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Effects of bovine SRS-A (SRS-Abov) on bovine respiratory tract and lung vasculature in vitro
European Journal o f Pharmacology, 44 (1977) 169--177
169
© Elsevier/North-Holland Biomedical Press
EFFECTS OF BOVINE SRS-A (SRS-A b°v) ON BOVINE R E S P I R A T O R Y T R A C T AND L U N G V A S C U L A T U R E IN V I T R O JOHN F. BURKA * and PETER EYRE Pharmacology Laboratory, Department o f Biomedical Sciences, University o f Guelph, Guelph, Ontario, Canada
Received 21 January 1977, revised MS received 23 March 1977, accepted 29 March 1977 J.F. BURKA and P. EYRE, Effects o f bovine SRS-A (SRS-A b°v) on bovine respiratory tract and lung vasculature in vitro, European J. Pharmacol. 44 (1977) 169--177. Evidence from isolated bovine pulmonary vessels and respiratory smooth muscles suggests that bovine slowreacting substance of anaphylaxis (SRS-Ab°v) may contribute to bronchoconstriction and pulmonary oedema during anaphylaxis in cattle. Bovine bronchus is sensitive to low concentrations of SRS-Ab°v and is considerably more sensitive than trachea. Contraction of the calf bronchus to SRS-Ab°v can be antagonized by sodium meclofenamate, suggesting prostaglandins may be involved in the contraction. Bovine pulmonary artery did not contract to SRS-A at any of the concentrations examined. None of the proposed SRS-A receptor antagonists, FPL 55712, PR-D-92-EA, or sodium meclofenamate, inhibit the effects of bovine SRS-A in bovine pulmonary vein. Bovine SRS°A
Anaphylaxis
Trachea
Bronchus
1. Introduction Slow-reacting substance of anaphylaxis (SRS-A) is released immunologically f r om bovine lung in vitro (Burka and Eyre, 1974b). The lung has been shown t o be an i m p o r t a n t 'shock organ' in immediate-type hypersensitivity o f cattle with resultant respiratory embarrassment (Aitken and Sanford, 1969; Eyre et al., 1973) and p u l m o n a r y o e d e m a (Wells et al., 1973). It was i m p o r t a n t to examine where SRS-A m a y be acting in the lung. To the present time, SRS-A has been shown to stimulate only a limited n u m b e r of s m o o t h muscle preparations. These include the guinea-pig ileum (Kellaway and Trethewie, 1940), guinea-pig trachea (Berry and Collier, 1964) and h u m a n bronchus (Brocklehurst, 1962). Intravenous administration of purified guinea-pig SRS-A to guinea pigs resulted in d o s e- d ep endent decrease in average
* Present address: Welicome Research Laboratories, Langley Court, Beckenham, Kent, England.
Pulmonary blood vessels
compliance (Drazen and Austen, 1974). Their results suggested t hat the effects of SRS-A were mainly in the smaller airways, whereas the increases in resistance and reduction in compliance induced by prostaglandin F2~ (PGF:a), histamine and 5 - h y d r o x y t r y p t a m i n e (5-HT) were a p p r o x i m a t e l y equally due to changes in the small and large airways. To test these findings in calves, 5-HT, histamine and purified bovine SRS-A (SRS-A b°v) were examined on isolated tracheal muscle and tertiary bronchi. Contraction of the p u l m o n a r y vein m ay occur during anaphylaxis, either as a result of an intrinsic response in the s m o o t h muscle (Schultz-Dale reaction: Eyre, 1971) a n d / o r due to the effects o f circulating mediators derived from 'shock' organs. The resultant increased alveolar capillary hydrostatic pressure and increased vessel permeability m ay give rise to p u l m o n a r y oedem a and congestion. Contraction o f the p u l m o n a r y artery in preference to the vein would t end to have the opposite e f f e c t and delay p u l m o n a r y oedema form at i on. The relative effects of
170 SRS-A on the pulmonary vein and artery were studied. Pharmaceutical research has recently developed c o m p o u n d FPL 55712, which appears to inhibit selectively SRS-A-induced contraction of the guinea-pig ileum (Augstein et al., 1973). Compound PR-D-92-EA (Possanza et al., 1975) has been suggested to block certain actions of SRS-A. Both these new compounds were examined on isolated bovine smooth muscles for their possible antagonism of the effects of SRS-A b°v. A non-steroidal antiinflammatory agent, sodium meclofenamate, is known to reduce anaphylactic bronchoconstriction in guinea pigs (Collier et al., 1968) and also SRS-A-induced contraction of human bronchus (Collier and Sweatman, 1968). We have already reported that meclofenamate protects calves from acute systemic anaphylaxis (Burka and Eyre, 1974a). Therefore it was also important to examine if meclofenamate was an antagonist of SRSA boy.
2. Materials and methods 2.1. Preparation o f S R S - A b°v
Lungs were obtained from male Guernsey or Jersey calves, 1--4 months old, which had been sensitized to horse plasma (Eyre et al., 1973). The animals were killed with intravenous pentobarbitone, the lungs removed within 5 min of death, placed in ice-cold saline and immediately transported to the laboratory. Tissue from the ventral border of the lung was chopped with scissors into pieces approximately 2--4 mm diameter. The chopped pieces were then placed in excess ice-cold Tyrode solution and left to 'wash' for at least 1 h at 4 °C. Portions of tissue (2 g) were then suspended in 25 ml conical flasks in 4.5 ml oxygenated Tyrode to which 0.5 ml horse plasma was added after 10 min pre-incubation. Pre-incubation (10 min) and incubation
J.F. BURKA, P. EYRE (20 min) was carried out at 37°C in a 'Dubn o f f ' shaking incubator. After incubation, the tissue and incubate were separated by filtration through gauze. The incubate was immediately mixed with 4 volumes absolute ethanol and maintained on ice for 30 min (Orange et al., 1973). The precipitated materials were sedimented at 20,000 g for 40 min. The supernatants were decanted and evaporated under vacuum at 60°C. The dry residue was dissolved in 25--50 ml 0.1 N NaOH and was incubated for 30 min at 37°C. The supernatant was filtered through gauze and applied to a water-washed 3 × 14 cm Amberlite XAD-8 (Rohm and Haas, Philadelphia, Pa.) column under upward flow at 5--7 ml/min. The column was washed with 2 volumes distilled water followed by 1 volume 80% ethanol. The water wash was re-applied to the column and similarly eluted with water and ethanol. The ethanol fractions were pooled and evaporated to dryness under negative pressure at 60°C. The dried extract was resuspended in 1--2 ml 80% ethanol and applied to a 1 × 8 cm silicic acid (Mallinckrodt, St. Louis, Mo.: 100 mesh) column washed in hexane. Elution was carried out by the consecutive addition of 20 ml of each of the following solvents: hexane, ethyl acetate, benzene, acetone, diethyl ether, n-butanol, methylene chloride, and 30 ml of ethanol : concentrated ammonia : water (6 : 3 : 1, v/v). Each fraction was evaporated to dryness under negative pressure at 60°C, stored at --30°C, and resuspended in distilled water or appropriate biological saline solution for bioassay. Only the last fraction was used for bioassay on the isolated bovine tissues. SRS-A in this fraction was free of prostaglandin El, E2 and F2~ as shown by thin-layer chromatography (Orange and Austen, 1969). The activity of SRS-A b°v was assayed on the isolated guinea-pig ileum. In Tyrode solution containing atropine (10 -6 g/ml), one unit of SRS-A b°v was arbitrarily taken to be the SRS-A-induced contraction of the guinea-pig ileum in the presence of mepyramine (10 -6 g/ml) equivalent to t h a t produced by 5 ng
PULMONARY EFFECTS OF SRS-Ab°v histamine base on the same preparation prior to the addition of mepyramine (Stechschulte et al., 1967). Contractions were recorded by means of a Narco isotonic detector/transducer and Physiograph Model 4 pen recorder (Narco Biosystems, Houston, Tx.). 2.2. Bioassay o f SRS-A b°v on isolated bovine pulmonary smooth muscles Healthy lung was obtained fresh, either from cattle killed at a local abattoir or from experimental calves (6--8 weeks old) killed with pentobarbitone. The principal pulmonary vein from the apical or cardiac lobe was carefully removed and placed in Krebs--Henseleit (1932) solution. The vein was cut into a single strip, approximately 3--5 mm in width and set up in a 10 ml isolated organ bath (Phipps and Bird, Richmond, Va.) at 37°C in Krebs solution mixed with 5% CO2 in oxygen, at a resting tension of 2.5 g. The principal pulmonary artery for the apical or Cardiac lobe was similarly prepared and maintained at a resting tension of 3.5 g. The m e t h o d employed for setting up the tracheal muscle was t h a t of Offermeier and Ari~ns (1966). A tracheal ring was dissected from the trachea just above the tracheo-bronchial bifurcation. The tracheal muscle was carefully dissected from the inside of the ring and bisected. The two muscle strips (5--7 mm in width) were attached end to end and set up in an isolated organ bath in Krebs solution as described for the pulmonary vessels, at a resting tension of 2.5 g. A terminal (tertiary) bronchus of the apical or cardiac lobe was carefully dissected, cut spirally into a single strip 3--5 mm in width, and set up in an isolated organ bath in Krebs solution, as previously described, under a resting tension of 2.5 g. Dose--response curves and threshold concentrations were determined for histamine and SRS-A b°v on the pulmonary vein, for histamine, 5-HT and SRS-A b°v on the bronchus, for histamine and 5-HT on the trachealis and pulmonary artery preparations. Only the
171 threshold concentrations of SRS-A b°v on the trachealis muscle were determined. SRS-A b°v was also applied to the pulmonary artery to a m a x i m u m concentration of 20 U/ml. The antagonists, sodium meclofenamate (20 and 100 pg/ml) (Collier and Sweatman, 1968), FPL 55712 (0.1, 0.5 and 5 p g / m l ) (Augstein et al., 1973), and PR-D-92-EA (20 and 100 /ag/ml) (Possanza et al., 1975) were examined on the actions of agonists on the pulmonary vein and bronchus. Each antagonist was left in contact for two minutes before agonists were retested. The effectiveness and specificity of each antagonist was estimated by determining the relative agonist 'dose ratio' (Gaddum et al., 1955). 2.3. Drugs Histamine diphosphate, 5-hydroxytryptamine creatinine sulphate and atropine sulphate were purchased from Nutritional Biochemicals Co., Cleveland, Ohio. The following drugs were gifts: mepyramine maleate (Poulenc Ltd., Montreal, Quebec), sodium meclofenamate (Parke, Davis & Co., Detroit, Michigan), FPL 55712 (Fisons Ltd., Loughborough, England) and PR-D-92-EA (PharmaResearch Canada Ltd., Pointe-Claire, Quebec).
3. Results Purified bovine SRS-A showed biological activity on the isolated pulmonary vein, trachealis muscle and bronchial preparations, but not on the pulmonary artery. Log dose-response curves to histamine, 5-HT and SRSA T M were obtained from 7 pulmonary vein preparations and 12 bronchi. Examples of each are shown in figs. 1 and 2 respectively. Only the lower segments o f the dose--response curves were obtained due to the limited a m o u n t of SRS-A available. Contractions induced by 5-HT, histamine and SRS-A b°v on the pulmonary vein and bronchus are shown in fig. 3. The threshold concentrations of each of
172
J.F. B U R K A , P. E Y R E
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Fig. 1. Log d o s e - - r e s p o n s e c u r v e s o f t h e c o n t r a c t i o n s induced by histamine and purified bovine SRS-A on isolated b o v i n e p u l m o n a r y vein. U n i t s o f c o n t r a c t i o n are a r b i t r a r i l y derived f r o m t h e l i n e a r m a g n i t u d e o f the recorded contraction. Standard errors of the m e a n s are i n d i c a t e d as vertical bars.
the agonists (5-HT, histamine and SItS-A) required to contract the 4 tissues are indicated in table 1. The bronchus was the most sensitive to SRS-A b°v o f the 4 bovine tissues studied (average threshold: 2.4 U/ml). In 2 bronchial samples the threshold was as low as 0.1 U/ml. The contraction induced by SRS-A b°v was sometimes preceded by a slight relaxation.
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Fig. 3. Contractions of isolated bovine pulmonary vein and bronchus to histamine, 5-HT and purified bovine SRS-A. Contractions of the pulmonary vein are cumulative. The time trace shows drug injections, and intervalsof 60 sec.
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Fig. 2. Log d o s e - - r e s p o n s e curves o f t h e c o n t r a c t i o n s i n d u c e d b y h i s t a m i n e , 5-HT a n d p u r i f i e d b o v i n e S R S - A o n isolated b o v i n e b r o n c h u s . U n i t s o f c o n t r a c t i o n are a r b i t r a r i l y derived f r o m t h e linear m a g n i t u d e o f t h e r e c o r d e d c o n t r a c t i o n . S t a n d a r d errors of t h e m e a n s are i n d i c a t e d as vertical bars.
PULMONARY EFFECTS OF SRS-Ab°v
173
TABLE 1 Threshold concentrations of bovine SRS-A, histamine and 5-HT to contract several bovine respiratory/pulmonary smooth muscles. Smooth muscle
SRS-A Pulmonary vein Pulmonary artery Trachea Bronchus
Number of animals
Agonist
9.3 U/ml (0.5--15) > 20 U/ml 18 U/ml (15--20) 2.4 U/ml (0.1--10)
Histamine
5-HT
68 ng/ml (5--150) 2100 ng/ml (300--3000) 533 ng/ml (100--1000) 1450 ng/ml (250-->20000)
Not done
7
15 ng/ml (10--20) 57 ng/ml (20--75) 33 ng/ml (5--75)
3
A latent period o f 4--7 min preceded contraction. A cumulative dose--response curve to SRS-A b°v could n o t be obtained owing to tachyphylaxis: a p p r o x i m a t e l y 3 0 m i n being required to recover the response. There was, however, n o cross-tachyphylaxis between histamine, 5-HT and SI~S-A b°v. The p u l m o n a r y artery was the least sensitive to SRS-A b°v, failing t o c o n t r a c t to concentrations o f SRS-A as high as 20 U/ml. Higher co n cen tr ations were n o t a t t e m p t e d . The artery was highly sensitive t o 5-HT however {threshold: 15 ng/ml), but n o t to histamine (threshold: 2100 ng/ml). The p u l m o n a r y vein was relatively sensitive to b o t h SRS-A b°v (threshold: 9.3 U / m l ) a n d histamine {threshold: 68 ng/ml). For comparison purposes, the p u l m o n a r y vein is very sensitive to 5-HT {threshold: 5 ng/ml) {Burka and Eyre, 1974c). Cumulative dose--response relationships could be established t o histamine and SRS-A as there was no t a c h y p h y laxis to either of these substances in the vein. The trachealis muscle was relatively insensitive to histamine and SRS-A b°v, but did n o t c o n t r a c t to m o d e r a t e concentrations of 5-HT {threshold: 57 ng/ml). This agrees with the observations o f Offermeier and Ari~ns (1966) who first showed evidence f or 5-HT receptors in the bovine tracheal muscle.
3 12
3.1. Inhibitor studies Sodium m e c l o f e n a m a t e , F P L 5 5 7 1 2 and PR-D-92-EA were examined as possible antagonists of SRS-A b°v and histamine on the p u l m o n a r y vein and SRS-A b°v, histamine and 5-HT on the bronchus. The results are tabulated in table 2. None of the drugs effectively antagonized histamine or SRS-A on the p u l m o n a r y vein. Sodium m e c l o f e n a m a t e at a concent rat i on r e c o m m e n d e d by Collier and Sweatman (1968) (20 pg/ml) showed no antagonism to any of t he agonists tested on bovine tissues. However, sodium m e c l o f e n a m a t e at 100 gg/ml selectively antagonized SRS-A b°v. The t y p e of antagonism could n o t be determined because SRS-A b°v in the presence of meclofenamate (100 pg/ml) induced a marked loss of t one of the muscle. In one preparation an a p p r o x i m a t e dose ratio of 5 was recorded, but it was n o t possible to re-establish a full dose--response curve due to insufficient SRS-A being available. Meclofenamate (100 pg/ml) showed no antihistaminic effect but slightly antagonized 5-HT which was n o t statistically significant (p > 0.05). FPL 55712 at all 3 concentrations examined did n o t antagonize histamine, 5-HT or SRS-A b°v on the bronchus. PR-D-92-EA
174
J.F. BURKA, P. EYRE
TABLE 2 Effects of inhibitors on contractions of bovine pulmonary vein and bronchus induced by histamine, 5-HT and bovine SRS-A. Smooth muscle
Pulmonary vein
Bronchus
Dose ratio of agonists
Meclofenamate 20 pg/ml 100/lg/ml FPL 55712 0.1//g/ml 0.5//g/ml 5.0/lg/mt PR-D-92-EA 20 pg/ml 100/lg/ml Meclofenamate 20 pg/ml 100 pg/ml FPL 55712 0.1/ag/ml 0.5 pg/ml 5.0 pg/ml PR-D-92-EA 20 pg/ml 100 pg/ml
Histamine
5-HT
SRS-A
1, 1, 1 1, 1, 1
n.d. 1 n.d.
1,3,1,1,1 t, 2, 1
1, 1, 1 2, 1, 1 2, 1, 1
n.d. n.d. n.d.
1,1,1 1,2,1,1 2, 2, 1
1, 1, 1 2, 2, 1, 1, 1
n.d. n.d.
1, 2, 7, 1 2, 1, 4, 1
1, 1, 1 2, 1, 1
1,1,1,1 t0,2,2, 1
1, 1 1, 1 1, 1, 1
1, 1, 1 1, 1, 1 1,1,1
1, 1, 1 1, 1, 1 1,1,1,1
1, 1,1 5, 6, >40, >40
1,1,1 1, 1, 1
1,1,1 1, 1, I
1 n.d. = not done. failed t o a n t a g o n i z e SRS-A b°v or 5-HT, b u t at a c o n c e n t r a t i o n o f 100 p g / m l a n t a g o n i z e d h i s t a m i n e o n l y . H i s t a m i n e in t h e p r e s e n c e o f P R - D - 9 2 - E A ( 1 0 0 p g / m l ) caused r e l a x a t i o n o f t h e b r o n c h i a l muscle. In 2 o u t o f 4 prep a r a t i o n s , tissue c o n t r a c t i l i t y was irrecoverable, even w i t h v e r y high c o n c e n t r a t i o n s o f histamine. In t h e t w o r e m a i n i n g tissues a h i s t a m i n e d o s e - - r e s p o n s e curve c o u l d be fully re-established in t h e p r e s e n c e o f PR-D-92-EA ( 1 0 0 p g / m l ) a n d dose ratios o f 5 and 6 were r e c o r d e d . T h e l a t t e r d o s e - - r e s p o n s e curves were parallel t o the d o s e - - r e s p o n s e curves in t h e a b s e n c e o f t h e drug, suggesting t h a t a n t a g o n i s m was c o m p e t i t i v e in n a t u r e .
4. Discussion E x a m i n a t i o n o f b o v i n e SRS-A o n isolated b o v i n e r e s p i r a t o r y a n d p u l m o n a r y vascular s m o o t h muscles helps t o clarify the poten-
1,1,1,1 >120, 5, >120
tial role o f SRS-A in a c u t e systemic anaphylaxis. B r o n c h o c o n s t r i c t i o n has long b e e n associated with a c u t e s y s t e m i c a n a p h y laxis in t h e guinea pig (Auer and Lewis, 1 9 1 0 ) . SRS-A is also i m p l i c a t e d in a n a p h y laxis in t h e rat ( S t o t l a n d a n d Share, 1 9 7 4 ) , and in a s t h m a in m a n (Orange and Langer, 1 9 7 3 ) . In t h e guinea pig, a n t i g e n - i n d u c e d b r o n c h o c o n s t r i c t i o n is associated with a decrease in p u l m o n a r y c o m p l i a n c e (Collier, 1 9 6 8 ) . Similarly, i.v. a d m i n i s t r a t i o n o f purified guinea-pig SRS-A t o guinea pigs results in a d o s e - d e p e n d e n t decrease in average p u b m o n a r y c o m p l i a n c e , suggesting t h a t SRS-A constricts t h e small airways (terminal b r o n c h i , b r o n c h i o l e s and alveoli) (Drazen and A u s t e n , 1 9 7 4 ) . H i s t a m i n e , 5 - H T a n d PGF2~ also increase resistance, which suggests t h a t these substances c o n s t r i c t b o t h the large and small airways. A similar p a t t e r n emerges in t h e b o v i n e
PULMONARY EFFECTS OF SRS-Ab°v respiratory tract. Previous studies in cattle have shown varying degrees of respiratory embarrassment in acute systemic anaphylaxis (Aitken and Sanford, 1969; Eyre et al., 1973). However, no direct evidence for bronchoconstriction has previously been shown and Schultz--Dale reactions on the bronchi are either feeble or absent (Eyre, 1971). This study gives the first direct evidence of a substance that contracts bovine bronchial smooth muscle at low concentrations (threshold: 2.4 U/ml). In contrast, the bovine trachea is less sensitive to SRS-A b°v (threshold: 18 U/ml). The sensitivities of the bronchus and trachea to 5-HT are similar (tresholds: 33 and 57 ng/ml respectively). Both tissues are very insensitive to histamine. However, histamine in vivo may cause tracheobronchial constriction by a vagal reflex mechanism (Eyre et al., 1973; Nadel, 1973). Our results appear to be consistent with in vivo results in the guinea pig (Drazen and Austen, 1974) where SRS-A specifically constricts small airways, whereas histamine and 5-HT affect small and large airways approximately equally. If sufficient SRS-A b°v could be made available for in vivo studied in calves, it would be interesting to study its effects on pulmonary compliance and resistance, to compare these to the changes in pulmonary compliance and resistance in acute systemic anaphylaxis, and to study inhibitor drugs. The contraction induced by SRS-A b°v on the bovine bronchus may not be entirely directly due to SRS-A as tachyphylaxis rapidly develops. This suggests t h a t SRS-A b°v may be either releasing another substance that contracts the muscle or acting on a SRS-A receptor with an extended recovery time. This is in contrast to human bronchial muscle which is also very sensitive to SRS-A (threshold: 2.2 U/ml), where no tachyphylaxis to SRS-A occurs (Math~ and Strandberg, 1971). There is no cross-tachyphylaxis between histamine, 5-HT and SRS-A T M in cattle, suggesting that these substances are quite distinct in their actions on particular receptors in the bronchial muscle.
175 Inhibitor studies further suggest a role for SRS-A b°v in bronchoconstriction in anaphylaxis of cattle. Sodium meclofenamate is of particular interest because this drug effectively protects calves during acute systemic anaphylaxis (Eyre et al., 1973; Burka and Eyre, 1974a), particularly in the control of respiratory symptoms (Aitken et al., 1975). A paradox already reported is that sodium meclofenamate markedly enhances the immunological release of SRS-A b°v from lung (Burka and Eyre, 1975). Therefore it was necessary to examine the possible antagonism of SRS-A b°v at the site of action. Meclofenamate was particularly effective in antagonizing SRS-Ab°V-induced contraction of the calf bronchus. It was not possible to determine the type of antagonism owing to the limited quantity of SRS-A b°v available, but it is also interesting that meclofenamate alone or meclofenamate and SRS-A b°v together induce a loss of tone of the muscle. Meclofenamate is a potent inhibitor of PG synthetase (Flower, 1974). A possible explanation is that meclofenamate and SRS-A may be acting on the tissue in such a way that endogenous prostaglandins required to maintain tone of the muscle (Frey and Schafer, 1974) are depleted and relaxation ensues. This explanation would be particularly applicable if SRS-A releases prostaglandins, particularly PGF2~, to contract the bronchus. Similar inhibition of contractions of human bronchial muscle induced by SRS-A and PGF2~ has been observed with aspirin and fenamates (Berry and Collier, 1964; Collier and Sweatman, 1968). These effects of meclofenamate on SRS-A b°vinduced contractions appear to be specific, despite the high concentration used, as the drug had little, if any, effect on the contractions induced by histamine and 5-HT, and had no measurable effect on the bronchial muscle per se. Further evidence to suggest that SRS-A b°v may be releasing another substance to contract the bronchus is the 4--7 min latent period between addition of SRS-A b°v to the bath and contraction of the muscle. This
176
latent period would seem to allow sufficient time for the formation and release of another substance, particularly prostaglandins (Piper and Vane, 1971). Dunlop and Smith (1975) have recently shown that there is a significant correlation between PGF2~ release and antigen-induced contraction of human bronchus and that indomethacin, while inhibiting synthesis of PGF2~, also reduces the antigeninduced contraction. Further experiments must be carried out to prove that PGF2~ is actually released from bovine bronchi on administration of SRS-A b°v. Neither FPL 55712 nor PR-D-92-EA antagonized the effects of bovine SRS-A on the bronchus, even though they have been reported to antagonize m o n k e y , human and guinea-pig SRS-A on the guinea-pig ileum (Augstein et al., 1973; Possanza et al., 1975). The species difference in the test tissue may afford one explanation for this. It is also possible that bovine SRS-A m a y be different in structure from SRS-A of other species because, even on the guinea-pig ileum, FPL 55712 is n o t as effective an antagonist of SRS-A b°v as of guinea-pig SRS-A. A dose ratio of approximately 4 was obtained for guinea-pig SRS-A with FPL 55712 (80 ng/ml) by Augstein et al. (1973), whereas the dose ratio for SRS-A b°v with FPL 55712 (100 ng/ml) is approximately 2.8 in guinea-pig ileum (Burka, unpublished results). It is interesting that a high concentration of PR-D-92-EA (100 pg/ml) antagonizes the effects of histamine on the bronchus, and that antagonism appears to be competitive. Inhibition of histamine by PR-D-92-EA has been observed previously on the guinea-pig ileum (Possanza et al., 1975). Pulmonary oedema is a principal pathological change observed in acute systemic anaphylaxis in calves (Wells et al., 1973). The present results confirm that the pulmonary vein is considerably more sensitive to histamine, 5-HT and SRS-A b°v (thresholds: 6 9 n g / m l , 5 ng/ml (Burka and Eyre, 1974c) and 9.3 U/ml respectively) than is the artery (thresholds: 2100 ng/ml, 15 ng/ml and > 2 0 U/ml
J.F. BURKA, P. EYRE
respectively). These results suggest that SRS-A might contribute to pulmonary oedema by contracting the pulmonary vein in preference to the artery, and thus raising capillary hydrostatic pressure. These experiments suggest that SRS-A b°v may contribute to bronchoconstriction and pulmonary oedema in immediate hypersensitivity reactions in the bovine species. It is now necessary to examine if SRS-A b°v can also induce bronchoconstriction and pulmonary oedema in calves in vivo.
Acknowledgements The authors are grateful to Mr. T.R. Deline for technical assistance. The work was supported in part by the Ontario Ministry of Agriculture and Food, by grant A5937 of the National Research Council of Canada and by grant OG3012 of Agriculture Canada. J.F. Burka was the recipient of an Ontario Graduate Scholarship.
References Aitken, M.M. and J. Sanford, 1969, Experimental anaphylaxis in cattle, J. Comp. Pathol. 79,131. Aitken, M.M., J. Sanford and D.P. Evans, 1975, Prophylaxis and treatment of experimental anaphylaxis in cattle by sodium meclofenamate, Res. Vet. Sci. 18, 41. Auer, J. and P.A. Lewis, 1910, The physiology of immediate reaction of anaphylaxis in the guinea-pig, J. Exptl. Med. 12,151. Augstein, J., J.B. Farmer, T.B. Lee, P. Sheard and M.L. Tattersall, 1973, Selective inhibitor of SRS-A, Nature New Biol. 245,215. Berry, P.A. and H.O.J. Collier, 1964, Bronchoconstrictor action and antagonism of a slow-reacting substance from anaphylaxis of guinea-pig isolated lung, Brit. J. Pharmacol. 23,201. Brocklehurst, W.E., 1962, Slow-reacting substance and related compounds, Progr. Allergy 6,539. Burka, J.P. and P. Eyre, 1974a, A study of prostaglandins and prostaglandin antagonists in relation to anaphylaxis in calves, Can. J. Physiol. Pharmacol. 52, 942. Burka, J.F. and P. Eyre, 1974b, The immunological release of slow-reacting substance of anaphylaxis from bovine lung, Can. J. Physiol. Pharmacol. 52, 1201.
PULMONARY EFFECTS OF SRS-A b°v Burka, J.F. and P. Eyre, 1974c, Studies of prostaglandins and prostaglandin antagonists on bovine pulmonary vein in vitro, Prostaglandins 6, 333. Burka, J.F. and P. Eyre, 1975, Modulation of the formation and release of bovine SRS-A in vitro by several anti-anaphylactic drugs, Intern. Arch. Allergy. Appl. Immunol. 49, 774. Collier, H.O.J., 1968, Humoral factors in bronchoconstriction, Sci. Basis Med. Ann. Rev. 308. Collier, H.O.J., G.W.L. James and P.J. Piper, 1968, Antagonism by fenamates and like-acting drugs of bronchoconstriction induced by bradykinin or antigen in the guinea pig, Brit. d. Pharmacol. 34, 76. Collier, H.O.J. and W.J.F. Sweatman, 1968, Antagonism by fenamates of prostaglandin F2~ and of slow-reacting substance on human bronchial muscle, Nature 219, 864. Drazen, J.M. and K.F. Austen, 1974, Effects of intravenous administration of SRS-A, histamine, bradykinin and prostaglandin F2~ on pulmonary mechanics in the guinea pig, J. Clin. Invest. 53, 1679. Dunlop, L.S. and A.P. Smith, 1975, Reduction of antigen-induced contraction of sensitized human bronchus in vitro by indomethacin, Brit. J. Pharmacol. 54,495. Eyre, P., 1971, Pharmacology of bovine pulmonary vein anaphylaxis in vitro, Brit. J. Pharmacol. 43, 302. Eyre, P., A.J. Lewis and P.W. Wells, 1973, Acute systemic anaphylaxis in the calf, Brit. J. Pharmacol. 47,504. Flower, R.J., 1974, Drugs which inhibit prostaglandin biosynthesis, Pharmacol. Reviews 26, 33. Frey, H.H. and A. Schafer, 1974, On the effects of prostaglandins E2, F2 ~ on bronchial tonus in cats, European J. Pharmacol. 29, 267. Gaddum, J.H., K.A. Hameed, D.E. Hathaway and F.F. Stephens, 1955, Quantitative studies of antagonists for 5-hydroxytryptamine, Q.J. Exptl. Physiol. 40, 49. Kellaway, C.H. and L.R. Trethewie, 1940, The liberation of a slow-reacting smooth muscle stimulating substance in anaphylaxis, Q. J. Exptl. Physiol. 30, 121.
177 Krebs, H.A. and K. Henseleit, 1932, Untersuchung fiber die Harnstoffbildung im Tierkorper, HoppeSeylers Z. Physiol. Chem. 210, 33. Math~, A.A. and K. Strandberg, 1971, Antagonism of slow reacting substance by polyphloretin phosphate on isolated human bronchi, Acta Physiol. Scand. 82, 460. Nadel, J.A., 1973, Neurophysiologic aspects of asthma, in: Asthma, eds. K.F. Austen and L.M. Lichtenstein (Academic Press, New York) p. 29. Offermeier, J. and E.J. Ari~ns, 1966, Serotonin I. Receptors involved in its action, Arch. Intern. Pharmacodyn. Therap. 164, 192. Orange, R.P. and K.F. Austen, 1969, Slow-reacting substance of anaphylaxis, Advan. Immunol. 10, 105. Orange, R.P. and H. Langer, 1973, Bronchial asthma: hyperreactivity of airways and target cells, in: Allergology--Proceedings of the 8th Congress of the International Association of Allergology, Tokyo, October 14--20, 1973 (Excerpta Medica, Amsterdam) p. 325. Orange, R.P., R.C. Murphy, H.L. Karnovsky and K.F. Austen, 1973, The physicochemical characteristics and purification of slow-reacting substance of anaphylaxis, J. Immunol. 110, 760. Piper, P.J. and J.R. Vane, 1971, The release of prostaglandins from lung and other tissues, Ann. N. Y. Acad. Sci. 180,363. Possanza, G.J., A. Bauen and P.B. Stewart, 1975, In vitro antagonism of the mediators of allergy by a benzopyrano-benzopyran carboxylic acid: PR-D92-EA, Intern. Arch. Allergy Appl. Immunol. 49, 789. Stechschulte, DIJ. K.F. Austen and K.J. Bloch, 1967, Antibodies involved in antigen-induced release of SRS-A in the guinea pig and rat, J. Exptl. Med. 125, 127. Stotland, L.M. and N.N. Share, 1974, Active bronchial anaphylaxis in the rat, Can. J. Physiol. Pharmacol. 52, 1114. Wells, P.W., P. Eyre and J.H. Lumsden, 1973, Hematological and pathological changes in acute systemic anaphylaxis in calves: effects of pharmacological agents, Can. J. Comp. Med. 3 7 , 1 1 9 .
169
© Elsevier/North-Holland Biomedical Press
EFFECTS OF BOVINE SRS-A (SRS-A b°v) ON BOVINE R E S P I R A T O R Y T R A C T AND L U N G V A S C U L A T U R E IN V I T R O JOHN F. BURKA * and PETER EYRE Pharmacology Laboratory, Department o f Biomedical Sciences, University o f Guelph, Guelph, Ontario, Canada
Received 21 January 1977, revised MS received 23 March 1977, accepted 29 March 1977 J.F. BURKA and P. EYRE, Effects o f bovine SRS-A (SRS-A b°v) on bovine respiratory tract and lung vasculature in vitro, European J. Pharmacol. 44 (1977) 169--177. Evidence from isolated bovine pulmonary vessels and respiratory smooth muscles suggests that bovine slowreacting substance of anaphylaxis (SRS-Ab°v) may contribute to bronchoconstriction and pulmonary oedema during anaphylaxis in cattle. Bovine bronchus is sensitive to low concentrations of SRS-Ab°v and is considerably more sensitive than trachea. Contraction of the calf bronchus to SRS-Ab°v can be antagonized by sodium meclofenamate, suggesting prostaglandins may be involved in the contraction. Bovine pulmonary artery did not contract to SRS-A at any of the concentrations examined. None of the proposed SRS-A receptor antagonists, FPL 55712, PR-D-92-EA, or sodium meclofenamate, inhibit the effects of bovine SRS-A in bovine pulmonary vein. Bovine SRS°A
Anaphylaxis
Trachea
Bronchus
1. Introduction Slow-reacting substance of anaphylaxis (SRS-A) is released immunologically f r om bovine lung in vitro (Burka and Eyre, 1974b). The lung has been shown t o be an i m p o r t a n t 'shock organ' in immediate-type hypersensitivity o f cattle with resultant respiratory embarrassment (Aitken and Sanford, 1969; Eyre et al., 1973) and p u l m o n a r y o e d e m a (Wells et al., 1973). It was i m p o r t a n t to examine where SRS-A m a y be acting in the lung. To the present time, SRS-A has been shown to stimulate only a limited n u m b e r of s m o o t h muscle preparations. These include the guinea-pig ileum (Kellaway and Trethewie, 1940), guinea-pig trachea (Berry and Collier, 1964) and h u m a n bronchus (Brocklehurst, 1962). Intravenous administration of purified guinea-pig SRS-A to guinea pigs resulted in d o s e- d ep endent decrease in average
* Present address: Welicome Research Laboratories, Langley Court, Beckenham, Kent, England.
Pulmonary blood vessels
compliance (Drazen and Austen, 1974). Their results suggested t hat the effects of SRS-A were mainly in the smaller airways, whereas the increases in resistance and reduction in compliance induced by prostaglandin F2~ (PGF:a), histamine and 5 - h y d r o x y t r y p t a m i n e (5-HT) were a p p r o x i m a t e l y equally due to changes in the small and large airways. To test these findings in calves, 5-HT, histamine and purified bovine SRS-A (SRS-A b°v) were examined on isolated tracheal muscle and tertiary bronchi. Contraction of the p u l m o n a r y vein m ay occur during anaphylaxis, either as a result of an intrinsic response in the s m o o t h muscle (Schultz-Dale reaction: Eyre, 1971) a n d / o r due to the effects o f circulating mediators derived from 'shock' organs. The resultant increased alveolar capillary hydrostatic pressure and increased vessel permeability m ay give rise to p u l m o n a r y oedem a and congestion. Contraction o f the p u l m o n a r y artery in preference to the vein would t end to have the opposite e f f e c t and delay p u l m o n a r y oedema form at i on. The relative effects of
170 SRS-A on the pulmonary vein and artery were studied. Pharmaceutical research has recently developed c o m p o u n d FPL 55712, which appears to inhibit selectively SRS-A-induced contraction of the guinea-pig ileum (Augstein et al., 1973). Compound PR-D-92-EA (Possanza et al., 1975) has been suggested to block certain actions of SRS-A. Both these new compounds were examined on isolated bovine smooth muscles for their possible antagonism of the effects of SRS-A b°v. A non-steroidal antiinflammatory agent, sodium meclofenamate, is known to reduce anaphylactic bronchoconstriction in guinea pigs (Collier et al., 1968) and also SRS-A-induced contraction of human bronchus (Collier and Sweatman, 1968). We have already reported that meclofenamate protects calves from acute systemic anaphylaxis (Burka and Eyre, 1974a). Therefore it was also important to examine if meclofenamate was an antagonist of SRSA boy.
2. Materials and methods 2.1. Preparation o f S R S - A b°v
Lungs were obtained from male Guernsey or Jersey calves, 1--4 months old, which had been sensitized to horse plasma (Eyre et al., 1973). The animals were killed with intravenous pentobarbitone, the lungs removed within 5 min of death, placed in ice-cold saline and immediately transported to the laboratory. Tissue from the ventral border of the lung was chopped with scissors into pieces approximately 2--4 mm diameter. The chopped pieces were then placed in excess ice-cold Tyrode solution and left to 'wash' for at least 1 h at 4 °C. Portions of tissue (2 g) were then suspended in 25 ml conical flasks in 4.5 ml oxygenated Tyrode to which 0.5 ml horse plasma was added after 10 min pre-incubation. Pre-incubation (10 min) and incubation
J.F. BURKA, P. EYRE (20 min) was carried out at 37°C in a 'Dubn o f f ' shaking incubator. After incubation, the tissue and incubate were separated by filtration through gauze. The incubate was immediately mixed with 4 volumes absolute ethanol and maintained on ice for 30 min (Orange et al., 1973). The precipitated materials were sedimented at 20,000 g for 40 min. The supernatants were decanted and evaporated under vacuum at 60°C. The dry residue was dissolved in 25--50 ml 0.1 N NaOH and was incubated for 30 min at 37°C. The supernatant was filtered through gauze and applied to a water-washed 3 × 14 cm Amberlite XAD-8 (Rohm and Haas, Philadelphia, Pa.) column under upward flow at 5--7 ml/min. The column was washed with 2 volumes distilled water followed by 1 volume 80% ethanol. The water wash was re-applied to the column and similarly eluted with water and ethanol. The ethanol fractions were pooled and evaporated to dryness under negative pressure at 60°C. The dried extract was resuspended in 1--2 ml 80% ethanol and applied to a 1 × 8 cm silicic acid (Mallinckrodt, St. Louis, Mo.: 100 mesh) column washed in hexane. Elution was carried out by the consecutive addition of 20 ml of each of the following solvents: hexane, ethyl acetate, benzene, acetone, diethyl ether, n-butanol, methylene chloride, and 30 ml of ethanol : concentrated ammonia : water (6 : 3 : 1, v/v). Each fraction was evaporated to dryness under negative pressure at 60°C, stored at --30°C, and resuspended in distilled water or appropriate biological saline solution for bioassay. Only the last fraction was used for bioassay on the isolated bovine tissues. SRS-A in this fraction was free of prostaglandin El, E2 and F2~ as shown by thin-layer chromatography (Orange and Austen, 1969). The activity of SRS-A b°v was assayed on the isolated guinea-pig ileum. In Tyrode solution containing atropine (10 -6 g/ml), one unit of SRS-A b°v was arbitrarily taken to be the SRS-A-induced contraction of the guinea-pig ileum in the presence of mepyramine (10 -6 g/ml) equivalent to t h a t produced by 5 ng
PULMONARY EFFECTS OF SRS-Ab°v histamine base on the same preparation prior to the addition of mepyramine (Stechschulte et al., 1967). Contractions were recorded by means of a Narco isotonic detector/transducer and Physiograph Model 4 pen recorder (Narco Biosystems, Houston, Tx.). 2.2. Bioassay o f SRS-A b°v on isolated bovine pulmonary smooth muscles Healthy lung was obtained fresh, either from cattle killed at a local abattoir or from experimental calves (6--8 weeks old) killed with pentobarbitone. The principal pulmonary vein from the apical or cardiac lobe was carefully removed and placed in Krebs--Henseleit (1932) solution. The vein was cut into a single strip, approximately 3--5 mm in width and set up in a 10 ml isolated organ bath (Phipps and Bird, Richmond, Va.) at 37°C in Krebs solution mixed with 5% CO2 in oxygen, at a resting tension of 2.5 g. The principal pulmonary artery for the apical or Cardiac lobe was similarly prepared and maintained at a resting tension of 3.5 g. The m e t h o d employed for setting up the tracheal muscle was t h a t of Offermeier and Ari~ns (1966). A tracheal ring was dissected from the trachea just above the tracheo-bronchial bifurcation. The tracheal muscle was carefully dissected from the inside of the ring and bisected. The two muscle strips (5--7 mm in width) were attached end to end and set up in an isolated organ bath in Krebs solution as described for the pulmonary vessels, at a resting tension of 2.5 g. A terminal (tertiary) bronchus of the apical or cardiac lobe was carefully dissected, cut spirally into a single strip 3--5 mm in width, and set up in an isolated organ bath in Krebs solution, as previously described, under a resting tension of 2.5 g. Dose--response curves and threshold concentrations were determined for histamine and SRS-A b°v on the pulmonary vein, for histamine, 5-HT and SRS-A b°v on the bronchus, for histamine and 5-HT on the trachealis and pulmonary artery preparations. Only the
171 threshold concentrations of SRS-A b°v on the trachealis muscle were determined. SRS-A b°v was also applied to the pulmonary artery to a m a x i m u m concentration of 20 U/ml. The antagonists, sodium meclofenamate (20 and 100 pg/ml) (Collier and Sweatman, 1968), FPL 55712 (0.1, 0.5 and 5 p g / m l ) (Augstein et al., 1973), and PR-D-92-EA (20 and 100 /ag/ml) (Possanza et al., 1975) were examined on the actions of agonists on the pulmonary vein and bronchus. Each antagonist was left in contact for two minutes before agonists were retested. The effectiveness and specificity of each antagonist was estimated by determining the relative agonist 'dose ratio' (Gaddum et al., 1955). 2.3. Drugs Histamine diphosphate, 5-hydroxytryptamine creatinine sulphate and atropine sulphate were purchased from Nutritional Biochemicals Co., Cleveland, Ohio. The following drugs were gifts: mepyramine maleate (Poulenc Ltd., Montreal, Quebec), sodium meclofenamate (Parke, Davis & Co., Detroit, Michigan), FPL 55712 (Fisons Ltd., Loughborough, England) and PR-D-92-EA (PharmaResearch Canada Ltd., Pointe-Claire, Quebec).
3. Results Purified bovine SRS-A showed biological activity on the isolated pulmonary vein, trachealis muscle and bronchial preparations, but not on the pulmonary artery. Log dose-response curves to histamine, 5-HT and SRSA T M were obtained from 7 pulmonary vein preparations and 12 bronchi. Examples of each are shown in figs. 1 and 2 respectively. Only the lower segments o f the dose--response curves were obtained due to the limited a m o u n t of SRS-A available. Contractions induced by 5-HT, histamine and SRS-A b°v on the pulmonary vein and bronchus are shown in fig. 3. The threshold concentrations of each of
172
J.F. B U R K A , P. E Y R E
10
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/ , /...
9 8
7 6 I
5
i
4
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ill l I l'i
lit)>) ÷IOO
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+l',)t) {Ig lal 1
t-i i i i t t l t l t l t l t l l l l l l RS°.\ ~0
+S
+S 11 m] I
I
1 5 SRS-A [U ml -~ ]
IO
50 IOO 2 0 0 HISTAMINE [n 9 ml -I]
Fig. 1. Log d o s e - - r e s p o n s e c u r v e s o f t h e c o n t r a c t i o n s induced by histamine and purified bovine SRS-A on isolated b o v i n e p u l m o n a r y vein. U n i t s o f c o n t r a c t i o n are a r b i t r a r i l y derived f r o m t h e l i n e a r m a g n i t u d e o f the recorded contraction. Standard errors of the m e a n s are i n d i c a t e d as vertical bars.
the agonists (5-HT, histamine and SItS-A) required to contract the 4 tissues are indicated in table 1. The bronchus was the most sensitive to SRS-A b°v o f the 4 bovine tissues studied (average threshold: 2.4 U/ml). In 2 bronchial samples the threshold was as low as 0.1 U/ml. The contraction induced by SRS-A b°v was sometimes preceded by a slight relaxation.
illi IE
llilliEl
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~iii S-EI~
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]~iiiiiilililllli
SR~-A
SR%-A
5
S
S iN
~iiililiilllillll SR~-A 7 ! ~q-I
Fig. 3. Contractions of isolated bovine pulmonary vein and bronchus to histamine, 5-HT and purified bovine SRS-A. Contractions of the pulmonary vein are cumulative. The time trace shows drug injections, and intervalsof 60 sec.
2 1
2
SRS-A[um'-'1
S
I0
20
50
S-"~In~ m'-' ]
IC,C,
200
500
I000
5000
.,S~AM,'.
Fig. 2. Log d o s e - - r e s p o n s e curves o f t h e c o n t r a c t i o n s i n d u c e d b y h i s t a m i n e , 5-HT a n d p u r i f i e d b o v i n e S R S - A o n isolated b o v i n e b r o n c h u s . U n i t s o f c o n t r a c t i o n are a r b i t r a r i l y derived f r o m t h e linear m a g n i t u d e o f t h e r e c o r d e d c o n t r a c t i o n . S t a n d a r d errors of t h e m e a n s are i n d i c a t e d as vertical bars.
PULMONARY EFFECTS OF SRS-Ab°v
173
TABLE 1 Threshold concentrations of bovine SRS-A, histamine and 5-HT to contract several bovine respiratory/pulmonary smooth muscles. Smooth muscle
SRS-A Pulmonary vein Pulmonary artery Trachea Bronchus
Number of animals
Agonist
9.3 U/ml (0.5--15) > 20 U/ml 18 U/ml (15--20) 2.4 U/ml (0.1--10)
Histamine
5-HT
68 ng/ml (5--150) 2100 ng/ml (300--3000) 533 ng/ml (100--1000) 1450 ng/ml (250-->20000)
Not done
7
15 ng/ml (10--20) 57 ng/ml (20--75) 33 ng/ml (5--75)
3
A latent period o f 4--7 min preceded contraction. A cumulative dose--response curve to SRS-A b°v could n o t be obtained owing to tachyphylaxis: a p p r o x i m a t e l y 3 0 m i n being required to recover the response. There was, however, n o cross-tachyphylaxis between histamine, 5-HT and SI~S-A b°v. The p u l m o n a r y artery was the least sensitive to SRS-A b°v, failing t o c o n t r a c t to concentrations o f SRS-A as high as 20 U/ml. Higher co n cen tr ations were n o t a t t e m p t e d . The artery was highly sensitive t o 5-HT however {threshold: 15 ng/ml), but n o t to histamine (threshold: 2100 ng/ml). The p u l m o n a r y vein was relatively sensitive to b o t h SRS-A b°v (threshold: 9.3 U / m l ) a n d histamine {threshold: 68 ng/ml). For comparison purposes, the p u l m o n a r y vein is very sensitive to 5-HT {threshold: 5 ng/ml) {Burka and Eyre, 1974c). Cumulative dose--response relationships could be established t o histamine and SRS-A as there was no t a c h y p h y laxis to either of these substances in the vein. The trachealis muscle was relatively insensitive to histamine and SRS-A b°v, but did n o t c o n t r a c t to m o d e r a t e concentrations of 5-HT {threshold: 57 ng/ml). This agrees with the observations o f Offermeier and Ari~ns (1966) who first showed evidence f or 5-HT receptors in the bovine tracheal muscle.
3 12
3.1. Inhibitor studies Sodium m e c l o f e n a m a t e , F P L 5 5 7 1 2 and PR-D-92-EA were examined as possible antagonists of SRS-A b°v and histamine on the p u l m o n a r y vein and SRS-A b°v, histamine and 5-HT on the bronchus. The results are tabulated in table 2. None of the drugs effectively antagonized histamine or SRS-A on the p u l m o n a r y vein. Sodium m e c l o f e n a m a t e at a concent rat i on r e c o m m e n d e d by Collier and Sweatman (1968) (20 pg/ml) showed no antagonism to any of t he agonists tested on bovine tissues. However, sodium m e c l o f e n a m a t e at 100 gg/ml selectively antagonized SRS-A b°v. The t y p e of antagonism could n o t be determined because SRS-A b°v in the presence of meclofenamate (100 pg/ml) induced a marked loss of t one of the muscle. In one preparation an a p p r o x i m a t e dose ratio of 5 was recorded, but it was n o t possible to re-establish a full dose--response curve due to insufficient SRS-A being available. Meclofenamate (100 pg/ml) showed no antihistaminic effect but slightly antagonized 5-HT which was n o t statistically significant (p > 0.05). FPL 55712 at all 3 concentrations examined did n o t antagonize histamine, 5-HT or SRS-A b°v on the bronchus. PR-D-92-EA
174
J.F. BURKA, P. EYRE
TABLE 2 Effects of inhibitors on contractions of bovine pulmonary vein and bronchus induced by histamine, 5-HT and bovine SRS-A. Smooth muscle
Pulmonary vein
Bronchus
Dose ratio of agonists
Meclofenamate 20 pg/ml 100/lg/ml FPL 55712 0.1//g/ml 0.5//g/ml 5.0/lg/mt PR-D-92-EA 20 pg/ml 100/lg/ml Meclofenamate 20 pg/ml 100 pg/ml FPL 55712 0.1/ag/ml 0.5 pg/ml 5.0 pg/ml PR-D-92-EA 20 pg/ml 100 pg/ml
Histamine
5-HT
SRS-A
1, 1, 1 1, 1, 1
n.d. 1 n.d.
1,3,1,1,1 t, 2, 1
1, 1, 1 2, 1, 1 2, 1, 1
n.d. n.d. n.d.
1,1,1 1,2,1,1 2, 2, 1
1, 1, 1 2, 2, 1, 1, 1
n.d. n.d.
1, 2, 7, 1 2, 1, 4, 1
1, 1, 1 2, 1, 1
1,1,1,1 t0,2,2, 1
1, 1 1, 1 1, 1, 1
1, 1, 1 1, 1, 1 1,1,1
1, 1, 1 1, 1, 1 1,1,1,1
1, 1,1 5, 6, >40, >40
1,1,1 1, 1, 1
1,1,1 1, 1, I
1 n.d. = not done. failed t o a n t a g o n i z e SRS-A b°v or 5-HT, b u t at a c o n c e n t r a t i o n o f 100 p g / m l a n t a g o n i z e d h i s t a m i n e o n l y . H i s t a m i n e in t h e p r e s e n c e o f P R - D - 9 2 - E A ( 1 0 0 p g / m l ) caused r e l a x a t i o n o f t h e b r o n c h i a l muscle. In 2 o u t o f 4 prep a r a t i o n s , tissue c o n t r a c t i l i t y was irrecoverable, even w i t h v e r y high c o n c e n t r a t i o n s o f histamine. In t h e t w o r e m a i n i n g tissues a h i s t a m i n e d o s e - - r e s p o n s e curve c o u l d be fully re-established in t h e p r e s e n c e o f PR-D-92-EA ( 1 0 0 p g / m l ) a n d dose ratios o f 5 and 6 were r e c o r d e d . T h e l a t t e r d o s e - - r e s p o n s e curves were parallel t o the d o s e - - r e s p o n s e curves in t h e a b s e n c e o f t h e drug, suggesting t h a t a n t a g o n i s m was c o m p e t i t i v e in n a t u r e .
4. Discussion E x a m i n a t i o n o f b o v i n e SRS-A o n isolated b o v i n e r e s p i r a t o r y a n d p u l m o n a r y vascular s m o o t h muscles helps t o clarify the poten-
1,1,1,1 >120, 5, >120
tial role o f SRS-A in a c u t e systemic anaphylaxis. B r o n c h o c o n s t r i c t i o n has long b e e n associated with a c u t e s y s t e m i c a n a p h y laxis in t h e guinea pig (Auer and Lewis, 1 9 1 0 ) . SRS-A is also i m p l i c a t e d in a n a p h y laxis in t h e rat ( S t o t l a n d a n d Share, 1 9 7 4 ) , and in a s t h m a in m a n (Orange and Langer, 1 9 7 3 ) . In t h e guinea pig, a n t i g e n - i n d u c e d b r o n c h o c o n s t r i c t i o n is associated with a decrease in p u l m o n a r y c o m p l i a n c e (Collier, 1 9 6 8 ) . Similarly, i.v. a d m i n i s t r a t i o n o f purified guinea-pig SRS-A t o guinea pigs results in a d o s e - d e p e n d e n t decrease in average p u b m o n a r y c o m p l i a n c e , suggesting t h a t SRS-A constricts t h e small airways (terminal b r o n c h i , b r o n c h i o l e s and alveoli) (Drazen and A u s t e n , 1 9 7 4 ) . H i s t a m i n e , 5 - H T a n d PGF2~ also increase resistance, which suggests t h a t these substances c o n s t r i c t b o t h the large and small airways. A similar p a t t e r n emerges in t h e b o v i n e
PULMONARY EFFECTS OF SRS-Ab°v respiratory tract. Previous studies in cattle have shown varying degrees of respiratory embarrassment in acute systemic anaphylaxis (Aitken and Sanford, 1969; Eyre et al., 1973). However, no direct evidence for bronchoconstriction has previously been shown and Schultz--Dale reactions on the bronchi are either feeble or absent (Eyre, 1971). This study gives the first direct evidence of a substance that contracts bovine bronchial smooth muscle at low concentrations (threshold: 2.4 U/ml). In contrast, the bovine trachea is less sensitive to SRS-A b°v (threshold: 18 U/ml). The sensitivities of the bronchus and trachea to 5-HT are similar (tresholds: 33 and 57 ng/ml respectively). Both tissues are very insensitive to histamine. However, histamine in vivo may cause tracheobronchial constriction by a vagal reflex mechanism (Eyre et al., 1973; Nadel, 1973). Our results appear to be consistent with in vivo results in the guinea pig (Drazen and Austen, 1974) where SRS-A specifically constricts small airways, whereas histamine and 5-HT affect small and large airways approximately equally. If sufficient SRS-A b°v could be made available for in vivo studied in calves, it would be interesting to study its effects on pulmonary compliance and resistance, to compare these to the changes in pulmonary compliance and resistance in acute systemic anaphylaxis, and to study inhibitor drugs. The contraction induced by SRS-A b°v on the bovine bronchus may not be entirely directly due to SRS-A as tachyphylaxis rapidly develops. This suggests t h a t SRS-A b°v may be either releasing another substance that contracts the muscle or acting on a SRS-A receptor with an extended recovery time. This is in contrast to human bronchial muscle which is also very sensitive to SRS-A (threshold: 2.2 U/ml), where no tachyphylaxis to SRS-A occurs (Math~ and Strandberg, 1971). There is no cross-tachyphylaxis between histamine, 5-HT and SRS-A T M in cattle, suggesting that these substances are quite distinct in their actions on particular receptors in the bronchial muscle.
175 Inhibitor studies further suggest a role for SRS-A b°v in bronchoconstriction in anaphylaxis of cattle. Sodium meclofenamate is of particular interest because this drug effectively protects calves during acute systemic anaphylaxis (Eyre et al., 1973; Burka and Eyre, 1974a), particularly in the control of respiratory symptoms (Aitken et al., 1975). A paradox already reported is that sodium meclofenamate markedly enhances the immunological release of SRS-A b°v from lung (Burka and Eyre, 1975). Therefore it was necessary to examine the possible antagonism of SRS-A b°v at the site of action. Meclofenamate was particularly effective in antagonizing SRS-Ab°V-induced contraction of the calf bronchus. It was not possible to determine the type of antagonism owing to the limited quantity of SRS-A b°v available, but it is also interesting that meclofenamate alone or meclofenamate and SRS-A b°v together induce a loss of tone of the muscle. Meclofenamate is a potent inhibitor of PG synthetase (Flower, 1974). A possible explanation is that meclofenamate and SRS-A may be acting on the tissue in such a way that endogenous prostaglandins required to maintain tone of the muscle (Frey and Schafer, 1974) are depleted and relaxation ensues. This explanation would be particularly applicable if SRS-A releases prostaglandins, particularly PGF2~, to contract the bronchus. Similar inhibition of contractions of human bronchial muscle induced by SRS-A and PGF2~ has been observed with aspirin and fenamates (Berry and Collier, 1964; Collier and Sweatman, 1968). These effects of meclofenamate on SRS-A b°vinduced contractions appear to be specific, despite the high concentration used, as the drug had little, if any, effect on the contractions induced by histamine and 5-HT, and had no measurable effect on the bronchial muscle per se. Further evidence to suggest that SRS-A b°v may be releasing another substance to contract the bronchus is the 4--7 min latent period between addition of SRS-A b°v to the bath and contraction of the muscle. This
176
latent period would seem to allow sufficient time for the formation and release of another substance, particularly prostaglandins (Piper and Vane, 1971). Dunlop and Smith (1975) have recently shown that there is a significant correlation between PGF2~ release and antigen-induced contraction of human bronchus and that indomethacin, while inhibiting synthesis of PGF2~, also reduces the antigeninduced contraction. Further experiments must be carried out to prove that PGF2~ is actually released from bovine bronchi on administration of SRS-A b°v. Neither FPL 55712 nor PR-D-92-EA antagonized the effects of bovine SRS-A on the bronchus, even though they have been reported to antagonize m o n k e y , human and guinea-pig SRS-A on the guinea-pig ileum (Augstein et al., 1973; Possanza et al., 1975). The species difference in the test tissue may afford one explanation for this. It is also possible that bovine SRS-A m a y be different in structure from SRS-A of other species because, even on the guinea-pig ileum, FPL 55712 is n o t as effective an antagonist of SRS-A b°v as of guinea-pig SRS-A. A dose ratio of approximately 4 was obtained for guinea-pig SRS-A with FPL 55712 (80 ng/ml) by Augstein et al. (1973), whereas the dose ratio for SRS-A b°v with FPL 55712 (100 ng/ml) is approximately 2.8 in guinea-pig ileum (Burka, unpublished results). It is interesting that a high concentration of PR-D-92-EA (100 pg/ml) antagonizes the effects of histamine on the bronchus, and that antagonism appears to be competitive. Inhibition of histamine by PR-D-92-EA has been observed previously on the guinea-pig ileum (Possanza et al., 1975). Pulmonary oedema is a principal pathological change observed in acute systemic anaphylaxis in calves (Wells et al., 1973). The present results confirm that the pulmonary vein is considerably more sensitive to histamine, 5-HT and SRS-A b°v (thresholds: 6 9 n g / m l , 5 ng/ml (Burka and Eyre, 1974c) and 9.3 U/ml respectively) than is the artery (thresholds: 2100 ng/ml, 15 ng/ml and > 2 0 U/ml
J.F. BURKA, P. EYRE
respectively). These results suggest that SRS-A might contribute to pulmonary oedema by contracting the pulmonary vein in preference to the artery, and thus raising capillary hydrostatic pressure. These experiments suggest that SRS-A b°v may contribute to bronchoconstriction and pulmonary oedema in immediate hypersensitivity reactions in the bovine species. It is now necessary to examine if SRS-A b°v can also induce bronchoconstriction and pulmonary oedema in calves in vivo.
Acknowledgements The authors are grateful to Mr. T.R. Deline for technical assistance. The work was supported in part by the Ontario Ministry of Agriculture and Food, by grant A5937 of the National Research Council of Canada and by grant OG3012 of Agriculture Canada. J.F. Burka was the recipient of an Ontario Graduate Scholarship.
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177 Krebs, H.A. and K. Henseleit, 1932, Untersuchung fiber die Harnstoffbildung im Tierkorper, HoppeSeylers Z. Physiol. Chem. 210, 33. Math~, A.A. and K. Strandberg, 1971, Antagonism of slow reacting substance by polyphloretin phosphate on isolated human bronchi, Acta Physiol. Scand. 82, 460. Nadel, J.A., 1973, Neurophysiologic aspects of asthma, in: Asthma, eds. K.F. Austen and L.M. Lichtenstein (Academic Press, New York) p. 29. Offermeier, J. and E.J. Ari~ns, 1966, Serotonin I. Receptors involved in its action, Arch. Intern. Pharmacodyn. Therap. 164, 192. Orange, R.P. and K.F. Austen, 1969, Slow-reacting substance of anaphylaxis, Advan. Immunol. 10, 105. Orange, R.P. and H. Langer, 1973, Bronchial asthma: hyperreactivity of airways and target cells, in: Allergology--Proceedings of the 8th Congress of the International Association of Allergology, Tokyo, October 14--20, 1973 (Excerpta Medica, Amsterdam) p. 325. Orange, R.P., R.C. Murphy, H.L. Karnovsky and K.F. Austen, 1973, The physicochemical characteristics and purification of slow-reacting substance of anaphylaxis, J. Immunol. 110, 760. Piper, P.J. and J.R. Vane, 1971, The release of prostaglandins from lung and other tissues, Ann. N. Y. Acad. Sci. 180,363. Possanza, G.J., A. Bauen and P.B. Stewart, 1975, In vitro antagonism of the mediators of allergy by a benzopyrano-benzopyran carboxylic acid: PR-D92-EA, Intern. Arch. Allergy Appl. Immunol. 49, 789. Stechschulte, DIJ. K.F. Austen and K.J. Bloch, 1967, Antibodies involved in antigen-induced release of SRS-A in the guinea pig and rat, J. Exptl. Med. 125, 127. Stotland, L.M. and N.N. Share, 1974, Active bronchial anaphylaxis in the rat, Can. J. Physiol. Pharmacol. 52, 1114. Wells, P.W., P. Eyre and J.H. Lumsden, 1973, Hematological and pathological changes in acute systemic anaphylaxis in calves: effects of pharmacological agents, Can. J. Comp. Med. 3 7 , 1 1 9 .