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Relaxation of the guinea-pig trachea induced by platelet-activating factor and by serotonin
European Journal of Pharmacology, 80 (1982) 29-35
29
Elsevier Biomedical Press
RELAXATION OF THE GUINEA-PIG TRACHEA INDUCED BY PLATELET-ACTIVATING FACTOR AND BY SEROTONIN ARTHUR PRANCAN *, JEAN LEFORT, MARY BARTON ** and B. BORIS VARGAFTIG ***
Unitb des Venins, D~partement de Physiopathologie Expbrimentale, Institut Pasteur, 28 Rue du Dr. Roux, Paris Cedex 15, France Received 22 September 1981, revised MS received 8 December 1981, accepted 11 January 1982
A. PRANCAN, J. LEFORT, M. BARTON and B.B. VARGAFTIG, Relaxation of the guinea-pig trachea induced by platelet-activating
factor and by serotonin, European J. Pharmacol. 80 (1982) 29-35. Platelet-activating factor (PAF-acether), a known platelet stimulant and bronchoconstrictor (in vivo), is a potential mediator of inflammation and thrombosis. However, all smooth muscle effects of PAF-acether described to date are indirect, relying upon intravascular platelet activation. Novel actions of PAF-acether and serotonin (5-HT) are presented here; these actions may lead to the development of a practical bioassay for PAF-acether and contribute to the understanding of the mechanism of action for both substances. PAF-acether, when added to a spiral cut guinea-pig trachea suspended in a tissue bath containing Krebs-Henseleit buffer, produced a dose-dependent loss of active tissue tension. The EDs0 for this effect of PAF-acether was 75 ng/ml. PAF-acether produced a maximal relaxation which was 68% of that produced by PGE I and the effect could not be modified by aspirin or propranoiol pretreatment. 5-HT, alone, contracted the guinea-pig trachea strip in a dose-dependent manner, but caused relaxation instead when methysergide was present. Aspirin, phenoxybenzamine and propranoloi did not alter this loss of active tissue tension. A similar observation was made in vivo using the guinea-pig bronchoconstriction model, in which PAF-acether as well as 5-HT given to methysergide-treated animals caused a decrease in intratracheal pressure. This action of PAF-acether may yield a suitable bioassay method which could facilitate routine measurements of the substance. Furthermore, the similarity in action of PAF-acether and of 5-HT on methysergide-treated animals leads one to speculate about the relationship between the two substances and their mechanism of action in smooth muscle. Guinea-pigs
Platelet-activating factor
Platelets
Methysergide
Serotonin
Trachea
1. Introduction
Platelet-activating factor (PAF-acether), l-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, stimulates platelet aggregation, which is independent of ADP and of the cyclooxygenase pathway (Cazenave et al., 1979, Chignard et al., 1979, 1980). It is also a potential mediator of immune (Benveniste et al., 1972) and non-immune (Vargaftig et
* On temporary leave from Department of Pharmacology, Rush University, 1753 West Congress Parkway, Chicago, Illinois 60612, U.S.A. ** Present address: Department of Family and Community Medicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033, U.S.A. *** To whom all correspondence should be addressed at Unit~ des Venins, the above address. 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
al., 1981a,b) inflammations. The identification of PAF-acether currently involves chemical, chromatographic and radiochemical tests, or the use of a bioassay on platelet activation. The bioassay requires fresh platelets when routine tests for the release of PAF-acether are made, and therefore, is often impractical. Until this time, PAF-acether has not been reported to influence any of the isolated tissues which are used to identify biologically active substances. In fact, most of the effects of PAF-acether are platelet-dependent and accordingly, it fails to affect isolated tissues in the absence of platelets. For this reason, PAF-acether, which is a potent bronchoconstrictor in vivo, will not contract the isolated guinea pig parenchymal lung strip (Vargaftig et al., 1981 a,b). In the search for an appropriate isolated tissue to be used for
30 routine bioassay of PAF-acether we noted, and report here, unexpected effects of this substance and of serotonin (5-HT) both in vivo and in vitro.
2. Materials and methods
2.1. Dru M
PAF-acether was obtained from Dr. J. Benveniste and Prof. J.J. Godfroid. It was stored in 95% ethanol at - 2 0 ° C and diluted in normal saline to obtain working concentrations. The final concentration of ethanol in the tissue bath was 0.0019% or less. PGE l, a gift from the Upjohn Company, was initially dissolved in ethanol, further dilutions being made with 20% Na2CO 3. Aspirin, as a soluble lysine salt (Aspegic®), was obtained from Laboratories Egic, Paris. Other drugs used in this study included: Acetylcholine chloride, arachidonic acid, phenoxybenzamine, histamine hydrochloride and serotonin creatine phosphate (Sigma); propranolol hydrochloride (Avlocardyl ®, ICI); mepyramine dihydrochloride (Rhone-Poulenc); and methysergide hydrogen bimaleate (Sandoz). 2.2. Isolated guinea pig. trachea preparation Tracheas were taken from Sprague-Dawley guinea pigs of either sex (370-450 g) which had been anesthetized with sodium pentobarbital (30 m g / k g i.p.). Each trachea was cut spirally and suspended under 2 g tension in a tissue bath containing oxygenated Krebs-Henseleit buffer (37°C), which was prepared without any receptor or enzyme antagonists. At least 2 h were allowed for tissue stabilization before PAF-acether, 5-HT and other substances were added directly to the bath in volumes of 20 #1 or less (16 ml bath volume). Changes in tension were recorded isometrically with a Statham UC3 transducer. Between tests, each trachea was washed three times with buffer and the next addition of a substance to the tissue took place only after the tension of the trachea had reached and maintained a stable plateau.
2.3. Testing the effects of PA F-acether and serotonin on the trachea PAF-acether was administered in single doses to the tissue bath so as to establish final concentrations of 5.1, 17, 51, 170 and 510 ng/ml. 5-HT was added alone in single dose concentrations of 4-400 n g / m l and also in the presence of 47 n g / m l of methysergide. Acetylcholine (100 n g / m l ) was used to test for the responsiveness of the trachea to a stimulus, and PGE I 0.1-1 # g / m l ) was used for evaluating the maximal loss of active tension by the tissue. Aspirin (1-20 /~g/ml) was added to the bath 10 min before PAF-acether or serotonin in order to test the involvement of prostaglandin synthesis in the response. Phenoxybenzamine (1.5 /~M) and propranolol (1 # g / m l ) pretreatment tested the involvement of adrenergic mediators and receptors in the response. 2.4. In vivo studies: bronchial resistance to inflation, intratracheal pressure Guinea-pigs ( n - - 6 ) were anesthetized with sodium pentobarbital (30 m g / k g i.p.) and artificially ventilated with a Palmer pump adjusted to deliver 1 ml air/100 g body weight at 72 strokes/rain. Propranolol (3 m g / k g i.p. plus. 1 m g / k g i.v.) was used to prevent interference by released bronchodilator catecholamines, and pancuronium ( 4 m g / k g i.v.) ensured muscle relaxation. One carotid artery was cannulated for the measurement of arterial blood pressure. The right jugular vein was cannulated as well and was used for all injections (Vargaftig et al., 1979). Bronchoconstriction was induced by histamine (1-3 /~g/kg) serotonin (0.5-2 ~ g / k g ) acetylcholine (10-20 ~ g / k g ) or PAF-acether (12-60 ng/kg) and recorded on a Beckman Dynograph by a slight modification of the Konzett-Rossler method (Lefort and Vargaftig, 1978). Injections were usually separated by an interval of 10 rain except for PAF-acether injections when intervals of 30-60 min were used. In addition to the above-mentioned parameters, intratracheal pressure was measured from an open tip catheter introduced into the trachea. For this purpose, the trachea was dissected towards the head after the animal had been
31
prepared for recording bronchoconstriction, an open-tip catheter was introduced caudally into the trachea and its caudal end firmly ligated (James, 1969).
220-
T
200-
-T
180-
2.5. Data
160-
The data are given as mean ± S.E.M. The data were tested in a semi-log dose-response plot for a straight line fit using linear regression. Statistical significance was evaluated using Student's t-test.
140-
1 T
!
T
120100-
±1
80-
3. Results 60-
3.1. The effect of PAF-acether on active trachea tension in vitro
I
40-
I
20-
The introduction of PAF-acether into the tissue bath containing a spiral-cut guinea pig trachea resulted in a dose-dependent loss of active tension, as shown in fig. 1. The entire dose-response series of tests using PAF-acether is summarized i n fig. 2. A semi-log dose-response plot of the dose-response data yielded a line with a correlation coefficient of 0.998. The EDs0 for PAF-acether on loss of active tension in the trachea was calculated as 75 n g / m l final concentration in the tissue bath. The isolated trachea was consistently responsive to PAF-acether. In every instance where the tissue contracted in response to acetylcholine or serotonin, or where it relaxed following P G E I, it was also responsive to PAF-acether. In 30% of the
2 .i, I
17o
1.80"
Fig. I. The effect of PAF-acether on an isolated guinea-pig trachea. Total tissue tension was 2 g. Doses of PAF-acether were applied individually and separated by a tissue wash. The tracings are superimposed to show the relationship between dose and effect. Scale: relaxation (g).
na12Jn-13 in-1 5.1
17
51
170
PAF-acether ( n g / m l )
Fig. 2. Dose-response: PAF-acether on isolated guinea-pig trachea tension. Total tissue tension was 2 g. All doses of PAF-acether were administered individually and separated by a tissue wash. All values are given as the mean loss of tissue tension - S.E.M. The n values represent the number of tracheas from different animals which were tested. Scale: relaxation (rag).
tracheas tested, however, there was evidence of a temporary refractoriness of the tissue to succeeding doses of PAF-acether. In these cases, the second response in a series was depressed to 60% of the original. The average maximal response of isolated trachea to PAF-acether was a 318.95 +- 120.72 mg loss of tension (n = 4), which was achieved with 510 n g / m l and higher concentrations. This response was 68% of the maximal effect observed when P G E I (200 n g / m l ) was added to the preparation (471.21 ± 90.28 mg, n = 9).
3.2. The effect of serotonin and methysergide on active trachea tension in vitro As seen in fig. 3, serotonin induced a concentration-dependent contraction of the isolated trachea
32
~r Control
~ " 5min / +500-
+400 100 nM methysergide +300-
t
5HT41JM)0.03
t
0.1
t
0.3
t
I
+200-
Fig. 3. Methysergide-inducedinversion of the contracting effect of serotonin on the guinea pig trachea. Total tissue tension was 2 g. Doses of 5-HT were applied individually and separated by a tissue wash. Methysergide was included in the buffer during the tests shown in the lower tracings. Scale of the upper panel: contraction and scale of the lower panel: relaxation.
strip. In the presence of methysergide, similar conc e n t r a t i o n s of 5 - H T produced d o s e - d e p e n d e n t relaxation. As s u m m a r i z e d in fig. 4, the dose-response relationship of serotonin to tracheal c o n t r a c t i o n was inverted to yield a progressive loss of tension w h e n methysergide was present.
3.3. Aspirin, propranolol or phenoxybenzamine pretreatment in vitro A s p i r i n p r e t r e a t m e n t ( 1 - 2 0 /~g/ml, 10 min) neither e l i m i n a t e d n o r d i m i n i s h e d the loss of trachea tension due to P A F - a c e t h e r or to serotonin in the presence of methysergide. W h e n using high doses of aspirin (10 / ~ g / m l or 20 t t g / m l ) , the aspirin alone caused a slow c o n t r a c t i o n of the p r e p a r a t i o n , which c o m p e t e d with the loss of tension caused by the test substances; this did not h a p p e n after the tissue reached a stable baseline tension. P r o p r a n o l o l (1 # g / m l ) did not interfere with the effect of either substance, a n d p h e n o x y b e n z a m i n e (1.5/~M) did not alter the response.
t -
100
:::::::::::::: n=7
:::.':~i4i":i:
METHYSERGIDE
(tO0 n M )
-r
_._5
0.03 0.1 0.3 ! DOSE OF SEROTONIN (jJM) Fig. 4. Dose-response: 5-HT plus methysergide relaxation of the guinea pig trachea. Total tissue tension was 2 g. All values are given as the mean change in tissue tension -~S.E.M. The n values represent the number of tracheas from different animals which were tested. A one-tailed unpaired Student's t-test was used to compare the effect of the same dose of 5-HT with and without methysergide. * P<:0.05. Scale: mg of tissue contraction (positive figures) or of tissue relaxation (negative figures).
3.4. In vivo studies 5 - H T (0.5-2 # g / k g ) , h i s t a m i n e ( 1 - 3 # g / k g ) a n d acetylcholine ( 1 0 - 2 0 /~g/kg) i n d u c e d the ex-
33
MEPY METHY
10 rain
20[ ,o
Agonists (l~kg ~)
5HT 0.5
H 1
Ach 10
5HT 1
5HT 1
H 1
Ach 10
Fig. 5. Interference of mepyramine (mepy) and of methysergide (methy) with the effects of 5-HT, histamine (H) and acetylcholine (AcH) in the guinea pig. The first panel shows the effects of 5-HT and ACH on intratracheal pressure, arterial blood pressure and bronchial resistance to inflation (tracings from top to bottom). The second and third panels show these responses after mepy and methy (0.2 mg/kg). All injections were i.v. pected b r o n c h o c o n s t r i c t i o n in the guinea pig (fig. 5). The effects o n tracheal pressure were less predictable since the a c c o m p a n y i n g variations of arterial b l o o d pressure interfered with the mea-
,
10 rain
,
surement. 5 - H T a n d h i s t a m i n e increased the intratracheal pressure with a secondary, small relaxing effect whereas acetylcholine had a relaxing effect, a c c o m p a n i e d b y s u d d e n a n d short-lasting
METHY
.,0[
0
Agonist
H 5HT PAF 5HT PAF 2 2 66 2 66 Fig. 6. Failure of methysergide to interfere with the effects of PAF-acether in the guinea-pig. The left-side panel shows the effects of histamine (H), PAF-acether (PAF) and 5-HT given at the doses indicated (/~g/kg for H and 5-HT; ng/kg for PAF-acether). After methysergide (methy, 0.2 mg/kg), as shown in the left-side panel, the effects of 5-HT on the intratracheal pressure were reversed, whereas its effects on bronchoconstriction, and those of PAF-acether on all parameters, were unaffected. Tracings from top to bottom: intratracheal pressure, arterial blood pressure and bronchial resistance to inflation. All injections were i.v.
34 hypotension. After mepyramine and methysergide (0.2 m g / k g of each) the effects of histamine were suppressed, those o f acetylcholine were unaffected whereas serotonin induced a reduction of the intratracheal pressure, unaccompanied by variations in arterial pressure (fig. 5). The administration of PAF-acether (12.5-132 ng/kg) was followed by the earlier described bronchoconstriction, accompanied by a short-lived contraction of the trachea which was followed by a long-lasting decrease of intratracheal pressure (fig. 6). This pattern was unchanged by methysergide (fig. 6) or by mepyramine (not shown).
4. Discussion
PAF-acether is the most active guinea pig platelet aggregating agent so far described (Vargaftig et al., 1980, 1981a,b). Its in vivo effects include bronchoconstriction in the guinea pig, and this effect was shown to be platelet-dependent and aspirin-resistant (Vargaftig et al., 1980). Since PAF-acether is released by macrophages (MenciaHuerta and Benveniste, 1979), which may participate in allergic reactions (Capron et al., 1975, 1977) including bronchoconstriction, it was suggested that PAF-acether might play a part in asthma (Vargaftig et al., 1981a,b). This is particularly relevant to the guinea-pig model for allergic bronchoconstriction which-is much used in the search for anti-asthma drugs. It is important to note that since the bronchial effects of Slow-Reacting Substance of Anaphylaxis (leukotrienes C4 and D4) are suppressed by aspirin (Berry and Collier, 1964; Vargaftig et al., 1981c), the effects of PAF-acether cannot be attributed to the release of these mediators. There is a need for an isolated tissue to be used for bioassaying PAF-acether, particularly for online studies of its release. Our present results indicate that the guinea pig trachea could be used as such a tissue, since it was concentration-dependently relaxed by PAF-acether. This effect was not due to the intramural formation of prostaglandins, because it was not inhibited by aspirin, not mediated by fl-adrenergic activation, since the effect was also not reduced by propranolol. Our data with
serotonin-induced relaxation of the tracheal strip, an effect uncovered when the usual contraction was inhibited by methysergide, are not sufficient evidence that PAF-acether interacts with a 'relaxing' receptor for 5-HT. Under different conditions, there was found a methysergide-induced unmasking of a 5-HT vasodilator effect on the nasal vessels (Vargaftig and Lefort, 1974). Since we observed (unpublished experiments) that PAFacether affects the tone of these vessels only in presence of circulating platelets, PAF-acether does not share a mechanism of action with 5-HT in this particular preparation. Alternatively, 5-HT might trigger the release of PAF-acether from the trachea, in which case one may speculate to what extent the release of 5-HT from platelets, such as occurs during migraine or after administration of reserpine, may lead to the formation of PAF-acether. Finally, a 'serotonin-releasing factor' was described in the plasma of migraine patients (Anthony and Lance, 1975), which might be related to PAF-acether. It thus appears speculative, but deserves further study, to suggest that the formation a n d / o r effects of PAF-acether and of 5-HT are related, and may account for at least part of the pathophysiology involving platelets, 5-HT and smooth muscles. Aspirin, mepyramine and methysergide form a synergistic combination which inhibits the bronchoconstriction (in vivo) and ex vivo platelet release reaction due to PAF-acether while individually the drugs in this group have no effect (Vargaftig et al., 1981a). It thus appears that PAF-acether, as a potential m e d i a t o r of anaphylaxis a n d / o r inflammation, interacts with established mediators, particularly the amines, for its overall in vivo effect. Use of the guinea pig trachea under conditions where the effects of other mediators are excluded may help to determine more precisely the relevance of the formation of PAF-acether under various conditions. In conclusion, PAF-acether and serotonin (after methysergide) have a similar direct action on the smooth muscle of the guinea pig trachea. Both of these substances have now been shown to decrease tension in the trachea in a dose-dependent fashion in vitro and in vivo. This action is independent of platelets, prostaglandin synthesis and adrenergic
35 r e c e p t o r a c t i v a t i o n . A p r a c t i c a l o u t c o m e o f this work may be the development of a suitable bioassay for P A F - a c e t h e r . W e b e l i e v e t h a t t h e s i m i l a r i t y in effects o f P A F - a c e t h e r a n d s e r o t o n i n ( a f t e r m e t h y s e r g i d e ) o n the t r a c h e a will p r o v i d e the b a c k g r o u n d for f u r t h e r studies o n the p r e c i s e mechanism of action of each substance.
Acknowledgements PAF-acether was kindly supplied by Drs. J. Benveniste and J.J. Godfroid. This study was supported by a grant from INSERM (PRC 121037). The authors wish to express their appreciation to Ms. De Borah McCaskill and Mrs. Marie-Christine Ferrand for assistance in preparing the manuscript.
References Anthony, M. and J.W. Lance, 1975, The role of serotonin in migraine, in: Modern Topics in Migraine, ed. J. Pearce (William Heinemann Medical Books, London) p. 107. Benveniste, J., P.M. Henson and C.G. Cochrane, 1972, Leukocyte-dependent histamine release from rabbit platelets: the role of lgE basophils, and a platelet-activating factor, J. Exp. Med. 136, 1356. 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, Br. J. Pharmacol. 23, 201. Capron, A., J.P. Dessaint, M. Capron and H. Bazin, 1975, Specific IgE antibodies in immune adherence of normal macrophages to Schistosoma mansoni schistosomules, Nature 253, 474. Capron, A., J.P. Dessaint, M. Joseph, R. Rousseaux, M. Capron and H. Bazin, 1977, Interaction between IgE complexes and macrophages in the rat: A new mechanism of macrophage activation, European J. lmmunol. 7, 315. Cazenave, J.P., J. Benveniste and J.F. Mustard, 1979, Aggrega-
tion of rabbit platelets by platelet-activating factor is independent of the release reaction and the arachidonate pathway and inhibited by membrane-active drugs, Lab. Invest. 3, 275. Chignard, M., J.P. Le Couedic, M. Tence, B.B. Vargaftig and J. Benveniste, 1979, The role of platelet-activating factor in platelet aggregation, Nature 279, 799. Chignard, M., J.P. Le Couedic, B.B. Vargaftig and J. Benveniste, 1980, Platelet-activating factor (PAF-acether) secretion from platelets. Effect of various aggregating agents, Br. J. Haematol. 46, 455. James, L.G.W., 1969, The use of the in vivo trachea preparation of the guinea-pig to assess drug action on lung, J. Pharm. Pharmacol. 21,379. Lefort, J. and B.B. Vargaftig, 1978, Role of platelets in aspirinsensitive bronchoconstriction in the guinea-pig; interactions with salicylic acid, Br. J. Pharmacol. 63, 35. Mencia-Huerta, J.M. and J. Benveniste, 1979, Platelet-activating factor and macrophages, I. Evidence for the release from rat and mouse peritoneal macrophages and not from mastocytes, European J. Immunol. 9, 409. Vargaftig, B.B., M. Chignard, J. Benveniste, J. Lefort and F. Wal, 198 la, Background and present status of research on platelet-activating factor (PAF-acether), Ann. N.Y. Acad. Sci. 370, 119. Vargaftig, B.B., M. Chignard, J.M. Mencia-Huerta, B. Arnoux and J. Benveniste, 1981b, Pharmacology of arachidonate metabolites and of platelet-activating factor (PAF-acether), in: Platelets in Biology and Pathology, Vol. II, ed. J.L. Gordon (North-Holland, Amsterdam) p. 373. Vargaftig, B.B. and J. Lefort, 1974, Pharmacological evidence for a vasodilator receptor to serotonin in the nasal vessels of the dog, European J. Pharmacol. 25, 216. Vargaftig, B.B., J. Lefort, M. Chignard and J. Benveniste, 1980, Platelet-activating factor induces a platelet dependent bronchoconstriction unrelated to the formation of prostaglandin derivatives, European J. Pharmacol. 65, 185. Vargaftig, B.B., J. Lefort, D. Joseph and F. Fouque, 1979, Mechanism of bronchoconstriction and of thrombocytopenia induced by collagen in the guinea-pig, European J. Pharmacol. 58, 273. Vargaftig, B.B., J. Lefort and R.C. Murphy, 1981c, Inhibition by aspirin of bronchoconstriction due to leukotrienes C4 and D4 in the guinea-pig, European J. Pharmacol. 72, 417.
29
Elsevier Biomedical Press
RELAXATION OF THE GUINEA-PIG TRACHEA INDUCED BY PLATELET-ACTIVATING FACTOR AND BY SEROTONIN ARTHUR PRANCAN *, JEAN LEFORT, MARY BARTON ** and B. BORIS VARGAFTIG ***
Unitb des Venins, D~partement de Physiopathologie Expbrimentale, Institut Pasteur, 28 Rue du Dr. Roux, Paris Cedex 15, France Received 22 September 1981, revised MS received 8 December 1981, accepted 11 January 1982
A. PRANCAN, J. LEFORT, M. BARTON and B.B. VARGAFTIG, Relaxation of the guinea-pig trachea induced by platelet-activating
factor and by serotonin, European J. Pharmacol. 80 (1982) 29-35. Platelet-activating factor (PAF-acether), a known platelet stimulant and bronchoconstrictor (in vivo), is a potential mediator of inflammation and thrombosis. However, all smooth muscle effects of PAF-acether described to date are indirect, relying upon intravascular platelet activation. Novel actions of PAF-acether and serotonin (5-HT) are presented here; these actions may lead to the development of a practical bioassay for PAF-acether and contribute to the understanding of the mechanism of action for both substances. PAF-acether, when added to a spiral cut guinea-pig trachea suspended in a tissue bath containing Krebs-Henseleit buffer, produced a dose-dependent loss of active tissue tension. The EDs0 for this effect of PAF-acether was 75 ng/ml. PAF-acether produced a maximal relaxation which was 68% of that produced by PGE I and the effect could not be modified by aspirin or propranoiol pretreatment. 5-HT, alone, contracted the guinea-pig trachea strip in a dose-dependent manner, but caused relaxation instead when methysergide was present. Aspirin, phenoxybenzamine and propranoloi did not alter this loss of active tissue tension. A similar observation was made in vivo using the guinea-pig bronchoconstriction model, in which PAF-acether as well as 5-HT given to methysergide-treated animals caused a decrease in intratracheal pressure. This action of PAF-acether may yield a suitable bioassay method which could facilitate routine measurements of the substance. Furthermore, the similarity in action of PAF-acether and of 5-HT on methysergide-treated animals leads one to speculate about the relationship between the two substances and their mechanism of action in smooth muscle. Guinea-pigs
Platelet-activating factor
Platelets
Methysergide
Serotonin
Trachea
1. Introduction
Platelet-activating factor (PAF-acether), l-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, stimulates platelet aggregation, which is independent of ADP and of the cyclooxygenase pathway (Cazenave et al., 1979, Chignard et al., 1979, 1980). It is also a potential mediator of immune (Benveniste et al., 1972) and non-immune (Vargaftig et
* On temporary leave from Department of Pharmacology, Rush University, 1753 West Congress Parkway, Chicago, Illinois 60612, U.S.A. ** Present address: Department of Family and Community Medicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033, U.S.A. *** To whom all correspondence should be addressed at Unit~ des Venins, the above address. 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
al., 1981a,b) inflammations. The identification of PAF-acether currently involves chemical, chromatographic and radiochemical tests, or the use of a bioassay on platelet activation. The bioassay requires fresh platelets when routine tests for the release of PAF-acether are made, and therefore, is often impractical. Until this time, PAF-acether has not been reported to influence any of the isolated tissues which are used to identify biologically active substances. In fact, most of the effects of PAF-acether are platelet-dependent and accordingly, it fails to affect isolated tissues in the absence of platelets. For this reason, PAF-acether, which is a potent bronchoconstrictor in vivo, will not contract the isolated guinea pig parenchymal lung strip (Vargaftig et al., 1981 a,b). In the search for an appropriate isolated tissue to be used for
30 routine bioassay of PAF-acether we noted, and report here, unexpected effects of this substance and of serotonin (5-HT) both in vivo and in vitro.
2. Materials and methods
2.1. Dru M
PAF-acether was obtained from Dr. J. Benveniste and Prof. J.J. Godfroid. It was stored in 95% ethanol at - 2 0 ° C and diluted in normal saline to obtain working concentrations. The final concentration of ethanol in the tissue bath was 0.0019% or less. PGE l, a gift from the Upjohn Company, was initially dissolved in ethanol, further dilutions being made with 20% Na2CO 3. Aspirin, as a soluble lysine salt (Aspegic®), was obtained from Laboratories Egic, Paris. Other drugs used in this study included: Acetylcholine chloride, arachidonic acid, phenoxybenzamine, histamine hydrochloride and serotonin creatine phosphate (Sigma); propranolol hydrochloride (Avlocardyl ®, ICI); mepyramine dihydrochloride (Rhone-Poulenc); and methysergide hydrogen bimaleate (Sandoz). 2.2. Isolated guinea pig. trachea preparation Tracheas were taken from Sprague-Dawley guinea pigs of either sex (370-450 g) which had been anesthetized with sodium pentobarbital (30 m g / k g i.p.). Each trachea was cut spirally and suspended under 2 g tension in a tissue bath containing oxygenated Krebs-Henseleit buffer (37°C), which was prepared without any receptor or enzyme antagonists. At least 2 h were allowed for tissue stabilization before PAF-acether, 5-HT and other substances were added directly to the bath in volumes of 20 #1 or less (16 ml bath volume). Changes in tension were recorded isometrically with a Statham UC3 transducer. Between tests, each trachea was washed three times with buffer and the next addition of a substance to the tissue took place only after the tension of the trachea had reached and maintained a stable plateau.
2.3. Testing the effects of PA F-acether and serotonin on the trachea PAF-acether was administered in single doses to the tissue bath so as to establish final concentrations of 5.1, 17, 51, 170 and 510 ng/ml. 5-HT was added alone in single dose concentrations of 4-400 n g / m l and also in the presence of 47 n g / m l of methysergide. Acetylcholine (100 n g / m l ) was used to test for the responsiveness of the trachea to a stimulus, and PGE I 0.1-1 # g / m l ) was used for evaluating the maximal loss of active tension by the tissue. Aspirin (1-20 /~g/ml) was added to the bath 10 min before PAF-acether or serotonin in order to test the involvement of prostaglandin synthesis in the response. Phenoxybenzamine (1.5 /~M) and propranolol (1 # g / m l ) pretreatment tested the involvement of adrenergic mediators and receptors in the response. 2.4. In vivo studies: bronchial resistance to inflation, intratracheal pressure Guinea-pigs ( n - - 6 ) were anesthetized with sodium pentobarbital (30 m g / k g i.p.) and artificially ventilated with a Palmer pump adjusted to deliver 1 ml air/100 g body weight at 72 strokes/rain. Propranolol (3 m g / k g i.p. plus. 1 m g / k g i.v.) was used to prevent interference by released bronchodilator catecholamines, and pancuronium ( 4 m g / k g i.v.) ensured muscle relaxation. One carotid artery was cannulated for the measurement of arterial blood pressure. The right jugular vein was cannulated as well and was used for all injections (Vargaftig et al., 1979). Bronchoconstriction was induced by histamine (1-3 /~g/kg) serotonin (0.5-2 ~ g / k g ) acetylcholine (10-20 ~ g / k g ) or PAF-acether (12-60 ng/kg) and recorded on a Beckman Dynograph by a slight modification of the Konzett-Rossler method (Lefort and Vargaftig, 1978). Injections were usually separated by an interval of 10 rain except for PAF-acether injections when intervals of 30-60 min were used. In addition to the above-mentioned parameters, intratracheal pressure was measured from an open tip catheter introduced into the trachea. For this purpose, the trachea was dissected towards the head after the animal had been
31
prepared for recording bronchoconstriction, an open-tip catheter was introduced caudally into the trachea and its caudal end firmly ligated (James, 1969).
220-
T
200-
-T
180-
2.5. Data
160-
The data are given as mean ± S.E.M. The data were tested in a semi-log dose-response plot for a straight line fit using linear regression. Statistical significance was evaluated using Student's t-test.
140-
1 T
!
T
120100-
±1
80-
3. Results 60-
3.1. The effect of PAF-acether on active trachea tension in vitro
I
40-
I
20-
The introduction of PAF-acether into the tissue bath containing a spiral-cut guinea pig trachea resulted in a dose-dependent loss of active tension, as shown in fig. 1. The entire dose-response series of tests using PAF-acether is summarized i n fig. 2. A semi-log dose-response plot of the dose-response data yielded a line with a correlation coefficient of 0.998. The EDs0 for PAF-acether on loss of active tension in the trachea was calculated as 75 n g / m l final concentration in the tissue bath. The isolated trachea was consistently responsive to PAF-acether. In every instance where the tissue contracted in response to acetylcholine or serotonin, or where it relaxed following P G E I, it was also responsive to PAF-acether. In 30% of the
2 .i, I
17o
1.80"
Fig. I. The effect of PAF-acether on an isolated guinea-pig trachea. Total tissue tension was 2 g. Doses of PAF-acether were applied individually and separated by a tissue wash. The tracings are superimposed to show the relationship between dose and effect. Scale: relaxation (g).
na12Jn-13 in-1 5.1
17
51
170
PAF-acether ( n g / m l )
Fig. 2. Dose-response: PAF-acether on isolated guinea-pig trachea tension. Total tissue tension was 2 g. All doses of PAF-acether were administered individually and separated by a tissue wash. All values are given as the mean loss of tissue tension - S.E.M. The n values represent the number of tracheas from different animals which were tested. Scale: relaxation (rag).
tracheas tested, however, there was evidence of a temporary refractoriness of the tissue to succeeding doses of PAF-acether. In these cases, the second response in a series was depressed to 60% of the original. The average maximal response of isolated trachea to PAF-acether was a 318.95 +- 120.72 mg loss of tension (n = 4), which was achieved with 510 n g / m l and higher concentrations. This response was 68% of the maximal effect observed when P G E I (200 n g / m l ) was added to the preparation (471.21 ± 90.28 mg, n = 9).
3.2. The effect of serotonin and methysergide on active trachea tension in vitro As seen in fig. 3, serotonin induced a concentration-dependent contraction of the isolated trachea
32
~r Control
~ " 5min / +500-
+400 100 nM methysergide +300-
t
5HT41JM)0.03
t
0.1
t
0.3
t
I
+200-
Fig. 3. Methysergide-inducedinversion of the contracting effect of serotonin on the guinea pig trachea. Total tissue tension was 2 g. Doses of 5-HT were applied individually and separated by a tissue wash. Methysergide was included in the buffer during the tests shown in the lower tracings. Scale of the upper panel: contraction and scale of the lower panel: relaxation.
strip. In the presence of methysergide, similar conc e n t r a t i o n s of 5 - H T produced d o s e - d e p e n d e n t relaxation. As s u m m a r i z e d in fig. 4, the dose-response relationship of serotonin to tracheal c o n t r a c t i o n was inverted to yield a progressive loss of tension w h e n methysergide was present.
3.3. Aspirin, propranolol or phenoxybenzamine pretreatment in vitro A s p i r i n p r e t r e a t m e n t ( 1 - 2 0 /~g/ml, 10 min) neither e l i m i n a t e d n o r d i m i n i s h e d the loss of trachea tension due to P A F - a c e t h e r or to serotonin in the presence of methysergide. W h e n using high doses of aspirin (10 / ~ g / m l or 20 t t g / m l ) , the aspirin alone caused a slow c o n t r a c t i o n of the p r e p a r a t i o n , which c o m p e t e d with the loss of tension caused by the test substances; this did not h a p p e n after the tissue reached a stable baseline tension. P r o p r a n o l o l (1 # g / m l ) did not interfere with the effect of either substance, a n d p h e n o x y b e n z a m i n e (1.5/~M) did not alter the response.
t -
100
:::::::::::::: n=7
:::.':~i4i":i:
METHYSERGIDE
(tO0 n M )
-r
_._5
0.03 0.1 0.3 ! DOSE OF SEROTONIN (jJM) Fig. 4. Dose-response: 5-HT plus methysergide relaxation of the guinea pig trachea. Total tissue tension was 2 g. All values are given as the mean change in tissue tension -~S.E.M. The n values represent the number of tracheas from different animals which were tested. A one-tailed unpaired Student's t-test was used to compare the effect of the same dose of 5-HT with and without methysergide. * P<:0.05. Scale: mg of tissue contraction (positive figures) or of tissue relaxation (negative figures).
3.4. In vivo studies 5 - H T (0.5-2 # g / k g ) , h i s t a m i n e ( 1 - 3 # g / k g ) a n d acetylcholine ( 1 0 - 2 0 /~g/kg) i n d u c e d the ex-
33
MEPY METHY
10 rain
20[ ,o
Agonists (l~kg ~)
5HT 0.5
H 1
Ach 10
5HT 1
5HT 1
H 1
Ach 10
Fig. 5. Interference of mepyramine (mepy) and of methysergide (methy) with the effects of 5-HT, histamine (H) and acetylcholine (AcH) in the guinea pig. The first panel shows the effects of 5-HT and ACH on intratracheal pressure, arterial blood pressure and bronchial resistance to inflation (tracings from top to bottom). The second and third panels show these responses after mepy and methy (0.2 mg/kg). All injections were i.v. pected b r o n c h o c o n s t r i c t i o n in the guinea pig (fig. 5). The effects o n tracheal pressure were less predictable since the a c c o m p a n y i n g variations of arterial b l o o d pressure interfered with the mea-
,
10 rain
,
surement. 5 - H T a n d h i s t a m i n e increased the intratracheal pressure with a secondary, small relaxing effect whereas acetylcholine had a relaxing effect, a c c o m p a n i e d b y s u d d e n a n d short-lasting
METHY
.,0[
0
Agonist
H 5HT PAF 5HT PAF 2 2 66 2 66 Fig. 6. Failure of methysergide to interfere with the effects of PAF-acether in the guinea-pig. The left-side panel shows the effects of histamine (H), PAF-acether (PAF) and 5-HT given at the doses indicated (/~g/kg for H and 5-HT; ng/kg for PAF-acether). After methysergide (methy, 0.2 mg/kg), as shown in the left-side panel, the effects of 5-HT on the intratracheal pressure were reversed, whereas its effects on bronchoconstriction, and those of PAF-acether on all parameters, were unaffected. Tracings from top to bottom: intratracheal pressure, arterial blood pressure and bronchial resistance to inflation. All injections were i.v.
34 hypotension. After mepyramine and methysergide (0.2 m g / k g of each) the effects of histamine were suppressed, those o f acetylcholine were unaffected whereas serotonin induced a reduction of the intratracheal pressure, unaccompanied by variations in arterial pressure (fig. 5). The administration of PAF-acether (12.5-132 ng/kg) was followed by the earlier described bronchoconstriction, accompanied by a short-lived contraction of the trachea which was followed by a long-lasting decrease of intratracheal pressure (fig. 6). This pattern was unchanged by methysergide (fig. 6) or by mepyramine (not shown).
4. Discussion
PAF-acether is the most active guinea pig platelet aggregating agent so far described (Vargaftig et al., 1980, 1981a,b). Its in vivo effects include bronchoconstriction in the guinea pig, and this effect was shown to be platelet-dependent and aspirin-resistant (Vargaftig et al., 1980). Since PAF-acether is released by macrophages (MenciaHuerta and Benveniste, 1979), which may participate in allergic reactions (Capron et al., 1975, 1977) including bronchoconstriction, it was suggested that PAF-acether might play a part in asthma (Vargaftig et al., 1981a,b). This is particularly relevant to the guinea-pig model for allergic bronchoconstriction which-is much used in the search for anti-asthma drugs. It is important to note that since the bronchial effects of Slow-Reacting Substance of Anaphylaxis (leukotrienes C4 and D4) are suppressed by aspirin (Berry and Collier, 1964; Vargaftig et al., 1981c), the effects of PAF-acether cannot be attributed to the release of these mediators. There is a need for an isolated tissue to be used for bioassaying PAF-acether, particularly for online studies of its release. Our present results indicate that the guinea pig trachea could be used as such a tissue, since it was concentration-dependently relaxed by PAF-acether. This effect was not due to the intramural formation of prostaglandins, because it was not inhibited by aspirin, not mediated by fl-adrenergic activation, since the effect was also not reduced by propranolol. Our data with
serotonin-induced relaxation of the tracheal strip, an effect uncovered when the usual contraction was inhibited by methysergide, are not sufficient evidence that PAF-acether interacts with a 'relaxing' receptor for 5-HT. Under different conditions, there was found a methysergide-induced unmasking of a 5-HT vasodilator effect on the nasal vessels (Vargaftig and Lefort, 1974). Since we observed (unpublished experiments) that PAFacether affects the tone of these vessels only in presence of circulating platelets, PAF-acether does not share a mechanism of action with 5-HT in this particular preparation. Alternatively, 5-HT might trigger the release of PAF-acether from the trachea, in which case one may speculate to what extent the release of 5-HT from platelets, such as occurs during migraine or after administration of reserpine, may lead to the formation of PAF-acether. Finally, a 'serotonin-releasing factor' was described in the plasma of migraine patients (Anthony and Lance, 1975), which might be related to PAF-acether. It thus appears speculative, but deserves further study, to suggest that the formation a n d / o r effects of PAF-acether and of 5-HT are related, and may account for at least part of the pathophysiology involving platelets, 5-HT and smooth muscles. Aspirin, mepyramine and methysergide form a synergistic combination which inhibits the bronchoconstriction (in vivo) and ex vivo platelet release reaction due to PAF-acether while individually the drugs in this group have no effect (Vargaftig et al., 1981a). It thus appears that PAF-acether, as a potential m e d i a t o r of anaphylaxis a n d / o r inflammation, interacts with established mediators, particularly the amines, for its overall in vivo effect. Use of the guinea pig trachea under conditions where the effects of other mediators are excluded may help to determine more precisely the relevance of the formation of PAF-acether under various conditions. In conclusion, PAF-acether and serotonin (after methysergide) have a similar direct action on the smooth muscle of the guinea pig trachea. Both of these substances have now been shown to decrease tension in the trachea in a dose-dependent fashion in vitro and in vivo. This action is independent of platelets, prostaglandin synthesis and adrenergic
35 r e c e p t o r a c t i v a t i o n . A p r a c t i c a l o u t c o m e o f this work may be the development of a suitable bioassay for P A F - a c e t h e r . W e b e l i e v e t h a t t h e s i m i l a r i t y in effects o f P A F - a c e t h e r a n d s e r o t o n i n ( a f t e r m e t h y s e r g i d e ) o n the t r a c h e a will p r o v i d e the b a c k g r o u n d for f u r t h e r studies o n the p r e c i s e mechanism of action of each substance.
Acknowledgements PAF-acether was kindly supplied by Drs. J. Benveniste and J.J. Godfroid. This study was supported by a grant from INSERM (PRC 121037). The authors wish to express their appreciation to Ms. De Borah McCaskill and Mrs. Marie-Christine Ferrand for assistance in preparing the manuscript.
References Anthony, M. and J.W. Lance, 1975, The role of serotonin in migraine, in: Modern Topics in Migraine, ed. J. Pearce (William Heinemann Medical Books, London) p. 107. Benveniste, J., P.M. Henson and C.G. Cochrane, 1972, Leukocyte-dependent histamine release from rabbit platelets: the role of lgE basophils, and a platelet-activating factor, J. Exp. Med. 136, 1356. 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, Br. J. Pharmacol. 23, 201. Capron, A., J.P. Dessaint, M. Capron and H. Bazin, 1975, Specific IgE antibodies in immune adherence of normal macrophages to Schistosoma mansoni schistosomules, Nature 253, 474. Capron, A., J.P. Dessaint, M. Joseph, R. Rousseaux, M. Capron and H. Bazin, 1977, Interaction between IgE complexes and macrophages in the rat: A new mechanism of macrophage activation, European J. lmmunol. 7, 315. Cazenave, J.P., J. Benveniste and J.F. Mustard, 1979, Aggrega-
tion of rabbit platelets by platelet-activating factor is independent of the release reaction and the arachidonate pathway and inhibited by membrane-active drugs, Lab. Invest. 3, 275. Chignard, M., J.P. Le Couedic, M. Tence, B.B. Vargaftig and J. Benveniste, 1979, The role of platelet-activating factor in platelet aggregation, Nature 279, 799. Chignard, M., J.P. Le Couedic, B.B. Vargaftig and J. Benveniste, 1980, Platelet-activating factor (PAF-acether) secretion from platelets. Effect of various aggregating agents, Br. J. Haematol. 46, 455. James, L.G.W., 1969, The use of the in vivo trachea preparation of the guinea-pig to assess drug action on lung, J. Pharm. Pharmacol. 21,379. Lefort, J. and B.B. Vargaftig, 1978, Role of platelets in aspirinsensitive bronchoconstriction in the guinea-pig; interactions with salicylic acid, Br. J. Pharmacol. 63, 35. Mencia-Huerta, J.M. and J. Benveniste, 1979, Platelet-activating factor and macrophages, I. Evidence for the release from rat and mouse peritoneal macrophages and not from mastocytes, European J. Immunol. 9, 409. Vargaftig, B.B., M. Chignard, J. Benveniste, J. Lefort and F. Wal, 198 la, Background and present status of research on platelet-activating factor (PAF-acether), Ann. N.Y. Acad. Sci. 370, 119. Vargaftig, B.B., M. Chignard, J.M. Mencia-Huerta, B. Arnoux and J. Benveniste, 1981b, Pharmacology of arachidonate metabolites and of platelet-activating factor (PAF-acether), in: Platelets in Biology and Pathology, Vol. II, ed. J.L. Gordon (North-Holland, Amsterdam) p. 373. Vargaftig, B.B. and J. Lefort, 1974, Pharmacological evidence for a vasodilator receptor to serotonin in the nasal vessels of the dog, European J. Pharmacol. 25, 216. Vargaftig, B.B., J. Lefort, M. Chignard and J. Benveniste, 1980, Platelet-activating factor induces a platelet dependent bronchoconstriction unrelated to the formation of prostaglandin derivatives, European J. Pharmacol. 65, 185. Vargaftig, B.B., J. Lefort, D. Joseph and F. Fouque, 1979, Mechanism of bronchoconstriction and of thrombocytopenia induced by collagen in the guinea-pig, European J. Pharmacol. 58, 273. Vargaftig, B.B., J. Lefort and R.C. Murphy, 1981c, Inhibition by aspirin of bronchoconstriction due to leukotrienes C4 and D4 in the guinea-pig, European J. Pharmacol. 72, 417.