Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, Rhode Island 02912
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ABSTRACT |
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We tested the hypothesis that strain is the primary mechanical signal in the mechanosensitive modulation of intracellular Ca2+ concentration ([Ca2+]i) in airway smooth muscle. We found that [Ca2+]i was significantly correlated with muscle length during isotonic shortening against 20% isometric force (Fiso). When the isotonic load was changed to 50% Fiso, data points from the 20 and 50% Fiso experiments overlapped in the length-[Ca2+]i relationship. Similarly, data points from the 80% Fiso experiments clustered near those from the 50% Fiso experiments. Therefore, despite 2.5- and 4-fold differences in external load, [Ca2+]i did not deviate much from the length-[Ca2+]i relation that fitted the 20% Fiso data. Maximal inhibition of sarcoplasmic reticular (SR) Ca2+ uptake by 10 µM cyclopiazonic acid (CPA) did not significantly change [Ca2+]i in carbachol-induced isometric contractions and isotonic shortening. CPA also did not significantly change myosin light-chain phosphorylation or force redevelopment when carbachol-activated muscle strips were quickly released from optimal length (Lo) to 0.5 Lo. These results are consistent with the hypothesis and suggest that SR Ca2+ uptake is not the underlying mechanism.
acetylcholine; calcium; intracellular calcium concentration; mechanotransduction; muscle length
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INTRODUCTION |
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AIRWAY SMOOTH MUSCLE CELLS function in a mechanically active environment and constantly undergo shortening and lengthening during lung ventilation. Recent findings indicate that muscle length significantly modulates muscarinic receptor-mediated phosphatidylinositol turnover, intracellular Ca2+ concentration ([Ca2+]), myosin light-chain phosphorylation, and active stress in airway smooth muscle (2, 18, 30). In theory, stress and strain can both be the mechanical signals involved in mechanosensitive modulation of smooth muscle activation. However, it is difficult to differentiate stress from strain during isometric contractions because the two change in parallel. Mechanical stress and strain can be temporarily uncoupled during isotonic shortening when the external load remains constant and muscle length decreases. In this study, we investigated the mechanical signals and the role of sarcoplasmic reticular Ca2+-ATPase during shortening-induced attenuation of airway smooth muscle activation as measured by intracellular [Ca2+].
External stress is transmitted to a smooth muscle cell via dense plaques on the cell membrane (7). Dense plaques are membrane structures similar to focal adhesions in nonmuscle cells (6). Pavalko et al. (22) have reported phosphorylation of the dense plaque proteins talin and paxillin during tracheal smooth muscle contractions. Tang et al. (26) have reported mechanosensitive tyrosine phosphorylation of paxillin and focal adhesion kinase in tracheal smooth muscle. Recently, the role of focal adhesions has been expanded from the structural understanding of cell integrity to the functional mediation of mechanotransduction (15, 29). It has been proposed that mechanical stress-dependent conformational change of the focal adhesion complex may be sufficient to regulate a whole array of protein kinase pathways, thereby regulating cell activation (5, 32). This proposed mechanism predicts that mechanical stress imposed on focal adhesions or dense plaques is the primary mechanical signal in mechanosensitive modulation, independent of cell deformation as a whole. In relation to smooth muscle shortening, this mechanism predicts that shortening-induced attenuation of intracellular [Ca2+] should be load dependent but relatively length independent.
Alternatively, strain-dependent membrane invagination and/or membrane internalization may be the primary mechanism(s) of mechanosensitive modulation (3, 17). Ellipsoidal geometry predicts that cell surface-to-volume ratio will decrease as a smooth muscle cell shortens from a more elongated shape to a more spherical shape. If some of the excess membrane and its associated signal transduction molecules become compartmentalized and inaccessible to extracellular agonists in a shortened smooth muscle cell, then the level of cell activation will be attenuated. Consistent with this theory, our recent findings suggest that the total number of functional G proteins and/or phospholipase C enzymes on the smooth muscle cell membrane may be regulated by mechanical strain (2). Therefore, we hypothesized that mechanical strain (muscle length) is the primary mechanical signal for shortening-induced inactivation of airway smooth muscle. In this study, we tested this hypothesis by measuring intracellular [Ca2+] and muscle length during isotonic shortening against different external loads. We then analyzed the data to differentiate the relative importance of muscle length and load in determining intracellular [Ca2+] during isotonic shortening.
Gunst (8) has measured intracellular [Ca2+] during isotonic shortening of electrically stimulated canine tracheal smooth muscle and found that intracellular [Ca2+] increased during isotonic shortening. In contrast, Mehta et al. (18) reported a decrease in intracellular [Ca2+] when acetylcholine-activated canine tracheal smooth muscle was released from optimal length (Lo) to 0.7 Lo. A comparison of these two studies is difficult because of the different means of cell activation and mechanical manipulations employed in these studies. Thus the mechanical signals involved in shortening-induced inactivation of muscarinic receptor-activated airway smooth muscle remain unknown. This study is designed to fill this gap in the understanding of mechanosensitive modulation.
Intracellular [Ca2+] is regulated by the sarcoplasmic reticulum (SR) and the cell membrane in airway smooth muscle cells (19, 28). Therefore, shortening-induced attenuation of intracellular [Ca2+] could be explained by an imbalance of Ca2+ fluxes across the SR, the cell membrane, or both. For example, muscle shortening could activate SR Ca2+ uptake, thereby attenuating intracellular [Ca2+]. Alternatively, muscle shortening could inactivate sarcolemmal Ca2+ influx, thereby attenuating intracellular [Ca2+]. Tanaka et al. (25) have reported stretch-induced release of Ca2+ from intracellular stores in cerebral arteries. This observation suggests the possibility that smooth muscle shortening may reverse this process by enhancing uptake of Ca2+ into intracellular stores. Therefore, we hypothesized that SR Ca2+ uptake is the mechanism that attenuates intracellular [Ca2+] during isotonic shortening of airway smooth muscle. We tested this hypothesis by studying the effect of inhibiting SR Ca2+-ATPase on intracellular [Ca2+] during isotonic shortening of airway smooth muscle.
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METHODS |
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Tissue preparation. Bovine tracheae were collected from a slaughterhouse and transported to the laboratory in cold (4°C) physiological salt solution (PSS) of the following composition (in mM): 140.1 NaCl, 4.7 KCl, 1.2 Na2HPO4, 2.0 MOPS (pH 7.4), 0.02 Na2EDTA, 1.2 MgSO4, 1.6 CaCl2, and 5.6 D-glucose. Adventitial and mucosal layers were carefully dissected away, and smooth muscle strips were prepared along the direction of muscle bundles in the circumferential direction, as described previously (10).
Isometric contraction experiments. One end of muscle strips was clamped to a stainless steel clip connected to a force transducer (Grass FT.03), and the other end was clamped to a stainless steel clip connected to a length manipulator (Narishige). Muscle strips were equilibrated for 2 h in PSS (pH 7.4 at 37°C), and the solution was bubbled with air. After a 2-h equilibration, muscle strips were adjusted to Lo for maximal active force development as described previously (10). Muscle strips at Lo were then stimulated by K+ depolarization with the use of K+-enriched PSS (K-PSS), a solution similar to PSS in composition except that 104.95 mM NaCl was substituted by an equimolar concentration of KCl. Active force developed in this contraction was recorded as F0, which was used as an internal standard to normalize active force induced by carbachol in subsequent contractions.
Ca2+-depletion and -repletion experiments. These experiments were performed to determine the concentration of cyclopiazonic acid (CPA) necessary for inhibiting Ca2+ uptake by the SR in bovine tracheal smooth muscle. The protocol of SR depletion and repletion was similar to that described by Bourreau et al. (4). Detailed descriptions of the protocol are included in the RESULTS (Fig. 6).
Isotonic shortening experiments. A computer-controlled lever system (Cambridge Technology 300B) was used in the isotonic shortening experiments. Each end of muscle strips was tied by a piece of silk and attached to the lever arm for length manipulation. A Pentium microprocessor-based computer sent a voltage to the lever system to control the maximum force that would be opposed by the lever arm against the muscle strips. When the force limit of the lever was greater than the force developed by muscle strips, the lever arm was maintained at a constant position, and muscle strips contracted isometrically. In isotonic shortening experiments, the force limit of the lever was set at a level lower than the isometric force developed by the muscle strip, and the muscle strip was shortened isotonically against the force limit of the lever. During the experiment, the computer displayed and stored the muscle length and force developed by muscle strips in 1-s intervals via an input/output board (Data Translation DT2801). Photons of aequorin luminescence were detected by the photomultiplier tube and measured by the counter board (Thorn EMI C660) in the computer. The main computer program was written in the C language, and the libraries for data acquisition were supplied by Data Translation and Thorn EMI.
Measurement of intracellular [Ca2+] using aequorin. Aequorin (Friday Harbor) was loaded into smooth muscle cells by use of a hyperpermeabilization method as described by Morgan and Morgan (20) and used in our previous studies (30, 31). Briefly, muscle strips held at Lo were incubated in a series of four solutions of the following compositions (in mM): 1) 30 min in 120 KCl, 2 MgCl2, 20 TES, 5 ATP, and 10 EGTA; 2) 120 min in 120 KCl, 2 MgCl2, 20 TES, 5 ATP, and 0.1 EGTA and 0.5 mg/ml aequorin; 3) 30 min in 120 KCl, 10 MgCl2, 20 TES, 5 ATP, and 0.1 EGTA; and 4) 120 min in CaCl2-free PSS. CaCl2 (1.6 mM) was added back gradually at the end of the incubation. All solutions were bubbled with air and cooled at 4°C during the aequorin-loading procedure. The subsequent aequorin experiments were done at 37°C in PSS. Aequorin-loaded smooth muscle strips were activated by 36 mM KCl (equimolar substitution of NaCl) instead of 109 mM KCl to induce the first contraction to conserve the amount of active aequorin. Aequorin-loaded muscle strips were then incubated for 1 h in PSS with or without CPA (in 0.1% DMSO), depending on the experiment. Muscle strips were then activated by 1 µM carbachol for 10 min, followed by isotonic shortening against 20, 50, or 80% of the isometric force induced by carbachol. Light emitted by aequorin was detected by a photomultiplier tube (Thorn EMI 9635QA). Anodal currents from the photomultiplier tube were converted to standard voltage pulses by an amplifier/discriminator (Thorn EMI AD1). The pulses were counted by the timer/counter board (Thorn EMI C660) installed in the computer. At the end of each experiment, all remaining active aequorin was discharged by cell lysis with 2% Triton X-100 and 10 mM CaCl2. Maximal luminescence (Lmax) at any given time (t) was determined by integrating aequorin luminescence (L) from time t to the end of the experiment. The ratio L/Lmax was used as a measure of intracellular [Ca2+]. The aequorin method has been found to be relatively insensitive to motion and length artifacts. Housmans et al. (13) have shown that motion and length had a negligible effect on the aequorin light signal. Similarly, using aequorin-loaded canine tracheal smooth muscle, Gunst (8) also found that the aequorin light signal was relatively insensitive to length changes.
Rapid-length-change experiments. Equilibrated muscle strips at Lo were incubated for 1 h in PSS containing CPA (in 0.1% DMSO) or 0.1% DMSO alone (control). After incubation, muscle strips were activated by 1 µM carbachol for 10 min and then rapidly released to 0.5 Lo. On the rapid length change, force typically fell to near zero and then redeveloped slowly over 60 min. CPA-treated and untreated muscle strips were frozen in acetone-dry ice slurry for the measurement of myosin light-chain phosphorylation.
Measurement of myosin light-chain phosphorylation.
Muscle strips were quickly frozen in acetone-dry ice slurry (78°C)
and then allowed to thaw back slowly to room temperature. Acetone-dried
tissues were homogenized in an aqueous solution containing 1% SDS,
10% glycerol, and 20 mM dithiothreitol on ice. The homogenate was then
analyzed by two-dimensional polyacrylamide gel electrophoresis (PAGE),
as described previously (10). Acetone-dry ice slurry has
been found to be as effective as 10% TCA-90% acetone-dry ice in
preserving myosin light-chain phosphorylation in muscle samples
(10). Tissue homogenate was first analyzed by isoelectric focusing (Pharmalyte 4-6.5, Pharmacia) in the presence of 8 M urea to
separate phosphorylated and unphosphorylated myosin light chains.
Sodium thioglycolate (5 mM) was included in the cathodal solution to
minimize protein oxidation. After isoelectric focusing, the tube gel
was transferred to a slab gel for SDS-PAGE to separate myosin light
chains from other proteins by molecular weight. At the end of
electrophoresis, the slab gel was stained by Coomassie blue and scanned
in a densitometer equipped with an integrator (Helena).
Unphosphorylated and phosphorylated myosin light chains appeared as two
spots of different isoelectric pH but similar molecular weight. Myosin
light-chain phosphorylation in moles of phosphate per mole of light
chain (mol Pi/mol LC) was calculated from the ratio of the
amount of phosphorylated myosin light chain to the total amount of
myosin light chain (sum of unphosphorylated and phosphorylated myosin
light chains).
Statistics. Data are shown in means ± SE; n represents the number of tracheal rings. Student's t-test was used for the comparison of two means (P < 0.05 considered significant). Correlation between two variables such as intracellular [Ca2+] and muscle length was analyzed by Pearson's correlation and linear regression analysis (P < 0.05 considered significant).
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RESULTS |
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Carbachol-induced intracellular
[Ca2+] and force during isometric
contraction at Lo.
As shown in Fig. 1, isometric
contraction of bovine tracheal smooth muscle is accompanied by a
transient and sustained increase in intracellular [Ca2+]
as measured by aequorin luminescence. Resting aequorin luminescence in
log(L/Lmax) was 5.38 ± 0.05. On the addition of 1 µM carbachol, log(L/Lmax) increased rapidly to a peak of
4.39 ± 0.14 and subsequently declined with time to a lower but
suprabasal level of
5.18 ± 0.02. When the muscle strip was held
at Lo, active force and aequorin luminescence
remained stable during the subsequent 240 s, the period required
for isotonic shortening experiments (data not shown).
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Intracellular [Ca2+] during
isotonic shortening against different external loads.
In the experiments shown in Fig. 2,
muscle strips were activated by 1 µM carbachol at
Lo for 600 s, and the external load was
then reduced rapidly to 20% isometric force (time 0 in Fig. 2A). As shown in Fig. 2B, muscle length first
recoiled instantaneously to the rapid change in load and then decreased
with time as muscle strips shortened isotonically against 20%
isometric force. Muscle length was 0.49 ± 0.04 Lo at 240 s after isotonic shortening
against 20% isometric force. Figure 2C shows the time
course of intracellular [Ca2+] as measured by aequorin
luminescence during isotonic shortening against 20% isometric force.
Because log(L/Lmax) is proportional to
log[Ca2+], aequorin luminescence is shown as change in
log(L/Lmax) from the prerelease value
[log(L/Lmax)] in this and subsequent figures. As shown
in Fig. 2C,
log(L/Lmax) increased at the time
of release and then decreased with time during isotonic shortening. The
average value and standard deviation of
log(L/Lmax)
during the last 30 s of shortening was
0.23 ± 0.03, which
was significantly different from zero. The negative value of
log(L/Lmax) indicates shortening-induced attenuation of
intracellular [Ca2+]. Figure 2D shows the
correlation between log(muscle length) and
log(L/Lmax)
during isotonic shortening against 20% isometric force. Correlation
analysis of the data indicated significant correlation between
log(L/Lmax) and log(muscle length). Linear regression
analysis of the data yielded a slope of 0.82 ± 0.03, which was
not significantly different from 1. Therefore, these data indicated a
linear dependence of aequorin luminescence and muscle length during
isotonic shortening against 20% isometric force.
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Effect of CPA on Ca2+ uptake by SR.
We performed the following experiments to determine the concentration
of CPA necessary for inhibiting Ca2+ uptake by the SR. The
procedure was similar to that described by Bourreau et al.
(4). As shown in Fig. 6, in
step 1, equilibrated muscle strips at
Lo were activated by K-PSS for 5 min to record the force (S0) of this control contraction. In step
2, muscle strips were allowed to relax in CaCl2-free
PSS containing 5 mM EGTA for 30 min to remove extracellular
Ca2+. In step 3, muscle strips were activated by
1 µM carbachol in CaCl2-free PSS containing 5 mM EGTA to
deplete the agonist-releasable Ca2+ from the SR. Muscle
strips typically developed a substantial but transient contraction in
this step. Steps 4 and 5 were the same as
steps 2 and 3 to further deplete the
agonist-releasable Ca2+ from the SR. The transient
contraction developed in step 4 was typically small,
indicating the depletion of agonist-releasable Ca2+ from
the SR. In step 6, muscle strips were treated with CPA in CaCl2-free PSS containing 5 mM EGTA for 1 h to inhibit
Ca2+-ATPase activity of the SR. In step 7,
muscle strips were activated by K-PSS (containing 1.6 mM
CaCl2) for 15 min to refill the SR with Ca2+.
In step 8, muscle strips were incubated in
CaCl2-free PSS containing 5 mM EGTA for 30 min to remove
extracellular Ca2+. Finally, in step 9, muscle
strips were activated by 1 µM carbachol in CaCl2-free PSS
containing 5 mM EGTA to induce a transient contraction. In the absence
of extracellular Ca2+, the contraction developed in
step 9 should be induced by Ca2+ release from
the SR. Therefore, if CPA inhibits Ca2+ refilling of the SR
in step 7, then CPA should inhibit force development in
step 9. As shown in Fig. 7,
CPA inhibited force development in step 9 with a
half-maximal concentration of ~0.4 µM and totally inhibited
contraction at 10 µM. Therefore, 10 µM CPA was used to investigate
the role of Ca2+ uptake by the SR during isotonic
shortening.
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Effect of CPA on carbachol-induced intracellular
[Ca2+] and force during isometric
contraction at Lo.
As shown in Fig. 8A, 10 µM
CPA increased the basal force of unstimulated muscle strips. Basal
force in CPA-treated muscle strips was 0.57 ± 0.02 F0, which was significantly higher than that (0.06 ± 0.01 F0) in untreated muscle strips. Carbachol-induced force in CPA-treated muscle strips was also significantly higher than
in untreated muscle strips at 60, 180, and 360 s, but steady-state forces developed by CPA-treated and untreated muscle strips at 600 s after the addition of carbachol were not statistically significant. Steady-state force at 600 s was 1.14 ± 0.03 F0
in CPA-treated muscle strips and 1.15 ± 0.02 F0 in
untreated muscle strips. As shown in Fig. 8B, aequorin
luminescence in CPA-treated and untreated muscle strips was similar
before and during carbachol-induced contraction. Basal values of
log(L/Lmax) in CPA-treated muscle strips (5.27 ± 0.09) and untreated muscle strips (
5.08 ± 0.05) at time
0 were not significantly different. Steady-state values of
log(L/Lmax) in CPA-treated muscle strips (
3.92 ± 0.16) and untreated muscle strips (
4.66 ± 0.09) at 600 s
were not significantly different.
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Effect of CPA on intracellular
[Ca2+] during isotonic shortening.
Figure 9A shows the
time courses of muscle length and intracellular [Ca2+] as
measured by aequorin luminescence during isotonic shortening against
20% isometric force. As shown in Fig. 9B, the changes in
muscle length of CPA-treated and untreated muscle strips were similar
during isotonic shortening. Statistical analysis of the two sets of
data indicated insignificant differences in muscle length at 30, 60, 90, 120, 150, 180, 210, and 240 s after isotonic shortening. As
shown in Fig. 9C, there were rapid bursts of aequorin luminescence immediately after isotonic release. The magnitude of the
rapid burst of aequorin luminescence was higher in CPA-treated than
untreated muscle strips. However, aequorin luminescence in CPA-treated
and untreated muscle strips during isotonic shortening was similar.
Statistical analysis of the two sets of data indicated insignificant
differences in log(L/Lmax) at 30, 60, 90, 120, 150, 180, 210, and 240 s after the start of isotonic shortening. Figure
9D shows the relation between
log(L/Lmax) and
log(muscle length) in CPA-treated and untreated muscle strips during
isotonic shortening. Linear regression analysis of the data yielded a
slope of 0.63 ± 0.15 in the CPA-treated muscle strips and
0.82 ± 0.03 in untreated muscle strips. The confidence intervals
of these two slopes overlapped, indicating that the two slopes were not significantly different.
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Effect of CPA on force redevelopment and myosin phosphorylation
after a rapid length change.
In these experiments, CPA-treated or untreated muscle strips were
activated by 1 µM carbachol at Lo for 10 min
and then released rapidly to 0.5 Lo. As shown in
Fig. 10A, active force fell
to zero after the rapid release and then redeveloped slowly with time. At 50 min after release, active force was 0.33 ± 0.05 F0 in CPA-treated muscle strips and 0.31 ± 0.05 F0 in untreated muscle. The forces redeveloped by
CPA-treated and untreated muscle strips at all measured times were not
significantly different. As shown in Fig. 10B, myosin
phosphorylation levels in CPA-treated and control muscle strips before
and after the rapid release were also similar. Before the rapid
release, myosin phosphorylation was 0.40 ± 0.05 mol Pi/mol LC in CPA-treated muscle strips, which was not
significantly different from the value (0.39 ± 0.05 mol
Pi/mol LC) in control muscle strips. After the rapid
release, myosin phosphorylation was 0.23 ± 0.03 mol
Pi/mol LC in CPA-treated muscle strips, which was not
significantly different from the value (0.23 ± 0.04 mol Pi/mol LC) in control muscle strips.
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DISCUSSION |
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Results from this study are consistent with the hypothesis that mechanical strain is the primary mechanical signal for shortening-induced attenuation of intracellular [Ca2+] in airway smooth muscle. As shown in Fig. 2D, intracellular [Ca2+] was significantly correlated with muscle length during isotonic shortening against 20% isometric force. This suggests that actual decrease in muscle length is necessary for the attenuation of intracellular [Ca2+]. This observation is also consistent with the findings of Harris and Warshaw (11) that isotonic shortening velocity of a single cell decreases with the extent of shortening. However, this observation does not exclude the possibility that mechanical stress may modulate the length-intracellular [Ca2+] relation during isotonic shortening. This possibility was tested by performing isotonic shortening experiments against different external loads. As shown in Fig. 5, data points from the 20 and 50% isometric force experiments overlapped in the length-intracellular [Ca2+] relationship. Similarly, data points from the 80% isometric force experiments also clustered close to the data points from the 50% isometric force experiments. Therefore, despite 2.5- and 4-fold increases in external load, intracellular [Ca2+] did not deviate much from the length-intracellular [Ca2+] relation that fitted the 20% isometric force data. These results are consistent with the hypothesis that mechanical strain, but not stress, is the primary mechanical signal for shortening-induced attenuation of intracellular [Ca2+] in airway smooth muscle. In contrast, mechanical stress appears to be sufficient to induce vascular smooth muscle contraction via myogenic mechanisms (27). An increase in transmural pressure leads to lung expansion during inspiration but leads to vasoconstriction in autoregulation of blood flow. Therefore, the different sensitivities of airway and vascular smooth muscle cells to mechanical stress appear to be well suited for their different physiological functions.
Gunst (8) has observed an increase in intracellular [Ca2+] during isotonic shortening in electrically stimulated canine tracheal smooth muscle. In this study, we observed an initial burst of aequorin luminescence at the time of isotonic release (Figs. 2-4), consistent with the findings of Gunst (8). However, we observed a decrease in intracellular [Ca2+] during the steady phase of muscle shortening. This observation is different from the findings of Gunst (8) on electrically stimulated canine tracheal smooth muscle. However, Mehta et al. (18) observed a similar decrease in intracellular [Ca2+] when acetylcholine-activated canine tracheal smooth muscle was released from Lo to 0.7 Lo. Therefore, intracellular [Ca2+] in electrically stimulated and receptor-activated airway smooth muscles appears to respond differently to muscle shortening.
The observed significant length dependence and insignificant load dependence of intracellular [Ca2+] may have implications on the underlying mechanisms. Ellipsoidal geometry predicts that cell surface-to-volume ratio will decrease as smooth muscle cells shorten from a more elongated shape to a more spherical shape. In theory, the excess membrane may invaginate or protrude in shortened smooth muscle cells. If some of the excess membrane and its associated signal transduction molecules become compartmentalized and inaccessible to extracellular agonists in a shortened smooth muscle cell, then the level of cell activation will be attenuated. This mechanism of membrane compartmentalization appears to be consistent with our recent findings that suggest mechanical strain may regulate the number of functional G proteins and/or phospholipase C enzymes on the cell membrane of a smooth muscle cell (2, 31). Similarly, this mechanism may also explain the attenuation of intracellular [Ca2+] during isotonic shortening of airway smooth muscle.
We also tested the hypothesis that sarcoplasmic reticular Ca2+ uptake may be the mechanism of shortening-induced attenuation of intracellular [Ca2+] in airway smooth muscle. CPA was used to inhibit sarcoplasmic reticular Ca2+ uptake using a protocol (Fig. 6) similar to that described by Bourreau et al. (4). The concentration-response relation found in this study (Fig. 7) was similar to that reported by Bourreau et al. except that the sensitivity to CPA appeared to be higher in bovine tracheal than canine tracheal smooth muscle. In this study, we found that 10 µM CPA maximally inhibited contraction of bovine tracheal smooth muscle (Fig. 7), whereas Bourreau et al. found that ~30 µM CPA maximally inhibited contraction of canine tracheal smooth muscle. Maximal inhibition of sarcoplasmic reticular Ca2+ uptake by 10 µM CPA appeared to increase basal intracellular [Ca2+] and basal force in unstimulated smooth muscle (Fig. 8). However, 10 µM CPA did not significantly change steady-state force or intracellular [Ca2+] in carbachol-activated smooth muscle during isometric contractions (Fig. 8). This finding is consistent with the observations of Amoako et al. (1) and Bourreau et al. (4) that CPA does not significantly affect steady-state isometric force induced by muscarinic receptor activation. In contrast, Janssen et al. (14) have reported CPA-dependent attenuation of acetylcholine-induced contractions and augmentation of KCl-induced contractions. However, it is not clear whether force has reached steady state in their experiments. Together, these observations suggest that sarcoplasmic reticular Ca2+ uptake does not play a critical role during the steady-state phase of isometric contractions (24).
We have hypothesized that shortening-induced increase in sarcoplasmic reticular Ca2+ uptake may be the mechanism of shortening-induced attenuation of intracellular [Ca2+] in airway smooth muscle. This hypothesis predicts that inhibition of sarcoplasmic reticular Ca2+ uptake by CPA should abolish the attenuation of intracellular [Ca2+] during muscle shortening. Contrary to this prediction, results from this study indicate that maximal inhibition of sarcoplasmic reticular Ca2+ uptake by CPA did not significantly change intracellular [Ca2+] during isotonic shortening (Fig. 9C). The time courses of muscle shortening and the length-intracellular [Ca2+] relations in CPA-treated and control smooth muscles were not significantly different (Fig. 9, B and D). Phosphorylation of the 20,000-Da myosin light chain is the central mechanism of smooth muscle contraction (9, 13). Additional experiments indicated that 10 µM CPA also did not significantly change myosin light-chain phosphorylation or force redevelopment in carbachol-activated smooth muscle before or after releasing of the muscle strips from Lo to 0.5 Lo (Fig. 10). In these experiments, postrelease measurements of myosin phosphorylation were made at 1 min after release. The relatively short time between the prerelease and postrelease measurements suggests that the observed changes in myosin phosphorylation after release reflected length dependence (18, 30) but not time dependence of myosin phosphorylation. The insensitivity of force development to 10 µM CPA suggests that 10 µM CPA does not have direct effects on cross-bridge cycling. Similar length-dependent changes in myosin phosphorylation have also been observed by Mehta et al. (18) on acetylcholine-activated canine tracheal smooth muscle after a change of muscle length from Lo to 0.7 Lo. As shown in Fig. 10, the length-dependent changes in myosin phosphorylation in CPA-treated and control smooth muscles were similar. Therefore, these findings suggest that the sarcoplasmic reticulum does not play an important role in shortening-induced attenuation of intracellular [Ca2+] in airway smooth muscle.
Because the sarcoplasmic reticulum has a finite capacity for storing Ca2+, it is perhaps not surprising that the sarcoplasmic reticulum does not regulate intracellular [Ca2+] during the steady-state phase of isometric contraction (Fig. 8). However, recent studies have suggested coupling between the emptying of the sarcoplasmic reticulum with the activation of sarcolemmal Ca2+ influx (23). Therefore, sarcoplasmic reticulum may indirectly regulate steady-state intracellular [Ca2+] by its coupled effect on sarcolemmal Ca2+ influx. Results from this study suggest that this mechanism is relatively unimportant in muscarinic receptor-activated smooth muscle. Mehta et al. (18) have proposed that mechanosensitive modulation of Ca2+ influx via mechanosensitive ion channels (16, 21) may be the mechanism of shortening-induced attenuation of intracellular [Ca2+] in muscarinic receptor-activated airway smooth muscle. Results from this study are consistent with this proposal.
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ACKNOWLEDGEMENTS |
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This study was supported by National Heart, Lung, and Blood Institute Grant HL-52714.
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FOOTNOTES |
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Address for reprint requests and other correspondence: C.-M. Hai, Div. of Biology and Medicine, Box G-B3, Brown Univ., Providence, RI 02912 (E-mail: Chi-Ming_Hai{at}brown.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 6 March 2000; accepted in final form 1 June 2000.
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