1 Unité de Pneumologie, In the guinea pig, tracheal smooth muscle
(TSM) exhibits intrinsic tone (IT). The active nature of IT suggests
that it could be influenced by muscle length and load. In the guinea
pig, IT is entirely suppressed by the cyclooxygenase inhibitor
indomethacin. IT could be measured as the difference between resting
tone before and after indomethacin addition. We examined, in
electrically stimulated TSM strips (n = 9), the influence of initial muscle length
(Li) on IT, the
relationship between IT and the maximum extent of relaxation
(
airway hyperreactivity; indomethacin; relaxation; mechanics
IN THE BASAL STATE, human and guinea pig airway smooth
muscle exhibits spontaneous tone (1, 4, 17). In vascular smooth muscle,
this spontaneous tone, called "intrinsic tone" (IT), has been
shown to contribute, along with the passive resting tone (PRT), to the
total resting tone (RT) (15, 16).
In guinea pig tracheal smooth muscle (TSM), IT generation involves the
release of prostanoids, inasmuch as indomethacin suppresses IT (1, 7,
9, 18). Moreover, spontaneous basal tone has been shown to increase
after immune sensitization to ovalbumin (17), suggesting a possible
role in the pathophysiology of airway hyperreactivity.
Previous reports (1, 9, 17) have shown that, in the guinea pig,
electrical field stimulation (EFS) of isolated TSM induces a biphasic
response: the initial contraction phase developed during EFS is
followed by a relaxation phase below baseline tone levels, followed by
a slow and gradual recovery of force. Both phases have been reported to
be tetrodotoxin sensitive, i.e., neurally mediated (9, 17). Moreover,
it has been established that the contraction phase results from the
activation of cholinergic mechanisms (9, 25). Conversely, the
relaxation phase results from the activation of both adrenergic and
nonadrenergic noncholinergic components (1, 9). Relaxation
has been attributed, at least in part, to a transient inhibition of IT
(1, 9, 17).
It has been demonstrated that initial muscle length
(Li) is an
important determinant of active isometric force (AF) in TSM (23). The
aim of our study was to analyze, in electrically stimulated guinea pig
TSM, the influence of
Li on IT. We
sought to determine whether the amplitude of relaxation accurately
quantified the amount of IT and measured the
AF-Li
relationship in the presence and absence of IT.
TSM Preparation
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
F1), and the influence of
indomethacin on active isometric force. When
Li decreased from
100 to 75% of optimal
Li, there was a
significant decrease in IT (from 12.0 ± 0.2 to 5.3 ± 0.1 mN; P < 0.001). Over the range
of Li studied,
F1 underestimated the amount of
IT, but there was a close linear relationship between
F1 and IT
(r = 0.9). Compared with the basal state, indomethacin increased active isometric force (from 9.5 ± 1.0 to 19.7 ± 2.0 mN at optimal
Li;
P < 0.001) and induced its length
dependency. In guinea pig TSM,
Li was an
important determinant of IT.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Electromagnetic Apparatus
The muscle strips were anchored to an electromagnetic lever cemented to a coil and suspended in the field of an electromagnet. The load applied to the TSM segment was determined by a servo-controlled current through the coil. The preload level, which determined the Li, was electronically held constant throughout the experiment. A photoelectric transducer measured the displacement of the lever induced by muscle shortening. The equivalent moving mass of the whole system was 150 mg and its compliance was 0.2 µm/mN. The system was linear up to 5 mm of muscle shortening (12). An adjustable electronic stop was set up to avoid muscle lengthening beyond Li when afterload was applied to the muscle. Two signals, force and length, were simultaneously recorded by a computer (IPC Dynasty LE), with a base time of 50 s. The software for calculating all the mechanical parameters was developed in our laboratory. The system is not auxotonic but enables us to measure both isometric and isotonic responses.Mechanical Parameters
Contraction phase. Classic mechanical parameters describing contraction in electrically stimulated TSM were obtained from fully isometric contractions. Total isometric force (TF; in mN) and maximum AF (in mN) were measured (Fig. 1).
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Relaxation phase. In electrically
stimulated guinea pig TSM, the contraction phase is followed by a
relaxation phase below baseline tone levels. Force then spontaneously
returns to preload levels in 3-4 min. During this phase of
relaxation, we measured the lowest measurable force
(F2; in mN) and the maximum
extent of force decay below preload
(
F1; in mN), i.e., the
difference between RT and
F2
(Fig. 1A).
Resting tone. RT (in mN) is defined as the tone developed by the muscle before the electrically induced contraction. In isolated TSM of the guinea pig, RT is divided into active (IT) and passive (PRT) components: RT = IT + PRT, measured in millinewtons. At micromolar concentrations, the cyclooxygenase inhibitor indomethacin is known to totally abolish IT (9, 17, 18). Thus indomethacin made it possible to directly measure the PRT of TSMs and thus to calculate IT. IT was calculated as RT at baseline (i.e., before indomethacin addition; Fig. 1A) minus PRT (determined after indomethacin addition; Fig. 1B).
Experimental Protocols
Influence of Li on IT and
F1.
To determine the effects of
Li on IT and
F1, mechanical parameters of
the isometric contraction were recorded at five different Li values ranging
from 100 to 75% of
Lo. These
different Li
values were obtained by reducing preload levels from 14 to 6 mN.
Successive measurements of RT and
F1 were performed in the
electrically induced isometric contractions before indomethacin
addition. Thereafter, the resting length of the TSM was replaced at
Lo, and
indomethacin (3 × 10
6
M) was added to the Krebs solution. After an equilibrium period of 30 min, the remaining resting tone (i.e., PRT) was measured at the same
corresponding Li
values as before indomethacin addition. IT was calculated as the RT at
baseline minus the RT after indomethacin (IT = RT
PRT).
Comparison of IT and F1.
To determine whether the amplitude of relaxation accurately
characterized IT in guinea pig TSM, baseline values of
F1 were compared with the
corresponding values of IT at different
Li values.
AF-Li relationship in presence and
absence of IT.
The influence of the amount of IT on AF was determined at five
Li values ranging
from 100 to 75% of
Lo. For each
Li value, AF was
measured before indomethacin addition. Thereafter, the resting length
of TSM was replaced at
Lo, and
indomethacin (3 × 106
M) was added to the Krebs solution. After an equilibration period of 30 min, AF was recorded at the same five
Li values as
before indomethacin addition.
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Statistical Analysis
Results are expressed as means ± SE. In all experiments, mean values were compared with analysis of variance and Student's paired t-test with the Bonferroni correction. In all cases, significance required a P value < 0.05. ![]() |
RESULTS |
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Mechanical Characteristics of TSM
At baseline, electrically stimulated guinea pig TSM exhibited a contraction phase followed by a phase of relaxation (Fig. 1A). The mechanical characteristics of TSM in the basal state are given in Table 1. At Lo, baseline values of AF and RT corresponded to 40 and 60% of TF, respectively. The effects of indomethacin are shown in Fig. 1B. As expected, indomethacin significantly reduced RT (Table 1) and abolished the phase of relaxation below the baseline tone level (Fig. 1B). Moreover, at Lo, indomethacin induced a 107% increase in AF compared with the baseline value (Table 1). There was no significant difference in TF after indomethacin addition (23.5 ± 1.0 vs. 21.7 ± 1.9 mN; P = 0.3; Table 1).
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Influence of Li on IT
Figure 3 depicts the relationship between Li and both IT and
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Comparison Between IT and
F1
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AF-Li Relationship in Presence and Absence of IT
Before indomethacin addition, the reduction in Li from 100 to 75% of Lo did not significantly modify AF (Fig. 5). After indomethacin addition, decreasing Li from 100 to 75% of Lo was associated with a progressive and significant reduction in AF (P < 0.001; Fig. 5). Moreover, compared with the basal state and for any Li value studied, AF was greater after indomethacin addition (Fig. 5).
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Influence of Muscle Afterload on Relaxation
Figure 2 shows a series of afterloaded contractions obtained at baseline. When the load was increased from preload up to isometric load, the maximum amplitude of muscle shortening decreased (Fig. 2A). Conversely, relaxation was not modified by this procedure (Fig. 2B). The quantitative results of the afterloaded contractions are given in Table 2. The increase in afterload induced a decrease in the maximum amplitude of muscle shortening. However, no variations in
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DISCUSSION |
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IT, which occurs spontaneously in guinea pig TSM, is totally abolished
by the cyclooxygenase inhibitor indomethacin (1, 7, 9, 18). In this
animal model, analysis of the effects of indomethacin on RT made it
possible to calculate the value of IT. Our results show that
1)
Li is a major
determinant of IT, 2)
F1 underestimates IT,
3)
F1 is not influenced by
afterload conditions, and 4)
indomethacin increases AF and induces its length dependency.
Our results pertain strictly to the animal species and experimental conditions used. The stimulation parameters were set at a pulse width of 10 ms, which is larger than that used in previous studies (9, 17). However, the mechanical properties and effects of indomethacin are similar to those previously reported.
The level of IT was a linear function of Li: as Li fell below Lo, IT declined linearly. Because Ca2+ plays a major role in regulating actomyosin interactions in TSM (2, 5, 23), it could be hypothesized that the mechanical effects induced by changes in Li reflect changes in the intracellular Ca2+ concentration and/or myofilament Ca2+ sensitivity. Li may influence both Ca2+ homeostasis and Ca2+ sensitivity of regulatory proteins such as G proteins and inhibitor proteins (20-22). In line with this hypothesis, previous authors have demonstrated a decrease at short length in both myoplasmic intracellular Ca2+ concentration (14) and Ca2+ sensitivity of myosin light chain kinase (6). The length dependency of IT may be compared with the phenomenon of reduced activation at short length demonstrated in both striated and smooth muscles (10, 23, 24). The mechanisms underlying the AF decrease with muscle length may be related to a reduction in cytosolic Ca2+ release at short length. Alternatively, an increase in prostaglandin release induced by TSM distension has been suggested (3). A decrease in myoplasmic prostaglandin content could be another putative hypothesis to explain the lower amount of IT measured at short Li.
Numerous studies (1, 9, 17) have shown that, in isolated guinea pig
TSM, a relaxation phase below baseline tone level follows the
electrically induced contraction phase. A relaxation phase below
baseline tone level has also been reported in isolated human TSM (8).
To determine whether F1 was a
good estimate of IT, we studied the relationship between
F1 and the amount of IT at
various Li
values. Our results showed that
F1 underestimated the amount of
IT; i.e.,
F1 represented
~80% of IT values. Thus IT was not totally abolished during
relaxation. However, there was a close linear relationship between
F1 and IT (Fig. 3). These results support the hypothesis that, in electrically stimulated TSM,
relaxation corresponds to a transient and incomplete inhibition of IT
but that
F1 does not accurately
quantify IT. The precise influence of IT on the relaxation process was
difficult to assess because of the simultaneous changes in
Li, IT, and
F1.
In guinea pig TSM, several mechanical and pharmacological studies have
analyzed initial force development and relaxation. Selective
anticholinergic drugs, such as atropine, have been shown to inhibit the
initial contraction phase without modifying the relaxation phase (1, 9,
11, 17). This suggests that F1
is independent of the cholinergic pathway. On the other hand, it has
been reported that the characteristics of EFS modulate both the
contraction and relaxation phases (1, 9). The effects of loading
conditions on
F1 (particularly
afterload level and muscle shortening length) have not been previously
reported. Our results show that, for a given preload,
F1 was not influenced by the
afterload level. This suggests that intracellular mechanisms underlying
relaxation remain uninfluenced by changes in muscle length
and/or load during the contraction phase.
In guinea pig TSM, indomethacin induces an increase in all the mechanical parameters of contraction. Muscle shortening, velocity of contraction, and AF are all increased by indomethacin. The mechanisms underlying the effect of indomethacin may involve variations in neurotransmitter release or in contraction regulation. The close relationship between IT and AF makes it difficult to study the effect of cyclooxygenase blockade on AF generation. Linden et al. (13) have recently demonstrated the role of the level of histamine-induced tone in the response to electrical stimulation. In this study, it has not been possible to examine IT because indomethacin was systematically added in all experiments. However, this study has demonstrated that, when a high histamine-induced tone is present, the response to EFS is relaxant. Conversely, when no tone is present, a contractile response to EFS is measured. The comparison between IT and histamine-induced tone is hazardous, but this result confirms that the level of tone is an important determinant of airway response to stimulation.
In striated muscle, it is well known that Li modulates AF (Frank-Starling relationship). Similarly, in dog TSM, in which IT is absent, AF declines with Li (23). Our results show that, in the presence of indomethacin, i.e., after the suppression of IT, AF significantly declines when Li falls below Lo. Conversely, before indomethacin addition, the reduction in Li from 100 to 75% of Lo is not associated with significant changes in AF (Fig. 5). It could be hypothesized that, in the absence of indomethacin, a given proportion of cross bridges are involved in the maintenance of IT. Consequently, the remaining cross bridges that could develop AF during the initial contraction phase may be less numerous before than after IT suppression. Recently, variations in TSM plasticity have been hypothesized to explain the length dependency of mechanical parameters in canine TSM (19). Variations in muscle length may induce variations in the number of contractile units. Similar phenomena may be hypothesized to explain the length dependency of both AF and IT in guinea pig TSM. Further studies are needed to elucidate the regulation of cross bridges involved in IT generation.
In conclusion, our results show that, in electrically stimulated guinea
pig TSM, Li
modulates IT and relaxation
(F1). Moreover, over the
range of Li
values studied, relaxation reflects a transient and incomplete
inhibition of IT. Finally, IT modulates the muscle length-AF relationship.
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ACKNOWLEDGEMENTS |
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We thank D. Chemla for helpful discussions and J. Kenneth Hilton for assistance in the preparation of the manuscript.
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FOOTNOTES |
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Address for reprint requests: Y. Lecarpentier, INSERM U451-LOA-ENSTA-Ecole Polytechnique, batterie de l'Yvette, 91125 Palaiseau Cedex, France.
Received 4 August 1997; accepted in final form 21 August 1998.
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