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Examination of the bronchoconstrictor response of guinea-pig isolated lung to β-phenylethylamine
Gen. Pharmac. Vol. 16, No. 4, pp. 371-378, 1985 Printed in Great Britain. All rights reserved
0306-3623/85 $3.00 +0.00 Copyright © 1985 Pergamon Press Ltd
EXAMINATION OF THE BRONCHOCONSTRICTOR RESPONSE OF GUINEA-PIG ISOLATED LUNG TO fl-PHENYLETHYLAMINE M. H. HAWTHORN, K. J. BROADLEY and C. J. GIBBON Department of Pharmacology, Welsh School of Pharmacy, University of Wales Institute of Science and Technology, P.O. Box 13, Cardiff CF1 3XF, U.K. (Tel: (0222) 42588) (Received 24 October 1984) fl-Phenylethylamine (PEA) produced a contraction of the guinea-pig isolated lung parenchymal strip and bronchoconstriction of perfused lungs. 2. Reserpine pretreatment had little effect on these responses indicating a substantial direct effect. 3. Phentolamine (10 -6 and 10-5 M) had minimal effect on PEA in the lung strip compared with that on noradrenaline, eliminating involvement of ~t-adrenoceptors. 4. PEA was unaffected by atropine (10 7 M) or a mepyramine (10 7M); metiamide (10 -4 M) mixture. The contraction was not therefore mediated via muscarinic or histaminergic receptors. 5-HT and dopamine receptors were also discounted. 5. Possible stimulation of a phenylethylaminergic receptor and its relevance to bronchial asthma is discussed. Abstraet--l.
INTRODUCTION
The indirectly acting sympathomimetic amine flphenylethylamine (PEA) is believed to produce its effects by promoting the release of noradrenaline from sympathetic neurones (Burn and Rand, 1958). In guinea-pig lung strips, however, a biphasic response curve is produced (Broadley et al., 1981), with the expected small fl-adrenoceptor-mediated relaxation occurring at low concentrations, followed by contraction. A similar contractile response o f rat and guinea-pig lung strips to another indirectly acting sympathomimetic amine, tyramine, has also been reported recently (Morcillo et al., 1984). This latter response does not, however, appear to be as pronounced as that for P E A (Broadley and Hawthorn, 1981; H a w t h o r n and Broadley, 1984), which suggests that the response to P E A may be mediated via a mechanism other than noradrenaline release. In support of this, there is a growing body of evidence that non-catecholic phenylethylamines may exert a direct action on a number of tissues, probably by interacting with adrenergic receptors (weak agonist action) (Antelman et al., 1977) or with more specific receptors of their own (Ungar et al., 1978). Recently, Hansen et al. (1980) have demonstrated a direct contractile effect of PEA on rat aortic strip which is mediated via ~-adrenoceptor stimulation. As vascular tissue may contribute to the responses of guinea-pig lung strip (Clayton et al., 1981), this work was carried out to initially determine if the site of action of P E A was vascular or parenchymal in origin and to examine the possible receptor type mediating the contractile response.
prepared as described by Lulich et al. (1976). Sections of lung tissue about 3 mm in width were dissected away from the peripheral margin of a lobe, with the axis of the strip cut parallel to the bronchus, but not including it. The strips were suspended in Krebs-bicarbonate solution (composition in mM: NaCI 118.4, KC14.7, CaC12.2H20 1.9, NaHCO~ 25, MgSO4"7H20 1.2, glucose 11.7, KH2PO 4 1.2), gassed with 5% CO2 in oxygen at 38°C. The tissues were incubated throughout with metanephrine (10-SM) to inhibit extraneuronal uptake. Responses were recorded isometrically using a Devices UF1 transducer (57 g sensitivity range) on a Devices (Lectromed) M19 polygraph. The lung strips were placed under a resting tension of 1.0-1.2 g and allowed to equilibrate for 40min with the bathing medium being changed at 10 min intervals before drug challenge.
MATERIALS AND METHODS
Lung strip preparations Guinea-pigs of either sex (300-500 g) were killed by a blow to the head and exsanguinated. Lung strips were 371 G.P. 1 6 / ~ E
Perfused lung preparations Guinea-pigs were killed as above and the thorax opened. The lungs were removed with approximately 2.5 cm of the trachea still attached. A plastic cannula was inserted into the cut end of the trachea and attached to the perfusion apparatus. The surfaces of the lung were scarified lightly with a hypodermic needle to facilitate loss of the perfusate from the alveolar spaces. The lungs were perfused via the airspaces with Krebsbicarbonate solution at a constant flow rate of 10 ml rainby use of a Watson-Marlow flow inducer (Type MHRE/ 30/T, 0.8 mm tube bore). The perfusion solution was aerated with 5% CO2 in oxygen before being passed into the flow inducer, warming coil (38°C) and tracheal cannula. The preparations were perfused throughout with propranolol (10-6 M) to inhibit fl-adrenoceptors and with metanephrine (10 -5 M), and left to stabilize for 10-20 min before drugs were added. Alterations in perfusion pressure (resting levels 5-25 mmHg; l mmHg~, 133Pa) arising from changes in pulmonary airway resistance were recorded on a Devices (Lectromed) M 19 polygraph by means of a pressure transducer (Bell & Howell, type 4-327-L221), situated at a side arm of the tracheal cannula. A Condon manometer was also included in the system in series with the transducer.
M. H. HAWTHORN et al.
372 05 -- Lung strips
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Contraction
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40 Norodrenaline
/~-Phenylethytomine (M) Fig
,8-Phenylethylomine
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1 M e a n (n = 4) concentration-response curves for
fl-phenylethylamine on the guinea-pig lung strip obtained before ((3) and in the presence (O) of propranolol (10 6 M).
o
I 10-10 10.9
10.8
10.7
10.6
I
I
I
10. 5
10-4
10.3
Agonist (tool)
Drug administration With the lung strip preparation, concentration-response curves were constructed by cumulative addition of the agonists to the bath. In the case of the perfused lung, drugs were injected sequentially as a bolus into the perfusion solution via the connecting rubber tubing immediately prior to entry into the tracheal cannula. Dose volumes were kept between 0.05 and 0.15ml. Maximum responses were assumed to have been achieved when an increase in concentration brought about no further increase in response, In lung strips, with this maximum concentration in the bath, carbachol (0.15 x 10-4 M) or histamine (5 x 10 5 M) were added to obtain the maximum contractile response of the tissue. Lung strips were then washed and when the resting tension had returned to control levels, the antagonist under study was added and left in contact with the tissue for 30 min before another cumulative concentration-response curve was constructed. Changes in sensitivity arising from repeating concentration-response curves were corrected for by performing control experiments which were identical except no antagonist was included. Changes in tension (lung strip) or perfusion pressure (lung) were measured at each agonist concentration. Concentration-response curves were plotted as a percentage of their own maximum from which the ECs0 value was calculated, or as a percentage of the carbachol or histamine maximum response. Geometric mean EC50 values and mean maximum responses were compared by Student's t-test. In some experiments tissues were taken from guinea-pigs that had received 0.5 mgkg ~ reserpine intraperitoneally 24 hr prior to killing.
Drugs used Atropine sulphate, carbacho[ (carbamylcholine) chloride, dopamine hydrochloride (Sigma), histamine acid phosphate
Fig. 2. Mean (n >/4) concentration-response curves for the increase in tension of lung strips (A) and increase in perfusion pressure of perfused lungs (B) in response to noradrenaline ((~) and ~-phenylethylamine (O). Responses are expressed as a percentage of a maximum response to carbachol. Propranolol (10 6M) and metanephrine (10 -5 M) were present throughout. (BDH), 5-hydroxytryptamine (creatinine sulphate complex) (Sigma), mepyramine maleate (May & Baker), ( + )-metanephrine hydrochloride (Sigma), metiamide (S K & F), (-)-noradrenatine bitartrate (Sigma), ~-phenylethylamine hydrochloride (Sigma), phentolamine mesylate (CibaGeigy), (+__)-propranolol hydrochloride (ICI) and reserpine (BDH). RESULTS
Effects of [3-phenylethylamine and noradrenaline on lung strips fl-Phenylethylamine ( P E A ) caused a n initial relaxa t i o n o f the lung strips followed at higher concentrations by c o n t r a c t i o n (Fig. 1). W h e n repeated in the presence of p r o p r a n o l o l ( 1 0 - 6 M ) , P E A caused only a contractile response a n d the c o n c e n t r a t i o n response curve for this response was displaced to the left (Fig. 1). F r o m this p o i n t on, all experiments were p e r f o r m e d t h r o u g h o u t in the presence o f p r o p r a n o l o l (10 -6 M). T h e m a x i m u m contractile response to P E A was 79.2 ___8.5% of the c a r b a c h o l - i n d u c e d m a x i m u m , a n d the m e a n (n = 4) ECs0 value was 2.38 x 10 4 M (Fig. 2A, T a b l e 1). P r e t r e a t m e n t with reserpine ( 0 . 5 m g k g ~ at 2 4 h r ) did n o t significantly affect
Table 1. Effects of reserpine pretreatment on responses of lung strip and perfused lung to fl-phenylethylamine Perfused lung Lung strip Maximum ECs0 Maximum ECs0 n (% carbachol max.) ( X 1(~ - 4 M ) n (% carbachol max) ( x 10-4 M) 4 63.1 _+ 10.2~% 4.53 5 Untreated 79.2 _+8.5% 2.38 (3.t5 6.49) (1.88-3.45) 4 60.8 +_ 11.4% 6.24 4 Reserpine pretreated 68.5 + 6.5% 11.34" (2.35 8.63) (9.42-13.17) Geometric mean ECs0 values with 95% confidence limits and mean maximum responses (+SEM) are shown. *Indicates a significant difference (P < 0.05) from the untreated value. Reserpine (0.5 mg kg -~) was administered at 24 hr.
373
Bronchoconstriction to fl-phenylethylamine
Table 2. Effect of noradrenaline on lung strip and perfused lung Maximum ECs0 (% carbachol max.) (x 10-TM) n Lung strip 42.6 + 6.3% 2.66 4 (0.81-9.17) Perfused lung 15.3 _+5.3% 1.67 4 (0.77 7.51) Geometric mean ECsovalues with 95% confidence limits and mean maximum responses (+ SEM) are shown.
(A)
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I
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(B)
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Effect of phentolamine on fl-phenylethylamine noradrenaline
80--
~
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in the perfused lung was significantly less (P < 0.05) than in the lung strip. Reserpine pretreatment had no significant effect (P >0.05) on the P E A maximum response ( 6 0 . 8 _ 11.4%) or on the mean (n = 4) ECs0 value (6.24 x 10 -4 M) (Fig. 3B, Table 1).
2c
o
I 10 . 7
10- 6
10-5
/3-Phenylethylamine
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(tool)
Concentration-response curves for P E A or noradrenaline were obtained in lung strips before and in the presence of phentolamine (10 _6 or 10 -5 M). There was a small shift of the P E A curves to the right (Fig. 4A), the mean (n = 8) ECs0 value being increased from 3.42 x 1 0 - 4 M to 4.61 x 10 4M by 10-6M phentolamine and from 1.61 × 1 0 - a M ( n - - 4 ) to 8.18 x 1 0 - 4 M by 1 0 - S M phentolamine. The corresponding concentration-ratios were 1.61 _ 0.25 and 5 . 3 4 _ 0.87 respectively (Table 4). Although significant (P < 0.05), these shifts were not as great as those induced by phentolamine on the concentration-
Fig. 3. Mean (n /> 4) concentration-response curves for the increase in tension of lung strips (A) and increase in perfusion pressure of perfused lungs (B) in response to fl-phenylethylamine. Tissues were taken from untreated guinea-pigs ((3) and from animals receiving reserpine (0.5 mg kg ~ i.p. at 24 hr) (0). Responses are expressed as a percentage of a maximum response to carbachol. Propranolol (10 -6 M) and metanephrine (10 5 M) were present throughout.
(P > 0.05) the m a x i m u m contraction (68.5 _+ 6.5%, n =4), although the mean ECs0 value of 11.34 × 10 -4 M was significantly greater (P < 0.05) than control values (Fig. 3A, Table 1). As with PEA, noradrenaline produced a contraction of the lung strip, the maximum response being 42.6 _ 6.3% (n = 4) of the carbachol maximum (Fig. 2A). This was significantly less (P < 0.05) than the maximum response produced by PEA. Noradrenaline was also significantly more potent (P < 0.05) than PEA, having a mean ECs0 value (n = 4) of 2.66 x 10 -7 M (Table 2).
Effects of fl-phenylethylamine and noradrenaline on the perfused lung Both PEA and noradrenaline produced bronchoconstriction of the perfused lung, as measured by an increase in perfusion pressure (Fig. 2B). The mean (n = 5) ECs0 values for noradrenaline and P E A were 1.67 x 1 0 - 7 M and 4.53 x 1 0 - 4 M respectively and the maxima were 15.3 _ 5.3% and 63.1 _ 10.2% of the carbachol maxima respectively (Fig. 2B, Tables 1 and 2). There was no significant difference (P > 0.05) between the PEA maxima in the perfused lung and lung strip, however, the m a x i m u m for noradrenaline
and
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Fig. 4. Mean (n >/4) concentration-response curves for the increase in tension of lung strips in response to (A) fl-phenylethylamine and (B) noradrenaline, obtained before (O) and in the presence of phentolamine, 10 -6 M ([]) and 10 -5 M (A). Responses are expressed as a percentage of their own maximum. Propranolol (10-6M) and metanephrine (10 -5 M) were present throughout.
M.H. HAWTHORNet al.
374
Table 3. Effect of phentolamine on responses to noradrenaline of lung strip Pre-antagonist
Post-antagonist
Phentolamine
Maximum ( ~ carbachol)
ECs0 ( × 10 6 M)
Maximum ( ~ carbachol)
EC~0 ( × 10 -6 M)
10-6M
49.9_+1.2
45.6_+2.6
10 -~ M
43.2 _+ 2.8
0.36 (0.07 1.57) 0.75 (0.11 2.81)
40.55* (20.6-93.2) 250* (140.7-480.1)
46.9 _+ 3.2
CR 112.5_+12.7
4
333.3 _+ 25.6
4
Geometric mean ECs0 values with 9 5 ~ confidence limits, mean maximum responses ( + SEM) and Concentration-ratios (CR) are shown. Pre-antagonist values are corrected from control experiments. *Indicates a significant difference (P < 0.05) from the pre-antagonist value.
response curves to noradrenaline (Fig. 4B). The mean (n = 4) ECs0 value for noradrenaline was increased from 0.36 × 10-6M to 40.55 × 10 6M by 10-6M phentolamine giving a mean concentration-ratio of 112.5_ 12.7 (Table 3) and from 0.75 × 10 -6 M to 250 × 10 - 6 M by 10 5 M phentolamine giving a mean (n = 4) concentration-ratio of 333.3 _ 25.6 (Table 3). These concentration-ratios were significantly greater (P < 0.05) than those obtained for the shifts of the PEA concentration-response curves at the corresponding concentrations of phentolamine.
Effects o f atropine on fl-phenylethylamine and carbachol Concentration-response curves for the contraction of lung strips by PEA were obtained before and in the presence of atropine ( 1 0 - 7 M ) and plotted as a percentage of the maximum response to histamine. The maximum responses obtained before (38.5 ___3.1~o) and in the presence of atropine (43.8 + 4.1~, n = 5) did not differ significantly (P > 0.05) (Table 4). Atropine was also without significant effect (P > 0.05) when the curves were plotted as a percentage of their own maximum, the mean ECs0 values before and after atropine being 3.41 x 10-4M and 5.75 x 10 -4 M respectively (Fig. 5A, Table 4), which yielded a concentration-ratio of 1.93 ___0.45. As with PEA, atropine (10-7M) had no effect on the maximum response to carbachol (expressed as a percentage of the histamine maximum), the preand post-atropine values being 41.0+1.1 and 45.5 _ 7.0~o respectively (n = 4) (Table 5). However, when expressed as a percentage of its own maximum response, the carbachol concentration-response
curve was shifted to the right (Fig. 5B), the mean ECs0 value increasing from 2.55 x 10-6M to 8.5 x 10-5 M (Table 5), giving a concentration-ratio of 34.3 + 4.5 (n = 4). Atropine had no significant (P > 0.05) effect on the maximum responses to histamine, which were 2.79 + 0.63 and 2.48 _ 0.61 g before and after atropine respectively in the tissues receiving PEA; and 1.90 + 0.21 and 1.42_ 0.13 g respectively in the tissues receiving carbachol.
Effects o f mepyramine and metiamide on fl-phenylethylamine and histamine A mixture of mepyramine ( 1 0 - 7 M ) and metiamide (10-aM) had no significant effect (P > 0.05) on the maximum responses to PEA, which were 43.5 + 4.3~ and 52.5 _ 6.5~o (n = 4) respectively of the carbachol maximum before and after the antagonists (Table 4). Mepyramine and metiamide were also without effect when the curves were expressed as a percentage of their own maxima, with no significant difference (P > 0.05) occurring between the mean ECs0 values of 6.71 x 10-4M before and 5.35 × 10-4M (n = 4 ) after the antagonists (Fig. 6A, Table 4). The concentration-ratio was 0.97 ___0.35. Unlike PEA, histamine concentration-response curves were displaced to the right by the mepyramine and metiamide mixture, with the mean ECs0 values being significantly increased (P < 0.05) from 2.3 x 10-6M to 14.8 x 10 5M (n = 4 ) (Fig. 6B, Table 5). These concentrations of mepyramine and metiamide had no significant effect (P > 0.05) on the maximum responses to histamine (presence, 1.73 + 0.3; absence, 1.49 + 0.53 g) or carbachol (presence, 0.38 + 0.07; absence, 0.40 ___0.04 g).
Table 4. Effects of antagonists on responses of lung strip to fl-phenylethylamine Pre-antagonist Antagonist Phentolamine 10-6M Phentolamine 10 -5 M Atropine 10 -7 M Mepyramine 10 -7 M + Metiamide l0 4M Methysergide 10 -7 M
Post-antagonist
Maximum response
ECs0 ( x 10-4M)
Maximum response
ECs0 ( × 10 4M)
49.9 + 5.4
3.42 (2.21 3.26) 1.61 (0.72-3.41) 3.41 (2.794.18) 6.71 (2.89-15.42)
57.8 __. 3.7
4.61" (1.88-7.31) 8.18" (1.35-5.22) 5.75 (2.36-14.0) 5.35 (2.7~10.87)
54.3 _ 6.5 38.5 _+ 3.1 43.5 + 4.3
61.2 _+ 3.5
2.67 (1.72~1.51)
51.6 _+ 9.3 43.8 _+4.1 52.5 _ 6.5
41.0 + 3.9*
2.66 (2.35-3.02)
CR
n
1.62 + 0.25
8
5.34 _ 0.87
4
1.93 + 0.45
5
0.97 + 0.35
4
1.21 -I-0.07
6
Geometric mean EC50 values with 9 5 ~ confidence limits, mean maximum responses (as a percentage of carbachol or histamine maxima) ( ± S E M ) and concentration-ratios (CR) are shown. Pre-antagonist values are corrected from control experiments. *Indicates a significant difference (P < 0.05) from the pre-antagonist value.
375
Bronchoconstriction to fl-phenylethylamine (A)
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(B)
80
/~-Phenylethylamine (M) 100
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80
60
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6O
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40 20 0
I 10-7
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Corboch0t (M)
Fig. 5. Mean (n t> 4) concentration-response curves for the increase in tension of lung strips in response to (A) fl-phenylethylamine and (B) carbachol, obtained before ( O ) and in the presence ( 0 ) of atropine (10-7 M). Responses are expressed as a percentage of their own maxima. Propranolol (10-6 M) and metanephrine (10- 5 M) were present throughout experiments with fl-phenylethylamine.
Effects of methysergide, 5-HT and dopamine Concentration-response curves to PEA were constructed before and in the presence of methysergide (10 -7 M ) which significantly reduced (P < 0.05) the maximum response from 61.2 _ 3.5~o to 41.0 +__3.9~o (n = 6) when expressed as a percentage of the carbachol maximum (Table 4). The maximum responses to carbachol obtained before (2.06 + 0.4 g) and after methysergide (2.04 _+ 0.29 g) were not significantly different (P > 0.05). When expressed as a percentage of their own maximum responses, the PEA curves were superimposed, with no significant difference (P > 0.05) between the mean ECs0 values (n = 6)
h */~___ 10-6 10-5 Histamine (M)
I ,10-4
L-I0-?
Fig. 6. Mean (n >/4) concentration-response curves for the increase in tension of lung strips in response to (A) fl-phenylethylamine and (B) histamine, obtained before ( O ) and in the presence ( 0 ) of mepyramine ( 1 0 - 7 M ) and metiamide (10 -4 M). Responses are expressed as a percentage of their own maxima. Propranolol (10 -6 M) and metanephrine (10-SM) were present throughout experiments with fl-phenylethylamine.
obtained before (2.67 x 10 - 4 M) and in the presence of methysergide (2.66 x 10 -4 M) (Table 4). It was not possible to examine the effect of methysergide upon 5-hydroxytryptamine, since only a small proportion of tissues responded and these yielded only small contractions of the lung strip. Similarly, dopamine failed to contract the lung strip in concentrations up to 10 4 M when examined in the presence of phentolamine (10 s M) and propranolol (10 -6 M ) . DISCUSSION
fl-Phenylethylamine exerted a dual effect upon the lung strip of the guinea-pig; an initial relaxation at low concentrations being followed by a contraction at higher concentrations. The relaxation was abolished by propranolol and therefore attributed to fl-adrenoceptor stimulation as reported previously (Broadley and Hawthorn, 1981). The predominant
Table 5. Effects of atropine (10 -7 M) on responses to carbachol and mepyramine (10 - 7 M) with metiamide (10 -4 M) on responses to histamine of lung strip Pre-antagonist Agonist Carbachol
Maximum (% histamine) 41.0 -+_1.1
Histamine
--
ECs0 ( x 10_6 M) 2.55 ( 1.87-3.48) 2.3 (0.68-7.5)
Post-antagonist Maximum (% histamine) 45.5 + 7.0 --
ECso ( x 10-s M) 8.5* (5.9-12.0) 14.8" (4.3-50.9)
CR 34.3 --F4.5
n 4
37.9 + 5.1
4
Geometric mean ECso values with 95% confidence limits, mean maximum responses (+_ SEM) and concentration-ratios (CR) are shown. Pre-antagonist values are corrected from control experiments. *Indicates a significant difference (P < 0.05) from the pre-antagonist value.
376
M.H. HAWTHORNet al.
response was therefore a contraction, indicative of bronchoconstriction, and this was further examined in the presence of propranolol throughout. Responses to PEA in other tissues have been shown to arise indirectly from the release of noradrenaline from sympathetic nerve terminals (Burn and Rand, 1958). After pretreatment with reserpine in a dose known to deplete catecholamines (Waud et al., 1958), there was a small shift of the PEA concentrationresponse curve to the right. However, the maximum contractile response was unaffected, indicating that only a small portion of this response could be attributed to an indirect effect. The majority of the response is therefore due to a direct action of PEA, a conclusion also drawn from the perfused lung, where reserpine was without effect upon PEA. A similar reserpine-resistant contractile response to PEA has been reported in rat aorta (Hansen et al., 1980) which appears to be mediated via ct-adrenoceptors since it was blocked by phentolamine. This does not appear to be the case in lung strips since although noradrenaline produced contractions of this tissue and was more potent than PEA, its maximum response was smaller. Further support for the conclusion that PEA is not acting directly via ~t-adrenoceptors in the lung strip comes from the use of phentolamine. Although the concentration-response curves for PEA were displaced to the right by 10 -6 M phentolamine (concentration-ratio 1.62) and 10-SM phentolamine (5.34), the degree of shift was substantially less than the shifts of noradrenaline by the same concentrations (112.5 and 333.3 respectively). This lack of antagonism of PEA by phentolamine is in contrast to the observations of Hansen et al. (1980). However, the antagonism observed may have been due to the excessively high concentration employed (10-4M) since Furchgott (1972) has demonstrated that concentrations of only 10 - 6 M phentolamine are needed to adequately antagonize ct-adrenoceptors. PEA appears to differ from the alternative indirectly acting sympathomimetic amine tyramine which we have shown to produce a weaker contractile response of the lung strip (Broadley and Hawthorn, 1981). This response has been shown to be substantially antagonized by reserpine pretreatment and by phentolamine, although not to the same extent as noradrenaline (Morcillo et al., 1984). The lung strip preparation has been criticised for not being truly representative of airway responses on the grounds that a vascular component may be involved (Clayton et al., 1981). To examine whether the contractile response of the lung strip to PEA is a vascular or airway response, isolated lungs were perfused via the trachea at a constant flow and the back pressure recorded. Any change in pressure could therefore be caused only by effects on airway tissue. As in the lung strip, both PEA and noradrenaline produced bronchoconstriction. However, the response to noradrenaline was much reduced when compared with the lung-strip response, but the effects of PEA were of similar magnitude in both tissues. Furthermore, reserpine pretreatment had no effect on the PEA response of the perfused lung, which indicates that PEA has a direct contractile effect on lung parenchymal tissue. In contrast, the contractile response to noradrenaline would appear to be mainly
vascular in origin, which agrees with the conclusion of Clayton et aL (1981). The small displacement by reserpine of the PEA concentration-response curves in the lung strip can probably be explained by a vascular component mediated via an indirect sympathomimetic effect. Since the contractions of the lung strip by PEA did not appear to be mediated via ~-adrenoceptors, the possibility was next examined that the response was produced via either muscarinic or histaminic receptors; both acetylcholine and histamine are known to contract the lung strip (Drazen and Schneider, 1978). However, atropine failed to affect either the maximum response to PEA or the ECs0 in a concentration that effectively antagonized carbachol. Similarly, the combined use of the H~ and H2 receptor antagonists mepyramine and metiamide respectively, had no effect on the PEA maximum response and ECs0 value, although the same concentrations antagonized histamine. The central effects of PEA have been attributed to a direct stimulation of either 5-hydroxytryptamine (5-HT) (Solviter et al., 1980) or dopamine receptors (Antelman et al., 1977). The guinea-pig lung strip has also been shown to be contracted by both 5-HT (Morcillo et al., 1984) and dopamine (Cortijo et al., 1984). In the present study, the response to 5-HT was inconsistent and weak compared with that to PEA. The 5-HT antagonist, methysergide, however reduced the maximum response to PEA indicative of a noncompetitive antagonism. This is unlikely to be due to antagonism at the 5-HT receptor since methysergide is a competitive antagonist in other tissues with no effect on the maximum responses (Apperley et al., 1980). A non-specific effect on the tissue can also be discounted since it did not reduce the maximum effect of carbachol. The precise cause of this reduced maximum remains to be established, however, PEA does not appear to be exerting its contractile effect via 5-HT receptors since methysergide failed to alter the ECs0 value and 5-HT itself failed to cause a contraction in the presence of ~- and fl-adrenoceptor blockade. Any contractile effects of dopamine observed previously in this preparation are mediated entirely via ct-adrenoceptors (Cortijo et al., 1984). Finally, having discounted the most likely recognized receptor systems, the possibility must be considered that PEA exerts its contractile effect via phenylethylaminergic receptors. There is evidence that such receptors exist in the central nervous system where they are involved in the analgesic (Giardina, 1974), behavioural (Antelman et al., 1977), hallucinogenic (Vogel, 1978) and cerebral vasoconstrictor properties (McCulloch and Harper, 1978) of PEA. Specific binding sites for PEA have also been identified in midbrain synaptosomes of the rat, cow and mouse (Ungar et aL, 1978), and in the periphery, PEA receptors have been suggested in pancreatic cells (Feldman and Lebovitz, 1971). The presence of such a receptor may also be proposed in the lung strip to explain the bronchoconstrictor effect of PEA. The concentration of PEA required to produce the response (10 5 M) is, however, relatively high and it is difficult to envisage a physiological role for such a receptor when the normal circulating levels of PEA are in the order of 10 -8 M (Saavedra, 1978; Manson,
Bronchoconstriction to fl-phenylethylamine 1979). Nevertheless, this study does raise the possibility of a pathophysiological involvement of P E A in bronchospasm associated with asthma. Airway sensitivity to bronchoconstrictors, which may include P E A , is raised in the asthmatic patient (Boushey et al., 1980) such that an elevation of plasma P E A may induce an asthmatic attack by the ingestion of dietary products rich in PEA, such as chocolate, cheese and wine (Chaytor et al., 1975; Koehler and Eitenmiller, 1978). Indeed, a similar hypothesis has been proposed for the precipitation of dietary migraine following consumption of PEA-containing foods (Sandler et al., 1974). SUMMARY
fl-Phenylethylamine (PEA) produced a biphasic effect on the guinea-pig isolated lung parenchymal strip. A small relaxation was followed at higher concentrations by a more pronounced contraction, indicative of brochoconstriction. The relaxation was fl-adrenoceptor-mediated since it was abolished by propranolol, which was therefore included throughout the study. The contraction was marginally reduced by reserpine pretreatment indicating that the response was predominantly direct. Phentolamine 0 0 -6 and l0 -5 M) produced small shifts 0 . 6 2 and 5.34) of the concentration-response curves which were substantially less than those for noradrenaline (112.5 and 333.3). The response was not therefore ct-adrenoceptor mediated. The perfused lung also yielded bronchoconstrictor responses to PEA, indicating that they were not of vascular origin. Atropine 0 0 -7 M) and a mixture of mepyramine (10 -7 M) and metiamide (10 -4 M) had no effect on the P E A contraction, which was not therefore due to stimulation of muscarinic and histaminergic receptors. Similarly, 5-HT and dopamine receptor stimulation was discounted. The proposal that P E A may be stimulating specific phenylethylaminergic receptors is made and the relevance to bronchial asthma is discussed. Acknowledgements--We are grateful to Ciba-Geigy, ICI
and SK & F for the gifts of drugs. REFERENCES
Antelman S. A., Edwards D. J. and Lin M. (1977) Phenylethylamine: Evidence for a direct post-synaptic dopamine-receptor stimulating action. Brain Res. 127, 317-324. Apperley E., Fenuik W., Humphrey P. P. A. and Levy G. P. (1980) Evidence for two types of excitatory receptor for 5-hydroxytryptamine in dog isolated vasculature. Br. J. Pharmac. 68, 215-224. Boushey H. A., Holtzman M. J., Sheller J. R. and Nadel J. A. (1980) Bronchial hyper-reactivity. Am. Rev. Resp. Dis. 121, 389-413. Broadley K. J. and Hawthorn M. H. (1981) Reserpineinduced supersensitivity to the fl-adrenoceptor-mediated responses of cardiac, respiratory and uterine tissues of the guinea-pig. Br. J. Pharmac. 73, 202-203P. Broadley K. J., Gibbon C. J. and Hawthorn M. H. (1981) A bronchoconstrictor response to fl-phenylethylamine of guinea-pig isolated lung. Br. J. Pharmac. 74, 980-981P. Burn J. H. and Rand M. J. (1958) The action of sympathomimetic amines in animals treated with reserpine. J. Physiol. 144, 314-336.
377
Chaytor J. P., Crathorne B. and Saxby M. J. (1975) The identification and significance of 2-phenylethylamine in foods. J. Sci. Fd. Agric. 26, 593-598. Clayton D. E., Busse W. W. and Buckner C. K. (1981) Contribution of vascular smooth muscle to contractile responses of guinea-pig isolated lung parenchymal strips. Eur. J. Pharmac. 70, 311-320. Cortijo J., Esplugues J., Morcillo E. J. and Perpifi~ M. (1984) Pharmacological analysis of the responsiveness of guinea-pig lung parenchymal strip to dopamine. Br. J. Pharmac. 83, 161-167. Drazen J. M. and Schneider M. W. (1978) Comparative responses of tracheal spirals and parenchymal strips to histamine and carbachol in vitro. J. clin. Invest. 61, 1441-1447. Feldman J. and Lebovitz H. (1971) The nature of the interaction of amines with the pancreatic beta cell to influence insulin secretion. J. Pharmac. exp. Ther. 179, 56-63. Furchgott R. F. (1972) The classification of adrenoceptors (adrenergic receptors). An evaluation from the standpoint of receptor theory. In Catecholamines: Handbook o f Experimental Pharmacology, Vol. 33 (Edited by Blaschko H. and Muscholl E.) pp. 283-335. SpringerVerlag, Berlin. Giardina W. J. (1974) Analgesic properties of phenylethylamine and phenylethanolamine in mice. Pharmacology 12, 1-7. Hansen T. R., Greenberg J. and Mosnaim A. D. (1980) Direct effect of phenylethylamine upon isolated vascular smooth muscle of the rat. Eur. J. Pharmac. 63, 95-101. Hawthorn M. H. and Broadley K. J. (1984) Reserpineinduced supersensitivity occurs for fl-adrenoceptormediated responses of heart and trachea but not of the uterus and lung. Eur. J. Pharmac. 105, 245-255. Koehler P. E. and Eitenmiller R. R. (1978) High pressure liquid chromatographic analysis of tyramine, phenylethylamine and tryptamine in sausage, cheese and chocolate. J. Fd. Sci. 43, 1245-1247. Lulich K. M., Mitchell H. W. and Sparrow M. P. (1976) The cat lung strip as an in vitro preparation of peripheral airways: A comparison of fl-adrenoceptor agonists, autocoids and anaphylactic challenge on the lung strip and trachea. Br. J. Pharrnac. 58, 71-79. McCulloch J. and Harper A. M. (1978) Phenylethylamine and cerebral circulation. In Current Concepts in Migraine Research (Edited by Green N.), pp. 85-93. Raven Press, New York. Manson M. F. (1979) fl-Phenylethylamine: Factors regulating its concentration and distribution in blood. Doctoral Thesis, Dept. Health Science, The Chicago Medical School. Morcillo E., Perpififi M. and Esplugues J. (1984) Responsiveness to tyramine in isolated lung parenchymal strip of guinea-pig and rat. Eur. J. Pharmac. 97, 13-19. Savedra J. M. (1978) fl-Phenylethylamine:--is this the biogenie amine related to neuropsychotic diseases? In Noncatecholic Phenylethylamines, Part I. Phenylethylamines: Biological Mechanisms and Clinical Aspects (Edited by Mosnaim A. D. and Wolf M. E.), pp. 139-152. Marcel Dekker, New York. Sandler M., Youdin M. B. H. and Hanington E. (1974) A phenylethylamine oxidising defect in migraine. Nature 250, 335-337. Solviter R. S., Cannon J. D. and Drust E. D. (1980) Serotonergie properties of fl-phenylethylamines in rats. Neuropharmacology 19, 1071-1074. Ungar F., Mosnaim A. D., Ungar B. and Wolf M. E. (1978) Preliminary studies of the sodium borohydride stabilizable binding of phenylethylamine and tyramine to brain preparations. Res. Commun, Chem. Path. Pharmac. 19, 427-431. Vogel W. H. (1978) Phenylethylamine and the conditional
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avoidance response. In Noncatecholic Phenylethylamines, Part I. Phenylethylamines: Biological Mechanisms and Clinical Aspects (Edited by Mosnaim A. D. and Wolf M. E.), pp. 371-388. Marcel Dekker, New York.
Waud D. R., Kattegada S. R. and Krayero O. (1958) Threshold dose and time course of norepinephrine depletion of the mammalian heart by reserpine. J. Pharmac. exp. Ther. 124, 340-346.
0306-3623/85 $3.00 +0.00 Copyright © 1985 Pergamon Press Ltd
EXAMINATION OF THE BRONCHOCONSTRICTOR RESPONSE OF GUINEA-PIG ISOLATED LUNG TO fl-PHENYLETHYLAMINE M. H. HAWTHORN, K. J. BROADLEY and C. J. GIBBON Department of Pharmacology, Welsh School of Pharmacy, University of Wales Institute of Science and Technology, P.O. Box 13, Cardiff CF1 3XF, U.K. (Tel: (0222) 42588) (Received 24 October 1984) fl-Phenylethylamine (PEA) produced a contraction of the guinea-pig isolated lung parenchymal strip and bronchoconstriction of perfused lungs. 2. Reserpine pretreatment had little effect on these responses indicating a substantial direct effect. 3. Phentolamine (10 -6 and 10-5 M) had minimal effect on PEA in the lung strip compared with that on noradrenaline, eliminating involvement of ~t-adrenoceptors. 4. PEA was unaffected by atropine (10 7 M) or a mepyramine (10 7M); metiamide (10 -4 M) mixture. The contraction was not therefore mediated via muscarinic or histaminergic receptors. 5-HT and dopamine receptors were also discounted. 5. Possible stimulation of a phenylethylaminergic receptor and its relevance to bronchial asthma is discussed. Abstraet--l.
INTRODUCTION
The indirectly acting sympathomimetic amine flphenylethylamine (PEA) is believed to produce its effects by promoting the release of noradrenaline from sympathetic neurones (Burn and Rand, 1958). In guinea-pig lung strips, however, a biphasic response curve is produced (Broadley et al., 1981), with the expected small fl-adrenoceptor-mediated relaxation occurring at low concentrations, followed by contraction. A similar contractile response o f rat and guinea-pig lung strips to another indirectly acting sympathomimetic amine, tyramine, has also been reported recently (Morcillo et al., 1984). This latter response does not, however, appear to be as pronounced as that for P E A (Broadley and Hawthorn, 1981; H a w t h o r n and Broadley, 1984), which suggests that the response to P E A may be mediated via a mechanism other than noradrenaline release. In support of this, there is a growing body of evidence that non-catecholic phenylethylamines may exert a direct action on a number of tissues, probably by interacting with adrenergic receptors (weak agonist action) (Antelman et al., 1977) or with more specific receptors of their own (Ungar et al., 1978). Recently, Hansen et al. (1980) have demonstrated a direct contractile effect of PEA on rat aortic strip which is mediated via ~-adrenoceptor stimulation. As vascular tissue may contribute to the responses of guinea-pig lung strip (Clayton et al., 1981), this work was carried out to initially determine if the site of action of P E A was vascular or parenchymal in origin and to examine the possible receptor type mediating the contractile response.
prepared as described by Lulich et al. (1976). Sections of lung tissue about 3 mm in width were dissected away from the peripheral margin of a lobe, with the axis of the strip cut parallel to the bronchus, but not including it. The strips were suspended in Krebs-bicarbonate solution (composition in mM: NaCI 118.4, KC14.7, CaC12.2H20 1.9, NaHCO~ 25, MgSO4"7H20 1.2, glucose 11.7, KH2PO 4 1.2), gassed with 5% CO2 in oxygen at 38°C. The tissues were incubated throughout with metanephrine (10-SM) to inhibit extraneuronal uptake. Responses were recorded isometrically using a Devices UF1 transducer (57 g sensitivity range) on a Devices (Lectromed) M19 polygraph. The lung strips were placed under a resting tension of 1.0-1.2 g and allowed to equilibrate for 40min with the bathing medium being changed at 10 min intervals before drug challenge.
MATERIALS AND METHODS
Lung strip preparations Guinea-pigs of either sex (300-500 g) were killed by a blow to the head and exsanguinated. Lung strips were 371 G.P. 1 6 / ~ E
Perfused lung preparations Guinea-pigs were killed as above and the thorax opened. The lungs were removed with approximately 2.5 cm of the trachea still attached. A plastic cannula was inserted into the cut end of the trachea and attached to the perfusion apparatus. The surfaces of the lung were scarified lightly with a hypodermic needle to facilitate loss of the perfusate from the alveolar spaces. The lungs were perfused via the airspaces with Krebsbicarbonate solution at a constant flow rate of 10 ml rainby use of a Watson-Marlow flow inducer (Type MHRE/ 30/T, 0.8 mm tube bore). The perfusion solution was aerated with 5% CO2 in oxygen before being passed into the flow inducer, warming coil (38°C) and tracheal cannula. The preparations were perfused throughout with propranolol (10-6 M) to inhibit fl-adrenoceptors and with metanephrine (10 -5 M), and left to stabilize for 10-20 min before drugs were added. Alterations in perfusion pressure (resting levels 5-25 mmHg; l mmHg~, 133Pa) arising from changes in pulmonary airway resistance were recorded on a Devices (Lectromed) M 19 polygraph by means of a pressure transducer (Bell & Howell, type 4-327-L221), situated at a side arm of the tracheal cannula. A Condon manometer was also included in the system in series with the transducer.
M. H. HAWTHORN et al.
372 05 -- Lung strips
lOO
CA)
,e- Phenylethylomine
Contraction
/
0.4
80 °~•
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60
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~c~ 0.3 c
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Propronolol
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/t /
NorodrenaI ~ L ine
"4:)'''0"-'~ ~
1
1
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20
E 0 ° o
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.
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10.8
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°
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10.2
Agonist (M)
o
~ 1oo I (B) 80 0.1
Relaxation I
10-7
[
I
10.6
10.5
I
10.4
I
10-3
40 Norodrenaline
/~-Phenylethytomine (M) Fig
,8-Phenylethylomine
o
1 M e a n (n = 4) concentration-response curves for
fl-phenylethylamine on the guinea-pig lung strip obtained before ((3) and in the presence (O) of propranolol (10 6 M).
o
I 10-10 10.9
10.8
10.7
10.6
I
I
I
10. 5
10-4
10.3
Agonist (tool)
Drug administration With the lung strip preparation, concentration-response curves were constructed by cumulative addition of the agonists to the bath. In the case of the perfused lung, drugs were injected sequentially as a bolus into the perfusion solution via the connecting rubber tubing immediately prior to entry into the tracheal cannula. Dose volumes were kept between 0.05 and 0.15ml. Maximum responses were assumed to have been achieved when an increase in concentration brought about no further increase in response, In lung strips, with this maximum concentration in the bath, carbachol (0.15 x 10-4 M) or histamine (5 x 10 5 M) were added to obtain the maximum contractile response of the tissue. Lung strips were then washed and when the resting tension had returned to control levels, the antagonist under study was added and left in contact with the tissue for 30 min before another cumulative concentration-response curve was constructed. Changes in sensitivity arising from repeating concentration-response curves were corrected for by performing control experiments which were identical except no antagonist was included. Changes in tension (lung strip) or perfusion pressure (lung) were measured at each agonist concentration. Concentration-response curves were plotted as a percentage of their own maximum from which the ECs0 value was calculated, or as a percentage of the carbachol or histamine maximum response. Geometric mean EC50 values and mean maximum responses were compared by Student's t-test. In some experiments tissues were taken from guinea-pigs that had received 0.5 mgkg ~ reserpine intraperitoneally 24 hr prior to killing.
Drugs used Atropine sulphate, carbacho[ (carbamylcholine) chloride, dopamine hydrochloride (Sigma), histamine acid phosphate
Fig. 2. Mean (n >/4) concentration-response curves for the increase in tension of lung strips (A) and increase in perfusion pressure of perfused lungs (B) in response to noradrenaline ((~) and ~-phenylethylamine (O). Responses are expressed as a percentage of a maximum response to carbachol. Propranolol (10 6M) and metanephrine (10 -5 M) were present throughout. (BDH), 5-hydroxytryptamine (creatinine sulphate complex) (Sigma), mepyramine maleate (May & Baker), ( + )-metanephrine hydrochloride (Sigma), metiamide (S K & F), (-)-noradrenatine bitartrate (Sigma), ~-phenylethylamine hydrochloride (Sigma), phentolamine mesylate (CibaGeigy), (+__)-propranolol hydrochloride (ICI) and reserpine (BDH). RESULTS
Effects of [3-phenylethylamine and noradrenaline on lung strips fl-Phenylethylamine ( P E A ) caused a n initial relaxa t i o n o f the lung strips followed at higher concentrations by c o n t r a c t i o n (Fig. 1). W h e n repeated in the presence of p r o p r a n o l o l ( 1 0 - 6 M ) , P E A caused only a contractile response a n d the c o n c e n t r a t i o n response curve for this response was displaced to the left (Fig. 1). F r o m this p o i n t on, all experiments were p e r f o r m e d t h r o u g h o u t in the presence o f p r o p r a n o l o l (10 -6 M). T h e m a x i m u m contractile response to P E A was 79.2 ___8.5% of the c a r b a c h o l - i n d u c e d m a x i m u m , a n d the m e a n (n = 4) ECs0 value was 2.38 x 10 4 M (Fig. 2A, T a b l e 1). P r e t r e a t m e n t with reserpine ( 0 . 5 m g k g ~ at 2 4 h r ) did n o t significantly affect
Table 1. Effects of reserpine pretreatment on responses of lung strip and perfused lung to fl-phenylethylamine Perfused lung Lung strip Maximum ECs0 Maximum ECs0 n (% carbachol max.) ( X 1(~ - 4 M ) n (% carbachol max) ( x 10-4 M) 4 63.1 _+ 10.2~% 4.53 5 Untreated 79.2 _+8.5% 2.38 (3.t5 6.49) (1.88-3.45) 4 60.8 +_ 11.4% 6.24 4 Reserpine pretreated 68.5 + 6.5% 11.34" (2.35 8.63) (9.42-13.17) Geometric mean ECs0 values with 95% confidence limits and mean maximum responses (+SEM) are shown. *Indicates a significant difference (P < 0.05) from the untreated value. Reserpine (0.5 mg kg -~) was administered at 24 hr.
373
Bronchoconstriction to fl-phenylethylamine
Table 2. Effect of noradrenaline on lung strip and perfused lung Maximum ECs0 (% carbachol max.) (x 10-TM) n Lung strip 42.6 + 6.3% 2.66 4 (0.81-9.17) Perfused lung 15.3 _+5.3% 1.67 4 (0.77 7.51) Geometric mean ECsovalues with 95% confidence limits and mean maximum responses (+ SEM) are shown.
(A)
F
8o~ 6oe g 4o m
]
_~
oL
I
I
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10. 5
(B)
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I
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(M)
q lOO-
Effect of phentolamine on fl-phenylethylamine noradrenaline
80--
~
60--
/./.
~ 4o ~
in the perfused lung was significantly less (P < 0.05) than in the lung strip. Reserpine pretreatment had no significant effect (P >0.05) on the P E A maximum response ( 6 0 . 8 _ 11.4%) or on the mean (n = 4) ECs0 value (6.24 x 10 -4 M) (Fig. 3B, Table 1).
2c
o
I 10 . 7
10- 6
10-5
/3-Phenylethylamine
I0 -4
I 10 .3
(tool)
Concentration-response curves for P E A or noradrenaline were obtained in lung strips before and in the presence of phentolamine (10 _6 or 10 -5 M). There was a small shift of the P E A curves to the right (Fig. 4A), the mean (n = 8) ECs0 value being increased from 3.42 x 1 0 - 4 M to 4.61 x 10 4M by 10-6M phentolamine and from 1.61 × 1 0 - a M ( n - - 4 ) to 8.18 x 1 0 - 4 M by 1 0 - S M phentolamine. The corresponding concentration-ratios were 1.61 _ 0.25 and 5 . 3 4 _ 0.87 respectively (Table 4). Although significant (P < 0.05), these shifts were not as great as those induced by phentolamine on the concentration-
Fig. 3. Mean (n /> 4) concentration-response curves for the increase in tension of lung strips (A) and increase in perfusion pressure of perfused lungs (B) in response to fl-phenylethylamine. Tissues were taken from untreated guinea-pigs ((3) and from animals receiving reserpine (0.5 mg kg ~ i.p. at 24 hr) (0). Responses are expressed as a percentage of a maximum response to carbachol. Propranolol (10 -6 M) and metanephrine (10 5 M) were present throughout.
(P > 0.05) the m a x i m u m contraction (68.5 _+ 6.5%, n =4), although the mean ECs0 value of 11.34 × 10 -4 M was significantly greater (P < 0.05) than control values (Fig. 3A, Table 1). As with PEA, noradrenaline produced a contraction of the lung strip, the maximum response being 42.6 _ 6.3% (n = 4) of the carbachol maximum (Fig. 2A). This was significantly less (P < 0.05) than the maximum response produced by PEA. Noradrenaline was also significantly more potent (P < 0.05) than PEA, having a mean ECs0 value (n = 4) of 2.66 x 10 -7 M (Table 2).
Effects of fl-phenylethylamine and noradrenaline on the perfused lung Both PEA and noradrenaline produced bronchoconstriction of the perfused lung, as measured by an increase in perfusion pressure (Fig. 2B). The mean (n = 5) ECs0 values for noradrenaline and P E A were 1.67 x 1 0 - 7 M and 4.53 x 1 0 - 4 M respectively and the maxima were 15.3 _ 5.3% and 63.1 _ 10.2% of the carbachol maxima respectively (Fig. 2B, Tables 1 and 2). There was no significant difference (P > 0.05) between the PEA maxima in the perfused lung and lung strip, however, the m a x i m u m for noradrenaline
and
100
(A)
80 60 40
2O
o
lO-r
10-6
10-5
10 - 4
/5'-- Phenylethylamine
100 --
10.3
I 10.2
(M )
(B)
80 6O 4O 2O 0
10-9
10-8
10 -7
10-6
Noradrenaline
10-5
10-4
10-3
(M)
Fig. 4. Mean (n >/4) concentration-response curves for the increase in tension of lung strips in response to (A) fl-phenylethylamine and (B) noradrenaline, obtained before (O) and in the presence of phentolamine, 10 -6 M ([]) and 10 -5 M (A). Responses are expressed as a percentage of their own maximum. Propranolol (10-6M) and metanephrine (10 -5 M) were present throughout.
M.H. HAWTHORNet al.
374
Table 3. Effect of phentolamine on responses to noradrenaline of lung strip Pre-antagonist
Post-antagonist
Phentolamine
Maximum ( ~ carbachol)
ECs0 ( × 10 6 M)
Maximum ( ~ carbachol)
EC~0 ( × 10 -6 M)
10-6M
49.9_+1.2
45.6_+2.6
10 -~ M
43.2 _+ 2.8
0.36 (0.07 1.57) 0.75 (0.11 2.81)
40.55* (20.6-93.2) 250* (140.7-480.1)
46.9 _+ 3.2
CR 112.5_+12.7
4
333.3 _+ 25.6
4
Geometric mean ECs0 values with 9 5 ~ confidence limits, mean maximum responses ( + SEM) and Concentration-ratios (CR) are shown. Pre-antagonist values are corrected from control experiments. *Indicates a significant difference (P < 0.05) from the pre-antagonist value.
response curves to noradrenaline (Fig. 4B). The mean (n = 4) ECs0 value for noradrenaline was increased from 0.36 × 10-6M to 40.55 × 10 6M by 10-6M phentolamine giving a mean concentration-ratio of 112.5_ 12.7 (Table 3) and from 0.75 × 10 -6 M to 250 × 10 - 6 M by 10 5 M phentolamine giving a mean (n = 4) concentration-ratio of 333.3 _ 25.6 (Table 3). These concentration-ratios were significantly greater (P < 0.05) than those obtained for the shifts of the PEA concentration-response curves at the corresponding concentrations of phentolamine.
Effects o f atropine on fl-phenylethylamine and carbachol Concentration-response curves for the contraction of lung strips by PEA were obtained before and in the presence of atropine ( 1 0 - 7 M ) and plotted as a percentage of the maximum response to histamine. The maximum responses obtained before (38.5 ___3.1~o) and in the presence of atropine (43.8 + 4.1~, n = 5) did not differ significantly (P > 0.05) (Table 4). Atropine was also without significant effect (P > 0.05) when the curves were plotted as a percentage of their own maximum, the mean ECs0 values before and after atropine being 3.41 x 10-4M and 5.75 x 10 -4 M respectively (Fig. 5A, Table 4), which yielded a concentration-ratio of 1.93 ___0.45. As with PEA, atropine (10-7M) had no effect on the maximum response to carbachol (expressed as a percentage of the histamine maximum), the preand post-atropine values being 41.0+1.1 and 45.5 _ 7.0~o respectively (n = 4) (Table 5). However, when expressed as a percentage of its own maximum response, the carbachol concentration-response
curve was shifted to the right (Fig. 5B), the mean ECs0 value increasing from 2.55 x 10-6M to 8.5 x 10-5 M (Table 5), giving a concentration-ratio of 34.3 + 4.5 (n = 4). Atropine had no significant (P > 0.05) effect on the maximum responses to histamine, which were 2.79 + 0.63 and 2.48 _ 0.61 g before and after atropine respectively in the tissues receiving PEA; and 1.90 + 0.21 and 1.42_ 0.13 g respectively in the tissues receiving carbachol.
Effects o f mepyramine and metiamide on fl-phenylethylamine and histamine A mixture of mepyramine ( 1 0 - 7 M ) and metiamide (10-aM) had no significant effect (P > 0.05) on the maximum responses to PEA, which were 43.5 + 4.3~ and 52.5 _ 6.5~o (n = 4) respectively of the carbachol maximum before and after the antagonists (Table 4). Mepyramine and metiamide were also without effect when the curves were expressed as a percentage of their own maxima, with no significant difference (P > 0.05) occurring between the mean ECs0 values of 6.71 x 10-4M before and 5.35 × 10-4M (n = 4 ) after the antagonists (Fig. 6A, Table 4). The concentration-ratio was 0.97 ___0.35. Unlike PEA, histamine concentration-response curves were displaced to the right by the mepyramine and metiamide mixture, with the mean ECs0 values being significantly increased (P < 0.05) from 2.3 x 10-6M to 14.8 x 10 5M (n = 4 ) (Fig. 6B, Table 5). These concentrations of mepyramine and metiamide had no significant effect (P > 0.05) on the maximum responses to histamine (presence, 1.73 + 0.3; absence, 1.49 + 0.53 g) or carbachol (presence, 0.38 + 0.07; absence, 0.40 ___0.04 g).
Table 4. Effects of antagonists on responses of lung strip to fl-phenylethylamine Pre-antagonist Antagonist Phentolamine 10-6M Phentolamine 10 -5 M Atropine 10 -7 M Mepyramine 10 -7 M + Metiamide l0 4M Methysergide 10 -7 M
Post-antagonist
Maximum response
ECs0 ( x 10-4M)
Maximum response
ECs0 ( × 10 4M)
49.9 + 5.4
3.42 (2.21 3.26) 1.61 (0.72-3.41) 3.41 (2.794.18) 6.71 (2.89-15.42)
57.8 __. 3.7
4.61" (1.88-7.31) 8.18" (1.35-5.22) 5.75 (2.36-14.0) 5.35 (2.7~10.87)
54.3 _ 6.5 38.5 _+ 3.1 43.5 + 4.3
61.2 _+ 3.5
2.67 (1.72~1.51)
51.6 _+ 9.3 43.8 _+4.1 52.5 _ 6.5
41.0 + 3.9*
2.66 (2.35-3.02)
CR
n
1.62 + 0.25
8
5.34 _ 0.87
4
1.93 + 0.45
5
0.97 + 0.35
4
1.21 -I-0.07
6
Geometric mean EC50 values with 9 5 ~ confidence limits, mean maximum responses (as a percentage of carbachol or histamine maxima) ( ± S E M ) and concentration-ratios (CR) are shown. Pre-antagonist values are corrected from control experiments. *Indicates a significant difference (P < 0.05) from the pre-antagonist value.
375
Bronchoconstriction to fl-phenylethylamine (A)
I
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4O
(A)
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a
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I0-4
I
10-3
10-2
lO-3
10-2
~ - Phenylethylamine (M)
®
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(B)
80
/~-Phenylethylamine (M) 100
(B)
•
80
60
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//
40 20
6O
0
I 10-7
40 20 0
I 10-7
10-6
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Fig. 5. Mean (n t> 4) concentration-response curves for the increase in tension of lung strips in response to (A) fl-phenylethylamine and (B) carbachol, obtained before ( O ) and in the presence ( 0 ) of atropine (10-7 M). Responses are expressed as a percentage of their own maxima. Propranolol (10-6 M) and metanephrine (10- 5 M) were present throughout experiments with fl-phenylethylamine.
Effects of methysergide, 5-HT and dopamine Concentration-response curves to PEA were constructed before and in the presence of methysergide (10 -7 M ) which significantly reduced (P < 0.05) the maximum response from 61.2 _ 3.5~o to 41.0 +__3.9~o (n = 6) when expressed as a percentage of the carbachol maximum (Table 4). The maximum responses to carbachol obtained before (2.06 + 0.4 g) and after methysergide (2.04 _+ 0.29 g) were not significantly different (P > 0.05). When expressed as a percentage of their own maximum responses, the PEA curves were superimposed, with no significant difference (P > 0.05) between the mean ECs0 values (n = 6)
h */~___ 10-6 10-5 Histamine (M)
I ,10-4
L-I0-?
Fig. 6. Mean (n >/4) concentration-response curves for the increase in tension of lung strips in response to (A) fl-phenylethylamine and (B) histamine, obtained before ( O ) and in the presence ( 0 ) of mepyramine ( 1 0 - 7 M ) and metiamide (10 -4 M). Responses are expressed as a percentage of their own maxima. Propranolol (10 -6 M) and metanephrine (10-SM) were present throughout experiments with fl-phenylethylamine.
obtained before (2.67 x 10 - 4 M) and in the presence of methysergide (2.66 x 10 -4 M) (Table 4). It was not possible to examine the effect of methysergide upon 5-hydroxytryptamine, since only a small proportion of tissues responded and these yielded only small contractions of the lung strip. Similarly, dopamine failed to contract the lung strip in concentrations up to 10 4 M when examined in the presence of phentolamine (10 s M) and propranolol (10 -6 M ) . DISCUSSION
fl-Phenylethylamine exerted a dual effect upon the lung strip of the guinea-pig; an initial relaxation at low concentrations being followed by a contraction at higher concentrations. The relaxation was abolished by propranolol and therefore attributed to fl-adrenoceptor stimulation as reported previously (Broadley and Hawthorn, 1981). The predominant
Table 5. Effects of atropine (10 -7 M) on responses to carbachol and mepyramine (10 - 7 M) with metiamide (10 -4 M) on responses to histamine of lung strip Pre-antagonist Agonist Carbachol
Maximum (% histamine) 41.0 -+_1.1
Histamine
--
ECs0 ( x 10_6 M) 2.55 ( 1.87-3.48) 2.3 (0.68-7.5)
Post-antagonist Maximum (% histamine) 45.5 + 7.0 --
ECso ( x 10-s M) 8.5* (5.9-12.0) 14.8" (4.3-50.9)
CR 34.3 --F4.5
n 4
37.9 + 5.1
4
Geometric mean ECso values with 95% confidence limits, mean maximum responses (+_ SEM) and concentration-ratios (CR) are shown. Pre-antagonist values are corrected from control experiments. *Indicates a significant difference (P < 0.05) from the pre-antagonist value.
376
M.H. HAWTHORNet al.
response was therefore a contraction, indicative of bronchoconstriction, and this was further examined in the presence of propranolol throughout. Responses to PEA in other tissues have been shown to arise indirectly from the release of noradrenaline from sympathetic nerve terminals (Burn and Rand, 1958). After pretreatment with reserpine in a dose known to deplete catecholamines (Waud et al., 1958), there was a small shift of the PEA concentrationresponse curve to the right. However, the maximum contractile response was unaffected, indicating that only a small portion of this response could be attributed to an indirect effect. The majority of the response is therefore due to a direct action of PEA, a conclusion also drawn from the perfused lung, where reserpine was without effect upon PEA. A similar reserpine-resistant contractile response to PEA has been reported in rat aorta (Hansen et al., 1980) which appears to be mediated via ct-adrenoceptors since it was blocked by phentolamine. This does not appear to be the case in lung strips since although noradrenaline produced contractions of this tissue and was more potent than PEA, its maximum response was smaller. Further support for the conclusion that PEA is not acting directly via ~t-adrenoceptors in the lung strip comes from the use of phentolamine. Although the concentration-response curves for PEA were displaced to the right by 10 -6 M phentolamine (concentration-ratio 1.62) and 10-SM phentolamine (5.34), the degree of shift was substantially less than the shifts of noradrenaline by the same concentrations (112.5 and 333.3 respectively). This lack of antagonism of PEA by phentolamine is in contrast to the observations of Hansen et al. (1980). However, the antagonism observed may have been due to the excessively high concentration employed (10-4M) since Furchgott (1972) has demonstrated that concentrations of only 10 - 6 M phentolamine are needed to adequately antagonize ct-adrenoceptors. PEA appears to differ from the alternative indirectly acting sympathomimetic amine tyramine which we have shown to produce a weaker contractile response of the lung strip (Broadley and Hawthorn, 1981). This response has been shown to be substantially antagonized by reserpine pretreatment and by phentolamine, although not to the same extent as noradrenaline (Morcillo et al., 1984). The lung strip preparation has been criticised for not being truly representative of airway responses on the grounds that a vascular component may be involved (Clayton et al., 1981). To examine whether the contractile response of the lung strip to PEA is a vascular or airway response, isolated lungs were perfused via the trachea at a constant flow and the back pressure recorded. Any change in pressure could therefore be caused only by effects on airway tissue. As in the lung strip, both PEA and noradrenaline produced bronchoconstriction. However, the response to noradrenaline was much reduced when compared with the lung-strip response, but the effects of PEA were of similar magnitude in both tissues. Furthermore, reserpine pretreatment had no effect on the PEA response of the perfused lung, which indicates that PEA has a direct contractile effect on lung parenchymal tissue. In contrast, the contractile response to noradrenaline would appear to be mainly
vascular in origin, which agrees with the conclusion of Clayton et aL (1981). The small displacement by reserpine of the PEA concentration-response curves in the lung strip can probably be explained by a vascular component mediated via an indirect sympathomimetic effect. Since the contractions of the lung strip by PEA did not appear to be mediated via ~-adrenoceptors, the possibility was next examined that the response was produced via either muscarinic or histaminic receptors; both acetylcholine and histamine are known to contract the lung strip (Drazen and Schneider, 1978). However, atropine failed to affect either the maximum response to PEA or the ECs0 in a concentration that effectively antagonized carbachol. Similarly, the combined use of the H~ and H2 receptor antagonists mepyramine and metiamide respectively, had no effect on the PEA maximum response and ECs0 value, although the same concentrations antagonized histamine. The central effects of PEA have been attributed to a direct stimulation of either 5-hydroxytryptamine (5-HT) (Solviter et al., 1980) or dopamine receptors (Antelman et al., 1977). The guinea-pig lung strip has also been shown to be contracted by both 5-HT (Morcillo et al., 1984) and dopamine (Cortijo et al., 1984). In the present study, the response to 5-HT was inconsistent and weak compared with that to PEA. The 5-HT antagonist, methysergide, however reduced the maximum response to PEA indicative of a noncompetitive antagonism. This is unlikely to be due to antagonism at the 5-HT receptor since methysergide is a competitive antagonist in other tissues with no effect on the maximum responses (Apperley et al., 1980). A non-specific effect on the tissue can also be discounted since it did not reduce the maximum effect of carbachol. The precise cause of this reduced maximum remains to be established, however, PEA does not appear to be exerting its contractile effect via 5-HT receptors since methysergide failed to alter the ECs0 value and 5-HT itself failed to cause a contraction in the presence of ~- and fl-adrenoceptor blockade. Any contractile effects of dopamine observed previously in this preparation are mediated entirely via ct-adrenoceptors (Cortijo et al., 1984). Finally, having discounted the most likely recognized receptor systems, the possibility must be considered that PEA exerts its contractile effect via phenylethylaminergic receptors. There is evidence that such receptors exist in the central nervous system where they are involved in the analgesic (Giardina, 1974), behavioural (Antelman et al., 1977), hallucinogenic (Vogel, 1978) and cerebral vasoconstrictor properties (McCulloch and Harper, 1978) of PEA. Specific binding sites for PEA have also been identified in midbrain synaptosomes of the rat, cow and mouse (Ungar et aL, 1978), and in the periphery, PEA receptors have been suggested in pancreatic cells (Feldman and Lebovitz, 1971). The presence of such a receptor may also be proposed in the lung strip to explain the bronchoconstrictor effect of PEA. The concentration of PEA required to produce the response (10 5 M) is, however, relatively high and it is difficult to envisage a physiological role for such a receptor when the normal circulating levels of PEA are in the order of 10 -8 M (Saavedra, 1978; Manson,
Bronchoconstriction to fl-phenylethylamine 1979). Nevertheless, this study does raise the possibility of a pathophysiological involvement of P E A in bronchospasm associated with asthma. Airway sensitivity to bronchoconstrictors, which may include P E A , is raised in the asthmatic patient (Boushey et al., 1980) such that an elevation of plasma P E A may induce an asthmatic attack by the ingestion of dietary products rich in PEA, such as chocolate, cheese and wine (Chaytor et al., 1975; Koehler and Eitenmiller, 1978). Indeed, a similar hypothesis has been proposed for the precipitation of dietary migraine following consumption of PEA-containing foods (Sandler et al., 1974). SUMMARY
fl-Phenylethylamine (PEA) produced a biphasic effect on the guinea-pig isolated lung parenchymal strip. A small relaxation was followed at higher concentrations by a more pronounced contraction, indicative of brochoconstriction. The relaxation was fl-adrenoceptor-mediated since it was abolished by propranolol, which was therefore included throughout the study. The contraction was marginally reduced by reserpine pretreatment indicating that the response was predominantly direct. Phentolamine 0 0 -6 and l0 -5 M) produced small shifts 0 . 6 2 and 5.34) of the concentration-response curves which were substantially less than those for noradrenaline (112.5 and 333.3). The response was not therefore ct-adrenoceptor mediated. The perfused lung also yielded bronchoconstrictor responses to PEA, indicating that they were not of vascular origin. Atropine 0 0 -7 M) and a mixture of mepyramine (10 -7 M) and metiamide (10 -4 M) had no effect on the P E A contraction, which was not therefore due to stimulation of muscarinic and histaminergic receptors. Similarly, 5-HT and dopamine receptor stimulation was discounted. The proposal that P E A may be stimulating specific phenylethylaminergic receptors is made and the relevance to bronchial asthma is discussed. Acknowledgements--We are grateful to Ciba-Geigy, ICI
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