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J Appl Physiol 86: 488-495, 1999;
8750-7587/99 $5.00
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Vol. 86, Issue 2, 488-495, February 1999

Effects of load and tone on the mechanics of isolated human bronchial smooth muscle

François-Xavier Blanc1, Sergio Salmeron2, Catherine Coirault1, Martin Bard2, Elie Fadel3, Elisabeth Dulmet4, Philippe Dartevelle3, and Yves Lecarpentier5

1 Laboratoire d'Optique Appliquée, Ecole Nationale Supérieure des Techniques Avancées, Ecole Polytechnique, Institut National de la Santé et de la Recherche Médicale Unité 451, 91125 Palaiseau Cédex; 2 Unité de Pneumologie-Service de Médecine Interne, Centre Hospitalier Universitaire de Bicêtre, Assistance Publique, Hôpitaux de Paris, 94275 Le Kremlin Bicêtre; 3 Service de Chirurgie Thoracique, Vasculaire, et Transplantation Cardiopulmonaire and 4 Service d'Anatomie Pathologique, Centre Chirurgical Marie Lannelongue, 92350 Le Plessis Robinson; and 5 Service d'Explorations Fonctionnelles Cardiovasculaires et Respiratoires, Centre Hospitalier Universitaire de Bicêtre, Unité de Formation et de Recherche Paris XI, 94275 Le Kremlin Bicêtre, France


    ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Isotonic and isometric properties of nine human bronchial smooth muscles were studied under various loading and tone conditions. Freshly dissected bronchial strips were electrically stimulated successively at baseline, after precontraction with 10-7 M methacholine (MCh), and after relaxation with 10-5 M albuterol (Alb). Resting tension, i.e., preload determining optimal initial length (Lo) at baseline, was held constant. Compared with baseline, MCh decreased muscle length to 93 ± 1% Lo (P < 0.001) before any electrical stimulation, whereas Alb increased it to 111 ± 3% Lo (P < 0.01). MCh significantly decreased maximum unloaded shortening velocity (0.045 ± 0.007 vs. 0.059 ± 0.007 Lo/s), maximal extent of muscle shortening (8.4 ± 1.2 vs. 13.9 ± 2.4% Lo), and peak isometric tension (6.1 ± 0.8 vs. 7.2 ± 1.0 mN/mm2). Alb restored all these contractile indexes to baseline values. These findings suggest that MCh reversibly increased the number of active actomyosin cross bridges under resting conditions, limiting further muscle shortening and active tension development. After the electrically induced contraction, muscles showed a transient phase of decrease in tension below preload. This decrease in tension was unaffected by afterload levels but was significantly increased by MCh and reduced by Alb. These findings suggest that the cross bridges activated before, but not during, the electrically elicited contraction may modulate the phase of decrease in tension below preload, reflecting the active part of resting tension.

airways; methacholine; albuterol


    INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

ALTHOUGH THE PHYSIOLOGICAL role of airway smooth muscle is not completely understood, it is well established that the maintenance of airway tone and volume is one of its most important functions. As airway smooth muscle is constantly subjected to changes in length and load during breathing (22), it regulates airflow to the lungs via shortening and elongation (17). It is thus important to measure not only isometric parameters, but also length and force changes during isotonic contraction and relaxation processes, to precisely study the mechanics of airway smooth muscle. Recent studies of isotonic mechanical properties of isolated human bronchial smooth muscle (HBSM) have been published (4, 14, 18, 24); all these studies deal with electrically stimulated muscles at baseline. Other studies have described the isometric properties of HBSM exposed to electrical field stimulation (EFS). In some of them, a transient decrease in tension below baseline was noticed after the initial EFS-elicited contraction, whether HBSM was studied under spontaneous tone (3, 8, 19, 27, 28, 35) or precontracted with histamine (8, 10, 19, 26, 28, 35), carbachol (9), or methacholine (MCh) (2). However, the mechanical characteristics and physiological significance of this phase of decline in tension below baseline remain poorly documented.

The purpose of the present study was to test the influence of load and tone changes on the isotonic and isometric mechanical properties of isolated HBSM. We hypothesized that an increase in basal tone, i.e., a precontraction, should act by increasing the number of active actomyosin cross bridges under resting conditions before the EFS-elicited contraction. If total resting tension (RT) is experimentally held constant, pharmacologically induced tone should increase the active part of RT, called the active RT or active "intrinsic tone" (11). Therefore, fewer cycling cross bridges should be available during the electrically induced contraction: muscle shortening and tension development should be limited. Conversely, removal of this tone should restore contractile parameters to baseline values by restoring more available cross bridges for the EFS-induced contraction. In this study we compared with baseline and under various loading conditions the effects of 1) the increase in tone with MCh and 2) the removal of this tone by addition of albuterol (Alb) on the three known EFS-elicited processes: contraction, relaxation, and decrease in tension below baseline.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Bronchial Smooth Muscle Preparation

Bronchial rings (generations 1-3, n = 9) were obtained from patients undergoing lobectomy or pneumonectomy to remove lung carcinoma (mean age 61 ± 5 yr). None of the patients had a history of atopy or asthma or was chronically treated with bronchodilators. Immediately after surgical resection, macroscopically tumor-free tissue was put into a 500-ml airtight container filled with Krebs-Henseleit (KH) solution bubbled with 95% O2-5% CO2. Composition of KH solution was (in mM) 118 NaCl, 4.7 KCl, 1.2 MgSO4, 1.1 KH2PO4, 24 NaHCO3, 2.5 CaCl2, and 4.5 glucose. After fast transport to the laboratory at room temperature (chilled solution was not used, because spontaneous muscle tone could be reduced during cooling) (19), bronchial rings were carefully dissected, freed from alveolar or ganglionic tissue, and longitudinally cut to obtain ~9 × 4 × 2-mm strips. Each preparation was then vertically suspended in a 100-ml organ bath containing the same KH solution bubbled with 95% O2-5% CO2 and maintained at 37°C, pH 7.4. While the lower end of the strip was held by a stationary clip at the bottom of the bath, the upper end was held in a spring clip linked to an electromagnetic lever system. Duration between surgical procedure and preparation setting never exceeded 1.5 h. Supramaximal EFS (30 V/cm, 50-Hz alternating current, 10-ms pulse duration, 12-s train duration) was provided every 5 min through two platinum electrodes arranged in parallel on either side of the preparation. Experiments were conducted after a 1-h equilibration period.

Electromagnetic Lever System

The electromagnetic device has been previously described for skeletal muscle experiments (6, 20). Briefly, it consists of an aluminum lever that is cemented to a coil suspended in the strong field of a permanent magnet. A force couple develops when an electric current passes through the coil. The vector moment of the force couple (M) acting on the coil at the fulcrum is given by Lorentz's law: M = B · n · l · d · I, where B is the magnetic induction, n is the number of turns, l is the length of the coil, d is the width of the coil, and I is the current intensity. At the tip of the aluminum lever of length L, the tangential force (F) is M/L. This force is always perpendicular to the lever. The vertical component of F (same direction as that of muscle force and shortening) does not change considerably, because it depends on the cosine of the angle alpha  due to the small displacement of the lever from its horizontal position during muscle shortening. Force measurement amplitude ranges from 0 to 140 mN. The error in measured force is <0.1%. The equivalent moving mass of the whole system is 155 mg. Its compliance is equal to 0.2 µm/mN. The displacement of the lever is measured by means of a photoelectric transducer that consists of an incandescent lamp, a miniature photodiode, and a preamplifier acting as a current-to-voltage converter. The light emitted by the lamp is modulated by the displacement of the lever, and current alterations in the photodiode are converted into voltage alterations. The linearity of the system ranges up to 5 mm of muscle shortening. The error in measured displacement is <0.5% of the full-scale deflection. A length-stop system allows muscle length changes between each protocol, so that preload remains constant throughout the whole experiment, except at the beginning of the procedure, when optimal initial muscle length (Lo) is determined.

All analyses were made from digital recordings obtained by means of a personal computer. Two signals were recorded: force and length. The recording speed was one analog-to-digital conversion of each signal every 1 ms. Total recording time ranged from 45 to 300 s. A homemade program was used to calculate mechanical parameters.

Mechanical Parameters

In isolated muscle terminology, preload determines resting initial muscle length and represents the RT that stretches the muscle before stimulation. Total RT is divided into active and passive components (16). During an EFS-elicited contraction, total tension is the sum of RT (preload) and active isometric tension (afterload).

At the end of the equilibration period, the resting length of the preparations was progressively increased: step by step, preloads ranging from 10 to 25 mN were applied 10 min before stimulation of muscle strips under fully isometric load. Lo was defined as the length at which the peak value of active isometric tension was measured. This procedure was used to assess the shallow maximum of the length-tension curve (23), and thus the optimal preload that was held constant until the end of the study. RT (mN/mm2) was defined as normalized preload. Mechanical parameters characterizing EFS-induced contraction-relaxation required at least three loading conditions to be measured (Fig. 1). Contraction 1 was loaded with preload only and abruptly clamped to zero load 4 ms after the onset of the electrical stimulus, according to the "zero-load clamp" technique (5). Contraction 2 was loaded with preload only. Contraction 3 was carried out against a heavy load that the muscle could not overcome, so its contraction was fully isometric.


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  		Fig. 1.   Mechanical parameters of electrically stimulated human bronchial smooth muscle. EFS, electrical field stimulation (30 V/cm, 50-Hz alternating current, 10-ms pulse duration, 12-s train duration). Top: shortening length (L) plotted vs. time. Lo, optimal initial muscle length; Vmax, maximal unloaded shortening velocity; Vr, maximum unloaded lengthening velocity of the contraction with preload only; Delta L, maximum extent of shortening of contraction with preload only. Bottom: muscle tension plotted vs. time. Contraction 1 was loaded with preload only and abruptly clamped to zero load 4 ms after onset of electrical stimulus. Contraction 2 was loaded with preload only. Contraction 3 was fully isometric. Top dashed line, preload level. During contraction phase, the following parameters were measured: Vmax, Delta L, peak isometric tension (Po), and positive peak of Po derivative (+dPo/dt). During relaxation phase, Vr and negative peak of Po derivative (-dPo/dt). During phase of tension fall below preload, Delta P2 was lowest measurable tension and Delta P1 was maximum extent of tension decrease below preload, so that Delta P1 was equal to preload, or resting tension (RT), minus Delta P2.

During the EFS-elicited contraction phase, we recorded (Fig. 1) maximum unloaded shortening velocity of contraction 1 (Vmax, Lo/s), maximum extent of muscle shortening of contraction 2 (Delta L, %Lo), peak isometric force of contraction 3 (Fo, mN) converted to peak isometric tension (Po, mN/mm2) when normalized per cross-sectional area, and positive peak of isometric tension derivative of contraction 3 (+dPo/dt, mN · mm-2 · s-1).

During the EFS-elicited relaxation phase, we recorded the maximum lengthening velocity of contraction 2 (Vr, Lo/s) and the negative peak of isometric tension derivative of contraction 3 (-dPo/dt, mN · mm-2 · s-1; Fig. 1).

After the contraction-relaxation process, tension fell below preload and spontaneously reverted to preload level in 3-4 min. During this phase of decrease in tension below preload, the lowest measurable tension was termed Delta P2 (mN/mm2). The maximum extent of decline in tension below preload (Delta P1, mN/mm2) was calculated as the difference between RT and Delta P2 (Fig. 1): Delta P1 = RT - Delta P2.

Experimental Protocols

Three protocols were successively carried out in an attempt to analyze the effects of load and tone changes on isolated HBSM mechanics.

Protocol 1. At Lo, isotonic and isometric mechanical parameters were recorded at baseline throughout the load continuum, i.e., during 6-10 EFS-elicited contractions against increasing loads, from zero load up to the fully isometric contraction.

Protocol 2. The muscarinic agonist MCh (MCh chloride, 10-7 M; Pharmacie Centrale des Hôpitaux de Paris) was added to the bath to precontract bronchial smooth muscle (MCh-induced tone). At 15 min after MCh addition, mechanical parameters were recorded at zero load, at preload only, and under isometric conditions.

Protocol 3. The beta 2-adrenoceptor agonist Alb (10-5 M; GlaxoWellcome) was added to the bath containing MCh to relax HBSM, i.e., to antagonize prior MCh-induced tone. Again, EFS was delivered 15 min after Alb addition, and mechanical parameters were recorded at zero load, at preload only, and under isometric conditions.

Inasmuch as MCh and Alb are light sensitive, protocols 2 and 3 were conducted under dim light. Between each protocol, bronchial strips were allowed to lengthen or shorten after drug addition, so that RT was held constant by means of the length-stop system. At the end of the study, cross-sectional area (mm2) was calculated from the ratio of muscle weight (mg) to muscle length at Lo (mm), with the assumption of a muscle density of 1 (32). The complete experimental procedure lasted ~5-6 h from preparation setting.

Statistical Analysis

Values are means ± SE. Statistical analyses were carried out by using ANOVA and Student's t-test for paired observations; P values were indicated after Bonferroni's correction was taken into account. P < 0.05 was considered statistically significant.


    RESULTS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Mechanical Parameters at Baseline

At baseline, electrically stimulated HBSM produced a contraction phase followed by a relaxation phase and a phase of decrease in tension below preload (Fig. 1). During the contraction phase, peak isometric force was 37.8 ± 4.6 mN, equivalent to Po reaching 7.2 ± 1.0 mN/mm2 when normalized per cross-sectional area. Mean cross-sectional area was 5.59 ± 0.73 mm2. Mean RT at Lo was 2.5 ± 0.4 mN/mm2. At Lo, Delta P1 represented 24 ± 7% of total RT. The Delta P1 index was not altered by the afterload applied during the contraction phase (Fig. 2): during the phase of decrease in tension below preload, all tension curves were superimposed regardless of the afterload level.


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  		Fig. 2.   Effects of afterload on transient phase of decrease in tension below preload observed in electrically stimulated human bronchial smooth muscle. Top: shortening length plotted vs. time. Bottom: muscle tension plotted vs. time. Top dashed line, preload level. Contractions were successively carried out against 6 increasing afterloads, from zero afterload (i.e., preload only, contraction 1) to full isometric contraction (contraction 6). During phase of decrease in tension below preload, Delta P2 was lowest measurable tension and Delta P1 was maximum extent of tension decrease below preload. Delta P1 and Delta P2 were unaltered by level of various afterloads applied during contraction phase.

Mechanical Parameters in MCh-Precontracted HBSM

Figure 3 illustrates the effects of 10-7 M MCh on initial muscle length before any EFS. Under preload determining Lo at baseline, MCh precontracted HBSM, i.e., reduced initial muscle length to 93 ± 1% Lo (P < 0.001). These length changes occurred during the first 30 s after MCh addition and reached a steady maximum after 8-10 min. Muscle length remained stable for >= 1 h.


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  		Fig. 3.   Effects of methacholine (MCh, 10-7 M) and albuterol (Alb, 10-5 M) on initial length of electrically stimulated human bronchial smooth muscle. A: individual results. B: means ± SE. Compared with baseline (B), MCh reduced initial muscle length, whereas Alb raised it beyond Lo. * P < 0.01; dagger  P < 0.001.

Effects of MCh on the EFS-elicited contraction phase, the relaxation phase, and the phase of decrease in tension below preload are illustrated by a typical experimental recording (Fig. 4). Compared with baseline, MCh decreased each contractile parameter. Isometric indexes Po (Fig. 5A) and +dPo/dt (Fig. 5B) decreased by 16 ± 3 and 23 ± 4% of their values at baseline, respectively, whereas isotonic indexes Vmax (Fig. 5C) and Delta L (Fig. 5D) both decreased by 30 ± 5%. During the EFS-elicited relaxation, MCh lowered Vr by 19 ± 5% (Fig. 6A), whereas -dPo/dt was not significantly modified (Fig. 6B). Finally, MCh markedly increased the extent of the phase of decrease in tension below preload in each tested muscle strip (Fig. 7A): Delta P1 was 85 ± 31% higher after MCh exposure than at baseline (Fig. 7B).


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  		Fig. 4.   Effects of MCh (10-7 M) on mechanical properties of electrically stimulated human bronchial smooth muscle. Typical recording of shortening length (L, top) and tension (P, bottom) are plotted vs. time. A: baseline. B: MCh exposure. RT determining Lo at baseline was experimentally held constant. Contraction 1 was loaded with preload only. Contraction 2 was fully isometric. In MCh-precontracted human bronchial smooth muscle, initial muscle length was reduced; during EFS-elicited contraction phase, Delta L decreased and Po was reduced; during phase of decrease in tension below preload, Delta P1 increased and Delta P2 decreased. Tension spontaneously reverted to preload level 3-4 min after onset of EFS.


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  		Fig. 5.   Effects of MCh (10-7 M) and Alb (10-5 M) on mechanical parameters of contraction phase of electrically stimulated human bronchial smooth muscle: Po, +dPo/dt, Vmax, and Delta L. B, baseline. Values are means ± SE. * P < 0.01; NS, nonsignificant.


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  		Fig. 6.   Effects of MCh (10-7 M) and Alb (10-5 M) on mechanical parameters of relaxation phase of electrically stimulated human bronchial smooth muscle: Vr and -dPo/dt. B, baseline. Values are means ± SE. ** P < 0.05.


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  		Fig. 7.   Effects of MCh (10-7 M) and Alb (10-5 M) on Delta P1 in electrically stimulated human bronchial smooth muscle. A: individual results. B: means ± SE. B, baseline. dagger  P < 0.001.

Mechanical Parameters in Alb-Relaxed HBSM

Figure 3 illustrates the effects of 10-5 M Alb on initial muscle length before any EFS. Under preload determining Lo at baseline, Alb relaxed precontracted HBSM; i.e., initial muscle length increased beyond Lo: mean muscle length was 111 ± 3% Lo after Alb exposure (P < 0.01; Fig. 3B).

Effects of Alb on the EFS-elicited contraction phase, the relaxation phase, and the phase of decrease in tension below preload are depicted in Figs. 5, 6, and 7, respectively. Alb restored all contractile indexes to baseline values (Fig. 5). During the relaxation phase, Alb did not change -dPo/dt (Fig. 6B), whereas Vr increased by 28 ± 9% compared with baseline values (Fig. 6A). The phase of decrease in tension below preload totally disappeared in five of nine muscles exposed to Alb, and Delta P1 was markedly decreased in the four other muscles (Fig. 7).


    DISCUSSION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

The present study describes the isotonic and isometric mechanical properties of electrically stimulated HBSM submitted to various tone and loading conditions. At preload determining Lo at baseline, our results showed that 1) contractile indexes were lowered when tone was increased with MCh and were restored to baseline values when this tone was removed with Alb, 2) isotonic relaxation was slower when tone was increased and faster when tone was reduced, whereas isometric relaxation was unaltered by tone changes, and 3) after the contraction-relaxation process, muscles exhibited a transient phase of decrease in tension below preload, which was unaltered by the afterload level applied during the contraction, greatly reinforced when tone was increased, and markedly reduced when tone was decreased.

There are relatively few quantitative data concerning the isotonic and isometric mechanical properties of isolated HBSM at baseline. In our study, values of the maximum shortening of the contraction with preload only (Delta L) and Vmax were comparable to those previously reported in HBSM (4, 14, 18, 24). The same is true for Po (18), but data are more difficult to compare because of the different methods used to normalize force parameters, even if the most suitable way to normalize force in airway smooth muscle is use of the ratio of muscle cross-sectional area to total tissue cross-sectional area (32). The values of Po, Delta L, and Vmax reported in HBSM are lower than those observed in canine (34), porcine (13), and rabbit (29) tracheal smooth muscle. Morphometric evaluations of muscle cross-sectional area have demonstrated the smaller amount of smooth muscle in HBSM strips (4, 14) than in porcine (13), guinea pig (25), and rat (25) tracheal smooth muscle. This may account for the lower isometric tension observed in HBSM. Moreover, a high percentage of connective tissue in HBSM may act as an important parallel elastic component. This may increase the load facing the muscle, limiting muscle-shortening ability (14). Jiang et al. (18) also suggested that the lower values of Delta L and Vmax in HBSM (which are independent of the method used to normalize force parameters) may reflect different properties of the actomyosin ATPase. Further studies are required to confirm these hypotheses.

At baseline, electrically stimulated muscles exhibited a transient phase of decrease in tension below preload after the contraction-relaxation process. Such a phase of decrease in tension below baseline has been previously reported in some but not all species. Electrically stimulated tracheal smooth muscles of dog (31, 34), pig (13), rabbit, and rat (personal observations) usually display no decrease in tension below baseline. Conversely, the phase of decrease in tension below preload has been extensively described in the airway smooth muscle of guinea pig at baseline (7). Recent studies have disclosed that nitric oxide is one of the intracellular agents mediating the phase of decrease in tension below baseline in HBSM (2, 3), whereas vasoactive intestinal peptide seems predominant in that of guinea pig (36). Data concerning the role of the endogenous prostaglandin E2 are still controversial in HBSM: some authors have reported a reduction in spontaneous tone with the cyclooxygenase inhibitor indomethacin (15), whereas others have noticed that baseline spontaneous tone is usually not affected by indomethacin addition (10, 11, 19). If prostaglandin E2 has a role, it may be a minor one in the phase of tension below preload observed in electrically stimulated HBSM.

To mechanically describe the phase of decrease in tension below preload, we proposed a calculated index, Delta P1, i.e., RT - Delta P2. At Lo, mean Delta P1 represented 24 ± 7% of RT. The decrease of tension below baseline spontaneously reverted to preload level in 3-4 min. This is consistent with an active phenomenon underlying the phase of decrease in tension below preload. Thus Delta P1 may partly reflect the active part of RT, in other words, part of active intrinsic tone (11). However, our experimental method does not make it possible to consider Delta P1 as an index of the entire intrinsic tone, inasmuch as one cannot exclude that a small part of Delta P2 is active. In our model we can only assert that the active part of RT was at least equal to 24 ± 7% of total RT at Lo. Furthermore, Delta P1 was unaltered by the level of afterload applied during the contraction (Fig. 2). This suggests that distinct intracellular mechanisms modulate the EFS-elicited contraction phase and the phase of decrease in tension below preload. The phase of decrease in tension below preload appeared to be insensitive to changes in general loading conditions affecting the muscle during EFS-elicited contraction.

To raise tone, MCh was chosen, because it is known to directly act on smooth muscle, mainly via M3 receptors (30), and to produce a stable contraction (2). Moreover, the main other pharmacological agent known to raise tone, i.e., histamine, has been shown to be particularly susceptible to oxidation when EFS is performed in KH oxygenated solution (12). In our experimental procedure, RT was held constant, so that MCh precontraction expressed itself by reducing HBSM initial length (Fig. 3). Moreover, MCh enhanced the amount of the active part of RT. Figure 7 shows that Delta P1 was indeed enhanced by MCh: the higher the active part of RT, the higher the decrease of tension below preload. This suggests that raising tone with MCh activates additional actomyosin cross bridges before the EFS-elicited contraction phase. The newly activated cross bridges might be slowly cycling rather than normally cycling, as the former is considered to be activated within 2 s of shortening (or force production) and a period of 15 min was allowed to elapse before the mechanical parameters were measured in MCh-precontracted muscles.

It would have been interesting to accurately specify the types of activated actomyosin cross bridges by performing quick-release and quick-stretch studies. In fact, such techniques enable measurement of instantaneous stiffness, which is known to reflect the elastic properties of the muscle series elastic component and to be directly proportional to the number of attached (or active) cross bridges (33). Such studies, although elegant in indirectly estimating the number of cycling cross bridges, do not make it possible to differentiate normally cycling from slowly cycling cross bridges. In the present study it was decided to work at constant preload throughout the experiment, thus allowing length changes after addition of MCh or Alb. We believe that additional changes in muscle length (e.g., quick release and quick stretch) would have hampered the interpretation of the mechanical results. This question should certainly be investigated further.

Compared with baseline and for a given RT set by the preload determining Lo at baseline, MCh lowered isotonic shortening and isometric tension (Fig. 5). As mentioned above, the number of the cross bridges activated before the EFS-elicited contraction phase was thought to have been increased by MCh. This would tend to reduce the number of the cross bridges available during the subsequent contraction-relaxation process: such a mechanism could explain lowered muscle shortening and tension development observed in MCh-precontracted HBSM during the EFS-elicited process. An alternative mechanism could be that the cross bridges activated by MCh (presumably slowly cycling cross bridges) act as an internal resistance to shortening, thus reducing velocity (33). Even after MCh exposure, Delta P1 was not affected by the various afterload levels applied during the contractile phase, confirming that the cross bridges formed during the EFS-elicited contraction (normally and slowly cycling cross bridges) did not modulate the phase of decrease in tension below preload.

For further insight into the role of tone changes on the mechanical behavior of electrically stimulated isolated HBSM, we abolished the previous pharmacologically induced tone and measured the same indexes in the same muscles. We chose a massive dose (10-5 M) of a widely used bronchorelaxant drug, Alb. We checked its effectiveness by measuring initial muscle length without changes in total RT. It seemed conceivable that any possible muscle lengthening occurring before EFS could result from a release of the additional cross bridges activated during MCh exposure. As expected, the major bronchorelaxant potency of Alb induced a lengthening of muscle strips beyond Lo (Fig. 3). With similar Alb concentrations, previous isometric studies have shown that the maximal EFS-elicited tension developed by isolated HBSM is markedly reduced (27). In contrast, the present results showed that peak isometric tension and +dPo/dt did not significantly differ from baseline if one allowed changes in initial muscle length (Fig. 5). It is important to consider that, in vivo, airway smooth muscle contracts and relaxes auxotonically: as the muscle shortens, load increases (21). Auxotonic loading increases the contractility of airway smooth muscle (21), which can offset, to some extent, the effect of increasing load. It seems unlikely that Alb could lead to a decrease in tension without changes in initial muscle length in vivo. Our experimental data demonstrated that Alb actually relaxed precontracted HBSM without any decline in its ability to develop isometric tension.

As expected, Alb restored Vmax and Delta L to baseline values (Fig. 5). This confirmed the role of tone changes in the variations of contractile parameters: contractile indexes were decreased by raising active intrinsic tone and were immediately and completely restored by removing MCh-induced tone. Interestingly, isotonic relaxation was faster when tone was decreased (Fig. 6A), whereas isometric relaxation was unaffected by tone changes (Fig. 6B). Further studies are needed to elucidate the underlying mechanisms.

The transient phase of decrease in tension below preload was totally abolished by Alb exposure in five of nine of the tested muscles and was markedly reduced for the remaining four muscles (Fig. 7). This observation indirectly reinforced the idea that Delta P1 represented a part of active RT: as Alb induced a muscle lengthening before EFS, the active component of RT was lowered; at the same time, Delta P1 was markedly lowered. On the other hand, the effects of Alb could not be attributed to changes in initial muscle length: at baseline, Delta P1 increased as initial muscle length increased (personal observation), so that Delta P1 should have been enhanced by Alb if it was markedly influenced by changes in muscle length.

Limitations of our experimental data need to be discussed. Each HBSM was obtained from a patient suffering from carcinoma. Only macroscopically tumor-free specimens were dissected, but we cannot assert that all HBSM were histologically tumor free. However, it is difficult to study the HBSM of tumor-free patients for ethical reasons. Moreover, a recent study has shown similar responses between the EFS-induced isometric contractions of HBSM resected for carcinoma and nondiseased (donor lung) specimens (1). To eliminate a time-induced artifact in our observed experimental data, it would have been useful to investigate a time-matched control group. However, none of the contractile mechanical parameters significantly differed from baseline at the end of protocol 3, i.e., Alb exposure (Fig. 5). Thus the contractile apparatus proved to be functional over a long period of time. Moreover, this type of preparation is often used in mechanical studies and is reputed to be stable for 4-6 h (33). Therefore, it seems unlikely that MCh-elicited changes could be attributed to time-induced damage.

In summary, at preload determining Lo at baseline, our study showed that 1) isotonic and isometric contractile parameters were reversibly lowered by increases in the active component of resting tone, 2) tone changes influenced the isotonic, but not the isometric, relaxation process, and 3) electrically stimulated HBSM exhibited a transient phase of decrease in tension below preload, which proved to be insensitive to afterload but greatly modulated by tone. This specific phase of tension below preload could represent at least part of the active RT. Our results suggest that in isolated HBSM the number of the cycling actomyosin cross bridges activated before, but not during, an electrically elicited contraction partly modulated the mechanical characteristics of the EFS-elicited contraction and the extent of decrease in tension below baseline.


    FOOTNOTES

Address for reprint requests: Y. Lecarpentier, INSERM Unité 451, LOA, ENSTA, Batterie de l'Yvette, 91125 Palaiseau Cédex, France.

Received 17 September 1997; accepted in final form 28 September 1998.


    REFERENCES
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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