Neuropeptides mediate the ozone-induced increase in the permeability of the tracheal mucosa in guinea pigs

Harumi Nishiyama1, Hirotada Ikeda1, Takeshi Kaneko1, Li Fu1, Makoto Kudo1, Takaaki Ito2, and Takao Okubo1

1 The First Department of Internal Medicine and 2 The First Department of Pathology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
Appendix
References

We examined the effects of acute exposure to ozone on the permeability of the tracheal mucosa and the contribution of neural pathways to the effects of ozone using horseradish peroxidase (HRP; mol wt 40,000) as a marker of lumen-to-blood transfer of a macromolecule in guinea pigs in vivo. Each guinea pig was anesthetized and exposed for 30 min to either ozone [0.5 or 3 parts/million (ppm)] or air. Immediately after exposure, a tracheal segment was isolated between two polyethylene cannulas in vivo and filled with HRP solution (50 mg/ml). Blood samples were drawn before and 10, 20, 30, and 40 min after the intratracheal instillation of HRP. The plasma levels of HRP in guinea pigs exposed for 30 min to 3 ppm of ozone, but not to 0.5 ppm of ozone, were significantly greater than those in guinea pigs exposed to air. Although the increased plasma HRP levels after exposure to 3 ppm of ozone were unaffected by propranolol or atropine, they were completely inhibited by pretreatment with capsaicin (50 mg/kg sc, injected in two doses). These results suggest that endogenous neuropeptides mediate the ozone-induced increase in the permeability of the tracheal mucosa in guinea pigs in vivo, but neither an adrenergic nor a cholinergic pathway appears to be involved.

horseradish peroxidase; capsaicin; model analysis

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
Appendix
References

THE INCREASE IN THE PERMEABILITY of the airway mucosa produced by irritants such as ozone (16, 20), nitrogen dioxide (23), and cigarette smoke (11, 12, 17) has been shown in laboratory animals and in humans with various methods. Among these irritants, cigarette smoke has been widely investigated for the mechanism by which it increases the permeability of the airway mucosa. Earlier studies (4, 25) have suggested that damage to epithelial tight junctions was the mechanism responsible for the observed increase in the permeability of the airway mucosa after exposure to cigarette smoke. However, later studies have refuted these previous studies and have shown that cigarette smoke exposure does not change the morphology (28) and integrity (27) of the tight junctions of the tracheal epithelium. It has been concluded that the mechanism for this increased permeability after cigarette smoke is alveolar epithelial cell injury of the bronchioloalveolar junctions (5).

Ozone, a principal component of photochemical smog, is known to cause airway inflammation (13). It is widely believed that the major sites of ozone-induced airway injury are the terminal bronchioles and adjacent centroacinar alveoli (3, 22, 26). It is thus suggested that these peripheral lesions are responsible for the increase in the permeability of the airway mucosa after ozone exposure. However, it has been shown that ozone exposure increases the permeability of the mucosa, even in the trachea (1). This suggests that the permeability of the airway mucosa increases without apparent epithelial injury, such as desquamation of the epithelium. Recently, Kaneko et al. (13) have found that neuropeptides are involved in the ozone-induced increase in airway microvascular leakage. It is likely that similar mechanisms participate in the ozone-induced increase in the permeability of the tracheal mucosa.

In this study, we sought to determine whether ozone exposure increases the permeability of the tracheal mucosa to horseradish peroxidase (HRP; mol wt 40,000) by using an isolated tracheal segment in vivo. We theoretically estimated the permeability of the tracheal wall from the tracheal mucosa to blood flow using the three-compartment model. We examined the morphological changes in the tracheal epithelium after ozone exposure, and we investigated the contribution of neural pathways in the ozone-induced increase in the permeability.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
Appendix
References

Animals. Male Hartley-strain guinea pigs (Japan SLC and Funabashi Farm, Shizuoka, Japan), 410-580 g, were anesthetized with pentobarbital sodium (40 mg/kg ip). A catheter (0.51-mm ID × 0.94-mm OD; Dow Corning, Midland, MI) filled with heparinized saline was inserted into the femoral vein of each guinea pig for the administration of drugs and the drawing of blood. As soon as the catheter had been inserted, the anesthetized guinea pig was exposed to ozone or filtered air.

Ozone exposure. Each anesthetized guinea pig was exposed to ozone in a 23.5-liter acrylic chamber. Ozone produced by an ozone generator (MOT-001A type, Nippon Ozone, Tokyo, Japan) was diluted with clean filtered air and delivered into the exposure chamber at a flow rate of 20 l/min. Ozone concentration in the chamber was monitored continuously with an ultraviolet ozone analyzer (model DY-1500, Nippon Ozone). Decomposition of the ozone was minimized by the use of Teflon tubing.

Preparation for measurement of the permeability of the tracheal mucosa. Immediately after the guinea pig had been exposed to ozone or air, a tracheal pouch was isolated between two polyethylene cannulas (disposable multipurpose tube, ATOM, Tokyo, Japan) in vivo (Fig. 1). The outside diameter of the cannula was about the same as the inside diameter of the guinea pig trachea.


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Fig. 1.   Experimental preparation for measuring permeability of tracheal mucosa between cannula 1 and cannula 2 (isolated tracheal pouch) in guinea pigs. Cannula 3 permitted spontaneous breathing. HRP, horseradish peroxidase.

In brief, tracheostomies were made at two sites, and a polyethylene cannula (Fig. 1, cannula 1) was inserted into the proximal part of the isolated trachea below the larynx. An L-shaped polyethylene cannula (Fig. 1, cannula 2) was then inserted into the distal part of the trachea above the sternum. Cannulas 1 and 2 were secured gently by suture with 3.0 surgical silk. To prevent the cannulas from advancing into the tracheal lumen, a site 4 mm from the end of cannulas 1 and 2 was marked with 2.0 surgical silk before cannulation. Another L-shaped small cannula (Fig. 1, cannula 3) was inserted to maintain spontaneous breathing. We completed this preparation within 15 min and washed the isolated tracheal lumen once with prewarmed (37°C) PBS. To minimize the changes in the volume of the tracheal pouch during the experiments, guinea pigs were kept in the supine position, and the upper jaw was fixed under anesthesia to extend the neck.

Measurement of the permeability of the tracheal mucosa to HRP. The permeability of the tracheal mucosa was measured by monitoring the appearance in the blood of HRP that had been instilled into the isolated tracheal pouch.

Each anesthetized guinea pig was exposed to either air or ozone for 30 min. Cannulas were then inserted as described in Preparation for measurement of the permeability of the tracheal mucosa, with preparation of the guinea pig requiring no more than 15 min. Fifteen minutes after inhalation exposure, HRP solution (50 mg/ml) in approximately twice the volume of the tracheal pouch was instilled very slowly through cannula 1 (Fig. 1) to fill the entire lumen of the isolated tracheal pouch and was left static for 40 min. The excess HRP remained in cannulas 1 and 2 to keep the concentration of HRP in the pouch as constant as possible. Blood samples (1 ml) were drawn before and 10, 20, 30, and 40 min after the instillation of HRP via the catheter previously inserted into the femoral vein, and the volume of blood withdrawn was replaced with heparinized saline. Blood samples were immediately centrifuged, and the plasma was stored frozen at -20°C until assayed.

Plasma HRP levels were normalized for the tracheal surface area between the ends of the two cannulas. The tracheal surface area was estimated from calculations based on the volume and length of the tracheal pouch measured after completion of the experiments.

Assay for HRP in serum. An ELISA was used to measure plasma HRP levels. Briefly, flat-bottomed Immulon 1 microtiter plates (Dynatech Laboratories, Alexandria, VA) were coated with rabbit anti-HRP diluted with carbonate buffer (pH 9.6). The plates were incubated overnight at 2-5°C and washed three times with PBS-0.05% Tween 20 before nonspecific binding was blocked with 2% bovine serum albumin in PBS. After incubation at room temperature for 1-2 h, the plates were washed three times with PBS-0.05% Tween 20. Aliquots of plasma samples diluted 1:2 in PBS were added to the plates. Standards for a range of HRP concentrations from 8 to 500 ng/ml were prepared by dilution in a mixture of PBS and normal guinea pig plasma (1:1) on the same plate. The plates were incubated for 3-4 h, then washed three times with PBS-0.05% Tween 20. Enzyme activity was measured with o-phenylenediamine dihydrochloride and 0.1% H2O2 as substrates in 0.05 M citrate buffer (pH 4.0). Color development was assessed at three or more time intervals, and unknowns were interpolated from a curve drawn from HRP standards incubated on the same microtiter plate.

Effect of acute ozone exposure on the permeability of the tracheal mucosa. To investigate the effect of exposure to ozone on the permeability of the tracheal mucosa, guinea pigs were divided into three exposure groups (each group consisting of 10 guinea pigs): 3 parts/million (ppm) of ozone for 30 min, 0.5 ppm of ozone for 30 min, and filtered air for 30 min. Immediately after exposure, the permeability of the tracheal mucosa was measured as described in Measurement of the permeability of the tracheal mucosa to HRP.

Effect of neural pathways on the ozone-induced permeability of the tracheal mucosa. To determine whether adrenergic and cholinergic pathways were involved in the ozone-induced increase in the permeability of the tracheal mucosa, guinea pigs were divided into three groups based on the administration of drugs: propranolol (1 mg/kg), atropine (1 mg/kg), and saline (vehicle). These drugs were administered intravenously via the femoral catheter. Ten minutes after the administration of a drug, each guinea pig was exposed for 30 min to either 3 ppm of ozone (propranolol treated, n = 6; atropine, n = 7; saline, n = 6) or filtered air (propranolol treated, n = 6; atropine, n = 7; saline, n = 6), and the permeability of the tracheal mucosa was then measured.

To determine whether endogenous neuropeptides from sensory nerves affect the ozone-induced increase in the permeability of the tracheal mucosa, other guinea pigs were assigned to one of two pretreatment groups: vehicle or capsaicin. Ten days later, when the effect of capsaicin was expected to be maximum (10), each guinea pig was exposed for 30 min to 3 ppm of ozone (capsaicin treated, n = 7; vehicle, n = 7) or to filtered air (capsaicin treated, n = 7; vehicle, n = 8), and the permeability of the tracheal mucosa was then measured.

Capsaicin treatment. To deplete neuropeptides from unmyelinated airway sensory nerves, capsaicin (total 50 mg/kg sc) was injected in two doses as previously described (2, 13). For the first dose, guinea pigs were anesthetized with pentobarbital sodium (30 mg/kg ip) and then sequentially treated with theophylline (2.5 mg/kg ip), 20 min later with salbutamol (0.6 mg/kg sc), and 10 min later with capsaicin (20 mg/kg sc, 12.5% solution in equal parts of 95% ethanol and Tween 80, diluted to 25 mg/ml with saline). For the second dose, 2 h later, more pentobarbital sodium was given (10-20 mg/kg ip), the salbutamol treatment was repeated, and capsaicin (30 mg/kg sc) was injected. If respiratory distress developed, the guinea pigs were given epinephrine (0.1 mg/kg sc). Control guinea pigs underwent the same procedure with vehicle.

Morphological analysis of epithelial cells. Eight guinea pigs were used for the light- and electron-microscopic observations. The guinea pigs were divided into two exposure groups: 3 ppm of ozone for 30 min (n = 4) and filtered air for 30 min (n = 4). After exposure to ozone or air, the tracheal pouch was isolated as described in Preparation for measurement of the permeability of the tracheal mucosa (Fig. 1) and then fixed with a one-half concentration of modified Karnovsky's fixative (2.5% glutaraldehyde and 2% paraformaldehyde in 0.04 M sodium cacodylate buffer) (15) as follows. The guinea pigs in each of the two groups (ozone exposure and air exposure) were further divided into two subgroups of two each according to the time point at which the tracheal pouch was fixed. In one subgroup, the fixative was introduced into the isolated tracheal pouch through cannula 1 (Fig. 1) immediately after isolation to examine the tracheal epithelium before the intratracheal instillation of HRP. In the other subgroup, the fixative was introduced into the isolated tracheal pouch 40 min after the intratracheal instillation of HRP to examine the effect of the HRP solution on the tracheal epithelium. In all guinea pigs, in conjunction with the intratracheal instillation of the fixative, retrograde perfusion via the abdominal aorta was performed with a rinsing solution of 0.15% sodium cacodylate buffer containing 0.025% heparin and 0.5% procaine hydrochloride, followed by the same fixative as that described above. Concurrently, the bilateral jugular veins were cut to allow drainage of the fixative. The trachea was then removed, and three specimens (each ~3 mm in length) were taken in a transverse fashion from the trachea to allow for easy orientation: 1) the upper part where the cannula was ligated to the trachea, 2) the central part, and 3) the lower part that was also ligated. Next, each specimen was cut into three longitudinal pieces (two anterior cartilaginous specimens and one posterior membranous specimen), which were immersed in the same fixative for 2 h and then postfixed in 1% OsO4. The specimens were dehydrated in a graded series of ethanol and propylene oxide and embedded in an epoxy resin mixture. All blocks from the central part of the trachea were sectioned at a thickness of 0.4 µm. One block from each ligated part was randomly selected and sectioned at a thickness of 0.4 µm for easy observation of the ligation site in a parallel plane to the axis of the trachea. At least three sections from each block were stained with toluidine blue and viewed by light microscopy. In addition, ultrathin sections were cut, mounted on naked grids, and then stained with uranyl acetate and lead citrate, followed by examination with a Hitachi H-600 transmission electron microscope at 75 kV.

In addition, to determine the epithelial surface changes induced by ozone exposure and experimental procedures, including treatment with HRP, eight guinea pigs were used for scanning-electron-microscopic observations. The guinea pigs were divided into four groups (n = 2 each): ozone exposure followed by HRP instillation, ozone exposure followed by PBS instillation, air exposure followed by HRP instillation, and air exposure followed by PBS instillation. Forty minutes after the instillation of HRP or PBS, a 5-mm-long specimen was removed from the central part of the tracheal pouch of the guinea pigs previously exposed for 30 min to ozone (3 ppm) or to air. Each specimen was cut into two longitudinal pieces (an anterior cartilaginous specimen and a posterior membranous specimen), which were gently washed with saline solution and fixed in a phosphate-buffered 2% glutaraldehyde solution (pH 7.3) for 1 day at 4°C. The specimens were then treated with 1% tannic acid for 2 h at room temperature and postfixed with 1% OsO4 for 1 h at 4°C. After dehydration with graded alcohols, the specimens were dried by the critical-point method, coated with gold, and observed with a Hitachi S-800 scanning electron microscope at 20 kV.

Drugs. The drugs used in this study were HRP (Zymed Laboratories, San Francisco, CA), rabbit anti-HRP (EY Laboratories, San Mateo, CA), o-phenylenediamine dihydrochloride, atropine, propranolol, capsaicin, salbutamol hemisulfate, theophylline, Tween 80 (all from Sigma, St. Louis, MO), pentobarbital sodium (Abbott Laboratories, North Chicago, IL), glutaraldehyde, sodium cacodylate (both from Nisshin EM, Tokyo, Japan), poly/bed 812 embedding media (Polysciences, Warrington, PA), and paraformaldehyde (Merck, Darmstadt, Germany).

Statistical analysis. The mean values of HRP concentration in the serum, normalized by tracheal surface area (in ng · ml-1 · cm2), are expressed as the arithmetic mean ± SE. Between-group differences in HRP concentration were assessed by a two-way repeated-measures analysis of variance followed by the contrast (means-to-regression coefficient comparisons). A level of P < 0.05 was accepted as statistically significant.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
Appendix
References

Effect of acute exposure to ozone on the permeability of the tracheal mucosa. During all of the experiments, the guinea pigs were maintained to breathe spontaneously under anesthesia and did not become hypoxemic (arterial PO2 < 60 mmHg), which was confirmed by arterial blood gas analysis.

Acute exposure for 30 min to 3 ppm of ozone, but not to 0.5 ppm of ozone, increased the permeability of the tracheal mucosa to HRP compared with filtered-air exposure (Fig. 2). In all three groups of guinea pigs, plasma levels of HRP had increased over time after HRP instillation. Plasma HRP levels in guinea pigs exposed to 3 ppm of ozone significantly exceeded those in guinea pigs exposed to filtered air. However, plasma HRP levels in guinea pigs exposed to 0.5 ppm of ozone were not statistically different from those in guinea pigs exposed to filtered air.


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Fig. 2.   Effect of acute ozone exposure on permeability of tracheal mucosa. Plasma levels of HRP during a 40-min sampling period in guinea pigs exposed for 30 min to 3 (n = 10; bullet ) and 0.5 (n = 10; black-triangle) parts/million (ppm) of ozone and air (n = 10; open circle ) are shown. Arrow, instillation of HRP solution into tracheal segment (time 0). * P < 0.05 compared with air-exposed group.

Effect of neural pathways on the ozone-induced permeability of the tracheal mucosa. Neither propranolol nor atropine affected the ozone-induced increase in the permeability of the tracheal mucosa to HRP (Fig. 3). In guinea pigs that received either propranolol or atropine and were then exposed to air, plasma levels of HRP were unchanged compared with vehicle-treated, air-exposed control animals. Plasma HRP levels in guinea pigs that received either propranolol or atropine and were then exposed to ozone were not significantly different from those in vehicle-treated, ozone-exposed control animals.


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Fig. 3.   Effects of adrenergic and cholinergic blockade on permeability of tracheal mucosa after a 30-min exposure to air or ozone. Shown are plasma levels of HRP in guinea pigs treated with intravenous propranolol (n = 6; ), atropine (n = 7; black-triangle), or saline (n = 6; bullet ) and then exposed to 3 ppm of ozone and plasma levels of HRP in guinea pigs treated with propranolol (n = 6; ), atropine (n = 7; triangle ), or saline (n = 6; open circle ) and then exposed to air. Significantly different (P < 0.05) compared with: § propranolol-pretreated, air-exposed group; dagger  atropine-pretreated, air-exposed group; * saline-pretreated, air-exposed group.

Pretreatment with capsaicin completely inhibited the ozone-induced increase in the permeability of the tracheal mucosa (Fig. 4). In vehicle-treated, ozone-exposed guinea pigs, plasma levels of HRP were significantly higher than those in air-exposed control animals. In capsaicin-treated, ozone-exposed guinea pigs, plasma HRP levels were significantly lower than those of vehicle-treated, ozone-exposed guinea pigs. There was no statistical difference between the plasma levels of HRP in capsaicin-treated, ozone-exposed guinea pigs and those in capsaicin-treated, air-exposed guinea pigs.


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Fig. 4.   Effect of capsaicin treatment on ozone-induced increase in permeability of tracheal mucosa. Shown are plasma levels of HRP in guinea pigs treated with capsaicin and then exposed for 30 min to 3 ppm of ozone (n = 7; ) and air (n = 7; ) and plasma levels of HRP in guinea pigs treated with vehicle and then exposed to ozone (n = 7; bullet ) and air (n = 8; open circle ). Significantly different (P < 0.05) compared with: * vehicle-treated, air-exposed group; § vehicle-treated, ozone-exposed group.

Morphological analysis of epithelium. Light-microscopic observation showed that there was neither desquamation of the epithelial cell layer nor subsequent denudation of the basement membrane in tracheal tissue from all parts irrespective of exposure and tracheal instillation (Fig. 5). Transmission-electron-microscopic observations also revealed no evidence of desquamation of epithelial cells in all groups (data not shown).


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Fig. 5.   Light micrographs of tracheal epithelium from isolated tracheal segments (toluidine blue; ×400). A: tracheal epithelium fixed immediately after a 30-min exposure to air. B: tracheal epithelium fixed 40 min after intratracheal instillation of HRP after a 30-min exposure to air. C: tracheal epithelium fixed immediately after a 30-min exposure to 3 ppm of ozone. D: tracheal epithelium fixed 40 min after intratracheal instillation of HRP after a 30-min exposure to 3 ppm of ozone. Desquamation of epithelial cell layer was not seen in all groups.

Scanning-electron-microscopic observations showed that the luminal surface structures of the tracheal mucosa exposed to ozone or air and treated with HRP or PBS after isolation were not different from each other, and no apparent cellular injury was observed. Micrographs of the tracheal surface structures from a guinea pig exposed to air and then instilled with PBS (Fig. 6A) and a guinea pig exposed to ozone and then instilled with HRP (Fig. 6B) are shown.


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Fig. 6.   Scanning electron micrographs (×5,000) of tracheal surface of guinea pigs exposed to air followed by PBS instillation (A) or to ozone (3 ppm for 30 min) followed by HRP instillation (B). No remarkable morphological changes are seen in ozone-treated mucosa.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
Appendix
References

In the present study, we demonstrated that the acute exposure to 3 ppm of ozone, but not to 0.5 ppm of ozone, for 30 min increases the permeability of the tracheal mucosa to HRP in guinea pigs in vivo. We found that capsaicin, a neuropeptide-depleting agent, completely inhibited the increase in the permeability induced by a 30-min exposure to 3 ppm of ozone. In contrast to capsaicin, the adrenergic and cholinergic blockers propranolol and atropine had no effect on the increase in the permeability. Our morphological observations revealed no evidence of either desquamation of the epithelial cells or subsequent denudation of the basement membrane.

We used a tracheal pouch system to assess more precisely the permeability of the tracheal mucosa. A previous study (1) had assessed the permeability of the tracheal mucosa, but its methods could not ensure that all tracers were equally distributed to the entire tracheal lumen or prevent some amount of tracer from being distributed to regions other than the trachea. The advantage of our technique is that the surface area for absorption can be estimated, and the distribution of the tracer in the airway lumen is restricted only to the trachea. Similar techniques have been shown to be useful with other animal subjects. In dogs, a segment of the trachea has been isolated with a double-balloon endotracheal tube and used for study to observe the recruitment of inflammatory cells and the release of mediators into the tracheal segment after the intratracheal administration of chemoattractants (18) and antigen (14). In sheep, an isolated tracheal segment was used to investigate the relationship between the change in tracheal blood flow and the clearance of 99mTc-diethylenetriaminepentaacetate from the tracheal lumen (9).

It is widely believed that the major sites of ozone-induced airway injury are the terminal bronchioles and adjacent centroacinar alveoli (3, 22, 26), and these peripheral lesions might be primarily responsible for the increase in the permeability of the airway mucosa after ozone exposure. However, our present study showed that ozone increases the permeability of the tracheal mucosa without any overt damage to the airway epithelium. Therefore, the routes by which HRP penetrates the tracheal mucosa after ozone exposure remain unclear. There may be two different pathways by which HRP penetrates the epithelial layer. One is a paracellular pathway and the other is a transcellular pathway. Regarding the transcellular pathway, HRP may enter by pinocytosis and/or diffusion. Any of these pathways can be affected by ozone exposure. However, we cannot exclude the possibility that a very small number of damaged tracheal epithelial cells, which could not be detected in our morphological observations, may function as the route for HRP penetration.

In this study, statistical analyses indicated that capsaicin pretreatment abolished the ozone-induced increase in the permeability of the tracheal mucosa. However, plasma levels of HRP in capsaicin-treated, ozone-exposed guinea pigs showed a tendency to exceed those in capsaicin-treated, air-exposed guinea pigs, but this was not statistically significant. It is possible that there is another mechanism(s) involved to a small extent in the ozone-induced increase in the permeability. Inflammatory mediators released in the airways by ozone exposure might be important. Platelet-activating factor and arachidonic acid products are thought to be potential mediators because cultured bronchial epithelial cell lines release platelet-activating factor (24) and arachidonic acid products (19) during exposure to ozone.

In contrast to our findings, there are two interesting reports by Burns and co-workers concerning the mechanism for the cigarette smoke-induced increase in the permeability of the airway mucosa, one showing that capsaicin treatment does not inhibit the cigarette smoke-induced increase in the permeability of the airway mucosa (6) and the other showing that urethan anesthesia prevents the cigarette smoke-induced increase in the permeability of the airway mucosa (7). There are two major differences in study designs that might explain these different findings. First, we used ozone instead of cigarette smoke to induce the changes in the permeability of the airway mucosa. Second, we measured the permeability of the tracheal mucosa instead of the entire airway, which may include damaged peripheral lesions. These peripheral lesions are thought to be primarily responsible for the increase in the permeability of the airway mucosa. In addition, in the former study (6), Burns et al. used juvenile guinea pigs, but we used adult guinea pigs. In the latter study (7), they used urethan for the anesthesia, which is a known anti-inflammatory agent (8), but we used pentobarbital sodium.

The permeability of HRP in the tracheal mucosa in our experiments was theoretically derived by using a three-compartment model as shown in the APPENDIX. In Table 1, the washin time constant (T) of HRP into the plasma, the HRP concentration at the plateau (Cpl), the excretion rate from the plasma (Fout), and the transfer rate of HRP through the tracheal mucosa (FTR) are shown for air breathing, 3 ppm of ozone, and 3 ppm of ozone with capsaicin pretreatment. T (= V/Fout, where V is plasma volume) is the time necessary for the HRP concentration to increase to (1 - 1/e) × 100 (= approximately 63%) of the final plateau value (21), which was obtained from the semilogarithmic plot of experimental data {HRP concentration at time t [C(t)]}. The HRP concentration in the tracheal pouch (Ctr) was 50 mg/ml. Cpl was the actual plasma concentration of HRP, and V was assumed to be 5% of the body weight. Fout was calculated as V/T, which is introduced from Eq. A3. The transfer rate of HRP through the tracheal mucosa was calculated from Ctr, Cpl, and Fout by Eq. A4 as 5.8 × 10-6 ml/min for air breathing. The permeability (Table 1), calculated from FTR, was 7.5 × 10-8 cm/s for the air-exposed animals, and this is comparable to the value (0.214 × 10-7 cm/s) of Wangensteen et al. (29) for the Dextran 20 in vitro experiment. The permeability value estimated from the permeability of mannitol (30), corrected for the difference in molecular weight, is also similar to our value. After a 30-min exposure to 3 ppm of ozone, the permeability of the tracheal mucosa became 3.5 times greater than that after air exposure. With capsaicin treatment, the permeability of the tracheal mucosa after ozone exposure became equivalent to that after air exposure.

                              
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Table 1.   Theoretical data calculated from curves in Figs. 2 and 4

This model is based on the following two assumptions: 1) HRP penetrates the tracheal wall in proportion to the concentration gradient, and 2) FTR is constant. This model neglects the complex structure and functions of the tracheal wall. Some of the HRP transport through the tracheal wall may be mediated by pinocytosis or bulk plasma flow from the capillaries into the tracheal tissue, and shunt flow from capillaries to the lymphatic system may occur in the tracheal tissue. It is also conceivable that some HRP may be trapped by loose connective tissue as it travels from the airway lumen into the blood, the effect of which is negligible when the HRP concentration has reached a steady state. A recent study by Kaneko et al. (13) has revealed that the extravasation of Evans blue dye in the trachea does not increase until 45 min after a 30-min exposure to ozone. Therefore, an increase in plasma flow from the tracheal capillaries is unlikely to have occurred during the measurement of permeability after exposure to ozone in this study.

In conclusion, the present study showed that acute exposure to 3 ppm of ozone for 30 min increased the permeability of the tracheal mucosa in guinea pigs in vivo. It was suggested that the increase in the permeability is mediated by endogenous neuropeptides but not by either an adrenergic or a cholinergic pathway.

    APPENDIX
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Abstract
Introduction
Methods
Results
Discussion
Appendix
References

To evaluate the permeability of the tracheal mucosa with our technique, we theoretically analyzed HRP kinetics by considering the three-compartment model shown in Fig. 7. Compartment I is the tracheal pouch in which the concentration of HRP (Ctr) is high and can be regarded as constant, Compartment II is the cardiovascular blood pool, and compartment III is the outside body or space where HRP is metabolized. C(t) is the plasma HRP concentration at time t, and V is the plasma volume. The concentration of HRP in compartment III (Cout) is assumed to be zero. Transfer rate of HRP from the tracheal space to the blood pool (FTR) is the amount of HRP penetrating through the tracheal wall per unit time and unit concentration difference. The excretion rate of HRP from the blood pool to the outside of the body is Fout. In this model, the amount of HRP in plasma is expressed as V · C(t), and the rate of its increase at time t is given by the time derivative of the HRP amount, d[V · C(t)]/dt, which is equal to the difference between the influx of HRP into the blood and the outflow from it, i.e., FTR[Ctr - C(t)] - Fout[C(t- Cout]. This relationship is arranged as follows
VdC(<IT>t</IT>)/d<IT>t</IT> = [(F<SUB>TR</SUB> ⋅ C<SUB>tr</SUB>) + (F<SUB>out</SUB> ⋅ C<SUB>out</SUB>)] − (F<SUB>TR</SUB> + F<SUB>out</SUB>) ⋅ C(<IT>t</IT>) (A1)
This differential equation is solved as follows
C(<IT>t</IT>) = <FR><NU>(F<SUB>TR</SUB> ⋅ C<SUB>tr</SUB>) + (F<SUB>out</SUB> ⋅ C<SUB>out</SUB>)</NU><DE>F<SUB>TR</SUB> + F<SUB>out</SUB></DE></FR> <FENCE>1 − <IT>e</IT><SUP> − <FENCE>(F<SUB>TR</SUB> + F<SUB>out</SUB>)/V</FENCE> ⋅ <IT>t</IT></SUP></FENCE> (A2)
where Cout is assumed to be zero and the concentration of HRP at time 0 is also zero.


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Fig. 7.   Schematic representation of HRP kinetics in this study. Compartment I, tracheal pouch in which concentration of HRP (Ctr) is kept constant; compartment II, cardiovascular blood pool; compartment III, outside body or space where HRP is metabolized. C(t), plasma HRP concentration at time t; V, plasma volume; Cout, HRP concentration in compartment III; FTR, amount of HRP penetrating through tracheal wall per unit time and unit concentration difference between tracheal pouch and serum; Fout, amount of HRP moving from serum to compartment III per unit time and concentration difference.

If FTR is small enough in comparison with Fout (FTR <<  Fout), Eq. A2 can be simplified as follows
<FR><NU>C(<IT>t</IT>)</NU><DE>C<SUB>tr</SUB></DE></FR> = <FR><NU>F<SUB>TR</SUB></NU><DE>F<SUB>out</SUB></DE></FR> <FENCE>1 − <IT>e</IT><SUP> − (F<SUB>out</SUB>/V) ⋅ <IT>t</IT></SUP></FENCE> (A3)
Clearly, the rate of increase in plasma HRP concentration is limited by the elimination rate from the plasma and not by the transfer rate of HRP through the tracheal mucosa. When C(t) reaches a plateau, as seen in Fig. 2 (i.e., t is large enough, rendering the exponential component to be zero), Eq. A3 becomes
C<SUB>pl</SUB>/C<SUB>tr</SUB> = F<SUB>TR</SUB>/F<SUB>out</SUB> (A4)
where the plasma HRP concentration at the plateau (Cpl) is proportional to the transfer rate of HRP through the tracheal mucosa when CTR and Fout stay constant. FTR is the transfer rate for the total surface area of the tracheal tube, so the permeability (in cm/s; transfer rate for unit area) was calculated by dividing FTR by the tracheal surface area and by 60, where 60 is 1 min (60 s).

In addition, the values in Table 1 were calculated as follows: the washin time constant was obtained from the semilogarithmic plot of the experimental data [C(t)], as stated in the text. Then, Fout was calculated as Fout = V/T, in which V was measured before and after the experiment by water filling. Finally, rearranging Eq. A4, FTR was obtained as FTR = Cpl/Ctr × Fout, in which Ctr is 50 mg/ml.

    ACKNOWLEDGEMENTS

We are greatly indebted to Dr. John Widdicombe (London, UK) for his review and invaluable suggestions in preparing the manuscript.

    FOOTNOTES

This work was supported in part by a grant from the Smoking Research Foundation of Japan.

Address for reprint requests: T. Okubo, The First Dept. of Internal Medicine, Yokohama City Univ. School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan.

Received 25 February 1997; accepted in final form 1 May 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
Appendix
References

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Am J Physiol Lung Cell Mol Physiol 275(2):L231-L238
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society




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