8-Bromo-cAMP decreases the Ca2+ sensitivity of airway smooth muscle contraction through a mechanism distinct from inhibition of Rho-kinase

Katsuaki Endou,1 Kunihiko Iizuka,1 Akihiro Yoshii,1 Hideo Tsukagoshi,1 Tamotsu Ishizuka,1 Kunio Dobashi,1 Tsugio Nakazawa,2 and Masatomo Mori1

1Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi; and 2Gunma University Faculty of Health Sciences, Gunma 371-8511, Japan

Submitted 21 August 2003 ; accepted in final form 26 April 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To clarify whether cyclic AMP (cAMP)/cAMP-dependent protein kinase (PKA) activation and Rho-kinase inhibition share a common mechanism to decrease the Ca2+ sensitivity of airway smooth muscle contraction, we examined the effects of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), a stable cAMP analog, and (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide dihydrochloride, monohydrate (Y-27632), a Rho-kinase inhibitor, on carbachol (CCh)-, guanosine 5'-O-(3-thiotriphosphate) (GTP{gamma}S)-, 4{beta}-phorbol 12,13-dibutyrate (PDBu)-, and leukotriene D4 (LTD4)-induced Ca2+ sensitization in {alpha}-toxin-permeabilized rabbit tracheal and human bronchial smooth muscle. In rabbit trachea, CCh-induced smooth muscle contraction was inhibited by 8-BrcAMP and Y-27632 to a similar extent. However, GTP{gamma}S-induced smooth muscle contraction was resistant to 8-BrcAMP. In the presence of a saturating concentration of Y-27632, PDBu-induced smooth muscle contraction was completely reversed by 8-BrcAMP. Conversely, PDBu-induced smooth muscle contraction was resistant to Y-27632. In the presence of a saturating concentration of 8-BrcAMP, GTP{gamma}S-induced Ca2+ sensitization was also reversed by Y-27632. The 8-BrcAMP had no effect on the ATP-triggered contraction of tracheal smooth muscle that had been treated with calyculin A in rigor solutions. The 8-BrcAMP and Y-27632 additively accelerated the relaxation rate of PDBu- and GTP{gamma}S-treated smooth muscle under myosin light chain kinase-inhibited conditions. In human bronchus, LTD4-induced smooth muscle contraction was inhibited by both 8-BrcAMP and Y-27632. We conclude that cAMP/PKA-induced Ca2+ desensitization contains at least two mechanisms: 1) inhibition of the muscarinic receptor signaling upstream from Rho activation and 2) cAMP/PKA's preferential reversal of PKC-mediated Ca2+ sensitization in airway smooth muscle.

calcium sensitization; cAMP; leukotriene D4


ALTHOUGH INTRACELLULAR calcium concentration Ca2+ ([Ca2+]i) is the primary regulator of smooth muscle contraction, Ca2+ sensitivity of the contractile apparatus can change in response to agonists. An increase and a decrease in muscle tension at a constant Ca2+ concentration are correspondingly referred to as Ca2+ sensitization and desensitization of smooth muscle contraction. These are believed to be the results of changes in the ratio of kinase and phosphatase activities toward the 20-kDa light chain of myosin (MLC20) (22, 25). We have demonstrated that receptor-dependent, G protein-mediated Ca2+ sensitization occurs in canine, rabbit, and human airway smooth muscles (28) and that a small G protein, RhoA, and its target protein, Rho-kinase, play a key role in G protein-mediated Ca2+ sensitization of smooth muscle contraction (26), especially in the sustained phase. Rho/Rho-kinase signaling increases phosphorylation of MLC20 through the inhibition of myosin light chain phosphatase (MLCP)-associated mechanisms, but it does not directly phosphorylate the MLC20 of airway smooth muscle in situ (13).

Adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents such as {beta}-adrenergic agonists and phosphodiesterase (PDE) inhibitors are most widely used clinically to relax airway smooth muscle. Elevated cAMP not only decreases net [Ca2+]i by enhancing Ca2+ extrusion to the extracellular space and Ca2+ sequestration to the [Ca2+]i store but also decreases the Ca2+ sensitivity of the contractile mechanism (16). We have demonstrated that (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide dihydrochloride, monohydrate (Y-27632), a Rho-kinase inhibitor, relaxed airway smooth muscle of guinea pigs, both in vitro and in vivo. The Y-27632 was less potent than salbutamol, a selective {beta}2-adrenergic agonist, but was more potent than theophylline, a PDE inhibitor (11). Nakahara et al. (21) reported that {beta}2-agonists and Y-27632 additively relaxed the bovine trachea. However, if cAMP/cAMP-dependent protein kinase (PKA) activation and Rho/Rho-kinase inhibition share at least one common mechanism for modulating Ca2+ sensitivity, Rho-kinase inhibitors may compete with cAMP/PKA-mediated relaxation in airway smooth muscle. Thus the mechanisms of these two pathways must be elucidated to clarify clinical applications.

To address this issue, we focused on the mechanisms of cAMP/PKA- and Rho/Rho-kinase-mediated changes in the Ca2+ sensitivity of airway smooth muscle contraction. In {alpha}-toxin-permeabilized rabbit tracheal smooth muscle, we examined the effects of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), a stable cAMP analog, and Y-27632 on carbachol (CCh)-, guanosine 5'-O-(3-thiotriphosphate) (GTP{gamma}S)-, and 4{beta}-phorbol 12,13-dibutyrate (PDBu)-induced Ca2+ sensitization. We wanted to determine the following: whether 8-BrcAMP blocks CCh- or GTP{gamma}S-induced Ca2+ sensitization in a similar manner to Y-27632; whether 8-BrcAMP affects myosin light chain kinase (MLCK)-associated mechanisms or MLCP-associated mechanisms; whether 8-BrcAMP reverses PDBu-induced Ca2+ sensitization in the presence of a saturating concentration of Y-27632, because PDBu-induced contraction is resistant to Y-27632 (6); whether the inhibitory effect of Y-27632 on GTP{gamma}S-induced Ca2+ sensitization is affected by 8-BrcAMP; and whether the Y-27632- and 8-BrcAMP-responsive mechanisms are involved in leukotriene D4 (LTD4)-induced Ca2+ sensitivity in human bronchial smooth muscle.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tissue preparation and isometric force measurement. The tissue preparation and force measurement have been reported elsewhere (9, 10, 12). We administered the anesthesia by placing the animals in an anesthetic chamber until the animals became anesthetized and unresponsive to corneal stimulation. When the tracheal tissue had been removed, the animals were killed by rapid exsanguination through the carotid artery, in accordance with the recommendations of the Animal Care and Experimentation Committee, Gunma University, Showa Campus. The airways were first cut longitudinally at the center of the cartilage opposite the smooth muscle. Small strips of tracheal smooth muscle were then carefully separated from connective tissue, epithelium, and cartilage with a razor blade under a binocular microscope.

Human bronchial smooth muscle was prepared from a macroscopically normal section of lung tissue that was obtained during surgery for lung cancer. Consent was obtained from each patient before surgery. The surgically resected tissue was put in ice-cold Dulbecco's modified Eagle's medium, and small bronchi with an outer diameter of 2–4 mm were carefully dissected as previously described (28). Cartilage was removed to the greatest extent possible. Small strips of rabbit tracheal smooth muscle (200–300 µm wide, 40–50 µm thick, 3 mm long) and human bronchial smooth muscle (150–200 µm wide, 20–30 µm thick, 3 mm long) were mounted on a bubble plate (400 ml per bubble), and isometric force development was measured with a force transducer (AE801; SensoNor, Horten, Norway). The developed force was normalized to an initial pCa 5.0 (10–5 M) response in the same strip (12, 28). Consent was obtained from each patient before surgery. These protocols were approved by the Institutional Review Board, Gunma University Faculty of Medicine, School of Medicine.

Solutions and permeabilization with {alpha}-toxin. The method of permeabilization with {alpha}-toxin has been described previously (12, 28). The trachea was permeabilized with {alpha}-toxin (16.4 µg/ml) for 30 min at 30°C. We added a Ca2+ ionophore, A-23187 (10 µM), to the trachea during {alpha}-toxin permeabilization to block the sarcoplasmic reticulum function. After permeabilization, all experiments except for that depicted in Fig. 5 were performed at 24°C (9, 10, 12).



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Fig. 5. Relaxation by Y-27632, 8-BrcAMP, or both in {alpha}-toxin-permeabilized smooth muscle with GTP{gamma}S- or PDBu-induced Ca2+ sensitization. After maximum contraction was achieved with pCa 5.0 at 24°C, the tracheal smooth muscle was contracted with pCa 5.0 at 10°C. When the submaximal contraction induced by pCa 6.5 became stable, PDBu (10 µM) or GTP{gamma}S (100 µM) was added to the strip, and then the tracheal smooth muscle was treated with PDBu (10 µM) or GTP{gamma}S (100 µM) to induce Ca2+ sensitization at pCa 4.5. After the contraction reached a peak, 8-BrcAMP (100 µM), Y-27632 (3 µM), or both were added to the strips, and the strips were incubated for 20 min. Then the strips were transferred in G10 containing PDBu or GTP{gamma}S, reagents, and ML-9 (100 µM). The kinetics of tracheal smooth muscle relaxation on PDBu-induced contraction (B) and GTP{gamma}S-induced contraction (C) with 8-BrcAMP ({circ}), Y-27632 ({square}), or both ({blacksquare}). *, ¶, # and {dagger}P < 0.05 vs. control ({bullet}, n = 4–6). Relative force was normalized to the initial pCa 5.0 (10–5 M) response in the same strip.

 
The normal relaxing solution (G1) contained (in mM) 74.1 potassium methanesulfonate, 2 Mg2+, 4.5 ATP (Mg2+ salt), 1 EGTA, 10 creatine phosphate, and 30 PIPES-KOH (pH 7.1 at 24°C, ionic strength 0.2). The same solution containing 10 mM EGTA rather than 1 mM EGTA and various amounts of calcium methanesulfonate was used to achieve the desired concentration of free Ca2+. According to Zimmermann et al. (29), we prepared EGTA (10 mM)-buffered ATP-free (rigor) Ca2+-free solution (G10 rigor) and ATP-free high Ca2+ solution (pCa 4.5 rigor). These rigor solutions contained 50 µM P1,P5-di(adenosine-5') pentaphosphate, an inhibitor of kinase activity. We obtained the desired concentration of free Ca2+ by mixing the G10 rigor and pCa 4.5 rigor solutions. All solutions contained ibuprofen (2 µM) to avoid cyclooxygenase activity that may attenuate airway smooth muscle tone.

Effect of 8-BrcAMP in ATP triggered Ca2+ sensitization. To clarify whether 8-BrcAMP-induced Ca2+ desensitization is due to inhibition of MLCK-associated mechanisms, we abolished the MLCP activity of tracheal smooth muscle with calyculin A (300 nM) in rigor solutions and then observed ATP (4.5 mM, Mg2+ salt)-triggered contraction. We knew that the rate of force would rise but the final amplitude would not, reflecting MLCK-associated mechanisms (13, 17, 19). After obtaining the maximum contraction at pCa 5.0, we relaxed the tracheal smooth muscle in G10 solution. To activate PKA, we incubated the tracheal smooth muscle with 8-BrcAMP (100 µM) in G10 (containing ATP) for 10 min. In the continued presence of 8-BrcAMP, the tracheal smooth muscle was quickly transferred to a G10 rigor solution containing calyculin A (300 nM) for 55 min to inactivate MLCP without contraction. After incubation of the tracheal smooth muscle in a pCa 5.0 rigor solution containing 8-BrcAMP and calyculin A for 5 min, ATP-triggered contraction was initiated. If 8-BrcAMP inhibits MLCK-dependent contraction, the rate of ATP-triggered contraction would be slowed by 8-BrcAMP. Time-matched, ATP-triggered experiments were carried out in the absence of 8-BrcAMP, as controls.

Comparison of relaxation rates between PKA activation and Rho inhibition of {alpha}-toxin-permeabilized tracheal smooth muscle. To compare the effects of PKA activation and Rho-kinase inhibition on MLCP-dependent contraction and relaxation in situ, we measured the relaxation rate of fully contracted tracheal smooth muscle in the presence of 8-BrcAMP (100 µM), Y-27632 (3 µM), or both at 10°C. The low temperature conditions were required for comparing relaxation rates, because the relaxation rate at 24°C was too fast to evaluate the reagent effects on relaxation (19, 20). The tracheal smooth muscle was fully contracted by high Ca2+ with PDBu or GTP{gamma}S and then relaxed by combined treatment with Ca2+ removal and 1-(5-chloronaphthalene-1-sulfonyl) homopiperadine-HCl (ML-9), an MLCK inhibitor. We measured the half-time of the relaxation rate (time required to reach 50% of maximum relaxation induced by a given inhibitor) in the absence or presence of 8-BrcAMP, Y-27632, or both (Fig. 5).

Inhibitory effect of 8-BrcAMP and Y-27632 on LTD4-induced Ca2+ sensitization in {alpha}-toxin-permeabilized human bronchial smooth muscle. When submaximum contraction induced by pCa 6.8 plus GTP (3 µM) was stable, LTD4 (1 µM) was added to the {alpha}-toxin-permeabilized human bronchial smooth muscle. At the peak of additional contractions, Y-27632 (3 µM), 8-BrcAMP (100 µM), or both were added to the strip.

Reagents. Staphylococcus aureus {alpha}-toxin was obtained from RBI (Natick, MA); P1,P5-di(adenosine-5') pentaphosphate was from Sigma (St. Louis, MO). The Y-27632 was a gift from Mitsubishi Pharma (Osaka, Japan). The Y-27632 was dissolved in distilled water to create a 10 mM stock solution, which was stored at –20°C until use. The GTP{gamma}S was purchased from Boehringer Mannheim (Indianapolis, IN). The 8-BrcAMP, calyculin A, and PDBu were purchased from Calbiochem (La Jolla, CA). The LTD4 was purchased from Sigma. All other chemicals were of reagent grade.

Statistical analysis. Data were normalized to the pCa 5.0 response measured before the reagent treatment of each strip and are shown as means ± SE of the indicated numbers of experiments. Data were compared by the Mann-Whitney U-test or Student's t-test with the Bonferroni correction for multiple comparisons. A P value of < 0.05 was considered to be statistically significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effects of 8-BrcAMP and Y-27632 on CCh- or GTP{gamma}S-induced Ca2+ sensitization in {alpha}-toxin-permeabilized tracheal smooth muscle. After obtaining the maximum contraction at pCa 5.0, we incubated the strip in G1 containing 8-BrcAMP, Y-27632, or saline for 20 min. In the continuous presence of the reagents, the tracheal smooth muscle was precontracted in a pCa 6.5 solution containing GTP (3 µM); CCh (100 µM) was then applied to the strip. In the experiments of GTP{gamma}S-induced Ca2+ sensitization, the tracheal smooth muscle was precontracted in a pCa 6.5 solution without GTP, followed by the application of GTP{gamma}S (10 µM) to the tracheal smooth muscle. As shown in Fig. 1A, CCh increased the contractile force from the steady-state level at pCa 6.5 (15.8 ± 2.2%) to 82.2 ± 5.0% (n = 6). Pretreatment with 8-BrcAMP dose dependently inhibited the agonist-induced smooth muscle contraction. The effect of 8-BrcAMP was saturated at 100 µM, and the extent of the maximum inhibition by 8-BrcAMP was comparable with that by Y-27632 at 100 µM. Similarly, GTP{gamma}S caused rapid contractions from 4.99 ± 1.3 to 97.4 ± 3.5% (n = 8) at pCa 6.5, and the GTP{gamma}S response was inhibited by Y-27632 (100 µM). The inhibitory effect of 8-BrcAMP was only partial in GTP{gamma}S-induced smooth muscle contraction, and the resultant contraction was 60.6 ± 9.1% in the presence of 8-BrcAMP at 300 µM (n = 6, Fig. 1B). Thus the GTP{gamma}S response was relatively more resistant to 8-BrcAMP.



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Fig. 1. Inhibitory effect of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) or Y-27632 on carbachol (CCh)- or guanosine 5'-O-(3-thiotriphosphate) (GTP{gamma}S)-induced Ca2+ sensitization. After a stable pCa 5.0 response was obtained, the {alpha}-toxin-permeabilized tracheal smooth muscle was incubated in G1 containing saline, 8-BrcAMP (0.1–300 µM), or Y-27632 (100 µM) for 20 min. In the continuous presence of reagents, the tracheal smooth muscle was precontracted in a pCa 6.5 solution containing GTP (3 µM); then CCh (100 µM) was applied to the strips (n = 3–6, A). In the experiments of GTP{gamma}S-induced Ca2+ sensitization, tracheal smooth muscle was precontracted in a pCa 6.5 solution, followed by the application of GTP{gamma}S (10 µM) to the trachea (n = 3–8, B). Relative force was normalized to saline.

 
Lack of effect of 8-BrcAMP on ATP-triggered contraction of calyculin A-treated tracheal smooth muscle. As shown in Fig. 2A, with or without 8-BrcAMP (100 µM), ATP elicited rapid contractions of tracheal smooth muscle that had been treated with calyculin A in the rigor solutions. The final force developments were not different between the two groups (80 ± 7.2% in the control, 80.9 ± 5.3% in the 8-BrcAMP-treated group, n = 4), and the values of T1/2 (time required to reach 50% of maximum force induced by a given stimulant) in the control and the 8-BrcAMP-treated strips were also comparable (63.1 ± 3.6 and 61.9 ± 7.6 s, respectively; n = 4). Thus 8-BrcAMP did not affect the MLCK-associated mechanisms.



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Fig. 2. Lack of effect 8-BrcAMP on kinase activity toward 20-kDa myosin light chain (MLC20). After obtaining the maximum contraction at pCa 5.0, we relaxed the tracheal smooth muscle in G10 solution. To activate PKA, we incubated the tracheal smooth muscle with 8-BrcAMP (100 µM) in G10 (containing ATP) for 10 min. In the continued presence of 8-BrcAMP, tracheal smooth muscle was quickly transferred to a G10 ATP-free (rigor) solution containing calyculin A (300 nM) for 55 min to inactivate myosin light chain phosphatase (MLCP) without contraction. After incubation of tracheal smooth muscle in a pCa 6.5 rigor solution containing 8-BrcAMP and calyculin A for 5 min, ATP-triggered contraction was initiated (A). The final force developments were not different between the 2 groups, and the values of the time required to reach 50% of maximum force induced by a given stimulant in the control strips ({circ}) and the 8-BrcAMP-treated strips ({bullet}) were also comparable (n = 4, B). Relative force was normalized to the initial pCa 5.0 (10–5 M) response in the same strip.

 
Involvement of a distinct mechanism between 8-BrcAMP- and Y-27632-induced decreases in Ca2+ sensitivity. To find a qualitative difference between 8-BrcAMP- and Y-27632-induced changes in Ca2+ sensitivity, we attempted inhibition of PDBu-induced Ca2+ sensitization by 8-BrcAMP in the presence of a saturating concentration of Y-27632. As shown in Fig. 3A, after obtaining the maximum contraction at pCa 5.0, we treated the tracheal smooth muscle with Y-27632 (30 µM) for 20 min, and then PDBu (10 µM)-induced Ca2+ sensitization was evoked at pCa 6.5. We verified that the concentration of Y-27632 was saturated, because an increase in the concentration of Y-27632 from 30 to 100 µM had no effect on force. In the saturated concentration of Y-27632, 8-BrcAMP dose dependently reversed contraction of the tracheal smooth muscle. Time-matched experiments were carried out in the absence of Y-27632 as controls. As shown in Fig. 3B, although Y-27632 showed a tendency to decrease PDBu-induced contractions, the contractions before the application of 8-BrcAMP were not statistically significant (P = 0.147, Mann-Whitney test), and the IC50 values with or without Y-27632 were comparable; the amplitude of the PDBu response and IC50 values for 8-BrcAMP in the Y-27632-treated group were 53.1 ± 7.1% and 5.24 ± 0.8 µM (n = 7), whereas those in the control group were 70.0 ± 8.1% and 8.21 ± 1.0 µM (n = 11), respectively.



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Fig. 3. Inhibition of 4{beta}-phorbol 12,13-dibutyrate (PDBu)-induced Ca2+ sensitization by 8-BrcAMP in the presence of Y-27632. After obtaining the maximum contraction at pCa 5.0, we treated tracheal smooth muscle with Y-27632 (30 µM) or saline for 20 min, and then PDBu (10 µM)-induced Ca2+ sensitization was evoked at pCa 6.5. After we verified that the concentration of Y-27632 was saturated, 8-BrcAMP dose dependently reversed contraction of the trachea (A), although Y-27632 tended to decrease PDBu-induced contractions. The contractions before 8-BrcAMP application were not statistically significant (P = 0.147, Mann-Whitney U-test), and the IC50 values with or without Y-27632 were also comparable; the amplitude of the PDBu response and the IC50 values for 8-BrcAMP in the Y-27632-treated group ({bullet}) were 53.1 ± 7.1% and 5.24 ± 0.8 µM (n = 7); those in the control group ({circ}) were 70.0 ± 8.1% and 8.21 ± 1.0 µM (n = 11), respectively (B). Relative force was normalized to the initial pCa 5.0 (10–5 M) response in the same strip.

 
Next, we verified inhibition of GTP{gamma}S-induced Ca2+ sensitization by Y-27632 in the presence of 8-BrcAMP (Fig. 4). The amplitude of the GTP{gamma}S response and IC50 values for Y-27632 in the 8-BrcAMP-treated group were 81.4 ± 7.0% and 3.06 ± 0.5 µM (n = 6), whereas those in the control group were 104.9 ± 7.0% and 2.24 ± 0.7 µM (n = 5), respectively.



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Fig. 4. Inhibition of GTP{gamma}S-induced Ca2+ sensitization by Y-27632 in the presence of 8-BrcAMP. After obtaining the maximum contraction at pCa 5.0, we treated the trachea with 8-BrcAMP (30 µM) for 20 min, and then GTP{gamma}S (10 µM)-induced Ca2+ sensitization was evoked at pCa 6.5. After we verified that the concentration of 8-BrcAMP was saturated, 8-BrcAMP dose dependently reversed contraction of the tracheal smooth muscle (A). The amplitude of the GTP{gamma}S response and the IC50 values for Y-27632 in the 8-BrcAMP-treated group ({bullet}) were 81.4 ± 7.0% and 3.06 ± 0.5 µM (n = 6); those in the control group ({circ}) were 104.9 ± 7.0% and 2.24 ± 0.7 µM (n = 5), respectively (B). Relative force was normalized to the initial pCa 5.0 (10–5 M) response in the same strip.

 
Time course of relaxation of {alpha}-toxin-permeabilized trachea in the presence of 8-BrcAMP, Y-27632, or both. We measured the relaxation rate of fully contracted tracheal smooth muscle in the presence of 8-BrcAMP, Y-27632, or both at 10°C to estimate the effect of MLCP-associated mechanisms. Figure 5 shows relaxation by ML-9/G10 after treatment with Y-27632, 8-BrcAMP, or both in {alpha}-toxin-permeabilized rabbit tracheal smooth muscle following PDBu- or GTP{gamma}S-induced Ca2+ sensitization. After obtaining the maximum contraction at pCa 5.0 at 10°C, we precontracted the strips in a pCa 6.5 solution containing PDBu (10 µM) or GTP{gamma}S (100 µM). The strips were then treated with a pCa 4.5 solution containing either PDBu or GTP{gamma}S and the inhibitors 8-BrcAMP (100 µM) or Y-27632 (3 µM) or both as indicated in Fig. 5. In the continuous presence of all reagents, the strips were moved into a G10 solution containing ML-9 (100 µM). The 8-BrcAMP accelerated the relaxation rate of PDBu-treated strips but not that of GTP{gamma}S-treated strips. In contrast, Y-27632 was effective in GTP{gamma}S-treated strips, but not in PDBu-treated strips (Table 1).


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Table 1. Effects of 8-BrcAMP, Y-27632, or both in {alpha}-toxin-permeabilized rabbit tracheal smooth muscle with PDBu-, GTP{gamma}S-, or LTD4-induced Ca2+ sensitization

 
Inhibitory effects of 8-BrcAMP and Y-27632 on LTD4-induced Ca2+ sensitization in {alpha}-toxin-permeabilized human bronchial smooth muscle. To estimate the relative contributions of PKA activation and Rho-kinase inhibition to LTD4-mediated Ca2+ sensitization, we treated strips of permeabilized human bronchial smooth muscle with 8-BrcAMP (100 µM), Y-27632 (3 µM), or both. Sensitization to Ca2+ was evoked with a saturated solution of 1 µM LTD4. The pCa 5.0-induced contractions before Ca2+ sensitization were 1.81 ± 0.17 mN in human bronchial smooth muscle (n = 8). As shown in Fig. 6, in the presence of GTP (3 µM), LTD4 (1 µM) induced an additional contraction at a fixed free Ca2+ concentration of pCa 6.8 in {alpha}-toxin-permeabilized human bronchial smooth muscle. The peak force achieved was 70.4 ± 4.7% (n = 8) of the initial contraction at pCa 5.0. The LTD4 response was reversed by 8-BrcAMP to 43.6 ± 3.7%, by Y-27632 to 33.7 ± 6.0%, and by their combination to 9.14 ± 2.3% (n = 4–8, Table 1).



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Fig. 6. Inhibitory effect of 8-BrcAMP and Y-27632 on leukotriene D4 (LTD4)-induced Ca2+ sensitization in human bronchial smooth muscle. In {alpha}-toxin-permeabilized human bronchial smooth muscle, after the maximum contraction at pCa 5.0 and the submaximum contraction from pCa 6.8 were obtained, LTD4 (1 µM) was added to strips. When LTD4 caused contraction became stable, 8-BrcAMP (100 µM) or Y-27632 (3 µM) or both were added to the strips. Developed force was normalized to the initial pCa 5.0 response (n = 4–8).

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
{beta}-Adrenergic agonists can be understood as a cascade involving activation of adenyl cyclase, elevation of cytoplasmic cAMP levels, and PKA activation leading to phosphorylation of target proteins. However, the precise mechanisms are still unknown. In Fig. 2, the contractile rate (T1/2) was comparable between 8-BrcAMP-treated strips and control strips. The T1/2 of the strips treated with a phosphatase inhibitor (such as microcystin-LR or calyculin A) is dependent on the MLCK-associated mechanisms in the presence of Ca2+ (13, 16). These results suggest that cAMP/PKA signaling preferentially affects MLCP-associated mechanisms of Ca2+ sensitization in rabbit tracheal smooth muscle.

The relaxing rate was mainly dependent on MLCP under our experimental conditions (19, 20). We considered that cellular events other than MLCK/MLCP-induced mechanisms might be involved in our experiment conditions. Previous studies have suggested that MLCK/MLCP-associated mechanisms play a key role in at least the initiation of contraction and early phase of maintenance of force (19, 20). Many cellular events other than MLCK/MLCP-induced mechanisms may be slower than changes in the phosphorylation state of myosin and thus rate limiting. However, precise time-course studies suggest that initiation of contraction occurred within milliseconds and that the early phase of maintenance of force occurred within several seconds (25). Furthermore, we have demonstrated that MLCK inhibition with wortmannin but not Rho-kinase inhibition with Y-27632 slowed force developments under the same experimental conditions (13). The final amplitudes were not different. Therefore, other cellular events appear to precede the development of maximum force by minutes.

The 8-BrcAMP accelerated the relaxation rate in PDBu-treated strips but not in GTP{gamma}S-treated strips (Fig. 5). In contrast, Y-27632 accelerated the relaxation rate in GTP{gamma}S-treated strips but not in PDBu-treated strips. It is difficult to assess cellular events that occur much faster than the force changes. A simple delay mechanism may explain the longer half-time of relaxation but not the shorter half-time of relaxation. This is why we decreased the temperature conditions in the relaxation experiments. These results suggest that cAMP/PKA signaling impairs the inhibition of MLCP-mediated responses by PKC signaling but not by the Rho/Rho-kinase signaling. Alternatively, this observation could have mechanistic implications; for example, one of many interpretations would be that 8-BrcAMP modulates activity of MLCP-associated mechanisms whereas Y-27632 modulates activation of MLCP-associated mechanisms, because 8-BrcAMP accelerates the rate of relaxation without changing the time of onset, whereas Y-27632 prolongs the onset of relaxing rate without changing its rate (Fig. 5, B and C).

Muscarinic receptor signaling for airway smooth muscle contraction contains Ca2+ sensitization mechanisms mediated by Rho/Rho-kinase (14, 28). Although CCh-induced Ca2+ sensitization was inhibited by both 8-BrcAMP and Y-27632 to a similar extent, GTP{gamma}S-induced Ca2+ sensitization was resistant to 8-BrcAMP. In permeabilized canine tracheal smooth muscle, both PDBu and acetylcholine induce Ca2+ sensitization (3, 4). Rho/Rho-kinase-mediated signaling may be distinct from the PKC system because the effects of saturating concentrations of GTP{gamma}S and PDBu were additive (9). Eto et al. (5) reported that PDBu-induced Ca2+ sensitization was partially inhibited by Y-27632 in {alpha}-toxin-permeabilized rabbit femoral artery. However, we previously reported only a minor contribution of PKC to GTP{gamma}S-induced Ca2+ sensitization in rabbit trachea. In our present study, PDBu-induced Ca2+ sensitization was not inhibited by Y-27632 (13), although the reason for the discrepancy is unknown.

In the present study, we demonstrated leukotriene-induced Ca2+ sensitization in human bronchial smooth muscle. Leukotrienes, as well as CCh, increased Ca2+ sensitivity in human bronchial smooth muscle, and leukotriene-induced Ca2+ sensitivity is reversed by 8-BrcAMP and Y-27632 (Fig. 6). Leukotrienes evoke a potent, sustained contraction of intact human airway smooth muscle (2, 24). Leukotrienes transiently increased Ca2+ sensitivity in {alpha}-toxin-permeabilized porcine tracheal smooth muscle (23). However, it is not known whether mechanisms of Ca2+ sensitization in the sustained contraction were evoked by leukotrienes. Leukotrienes increased contractile force by ~25%, and the LTD4-induced Ca2+ sensitization was inhibited by 8-BrcAMP and Y-27632 together in an additive manner. This is the first report stating that Ca2+ sensitization is present in human bronchial smooth muscle contraction induced by leukotrienes and that mechanisms of both 8-BrcAMP sensitivity and Y-27632 sensitivity are involved. The LTD4-induced contraction in human bronchial smooth muscle is at least in part independent of increases in Ca2+ sensitivity through activation of PKC (1). Thus inhibition of Rho/Rho-kinase signaling may become a second-line bronchodilator (in addition to {beta}2-stimulants) for resolving limited airflow in asthma. Further studies are required to determine the relative contributions of Rho/Rho-kinase and PKC/CPI-17 in human tissue (27).

In smooth muscle, receptor-dependent, trimetric G protein-mediated Ca2+ sensitization was inhibited by both 8-BrcAMP and Y-27632 (Fig. 1B). Theoretically, GTP{gamma}S may activate both trimetric and monometric G proteins, resulting in direct activation of Rho/Rho-kinase signaling and indirect activation of trimetric G protein-mediated PKC signaling. However, practically, PKC plays a minor role in GTP{gamma}S-induced Ca2+ sensitization in rabbit tracheal smooth muscle (13). Hepler et al. (8) showed that purified Gq had only a slight binding affinity to GTP{gamma}S. Additionally, AlF4 is known to activate the trimetric G protein. Contraction induced by AlF4 but not that induced by GTP{gamma}S was insensitive to GDP{beta}S (15, 28). These findings suggest that GTP{gamma}S mainly activates small G proteins, although the precise mechanism is still unknown.

The next question was why 8-BrcAMP effectively blocked CCh- or LTD4-induced Ca2+ sensitization, in which PKC signaling might be involved. In fibroblasts, 8-BrcAMP blocks Rho activation by phosphorylation of heterotrimetric G proteins (7, 18). Thus the cAMP/PKA system may inhibit CCh- and leukotriene-induced Ca2+ sensitization through two steps. First, the cAMP/PKA system may inhibit the muscarinic receptor signaling upstream of Rho activation. Second, the cAMP/PKA system might be present downstream of the PKC pathway (Fig. 7).



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Fig. 7. Signaling pathway for Ca2+ sensitization in smooth muscle. Activation of the muscarinic receptor (M3) initiates signaling through the illustrated cascades that inhibit MLCP, increasing MLC20 phosphorylation and contraction. The Rho/Rho-kinase and PKC/CPI-17 pathways inhibit the phosphorylation of MLCP and enhance the Ca2+ sensitivity of myosin phosphorylation. GTP{gamma}S increases the activity of G-binding proteins, including the heterotrimetric G protein and small G proteins such as Rho/Rho-kinase. PDBu increases the activity of PKC. GTP{gamma}S-induced Ca2+ sensitization is inhibited by Y-27632, but not by 8-BrcAMP. On the other hand, PDBu-induced Ca2+ sensitization was inhibited 8-BrcAMP, but not by Y-27632. Thus 8-BrcAMP has the potential to affect the PKC/CPI-17 pathway and upstream of the Rho/Rho-kinase pathway.

 
In conclusion, cAMP/PKA preferentially reverses PKC-mediated Ca2+ sensitization to Rho/Rho-kinase-mediated Ca2+ sensitization. Activation of the MLCP-associated system is a mechanism of cAMP/PKA-induced Ca2+ desensitization, and this is distinct from Rho/Rho-kinase inhibition in airway smooth muscle. Human bronchial smooth muscle has Ca2+ sensitivity mechanisms that respond not only to contractile agonists but also to cAMP/PKA elevating agents.


    ACKNOWLEDGMENTS
 
The authors thank Y. Ohtani for preparing human lung tissue samples.


    FOOTNOTES
 

Address for reprint requests and other correspondence: K. Dobashi, Dept. of Medicine and Molecular Science, Gunma Univ. Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan (E-mail: dobashik{at}med.gunma-u.ac.jp)

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.


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