environmental tobacco smoke ( ets ) is an environmental trigger that leads to airway inflammation and airway hyperresponsiveness ( ahr ) in susceptible individuals and animals [ 15 ] .
epidemiological studies show that the probability of developing or exacerbating asthma increases in ets exposure patients [ 310 ] .
the nervous system , including the nerves supplying the airways , is highly susceptible to environmental influences .
the airways are innervated through autonomic and sensory nerve fibers [ 12 , 13 ] .
sensory innervation in the airways is important in the pathogenesis of inflammation associated with asthma .
substance p ( sp ) , a member of the tachykinin family , releases from sensory nerve fibers and has potent effects on airway smooth muscle tone , vascular permeability to protein and mucus secretion [ 14 , 15 ] .
recent studies have shown that sp plays an important role in antigen- and irritant - induced ahr [ 2 , 16 , 17 ] .
furthermore , the studies including ours found that increased sp in the airway is involved in cigarette smoke exposure - induced ahr and airway inflammation [ 2 , 1825 ] . however , the role of sp in cigarette smoke exposure - enhanced airway responsiveness is not clear .  
recent studies found that sp also acts as a neuromodulator increasing the tracheal smooth muscle contractions to electrical field stimulation ( efs ) [ 2630 ] .
both of watson and hall 's studies found that exogenous and endogenous sp facilitated efs - induced tracheal contractions , but had no effect on contractions induced exogenous ach [ 26 , 27 ] .
nadel 's group further showed that sp potentiated airway smooth contraction to efs in a concentration - dependent fashion [ 28 , 29 ] .
tournoy et al . also found that efs - induced airway smooth muscle contraction was decreased in nk1 receptor knockout mice compared with wild - type control .
all of these studies suggest that sp enhances efs - induced airway smooth contraction due to increasing the release of ach ( figure 1 ) .
thus , we hypothesized that ets exposure increases sp from sensory nerves or changes nk receptor , which facilitates ach release from cholinergic nerve and enhances airway smooth muscle responses ( figure 1 ) . the enhanced airway smooth muscle response may contribute to increased susceptibility to ahr .
the present experiments characterize the role of sp on ets - enhanced airway smooth muscle responses .
icr mice ( harlan , indianapolis , in , usa ) were housed with access to food and water ad libitum in an fda - approved facility .
all procedures were performed in accordance with the recommendations of the guide for the care and use of laboratory animals , published by the national institutes of health , and the protocols were approved by the wvu animal care and use committee no .
the animals were treated humanely and with regard for alleviation of suffering . by classical definition ,
environmental tobacco smoke ( ets ) is a diluted mixture of the smoke given off by the burning end of a tobacco product ( side stream smoke , ~85% ) and the smoke exhaled by smokers ( mainstream smoke , ~15% ) .
based on previous ets exposure studies [ 1 , 31 , 32 ] , we used sidestream tobacco smoke ( ss ) as a surrogate to ets .
the two - month - older mice were exposed to either ss or filtered air ( fa ) for 6  h / day for 1 day or 5 consecutive days .
the trachea and lung tissues were collected 16  h after ss or fa exposure . a major goal of the current study was to understand possible neuronal mechanisms by ss that affect lung function .
the ss exposure protocol and methods used in this study have been described in our recent publication .
briefly , mice were randomly placed in an exposure chamber ( bioclean , duoflo model h 5500 , lab products inc ) that measured 1.92  1.92  0.97  m ( 3.58  m ) .
sidestream smoke from marlboro filter cigarettes ( phillip morris , richmond , va , usa ) was introduced into the exposure chamber at a rate of four cigarettes every 15 minutes for 6 hours using a smoking machine ( rm 1/g , heinr borgwaldt gmbh , hamburg , germany ) . at the end of the 6 h exposure period ,
the exhaust fan unit was turned on to rapidly lower the level of smoke in the exposure chamber .
the concentrations of carbon monoxide in the exposure chamber were monitored and kept to an average of about 50 parts per million ( ppm ) , relative humidity was about 50% , and temperature was about 23c .
total suspended particulate concentration was about 1.1  mg / m , similar to exposure levels used by others to approximate the cloud of particulates surrounding a person during active smoking .  
fresh tracheas from mice 16  h after fa or ss exposure were cut into 3  mm wide rings , mounted in holders , and maintained in gassed ( 95% o2 - 5% co2 ) modified krebs - henseleit solution at 37c with a composition ( in  mm ) as follows : 113  nacl , 4.8  kcl , 2.5  cacl2 , 1.2  mgso4 , 24  nahco3 , 1.2  kh2po4 , and 5.7 glucose , ph 7.4 .
each tracheal ring was loaded into a pair of stainless - steel hooks , suspended in tissue holders .
each holder was anchored in a 10-ml water - jacketed organ bath , and the top steel hook was attached to a force - displacement transducer connected to a recorder ( gould instruments , valley view , oh , usa ) .
the rings were equilibrated for 60  min at a resting tension of 0.25  g , determined by preliminary studies , during which time the modified krebs - henseleit solution in the baths was changed every 15  min .
after equilibration , methacholine ( mch ) responses were constructed by adding 10  m mch to the bath and sp cumulative concentration - response curves were constructed by adding a series of concentrations of sp to the bath in half - log - increment concentrations ranging from 10 to 10  m. after concentration - response curves were completed , electrical - field - stimulation- ( efs- ) induced responses were obtained with a stimulator ( model s48 , grass instruments , richmond , va , usa ) .
frequency - response curves were constructed by increasing the frequency from 0.3 to 30  hz using a submaximum voltage of 120  v , 0.2-ms pulse duration , and 10  s train duration . between each stimulation period ,
efs - induced contractions were normalized by the percent response of each tissue to 10  m mch . in some experiments , atropine ( 10  m )
was added to krebs solution to verify that the responses elicited by efs were mediated by the release of ach . to determine the possible involvement of endogenously released sp , some experiments were given cp-99994 ( final concentration 10  m ) , sp receptor antagonist , which was incubated at least 30  min before addition of sp or efs .
the procedures for immunohistochemical quantification of airway nerves have been described previously [ 1 , 33 , 34 ] .
briefly , in a separate group of mice - exposed using the same ss exposure protocol , tracheas were removed 16  h after ss or fa exposure , fixed in picric acid - formaldehyde fixative for 3  h , and rinsed three times with a 0.1  m phosphate - buffered saline containing 0.15% triton x-100 .
the tracheas were dissected and frozen in isopentane , cooled with liquid nitrogen , and stored in airtight bags at 80c .
the tracheas were oriented with the dorsal surface uppermost so the tracheal muscle would be sectioned in a coronal plane .  
cryostat sections ( 12  m thick ) were collected on gelatin - coated coverslips and dried at room temperature .
the cryostat sections on coated coverslips were covered with sp antibody diluted 1  :  100 , incubated at 4c for overnight , and rinsed with a 1% bovine serum albumin + phosphate - buffered saline containing 0.15% triton x-100 three times , with 5  min being allowed for each rinse .
the sections were then covered with fluorescein isothiocyanate - labeled goat antirabbit antibody diluted 1  :  100 , incubated at 37c for 45  min , and rinsed .
after all immunohistochemical procedures were conducted , the coverslips were mounted with fluoromount and observed with a fluorescence microscope equipped with fluorescein ( excitation wavelengths 455500  nm , emission wavelengths > 510  nm ) .
controls consisted of testing the specificity of primary antiserum by absorption with 1  g / ml of the specific antigen .
nonspecific background labeling was determined by omission of primary antiserum .   for measuring nerve fiber density in tracheal smooth muscle , we collected images of control , sp nerve fibers in series under the zeiss lsm 510 confocal microscope .
a series of images representing all of the tracheal smooth muscles in a section was collected in digital files and saved to an internal database and measured using optimus software .
we selected regions of smooth muscle using the rhodamine channel to avoid possible bias created by the presence or absence of nerve fibers .
the smooth muscle regions were outlined to measure total cross - sectional area of smooth muscle .
the microscope was then switched to reveal nerve fibers in the fluorescein channel , and the image was digitally captured .
then nerve fiber area was standardized to the total cross - sectional area of smooth muscle .
the final value of nerve fiber density is expressed as % of dividing the sp nerve fiber area by the total area of smooth muscle .
at least 10 measurements were made for each section , and 15 sections were measured in each animal .  
the concentration of sp ( 392500  pg / ml ) in each sample was assayed using the sp immunoassay system ( r&d systems , minneapolis , mn , usa ) according to manufacturer 's instructions .
all samples were running in duplicate . unless otherwise stated , values are means  se .
contractions elicited by efs and sp are expressed as a percentage of the contraction elicited by mch 10  m. the half - maximal effective concentration ( ec50 ) values for sp were calculated using a four - parameter logistic curve fit ( sigmoidal , sigmaplot 2000 ) and were presented with 95% confidence intervals .
force development was expressed by normalizing force ( g ) divided by the wet weight of the tissue .
nerve fiber density was expressed as % area of sp - immunoreactive nerve fibers in the total area of the smooth muscle .
statistical analyses of immunohistochemistry and ec50 were performed using student 's t - test . statistical analysis of efs was performed using two - way repeated - measures analysis of variance .
one factor between the groups was ss exposure ; the other factor within the group was efs or sp effect . when the main effect was considered significant at p < 0.05 , pairwise comparisons were made with a post hoc analysis ( fisher 's least significant difference ) .
p < 0.05 was considered significant , and n represented the number of animals studied .  
mch chloride and atropine sulfate were obtained from sigma chemical ( st . louis , mo , usa ) .
sp and sp antibody were obtained from peninsula ( belmont , ca , usa ) .
fluorescein isothiocyanate - labeled goat antirabbit antibody was obtained from icn immunobiologicals ( costa mesa , ca , usa ) .
the initial experiments were intended to find if ss exposure increases airway smooth muscle contraction to mch .
there was no significant difference in the tracheal smooth muscle contraction to mch ( 10  m ) between 1 day - ss - exposed ( n = 16 ) mice and fa exposed mice ( n = 16 ) ( figure 2 ) . although 5 days of ss exposure slightly increased smooth muscle contraction to mch , there was no significant difference in airway responses to mch between 5 days of ss - exposed ( n = 30 ) mice and fa exposed mice ( n = 30 ) ( figure 2 ) .  
the next experiments were intended to figure out the effect of ss on airway responsiveness to sp and efs .
the cumulative dose - response curve for sp was not significantly shifted to the left after 1 day of ss exposure ( figure 3(a ) ) .
there is no significant difference between ec50 values in ss exposure ( 6.17  0.11 ) and fa exposure ( 5.96  0.14 ) .
also the frequency - response curve to efs was not significantly altered 1 day of ss exposure ( figure 3(b ) ) .
however , the cumulative dose - response curve for sp was markedly shifted to the left after 5 days exposure to ss ( figure 3(c ) ) , there is significant difference between ec50 values in ss exposure ( 6.65  0.09 ) and fa exposure ( 6.01  0.11 ) . also , a leftward shift in the frequency - response curve to efs was observed after 5 days of ss exposure ( figure 3(d ) ) , and contractions produced by efs at 10  hz and 30  hz were significantly increased after 5 days of ss exposure .
the pretreatment with atropine ( final concentration 10  m ) totally abolished efs - induced airway smooth muscle contraction in both fa and ss - exposed mice ( figures 3(b ) and 3(d ) ) , indicating that efs - induced airway smooth muscle contraction mainly involves endogenously acetylcholine ( ach ) release .   to ensure sufficient reduction of sp effects , the effective dosage of nk1 receptors antagonist , cp-99994 , was tested first . in separate experiments ,
the different concentrations of cp-99994 were tested by sp cumulative doses ( 10  m to 10  m ) .
the sp cumulative dose - response curves were significantly shifted to the right in a concentration - dependent manner by cp-99994 ( figure 4 ) .
ec50 values for sp are 6.04  0.14 ( control ) , 5.83  0.12 ( cp-99994 10  m ) , 5.64  0.11 ( cp-99994 10  m ) , and 5.56  0.11 ( cp-99994 10  m ) .
there is significant difference in ec5 between control and cp-99994 10  m. the combination of cp-99994 ( 10  m ) and nk2 receptors antagonist sr48968 ( 10  m ) virtually abolished sp - induced airway smooth muscle contraction , indicating that cp-99994 10  m is effective dose to block sp - induced airway smooth muscle contraction .
thus , next experiment , cp-99994 10  m was chosen to test the role of sp on 5 of days ss - enhanced airway smooth muscle responses .
cp-99994 ( final concentration 10  m ) was incubated at least 30  min before using sp or efs .
cumulative concentration  response curve for sp and the efs - stimulated contractions in saline pretreated groups after 5 of days ss exposure ( figures 5(a ) and 5(c ) ) demonstrated similar changes as those described above , 5 days of ss exposure .
the contractions produced by efs at 10  hz and 30  hz were significantly increased in saline pretreated groups after 5 days of ss exposure .
the tracheal smooth muscle contractions to efs at 10 and 30  hz were not significantly altered in the cp-99994 treatment group ( 10  hz : 21.44  2.54% ; 30  hz : 25.8  g  2.31% ) compared with the saline group ( 10  hz : 23.36  2.28% ; 30  hz : 27.89  2.29% ) in fa exposed mice .
however , cp-99994 significantly inhibited the ss - enhanced smooth muscle responses to efs at 10 and 30  hz ( figure 5(d ) ) .
there are no significant differences in efs - induced tracheal smooth muscle contractions between 5 of days ss - exposed mice and fa exposed mice after cp-99994 treatment . also , the enhanced airway responses to sp were total abolished by cp-99994 treatment ( figure 5(b ) ) .  
sp nerve fibers in the tracheal smooth muscle of mice were analyzed based on the immunohistochemical localization by fluorescein .
the sp nerve fibers were mainly localized on tracheal smooth muscle and epithelium ( figure 6(a ) ) .
there is no significant difference in density of sp nerve fibers in the airway smooth muscle between 1 day of ss exposure and fa exposure ( figures 6(a ) and 6(b ) ) .
however , the density of sp nerve fibers in the airway smooth muscle was significantly increased after 5 days of ss exposure ( figures 6(a ) and 6(b ) ) .
these findings suggest that 5 days of ss exposure increases sp content in tracheal smooth muscle .
elisa data showed that sp levels in lung were significantly elevated in 5 days of ss - exposed mice compared with fa exposure ( figure 6(c ) ) .
the results obtained from the current study show that exposure to ss significantly enhances tracheal smooth muscle responsiveness in the mice , as evidenced by elevated contractility to sp and efs .
the elevation of airway smooth muscle responses by ss exposure was attenuated by treatment with a nk1 receptor antagonist , indicating that sp played a key role in the enhancement of smooth muscle contractile responses .
our previous study showed that exposure to irritant changes sp airway innervation and enhances tracheal smooth muscle responsiveness [ 25 , 35 ] , suggesting that sp may contribute to irritant - enhanced smooth muscle responsiveness .
the current results from immunohistochemistry found that ss exposure changes sp innervation of tracheal smooth muscle , directly indicating that increased sp level in airway involved ss - enhanced smooth muscle responsiveness .
sp localized in the peripheral endings of sensory nerves innervating the lung and airways originates in nerve cell bodies located in sensory ganglia [ 37 , 38 ] .
stimulation of sensory nerves by inhalation of irritants is known to trigger the release of sp from afferent endings [ 2 , 25 , 39 , 40 ] .
the data in the present study found that ss - enhanced - airway smooth muscle contractions to efs were attenuated by sp receptor antagonist , directly indicating that sp is involved .
however , the role of sp in smoke exposure - enhanced airway responsiveness is not clear .
recent studies found that sp acts as a neuromodulator altering airway responsiveness to efs [ 2630 ] .
watson and hall 's studies found that exogenous and endogenous sp facilitated electrical field stimulation ( efs)-induced tracheal contractions , but had no effect on contractions induced exogenous ach [ 26 , 27 ] .
nadel 's group further showed that sp potentiated airway smooth contraction to efs in a concentration - dependent fashion [ 28 , 29 ] .
thus , one possible explanation for our finding is that enhanced sp in airway after ss exposure acted as mediator , which altered airway responsiveness to efs .
our result found that administration of atropine completely blocks airway smooth muscle responsiveness to efs in both fa and ss - exposed animals , demonstrating that smooth muscle contraction to efs in mice trachea is entirely atropine sensitive and totally depends on endogenous ach release from parasympathetic nerve terminals .
it also indicates that there is no sp release in efs - induced airway responses .
furthermore , our mch experiments found that ss exposure did not increase airway contractions to mch and suggested enhanced sensitivity of airway smooth muscle was not involved in ss exposure .
thus the logical explanation is that enhanced sp acts as a neuromodulator increasing the release of acetylcholine ( ach ) from parasympathetic nerve .
although the exact mechanism of enhanced ach release from parasympathetic nerve was not determined in the present study , the finding that the sp receptor antagonist cp-99994 significantly attenuated the effect of ss on efs responses in trachea suggests the idea that ss exposure induces sp upregulation .
the enhancing sp may associate with nk1 receptor on the surface of cholinergic neurons , which alters the sensitivity of cholinergic neurons or activates cholinergic neurons and facilitates ach release from cholinergic nerve .  
our data also found that contractions of airway smooth muscle to efs at 10 and 30  hz were slightly decreased in the cp-99994 treatment group ( 10  hz : 21.44  2.54% ; 30 hz : 25.89  2.31% ) compared with the saline treatment group ( 10  hz : 23.36  2.28% ; 30  hz : 27.89  2.29% ) after fa exposure .
study that efs - induced airway smooth muscle contraction was decreased in nk1 receptor knockout mice compared with of nk1 receptor wild - type controls   supports that normal level of sp in airway smooth muscle involved airway responses to efs , although there is no direct effect of sp on efs - induced airway responses .
the present experiment also showed that sp dose responses are enhanced after 5 days ss exposure .
one possibility is that sensitivity of airway smooth muscle to sp is enhanced by ss exposure .
however , it is unlikely that the sensitivity of airway smooth muscle was enhanced due to sensitivity of airway smooth muscle to mch that was not significantly changed .
alternatively , an altered density and/or expression of the nk receptors or properties of nk receptors ( e.g. , affinity ) on the airway smooth muscle may be involved by ss exposure .
our experiments used three concentrations of cp 99994 and detected that even high - dose cp 99994 ( 1  m ) only partially but incomplete inhibited exogenous sp - induced airway contraction , suggesting that mice airway may contain a heterogeneous population of receptors .
furthermore , our experiment uses combination with nk1 and nk2 receptor antagonist ( sr 48968 ) treatment and found that combination with nk1 and nk2 receptor antagonist treatment virtually eliminated the response to sp in airway , indicating both nk1 and nk2 receptors mediate the contractile response to sp .
thus , the possible explanation is that ss exposure alters density and/or expression of the nk receptors or properties of nk receptors ( e.g. , affinity ) on the airway smooth muscle , which affect airway repose to sp .   the previous studies including ours have found that childhood exposure to ets is a significant risk factor for exacerbation of asthma , but studies of smoking in adults are less conclusive .
there are evidence that mainstream cigarette smoking exposure may decrease the incidence of some chronic inflammatory and have a beneficial effect of smoking on airway responsiveness [ 4144 ] .
these studies found that exposure to mainstream cigarette smoke attenuates airway responses to ach , methacholine [ 4143 ] and neurokinin a , also decreased airway inflammation comparing with nonsmoking animals .
however , it has also been reported that mainstream cigarette smoking or ets exposure can enhance allergic airway inflammation [ 45 , 46 ] and increase airway responses to ach [ 45 , 47 ] , endothelin [ 48 , 49 ] .
the low levels of ets or mainstream cigarette smoke promote allergic sensitization and increase airway responsiveness [ 46 , 50 ] , whereas high levels of mainstream cigarette smoke exposure associated with direct smoking suppress allergic airway inflammation and airway hyperresponsiveness [ 4143 , 50 ] . in our present experiment , we used low levels of sidestream tobacco smoke ( ss ) as a surrogate to ets instead of mainstream cigarette smoke , and found ss exposure significantly enhances tracheal smooth muscle responsiveness sp and efs , supporting that low levels of ets enhance the development of allergic airway inflammation .  
one of the significant findings in the current results from immunohistochemistry found that ss enhances sp expression in airway nerves .
these data provide direct evidence that sp nerve fibers in airway smooth muscles were increased by 5 days of ss exposure .
clinic data demonstrated that sp nerve fiber density was increased in airway smooth muscle of severe asthmatics .
a more recent study showed that the sp nerve fiber density was increased in airway epithelium from human subjects with persistent nonproductive cough .
these studies show that increased levels of sp in human airway sensory nerves may contribute to alteration of airway responses .   in conclusion ,
our results show that 5 days of ss exposure increases sp nerve fibers innervating tracheal smooth muscle . at the same time , smooth muscle responses to efs and sp are increased in tracheal segments .
administration of cp-99994 , an antagonist of the nk1 receptor , attenuates the ss exposure - enhanced tracheal smooth muscle responses to efs , indicating that the enhanced responses are dependent on sp increase from sensory nerves or change of nk receptors density in the airway smooth muscle .
combining immunohistochemistry finding that sp nerve fibers were increased in tracheal smooth muscle after 5 days of ss exposure , the current study suggests that the increased sp production by ss exposure may contribute ss - enhanced smooth muscle responsiveness in mice trachea .
 environmental tobacco smoke ( ets ) is an environmental trigger that leads to airway inflammation and airway hyperresponsiveness ( ahr ) in susceptible individuals and animals , but the underlying mechanism is not fully understood . 
 substance p ( sp ) release from sensory nerve fibers has been linked to ahr . 
 the present experiments characterize the role of sp in tracheal smooth muscle on ets - increased airway responses . 
 the mice were exposed to either sidestream tobacco smoke ( ss ) , a surrogate to ets , or filtered air ( fa ) for 1 day or 5 consecutive days . 
 contractions of tracheal smooth muscle to sp and electrical field stimulation ( efs ) were not significantly altered in 1 of day ss - exposed mice . 
 however , 5 of days ss exposure significantly increased airway smooth muscle contractions to sp and efs . 
 administration of cp-99994 , an antagonist of the neurokinin ( nk)1 receptor , attenuates the ss exposure - enhanced tracheal smooth muscle responses to efs . 
 furthermore , the immunohistochemistry showed that sp nerve fibers were increased in tracheal smooth muscle after 5 of days ss exposure . 
 these results suggest that the increased sp production may contribute to ss - enhanced smooth muscle responsiveness in mice trachea . 
1. Introduction
2. Material and Methods 
3. Results
4. Discussion