Epithelium-dependent regulation of airways smooth muscle function. A histamine-nitric oxide pathway.

The airway epithelium is responsible for the production of a number of arachidonic acid and non-prostanoid inhibitory factors. Epithelium synthesises nitric oxide (NO) which may be important in regulating the function of airways smooth muscles. We studied in vitro the effect of histamine (100 nM-100 microM) which increases the NO release on rabbit airway smooth muscles induced by 80 mM KC1 in the presence or not of 10(-5) Methylene blue (MB) (inactivator of guanylate cyclase) or N(G)-monomethyl L-arginine (L-NMMA), a NOS inhibitor. All experiments were done in tracheal muscle strips from 28 rabbits with epithelium and after epithelium removal. The additional use of histamine (1 microM) on KC1 contraction induced a relaxation of 10% of the initial contraction. The additional use of L-NMMA decreased the relaxation to 5% of initial contraction. MB rather than L-NMMA increased the contraction significantly (p<0.01). Epithelium removal increased the contraction induced by KC1 (80 mM) and histamine (1 microM) by about 30% (p<0.001). NO release especially from epithelium regulates the airways smooth muscle functions. Damage to the epithelium may contribute to an increase in airways sensitivity, observed in asthma.


Introduction
Bronchial asthma is an inflammatory disorde r involving a varie ty of c ells and mediators. The denudation of the e pithelium, the muc ous p lugging, the c ollagen deposition be ne ath the epithelial baseme nt me mbrane , the edema of submuc osa and the infiltration of e osinophils have be en re cognize d to be major features in many patie nts w ith status asthmaticus. 1 Although it w as be lieved that airw ay epithelial ce lls functione d as a passive prote c tive barrier, now it is cle ar that the se ce lls may also p artic ipate in the inflammatory re sponse. Epithe lial ce lls may re lease me diators and produc e nitric ox ide (NO) w hich may amplify asthmatic inflammation in the airw ays. 2 NO is a ne urotransmitter of inhibitory nonadre ne rgic non-cholinergic ne rves w ith a short half life . 3 Mediators such as histamine effect NO synthesis by modulating nitric ox ide synthas es (NOS). These factors most like ly affec t NO by modulating intrace llular Ca + + , w hich re gulates the cons titutiv e NOS re sulting in a rapid inc re ase in NO, w he re as the cytokines incre ase NO more slow ly by incre asing mRNA transcription for the induc ible NOS. Stimulation of the e pithelial laye r by 0.1 m M histamine inc re as ed the re le as e of NO 3 -4-fold c ompared to basal le vels. 4 In this study w e inve stigate d the role of the epithe lium, spe cifically the pathw ay of NO in the re gulation of airw ays smooth muscle functions.

Methods
Portions of tracheas w e re obtaine d from rabbits (1-2 kg body w eight) that had be e n anaesthe tize d w ith pentothal. Muscle strips (2 -3 mm) take n from the tracheas w ere sup erfused lume nal side up under 1 g of te nsion in a bathing chamber that w as continuously perfused w ith Kerbs solution w ith the follow ing c omposition (in mM): Na + 137, Mg 2+ 1.1, K + 5.9, Cl -123, Ca 2 + 2, H 2 PO -4 1.2, HCO -3 24.9, gluc ose 9.6 (gassed w ith 95% O 2 and 5% CO 2 , pH 7.4, 37°C). Change s in te nsion w e re re corde d on a Grass FTO3C forc e displace ment transduc er and displayed on a Univers al osc illograph (Harvard) re c order. We studied the effect of histamine (100 nM-100 m M) on c ontrac tions induc ed by elevating the ex trac ellular [K + ] o conc entration to 80 mM, by isosmotic substitution w ith [Na + ] o in the pre senc e (or not) of 10 -5 Me thyle ne blue (MB) (inactivator of guanylate cyclase) or N(G)-monome thyl L-arginine (L-NMMA), a NOS inhibitor. All these ex periments w e re done on trache al strips be fore and afte r e pithe lium re moval. We use d trache al muscle strips from 28 rabbits, se ven for each se parate ex pe riment. A t-te st w as use d to de te c t differe nc es be tw e en groups.

Results
The re sults of our study (see Fig. 1) show ed that the additional use of histamine (1 m M) for KCl contraction induce d a re lax ation of 10% of the initial c ontrac tion. The additional use of L-NMMA decre as ed the re lax ation to 5% of initial contraction. De spite this data the use of MB inste ad of L-NMMA inc re ased the c ontraction significantly (p< 0.01). Epithe lium re moval inc re ased the contraction induced from KCl (80 mM) and histamine (1 mM) by about 30% (p< 0.001).

Discussion
The aim of this study is the simultaneous use of histamine and KCl. Histamine induce d an inc re as e in intra ce llular Ca + + via re cep tor-op erated channels and KCl via voltage -de pendent channe ls. In ex c itable c ells like airw ays smooth muscle Ca + + influx is gene rally acc omp lished by voltage depe ndent Ca + + channe ls. In non-ex c itable cells like epithe lial cells in the airw ays, voltage -ope rate d Ca + + channels are not ex pre ssed. In these c ells store -operated Ca + + entry, w he re Ca + + influx is spe cific ally controlle d by the filling state of the intra ce llular Ca + + store s, is the pre dominant pathw ay. 5 The intra ce llular Ca + + conc entration of epithelium re sponse to agonists such as histamine is biphasic c onsisting of an initial transie nt rise w hich is indepe nde nt of ex tracellular Ca + + follow ed by sustaine d ele vation mediated by Ca + + influx . The re is a close correlation be tw e en intra ce llular Ca + + mobilization and NO re lease in epithe lial cells stimulated w ith histamine. NOs ac tivate guanylate cyclase, raise c-GMP le vels and induc e re lax ation of the isolated airw ay smooth muscle. 6 In our study the ele vation of e pithelial NO production induc ed from histamine in addition to ac ute intra ce llular incre ase of airw ays smooth muscle Ca + + , stimulated cytosolic guanylate c yclase to ge ne rate cGMP. The above inte rac tion be tw een airw ays epithelium and muscles is the main re as on for the re lax ation. In our in v itro study the use of L-NMMA inhibits NOS and the ep ithe lial produc tion of NO. The pathw ay of re lax ation is disrupte d and the pre vious re lax ation is re duc ed. MB inac tivates guanylate cyclase in the airw ay muscles w hich re sults in an end to the re lax ation proc ess and an inc re ase of initial contraction. How e ver, the re moval of ep ithe lium induc ed the pre vious c ontrac tion further. In man damage to epithelial ce lls could affec t airw ays home ostasis , be c ause the epithelial layer inte racts w ith smooth muscles. The production of nitric ox ide is decre as ed in guine a pig airw ays afte r a viral infe ction and is assoc iated w ith hyper-re sponsivene ss. Epithelial c ells re lease other re lax ing factors such as prostaglandin E 2 and metabolize inflammatory me diato rs. 5 Epithe lial injury and bronchial hyper-re sponsivene ss are commonly associate d w ith airw ay dise ase s such as as thma and are considere d to oc cur as a re sult of inflammatory change s in the airw ay w all. Cortic oste roids w hich have assume d a dominant role in asthma therapy, direc tly dec re ase production of NO from airw ay epithelial c ells but indire ctly atte nuate TNF-a and IL-1b re le as e from macrophage s and re duc e epithelial NO production. 7 How ever the pathw ay of endoge nous NO w hich be gins from epithe lium re c eptors of me diators in addition to rapid inc re as e of Ca + + intrac ellularly is a main physiologic route of re lax ation in airw ays smooth muscles.
In summary the epithelium modulates other struc tural compone nts of the airw ays such as airw ays smooth muscles by a numbe r of mechanisms including NO production. Damage to the epithelium may c ontribute to the incre as e in airw ays se nsitivity observe d in as thma. More ex te nsive e valuation of ne w therapie s w hich modulate airw ay epithe lial function together w ith anti-inflammatory propertie s may be a target for as thma treatme nt.