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Release of slow reacting substance from the guinea-pig lung by phospholipases A2 of Vipera russelli snake venom
Toxlrnn, Vol . 22, No. 3, pp. 339-372, 1984 . Printed in Chat Hrlhin .
0041-0101/84 53 .00+ .00 . © 1984 Pergamon Prep Ltd .
RELEASE OF SLOW REACTING SUBSTANCE FROM THE GUINEA-PIG LUNG BY PHOSPHOLIPASES A2 OF VIPERA R USSELLI SNAKE VENOM HUEI-CHEN HUANG Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan, R.O .C . (AcceptedJor publication 2 November 1983) H.-C. Huuvc. Release of slow reacting substance from the guinea-pig lung by phospholipases A, of Vlperu russelli snake venom. Taxiton 22, 359 -372, 1984. - Phospholipases A, (PLA,) of Vlpera rusAelli venom were isolated by column chromatography . The ability of PLA, fractions to release slow reacting substance (SRS) was studied in We guinea-pig lung perfused with Krebs' solution . The relationship between the perfusion pressure change produced by PLA, and SRS release was also studied. Two PLA, fractions (II-S and III-3; 3 -100 Ftg), injected into the lung increased the perfusion pressure and released SRS. Pretreatment of the lung with indomethacin (10 Ftg) reduced the pressure iesponse induced by the PLA, fractions. T'he SRS released in the lung effluent by PLA, was identified by bioassay as a mixture of thromboxane A, (TXA,), prostacyclin (PGI,) and leukotrienes . TXA, and PGI, release was also quantitatedby radioimmunoassay of the degradation products TXB, and 6keto-PGF , respectively. There was a positive linear correlation between the pressure increases and the ratios of TXB, to ~keto-PGF (r=0 .87) . It appears that the relative amounts of TXA,,and PGI, released determine the effects of PLA, fractions on the guinea-pig lung . The release of arachidonic acid metabolites, prostaglandins and leukotrienes may account for pan of the hypotensive action of PLA, . INTRODUCTION
have indirect effects due to the release or formation of pharmacologically highly active autacoids (ROTHSCHILD and ROTHSCHILD, 1979). Autacoids such as histamine, 5-hydroxytryptamine, kinins, slow reacting substance (SRS) and prostaglandins have been associated with a number of pathologic processes involving blood pressure, pulmonary function, vascular permeability and visceral smooth muscle tone. SRS was described by FELDBERG and KELLAWAY (1938) as a smooth muscle contracting factor appearing in the perfusate of guinea-pig lung following treatment with cobra venom. SRS, as well as histamine, is released from the lungs of nonsensitized guinea-pigs by various preparations (heat-treated or heparin-treated) from cobra (N. ngja) venom. This effect has been suggested to be due to phospholipase Az (PLA=) present in the venom (FELDBERG and ICELLAWAY, 1938 ; SCHÜTZ and VOGT, 1961 ; PHILLIPS and MIDDLETON, 1965). Chromatographic analysis of SRS formed by cobra venom showed it to be a mixture of hydroxylated, unsaturated fatty acids belonging to the prostaglandin family (VOLT et al ., 1969). By means of thin layer chromatography, DAMERAU et al. (1975) suggested that SRS activity is primarily due to a species of prostaglandin E. According to VOLT (1969) and DAMERAU et al. (1975), snake venoms supply the enzyme (PLA,) required for the first step in prostaglandin (PG) formation by releasing unsaturated fatty acids from tissue phospholipids . Tissue enzymes, in a secondary phase, transform them into pharmacologically active prostaglandins . PGs are the cyclooxygenase products of SNAKE VENOMS
359
36 0
HUEI-CHEN HUANG
arachidonic acid and have biological activity on pulmonary smooth muscle and other tissues. Slow reacting substance of anaphylaxis (SRS-A) is produced from sensitized guinea-pig lungs and has been determined to be leukotrienes (MORRIS et al., 1979, LEwts et al., 1980). Leukotrienes are the lipoxygenase products of arachidonic acid and also have potent biological activity on pulmonary and other tissues. The components of SRS released from guinea-pig lungs by snake venom remain to be identified . Vipers russelli venom contains several isoenzymes of PLAN (~ALACH et al., 1971). PLA2 of Y. russelli venom affects membrane and granule phospholipids of red blood cells and platelets in a manner similar to PLAz of N. ngja venom (BRADLOW and MARCUS, 1966). In our preliminary tests, SRS was also released by PLA, fractions of V. russelli venom from guinea-pig lungs, similar to that induced by cobra venom. In the present study, the ability of PLAZ fractions to release SRS was studied in guinea-pig lungs perfused with Krebs' solution and the components of SRS were identified . PLAz caused a hypotensive effect accompanied by an increase in pulmonary pressure (CHID et al., 1968). The possible involvement of SRS release in the hypotensive effect of PLAN was also studied.
Phospholipase A, fractions
MATERIALS AND METHODS
Vipers rttsselli venom was purchased from Astik Farm and Laboratory, Bombay, India. Crude venom was appliedto a chromatography column (2 .5 x 72 cm) of Sephadex G-75 (Pharmacia, Uppsala, Sweden). The elution was carried out with 0.03 M ammonium acetate (pH 6.5). Two PLA, fractions, Frs. II and III, were subjected to refractionation by CM-Sephadex C-50 (Pharmacis, Uppsala, Sweden) column chromatography (1 .8 x 80 cm). The gradient elution was carried out with ammonium acetate buffer, 0.05 M (pH 5 .0) to 1 .5 M (pH 8.5) (Hunnc and LEE, 1984). The biologically active subfractions (II-5, II-7, III-3 and III) were collected and lyophilized. These subfractions had both neurotoxic and hypotensive actions. The purity, PLA, activity and general pharmacological properties of these fractions have been presented in a previous paper (HuAtvc and LEE, 1984).
Perfusion of guineapig lungs
Guinea-pigs weighing 200-300 g were killed by cervical dislocation. The thoracic cavity was rapidly opened, the pericardium incised and 1 ml of Krebs' solution containing 100 units of heparin was injected intracardially. The left heart was cut away and the lung was removed. The pulmonary artery was cannulated and the blood was flushed out with Krebs' solution . The trachea was then cannulated and the lung was attached to the perfusion apparatus by a pulmonary artery cannula. Krebs' solution, gassed continuously with 95% O, and S% CO, and warmed to 37°C, was then pumped through the lung at the rate of 7 ml/min . The lung was aeratedviathetracheal cannula by addition of 10-20 ml of air to fully extend the alveoli, after which the tracheal cannula was clamped. The effluent from the lung superfused the tissues used for bioassay (VANE, 1969). Perfusion pressure was measured with a pressure transducer (Statham P23AC) attached to a Grass 79D polygraph (Grass Instrument Co ., Quinsy, MA, U.S .A .) . PLA, in 0.1 ml saline was injected into the Krebs' solution through the pulmonary artery cannula.
Bioassay technique
The effluent from the lung superfused a number of isolated muscle strips, including chick rectum (MnNN and WEST, 1950), rat aortic strip (FtntcHCO~rr, 1960), rat colon (REOOLI and VANE, 1964) and guinea-pig ileum (S~rECxscttuL~rE et al., 1967). The assay tissues were treated with continuous infusion (2 ml/min) of a mixture of antagonists (BLOCx et al., 1975) and indomethacin to give the following final concentration of the active bases: atropine sulfate 0.2 ~g/ml; diphenhydramine-HC10 .2 ~g/ml; cimetidine-HC10.2 ~g/ml; methysergide maleate 0.1 4~g/ml ; phenoxybenzaR+~ne-HCl 0.1 pg/ml; propranolol HCl 2 kg/ml; indomethacin 1 ~g/ml . PGE-like substance was detected by contraction of the chick rectum, PGF-like substance by contraction of the rat colon (F~xxEtxA and VANE, 1967), TXA,-like substance by contraction of the rat aortic strip (WALLACH SIId BROWN, 1981) and leukotriene (SRS-A)-like substance by contraction of guinea-pig ileum (S~rECxscrtuLrE et al., 1967). One unit of SRS-A was defined as the amount giving the same response as 5 ng of histamine. PGI,-like activity could also be detected on the rat colon. The response to PGI,-like substance was cessation of spontaneous contraction, accompanied in some cases by a relaxation .
Radioimmtmoassays of thromboxane B, and 6-keto-prostaglandin F
TXA, and PGI, released in the lung effluent were quantitated by radioimmunoassays of the degradation products, TXB, and 6-keto-PGF , respectively (SHiaourA et al., 1979). After passage over the assay tissues, the lung
Release of Slow Reacting Substance by Phospholipases A,
36 1
effluents (1 min per tube) were collected serially. TXB, and 6-keto-PGF in control tubes collected before the injection of the PLA, fractions and sample tubes collected at 1, 2, 4 and 6 min afterthe injection of PLA, fractions were determined by radioimmunoassay. Twenty to one-hundred microliters of the effluent from each tube were used. The mixture of the antiserum, tritiated antigen (New England Nuclear, Boston, MA, U.S .A .) and the sample or the standard cold antigen were vortexed and incubated in tubes at 4°C for 16 hr . The antibody-bound and free antigen were then separated by mixing with dextran-coated charcoal followed by centrifugation . The radioactivity of the bound tritiated antigen was measured by means of a liquid scintillation counter (Beckman Instruments, Inc., Palo Alto, CA, U.S .A.) using "Aquasol" scintillator (New England Nuclear) . Per cent cross reaction of the TXB, antiserum (New England Nuclear) used was 0.2 for PGE, and <0 .2 for PGA,, PGF and 6-keto-PGF,° , respectively . Per cent cross reaction of the 6-keto-PGF antiserum (New England Nuclear) used was 2.7 for PGF , 2.0 for PGE <0 .1 for TXB, and PGA, and <0 .3 for PGA respectively . Statistics Means, S.E .M. and correlation coefficients were calculated aaording to standard statistical methods. Differences in means were assessed by the Student's t-test . P values of less than 0.05 were accepted as being statistically significant . Drugs Indomethacin, dl-propranolol-HCl and atropine sulfate were purchased from Sigma, St . Louis, MO, U.S .A . Cimetidine-HCl and phenoxybenzamine-HCl were purchased from Smith Kline and French Labs, Philadelphia, PA, U.S .A. Diphenhydramine-HCI was purchased from Taiwan Tanabe SeiyakuCo ., Taipei, Taiwan . Methysergide meleate was kindly supplied by Sandoz, Basle, Switzerland . FPL55712 (Sodium 7-{3-(4acetyl-3-hydroxy-2-propylphenoxy}2-hydroxy propoxy}-4-oxo-g-propyl~H-l-benzopyran-2~arboxylate ; C H O,Na) was kindly supplied by Fison Co ., Leicestershire, U.K . RESULTS
Effects on the lung perfusion pressure
The basal perfusion pressure (means t S .E.M.) in guinea-pig lungs was 25 ± 3 mmHg
(n = 25). Bolus injection of PLAN fractions from V. russelli venom into the pulmonary
circulation increased the perfusion pressure . The effluent from the lung which superfused the guinea-pig ileum induced a contraction. The typical response to Fr. III-3 is illustrated in Fig. 1 . In a screening test, the main subfractions Frs. II-5, II-7, III-3 and III-6 were more potent in increasing the perfusion pressure than were the other PLAN subfractions . The response of slow reacting substa~lce appeared at 1- 3 min after venom application in the nonsensitized lung (MIDDLETON and PHILLIPS, 1962). Either the maximal pressure increase (peak App) or the pressure increase at 2 min after PLA, application (App 2 min) induced by PLAz fractions were generally dose-dependent. Even when the App did not increase further as the dose was increased, the duration of response was prolonged (Table 1). Tachyphylaxis was observed on a subsequent injection 30 min after the first injection of PLA= fractions. Edema formation was found in the perfused lung after the injection of PLAN fractions. When the lung preparation was perfused continuously for 30 min after a bolus injection of Fr . III-3 (10 pg), the basal weight of the lung increased by 46% from 2.6 t 0.2 g (n = 5) to 3 .8 t 0.1 g (n = 5). PLA, may have increased the vascular permeability in the pulmonary vascular bed. Both the peak App and App 2 min were reduced by pretreating the lung with indomethacin (10 fig) at 1 min before PLA, application (Table 2). The effect of Fr. III-3 on rat lungs was compared to that on guinea-pig lungs (Table 1), with the rat lung effect being less marked . The time to maximal pressure response (peak time) was 0.8 t 0.2 min and the maximal pressure increase was 1 .50 t 1 .22 mmHg for rat lungs induced by Fr. III-3 (10 ~tg) . Prostaglandin-like substances released
Injection of Fr. II-5 (10 Ng) into the Krebs' solution perfusiog the guinea-pig lung caused an increase in the perfusion pressure and resulted in the appearance in the lung effluent
362
HUEZ-CHEN HUANG 2 min
A
B lg Fr. III-~ 10 ~tg TL F1G. 1 . RELEASE OF SLOW REACTING SUBSTANCE FROM THE PERFUSED GUINEA-PIG LUNGS INDUCED HY
Fr . III-3 . (A) Perfusion pressure response to Fr. III-3 . Fr . III-3 (10 Ng), injected as a bolus into the pulmonary circulation, produced an increase in perfusion pressure. (B) Response of guinea-pig ileum superfused with the effluent from the lung. The material released in the effluent by Fr . III-3 contracted the ileum . TL, Fr. III-3 was injected through the lung and not applied directly to the ileum .
of substances which contracted the chick rectum and the rat aortic strip (Fig. 2) . Pretreatment of the perfused lung with a bolus injection of indomethacin (10 kg), a cyclooxygenase inhibitor, reduced the pressure response . The contraction response of the rat aortic strip was also significantly reduced by indomethacin, indicating the presence of TXA, in the effluent (Fig. 2C, Table 3) . The contraction of chick rectum was not changed significantly by indomethacin, indicating the absence of PGE in the effluent (Fig. 2B, Table 3). Injection of Fr . III-3 (30 Fig) also caused an increase in the perfusion pressure and resulted in the appearance of substances which contracted the chick rectum and the rat aortic strip (Fig. 3). Pretreatment with indomethacin (10 pg) reduced the pressure response. The contraction response of the rat aortic strip was significantly increased by indomethacin (Fig. 3C, Table 3). This may be due to simultaneous release of vasodilator PG, which was inhibited by indomethacin, and release of leukotriene, which was potentiated by indomethacin . The contraction of the chick rectum was not changed significantly by indomethacin, indicating the absence of PGE (Fig . 3B, Table 3). The effects of the lung effluent on the rat colon were very variable and no conclusive results were obtained.
Release of Slow Reacting Substance by Phospholipases A,
363
TABLE 1 . EFFECT'S OF CRUDE VENOM AND PLA, FRACTIONS ON THE PERFUSION PRESSURE OF GUINEA-PIG LUNGS
Fraction Crude venom Fr . II-S Fr .I1-7 Fr . III-3
Dose (Ng) 10 30 100 10 30 100 30 100
n 3 12 5 10 3 3 8 3 3 S 7
Time to peak App (min) 1 .5 t 0.5 0 .ßt0 .2 1 .1 t 0.3 2.0 t 0.2 1 .7t0 .2 1 .S t 0.0 1 .4t0 .2 1 .0 t 0.0 0.7 t 0.1 O.St0 .3 0.8t0.2 0.8f0 .1 0.9t0.2 0.3*0 .0
Perfusion pressure Peak App (mmHg) 1.50 t 0.30* 3 .2310.31* 6 .00 f 1 .36* 3.06 t 0.30* 4.OOt0.38* 4.SO t 0.33* 2.30t0 .43* 6.00 t 0.71* 4.67 t 0.54* 4.73t0.92* 3.73t0.48* 3.30t0.71* 3 .74t1 .09* 7.33t0.33*
Duration (min) 1 .3 t 0.3 3 .210 .6 5 .0 t 2.5 7.6 t 1.6 6.Of0 .7 18 .0 t 0.0 2.0t0 .3 8.0 t 1.4 4.7 t 0.2 3.7f1 .2 20.6t4.6 6.2t0 .9 9.3t2.3 13 .0t1 .3
App 2 min (mmHg) 1.50 t 0.50* 2.5010.35* 5.00 * 1 .23* 2.83 t 0.33* 4.OOt0.58* 4.SO t 0.35* 2.12*0.37* S.SO t 0.33* 3.00 * 0.47* 3.OOf0.13* 4.12*0.63* 3.00*0.47* 3.10t0.86* 6.67f0.27*
3 10 30 Fr .III-6 3 3 10 7 30 3 Values are the mean t S.E .M . Peak App denotes maximal increase in perfusion pressure induced by venom or PLA,-fractions . App 2 min denotes increase in perfusion pressure at 2 min after the injection of PLA, fractions. The basal perfusion pressure of guinea-pig lung during the control period was 23 t 3 mmHg (n=25) . *PG0 .05 when venom or PLA, fraction-treated preparations were compared to controls . All data of peak App and App 2 min were analyzed by a paired t-test.
Release of a leukotriene-like substances Leukotriene-like substances in the lung effluent were detected by superfusing the guineapig ileum. The ileum was treated with a continuous infusion of a mixture of antagonists and FPL55712 (10 ng/ml) . At 0.5 min before injection of the PLA, fractions, an additional dose of FPL55712 (10 ng) was superfused directly onto the ileum. FPL55712, a specific antagonist of leukotrienes C and D, reduced the contractile response of the ileum, indicating the presence of leukotriene C and/or D in the lung effluent treated with Frs. II-5 and III-3 (Table 4). The per cent inhibition by FPL55712 was 75% for Fr. II-5 and 66% for Fr . III-3 respectively . Release of histamine The contractile response of the guinea-pig ileum to the effluent from the lung treated with Fr. III-3 (10 ~tg) was reduced significantly by antihistaminics, indicating the presence of histamine in the effluent. Antihistaminics (diphenhydramine 0.2 Ng/ml and cimetidine 0.2 Ng/ml) superfused directly and continuously onto the ileum reduced the maximal tension of the ileum by 32% from 0 .47 ± 0.01 g to 0.32 t 0.01 g (P~0.05) . Radioimmunoassay of thromboxane B= and 6-ketoprostaglandin F Thromboxane Az (TXAs) and prostacyclin (PGI,) released by PLAz fractions were also quantitated by the radioimmunoassay of the degradation products TXB~ and 6-ketoPGF1Q, respectively . Figure 4 shows the time courses and dose - response relationships of TXB, release induced by Frs . II-5 and III-3 (3 - 30 Ftg). Figure S shows the time courses and dose - response relationships of the 6-keto-PGF release induced by Frs. II-5 and III-3 (3 - 30 lig) . All ri,A2 fractions tested, Frs. II-S, II-7, III-3 and III , induced the release
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Fio . 2. BFFHCr of Fr . II-3 ox 1tm al~IewTION of PnosrwoLwHnn~-Lna: sussrwtlc8 uv n~ PsAPUSen OUINEA-PIO LUNG AND rIS MODIFICA710N nY INDOMSI'HAC1N . (A) Perfusion pressure response to Fr . II-3 . A bolus injection of Fr . II-S (10 ~ through the lung (TL) increased the perfusion pressure pelt hand extion). Indomethsdn pNDO ; 10 ~ igjected through the lung (fL) 1 min before Fr . II-3 application produced a significant attenuation of the pressure response to Fr. ü-3 (right hand section). pi) Responses of chick rectum (CR) and of (C) rat aortic strip (RA) superfused with effluent from the lung are shown . The substances rdeased in the effluent by Fr. II-S contracted CR and RA peR hand radon) . The contraction response on RA was significantly reduced by pmt with i hadn pNDO), indicating the preseace of thromboxsne A,-like material in the effluent . TL, Fr . II-3 and 1ND0 were igjected through the lung and not appüod directly to the assay tissue .
Twlu.a 3 . RHSPONSHS OF CHICIC AHCR7M AND aAT AORTIC SfA1P SUPERPUSCD wrI'H TI~ EFFLUF.Nr FAOM Tf~ PLA, FAACI'ION~TIMULAT'® OUINEA-PIO LUNCi Fraction
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Increase in tension at 2 min (g) Chick rectum Rat aortic strip 0.70 t 0.29 1 .10 f 0.14 1 .70 t 0.43 1 .73 f 0.03
0 .23 t 0 .07 Ot 0.28 t 0 .02 0 .30 t 0 .07t
Values are the mean t S.B.M. PLA, fractions or indomethacin were igjated as a bolus through the hlng . The hmg effluent superfused the assay tissues . "Response of the assay tissue to the effluent from the indomethacin-pretreated guinea-pig hung . tPC0 .03 when indomethadn-pretreated preparations were compared to controls .
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Fr . III-3 ON THE GENERATION OF PROSTAGLANDIN-LIKE SUBSTANCE IN THE PERFUSED GUINEA-PIG LUNG AND ITS MODIFICATION HY INDOMETHACIN .
Perfusion pressure response to Fr . III-3 . A bolus injection of Fr . III-3 (30 pg) through the lung (TL) increased the perfusion pressure (left hand section) . Indomethacin (INDO; 10 pg) injected through the lung (TL) 1 min before Fr . III-3 injection produced a significant attenuation of the pressure response to Fr . III-3 (right hand section) . (B) Responses of chick rectum (CR) and of (C) rat aortic strip (RA) superfused with effluent from the lung are shown. The substances released in the effluent by Fr . III-3 contracted CR and RA (left hand section) . The contraction response on RA was significantly increased by pretreatment with INDO. TL, Fr. III-3 and INDO were injected through the lung and not applied directly to the assay tissue . (A)
TABLE 4 . RESPONSE OF GUINEA-P1G ILEUM SUPERFUSED WITH THE EFFLUENT FROM THE PLA, FRACTION-STIMULATED GUINEA-PIG LUNG
FPL 55712 Dose treated n Increase in tension at 2 min (g) (10 ng) (hg) Fr . II-5 10 3 0.60 t 0.07 10 +' 3 O .15t0.11t Fr . III-3 10 3 0.38 f 0.06 10 +' 3 0.13 t O.OSt Values are the mean t S.E .M . FPL 55712 (10 ng/ml) contained in a mixture of antagonists was continuously infused onto the ileum and an additional dose (10 ng) was superfused directly onto the ileum at 0.5 min before the injection of PLA, fractions. 'Response of the guinea-pig ileum pretreated with FPL 55712. tP~0.05 when FPL55712-pretreated preparations were compared to controls . Fraction
Release of Slow Reacting Substance by Phospholipases A,
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Fr . III-3 (B) ON TFn: LEVEL OF THROMBOXANE B, IN TIC EFFLUENT OF GUINEA-PIG LUNG .
Fr . II-S and Fr . III-3 (3 Ng - 30 Ng) were injected as a bolus injection through the lung . The lung effluents (1 min per tube) were collected serially. Thrombosane B, (TXB,) in a control tube collected before PLA, fraction injection (0 min) and sample tubes collected at 1, 2, 4 and 6 min after PLA, fraction injection were determined by radioimalunoassay. Each column represents the amount (ng/ml) of TXB, in the effluent . Each bar represents the S .E .M . of at least three determinations .'P<0.05 when compared to the control period . 0 min denotes the control period before injection. 1, 2, 4 and 6 min denote time after injection of PLA, fractions.
of TXB1 and Crketo-PGF from the perfused guinea-pig lung. The levels of TXB, and 6-keto-PGF measured 2 min after the injection of PLA, fractions are shown in Table 5. As shown in Fig . 6, there was a positive linear correlation between the pressure increases (App) induced by PLAz fractions and the ratios of TXB2 to Cz^keto-PGF, Q (TXB,/6-keto-PGF ) released by PLAN fractions (r=0.87). Both App and the amount of TXB, and 6-keto-PGF,Q were measured 2 min after the injection of PLA 1 fractions. DISCUSSION
PI :A= induced an increase in the lung perfusion pressure accompanied by a release of prostaglandin-like and leukotriene-like substances. The pressure response and TXA, and
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FIG. S. EFFECTS of Fr . II-S (A) AND Fr . III-3 (B) ON THE LEVEL OF o-KETO-PROSTAGLANDIN F wTHE EFFLUENT OF GUINEA-PIG LUNG Fr. II-S and Fr . Ill-3 (3 -30 Ng) were injected as a bolus injection through the lung . The lung effluents (1 min per tube) were collected serially . Crketo-prostaglandin F (6-kao-l?GF ) in a control tube collected before PLA, fraction injection (0 min) and sample tubes collected at 1, 2, 4 and 6 min after PLA, fraction injection were determined by radioimmunoassay . Each column represents the amount (ng/ml) of rrketo-PGF in the effluent . Each bar represents the S.E .M . of at least three determinations . "P<0.05 when compared to the control period . 0 min denotes the control period before injection . 1, 2, 4 and 6 min denote time after injection of PLA, fractions . TABLE S. EFFECTS OF PLA, FRACTIONS ON THROMBOXANE B, AND 6-KET0 .PROSTAGLANDIN F RELEASE 1N GUINEA-PIO LUNAS Fraction Control Fr . II-5 Fr . II-7 Fr . III-3 Fr . III-6
Dose lf+g) 10 30 30 10 30 10
TXB,
(n8/ml) 0.20 t 0.01 3 .11 t 0.4T 3.58 t 0.48' 7.00 t 0.00' 3 .78 t 0.33' 29 .75 t 1.9T 2.35 t 0.15'
6-keto-PGF 0.08 0.75 0.76 2.70 1 .48 5 .53 0.35
t 0.02 t 0.08' t 0.04' t 0.11' f 0.18' t 0.8T t 0 .11'
Values are the mean t S.E .M . of at least 3 determinations . The levels of TXB, and 6-keto-PGF were measured 2 min after the injection of each PLA, fraction . 'P'<0.05 when sample effluents collected after the injection of PLA, fraction were compared to effluent collected in the control period .
Release of Slow Reacting Substance by Phospholipases A,
369
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5
3
TXBZ/6-keto-PGFI a COAAELATION BETWEEN THE INCREASE IN PERFUSION PRESSURE (App) AND TfIE RATIO OF THROMBOxwNe B, To 6-x~ro-PROSrwaLArmw F (TJ{B,/CrtcE'ro-PGF ) IN THE EFFLUENT OF PLA,-STIMULATED GUINEA-PIG LUNG . Both App and the ratio T7LB,/(rketo-PßF were meaaurod 2 min after injection of PLA, fractions. FIG. G.
Each point is the mean of at least three determinations from either Fr . II-5, II-7, III-3 or III at a dose between 10 and 30 Ng .
PGI= release induced by PLA= fractions were generally dose-dependent . It is suggested that PLA, fractions from V, russelli venom released arachidonic acid from membrane phospholipids, which was transformed to TXAz and PGI, by cyclooxygenase or to leukotrienes by lipoxygenase . Injection of Fr . III-3 induced less pressure response in rat lungs than in guinea-pig lungs. Similarly, arachidonic acid perfused through the pulmonary circulation of guinea-pig lungs was metabolised to more myotropic metabolites than it was in rat or human lungs (ALLIBAIDI and BAKHLE, 1980). These results suggest that there may be important qualitative and quantitative differences in the activation of exogenous or endogenous arachidonic acid between guinea-pig lungs and rat lungs. According to DAMRAU et al. (1975), the main active principle formed in the cobra venom perfused guinea-pig lung (SRS-C) was PGE. By studying the effect of lung effluent on rat aortic strip in our experiments, we determined that application of Fr . II-5 was followed by a release of TXAs from the guinea-pig lung . Since the effect on the chick rectum was not significantly reduced by indomethacin, it is more likely that TXA=, and not PGE, is the major cyclooxygenase product released by Fr . II-5 . TXA3 has potent blood vessel and airway constrictive properties (SPAxtvI-IAxE et al., 1981). From the data obtained from radioimmunoassay of 6-keto-PGF , PGIs was also markedly released by Fr. III-3 and slightly by Fr. II-5 . The release of more PGIz induced by Fr. III-3 may have an opposing effect to TXA,, attenuating the vasoconstrictor action of TXAs on the pulmonary vascular muscle . PGIz has potent vasodilatory and bronchodilatory properties (SPANIVI-IAI{E et al ., 1981). There was a positive linear correlation between the pressure responses induced and the ratios of TXB, to 6-keto-PGF released by various PLA, fractions. It appears that the relative proportion of two different arachidonic acid metabolites released determines the effects of some PLA, fractions in the perfused lung . FPL55712, a specific antagonist of leukotrienes C and D, inhibited the contraction of the glûnea-pig ileum to the effluent from a lung which had been treated with PLA= at a concentration of 10 ng/ml, a concentration which is known to antagonize the contraction of ileum to SRS-A, without affecting the responses to other contractile agents (AUGUSTEIN
370
HUEZ-CHEN HUANG
et al ., 1973). The results indicated that both Frs. II-5 and III-3 released leukotriene C and/or D. Leukotrienes are most active in contracting smooth muscle of lungs (SPANNHAKE et al., 1981). In the present study, the SRS released by PLAN fractions of Russell's viper venom from the perfused lung was identified as a mixture of thromboxane A~, prostacyclin (PGI,) and leukotriene C and/or D. Injection of PLAN fractions from Russell's viper venom increased the perfusion pressure, indicating that the contribution of the contractor substances released was more than that of the dilator substances . Pretreatment with indomethacin reduced the pressure response, indicating that the contribution of the cyclooxygenase products was more than that of the lipoxygenase products . Examination of the activity of PLAN fractions on release of arachidonic acid metabolites from the perfused lung revealed a similar effect, but some quantitative differences . It may be due to the different substrate specificities of PLAN fractions. Similarly, SHUKLA and HANAHAN (1982) showed that acidic and basic PLA, of A. halys blomhoffü can also identify the different domains of phosphatidylcholine in human erythrocyte membranes. The latent period of SRS-C released by snake venom in the nonsensitized lung was 1- 3 min (MIDDLETON and PHILLIPS, 1962). The histamine effect peaked at 10-15 sec and terminated within 1 min (SMEDEGARD et al., 1982). In the present study, PLA, fractions of Russell's viper venom released TXA2, PGh, leukotrienes and also histamine from the perfused guinea-pig lungs. All these autacoids increase vascular permeability and may result in edema formation in the perfused lung . Histamine and PGI 2 have direct vasodilatory properties . Recently, leukotriene was also shown to have a prolonged hypotensive effect in guinea-pig and monkey (DRAZEN et al ., 1980 ; PIPER et al ., 1981 ; SMEDEGXRD et al., 1982). Therefore, part of the hypotensive action of PLA fractions may be due to the actions of histamine, PGI, and leukotriene released . There are some reports which provide evidence for the involvement of endogenous PGlike substance in the hypotensive action of antihypertensive drugs, including vasodilators (HAEUSLER and GEROLD, 1979 ; REIMANN et al ., 1981), lI-adrenoceptor blocking agents (JACKSON et al ., 1979 ; GRAHAM et al ., 1982) and diuretics (SCRIABINE et al., 1979). In our results, PLA, fractions of Russell's viper venom increased lung perfusion pressure, which may restrict blood return to the heart and lead to a decrease in cardiac output and induce a hypotensive effect indirectly . For Fr . II-5, the hypotensive action may be mediated by pulmonary vasoconstriction . For Fr . III-3, more PGh was released, which may cause vasodilatoon in the periphery, combined with pulmonary vasoconstriction, to induce a greater hypotensive effect . Further investïgation on the release of arachidonic acid metabolites in other vascular beds by PLAN is necessary for determining the relationship between the hypotensive action of PLA2 and the release of arachidonic acid metabolites.
I gratefully acknowledge the valuable advise and constructive criticism from Dr . C. Y. Lam, Professor of Pharmacology, College of Medicine, National Taiwan University .
Acknowledgements -
REFERENCES At .-Uentnt, F. and BwxHLe, Y. S. (1980) Difference in biological activation of arachidonic acid in perfused lungs from guinea-pig, rat and man. Eur. J. Pharmac. 62, 89. AuousTenv, J., FwxMSa, J. B., LF,$, T. B., Stte~.xn, P. and TATTERSALL, M. L. (1973) Selective inhibitor of slow reacting substance of anaphylaxis . Nature, Nrw Bio% 245, 215. BLOCK, A. J., F~nveexc, H., Hstmwcznvsxn-CeDRO, K. and Vn1vE, J. R. (1975) Anoxia-induced release of prostaglandins in rabbit isolated hearts . Circulation Res. 36, 34.
Release of Slow Reacting Substance by Phospholipases A,
37 1
BRAntow, B. A. and Mwxcus, A. J. (1966) Action of snake venom phospholipase A on isolated platelet membranes. Proc . Soc. exp. Biol. 123, 889. CHIU, T. H., LEE, C. Y. and LEE, S. Y. (1968) Hemodynamic effects of cardiotoxin isolated from Formosan cobra venom. J. Formosan mad. Ass. 67, 557. DAMERAU, B., LEae, L., OLDIGS, H.-D. and Voor, W. (1975) Histamine release, formation of prostaglandinlike activity (SRS-G) and mast cell degranulation by the direct lyric factor (DLF) and phospholipase A of cobra venom . Naunyn-Schmiedebergs Archs Phormae. 287, 141 . DRwzEx, J. M., AUSTEx, K. F., L1:WIS, R. A., CtARx, D. A., Goro, G., MARFAT, A. and CoREV, E. J. (1980) Comparative airway and vascular activities of leukotrienes C-1 and D in vivo and in vitro. Proc . natn . Acad. Sci. U.S.A . 77, 4354 . FELDBERG, W. and KELIAWAY, C. H. (1938) Liberation of histamine and formation of lysolecithin-like substances by cobra venom. J. Physiol., Lond. 94, 187. FERREtRw, S. H. and VANE, J. R. (1%7) Prostaglandins : their disappearance from and release into the circulation. Nature, Lond. 216, 868. Fuacttoo~I-r, R. F. (1960) Spiral~tlt strip of rabbit aorta for in vitro studies of responses of arterial smooth muscle . In : Methods in Medical Research, Vol. 8, p. 177 (SAUNER, H. D., Ed .) . Chicago: The Yearbook Publishers . GRwIIwM, R. M., CAMPBELL, W. B. and JACxsoN, E. K. (1982) Effects of short term beta blockade on blood pressure, plasma thromboxane B,, and plasma and urinary prostaglandins E, and F in normal subjects . Clin . Pharmac. Ther. 31, 324. HAEUSLER, G. 8nd GEROLD, M. (1979) Increased levels of prostaglandin-like material in the canine blood during arterial hypotension produced by hydralazine, dihydralazine and minoxidil. Naunyn-Schmiedebergs Archs Aharmac. 310, 155. Huwxa, H. C. and LEE, C. Y. (1984) Isolation and pharmacological properties of phospholipases A, from Vipers russelli (Russell's viper) snake venom. Toxicon 22, 207. Jwcxsox, E. K., CAMPBELL, W. B. and C1.ARKE, W. G. (1979) Inhibition of angiotensin II enhancement of adrenergic transmission by propranolol: an antihypertensive mechanism. Fedn Proc . Fedn Am . Socs exp. Biol. 38, 678. LEWIS, R. A., AusTEnt, K. F., DRwzEx, J. M., CtARtc, D. A., MARFAT, A. and COREY, E. J. (1980) Slow reacting substances of anaphylaxis : identification of leukotrienes C-1 and D from human and rat sources. Proc . nain . Acad. Sci. U.S.A . 77, 3710 . MANN, M. and Wes'r, G. B. (1950) The nature of hepatic and splenic sympathie. Br . J. Pharmac. Chemother. S, 173. MIDDLETON, E., JR . eIId PHILLIPS, G. B. (1%2)1?istribution and properties of anaphylactic and venom induced, slow-reacting substance in guinea-pigs . J. clin . Invest. 41, 1385 . MORRIS, H. R., PIPER, P. J., TAYLOR, G. W. and TIPPINS, M. R. (1979) Comparative studies on immunologicafy produced slow-reacting substances from man, guinea-pig and rat. Br. J. Pharmac. 67, 179. PHILLIPS, G. B. and Mmnttrrox, E., JR. (1%5) Release of histamine and slow reacting substance activities from guinea-pig . Proc. Soc. exp. Biol. 119, 465. PIPER, P. J., SwMxoux, M. N., TIPPINS, J. R., MORRIS, H. R., JONFS, C. M. and TAYLOR, G. M. (1981) SRSA and SRS: their structure, biosynthesis and actions. Archs Allergy appl. Immun. 66 Suppl. 1, 107. REOOLI, D. and VANE, J. R. (1964) A sensitive method for the assay of angiotensin . Br. J. Pharmac. 23, 351 . REIMANN, I. W., RATae, R. D., WtssER, H. and FttöucH, J. C . (1981) Are prostaglandins involved in the antihypertensive effect of dihydralazine7 Clin . Sci. 61, 3195 . RoTescxltn, A. M. and Ronascrlltn, Z. (1979) Liberation of pharmacologically active substances by snake venoms . In: Spoke Venoms. Handbook Jor Experimental Pharmacology, Vol. 52, p. 591 (LEE, C. Y., Ed .) . Berlin : Springer-Verlag . SALwctI, J. L, TURINI, P., SPJJO, G., HAUIiER, J. and SINGER, T. P. (1971) PhosphoGpase A of snake venoms . I. Isolation and molecular properties of isoenzyme from Naja naja and Vipers rwsselli venom. J. biol. Chem . 246, 331. Sexürz, R. M. and VoaT, W. (1961) Über die Natur der durch Cobragift in durchstrSmten Meerschweinchenlungen freigesetzten "Slow Reacting Substance" . Naunyn-Schmiedebergs Arch . exp. Path . Pharmak. 240, 500. SCRIAHINE, A., Ww~tsox, L. S., Russo, H. F., LuDnEx, C. T., SwEEr, C. S., FANELLI, G. M., JR., BOHIDAR, N. R. and $TONE, C. A. (1979) Diuretic and antihypertensive effects of 2-amino{thyl-4-(l,l~iamethyl ethyl){riodonphenol hydrochloride (MK447). J. Pharmac . exp. Ther. 208, 148. SfnBOUTw, Y ., lxwow, Y., TBRASFiITA, Z., NISIiIKAWA, K., Ktxuctu, S. and St~tIMwMOTO, K. (1979) AegiotensinII-stimulated release of thromboxane A, and prostacyclin (PGI,) in isolated perfused kidneys of spontaneously hypertensive rats . Biochem. Pharmac. 28, 3601 . StIUICLA, S. D. and Hwxwtlwx, D. J. (1982) Identification of domains of phosphatidylcholine in human erythrocyte plasma membranes. J. biol. Chem. 257, 2908 . St~nEaARn, G., HEnQvtsr, P., DwxLEx, S. E., Rt vENxsE, B., HwMMwRSTöRM, S. and SwMUetssox, B. (1982) Leukotriene C, affects pulmonary and cardiovascular dynamics in monkey . Nature 295, 327. SPwxxrtwxE, E. W., HYMAN, A. L. and Kwnowtrz, P. J. (1981) Bronchoactive metabolites of arachidonic acid
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and their role in airway function . Prostaglandins 22, 1013 .
STECHSCHULTE, D . J ., AustErv, K . F . and B~ocx, K . J . (1967) Antibodies involved in antigen-induced release of slow reacting substance of anaphylaxis (SRS-A) in the guinea-pig and rat . J. exp. Med. 1Z5, 127 . VANE, J . R. (1969) The release and fate of vasoactive hormones in the circulation . Br. J. Pharmac . 35, 209 . Voct, W . (1969) Slow reacting substances . In : Cellular and Humoral Merhonisms in Anaphylaxis and Allergy,
p . 187 (Mount, H . Z ., Ed .) . Basel : Karger .
Voct, W., MEYER, U ., KUNZE, H ., Lth-r, E . end BABILLI, S . (1969) Entstehung von SRS-C in der durchstrbmten
Meetschweinchenlunge durch Phospholipase A . Identifizierung mit Prostaglandin . Naunyn-Schmiedebergs Arch . exp. Path . Pharmak . 262, 124 . WALLACH, D . P . and BROWN, V . J . R. (1981) Studies on the arachidonic acid cascade - I . Inhibition of phospholipase A, in vitro and in vivo by several novel series of inhibitor compounds . Biochem. Pharmac. 30, 131 S .
0041-0101/84 53 .00+ .00 . © 1984 Pergamon Prep Ltd .
RELEASE OF SLOW REACTING SUBSTANCE FROM THE GUINEA-PIG LUNG BY PHOSPHOLIPASES A2 OF VIPERA R USSELLI SNAKE VENOM HUEI-CHEN HUANG Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan, R.O .C . (AcceptedJor publication 2 November 1983) H.-C. Huuvc. Release of slow reacting substance from the guinea-pig lung by phospholipases A, of Vlperu russelli snake venom. Taxiton 22, 359 -372, 1984. - Phospholipases A, (PLA,) of Vlpera rusAelli venom were isolated by column chromatography . The ability of PLA, fractions to release slow reacting substance (SRS) was studied in We guinea-pig lung perfused with Krebs' solution . The relationship between the perfusion pressure change produced by PLA, and SRS release was also studied. Two PLA, fractions (II-S and III-3; 3 -100 Ftg), injected into the lung increased the perfusion pressure and released SRS. Pretreatment of the lung with indomethacin (10 Ftg) reduced the pressure iesponse induced by the PLA, fractions. T'he SRS released in the lung effluent by PLA, was identified by bioassay as a mixture of thromboxane A, (TXA,), prostacyclin (PGI,) and leukotrienes . TXA, and PGI, release was also quantitatedby radioimmunoassay of the degradation products TXB, and 6keto-PGF , respectively. There was a positive linear correlation between the pressure increases and the ratios of TXB, to ~keto-PGF (r=0 .87) . It appears that the relative amounts of TXA,,and PGI, released determine the effects of PLA, fractions on the guinea-pig lung . The release of arachidonic acid metabolites, prostaglandins and leukotrienes may account for pan of the hypotensive action of PLA, . INTRODUCTION
have indirect effects due to the release or formation of pharmacologically highly active autacoids (ROTHSCHILD and ROTHSCHILD, 1979). Autacoids such as histamine, 5-hydroxytryptamine, kinins, slow reacting substance (SRS) and prostaglandins have been associated with a number of pathologic processes involving blood pressure, pulmonary function, vascular permeability and visceral smooth muscle tone. SRS was described by FELDBERG and KELLAWAY (1938) as a smooth muscle contracting factor appearing in the perfusate of guinea-pig lung following treatment with cobra venom. SRS, as well as histamine, is released from the lungs of nonsensitized guinea-pigs by various preparations (heat-treated or heparin-treated) from cobra (N. ngja) venom. This effect has been suggested to be due to phospholipase Az (PLA=) present in the venom (FELDBERG and ICELLAWAY, 1938 ; SCHÜTZ and VOGT, 1961 ; PHILLIPS and MIDDLETON, 1965). Chromatographic analysis of SRS formed by cobra venom showed it to be a mixture of hydroxylated, unsaturated fatty acids belonging to the prostaglandin family (VOLT et al ., 1969). By means of thin layer chromatography, DAMERAU et al. (1975) suggested that SRS activity is primarily due to a species of prostaglandin E. According to VOLT (1969) and DAMERAU et al. (1975), snake venoms supply the enzyme (PLA,) required for the first step in prostaglandin (PG) formation by releasing unsaturated fatty acids from tissue phospholipids . Tissue enzymes, in a secondary phase, transform them into pharmacologically active prostaglandins . PGs are the cyclooxygenase products of SNAKE VENOMS
359
36 0
HUEI-CHEN HUANG
arachidonic acid and have biological activity on pulmonary smooth muscle and other tissues. Slow reacting substance of anaphylaxis (SRS-A) is produced from sensitized guinea-pig lungs and has been determined to be leukotrienes (MORRIS et al., 1979, LEwts et al., 1980). Leukotrienes are the lipoxygenase products of arachidonic acid and also have potent biological activity on pulmonary and other tissues. The components of SRS released from guinea-pig lungs by snake venom remain to be identified . Vipers russelli venom contains several isoenzymes of PLAN (~ALACH et al., 1971). PLA2 of Y. russelli venom affects membrane and granule phospholipids of red blood cells and platelets in a manner similar to PLAz of N. ngja venom (BRADLOW and MARCUS, 1966). In our preliminary tests, SRS was also released by PLA, fractions of V. russelli venom from guinea-pig lungs, similar to that induced by cobra venom. In the present study, the ability of PLAZ fractions to release SRS was studied in guinea-pig lungs perfused with Krebs' solution and the components of SRS were identified . PLAz caused a hypotensive effect accompanied by an increase in pulmonary pressure (CHID et al., 1968). The possible involvement of SRS release in the hypotensive effect of PLAN was also studied.
Phospholipase A, fractions
MATERIALS AND METHODS
Vipers rttsselli venom was purchased from Astik Farm and Laboratory, Bombay, India. Crude venom was appliedto a chromatography column (2 .5 x 72 cm) of Sephadex G-75 (Pharmacia, Uppsala, Sweden). The elution was carried out with 0.03 M ammonium acetate (pH 6.5). Two PLA, fractions, Frs. II and III, were subjected to refractionation by CM-Sephadex C-50 (Pharmacis, Uppsala, Sweden) column chromatography (1 .8 x 80 cm). The gradient elution was carried out with ammonium acetate buffer, 0.05 M (pH 5 .0) to 1 .5 M (pH 8.5) (Hunnc and LEE, 1984). The biologically active subfractions (II-5, II-7, III-3 and III) were collected and lyophilized. These subfractions had both neurotoxic and hypotensive actions. The purity, PLA, activity and general pharmacological properties of these fractions have been presented in a previous paper (HuAtvc and LEE, 1984).
Perfusion of guineapig lungs
Guinea-pigs weighing 200-300 g were killed by cervical dislocation. The thoracic cavity was rapidly opened, the pericardium incised and 1 ml of Krebs' solution containing 100 units of heparin was injected intracardially. The left heart was cut away and the lung was removed. The pulmonary artery was cannulated and the blood was flushed out with Krebs' solution . The trachea was then cannulated and the lung was attached to the perfusion apparatus by a pulmonary artery cannula. Krebs' solution, gassed continuously with 95% O, and S% CO, and warmed to 37°C, was then pumped through the lung at the rate of 7 ml/min . The lung was aeratedviathetracheal cannula by addition of 10-20 ml of air to fully extend the alveoli, after which the tracheal cannula was clamped. The effluent from the lung superfused the tissues used for bioassay (VANE, 1969). Perfusion pressure was measured with a pressure transducer (Statham P23AC) attached to a Grass 79D polygraph (Grass Instrument Co ., Quinsy, MA, U.S .A .) . PLA, in 0.1 ml saline was injected into the Krebs' solution through the pulmonary artery cannula.
Bioassay technique
The effluent from the lung superfused a number of isolated muscle strips, including chick rectum (MnNN and WEST, 1950), rat aortic strip (FtntcHCO~rr, 1960), rat colon (REOOLI and VANE, 1964) and guinea-pig ileum (S~rECxscttuL~rE et al., 1967). The assay tissues were treated with continuous infusion (2 ml/min) of a mixture of antagonists (BLOCx et al., 1975) and indomethacin to give the following final concentration of the active bases: atropine sulfate 0.2 ~g/ml; diphenhydramine-HC10 .2 ~g/ml; cimetidine-HC10.2 ~g/ml; methysergide maleate 0.1 4~g/ml ; phenoxybenzaR+~ne-HCl 0.1 pg/ml; propranolol HCl 2 kg/ml; indomethacin 1 ~g/ml . PGE-like substance was detected by contraction of the chick rectum, PGF-like substance by contraction of the rat colon (F~xxEtxA and VANE, 1967), TXA,-like substance by contraction of the rat aortic strip (WALLACH SIId BROWN, 1981) and leukotriene (SRS-A)-like substance by contraction of guinea-pig ileum (S~rECxscrtuLrE et al., 1967). One unit of SRS-A was defined as the amount giving the same response as 5 ng of histamine. PGI,-like activity could also be detected on the rat colon. The response to PGI,-like substance was cessation of spontaneous contraction, accompanied in some cases by a relaxation .
Radioimmtmoassays of thromboxane B, and 6-keto-prostaglandin F
TXA, and PGI, released in the lung effluent were quantitated by radioimmunoassays of the degradation products, TXB, and 6-keto-PGF , respectively (SHiaourA et al., 1979). After passage over the assay tissues, the lung
Release of Slow Reacting Substance by Phospholipases A,
36 1
effluents (1 min per tube) were collected serially. TXB, and 6-keto-PGF in control tubes collected before the injection of the PLA, fractions and sample tubes collected at 1, 2, 4 and 6 min afterthe injection of PLA, fractions were determined by radioimmunoassay. Twenty to one-hundred microliters of the effluent from each tube were used. The mixture of the antiserum, tritiated antigen (New England Nuclear, Boston, MA, U.S .A .) and the sample or the standard cold antigen were vortexed and incubated in tubes at 4°C for 16 hr . The antibody-bound and free antigen were then separated by mixing with dextran-coated charcoal followed by centrifugation . The radioactivity of the bound tritiated antigen was measured by means of a liquid scintillation counter (Beckman Instruments, Inc., Palo Alto, CA, U.S .A.) using "Aquasol" scintillator (New England Nuclear) . Per cent cross reaction of the TXB, antiserum (New England Nuclear) used was 0.2 for PGE, and <0 .2 for PGA,, PGF and 6-keto-PGF,° , respectively . Per cent cross reaction of the 6-keto-PGF antiserum (New England Nuclear) used was 2.7 for PGF , 2.0 for PGE <0 .1 for TXB, and PGA, and <0 .3 for PGA respectively . Statistics Means, S.E .M. and correlation coefficients were calculated aaording to standard statistical methods. Differences in means were assessed by the Student's t-test . P values of less than 0.05 were accepted as being statistically significant . Drugs Indomethacin, dl-propranolol-HCl and atropine sulfate were purchased from Sigma, St . Louis, MO, U.S .A . Cimetidine-HCl and phenoxybenzamine-HCl were purchased from Smith Kline and French Labs, Philadelphia, PA, U.S .A. Diphenhydramine-HCI was purchased from Taiwan Tanabe SeiyakuCo ., Taipei, Taiwan . Methysergide meleate was kindly supplied by Sandoz, Basle, Switzerland . FPL55712 (Sodium 7-{3-(4acetyl-3-hydroxy-2-propylphenoxy}2-hydroxy propoxy}-4-oxo-g-propyl~H-l-benzopyran-2~arboxylate ; C H O,Na) was kindly supplied by Fison Co ., Leicestershire, U.K . RESULTS
Effects on the lung perfusion pressure
The basal perfusion pressure (means t S .E.M.) in guinea-pig lungs was 25 ± 3 mmHg
(n = 25). Bolus injection of PLAN fractions from V. russelli venom into the pulmonary
circulation increased the perfusion pressure . The effluent from the lung which superfused the guinea-pig ileum induced a contraction. The typical response to Fr. III-3 is illustrated in Fig. 1 . In a screening test, the main subfractions Frs. II-5, II-7, III-3 and III-6 were more potent in increasing the perfusion pressure than were the other PLAN subfractions . The response of slow reacting substa~lce appeared at 1- 3 min after venom application in the nonsensitized lung (MIDDLETON and PHILLIPS, 1962). Either the maximal pressure increase (peak App) or the pressure increase at 2 min after PLA, application (App 2 min) induced by PLAz fractions were generally dose-dependent. Even when the App did not increase further as the dose was increased, the duration of response was prolonged (Table 1). Tachyphylaxis was observed on a subsequent injection 30 min after the first injection of PLA= fractions. Edema formation was found in the perfused lung after the injection of PLAN fractions. When the lung preparation was perfused continuously for 30 min after a bolus injection of Fr . III-3 (10 pg), the basal weight of the lung increased by 46% from 2.6 t 0.2 g (n = 5) to 3 .8 t 0.1 g (n = 5). PLA, may have increased the vascular permeability in the pulmonary vascular bed. Both the peak App and App 2 min were reduced by pretreating the lung with indomethacin (10 fig) at 1 min before PLA, application (Table 2). The effect of Fr. III-3 on rat lungs was compared to that on guinea-pig lungs (Table 1), with the rat lung effect being less marked . The time to maximal pressure response (peak time) was 0.8 t 0.2 min and the maximal pressure increase was 1 .50 t 1 .22 mmHg for rat lungs induced by Fr. III-3 (10 ~tg) . Prostaglandin-like substances released
Injection of Fr. II-5 (10 Ng) into the Krebs' solution perfusiog the guinea-pig lung caused an increase in the perfusion pressure and resulted in the appearance in the lung effluent
362
HUEZ-CHEN HUANG 2 min
A
B lg Fr. III-~ 10 ~tg TL F1G. 1 . RELEASE OF SLOW REACTING SUBSTANCE FROM THE PERFUSED GUINEA-PIG LUNGS INDUCED HY
Fr . III-3 . (A) Perfusion pressure response to Fr. III-3 . Fr . III-3 (10 Ng), injected as a bolus into the pulmonary circulation, produced an increase in perfusion pressure. (B) Response of guinea-pig ileum superfused with the effluent from the lung. The material released in the effluent by Fr . III-3 contracted the ileum . TL, Fr. III-3 was injected through the lung and not applied directly to the ileum .
of substances which contracted the chick rectum and the rat aortic strip (Fig. 2) . Pretreatment of the perfused lung with a bolus injection of indomethacin (10 kg), a cyclooxygenase inhibitor, reduced the pressure response . The contraction response of the rat aortic strip was also significantly reduced by indomethacin, indicating the presence of TXA, in the effluent (Fig. 2C, Table 3) . The contraction of chick rectum was not changed significantly by indomethacin, indicating the absence of PGE in the effluent (Fig. 2B, Table 3). Injection of Fr . III-3 (30 Fig) also caused an increase in the perfusion pressure and resulted in the appearance of substances which contracted the chick rectum and the rat aortic strip (Fig. 3). Pretreatment with indomethacin (10 pg) reduced the pressure response. The contraction response of the rat aortic strip was significantly increased by indomethacin (Fig. 3C, Table 3). This may be due to simultaneous release of vasodilator PG, which was inhibited by indomethacin, and release of leukotriene, which was potentiated by indomethacin . The contraction of the chick rectum was not changed significantly by indomethacin, indicating the absence of PGE (Fig . 3B, Table 3). The effects of the lung effluent on the rat colon were very variable and no conclusive results were obtained.
Release of Slow Reacting Substance by Phospholipases A,
363
TABLE 1 . EFFECT'S OF CRUDE VENOM AND PLA, FRACTIONS ON THE PERFUSION PRESSURE OF GUINEA-PIG LUNGS
Fraction Crude venom Fr . II-S Fr .I1-7 Fr . III-3
Dose (Ng) 10 30 100 10 30 100 30 100
n 3 12 5 10 3 3 8 3 3 S 7
Time to peak App (min) 1 .5 t 0.5 0 .ßt0 .2 1 .1 t 0.3 2.0 t 0.2 1 .7t0 .2 1 .S t 0.0 1 .4t0 .2 1 .0 t 0.0 0.7 t 0.1 O.St0 .3 0.8t0.2 0.8f0 .1 0.9t0.2 0.3*0 .0
Perfusion pressure Peak App (mmHg) 1.50 t 0.30* 3 .2310.31* 6 .00 f 1 .36* 3.06 t 0.30* 4.OOt0.38* 4.SO t 0.33* 2.30t0 .43* 6.00 t 0.71* 4.67 t 0.54* 4.73t0.92* 3.73t0.48* 3.30t0.71* 3 .74t1 .09* 7.33t0.33*
Duration (min) 1 .3 t 0.3 3 .210 .6 5 .0 t 2.5 7.6 t 1.6 6.Of0 .7 18 .0 t 0.0 2.0t0 .3 8.0 t 1.4 4.7 t 0.2 3.7f1 .2 20.6t4.6 6.2t0 .9 9.3t2.3 13 .0t1 .3
App 2 min (mmHg) 1.50 t 0.50* 2.5010.35* 5.00 * 1 .23* 2.83 t 0.33* 4.OOt0.58* 4.SO t 0.35* 2.12*0.37* S.SO t 0.33* 3.00 * 0.47* 3.OOf0.13* 4.12*0.63* 3.00*0.47* 3.10t0.86* 6.67f0.27*
3 10 30 Fr .III-6 3 3 10 7 30 3 Values are the mean t S.E .M . Peak App denotes maximal increase in perfusion pressure induced by venom or PLA,-fractions . App 2 min denotes increase in perfusion pressure at 2 min after the injection of PLA, fractions. The basal perfusion pressure of guinea-pig lung during the control period was 23 t 3 mmHg (n=25) . *PG0 .05 when venom or PLA, fraction-treated preparations were compared to controls . All data of peak App and App 2 min were analyzed by a paired t-test.
Release of a leukotriene-like substances Leukotriene-like substances in the lung effluent were detected by superfusing the guineapig ileum. The ileum was treated with a continuous infusion of a mixture of antagonists and FPL55712 (10 ng/ml) . At 0.5 min before injection of the PLA, fractions, an additional dose of FPL55712 (10 ng) was superfused directly onto the ileum. FPL55712, a specific antagonist of leukotrienes C and D, reduced the contractile response of the ileum, indicating the presence of leukotriene C and/or D in the lung effluent treated with Frs. II-5 and III-3 (Table 4). The per cent inhibition by FPL55712 was 75% for Fr. II-5 and 66% for Fr . III-3 respectively . Release of histamine The contractile response of the guinea-pig ileum to the effluent from the lung treated with Fr. III-3 (10 ~tg) was reduced significantly by antihistaminics, indicating the presence of histamine in the effluent. Antihistaminics (diphenhydramine 0.2 Ng/ml and cimetidine 0.2 Ng/ml) superfused directly and continuously onto the ileum reduced the maximal tension of the ileum by 32% from 0 .47 ± 0.01 g to 0.32 t 0.01 g (P~0.05) . Radioimmunoassay of thromboxane B= and 6-ketoprostaglandin F Thromboxane Az (TXAs) and prostacyclin (PGI,) released by PLAz fractions were also quantitated by the radioimmunoassay of the degradation products TXB~ and 6-ketoPGF1Q, respectively . Figure 4 shows the time courses and dose - response relationships of TXB, release induced by Frs . II-5 and III-3 (3 - 30 Ftg). Figure S shows the time courses and dose - response relationships of the 6-keto-PGF release induced by Frs. II-5 and III-3 (3 - 30 lig) . All ri,A2 fractions tested, Frs. II-S, II-7, III-3 and III , induced the release
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Fio . 2. BFFHCr of Fr . II-3 ox 1tm al~IewTION of PnosrwoLwHnn~-Lna: sussrwtlc8 uv n~ PsAPUSen OUINEA-PIO LUNG AND rIS MODIFICA710N nY INDOMSI'HAC1N . (A) Perfusion pressure response to Fr . II-3 . A bolus injection of Fr . II-S (10 ~ through the lung (TL) increased the perfusion pressure pelt hand extion). Indomethsdn pNDO ; 10 ~ igjected through the lung (fL) 1 min before Fr . II-3 application produced a significant attenuation of the pressure response to Fr. ü-3 (right hand section). pi) Responses of chick rectum (CR) and of (C) rat aortic strip (RA) superfused with effluent from the lung are shown . The substances rdeased in the effluent by Fr. II-S contracted CR and RA peR hand radon) . The contraction response on RA was significantly reduced by pmt with i hadn pNDO), indicating the preseace of thromboxsne A,-like material in the effluent . TL, Fr . II-3 and 1ND0 were igjected through the lung and not appüod directly to the assay tissue .
Twlu.a 3 . RHSPONSHS OF CHICIC AHCR7M AND aAT AORTIC SfA1P SUPERPUSCD wrI'H TI~ EFFLUF.Nr FAOM Tf~ PLA, FAACI'ION~TIMULAT'® OUINEA-PIO LUNCi Fraction
Dose (~
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10 10 30 30
Fr . III-3
Indomethacin treated (10 wg) +' +"
I rt 3 3 3 3
Increase in tension at 2 min (g) Chick rectum Rat aortic strip 0.70 t 0.29 1 .10 f 0.14 1 .70 t 0.43 1 .73 f 0.03
0 .23 t 0 .07 Ot 0.28 t 0 .02 0 .30 t 0 .07t
Values are the mean t S.B.M. PLA, fractions or indomethacin were igjated as a bolus through the hlng . The hmg effluent superfused the assay tissues . "Response of the assay tissue to the effluent from the indomethacin-pretreated guinea-pig hung . tPC0 .03 when indomethadn-pretreated preparations were compared to controls .
HUEI-CHEN HUANG
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CONTROL
Fr.lll-3 ~~9 FIG .
3.
EFFECT OF
INDOMETHACJN
INDO. Fr.llF3 0J~9 ~N9
Fr . III-3 ON THE GENERATION OF PROSTAGLANDIN-LIKE SUBSTANCE IN THE PERFUSED GUINEA-PIG LUNG AND ITS MODIFICATION HY INDOMETHACIN .
Perfusion pressure response to Fr . III-3 . A bolus injection of Fr . III-3 (30 pg) through the lung (TL) increased the perfusion pressure (left hand section) . Indomethacin (INDO; 10 pg) injected through the lung (TL) 1 min before Fr . III-3 injection produced a significant attenuation of the pressure response to Fr . III-3 (right hand section) . (B) Responses of chick rectum (CR) and of (C) rat aortic strip (RA) superfused with effluent from the lung are shown. The substances released in the effluent by Fr . III-3 contracted CR and RA (left hand section) . The contraction response on RA was significantly increased by pretreatment with INDO. TL, Fr. III-3 and INDO were injected through the lung and not applied directly to the assay tissue . (A)
TABLE 4 . RESPONSE OF GUINEA-P1G ILEUM SUPERFUSED WITH THE EFFLUENT FROM THE PLA, FRACTION-STIMULATED GUINEA-PIG LUNG
FPL 55712 Dose treated n Increase in tension at 2 min (g) (10 ng) (hg) Fr . II-5 10 3 0.60 t 0.07 10 +' 3 O .15t0.11t Fr . III-3 10 3 0.38 f 0.06 10 +' 3 0.13 t O.OSt Values are the mean t S.E .M . FPL 55712 (10 ng/ml) contained in a mixture of antagonists was continuously infused onto the ileum and an additional dose (10 ng) was superfused directly onto the ileum at 0.5 min before the injection of PLA, fractions. 'Response of the guinea-pig ileum pretreated with FPL 55712. tP~0.05 when FPL55712-pretreated preparations were compared to controls . Fraction
Release of Slow Reacting Substance by Phospholipases A,
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Fr . III-3 (B) ON TFn: LEVEL OF THROMBOXANE B, IN TIC EFFLUENT OF GUINEA-PIG LUNG .
Fr . II-S and Fr . III-3 (3 Ng - 30 Ng) were injected as a bolus injection through the lung . The lung effluents (1 min per tube) were collected serially. Thrombosane B, (TXB,) in a control tube collected before PLA, fraction injection (0 min) and sample tubes collected at 1, 2, 4 and 6 min after PLA, fraction injection were determined by radioimalunoassay. Each column represents the amount (ng/ml) of TXB, in the effluent . Each bar represents the S .E .M . of at least three determinations .'P<0.05 when compared to the control period . 0 min denotes the control period before injection. 1, 2, 4 and 6 min denote time after injection of PLA, fractions.
of TXB1 and Crketo-PGF from the perfused guinea-pig lung. The levels of TXB, and 6-keto-PGF measured 2 min after the injection of PLA, fractions are shown in Table 5. As shown in Fig . 6, there was a positive linear correlation between the pressure increases (App) induced by PLAz fractions and the ratios of TXB2 to Cz^keto-PGF, Q (TXB,/6-keto-PGF ) released by PLAN fractions (r=0.87). Both App and the amount of TXB, and 6-keto-PGF,Q were measured 2 min after the injection of PLA 1 fractions. DISCUSSION
PI :A= induced an increase in the lung perfusion pressure accompanied by a release of prostaglandin-like and leukotriene-like substances. The pressure response and TXA, and
HUEI-CHEN HUANG
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FIG. S. EFFECTS of Fr . II-S (A) AND Fr . III-3 (B) ON THE LEVEL OF o-KETO-PROSTAGLANDIN F wTHE EFFLUENT OF GUINEA-PIG LUNG Fr. II-S and Fr . Ill-3 (3 -30 Ng) were injected as a bolus injection through the lung . The lung effluents (1 min per tube) were collected serially . Crketo-prostaglandin F (6-kao-l?GF ) in a control tube collected before PLA, fraction injection (0 min) and sample tubes collected at 1, 2, 4 and 6 min after PLA, fraction injection were determined by radioimmunoassay . Each column represents the amount (ng/ml) of rrketo-PGF in the effluent . Each bar represents the S.E .M . of at least three determinations . "P<0.05 when compared to the control period . 0 min denotes the control period before injection . 1, 2, 4 and 6 min denote time after injection of PLA, fractions . TABLE S. EFFECTS OF PLA, FRACTIONS ON THROMBOXANE B, AND 6-KET0 .PROSTAGLANDIN F RELEASE 1N GUINEA-PIO LUNAS Fraction Control Fr . II-5 Fr . II-7 Fr . III-3 Fr . III-6
Dose lf+g) 10 30 30 10 30 10
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(n8/ml) 0.20 t 0.01 3 .11 t 0.4T 3.58 t 0.48' 7.00 t 0.00' 3 .78 t 0.33' 29 .75 t 1.9T 2.35 t 0.15'
6-keto-PGF 0.08 0.75 0.76 2.70 1 .48 5 .53 0.35
t 0.02 t 0.08' t 0.04' t 0.11' f 0.18' t 0.8T t 0 .11'
Values are the mean t S.E .M . of at least 3 determinations . The levels of TXB, and 6-keto-PGF were measured 2 min after the injection of each PLA, fraction . 'P'<0.05 when sample effluents collected after the injection of PLA, fraction were compared to effluent collected in the control period .
Release of Slow Reacting Substance by Phospholipases A,
369
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3
TXBZ/6-keto-PGFI a COAAELATION BETWEEN THE INCREASE IN PERFUSION PRESSURE (App) AND TfIE RATIO OF THROMBOxwNe B, To 6-x~ro-PROSrwaLArmw F (TJ{B,/CrtcE'ro-PGF ) IN THE EFFLUENT OF PLA,-STIMULATED GUINEA-PIG LUNG . Both App and the ratio T7LB,/(rketo-PßF were meaaurod 2 min after injection of PLA, fractions. FIG. G.
Each point is the mean of at least three determinations from either Fr . II-5, II-7, III-3 or III at a dose between 10 and 30 Ng .
PGI= release induced by PLA= fractions were generally dose-dependent . It is suggested that PLA, fractions from V, russelli venom released arachidonic acid from membrane phospholipids, which was transformed to TXAz and PGI, by cyclooxygenase or to leukotrienes by lipoxygenase . Injection of Fr . III-3 induced less pressure response in rat lungs than in guinea-pig lungs. Similarly, arachidonic acid perfused through the pulmonary circulation of guinea-pig lungs was metabolised to more myotropic metabolites than it was in rat or human lungs (ALLIBAIDI and BAKHLE, 1980). These results suggest that there may be important qualitative and quantitative differences in the activation of exogenous or endogenous arachidonic acid between guinea-pig lungs and rat lungs. According to DAMRAU et al. (1975), the main active principle formed in the cobra venom perfused guinea-pig lung (SRS-C) was PGE. By studying the effect of lung effluent on rat aortic strip in our experiments, we determined that application of Fr . II-5 was followed by a release of TXAs from the guinea-pig lung . Since the effect on the chick rectum was not significantly reduced by indomethacin, it is more likely that TXA=, and not PGE, is the major cyclooxygenase product released by Fr . II-5 . TXA3 has potent blood vessel and airway constrictive properties (SPAxtvI-IAxE et al., 1981). From the data obtained from radioimmunoassay of 6-keto-PGF , PGIs was also markedly released by Fr. III-3 and slightly by Fr. II-5 . The release of more PGIz induced by Fr. III-3 may have an opposing effect to TXA,, attenuating the vasoconstrictor action of TXAs on the pulmonary vascular muscle . PGIz has potent vasodilatory and bronchodilatory properties (SPANIVI-IAI{E et al ., 1981). There was a positive linear correlation between the pressure responses induced and the ratios of TXB, to 6-keto-PGF released by various PLA, fractions. It appears that the relative proportion of two different arachidonic acid metabolites released determines the effects of some PLA, fractions in the perfused lung . FPL55712, a specific antagonist of leukotrienes C and D, inhibited the contraction of the glûnea-pig ileum to the effluent from a lung which had been treated with PLA= at a concentration of 10 ng/ml, a concentration which is known to antagonize the contraction of ileum to SRS-A, without affecting the responses to other contractile agents (AUGUSTEIN
370
HUEZ-CHEN HUANG
et al ., 1973). The results indicated that both Frs. II-5 and III-3 released leukotriene C and/or D. Leukotrienes are most active in contracting smooth muscle of lungs (SPANNHAKE et al., 1981). In the present study, the SRS released by PLAN fractions of Russell's viper venom from the perfused lung was identified as a mixture of thromboxane A~, prostacyclin (PGI,) and leukotriene C and/or D. Injection of PLAN fractions from Russell's viper venom increased the perfusion pressure, indicating that the contribution of the contractor substances released was more than that of the dilator substances . Pretreatment with indomethacin reduced the pressure response, indicating that the contribution of the cyclooxygenase products was more than that of the lipoxygenase products . Examination of the activity of PLAN fractions on release of arachidonic acid metabolites from the perfused lung revealed a similar effect, but some quantitative differences . It may be due to the different substrate specificities of PLAN fractions. Similarly, SHUKLA and HANAHAN (1982) showed that acidic and basic PLA, of A. halys blomhoffü can also identify the different domains of phosphatidylcholine in human erythrocyte membranes. The latent period of SRS-C released by snake venom in the nonsensitized lung was 1- 3 min (MIDDLETON and PHILLIPS, 1962). The histamine effect peaked at 10-15 sec and terminated within 1 min (SMEDEGARD et al., 1982). In the present study, PLA, fractions of Russell's viper venom released TXA2, PGh, leukotrienes and also histamine from the perfused guinea-pig lungs. All these autacoids increase vascular permeability and may result in edema formation in the perfused lung . Histamine and PGI 2 have direct vasodilatory properties . Recently, leukotriene was also shown to have a prolonged hypotensive effect in guinea-pig and monkey (DRAZEN et al ., 1980 ; PIPER et al ., 1981 ; SMEDEGXRD et al., 1982). Therefore, part of the hypotensive action of PLA fractions may be due to the actions of histamine, PGI, and leukotriene released . There are some reports which provide evidence for the involvement of endogenous PGlike substance in the hypotensive action of antihypertensive drugs, including vasodilators (HAEUSLER and GEROLD, 1979 ; REIMANN et al ., 1981), lI-adrenoceptor blocking agents (JACKSON et al ., 1979 ; GRAHAM et al ., 1982) and diuretics (SCRIABINE et al., 1979). In our results, PLA, fractions of Russell's viper venom increased lung perfusion pressure, which may restrict blood return to the heart and lead to a decrease in cardiac output and induce a hypotensive effect indirectly . For Fr . II-5, the hypotensive action may be mediated by pulmonary vasoconstriction . For Fr . III-3, more PGh was released, which may cause vasodilatoon in the periphery, combined with pulmonary vasoconstriction, to induce a greater hypotensive effect . Further investïgation on the release of arachidonic acid metabolites in other vascular beds by PLAN is necessary for determining the relationship between the hypotensive action of PLA2 and the release of arachidonic acid metabolites.
I gratefully acknowledge the valuable advise and constructive criticism from Dr . C. Y. Lam, Professor of Pharmacology, College of Medicine, National Taiwan University .
Acknowledgements -
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Release of Slow Reacting Substance by Phospholipases A,
37 1
BRAntow, B. A. and Mwxcus, A. J. (1966) Action of snake venom phospholipase A on isolated platelet membranes. Proc . Soc. exp. Biol. 123, 889. CHIU, T. H., LEE, C. Y. and LEE, S. Y. (1968) Hemodynamic effects of cardiotoxin isolated from Formosan cobra venom. J. Formosan mad. Ass. 67, 557. DAMERAU, B., LEae, L., OLDIGS, H.-D. and Voor, W. (1975) Histamine release, formation of prostaglandinlike activity (SRS-G) and mast cell degranulation by the direct lyric factor (DLF) and phospholipase A of cobra venom . Naunyn-Schmiedebergs Archs Phormae. 287, 141 . DRwzEx, J. M., AUSTEx, K. F., L1:WIS, R. A., CtARx, D. A., Goro, G., MARFAT, A. and CoREV, E. J. (1980) Comparative airway and vascular activities of leukotrienes C-1 and D in vivo and in vitro. Proc . natn . Acad. Sci. U.S.A . 77, 4354 . FELDBERG, W. and KELIAWAY, C. H. (1938) Liberation of histamine and formation of lysolecithin-like substances by cobra venom. J. Physiol., Lond. 94, 187. FERREtRw, S. H. and VANE, J. R. (1%7) Prostaglandins : their disappearance from and release into the circulation. Nature, Lond. 216, 868. Fuacttoo~I-r, R. F. (1960) Spiral~tlt strip of rabbit aorta for in vitro studies of responses of arterial smooth muscle . In : Methods in Medical Research, Vol. 8, p. 177 (SAUNER, H. D., Ed .) . Chicago: The Yearbook Publishers . GRwIIwM, R. M., CAMPBELL, W. B. and JACxsoN, E. K. (1982) Effects of short term beta blockade on blood pressure, plasma thromboxane B,, and plasma and urinary prostaglandins E, and F in normal subjects . Clin . Pharmac. Ther. 31, 324. HAEUSLER, G. 8nd GEROLD, M. (1979) Increased levels of prostaglandin-like material in the canine blood during arterial hypotension produced by hydralazine, dihydralazine and minoxidil. Naunyn-Schmiedebergs Archs Aharmac. 310, 155. Huwxa, H. C. and LEE, C. Y. (1984) Isolation and pharmacological properties of phospholipases A, from Vipers russelli (Russell's viper) snake venom. Toxicon 22, 207. Jwcxsox, E. K., CAMPBELL, W. 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Meetschweinchenlunge durch Phospholipase A . Identifizierung mit Prostaglandin . Naunyn-Schmiedebergs Arch . exp. Path . Pharmak . 262, 124 . WALLACH, D . P . and BROWN, V . J . R. (1981) Studies on the arachidonic acid cascade - I . Inhibition of phospholipase A, in vitro and in vivo by several novel series of inhibitor compounds . Biochem. Pharmac. 30, 131 S .