPL 55712 (2juM) the GPISM no authentic (LTC4; 30 ng), with those to AA longer responded to LTC4 but still contracted to PGE2 and ionophore given through the lung suggests that a showing the expected specificity of antagonism. leukotriene-like substance was produced by either However, on the GPT, contractions to both LTC4 and stimulus in addition to TXA2. The assay tissues did not PGE2 were abolished by this antagonist. At lower respond to either AA or ionophore given directly to the concentrations (0.5 AM- FPL 55712 neither tissues (not shown). The same pattern of responses was IpM), abolished LTC4 contractions nor differentiated bet- observed in another four experiments. T.L. = injection ween LTC4 and PGE2 on the GPT (data not shown). through the lung; O.T. = injection over tissues. At this stage, it appeared that there were considera- ble amounts ofbioassayable LT-LS released from lung tion ofguinea-pig lung, indicating release of a LT-LS. by ionophore stimulation either by injection or in- The results so far suggest that although the LT-LS fusions. We had already found very low levels of produced by ionophore met all three criteria, that immunoreactive LTC4 (and LTB4) in the effluent produced by AA or bradykinin behaved like a cyclo- following infusions of ionophore A23187 (see above) oxygenase product but was still antagonized by and to confirm the discrepancy between RIA and 60 Y.S. BAKHLE et al.

Delay - -Delayr- - - Delay 5 min 8 min GPT _IA - GPT - OT. LTC4 23 ng LTC4 46 ng FPL 55712 2 p.M T.L. A23187 2.5 tg GPT--` RIA lOng 39 ng < 1.5 ng O.T. cTC, LTC4 E2 E2 T.L. 45ng 60 Figure 8 Lack of correlation of measurement of leuk- ng A 30ng 150ng A23187 otriene C4 (LTC4) by radioimmunoassay with bioassay 50 ,ug 5 ,ug estimates ofleukotriene-like substance (LT-LS) produced Figure 6 Effect of FPL 55712, a leukotriene antagonist by guinea-pig isolated lung. Effluent from lung perfused on the leukotriene-like substance (LT-LS) produced from with Krebs solution containing indomethacin (5.6 tLM) guinea-pig lung stimulated by injections of arachidonic superfused a single guinea-pig trachea (GPT). The first acid (AA) and A23 187. After the first injection of two contractions were induced by known amounts of authentic LTC4 (45 ng) directly to the assay tissues, the LTC4 (23 ng and 46 ng respectively) given directly over infusion of FPL 55712 (2 gM) over the lower guinea-pig the assay tissues (O.T.) and the third contraction followed trachea (GPT) was started and this infusion prevented the an injection of A23187 (2.5 jug) into the lung (T.L.). On lower tissue from responding to the second, larger, dose the bottom the amounts of immunoreactive LTC4 (10 ng of LTC4 (60 ng). Injections ofAA (50 jg) or ofionophore and 39 ng respectively) recovered by radioimmunoassay (5 jug) through the lung caused contractions of the upper of the effluent are shown. untreated GPT whereas the lower FPL-treated GPT remained unresponsive. The same pattern of responses was observed in four other experiments. T.L. = injection started 2 min before injections and continued untiljust through the lung; O.T. = injection over tissues; after the peak of the assay tissue response. The E2 = . fractions were then assayed by RIA for LTC4. As shown in Figure 8, the first two tissue responses are to direct injections of LTC4 (23 and 46 ng) and below the bioassay for injections of ionophore, a combined experimental trace is shown the amount of LTC4 bioassay and RIA experiment was performed. Only recovered (10 and 39 ng, respectively). The third one GPT was superfused with lung effluent and the response was to ionophore injection through an superfusate was collected in 1 min fractions im- indomethacin-treated lung and, although the response mediately below the assay tissue. Collections were was clearly greater than that to 23 ng and almost as large as that to 46 ng, less than 1.5 ng LTC4 was detected by RIA. Thus, either with injections or FPL 55712 infusions the majority ofthe bioassayable LT-LS does not appear to be immunoreactive LTC4. 5 min GPT Discussion Our experiments show that the eicosanoid metabolites of AA that are found in the effluent from GPISM guinea-pig isolated lung vary with the nature of the stimulus. Differences between exogenous and en- PGE2 LTC4 PGE2 LfC4 PGE2 dogenous metabolism of AA have already been des- 50 ng 30 ng 50 ng 30 ng 50 ng cribed, using bradykinin and AA stimulation in other Figure 7 Effect of FPL 55712 (2 jM) on the contractions isolated organs (Isakson et al., 1976; Needleman et al., induced by prostaglandin E2 (PGE2; 50ng) and leuk- 1979). We show here in guinea-pig isolated lung that otriene C4 (LTC4; 30 ng) on guinea-pig trachea (GPT) qualitative differences can be readily demonstrated in and guinea-pig ileum longitudinal smooth muscle the TXB2:6-oxo-PGFI. ratio; for instance, bradykinin (GPISM) preparations. GPT and GPISM strips were was the only stimulus to yield more 6-oxo-PGF1. than superfused with Krebs solution containing indomethacin TXB2 and both the other two stimuli favoured TXB2 (5.6 pM). The first and third contraction were induced by standard doses of PGE2 (50 ng). The second contraction release. The balance between 6-oxo-PGFI,. and TXB2 was induced by a known amount of LTC4 (30 ng). The is not simply related to endogenous vs. exogenous same pattern of responses was observed in four other substrate as the two extremes of our range of stimuli experiments. (bradykinin and ionophore) both induce eicosanoid STIMULUS-DEPENDENT EICOSANOID RELEASE BY GUINEA-PIG LUNG 61 production from endogenous AA. The apparent inef- relevant to note that synthesis of SRS-A (probably a ficiency of cyclo-oxygenase in guinea-pig lung in mixture of LTC4 and LTD4) by lung in response to metabolizing exogenous AA (only 2% of the AA ionophore stimulation has already been demonstrated infused was converted into TXB2, 6-oxo-PGF1. and (Piper & Seale, 1979). PGE2) is commonly observed (Al-Ubaidi & Bakhle, It is interesting that the other two stimuli (AA and 1980; Jose & Seale, 1979) and may be due to competi- bradykinin) did not cause the formation of a leuk- tion for the exogenous substrate between cyclo- otriene-like substance, since in the lung parenchymal oxygenase and incorporation into . strip of the guinea-pig, incubation with AA leads to It has been suggested that PGI2 is released mainly by the formation of LTC4 and LTD4 (Burka & Saad, the vasculature (endothelial cells) and TXA2 by the 1984). This discrepancy may be due to access to a parenchymal cells in lung (Gryglewski et al.,1978). different range of cell types in the two preparations or Bradykinin has already been shown to increase to differences in substrate availability or preferentially 6-oxo-PGFI., synthesis in relation to activation. that of TXB2 in lung and vascular tissues of several Our experiments have also illustrated very clearly species (Saldeen & Saldeen, 1983), although in the lung that the leukotriene antagonist FPL 55712 lacks specimens used by these authors the pulmonary vessels specificity on the GPT in so far as it also inhibits could have been the source of both eicosanoids. contractions due to PGE2. The specificity of this Furthermore, in cultured endothelial cells, the ratio of antagonist on the tissue originally used, the GPISM 6-oxo-PGFI. to TXB2 was always in favour of the (Augstein et al., 1973), was confirmed. Welton et al. former whether basal or stimulated formation of (1981), showed that FPL 55712, at higher concentra- eicosanoids was measured (Ingerman-Wojenski et al., tions (2-10I1M), also inhibits thromboxane synthase 1981; Crutchley et al., 1983). In rabbit pulmonary in human microsomes and antigen-induced artery, the ratio is almost identical with that found histamine release from actively sensitized guinea-pig here (Salzman et al., 1980). Thus, it is likely that the lung fragments. The more general inference from these activity of bradykinin in our preparation is on the results is that the specificity of antagonists should not pulmonary vascular endothelial cells. It is interesting be assumed for tissues other than those on which it has that in isolated perfused kidney, bradykinin favoured been demonstrated. Furthermore, contractions of the the synthesis of PGE2 whereas AA favoured that of GPT reversible by FPL 55712 cannot be taken in PGI2 (Needleman et al., 1979). isolation as evidence of leukotriene production. The response to the calcium ionophore A23187 The measurement of PGI2 and TXA2 synthesis in exhibited two interesting characteristics - the delay in the lung by monitoring the release of their stable the formation of eicosanoids and the presence of a hydrolysis products, 6-oxo-PGF1, and TXB2 respec- bioactive LT-LS, probably LTD4. The delay may tively, underestimates their total synthesis since both represent the stimulation of a population of cells products can be metabolized by the lung (Dawson et physically distant from the pulmonary vessels or one al., 1976). The degree ofmetabolism apparently varies with relatively difficult access, for instance, cells in the if the lung is stimulated with different agents (Boot et major airways further from the alveoli. In this ins- al., 1978; Robinson et al., 1984) and consequently it tance, there would be a delay both in the stimulus is impossible to extrapolate from our data to total arriving at the cells and in the eicosanoids formed synthesis of either PGI2 or TXA2. However, diffusing to the pulmonary vessels and appearing in measurement of 6-oxo-PGF1. and TXB2 probably lung effluent. Alternatively, the delay could be due to provides a valid assessment of the biologically active the several steps (for instance, enzymic reactions or prostacyclin and TXA2 in the perfusate, since it does cell-cell interactions) involved in the ionophore- correlate with the bioassay data. stimulated eicosanoid production. Overall, our results suggest that the profile of In relation to the release of a leukotriene-like eicosanoid production in an intact tissue is not only substance, our criteria for identifying it as LT-LS were species-dependent (Al-Ubaidi & Bakhle, 1980; Ally et all met, but the responses of the bioassay tissues were al., 1980) and cell-dependent (Salmon & Flower, greatly in excess of the immunoreactive LTC4 1979), but is also stimulus-dependent. In lung the measured in effluents. The LT-LS could therefore be stimulus-dependent response probably reflects the either LTD4 or LTE4, which are both capable of different populations of cells excited by each stimulus contracting the assay tissues, although their potency is and, by extension, the different mixture of lipoxygen- different (Lewis et al., 1981). Since LTC4 is rapidly ase, cyclo-oxygenase, PGI2 and TXA2 synthases in metabolized to LTD4 by guinea-pig lung homogenates those populations. The finding that bradykinin (Sirois & Brousseau, 1983) and LTD4 is the major produced more 6-oxo-PGF1,, than TXB2 may reflect a component of guinea-pig slow-reacting substance of selective stimulation of the vascular compartment (SRS-A; Morris et al., 1982), in our whereas the release induced by the ionophore A23187 experiments the LT-LS is most probably LTD4. It is indicates an effect on other cell type compartments. 62 Y.S. BAKHLE et al.

References

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