In vivo airway reactivity: predictive value of morphological estimates of ainvay smooth muscle'
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J. G. MARTIN,^ A. OPAZO-%AEZ, T. Du,R. ~ P P E W AND , D. HeEHDELMAN Meakins-Christie Laboratories, McGill University a d the Respimtory Health Network of Centres of Excellence, Montrtal, Quibec, Canada Received July 22, 1991 A., Du, T., T~PPEW, R., and EIBBLMAN, H.1992. In vivo airway reactivity: predictive value MARTIN,J. G., OPAZO-SAEZ, of morphological estimates of airway smooth muscle. Can. J. Physiol. Pharmacol. '70: 597-601. Airway responsiveness to methacholine and other bronchoconsgrictors is highly variable within and among species. The aim of the experiments in this report was to evaluate the importance of the quantity of airway smooth muscle as a determinant of intra- and inter-species variability in airway responsiveness. To do this we established concentration -response curves to methacholine in a sample of normal guinea pigs as well as in rat, rabbit, and dog. After challenge we excised the lungs for the quantitation of smooth muscle by morphometry. Animals were anesthetized with pentobarbital and mechanically ventilated using a Hawlard ventilator. Aerosols of methacholine were administered in progressively doubling concentrations from 0.0625 to 256 m g i d for a period of 30 s for each concentration. The maxim1 response, determined from pulmonary resistance ( R g , and the concentration of methacholine required to effect 50% of the maxim1 R, were determined. After provmation testing the lungs were removed and fixed with 10% F o m l i n . Midsagittal sections and piuahilar sections were stained with hemtoxylin-phloxine-saffron for microscopic examination of smooth muscle. The images of all airways in the sections were traced using a camera lucida side-am attachment and digitized using commercial software. The area of the airway wall occupied by smooth muscle was determined and standardized for airway size by dividing it by the square of the epithelial basement membrane length. The variability in airway smooth muscle in the intraparenchymal airways was significantly greater between than within individual guinea pigs (n = 113). This was mot true of extraparenchyd airways. There was a significant relationship between the quantity of airway smooth muscle in the intraparenchymal cartilaginous airways and the EC,, but not the maximal value of resistance (I?-). In contrast there was a statistically significant positive correlation between R,, and airway smooth muscle for all species. There was also a significant inverse cornlation between EC,, and airway smooth muscle for all species. We conclude that airway smooth muscle appears to be an important determinant of i n t e r - a n i d differences in sensitivity s f guinea pigs to aerosolized methacholine. Smooth muscle also appears to be a determinant of interspecies differences in both sensitivity and maxim1 responses to methacholine. Key words: airways responsiveness, mechmical determinants, limited bronchoconstriction, methacholine, morphometry. MARTIN,J. G., O P A ~ - S A E Z A., , Du, T., TEPPER,R.,et EHBEEMAN, D.H.1992. In vivo airway reactivity: predictive value of morphological estimates of airway smooth muscle. Can. $. Physiol. Pharmacol. 70 : 597-601. La rCactivit6 des voies akriemes B la mCkkacholine et autres bronchoconstrict~rsvarie considCrablement & 19intCrieurd'une meme espbce et entre les espkces. Le but des prdsentes expkriences a CtC d'kvaluer quel p i n t la quantitk de muscle Bisse bronchique p u t expliquer la variabilitk intra- et inter-espbces dans la rCactivit6 des voies aCriennes. Pour ce faire, nous avons a de cobayes nomaux, ainsi que chez le Ctabli les courbes de concentration-rCponse B la mCthacholine dans ~ u Cchantillon rat, le Hapin et le chien. Aprhs lqCpreuve,les pumons ont CtC excis6s afin d'andyser le muscle Hisse par morphomktrie. k s animux ont $tC anesthCsi6s au pentobarbitid et ventHICs mbcaniquement en atilisant un ventilateur Hawlard. De la mChacholine a CtC administrke sous f o m e d'aCroso1, en doublant progressivement les concentrations de 0,0425 B 256 m g / d pendant une @riode de 30 s pour chacune des concentrations. La r6pnse m x i m d e a CtC Ctablie I? partir de la rksistance pulmonaire (R,) et on a dCteminC la concentration de mkthacholine rquise pour atteindre 50% de la R, m a w i d e . A p r b I'Cpreuve, les poumans ont CtC prClevCs et fixCs avec 10% de Fomdin. k s sections sagittales mMianes et les sections parahihires omt 6tC colorCes avec les substances h6matoxyline-phlowine-safran pour un examen micrsscopique du muscle lisse. Les images de toutes les voies atriennes des sections ont CtC tracks en utilisant une chambre cilaire, puis elles ont CtC nudrisCes. La zone de la paroi bronchique mcup5e par le muscle lisse a CtC dCteminCe et n o r d i s C e pour tenir compte de la dimension des voies akriennes, en la divisant par le can6 de la longueur de la membrane basale de 1'6pith6lium. La variabilitC du muscle lisse bronchique &ns les voies aCriennes intraparenchymteuses a CtC significativernent plus grande inter-cobaye qu9intracobaye (n = 131, ce qui n'a pas CtC le cas pour les voies extraparenchymteuses. %Iy a eu une relation significative entre la qaantitC de muscle lisse bronchiqae dans les voies akriennes cart!agineuses intraparenchymakuses et l'ECS0, relation qui n9apas Ct6 obsewrCe pour la vdeur maximale de rksistance (R,,,). A 190ppsC,il y a eu une comklation positive, statistiqaement significative, entre la Rm,, et le muscle lisse de voies aCriennes chez toutes les esp2ces. Il y a eu aussi une corrklatisn inverse significative entre l'EC,, et le muscle lisse des voies aCriennes chez toutes les es&es. Nous concluons que le muscle lisse des voies akriemes semble &re an important ddteraninant des diffkrences interanides dans la seneibilitk des cobayes h la m6thacholine en aCrosol. Le muscle lisse semble aussi Ctre un dktermimnt des differences interespkes, a n t pour ce qui est de la sensibilitk que des rCponses mzpximles B la mCthacholine. Moa cl&s : rkactivitd des voies akriennes, determinants mkcaniques, bronchoconstriction limitbe, mChcholine, morphomCtrie. [Tmduit par la r&action]
IThis paper was presented at the CFBS Symposium on Force Generation in Airway Smooth Muscle: Morphologicd and Biochemical Basis for Comparisons, held in Kingston, Ontario, Canada, June 10, 1991, and has undergone the Journal's peer review. 'Correspondence may be sent to the author at the following address: Meakins-Christie Laboratories, McGill University9 3626 St. Urbain Street, MontrCal, Quebec, Canada H2X 2B%. Rinted in Canada I Impdm6 au Canada
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CAN. J.
PHYSIQL.PPIAWEVIACOL. VQL.70, 1992
Introduction ~h~ capacity of the airnays to narrow in response to inhaled ~ronc~ocomtr~c~ve substancessuch as kS&ne, methacholhe, and peptide leukotrienes, as well as certain physical stimuli such as cold dry air, has been the subject of intense interest over the past 10 years. This capacity, termed reactivity or responsiveness, is Very variable from to animal in all species so far examined (Bai et al. 1987; Douglas et al. 1977; ~ ~ et al. l 985;b Snapper ~ et d. ~ 1978), including humans (Cockcroft et al. 1977; Mdo et ale 1983). The physiological implications of airway responsiveness are not clear, although the mechanisms of airway evoked by various non-imunoloaicd stimuli be of relevance to the fine tuning of ventilatioi -perfusion ratios within the lung or to the stabifization of the ai&ays in circumstances in which they are exposed to high trmsmural pressures such as those that occur during exercise hyperpnea. While problems deriving from hyporesponsive airways await description, the importance of excessive airway responsiveness for disease has long been recognized. Indeed, the major thrust of research into understanding the regulation of airway caliber has been motivated by the need to treat the airway diseases characterized by hyperrespnsiveness. Mechanical deternitants %i$ responsiveness The measurement of airways responsiveness by provocation testing with b r ~ n ~ h ~ ~ ~ n ~involves t r i ~ t oa rmultistep s process from the inhalation of the aerosol of agonist to the final measurement of airway narrowing as an increase in pulmomry resistance or reduction in maximum expiratory airflow. Many of the potentially important steps have been discussed by MacHem (1985) and Moreno et al. (1986). MacHem proposed that shortening of the airway smooth muscle was limited in vivo by the impedance imposed by the lung parenchyma through its attachments to the outer aspect of the airway wall (MacMem 1985). Recent experimental findings support the role of an impedance to shortening that is related to lung volume (Ding 1988). It is probable, although not et d. 1987; Sly et daH. directly proven, that lung elastic recoil is an important determinant of maximal airway narrowing. As a corollary the force of contraction of airway smooth muscle also swans likely to be an important determinant of airway narrowing. Since the force of contraction of muscle is related to its cross-sectional area, morphometric approaches have promise for resolving the issue of whether the quantity of smooth muscle is related to in vivs airways responsiveness. Previous reports provide support for the notion that the quantity of airway smooth muscle may be important. Several investigators have described increases in airway smooth muscle in subjects suffering from asthma (Dunnill et al. 1969; Hossain 1973; Soboyna 1984), the condition for which airway hyperresponsiveness is well known. However, the plasticity of smooth muscle, at least in vitro, suggests the possibility that muscle may increase as a result of rather than be the cause of asthma. Recently, it has been reported that histamine, an important mediator in allergic responses, can be mitogenic for aimay smooth muscle in culture (Paneteieri et d. 1990). It seems likely that other substances that stimulate protein kinase C , the step that appears to be important in histamine's actions, will also prove to act similarly on airway smooth muscle. It is plausible that muscle may increase in the airways in asthma because of local release of mediators from infl There is no information as to the role that airway smooth
muscle plays in determining Away responsiveness prior to the development of disease. For obvious reasons it is difficult to explore the relationship between the quantity of airway smooth muscle and h e responsiveness in healthy human subjects, but the similarity between the distribution of airway responsiveness in humans and various animal suggests that the mechanisms that determine variability in responsiveness may be the same. Therefore it is a plausible hypothesis that quantitative differences in smooth among may be an important cause of variability in responsiveness. We also hypothesized that genetic factors influence the quantity of smooth muscle and may contribute to intra- and inter-species - - -differences in responsiveness-
Methods and results To address these questions we have studied the role of airway smooth muscle in responsiveness by looking for structure -hnction correlations. Measurements of responsiveness to methacholine were made on a number of animal species m d morphometry was used to detemine the quantity of aimay smooth muscle. We examined the influence of aimay smooth muscle on both the sensitivity of the mimds to methacholine as well as on the maximal response that could be induced. Airway smooth muscle as a klletemimnt of strain-redated aliflerences i t respsnsiveness a m n g highly inbred animals To detemine the contribution of genetic constitution to airways responsiveness we studied commercially available highly inbred rats. Although developed for other purposes, these strains provide a convenient source of a n i d s whose genetic composition is relatively homogeneous within strains so that it is possiMe to test the hypothesis that airway responsiveness is genetically determined. If genetic factors play a role in determining responsiveness, then the. intra-strain variability in responsiveness should be less than the inter-strain variability. TO test this hypothesis we studied 8- to 10-week-old rats from seven different highly inbred strains of rat, Lewis, ACI, Brown Norway (BN), Wistar-Fur& (WF), Buffalo (Buf), Wistar -Kyoto (WKY), and three separate batches of F34-4. Animals were anesthetized and endotrachedly intubated. The peak value of pulmonary resistance (ItL) was measured following progressively doubling concentrations of inhaled aerosolized methacholine (MCh) , ranging from 0.25 to 64 mg/mL. The methods have k n previously described in detail @idelman et al. 1988). The concentration of MCh required to double RL (EC2CB(3RL) was calculated as an index of responsiveness. Of the strains examined, the Lewis rats had the highest mean EC200RLin response to MCh (6.82 mgImE) and the F 3 4 had the lowest value (0.35 rng1n-L). The other strains had intermediate values (Fig. I). The inter-strain variability in responsiveness was sipificmtly greater than the htra-strain variability. Batch to batch reproducibility of responsiveness was observed in the F 3 U mimds. These results are consistent with the concept that genetic constitution is an important determinant of responsiveness. We next examined the role of the quantity of aimay smooth muscle as a cause of the observed differences in responsiveness. To do this we selected further samples of Lewis ( t = 8) and F344 (n = 11) animals for the determination of smooth muscle; these strains showed the greatest differences in responsiveness. The lungs were removed and fixed at 25 c d 2 0
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MARTIN ET AE.
1.O F344
FIG. 1 . Tlae concentration of methacholine required to effect a doubling of the pulmonary resistance (EC,&d is shown for seven different highly inbred rat strains, Buf (Buffalo), WF (Wistar-Furth), BN (Brown Noway), Lew (Lewis), ACI, WKY (Wistslr-Kyoto), and three batches of F344 (Fisher). The number of animals studied is indicated in parentheses for each strain. There was a significantly greater inter-strain variability in responsiveness than intra-strain.
(1 cmH20 = 98.1 Pa) of pressure with 18% Formdin for 24 -48 h. Histologicd slides of midsagittal sections were prep a r d for morphomettdc measurements. The quantity of airway smooth muscle in intraparenchymd airways was determined by pirat-counthg performed by a single observer who was unaware of the p u p s e of the study. F344 animals had more airway smooth muscle (3.22 + 0.176 96 of total lung tissue) than the Lewis a n i d s (2.48 0.185%; mean %EM,p g0.001; Fig. 2). There was a correlation between the EC208REand the quantity of airway smooth muscle, but the relationship was not present within each strain. We conclude that certain highly inbred strains of rat exhibit characteristic degrees of responsiveness and that the quantity of airway smobth muscle may be an important determinant of this responsiveness.
+
+
Airway smooth muscle and airway responsiveness in outbred animals Guinea pigs show a large variability in airway responsiveness m o n g animals (Douglas et al- 1974; Hulbert et al. 1985). In this regard they differ from highly inbred rats and are suitable for evaluating the role of airway smooth muscle as a cause of differences in airway responsiveness among different animals within a species. To do this we constructed complete concentration -response curves to methacholine on 13 anesthetized and tracheostoarnized guinea pigs. Animds were mechanically ventilated using a rodent ventilator at settings of 60 breathstfin and a tidal volume of 5 mL/kg. Aerosols of methacholine were administered in progressively doubling concentrations from 0.0625 to 256 mg/mL. Responses were calculated from RL. The maximal change in RL (iR,,,) and the concentration of MCh required t~ effect 50% of the change from baseline to maximum (EC50) were determined. After measurements of responsiveness the lungs were removed and fixed with Formalin as above, and midsagittal, parahgar, and hilar sections were stained for morphometry. A1 airways that were cut transversely (ratio of the major to Hainor axis less than 2) were analyzed using a camera lucida. The images of the airway and the smooth muscle were traced and digitized by a single observer (A.0-%)blinded to the results of provocation tests. The quantity of airway smooth
Lewis
FIG.2. The quantity of airway smooth muscle determined by pintcounting and expressed as a percentage of total lung tissue is significantly greater in the F344 than Lewis rats. (Reproduced from Eidelm n et al. 1991, with permission).
muscle was expressed as an area that was standardized for airway size by dividing by the square of the length s f the epithelial basement membrane. There was substantial variability in both the Rm, and ECSQ among animals. The Rm, ranged from 1.24 to 4.52 cmH20 mL-I s, a 3.6-fold range of responses, and the ECS0varied from 0.W to 22.63 mg/mL, a 250-fold variability. There was a significant difference in the quantity of airway smooth muscle in the intraparenchymal airways among different guinea pigs. This was not the case, however, for extraparenchyd airways. There was no correlation between the Rm, m d the quantity of airway smooth muscle in either the cartilaginous or the noncartilaginous intraparenchymal airways. There was a statistically significant inverse correlation between the ECso and the quantity of airway smooth muscle in intraparenchymal cartilaginous airways ( r = -0.59 1 , p < 0.05). We conclude that airway smooth muscle may have a role in determining interanimal differences in sensitivity but not maximal responses among guinea pigs. a
Airway smooth muscle as a determinant of inter-species diflerences in responsiveness Part of the difficulty in demonstrating the importance of airway smooth muscle to responsiveness in guinea pigs is that the variability in smooth muscle is not very large, and indeed in the extraparenchymal airways, airway smooth muscle does not vary in a statistically significant fashion m o n g different animals. For this reason we decided to examine the relationship between airway smooth muscle and responsiveness among a number of species. We anticipated greater differences in the quantity of airway smooth muscle between species than within species, which should enhance the possibility of demonstrating a relationship between muscle and responsiveness. We constructed complete concentration -response curves to inhaled MCh on rat (n = 3), rabbit (n = 6 ) , and dog (n = I). The quantity of airway smooth muscle was determined by digitization using an identical protocol to that described above for the guinea pigs. Taken together with the guinea pigs there was a substantially greater range of sensitivity as well as maximal responses to methacholine. Rats had the least sensitivity and d s o the smallest maximal responses to methacholine, whereas the guinea pigs had both the greatest responses and the greatest sensitivity to the drug. There were statistically sig-
CAN. J. PHYSIOL. P M W A C O L . VOL. 70, 1992 @
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PIG.3. The logsarithmiedlytransformed vdues of the concentration ~f mehcholine required to effect 50% of the maxim1 change in RL (log ECSO)are significantly inversely correlated with the quantity of airway smooth muscle, expressed as the median value for each animal.
FIG.4. The logarithmidly transfsmd mxirnd response of each animal (log ha,), expressed as a percentage s f the baseline value, is significantly positively correlated with the quantity of airway s w o h muscle. The quantity s f muscje is expressed as the median value for each animal.
nificant relatiomhips between the Bog ECS0md the log and the quantity sf airway smooth muscle in d1 airways (r = -0.535 and 0.667, respectively; Figs. 3 and 4).
altering lung volume alter wpbafimd responses but not sensitivity to agonists in human subjects and animals (Ding et d. 1987; Sly et id. B988), suggesting that these two characteristics are independently determined. The finding that the impedance to shortening of the airway smooth muscle influences maximal constriction suggests that the quantity s f airway s m w h muscle, which determines the force available to came airway narrowing, should dso affect the maximal response. One would also predict an alteration in the maximal response but not the sensitivity based on pharmcologic principles (Moreno et al. 1986). Therefore we were surprised that here was no relationship between the Elm,, and airway smooth muscle in the guinea pigs. The absence of a relationship between airway smooth muscle and the R,, in the guinea pig may simply reflect the relatively smdl digerewce in maximal responses elicited in these animals. This conclusion is suppofied by the significant relationship present in the several species that we studied, among which a larger range of responses was observed. An alternative explanation is suggested by the curvilinear relationship between muscle and maximal responses: the Itm,, appears to be independent of the quantity of muscle above a certain value. The correlation between airway smooth muscle and ECSois also a surprising finding that is not expected on theoretical grounds. This relationship was present both among the guinea pigs and dso other species. However, the prediction that the maximal response and not the sensitivity should relate to muscle is based on the notion of a graded constriction of the airways in response to increasing concentrations of agonist. This model may be an incorrect one, as here is evidence of heterogeneity of bronchoc~nstrictionfrom studies of ventilation distribution (Wagner et d. 1978) as well as direct evidence from fast-freezing during br0nchoconstricti6~~~ (Opmo-Saez et d. 1991). Since responses to inhaled agonists are greater in the large than s d l airways because of central deposition of dm$, it is more likely that relationships between muscle and responsiveness would emerge for these airways, rather than smaller
Diserassiasn We confirmed that airway responsiveness to mehchsline was very variable among mimd species and dso within species when tihe anirnds were not highly inbred. The variability in responsiveness was less within highly inbred strains sf rat than between the strains, consistent with the notion that differences in responsiveness are genetically determined. Part sf the variability in measures of responsiveness appears to be attributable to differences in airway smooth muscle. Differences in respnsiveness among anirnds of a single outbred species, such as the guinea pig, albeit substmtid, are less clewly related to the quantity of airway smooth muscle than differences mong species. The strongest relationship between muscle and responsiveness emerges when interspecies differences are considered. The finding of strain-related responsiveness is consistent with the hypothesis that airway respnsiveness is in part determined by genetic factors. Pauwels et d. (1985) have previously reported similar findings among strains of inbred rats, e of genetic factors in responsiveness suggesting the i m p in rats. The pattern of inheritance of responsiveness to cholinergic agonists has dso been studied in mice and is reportedly consistent with single gene transmission sf differences in responsiveness (Levitt and Mitzner 1988). It has dso been reported that differences in responsiveness can be trammigted by selective breeding of guinea pigs (Takino et d, 1971). The airway hyperresponsiveness found among Basenji greyhound dogs dso appears $0 be a heritable characteristic (Hirshmm et d. 1984). Airway respnsiveness can be dtered in two ways. There cm be an alteration of the maximal respnse that can be induced or the sensitivity to agonist may be dtered. There is considerable evidence that h e maximal response is determined by mechanicd factors. Changes in lung elastic recoil caused by
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MARTIN BT AL.
ones. Consistent with this idea, the relationship between sensitivity and muscle was present ody for the cartilaginous airways in the guinea pig. However, for all s p i e s the relationship between either Rm, and muscle or sensitivity md muscle was not significantly strengthened by considering ody the large airways. Differences in sensitivity between the Lewis md F344 rats were dso associated with differences in muscle; there was a greater quantity of muscle in the more responsive F344 an Unfortunately, we did not construct complete concentrationresponse curves to MCR in these rats and cannot comment on the relationship of muscle to the maximal responses. It is quite possible that the differences in E C 2 d L between Lewis md H34-4 could be entirely due to differences in the Itm, and not sociated with my changes in sensitivity. It would ,however, in view of eke rather consistent changes with changes in muscle that we Rave found. In conclusion, differences in responsiveness a p p m to be geneticdly determined to some extent. These differences me in part explained by differences in smooth muscle. It is possithe quantity of airway smooth ble that inherited differen muscle may dso be an im t determinant of differences in responsiveness mong hurnm subjects. The fact that there are important differences in responsiveness mong animals that are not accounted for by muscle implies that mechzanisms such as differences in receptor density or affinity, intracellular signalling, or the contractile apparatus d w need to be considered.
Acknowledgement This work was supported by the Medicd Research Council of Canada (grant no. MA 7852). Bai, T. R., MacMem, P. T., and Martin, J. G. 1987. Airway responses to aerosolized methacholine in the cat. Am. Rev. Respir. Dk. 135: 190-193. Cwkcrofi, D. W., ~~, B. N., Meflon, %. A., md Hagreave, H.F. 1977. Bronchid reactivity to inhaled his ne: a method and clinical survey. J. Clin. Allergy, 7: 235-243. Ding, D. %. ,Martin, J. G., and MacUem, P. T o4987. Effects of lung volume on maximal mebchsline induced bronchoconstrictisn in m o m 1 h u m n subjects. J. Appl. Physiol. 62: 1324- 1330. Douglas, S. S., Ridgway , P., and Brink, C. 19%%* Airway responses of gRe guinea pig in vivo and in vitro. J. Phamcol. Exp. Ther. 202: 116- 124. h d l , M. S., Masamella, G. R., and Anderson, J. A. 1%9. A comparison of the quantitative anatomy of the bronchi in normal subjects, in status asthmaticus, in chronic bronchitis and in emphysema. Thorax, 24: 176 - 179.
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Eidelman, D. H., Bellofiore, S., and Martin, J. G. 1988. Late airway responses to antigen in sensitbed inbred rats. Am.Rev. Respir. Dis. 137: 1833 - 1837. Eidelman, D. H., Dimaria, G. V., Bellofisre, S., Wang, M. S., R. D., and Martin, J. G. 1991. Strain-related differences smooth muscle and airway responsiveness in the rat. Am. Rev. Respir. Dis. 144: 792-796. Hirshman C. A., Malloy, A., and Downes, H. 1984. Basenjigreyhound dog model of asthma: reactivity to Ascaris suum, citric acid, and methacholine. J. Appl. Physiol. 56: 1272 - 1277. Hossain, S. 19'73. Quantitative measurement sf bronchial muscle in men with asthma. Am. Rev. Respir. Dis. 1Q7: 99 - 189. ,T., Wiggs, B., Pare, P. D., zarnd Hogg, J. C. 1985. Histamine dose -response curves in guinea pigs. J . Appl. Physiol 58: 625 -634. k v i t t , R. C., and Mitzner, W. 1988. Expression of airway hyperreactivity to acetylcholine as a simple recessive trait in mice. FASEB J. 2: 2685 -2608. Macklem, P. T. 1985. Bronchial hyporespansiveness. Chest, 87: 158s - 159s. Malo, J. E., Pineau, E., Cartier, A., and Martin, R. R. 1983. Reference values of the prsvomtive concentrations of methacholine that cause 6% and 28% changes in forced expiratory volume in awe population. Am. Rev. Respir. Dis. 128: 8- 11. Moreno, R. H., Hogg, J. C.,and Pare, P. D. 1986. Mechanics of airway narrowing. Am. Rev. Respir. Dis. 133: 1171 - 4 180. Opazo-Saez, A. M., Wang, M. S., a d Martin, J.G. 1991. Distribution of airway narrowing in methacholine (MCh)-induced bronchoconstriction in the guinea pig: a morphometric study. Am. Rev. Respir. Dis. 143: A558. Panettieri, R. A., Yadvish, P. A., Kelly, A. M., Rubinstein, N. A., and Katlikoff, M. I. 1990. Histamine stimulates proliferation of airway smooth muscle and induces c-fos expression. Am. S. Physiol. 259: E365 -E371. Pauwels, R., Van De Straeten, M., Weyne, J., and Bazin, H. 1985. Genetic factors in ncen-specific bronchid reactivity in rats. Eur. J. Respir. Dis. 66: 98- 104. Sly, P. D., Brown, K. A., Bates, J. H. T., Macklern, P. T.,MilicBmili, J., and Martin, J. @. 1988. Effects of lung volume on interrupter resistance in cats challenged with methacholine. J. Appl. Physiol. 64: 360-366. Snapper, J. R., Brazen, J. M., Lorhg, S. H., Schneider, V., and Bngrm, R. H., Jr. 1978. Distribution of pulmonary responsiveness t s aerosol histamine in dogs. J. Appi. Physiol. 44: 738 -742. Soboyna, R. E. 1984. Quantitative stmcturd alterations in longstanding allergic asthma. Am. Rev. Respir. Dis. 130: 289 -292. Tahno, Y., Sugahara, K.,and Horino, I. 1971. TWOlines of guinea pigs sensitive and nonsensitive to chemical mediators and anaphylaxis. J . Allergy, 47: 24'7 -26 1. Wagner, P. D., Dantzker, D. R., Bacovoni, V. E., T o d i n , W. C., and West. J. B. 1978. Ventilation-perfusion unqudity in asymptomatic asthma. Am. Rev. Respir. Dis. 118: 511 -524.
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J. G. MARTIN,^ A. OPAZO-%AEZ, T. Du,R. ~ P P E W AND , D. HeEHDELMAN Meakins-Christie Laboratories, McGill University a d the Respimtory Health Network of Centres of Excellence, Montrtal, Quibec, Canada Received July 22, 1991 A., Du, T., T~PPEW, R., and EIBBLMAN, H.1992. In vivo airway reactivity: predictive value MARTIN,J. G., OPAZO-SAEZ, of morphological estimates of airway smooth muscle. Can. J. Physiol. Pharmacol. '70: 597-601. Airway responsiveness to methacholine and other bronchoconsgrictors is highly variable within and among species. The aim of the experiments in this report was to evaluate the importance of the quantity of airway smooth muscle as a determinant of intra- and inter-species variability in airway responsiveness. To do this we established concentration -response curves to methacholine in a sample of normal guinea pigs as well as in rat, rabbit, and dog. After challenge we excised the lungs for the quantitation of smooth muscle by morphometry. Animals were anesthetized with pentobarbital and mechanically ventilated using a Hawlard ventilator. Aerosols of methacholine were administered in progressively doubling concentrations from 0.0625 to 256 m g i d for a period of 30 s for each concentration. The maxim1 response, determined from pulmonary resistance ( R g , and the concentration of methacholine required to effect 50% of the maxim1 R, were determined. After provmation testing the lungs were removed and fixed with 10% F o m l i n . Midsagittal sections and piuahilar sections were stained with hemtoxylin-phloxine-saffron for microscopic examination of smooth muscle. The images of all airways in the sections were traced using a camera lucida side-am attachment and digitized using commercial software. The area of the airway wall occupied by smooth muscle was determined and standardized for airway size by dividing it by the square of the epithelial basement membrane length. The variability in airway smooth muscle in the intraparenchymal airways was significantly greater between than within individual guinea pigs (n = 113). This was mot true of extraparenchyd airways. There was a significant relationship between the quantity of airway smooth muscle in the intraparenchymal cartilaginous airways and the EC,, but not the maximal value of resistance (I?-). In contrast there was a statistically significant positive correlation between R,, and airway smooth muscle for all species. There was also a significant inverse cornlation between EC,, and airway smooth muscle for all species. We conclude that airway smooth muscle appears to be an important determinant of i n t e r - a n i d differences in sensitivity s f guinea pigs to aerosolized methacholine. Smooth muscle also appears to be a determinant of interspecies differences in both sensitivity and maxim1 responses to methacholine. Key words: airways responsiveness, mechmical determinants, limited bronchoconstriction, methacholine, morphometry. MARTIN,J. G., O P A ~ - S A E Z A., , Du, T., TEPPER,R.,et EHBEEMAN, D.H.1992. In vivo airway reactivity: predictive value of morphological estimates of airway smooth muscle. Can. $. Physiol. Pharmacol. 70 : 597-601. La rCactivit6 des voies akriemes B la mCkkacholine et autres bronchoconstrict~rsvarie considCrablement & 19intCrieurd'une meme espbce et entre les espkces. Le but des prdsentes expkriences a CtC d'kvaluer quel p i n t la quantitk de muscle Bisse bronchique p u t expliquer la variabilitk intra- et inter-espbces dans la rCactivit6 des voies aCriennes. Pour ce faire, nous avons a de cobayes nomaux, ainsi que chez le Ctabli les courbes de concentration-rCponse B la mCthacholine dans ~ u Cchantillon rat, le Hapin et le chien. Aprhs lqCpreuve,les pumons ont CtC excis6s afin d'andyser le muscle Hisse par morphomktrie. k s animux ont $tC anesthCsi6s au pentobarbitid et ventHICs mbcaniquement en atilisant un ventilateur Hawlard. De la mChacholine a CtC administrke sous f o m e d'aCroso1, en doublant progressivement les concentrations de 0,0425 B 256 m g / d pendant une @riode de 30 s pour chacune des concentrations. La r6pnse m x i m d e a CtC Ctablie I? partir de la rksistance pulmonaire (R,) et on a dCteminC la concentration de mkthacholine rquise pour atteindre 50% de la R, m a w i d e . A p r b I'Cpreuve, les poumans ont CtC prClevCs et fixCs avec 10% de Fomdin. k s sections sagittales mMianes et les sections parahihires omt 6tC colorCes avec les substances h6matoxyline-phlowine-safran pour un examen micrsscopique du muscle lisse. Les images de toutes les voies atriennes des sections ont CtC tracks en utilisant une chambre cilaire, puis elles ont CtC nudrisCes. La zone de la paroi bronchique mcup5e par le muscle lisse a CtC dCteminCe et n o r d i s C e pour tenir compte de la dimension des voies akriennes, en la divisant par le can6 de la longueur de la membrane basale de 1'6pith6lium. La variabilitC du muscle lisse bronchique &ns les voies aCriennes intraparenchymteuses a CtC significativernent plus grande inter-cobaye qu9intracobaye (n = 131, ce qui n'a pas CtC le cas pour les voies extraparenchymteuses. %Iy a eu une relation significative entre la qaantitC de muscle lisse bronchiqae dans les voies akriennes cart!agineuses intraparenchymakuses et l'ECS0, relation qui n9apas Ct6 obsewrCe pour la vdeur maximale de rksistance (R,,,). A 190ppsC,il y a eu une comklation positive, statistiqaement significative, entre la Rm,, et le muscle lisse de voies aCriennes chez toutes les esp2ces. Il y a eu aussi une corrklatisn inverse significative entre l'EC,, et le muscle lisse des voies aCriennes chez toutes les es&es. Nous concluons que le muscle lisse des voies akriemes semble &re an important ddteraninant des diffkrences interanides dans la seneibilitk des cobayes h la m6thacholine en aCrosol. Le muscle lisse semble aussi Ctre un dktermimnt des differences interespkes, a n t pour ce qui est de la sensibilitk que des rCponses mzpximles B la mCthacholine. Moa cl&s : rkactivitd des voies akriennes, determinants mkcaniques, bronchoconstriction limitbe, mChcholine, morphomCtrie. [Tmduit par la r&action]
IThis paper was presented at the CFBS Symposium on Force Generation in Airway Smooth Muscle: Morphologicd and Biochemical Basis for Comparisons, held in Kingston, Ontario, Canada, June 10, 1991, and has undergone the Journal's peer review. 'Correspondence may be sent to the author at the following address: Meakins-Christie Laboratories, McGill University9 3626 St. Urbain Street, MontrCal, Quebec, Canada H2X 2B%. Rinted in Canada I Impdm6 au Canada
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CAN. J.
PHYSIQL.PPIAWEVIACOL. VQL.70, 1992
Introduction ~h~ capacity of the airnays to narrow in response to inhaled ~ronc~ocomtr~c~ve substancessuch as kS&ne, methacholhe, and peptide leukotrienes, as well as certain physical stimuli such as cold dry air, has been the subject of intense interest over the past 10 years. This capacity, termed reactivity or responsiveness, is Very variable from to animal in all species so far examined (Bai et al. 1987; Douglas et al. 1977; ~ ~ et al. l 985;b Snapper ~ et d. ~ 1978), including humans (Cockcroft et al. 1977; Mdo et ale 1983). The physiological implications of airway responsiveness are not clear, although the mechanisms of airway evoked by various non-imunoloaicd stimuli be of relevance to the fine tuning of ventilatioi -perfusion ratios within the lung or to the stabifization of the ai&ays in circumstances in which they are exposed to high trmsmural pressures such as those that occur during exercise hyperpnea. While problems deriving from hyporesponsive airways await description, the importance of excessive airway responsiveness for disease has long been recognized. Indeed, the major thrust of research into understanding the regulation of airway caliber has been motivated by the need to treat the airway diseases characterized by hyperrespnsiveness. Mechanical deternitants %i$ responsiveness The measurement of airways responsiveness by provocation testing with b r ~ n ~ h ~ ~ ~ n ~involves t r i ~ t oa rmultistep s process from the inhalation of the aerosol of agonist to the final measurement of airway narrowing as an increase in pulmomry resistance or reduction in maximum expiratory airflow. Many of the potentially important steps have been discussed by MacHem (1985) and Moreno et al. (1986). MacHem proposed that shortening of the airway smooth muscle was limited in vivo by the impedance imposed by the lung parenchyma through its attachments to the outer aspect of the airway wall (MacMem 1985). Recent experimental findings support the role of an impedance to shortening that is related to lung volume (Ding 1988). It is probable, although not et d. 1987; Sly et daH. directly proven, that lung elastic recoil is an important determinant of maximal airway narrowing. As a corollary the force of contraction of airway smooth muscle also swans likely to be an important determinant of airway narrowing. Since the force of contraction of muscle is related to its cross-sectional area, morphometric approaches have promise for resolving the issue of whether the quantity of smooth muscle is related to in vivs airways responsiveness. Previous reports provide support for the notion that the quantity of airway smooth muscle may be important. Several investigators have described increases in airway smooth muscle in subjects suffering from asthma (Dunnill et al. 1969; Hossain 1973; Soboyna 1984), the condition for which airway hyperresponsiveness is well known. However, the plasticity of smooth muscle, at least in vitro, suggests the possibility that muscle may increase as a result of rather than be the cause of asthma. Recently, it has been reported that histamine, an important mediator in allergic responses, can be mitogenic for aimay smooth muscle in culture (Paneteieri et d. 1990). It seems likely that other substances that stimulate protein kinase C , the step that appears to be important in histamine's actions, will also prove to act similarly on airway smooth muscle. It is plausible that muscle may increase in the airways in asthma because of local release of mediators from infl There is no information as to the role that airway smooth
muscle plays in determining Away responsiveness prior to the development of disease. For obvious reasons it is difficult to explore the relationship between the quantity of airway smooth muscle and h e responsiveness in healthy human subjects, but the similarity between the distribution of airway responsiveness in humans and various animal suggests that the mechanisms that determine variability in responsiveness may be the same. Therefore it is a plausible hypothesis that quantitative differences in smooth among may be an important cause of variability in responsiveness. We also hypothesized that genetic factors influence the quantity of smooth muscle and may contribute to intra- and inter-species - - -differences in responsiveness-
Methods and results To address these questions we have studied the role of airway smooth muscle in responsiveness by looking for structure -hnction correlations. Measurements of responsiveness to methacholine were made on a number of animal species m d morphometry was used to detemine the quantity of aimay smooth muscle. We examined the influence of aimay smooth muscle on both the sensitivity of the mimds to methacholine as well as on the maximal response that could be induced. Airway smooth muscle as a klletemimnt of strain-redated aliflerences i t respsnsiveness a m n g highly inbred animals To detemine the contribution of genetic constitution to airways responsiveness we studied commercially available highly inbred rats. Although developed for other purposes, these strains provide a convenient source of a n i d s whose genetic composition is relatively homogeneous within strains so that it is possiMe to test the hypothesis that airway responsiveness is genetically determined. If genetic factors play a role in determining responsiveness, then the. intra-strain variability in responsiveness should be less than the inter-strain variability. TO test this hypothesis we studied 8- to 10-week-old rats from seven different highly inbred strains of rat, Lewis, ACI, Brown Norway (BN), Wistar-Fur& (WF), Buffalo (Buf), Wistar -Kyoto (WKY), and three separate batches of F34-4. Animals were anesthetized and endotrachedly intubated. The peak value of pulmonary resistance (ItL) was measured following progressively doubling concentrations of inhaled aerosolized methacholine (MCh) , ranging from 0.25 to 64 mg/mL. The methods have k n previously described in detail @idelman et al. 1988). The concentration of MCh required to double RL (EC2CB(3RL) was calculated as an index of responsiveness. Of the strains examined, the Lewis rats had the highest mean EC200RLin response to MCh (6.82 mgImE) and the F 3 4 had the lowest value (0.35 rng1n-L). The other strains had intermediate values (Fig. I). The inter-strain variability in responsiveness was sipificmtly greater than the htra-strain variability. Batch to batch reproducibility of responsiveness was observed in the F 3 U mimds. These results are consistent with the concept that genetic constitution is an important determinant of responsiveness. We next examined the role of the quantity of aimay smooth muscle as a cause of the observed differences in responsiveness. To do this we selected further samples of Lewis ( t = 8) and F344 (n = 11) animals for the determination of smooth muscle; these strains showed the greatest differences in responsiveness. The lungs were removed and fixed at 25 c d 2 0
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MARTIN ET AE.
1.O F344
FIG. 1 . Tlae concentration of methacholine required to effect a doubling of the pulmonary resistance (EC,&d is shown for seven different highly inbred rat strains, Buf (Buffalo), WF (Wistar-Furth), BN (Brown Noway), Lew (Lewis), ACI, WKY (Wistslr-Kyoto), and three batches of F344 (Fisher). The number of animals studied is indicated in parentheses for each strain. There was a significantly greater inter-strain variability in responsiveness than intra-strain.
(1 cmH20 = 98.1 Pa) of pressure with 18% Formdin for 24 -48 h. Histologicd slides of midsagittal sections were prep a r d for morphomettdc measurements. The quantity of airway smooth muscle in intraparenchymd airways was determined by pirat-counthg performed by a single observer who was unaware of the p u p s e of the study. F344 animals had more airway smooth muscle (3.22 + 0.176 96 of total lung tissue) than the Lewis a n i d s (2.48 0.185%; mean %EM,p g0.001; Fig. 2). There was a correlation between the EC208REand the quantity of airway smooth muscle, but the relationship was not present within each strain. We conclude that certain highly inbred strains of rat exhibit characteristic degrees of responsiveness and that the quantity of airway smobth muscle may be an important determinant of this responsiveness.
+
+
Airway smooth muscle and airway responsiveness in outbred animals Guinea pigs show a large variability in airway responsiveness m o n g animals (Douglas et al- 1974; Hulbert et al. 1985). In this regard they differ from highly inbred rats and are suitable for evaluating the role of airway smooth muscle as a cause of differences in airway responsiveness among different animals within a species. To do this we constructed complete concentration -response curves to methacholine on 13 anesthetized and tracheostoarnized guinea pigs. Animds were mechanically ventilated using a rodent ventilator at settings of 60 breathstfin and a tidal volume of 5 mL/kg. Aerosols of methacholine were administered in progressively doubling concentrations from 0.0625 to 256 mg/mL. Responses were calculated from RL. The maximal change in RL (iR,,,) and the concentration of MCh required t~ effect 50% of the change from baseline to maximum (EC50) were determined. After measurements of responsiveness the lungs were removed and fixed with Formalin as above, and midsagittal, parahgar, and hilar sections were stained for morphometry. A1 airways that were cut transversely (ratio of the major to Hainor axis less than 2) were analyzed using a camera lucida. The images of the airway and the smooth muscle were traced and digitized by a single observer (A.0-%)blinded to the results of provocation tests. The quantity of airway smooth
Lewis
FIG.2. The quantity of airway smooth muscle determined by pintcounting and expressed as a percentage of total lung tissue is significantly greater in the F344 than Lewis rats. (Reproduced from Eidelm n et al. 1991, with permission).
muscle was expressed as an area that was standardized for airway size by dividing by the square of the length s f the epithelial basement membrane. There was substantial variability in both the Rm, and ECSQ among animals. The Rm, ranged from 1.24 to 4.52 cmH20 mL-I s, a 3.6-fold range of responses, and the ECS0varied from 0.W to 22.63 mg/mL, a 250-fold variability. There was a significant difference in the quantity of airway smooth muscle in the intraparenchymal airways among different guinea pigs. This was not the case, however, for extraparenchyd airways. There was no correlation between the Rm, m d the quantity of airway smooth muscle in either the cartilaginous or the noncartilaginous intraparenchymal airways. There was a statistically significant inverse correlation between the ECso and the quantity of airway smooth muscle in intraparenchymal cartilaginous airways ( r = -0.59 1 , p < 0.05). We conclude that airway smooth muscle may have a role in determining interanimal differences in sensitivity but not maximal responses among guinea pigs. a
Airway smooth muscle as a determinant of inter-species diflerences in responsiveness Part of the difficulty in demonstrating the importance of airway smooth muscle to responsiveness in guinea pigs is that the variability in smooth muscle is not very large, and indeed in the extraparenchymal airways, airway smooth muscle does not vary in a statistically significant fashion m o n g different animals. For this reason we decided to examine the relationship between airway smooth muscle and responsiveness among a number of species. We anticipated greater differences in the quantity of airway smooth muscle between species than within species, which should enhance the possibility of demonstrating a relationship between muscle and responsiveness. We constructed complete concentration -response curves to inhaled MCh on rat (n = 3), rabbit (n = 6 ) , and dog (n = I). The quantity of airway smooth muscle was determined by digitization using an identical protocol to that described above for the guinea pigs. Taken together with the guinea pigs there was a substantially greater range of sensitivity as well as maximal responses to methacholine. Rats had the least sensitivity and d s o the smallest maximal responses to methacholine, whereas the guinea pigs had both the greatest responses and the greatest sensitivity to the drug. There were statistically sig-
CAN. J. PHYSIOL. P M W A C O L . VOL. 70, 1992 @
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A Rats
Rabbits e Dog
Rabbits
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( Median )
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0.010
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PIG.3. The logsarithmiedlytransformed vdues of the concentration ~f mehcholine required to effect 50% of the maxim1 change in RL (log ECSO)are significantly inversely correlated with the quantity of airway smooth muscle, expressed as the median value for each animal.
FIG.4. The logarithmidly transfsmd mxirnd response of each animal (log ha,), expressed as a percentage s f the baseline value, is significantly positively correlated with the quantity of airway s w o h muscle. The quantity s f muscje is expressed as the median value for each animal.
nificant relatiomhips between the Bog ECS0md the log and the quantity sf airway smooth muscle in d1 airways (r = -0.535 and 0.667, respectively; Figs. 3 and 4).
altering lung volume alter wpbafimd responses but not sensitivity to agonists in human subjects and animals (Ding et d. 1987; Sly et id. B988), suggesting that these two characteristics are independently determined. The finding that the impedance to shortening of the airway smooth muscle influences maximal constriction suggests that the quantity s f airway s m w h muscle, which determines the force available to came airway narrowing, should dso affect the maximal response. One would also predict an alteration in the maximal response but not the sensitivity based on pharmcologic principles (Moreno et al. 1986). Therefore we were surprised that here was no relationship between the Elm,, and airway smooth muscle in the guinea pigs. The absence of a relationship between airway smooth muscle and the R,, in the guinea pig may simply reflect the relatively smdl digerewce in maximal responses elicited in these animals. This conclusion is suppofied by the significant relationship present in the several species that we studied, among which a larger range of responses was observed. An alternative explanation is suggested by the curvilinear relationship between muscle and maximal responses: the Itm,, appears to be independent of the quantity of muscle above a certain value. The correlation between airway smooth muscle and ECSois also a surprising finding that is not expected on theoretical grounds. This relationship was present both among the guinea pigs and dso other species. However, the prediction that the maximal response and not the sensitivity should relate to muscle is based on the notion of a graded constriction of the airways in response to increasing concentrations of agonist. This model may be an incorrect one, as here is evidence of heterogeneity of bronchoc~nstrictionfrom studies of ventilation distribution (Wagner et d. 1978) as well as direct evidence from fast-freezing during br0nchoconstricti6~~~ (Opmo-Saez et d. 1991). Since responses to inhaled agonists are greater in the large than s d l airways because of central deposition of dm$, it is more likely that relationships between muscle and responsiveness would emerge for these airways, rather than smaller
Diserassiasn We confirmed that airway responsiveness to mehchsline was very variable among mimd species and dso within species when tihe anirnds were not highly inbred. The variability in responsiveness was less within highly inbred strains sf rat than between the strains, consistent with the notion that differences in responsiveness are genetically determined. Part sf the variability in measures of responsiveness appears to be attributable to differences in airway smooth muscle. Differences in respnsiveness among anirnds of a single outbred species, such as the guinea pig, albeit substmtid, are less clewly related to the quantity of airway smooth muscle than differences mong species. The strongest relationship between muscle and responsiveness emerges when interspecies differences are considered. The finding of strain-related responsiveness is consistent with the hypothesis that airway respnsiveness is in part determined by genetic factors. Pauwels et d. (1985) have previously reported similar findings among strains of inbred rats, e of genetic factors in responsiveness suggesting the i m p in rats. The pattern of inheritance of responsiveness to cholinergic agonists has dso been studied in mice and is reportedly consistent with single gene transmission sf differences in responsiveness (Levitt and Mitzner 1988). It has dso been reported that differences in responsiveness can be trammigted by selective breeding of guinea pigs (Takino et d, 1971). The airway hyperresponsiveness found among Basenji greyhound dogs dso appears $0 be a heritable characteristic (Hirshmm et d. 1984). Airway respnsiveness can be dtered in two ways. There cm be an alteration of the maximal respnse that can be induced or the sensitivity to agonist may be dtered. There is considerable evidence that h e maximal response is determined by mechanicd factors. Changes in lung elastic recoil caused by
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MARTIN BT AL.
ones. Consistent with this idea, the relationship between sensitivity and muscle was present ody for the cartilaginous airways in the guinea pig. However, for all s p i e s the relationship between either Rm, and muscle or sensitivity md muscle was not significantly strengthened by considering ody the large airways. Differences in sensitivity between the Lewis md F344 rats were dso associated with differences in muscle; there was a greater quantity of muscle in the more responsive F344 an Unfortunately, we did not construct complete concentrationresponse curves to MCR in these rats and cannot comment on the relationship of muscle to the maximal responses. It is quite possible that the differences in E C 2 d L between Lewis md H34-4 could be entirely due to differences in the Itm, and not sociated with my changes in sensitivity. It would ,however, in view of eke rather consistent changes with changes in muscle that we Rave found. In conclusion, differences in responsiveness a p p m to be geneticdly determined to some extent. These differences me in part explained by differences in smooth muscle. It is possithe quantity of airway smooth ble that inherited differen muscle may dso be an im t determinant of differences in responsiveness mong hurnm subjects. The fact that there are important differences in responsiveness mong animals that are not accounted for by muscle implies that mechzanisms such as differences in receptor density or affinity, intracellular signalling, or the contractile apparatus d w need to be considered.
Acknowledgement This work was supported by the Medicd Research Council of Canada (grant no. MA 7852). Bai, T. R., MacMem, P. T., and Martin, J. G. 1987. Airway responses to aerosolized methacholine in the cat. Am. Rev. Respir. Dk. 135: 190-193. Cwkcrofi, D. W., ~~, B. N., Meflon, %. A., md Hagreave, H.F. 1977. Bronchid reactivity to inhaled his ne: a method and clinical survey. J. Clin. Allergy, 7: 235-243. Ding, D. %. ,Martin, J. G., and MacUem, P. T o4987. Effects of lung volume on maximal mebchsline induced bronchoconstrictisn in m o m 1 h u m n subjects. J. Appl. Physiol. 62: 1324- 1330. Douglas, S. S., Ridgway , P., and Brink, C. 19%%* Airway responses of gRe guinea pig in vivo and in vitro. J. Phamcol. Exp. Ther. 202: 116- 124. h d l , M. S., Masamella, G. R., and Anderson, J. A. 1%9. A comparison of the quantitative anatomy of the bronchi in normal subjects, in status asthmaticus, in chronic bronchitis and in emphysema. Thorax, 24: 176 - 179.
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Eidelman, D. H., Bellofiore, S., and Martin, J. G. 1988. Late airway responses to antigen in sensitbed inbred rats. Am.Rev. Respir. Dis. 137: 1833 - 1837. Eidelman, D. H., Dimaria, G. V., Bellofisre, S., Wang, M. S., R. D., and Martin, J. G. 1991. Strain-related differences smooth muscle and airway responsiveness in the rat. Am. Rev. Respir. Dis. 144: 792-796. Hirshman C. A., Malloy, A., and Downes, H. 1984. Basenjigreyhound dog model of asthma: reactivity to Ascaris suum, citric acid, and methacholine. J. Appl. Physiol. 56: 1272 - 1277. Hossain, S. 19'73. Quantitative measurement sf bronchial muscle in men with asthma. Am. Rev. Respir. Dis. 1Q7: 99 - 189. ,T., Wiggs, B., Pare, P. D., zarnd Hogg, J. C. 1985. Histamine dose -response curves in guinea pigs. J . Appl. Physiol 58: 625 -634. k v i t t , R. C., and Mitzner, W. 1988. Expression of airway hyperreactivity to acetylcholine as a simple recessive trait in mice. FASEB J. 2: 2685 -2608. Macklem, P. T. 1985. Bronchial hyporespansiveness. Chest, 87: 158s - 159s. Malo, J. E., Pineau, E., Cartier, A., and Martin, R. R. 1983. Reference values of the prsvomtive concentrations of methacholine that cause 6% and 28% changes in forced expiratory volume in awe population. Am. Rev. Respir. Dis. 128: 8- 11. Moreno, R. H., Hogg, J. C.,and Pare, P. D. 1986. Mechanics of airway narrowing. Am. Rev. Respir. Dis. 133: 1171 - 4 180. Opazo-Saez, A. M., Wang, M. S., a d Martin, J.G. 1991. Distribution of airway narrowing in methacholine (MCh)-induced bronchoconstriction in the guinea pig: a morphometric study. Am. Rev. Respir. Dis. 143: A558. Panettieri, R. A., Yadvish, P. A., Kelly, A. M., Rubinstein, N. A., and Katlikoff, M. I. 1990. Histamine stimulates proliferation of airway smooth muscle and induces c-fos expression. Am. S. Physiol. 259: E365 -E371. Pauwels, R., Van De Straeten, M., Weyne, J., and Bazin, H. 1985. Genetic factors in ncen-specific bronchid reactivity in rats. Eur. J. Respir. Dis. 66: 98- 104. Sly, P. D., Brown, K. A., Bates, J. H. T., Macklern, P. T.,MilicBmili, J., and Martin, J. @. 1988. Effects of lung volume on interrupter resistance in cats challenged with methacholine. J. Appl. Physiol. 64: 360-366. Snapper, J. R., Brazen, J. M., Lorhg, S. H., Schneider, V., and Bngrm, R. H., Jr. 1978. Distribution of pulmonary responsiveness t s aerosol histamine in dogs. J. Appi. Physiol. 44: 738 -742. Soboyna, R. E. 1984. Quantitative stmcturd alterations in longstanding allergic asthma. Am. Rev. Respir. Dis. 130: 289 -292. Tahno, Y., Sugahara, K.,and Horino, I. 1971. TWOlines of guinea pigs sensitive and nonsensitive to chemical mediators and anaphylaxis. J . Allergy, 47: 24'7 -26 1. Wagner, P. D., Dantzker, D. R., Bacovoni, V. E., T o d i n , W. C., and West. J. B. 1978. Ventilation-perfusion unqudity in asymptomatic asthma. Am. Rev. Respir. Dis. 118: 511 -524.
