INTRODUCTION

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A GREATER AIRWAY RESPONSIVENESS in juveniles compared with adults of many species, including humans, has been reported in various studies (7, 11, 23). The mechanisms of this age-related change remain incompletely understood and may involve differences in airway smooth muscle (ASM) function, airway and lung structure, and airway geometry. Although species differences have been shown, results obtained by using in vitro studies of airway tissue of different ages suggest that a central role may be played by maturational changes in ASM (5, 30).

Both pre- and postreceptor maturational changes have been identified in the mechanisms leading to the ASM contractile response, e.g., increased acetylcholinesterase activity (16), reduced muscarinic-receptor density and change in their subtypes ratio (29), changed receptor-G protein coupling (6), and reduced second-messenger generation (19). However, it remains controversial whether maturational changes occur in the overall response of the ASM; in fact, normalization by different parameters, i.e., myosin content, tissue cross-sectional area (CSA), or ASM CSA, has been shown to alter conclusions about force productions at different ages (5). Moreover, ASM contribution to airway responsiveness is influenced by several factors that, in opposing the generated force, modulate ASM shortening (21, 22) and reduction of the airway lumen (12). Therefore, because these factors affect the relationship between production of force and shortening, one needs to take them into account to extrapolate the observations on maturation of in vitro ASM cellular properties to in vivo airway responsiveness.

At any given amount of force produced by ASM in vivo, the corresponding shortening will depend on the opposing passive forces (e.g., due to tissue stiffness, compression, deformation, and elastic recoil) preexisting and developing in the airways. Indeed, while the muscle shortens, both internal and external structural alterations occur that progressively increase forces opposing further shortening (12, 22, 27). Different elastic properties of ASM cells themselves or of the adjacent tissue may therefore confer to the airways substantial differences in shortening capacity and velocity (22). This is conceivably of crucial importance when airway responsiveness during maturation is considered.

In the present study we sought to investigate the relationship between force production and shortening velocity during maturation. As a maturational model we used three age groups of guinea pigs: 1 wk, 3 wk, and 3 mo old. Experiments were conducted on tracheal strips in which we measured maximum amplitude and rate of force generation as well as maximum velocity of shortening and maximum power. To analyze tissue factors opposing shortening velocity, we used a modified form recently developed for ASM (28) of the ratio a/b obtained from the constants a and b of Hill's equation; in fact, this parameter has been shown to be a valid index of internal factors affecting shortening velocity of unloaded ASM (22). Because variability of smooth muscle content may occur at different ages, in cross sections of the trachea and of some of the strips we also measured the area of smooth muscle and of the total cross section to validate our method of normalization of the active tension.

	METHODS

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Animals. Hartley guinea pigs (Sasco, Omaha, NE; Charles River Laboratories, Wilmington, MA) were employed for the present investigation. Three age groups were used: 1-wk-old guinea pigs [1 wk, n = 20, 148 ± 22 (SD) g, 8.8 ± 2.0 days old], 3-wk-old guinea pigs (3 wk, n = 18, 255 ± 27 g, 22.5 ± 2.8 days old), and 3-mo-old guinea pigs (adult, n = 29, 649 ± 125 g, 89 ± 31 days old). Only male animals were used for the 3 wk and adult groups.

Tissue preparation. After loose connective tissue was cleaned away, one or two intact trachealis strips were dissected under a dissecting microscope (SZH10 Olympus stereomicroscope) from transverse sections of the trachea so that parallel-fibered strips were obtained. Trachealis strips were ~0.5-1 mm in width and were dissected with ~2-mm cartilaginous attachments at both ends. One cartilaginous end was clamped in a phosphor-bronze clip at the bottom of a 80-ml double-jacketed organ bath with K-H solution prepared as described in Animals (PO₂ 600 Torr, PCO₂ 40 Torr, pH 7.4, 37°C) and 10⁵ M indomethacin. The other end was fixed to the transducer tip of an electromagnetic lever system with 4-0 braided silk surgical thread inserted through the cartilage, so that the two cartilage pieces constituted the holders of the muscle via their natural structural connections. All the preparative procedures were performed in KH solution buffered to pH 7.35-7.45 by continuous aeration with 95% O₂-5% CO₂.

Electromagnetic lever system. The system consists of the original apparatus by Brutsaert et al. (2) to which new electronic components have been added to improve its computerized control (Qjin Design, Winnipeg, MB). For the parts relevant to the present work, it consists of a force-displacement transducer, an electronic controller unit that controls the transducer performances, a power supply-stimulator, an analog-digital interface, and a computer system with dedicated software that allows data acquisition and analysis. The total compliance of the lever system is 0.2 µm/mN, its total equivalent moving mass is 225 mg, and its time resolution is 2 ms. The average total compliance in our experimental setup and conditions was 5 µm/mN (mainly due to the silk thread), a value that did not considerably affect measurement of the variables under investigation. The resolution of the transducer is 0.1 mN for force recording and 0.002 mm for displacement recording. The controller unit allows application to the muscle of a load ranging from a minimum of 0 mN to a maximum of 150 mN at steps of 0.1 mN. It also permits abrupt changes of the load applied to the strip, which produces, when appropriate pre- and afterloads are used, an instantaneous switch from isometric to isotonic conditions (see Force-velocity studies). The voltage signal from the transducer is converted into a digital signal by a RTD1000 computer board with a maximum throughput of 25 kHz (Real Time Device, State College, PA). The computer program used for data acquisition and analysis (Cunningham Engineering, Lethbridge, AB) allows a maximum sampling frequency of 1,000 Hz, and it controls the stimulus duration and the application of a force clamp at a given desired time. Data acquisition was carried out at 75 Hz to obtain enough data points for a good resolution of both the isometric traces and the slow transients in the quick-release experiments described in Active stress generation and Force-velocity studies.

Active stress generation. After equilibration for 90 min, supramaximal electrical field stimulation (18 V, 60 Hz, 400 mA/cm²) was effected by wire platinum electrodes positioned on both sides of the strip. A partial length-tension curve was elicited by stretching the strips at increasing length and recording the isometric response to electrical field stimulation so that the optimal muscle length (l_o) was identified and used in the subsequent studies.

Force-velocity studies. Preparations and stimulus parameters for these experiments were the same as for Active stress generation. The strips were stretched to l_o, and stimulus duration was kept to the minimum time, i.e., 5 s, to allow application of load clamp. Outputs from the force-displacement transducer were recorded simultaneously for force and length vs time. To elicit a force-velocity (FV) curve the quick-release load-clamp technique was employed. Every 8 min a stimulus was triggered, and the muscle contracted isometrically. At 2.5 s after the onset of the stimulus, when mainly rapidly cycling cross bridges operate (4), load clamps to various afterloads were applied by the electronic controller abruptly (within 3 ms) changing conditions from isometric to isotonic. The quick release resulted in a rapid transient due to shortening of the smooth muscle series elastic component followed by two or three critically damped (to minimize them) oscillations due to the sudden change in load. This rapid transient lasted for ~80 ms and was not analyzed. A slow transient followed, and the maximal slope of this transient (which occurred at ~170 ms after the release) was computed and identified as the maximum velocity of shortening for each given afterload. Afterloads were applied in a random order to minimize time-dependent and history-related effects on shortening velocity. A minimum of 13 different afterloads were used in a single experiment; those experiments in which we could not obtain data with afterloads lower than 0.4 P_o were rejected.

Histology and planimetry. Although we could perform an accurate direct measurement of the strip CSA, the percentage of smooth muscle in the different age groups could be different. Therefore, we measured the area occupied by smooth muscle in 9, 5, and 10 transverse sections of the trachea from 1 wk, 3 wk, and adult animals, respectively, as well as in 4, 3, and 4 cross sections of strips from the three age groups, respectively, processed for histology and planimetry at the end of the experiment. For this purpose, strips were fixed at l_o in 10% Formalin while still connected to the lever. After fixation, strips were embedded in paraffin and cross sections (obtained by stratified random sampling, 5 µm thick) were stained with hematoxylin and eosin. Transverse sections of the trachea were obtained by fixing tracheal rings in 10% Formalin and then processing them as described above for the strips.

View larger version (118K): [in this window] [in a new window]   Fig. 1.   Histological slide illustrating cross section of a tracheal strip (A) and paries membranaceus in a transverse section of the trachea (B). Rectangle in B shows area used to measure ratio of smooth muscle in transverse sections of the trachea. w, Width of strip cross section; t, thickness of strip cross section.

Drugs and chemicals. The drugs used were pentobarbital sodium (Abbot Laboratories, Chicago, IL), indomethacin (Sigma Chemical, St. Louis, MO), and 10% neutral buffered Formalin (Trend Scientific, St. Paul, MN).

Data analysis. Data are expressed as means ± SE, except when differently indicated. Confidence intervals are reported in figure legends. Statistical analyses performed were ANOVA and Fisher's post hoc least significant difference (PLSD) test to find out which groups were responsible for differences showed by ANOVA. The software employed was the StatView II statistical package (Abacus Concepts, Berkeley, CA). P < 0.05 was considered significant.

	RESULTS

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Planimetric data. Comparison of CSA values obtained from histological cross sections and from measurements of nonfixed strips showed that the shrinkage effect was very pronounced on the tissue of the paries membranaceus (41.6 ± 7.3, 51.7 ± 10.2, and 50.9 ± 3.7% in 1 wk, 3 wk, and adult animals, respectively), but it was not significantly different ftom strips of animals of different ages.

Active stress generation. The maximum stress developed in response to electrical field stimulation, expressed as "tissue stress" (P_o/CSA), did not show statistically significant differences, although it slightly increased with age. Figure 2A shows the mean values of maximum active tissue stress obtained by using corrected CSA values as described in Planimetric data. The resting tension needed to keep the strips at their l_o was 4.7 ± 1.1, 6.8 ± 0.7, and 7.8 ± 2.8 mN/mm² for 1 wk, 3 wk, and adult animals, respectively; no significant difference was revealed by ANOVA.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   A: maximum tissue stress generated by electrical field stimulation in tracheal strips from guinea pigs of different ages. B: maximum rate of stress generation in tracheal strips from guinea pigs of different ages. 1 wk, 3 wk, adult: 1-wk-old, 3-wk-old, and 3-mo-old guinea pigs, respectively; Po, maximum stress generated by electrical field stimulation. Values are means ± SE for ten 1 wk, eleven 3 wk, and fourteen adult animals. The 95% confidence intervals for 1 wk, 3 wk, and adult animals, respectively, are as follows: stress (A) = 12.77-21.53, 10.78-27.84, and 16.81-26.83; rate of stress generation (B) = 0.334-0.464, 0.411-0.491, and 0.457-0.571. Rate of stress generation in strips from adult was greater than from 1 wk animals [* P < 0.05 by ANOVA and Fisher's post hoc least significant difference (PLSD) test].

FV studies. Figure 3A shows the V_o as obtained from force-velocity experiments performed at 2.5 s from the onset of the electrical stimulation. In strips from 1 wk and 3 wk guinea pig V_o was three- to fivefold greater compared with the values of adult animals (P < 0.05 by ANOVA and Fisher's PLSD test). The full FV relationship for each age group is shown in Fig. 3B, in which the shortening velocity has been computed as the average of the single best-fit FV curves.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   A: maximum shortening velocity at zero load (Vo) in tracheal strips from guinea pigs of different ages. lo, Optimal length. In strips from 1 wk and 3 wk animals, Vo was 3- to 5-fold greater compared with that from adult animals (* P < 0.05 by ANOVA and Fisher's PLSD test). B: force-velocity curves calculated as average of single curves obtained by fitting experimental data with a modification of Hill's equation for airway smooth muscle (see text and Ref. 28). C: maximum power (PV) in tracheal strips from guinea pigs of different ages. PV in strips from 3 wk guinea pigs was 2- to 3-fold greater than that in strips from 1 wk and adult guinea pigs (P < 0.01 by ANOVA, * P < 0.05 by Fisher's PLSD test). Values are means ± SE for seven 1 wk, five 3 wk, and fifteen adult animals. The 95% confidence intervals for 1 wk, 3 wk, and adult animals, respectively, are as follows:. Vo (A) = 0.159-3.429, 0.104-4.994, and 0.363-0.743; PV (C) = 0.035-0.134, 0.201-0.341, and 0.039-0.067.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Internal resistance to shortening (Rsi) in tracheal strips from guinea pigs of different ages. Values are means ± SE for seven 1 wk, five 3 wk, and fifteen adult. The 95% confidence intervals for 1 wk, 3 wk, and adult animals, respectively, are as follows: 3.07-37.37, 0.85-23.41, and 27.26-59.34. Rsi in strips from adult animals was 2- to 3-fold greater than in strips from 1 wk and 3 wk guinea pigs (* P < 0.05 by ANOVA and Fisher's PLSD test).

	DISCUSSION

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In the present study we investigated the relationship between force production and shortening velocity in tracheal strips from 1-wk-old, 3-wk-old, and 3-mo-old guinea pigs. By planimetric analysis, we showed that to calculate the strip CSA on the basis of a simple measurement of width and thickness of the strip is appropriate to express and compare stress values from strips of animals of different ages. The rate of stress generation, but not the maximum tissue stress, increased significantly with age, whereas V_o and the maximum power were significantly lower in strips from adult animals. A significant increase of the Rsi was also found in strips from adult guinea pigs. This last finding may explain the reduction of shortening velocity with age and may represent a simple mechanism to maintain optimal regulation of airway lumen by balancing opposing factors involved in ASM contractility.

The interest in studying how ASM contractility varies with age arose from the general observation that in vivo airway response to stimulatory agents decreases gradually from childhood to adulthood (3, 11). To study the ontogeny of ASM contractility is of potential relevance to asthma and may elucidate the differences existing between asthma in children and adults (3). However, some inconsistencies among both in vivo and in vitro studies have so far reduced the impact of these investigations.

Most of the in vitro studies reported in the literature on ASM development have been conducted in isometric conditions, assuming force generation to be the main index of contractile function. In strips of isolated tracheal smooth muscle from dogs (5) and sheep (17), the force production per unit CSA of smooth muscle (smooth muscle stress) increased with age. In contrast, by using tracheal strips without removing epithelium and connective tissue, the force production per unit CSA of the whole tissue (tissue stress) has been shown not to change in guinea pig (1) and to increase early and then decrease with age in swine (14, 18, 25).

Inconsistent results exist also among those studies that aimed to examine the maturation of airway responsiveness in vivo (30). This has been attributed to species differences as well as to different maturation of the response to specific agonists. If we restrict our focus to cholinergic agonists, an increased responsiveness with age has been reported in sheep (20), whereas a decline in responsiveness has been reported in dog (5), rabbit (23, 26), and guinea pig (9). From these reports, it is manifest that there is no strict correlation between maturational changes of bronchial responsiveness observed in vivo and in vitro. Nonetheless, the relevance of ASM in these changes is strongly suggested by the finding that the maturational difference in methacholine-induced increase in airway resistance is primarily caused by greater airway narrowing in immature animals (23). To explain why in vitro data do not always match those obtained in vivo, one must consider the several factors that contribute to in vivo bronchoconstriction and that translate force production by ASM into lumen reduction (12). In particular, it is of interest to evaluate how shortening of ASM occurs at different ages, considering that this is a closely related parameter to in vivo bronchospasm. It becomes therefore important to understand the link between force, shortening, and bronchospasm and to analyze which factors may determine variation of ASM shortening at a given amount of force production.

Stress in different preparations, such as those from animals of different ages, would be strongly influenced by a variation in the ratio of smooth muscle to total tissue in the CSA. Tepper et al. (26) have shown that "the relationship between smooth muscle and airway size" is similar in immature and mature rabbits. Our study shows that the ratio of smooth muscle to total tissue in guinea pig trachea does not vary with age. Similarly, in airways of different sizes from pigs, neither the ratio of smooth muscle nor the size of smooth muscle cells varied with age (15). These data imply that smooth muscle stress and tissue stress vary in a parallel fashion during maturation and therefore that the different in vitro results reported in sheep and dogs compared with those in guinea pigs and pigs are not due to the stress parameter employed.

Conceivably, during development, differences between species may be determined by influences exerted on force production by nonmuscle tissue, e.g., the epithelium. The removal of the epithelium did not alter force production in an in situ swine tracheal preparation (14), but it abolished maturational differences in guinea pigs (24). Moreover, our laboratory (13) has recently observed in guinea pigs an age-related increase in epithelial secretion of inhibitory PGE₂ accompanied by decreases in contractile thromboxane B₂ and leukotriene C₄. The different results discussed above may therefore be due, at least in part, to the respective absence and presence of the epithelium. We suggest that the integrity of the epithelium should be preserved when the aim is, as in the present work, to compare the overall contractile response at different ages. Nonetheless, studies on isolated tissues are needed to elucidate the specific contribution of each component to the response.

Force generation by ASM in vivo results in shortening, the extent of which may vary according to specific conditions and properties of the airway and lung tissue. For a contracting muscle to stop shortening or not to produce it, a force must exist or develop that opposes the force that muscle actively produces. The development of forces opposing shortening results in gradual reduction of shortening velocity down to zero (when maximum shortening is achieved). Many factors contribute to this reduction of shortening velocity (21) and, taken together, can be quantified as the Rsi. As described in METHODS, for zero-load conditions at any given time during contraction, a valid value for the Rsi can be obtained by using the constants a and b derived from Hill's equation (22). Although no direct evidence has so far been accumulated, structural counterparts to this parameter are conceivably those intracellular components, e.g., noncontractile and cytoskeletal proteins, that confer viscoelastic properties to smooth muscle cells and are compressed during shortening. Changes of their properties during aging may differentially affect shortening capacity, limiting its potential increase because of the maturational increase in contractility. Alterations in lung extracellular matrix components have been reported during normal development in primates, with the interstitial matrix becoming increasingly organized and proteoglycans diminishing with maturation (10). Our data show a remarkable increase of the Rsi after the first month of life in the guinea pig. Because we found that the amount of maximum force that could be produced in the same animals increased slightly with age, whereas V_o decreased, we attribute the latter to the change observed in Rsi. This conclusion is reinforced by our finding of a significant increase with age of the maximum rate of force generation. In fact, maximum production of force is attained late during contraction, when mainly latch bridges operate. In contrast the maximum rate of force generation occurs early during contraction, when mainly fast cycling cross bridges operate, and depends on the actomyosin ATPase activity of the cross bridges. Shortening velocity also reflects ATPase activity so that these two parameters are more closely related. Moreover, we measured shortening velocity at 2.5 s from the onset of the stimulus, when, because of the different values of maximum rate of force production, a greater difference in stress exists among our age groups compared with that at 10 s, with stress increasing with age. Following these considerations, the inverse relationship between force production and V_o is even more striking, suggesting that a dramatic increase of resistive forces must occur during development to oppose the force actively produced by the ASM.

We have so far considered the maximum possible velocity of shortening (obtained by extrapolation at zero load) as a parameter to compare ASM contractile function at different ages. However, we cannot exclude that the ontogenetic differences in velocity of shortening may also vary as a function of the afterload applied to the ASM. From the analysis of the full FV curves shown in Fig. 3, it appears evident that the velocity of shortening has a similar ontogeny independent from the afterload, with a tendency to a stronger difference between the 3 wk and the other two age groups at intermediate afterloads. The maximum power was attained at values of afterloads of 40-50% P_o in all the three age groups so that this parameter gives us an index of ASM contractile function when the load is in a range conceivably close to physiological conditions. Considerations similar to those taken into account for V_o apply to maximum power. Because a maximum power is attained with similar values of afterloads, the differences observed among our three age groups imply a maturational change of resistive forces, i.e., the Rsi.

Our findings show an unchanged maximum amount of force production and an increase of the rate of force generation with age, associated to a decrease in V_o. By contrast, Ikeda et al. (8) found a reduction of the force production and an increase of V_o with age in isolated ASM from pig trachealis. We want to point out that in that study, similar to our results, when force is elevated shortening velocity is decreased and when V_o is increased the index of Rsi is decreased. This confirms that changes occurring with age in the airway tissue may alter the shortening resulting from a given amount of force. Moreover, it is force that matches the reduced in vivo airway responsiveness observed with age in pig, whereas in our results in guinea pig it is V_o which shows conformity with the change of responsiveness in vivo. This raises the question of which factors affect translation of ASM shortening into lumen reduction. In the study by Ikeda et al. (8), strips of isolated ASM were employed. It remains to be determined whether similar results would have been obtained if intact tracheal strips had been used. Indeed, the results reported by Ikeda et al. could also be explained on the basis of species ontogenetic differences in the nonmuscle factors that may limit in older animal the reduction of airway lumen by affecting ASM load, e.g., extracellular matrix and elastic elements in series to the muscle (9, 12). If in pig the load imposed by cartilage- and/or connective tissue-smooth muscle interdependence increased substantially with age, the intrinsic greater shortening velocity reported in adult animal would indeed be affected and the bronchospasm reduced, so explaining the reduced in vivo bronchial reactivity in older animals.

In conclusion, we have shown a different ontogenetic time course of the rate of force generation, which increases gradually with maturation, and of the stiffness of the Rsi, which increases only later. This may explain the reduced maximum velocity of shortening we found in adult compared with younger animals, because the events occurring during shortening are structural modifications secondary to changes in the equilibrium of applied forces. We suggest that this may constitute a mechanism to balance the forces operating during bronchoconstriction to maintain an optimal regulation of airway lumen; i.e., the increase of the Rsi with age would counteract the increase of force production to reduce shortening velocity and eventually bronchospasm. We also suggest that the elevated ASM shortening velocity manifest early during growth may contribute to the greater incidence of airway hyperresponsiveness reported in children and juvenile animals.