Mechanical signals and mechanosensitive modulation of intracellular [Ca2+] in smooth muscle

Steven S. An and Chi-Ming Hai

Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, Rhode Island 02912


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Development of carbachol-induced isometric contraction at optimal length. Time courses of force (A) and intracellular Ca2+ concentration ([Ca2+]) as measured by aequorin luminescence (B) during 600 s of isometric contraction induced by 1 µM carbachol. A: force is expressed as fraction of the force (F0) during the initial control contraction. B: aequorin luminescence (L) is normalized by total aequorin luminescence (Lmax) at each time point. Time 0 represents the time when carbachol was added. Symbols and vertical bars represent means ± SE (n = 4).

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 [Delta log(L/Lmax)] in this and subsequent figures. As shown in Fig. 2C, Delta log(L/Lmax) increased at the time of release and then decreased with time during isotonic shortening. The average value and standard deviation of Delta 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 Delta log(L/Lmax) indicates shortening-induced attenuation of intracellular [Ca2+]. Figure 2D shows the correlation between log(muscle length) and Delta log(L/Lmax) during isotonic shortening against 20% isometric force. Correlation analysis of the data indicated significant correlation between Delta 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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Fig. 2.   Isotonic shortening against 20% isometric force. Time courses of force (A), muscle length (B), and aequorin-measured intracellular [Ca2+] (C) during 240 s of isotonic shortening against 20% isometric force subsequent to the initial 600 s of isometric contraction. In these experiments, muscle strips were activated isometrically at optimal length (Lo) for 600 s as shown in Fig. 1 and were then allowed to shorten against 20% isometric force for another 240 s. Time 0 is equivalent to 600 s in Fig. 1. A: force is expressed as percent isometric force. B: muscle length is expressed as fraction of Lo. C: aequorin luminescence is expressed as a change from (Delta ) the level immediately before isotonic release. D: Delta log(L/Lmax) is plotted against log(muscle length) for correlation analysis. Symbols and vertical bars represent means ± SE (n = 5).

Figure 3 shows the time courses of muscle length and aequorin luminescence during isotonic shortening against 50% isometric force. These data were similar to those shown in Fig. 2 except that the extent of shortening and Delta log(L/Lmax) were smaller during isotonic shortening against 50 than 20% isometric force. As shown in Fig. 3B, muscle length decreased to 0.83 ± 0.02 Lo after 240 s of isotonic shortening against 50% isometric force. As shown in Fig. 3C, during the last 30 s of shortening (211-240 s), the average value and standard deviation of Delta log(L/Lmax) was 0.0009 ± 0.0107, which was not significantly different from zero. Therefore, the 50% decrease in force and 17% decrease in muscle length were not associated with detectable changes in intracellular [Ca2+] as measured by aequorin luminescence. Figure 3D shows the relation between Delta log(L/Lmax) and log(muscle length) during isotonic shortening against 50% isometric force.


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Fig. 3.   Isotonic shortening against 50% isometric force. These experiments (A-D) are similar to those shown in Fig. 2, except for the difference in isotonic load. Symbols and vertical bars represent means ± SE (n = 6).

Figure 4 shows the time courses of muscle length and aequorin luminescence during isotonic shortening against 80% isometric force. These data were similar to those shown in Figs. 2 and 3, except that the extent of shortening and Delta log(L/Lmax) were the smallest. As shown in Fig. 4B, muscle length decreased to 0.96 ± 0.01 Lo at 240 s after isotonic shortening against 80% isometric force. As shown in Fig. 4C, during the last 30 s of shortening (211-240 s), the average value and standard deviation of Delta log(L/Lmax) was -0.012 ± 0.022, which was not significantly different from zero. Therefore, the 20% decrease in force and 4% decrease in muscle length were not associated with detectable changes in intracellular [Ca2+] as measured by aequorin luminescence. Figure 4D shows the relation between Delta log(L/Lmax) and log(muscle length) during isotonic shortening against 80% isometric force.


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Fig. 4.   Isotonic shortening against 80% isometric force. These experiments (A-D) are similar to those shown in Fig. 2, except for the difference in isotonic load. Symbols and vertical bars represent means ± SE (n = 5).

To determine whether the relationship between Delta log(L/Lmax) and log(muscle length) was load dependent, data from the three different isotonic shortening experiments (Figs. 2D, 3D, and 4D) were plotted on the same graph. As shown in Fig. 5, the three clusters of data points during shortening against 20, 50, and 80% isometric force appeared to be positioned on a single relationship. For example, at a log(muscle length) value of -0.08 (equivalent to 0.83 Lo), the values of Delta log(L/Lmax) during isotonic shortening against 20 and 50% isometric force were similar despite the 2.5-fold difference in external load.


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Fig. 5.   Length dependence of aequorin-measured intracellular [Ca2+] during isotonic shortening against 20% isometric force, 50% isometric force, and 80% isometric force. Symbols and vertical bars represent means ± SE (n = 5-6). Correlation between Delta log(L/Lmax) and log(muscle length) was analyzed by linear regression (solid line). Data points presented were taken from Figs. 2D, 3D, and 4D.

As shown in Fig. 5, there were some rapid bursts of aequorin luminescence at the time of isotonic release that were higher than what would be predicted by the data during the steady-state phase of isotonic shortening. The magnitude of these rapid bursts of aequorin luminescence appeared to be inversely proportional to isotonic load, as indicated by the muscle length at the time of release in Fig. 5. This inverse relationship was also evident in the time courses of aequorin luminescence during isotonic shortening as shown in Figs. 2C, 3C, and 4C. Gunst (8) has suggested that the burst of aequorin luminescence represented shortening-induced release of Ca2+ from contractile or regulatory proteins. However, we cannot rule out the alternative that the spikes in luminescence on release could be an artifact of superficial cell injury during the rapid elastic recoil of the muscle at the start of muscle shortening.

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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Fig. 6.   Experimental protocol for investigating the effect of cyclopiazonic acid (CPA) on Ca2+ uptake by the sarcoplasmic reticulum (SR). Step 1 was used to induce a control contraction, and the force (S0) developed in this contraction was used to normalize the forces developed in subsequent contractions. Steps 2-5 were used to deplete the carbachol-releasable pool of Ca2+ from the SR using 1 µM carbachol and 5 mM EGTA. PSS, physiological salt solution. Step 6 was used to pretreat muscle strips with CPA or DMSO, and step 7 was used to induce Ca2+ uptake by the SR using K+-enriched PSS (KPSS; containing 1.6 mM CaCl2). Step 8 was used to remove extracellular Ca2+ using EGTA, and step 9 was used to induce Ca2+ release from the SR using 1 µM carbachol and EGTA. The peak force developed during the transient contraction in step 9 reflected the amount of Ca2+ taken up by the SR in step 7. [CPA], concentration of CPA.



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Fig. 7.   Concentration dependence of CPA-induced inhibition of carbachol-induced contraction in tissues after Ca2+ depletion and Ca2+ repletion of the SR (see step 9, Fig. 6). Active force is expressed as fraction of the force (S0) developed in the first control contraction. Symbols and vertical bars represent means ± SE (n = 4-5). Data points were fitted by a polynomial function (solid line) for estimating concentrations of CPA at which 50 and 100% force were inhibited.

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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Fig. 8.   Effect of CPA on isometric contraction at Lo. Time courses of active force (A) and intracellular [Ca2+] (B) as measured by aequorin luminescence in CPA-treated and untreated muscle strips. Equilibrated muscle strips were preincubated in PSS containing 10 µM CPA and then activated by 1 µM carbachol in the presence of 10 µM CPA. Time 0 represents the time when carbachol was added to the solution. Active force is expressed as a fraction of the active force (F0) induced by K+ depolarization at the start of the experiment. Aequorin luminescence is expressed in log(L/Lmax), which is proportional to log[Ca2+]. Symbols and vertical bars represent means ± SE (n = 4).

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 Delta 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 Delta 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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Fig. 9.   Effect of CPA on isotonic shortening against 20% isometric force. Time courses of force (A), muscle length (B), and intracellular [Ca2+] (C) as measured by aequorin luminescence during isotonic shortening of CPA-treated and untreated muscle strips. In these experiments, equilibrated muscle strips held at Lo were activated by 1 µM carbachol in the presence or absence of 10 µM CPA for 10 min and then released rapidly to an external load equal to 20% of the isometric force. C: aequorin luminescence is expressed as a change in log from the level before isotonic release. D: length dependencies of intracellular [Ca2+] in CPA-treated and control muscle strips. Symbols and vertical bars represent means ± SE (n = 4-5).

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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Fig. 10.   Force redevelopment (A) and myosin phosphorylation (B) in CPA-treated and untreated smooth muscle after a rapid change in muscle length from Lo to 0.5 Lo. In these experiments, equilibrated muscle strips at Lo were incubated in PSS with or without 10 µM CPA for 1 h, activated by 1 µM carbachol for 10 min in the same solution, and then rapidly released to 0.5 Lo. LC, light chain. Data are presented as means ± SE (n = 7-12).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

This study was supported by National Heart, Lung, and Blood Institute Grant HL-52714.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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