Airway responsiveness: role of inflammation, epithelium damage and smooth muscle tension.

The purpose of this study was the effect of epithelium damage on mechanical responses of airway smooth muscles under different resting tension. We performed acetylcholine (ACh) (10(-5) M)-induced contraction on tracheal strips from 30 rabbits in five groups (0.5, 1, 1.5, 2 and 2.5 g) before and after epithelium removal. At low resting tension (0.5-1.5 g), the epithelium removal decreased the ACh-induced contractions. At 2 g resting tension, the epithelium removal increased the ACh-induced contractions of airways with intact epithelium about 20%. At 2.5 g resting tension, the elevation of contraction is about 25% (P<0.01). Consequently, after epithelium loss, the resting tension determines the airway smooth muscles responsiveness. In asthma, mediators such as ACh act on already contracted inflammatory airways, which results in additional increase of contraction. In contrast, low resting tension, a condition that simulates normal tidal breathing, protects from bronchoconstriction even when the epithelium is damaged.


Introduction
The effects of lung-volume change s on airw ay tone s can be mimicked in isolate d bronchial segments in v itro , in w hich inflation -deflatio n cycles dec re ase the trans mural p re ssure of contrac te d bronc hi. 1 Studie s w ith pre paratio ns of bronchial smooth muscles have been pre sente d in the lite rature . A large range of re sting te ns ions (0.5-2.5 g) w as use d in the se ex pe riments. 2 Resting te nsion dete rmine s the re sponsive ne ss of airw ay smooth muscle s to c ontrac tile and re lax ant age nts . In normal human subjects, de ep insp iratio n de cre ase s airw ay re sistanc e. 3 Fis h e t a l. suggeste d that the major proble m in asthma might be an impairme nt in the ability of inspiratio n to stre tch the airw ay smooth muscle. 4 Cyc lic stre tch of airw ay epithe lium also plays a ke y role in re gulating inflammatory airw ay dis eases inc luding bronchial asthma, in w hich airw ay mechanic s are alte re d. 5 In this study, w e e valuate d the prop ertie s of airw ay smooth muscle unde r diffe re nt re sting te nsions. First, w e re corde d the mechanic al re sponses of smooth muscles during c ontrac tion w ith ac etylcholine (ACh) and, se cond, w e re moved epithe lium and AChinduc ed contrac tions in a range of re sting te nsion w ere also re c orde d.

Materials and methods
Portio ns of trac heas w e re obtaine d from 30 rabbits (1-2 kg body w e ight) that have been pre viously anae sthetiz ed w ith pe ntothal (dose, 20 mg/kg intravenously, slow ly). Afte r immediate ex cision, the trache as w e re placed in Kre bs buffer (pH 7.4, 37°C) w ith the follow ing compositio n: Na + , 137 mM; Mg 2+ , 1.1 mM; K + , 5.9 mM; Ca 2+ , 2.0 mM; Cl -, 123.0 mM; H 2 PO 4 -, 1.2 mM; HCO 3 -, 24.9 mM; gluc ose, 9.6 mM. The solution w as gas sed w ith 95% O 2 and 5% CO 2 . Muscle strip s (2-3 mm) take n from the trac he as w ere supe rfuse d unde r 1 g of te nsion, in a bathing chamber that w as continuo usly perfuse d w ith Kre bs solution. Change s in te nsion w ere re c orde d on a Grass FT03C forc e displac eme nt trans duce r and disp laye d on a Unive rs al oscilograph (Harvard) re c orde r.
A single bolus dose of 0.2-0.5 ml of 10 -1 M ACh w as give n to each muscle strip , to induc e c ontrac tion. The pre paration w as the n w ashed re pe ate dly and left until te nsion had re turne d to base line . Each muscle strip w as equilibrate d at a re sting load (1 g) for at least 60 min before starting the ex perime nt.
We used ACh (10 -5 M) to induc e c ontrac tions at diffe re nt re sting te nsions (0.5, 1, 1.5, 2 and 2.5 g). Each muscle strip in the same re sting te ns ion w as contrac te d by ACh before and afte r epithelium re moval. Ex perime nts from six rabbits w ere performe d for each re sting te nsion. Epithe lium w as re moved mechanically. ACh w as purchas ed from Sigma. Figure 1 show s that e pithe lium re moval inc re as es the ACh-induc ed c ontrac tion at high re sting te ns ion. At 2.5 g re sting te nsion, the inc re as e of c ontrac tion is 24.45% (P< 0.04). At low re sting te ns ion, unde r 1.5 g, the e pithelium re moval dec re ases the ACh-induc e d contrac tions . The highe st de cre ase w as re corde d at 1 g re sting te nsion (72.30%) (P< 0.01).

Discussion
Our re sults show ed that re sting te ns ion alte rs airw ay smooth muscle hyper-re sponsive ne ss. Ep ithe lium re moval inc re ase s airw ay smooth muscle contrac tion only in high re sting te nsion. The influe nce of low re sting te nsion on epithe lial damage has a prote c tive effect on airw ays muscle contrac tion.
In our ex perime nts, ACh induc e s diffe re nt contractions on diffe re nt airw ay smooth muscle re sting te nsion. Rece nt artic le s show e d that te nsion ap plie d to airw ay smooth muscle s at the start of an isome tric in vitro ex pe rime nt is an important fac tor that dete rmine s re spons ive ne ss. 6,7 Airw ay epithelium also mode rate s the re sponsive ne ss of unde rlying smooth muscles. Hype r-re activ ity of the airw ays is associate d w ith damage of the e pithe lium. 8 Intac t epithe lium has a prote ctive e ffec t because it re lease s re lax ant substanc es such as prostano ids and nitric ox ide . 9 Bronchial as thma is charac te rize d by epithe lium damage and airw ay smooth muscle contrac tion. Furthe r c ontrac tion, induc ed by re lease d me diato rs, depe nds on the pre sente d finding s. Endothelin-1, a 21-amino -acid pe ptide that has be en ide ntifie d in trac he al e pithe lial ce lls, has a dual action on guine a-pig is olate d trac he a. It evoke s c ontrac tions at low re sting tone , w he re as it induc es re lax atio ns at higher re sting.
Re sting te nsion see ms to influe nce the magnitude of non-adre ne rgic non-choline rgic (NANC) re spons es. Nitric ox ide is produc ed in the airw ays, mainly from epithe lium, and is the main me diato r of the NANC syste m. 10 Airw ay e pithelial cells metabolize arac hidonic acid to biologically active eic osanoids, w hich contrib ute to re gulatio n of airw ay smooth muscle tone . 8 Cyc lic stre tch of airw ays causes rapid inhibitio n of pros tanoid synthe sis. 5 All these data may have important implicatio ns for the pathogene sis of as thma, in w hich airw ay me chanic s are alte re d. In our study, the re sting te nsion affe ct the magnitude of contrac tion in the loss of epithelium. Whe n the re sting te nsion is low (normal c onditio n), the damage of e pithe lium se ems to induc e additional re lax atio n. When the re sting te nsion is high (bronchocons tric tion, such as an asthma attac k), the damage of epithe lium inc re ase s the airw ays smooth muscle contrac tion.
A few years ago, Skloot e t a l. show e d that airw ay hyper-re sponsive ne ss in asthma is a proble m of 'limite d smooth muscle re lax atio n' w ith inspiration. 11 This observatio n is base d on the sp eculation of Gre en and Mead that in patie nts w ith asthma, max imal insp iratio n is unable to dilate the airw ays complete ly. 12 It is w e ll know n that max imal ins piratio n ele vate s the airw ay re sting te ns ion. The inte rac tion betw e en high re sting te nsion and damage of epithelium w hich happen in asthma attac k se em to be the re al c ause for 'limite d smooth muscle re lax atio n'.
In normal humans subjec te d to bronchoc ons trictions, de ep ins piratio n dec re ases airw ay re sistanc e and inc re ases ex pirator y flow. 13 Normal tidal bre athing also plays an important role in limiting airw ay re spons ive ne ss.
Lung-volume change s have an important effe ct on airw ay tone and the airw ay re sponse to bronchoconstric tors. 14 Airw ay smooth muscle unde rgoe s c onstant stre tch and re lax atio n during the re spirato ry cycle. The re sponses of airw ays to a varie ty of stimuli are modulate d by the e pithelium. 15,16 In bronchial asthma, all the normal me chanis ms are impaire d. Additio nally, an asthmatic airw ay has inc re ased thickne ss of the smooth muscle laye r, c ollagen depositio n bene ath the bas eme nt me mbrane and, e spe cially, variab le inflammatory produc ts in the w all. 17,18 In c onc lusion, mediators of asthma, such as ac etylcholine , induc e more c ontrac tion w hen ac ting on alre ady c ontrac te d airw ays w ith e pithe lium damage , a conditio n mimicking asthma attac k. 19 De spite this vic ious c irc le of asthmatic airw ay, in physiologic conditio ns, normal tidal bre athing prote c t from bronchoconstric tion e ven w he n the e pithe lium is damage d.