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Oxygen radicals in the nonvagal component of noncholinergic airway constriction
E
ELSEVIER
Respiration Physiology 104 (1996) 213-220
Oxygen radicals in the nonvagal component of noncholinergic airway constriction H . - Q . Z h a n g a, H . - H . Tai b, Y.-L. L a i a,c,* a Division of Pharmacology and Experimental Therapeutics, Lexington, KY 40536, USA h Division of Medicinal Chemistry and Pharmaceutics, College of Pharmacy, University of Kentucky., Lexington, KY 40536, USA c Department of Physiology, College of Medicine, National Taiwan University, No. 1, Sec. 1, Jen-Ai Road, Taipei, Taiwan, ROC Accepted 15 January 1996
Abstract
To test the hypothesis that oxygen radicals play an important role in the nonvagal component of the noncholinergic bronchoconstriction in vivo, 37 guinea pigs weighing 329 -t- 8 g were randomly divided into five groups: group 1, vagotomy; group 2, vagotomy + CAT (catalase); group 3, vagotomy -t- SOD (superoxide dismutase); group 4, vagotomy + PBN (ot-phenyl-N-tert-butyl nitrone); and group 5, capsaicin pretreatment. CAT, SOD, and PBN are antioxidants. Each animal was anesthetized, paralyzed, artificially ventilated, and pretreated with atropine and phenoxybenzamine. Immediately after acute capsaicin challenge, animals in group 1 exhibited decreases in maximal expiratory flow, dynamic respiratory compliance, and total lung capacity, as well as an increase in functional residual capacity, indicating noncholinergic airway constriction. The bronchoconstriction was significantly ameliorated by SOD and PBN, and it was almost abolished by capsaicin pretreatment. Thirty minutes after acute capsaicin challenge, there was a significant decrease in airway NEP activity and an increase in lung substance P level in group 1 but not in other groups. These results indicate that nonvagal component of noncholinergic bronchoconstriction is partially modulated by oxygen radicals.
Keywords: Mammals, guinea pigs; Mediators, NEE tachykinins; Oxygen radicals, bronchoconstriction: Pharmacological agents, capsaicin; Upper airways, bronchoconstriction, oxygen radicals
1. I n t r o d u c t i o n
The vagus nerves have been considered to play an important role in the airway hyperresponsiveness (Simonsson et al., 1967). It was reported that the vagus nerves contain many afferent substance P (SP)-immunoreactive nerve fibres (Lundberg et al., 1978). Lundberg et al. (1983) indicated that all SP-immunoreactive nerves in trachea and lung are * Corresponding author. Tel.: 886-(0)2-3938235. Fax: 886-(0)23938235.
afferent and capsaicin-sensitive, and a major portion (90%) of the SP produced in the sensory nervecell bodies in the nodose ganglion is transported into peripheral vagal branches. Tradition holds that the airway is mainly innervated with vagus nerves (Richardson and Ferguson, 1980). Data in the literature suggest, however, that both vagal and nonvagal components significantly contribute to the excitatory noncholinergic system. Using bilateral vagotomy and local capsaicin treatment-induced vagal dysfunction, we previously separated noncholinergic bronchoconstriction into vagal and nonvagal components (Zhang
0034-5687/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PII S 0 0 3 4 - 5 6 8 7 ( 9 6 ) 0 0 0 0 4 - 7
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and Lai, 1993). We found that the nonvagal component contributes 46% of the total constriction (Zhang and Lai, 1993). Lundberg et al. (1983) also showed that about 60% of the capsaicin-induced substance P release is from the vagus nerves and the remaining release is nonvagal in origin. Our previous data indicate that oxygen radicals play an important role in noncholinergic airway constriction induced by acute capsaicin challenge (Lai, 1990), exsanguination (Zhang and Lai, 1994), and hyperventilation (Fang and Lai, 1993). Accordingly, it is important to explore whether the bronchoconstriction caused by the nonvagal component is modulated by oxygen radicals. The purpose of this paper was to test the role of oxygen radicals in the nonvagal component of the noncholinergic bronchoconstriction. Several antioxidants including catalase (CAT), superoxide dismutase (SOD) and Ntert-butyl-c~-phenylnitrone (PBN) were used to antagonize oxygen radicals. We used CAT and SOD previously. Carney et al. (1991) demonstrated that PBN diminished production of oxygen radicals in brain. In this study, we tested whether PBN is an effective antioxidant in airways. 2. Material and methods 2.1. Animal preparations
Thirty seven Hartley strain guinea pigs weighing 329 4- 8 g were randomly divided into five groups: group 1, vagotomy (n = 8); group 2, vagotomy 4- CAT (n = 7); group 3, vagotomy + SOD (n = 8); group 4, vagotomy 4- PBN (n = 6); and group 5, systemic capsaicin pretreatment (n = 8). In group 1, each animal was bilaterally vagotomized under anesthesia with sodium pentobarbital (30-50 mg/kg, i.p.). The cervical vagus nerve was exposed and about 2 cm of the nerve was cut. Simultaneous bilateral vagotomy can easily cause the death of animals due to gastrointestinal disturbance. This risk, however, can be avoided by cutting the two vagus nerves one week apart. About half of vagotomized guinea pigs the right vagus nerve was cut first, then the left vagus nerve was cut one week later. Under the same conditions in another 50% animals, the left vagus nerve was first cut, the right one a week later. Three weeks after the initial vago-
tomy, animals were anesthetized and their airway functions were tested before and after acute capsaicin challenge. No significant differences in any capsaicin-induced respiratory parameters (expressed as percent of baseline value) between these two sequences of vagotomy were found. Each vagotomized animal received an intratracheal instillation of 0.75 ml saline 30 rain before intravenous injection of capsaicin. In groups 2 and 3, CAT (400,000 U) or SOD (120,000 U) was administrated. Our previous experiment indicated that the above doses of CAT and SOD were sufficient (Zhang and Lai, 1994). Each in 0.75 ml saline solution, CAT or SOD was intratracheally instilled in each guinea pig 30 min before acute capsaicin challenge. In group 4, PBN (32 mg/kg, i.p) was treated with twice daily for 14 days (Carney et al., 1991) before acute capsaicin challenge. To deplete tachykinins in group 5, a 5day chronic capsaicin pretreatment, consisting of two consecutive daily doses of 50 mg/kg and three consecutive daily doses of 100 mg/kg capsaicin administered subcutaneously, was begun 9 days before the study. Especially during the first 1-3 days the injections were administered in small doses, as many as l0 times per day with a 1 h interval between any two injections. Each animal was anesthetized with a combination of sodium pentobarbital (15 mg/kg), fentanyl (0.2 mg/kg), and droperidol (10 mg/kg) before the capsaicin was subcutaneously administered. 2.2. Functional testing o f bronchial constriction
On the day of the study, each animal was anesthetized with sodium pentobarbital (35 mg/kg), paralyzed with gallamine triethiodide (4 mg/kg, i.v.), and artificially ventilated with a tidal volume of 6 ml/kg at a frequency of 60/min. These ventilatory variables were found to maintain arterial blood gases and pH within the normal range (Ray et al., 1988). No positive end-expiratory pressure was added during artificial ventilation. All animals were treated with atropine (0.2 mg/kg) and phenoxybenzamine (0.5 mg/kg) via intramuscular injection 15 min before the experiment. These doses of atropine and phenoxybenzamine were sufficient to suppress the cholinergic and ~-adrenergic nervous systems, respectively (Ko and Lai, 1988). Each anesthetized-paralyzed and artificially ventilated guinea pig was placed
H.-Q. Zhang et aL /Respiration Physiology 104 (1996) 213-220
supine inside a whole-body plethysmograph. The flow rate was monitored with a Statham PM15 differential pressure transducer as the pressure dropped across three layers of 325-mesh wire screen in the plethysmograph wall. Lung volume change was obtained via integration of flow. Airway opening pressure (Pao) was measured with a Statham PM131 pressure transducer. To induce airway constriction via activation of afferent C-fibers, capsaicin was intravenously injected into the jugular vein of each animal (16 #g/kg, bolus injection). Before (baseline) and after acute capsaicin challenge, the maximal expiratory flow-volume (MEFV) maneuver was performed to evaluate functional changes in airway dimension according to a previous method (Lai, 1988). Briefly, three times the lungs were inflated to total lung capacity (TLC, lung volume at Pao = 30 cmH20). At peak volume during the third inflation, the inflation valve was shut off and immediately another solenoid valve for deflation was turned on automatically. The deflation valve was connected to a 20-L container which had a pressure of - 4 0 cmH20 (subatmospheric). The negative pressure of 40 cmH20 produced the maximal expiratory flow (Vmax)- The MEFV plot was stored on a cathode ray storage oscilloscope (V-134, Hitachi). During artificial ventilation (between the interval of the MEFV maneuvers), compliance of the respiratory system (Crs) was obtained as the ratio of tidal volume/Pao difference between end-inspiration and end-expiration, The general experimental protocol was that the MEFV maneuver was performed before as well as 1, 5, 10, 15, 20, and 30 min after acute capsaicin challenge. To evaluate the change in airway dimension, "¢max at 50% baseline TLC (Vmax50) and dynamic compliance of the respiratory system (Crs) were used as indicators of bronchoconstriction. Before and after each MEFV maneuver, functional residual capacity (FRC) was determined using a modified neon dilution method (Lai, 1988). 2.3. Measurement of NEP activi~
At the end of the above experiment we excised airway tissues (main stem bronchus and all dissectible bronchial tree) from the left lung of each animal. The bronchial tissues were washed with physiological saline and stored at -70°C for later analysis.
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A cell-free extract of bronchial tissues was made by following the procedures of Haxhiu-Poskurica et al. (1992). The frozen tissues (0.06 g) were thawed and sonicated in 50 mM Tris-HC1 buffer (3 ml), pH 7.4, at 4°C for 30 sec. The residue was removed from the extract by centrifuge at 17,500 g for 15 min at 4°(7. The supernatants were used for enzyme analysis. Neutral endopeptidase activity was determined by following the procedures of Orlowski and Wilk (1981), modified by Haxhiu-Poskurica et al. (1992). The reaction mixture (250 gl) consisted of 10-50 #1 of tissue extract, 1.25 mM (final concentration) substrate, i.e., glutaryl-alanine-phenylalanine4-methoxy-2-naphthylamide, aminopeptidase M (10 #g), and 50 mM Tris-HC1 buffer, pH 7.4. The mixture was incubated at 37°C for 60 min. Reaction was stopped by adding 1 ml of 10% trichloroacetic acid and then 1 ml of 0.005% (wt/vol) fast garnet (GBC) (a stabilized diazonium salt). Under this incubation condition, the hydrolysis of the substrate was proportional to enzyme concentration and time, reflecting steady-state kinetics and linearity (Orlowski and Wilk, 1981). The amount of 2-naphthylamine formed during the reaction was determined by spectrophotometer (Beckman Model 35) at 530 nm. Specific activity of NEP was expressed in picomoles per milligram tissue protein per hour. Protein concentration was determined by the method of Lowry et al. (1951), with bovine serum albumin (BSA) as the standard. 2.4. Measurement of substance P of lung tissue
At the end of the above experiment the right lung was excised, and washed with physiological saline and stored at -70°C for later analysis. SP of lung tissue was extracted via the procedures of Saria et al. (1988). The frozen tissue (0.3 g) were thawed and cut into pieces, suspended in 10 vol 2 N acetic acid, and boiled for 10 min. Then the samples were homogenized with sonicator (Model W-375). The homogenates were transferred to polypropylene tubes and centrifuged for 20 min at 3000 g. The supernatants were used for analysis of lung SP using enzyme immunoassay (EIA) (Tai, 1992). A 96microwell plate was first coated with protein A diluted in coating buffer, pH 9.6. Diluted SP antibodies were added to each well. Subsequently, SP standards
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H.-Q. Zhang et al./Respiration Physiology 104 (1996) 213-220
Table 1 Body weight and respiratory variables during the baseline period in five groups of guinea pigs Group
n
Body wt. (g)
TLC (ml)
FRC (ml)
Crs (ml/cmH20)
VmaxS0 (ml/sec)
Vagotomy Vagotomy 4- CAT Vagotomy 4- SOD Vagotomy -+- PBN Capsaicin pretreatment
8 7 8 6 8
328 330 329 329 330
10.4 10.6 10.5 10.6 10.3
2.4 2.3 2.4 2.5 2.4
0.22 0.22 0.19 0.22 0.21
93 95 95 91 92
444± 4-
8 9 9 7 8
4+ + 4±
0.4 0.3 0.2 0.2 0.2
4- 0.3 2_ 0.2 :L 0.1 4- 0.1 4- 0.2
44444-
0.02 0.01 0.01 0.02 0.01
4-4-4 -F 5 4- 5 4- 6 4- 4
Values are mean ± SE; TLC = total lung capacity: FRC = functional residual capacity: Crs = dynamic respiratory compliance; and Vma×5o = maximal expiratory flow at 50% baseline TLC; CAT = catalase: SOD = superoxide dismutase; and PBN = o~-phenyl-N-tertbutyl nitrone.
or unknown samples, in duplicate, were also added to each well. Diluted SP-enzyme (horseradish peroxidase) conjugate was delivered to each well. The plate was incubated at 37°C for 1-2 h with light shaking (Kjanke and Konkel TYP VX7). After washing three times with washing buffer, substrate (K-Blue Substrate) was added to each well. After having stood at 37°C for 30 rain the plate was read in a microwell reader (Model LE 310) at 650 nm. SP concentration in unknown samples was obtained from the simultaneously obtained absorbance-SP concentration standard curve. All values are reported as means -4- SE. Analysis of variance was used to establish the statistical significance of differences among groups. If significant differences among groups were obtained, Dunnett's test was used to differentiate differences between experimental and control groups. We employed Scheffe's test to analyze significance of differences between any two experimental groups. Differences were considered significant if P < 0.05.
100'
A
80"
g
.>E 60 I~
--
Vagotomy + CAT
z,
Vagotomy + SOD
-¢ '
Vagotomy + PBN Capsaicin pretreatment
40
-
1'0
"
1'5
Time
3. Results Body weight and respiratory variables during the baseline period (before acute capsaicin challenge) are shown in Table 1. No significant differences in any respiratory variable between groups were found. In vagotomy animals, acute capsaicin challenge (16 #g/kg) induced an immediate (at 1 min) decreases in V .... 5o (Fig. 1) and Cr, (Fig. 2), indicating moderate to severe bronchial spasm. This airway constriction was gradually attenuated with time. However, capsaicin-induced changes in VmaxS0 and Cr~ were significantly ameliorated by SOD, PBN and a systemic capsaicin pretreatment at 1-30 rain
,
20
-
i
25
-
,
30
(rain)
Fig. 1. Capsaicin-induced changes in the maximal expiratory flow rate at 50% baseline TLC (Vm:ixs0) in five groups of guinea pigs. All changes were expressed as a percent of the baseline (zero time) values. Time indicates the time period after the bolus injection of capsaicin (16 #g/kg) via the jugular vein. CAT = catalase: SOD = superoxide dismutase: PBN = c~-phenyl-N-tertbutyl nitrone. Statistical differences between groups (p < 0.05): a = compared to the vagotomy group; b = compared to the capsaicin pretreatment group•
(Fig. 1). The most efficient way to prevent the noncholinergic bronchoconstriction was a systemic capsaicin pretreatment to deplete tachykinins. In addition, an acute capsaicin challenge caused alterations in lung volumes. At one minute after an acute cap-
H.-Q. Zhang et al./ Respiration Physiology 104 (1996) 213-220 a
a
100
a
a
Table 2 Airway neutral endopeptidase(NEP) activityand lung substance P level of guinea pigs at 30 min after acute capsaicin challenge
bc
Group
90
Vagotomy Vagotomy + CAT Vagotomy + SOD Vagotomy + PBN Capsaicin pretreatment
8(1
¢0
Vagotomy
70"
60"
•
Vagotomy + CAT
.,t
Vagotomy + SOD
,t
Vagotomy + PBN
.... 0
-
,
5
-
,
10
-
.
1'5
Time
,
20
n
8 7 8 6 8
NEP
Substance P
(pmol/mg protein/h)
(pmol/g tissue)
28.0 40.9 42.5 41.9 37.4
0.94 0.65 0.38 0.17 0.05
± 4444-
1.2 1.0 a 0.6 a'b 0.4 a 1.6a
-4- 0.1 :[: 0.1 b -4- 0. I a 4- 0.1 a 4- 0.04 a
Values are means + SE; CAT : catalase; SOD = superoxide dismutase; PBN = a-phenyl-N-tert-butyl nitrone. Statistical differences between groups (P < 0.05): acompared to the vagotomy group; bcompared to the systemic capsaicin pretreatment group.
Capsaicin pretreatment
50
217
.
,
25
.
,
30
(rain)
Fig. 2. Capsaicin-induced changes in the compliance of the respiratory system (Cry) in five groups of guinea pigs. All changes were expressed as a percent of the baseline (zero time) values. Time indicates the time period after the bolus injection of capsaicin (16 /lg/kg) via the jugular vein. CAT = catalase; SOD = superoxide dismutase; PBN = a-phenyl-N-tert-butyl nitrone. Statistical differences between groups (P < 0.05): a = compared to the vagotomy group; b = compared to the capsaicin pretreatment group; c = compared to the vagotomy + SOD and the vagotomy 4- PBN groups.
saicin challenge, TLC decreased to 86.1 + 0.6% of the baseline value in the vagotomy group. The decrease was significantly attenuated by SOD (89.1 -4- 0.8% of baseline), PBN (90.0 -+- 0.6% of baseline), and by capsaicin pretreatment (96.1 4- 0.8% of baseline). On the other hand, FRC increased to 124 + 5.1% of baseline in the vagotomy group at one minute after acute capsaicin challenge. The increase was significantly attenuated by SOD (109.0 -4- 0.7% of baseline), PBN (108.9 -4- 0.6% of baseline), and by systemic capsaicin pretreatment (105.0 ± 0.7% of baseline). Compared to the vagotomy group, however, the CAT group did not significantly change acute capsaicin-induced alteration in either ~/max50, Cry, TLC, or FRC. Bronchial NEP activity and lung SP level at 30
min after acute capsaicin challenge are listed in Table 2. Compared to the vagotomy group, there was a significant increase in bronchial NEP activity in all experimental groups (groups 2-5). On the other hand, lung tissue SP level showed a significant decrease in all experimental groups (Table 2). 4. Discussion
In the anesthetized-paralyzed and vagotomized guinea pig, an intravenous injection of a potent capsaicin (16 #g/kg) immediately induced marked decreases in Vm~50, Crs, and TLC, as well as a significant increase in FRC. After the peak change at 1 min these parameters gradually returned toward the baseline values. SOD, PBN, and systemic capsaicin pretreatment significantly attenuated the acute capsaicin challenge-induced respiratory changes. In addition, SOD, PBN, and capsaicin pretreatment significantly increased airway NEP activity and decreased lung substance P level. Several features of these noncholinergic respiratory changes after vagal dysfunction will now be discussed as follows.
4.1. Oxygen radicals and the nonvagal component of the noncholinergic airway constriction Acute capsaicin challenge significantly decreased VmaxS0, Crs and TLC, but markedly increased FRC in the vagotomy group. These alterations indicate capsaicin challenge-induced noncholinergic airway constriction. We previously showed that noncholin-
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H.-Q. Zhang et al./Respiration Physiology 104 (1996) 213-220
ergic airway constriction can be separated into vagal and nonvagal components (Zhang and Lai, 1993). At 1 min after injection of resiniferatoxin or capsaicin, about 36-51% of the airway constriction is due to the vagal component, while about 49-64% airway constriction is due to the nonvagal component (Zhang and Lai, 1993). As in vagus-intact animals (Zhang and Lai, 1993), we found similar patterns, although with a smaller degree, of acute capsaicin challenge-induced alterations in respiratory parameters in vagotomized animals. Since this constriction occurred in vagotomized animals, the noncholinergic activation should be due to the nonvagal component alone. Hydrogen peroxide and superoxide can be scavenged by CAT and SOD, respectively. In addition, PBN can also antagonize oxygen radicals. The major new finding to emerge from the present study is that antioxidants (SOD and PBN) significantly decreased the nonvagal, noncholinergic airway constriction. Intratracheal instillation of SOD and intraperitoneal administration of PBN significantly ameliorated all the above acute capsaicin-induced changes. At 1 min after acute capsaicin challenge, compared to the vagotomy group, the decrease in VmaxS0 was ameliorated about 25.2%, 31.5%, or 31.7% by CAT, SOD, or PBN. These magnitudes of attenuation are markedly larger than those in vagus-intact animals (Lai, 1990), in which acute intravenous CAT and SOD did not significantly alter capsaicin-induced airway constriction. The effective action of antioxidants in this study may be related to their intratracbeal application. Compared to the marked intratracheal effects of CAT on noncholinergic airway constriction in vagus-intact animals (Zhang and Lai, 1993), no significant effect of CAT in this study could be due to a smaller degree of airway constriction in vagotomized animals. Other reasons may include variable responses in animals and different stimuli (capsaicin vs. exsanguination) to activate afferent C-fibers. Collectively, our data suggest that oxygen radicals play an important role in capsaicin-induced nonvagal, noncholinergic airway constriction. It was demonstrated that oxygen radicals cause airway constriction (Katsumata et al., 1990). After capsaicin pretreatment, however, acute capsaicin challenge did not induce airway constriction. This result can be interpreted as that acute capsaicin chal-
lenge does not cause an increase in the production of oxygen radicals or that the increased oxygen radicals alone cannot induce airway constriction in capsaicinpretreated animals. We are in favor of the latter interpretation. Since oxygen radicals activate afterent C-fibers (Stahl et al., 1993), it is possible that the radicals cause the observed airway constriction indirectly via tachykinin release. Further studies are needed to delineate the underlying mechanism. 4.2. Tachykinins and NEP in the nonvagal component of the noncholinergic airway constriction Chronic bilateral cervical vagotomy was used to cause degeneration of vagus nerves (Daly et al., 1953) which, in turn, decreased substance P concentration in airways and lungs (Lundberg et al., 1983). Similarly in our studies, the lung substance P decreased to a very low level (0.9 pmol/g) in the vagotomy group compared to the control group with intact vagus nerves (16-7 pmol/g) (Zhang and Lai, 1994). The degree of airway constriction, however, seems not linearly related to the lung substance P level. For example, acute capsaicin challenge (16 #g/kg) produced about 95% and 55% decreases in Vma× in the control (Zhang and Lai, 1993) and the vagotomy (this study) groups, respectively. As mentioned above, their respective lung substance P levels are 6-7 pmol/g (Zhang and Lai, 1994) and 0.9 pmol/g (this study). If we assume that the released substance P is proportional to its lung content, the concentration-constriction relationship is not a linear but an exponential one. This type of exponential concentration-response relation of substance P was found by Martins et al. (1991) in guinea pig lungs. Sources of nonvagal tachykinins may be released mainly from the sympathetic nerves (Gamse et al., 1981), which may originate from the spinal cord (Lundberg et al., 1983). Johnson et al. (1985) suggested that NEP activity exists in the epithelium, submucosal glands, nerves of the airway, lung and airway smooth muscle. NEP in the airway normally degrades tachykinins which, in turn, results in a decreased contractile response of the airway smooth muscle (Dusser et al., 1988). In this study, antioxidants increased airway NEP activity and decreased lung substance P level (Table 2). This may indicate that antioxidants can pre-
H.-Q. Zhang et al./ Respiration Physiology 104 (19961 213-220
v e n t the inactivation o f N E P by o x y g e n radicals. C o n s e q u e n t l y , a portion o f the d e c r e a s e in substance P f o l l o w i n g a n t i o x i d a n t t r e a t m e n t should be related to the i n c r e a s e d N E P activity. O t h e r portion o f the d e c r e a s e in substance P after a n t i o x i d a n t administration m a y be due to a d e c l i n e in the b r e a k d o w n o f the t a c h y k i n i n p r e c u r s o r during activation o f afferent fibers. T h i s possibility was d i s c u s s e d in a p r e v i o u s p a p e r ( Z h a n g and Lai, 1995). F u r t h e r m o r e , chronically a d m i n i s t e r e d P B N m i g h t d e c r e a s e substance P level through attenuation o f synthesis and/or axonal transport (in n o n v a g a l fibers) o f tachykinins. A c c o r d i n g to our data in Table 2, two factors can a c c o u n t for the attenuated n o n c h o l i n e r g i c airw a y c o n s t r i c t i o n f o l l o w i n g a n t i o x i d a n t treatment: ele v a t e d N E P activity and d e c r e a s e d t a c h y k i n i n level. S i n c e the b r o n c h i a l r e s p o n s e to t a c h y k i n i n is inv e r s e l y related to N E P a c t i v i t y ( D u s s e r et al., 1988), the i n c r e a s e d N E P activity should r e d u c e a i r w a y c o n s t r i c t i o n e v e n w i t h no c h a n g e in the substance P level. T h e l o w e r substance P level after antioxidant should r e d u c e further the n o n c h o l i n e r g i c a i r w a y constriction.
Acknowledgements T h e authors g r a t e f u l l y a c k n o w l e d g e the t e c h n i c a l assistance o f W a n g Min. T h e w o r k was supported by the N a t i o n a l Heart, L u n g and B l o o d Institute Grant H L - 4 0 3 6 9 , and by the N a t i o n a l S c i e n c e C o u n c i l o f the R e p u b l i c o f C h i n a ( N S C 8 4 - 2 3 3 1 - B - 0 0 2 - 3 1 4 ) .
References Carney, J.M., EL. Starke-Reed, C.N. Oliver, R.W. Landum, M.S, Cheng, J.F. Wu and R.A. Floyd (19911. Reversal of age-related increase in brain protein oxidation, decrease in enzyme activity, and loss in temporal and spatial memory by chronic administration of the spintrapping compound Ntert-butyl-~-phenylnitrone. Proc. Natl. Acad. Sci. USA 88: 3633-3636. Daly, M., D. Burgh and D.H.L. Evans (1953). Functional and histological changes in the vagus nerve of the cat after degenerative section at various levels. J. Physiol. (London) 120: 579-595. Dusser, D.J., E. Umeno, RD. Graf, T. Djokic, D.E. Borson and J.A. Nadel (19881. Airway neutral endopeptidase-like enzyme modulates tachykinin-induced bronchoconstriction in vivo. J. Appl. Physiol. 65: 2585-2591. Fang, Z.X. and Y.-L. Lai (1993). Oxygen radicals in bronchocon-
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striction of guinea pigs elicited by isocapnic hyperpnea. J. Appl. Physiol. 74: 627~633. Gamse, R., A. Wax, R.E. Zigmond and S.E. Leeman (1981). Immunoreactive substance P in sympathetic ganglia: distribution and sensitivity towards capsaicin. Neuroscience 6: 437-441. Haxhiu-Poskurica, B., M.A. Haxhiu, G.K. Kumar, M.J. Miller and R.J. Martin (19921. Tracheal smooth muscle responses to substance P and neurokinin A in the piglet. J. Appl. Physiol. 72: 1090-1095. Johnson, A.R., J. Ashton, W.W. Schulz and E.G. Erdos (1985). Neutral metalloendopeptidase in human lung tissue and cultured cells. Am. Rev. Respir. Dis. 132: 564-568. Katsumata, U., M. Miura, M. Ichinose, K. Kimura, T. Takahashi, H. Inoue and T. Takishima (1990). Oxygen radicals produce airway constriction and hyperresponsiveness in anesthetized cats. Am. J. Respir. Dis. 141: 1158-1161. Ko, W.C. and Y.-L. Lai (1988). Cyclic GMP affecting the tracheal nonadrenergic noncholinergic inhibitory system. Respir. Physiol. 73: 351-362. Lai, Y.-L. (1988). Maximal expiratory flow in the guinea-pig. Lung 166: 303-313. Lai, Y.-L. (1990). Oxygen radicals in capsaicin-induced bronchoconstriction. J. Appl. Physiol. 68: 568-573. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall (19511. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 265-275. Lundberg, J.M., T. Hokfelt, G. Nilsson, L. Terenius, J.E Rehfeld, R. Elde and S. Said (19781. Peptide neurons in the vagus, splanchnic and sciatic nerves. Acta Physiol. Scand. 104: 499501. Lundberg, J.M., E. Brodin and A. Saria (19831. Effects and distribution of vagal capsaicin-sensitive substance P neurons with special reference to the trachea and lungs. Acta Physiol. Scand. 119: 243-252. Martins, M.A., S.A. Shore and J.M. Drazen (19911. Capsaicininduced release of tachykinins: effects of enzyme inhibitors. J. Appl. Physiol. 70: 1950-1956. Orlowski, M. and S. Wilk (1981). Purification and specificity of membrane-bound metalloendopeptidase from bovine pituitaries. Biochemistry 20: 4942-4950. Ray, D.W., C. Hernandez, N. Munoz, A.R. Left and J. Solway (19881. Bronchoconstriction elicited by isocapnic hyperpnea in guinea pigs. J. Appl. Physiol. 65: 934-939. Richardson, J.B. and C.C. Ferguson (19801. Morphology of the airways. In: Physiology and Pharmacology of the Airways, edited by J.A. Nadel, New York, Marcel Dekker, pp. 1-30. Saria, A., C.R. Martling, Z. Yan, E. Theodorsson-Norheim, R. Gamse and J.M. Lundberg (19881. Release of multiple tachykinins from capsaicin-sensitive sensory nerves in the lung by bradykinin, histamine, dimethylphenyl piperazinium, and vagal nerve stimulation. Am. Rev. Respir. Dis. 137: 13301335. Simonsson, B.G, EM. Jacobs and J.A. Nadel (1967). Role of autonomic nervous system and the cough reflex in the increased responsiveness of airways in patients with obstructive airway disease. J. Clin. Invest. 46: 1812-1818. Stahl, G.L., H.-L. Pan and J.C. Longhurst (19931. Activation of
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ischemia- and reperfusion-sensitive abdominal visceral C-fiber afferents. Role of hydrogen peroxide and hydroxyl radicals. Circ. Res. 72: 1266-1275. Tai, H.H. (1992). Enzyme immunoassay. In: Encyclopedia of Pharmaceutical Technology, Volume 5, edited by J. Swarbrick and J.C. Boylan, New York, Marcel Dekker, pp. 201-233. Zhang, H.-Q. and Y.-L. Lai (1993). Role of vagus nerves in resiniferatoxin-induced bronchoconstriction of guinea pigs.
Respir. Physiol. 94: 285-295. Zhang, H.-Q. and Y.-L. Lai (1994). Intratracheal antioxidants attenuate exsanguination-induced bronchoconstriction in guinea pigs. J. Appl. Physiol. 76: 553-559. Zhang, H.-Q. and Y.-L. Lai (1995). Age-dependent mechanism in guinea pig bronchoconstriction induced by exsanguination. Respir. Physiol. 90: 361-369.
ELSEVIER
Respiration Physiology 104 (1996) 213-220
Oxygen radicals in the nonvagal component of noncholinergic airway constriction H . - Q . Z h a n g a, H . - H . Tai b, Y.-L. L a i a,c,* a Division of Pharmacology and Experimental Therapeutics, Lexington, KY 40536, USA h Division of Medicinal Chemistry and Pharmaceutics, College of Pharmacy, University of Kentucky., Lexington, KY 40536, USA c Department of Physiology, College of Medicine, National Taiwan University, No. 1, Sec. 1, Jen-Ai Road, Taipei, Taiwan, ROC Accepted 15 January 1996
Abstract
To test the hypothesis that oxygen radicals play an important role in the nonvagal component of the noncholinergic bronchoconstriction in vivo, 37 guinea pigs weighing 329 -t- 8 g were randomly divided into five groups: group 1, vagotomy; group 2, vagotomy + CAT (catalase); group 3, vagotomy -t- SOD (superoxide dismutase); group 4, vagotomy + PBN (ot-phenyl-N-tert-butyl nitrone); and group 5, capsaicin pretreatment. CAT, SOD, and PBN are antioxidants. Each animal was anesthetized, paralyzed, artificially ventilated, and pretreated with atropine and phenoxybenzamine. Immediately after acute capsaicin challenge, animals in group 1 exhibited decreases in maximal expiratory flow, dynamic respiratory compliance, and total lung capacity, as well as an increase in functional residual capacity, indicating noncholinergic airway constriction. The bronchoconstriction was significantly ameliorated by SOD and PBN, and it was almost abolished by capsaicin pretreatment. Thirty minutes after acute capsaicin challenge, there was a significant decrease in airway NEP activity and an increase in lung substance P level in group 1 but not in other groups. These results indicate that nonvagal component of noncholinergic bronchoconstriction is partially modulated by oxygen radicals.
Keywords: Mammals, guinea pigs; Mediators, NEE tachykinins; Oxygen radicals, bronchoconstriction: Pharmacological agents, capsaicin; Upper airways, bronchoconstriction, oxygen radicals
1. I n t r o d u c t i o n
The vagus nerves have been considered to play an important role in the airway hyperresponsiveness (Simonsson et al., 1967). It was reported that the vagus nerves contain many afferent substance P (SP)-immunoreactive nerve fibres (Lundberg et al., 1978). Lundberg et al. (1983) indicated that all SP-immunoreactive nerves in trachea and lung are * Corresponding author. Tel.: 886-(0)2-3938235. Fax: 886-(0)23938235.
afferent and capsaicin-sensitive, and a major portion (90%) of the SP produced in the sensory nervecell bodies in the nodose ganglion is transported into peripheral vagal branches. Tradition holds that the airway is mainly innervated with vagus nerves (Richardson and Ferguson, 1980). Data in the literature suggest, however, that both vagal and nonvagal components significantly contribute to the excitatory noncholinergic system. Using bilateral vagotomy and local capsaicin treatment-induced vagal dysfunction, we previously separated noncholinergic bronchoconstriction into vagal and nonvagal components (Zhang
0034-5687/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PII S 0 0 3 4 - 5 6 8 7 ( 9 6 ) 0 0 0 0 4 - 7
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and Lai, 1993). We found that the nonvagal component contributes 46% of the total constriction (Zhang and Lai, 1993). Lundberg et al. (1983) also showed that about 60% of the capsaicin-induced substance P release is from the vagus nerves and the remaining release is nonvagal in origin. Our previous data indicate that oxygen radicals play an important role in noncholinergic airway constriction induced by acute capsaicin challenge (Lai, 1990), exsanguination (Zhang and Lai, 1994), and hyperventilation (Fang and Lai, 1993). Accordingly, it is important to explore whether the bronchoconstriction caused by the nonvagal component is modulated by oxygen radicals. The purpose of this paper was to test the role of oxygen radicals in the nonvagal component of the noncholinergic bronchoconstriction. Several antioxidants including catalase (CAT), superoxide dismutase (SOD) and Ntert-butyl-c~-phenylnitrone (PBN) were used to antagonize oxygen radicals. We used CAT and SOD previously. Carney et al. (1991) demonstrated that PBN diminished production of oxygen radicals in brain. In this study, we tested whether PBN is an effective antioxidant in airways. 2. Material and methods 2.1. Animal preparations
Thirty seven Hartley strain guinea pigs weighing 329 4- 8 g were randomly divided into five groups: group 1, vagotomy (n = 8); group 2, vagotomy 4- CAT (n = 7); group 3, vagotomy + SOD (n = 8); group 4, vagotomy 4- PBN (n = 6); and group 5, systemic capsaicin pretreatment (n = 8). In group 1, each animal was bilaterally vagotomized under anesthesia with sodium pentobarbital (30-50 mg/kg, i.p.). The cervical vagus nerve was exposed and about 2 cm of the nerve was cut. Simultaneous bilateral vagotomy can easily cause the death of animals due to gastrointestinal disturbance. This risk, however, can be avoided by cutting the two vagus nerves one week apart. About half of vagotomized guinea pigs the right vagus nerve was cut first, then the left vagus nerve was cut one week later. Under the same conditions in another 50% animals, the left vagus nerve was first cut, the right one a week later. Three weeks after the initial vago-
tomy, animals were anesthetized and their airway functions were tested before and after acute capsaicin challenge. No significant differences in any capsaicin-induced respiratory parameters (expressed as percent of baseline value) between these two sequences of vagotomy were found. Each vagotomized animal received an intratracheal instillation of 0.75 ml saline 30 rain before intravenous injection of capsaicin. In groups 2 and 3, CAT (400,000 U) or SOD (120,000 U) was administrated. Our previous experiment indicated that the above doses of CAT and SOD were sufficient (Zhang and Lai, 1994). Each in 0.75 ml saline solution, CAT or SOD was intratracheally instilled in each guinea pig 30 min before acute capsaicin challenge. In group 4, PBN (32 mg/kg, i.p) was treated with twice daily for 14 days (Carney et al., 1991) before acute capsaicin challenge. To deplete tachykinins in group 5, a 5day chronic capsaicin pretreatment, consisting of two consecutive daily doses of 50 mg/kg and three consecutive daily doses of 100 mg/kg capsaicin administered subcutaneously, was begun 9 days before the study. Especially during the first 1-3 days the injections were administered in small doses, as many as l0 times per day with a 1 h interval between any two injections. Each animal was anesthetized with a combination of sodium pentobarbital (15 mg/kg), fentanyl (0.2 mg/kg), and droperidol (10 mg/kg) before the capsaicin was subcutaneously administered. 2.2. Functional testing o f bronchial constriction
On the day of the study, each animal was anesthetized with sodium pentobarbital (35 mg/kg), paralyzed with gallamine triethiodide (4 mg/kg, i.v.), and artificially ventilated with a tidal volume of 6 ml/kg at a frequency of 60/min. These ventilatory variables were found to maintain arterial blood gases and pH within the normal range (Ray et al., 1988). No positive end-expiratory pressure was added during artificial ventilation. All animals were treated with atropine (0.2 mg/kg) and phenoxybenzamine (0.5 mg/kg) via intramuscular injection 15 min before the experiment. These doses of atropine and phenoxybenzamine were sufficient to suppress the cholinergic and ~-adrenergic nervous systems, respectively (Ko and Lai, 1988). Each anesthetized-paralyzed and artificially ventilated guinea pig was placed
H.-Q. Zhang et aL /Respiration Physiology 104 (1996) 213-220
supine inside a whole-body plethysmograph. The flow rate was monitored with a Statham PM15 differential pressure transducer as the pressure dropped across three layers of 325-mesh wire screen in the plethysmograph wall. Lung volume change was obtained via integration of flow. Airway opening pressure (Pao) was measured with a Statham PM131 pressure transducer. To induce airway constriction via activation of afferent C-fibers, capsaicin was intravenously injected into the jugular vein of each animal (16 #g/kg, bolus injection). Before (baseline) and after acute capsaicin challenge, the maximal expiratory flow-volume (MEFV) maneuver was performed to evaluate functional changes in airway dimension according to a previous method (Lai, 1988). Briefly, three times the lungs were inflated to total lung capacity (TLC, lung volume at Pao = 30 cmH20). At peak volume during the third inflation, the inflation valve was shut off and immediately another solenoid valve for deflation was turned on automatically. The deflation valve was connected to a 20-L container which had a pressure of - 4 0 cmH20 (subatmospheric). The negative pressure of 40 cmH20 produced the maximal expiratory flow (Vmax)- The MEFV plot was stored on a cathode ray storage oscilloscope (V-134, Hitachi). During artificial ventilation (between the interval of the MEFV maneuvers), compliance of the respiratory system (Crs) was obtained as the ratio of tidal volume/Pao difference between end-inspiration and end-expiration, The general experimental protocol was that the MEFV maneuver was performed before as well as 1, 5, 10, 15, 20, and 30 min after acute capsaicin challenge. To evaluate the change in airway dimension, "¢max at 50% baseline TLC (Vmax50) and dynamic compliance of the respiratory system (Crs) were used as indicators of bronchoconstriction. Before and after each MEFV maneuver, functional residual capacity (FRC) was determined using a modified neon dilution method (Lai, 1988). 2.3. Measurement of NEP activi~
At the end of the above experiment we excised airway tissues (main stem bronchus and all dissectible bronchial tree) from the left lung of each animal. The bronchial tissues were washed with physiological saline and stored at -70°C for later analysis.
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A cell-free extract of bronchial tissues was made by following the procedures of Haxhiu-Poskurica et al. (1992). The frozen tissues (0.06 g) were thawed and sonicated in 50 mM Tris-HC1 buffer (3 ml), pH 7.4, at 4°C for 30 sec. The residue was removed from the extract by centrifuge at 17,500 g for 15 min at 4°(7. The supernatants were used for enzyme analysis. Neutral endopeptidase activity was determined by following the procedures of Orlowski and Wilk (1981), modified by Haxhiu-Poskurica et al. (1992). The reaction mixture (250 gl) consisted of 10-50 #1 of tissue extract, 1.25 mM (final concentration) substrate, i.e., glutaryl-alanine-phenylalanine4-methoxy-2-naphthylamide, aminopeptidase M (10 #g), and 50 mM Tris-HC1 buffer, pH 7.4. The mixture was incubated at 37°C for 60 min. Reaction was stopped by adding 1 ml of 10% trichloroacetic acid and then 1 ml of 0.005% (wt/vol) fast garnet (GBC) (a stabilized diazonium salt). Under this incubation condition, the hydrolysis of the substrate was proportional to enzyme concentration and time, reflecting steady-state kinetics and linearity (Orlowski and Wilk, 1981). The amount of 2-naphthylamine formed during the reaction was determined by spectrophotometer (Beckman Model 35) at 530 nm. Specific activity of NEP was expressed in picomoles per milligram tissue protein per hour. Protein concentration was determined by the method of Lowry et al. (1951), with bovine serum albumin (BSA) as the standard. 2.4. Measurement of substance P of lung tissue
At the end of the above experiment the right lung was excised, and washed with physiological saline and stored at -70°C for later analysis. SP of lung tissue was extracted via the procedures of Saria et al. (1988). The frozen tissue (0.3 g) were thawed and cut into pieces, suspended in 10 vol 2 N acetic acid, and boiled for 10 min. Then the samples were homogenized with sonicator (Model W-375). The homogenates were transferred to polypropylene tubes and centrifuged for 20 min at 3000 g. The supernatants were used for analysis of lung SP using enzyme immunoassay (EIA) (Tai, 1992). A 96microwell plate was first coated with protein A diluted in coating buffer, pH 9.6. Diluted SP antibodies were added to each well. Subsequently, SP standards
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H.-Q. Zhang et al./Respiration Physiology 104 (1996) 213-220
Table 1 Body weight and respiratory variables during the baseline period in five groups of guinea pigs Group
n
Body wt. (g)
TLC (ml)
FRC (ml)
Crs (ml/cmH20)
VmaxS0 (ml/sec)
Vagotomy Vagotomy 4- CAT Vagotomy 4- SOD Vagotomy -+- PBN Capsaicin pretreatment
8 7 8 6 8
328 330 329 329 330
10.4 10.6 10.5 10.6 10.3
2.4 2.3 2.4 2.5 2.4
0.22 0.22 0.19 0.22 0.21
93 95 95 91 92
444± 4-
8 9 9 7 8
4+ + 4±
0.4 0.3 0.2 0.2 0.2
4- 0.3 2_ 0.2 :L 0.1 4- 0.1 4- 0.2
44444-
0.02 0.01 0.01 0.02 0.01
4-4-4 -F 5 4- 5 4- 6 4- 4
Values are mean ± SE; TLC = total lung capacity: FRC = functional residual capacity: Crs = dynamic respiratory compliance; and Vma×5o = maximal expiratory flow at 50% baseline TLC; CAT = catalase: SOD = superoxide dismutase; and PBN = o~-phenyl-N-tertbutyl nitrone.
or unknown samples, in duplicate, were also added to each well. Diluted SP-enzyme (horseradish peroxidase) conjugate was delivered to each well. The plate was incubated at 37°C for 1-2 h with light shaking (Kjanke and Konkel TYP VX7). After washing three times with washing buffer, substrate (K-Blue Substrate) was added to each well. After having stood at 37°C for 30 rain the plate was read in a microwell reader (Model LE 310) at 650 nm. SP concentration in unknown samples was obtained from the simultaneously obtained absorbance-SP concentration standard curve. All values are reported as means -4- SE. Analysis of variance was used to establish the statistical significance of differences among groups. If significant differences among groups were obtained, Dunnett's test was used to differentiate differences between experimental and control groups. We employed Scheffe's test to analyze significance of differences between any two experimental groups. Differences were considered significant if P < 0.05.
100'
A
80"
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--
Vagotomy + CAT
z,
Vagotomy + SOD
-¢ '
Vagotomy + PBN Capsaicin pretreatment
40
-
1'0
"
1'5
Time
3. Results Body weight and respiratory variables during the baseline period (before acute capsaicin challenge) are shown in Table 1. No significant differences in any respiratory variable between groups were found. In vagotomy animals, acute capsaicin challenge (16 #g/kg) induced an immediate (at 1 min) decreases in V .... 5o (Fig. 1) and Cr, (Fig. 2), indicating moderate to severe bronchial spasm. This airway constriction was gradually attenuated with time. However, capsaicin-induced changes in VmaxS0 and Cr~ were significantly ameliorated by SOD, PBN and a systemic capsaicin pretreatment at 1-30 rain
,
20
-
i
25
-
,
30
(rain)
Fig. 1. Capsaicin-induced changes in the maximal expiratory flow rate at 50% baseline TLC (Vm:ixs0) in five groups of guinea pigs. All changes were expressed as a percent of the baseline (zero time) values. Time indicates the time period after the bolus injection of capsaicin (16 #g/kg) via the jugular vein. CAT = catalase: SOD = superoxide dismutase: PBN = c~-phenyl-N-tertbutyl nitrone. Statistical differences between groups (p < 0.05): a = compared to the vagotomy group; b = compared to the capsaicin pretreatment group•
(Fig. 1). The most efficient way to prevent the noncholinergic bronchoconstriction was a systemic capsaicin pretreatment to deplete tachykinins. In addition, an acute capsaicin challenge caused alterations in lung volumes. At one minute after an acute cap-
H.-Q. Zhang et al./ Respiration Physiology 104 (1996) 213-220 a
a
100
a
a
Table 2 Airway neutral endopeptidase(NEP) activityand lung substance P level of guinea pigs at 30 min after acute capsaicin challenge
bc
Group
90
Vagotomy Vagotomy + CAT Vagotomy + SOD Vagotomy + PBN Capsaicin pretreatment
8(1
¢0
Vagotomy
70"
60"
•
Vagotomy + CAT
.,t
Vagotomy + SOD
,t
Vagotomy + PBN
.... 0
-
,
5
-
,
10
-
.
1'5
Time
,
20
n
8 7 8 6 8
NEP
Substance P
(pmol/mg protein/h)
(pmol/g tissue)
28.0 40.9 42.5 41.9 37.4
0.94 0.65 0.38 0.17 0.05
± 4444-
1.2 1.0 a 0.6 a'b 0.4 a 1.6a
-4- 0.1 :[: 0.1 b -4- 0. I a 4- 0.1 a 4- 0.04 a
Values are means + SE; CAT : catalase; SOD = superoxide dismutase; PBN = a-phenyl-N-tert-butyl nitrone. Statistical differences between groups (P < 0.05): acompared to the vagotomy group; bcompared to the systemic capsaicin pretreatment group.
Capsaicin pretreatment
50
217
.
,
25
.
,
30
(rain)
Fig. 2. Capsaicin-induced changes in the compliance of the respiratory system (Cry) in five groups of guinea pigs. All changes were expressed as a percent of the baseline (zero time) values. Time indicates the time period after the bolus injection of capsaicin (16 /lg/kg) via the jugular vein. CAT = catalase; SOD = superoxide dismutase; PBN = a-phenyl-N-tert-butyl nitrone. Statistical differences between groups (P < 0.05): a = compared to the vagotomy group; b = compared to the capsaicin pretreatment group; c = compared to the vagotomy + SOD and the vagotomy 4- PBN groups.
saicin challenge, TLC decreased to 86.1 + 0.6% of the baseline value in the vagotomy group. The decrease was significantly attenuated by SOD (89.1 -4- 0.8% of baseline), PBN (90.0 -+- 0.6% of baseline), and by capsaicin pretreatment (96.1 4- 0.8% of baseline). On the other hand, FRC increased to 124 + 5.1% of baseline in the vagotomy group at one minute after acute capsaicin challenge. The increase was significantly attenuated by SOD (109.0 -4- 0.7% of baseline), PBN (108.9 -4- 0.6% of baseline), and by systemic capsaicin pretreatment (105.0 ± 0.7% of baseline). Compared to the vagotomy group, however, the CAT group did not significantly change acute capsaicin-induced alteration in either ~/max50, Cry, TLC, or FRC. Bronchial NEP activity and lung SP level at 30
min after acute capsaicin challenge are listed in Table 2. Compared to the vagotomy group, there was a significant increase in bronchial NEP activity in all experimental groups (groups 2-5). On the other hand, lung tissue SP level showed a significant decrease in all experimental groups (Table 2). 4. Discussion
In the anesthetized-paralyzed and vagotomized guinea pig, an intravenous injection of a potent capsaicin (16 #g/kg) immediately induced marked decreases in Vm~50, Crs, and TLC, as well as a significant increase in FRC. After the peak change at 1 min these parameters gradually returned toward the baseline values. SOD, PBN, and systemic capsaicin pretreatment significantly attenuated the acute capsaicin challenge-induced respiratory changes. In addition, SOD, PBN, and capsaicin pretreatment significantly increased airway NEP activity and decreased lung substance P level. Several features of these noncholinergic respiratory changes after vagal dysfunction will now be discussed as follows.
4.1. Oxygen radicals and the nonvagal component of the noncholinergic airway constriction Acute capsaicin challenge significantly decreased VmaxS0, Crs and TLC, but markedly increased FRC in the vagotomy group. These alterations indicate capsaicin challenge-induced noncholinergic airway constriction. We previously showed that noncholin-
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H.-Q. Zhang et al./Respiration Physiology 104 (1996) 213-220
ergic airway constriction can be separated into vagal and nonvagal components (Zhang and Lai, 1993). At 1 min after injection of resiniferatoxin or capsaicin, about 36-51% of the airway constriction is due to the vagal component, while about 49-64% airway constriction is due to the nonvagal component (Zhang and Lai, 1993). As in vagus-intact animals (Zhang and Lai, 1993), we found similar patterns, although with a smaller degree, of acute capsaicin challenge-induced alterations in respiratory parameters in vagotomized animals. Since this constriction occurred in vagotomized animals, the noncholinergic activation should be due to the nonvagal component alone. Hydrogen peroxide and superoxide can be scavenged by CAT and SOD, respectively. In addition, PBN can also antagonize oxygen radicals. The major new finding to emerge from the present study is that antioxidants (SOD and PBN) significantly decreased the nonvagal, noncholinergic airway constriction. Intratracheal instillation of SOD and intraperitoneal administration of PBN significantly ameliorated all the above acute capsaicin-induced changes. At 1 min after acute capsaicin challenge, compared to the vagotomy group, the decrease in VmaxS0 was ameliorated about 25.2%, 31.5%, or 31.7% by CAT, SOD, or PBN. These magnitudes of attenuation are markedly larger than those in vagus-intact animals (Lai, 1990), in which acute intravenous CAT and SOD did not significantly alter capsaicin-induced airway constriction. The effective action of antioxidants in this study may be related to their intratracbeal application. Compared to the marked intratracheal effects of CAT on noncholinergic airway constriction in vagus-intact animals (Zhang and Lai, 1993), no significant effect of CAT in this study could be due to a smaller degree of airway constriction in vagotomized animals. Other reasons may include variable responses in animals and different stimuli (capsaicin vs. exsanguination) to activate afferent C-fibers. Collectively, our data suggest that oxygen radicals play an important role in capsaicin-induced nonvagal, noncholinergic airway constriction. It was demonstrated that oxygen radicals cause airway constriction (Katsumata et al., 1990). After capsaicin pretreatment, however, acute capsaicin challenge did not induce airway constriction. This result can be interpreted as that acute capsaicin chal-
lenge does not cause an increase in the production of oxygen radicals or that the increased oxygen radicals alone cannot induce airway constriction in capsaicinpretreated animals. We are in favor of the latter interpretation. Since oxygen radicals activate afterent C-fibers (Stahl et al., 1993), it is possible that the radicals cause the observed airway constriction indirectly via tachykinin release. Further studies are needed to delineate the underlying mechanism. 4.2. Tachykinins and NEP in the nonvagal component of the noncholinergic airway constriction Chronic bilateral cervical vagotomy was used to cause degeneration of vagus nerves (Daly et al., 1953) which, in turn, decreased substance P concentration in airways and lungs (Lundberg et al., 1983). Similarly in our studies, the lung substance P decreased to a very low level (0.9 pmol/g) in the vagotomy group compared to the control group with intact vagus nerves (16-7 pmol/g) (Zhang and Lai, 1994). The degree of airway constriction, however, seems not linearly related to the lung substance P level. For example, acute capsaicin challenge (16 #g/kg) produced about 95% and 55% decreases in Vma× in the control (Zhang and Lai, 1993) and the vagotomy (this study) groups, respectively. As mentioned above, their respective lung substance P levels are 6-7 pmol/g (Zhang and Lai, 1994) and 0.9 pmol/g (this study). If we assume that the released substance P is proportional to its lung content, the concentration-constriction relationship is not a linear but an exponential one. This type of exponential concentration-response relation of substance P was found by Martins et al. (1991) in guinea pig lungs. Sources of nonvagal tachykinins may be released mainly from the sympathetic nerves (Gamse et al., 1981), which may originate from the spinal cord (Lundberg et al., 1983). Johnson et al. (1985) suggested that NEP activity exists in the epithelium, submucosal glands, nerves of the airway, lung and airway smooth muscle. NEP in the airway normally degrades tachykinins which, in turn, results in a decreased contractile response of the airway smooth muscle (Dusser et al., 1988). In this study, antioxidants increased airway NEP activity and decreased lung substance P level (Table 2). This may indicate that antioxidants can pre-
H.-Q. Zhang et al./ Respiration Physiology 104 (19961 213-220
v e n t the inactivation o f N E P by o x y g e n radicals. C o n s e q u e n t l y , a portion o f the d e c r e a s e in substance P f o l l o w i n g a n t i o x i d a n t t r e a t m e n t should be related to the i n c r e a s e d N E P activity. O t h e r portion o f the d e c r e a s e in substance P after a n t i o x i d a n t administration m a y be due to a d e c l i n e in the b r e a k d o w n o f the t a c h y k i n i n p r e c u r s o r during activation o f afferent fibers. T h i s possibility was d i s c u s s e d in a p r e v i o u s p a p e r ( Z h a n g and Lai, 1995). F u r t h e r m o r e , chronically a d m i n i s t e r e d P B N m i g h t d e c r e a s e substance P level through attenuation o f synthesis and/or axonal transport (in n o n v a g a l fibers) o f tachykinins. A c c o r d i n g to our data in Table 2, two factors can a c c o u n t for the attenuated n o n c h o l i n e r g i c airw a y c o n s t r i c t i o n f o l l o w i n g a n t i o x i d a n t treatment: ele v a t e d N E P activity and d e c r e a s e d t a c h y k i n i n level. S i n c e the b r o n c h i a l r e s p o n s e to t a c h y k i n i n is inv e r s e l y related to N E P a c t i v i t y ( D u s s e r et al., 1988), the i n c r e a s e d N E P activity should r e d u c e a i r w a y c o n s t r i c t i o n e v e n w i t h no c h a n g e in the substance P level. T h e l o w e r substance P level after antioxidant should r e d u c e further the n o n c h o l i n e r g i c a i r w a y constriction.
Acknowledgements T h e authors g r a t e f u l l y a c k n o w l e d g e the t e c h n i c a l assistance o f W a n g Min. T h e w o r k was supported by the N a t i o n a l Heart, L u n g and B l o o d Institute Grant H L - 4 0 3 6 9 , and by the N a t i o n a l S c i e n c e C o u n c i l o f the R e p u b l i c o f C h i n a ( N S C 8 4 - 2 3 3 1 - B - 0 0 2 - 3 1 4 ) .
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