Relationship between force and regulatory myosin light chain phosphorylation in airway smooth muscle

Tetsuya Kai1,2, Hayashi Yoshimura1, Keith A. Jones1, and David O. Warner1,3

Departments of 1 Anesthesiology and 3 Physiology and Biophysics, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905; and 2 Department of Anesthesiology and Critical Care Medicine, Faculty of Medicine, Kyushu University, Fukuoka 812-8582, Japan


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ABSTRACT
INTRODUCTION
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We tested the hypothesis that increases in force at a given cytosolic Ca2+ concentration (i.e., Ca2+ sensitization) produced by muscarinic stimulation of canine tracheal smooth muscle (CTSM) are produced in part by mechanisms independent of changes in regulatory myosin light chain (rMLC) phosphorylation. This was accomplished by comparing the relationship between rMLC phosphorylation and force in alpha -toxin-permeabilized CTSM in the absence and presence of acetylcholine (ACh). Forces were normalized to the contraction induced by 10 µM Ca2+ in each strip, and rMLC phosphorylation is expressed as a percentage of total rMLC. ACh (100 µM) plus GTP (1 µM) significantly shifted the Ca2+-force relationship curve to the left (EC50: 0.39 ± 0.06 to 0.078 ± 0.006 µM Ca2+) and significantly increased the maximum force (104.4 ± 4.8 to 120.2 ± 2.8%; n = 6 observations). The Ca2+-rMLC phosphorylation relationship curve was also shifted to the left (EC50: 1.26 ± 0.57 to 0.13 ± 0.04 µM Ca2+) and upward (maximum rMLC phosphorylation: 70.9 ± 7.9 to 88.5 ± 5.1%; n = 6 observations). The relationships between rMLC phosphorylation and force constructed from mean values at corresponding Ca2+ concentrations were not different in the presence and absence of ACh. We find no evidence that muscarinic stimulation increases Ca2+ sensitivity in CTSM by mechanisms other than increases in rMLC phosphorylation.

caldesmon; calponin; cytoskeleton


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INTRODUCTION
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CONTRACTION OF SMOOTH MUSCLE is associated with increases in cytosolic Ca2+ concentration ([Ca2+]i), which binds to calmodulin and increases myosin light chain kinase activity, regulatory myosin light chain (rMLC) phosphorylation, and actomyosin cross-bridge cycling rate (22, 24). The relationship between [Ca2+]i and rMLC phosphorylation is further regulated by agonist-induced inhibition of smooth muscle protein phosphatase activity via G protein-coupled mechanisms, which produces a leftward shift of the Ca2+-force relationship (i.e., Ca2+ sensitization) (24). Several studies have suggested that mechanisms other than phosphorylation of rMLC may also regulate force in smooth muscle (17). Two proposed mechanisms include 1) those involving thin filament-associated proteins such as caldesmon and calponin that may control actomyosin interactions (5, 39) and 2) reorganization of the cytoskeleton to optimize transduction of force from contractile elements to the environment (14, 30, 38). Evidence for the former mechanism in vascular smooth muscle is now fairly compelling. However, the functional importance of such mechanisms in the airway remains unclear.

We tested the hypothesis that the Ca2+ sensitization produced by muscarinic stimulation of canine tracheal smooth muscle (CTSM) is produced in part by other mechanisms in addition to changes in rMLC phosphorylation. We reasoned that if these mechanisms were activated by muscarinic stimulation, the relationship between rMLC phosphorylation and the force generated by changing [Ca2+]i would differ in the absence and presence of acetylcholine (ACh). An alpha -toxin-permeabilized CTSM preparation was developed and used to test this hypothesis.


    MATERIALS AND METHODS
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Tissue preparation. After we received approval from the Institutional Animal Care and Use Committee, mongrel dogs (20-25 kg) of either sex were anesthetized with an intravenous injection of pentobarbital sodium (50 mg/kg) and killed by exsanguination. The tracheae were excised and immersed in chilled physiological salt solution (PSS) of the following composition (in mM): 110.5 NaCl, 25.7 NaHCO3, 5.6 dextrose, 3.4 KCl, 2.4 CaCl2, 1.2 KH2PO4, and 0.8 MgSO4. The adventitia and mucosa were removed after the visceral side of cartilage was cut, then connective tissues were carefully removed under microscopic observation and made into muscle strips of 0.1-0.2 mm width, approx 1 cm length, and 0.2-0.4 mg wet weight. All tissue preparation was done in the PSS aerated with 94% O2 and 6% CO2.

Isometric force measurements. Muscle strips were mounted in 0.1-ml quartz cuvettes and continuously superfused at 1.2 ml/min with PSS (37°C) aerated with 94% O2 and 6% CO2. One end of each strip was anchored with stainless steel microforceps to a metal rod fixed to a micromanipulator, and the other end was attached with stainless steel microforceps to a calibrated force transducer (model KG4, Scientific Instruments, Heidelberg, Germany). The initial gap between microforceps (i.e., initial muscle length) was set at 5 mm. During a 3-h equilibration period, the length of each muscle strip was increased after repeated isometric contractions (of 1-2 min duration) induced by 1 µM ACh until isometric force was maximal (optimal length). Each muscle strip was maintained at this optimal length for the remainder of the experiment. These tissues produced maximal isometric forces of 1-3 mN when stimulated with 1 µM ACh. Once optimal length had been set, subsequent experimental protocols were performed at room temperature (25°C) to minimize deterioration of the experimental preparation.

Permeabilization procedure. The muscle strips were permeabilized with Staphylococcus aureus alpha -toxin (24, 33). alpha -Toxin creates pores in the plasma membrane of the smooth muscle cell, thus allowing substances of small molecular weight such as Ca2+ to freely diffuse across the cell membrane. Accordingly, [Ca2+]i can be manipulated and controlled by changing the concentration of Ca2+ ([Ca2+]) in the solution bathing the smooth muscle strip. The larger cellular proteins necessary for contraction are preserved. Additionally, the membrane receptor-coupled mechanisms that modulate Ca2+ sensitivity remain intact and can be activated.

Muscle strips were treated for 20 min with 2,500 U/ml of alpha -toxin in the relaxing solution. The relaxing solution was prepared with the use of the algorithm of Fabiato and Fabiato (6): 7.5 mM MgATP, 4 mM EGTA, 20 mM imidazole, 1 mM dithiothreitol (DTT), 1 mM free Mg2+, 1 nM free Ca2+, 10 mM creatine phosphate, and 0.1 mg/ml of creatine phosphokinase. Ionic strength was kept constant at 200 mM by adjusting the concentration of potassium acetate. The pH was adjusted to 7.0 at 25°C with KOH. The Ca2+ ionophore A-23187 (10 µM) was added to the relaxing solution and all subsequent experimental solutions to deplete the sarcoplasmic reticulum Ca2+ stores (26). Solutions of varying free [Ca2+] values were also prepared with the use of the Fabiato algorithm (6) with the components other than Ca2+ being the same as described above.

Based on preliminary studies, the following procedure was devised to provide stable, reproducible contractions. After the permeabilization procedure, the strips were washed with relaxing solution without alpha -toxin for 60 min. Strips were then stimulated with 10 µM Ca2+ for 5 min to obtain a maximum Ca2+ contraction. Then strips were superfused with relaxing solution containing 5 mM inorganic phosphate for 5 min to speed relaxation by accelerating the rate of cross-bridge detachment (18). Thereafter, the strips were superfused with relaxing solution for 5 min to remove inorganic phosphate. This sequence was repeated three times, after which responses to 10 µM Ca2+ were reproducible (preliminary data not shown); all subsequent force measurements were normalized to the third contraction. During experimental protocols, whenever ACh was applied to stimulate muscarinic receptors, 1 µM GTP was also included to ensure adequate functioning of guanine nucleotide-binding proteins. In preliminary studies, we confirmed that 1 µM GTP had no significant effect on the contraction induced by free Ca2+ alone, whereas it potentiated ACh-induced Ca2+ sensitization (data not shown).

Fura 2 fluorescence measurements. The fluorescence measurements of the Ca2+ indicator dye fura 2 were performed to document that [Ca2+]i is constant in alpha -toxin-permeabilized preparation during activation of the membrane receptor-coupled mechanisms that modulate myofilament Ca2+ sensitivity. Muscle strips set at optimal length were circulatingly superfused with aerated (94% O2 and 6% CO2) PSS containing 5 µM of fura 2-AM) for 3 h at room temperature. Fura 2-AM was dissolved in DMSO, resulting in a final concentration of 0.1%. After washout of fura 2, strips were then permeabilized with alpha -toxin as described. The nonfluorescent analog of A-23187, 4-bromo A-23187, was used to deplete intracellular Ca2+ stores. Fura 2 fluorescence intensity was measured by a photometric system (model ph2, Scientific Instruments) that measures optical and mechanical parameters of isolated tissue simultaneously (15). Details of our system have been described elsewhere (21). The fluorescence intensities of 500-nm emission light due to excitation at 340-nm (F340) and 380-nm (F380) wavelengths were measured, and the ratio of F340 to F380 (F340/F380) was used as an index of [Ca2+]i.

rMLC phosphorylation measurements. Samples for rMLC phosphorylation measurements were prepared separately according to the same procedures as those for force measurements, but they were incubated in wells at approximately optimal length instead of being superfused. Preliminary experiments revealed that muscle length does not affect rMLC phosphorylation under these conditions. After the experimental protocols, muscle strips were flash-frozen with dry ice-cooled acetone containing 10% (wt/vol) trichloroacetic acid (TCA) and 10 mM DTT (16). Strips were then allowed to warm to room temperature in the same solution. After TCA was washed out with acetone-DTT, the strips were then allowed to dry. Dry weight of the strips was 0.07-0.13 mg. rMLC was extracted as described by Gunst et al. (12), and phosphorylation was determined by glycerol-urea gel electrophoresis followed by Western blotting as previously described (1). Unphosphorylated and phosphorylated bands of rMLC were visualized by phosphorimaging analysis (PhosphorImager, Molecular Dynamics, Sunnyvale, CA), and fractional phosphorylation was calculated as the density ratio of the sum of mono- and diphosphorylated rMLC to total rMLC with ImageQuant software (Molecular Dynamics).

Materials. The polyclonal affinity-purified rabbit anti-rMLC antibody for Western blotting was the generous gift of Dr. Susan J. Gunst (Departments of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis, IN). 125I-labeled protein A was purchased from NEN Life Science Products (Boston, MA). alpha -Toxin was purchased from Calbiochem (La Jolla, CA). ATP disodium salt was purchased from Research Organics (Cleveland, OH). Fura 2-AM and 4-bromo A-23187 were purchased from Molecular Probes (Eugene, OR). TCA was purchased from Fisher Scientific (Fair Lawn, NJ). Other drugs and chemicals used for electrophoresis were purchased from Bio-Rad (Hercules, CA). All other drugs and chemicals were purchased from Sigma (St. Louis, MO). A-23187 and 4-bromo A-23187 were dissolved in DMSO (0.05% final concentration). All other drugs and chemicals were prepared in distilled filtered water.

Statistical analysis. Data are expressed as means ± SD; n is the number of observations. Forces are expressed as percentage of the force induced by 10 µM Ca2+, which was determined in each individual strip before the experimental protocol. Time-dependent changes in rMLC phosphorylation were assessed by paired t-test. Ca2+ concentration-response curves were compared by nonlinear regression analysis with the method of Meddings et al. (28) with the four-parameter logistic model described by DeLean et al. (3). P < 0.05 was considered significant.


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Characterization of permeabilized preparation. Interpretation of the data obtained with the permeabilized smooth muscle preparation required that at least two conditions were satisfied: 1) [Ca2+]i remained constant during the receptor stimulation, and 2) the mechanisms that modulate Ca2+ sensitivity were not destroyed by the permeabilization procedure. Therefore, it was first necessary to confirm that these two conditions were satisfied in our alpha -toxin-permeabilized CTSM preparation.

Experiments were conducted to corroborate that [Ca2+]i is constant during muscarinic receptor stimulation as measured by fura 2, whereas force is enhanced. Muscle strips were loaded with fura 2 and then permeabilized with alpha -toxin. After control contractions with 10 µM Ca2+, strips were superfused with solutions containing 0.18 µM free Ca2+ and then with solutions containing 0.18 µM free Ca2+ and 10 µM ACh plus 1 µM GTP.

Increasing the free [Ca2+] in the perfusate from 1 nM to 0.18 µM caused sustained increases in F340/F380 and force (Fig. 1). The subsequent addition of 10 µM ACh plus 1 µM GTP to the perfusate caused an increase in force but no change in F340/F380. Similar observations were made in tissues obtained from three other dogs. These observations demonstrate that, in this preparation, the cell membrane is permeable to Ca2+, [Ca2+]i is constant during receptor stimulation with ACh, and the cellular constituents and biochemical mechanisms necessary for contraction are maintained, including Ca2+ sensitizing mechanisms activated by ACh.


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Fig. 1.   Representative tracing showing time course for changes in fura 2 fluorescence ratio [340- to 380-nm fluorescence (F340/F380); top] and isometric force (bottom) in an alpha -toxin-permeabilized canine tracheal smooth muscle (CTSM) strip. After a control response to 10 µM Ca2+, the strip was first activated by 0.18 µM Ca2+ and then by 0.18 µM Ca2+ and 10 µM ACh plus 1 µM GTP. Note that F340/F380 did not change with Ca2+ sensitization produced by ACh.

Time course of rMLC phosphorylation. To construct the force-rMLC phosphorylation relationship, we first investigated the time course of rMLC phosphorylation. Two sets of six permeabilized muscle strips were prepared from each of five dogs. Five muscle strips from one set were stimulated with 0.32 µM free Ca2+ alone, and five muscle strips from the second set were stimulated with 0.32 µM free Ca2+ in the presence of 100 µM ACh plus 1 µM GTP. Then the five muscle strips from each set were flash-frozen for rMLC phosphorylation measurements after stimulation for 1, 2.5, 5, 10, or 15 min. The sixth muscle strip from each set was not stimulated and was flash-frozen in relaxing solution for baseline measurements. This [Ca2+] produces approximately half of the maximum contraction induced by Ca2+ alone, and this ACh concentration produces maximal Ca2+ sensitization as determined by preliminary studies (data not shown).

Stimulation with 0.32 µM free Ca2+ alone or 0.32 µM free Ca2+ in the presence of 100 µM ACh plus 1 µM GTP significantly increased rMLC phosphorylation, which reached a plateau within 5 min and was sustained until at least 15 min (Fig. 2). rMLC phosphorylation values were significantly increased by ACh plus GTP.


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Fig. 2.   Time course of changes in regulatory myosin light chain phosphorylation (rMLC-P) induced by 0.32 µM free Ca2+ alone (open circle ) and by 0.32 µM free Ca2+ in the presence of 100 µM ACh plus 1 µM GTP () in alpha -toxin-permeabilized CTSM strips. Values are means ± SD; n = 5 observations.

Ca2+-force relationship. A pair of permeabilized strips were prepared for force measurement. After control contractions induced by 10 µM free Ca2+, strips were superfused with solutions in which [Ca2+] was incrementally increased from 1 nM to 100 µM at 5-min intervals. The solution superfusing one strip of the pair contained 100 µM ACh plus 1 µM GTP, and the other one did not contain any of these compounds (Ca2+ alone). Thus free Ca2+-force response curves were constructed in both the absence (Ca2+ alone) and presence of 100 µM ACh plus 1 µM GTP (Fig. 3).


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Fig. 3.   Free Ca2+ concentration-force relationship curve of alpha -toxin-permeabilized CTSM in the absence (Ca2+ alone; open circle ) and presence of 100 µM ACh plus 1 µM GTP (). Force is expressed as a percentage of the control response to 10 µM Ca2+ in each individual strip. Values are means ± SD; n = 6 observations.

ACh plus GTP had no effect on baseline force at 1 nM free Ca2+. ACh plus GTP caused a leftward and upward shift of the Ca2+-force relationship curve, significantly decreasing the EC50 for free Ca2+ from 0.39 ± 0.06 to 0.078 ± 0.006 µM and significantly increasing the maximum force from 104.4 ± 4.8 to 120.2 ± 2.8% (n = 6).

Ca2+-rMLC phosphorylation relationship. Two sets of eight permeabilized muscle strips were prepared in wells. After a 60-min equilibration period in relaxing solution and repetitive stimulation with 10 µM Ca2+ (according to the protocol followed for force measurements), strips of each set were immersed in solutions of varying free [Ca2+] values for 10 min, then flash-frozen for rMLC phosphorylation measurements. The solutions for one set also contained 100 µM ACh plus 1 µM GTP. Thus free Ca2+-rMLC phosphorylation relationships were constructed in both the absence (Ca2+ alone) and presence of 100 µM ACh plus 1 µM GTP (Fig. 4).


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Fig. 4.   Free Ca2+ concentration-rMLC-P relationship curve of the alpha -toxin-permeabilized CTSM in the absence (Ca2+ alone; open circle ) and presence of 100 µM ACh plus 1 µM GTP (). Values are means ± SD; n = 6 observations.

ACh plus GTP had no effect on baseline rMLC phosphorylation at 1 nM free Ca2+. ACh plus GTP caused a leftward and upward shift of the free Ca2+-rMLC phosphorylation relationship curve, significantly decreasing the EC50 for free Ca2+ from 1.26 ± 0.57 to 0.13 ± 0.04 µM and significantly increasing the maximum rMLC phosphorylation from 70.9 ± 7.9 to 88.5 ± 5.1% (n = 6).

rMLC phosphorylation-force relationship. The relationships between rMLC phosphorylation and force were then constructed from their mean values at corresponding free [Ca2+] values (Fig. 5). The rMLC phosphorylation-force relationships in the absence and presence of ACh plus GTP were similar.


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Fig. 5.   rMLC-P-force relationship generated by changing the Ca2+ concentration of the alpha -toxin-permeabilized CTSM in the absence (Ca2+ alone; open circle ) and presence of 100 µM ACh plus 1 µM GTP (). Values are means ± SD.


    DISCUSSION
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ABSTRACT
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In alpha -toxin-permeabilized CTSM, ACh caused a leftward and upward shift in both the Ca2+-force and Ca2+-rMLC phosphorylation relationships, decreasing the EC50 for Ca2+ and increasing the maximum response. The rMLC phosphorylation-force relationships in the presence and absence of ACh were nearly identical, demonstrating that the increase in force produced by ACh was according to that predicted by the rMLC phosphorylation-force relationship generated by Ca2+ stimulation alone.

It is well established that receptor agonists such as ACh increase Ca2+ sensitivity in smooth muscle by inhibiting smooth muscle protein phosphatases via pathways involving one or more G proteins (for recent review, see Ref. 37). The resulting increase in rMLC phosphorylation increases actomyosin ATPase activity and force. However, several studies (8, 31, 36, 39) in a variety of smooth muscle types have shown that the relationship between rMLC phosphorylation, ATPase activity, and force can change, implying that other mechanisms may also regulate force. Specifically in airway smooth muscle, muscarinic stimulation of intact CTSM exposed to varying extracellular [Ca2+] values appears to affect the relationship between rMLC phosphorylation and force (7, 8). For example, Gerthoffer et al. (9) found that CTSM developed less force for a given level of rMLC phosphorylation when muscles were activated with carbachol compared with KCl-induced depolarization. Dissociation between actomyosin ATPase activity (or maximal shortening velocity) and rMLC phosphorylation has been observed in both intact (13) and Triton X-skinned (19) CTSM. For example, Jones et al. (19) observed that increases in force and rMLC phosphorylation produced by Ca2+ stimulation of Triton X-skinned CTSM are sustained (similar to the present findings in an alpha -toxin preparation), whereas actomyosin ATPase activity decreases with time. Similar findings have been observed in a variety of other smooth muscles (27).

At least two classes of mechanism have been proposed to explain regulation of force in smooth muscle independent of changes in rMLC phosphorylation. The first postulates an independent regulatory system that is responsible for force maintenance after initial rapid force development (dependent on rMLC phosphorylation) (5, 39). Current candidates include the thin filament-associated proteins caldesmon and calponin, based on evidence obtained in several smooth muscle types. Although evidence for their role in contraction of vascular smooth muscle under some circumstances is now fairly well established (5, 17, 39), evidence for their functional role in airway smooth muscle is limited. Both proteins are phosphorylated in response to muscarinic stimulation of intact CTSM (10, 35). As in studies of other types of smooth muscle, phosphorylation of these proteins releases inhibition of actin filament motility assays, indicating a permissive role on actomyosin ATPase activity (35). In support of a functional role in situ, addition of activated mitogen-activated protein (MAP) kinase to Triton X-skinned CTSM potentiates Ca2+-induced contraction, suggesting that a substrate for MAP kinase (such as caldesmon) may regulate force (10). Others (4) suggest that protein tyrosine phosphorylation is important for regulating Ca2+ sensitization of force in smooth muscle.

A second proposal concerns the possibility that the actin cytoskeleton may remodel in response to contractile stimuli to optimize transduction of force from contractile elements to the environment. This idea is supported by the peculiar mechanical plasticity of airway smooth muscle explored in the work of Gunst et al. (14) and Mehta et al. (30). For example, using changes in length to perturb the cytoskeleton, they (30) demonstrated that shortening of actively contracted airway smooth muscle depresses contractility without changing [Ca2+]i or rMLC phosphorylation. They speculated that this acute change in Ca2+ sensitivity may be caused by the inability of the cytoskeleton to adjust to changes in cell length during activation. In further studies, Pavalko et al. (34) also found that several different proteins involved with the actin cytoskeleton (such as paxillin and focal adhesion kinase) are tyrosine phosphorylated during airway smooth muscle contraction. Tyrosine phosphorylation of paxillin is stimulated in CTSM by muscarinic stimulation independently of changes in [Ca2+]i (29). Thus force may be increased for a given rMLC phosphorylation with activation of these systems by muscarinic stimulation. Further support for this concept in airway smooth muscle comes from the recent observation (20) that inhibition of actin turnover increases the tension cost of developed force (the ATPase activity needed to maintain force) in CTSM and interferes with force maintenance.

Given these observations, we speculated that at least part of the increased Ca2+ sensitivity produced by membrane receptor activation might be mediated by these putative mechanisms. After developing an alpha -toxin-permeabilized CTSM preparation, we found that maximal ACh stimulation did not affect the rMLC phosphorylation-force relationship measured in the absence of ACh stimulation by changing [Ca2+]. If any mechanisms in addition to rMLC phosphorylation contributed to receptor-stimulated increases in Ca2+ sensitivity, the relationship should have shifted to the left. At the "midrange" of rMLC phosphorylation measurements, where such effects should be visible, the relationship rather tended to be right shifted (Fig. 5). Thus increases in the Ca2+ sensitivity produced by acute muscarinic stimulation can be accounted for entirely by increases in rMLC phosphorylation. This result is consistent with that of Kitazawa et al. (25) in permeabilized vascular smooth muscle. It is possible that the permeabilization procedure itself may have altered or destroyed rMLC phosphorylation-independent mechanisms. We regard this as unlikely because evidence for them is found even in Triton X-skinned CTSM (10), a more stringent permeabilization procedure. However, we cannot exclude the possibility that the membrane pores produced by alpha -toxin might interfere with these mechanisms.

The maximum rMLC phosphorylation values observed in the present study exceed those previously reported by our laboratory and others in beta -escin- or Triton X-permeabilized CTSM (19, 21) or in intact CTSM during maximal muscarinic stimulation (~60%) (2). To our knowledge, there are no prior reports of rMLC phosphorylation values in alpha -toxin-permeabilized airway smooth muscle, although our values approximate those found in other types of smooth muscle permeabilized with alpha -toxin (23). Compared with other permeabilization methods, alpha -toxin, which assembles on cell membranes to form transmembrane heptameric channels (11), may better preserve cellular integrity. For example, alpha -toxin-permeabilized CTSM yields reproducible and stable responses over several hours, behavior not observed in beta -escin- or Triton X-treated muscles. Regarding comparisons of rMLC phosphorylation values with maximally stimulated intact CTSM, we note that 1) [Ca2+]i in intact muscle stimulated with maximal ACh (at most 1 µM) is less than the free [Ca2+] in maximally stimulated permeabilized muscle (10 µM) so that the conditions are not strictly comparable, and 2) these permeabilized experiments have been conducted at room temperature. In ongoing work, we also performed experiments at 37°C in the alpha -toxin preparation and found that maximum rMLC phosphorylation values are ~60%, similar to those found in intact muscle. This finding may be anticipated from the relative temperature dependence of the activities of myosin light chain kinase and smooth muscle phosphatases (32). It is not known whether room temperature conditions would modulate the functional significance of any rMLC phosphorylation-independent mechanisms, although most prior work providing evidence for such mechanisms was also conducted at room temperature.

The lack of evidence for mechanisms in addition to rMLC phosphorylation in our study implies either that these mechanisms are not functionally significant in CTSM under the conditions of study (maximal muscarinic stimulation) or that they are present but are regulated by receptor-coupled mechanisms that are secondary to changes in [Ca2+]i; i.e., they do not affect agonist-induced Ca2+ sensitization per se. Because we used the rMLC phosphorylation-force relationship constructed by increasing [Ca2+] as a basis for comparison, we would not detect their existence if they were regulated only via changes in [Ca2+] alone. Indeed, Tang et al. (38) found that paxillin was tyrosine phosphorylated during contraction of CTSM produced by KCl (i.e., by increases in [Ca2+]i alone without receptor stimulation). However, paxillin and focal adhesion kinase were also tyrosine phosphorylated by muscarinic stimulation in the absence of intracellular Ca2+, suggesting that cytoskeletal proteins may be regulated independently of changes in [Ca2+]i by a process sensitive to changes in muscle length. Studies of MAP kinase activation, caldesmon phosphorylation, and calponin phosphorylation in smooth muscle have not been performed under constant [Ca2+]i conditions. We also cannot exclude that other conditions, such as stimulation with different agonists or chronic (vs. acute) stimulation would reveal evidence for rMLC phosphorylation-independent mechanisms.

In summary, we find no evidence that muscarinic stimulation increases Ca2+ sensitivity in CTSM by mechanisms other than increases in rMLC phosphorylation.


    ACKNOWLEDGEMENTS

This study was supported in part by Grants HL-45532 and HL-54757 from the National Heart, Lung, and Blood Institute and grants from the Mayo Foundation.


    FOOTNOTES

Address for reprint requests and other correspondence: D. O. Warner, Anesthesia Research, Mayo Clinic and Foundation, 200 First St. SW, Rochester, MN 55905 (E-mail: warner.david{at}mayo.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. §1734 solely to indicate this fact.

Received 1 November 1999; accepted in final form 15 February 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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