Pulmonary Pharmacology (1991) 2, 106-113 © 1991 Longman Group UK Ltd
The Effect of Hydrogen Peroxide on Smooth Muscle Tone, Mucus Secretion and Epithelial Albumin Transport of the Ferret Trachea in vitro T . Morikawa, S . E . Webber, J . G . Widdicombe Department of Physiology, St George's Hospital Medical School, Cranmer Terrace, London SW17 ORE SUMMARY. The effect of hydrogen peroxide (H2 0 2 ) was examined on baseline and on methacholine- and phenylephrine-stimulated smooth muscle tone, mucus volume and lysozyme outputs, and epithelial albumin transport of the ferret whole trachea in vitro. H 2 0 2 (10 ttM-10 mM) had no significant effect on tracheal smooth muscle tone but produced concentration-dependent increases in mucus volume, lysozyme and albumin outputs . The potential difference (P.D .) across the trachea was not changed by H2 0 2 . Exposure of the trachea to H2 0 2 (1 mM) for 2 h reduced the smooth muscle contractions and lysozyme outputs due to methacholine (1 µM) and phenylephrine (10 ttM) • Methacholine-induced albumin output was significantly increased by H 2 0 2 but that due to pbenylephrine was not significantly affected . Exposure to H2 0 2 had no significant effect on the mucus volume output produced by methacholine or phenylephrine . Thus H2 0 2 directly stimulates submucosal gland secretion, including secretion from serous cells, and epithelial albumin transport across the ferret trachea but has no effect on trachea] smooth muscle tone . H20 2 reduces methacholine- and phenylephrine-induced smooth muscle contractions and serous cell secretion . H 2 02 causes hyperresponsiveness of albumin output to methacholine but not to phenylephrine .
ever the direct effects of active oxygen species on airway mucus secretion and epithelial functions have not been studied . The ferret in vitro whole trachea preparation 12 .11 allows the simultaneous measurement of tracheal smooth muscle tone and mucus secretion . It has few secretory cells in the epithelium 14 and so the major source of mucus is from mucous and serous cells in the submucosal glands . Lysozyme is located to serous but not mucous cells 15 and so is a good marker for secretion from these cells . Albumin is actively transported across the ferret tracheal epithelium-12 We have investigated the effect of H 2 O 2 on the tracheal smooth muscle tone, mucus volume and lysozyme outputs and albumin transport across the ferret trachea . Mucosal potential difference (P.D .), which reflects epithelial integrity and ion transport across epithelial cells, was also measured . Since H202 may alter the responsiveness of the airway tissues to other mediators, we examined the effect of H 2 0 2 on the responses to methacholine (muscarinic agonist) and phenylephrine (a-adrenergic agonist) .
During bacterial infection of the lungs, oxygen derived free radicals are important components of the oxygen-dependent killing of ingested microbes by phagocytes . Alveolar macrophages,' neutrophils' and eosinophils' release highly reactive oxygen metabolites including the superoxide anion (0 2 - ), hydrogen peroxide (H 2 0 2) and the hydroxyl radical (OH • ) . However in addition to their beneficial antibacterial action these products are released into the extracellular space and are important mediators of cell and tissue injury during inflammatory processes including those in the airways . Effects of oxygen-derived free radicals on airway smooth muscle have been reported . OH • contracts guinea-pig tracheal smooth muscle ." Inhalation of xanthine/xanthine oxidase (a free radical generating system) contracts airway smooth muscle in anaesthetized cats' and H 20 2 contracts canine lung parenchyma and bovine tracheal smooth muscle .' It induces variable contractions of guinea-pig tracheal smooth muscle .' Pulmonary macrophages and monocytes synthesize mucus secretagogues which stimulate mucus secretion during phagocytosis .' , ' The synthesis of these secretagogues may be stimulated by oxygen radicals which are also released by leukocytes . l o Oxidants also increase the permeability of paracellular pathways in a cultured kidney epithelium ." How-
METHODS The ferret in vitro trachea Ferrets of either sex, weighing 0 .5-1 .5 kg, were anaesthetised by i .p. sodium pentobarbitone (Sagatal, May & Baker, 50 mg .kg -1 ) . The trachea was exposed and cannulated about 5 mm below the larynx with a perspex cannula containing a conical collecting well . 12
Correspondence to: Tsuguo Morikawa M .D ., Department of Physiology, St George's Hospital Medical School, Cranmer Terrace, London SWl7 ORE, UK . 106
H20 2 on Ferret Trachea in vitro
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 KrebsHenseleit solution was (mM) : NaCl 120 .8, KCl 4 .7, KH 2 PO 4 1 .2, MgSO 4 7H20 1 .2, NaHCO 3 24 .9, CaCl2 2 .4, glucose 5 .6 . The buffer was maintained at 37°C and gassed with 95% 0 2 /5% CO 2 . 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 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 using 0 .5 ml distilled H 2 0 . 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 of the samples . 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 µl.min -1 (assuming 1 g of secretion is equivalent to 1 m1 16 ) 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 recorded on the pen-recorder, thus enabling assessment of changes in smooth muscle tone of the trachea during mucus collection . The electrical potential difference (P .D.) across the tracheal wall was measured using two calomel reference electrodes . These were filled with 3 .8 M KCl and placed in separate beakers of the same solution . Electrical contact was made with the preparation using two agar bridges . These were constructed from polyethylene tubing (0 .5 mm 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 via a high input impedance buffer amplifier (> 10 9 MSI) and 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 0 V . Any residual voltage measured here was subtracted from subsequent measurements of potential difference made in the preparation . Before the start of an experiment each trachea was allowed to equilibrate for 30 min, and during this time changes of bathing medium were made every 5 min. Assay for lysozyme The lysozyme concentrations of the mucus samples were measured using 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 Micrococcus lysodeikticus of 3 mg.ml -1 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 (0.5-100 ng .ml -1 ) were incubated in duplicate in 1 .5 ml potassium phosphate buffer (50 mM, pH 7 .4) containing Micrococcus lysodeikticus (0 .3 mg.ml -1 ), sodium azide (1 mg.ml -1 ) and bovine serum albumin (BSA, 1 mg .ml -1 ). 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 -1 ) 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 µl 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 VI 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 H 2 02 on the transport of albumin across the ferret trachea, BSA was added to the buffer bathing the submucosal surface of the _1. Fluorescent trachea in a concentration of 4 mg.m l _ 1) was also added to the BSA (0 .02-0.03 mg.ml
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, using an excitation wavelength of 550 nm and an emission wavelength of 490 nm. The fluorescent albumin concentration of the mucus samples was estimated from a standard curve relating fluorescence (arbitrary units) to the concentration of fluorescent albumin (range 25 ng .ml -1 to 3 µg.ml -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 . Experimental protocol Two sets of experiments were performed . In the first set the effects of H 202 (10 µM-10 mM) on tracheal smooth muscle tone and mucus volume, lysozyme and albumin outputs and P .D . were investigated . In the second set of experiments, the effects of a single concentration of H 202 (1 mM) on the responsiveness of the tracheal smooth muscle, mucus secretion and albumin transport to methacholine (1 AM) and phenylephrine (10 µM) were examined. These concentrations of methacholine and phenylephrine produce 60-70% of their respective maximum responses for intraluminal pressure changes and mucus volume, lysozyme and albumin outputs . 11,12 H202 was added to the external buffer, because it is impossible to measure mucus volume output in this experimental preparation if Krebs-Henseleit buffer containing H202 is added intraluminally . In the first set of experiments, after a 30 min control period, the trachea was exposed to methacholine (1 µM) for 30 min to test responses of the measured variables. The trachea was then washed twice and left for two control periods of 30 min . Three or four different concentrations of H 202 were then added to the submucosal buffer bathing the trachea in a random sequence . Each concentration was left in contact with the trachea for 30 min and during this time any changes in smooth muscle tone were recorded . After 30 min the secretion produced was withdrawn and processed as described above . The trachea was then washed twice and fresh buffer containing no H 202 was placed in the organ bath . Between two and four control periods of 30 min were allowed between additions of H 202, depending on how quickly the mucus volume output returned to the baseline level . Albumin is an antioxidant 17 and its presence in the submucosal buffer may alter the responsiveness of the trachea to H 202 . Therefore, in half of these
experiments albumin was omitted from the submucosal buffer and the results obtained with H202 were compared to those when albumin was present . In the second set of experiments, after a 30 min control period the trachea was exposed to either methacholine (1 AM) or phenylephrine (10 AM) for 30 min to test responses of the measured variables . The trachea was then washed twice and left for two control periods of 30 min. The buffer surrounding the trachea was then replaced with buffer containing H 202 (1 mM) . This buffer was left in contact with the trachea for 2 h and replenished every 30 min . During the third period of 30 min, methacholine (1 AM) or phenylephrine (10 µM) was added to the buffer containing H202 . After removal of the H 202 four or five control periods of 30 min were allowed . The responses to methacholine (1 AM) or phenylephrine (10 AM) were then redetermined . In separate experiments three consecutive responses to methacholine or phenylephrine were determined over the same time course without exposure to H 202. These experiments were performed to determine if the responsiveness of the trachea changed with time in the absence of H 202. 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 then sections (5 µm thick) were cut and prepared for light microscopy with hematoxylin and eosin staining . Analysis of results For the first set of experiments, the concentrationresponse curves for the effects of H 202 on intraluminal pressure and mucus volume, lysozyme and albumin outputs were obtained by pooling results from separate experiments and are expressed in absolute terms. The data obtained during the 30 min exposure to H 202 were compared with baseline values using one-way ANOVA . For the second set of experiments, the responses to methacholine (1 AM) or phenylephrine (10 AM) before and after exposure to H 202 (1 mM) have been expressed in absolute terms after subtracting the values obtained in the previous control periods . Differences between responses to methacholine and phenylephrine before and after exposure to H 202 were analysed by Student's t-test for paired data . Results are given as means ±s .e .m.
RESULTS Baseline values and responses to methacholine The baseline mucus volume, lysozyme and albumin outputs before the addition of any drugs were
H 20 2 on Ferret Trachea in vitro
0.04±0.02 µl .min -1 (n = 8), 64±25 ng .min -1 (n = 8) and 1 .00±0 .38 tg.min -1 (n=4), respectively. The P.D. before the addition of any drugs was -9.3±0 .6mV (n=8) . Methacholine (1 µM) increased intratracheal pressure by 84±5 mm H 2 O. It also increased mucus volume, lysozyme and albumin outputs to 0 .38±0 .06 µl .min -1 , 388± 73 ng .min- 1 and 3 .8±0 .43 µg .min -1 , respectively but did not significantly change the P .D. (-7 .9± 1 .7 mV). Direct effects of H 20 2 The addition of albumin to the submucosal buffer did not affect the responses of intraluminal pressure, mucus volume output, lysozyme output or P .D. to H2 02 . Therefore, results were pooled from experiments with and without albumin . H202 (10 µM-10 mM) had no significant effect on intraluminal pressure (Fig. la). However, H 2 02 produced weak concentration-dependent increases in mucus volume, lysozyme and albumin outputs (Fig . lb,c,d) . Because of the variability between animals the increase in mucus volume, lysozyme and albumin outputs was significant only at the highest concentration of H 2 0 2 where the responses were about 180%, 73% and 460% of the responses to
methacholine (1 gM), respectively . P.D. was not significantly changed throughout the experiment .
Effects of H2 02 on responses to methacholine and phenylephrine Intraluminal pressure Methacholine (1 µM) and phenylephrine (10 µM) increased intraluminal pressure before exposure of the trachea to H 2 0 2 (1 mM) by 95 ± 7 mm H20 (n = 8) and 55 ± 6 mm H 2 0, respectively (Table 1) . During exposure of the trachea to H 20 2 , the increase in intraluminal pressure to methacholine was not significantly changed but the response to phenylephrine was significantly reduced . Two hours after exposure of the trachea to H 20 2 , the intraluminal pressure responses to methacholine and phenylephrine were both significantly reduced by 55% and 89%, respectively. Mucus volume output Methacholine (1 gM) and phenylephrine (10 µM) increased mucus volume output before exposure of the trachea to H 2 0 2 (1 mM) by 0 .80±0 .13 gl .min -1 and 0.56±0 .12 pl .min -1 , respectively . These responses
0.6 0.5 0.4 0.3 -
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25 20 15 -
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Fig . 1-Concentration-response curves for the effects of H 20 2 on (a) intraluminal pressure, (b) mucus volume, (c) lysozyme and (d) albumin outputs . Horizontal dotted lines indicate the baseline values . Points are mean±s .e.m . (n=8(a,b,c), n=4(d)) . * and ** indicates p