Calcium sensitization produced by G protein activation in airway smooth muscle

Hayashi Yoshimura, Keith A. Jones, William J. Perkins, Tetsuya Kai, and David O. Warner

Department of Anesthesiology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We determined whether activation of G proteins can affect the force developed for a given intracellular Ca2+ concentration ([Ca2+]; i.e., the Ca2+ sensitivity) by mechanisms in addition to changes in regulatory myosin light chain (rMLC) phosphorylation. Responses in alpha -toxin-permeabilized canine tracheal smooth muscle were determined with Ca2+ alone or in the presence of ACh, endothelin-1 (ET-1), or aluminum fluoride (AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>; acute or 1-h exposure). Acute exposure to each compound increased Ca2+ sensitivity without changing the response to high [Ca2+] (maximal force). However, chronic exposure to AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>, but not to chronic ACh or ET-1, increased maximal force by increasing the force produced for a given rMLC phosphorylation. Studies employing thiophosphorylation of rMLC showed that the increase in force produced by chronic AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> exposure required Ca2+ during activation to be manifest. Unlike the acute response to receptor agonists, which is mediated solely by increases in rMLC phosphorylation, chronic direct activation of G proteins further increases Ca2+ sensitivity in airways by additional mechanisms that are independent of rMLC phosphorylation.

regulatory myosin light chain phosphorylation; aluminum fluoride; remodeling; airway inflammation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

STIMULATION OF SMOOTH MUSCLE by contractile agonists increases cytosolic Ca2+ concentration ([Ca2+]i). Ca2+ binds to calmodulin and increases myosin light chain kinase activity, regulatory myosin light chain (rMLC) phosphorylation, actomyosin cross-bridge cycling rate, and force (27). The relationship between [Ca2+]i and rMLC phosphorylation further depends on agonist-induced inhibition of smooth muscle protein phosphatase (SMPP) activity, which increases the amount of force produced for a given [Ca2+]i (i.e., the Ca2+ sensitivity; see Refs. 28 and 29). Membrane receptors that inhibit SMPP are coupled to these enzymes via a cascade of heterotrimeric and monomeric GTP-binding regulatory proteins (G proteins; see Ref. 40). Several studies have suggested that mechanisms in addition to rMLC phosphorylation may also regulate force in smooth muscle (18). Proposed mechanisms include those thought to regulate actomyosin cross-bridge interaction, such as the actin-associated proteins such as caldesmon and calponin (10, 44), and others such as remodeling of the smooth muscle cytoarchitecture to optimize the transduction of ATPase activity to force (15, 23, 31, 33, 41).

If these additional regulatory mechanisms are functionally important and can be activated by receptor agonists, then the relationship between rMLC phosphorylation and force at various [Ca2+]i levels in the presence and absence of receptor activation should differ. In a recent study, we showed that acute muscarinic stimulation did not alter this relationship in permeabilized canine tracheal smooth muscle (CTSM), providing no evidence for such mechanisms in this preparation (26). Given this result, we questioned whether there are any conditions where Ca2+ sensitivity can be regulated by rMLC-independent mechanisms mediated by G proteins in airway smooth muscle. We speculated that if such mechanisms exist, they may require chronic stimulation to become apparent. For example, any reorganization of the cytoskeleton may take some period of time to occur (23, 31). Such chronic changes may be relevant to clinical states such as asthma, in which CTSM may be chronically exposed to inflammatory mediators and other contractile stimuli (35, 37).

The purpose of the present study was to determine if chronic activation of G proteins in CTSM, produced by either receptor activation or direct stimulation with aluminum fluoride (AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>), could regulate Ca2+ sensitivity independently of changes in rMLC phosphorylation.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental techniques. After Institutional Animal Care and Use Committee approval, we anesthetized mongrel dogs (15-20 kg) of either sex with an intravenous injection of pentobarbital sodium (30 mg/kg) and exsanguinated the dogs. The trachea was 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. Fat, connective tissue, and the epithelium were removed with tissue forceps and scissors under microscopic observation to make muscle strips of 0.1-0.2 mm width, 1 cm length, and 0.2-0.3 mg wet weight.

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

Permeabilization procedure. CTSM strips were permeabilized with 2,500 U/ml Staphylococcus aureus alpha -toxin for 20 min at 25°C in relaxing solution (28, 34). The composition of the relaxing solution was as follows [using the algorithm of Fabiato and Fabiato (11)]: 7.5 mM MgATP, 4 mM EGTA, 20 mM imidazole, 1 mM dithiothreitol (DTT), 1 nM free Ca2+, 10 mM phosphocreatine, and 0.1 mg/ml 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 or HCl. After the permeabilization procedure, strips were washed with relaxing solution without alpha -toxin for 10 min. 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 and to maintain Ca2+ concentration ([Ca2+]). Solutions of varying free [Ca2+] used in the subsequent experiment were also prepared using the algorithm of Fabiato and Fabiato (11). After permeabilization, the strips were contracted repeatedly with 10 µM Ca2+ until reproducible responses were obtained, as previously described (26). For rMLC thiophosphorylation studies, low-Ca2+ rigor solution contained (in mM) 85 K+, 0.01 free Mg2+, 4 EGTA, 20 imidazole, 1 DTT, and 1 nM free Ca2+. High-Ca2+ rigor solutions contained (in mM) 85 K+, 0.1 free Mg2+, 4 EGTA, 20 imidazole, 1 DTT, and 10 µM free Ca2+.

rMLC phosphorylation measurements. Samples for rMLC phosphorylation measurements were prepared separately according to the same procedures as for force measurements but were incubated in wells (without determination of optimal length) instead of being superfused. The strips were pinned at both ends to maintain isometric conditions. After permeabilization, all conditions, including repeated contraction with 10 µM Ca2+, were identical to those present in the strips used to measure isometric force responses. At appropriate times in the experimental protocols, muscle strips were flash-frozen with dry ice-cooled acetone containing 10% (wt/vol) TCA and 10 mM DTT (17). Strips were then allowed to warm to room temperature in the same solution. After TCA was washed out with acetone containing 10 mM DTT, strips were allowed to dry. The dry weight of the strips was 0.07-0.13 mg. rMLC was extracted as described by Gunst et al. (14), and phosphorylation was determined by glycerol-urea gel electrophoresis followed by Western blotting as previously described (5). Unphosphorylated and phosphorylated bands of rMLC were visualized by phosphorimage analysis (Cyclone storage phosphor system; Packard Instrument, Downers Grove, IL), and fractional phosphorylation was calculated as the density ratio of the sum of mono- and diphosphorylated rMLC to total rMLC using OptiQuant software (version 3.0; Packard Instrument).

Materials. The polyclonal affinity-purified rabbit anti-20-kDa rMLC antibody was a generous gift from Dr. Susan J. Gunst (Dept. of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis, IN). Synthetic endothelin-1 (ET-1) was obtained from Phoenix Pharmaceuticals (Mountain View, CA). All other drugs and chemicals were purchased from Sigma Chemical (St. Louis, MO). A-23187 was dissolved in dimethyl sulfoxide (DMSO); in all experimental solutions, the final DMSO concentration did not exceed 0.1%. At this concentration, DMSO had no effect on isometric force or rMLC phosphorylation induced by free Ca2+ alone (data not shown). All other drugs and chemicals were prepared in nanopure filtered water.

Statistical analysis. Data are expressed as means ± SD; n is the number of dogs. Isometric force is expressed as a percentage of the maximal response to 10 µM Ca2+. Comparisons between means were performed with paired t-tests as appropriate. Parameters for concentration-response curves were calculated using nonlinear regression fit to a three-parameter Hill equation (Sigma Stat; Jandel Scientific, San Rafael, CA), and parameter coefficients were compared using paired t-tests. In all cases, a P value <0.05 was taken as significant.

We compared the relationship between rMLC phosphorylation and force in the presence and absence of chronic AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> stimulation. Because data for these parameters were collected from two separate experiments using separate animals (i.e., were unpaired), the following procedure was employed. A data set was created that contained data for all possible pairings of animals from the two experiments (i.e., each animal from the first experiment was matched with each animal from the second experiment). Fifty random bootstrap samples of size n = 6 were drawn with replacement from the data set of all possible pairings. For each sample, a linear regression analysis was performed for each treatment group using force and rMLC phosphorylation as dependent and independent variables, respectively. A paired t-test was then used to compare the fitted parameters of the regression lines between treatment groups to determine if the relationship between rMLC phosphorylation and force differed.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of acute application of ACh, ET-1, or AlF<UP><SUB>4</SUB><SUP><UP>−</UP></SUP></UP> on Ca2+ sensitivity. Four alpha -toxin-permeabilized CTSM strips were stimulated with increasing concentrations of free Ca2+ (1 nM to 100 µM). In three strips, 100 µM ACh or 100 nM ET-1 or AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> (2 mM NaF + 20 µM AlCl3) was added 5 min before the first increment in [Ca2+] (acute exposure). These concentrations of ACh, ET-1, and AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> were chosen as producing maximal responses (preliminary data not shown). During all experimental protocols, when ACh, ET-1, or AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> was applied, 1 µM GTP was applied concomitantly. In preliminary studies, we confirmed that 1 µM GTP had no significant effect on the contraction induced by free Ca2+ alone (data not shown). Acute exposure to ACh, ET-1, or AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> markedly increased Ca2+ sensitivity (Fig. 1), decreasing the [Ca2+] required to produce half-maximal force (EC50; Fig. 1A and Table 1). In contrast, maximal isometric force developed to high [Ca2+] was not affected significantly. Neither the maximal force nor the EC50 values differed among stimulation with ACh, ET-1, or AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> (Table 1). Thus acute exposure to ACh, ET-1, or AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> produced similar degrees of Ca2+ sensitization (as shown by decreases in EC50) without increasing the maximal force developed in response to high [Ca2+].


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Fig. 1.   Effect of 100 µM ACh, 100 nM endothelin-1 (ET-1), and AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> (2 mM NaF + 20 µM AlCl3) on free Ca2+ concentration-response curves; control strips received only Ca2+. A: results obtained with acute exposure to the compounds. B: results obtained after chronic (1-h) exposure to the compounds. Values are means ± SD; n = 6 dogs for each condition. Force is expressed as a percentage of the response to 10 µM Ca2+ determined in each strip before each experiment.


                              
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Table 1.   Coefficients for nonlinear regression of concentration-response relationships

Effect of chronic application of ACh, ET-1, or AlF<UP><SUB>4</SUB><SUP><UP>−</UP></SUP></UP> on Ca2+ sensitivity. Four alpha -toxin-permeabilized strips were stimulated with increasing concentrations of free Ca2+ (1 nM to 100 µM). Three strips were exposed to 100 µM ACh or 100 nM ET-1 or AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> (2 mM NaF + 20 µM AlCl3) beginning 1 h before the first increment in [Ca2+] (chronic exposure). The fourth strip served as a control for the effects of time. Chronic (1-h) exposure to ACh, ET-1, or AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> increased Ca2+ sensitivity as shown by a significant decrease in EC50 for Ca2+ (Fig. 1B and Table 1). This duration of chronic exposure produced maximal effects in preliminary studies (data not shown). The change in EC50 produced by chronic exposure was similar to that produced by acute exposure to ACh and ET-1 (Table 1). However, chronic exposure to AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> produced a significantly greater decrease in EC50 compared with acute exposure (Table 1). The maximal force developed at high [Ca2+] was not significantly affected by chronic exposure to ACh or ET-1 (Fig. 1B and Table 1), as found with acute exposure. In contrast, chronic exposure to AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> significantly increased maximal force (Table 1). Thus receptor agonists (ACh and ET-1) had similar effects on Ca2+ sensitivity whether applied acutely or chronically, whereas chronic, but not acute, application of AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> increased maximal force developed in response to high [Ca2+].

Effect of chronic AlF<UP><SUB>4</SUB><SUP><UP>−</UP></SUP></UP> exposure on the rMLC phosphorylation-force relationship. In preliminary experiments, we found that stimulation with 0.3 µM free Ca2+ alone or 0.3 µM free Ca2+ in the presence of AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> produced increases in rMLC phosphorylation that were stable within 5 min and that were sustained for at least 15 min (data not shown). Fourteen permeabilized CTSM strips were prepared from each of six dogs. One strip was not stimulated and was flash-frozen in relaxing solution for baseline rMLC phosphorylation measurements. Six muscle strips were exposed to varying free [Ca2+] levels (0.1, 0.3, 1, 3, 10, or 100 µM) for 10 min before being frozen. The remaining seven muscle strips were exposed to AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> (2 mM NaF + 20 µM AlCl3) in relaxing solution for 1 h (chronic exposure). They were then stimulated with the same varying free [Ca2+] levels (0.03, 0.1, 0.3, 1, 3, or 10 µM) in the continuing presence of AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> for 10 min before being frozen. In additional experiments, the effects of acute exposure to AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> were also determined for a more limited range of free [Ca2+] levels (0.3, 1, and 3 µM).

rMLC phosphorylation increased as free [Ca2+] increased in a concentration-dependent manner (Fig. 2). Treatment with AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> (1-h exposure) markedly increased the sensitivity of rMLC phosphorylation to Ca2+, decreasing the [Ca2+] required to produce half-maximal rMLC phosphorylation from 0.42 ± 0.20 to 0.096 ± 0.015 µM. For [Ca2+] at which the effects of acute AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> exposure were also studied (0.3, 1, and 3 µM), there was no significant difference between rMLC phosphorylation measured during acute and chronic AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> exposure (Fig. 2). When combined with the force data obtained in the previous protocol, the relationship between rMLC phosphorylation and force could be plotted (Fig. 3). Chronic, but not acute, AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> exposure clearly increased the force developed for a given rMLC phosphorylation, an effect especially pronounced at high levels of rMLC phosphorylation. When analysis was performed using the paired t-test to compare the fitted slope of the regression line between the groups according to the procedure described above, a significant difference was detected for 98% of the bootstrap samples. When the regression was used to calculate the force produced by 70% rMLC phosphorylation, a significant difference was detected for 96% of the bootstrap samples. Thus chronic, but not acute, exposure to AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> increased Ca2+ sensitivity in CTSM by mechanisms in addition to increases in rMLC phosphorylation.


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Fig. 2.   Relationship between Ca2+ concentration ([Ca2+]; 1 nM to 100 µM) and regulatory myosin light chain (rMLC) phosphorylation in the absence (Ca2+ alone) and presence of AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> (2 mM NaF + 20 µM AlCl3) applied either acutely (acute) or for 1 h (chronic) to permeabilized canine tracheal smooth muscle. Values are means ± SD; n = 7 for Ca2+ alone and AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>, chronic; n = 5 for AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>, acute.



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Fig. 3.   Relationship between rMLC phosphorylation and force (points plotted at common [Ca2+] from data presented in Figs. 2 and 3) in the absence (Ca2+ alone) and presence of AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> (2 mM NaF + 20 µM AlCl3) applied either acutely (acute) or for 1 h (chronic) to permeabilized canine tracheal smooth muscle. Values are means ± SD; n = 6-7 for Ca2+ alone and AlF <UP><SUB>4</SUB><SUP>−</SUP></UP>, chronic; n = 5-6 for AlF <UP><SUB>4</SUB><SUP>−</SUP></UP>, acute.

Dependence of AlF<UP><SUB>4</SUB><SUP><UP>−</UP></SUP></UP> effects on G proteins. We wanted to confirm that the effects of chronic AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> exposure on Ca2+ sensitivity were indeed produced by G protein activation and not by some other nonspecific effect of AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>. We exploited a prior observation by Gong et al. (13), who noted that chronic exposure of permeabilized smooth muscle to guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) inhibits G protein-mediated responses.

Four strips were permeabilized, and responses to both 10 µM Ca2+ (defined as a maximal response) and 0.18 µM Ca2+ with 100 µM ACh were sequentially determined (see Fig. 4 for representative protocol). Pairs of strips were then incubated with relaxing solution alone (control) or with 10 µM GTPgamma S in relaxing solution for 6 h, a length of time sufficient for the maximal effect in preliminary studies. After incubation, the strips were washed in relaxing solution, and the responses to AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> or 100 µM ACh in 0.18 µM Ca2+ were determined. The responses of the alpha -toxin preparation were stable over the period of study as shown by the reproducibility of responses to 10 µM Ca2+ (103 ± 5% of the initial maximal Ca2+ response, n = 4) and 100 µM ACh with 0.18 µM Ca2+ (which produced 69 ± 8 and 71 ± 7% of the initial maximal Ca2+ response before and after the 6-h period, respectively) in control strips (Fig. 4). In contrast, 6 h of exposure to GTPgamma S significantly inhibited contraction produced by both ACh (from 71 ± 7 to 18 ± 7% of maximal; Fig. 4A) and AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> (from 79 ± 15 to 27 ± 15% of maximal; Fig. 4B). These values after GTPgamma S exposure were similar to those produced by 0.18 µM Ca2+ alone (19 ± 11 and 22 ± 5% of maximal for ACh and AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>, respectively). Responses to maximal Ca2+ alone were not affected by GTPgamma S (98 ± 2% of the initial response). These results suggest that 6 h of exposure to GTPgamma S completely abolishes Ca2+ sensitization responses to the G protein-coupled muscarinic receptor and that acute sensitization produced by AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> is mediated entirely by G proteins.


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Fig. 4.   Representative experiments demonstrating that prolonged exposure to guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) inhibits G protein-coupled responses in permeabilized smooth muscle. In A and B, a maximal response to 10 µM Ca2+ was first obtained, followed by sequential activation with 0.18 µM Ca2+ and 100 µM ACh, demonstrating Ca2+ sensitization produced by muscarinic stimulation. After 6 h of incubation with (GTPgamma S) or without (control) 10 µM GTPgamma S, the strips were sequentially activated with 0.3 µM Ca2+ and 100 µM ACh (A) or 0.3 µM Ca2+ and AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> (2 mM NaF + 20 µM AlCl3; B). Treatment with GTPgamma S abolished responses to both ACh and AlF <UP><SUB>4</SUB><SUP>−</SUP></UP> without affecting the response to Ca2+ alone.

A similar protocol was conducted in another series of strips, this time exposing strips treated with and without GTPgamma S to AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> during the last hour of the 6-h incubation (to duplicate the chronic AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> exposure studied in earlier protocols). Four strips were permeabilized, and responses to both 10 and 0.18 µM Ca2+ with 100 µM ACh were sequentially determined (see Fig. 5 for representative protocol). Pairs of strips were then incubated in 1 nM free Ca2+ with and without 10 µM GTPgamma S for 6 h. In one strip of each pair, AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> (2 mM NaF + 20 µM AlCl3) was added during the last hour of incubation and was left in solutions during subsequent contractions. After incubation, the strips were washed in Ca2+-free solution, and the responses to 10 µM Ca2+ were determined. As before, 6 h of GTPgamma S did not significantly affect responses to maximal Ca2+ (data not shown). However, after treatment with GTPgamma S, chronic (1-h) AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> no longer increased the response to 10 µM Ca2+ (98 ± 13 and 127 ± 18% of the initial response to 10 µM Ca2+ in treated and untreated strips, respectively, n = 5, P < 0.001; Fig. 5). These results suggest that the effect of chronic AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> exposure to increase the response to maximal Ca2+ is indeed mediated by G protein activation.


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Fig. 5.   Representative experiment using prolonged exposure to GTPgamma S to determine if the effects of chronic AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> are mediated via G proteins. A maximal response to 10 µM Ca2+ was first obtained, followed by sequential activation with 0.18 µM Ca2+ and 100 µM ACh, demonstrating Ca2+ sensitization produced by muscarinic stimulation. Strips were incubated for 6 h with (GTPgamma S) or without (control) 10 µM GTPgamma S, and AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> (2 mM NaF + 20 µM AlCl3) was added during the last hour of incubation and left in solutions during subsequent contraction with 10 µM Ca2+. Treatment with GTPgamma S abolished the ability of chronic (1-h) incubation with AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> to increase maximal force.

AlF<UP><SUB>4</SUB><SUP><UP>−</UP></SUP></UP> effects on thiophosphorylated CTSM. The aims of this protocol were to confirm that increases in force produced by chronic AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> were independent of changes in rMLC phosphorylation and to determine if the effect of AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> depended on Ca2+. Four strips were permeabilized with alpha -toxin, and the response to 10 µM Ca2+ was determined. After exposure to low-Ca2+ rigor solution for 20 min to remove ATP, the strips were incubated with a high-Ca2+ rigor solution containing 2.1 mM magnesium adenosine 5'-O-(3-thiotriphosphate) for 20 min. Preliminary experiments showed that this regimen produced >95% rMLC thiophosphorylation (data not shown). The strips were then washed with low-Ca2+ rigor solution for 20 min with and without AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> (2 mM NaF + 20 µM AlCl3). After this period, the strips were exposed to 7.5 mM ATP (to activate cross-bridge cycling) in low (1 nM, one pair)- or high (10 µM, one pair)-Ca2+ solution, and the force response was determined 10 min after activation.

Activation of thiophosphorylated strips by ATP produced force similar to the initial response to 10 µM Ca2+ (106 ± 20 and 105 ± 22% of initial response for low- and high-Ca2+ conditions, respectively, n = 6). When cross-bridge cycling was initiated by ATP in the presence of low [Ca2+] conditions (1 nM), prior exposure to AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> did not affect developed force (100 ± 17% of the initial response). However, when the strips were activated in the presence of 10 µM Ca2+, AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> significantly increased the force response (to 127 ± 29% of the initial response, P < 0.05). Thus the increase in maximal force produced by AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> observed in prior experiments was also present under conditions of maximal thiophosphorylation but was manifest only at high [Ca2+].


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The major finding of this study in alpha -toxin-permeabilized CTSM is that chronic (1-h) but not acute exposure to AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> increases Ca2+ sensitivity by mechanisms in addition to rMLC phosphorylation, producing a significant increase in the maximal force developed in response to high [Ca2+]. This effect was not observed after chronic stimulation with G protein-coupled receptor agonists.

We confirmed prior observations that the acute activation of G protein-coupled receptors produces Ca2+ sensitization in permeabilized airway smooth muscle, decreasing the EC50 for [Ca2+] without significantly changing maximal force (21). Prior studies suggest that receptor binding triggers dissociation of a heterotrimeric G protein in airway smooth muscle phosphorylation. These subunits in turn activate a monomeric G protein that subsequently inhibits SMPPs and thus increases rMLC (20, 25, 40). Some evidence suggests that kinases activated by monomeric G proteins, such as Rho-associated kinase, can also regulate Ca2+ sensitivity produced by receptor activation by directly phosphorylating rMLC (30), although evidence for this mechanism is lacking in airway smooth muscle (20). Recently, integrin-linked kinase has been shown to phosphorylate rMLC in the absence of Ca2+ (9); its possible role in agonist-induced Ca2+ sensitization is unknown. We found that fluoroaluminate complexes (comprised mainly of AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>) produced acute Ca2+ sensitization very similar to that produced by maximal stimulation using receptor agonists. The structural similarity of AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> to phosphate allows it to bind next to the beta -phosphate of GDP and mimic the terminal gamma -phosphate of GTP (3). This eliminates the normal requirement for GDP-GTP exchange to cause conformational change and consequent activation of G proteins. The bound AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> cannot be hydrolyzed, so the G protein complex is maintained in its active, dissociated state, thus interrupting its normal cycling through GDP- and GTP-bound states. AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> is considered to specifically activate heterotrimeric G proteins (24), although it has been suggested that fluoroaluminates may also activate monomeric G proteins, such as Rho, in smooth muscle (3).

In contrast to acute activation, chronic (1-h) activation of G proteins with AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> significantly increased the maximal force produced in response to high [Ca2+]. We confirmed that the effects of both acute and chronic exposure to AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> are mediated by G proteins by noting that these responses were abolished by chronic exposure to GTPgamma S. As first noted by Gong et al. (13) in rabbit portal vein, chronic exposure of permeabilized smooth muscle to GTPgamma S inhibits G protein-mediated responses, including responses to AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> and receptor agonists. The mechanisms producing this effect are unclear but are not related to expression of G proteins and may involve either the function of monomeric G proteins such as Rho or sites further downstream in the cascade. The reason why chronic stimulation of G proteins by AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>, which presumably activates the same downstream effectors, actually augments the responses is unknown. In preliminary studies, we observed inhibition of G protein receptor-coupled responses after 1 h of GTPgamma S exposure in preliminary experiments (data not shown), although 6 h of exposure were required for maximal inhibition. The ability of GTPgamma S to abolish the effects of chronic AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> and the lack of effect of AlF <UP><SUB>4</SUB><SUP>−</SUP></UP> in thiophosphorylated strips under low-Ca2+ conditions exclude mechanisms such as direct actions on the myosin head (6, 7). We also performed further studies in Triton X-skinned CTSM in which G protein activity is abolished and found no effect of AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> (data not shown).

We cannot explain why chronic exposure to AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>, but not to ACh or ET-1 (which should also chronically activate G proteins), increases maximal force. Chronic receptor stimulation can produce downregulation of responses that could offset increases in sensitivity, although the receptor reserve for muscarinic receptors in canine airway is high (16). The kinetics of the G protein activation differ between receptor and AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> activation; normal cycling through GTP- and GDP-bound states during receptor occupancy is interrupted by AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>, although it is not immediately apparent how this would make a difference. The most likely explanation is that AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> activates G proteins in addition to those coupled to muscarinic or ET receptors. Candidates include receptors coupled to inflammatory mediators such as cytokines and cysteinyl leukotrienes, which in other tissues appear to be associated with G proteins (1, 2). However, their existence and possible functional role in the canine airway remain unknown. At present, we can conclude only that G protein signaling pathways exist in CTSM that, when chronically stimulated, can increase the maximal force produced by high [Ca2+].

According to the observed relationship between rMLC phosphorylation and force, this increase in maximal force is produced by mechanisms in addition to rMLC phosphorylation. It should be noted that, although the strips used to measure rMLC phosphorylation were pinned at both ends to maintain isometric conditions, optimal length was not determined in these strips, in contrast to the strips used to measure force responses. Even though all strips were otherwise treated in the same manner after permeabilization, this represents a possible limitation to the interpretation of our phosphorylation data. Indeed, in intact (nonpermeabilized) airway smooth muscle, muscle length affects rMLC phosphorylation via effects on intracellular [Ca2+] (33). However, in preliminary data obtained for prior studies (5, 17, 22, 26), we showed that muscle length does not affect rMLC phosphorylation in permeabilized airway smooth muscle in which intracellular [Ca2+] is controlled. Thus this factor should not influence our conclusions. We confirmed that these mechanisms are independent of rMLC phosphorylation by showing that chronic AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> also increases maximal force in the presence of maximal thiophosphorylation of the rMLC. These experiments further demonstrated that Ca2+ is required for increases in maximal force to occur, although it is of interest that the chronic incubation with AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> necessary to produce these increases was performed under low [Ca2+] conditions (1 nM). Also, this finding shows that we could indeed measure very high values of rMLC phosphorylation in these permeabilized tissues, consistent with our prior experience (26). Thus an inability to detect high rMLC phosphorylation values does not limit our conclusions. We previously demonstrated that increases in Ca2+ sensitivity produced by acute exposure to ACh were produced exclusively by increases in rMLC phosphorylation (26). Increases in sensitivity produced by acute exposure to AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> can also be explained entirely by increases in rMLC phosphorylation (Fig. 3). Thus these rMLC phosphorylation-independent mechanisms appear to be specific to chronic G protein activation by AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>.

At least two classes of mechanisms may regulate force in smooth muscle independent of changes in rMLC phosphorylation. Some have proposed an independent regulatory system that is responsible for force maintenance after initial rapid force development (10, 44). Current candidates include the actin-associated proteins caldesmon and calponin, based on evidence obtained in several types of smooth muscle. A role for these proteins in vascular smooth muscle under some circumstances has been demonstrated (10, 44), although evidence for their functional role in airway smooth muscle is more limited. Both proteins are phosphorylated in response to muscarinic stimulation of intact CTSM (12, 38), although whether this can occur with stimulation under constant [Ca2+]i conditions in permeabilized preparations is not known. Others have suggested that the actin cytoskeleton may remodel in response to contractile stimuli to more efficiently transduce force from contractile elements to the cytoskeleton. Several different proteins involved with the actin cytoskeleton, including paxillin and focal adhesion kinase, are tyrosine phosphorylated during airway smooth muscle contraction (36) independent of changes in [Ca2+]i in the case of paxillin (32). We have recently shown that the kinetics of actin polymerization are also a potential regulatory mechanism in CTSM (23), and muscarinic control of actin polymerization has been demonstrated in cultured airway smooth muscle cells (42). Further studies will be necessary to determine which (if any) of these mechanisms might explain the increase in maximal force produced by chronic activation of heterotrimeric G proteins.

The possible physiological significance of these observations remains to be determined. As discussed above, it is possible that activation of G proteins not coupled to ACh or ET-1 by mediators such as leukotrienes and cytokines produces these effects (1, 2). Parris et al. (35) have shown that chronic tumor necrosis factor-alpha (TNF-alpha ) exposure activates a Ca2+ sensitization pathway in guinea pig bronchial smooth muscle, although this effect appears to depend on increases in rMLC phosphorylation. Hotta et al. (19) found that chronic exposure of cultured airway smooth muscle to TNF-alpha increased G protein expression and enhanced some responses to muscarinic stimulation. Airway smooth muscle may be chronically exposed to a variety of inflammatory mediators in diseases such as asthma, which are characterized by airway inflammation (2). Any resultant increases in Ca2+ sensitivity might contribute to clinical hyperreactivity (39). In particular, increases in maximal force produced by airway smooth muscle could contribute to the lack of plateau in the histamine dose-response relationship noted in asthmatic subjects, producing an increase in maximal airway narrowing (43). Studies of isolated airway smooth muscle have found inconsistent effects on maximal force when comparing muscle from asthmatic subjects (or from sensitized models) with normal subjects (4, 8). Beyond the technical challenges involved in comparing maximal force among airways (21), our results suggest that another source of these discrepancies may be that regulation of maximal force can be a relatively rapid process (<= 1 h in our studies). Thus any changes present in vivo may be diminished by several hours of in vitro studies in tissue baths where inflammatory mediators are not present. Finally, we cannot exclude the possibility that the pattern of G protein activation produced by chronic AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> exposure is never achieved in vivo.

In conclusion, we demonstrate that, unlike the acute response to receptor agonists, which is mediated solely by rMLC phosphorylation, chronic direct activation of G proteins further increases Ca2+ sensitivity in CTSM by additional mechanisms that are independent of rMLC phosphorylation. If also present in vivo, these mechanisms may be relevant to the development of airway hyperactivity.


    ACKNOWLEDGEMENTS

We thank K. Street for technical assistance.


    FOOTNOTES

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

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. Section 1734 solely to indicate this fact.

Received 17 January 2001; accepted in final form 1 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 281(3):L631-L638
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