Br. J. Pharmacol. (1992), 105, 230-237
(D Macmillan Press Ltd, 1992
PAF-induced muscarinic cholinoceptor hyperresponsiveness of ferret tracheal smooth muscle and gland secretion in vitro S.E. Webber, T. Morikawa & J.G. Widdicombe Department of Physiology, St George's Hospital Medical School, Cranmer Terrace, London, SW17 ORE The effects of exposure of the ferret trachea in vitro to platelet activating factor (PAF) were examined methacholine-induced smooth muscle contraction, mucus volume and lysozyme outputs, and albumin transport across the tracheal epithelium. 2 Methacholine (0.1-301uM) produced concentration-dependent increases in tracheal smooth muscle tone and mucus volume, lysozyme and albumin outputs from the trachea. 3 The concentration-response curves for methacholine-induced smooth muscle contraction, mucus volume and lysozyme outputs were all shifted upwards after exposure of the trachea to PAF (1 uM) with a significant increase in maximum response for each variable. The EC50 values for methacholine-induced smooth muscle contraction and mucus volume output were significantly reduced after PAF exposure suggesting an increase in the potency of methacholine. The concentration-response curve for methacholine-induced albumin output was shifted downwards after PAF exposure with a greatly reduced maximum but no change in the EC50 for methacholine. 4 PAF-induced hyperresponsiveness of methacholine-induced smooth muscle contraction, mucus volume and lysozyme outputs was not affected by indomethacin, FPL55712, or mepyramine and cimetidine, but was prevented by catalase and superoxide dismutase (SOD), and by WEB2086. Similarly, PAFinduced inhibition of methacholine-stimulated albumin output was prevented by catalase and SOD, and by WEB2086. 5 We conclude that PAF induces hyperresponsiveness of ferret tracheal smooth muscle and submucosal gland secretion (including lysozyme secretion from serous cells) to methacholine. This hyperresponsiveness is probably produced by receptor-mediated release of oxygen free-radicals. The inhibition of methacholine-induced albumin flux suggests a loss of epithelial function which is also probably mediated by release of free-radicals. The mechanism by which the free-radicals produce the changes in responsiveness to methacholine, and the cellular source of the free-radicals, remain to be established. Keywords: Platelet activating factor; methacholine; mucus; smooth muscle; albumin; catalase; superoxide dismutase; WEB2086 1
on
Introduction
Methods
In the previous paper we described the direct effects of platelet activating factor (PAF) on ferret tracheal smooth muscle, submucosal gland secretion, epithelial albumin transport and potential difference (PD) across the trachea (Webber et al., 1992). Perhaps the most important property of PAF is its ability to induce a potent and long-lasting hyperresponsiveness of airway smooth muscle in vivo (Mazzoni et al., 1985; Cuss et al., 1986; Robertson & Page, 1987). This hyperresponsiveness, like PAF-induced bronchoconstriction, may be dependent on the presence of circulating platelets (Mazzoni et al., 1985) and may involve the release of mediators from eosinophils (Sanjar et al., 1990). However, whether PAF can induce a hyperresponsiveness of airway smooth muscle in vitro, predominantly in the absence of circulating inflammatory cells, has not been studied systematically. Furthermore, it is not known whether PAF changes the responsiveness of other airway effector tissues such as submucosal glands and epithelium in a way similar to that with airway smooth muscle. The muscarinic agonist, methacholine, potently contracts tracheal smooth muscle and stimulates submucosal gland secretion from the ferret trachea in vitro (Webber & Widdicombe, 1987). Albumin is actively transported across the ferret tracheal epithelium and this transport is stimulated by methacholine (Webber & Widdicombe, 1989). In the present study we have investigated the effect of exposing the ferret trachea in vitro to PAF on the responses of the smooth muscle, submucosal gland secretion (using lysozyme as a specific marker for serous cells) and epithelial albumin transport produced by methacholine.
The description of the ferret trachea in vitro and of the assays for lysozyme and albumin were described in detail in the previous paper (Webber et al., 1992).
Experimental protocol In the first set of experiments the effects of a single concentration of PAF (1 uM) on concentration-response curves for methacholine-induced tracheal smooth muscle contraction and mucus volume, lysozyme and albumin outputs was examined. In the second set of experiments we investigated the effect of increasing PAF concentrations (0.1-10pM) on the responses to a single concentration of methacholine (1 pM). In the first set of experiments, after an initial 30min control period, three or four different concentrations of methacholine, covering at least a tenfold concentration range, were added to the submucosal buffer bathing the trachea singly and in random sequence. Each concentration was left in contact with the trachea for 30min and during this time any increase in smooth muscle tone was recorded. The trachea was then washed twice and fresh buffer containing no methacholine was placed in the organ bath. One or two control periods of 30 min were allowed between addition of methacholine, depending on how quickly the secretion rate returned to a basal level. After three or four concentrations of methacholine had been added to the trachea, the buffer surrounding the trachea was replaced with buffer containing PAF (1 uM). This buffer was left in contact with the trachea for 1 h and was replaced every 30 min. Any change in smooth muscle tone during this period
PAF-INDUCED HYPERRESPONSIVENESS IN VITRO
231
was recorded, and any mucus produced was withdrawn at the end of each 30min. After' a 30min control period without PAF the same three or four concentrations of methacholine were added to the trachea in the same sequence as before PAF exposure. Methacholine was added to the trachea in a buffer which did not contain PAF. All mucus samples were assayed for lysozyme and albumin (Webber et al., 1992). In the second set of experiments, tracheae were set up in pairs, in separate organ baths. One trachea was exposed twice to methacholine (1uM) for 30min with one or two control periods of 30min between each addition of methacholine. Any changes in smooth muscle tone or mucus volume output produced by methacholine were recorded. After methacholine (1pM) had been added to the trachea twice, the buffer surrounding the trachea was replaced with buffer containing PAF (0.1 pM). This buffer was left in contact with the trachea for 1 h. The trachea was then washed and left for a 30 min control period in buffer containing no PAF. Methacholine (1 M) was then added to the trachea and the changes in smooth muscle tone and mucus volume output were recorded. The procedure described above was repeated twice with the trachea being exposed to PAF (1 M) then PAF (10pM) and responses to methacholine (1pM) after each PAF exposure were recorded. The second trachea acted as a control. Exactly the same procedure as described for the test trachea was carried out except that the second trachea was exposed not to PAF but to buffer containing no PAF.
and maximum responses (+95% confidence limits) for methacholine were estimated by fitting concentration-response curves to the data points by a computerised, non-linear, least squares estimate. ECQ0 is the concentration of methacholine which produced 50% of the maximum response to methacholine. Differences between ECQ0 values or maximum responses for methacholine before and after exposure of the trachea to PAF were analysed for statistical significance by Student's unpaired t test. In the second set of experiments differences between control responses (means + s.e.mean) to methacholine and responses to methacholine after PAF were analysed for significance by Student's paired t test. The effects of antagonists on the PAF-induced changes in responsiveness to methacholine were also analysed for signifiance by Student's paired t test. In all cases P < 0.05 was taken as significant.
Effects of antagonists To examine the effects of antagonists on PAF-induced changes in responsiveness to methacholine, tradheae were again set up in pairs. Exactly the same protocol was used for both tracheae except the buffer used for the test trachea contained one of the antagonists. Both tracheae were exposed to methacholine (1 pM) for 30 min and any changes in smooth
Mucus volume output The mucus volume output in control periods before addition of methacholine was 0.07 + 0.05 ,ul min' (n = 24). Methacholine produced a concentration-dependent increase in mucus volume output from this control level (Figure 1). This concentration-response curve for methacholine was shifted upwards and leftwards after exposure of the trachea to PAF (1 pM) with a significantly reduced ECQ0 value (3.3 fold, Table 1) and a significantly increased maximum response (38%, Table 1). Lysozyme output The lysozyme output in control periods before addition of methacholine was 16 + 11ngmin-1 (n = 24). Methacholine produced a concentration-dependent increase in lysozyme output from these control levels (Figure 2). This concentration-response curve for methacholine was shifted upwards after exposure of the trachea to PAF with a significantly increased maximum response (73%, Table 1), but no significant change in the ECQ0 (Table 1). Albumin output The albumin output in controls before methacholine was 0.32 + 0.14 pgmin-' (n = 24). Methacholine produced a concentration-dependent increase in albumin output from this control level (Figure 2). After exposure of the trachea to PAF, the maximum response for methacholine was greatly reduced (50%, Table 1) but there was no significant change in the ECQ0 value (Table 1). Concentration-related effects of PAF (0.1-JOpM) on responses to methacholine (IpM) Baseline values in control and test tracheae The baseline mucus volume output in control tracheae (tracheae which did
muscle tone or mucus volume output were recorded. After two control periods the tracheae were then exposed to PAF (1 pM) for 1 h. The tracheae were then washed thoroughly and left for a further control period of 30min. The responsiveness of the tracheae to methacholine (1 pM) was then re-assessed. The antagonists tested were indomethacin (1pM), FPL55712 (1pM), a combination of mepyramine (1 M) and cimetidine (10uM), a combination of catalase (500 u ml 1) and superoxide dismutase (40 u ml- 1), and WEB 2086 (10pM). An exposure time for PAF of 1 h was used in all experiments because this was shown to be optimal in preliminary studies. Thus, the maximum degree of hyperresponsiveness occurred with a 1 h exposure and there was no further increase in the effect of PAF with exposure times greater than 1 h. For example, in the case of smooth muscle tone, the increase in response to methacholine (1 pM) was 42 + 5% (n = 6) with a 30 min exposure to PAF, 71 + 6% (n = 6) with a 1 h exposure and 68 + 4% (n = 6) with a 2 h exposure.
Analysis of results The concentration-response curves and bar charts presented in Results were obtained by pooling the results from groups of experiments. In the first set of experiments, the EC50 values Table 1
Results Effect of PAF (1 pM) on concentration-response curvesfor met hacholine Intraluminal pressure Methacholine (0.1-3O0pM) produced a concentration-dependent increase in intraluminal pressure (Figure 1). This concentration-response curve was shifted upwards and leftwards after exposure of the trachea to PAF (1 pm, Figure 1) with a significantly reduced ECQ0 value (3 fold, Table 1) and a significant increase in maximum response (32%, Table 1).
ECQ0 values and maximum responses (±95% confidence limits) for methacholine before and after exposure of the trachea to
PAF (1 uM)
EC50 (M) Before PAF Intraluminal pressure (mmH20) Mucus volume output (pulmin-') Lysozyme output (ng min -) Albumin output (ug min - 1) *
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0.6 (0.4-0.8)* 0.4 (0.3-0.5)* 1.0 (0.8-1.2) 1.0 (0.7-1.3)
127 (121-133) 1.62 (1.56-1.68) 395 (380-410) 2.43 (2.33-2.53)
168 (162-174)* 2.24 (2.21-2.27)* 570 (520-620)* 1.22 (1.18-1.26)*
Significantly different (P < 0.05) from value before exposure to PAF using Student's unpaired t test.
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not receive PAF) before addition of any drugs was 0.06 + to (n = 6), compared 0.04 + 0.04 PI min-1 0.03 p1mmin- ' (n = 6) in test tracheae (tracheae before exposure to PAF). Similarly the baseline lysozyme outputs in control and test tracheae were 27 + 12 and 21 + 6 ng min-1 respectively, and the baseline albumin outputs were 0.18 + 0.09 and 0.27 + 0.16pjugmin'- respectively. The PD's across control and test tracheae before addition of drugs were -8.2 + 0.4 and -7.9 + 0.6 mV respectively. There are no significant differences between baseline values in control and test tracheae for any of these variables.
Intraluminal pressure Methacholine (1 pM) increased intraluminal pressure by 75 + 5 mmH2O (n = 6) in control tracheae and by 63 + 9 mmH2O in test tracheae. These responses are not significantly different. Exposure of the trachea to PAF (0.1-lOpM) produced a concentration-dependent enhancement of the increase in intraluminal pressure produced by methacholine (Figure 3). By contrast there was no significant change in the tracheal smooth muscle response to methacholine in control tracheae not exposed to PAF (Figure 3). Mucus volume output Methacholine (I pM) increased mucus volume output from 0.06 + 0.04 and 0.05 + 0.04up1 min' to
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1.05 + 0.15 and 1.13 + 0.l9pl-' in control and test tracheae respectively. The responses in control and test tracheae were not significantly different. Exposure of the trachea to PAF (0.1-10 pM) produced a concentration-dependent enhancement of the methacholine-induced increase in mucus volume output (Figure 3). By contrast, there was no change in the methacholine-induced increase in mucus volume output in control tracheae not exposed to PAF (Figure 3).
Lysozyme output Methacholine (1 pM) increased lysozyme output from 23 + 6 and 21 + 9ngmin1 to 214 + 80 and 158 + 39 ng min-1 in control and test tracheae respectively. These responses to methacholine are not significantly different. PAF (0.1-10 uM) concentration-dependently enhanced the methacholine-induced increase in lysozyme output (Figure 4). There was no change in methacholine-induced lysozyme output in the control tracheae not exposed to PAF (Figure 4).
Albumin output Methacholine (1 pM) increased albumin output from 0.23 + 0.07 and 0.33 + 0.06pgmin-' to 5.5 + 1.9 and 6.1 + 1.6pgmin-m in control and test tracheae respectively. These responses to methacholine are not significantly different. PAF (0.1-10pM) produced a concentration-
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dependent inhibition of the methacholine-induced increase in albumin output (Figure 4b). There was no change in methacholine-induced albumin output in control tracheae (Figure 4). The concentration-related effects of PAF are not simply due to an additive effect of the drug since in preliminary experiments, repeated exposure to the same concentration of PAF resulted in the same increase in responsiveness to methacholine. For example, in the case of smooth muscle, the was (1 gM) methacholine to control response +68 + 6mmH20 (n = 6); this was increased by 72 + 4,
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Figure 4 Concentration-response effects of tracheal exposure to PAF (0.1-1OpM) on (a) the increase in lysozyme and (b) albumin output produced by methacholine (1pM). Responses to methacholine have been expressed as a fraction of the first control response to methacholine. (i) Control tracheae not exposed to PAF; (ii) tracheae exposed to PAF. (El) Control responses to methacholine; responses to methacholine after exposure to PAF, 0.1 fIM (0), 1 gM (f) and 10PM (0). Responses are the means of six determinations with s.e.means shown by vertical lines. * Significantly different (P < 0.05) from first control to methacholine.
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Effects of antagonists None of the antagonists themselves had any significant effect on the responses produced by methacholine; the smooth muscle contraction, and mucus volume, lysozyme and albumin outputs produced by methacholine in control and test tracheae were not significantly different (Table 2). FPL55712 (7-[(4-acetyl-3-hydroxy-2Indomethacin, propylphenoxy)- 2- hydroxypropoxy] -4 oxo 8 -propyl -4H 1 benzopyran-2-carboxylic acid) and mepyramine with cimetidine had no effect on the PAF-induced increase in smooth -
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Table 2 The effect of pharmacological antagonists on the responses to methacholine (1 PM) Antagonist
Indomethacin FPL55712 Mepyramine + cimetidine Catalase + SOD WEB2086
Luminal pressure (mmH2O) + Antagonist Control 73 + 8 68 + 2 64+3 70 + 9 75 + 6
71 60 70 68 73
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+ 0.05
+ 0.14 + 0.11 + 0.20 + 0.09
0.56 + 0.20 0.51 + 0.06 0.42 + 0.08 0.50 + 0.15 0.47 + 0.03
Lysozyme output (ngmin-1) Control + Antagonist 123 206 215 316 84
+ 35 +46 ± 22 + 28 +9
Values given are means + s.e.means with n = 6. None of the antagonists has had any effect and albumin outputs produced by methacholine. SOD: superoxide dismutase.
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muscle contraction, mucus volume and lysozyme output to methacholine, or on the PAF-induced inhibition of methacholine-induced albumin output (Table 3). A combination of catalase and SOD significantly inhibited the PAFinduced increase in responsiveness of smooth muscle, mucus volume and lysozyme, and the decreased albumin output to methacholine (Figure 5). Similarly the PAF-antagonist WEB 2086 (3-(4-(2-chlorophenyl)-9-methyl-6H-thieno-(3,2-t) (1,2,4)-triazolo-(4,3-a) (1,4)-diazepine-2-yl)-1-(4-morpholinyl)-1propanone) abolished the changes in methacholine-induced responsiveness produced by PAF (Figure 6).
responsiveness is also not due to release of cyclo-oxygenase products, leukotrienes C4 and D4, or histamine since indomethacin, FPL55712 and, mepyramine and cimetidine were without effect. In contrast, the hyperresponsiveness was significantly attenuated by catalase and SOD suggesting that it is probably mediated, at least in part, by the release of oxygen free-radicals. This free-radical release is likely to be mediated by specific PAF-receptors since the hyperresponsiveness was also abolished by WEB2086. Leukotriene D4-induced hyperresponsiveness of guinea-pig tracheal smooth muscle to histamine is also prevented by pretreatment with SOD (Weiss & Bellino, 1986). Direct application of oxygen free-radicals also induces hyperresponsiveness of cat airway smooth muscle to
Discussion
duces hyperresponsiveness of rat bronchi to electrical field stimulation in vitro (Szarek & Schmidt, 1990). In contrast, there was no hyperresponsiveness to histamine of guinea-pig trachea in vitro after exposure to H202 (Rhoden & Barnes, 1989). The reason for these conflicting reports is not clear but may represent a species difference. In the present study there was also an increase in the responses of the submucosal glands (mucus volume and lysozyme output) to methacholine after PAF exposure. The concentration-response curves for methacholine-induced mucus volume and lysozyme output were both shifted upwards after PAF exposure and the maximum responses were increased. There was also an increase in the potency of methacholine in stimulating mucus volume output. There was no increase in potency for lysozyme output, but at each concentration of methacholine the response was significantly enhanced after PAF exposure. Thus PAF induces a hyperresponsiveness of ferret submucosal gland secretion to methacholine and since lysozyme output was also increased at least part of this hyperresponsiveness is secretion from serous cells. As with tracheal smooth muscle the degree of hyperresponsiveness was dependent on the concentration of PAF that the trachea was exposed to and no hyperresponsiveness of submucosal gland secretion was seen in tracheae not exposed to PAF. As with smooth muscle, the enhanced response to methacholine produced by PAF cannot be due to an additive effect of this mediator as PAF itself has no effect on baseline mucus volume or lysozyme output. The hyperresponsiveness of submucosal gland secretion after PAF is a novel response which has not previously been reported even in vivo in the presence of platelets. The submucosal gland secretion hyperresponsiveness, like that of the smooth muscle, is not due to release of cyclo-oxygenase products, leukotrienes or histamine as the respective antagonists were without effect, but is mediated by PAF receptor-induced release of oxygen radicals since it was prevented by catalase and SOD, and by WEB2086. There have been no previous studies on the effects of free-radicals on mucus secretion. Methacholine produced a concentration-dependent increase in the output of albumin into the ferret tracheal lumen suggesting stimulation of epithelial albumin transport. The concentration-response curve for methacholine-induced albumin output was shifted downwards after PAF exposure with a greatly reduced maximum response. Although there was no change in the potency of methacholine, at each concentration of methacholine albumin output was significantly
acetylcholine in vivo (Katsumata et al., 1990), and H202
PAF induces a non-selective and long lasting increase in bronchial smooth muscle responsiveness to several mediators including histamine, methacholine and 5-hydroxytryptamine in intact guinea-pigs (Mazzoni et al., 1985; Robertson & Page, 1987; Fitzgerald et al., 1987), dogs (Chung et al., 1986), sheep (Christman et al., 1987) and healthy human subjects (Cuss et al., 1986). The hyperresponsiveness to intravenous PAF in guinea-pigs seems to depend on platelets, since platelet depletion with specific cytotoxic agents abrogates the hyperresponsiveness (Mazzoni et al., 1985). However the hyperresponsiveness to inhaled PAF in guinea-pigs is plateletindependent (Lefort et al., 1984; Sanjar et al., 1990), whereas that in rabbits is again dependent on platelets (Coyle et al., 1990). Clearly there are species and route of administration differences in the role of platelets in PAF-induced smooth muscle hyperresponsiveness. The exact mechanism by which PAF produces hyperresponsiveness of airway smooth muscle in vivo is unclear but may involve the release of thromboxane from inflammatory cells (Chung et al., 1986), or other mediators from eosinophils (Coyle et al., 1990). In plateletdependent responses it may be the platelets themselves that are regulating the influx and activation of other inflammatory cells such as the eosinophil (Coyle et al., 1990). In the present in vitro study, the concentration-response curve for methacholine-induced tracheal smooth muscle contraction was shifted to the left and upwards after exposure to PAF with an increase in both the potency of methacholine and the maximum contraction produced by methacholine. Thus, PAF induces a hyperresponsiveness of ferret tracheal smooth muscle to methacholine in vitro. This enhancement in response to methacholine is unlikely to be due to an additive effect of PAF as PAF has a direct relaxant effect on the ferret tracheal smooth muscle in vitro (Webber et al., 1992). This hyperresponsiveness depends on the concentration of PAF that the trachea is exposed to and no change in responsiveness to methacholine was seen in tracheae which were not exposed to PAF. PAF also induces increased tracheal responsiveness to parasympathetic stimulation in the dog, and this may be due to a direct hyperresponsiveness to acetylcholine (Bethel et al., 1989). In contrast to the in vivo studies described above, the hyperresponsiveness to PAF in vitro cannot depend on platelets; it is also unlikely to be due to release of mediators from inflammatory cells since histological analysis showed there are very few such cells in the in vitro trachea. The hyper-
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reduced after PAF exposure. Thus PAF exposure inhibits the stimulation of epithelial albumin transport produced by methacholine. This loss of epithelial function is consistent with the reduction in PD across the trachea seen with PAF (Webber et al., 1992). As with the reduction in PD, and the smooth muscle and submucosal gland hyperresponsiveness, the PAFinduced inhibition of albumin output to methacholine was attenuated by catalase and SOD, and abolished by WEB2086 again suggesting the involvement of receptor-mediated release of free-radicals.
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Figure 6 The effects of WEB2086 on (a) the changes in responsiveness of tracheal smooth muscle and (b) mucus, (c) lysozyme and (d) albumin outputs to methacholine produced by PAF (1 FM). (i) Control tracheae where WEB2086 was absent; (ii) test tracheae where WEB2086 was present. (l) Control response to methacholine; (U) response to methacholine after exposure to PAF. All responses to methacholine have been expressed as a fraction of the first control response to methacholine. n = 6. * Significant change in response to methacholine after PAF. t Significant inhibition by WEB2086 of the change in responsiveness produced by PAF.
In summary, the addition of PAF to the ferret trachea in vitro leads to the production of a cyclo-oxygenase product which immediately relaxes the smooth muscle and oxygen free-radicals which reduce the PD indicating a change in epithelial function (Webber et al., 1992). Neither the cyclooxygenase product or the free-radicals have any effect on basal submucosal gland secretion or epithelial albumin transport (Webber et al., 1992). However, the results from the study described here suggest the free-radicals induce a hyperresponsiveness of submucosal gland secretion as well as
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smooth muscle contraction to the muscarinic agonist methacholine; but they inhibit methacholine-stimulated epithelial albumin transport. The hyperresponsiveness of smooth muscle and submucosal gland secretion occurs in vitro in the absence of circulating platelets or other inflammatory cells demonstrating that these cells are not essential for PAF-induced changes
237
in responsiveness. The cellular source of the dilator cyclooxygenase product and the free-radicals is not known but the epithelium is a likely candidate. Similarly the exact mechanism behind the free radical-induced changes in responsiveness to methacholine are not known.
References BETHEL, R.A., CURTIS, S.P., LIEN, D.C., IRVIN, C.G., WORTHEN, G.S.,
LEFF, A.B. & HENSON, P.M. (1989). Effect of PAF on parasympathetic contraction of canine airways. J. Appl. Physiol., 66, 26292634. CHRISTMAN, B.W., LEFFERTS, P.L. & SNAPPER, J.P. (1987). Effect of Platelet-activating factor on aerosol histamine responses in awake sheep. Am. Rev. Respir. Dis., 135, 1267-1270. CHUNG, K.F., AIZCIWA, H., LEIKAUF, G.D., UEKI, I.F., EVANS, T.W. &
NADEL, J.A. (1986). Airway hyperresponsiveness induced by platelet-activating factor: role of thromboxane generation. J. Pharmacol. Exp. Ther., 236, 580-584. COYLE, A.J., SPINA, D. & PAGE, C.P. (1990). PAF-induced bronchial hyperresponsiveness in the rabbit: contribution of platelets and airway smooth muscle. Br. J. Pharmacol., 101, 31-38. CUSS, F.M., DIXON, C.M.S. & BARNES, P.J. (1986). Effects of inhaled platelet-activating factor on pulmonary function and bronchial responsiveness in man. Lancet, ii, 189-192. FITZGERALD, M.F., LEES, I., PARENTE, L. & PAYNE, A.N. (1987). Exposure to Paf-acether aerosol induces airway hyperresponsiveness to 5-HT in guinea-pigs. Br. J. Pharmacol., 90, 112P. KATSUMATA, U., MIURA, M., ICHINOSE, M., KIMURA, K., TAKAHASHI, T., INOUE, H. & TAKISHIMA, T. (1990). Oxygen radicals
produce airway constriction and hyperresponsiveness in anesthetised cats. Am. Rev. Respir. Dis., 141, 1158-1161. LEFORT, J., ROTILIO, D. & VARGAFTIG, B.B. (1984). The plateletindependent release of thromboxane A2 by Paf-acether from guinea-pig lungs involves mechanisms distinct from those for leukotriene. Br. J. Pharmacol., 82, 565-575. MAZZONI, I., MORLEY, J., PAGE, C.P. & SANJAR, S. (1985). Induction of airway hyperreactivity by platelet-activating factor in the guinea-pig. J. Physiol., 365, 107P.
RHODEN, K.J. & BARNES, P.J. (1989). Effect of hydrogen peroxide on guinea-pig tracheal smooth muscle in vitro: role of cyclooxygenase and airway epithelium. Br. J. Pharmacol., 98, 325-330. ROBERTSON, D.N. & PAGE, C.P. (1987). Effect of platelet agonists on airway reactivity and intrathoracic platelet accumulation. Br. J. Pharmacol., 92, 105-111. SANJAR, S., AOKI, S., BOUBEKEUR, K., CHAPMAN, I.D., SMITH, D.,
KINGS, M.A. & MORLEY, J. (1990). Eosinophil accumulation in pulmonary airways of guinea-pigs induced by exposure to an aerosol of platelet-activating factor: effect of anti-asthma drugs. Br. J. Pharmacol., 99, 267-272. SZAREK, J.L & SCHMIDT, N.L. (1990). Hydrogen peroxide-induced potentiation of contractile responses in isolated rat airways. Am. J. Physiol., 258, L232-L237. WEBBER, S.E. & WIDDICOMBE, J.G. (1987). The effect of VIP on smooth muscle tone and mucus secretion from the ferret trachea. Br. J. Pharmacol., 91, 139-148. WEBBER, S.E. & WIDDICOMBE, J.G. (1989). The transport of albumin across the ferret in-vitro trachea. J. Physiol., 408, 457-472. WEBBER, S.E., MORIKAWA, T. & WIDDICOMBE, J.G. (1992). Plateletactivating factor relaxes ferret tracheal smooth muscle and reduces transepithelial potential difference in vitro. Br. J. Pharmacol., 105, 223-229. WEISS, E.B. & BELLINO, J.R. (1986). Leukotriene-associated toxic oxygen metabolites induce airway hyperreactivity. Chest, 89, 709716.
(Received July 25, 1990 Revised August 10, 1991 Accepted September 20, 1991)
(D Macmillan Press Ltd, 1992
PAF-induced muscarinic cholinoceptor hyperresponsiveness of ferret tracheal smooth muscle and gland secretion in vitro S.E. Webber, T. Morikawa & J.G. Widdicombe Department of Physiology, St George's Hospital Medical School, Cranmer Terrace, London, SW17 ORE The effects of exposure of the ferret trachea in vitro to platelet activating factor (PAF) were examined methacholine-induced smooth muscle contraction, mucus volume and lysozyme outputs, and albumin transport across the tracheal epithelium. 2 Methacholine (0.1-301uM) produced concentration-dependent increases in tracheal smooth muscle tone and mucus volume, lysozyme and albumin outputs from the trachea. 3 The concentration-response curves for methacholine-induced smooth muscle contraction, mucus volume and lysozyme outputs were all shifted upwards after exposure of the trachea to PAF (1 uM) with a significant increase in maximum response for each variable. The EC50 values for methacholine-induced smooth muscle contraction and mucus volume output were significantly reduced after PAF exposure suggesting an increase in the potency of methacholine. The concentration-response curve for methacholine-induced albumin output was shifted downwards after PAF exposure with a greatly reduced maximum but no change in the EC50 for methacholine. 4 PAF-induced hyperresponsiveness of methacholine-induced smooth muscle contraction, mucus volume and lysozyme outputs was not affected by indomethacin, FPL55712, or mepyramine and cimetidine, but was prevented by catalase and superoxide dismutase (SOD), and by WEB2086. Similarly, PAFinduced inhibition of methacholine-stimulated albumin output was prevented by catalase and SOD, and by WEB2086. 5 We conclude that PAF induces hyperresponsiveness of ferret tracheal smooth muscle and submucosal gland secretion (including lysozyme secretion from serous cells) to methacholine. This hyperresponsiveness is probably produced by receptor-mediated release of oxygen free-radicals. The inhibition of methacholine-induced albumin flux suggests a loss of epithelial function which is also probably mediated by release of free-radicals. The mechanism by which the free-radicals produce the changes in responsiveness to methacholine, and the cellular source of the free-radicals, remain to be established. Keywords: Platelet activating factor; methacholine; mucus; smooth muscle; albumin; catalase; superoxide dismutase; WEB2086 1
on
Introduction
Methods
In the previous paper we described the direct effects of platelet activating factor (PAF) on ferret tracheal smooth muscle, submucosal gland secretion, epithelial albumin transport and potential difference (PD) across the trachea (Webber et al., 1992). Perhaps the most important property of PAF is its ability to induce a potent and long-lasting hyperresponsiveness of airway smooth muscle in vivo (Mazzoni et al., 1985; Cuss et al., 1986; Robertson & Page, 1987). This hyperresponsiveness, like PAF-induced bronchoconstriction, may be dependent on the presence of circulating platelets (Mazzoni et al., 1985) and may involve the release of mediators from eosinophils (Sanjar et al., 1990). However, whether PAF can induce a hyperresponsiveness of airway smooth muscle in vitro, predominantly in the absence of circulating inflammatory cells, has not been studied systematically. Furthermore, it is not known whether PAF changes the responsiveness of other airway effector tissues such as submucosal glands and epithelium in a way similar to that with airway smooth muscle. The muscarinic agonist, methacholine, potently contracts tracheal smooth muscle and stimulates submucosal gland secretion from the ferret trachea in vitro (Webber & Widdicombe, 1987). Albumin is actively transported across the ferret tracheal epithelium and this transport is stimulated by methacholine (Webber & Widdicombe, 1989). In the present study we have investigated the effect of exposing the ferret trachea in vitro to PAF on the responses of the smooth muscle, submucosal gland secretion (using lysozyme as a specific marker for serous cells) and epithelial albumin transport produced by methacholine.
The description of the ferret trachea in vitro and of the assays for lysozyme and albumin were described in detail in the previous paper (Webber et al., 1992).
Experimental protocol In the first set of experiments the effects of a single concentration of PAF (1 uM) on concentration-response curves for methacholine-induced tracheal smooth muscle contraction and mucus volume, lysozyme and albumin outputs was examined. In the second set of experiments we investigated the effect of increasing PAF concentrations (0.1-10pM) on the responses to a single concentration of methacholine (1 pM). In the first set of experiments, after an initial 30min control period, three or four different concentrations of methacholine, covering at least a tenfold concentration range, were added to the submucosal buffer bathing the trachea singly and in random sequence. Each concentration was left in contact with the trachea for 30min and during this time any increase in smooth muscle tone was recorded. The trachea was then washed twice and fresh buffer containing no methacholine was placed in the organ bath. One or two control periods of 30 min were allowed between addition of methacholine, depending on how quickly the secretion rate returned to a basal level. After three or four concentrations of methacholine had been added to the trachea, the buffer surrounding the trachea was replaced with buffer containing PAF (1 uM). This buffer was left in contact with the trachea for 1 h and was replaced every 30 min. Any change in smooth muscle tone during this period
PAF-INDUCED HYPERRESPONSIVENESS IN VITRO
231
was recorded, and any mucus produced was withdrawn at the end of each 30min. After' a 30min control period without PAF the same three or four concentrations of methacholine were added to the trachea in the same sequence as before PAF exposure. Methacholine was added to the trachea in a buffer which did not contain PAF. All mucus samples were assayed for lysozyme and albumin (Webber et al., 1992). In the second set of experiments, tracheae were set up in pairs, in separate organ baths. One trachea was exposed twice to methacholine (1uM) for 30min with one or two control periods of 30min between each addition of methacholine. Any changes in smooth muscle tone or mucus volume output produced by methacholine were recorded. After methacholine (1pM) had been added to the trachea twice, the buffer surrounding the trachea was replaced with buffer containing PAF (0.1 pM). This buffer was left in contact with the trachea for 1 h. The trachea was then washed and left for a 30 min control period in buffer containing no PAF. Methacholine (1 M) was then added to the trachea and the changes in smooth muscle tone and mucus volume output were recorded. The procedure described above was repeated twice with the trachea being exposed to PAF (1 M) then PAF (10pM) and responses to methacholine (1pM) after each PAF exposure were recorded. The second trachea acted as a control. Exactly the same procedure as described for the test trachea was carried out except that the second trachea was exposed not to PAF but to buffer containing no PAF.
and maximum responses (+95% confidence limits) for methacholine were estimated by fitting concentration-response curves to the data points by a computerised, non-linear, least squares estimate. ECQ0 is the concentration of methacholine which produced 50% of the maximum response to methacholine. Differences between ECQ0 values or maximum responses for methacholine before and after exposure of the trachea to PAF were analysed for statistical significance by Student's unpaired t test. In the second set of experiments differences between control responses (means + s.e.mean) to methacholine and responses to methacholine after PAF were analysed for significance by Student's paired t test. The effects of antagonists on the PAF-induced changes in responsiveness to methacholine were also analysed for signifiance by Student's paired t test. In all cases P < 0.05 was taken as significant.
Effects of antagonists To examine the effects of antagonists on PAF-induced changes in responsiveness to methacholine, tradheae were again set up in pairs. Exactly the same protocol was used for both tracheae except the buffer used for the test trachea contained one of the antagonists. Both tracheae were exposed to methacholine (1 pM) for 30 min and any changes in smooth
Mucus volume output The mucus volume output in control periods before addition of methacholine was 0.07 + 0.05 ,ul min' (n = 24). Methacholine produced a concentration-dependent increase in mucus volume output from this control level (Figure 1). This concentration-response curve for methacholine was shifted upwards and leftwards after exposure of the trachea to PAF (1 pM) with a significantly reduced ECQ0 value (3.3 fold, Table 1) and a significantly increased maximum response (38%, Table 1). Lysozyme output The lysozyme output in control periods before addition of methacholine was 16 + 11ngmin-1 (n = 24). Methacholine produced a concentration-dependent increase in lysozyme output from these control levels (Figure 2). This concentration-response curve for methacholine was shifted upwards after exposure of the trachea to PAF with a significantly increased maximum response (73%, Table 1), but no significant change in the ECQ0 (Table 1). Albumin output The albumin output in controls before methacholine was 0.32 + 0.14 pgmin-' (n = 24). Methacholine produced a concentration-dependent increase in albumin output from this control level (Figure 2). After exposure of the trachea to PAF, the maximum response for methacholine was greatly reduced (50%, Table 1) but there was no significant change in the ECQ0 value (Table 1). Concentration-related effects of PAF (0.1-JOpM) on responses to methacholine (IpM) Baseline values in control and test tracheae The baseline mucus volume output in control tracheae (tracheae which did
muscle tone or mucus volume output were recorded. After two control periods the tracheae were then exposed to PAF (1 pM) for 1 h. The tracheae were then washed thoroughly and left for a further control period of 30min. The responsiveness of the tracheae to methacholine (1 pM) was then re-assessed. The antagonists tested were indomethacin (1pM), FPL55712 (1pM), a combination of mepyramine (1 M) and cimetidine (10uM), a combination of catalase (500 u ml 1) and superoxide dismutase (40 u ml- 1), and WEB 2086 (10pM). An exposure time for PAF of 1 h was used in all experiments because this was shown to be optimal in preliminary studies. Thus, the maximum degree of hyperresponsiveness occurred with a 1 h exposure and there was no further increase in the effect of PAF with exposure times greater than 1 h. For example, in the case of smooth muscle tone, the increase in response to methacholine (1 pM) was 42 + 5% (n = 6) with a 30 min exposure to PAF, 71 + 6% (n = 6) with a 1 h exposure and 68 + 4% (n = 6) with a 2 h exposure.
Analysis of results The concentration-response curves and bar charts presented in Results were obtained by pooling the results from groups of experiments. In the first set of experiments, the EC50 values Table 1
Results Effect of PAF (1 pM) on concentration-response curvesfor met hacholine Intraluminal pressure Methacholine (0.1-3O0pM) produced a concentration-dependent increase in intraluminal pressure (Figure 1). This concentration-response curve was shifted upwards and leftwards after exposure of the trachea to PAF (1 pm, Figure 1) with a significantly reduced ECQ0 value (3 fold, Table 1) and a significant increase in maximum response (32%, Table 1).
ECQ0 values and maximum responses (±95% confidence limits) for methacholine before and after exposure of the trachea to
PAF (1 uM)
EC50 (M) Before PAF Intraluminal pressure (mmH20) Mucus volume output (pulmin-') Lysozyme output (ng min -) Albumin output (ug min - 1) *
1.8 1.3 1.2 0.9
(1.6-2.0) (1.1-1.5) (0.9-1.5) (0.6-1.2)
Maximum response
After PAF
Before PAF
After PAF
0.6 (0.4-0.8)* 0.4 (0.3-0.5)* 1.0 (0.8-1.2) 1.0 (0.7-1.3)
127 (121-133) 1.62 (1.56-1.68) 395 (380-410) 2.43 (2.33-2.53)
168 (162-174)* 2.24 (2.21-2.27)* 570 (520-620)* 1.22 (1.18-1.26)*
Significantly different (P < 0.05) from value before exposure to PAF using Student's unpaired t test.
232
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not receive PAF) before addition of any drugs was 0.06 + to (n = 6), compared 0.04 + 0.04 PI min-1 0.03 p1mmin- ' (n = 6) in test tracheae (tracheae before exposure to PAF). Similarly the baseline lysozyme outputs in control and test tracheae were 27 + 12 and 21 + 6 ng min-1 respectively, and the baseline albumin outputs were 0.18 + 0.09 and 0.27 + 0.16pjugmin'- respectively. The PD's across control and test tracheae before addition of drugs were -8.2 + 0.4 and -7.9 + 0.6 mV respectively. There are no significant differences between baseline values in control and test tracheae for any of these variables.
Intraluminal pressure Methacholine (1 pM) increased intraluminal pressure by 75 + 5 mmH2O (n = 6) in control tracheae and by 63 + 9 mmH2O in test tracheae. These responses are not significantly different. Exposure of the trachea to PAF (0.1-lOpM) produced a concentration-dependent enhancement of the increase in intraluminal pressure produced by methacholine (Figure 3). By contrast there was no significant change in the tracheal smooth muscle response to methacholine in control tracheae not exposed to PAF (Figure 3). Mucus volume output Methacholine (I pM) increased mucus volume output from 0.06 + 0.04 and 0.05 + 0.04up1 min' to
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1.05 + 0.15 and 1.13 + 0.l9pl-' in control and test tracheae respectively. The responses in control and test tracheae were not significantly different. Exposure of the trachea to PAF (0.1-10 pM) produced a concentration-dependent enhancement of the methacholine-induced increase in mucus volume output (Figure 3). By contrast, there was no change in the methacholine-induced increase in mucus volume output in control tracheae not exposed to PAF (Figure 3).
Lysozyme output Methacholine (1 pM) increased lysozyme output from 23 + 6 and 21 + 9ngmin1 to 214 + 80 and 158 + 39 ng min-1 in control and test tracheae respectively. These responses to methacholine are not significantly different. PAF (0.1-10 uM) concentration-dependently enhanced the methacholine-induced increase in lysozyme output (Figure 4). There was no change in methacholine-induced lysozyme output in the control tracheae not exposed to PAF (Figure 4).
Albumin output Methacholine (1 pM) increased albumin output from 0.23 + 0.07 and 0.33 + 0.06pgmin-' to 5.5 + 1.9 and 6.1 + 1.6pgmin-m in control and test tracheae respectively. These responses to methacholine are not significantly different. PAF (0.1-10pM) produced a concentration-
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dependent inhibition of the methacholine-induced increase in albumin output (Figure 4b). There was no change in methacholine-induced albumin output in control tracheae (Figure 4). The concentration-related effects of PAF are not simply due to an additive effect of the drug since in preliminary experiments, repeated exposure to the same concentration of PAF resulted in the same increase in responsiveness to methacholine. For example, in the case of smooth muscle, the was (1 gM) methacholine to control response +68 + 6mmH20 (n = 6); this was increased by 72 + 4,
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Figure 4 Concentration-response effects of tracheal exposure to PAF (0.1-1OpM) on (a) the increase in lysozyme and (b) albumin output produced by methacholine (1pM). Responses to methacholine have been expressed as a fraction of the first control response to methacholine. (i) Control tracheae not exposed to PAF; (ii) tracheae exposed to PAF. (El) Control responses to methacholine; responses to methacholine after exposure to PAF, 0.1 fIM (0), 1 gM (f) and 10PM (0). Responses are the means of six determinations with s.e.means shown by vertical lines. * Significantly different (P < 0.05) from first control to methacholine.
+
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Effects of antagonists None of the antagonists themselves had any significant effect on the responses produced by methacholine; the smooth muscle contraction, and mucus volume, lysozyme and albumin outputs produced by methacholine in control and test tracheae were not significantly different (Table 2). FPL55712 (7-[(4-acetyl-3-hydroxy-2Indomethacin, propylphenoxy)- 2- hydroxypropoxy] -4 oxo 8 -propyl -4H 1 benzopyran-2-carboxylic acid) and mepyramine with cimetidine had no effect on the PAF-induced increase in smooth -
-
-
-
234
S.E. WEBBER et al.
Table 2 The effect of pharmacological antagonists on the responses to methacholine (1 PM) Antagonist
Indomethacin FPL55712 Mepyramine + cimetidine Catalase + SOD WEB2086
Luminal pressure (mmH2O) + Antagonist Control 73 + 8 68 + 2 64+3 70 + 9 75 + 6
71 60 70 68 73
+ + + + +
4 7 3 5 5
Mucus volume output (l min -1) Control + Antagonist 0.56 0.42 0.40 0.65 0.44
+ 0.05
+ 0.14 + 0.11 + 0.20 + 0.09
0.56 + 0.20 0.51 + 0.06 0.42 + 0.08 0.50 + 0.15 0.47 + 0.03
Lysozyme output (ngmin-1) Control + Antagonist 123 206 215 316 84
+ 35 +46 ± 22 + 28 +9
Values given are means + s.e.means with n = 6. None of the antagonists has had any effect and albumin outputs produced by methacholine. SOD: superoxide dismutase.
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132 252 192 357 86
Albumin output (pg min-') Control + Antagonist
+ 33
+ 54 + 23 + 17 +5
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lysozyme
muscle contraction, mucus volume and lysozyme output to methacholine, or on the PAF-induced inhibition of methacholine-induced albumin output (Table 3). A combination of catalase and SOD significantly inhibited the PAFinduced increase in responsiveness of smooth muscle, mucus volume and lysozyme, and the decreased albumin output to methacholine (Figure 5). Similarly the PAF-antagonist WEB 2086 (3-(4-(2-chlorophenyl)-9-methyl-6H-thieno-(3,2-t) (1,2,4)-triazolo-(4,3-a) (1,4)-diazepine-2-yl)-1-(4-morpholinyl)-1propanone) abolished the changes in methacholine-induced responsiveness produced by PAF (Figure 6).
responsiveness is also not due to release of cyclo-oxygenase products, leukotrienes C4 and D4, or histamine since indomethacin, FPL55712 and, mepyramine and cimetidine were without effect. In contrast, the hyperresponsiveness was significantly attenuated by catalase and SOD suggesting that it is probably mediated, at least in part, by the release of oxygen free-radicals. This free-radical release is likely to be mediated by specific PAF-receptors since the hyperresponsiveness was also abolished by WEB2086. Leukotriene D4-induced hyperresponsiveness of guinea-pig tracheal smooth muscle to histamine is also prevented by pretreatment with SOD (Weiss & Bellino, 1986). Direct application of oxygen free-radicals also induces hyperresponsiveness of cat airway smooth muscle to
Discussion
duces hyperresponsiveness of rat bronchi to electrical field stimulation in vitro (Szarek & Schmidt, 1990). In contrast, there was no hyperresponsiveness to histamine of guinea-pig trachea in vitro after exposure to H202 (Rhoden & Barnes, 1989). The reason for these conflicting reports is not clear but may represent a species difference. In the present study there was also an increase in the responses of the submucosal glands (mucus volume and lysozyme output) to methacholine after PAF exposure. The concentration-response curves for methacholine-induced mucus volume and lysozyme output were both shifted upwards after PAF exposure and the maximum responses were increased. There was also an increase in the potency of methacholine in stimulating mucus volume output. There was no increase in potency for lysozyme output, but at each concentration of methacholine the response was significantly enhanced after PAF exposure. Thus PAF induces a hyperresponsiveness of ferret submucosal gland secretion to methacholine and since lysozyme output was also increased at least part of this hyperresponsiveness is secretion from serous cells. As with tracheal smooth muscle the degree of hyperresponsiveness was dependent on the concentration of PAF that the trachea was exposed to and no hyperresponsiveness of submucosal gland secretion was seen in tracheae not exposed to PAF. As with smooth muscle, the enhanced response to methacholine produced by PAF cannot be due to an additive effect of this mediator as PAF itself has no effect on baseline mucus volume or lysozyme output. The hyperresponsiveness of submucosal gland secretion after PAF is a novel response which has not previously been reported even in vivo in the presence of platelets. The submucosal gland secretion hyperresponsiveness, like that of the smooth muscle, is not due to release of cyclo-oxygenase products, leukotrienes or histamine as the respective antagonists were without effect, but is mediated by PAF receptor-induced release of oxygen radicals since it was prevented by catalase and SOD, and by WEB2086. There have been no previous studies on the effects of free-radicals on mucus secretion. Methacholine produced a concentration-dependent increase in the output of albumin into the ferret tracheal lumen suggesting stimulation of epithelial albumin transport. The concentration-response curve for methacholine-induced albumin output was shifted downwards after PAF exposure with a greatly reduced maximum response. Although there was no change in the potency of methacholine, at each concentration of methacholine albumin output was significantly
acetylcholine in vivo (Katsumata et al., 1990), and H202
PAF induces a non-selective and long lasting increase in bronchial smooth muscle responsiveness to several mediators including histamine, methacholine and 5-hydroxytryptamine in intact guinea-pigs (Mazzoni et al., 1985; Robertson & Page, 1987; Fitzgerald et al., 1987), dogs (Chung et al., 1986), sheep (Christman et al., 1987) and healthy human subjects (Cuss et al., 1986). The hyperresponsiveness to intravenous PAF in guinea-pigs seems to depend on platelets, since platelet depletion with specific cytotoxic agents abrogates the hyperresponsiveness (Mazzoni et al., 1985). However the hyperresponsiveness to inhaled PAF in guinea-pigs is plateletindependent (Lefort et al., 1984; Sanjar et al., 1990), whereas that in rabbits is again dependent on platelets (Coyle et al., 1990). Clearly there are species and route of administration differences in the role of platelets in PAF-induced smooth muscle hyperresponsiveness. The exact mechanism by which PAF produces hyperresponsiveness of airway smooth muscle in vivo is unclear but may involve the release of thromboxane from inflammatory cells (Chung et al., 1986), or other mediators from eosinophils (Coyle et al., 1990). In plateletdependent responses it may be the platelets themselves that are regulating the influx and activation of other inflammatory cells such as the eosinophil (Coyle et al., 1990). In the present in vitro study, the concentration-response curve for methacholine-induced tracheal smooth muscle contraction was shifted to the left and upwards after exposure to PAF with an increase in both the potency of methacholine and the maximum contraction produced by methacholine. Thus, PAF induces a hyperresponsiveness of ferret tracheal smooth muscle to methacholine in vitro. This enhancement in response to methacholine is unlikely to be due to an additive effect of PAF as PAF has a direct relaxant effect on the ferret tracheal smooth muscle in vitro (Webber et al., 1992). This hyperresponsiveness depends on the concentration of PAF that the trachea is exposed to and no change in responsiveness to methacholine was seen in tracheae which were not exposed to PAF. PAF also induces increased tracheal responsiveness to parasympathetic stimulation in the dog, and this may be due to a direct hyperresponsiveness to acetylcholine (Bethel et al., 1989). In contrast to the in vivo studies described above, the hyperresponsiveness to PAF in vitro cannot depend on platelets; it is also unlikely to be due to release of mediators from inflammatory cells since histological analysis showed there are very few such cells in the in vitro trachea. The hyper-
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reduced after PAF exposure. Thus PAF exposure inhibits the stimulation of epithelial albumin transport produced by methacholine. This loss of epithelial function is consistent with the reduction in PD across the trachea seen with PAF (Webber et al., 1992). As with the reduction in PD, and the smooth muscle and submucosal gland hyperresponsiveness, the PAFinduced inhibition of albumin output to methacholine was attenuated by catalase and SOD, and abolished by WEB2086 again suggesting the involvement of receptor-mediated release of free-radicals.
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236
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smooth muscle contraction to the muscarinic agonist methacholine; but they inhibit methacholine-stimulated epithelial albumin transport. The hyperresponsiveness of smooth muscle and submucosal gland secretion occurs in vitro in the absence of circulating platelets or other inflammatory cells demonstrating that these cells are not essential for PAF-induced changes
237
in responsiveness. The cellular source of the dilator cyclooxygenase product and the free-radicals is not known but the epithelium is a likely candidate. Similarly the exact mechanism behind the free radical-induced changes in responsiveness to methacholine are not known.
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LEFF, A.B. & HENSON, P.M. (1989). Effect of PAF on parasympathetic contraction of canine airways. J. Appl. Physiol., 66, 26292634. CHRISTMAN, B.W., LEFFERTS, P.L. & SNAPPER, J.P. (1987). Effect of Platelet-activating factor on aerosol histamine responses in awake sheep. Am. Rev. Respir. Dis., 135, 1267-1270. CHUNG, K.F., AIZCIWA, H., LEIKAUF, G.D., UEKI, I.F., EVANS, T.W. &
NADEL, J.A. (1986). Airway hyperresponsiveness induced by platelet-activating factor: role of thromboxane generation. J. Pharmacol. Exp. Ther., 236, 580-584. COYLE, A.J., SPINA, D. & PAGE, C.P. (1990). PAF-induced bronchial hyperresponsiveness in the rabbit: contribution of platelets and airway smooth muscle. Br. J. Pharmacol., 101, 31-38. CUSS, F.M., DIXON, C.M.S. & BARNES, P.J. (1986). Effects of inhaled platelet-activating factor on pulmonary function and bronchial responsiveness in man. Lancet, ii, 189-192. FITZGERALD, M.F., LEES, I., PARENTE, L. & PAYNE, A.N. (1987). Exposure to Paf-acether aerosol induces airway hyperresponsiveness to 5-HT in guinea-pigs. Br. J. Pharmacol., 90, 112P. KATSUMATA, U., MIURA, M., ICHINOSE, M., KIMURA, K., TAKAHASHI, T., INOUE, H. & TAKISHIMA, T. (1990). Oxygen radicals
produce airway constriction and hyperresponsiveness in anesthetised cats. Am. Rev. Respir. Dis., 141, 1158-1161. LEFORT, J., ROTILIO, D. & VARGAFTIG, B.B. (1984). The plateletindependent release of thromboxane A2 by Paf-acether from guinea-pig lungs involves mechanisms distinct from those for leukotriene. Br. J. Pharmacol., 82, 565-575. MAZZONI, I., MORLEY, J., PAGE, C.P. & SANJAR, S. (1985). Induction of airway hyperreactivity by platelet-activating factor in the guinea-pig. J. Physiol., 365, 107P.
RHODEN, K.J. & BARNES, P.J. (1989). Effect of hydrogen peroxide on guinea-pig tracheal smooth muscle in vitro: role of cyclooxygenase and airway epithelium. Br. J. Pharmacol., 98, 325-330. ROBERTSON, D.N. & PAGE, C.P. (1987). Effect of platelet agonists on airway reactivity and intrathoracic platelet accumulation. Br. J. Pharmacol., 92, 105-111. SANJAR, S., AOKI, S., BOUBEKEUR, K., CHAPMAN, I.D., SMITH, D.,
KINGS, M.A. & MORLEY, J. (1990). Eosinophil accumulation in pulmonary airways of guinea-pigs induced by exposure to an aerosol of platelet-activating factor: effect of anti-asthma drugs. Br. J. Pharmacol., 99, 267-272. SZAREK, J.L & SCHMIDT, N.L. (1990). Hydrogen peroxide-induced potentiation of contractile responses in isolated rat airways. Am. J. Physiol., 258, L232-L237. WEBBER, S.E. & WIDDICOMBE, J.G. (1987). The effect of VIP on smooth muscle tone and mucus secretion from the ferret trachea. Br. J. Pharmacol., 91, 139-148. WEBBER, S.E. & WIDDICOMBE, J.G. (1989). The transport of albumin across the ferret in-vitro trachea. J. Physiol., 408, 457-472. WEBBER, S.E., MORIKAWA, T. & WIDDICOMBE, J.G. (1992). Plateletactivating factor relaxes ferret tracheal smooth muscle and reduces transepithelial potential difference in vitro. Br. J. Pharmacol., 105, 223-229. WEISS, E.B. & BELLINO, J.R. (1986). Leukotriene-associated toxic oxygen metabolites induce airway hyperreactivity. Chest, 89, 709716.
(Received July 25, 1990 Revised August 10, 1991 Accepted September 20, 1991)
