Involvement of Ca 2 Signaling in the Synergistic Effects Between Muscarinic Receptor Antagonists and Β2-Adrenoceptor Agonists in Airway Smooth Muscle

International Journal of Molecular Sciences

Article Involvement of Ca2+ Signaling in the Synergistic Effects between Muscarinic Receptor Antagonists and β2-Adrenoceptor Agonists in Airway Smooth Muscle

Kentaro Fukunaga 1, Hiroaki Kume 2,*, Tetsuya Oguma 1, Wataru Shigemori 1, Yuji Tohda 2, Emiko Ogawa 1 and Yasutaka Nakano 1 1 Division of Respiratory Medicine, Department of Internal Medicine, Shiga University of Medical Science, Seta, Tsukinowa-cho, Otsu, Shiga 520-2192, Japan; [email protected] (K.F.); [email protected] (T.O.); [email protected] (W.S.); [email protected] (E.O.); [email protected] (Y.N.) 2 Department of Respiratory Medicine and Allergology, Kindai University Faculty of Medicine, 377-2 Ohnohigashi, Osakasayama, Osaka 589-8511, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-72-366-0221 (ext. 3602); Fax: +81-72-367-7772

Academic Editor: Kathleen Van Craenenbroeck Received: 1 August 2016; Accepted: 8 September 2016; Published: 21 September 2016

Abstract: Long-acting muscarinic antagonists (LAMAs) and short-acting β2-adrenoceptor agonists (SABAs) play important roles in remedy for COPD. To propel a translational research for development of bronchodilator therapy, synergistic effects between SABAs with LAMAs were examined focused on Ca2+ signaling using simultaneous records of isometric tension and F340/F380 in fura-2-loaded tracheal smooth muscle. Glycopyrronium (3 nM), a LAMA, modestly reduced methacholine (1 µM)-induced contraction. When procaterol, salbutamol and SABAs were applied in the presence of glycopyrronium, relaxant effects of these SABAs are markedly enhanced, and percent inhibition of tension was much greater than the sum of those for each agent and those expected from the BI theory. In contrast, percent inhibition of F340/F380 was not greater than those values. Bisindolylmaleimide, an inhibitor of protein kinase C (PKC), signiﬁcantly increased the relaxant effect of LAMA without 2+ + reducing F340/F380. Iberiotoxin, an inhibitor of large-conductance Ca -activated K (KCa) channels, signiﬁcantly suppressed the effects of these combined agents with reducing F340/F380. In conclusion, combination of SABAs with LAMAs synergistically enhances inhibition of muscarinic contraction 2+ 2+ via decreasing both Ca sensitization mediated by PKC and Ca dynamics mediated by KCa channels. PKC and KCa channels may be molecular targets for cross talk between β2-adrenoceptors and muscarinic receptors.

Keywords: cross talk; Ca2+ dynamics; Ca2+ sensitization; large-conductance Ca2+-activated K+ channel; protein kinase C; tracheal smooth muscle; tiotropium; procaterol; COPD

1. Introduction Chronic obstructive pulmonary disease (COPD) is characterized by irreversible narrowing of the small airways and decreased elastic recoil of the lung due to destruction of the alveoli [1,2]. As a result, hyperinflation occurs, causing dyspnea during exercise, and decreasing exercise tolerability in patients with COPD [3,4]. Although the fundamental pathophysiology of COPD is considered to be neutrophilic inflammation in the airways, there are currently no specific anti-inflammatory agents for this disease. Inhaled bronchodilators, such as muscarinic receptor antagonists and β2-adrenoceptor agonists, are widely used clinically to reduce symptoms (shortness of breath, wheezing), augment lung function (forced expiratory volume in 1 second, inspiratory capacity), and reduce exacerbations and hospitalizations, improving quality of life.

Int. J. Mol. Sci. 2016, 17, 1590; doi:10.3390/ijms17091590 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2016, 17, 1590 2 of 18

Inhalation of muscarinic receptor antagonists [5,6] and β2-adrenoceptor agonists [7,8] is used in the management of COPD. However, a single type of bronchodilator may be insufficiently effective for this disease. A recent COPD guideline states that a combination of bronchodilators of different pharmacological classes may improve effectiveness and decrease the risk of adverse reactions compared to increasing the dose of a single bronchodilator [2]. Long-acting β2-adrenoceptor agonists (LABAs) and long-acting muscarinic receptor antagonists (LAMAs) are two types of bronchodilators widely used as therapy for this disease, and there is a pharmacological rationale for the use of a combination of these agents [9–12]. Recent studies in patients with COPD have also demonstrated that the LABA/LAMA combination is more effective in improving symptoms and lung function, and reducing exacerbations, than monotherapy with either agent [13–16]. In airway smooth muscle, LABAs and LAMAs act synergistically, enhancing inhibition of muscarinic contraction more than the sum of the effects of each agent [9,10]. Cross talk between these two receptors may contribute to this synergistic effect in airway smooth muscle. Large-conductance 2+ + Ca -activated K (KCa) channels are regulated by Gs proteins coupled to β2-adrenoceptors [17–19] and Gi proteins coupled to muscarinic M2 receptors [18–21]. Since the functional antagonism between β2-adrenoceptor and muscarinic receptor action converges on KCa channels [22], this synergism may 2+ 2+ be caused by Ca dynamics (changes in concentration of intracellular Ca ) via KCa channel activity (Figure1)[ 23,24]. However, little is currently known about the involvement of Ca2+ sensitization in the mechanism underlying this synergy between these two G protein-coupled receptors (GPCRs), such as β2-adrenoceptors and muscarinic receptors. Various contractile agents such as acetylcholine cause smooth muscle contraction via GPCRs. Airway smooth muscle tone is regulated by Ca2+ dynamics and Ca2+ sensitization. The Ca2+ dynamics is a mechanism of airway smooth muscle contraction via activation of a Ca2+/calmodulin-dependent myosin light chain kinase and increase intracellular Ca2+ concentration via a Ca2+ influx through Ca2+ channels (Ca2+-dependent contraction). In contrast, since Phosphorylation (inactivation) of myosin phosphatase (MP) causes contraction as intracellular Ca2+ level unchanged, MP inhibition contributes to Ca2+ sensitization (Ca2+-independent contraction) [25]. MP is phosphorylated by Rho-kinase, which is an effector of the small G protein RhoA [26–28] and by C-kinase-potentiated protein phosphatase-1 inhibitor (CPI-17), which is another potential mediator regulated by protein kinase C (PKC) (Figure1)[ 29–31]. Ca2+ signaling in airway smooth muscle plays an essential role in the pathophysiology of asthma and COPD [9,10,32–37]. Inhalation of rapid- and short-acting bronchodilators such as short-acting β2-adrenoceptor agonists (SABAs) is useful for symptom relief in patients with COPD. Because of the synergistic effect of these two agents on bronchodilation, pre-administration of LAMAs may be effective for as-needed use of SABAs, such as salbutamol and procaterol. Therefore, there is a need for the development of pharmacological treatments for COPD that exploit the synergistic effects between LAMAs and SABAs. This study was designed to determine whether procaterol and salbutamol (SABAs) and glycopyrronium (a LAMA) act synergistically to relax contracted airway smooth muscle. We promoted this study on the assumption of involvement of the major pathway in Ca2+ dynamics and Ca2+ sensitization, which play a key role of contraction of airway smooth muscle. In particular, mechanisms underlying this phenomenon were examined with a focus on Ca2+ signaling, 2+ 2+ specifically Ca dynamics mediated by KCa channels and Ca sensitization mediated by Rho-kinase and PKC in airway smooth muscle. The functional antagonism between β2-adrenergic and muscarinic action converges on KCa channel currents, and Rho-kinase and PKC play key role in muscarinic contraction of airway smooth muscle. Int. J. Mol. Sci. Sci. 20162016,, 1717,, 1590 1590 3 of 18

Figure 1. The intracellular mechanisms of the synergistic effects between β2-adrenoceptor agonists Figure 1. The intracellular mechanisms of the synergistic effects between β2-adrenoceptor agonists and muscarinic receptorreceptor antagonists.antagonists. NotNot onlyonly CaCa2+2+ sensitization butbut alsoalso CaCa2+2+ dynamics contributes to the synergistic effect of β2-adrenoceptor agonists and muscarinic receptor antagonists (cross talk to the synergistic effect of β2-adrenoceptor agonists and muscarinic receptor antagonists (cross talk 2 2+ 2+2+ between ββ2-adrenoceptors-adrenoceptors and muscarinicmuscarinic receptors).receptors). In In Ca dynamic,dynamic, Ca Ca influxinﬂux by by VDCE is involved in thisthis phenomenon.phenomenon. VDCE VDCE is regulated by membrane potential via KCa channelchannel activity, activity, which is augmentedaugmented byby GGss coupledcoupled toto ββ22-adrenoceptor, in contrast, attenuated by GGii coupled to 2+2+ muscarinic M2 receptors (dual regulationregulation byby GG proteins).proteins). IncreasedIncreased intracellularintracellular CaCa concentrationconcentration causes contraction by activation of of MLCK MLCK via via Ca Ca2+2+/CAM/CAM processes processes (Ca (Ca2+-dependent2+-dependent contraction). contraction). In 2+ 2+ InCa Ca sensitization,sensitization, PKC PKC is isinvolved involved inin this this phenomenon. phenomenon. PKC PKC is is activated activated by by muscarinic MM33 receptors. PKC inhibits MP activity via CPI-17 processes. Inactivation of MP causes contraction by increased sensitivitysensitivity toto intracellularintracellular CaCa2+2+ (Ca2+-independent-independent contraction). contraction). CPI-17 CPI-17 MP MP is is activated by

PKC, in contrast, inhibited by PKA. KKCaCa channels and CPI-17 are key molecules for this synergisticsynergistic effect viavia the the cross cross talk talk between between these these two receptors.two receptors. This synergism This synergism may be causedmay be by caused Ca2+ dynamics by Ca2+ 2+ 2+ (tonedynamics with changes(tone with in concentration changes in ofconcentration intracellular Ca of )intracellular via KCa channel Ca activity) via reciprocallyKCa channel regulated activity 2+ 2+ byreciprocally G proteins regulated (Gs and Gbyi), G and proteins caused (G bys Caand Gsensitizationi), and caused (tone by without Ca sensitization changes in (tone concentration without 2+ 2+ ofchanges intracellular in concentration Ca ) via of CPI-17 intracellular reciprocally Ca ) regulatedvia CPI-17 byreciprocally protein kinases regulated (PKA by andprotein PKC). kinases KCa: 2+ + 2+ large-conductance(PKA and PKC). K CaCa: large-conductance-activated K channels. Ca2+-activated VDCE: voltage-dependent K+ channels. VDCE: Ca voltage-dependententry. ACh: acetylcolin, Ca2+ LAMA:entry. ACh: long-acting acetylcolin, muscarinic LAMA: receptorlong-acting antagonist. muscarinic AC: receptor adenylate antagonist. cyclase. PKA:AC: adenylate protein kinase cyclase. A. PKC:PKA: proteinprotein kinasekinase C. A. CPI-17: PKC: protein C-kinase-potentiated kinase C. CPI-17: protein C-kinase-potentiated phosphatase-1 inhibitor. protein CaM: phosphatase-1 calmodulin. MLCK:inhibitor. myosin CaM: lightcalmodulin. chain kinase. MLCK: MP: myosin myosin light phosphatase. chain kinase. MP: myosin phosphatase.

2. Results Results

2.1. Inhibitory Effects of Procaterol, Salbutamol, and Glycopyrronium onon TensionTension andand IntracellularIntracellular CaCa2+2+ Concentration in Contracted MuscleMuscle When procaterol procaterol (0.1–10 (0.1–10 nM) nM) and and salbutamol salbutamol (1–100 (1–100 nM) were nM) cumulatively were cumulatively applied appliedto strips toof stripstracheal of smooth tracheal muscle smooth pre-contracted muscle pre-contracted with MCh with (always MCh (always 1 μM throughout 1 µM throughout this study), this study), both bothinhibited inhibited MCh-induced MCh-induced contraction contraction and and reduced reduced F340/F380 F340/F380 in ina concentration-dependent a concentration-dependent manner. manner. However, when almost complete relaxation was achieved at thethe maximummaximum concentrationsconcentrations tested (procaterol, 10 10 nM; nM; SB, SB, 100 100 nM), nM), F340/F380 F340/F380 was was still still higher higher than than at the at thebasal basal level level (Figure (Figure 2a,b).2a,b). The Theconcentration–inhibition concentration–inhibition curve curve of procaterro of procaterroll and and salbutamol salbutamol for for MCh-induced MCh-induced tension tension was signiﬁcantlysignificantly dissociated dissociated from from the the curve curve for for F340/F380 F340/F380 (n =( n5, =p < 5, 0.05),p < and 0.05), the and reduction the reduction in tension in tensionwas significantly was signiﬁcantly greater than greater the thanreduction the reduction in F340/F380 in F340/F380 at each concentration at each concentration ≥1 nM (procaterol)≥1 nM (procaterol)and ≥10 nM and (salbutamol)≥10 nM (salbutamol) (Figure 2c,d). (Figure However,2c,d). However, when glycopyrronium when glycopyrronium (0.3–10 (0.3–10 nM) nM)was wascumulatively cumulatively applied applied to the to thestrips strips of oftracheal tracheal smooth smooth muscle muscle in in the the same same way, way, glycopyrronium inhibited MCh-induced contraction and reduced F340/F380 in in a a concentration-dependent manner. manner. The concentration–inhibition curve of glycopyrronium for MCh-induced tension was also

Int. J. Mol. Sci. 2016, 17, 1590 4 of 18

Int. J. Mol. Sci. 2016, 17, 1590 4 of 18 The concentration–inhibitionInt. J. Mol. Sci. 2016, 17, 1590 curve of glycopyrronium for MCh-induced tension was also dissociated4 of 18 fromdissociated the curve forfrom F340/F380 the curve ( nfor= 5,F340/F380p < 0.05), ( butn = the5, p reduction < 0.05), but in F340/F380the reduction was in signiﬁcantly F340/F380 was greater thansignificantlydissociated that in tension fromgreater at the each than curve concentrationthat for in tensionF340/F380 (0.1–3at each (n nM)=concentration 5, p (Figure < 0.05),3). (0.1–3but the nM) reduction (Figure 3). in F340/F380 was significantly greater than that in tension at each concentration (0.1–3 nM) (Figure 3).

Figure 2. Involvement 2+of Ca2+ dynamics 2+and Ca2+ sensitization in the relaxant effect of Figure 2. Involvement of Ca dynamics and Ca sensitization in the relaxant effect of β2-adrenoceptor 2+ 2+ agonists.βFigure2-adrenoceptor (a ,b2.) TypicalInvolvement agonists. samples (ofa, b of)Ca Typical continuous dynamics samples recording and of continuousCa of tensionsensitization recording and F340/F380in of thetension relaxant demonstrating and F340/F380effect of the demonstratingβ2-adrenoceptor the agonists. inhibitory (a,b ) effectTypical of samplesprocaterol of continuous(0.1–30 nM) recording (a) and ofsalbutamol tension and (1–100 F340/F380 nM) inhibitory effect of procaterol (0.1–30 nM) (a) and salbutamol (1–100 nM) (b) on MCh (1 µM)-induced (demonstratingb) on MCh (1 the μM)-induced inhibitory effectsmooth of muscleprocaterol contraction; (0.1–30 nM)(c,d )( aConcentration–response) and salbutamol (1–100 curve nM) smooth muscle contraction; (c,d) Concentration–response curve for procaterol (0.1–10 nM) (c) and for(b) procaterolon MCh (0.1–10(1 μM)-induced nM) (c) and smooth salbutamol muscle (1–100 contraction; nM) (d) in tension(c,d) Concentration–response (□) and F340/F380 (■) in 1curve μM salbutamol (1–100 nM) (d) in tension ( ) and F340/F380 ( ) in 1 µM MCh-precontracted smooth MCh-precontractedfor procaterol (0.1–10 smooth nM) (c )muscle. and salbutamol Resting (1–100state tension nM) (d )and in tension F340/F380 (□) and were F340/F380 taken as (■ )0%, in 1and μM muscle.thoseMCh-precontracted Resting in each state MCh-stimulated tension smooth and muscle. F340/F380state were Resting taken were state takenas 100%.tension as 0%,MCh, and and methacholine;F340/F380 those in eachwere PRO, MCh-stimulated taken procaterol; as 0%, andSB, state weresalbutamol.those taken in as each 100%. **** MCh-stimulated p MCh,< 0.0001; methacholine; *** p state< 0.001; were PRO,** p taken < 0.01; procaterol; as * p100%. < 0.05. SB,MCh, salbutamol. methacholine; **** pPRO,< 0.0001; procaterol; *** p < SB, 0.001; ** p

Figure 3. Involvement of intracellular Ca2+ dynamics in the relaxant effect of glycopyrronium. Figure(Figurea) Typical 3. Involvement3. Involvementsample of continuous of of intracellular intracellular recordingCa Ca2+ 2+of dynamics tension and in in F340/F380the the relaxant relaxant demonstrating effect effect of ofglycopyrronium. glycopyrronium.the inhibitory (a) Typicaleffect(a) Typical of sample glycopyrronium sample of continuous of continuous (0.3–10 recording recordingnM) on ofMCh of tension te nsion(1 μ andM)-induced and F340/F380F340/F380 contraction; demonstrating demonstrating (b) Concentration– the the inhibitory inhibitory effectresponseeffect of glycopyrronium of curveglycopyrronium for glycopyrronium (0.3–10 (0.3–10 nM) onnM) (0.1–10 MCh on (1nM)MChµM)-induced in (1 tension μM)-induced (□ contraction;) and contraction;F340/F380 (b) Concentration–response(■ ()b induced) Concentration– by MCh (1response μM). GB, curve glycopyrronium for glycopyrronium bromide; (0.1–10 MCh, methacholine.nM) in tension ** ( □p )< and 0.01; F340/F380 * p < 0.05. (■) induced by MCh curve for glycopyrronium (0.1–10 nM) in tension () and F340/F380 () induced by MCh (1 µM). (1 μM). GB, glycopyrronium bromide; MCh, methacholine. ** p < 0.01; * p < 0.05. GB, glycopyrronium bromide; MCh, methacholine. ** p < 0.01; * p < 0.05.

Int. J. Mol. Sci. 2016, 17, 1590 5 of 18

2.2. Effects of Procaterol and Salbutamol Combined with Glycopyrronium on Tension and Intracellular Ca2+ Concentration in Contracted Muscle Glycopyrronium (3 nM) reduced MCh-induced tension and F340/F380 by 7.2% ± 4.5% and 23.6% ± 4.0% (n = 5), respectively; procaterol (1 nM) reduced them by 37.0% ± 9.1% and 32.9% ± 5.4% (n = 5), respectively. When procaterol (1 nM) was applied to the MCh-precontracted strips in the presence of glycopyrronium (3 nM), inhibition of contraction and decrease in F340/380 were markedly enhanced, to 73.9% ± 8.2% and 55.3% ± 1.5% (n = 5), respectively (Figure4a,b). Under these conditions, inhibition of contraction was considerably greater than the sum of the effects of each agent (44.2% ± 10.3%, n = 5, p < 0.0001; Figure4b), and the values predicted by the Bliss independence (BI) theory (41.5% ± 8.8%, n = 5, p < 0.0001; Figure4b). A detailed discussion of the BI theory can be found in the method section. In contrast, reduction of F340/F380 was not significantly greater than the sum of the effect of each agent (59.1% ± 8.7%, n = 5; Figure4b) or the expected value by BI theory (50.4% ± 6.2%, n = 5; Figure3b). Next, we lowered the concentration of procaterol to 0.1 and 0.3 nM. At 0.1 nM, procaterol inhibited MCh-induced contraction by 3.4% ± 2.4% and reduced F340/F380 by 4.5% ± 3.2% (n = 5). However, in the presence of glycopyrronium (3 nM), those values were augmented to 31.2% ± 13.8% and 26.9% ± 8.1% (n = 5), respectively (Figure4c). Under these conditions, percent inhibition of contraction was also much greater than the sum of the values for each agent (10.6% ± 5.6%, n = 5, p < 0.001; Figure4c) and the expected value (10.3% ± 5.4%, n = 5, p < 0.01; Figure4c). In contrast, percent inhibition of F340/F380 was not increased significantly more than the sum of the values for each agent (30.8% ± 5.9%, n = 5; Figure4c) or the expected values (29.6% ± 5.2%, n = 5; Figure4c). Percent inhibition of contraction and F340/F380 for procaterol (0.3 nM) in MCh-precontracted tissue were 13.1% ± 11.0% and 11.4% ± 3.6% (n = 5), respectively. When procaterol (0.3 nM) was applied in the presence of glycopyrronium (3 nM), those values were augmented to 46.0% ± 7.7% and 38.5% ± 9.7% (n = 5), respectively (Figure4d). Percent inhibition of contraction by the two agents in combination was considerably greater than the sum of the values for each agent (20.3% ± 11.2%, n = 5, p < 0.001; Figure4d) and the expected value (19.4% ± 10.4%, n = 5, p < 0.001; Figure4d). In contrast, percent inhibition of F340/F380 was not significantly greater for the combination than the sum of the values for each agent (41.1% ± 5.3%, n = 5; Figure4d) or the expected value (36.5% ± 2.3%, n = 5; Figure4d). Furthermore, we examined the interaction between glycopyrronium and salbutamol, another SABA, in the same way. Salbutamol (10 nM) inhibited MCh-induced contraction and reduced F340/F380 by 60.8% ± 10.6% and 59.8% ± 8.0% (n = 5), respectively. When salbutamol (10 nM) was applied in the presence of glycopyrronium (3 nM), contraction was inhibited by 85.4% ± 6.2% and F340/F380 was reduced by 60.4% ± 8.4% (n = 5; Figure5a,b). The inhibition of contraction by salbutamol (10 nM) combined with glycopyrronium (3 nM) was significantly greater than the sum of the values for each agent (68.0% ± 9.3%, n = 5, p < 0.01; Figure5b) and the expected value by BI theory (63.8% ± 8.9%, p < 0.01; Figure4b). However, the combination of agents decreased F340/F380 much by less than the sum of each agent (86.1% ± 6.3%, n = 5, p < 0.0001; Figure5b) or the expected value ( 70.5% ± 5.0%, p < 0.05; Figure5b). The inhibitory effects of salbutamol (3 nM) on these measures were 24.6% ± 14.7% and 29.5% ± 7.0% (n = 5), respectively. When salbutamol (3 nM) was applied in the presence of glycopyrronium (3 nM), contraction was inhibited by 56.7% ± 15.3% and F340/F380 was reduced by 45.7% ± 10.9% (n = 5; Figure5c). Contraction was inhibited more than the sum of the effects of each agent (31.8% ± 13.7%, n = 5, p < 0.01; Figure5c) and the expected value (30.2% ± 12.7%, p < 0.01; Figure5c), but reduction of F340/F380 was not significantly greater than the sum the effects of each agent (55.8% ± 8.1%, n = 5; Figure5c) or expected value (48.1% ± 5.9%; Figure5c). Int. J. Mol. Sci. 2016, 17, 1590 6 of 18 Int. J. Mol. Sci. 2016, 17, 1590 6 of 18

Figure 4. Effects of combination of procaterolprocaterol andand glycopyrroniumglycopyrronium onon tensiontension andand intracellularintracellular CaCa2+2+ concentration inducedinduced by by muscarinic muscarinic activation. activation. (a) Typical (a) Typical traces oftraces tension of (tensionupper side (upperand F340/F380side and (F340/F380lower side ()lower showing side the) showing inhibitory the effect inhibitory of procaterol effect (1of nM)procaterol in the presence(1 nM) in of glycopyrroniumthe presence of (3glycopyrronium nM) on MCh (1 (3µ M)-inducednM) on MCh contraction; (1 μM)-induced (b–d) Percentcontraction; inhibition (b–d of) Percent tension inhibition (white columns) of tension and F340/F380(white columns) (black columns)and F340/F in380 1 µ M(black MCh-induced columns) contractionin 1 μM MCh-induced under the experimental contraction conditions under the of 1experimental nM procaterol conditions (b); 0.1 nM of 1 procaterol nM procaterol (c); and (b 0.3), 0.1 nM nM procaterol procaterol (d )(c in), and the presence0.3 nM procaterol of glycopyrronium (d) in the (3presence nM). Left of glycopyrronium, sum of percent (3 inhibition nM). Left, of sum tension of percent and F340/F380inhibition of by tension the two and agents; F340/F380Center by, the BI, expectedtwo agents; percent Center inhibition, BI, expected of tension percent and inhibition F340/F380 of tens calculatedion and by F340/F380 the Bliss calculated independence by the theory; Bliss Rightindependence, percent inhibitiontheory; Right of tension, percent and F340/F380inhibition withof tension the two an agentsd F340/F380 in combination. with the PRO, two procaterol;agents in GB,combination. glycopyrronium PRO, procaterol; bromide; MCh, GB, glycop methacholine.yrronium **** bromide;p < 0.0001; MCh, *** pmethacholine.< 0.001; ** p < **** 0.01. p < 0.0001; *** p < 0.001; ** p < 0.01. 2.3. Effects of Procaterol Combined with Tiotropium on Tension and Intracellular Ca2+ Concentration in Contracted2.3. Effects Muscleof Procaterol Combined with Tiotropium on Tension and Intracellular Ca2+ Concentration in Contracted Muscle We investigated whether the same synergistic effect was shown when tiotropium administered in combinationWe investigated with procaterol. whether the same synergistic effect was shown when tiotropium administered in combinationTiotropium with (1 nM)procaterol. reduced MCh-induced tension and F340/F380 by 2.9% ± 3.1% and 11.3%Tiotropium± 3.6% (n =(1 5), nM) respectively. reduced MCh-induced When procaterol tension (1 nM)and wasF340/F380 applied by to 2.9% the MCh-precontructed± 3.1% and 11.3% ± strips3.6% ( inn = the 5),presence respectively. of tiotropium When procaterol (1 nM), inhibition (1 nM) wa ofs contructionapplied to the and MCh-precontructed decrease in F340/F380 strips were in markedlythe presence enhanced, of tiotropium to 55.6% ±(1 7.1%nM), and inhibition 35.5% ± 5.5%of contruction (n = 5), respectively and decrease (Figure in6 a,b).F340/F380 Under thesewere conditions,markedly enhanced, inhibition to of55.6% contraction ± 7.1% and was 35.5% considerably ± 5.5% (n greater= 5), respectively than the sum(Figure of the6a,b). effects Under of these each agentconditions, (39.9% inhibition± 7.8%, n of= 5,contractp < 0.01;ion Figure was considerably6b), and the expected greater than value the ( 38.9% sum ±of7.9% the ,effectsn = 5, pof< each 0.01; Figureagent (39.9%6b). In ± contrast, 7.8%, n reduction= 5, p < 0.01; of F340/F380Figure 6b), wasand signiﬁcantlythe expected greatervalue (38.9% than the ± 7.9%, sum ofn the= 5, effectp < 0.01; of eachFigure agent 6b). (44.1%In contrast,± 7.6%, reductionn = 5, p of< 0.05,F340/F380 Figure was6b), butsignificantly that value greater was not than signiﬁcant the sum greater of the thaneffect the of expectedeach agent value (44.1% (38.1% ± 7.6%,± 3.8%, n = 5,n p= < 5, 0.05, Figure Figure6b). At6b), 0.3 but nM that procaterol, value was in not the significant presence of greater tiotropium than (1the nM), expected inhibition value of (38.1% contraction ± 3.8%, and n = decrease 5, Figure in 6b). F340/F380 At 0.3 werenM procaterol, augmented in to the42.6% presence± 16.4% of andtiotropium 26.0% ±(114.4% nM), (ninhibition= 5, Figure of6 c),contraction respectively. and Underdecrease these in conditions,F340/F380 percentwere augmented inhibition ofto contraction42.6% ± 16.4% was and also 26.0% much ± greater14.4% ( thann = 5, the Figure sum of6c), the respectively. values for eachUnder agent these (16.0% conditions,± 12.4% percent, n = 4, pinhibition< 0.05; Figure of contraction6c) and the was expected also much value greater (17.4% than± the14.8%, sumn of= 4,thep values< 0.05; for Figure each6 c).agent In contrast,(16.0% ± 12.4%, n = 4, p < 0.05; Figure 6c) and the expected value (17.4% ± 14.8%, n = 4, p < 0.05; Figure 6c). In contrast, percent inhibition of F340/F380 was not increased significantly more than the sum of the values for each agent (22.6% ± 3.2%, n = 4; Figure 6c) or the expected values (21.4% ± 2.9%, n = 4).

Int. J. Mol. Sci. 2016, 17, 1590 7 of 18 percent inhibition of F340/F380 was not increased signiﬁcantly more than the sum of the values for each agentInt. (22.6%J. Mol. Sci.± 20163.2%,, 17, 1590n = 4; Figure6c) or the expected values (21.4% ± 2.9%, n = 4). 7 of 18

Figure 5. Effects of salbutamol and glycopyrronium in combination on tension and intracellular Ca2+ Figure 5. Effects of salbutamol and glycopyrronium in combination on tension and intracellular Ca2+ concentration induced by muscarinic activation. (a) Typical sample record of tension (upper side) concentrationand F340/F380 induced (lower by muscarinic side) showing activation. the inhibitory (a) Typicaleffect of sa samplelbutamol record (10 nM) of in tension the presence (upper of side) and F340/F380glycopyrronium (lower side(3 nM)) showing against 1 theμM inhibitoryMCh-induced effect contraction; of salbutamol (b,c) Percent (10 inhibition nM) in the of tension presence of glycopyrronium(white columns) (3 nM) and against F340/F 1380µM (black MCh-induced columns) in 1 contraction; μM MCh-precontracted (b,c) Percent tissue inhibition incubated ofwith tension (white columns)3 nM salbutamol and F340/F380 (b) and 10 (blacknM salbutamol columns) (c) in in the 1 µ presenceM MCh-precontracted of glycopyrronium tissue (3 nM). incubated Left, sum with 3 nM salbutamolof percent ( binhibition) and 10 of nM tensio salbutamoln and F340/F380 (c) in by the the presence two agents; of glycopyrroniumCenter, expected percent (3 nM). inhibitionLeft, sum of of tension and F340/F380 calculated by the Bliss independence theory; Right, percent inhibition of percent inhibition of tension and F340/F380 by the two agents; Center, expected percent inhibition tension and F340/F380 with the two agents in combination. SB, salbutamol; GB, glycopyrronium of tension and F340/F380 calculated by the Bliss independence theory; Right, percent inhibition of bromide; MCh, methacholine; BI, Bliss independence. **** p < 0.0001;; ** p < 0.01; * p < 0.05. tension and F340/F380 with the two agents in combination. SB, salbutamol; GB, glycopyrronium bromide;2.4. Role MCh, of Ca methacholine;2+ Sensitization BI,in the Bliss Combined independence. Effects of Procaterol **** p < 0.0001; and Glycopyrronium ** p < 0.01; * p < 0.05. From results of our experiments (Figures 4–6), synergistic effect of LAMAs and SABAs were 2+ 2.4. Role ofinvolved Ca Sensitization not only Ca in2+ dynamics the Combined but also Effects Ca2+ of sensitization. Procaterol and Therefore, Glycopyrronium we investigated which RhoA/Rho kinase or PKC processes were involved more strongly in this synergistic effect. From resultsBisindolylmaleimide of our experiments (10 μM), (Figures a potent4–6 ),inhibito synergisticr of PKC, effect caused of LAMAs modest anddecreases SABAs in were 2+ 2+ involvedMCh-induced not only Ca tensiondynamics (2.7% ± 3.7%, but n also = 5) Caand F340/F380sensitization. (14.0% ± Therefore,6.6%, n = 5; Figure we investigated 7b, left panel), which RhoA/Rhoas did kinase 3 nM orglycopyrronium PKC processes (7.2% were ± 4.5% involved and 26.3% more ± 4.0%, strongly respectively; in this see synergistic Figures 3 and effect. 4). When Bisindolylmaleimideglycopyrronium (3 (10nM)µ wasM), applied a potent with inhibitor bisindolylmaleimide of PKC, caused (10 modestμM), contraction decreases was in inhibited MCh-induced tension (2.7%by 20.4%± ±3.7%, 10.7% nand= F340/F380 5) and F340/F380was reduced (14.0%by 19.9%± ± 6.6%,9.7% (nn = =5; Figure 5; Figure 7b, right7b, leftpanel). panel), The relaxant effect of glycopyrronium was increased in the presence of bisindolylmaleimide, and as did 3 nM glycopyrronium (7.2% ± 4.5% and 26.3% ± 4.0%, respectively; see Figures3 and4). the combination was more effective than the sum of the values for each agent (Figure 7b; p < 0.01). µ When glycopyrroniumIn contrast, the (3inhibitory nM) was effect applied of glycop withyrronium bisindolylmaleimide on F340/F380 was (10 notM), increased contraction by was inhibitedbisindolylmaleimide, by 20.4% ± 10.7% andand F340/F380was not greater was in reduced combination by 19.9%than the± sum9.7% of ( nthe= values 5; Figure for 7eachb, right agent panel). The relaxant(Figure effect 7b). Inof tension glycopyrronium and F340/F380, the was effects increased of adding in bisindolylmaleimide the presence of to bisindolylmaleimide, glycopyrronium and thewere combination consistent waswith those more of effective adding procaterol than the and sum salbutamol of the to values glycopyrronium for each (see agent Figures (Figure 4 7b; p < 0.01).and In 5). contrast, the inhibitory effect of glycopyrronium on F340/F380 was not increased by bisindolylmaleimide, and was not greater in combination than the sum of the values for each agent (Figure7b). In tension and F340/F380, the effects of adding bisindolylmaleimide to Int. J. Mol. Sci. 2016, 17, 1590 8 of 18 glycopyrronium were consistent with those of adding procaterol and salbutamol to glycopyrronium

(see FiguresInt. J.4 Mol. and Sci.5 ).2016, 17, 1590 8 of 18

2+ Figure 6.FigureEffects 6. Effects of combination of combination of procaterolof procaterol and and tiotropiumtiotropium on on tension tension and and intracellularintracellular Ca Ca2+ concentration induced by muscarinic activation. (a) A typical sample record of tension (upper side) concentration induced by muscarinic activation. (a) A typical sample record of tension (upper side) and and F340/F380 (lower side) showing the inhibitory effect of procaterol (1 nM) in the presence of F340/F380tiotropium (lower side(1 nM)) showing against 1 theμM inhibitoryMCh-induced effect contraction; of procaterol (b,c) Percent (1 nM) inhibition in the presence of tension of (white tiotropium (1 nM) againstcolumns) 1 µandM F340/F380 MCh-induced (black contraction;columns) in 1 (μbM,c )MCh-precontracted Percent inhibition tissue of tensionincubated (white with 1 columns)nM and F340/F380procaterol (black (b, n = columns) 5) and 0.3 nM in 1procaterolµM MCh-precontracted (c, n = 4) in the presence tissue of tiotropium incubated (1 withnM). Left 1 nM, sum procaterol of (b, n = 5)percent and 0.3 inhibition nM procaterol of tension ( cand, n F340/F380= 4) in the by presencethe two agents; of tiotropium Center, expected (1 nM). percentLeft inhibition, sum of of percent inhibitiontension of tension and F340/F380 and F340/F380 calculated by by the the two Bliss agents; independenceCenter, expectedtheory; Right percent, percent inhibition inhibition of of tension tension and F340/F380 with the two agents in combination. PRO, procaterol; TIO, tiotropium; MCh, and F340/F380 calculated by the Bliss independence theory; Right, percent inhibition of tension and methacholine; BI, Bliss independence. ** p < 0.01; * p < 0.05. F340/F380 with the two agents in combination. PRO, procaterol; TIO, tiotropium; MCh, methacholine; BI, Bliss independence.When procaterol ** (0.3p < 0.01;nM) and * p < glycopyrronium 0.05. (3 nM) were applied together in the presence of bisindolylmaleimide (10 μM), percent inhibition of tension was almost the same as in the Whenabsence procaterol of bisindolylmaleimide (0.3 nM) and glycopyrronium (Figure 7c). Howeve (3r, nM) reduction were appliedof F340/F380 together by procaterol in the presence with of glycopyrronium in the presence of bisindolylmaleimide was markedly less than in its absence bisindolylmaleimide (10 µM), percent inhibition of tension was almost the same as in the absence of (p < 0.01; Figure 7c). bisindolylmaleimideY-27632 (1 (Figure μM), a7 c).selective However, inhibitor reduction of Rho-kinase, of F340/F380 caused bysmall procaterol decreases within MCh-induced glycopyrronium in the presencetension of(5.1% bisindolylmaleimide ± 4.1%, n = 5) and wasF340/F380 markedly (5.0% less± 6.8%, than n in= its5; Figure absence 8a; ( pleft< 0.01;panel). Figure When7 c). Y-27632glycopyrronium (1 µM), a (3 selective nM) was inhibitorapplied with of Y-27632 Rho-kinase, (1 μM), causedcontraction small was decreasesreduced by 12.0% in MCh-induced ± 4.9% tension (5.1%(n = 5) ±and4.1%, F340/F380n = by 5) 26.5% and ± F340/F380 8.9% (n = 5; (5.0% Figure± 8a,6.8%, right panel).n = 5; The Figure relaxant8a; effect left of panel). When glycopyrroniumglycopyrronium in (3the nM) presence was of appliedY-27632 was with greater Y-27632 than without (1 µM), it, but contraction the combined was effect reduced was by not significantly greater than the sum of the effects of each agent (Figure 8a). Reduction in F340/F380 ± ± 12.0% 4.9%by glycopyrronium(n = 5) and F340/F380was also not bysignificantly 26.5% grea8.9%ter (inn =the 5; presence Figure 8ofa, Y-27632 right panel). than the The sum relaxant of effect of glycopyrroniumeach agent’s effects in(Figure the presence8a). Furthermore, of Y-27632 the effect was greaterof adding than Y-27632 without to glycopyrronium it, but the combined on effect wasMCh-induced not signiﬁcantly tension greater and F340/F380 than the was sum not of theconsis effectstent with of each that agent of adding (Figure procaterol8a). Reduction and in F340/F380salbutamol by glycopyrronium to glycopyrronium was (s alsoee Figures not signiﬁcantly 4 and 5). When greater procater in theol (0.3 presence nM) was of applied Y-27632 with than the sum of eachglycopyrronium agent’s effects (3 nM) (Figure in the8 a).presence Furthermore, of Y-27632the (1 μ effectM), inhibition of adding of MCh-induced Y-27632 to glycopyrronium contraction was almost the same as in the absence of Y-27632 (Figure 8b). Percent inhibition of F340/F380 for on MCh-induced tension and F340/F380 was not consistent with that of adding procaterol and procaterol with glycopyrronium in the presence of Y-27631 was markedly greater than in its absence salbutamol(p < to0.05; glycopyrronium Figure 8b). (see Figures4 and5). When procaterol (0.3 nM) was applied with glycopyrronium (3 nM) in the presence of Y-27632 (1 µM), inhibition of MCh-induced contraction was almost the same as in the absence of Y-27632 (Figure8b). Percent inhibition of F340/F380 for procaterol with glycopyrronium in the presence of Y-27631 was markedly greater than in its absence (p < 0.05; Figure8b). Int. J. Mol. Sci. 2016, 17, 1590 9 of 18 Int. J. Mol. Sci. 2016, 17, 1590 9 of 18

Figure 7. Involvement of protein kinase C-induced Ca2+ sensitization in synergy between Figure 7. Involvement of protein kinase C-induced Ca2+ sensitization in synergy between β2-adrenoceptor agonists and muscarinic receptor antagonists. (a) Typical record of tension (upper β -adrenoceptor agonists and muscarinic receptor antagonists. (a) Typical record of tension (upper side) 2 side) and F340/F380 (lower side) of the inhibitory effect in combination with procaterol (0.3 nM) and and F340/F380glycopyrronium (lower (3 sidenM) )in of the the presence inhibitory of bisindolylmaleimide effect in combination (10 μM) against with procaterol1 μM MCh-induced (0.3 nM) and glycopyrroniumcontraction. (3 In nM) each in panel, the presence percent inhibition of bisindolylmaleimide of tension is represented (10 µM) by against white 1columns,µM MCh-induced and contraction.F340/F380 In is eachrepresented panel, by percentblack columns; inhibition (b) Left, of percent tension inhibition is represented in tension and by F340/F380 white by columns, and F340/F380bisindolylmaleimide is represented (10 μ byM); black Center columns; , sum of ( bpercent) Left, percentinhibition inhibition of tension inand tension F340/F380 and with F340/F380 by bisindolylmaleimidebisindolylmaleimide (10 (10µ μM);M) andCenter glycopyrronium, sum of percent (3 nM); inhibitionRight, percent of tensioninhibition and of tension F340/F380 and with F340/F380 with combination of bisindolylmaleimide (10 μM) and glycopyrronium (3 nM); (c) Left, bisindolylmaleimide (10 µM) and glycopyrronium (3 nM); Right, percent inhibition of tension and sum of percent inhibition of tension and F340/F380 with procaterol (0.3 nM) and glycopyrronium F340/F380(3 nM); with Center combination, percent inhibition of bisindolylmaleimide of tension and F340/F380 (10 µ inM) combination and glycopyrronium with procaterol (3 (0.3 nM); nM) ( c) Left, sum ofand percent glycopyrronium inhibition (3 of nM); tension Right and, percent F340/F380 inhibitionwith of tension procaterol and F340/F380 (0.3 nM) with and combination glycopyrronium of (3 nM); Centerprocaterol, percent (0.3 nM) inhibition and glycopyrronium of tension (3 and nM F340/F380) in the presence in combination of bisindolylmaleimide with procaterol (10 μM). (0.3 nM) and glycopyrroniumGB, glycopyrronium (3 nM); bromide;Right ,PRO, percent procaterol; inhibition MC ofh, tensionmethacholine; and F340/F380 BIS, bisindolylmaleimide. with combination of procaterol**** (0.3p < 0.0001; nM) ** and p < 0.01; glycopyrronium * p < 0.05. (3 nM) in the presence of bisindolylmaleimide (10 µM). GB, glycopyrronium bromide; PRO, procaterol; MCh, methacholine; BIS, bisindolylmaleimide. 2.5. Role of Large-Conductance Ca2+-Activated K+ Channels in Ca2+ Dynamics Due to Procaterol and ****Salbutamolp < 0.0001; with ** p Glycopyrronium< 0.01; * p < 0.05.

Described above, KCa channel activity plays an important role in the functional antagonism 2.5. Role of Large-Conductance Ca2+-Activated K+ Channels in Ca2+ Dynamics due to Procaterol and between β2-adrenoceptors and muscarinic receptors [22]. Therefore, we investigated whether KCa Salbutamol with Glycopyrronium channel was involved in synergistic effect of LAMA and SABAs. In the presence of iberiotoxin, a selective antagonist of KCa channels, procaterol or salbutamol with glycopyrronium was applied to Described above, KCa channel activity plays an important role in the functional antagonism MCh-precontracted tissue (Figure 9a). Iberiotoxin (30 nM) caused modest increases in MCh-induced between β -adrenoceptors and muscarinic receptors [22]. Therefore, we investigated whether K tension2 and F340/F380, to 111.5% ± 3.6% and 126.5% ± 13.4%, respectively. When procaterol (1 nM) Ca channelwas was applied involved with inglycopyrronium synergistic effect(3 nM) of in LAMA the presence and SABAs.of iberiotoxin In the (30 presencenM), inhibition of iberiotoxin, of a selectivecontraction antagonist and F340/F380 of KCa channels, were markedly procaterol decreased, or salbutamol to 34.7% ± 14.6% with ( glycopyrroniump < 0.001) and 32.2% was ± 10.8% applied to MCh-precontracted(p < 0.01), respectively, tissue (Figure compared9a). to Iberiotoxin the values (30in the nM) absence caused iberiotoxin modest (Figure increases 9b). in Moreover, MCh-induced tensionwhen and F340/F380,salbutamol (10 to nM) 111.5% and glycopyrronium± 3.6% and (3 126.5% nM) were± 13.4%, applied respectively.in the same way, When values procaterol of (1 nM) waspercent applied inhibition with in glycopyrroniumcontraction and F340/F380 (3 nM) by in salbutamol the presence (10 nM) of iberiotoxin with glycopyrronium (30 nM), (3 inhibition nM) of in the presence of iberiotoxin (30 nM) were also markedly decreased to 36.5% ± 8.8% (p < 0.001) contraction and F340/F380 were markedly decreased, to 34.7% ± 14.6% (p < 0.001) and 32.2% ± 10.8% and 36.2% ± 19.6% (p < 0.05), respectively, compared to those values in the absence of iberiotoxin (p < 0.01),(Figure respectively, 9c). compared to the values in the absence iberiotoxin (Figure9b). Moreover, when salbutamol (10 nM) and glycopyrronium (3 nM) were applied in the same way, values of percent inhibition in contraction and F340/F380 by salbutamol (10 nM) with glycopyrronium (3 nM) in the presence of iberiotoxin (30 nM) were also markedly decreased to 36.5% ± 8.8% (p < 0.001) and 36.2% ± 19.6% (p < 0.05), respectively, compared to those values in the absence of iberiotoxin (Figure9c). Int. J. Mol. Sci. 2016, 17, 1590 10 of 18 Int. J. Mol. Sci. 2016, 17, 1590 10 of 18 Int. J. Mol. Sci. 2016, 17, 1590 10 of 18

2+ 2+ FigureFigure 8. Y-27632-induced 8. Y-27632-induced Ca Casensitization sensitization is is not not involvedinvolved inin synergysynergy between ββ2-adrenoceptor2-adrenoceptor Figure 8. Y-27632-induced Ca2+ sensitization is not involved in synergy between β2-adrenoceptor agonistsagonists and muscarinicand muscarinic receptor receptor antagonists. antagonists. In each In panel, each percentpanel, inhibitionpercent inhi ofbition tension of is tension represented is agonists and muscarinic receptor antagonists. In each panel, percent inhibition of tension is by whiterepresented columns, by white and F340/F380columns, and is representedF340/F380 is represented by black columns. by black (columns.a) Left, percent(a) Left, inhibitionpercent represented by white columns, and F340/F380 is represented by black columns. (a) Left, percent of tensioninhibition and ofF340/F380 tension and withF340/F380 Y-27632 with (1 Y-27632µM); Center(1 μM);, Center sum of, sum percent of percent inhibition inhibition of tensionof tension and inhibition of tension and F340/F380 with Y-27632 (1 μM); Center, sum of percent inhibition of tension F340/F380and F340/F380 with Y-27632 with Y-27632 (1 µM) (1 μ andM) and glycopyrronium glycopyrronium (3 (3 nM); nM);Right Right,, percentpercent inhibiti inhibitionon of of tension tension andand F340/F380 F340/F380 with with Y-27632 combination (1 μM) of and Y-27632 glycopyrronium (1 μM) and (3 glycopyrroniumnM); Right, percent (3 nM); inhibiti (b)on Left of ,tension sum of and F340/F380 with combination of Y-27632 (1 µM) and glycopyrronium (3 nM); (b) Left, sum of andpercent F340/F380 inhibition with of combination tension and of F340/F380 Y-27632 (1with μM) pr andocaterol glycopyrronium (0.3 nM) and (3 glycopyrronium nM); (b) Left, sum(3 nM); of percent inhibition of tension and F340/F380 with procaterol (0.3 nM) and glycopyrronium (3 nM); percentCenter ,inhibition percent inhibition of tension of and tension F340/F380 and F340/F380 with procaterol with combination (0.3 nM) and of glycopyrronium procaterol (0.3 nM)(3 nM); and Center, percent inhibition of tension and F340/F380 with combination of procaterol (0.3 nM) and Centerglycopyrronium, percent inhibition (3 nM); Rightof tension, percent and inhibitionF340/F380 ofwi tensionth combination and F340/F380 of procaterol with combination (0.3 nM) and of glycopyrronium (3 nM); Right, percent inhibition of tension and F340/F380 with combination of glycopyrroniumprocaterol (0.3 (3nM) nM); and Right glycopyrronium, percent inhibition (3 nM) of tensionin the andpresence F340/F380 of Y-27632 with combination (1 μM). GB,of µ procaterolprocaterolglycopyrronium (0.3 nM)(0.3 andnM) bromide; glycopyrroniumand PRO,glycopyrronium procaterol; (3 nM) MCh,(3 in thenM) methacholine. presence in the ofpresence Y-27632*** p < 0.001; of (1 Y-27632 M).** p < GB, 0.01; (1 glycopyrronium * μp M).< 0.05. GB, bromide;glycopyrronium PRO, procaterol; bromide; MCh, PRO, methacholine. procaterol; MCh, *** methacholine.p < 0.001; ** p ***< 0.01;p < 0.001; * p < ** 0.05. p < 0.01; * p < 0.05.

Figure 9. Involvement of large-conductance Ca2+-activated K+ channel in synergy between Figure 9. Involvement of large-conductance Ca2+-activated K+ +channel in synergy between Figureβ2-adrenoceptor 9. Involvement agonists of and large-conductance muscarinic receptor Ca antagonists.-activated (a) K Typicalchannel record in of synergy tension ( betweenupper β2-adrenoceptor agonists and muscarinic receptor antagonists. (a) Typical record of tension (upper β2-adrenoceptorside) and F340/F380 agonists (lower and side muscarinic) showing the receptor inhibitory antagonists. effect of a combination (a) Typical of procaterol record of (1 tension nM) (uppersideand side) andglycopyrronium) andF340/F380 F340/F380 (lower (3 (nM) lowerside )in showing sidethe )presen showing the inhibitoryce of the iberiotoxin inhibitory effect of (30a effect combination nM) of aon combination MCh of procaterol (1 μM)-induced of procaterol(1 nM) (1 nM)andcontraction; andglycopyrronium glycopyrronium (b) Percent (3 nM)inhibition (3 nM)in the in of thepresen tension presencece (whiteof iberiotoxin of iberiotoxincolumn) (30and (30nM) F340/F380 nM) on onMCh (black MCh (1 (1μcolumn)M)-inducedµM)-induced with contraction; (b) Percent inhibition of tension (white column) and F340/F380 (black column) with contraction;combination (b) Percentof procaterol inhibition (1 nM) and of tension glycopyrronium (white column) (3 nM) in and the absence F340/F380 (left) (black or presence column) (right with) combination of procaterol (1 nM) and glycopyrronium (3 nM) in the absence (left) or presence (right) combinationof iberiotoxin of procaterol (30 nM); ( (1c) nM)Percent and inhibition glycopyrronium of tension (3 (whi nM)tecolumn) in the absence and F340/F380 (left) or (black presence column) (right ) of iberiotoxin (30 nM); (c) Percent inhibition of tension (whitecolumn) and F340/F380 (black column) of iberiotoxinwith combination (30 nM); of ( csalbutamol) Percent inhibition(10 nM) an ofd glycopyrronium tension (whitecolumn) (3 nM) in and the absence F340/F380 (left (black) or presence column) with combination of salbutamol (10 nM) and glycopyrronium (3 nM) in the absence (left) or presence with( combinationright) of iberiotoxin of salbutamol (30 nM). (10 IbTX, nM) iberiotoxin; and glycopyrronium GB, glycopyrronium (3 nM) in bromide; the absence PRO, ( leftprocaterol;) or presence SB, (right) of iberiotoxin (30 nM). IbTX, iberiotoxin; GB, glycopyrronium bromide; PRO, procaterol; SB, (rightsalbutamol;) of iberiotoxin MCh, methacholine. (30 nM). IbTX, **** iberiotoxin;p < 0.0001; *** GB,p < 0.001; glycopyrronium ** p < 0.01; * p bromide;< 0.05. PRO, procaterol; salbutamol; MCh, methacholine. **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05. SB, salbutamol; MCh, methacholine. **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05. Int. J. Mol. Sci. 2016, 17, 1590 11 of 18

3. Discussion In the present study, GB (a LAMA) synergistically enhanced the inhibitory effect of procaterol and salbutamol (SABAs) on muscarinic contraction in airway smooth muscle via Ca2+ dynamics due 2+ to an inhibition in KCa channel activity, and Ca sensitization due to an increase in PKC activity. These results demonstrate that this synergistic effect of combination between a SABA and a LAMA is mediated by the intracellular processes of β2-adrenoceptors and muscarinic receptors shown in Figure1. We also observed similar synergistic effects between procaterol and tiotropium (another LAMA) in airway smooth muscle (Figure6). These results demonstrate that pre-administration of a LAMA causes a marked augmentation in the relaxant action of SABAs, and that Ca2+ signaling due to both Ca2+ dynamics and Ca2+ sensitization contributes to this synergistic effect (cross talk between β2-adrenoceptors and muscarinic receptors). Recent reports have demonstrated that indacaterol, a LABA, synergistically augments the inhibitory effects of glycopyrronium against muscarinic contraction in airway smooth muscle [9,10]. Moreover, synergistic effects are similarly observed between olodaterol, another LABA, and tiotropium [38]. Therefore, synergistic effects against muscarinic contraction may be a ubiquitous phenomenon between β2-adrenoceptor agonists and muscarinic receptor antagonists. Ca2+ signaling, mediated by Ca2+ dynamics via various channels and by Ca2+ sensitization due to the inactivation of MP via RhoA/Rho-kinase and PKC/CPI-17, contributes to airway smooth muscle tone induced by contractile agents via GPCRs, and other receptors [26–37,39–43]. The inhibitory effect of procaterol and salbutamol on MCh-induced contraction is mediated by reducing intracellular Ca2+ concentration; however, the reduction in contraction is greater than the reduction in intracellular Ca2+ concentration (Figure2). These results are consistent with those shown in a previous report using isoprenaline, a SABA [43], suggesting that procaterol and salbutamol relax airway smooth muscle by reducing both Ca2+ dynamics and Ca2+ sensitization, like isoprenaline. Conversely, glycopyrronium inhibited MCh-induced contraction while also reducing intracellular Ca2+ concentration, although the contraction was reduced less than intracellular Ca2+ concentration, unlike the SABAs in this study (Figure3). The relationship between tension and F340/F380 for glycopyrronium was consistent with that for SKF96365, a non-selective inhibitor of Ca2+ influx [43]. These results suggest that muscarinic receptor antagonists inhibit muscarinic contraction of airway smooth muscle by reducing Ca2+ dynamics. The role of Ca2+ signaling (dynamics and sensitization) in the inhibition of muscarinic contraction is not identical between β2-adrenoceptor activation and muscarinic receptor suppression (Figures2 and3). We also examined the effects of a combination of SABAs with glycopyrronium on MCh-induced tension and intracellular Ca2+ concentration. When the SABAs procaterol (0.1–1 nM) and salbutamol (1–10 nM) were applied in the presence of glycopyrronium, the relaxant effect was greater than the sum of the effects of the equivalent concentrations of each agent (Figures4 and5), similar to the combined relaxant effect of indecaterol (a LABA) and glycopyrronium shown in previous reports [9,10]. These results demonstrate that pre-exposure to a lower concentration of glycopyrronium markedly enhances the effect of SABAs. To confirm these synergistic effects between SABAs and glycopyrronium, the inhibitory effect of these agents on tension and F340/F380 was evaluated using the BI theory, used previously to evaluate the synergistic effect of bronchodilators [44,45]. The main assumption of this model is that two or more agents act independently in terms of their sites of action [46,47]. The theory is represented by a very simple mathematical equation, and experimental data (from single points to entire dose–response curves) are applied to the equation to investigate synergy [12,46,47]. The relaxant effects of the SABAs procatrol (0.1–1 nM) and salbutamol (1–10 nM) in combination with glycopyrronium are greater than the expected values calculated by BI theory in equivalent concentrations of each agent (Figures4 and5). These results demonstrate that the combination of SABAs with glycopyrronium synergistically inhibits muscarinic contraction. However, the reduction in F340/F380 ratio by procaterol (0.1–1 nM) and salbutamol (1–10 nM) in the presence of glycopyrronium is not greater than the sum of the effect of equivalent concentrations of each agent or the values Int. J. Mol. Sci. 2016, 17, 1590 12 of 18 calculated by BI theory in equivalent concentrations of each agent (Figures4 and5). These results demonstrate that an inhibition in Ca2+ sensitization contributes to this synergistic effect of SABAs with LAMAs against muscarinic contraction in airway smooth muscle. As described above, given that contractile agonists, which stimulate GPCRs, activate RhoA and PKC, this indicates that Ca2+ sensitization via RhoA/Rho-kinase or PKC/CPI-17 processes is involved in the mechanism underlying the synergistic effect between these receptors. The inhibitory effect of a combination of SABAs with glycopyrronium in tension and F340/F380 is mimicked by the addition of bisindolylmaleimide, a PKC antagonist, to glycopyrronium, but not by the addition of Y-27632, a Rho-kinase antagonist (Figures5 and6). These results demonstrate that an inhibition in PKC-induced Ca2+ sensitization is involved in this synergistic effect, whereas Rho-kinase-induced Ca2+ sensitization is not. This phenomenon is similar to that observed in a previous report showing that the SABA isoprenaline acts by inhibiting Ca2+ sensitization independently of RhoA/Rho-kinase processes [43]. Because muscarinic receptor agonists act on PKC, sensitivity to intracellular Ca2+ is augmented via activation of CPI-17, independent of RhoA/Rho-kinase processes. Activation of β2-adrenoceptors by binding of agonists such as SABA causes an increase of PKA activity. CPI-17 may be inhibited by PKA-induced phosphorylation. As a result, PKC-induced Ca2+ sensitization is attenuated by PKA [48]. Although it is still unclear about involvement of β2-adrenergic action in PKC-induced Ca2+ sensitization mediated by contractile agonists in airway smooth muscle, a recent report has demonstrated that PKA suppresses (phosphorylates) CPI-17 in endothelial cells [49]. This finding may support our data that β2-adrenergic relaxation is mediated by an inhibition of PKC-induced Ca2+ sensitization. Hence, PKC could be a target protein for the cross talk based 2+ on Ca sensitization between β2-adrenoceptors and muscarinic receptors. From results of our study, SABA may attenuate PKC-induced Ca2+-sensitization via activation of PKA. To clarify this phenomenon, further investigation will be needed. Although muscarinic receptor activation causes contraction by increasing both Ca2+ dynamics and Ca2+ sensitization [26,43], muscarinic blockade is mediated by affecting Ca2+ dynamics (reducing intracellular Ca2+ concentration), not by affecting Ca2+ sensitization (Figure3). Hence, muscarinic receptor antagonists might enhance the relaxant effects of SABAs via mechanisms other than muscarinic 2+ 2+ blockade-induced Ca dynamics. LAMAs may act on β2-adrenoceptors via PKC-induced Ca sensitization, leading to this synergistic effect between these two receptors [38]. Given that KCa channel activity plays an important role in the functional antagonism between β2-adrenoceptors and muscarinic receptors [22], intracellular processes concerning these channels may be involved in this synergistic effect [17,18,20,22,50]. The G protein/KCa channel processes may contribute to an increase in response to muscarinic receptor agonists (airway hyperresponsiveness) [22,23] and a decrease in response to β2-adrenoceptor agonists (β2-adrenoceptor desensitization) [51,52]. We examined involvement of these processes in this synergistic effect. In the presence of iberiotoxin, a selective KCa channel inhibitor, the relaxant effects of the SABA/glycopyrronium combination were markedly attenuated with an increase in F340/F380 (Figure7). As shown in Figures4–6, Ca 2+ dynamics might not be involved in the synergistic effect between SABAs and glycopyrronium. However, these results indicate that a reduction in intracellular 2+ Ca concentration by KCa channel activation also causes this synergistic effect between these two receptors (Figure1), consistent with the combination of indacaterol with glycopyrronium [ 9,10]. Because the outward currents are markedly increased by KCa channel opening, the membrane potential is hyperpolarized, leading to decreasing voltage-dependent Ca2+ entry (VDCE), such as L-type 2+ voltage-dependent Ca channels [9,10,23] (Figure1). Therefore, an inhibition in the G i/KCa channel inhibitory linkage and an augmentation in the Gs/KCa channel stimulatory linkage are thought to be another functional intracellular mechanism of this synergistically relaxant effect of combination of β2-adrenoceptor agonists with muscarinic receptor antagonists. KCa channels also could be a target protein for the cross talk based on Ca2+ dynamics between these two GPCRs. Int. J. Mol. Sci. 2016, 17, 1590 13 of 18

4. Experimental Section

4.1. Tissue Preparation and Bathing Solution Tracheae were excised from male Hartley guinea pigs (300–350 g) after intraperitoneal injection of pentobarbital (150 mg/kg). The tracheal rings were opened by cutting longitudinally at the cartilaginous region, and the epithelium was peeled off carefully. Normal bathing solution (in mM: NaCl 137, KHCO3 5.9, CaCl2 2.4, MgCl2 1.2, and glucose 11.8) bubbled with a mixture of 99% O2 and 1% CO2 (pH 7.4) was perfused at a constant ﬂow of 4 mL/min. Organ bath temperature was maintained at 37 ◦C. The animal protocols used in this work were evaluated and approved by the Research Center for Animal Life Science Committee, Shiga University of Medical Science (identiﬁcation code: 2013-4-1, 15 April 2013).

4.2. Isometric Tension Recording and Measurement of Fura-2 Fluorescence This was conducted as described previously [26,43,53]. In brief, muscle strips were incubated with 15 µM fura-2/AM for about 2 h at room temperature (22–24 ◦C). The non-cytotoxic detergent Pluronic F-127 (0.01% w/v) was added to increase the solubility of fura-2/AM. After the fura-2/AM had penetrated the tissue, the experimental chamber was filled with normal bathing solution. Isometric tension and fura-2 fluorescence of muscle strips were measured simultaneously, using a displacement transducer and a spectrofluorometer (CAF-110, Japan Spectroscopic, Tokyo, Japan). The mucosal side of the smooth muscle was exposed to excitation light, and the intensities of excitation fluorescence at 340 nm and 380 nm were measured after background subtraction. Because the dissociation constant of fura-2 for Ca2+ in smooth muscle cytoplasm was shown to be different from that measured in vitro, the absolute amount of intracellular Ca2+ concentration was not calculated [54]. Therefore, the ratio of F340 to F380 (F340/F380) was used as a relative indicator of intracellular Ca2+ concentration. Methacholine (MCh, 1 µM) was administered for 5 min at 10 min intervals until the control response to 1 µM MCh was established, and then the experiment was started. Muscle tension and F340/F380 in the resting state were taken as 0%, and the value of percent inhibition of tension and F340/F380 were measured taking the control response to MCh (1 µM) as 100%. Indomethacin (2 µM) was administered throughout the experiments to abolish resting tone.

4.3. Experimental Protocol To examine the relationship between muscle tone and intracellular Ca2+ concentration in the presence of β2-adrenoceptor agonists or muscarinic receptor antagonists, procaterol (0.1–10 nM), salbutamol (1–100 nM) or glycopyrronium (0.3–10 nM) were each administered cumulatively to tissue precontracted with MCh (1 µM throughout the study). To examine this relationship in the presence of a combination of β2-adrenoceptor agonists and muscarinic receptor antagonists, procaterol (0.1–1 nM) and salbutamol (1–10 nM) were added to MCh-precontracted tissue in the presence of glycopyrronium (3 nM), and procaterol (0.3–1 nM) was added to MCh-precontracted tissue in the presense of tiotropium 2+ (1 nM). To investigate the role of Ca dynamics due to KCa channel activity in the synergistic effects of β2-adrenoceptor agonists and muscarinic receptor antagonists, procaterol and glycopyrronium were added together to MCh-precontracted tissue, in the presence of iberiotoxin (30 nM), a selective inhibitor of KCa channels, which are densely distributed on airway smooth muscle cell membrane [55]. Moreover, to investigate the role of Ca2+ sensitization due to Rho-kinase and PKC in the synergistic effects of β2-adrenoceptor agonists and muscarinic receptor antagonists, procaterol and glycopyrronium were both added to MCh-precontracted tissue in the presence of Y-27632 (1 µM), a selective inhibitor of Rho-kinase, and bisindolylmaleimide (10 µM), a potent inhibitor of PKC. Each reagent at each concentration was administered for 15 min (except MCh). Time-matched control tissues were treated similarly to the test tissues, but exposed continuously to the normal bathing solution (sham incubation) instead of procaterol, salbutamol, iberiotoxin, Y-27632, or bisindolylmaleimide. Int. J. Mol. Sci. 2016, 17, 1590 14 of 18 Int. J. Mol. Sci. 2016, 17, 1590 14 of 18 4.4. Analysis of Synergistic Effect 4.4. Analysis of Synergistic Effect The synergistic effect of glycopyrronium and procaterol or salbutamol and that of tiotropium The synergistic effect of glycopyrronium and procaterol or salbutamol and that of tiotropium and procaterol on contracted airway smooth muscle and change in intracellular Ca2+ concentration and procaterol on contracted airway smooth muscle and change in intracellular Ca2+ concentration was evaluated using the BI theory. This model assumes that two or more agents act independently, was evaluated using the BI theory. This model assumes that two or more agents act independently, with different modes and sites of action [46], and is expressed by the following equation: with different modes and sites of action [46], and is expressed by the following equation: E(x,E(x, y )y) = = E(E(xx)) ++ E(Ey)( y−) E(−xE) (×x )E(×y)E (y) (1)(1) wherewhere E E is is the the fractional fractional effect, effect, and and xx andand yy areare the the doses doses of of two two compounds compounds in in a a combination combination experiment.experiment. If If the the combined combined experimental experimental value value is is higher higher than than the the expected expected value, value, the the interaction interaction is is synergistic.synergistic. If If it it is is lower, lower, the the interaction interaction is is antagonistic antagonistic [46,47]. [46,47].

4.5.4.5. Materials Materials MCh,MCh, indomethacin, indomethacin, procaterol, salbutamol,salbutamol, and and glycopyrronium glycopyrronium were were obtained obtained from from Wako Wako pure pureChemical Chemical Industries Industries (Osaka, (Osaka Japan)., Japan). iberiotoxin iberiotoxin was obtainedwas obtained from Peptidefrom Peptide Institute Institute (Osaka, (Osaka, Japan). Japan).Tiotropium, Tiotropium, bisindolylmaleimide bisindolylmaleimide and Y-27632 and Y-2763 were2 obtainedwere obtained from Sigmafrom Sigma (St. Louis, (St. Louis, MO, USA).MO, USA).Fura 2-AMFura 2-AM was obtained was obtained from Dojinfrom Do Laboratoriesjin Laboratories (Kumamoto, (Kumamoto, Japan). Japan).

4.6.4.6. Statistical Statistical Analysis Analysis AllAll data data areare expressedexpressed as as the the mean mean± standard ± standard deviation deviation (SD). (SD). Statistical Statistical signiﬁcance significance was assessed was assessedby t-test by or Student’st-test or Student’s unpaired unpairedt-test and t-test one-way and analysisone-way of analysis variance. of pvariance.< 0.05 was p < considered0.05 was consideredsigniﬁcant. significant. Statistical Statistical analyses analyses were performed were performed using JMP using version JMP version 11 (SAS 11 Institute(SAS Institute Inc., Inc., Cary, Cary,NC, USA).NC, USA).

FigureFigure 10. ClinicalClinical effectivenesseffectiveness of of Combination Combination of β 2of-adrenoceptor β2-adrenoceptor agonists agonists and muscarinic and muscarinic receptor receptorantagonists antagonists is in the treatmentis in the fortreatment COPD viafor CaCOPD2+ signaling via Ca (Ca2+ signaling2+ dynamics (Ca and2+ dynamics Ca2+ sensitization). and Ca2+

sensitization).Since addition Since of β 2-adrenoceptoraddition of β2 agonists-adrenoceptor to muscarinic agonists receptor to muscarinic antagonists receptor markedly antagonists enhance markedlyan inhibition enhance of airway an inhibition smooth muscleof airway contraction, smooth muscle combination contraction, of these combination two agents of are these useful two to agentssuppression are useful of excessive to suppression stimulation of toexcessive muscarinic stimulation receptors to induced muscarinic by acetylcholine receptors induced production by acetylcholinein the airways, production which is anin fundamentalthe airways, characteristicwhich is an fundamenta for COPD. Thisl characteristic phenomenon for isCOPD. due to This cross 2+ 2+2+ phenomenontalk between is these due two to receptorscross talk via between KCa channel-induced these two receptors Ca dynamics via KCa andchannel-induced PKC-induced Ca Ca dynamicssensitization and in PKC-induced airway smooth Ca muscle.2+ sensitization Therefore, in thisairway combination smooth therapymuscle. leadsTherefore, to reducing this combinationsymptoms suchtherapy as dyspnea leads to onreducing exertion symptoms and frequency such as of dyspnea exacerbations, on exertion and to and improving frequency health of exacerbations,status and lung and function to improving in patients health with status COPD. and lung function in patients with COPD.

5. Conclusions

Int. J. Mol. Sci. 2016, 17, 1590 15 of 18

5. Conclusions

The combination of β2-adrenoceptor agonists with muscarinic receptor antagonists causes a synergistic inhibition of muscarinic contraction in airway smooth muscle. Ca2+ signaling via 2+ 2+ PKC-induced Ca sensitization and KCa channel-induced Ca dynamics play a functional role in this synergistic effect (Figure1). K Ca channels and PKC are key protein for cross talk between β2-adrenoceptors and muscarinic receptors (Figure 10). Since it is generally considered that acetylcholine production (both neural and non-neural) in the airways is an essential pathophysiology for COPD, the effectiveness of therapy for this disease is depend on inhibitory action against excessive muscarinic stimulation. Hence, combination between these two agents is beneﬁcial to reducing symptoms and exacerbations, and to improving health status and lung function (Figure 10). Our results provide further evidence that the combination of β2-adrenoceptor agents and anti-cholinergic agents are a promising advancement in bronchodilator therapy for COPD and asthma, and that PKC and KCa channels may be novel targets for bronchodilator research and development.

Acknowledgments: This work is supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (H.K. #25461201), and by Otsuka Pharmaceuticals (Tokyo, Japan). Author Contributions: Kentaro Fukunaga, Hiroaki Kume and Tetsuya Oguma conceived and designed the experiments; Kentaro Fukunaga and Wataru Shigemori performed the experiments; Kentaro Fukunaga and Hiroaki Kume wrote the paper; Yasutaka Nakano, Emiko Ogawa and Yuji Tohda contributed reagents/materials/analysis tools. Conflicts of Interest: This work was partly supported by Otsuka Pharmaceutical Co., Ltd. (Tokyo, Japan) (Hiroaki Kume, Tetsuya Oguma, Yasutaka Nakano). In addition, this study was funded, in part, by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (Hiroaki Kume #25461201). Otsuka Pharmaceutical Co., Ltd. (Tokyo, Japan) had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

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