Dynamic cytosolic Ca²⁺ and force responses to muscarinic stimulation in airway smooth muscle

Abstract

During agonist stimulation of airway smooth muscle (ASM), agonists such as ACh induce a transient increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt), which leads to a contractile response [excitation-contraction (E-C) coupling]. Previously, the sensitivity of the contractile response of ASM to elevated [Ca²⁺]_cyt (Ca²⁺ sensitivity) was assessed as the ratio of maximum force to maximum [Ca²⁺]_cyt. However, this static assessment of Ca²⁺ sensitivity overlooks the dynamic nature of E-C coupling in ASM. In this study, we simultaneously measured [Ca²⁺]_cyt and isometric force responses to three concentrations of ACh (1, 2.6, and 10 μM). Both maximum [Ca²⁺]_cyt and maximum force responses were ACh concentration dependent, but force increased disproportionately, thereby increasing static Ca²⁺ sensitivity. The dynamic properties of E-C coupling were assessed in several ways. The temporal delay between the onset of ACh-induced [Ca²⁺]_cyt and onset force responses was not affected by ACh concentration. The rates of rise of the ACh-induced [Ca²⁺]_cyt and force responses increased with increasing ACh concentration. The integral of the phase-loop plot of [Ca²⁺]_cyt and force from onset to steady state also increased with increasing ACh concentration, whereas the rate of relaxation remained unchanged. Although these results suggest an ACh concentration-dependent increase in the rate of cross-bridge recruitment and in the rate of rise of [Ca²⁺]_cyt, the extent of regulatory myosin light-chain (rMLC₂₀) phosphorylation was not dependent on ACh concentration. We conclude that the dynamic properties of [Ca²⁺]_cyt and force responses in ASM are dependent on ACh concentration but reflect more than changes in the extent of rMLC₂₀ phosphorylation.

INTRODUCTION

In smooth muscle, agonist-induced transient elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) leads to a contractile response [excitation-contraction (E-C) coupling] in a concentration-dependent fashion (force/Ca²⁺ response). In airway smooth muscle (ASM) cells, ACh induces localized oscillations in [Ca²⁺]_cyt that are temporally and spatially integrated and transduced to generate force (1–3). The dynamic nature of [Ca²⁺]_cyt regulation is evident by spontaneous, small-amplitude transient elevations in [Ca²⁺]_cyt that are localized within ASM cells (Ca²⁺ sparks). With agonist stimulation, these Ca²⁺ sparks fuse into larger-amplitude-propagating [Ca²⁺]_cyt oscillations. With increasing ACh concentration, the amplitude of the localized [Ca²⁺]_cyt transient is not affected; however, both the frequency and propagation velocity of [Ca²⁺]_cyt oscillations increase. With spatial and temporal summation of localized [Ca²⁺]_cyt oscillations, the global [Ca²⁺]_cyt response of the ASM cell increases with ACh concentration. The time course of this spatial/temporal summation of [Ca²⁺]_cyt oscillations within ASM cells is on the order of 500–800 ms. Pulsing the release of caged Ca²⁺ within ASM results in the fusion of force responses as the interval between caged Ca²⁺ is shortened, similar to the force-frequency response observed in skeletal muscle (1–3).

The steady-state relationship between [Ca²⁺]_cyt and force has been directly assessed in permeabilized ASM, to determine Ca²⁺ sensitivity (4, 5). In intact ASM, steady-state Ca²⁺ sensitivity has been assessed by determining the ratio of maximum force to maximum [Ca²⁺]_cyt responses (6–9). However, the assessment of Ca²⁺ sensitivity as the ratio of maximum force to maximum [Ca²⁺]_cyt does not account for the temporal differences in these responses and the dynamic nature of E-C coupling in ASM (4, 6, 7). It is widely accepted that in smooth muscle, E-C coupling involves activation of an intracellular signaling cascade that includes 1) agonist-induced elevation of [Ca²⁺]_cyt, 2) mobilization of calmodulin (CaM) from internal binding sites, 3) binding of Ca²⁺ to CaM, 4) Ca²⁺-CaM activation of myosin light-chain kinase (MLCK), 5) phosphorylation of the regulatory myosin light chain (rMLC₂₀), 6) cross-bridge recruitment and cycling, and 7) dephosphorylation of rMLC₂₀ via MLC phosphatase (Fig. 1) (2, 10–12). In this canonical signaling cascade, the major contribution to the temporal delay in the onset of force generation in ASM is the mobilization of intracellular CaM (500–800 ms), whereas phosphorylation of rMLC₂₀ and cross-bridge recruitment are much faster (50–90 ms) (2). Thus, if sensitivity of the force response to the elevation of [Ca²⁺]_cyt is dependent on the delay introduced by rMLC₂₀ phosphorylation and cross-bridge recruitment, this should be reflected by the delay between onset of the [Ca²⁺]_cyt response and the onset of the force response.

Figure 1.

Agonist-induced activation of ligand-gated or voltage-gated receptors channels triggers an increase in [Ca²⁺]_cyt concentration. The increased Ca²⁺ binds to calmodulin (CaM) that activates myosin light-chain kinase (MLCK) mediating phosphorylation of rMLC₂₀, which in turn allows cross-bridge (actin and myosin interaction) recruitment. When cross bridges are formed, force is generated and transferred to the sarcolemma through the cortical cytoskeleton (actin filaments, dense bodies, and intermediate filaments) that attaches the contractile units to the plasma membrane. In turn, the plasma membrane of ASM cells attaches to the extracellular matrix (ECM). The hydrolysis of ATP occurs during cross-bridge cycling. The increase in [Ca²⁺]_cyt concentration also increases G- to F-actin polymerization, which contributes to the number of contractile units. Dephosphorylation of rMLC₂₀ is regulated by myosin light-chain phosphatase (MLCP) and is believed to play a role in modulating the Ca²⁺ sensitivity of force generation in ASM. ASM, airway smooth muscle; [Ca²⁺]_cyt, cytosolic Ca²⁺ concentration; rMLC, regulatory myosin light chain.

The temporal delay between ACh-induced elevation of [Ca²⁺]_cyt and the onset of force generation and the rate of force development in ASM is also affected by internal and external loading of cross bridges (Fig. 1). Internal loading of cross bridges is driven by the tethering of filamentous actin to the cortical cytoskeleton of ASM cells. Time-varying changes in loading are reflected by cross-bridge cycling rate and the rate of ATP hydrolysis. In permeabilized ASM, Ca²⁺ activation induces a rapid increase in ATP hydrolysis rate that reaches a maximum after ∼90 s before decreasing to a steady state level by 5 min (13). External loading is influence by the tethering of the ASM cell to the extracellular matrix (ECM). It is well documented that agonist stimulation can affect the tethering of actin to the cortical cytoskeleton of ASM cells and, thus, can influence the rate of force development. This effect of ACh is not thought to be influenced by [Ca²⁺]_cyt but is modulated by RhoA signaling that also affects dephosphorylation of rMLC₂₀ (14, 15).

Given the time-varying nature of both the [Ca²⁺]_cyt and force responses to ACh stimulation in ASM, an assessment of the sensitivity of the force response to the elevation of [Ca²⁺]_cyt (i.e., Ca²⁺ sensitivity) in ASM should consider the dynamic relationship between elevated [Ca²⁺]_cyt and the force response. This can be accomplished by simultaneous measurements of [Ca²⁺]_cyt and force responses to agonist stimulation and by using phase-loop plots previously used to determine dynamic Ca²⁺ sensitivity in cardiomyocytes (16–19). We hypothesized that phase-loop plots of [Ca²⁺]_cyt and force responses to ACh stimulation would reflect the dynamic nature of Ca²⁺ sensitivity in ASM.

MATERIALS AND METHODS

Preparation of Porcine Airway Smooth Muscle Strips

Porcine tracheas from both male and female pigs were obtained from a local abattoir, which was exempt from Institutional Animal Care and Use Committee (IACUC) approval. Tracheas were kept in ice-cold physiological saline solution (PSS; composition in mM: 118.99 NaCl, 1.17 MgSO₄, 1.18 KH₂PO₄, 4.7 KCl, 2.5 CaCl₂, 0.03 EDTA, 5.5 dextrose, 25 HEPES, pH 7.4) during transport. From the tracheas, ASM strips were isolated as previously described (2, 4, 20–23). Briefly, ASM strips (dimensions: 0.5–1 × 3–5 mm) were dissected with fine tweezers from tracheal rings placed under a binocular microscope. From the same tracheal ring, two ASM strips were dissected and used to simultaneously measure [Ca²⁺]_cyt and force responses to different ACh concentrations (0, 1, 2.6, 10 µM) selected based on a previous study characterizing the ACh concentration/force response in porcine ASM (23). To examine whether changes in rMLC₂₀ phosphorylation or actin polymerization affect the dynamic relationship between elevated [Ca²⁺]_cyt and the force response, ASM strips were incubated with either 0.1 µM calyculin A (phosphatase inhibitor) or 1 µM cytochalasin D (cyto-D, actin polymerization inhibitor) for 30 min in PSS (25°C) before ACh stimulation. In addition, four adjacent ASM strips were exposed to the different ACh concentrations for 30 s (time required to achieve maximum force and rMLC₂₀ phosphorylation) and snap-frozen in 10% trichloroacetic acid (TCA)/10 mM dithiothreitol (DTT) in prechilled acetone and stored at −80°C for measurement of MLC₂₀ phosphorylation.

Simultaneous Measurements of ACh-Induced [Ca²⁺]_cyt and Force Responses

[Ca²⁺]_cyt and isometric force responses to ACh stimulation (0, 1, 2.6, 10 µM) in ASM strips were simultaneously measured as previously described (4, 20–23) (Fig. 1A). These three ACh concentrations were selected based on a previous study in which we determined the EC₅₀ for muscarinic stimulation in porcine ASM (23). The ASM strips were incubated for 4 h in PSS (25°C) containing the acetoxymethyl ester of fura-2 (fura-2 AM; 5 µM) dissolved in dimethyl sulfoxide (DMSO) and also containing 0.02% pluronic (F-127; Molecular Probes) to facilitate efficient loading of fura-2 AM into the cytosol. During fura-2 AM incubation, the strips were gently agitated using a shaker to facilitate better diffusion of fura-2 AM into the cytosol. Following fura-2 AM loading, the strips were mounted in a 0.2-mL quartz cuvette on a Guth Muscle Research System (23, 24) and continually superfused with PSS aerated with 95% O₂ and 5% CO₂ for 30 min to wash out excess fura-2 AM. One end of each strip was connected via stainless steel microforceps to a micrometer (Mitutoyo; to adjust preload to 10 mN) to adjust muscle length, and the other end was connected to an isometric force transducer (KG4; Scientific Instruments, Germany) using stainless steel microforceps.

To determine [Ca²⁺]_cyt, fura-2 fluorescence was measured ratiometrically as previously described (8, 25). Fura-2 fluorescence in the ASM strips was excited using a mercury high-pressure lamp (75 W) as a light source with the wavelengths of excitation light alternatively restricted to 340 nm and 380 nm using a rotating filter wheel alternating between wavelengths every 2 ms. At each excitation wavelength, emitted fluorescence was detected at 510 nm using a photomultiplier tube (PMT) equipped with a bandpass filter (>500 nm). The ratio of emitted fluorescence at 340 nm and 380 nm excitation wavelengths (F₃₄₀/F₃₈₀) was used to determine [Ca²⁺]_cyt based on a calibration equation described by Grynkiewicz et al. (9, 23, 25, 26).

To measure isometric force, the length of ASM strips was adjusted during a 1-h equilibration period until a passive tension of 10 mN was maintained. Thereafter, both ASM strips (duplicate measures) were stimulated with progressively increasing concentrations of ACh (0, 1, 2.6, 10 µM), with an exposure time of 5 min at each concentration. Between each incremental increase in ACh concentration, there was a 5-min period washout with normal PSS before exposure to the next higher ACh concentration. At each ACh concentration, [Ca²⁺]_cyt and force responses were digitized (1 kHz sampling rate) using LabChart8 (AD Instruments, Colorado Springs) and subsequently analyzed using GraphPad Prism 8 (San Diego, CA). The [Ca²⁺]_cyt and force responses from the two strips were then averaged to obtain single measurements per animal.

Measurement of rMLC₂₀ Phosphorylation in Response to ACh Stimulation

The extent of rMLC₂₀ phosphorylation was determined in ASM strips as previously described (23). Briefly, phosphorylated rMLC₂₀ was assayed using a Phos-tag sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel with Zn²⁺ (27, 28) (Wako Chemicals Inc., Richmond, VA) and standard Western blotting. From each trachea, one isolated ASM strip was not exposed to ACh and was thus used to determine the baseline level of rMLC₂₀ phosphorylation. Three adjacent ASM strips were stimulated with 1, 2.6, or 10 µM ACh for 30 s, a period previously found to induce maximum force and rMLC₂₀ phosphorylation (29). After 30 s, the ASM strips were flash-frozen in 10% trichloroacetic acid (TCA)/10 mM dithiothreitol (DTT) in prechilled acetone. For protein extraction, the strips were washed with 10 mM DTT in acetone to remove TCA. Samples were then minced and ground in 2% SDS, 50 mM DTT, 50 mM Bis-Tris pH 6.8, 1 tab Roche EDTA-free protease inhibitor, 1 tab Roche PhosStop phosphatase inhibitor, 5% glycerol, and 0.01% bromophenol blue. Electrophoresis of the Phos-tag gels was performed at 20 mA for 1 h 50 min in running buffer [pH 7.4; 100 mM Tris-base, 100 mM 3-morpholinopropane-1-sulfonic acid (MOPS), 0.1% SDS, 5 mM sodium bisulfate] at room temperature. The gel was then immersed in transfer buffer [25 mM Tris, 192 mM glycine, 10% methanol (v/v)] containing 10 mM EDTA to remove Zn²⁺ for 30 min, and proteins were transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was fixed with 0.5% formaldehyde in PBS for 45 min (27) and blocked with 5% dry milk in Tris-buffered saline-Tween 20 (TBST). Using a standard Western blotting technique, the membrane was incubated with a primary antibody (1:2,000 dilution, rabbit polyclonal anti-rMLC₂₀; sc-15370; Santa Cruz, CA) and a secondary antibody (1:10,000 dilution, goat anti-rabbit IgG-horseradish peroxidase conjugate; Santa Cruz, CA). Antibodies were validated in a previous study (23). Unphosphorylated rMLC₂₀ and phosphorylated rMLC₂₀ (p-rMLC₂₀) were detected by enhanced chemiluminescence (ECL, SuperSignal West Dura Extended Duration Substrate; Thermo Scientific, Rockford, IL) and imaged on the ChemiDoc MP Image System (Bio-Rad) and analyzed using an Image-Lab-Software (v. 6.0.1, Bio-Rad) (Fig. 5C).

Figure 5.

The integral of the rising phase (steps a→c) of the phase-loop plots of ACh-induced [Ca²⁺]_cyt and force increased with ACh concentrations (1, 2.6, and 10 µM). A: representative tracings of phase-loop plots at the three ACh concentrations (1, 2.6, 10 µM) are presented. B: the integral of the rising phase (steps a→c) of the phase-loop plots of ACh-induced [Ca²⁺]_cyt and force are summarized and were found to increase significantly with increasing ACh concentration [*P < 0.05, n = 6 (number of animals)]. C: relaxation rates of the phase-loop plots (steps c→a) were comparable across the three ACh concentrations [P > 0.05, n = 6 (number of animals)]. [Ca²⁺]_cyt, cytosolic Ca²⁺ concentration.

Statistical Analysis

For the analysis of [Ca²⁺]_cyt and force responses and rMLC₂₀ phosphorylation, porcine tracheas from six animals were used (n = 6) and six adjacent ASM strips were dissected. Two ASM strips from each animal were used for simultaneous measurements of [Ca²⁺]_cyt and force responses to 1, 2.6, or 10 µM ACh stimulation and to 1 µM ACh stimulation with or without cyto-D or calyculin A treatment. The two independent measurements of [Ca²⁺]_cyt and force responses to ACh were then averaged to provide a single measurement per animal. In a separate set of four adjacent ASM strips, the extent of rMLC₂₀ phosphorylation induced by ACh was determined at four concentrations (0, 1, 2.6, 10 µM ACh). Results were analyzed using a one-way repeated-measures ANOVA (JMP Pro software; JMP, RRID: SCR_014242). Data were presented as medians, and interquartile range (IQR) is represented as a box plot whisker. Significance was considered at P < 0.05.

RESULTS

ACh-Induced [Ca²⁺]_cyt and Force Responses

From two porcine ASM strips from each animal, [Ca²⁺]_cyt and isometric force responses to ACh stimulation were simultaneously measured at three ACh concentrations (1, 2.6, 10 µM). In all cases, the [Ca²⁺]_cyt and isometric force responses were comparable between the two ASM strips. At each ACh concentration, a robust biphasic [Ca²⁺]_cyt response was induced with a delayed and slower but sustained force response (Fig. 2A). The maximum of both the [Ca²⁺]_cyt and force responses increased with increasing ACh concentrations (P < 0.05; Fig. 2B). Compared with the max [Ca²⁺]_cyt response induced by 1 µM ACh, the response of maximum [Ca²⁺]_cyt to 10 µM ACh increased by ∼25% (510–660 nM). In contrast, the maximum force response induced by 10 µM ACh (5.8 N·cm⁻²) was ∼41% greater than that induced by 1 µM ACh (3.4 N·cm⁻²). As a result of the disproportionately greater effect of increasing ACh concentration on maximum force as compared with maximum [Ca²⁺]_cyt response, the ratio of maximum force to maximum [Ca²⁺]_cyt increased by ∼18% from 1 to 10 µM ACh stimulation (P < 0.01; Fig. 2C). These results indicate that static Ca²⁺ sensitivity of force generation in ASM is dependent on ACh concentration.

Figure 2.

Maximum [Ca²⁺]_cyt, maximum force, and static Ca²⁺ sensitivity of ASM increased with increasing ACh stimulation. A: representative tracings of simultaneous measurements of [Ca²⁺]_cyt and force responses of ASM to increasing levels of ACh stimulation (1, 2.6, and 10 µM). B: both maximum [Ca²⁺]_cyt and maximum force responses [summarized as medians and interquartile range (IQR)] increased with increasing ACh stimulation. C: static Ca²⁺ sensitivity of the ASM force response, as reflected by the ratio of maximum [Ca²⁺]_cyt to maximum force, increased with increasing ACh concentration. Data were analyzed using a one-way repeated-measures ANOVA. Significance was considered at P < 0.05 [*compared with 1 µM ACh; n = 6 (number of animals)]. ASM, airway smooth muscle; [Ca²⁺]_cyt, cytosolic Ca²⁺ concentration.

Temporal Delay between the Onset of ACh-Induced [Ca²⁺]_cyt and Force Responses

From the simultaneous recorded [Ca²⁺]_cyt and isometric force responses to ACh stimulation in ASM, the temporal delay between the onset of ACh-induced [Ca²⁺]_cyt and force responses was determined (Fig. 3A). The ACh-induced elevation of [Ca²⁺]_cyt always preceded the force response by ∼3–5 s (Fig. 3A). The fura-2 fluorescent signal of [Ca²⁺]_cyt was noisier, affecting the signal-to-noise level and detection of the onset of the ACh-induced response. Yet, the signal-to-noise ratio of the [Ca²⁺]_cyt responses was less than 10%, which was sufficient to determine the onset of the [Ca²⁺]_cyt response within 10 ms at the 1 kHz sampling rate. The noise level of the mechanical force signal was much lower, and thus, it did not affect the detection of the ACh force response. Importantly, with increasing ACh concentrations, there was no change in the temporal delay between the onset of [Ca²⁺]_cyt and force responses (Fig. 3B). From the simultaneous recorded [Ca²⁺]_cyt and isometric force responses to ACh stimulation in ASM, force at maximum [Ca²⁺]_cyt response was determined (Fig. 3C). With increasing ACh concentrations, there was no change in force at maximum [Ca²⁺]_cyt response (Fig. 3D).

Figure 3.

The temporal delay between the onset of ACh-induced [Ca²⁺]_cyt and force responses was independent of ACh concentration. A: representative tracings of [Ca²⁺]_cyt (blue) and force (red) response of ASM to ACh stimulation. B: the temporal delay (t_d) between the onset of [Ca²⁺]_cyt and force responses was independent of ACh concentration. C: representative tracings of [Ca²⁺]_cyt (blue) and force (red) response of ASM to stimulation of increasing ACh concentration (1, 2.6 and 10 μM) to determine specific force at maximum [Ca²⁺]_cyt. D: force at maximum [Ca²⁺]_cyt was independent of ACh concentration. Results were analyzed using a one-way repeated-measures ANOVA, n = 6 (number of animals), and summarized as medians and interquartile range (IQR). ASM, airway smooth muscle; [Ca²⁺]_cyt, cytosolic Ca²⁺ concentration.

Rate of Rise of ACh-Induced [Ca²⁺]_cyt and Force Responses

The temporal coupling of the ACh-induced [Ca²⁺]_cyt and force responses was determined by calculating the first derivatives of [Ca²⁺]_cyt and force responses (i.e., d[Ca²⁺]_cyt/dt and dForce/dt) (Fig. 4A). Figure 4B shows a summary of the rate of rise in [Ca²⁺]_cyt and force in response to ACh concentrations (1, 2.6, 10 µM). Taking the first derivative of [Ca²⁺]_cyt and force responses in response to ACh concentrations allowed for objective measures of how fast the response curves changed.

Figure 4.

The rates of rise of both the [Ca²⁺]_cyt and force responses increased with increasing ACh concentration. A: representative tracings of the first derivatives of [Ca²⁺]_cyt (solid lines) and force (dashed lines) responses to three concentrations of ACh (1, 2.6, and 10 µM). B: the maximum d[Ca²⁺]_cyt/dt and maximum dF/dt at the three ACh concentrations are summarized as medians and interquartile range (IQR). Force responses accelerated after ACh stimulation and the maximum acceleration rates depended on ACh concentration (*P < 0.05, n = 6). Results were analyzed using a one-way repeated-measures ANOVA; n = 6 (number of animals). [Ca²⁺]_cyt, cytosolic Ca²⁺ concentration.

Integral of the Phase-Loop Plots of ACh-Induced [Ca²⁺]_cyt and Force Responses

To determine the dynamic Ca²⁺ sensitivity of E-C coupling during force generation in ASM, the integral of the phase-loop plots of the ACh-induced [Ca²⁺]_cyt and force responses (steps a→c in Fig. 5A) was calculated. With increasing ACh concentration (1, 2.6, 10 µM), the integral of the rising phase of the phase-loop plot significantly increased (P < 0.05, Fig. 5B).

Relaxation Rate of the Phase-Loop Plots of ACh-Induced [Ca²⁺]_cyt and Force Responses

To determine the dynamic Ca²⁺ sensitivity of force relaxation in ASM, the relaxation rate of ACh-induced [Ca²⁺]_cyt and force responses was determined (steps c→a in Fig. 5A). With increasing ACh stimulation, there was no significant difference in the relaxation rate (Fig. 5C).

Phosphorylation of rMLC₂₀ in Response to ACh Stimulation

In a separate set of experiments, the extent of rMLC₂₀ phosphorylation was assayed using Western blotting in ASM strips treated at the four different ACh concentrations (0, 1, 2.6,10 µM) for 30 s, in which maximum phosphorylation of rMLC₂₀ was known to occur (4). The extent of rMLC₂₀ phosphorylation was determined by the ratio of sum of double and single p-rMLC₂₀ to the total double, single, and un p-rMLC₂₀: (double + single p-rMLC₂₀)/(double + single + un p-rMLC₂₀) × 100 (%) (Fig. 6). The result of the extent of rMLC₂₀ phosphorylation was comparable across the three ACh concentrations (1, 2.6, 10 µM; P > 0.05, ASM strips = 24; Fig. 6, B and C), although these three ACh concentrations significantly increased the extent of rMLC₂₀ phosphorylation compared with 0 µM ACh concentration (P < 0.05, ASM strips = 24; Fig. 6C). As a result, the extent of rMLC₂₀ phosphorylation stimulated with three ACh concentrations was not ACh concentration dependent. These results are consistent with previous studies that reported that changes in rMLC₂₀ phosphorylation are not dynamic and do not reflect dynamic changes in the relationship between [Ca²⁺]_cyt and force generation (4, 22, 23, 30).

Figure 6.

A: force generated by ASM after 30 s of ACh stimulation increased with ACh concentration (1, 2.6, 10 µM) [*P < 0.05, n = 6 (number of animals)]. B: representative Western blots in which phosphorylated (single and double p-rMLC₂₀) and unphosphorylated (un-p-rMLC₂₀) rMLC₂₀ were separated using modified Phos-tag gels. C: compared with baseline, the extent of rMLC₂₀ phosphorylation increased with ACh stimulation [*P < 0.05, n = 6 (number of animals)], but rMLC₂₀ phosphorylation did not change across the three ACh concentrations (1, 2.6, 10 µM) [P > 0.05, n = 6 (number of animals)]. ASM, airway smooth muscle; rMLC, regulatory myosin light chain.

Effects of Calyculin A on ACh-Induced [Ca²⁺]_cyt and Force Responses

To determine the role of phosphatase inhibition on ACh-induced [Ca²⁺]_cyt and force responses and Ca²⁺ sensitivity, the effects of the phosphatase inhibitor calyculin A were examined. Although calyculin A transiently increased rMLC phosphorylation (Fig. 7, A and B) and force generation (Fig. 7C) induced by ACh stimulation (after 30 s), calyculin A had no effect on the dynamic relationship between [Ca²⁺]_cyt and force responses (Fig. 7D). Similarly, calyculin A had no effect on the temporal delay from the onset of [Ca²⁺]_cyt to the onset of force responses (Fig. 8A) and the integral and relaxation rate of the phase-loop plot of ACh-induced [Ca²⁺]_cyt and force responses (Fig. 8, B–D).

Figure 7.

Force generated by ASM after 30 s of ACh stimulation with 1 µM ACh [P < 0.05, n = 6 (number of animals)] in the presence and absence of calyculin A. A: representative Western blots in which phosphorylated (single and double p-rMLC₂₀) and unphosphorylated (un-p-rMLC₂₀) rMLC₂₀ were separated using modified Phos-tag gels. B: compared with baseline, the extent of rMLC₂₀ phosphorylation increased with ACh stimulation [*P < 0.05, n = 6 (number of animals)], and rMLC₂₀ phosphorylation by calyculin A transiently increased higher than control at 30 s with 1 µM ACh stimulation [#P < 0.05, n = 6 (number of animals)], but no further changes at 2 and 4 min. C: calyculin A significantly increased maximum force but not maximum [Ca²⁺]_cyt compared with control [*P < 0.05, n = 6 (number of animals)]. D: there was no difference in maximum dF/dt between control and calyculin A. ASM, airway smooth muscle; [Ca²⁺]_cyt, cytosolic Ca²⁺ concentration; rMLC, regulatory myosin light chain.

Figure 8.

The temporal delay between the onset of [Ca²⁺]_cyt and force responses to 1 µM ACh was not affected by calyculin A (A). Representative tracings of the phase-loop plot of ACh-induced [Ca²⁺]_cyt and force responses in control (gray) and calyculin A (black) (B). Calyculin A had no effect on the integral and relaxation rate of the phase-loop plot [C and D; P > 0.05, n = 6 (number of animals)]. [Ca²⁺]_cyt, cytosolic Ca²⁺ concentration.

Effects of Cyto-D on ACh-Induced [Ca²⁺]_cyt and Force Responses

In a separate set of experiments, the effects of the inhibitor of actin polymerization cyto-D on ACh-induced [Ca²⁺]_cyt and force responses were explored. Cyto-D treatment had no effect on rMLC phosphorylation as compared with control (untreated) ASM strips (Fig. 9, A and B), but force generation was significantly decreased (Fig. 9C). Treatment with cyto-D also decreased the rate of rise of force (Fig. 9D). Cyto-D had no effect on the temporal delay from the onset of [Ca²⁺]_cyt to the onset of force responses (Fig. 10A). By contrast, the integral of the phase-loop plot of ACh-induced [Ca²⁺]_cyt and force response was significantly decreased (Fig. 10, B and C), but cyto-D did not affect the relaxation rate (Fig. 10, B and D).

Figure 9.

Force generated by ASM after 30 s of ACh stimulation with 1 µM ACh in the presence and absence of cyto-D. A: representative Western blots in which phosphorylated (single and double p-rMLC₂₀) and unphosphorylated (un-p-rMLC₂₀) rMLC₂₀ were separated using modified Phos-tag gels. B: compared with baseline, the extent of rMLC₂₀ phosphorylation increased with 1 µM ACh stimulation (*P < 0.05, n = 6), but rMLC₂₀ phosphorylation by cyto-D remained the same as control with 1 µM ACh stimulation during the time course (P > 0.05, n = 6). C: cyto-D significantly decreased the maximum force (*P < 0.05, n = 6) but not the maximum [Ca²⁺]_cyt response induced by 1 µM ACh compared with control. D: there was a significant decrease in maximum dF/dt in cyto-D compared with control [*P < 0.05, n = 6]. ASM, airway smooth muscle; [Ca²⁺]_cyt, cytosolic Ca²⁺ concentration; cyto-D, cytochalasin D; rMLC, regulatory myosin light chain.

Figure 10.

The temporal delay between the onset of [Ca²⁺]_cyt and force responses induced by 1 µM ACh was not affected by Cyto-D (A). Representative tracings of the phase-loop plot of ACh-induced [Ca²⁺]_cyt and force responses in control (gray) and Cyto-D (black) (B). However, compared with control, treatment with cyto-D significantly decreased the integral of the phase-loop plot of ACh-induced [Ca²⁺]_cyt and force responses (*P < 0.05, n = 6) (C) but had no effect on relaxation rate (P > 0.05, n = 6) (D). [Ca²⁺]_cyt, cytosolic Ca²⁺ concentration; cyto-D, cytochalasin D.

DISCUSSION

The present study demonstrates that both [Ca²⁺]_cyt and isometric force responses are dependent on ACh concentration, with a disproportionately greater force compared with [Ca²⁺]_cyt response resulting in an increase in static Ca²⁺ sensitivity (measured as the ratio of maximum force to maximum [Ca²⁺]_cyt). However, the dynamics of [Ca²⁺]_cyt and force responses are also divergent, reflecting the dynamic nature of E-C coupling. Thus, using measurements of maximum [Ca²⁺]_cyt and maximum isometric force responses to estimate Ca²⁺ sensitivity does not reflect the dynamic nature of E-C coupling. This study emphasizes the importance of exploring the non-steady-state properties of E-C coupling in ASM by measuring the temporal delay and rising phase of [Ca²⁺]_cyt and isometric force response to increasing concentrations of ACh stimulation. We found that the temporal delay between [Ca²⁺]_cyt and force responses was not dependent on ACh concentration. In contrast, the rising phase (first derivatives) of both the [Ca²⁺]_cyt and force responses was ACh concentration dependent. The integral of the phase-loop plot of [Ca²⁺]_cyt and force from onset to the steady-state phase was also dependent on ACh concentration, whereas the relaxation rate was similar across all ACh concentrations. It is commonly thought that changes in Ca²⁺ sensitivity in ASM are mediated by the canonical rMLC₂₀ phosphorylation pathway triggered by transient elevation of [Ca²⁺]_cyt (Fig. 1). In particular, it is widely held that agonist-induced activation of RhoA signaling inhibits MLCP and thereby promotes an increase in rMLC₂₀ phosphorylation (10, 31, 32). However, we did not find that the extent of rMLC₂₀ phosphorylation increased with increasing ACh concentration. Moreover, the time course of dynamic E-C coupling in ASM is inconsistent with regulation through the rMLC₂₀ phosphorylation pathway. Thus, we conclude that ACh-dependent rMLC₂₀ phosphorylation does not primarily drive the dynamic properties of the [Ca²⁺]_cyt and force responses in ASM.

Dynamic Properties of [Ca²⁺]_cyt and Force Responses to ACh Stimulation

In ASM, ACh stimulation induced a transient elevation in [Ca²⁺]_cyt with a force response delayed by several hundred milliseconds (2). This time course is consistent with Ca²⁺-induced activation of rMLC₂₀ phosphorylation and recruitment of actin-myosin cross bridges (Fig. 1). The first derivative of [Ca²⁺]_cyt and force responses allows for objective measures of the dynamic responses during the initial activating phase of E-C coupling (Fig. 3, A and B). The rates of rise of both the [Ca²⁺]_cyt and force responses significantly increased as ACh concentration increased; however, the rate of rise of the [Ca²⁺]_cyt response was steeper than that of the force response.

Rate of rise of [Ca²⁺]_cyt response.

In ASM, the [Ca²⁺]_cyt response to ACh stimulation reflects several sources of Ca²⁺ influx into the cytosolic compartment. ACh stimulation results in membrane depolarization and Ca²⁺ influx via the opening of L-type, voltage-gated Ca²⁺ channels (7, 33). However, the transient elevation of [Ca²⁺]_cyt induced by ACh mainly reflects Ca²⁺ release from the endoplasmic reticulum through inositol 1,4,5-triphosphate receptor (IP₃R) and ryanodine receptor (RyR) channels (34–38). Furthermore, both IP₃R and RyR channels are Ca²⁺ sensitive; thus, Ca²⁺-induced sarcoplasmic reticulum (SR) Ca²⁺ release (CICR) occurs, accelerating the elevation of [Ca²⁺]_cyt (34–38). The extent of membrane depolarization and phospholipase C (PLC)-dependent production of IP₃ are both directly affected by ACh concentration. In contrast, RyR channels are primarily opened in response to CICR, although ACh-dependent production of cyclic ADP ribose (cADPR) can modulate CICR (39–42). Thus, the effect of increasing ACh concentration on both IP₃R and RyR channel-mediated Ca²⁺ influx may underlie the accelerating [Ca²⁺]_cyt response via CICR.

In the present study, global elevation of [Ca²⁺]_cyt was measured, but this global response reflects the spatial-temporal integration of propagating localized [Ca²⁺]_cyt oscillations (1–3). The amplitude of localized [Ca²⁺]_cyt oscillations is not affected by increasing ACh concentration. Instead, both the frequency and propagation velocity of [Ca²⁺]_cyt oscillations increase with increasing ACh concentration. As a result, the global [Ca²⁺]_cyt response of ASM cells increases with ACh concentration. Importantly, the time course of this spatial/temporal summation of [Ca²⁺]_cyt oscillations within ASM cells is on the order of 500–800 ms. When the interval of pulsed caged release of Ca²⁺ within ASM is reduced to ∼1 s, the resulting force responses are fused (1–3).

Rate of rise of force response.

The rate of rise of force generated in response to ACh stimulation depends on a combination of several factors including [Ca²⁺]_cyt-induced rMLC₂₀ phosphorylation, cross-bridge recruitment, load-dependent cross-bridge cycling (and ATP hydrolysis), the addition of de novo contractile units due to actin and myosin polymerization, and the tethering of filamentous actin to the cortical cytoskeleton for force translation to the extracellular matrix (ECM) (Fig. 1) (2, 13, 15, 43–50). As mentioned, the rate of rise of [Ca²⁺]_cyt induced by ACh depends on ACh concentration. However, the rate of rise of ACh-induced force is significantly slower than the dynamic response of [Ca²⁺]_cyt, suggesting that the dynamic response of force generation in ASM is not entirely dependent on [Ca²⁺]_cyt-induced rMLC₂₀ phosphorylation and cross-bridge recruitment. In the canonical MLCK-mediated rMLC₂₀ phosphorylation signaling cascade, elevated [Ca²⁺]_cyt binds to CaM, which activates MLCK, leading to phosphorylation of rMLC₂₀ and cross-bridge recruitment (Fig. 1) (2, 10–12). The extent of rMLC₂₀ phosphorylation is also regulated by dephosphorylation of rMLC₂₀ via MLC phosphatase (MLCP), which is considered [Ca²⁺]_cyt independent. As a result, the phosphorylation of rMLC₂₀ reflects the balance between MLCK and MLCP activities, with Ca²⁺-dependent and -independent mechanisms. In this signaling cascade, the major contributor to the temporal delay in force generation in ASM is the mobilization of intracellular CaM, whereas phosphorylation of rMLC₂₀ and cross-bridge recruitment are much faster (2). The total time course for the rMLC₂₀ signaling pathway in ASM is ∼900 ms, and with increasing rMLC₂₀ phosphorylation, more cross bridges are recruited, thereby increasing force (Fig. 1). However, the rate of rise of ACh-induced force observed in the present study indicated that addition of contractile units via actin/myosin polymerization and the tethering of filamentous actin to the cortical cytoskeleton for force translation are major determinants.

Previously, it was suggested that Ca²⁺ sensitivity in ASM depends on the extent of rMLC₂₀ phosphorylation, primarily through rho kinase (ROCK)-dependent inhibition of MLC phosphatase activity resulting in increased net phosphorylation of rMLC₂₀ (6, 9, 10, 21, 22, 31, 32, 51–53). However, in the present study and in previous studies, we and others found that the extent of rMLC₂₀ phosphorylation induced by ACh stimulation does not change with time, and thus, it does not reflect dynamic changes in the relationship between [Ca²⁺]_cyt and force generation (4, 15, 22, 23, 30, 54, 55). Moreover, we found that the extent of rMLC₂₀ phosphorylation was not dependent on ACh concentration. Thus, [Ca²⁺]_cyt-dependent phosphorylation of rMLC₂₀ is necessary for cross-bridge recruitment and initiation of a force response but does not regulate the time course or amplitude of the force response. Instead, the time course and amplitude of the force response are likely due to the number of contractile units that can translate force through the cortical cytoskeleton of ASM cells to the ECM. Using the phosphatase inhibitor calyculin A, we found that the extent of rMLC₂₀ phosphorylation induced by ACh stimulation was increased after 30 s. Maximum force response was also increased at 30 s, whereas maximum [Ca²⁺]_cyt response was unchanged. However, calyculin A had no effect on the temporal delay from the onset of [Ca²⁺]_cyt to the onset of force responses. The integral of the phase-loop plot of ACh-induced [Ca²⁺]_cyt and force response and the relaxation rate were also unaffected by calyculin A. Moreover, we found that cyto-D, an inhibitor of actin polymerization, significantly decreased maximum force response to ACh stimulation but had no effect on maximum [Ca²⁺]_cyt response or rMLC phosphorylation as previously reported (13, 54). However, the integral of the phase-loop plot of ACh-induced [Ca²⁺]_cyt and force response was significantly decreased with cyto-D treatment. These results further indicate that ACh-dependent rMLC₂₀ phosphorylation does not underlie the dynamic properties of the [Ca²⁺]_cyt and force responses in ASM.

In addition, Gunst et al. (43) reported that ACh-induced ASM force is regulated by RhoA-mediated actin polymerization rather than rMLC₂₀ phosphorylation (14, 15). In ASM cells, agonist-induced RhoA activation enhances the assembly of the adhesome within the cortical cytoskeleton at the cell membrane (30). In this case, the extent of actin polymerization and tethering to the cortical cytoskeleton increases with time and ACh concentration (23).

Temporal Delay between the Onset of ACh-Induced [Ca²⁺]_cyt and Force Responses

The ACh-induced elevation of [Ca²⁺]_cyt preceded the force response in ASM, but this temporal delay did not depend on ACh concentration. In a previous study, we attributed the temporal delay between the onset of [Ca²⁺]_cyt and the force responses to the mobilization of CaM, activation of MLCK phosphorylation of rMLC₂₀, and recruitment of cross bridges (2). The results of the present study also demonstrated that increasing ACh concentration had no significant effect on the temporal delay between the onset of [Ca²⁺]_cyt and the force responses (Fig. 3B) or on the extent of rMLC₂₀ phosphorylation (Fig. 6C). These results suggest that temporal delays due to the signaling cascade that regulate rMLC₂₀ phosphorylation and cross-bridge recruitment are independent of ACh concentration.

Integral of Phase-Loop Plots of [Ca²⁺]_cyt and Force Responses to ACh Stimulation

Phase-loop plots have been previously used to determine Ca²⁺ sensitivity in cardiomyocytes (17, 56) and cardiac muscle contraction (25) based on the shift of the relaxing phase at the EC₅₀ for [Ca²⁺]_cyt in the plot (19). However, in contrast to cardiomyocytes, the results of the present study suggest that Ca²⁺ sensitivity of ASM force is better reflected by the integral of the rising phase of the phase-loop plot of ACh-induced [Ca²⁺]_cyt and force responses rather than the relaxation rate or the shift in the phase-loop plot at the EC₅₀ for [Ca²⁺]_cyt. The integral of the rising phase of the ACh-induced [Ca²⁺]_cyt and force phase-loop plot significantly increased with increasing ACh concentration. However, the rates of relaxation of the phase-loop plot of [Ca²⁺]_cyt and force remained unchanged with increasing ACh concentration.

We conclude that dynamic properties of E-C coupling in ASM provide valuable information on how agonists or pharmacological/molecular manipulations affect Ca²⁺ sensitivity of ASM force generation. Our results indicate that the dynamic acceleration of ACh-induced force is not dependent on rMLC₂₀ phosphorylation but reflects the recruitment of additional contractile units and/or force translation through the cortical cytoskeleton to the ECM.

GRANTS

This work was supported by NIH National Heart, Lung, and Blood Institute Grant R01HL157984 to G.C.S.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

P.F.D. and G.C.S. conceived and designed research; Y.H. and G.M.A. performed experiments; Y.H., P.F.D., G.M.A., and G.C.S. analyzed data; Y.H., P.F.D., G.M.A., and G.C.S. interpreted results of experiments; Y.H., P.F.D., and G.C.S. prepared figures; Y.H., P.F.D., G.M.A., and G.C.S. drafted manuscript; Y.H., P.F.D., G.M.A., and G.C.S. edited and revised manuscript; Y.H., P.F.D., G.M.A., and G.C.S. approved final version of manuscript.