Br. J. Pharmacol. (1992), 105, 223-229

C) Macmillan Press Ltd, 1992

Platelet-activating factor relaxes ferret tracheal smooth muscle and reduces transepithelial potential difference in vitro S.E. Webber, T. Morikawa & J.G. Widdicombe Department of Physiology, St George's Hospital Medical School, Cranmer Terrace, London, SW17 ORE 1 The effects of platelet activating factor (PAF) were examined on the smooth muscle tone, mucus volume, lysozyme and albumin outputs and potential difference (PD) across the ferret tracheal wall. 2 PAF (O.1-1OpuM) had no direct effect on mucus volume, lysozyme or albumin output from the ferret trachea. PAF produced concentration-dependent relaxations of the tracheal smooth muscle and reductions in PD across the tracheal wall. There was no change in the histological appearance of the trachea after exposure to PAF. 3 The PAF-induced smooth muscle relaxation was not affected by FPL55712, a combination of mepyramine and cimetidine, or by a combination of the oxygen free-radical scavengers catalase and superoxide dismutase (SOD); but was abolished by indomethacin or the PAF-receptor antagonist WEB2086. 4 The PAF-induced reduction in PD was not affected by indomethacin, FPL55712 or mepyramine and cimetidine, but was prevented by catalase and SOD, and by WEB2086. 5 We conclude that PAF relaxes ferret tracheal smooth muscle in vitro by receptor-mediated release of a bronchodilator prostaglandin, possibly PGE2. PAF also reduces PD across the trachea suggesting changes in epithelial function; however, there is no histological epithelial damage after PAF. The reduction in PD with PAF is probably produced by receptor-mediated release of oxygen free-radicals. The cellular source of these free-radicals and of the dilator prostaglandin is unclear. Keywords: Platelet activating factor; trachea; mucus; smooth muscle; albumin; potential difference

Introduction

Methods

Platelet-activating factor (PAF) 1-O-alkyl-2-acetyl-sn-glycero3-phosphocholine (Benveniste et al., 1979) is a derivative of phosphatidyicholine which is released from a variety of inflammatory cells including basophils, mast cells, platelets, macrophages and neutrophils (Camussi et al., 1981; Benveniste et al., 1982). When released, PAF has potent effects on the airways including bronchoconstriction in vivo in both experimental animals and man (Morley et al., 1984; Cuss et al., 1986) increased microvascular permeability (O'Donnell & Barnett, 1987; Evans et al., 1987) and chemotaxis and activation of inflammatory cells (Morley & Page, 1984). In contrast to its bronchoconstrictor action in vivo, PAF does not contract airway smooth muscle in vitro; it either has no effect (Schellenberg, 1987) or produces a relaxation (Brunelleschi et al., 1987), suggesting the in vivo action is indirect and possibly occurs predominantly through the release of bronchoconstrictor mediators from platelets (Schellenberg, 1987). The effects of PAF on airway mucus secretion have not been examined in detail, although high doses of PAF increase the secretion of radiolabelled macromolecules from ferret trachea in vivo (Hahn et al., 1985) and explants of human airways (Goswami et al., 1987; Rogers et al., 1990; Lundgren et al., 1990). The ferret whole trachea in vitro is a preparation which

The ferret trachea in vitro

allows the simultaneous assessment of smooth muscle tone and submucosal gland secretion (Webber & Widdicombe, 1987; Webber, 1989). Lysozyme is a bactericidal enzyme localized specifically to serous cells (Tom-Moy et al., 1983) and is a useful marker for secretion from these cells. Albumin is actively transported across the ferret tracheal epithelium in vitro (Webber & Widdicombe, 1989) and the measurement of this transport together with the measurement of potential difference (PD) across the tracheal wall provides a good indication of epithelial function and integrity. In the present study we have investigated the effects of PAF on tracheal smooth muscle tone, submucosal gland secretion, epithelial albumin transport, and transepithelial PD in the ferret trachea in vitro.

Ferrets of either sex, weighing 0.5-1.5kg, were anaesthetized by an intraperitoneal injection of sodium pentobarbitone (Sagatal, May & Baker, 50mg kg- 1). The trachea was exposed and cannulated about 5 mm below the larynx with a perspex cannula containing a conical collecting well (Webber & Widdecombe, 1987). The ferret was then killed with an overdose of sodium pentobarbitone injected into the heart. The chest was opened along the midline and the trachea exposed to the carina, cleared of adjacent tissue, removed and cannulated just above the carina. The trachea was mounted, laryngeal end down, in a jacketed organ bath with Krebs-Henseleit buffer restricted to the submucosal side. The composition of the Krebs-Henseleit solution was (mM): NaCl 120.8, KCI 4.7, KH2PO4 1.2, MgSO47H2O 1.2, NaHCO3 24.9, CaCl2 2.4, glucose 5.6. The buffer was maintained at 370C and gassed with 95% 02:5% CO2. The lumen of the trachea remained air-filled. Secretions were carried by gravity and mucociliary transport to the lower cannula, where they pooled and could be withdrawn periodically into a polyethylene catheter which was inserted into the lower cannula to form an airtight seal. The catheters containing the secretions were sealed at both ends with bone wax, numbered and stored frozen until required. After defrosting, the secretions were washed out of the catheters into labelled plastic vials with 0.5ml distilled H20. The vials were frozen and stored for use in the albumin and lysozyme assays. Preliminary experiments had shown that frozen storage for up to 6 months does not affect the enzymatic activity of lysozyme or the albumin content. Secretion volumes were estimated by the differences in weights of the catheters with secretions and dried without secretions, and the secretion rates were expressed as plminm (assuming 1 g of secretion is equivalent to 1 ml). During an experiment the carinal cannula was attached to a pressure transducer which was connected to a pen-recorder. Changes in smooth muscle tone produced changes in tracheal pressure which were registered by the pressure transducer and

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recorded on the pen-recorder, thus enabling assessment of changes in smooth muscle tone of the trachea during mucus collection. The electrical potential difference (PD) across the tracheal wall was measured with two calomel reference electrodes. These were filled with 3.8 M KCI and placed in separate beakers of the same solution. Electrical contact was made with the preparation by use of two agar bridges. These were constructed from polyethylene tubing (0.5mm internal diameter) filled with 3.8 M KCl in 2.5% w/v agar solution. One bridge was placed in the buffer on the submucosal side of the trachea and the second inserted into a second hole in the perspex cannula used to collect the mucus. Electrical contact between this bridge and the tracheal luminal wall was maintained by the mucus collecting in the perspex cannula. Output from the two electrodes was into a high input impedance buffer amplifier and then displayed on a digital voltmeter. The two agar bridges were initially placed together in 0.15 M NaCl to confirm that this produced a stable potential difference close to 0V. Any residual voltage measured here was subtracted from subsequent measurements of potential difference made in the preparation.

Assayfor lysozyme The lysozyme concentrations of the mucus samples were measured by a turbidimetric assay which relies on the ability of lysozyme to break down the cell wall of the bacterium Micrococcus lysodeikticus. Addition of lysozyme to a solution of the bacteria reduces the turbidity of the solution, thereby leading to a fall in optical density (OD) measured at 450 nm. A stock suspension of M. lysodeikticus of 3 mg ml1 was prepared. When diluted 10 fold (the dilution in the assay) this suspension gives an OD of approximately 0.6 at 450 nm. To produce a standard curve, various concentrations of hen egg white lysozyme (0.5 to 100ngml-') were incubated in duplicate in 1.5 ml potassium phosphate buffer (50 mM, pH 7.4) containing M. lysodeikticus (0.3 mgml- 1), sodium azide (1 mgmlP 1) and bovine serum albumin (BSA, 1 mgmlP'). The BSA was included in the assay for its protein stabilizing effects and the sodium azide was added to prevent the growth of bacteria in the incubating solutions. The reaction mixtures were incubated for 18 h at 37°C. After incubation the OD of each solution was measured at a wavelength of 450 nm with potassium phosphate buffer pH 7.4 containing BSA (1 mg ml -) as a blank. The standard curve was constructed by plotting the fall in OD (reduction in turbidity) against the concentration of lysozyme in the solution. To estimate the concentration of lysozyme in a mucus sample, 20,ul of sample were incubated in 1.5 ml potassium phosphate buffer (50 mm, pH 7.4), exactly as described above for the known concentrations of lysozyme used in the preparation of the standard curve. The lysozyme concentrations (equivalent to hen egg white lysozyme) of the 20,p1 samples and hence of the original mucus samples were estimated from the standard curve. The rate of output of lysozyme was then calculated by dividing the total amount of lysozyme in a mucus sample by the time over which the sample accumulated.

Albumin transport To examine the effect of PAF on methacholine-induced transport of albumin across the ferret trachea, BSA was added to the buffer bathing the submucosal surface of the trachea in a concentration of 4mg ml-'. Fluorescent BSA (0.020.03mgml 1) was also added to the buffer as a marker and enabled an estimate to be made of the total amount of albumin which appeared in the mucus samples. The fluorescence of the mucus samples was measured with a fluorimeter, at an excitation wavelength of 490 nm and an emission wavelength of 550nm. The fluorescent albumin concentration of the mucus samples was estimated from a stan-

dard curve relating fluoresence (arbitrary units) to the concentration of fluorescent albumin (range 25ngml-1 to 3,ugml-1). The total concentration of albumin in the mucus samples was obtained by multiplying the fluorescent albumin concentration (estimated from the standard curve) by the ratio of non-fluorescent to fluorescent albumin used in the experiment. The rate of output of albumin was determined by dividing the total amount of albumin in a mucus sample by the time over which that sample accumulated. Previous studies have shown that the measurement of fluorescence provides an accurate indication of the total albumin concentration in the mucus samples (Webber & Widdicombe, 1989).

Experimental protocol Tracheae were set-up in pairs. After a 30min equilibration period the control PD across both tracheae was recorded and any mucus produced was removed and processed as described above. All tracheae were then exposed to three concentrations of PAF (0.1, 1 and 1OMm) singly and in a random sequence. Each concentration of PAF was left in contact with the trachea for 30min and during this time any change in intraluminal pressure was recorded. After 30min the PD was recorded and any secretion produced was withdrawn and processed. The tracheae were then washed twice and fresh buffer containing no PAF was placed in the organ bath. Three control periods of 30min were allowed between each addition of PAF in order for the PD to recover towards a value similar to that before addition of PAF. After three concentrations of PAF had been added the tracheae were allowed to rest for four periods of 30min. The buffer surrounding one of the tracheae (test trachea) was then replaced with buffer containing one of the pharmacological antagonists. This buffer was left in contact with the trachea for 30min and any change in PD or intraluminal pressure was recorded. Any mucus produced after 30 min was withdrawn. The other trachea (control trachea) was bathed by buffer containing no antagonist. PAF (1I M) was then added to both tracheae for 30 min. The control trachea was exposed to PAF in normal buffer and the test trachea to PAF in buffer containing the antagonist. Any changes in PD and intraluminal pressure produced by PAF were recorded and any mucus produced was collected at the end of the 30 min. This was followed by three control periods with no PAF present in order for the PD to recover to a value not significantly different from that before PAF. The antagonists tested were the cyclo-oxygenase inhibitor indomethacin (1 ,M), the leukotriene receptor antagonist FPL55712 (1 pM), a combination of the H1-antagonist mepyramine (1p M) and the H2-antagonist cimetidine (10pM), a combination of the oxygen free radical scavengers catalase (500 u ml -) and superoxide dismutase (40uml- 1), and the PAF-receptor antagonist

WEB2086 (10pM). Only one antagonist or combination of antagonists was tested on each test trachea.

Analysis of results The concentration-response curves and bar charts presented in Results were obtained by pooling the results from a group of experiments. All values shown are means + s.e.means. Differences between PAF-induced responses and baseline values were analysed for statistical significance using a one-way analysis of variance. Differences between PAF-induced responses in control tracheae and those obtained in the presence of a pharmacological antagonist were analysed by Student's unpaired t test. In all cases P < 0.05 was taken as significant.

Histology In each experiment samples of trachea were taken for histological examination before and at the end of experiments. These samples were fixed in formal saline and sections (5 pm thick) were cut and prepared for light microscopy with hae-

PAF-INDUCED TRACHEAL SMOOTH MUSCLE RELAXATION

matoxylin and eosin staining. Changes in the thickness of the epithelium were assessed by measuring the depth of the epithelium (including basement membrane) at ten points selected at random in each of twelve pre- and post-PAF samples. Changes in gland and acini number were assessed by manual counting in twelve pre- and post-PAF samples.

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Results

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The basal mucus volume, lysozyme and albumin outputs before the first addition of PAF in all tracheae were (n = 24) 0.04 + 0.03 ul min 1, 25 + 13ng min-' and 0.24 + 0.07/,gmin-m respectively. The basal PD before addition of PAF was -8.6 + 0.5 mV.

Effects of PAF PAF (0.1-lOpM) had no significant effect on basal mucus volume output, lysozyme output or albumin output (Figure 1). PAF produced a concentration-dependent reduction in intraluminal tracheal pressure (Figure 2) indicating tracheal smooth muscle relaxation. PAF also produced a concentration-dependent reduction in the negativity of the PD across the trachea (Figure 3). These PD changes produced by PAF were long-lasting and the PD was still depressed in the control period after PAF-removal (Figure 3). However, the PD recovered before the next addition of PAF (Figure 3) and,

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because the concentrations of PAF were applied in a random sequence, there are no significant differences between the control values for PD before addition of each concentration of PAF (Figure 3).

Effects of antagonists on responses to PAF None of the antagonists had any significant effect on tracheal intraluminal pressure. Furthermore no antagonist affected basal mucus volume, lysozyme or albumin outputs, or the PD across the trachea (Table 1). PAF (1 gM) reduced intraluminal tracheal pressure in all control tracheae (not exposed to an antagonist, Table 2). This reduction in intraluminal pressure was not significantly different in test tracheae exposed to FPL55712 (7- [3 -(4-acetyl- 3 -hydroxy- 2-propylphenoxyl- 2hydroxypropoxy]-4-oxo-8-propyl-4H- 1 -benzopyran-2-carboxylic acid), mepyramine and cimetidine, and catalase and SOD (Table 2). However, the PAF-induced smooth muscle relaxation was virtually abolished in test tracheae exposed to indomethacin or the PAF-antagonist WEB2086 (3-(4-(2chlorophenyl) - 9 - methyl - 6H - thieno - (3,2-0X(1,2,4) - triazolo (4,3-aX1,4)-diazepine-2-yl)-1-(4-morpholinyl)-1-propanone) (Table 2). It is possible that the tracheal smooth muscle is not able to relax after indomethacin or WEB2086. However, in separate experiments the fl2-adrenoceptor agonist salbutamol produced reductions in intraluminal pressure of 52 + 4 and

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Platelet-activating factor relaxes ferret tracheal smooth muscle and reduces transepithelial potential difference in vitro.

1. The effects of platelet activating factor (PAF) were examined on the smooth muscle tone, mucus volume, lysozyme and albumin outputs and potential d...
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