Mechanism of H202-induced smooth muscle JANG

B. GUPTA

AND KAILASH

modulation

of airway

PRASAD

Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N 0 WO, Canada Prasad. Mechanism of Gupta, Jang B., and Kailash H,O,-induced modulation of airway smooth muscle. Am. J. Physiol. 263 (Lung Cell. Mol. Physiol. 7): L714-L722, 1992.We investigated the effects of H202 generated by glucose (G) and glucose oxidase (GO) on the isolated rabbit tracheal smooth muscle suspended in Krebs-Ringer solution. H,O, generated by G + GO was measured with luminol-dependent chemiluminescence. G + GO in the concentrations of IX (1.80 PM G, 0.075 U/ml GO) and 2, 4, and 8~ generated 1.35, 3.2, 6.10, and 6.00 PM of H202, respectively. H202 produced relaxation of rabbit tracheal smooth muscle, relaxed acetylcholine (ACh)-precontracted muscle, and reduced muscle responsiveness to ACh. These effects were concentration dependent. H202, however, produced contraction of guinea pig tracheal smooth muscle. Catalase completely inhibited the H,O,-induced relaxation of ACh-precontracted tracheal smooth muscle. H,02-induced relaxation was greater in preparations with intact epithelium (65%) than in those denuded of epithelium (40%). The relaxant effects of H202 in the presence of an inhibitor of nitric oxide synthesis (NC:-monomethyl-L-arginine), an inhibitor of guanylate cyclase (methylene blue), an inhibitor of cyclooxygenase (indomethacin), and an ATP-sensitive K+ channel blocker (glipizide) were 44,44,39, and 48%, respectively. H,O,-induced relaxation in the presence of indomethacin in preparations with denuded epithelium was 29%. These results suggest that H202induced relaxation of tracheal smooth muscle is partly epithelium dependent and is mediated by inhibitory arachidonic acid metabolites, epithelium-derived relaxing factor (nitric oxide), ATP-sensitive K+ channels, and the synthesis and release of prostaglandins from epithelium and the underlying smooth muscle. tracheal smooth muscle; N’“-monomethyl-L-arginine; glipizide; indomethacin; acetylcholine; hydrogen peroxide chemiluminescence; catalase; hydrogen peroxide INFLAMMATION is widely accepted as the mechanism of bronchial hyperresponsiveness and as an important factor in pathophysiology of asthma (11, 13). Pathophysiological changes in asthma are very likely produced by the release of various mediators from the inflammatory cells in the airways or in blood. Inflammation of the airways is associated with infiltration by phagocytic cells, which, when activated, release reactive oxygen species [superoxide anion (OF), hydrogen peroxide (H,O,), hydroxyl radicals (*OH), and hypochlorous acid (HOCl)], and these may be involved in the pathophysiology of asthma. There are various sources of reactive oxygen species, so-called oxygen free radicals (OFRs), including polymorphonuclear (PMN) leukocytes, eosinophils, mast cells, and macrophages (1, 15). OFRs also have been implicated in the pathophysiology of various cardiovascular diseases (25, 35-38). The effects of OFRs on the responsiveness of airway smooth muscle are uncertain. Stewart et al. (47) showed that 0; generated from xanthine and xanthine oxidase had no effect on bovine trachealis muscle. H202 produces AIRWAY

L714

1040-0605/92

$2.00 Copyright

0

contraction in bovine and guinea pig airway smooth muscle (4,473. In rat the effect of Hz02 is transient and ~20% of the reference contraction produced by electrical stimulation (48). The mechanism of action of H202 on airway smooth muscle is unknown. The contractile effect of H202 is greatly enhanced in guinea pig trachealis preparations denuded of epithelium (4), which suggests that release of epithelium-derived relaxing factor (EpDRF) from the epithelium may occur. Interaction between active substances and the epithehum of the airways is becoming increasingly important in defining the responsiveness of the airway to various stimuli (20, 32). Several studies (17, 23, 32) have shown that removal of epithelium in isolated airways from different animals augmented the responses to bronchoconstrictor agents. These observations suggest that the airway epithelium releases inhibitory factor(s) that partially antagonizes activation of airway smooth muscle by bronchoconstrictor agents. However, the nature of the EpDRF has not been defined, and there is still debate as to whether it is nitric oxide (NO) (28, 32, 50) or epithelium-derived hyperpolarizing factor that is distinct from NO (6, 32, 40). Epithelium-derived hyperpolarizing factor has been proposed to open ATP-sensitive K+ channels and to activate a Na+-K+ pump in the smooth muscle (19, 40, 46). Epithelium has also been reported to produce a variety of arachidonic acid metabolites, including prostaglandin (PG) EB, a potent smooth muscle relaxant (9, 14, 16). We investigated the role of H20z in modulation of the airway smooth muscle tone by studying the effects of Hz02 on the tracheal smooth muscle with intact or denuded of epithelium. We also determined if the Hz02induced response is mediated through NO, ATP-sensitive K+ channel, guanylate cyclase, or arachidonic acid metabolites by use of specific inhibitors. METHODS New Zealand White rabbits weighing 2.5-3.5 kg and guinea pigs weighing 350-450 g were each stunned by a sharp blow to the head. The tracheas were rapidly excised, gently cleaned of debris and blood, and placed in an ice-cold Krebs-Ringer solution of the following composition (in meq/l): 140 Na, 4.6 K, 4.9 Ca, 2.3 Mg, 21.91 HCOn, 3.48 P04, 2.32 S04, 125 Cl, and 5 mM glucose (pH 7.4). Tracheal chains were prepared by cutting longitudinally through the cartilaginous portion of the tracheal rings. Three tracheal rings were tied at the cartilaginous ends of the rings to form a chain. One end was attached to a stainless steel wire stirrup at the base of a IO-ml organ bath filled with Krebs-Ringer solution, and the free end was attached to a Grass FT-03 force displacement transducer attached to a Grass Polygraph Recorder for recording the contraction of tracheal muscle. The solution in the bath at 37°C was constantly bubbled with a mixture of 95% O,-5% CO,. All preparations were allowed to equilibrate for 60 min at a resting tension of 1.5 g and were

1992 The American

Physiological

Society

Downloaded from www.physiology.org/journal/ajplung at Macquarie Univ (137.111.162.020) on February 13, 2019.

HYDROGEN

PEROXIDE

washed at 15min intervals. The resting tension was then adjusted to 1.0 g. This resting tension represented the optimal resting length of the muscle at which acetylcholine (ACh) produced maximal contraction. Optimal length was determined by using length-tension response to ACh. The dose-response curve was determined to select a concentration of ACh (0.5 pg/ml) that produced 80% maximal contraction. This concentratoin of ACh was used in all experiments. The response of the preparation to ACh was recorded. This was repeated till identical responses were obtained. The preparation was then washed at 5-min intervals until the tension reached the resting level. All the tensions were measured in milligrams. The responses to glucose and glucose oxidase (G + GO) in ACh-precontracted tracheal preparations were expressed as percentage of ACh response. Deepithelialization

of Trachea

The epithelial layer was removed by gentle rubbing with a cotton ball moistened with Krebs-Ringer solution (19). Presence or absence of epithelium was confirmed by histological examination. Tracheal rings with intact epithelium or those denuded of epithelium were prepared for histological study by fixing with 10% buffered Formalin. The tissue was then frozen in optimal cutting temperature compound (Miles, Diagnostic Division, Elhart, IN), and a IO-pm section was cut. The tissue was stained with hematoxylin and eosin. After dehydration with graded alcohol concentrations, the tissue was cleared in xylene. The tissue was mounted in permanent mounting media, and histological examination by light microscopy was performed. Measurement of Glucose and Glucose Oxidase-Induced H202 H202 generated by G + GO was measured by a modified method of Trinder (49) and Misra and Squatrito (31). The oxidation of glucose by glucose oxidase produces gluconic acid and H,O,. The latter is measured by means of luminol-dependent chemiluminescence in the presence of peroxidase. Luminol is oxidized by H,O, and peroxidase to a luminol radical, which results in formation of an endoperoxide that decomposes to yield an electronically excited 3-aminopthalate dianion that emits light on returning to its ground state. The chemiluminescence of the sample is converted to concentration of H,O, from the chemiluminescence of a standard amount of H,O,. Various concentrations of glucose (0.72-14.4 PM) and glucose oxidase (0.03-0.6 U/ m 1) were used in the presence of horseradish peroxidase (final concentration, 1 hg/ml) and luminol (lo+ M). H202-induced chemiluminescence was calculated from the difference in chemiluminescence in the absence and in the presence of catalase (500 U/ml). The chemiluminescence was measured in millivolts with the use of an LKB Wallace 1251 luminometer. All measurements were made in triplicate. H202 Generation Four concentrations (1,2,4, and 8~) of G + GO were used to generate H,02: lx, 1.8 PM glucose and 0.075 U/ml glucose oxidase; 2x, 3.6 PM glucose and 0.15 U/ml glucose oxidase; 4x, 7.2 PM glucose and 0.3 U/ml glucose oxidase; and 8X, 14.4 PM glucose and 0.6 U/ml glucose oxidase. All solutions were made in glucose-free Krebs-Ringer buffer solution, and 0.2-0.6 ml of solutions were added to the myobath to elicit responses. Protocol Ten sets of studies were made: Group I. The effects of three concentration (1, 2, and 4x) of G + GO were studied on the basal tone of the tracheal preparation for 20 min. Group II. This study was designed to examine the ability of H202 to relax ACh-precontracted tracheal preparation. The ef-

AND

AIRWAY

TONE

L715

fects of ACh were observed for 20 min, and the preparations were then washed until the tension returned to pretreatment level. ACh was then added, and 2.5 min later four concentrations (1,2,4, and 8~) of G + GO were added, one at a time, and the effects were observed for a total period of 20 min. Contractile responses to ACh with or without G + GO were compared. Group 111. This study was designed to determine the ability of H202 to affect the contractile response of the preparation to ACh. The preparations were exposed to three different concentrations (I, 2, and 4x) of G + GO, and 5 min later ACh was added to the bath, and the response was observed for 20 min. Group IV. This study was designed to determine whether the relaxant effect of G + GO is mediated through H,O,. Effects of 4~ concentration of G + GO on the ACh-precontracted preparation, in the presence and absence of catalase (H,Oz metabolizer), were determined. Catalase (500 U/ml) was added to the bath containing tracheal preparation 5 min prior to addition of ACh, and G + GO was added 2.5 min after ACh. The effects were observed for 20 min. Group V. This study was designed to evaluate the role of epithelium in H,O,-induced relaxation of ACh-precontracted preparation. The effects of 4~ concentration of G + GO on ACh-precontracted tracheal preparations denuded of epithelium were determined. Group VI. This study was designed to determine whether H,O,-induced relaxation is mediated through NO. Effects of 4~ concentration of G + GO on the ACh-precontracted tracheal preparation in the presence or absence of NG-monomethyl+ arginine (L-NMMA) were measured. L-NMMA inhibits synthesis of NO from L-arginine (42). L-NMMA (0.25 mM) was added to the bath 20 min prior to addition of ACh. G + GO was added 2.5 min after addition of the ACh. Group VII. This study was designed to determine the role of guanosine 3’,5’-cyclic monophosphate (cGMP). The effects of G + GO on the ACh-precontracted preparation in the presence or absence of methylene blue (10m5 M) were measured. Methylene blue inhibits cGMP synthesis (30). It was added to the bath 20 min prior to addition of ACh. Group VIII. This study was designed to measure the role of epithelium-derived hyperpolarizing factor. The effects of G + GO in the presence or absence of glipizide (lo-” M), an inhibitor of ATP-sensitive K+ channel (39), were measured. Group IX. This study was designed to determine the role of arachidonic acid metabolites. The effects of G + GO in the presence and the absence of indomethacin ( 10e5 M), an inhibitor of cyclooxygenase (48), were measured. Indomethacin was added to the bath 30 min prior to addition of ACh. Group X. Contrary to the earlier reports (4, 47), H,O, produced relaxation of rabbit tracheal preparation. To find out whether this is a peculiarity of the rabbit, the effects of three concentrations (1, 2, and 4x) of G + GO on the tracheal preparation of guinea pig were also studied. Histamine (1 pg/ml) was used to produce reference contraction. G + GO-induced contraction was expressed as precent of reference contraction produced by histamine. Statistical

Analysis

The results were expressed as means t SE. The data were analyzed by two-way analysis of variance using repeated measures (BMDP statistical software, University of California, Berkeley, CA) followed by the least-significant differences (12). P < 0.05 was considered significant.

RESULTS Measurement of H202 Generated by G + GO G + GO produced a time-dependent increase in the chemiluminescence that plateaued at - 16 min (Fig. 1).

Downloaded from www.physiology.org/journal/ajplung at Macquarie Univ (137.111.162.020) on February 13, 2019.

L716

HYDROGEN 400

GCGO

r

o

CAT + G+GO

PEROXIDE

AND

AIRWAY

m

L

*I



4

1



1



8 12 TIME (Min)

11

I

16

20

Preparation

Effects of three concentrations of G + GO on the baseline tension of tracheal preparations are summarized in Fig. 3. At all concentrations, G + GO produced significant relaxation within 5-10 min that progressed for another 15-min period of observation. This relaxation was concentration dependent. The effects of ACh on the tracheal preparation and four concentrations of G + GO in ACh-precontracted tracheal preparations are summarized in Fig. 4. ACh produced a maximal contractile response within 2.5 min that was maintained during a 20-min period of observation. 400

F

E -1.5

-

G+GO(p[)

v

*a

I

I

I

L

5

0

The chemiluminescence was abolished in the presence of catalase, indicating that it was due to HzOz (Fig. 1). These findings were consistent (6 experiments). There was progressive increase in the peak chemiluminescence with increasing concentrations of G + GO up to 4~ concentration (Fig. 2). Further increase in the concentrations of G + GO did not produce any further increase in the peak chemiluminescence. There was a linear relationship between peak chemiluminescence and various concentrations of standard HZOZ. From the chemiluminescence of known concentrations of H202, the concentrations of HzOz generated by G + GO were determined. G + GO in the concentrations of 1,2,4, and 8~ generated 1.35, 3.20, 6.10, and 6.00 PM of H202, respectively.

.

G+cO(u[)

0

*a

1 *a

0.5

'3

-2.0

Fig. 1. Typical chemiluminescent activity of H,O, generated by 4~ concentration of glucose plus glucose oxidase (G + GO) in absence or presence of catalase (CAT). Note peak activity at 16 min. Also note prevention of chemiluminescent activity of G + GO in presence of CAT.

Effect on Tracheal

G+GO(lX)

1.0

1

0

TONE

I

I

I

I

I

20

15

10 TIME (Min)

Fig. 3. Effects of various concentrations of G + GO on tracheal preparation. Each point represents mean of: SE ments). * P < 0.05, comparison of values at various time those before drug treatment (0 min) in respective groups. vs. 2 or 4X.

basal tone of (n = 8 experiintervals with a P < 0.05, IX

Relaxation was dependent on the concentrations of G + GO in the range of 1-4~. A concentration of 8~ produced relaxation that was similar to that produced by the 4~ concentration (65%). Relaxation was progressive, reaching a maximum at 20 min. Because the 4~ concentration produced maximum effect, it was used for the study of the mechanism of the relaxant effect of H202. Experiments were also conducted to determine whether the responses to ACh are affected by the presence of HZ02. The results of ACh response in absence and presence of three concentrations (1,2, and 4x) of G + GO are summarized in Fig. 5. The response of ACh decreased progressively with time in a manner dependent on the concentrations of G + GO. Effects of Catalase

Effects of 4~ concentration of G + GO on the AChprecontracted tracheal preparations in the absence or presence of catalase are summarized in Fig. 6. Catalase did not affect the basal tone or the ACh response. However, pretreatment with catalase completely prevented Ach (o.ep&d) 0 ACh + G+GO (2X) ACh + G+GO (1X) l ACH + G+GO (4X) o ACh + G+GO (8X) 120

v v

r

r

01 0



’ 5



’ 10 TIME

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2.4

4.8

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0

0.1

0.2

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0.4

12.0 0.5

14.4 0.6

Glucose

(@A)

Gluco:e Oxidase (u/m0

Fig. 2. Effect of various concentrations of G + GO on peak nescence. Results are expressed as mean & SE. Each point mean of 6 experiments.

chemilumion curve is



’ 15



’ 20

(Min)

Fig. 4. Effect of various concentrations (1, 2, 4, 8X) of G + GO on ACh-precontracted tracheal preparations. Responses are expressed as % change from maximal ACh response taken as 100%. Each point on curve represents mean t SE from 7 separate preparations, except 4~ of G + GO, which is mean rfr: SE from 20 experiments. * P < 0.05, comparison of values at various times with respect to values before exposure to various concentrations of G + GO in respective gr0ups.t P < 0.05, ACh vs. various concentrations (1, 2,4, 8x,) of G + GO. a P < 0.05, IX vs. 2, 4, and 8~.

Downloaded from www.physiology.org/journal/ajplung at Macquarie Univ (137.111.162.020) on February 13, 2019.

HYDROGEN ACh (O.Spg/ml) G+GO + ACh(2X) 160

o v

G+GO + ACB (1X) G+GO + ACh (4X)

PEROXIDE

AND

AIRWAY

ACh

l

160

o

L717

TONE o (EJ) ACh+C+GO

(ED)ACh+G+GO

l

D

-

r

-40"""""'

0

t -5

0

5 TIME (Min)

10

J 20

15

Fig. 5. Effects of ACh in absence and presence of 3 concentrations of G + GO on tension of tracheal preparations. ACh was added 5 min after addition of G + GO. Results are expressed as % change from control value (0) at -5 min. ACh was added 5 min after addition of G + GO at 0 min. Each point on curve prepresents mean t SE (n. = 8). “f P < 0.05, ACh vs. 1, 2, or 4x. aP c 0.05, IX vs. 2 or 4x.

the relaxant effect of G + GO. The results suggest that G + GO-induced relaxation is mediated through H202. Effect on Deepithelialized

Tracheal Preparation

The effects of 4~ concentration of G + GO on the ACh-precontracted preparations with intact or denuded of epithelium are summarized in Fig. 7. ACh produced a maximal contractile response within 2.5 min that was maintained for a 2O-min period of observation. A significant relaxation was produced by preparations with intact epithelium. G + GO produced relaxation of ACh-precontracted preparation denuded of epithelium. However, in preparations denuded of epithelium, the extent of relaxation was lower than that in preparations with intact epithelium (40 vs. 65%). Effects of L-NMMA

The effect of G + GO in the presence or absence of L-NMMA in tracheal preparation with intact epithelium is summarized in Fig. 8. Pretreatment with L-NA4MA ACh

o ACh+G+GO

l

CAT+ACh+G+GO

o

I

I

0

I

5

I

I

!

I

,

15

10 TIME (Min)

20

Fig. 7. Effects of 4~ concentration of G + GO on ACh-precontracted tracheal preparations with intact epithelium (EI) and denuded of epithelium (ED). Responses are expressed as % change from maximal ACh response taken as 100%. Each point on curve represents mean & SE from 7 experiments. * P < 0.05, comparison of values at various times with respect to values before exposure to G + GO in respective groups. t P < 0.05, ACh vs. (EI) ACh + G + GO or (ED) ACh + G + GO. a P < 0.05, (EI) ACh + G + GO vs. (ED) ACh + G + GO.

reduced the relaxant effect of G + GO by 32.3% (i.e., from 65 to 44%). The results suggest a partial involvement of NO in the G + GO-induced relaxation. Effect of Methylene Blue

The effects of G + GO in the presence or absence of methylene blue in tracheal preparations with intact epithelium are summarized in Fig. 9. Pretreatment with methylene blue reduced the relaxant effect of G + GO by 32.3% (from 65 to 44%), indicating the involvement of cGMP in the G + GO-induced relaxation. Effect of Glipizide

The effects of G + GO in the presence or absence of glipizide in tracheal preparations with intact epithelium are summarized in Fig. 10. The relaxant effect of G + GO was significantly reduced (from 65 to 48%) in the presence of glipizide, suggesting a role of ATP-sensitive K+ -

160

ACh

o ACh+G+GO

l

L-NMMA+ACh+G+GO

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160 r

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Fig. 6. Effects of 4~ concentration of G + GO in absence or presence of CAT on ACh-precontracted tracheal preparations. Responses are expressed as percentage change from maximal ACh responses taken as 100%. Each point on curve represents mean & SE (n = 8). * P < 0.05, comparison of values at various times with respect to values before exposure to G + GO in respective groups. “f P < 0.05, ACh vs. ACh + G +GOorCAT+ACh+G+G0.aP

Mechanism of H2O2-induced modulation of airway smooth muscle.

We investigated the effects of H2O2 generated by glucose (G) and glucose oxidase (GO) on the isolated rabbit tracheal smooth muscle suspended in Krebs...
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