Bovine Tracheal Smooth Muscle Research Articles

Purification of Myosin from Bovine Tracheal Smooth Muscle, Filament Formation and Endogenous Association of Its Regulatory Complex.

Dynamic regulation of myosin filaments is a crucial factor in the ability of airway smooth muscle (ASM) to adapt to a wide length range. Increased stability or robustness of myosin filaments may play a role in the pathophysiology of asthmatic airways. Biochemical techniques for the purification of myosin and associated regulatory proteins could help elucidate potential alterations in myosin filament properties of asthmatic ASM. An effective myosin purification approach was originally developed for chicken gizzard smooth muscle myosin. More recently, we successfully adapted the procedure to bovine tracheal smooth muscle. This method yields purified myosin with or without the endogenous regulatory complex of myosin light chain kinase and myosin light chain phosphatase. The tight association of the regulatory complex with the assembled myosin filaments can be valuable in functional experiments. The purification protocol discussed here allows for enzymatic comparisons of myosin regulatory proteins. Furthermore, we detail the methodology for quantification and removal of the co-purified regulatory enzymes as a tool for exploring potentially altered phenotypes of the contractile apparatus in diseases such as asthma.

Second M3 muscarinic receptor binding site contributes to bronchoprotection by tiotropium.

The bronchodilator tiotropium binds not only to its main binding site on the M3 muscarinic receptor but also to an allosteric site. Here, we have investigated the functional relevance of this allosteric binding and the potential contribution of this behaviour to interactions with long-acting β-adrenoceptor agonists, as combination therapy with anticholinergic agents and β-adrenoceptor agonists improves lung function in chronic obstructive pulmonary disease. ACh, tiotropium, and atropine binding to M3 receptors were modelled using molecular dynamics simulations. Contractions of bovine and human tracheal smooth muscle strips were studied. Molecular dynamics simulation revealed extracellular vestibule binding of tiotropium, and not atropine, to M3 receptors as a secondary low affinity binding site, preventing ACh entry into the orthosteric binding pocket. This resulted in a low (allosteric binding) and high (orthosteric binding) functional affinity of tiotropium in protecting against methacholine-induced contractions of airway smooth muscle, which was not observed for atropine and glycopyrrolate. Moreover, antagonism by tiotropium was insurmountable in nature. This behaviour facilitated functional interactions of tiotropium with the β-agonist olodaterol, which synergistically enhanced bronchoprotective effects of tiotropium. This was not seen for glycopyrrolate and olodaterol or indacaterol but was mimicked by the interaction of tiotropium and forskolin, indicating no direct β-adrenoceptor-M3 receptor crosstalk in this effect. We propose that tiotropium has two binding sites at the M3 receptor that prevent ACh action, which, together with slow dissociation kinetics, may contribute to insurmountable antagonism and enhanced functional interactions with β-adrenoceptor agonists.

Phosphodiesterase 9 (PDE9) regulates bovine tracheal smooth muscle relaxation.

The present study was designed to clarify phosphodiesterase 9 (PDE9) expression in bovine tracheal smooth muscle tissue, and to elucidate that PDE9 may contribute to the regulation of airway relaxation. PDE9 mRNA expression was detected in bovine tracheal smooth muscle. Sodium nitroprusside (an NO donor) and BAY 73-6691 (a selective PDE9 inhibitor) reduced high K+- and carbachol-induced contraction. BAY 73-6691 relaxed tracheal tissue on the same level with vardenafil (a selective PDE5 inhibitor). These results support our hypothesis that PDE9 plays functional role in the tracheal smooth muscle relaxation. PDE9 inhibitors are expected to be a novel target of the add-on treatment of airway hyperresponsiveness.

Imidazole-induced contractions in bovine tracheal smooth muscle are not dependent on the cAMP pathway.

The mechanism of imidazole-induced contraction on the bovine tracheal smooth muscle was investigated. Imidazole induced muscle contraction in a concentration-dependent manner on bovine, porcine and guinea-pig tracheas, but not in rat or mouse. In bovine tracheas, imidazole was cumulatively applied and induced muscle tension and increasesd intracellular Ca2+ level in a concentration -dependent manner. Imidazole, even at 300 µM, the concentration at which maximum contractile response occurs, did not significantly increase in cAMP content relative to control. Atropine inhibited imidazole-induced contraction at a concentration- dependent manner and pretreatment of hemicholinium-3 almost abolished imidazole-induced contraction. Conversely, pretreatment of tripelennamine, indomethacin or tetrodotoxin did not affect imidazole-induced contraction. Acetylcholine or eserine induced contraction in bovine, porcine, guinea pig, rat and mice trachea in a concentration-dependent manner. However, there was little difference in the rank order of maximum contraction of these agents. Imidazole-induced contraction was greater in bovine trachea compared to the other species tested. Further, cAMP did not appear to play a role in imidazole-induced contraction, suggesting other mechanisms, such as the release of endogenous acetylcholine.

Physiological vs. pharmacological signalling to myosin phosphorylation in airway smooth muscle.

Smooth muscle myosin regulatory light chain (RLC) is phosphorylated by Ca2+ /calmodulin-dependent myosin light chain kinase and dephosphorylated by myosin light chain phosphatase (MLCP). Tracheal smooth muscle contains significant amounts of myosin binding subunit 85 (MBS85), another myosin phosphatase targeting subunit (MYPT) family member, in addition to MLCP regulatory subunit MYPT1. Concentration/temporal responses to carbachol demonstrated similar sensitivities for bovine tracheal force development and phosphorylation of RLC, MYPT1, MBS85 and paxillin. Electrical field stimulation releases ACh from nerves to increase RLC phosphorylation but not MYPT1 or MBS85 phosphorylation. Thus, nerve-mediated muscarinic responses in signalling modules acting on RLC phosphorylation are different from pharmacological responses with bath added agonist. The conditional knockout of MYPT1 or the knock-in mutation T853A in mice had no effect on muscarinic force responses in isolated tracheal tissues. MLCP activity may arise from functionally shared roles between MYPT1 and MBS85, resulting in minimal effects of MYPT1 knockout on contraction. Ca2+ /calmodulin activation of myosin light chain kinase (MLCK) initiates myosin regulatory light chain (RLC) phosphorylation for smooth muscle contraction with subsequent dephosphorylation for relaxation by myosin light chain phosphatase (MLCP) containing regulatory (MYPT1) and catalytic (PP1cδ) subunits. RLC phosphorylation-dependent force development is regulated by distinct signalling modules involving protein phosphorylations. We investigated responses to cholinergic agonist treatment vs. neurostimulation by electric field stimulation (EFS) in bovine tracheal smooth muscle. Concentration/temporal responses to carbachol demonstrated tight coupling between force development and RLC phosphorylation but sensitivity differences in MLCK, MYPT1 T853, MYPT1 T696, myosin binding subunit 85 (MBS85), paxillin and CPI-17 (PKC-potentiated protein phosphatase 1 inhibitor protein of 17 kDa) phosphorylations. EFS increased force and phosphorylation of RLC, CPI-17 and MLCK. In the presence of the cholinesterase inhibitor neostigmine, EFS led to an additional increase in phosphorylation of MYPT1 T853, MYPT1 T696, MBS85 and paxillin. Thus, there were distinct pharmacological vs. physiological responses in signalling modules acting on RLC phosphorylation and force responses, probably related to degenerate G protein signalling networks. Studies with genetically modified mice were performed. Expression of another MYPT1 family member, MBS85, was enriched in mouse, as well as bovine tracheal smooth muscle. Carbachol concentration/temporal-force responses were similar in trachea from MYPT1SM+/+ , MYPT1SM-/- and the knock-in mutant mice containing nonphosphorylatable MYPT1 T853A with no differences in RLC phosphorylation. Thus, MYPT1 T853 phosphorylation was not necessary for regulation of RLC phosphorylation in tonic airway smooth muscle. Furthermore, MLCP activity may arise from functionally shared roles between MYPT1 and MBS85, resulting in minimal effects of MYPT1 knockout on contraction.

Regulation of actin dynamics by WNT-5A: implications for human airway smooth muscle contraction.

A defining feature of asthma is airway hyperresponsiveness (AHR), which underlies the exaggerated bronchoconstriction response of asthmatics. The role of the airway smooth muscle (ASM) in AHR has garnered increasing interest over the years, but how asthmatic ASM differs from healthy ASM is still an active topic of debate. WNT-5A is increasingly expressed in asthmatic ASM and has been linked with Th2-high asthma. Due to its link with calcium and cytoskeletal remodelling, we propose that WNT-5A may modulate ASM contractility. We demonstrated that WNT-5A can increase maximum isometric tension in bovine tracheal smooth muscle strips. In addition, we show that WNT-5A is preferentially expressed in contractile human airway myocytes compared to proliferative cells, suggesting an active role in maintaining contractility. Furthermore, WNT-5A treatment drives actin polymerisation, but has no effect on intracellular calcium flux. Next, we demonstrated that WNT-5A directly regulates TGF-β1-induced expression of α-SMA via ROCK-mediated actin polymerization. These findings suggest that WNT-5A modulates fundamental mechanisms that affect ASM contraction and thus may be of relevance for AHR in asthma.

Regulation Of Actin Dynamics By WNT‐5A: Implications For Human Airway Smooth Muscle Contraction

An important pathophysiological feature of asthma is airway hyperresponsiveness (AHR), characterized by exaggerated bronchoconstriction in which the airway smooth muscle (ASM) is fundamentally involved. How the ASM in asthmatics differs from that in non‐asthmatics is a current focus for research. We have shown previously that WNT‐5A is increasingly expressed in ASM of mild to moderate asthmatics. WNT proteins have been implicated in the regulation of cytoskeletal filaments and microtubules, thus, we tested the hypothesis that WNT‐5A is a determinant of the contractile response of ASM through modulation of the cytoskeleton.Using bovine tracheal smooth muscle strips, we found that following pre‐incubation with recombinant WNT‐5A (500 ng/mL, 48 h), maximum isometric tension induced by histamine was increased (Emax = 1,67 fold of control, p < 0.01). We used immortalized human ASM cells to study the underlying mechanisms. In myocytes loaded with the ratiometric calcium indicator, Fura‐2, we found that neither direct exposure to WNT‐5A nor pre‐incubation (48 h) had any effect on calcium flux handling. Moreover, WNT‐5A treatment for 24 h did not yield any detectable change in mRNA expression of the major calcium channels or receptors (IP3R, SERCA2 and RyR1/2/3). However, while direct exposure to WNT‐5A (10–200 ng/mL, 24 h) had no effect on total alpha smooth muscle actin (α‐SMA) protein, it did lead to increased abundance of filamentous actin (labelled with fluorochrome‐conjugated phalloidin). These changes, which were visible after 2 hours, could be completely prevented by treatment with the Rho kinase inhibitor Y27632 (1 uM). Interestingly, ASM cells serum‐deprived for 7 days to adopt a contractile phenotype also showed greater WNT‐5A protein expression (1,99 fold of control, p < 0.05) compared to the proliferative phenotype (serum‐fed cells). We next assessed whether TGF‐β1 played an upstream role in the WNT‐5A response. We found that WNT‐5A siRNA not only blocked TGF‐β1‐induced actin polymerization, but could also abrogate TGF‐β1‐induced accumulation of α‐SMA protein. In line with this contention, adding WNT‐5A on top of TGF‐β1 synergistically induced α‐SMA protein abundance (2,21 fold of TGF‐β, p < 0.01).In summary, WNT‐5A promotes increased agonist‐induced airway smooth muscle contraction, most likely by increasing actin polymerization and actin expression in concerted action with TGF‐β1. These results may be relevant in the understanding of AHR and warrant further investigation.Support or Funding InformationThis work was financially supported by a NWO Vidi grant, 016.126.307.

A novel PDE1A coupled to M2AChR at plasma membranes from bovine tracheal smooth muscle

Muscarinic antagonists, via muscarinic receptors increase the cAMP/cGMP levels at bovine tracheal smooth muscle (BTSM) through the inhibition of phosphodiesterases (PDEs), displaying a similar behavior of vinpocetine (a specific-PDE1 inhibitor). The presence of PDE1 hydrolyzing both cyclic nucleotides in BTSM strips was revealed. Moreover, a vinpocetine and muscarinic antagonists inhibited PDE1 located at plasma membranes (PM) fractions from BTSM showing such inhibition, an M2AChR pharmacological profile. Therefore, a novel Ca2+/CaM dependent and vinpocetine inhibited PDE1 was purified and characterized at PM fractions from BTSM. This PDE1 activity was removed from PM fractions using a hypotonic buffer and purified some 38 fold using two columns (Q-Sepharose and CaM-agarose). This PDE1 was stimulated by CaM and inhibited by vinpocetine showing two bands in PAGE-SDS (56, 58 kDa) being the 58 kDa identified as PDE1A by Western blotts. This PDE1A activity was assayed with [3H]cGMP and [3H]cAMP exhibiting a higher affinity as Km (μM) for cGMP than cAMP but being close values with Vmax cAMP/cGMP ratio of 1.5. The co-factor Mg2+ showed similar K(A) (mM) for both cyclic nucleotides. Vinpocetine showed similar inhibition concentration 50% (IC50 of 4.9 and 4.6 μM) for cAMP and cGMP, respectively. CaM stimulated the cyclic nucleotides hydrolysis by PDE1A exhibiting similar activation constant as K(CaM), in nM range. The original finding was the identification and purification of a vinpocetine and muscarinic antagonist-inhibited and CaM-activated PM-bound PDE1A, linked to M2AChR. A model of this novel signal transducing cascade for the regulation of cyclic nucleotides levels at BTSM is proposed.

Muscarinic drugs regulate the PKG-II-dependent phosphorylation of M3 muscarinic acetylcholine receptors at plasma membranes from airway smooth muscle

Muscarinic agonists induce the activation of the airway smooth muscle (ASM) leading to smooth muscle contraction, important in asthma. This activation is mediated through M2/M3 muscarinic acetylcholine receptors (mAChRs). Muscarinic receptor activity, expressed as [3H]QNB binding at plasma membranes from bovine tracheal smooth muscle (BTSM), increased with cGMP and was augmented significantly cGMP plus ATP but diminished with the PKG-II inhibitor, Sp-8-pCPT-cGMPS. The [3H]-QNB binding was accelerated by okadaic acid, (OKA), a protein phosphatase (PPase) inhibitor. These two results indicated the involvement of a membrane-bound PPase. Moreover, a cGMP-dependent-[32P]γATP phosphorylation of plasma membranes from BTSM was stimulated at low concentrations of muscarinic agonist carbamylcholine (CC). However, higher amounts of CC produced a significant decrement of [32P]-labeling. A selective M3mAChR antagonist, 4-DAMP produced a dramatic inhibition of the basal and CC-dependent [32P]-labeling. The [32P] labeled membrane sediments were detergent solubilized and immunoprecipitated with specific M2/M3mAChR antibodies. The M3mAChR immuno-precipitates exhibited the highest cGMP-dependent [32P]-labeling, indicating it is a PKG-II substrate. Experiments using synthetic peptides from the C-terminal of the third intracellular loop (i3) of both M2mAChR (356–369) and M3mAChR (480–493) as external PKG-II substrates resulted in the i3M3-peptide being heavily phosphorylated. These results indicated that PKG-II phosphorylated the M3mAChR at the i3M3 domain (480MSLIKEKK485), suggesting that Ser481 may be the target. Finally, this phosphorylation site seems to be regulated by a membrane-bound PPase linked to muscarinic receptor. These findings are important to understand the role of M3mAChR in the patho-physiology of ASM involved in asthma and COPD.

Cyclic GMP regulates M3AChR activity at plasma membranes from Airway smooth muscle

Muscarinic acetylcholine receptors MAChRs from Bovine Tracheal Smooth Muscle (BTSM) plasma membranes are responsible for the cGMP rise and signal-amplitude peaks associated with smooth muscle contraction present in bronchial asthma. These MAChRs bind [(3)H]QNB and exhibit the classic G Protein Coupled-Receptor (GPCR) behavior towards muscarinic agonist and antagonists that is sensitive to sensitive to GTP analogs. Interestingly, the [(3)H]QNB binding activity was stimulated by cGMP and ATP, and was enhanced by IBMX and Zaprinast, inhibitors of cGMP-PDE. Cyclic GMP plus ATP affected the agonist-antagonist muscarinic binding activities. Thus, the high affinity agonist (Carbamylcholine) binding sites disappeared, whereas, 4-DAMP, a M3 selective antagonist displayed an additional high affinity-binding site. In contrast, non-selective (atropine) and M2-selective (methoctramine and gallamine) antagonists revealed one low binding site. Moreover, the 4-DAMP-mustard alkylation of the MAChRs blocked the cGMP effect indicating that the M3AChR is the main receptor target of cGMP. Interestingly, these cGMP effects were potentiated by an activator (Sp-8-pCPT-cGMPS), and diminished by an inhibitor (Rp-8-pCPT-CGMPS), of cGMP-dependent protein kinase (PKG-II), which was detected by Western blotting using specific PKG II antibodies. Finally, plasma membrane M3AChRs were phosphorylated in a cGMP-dependent manner and this novel post-translational reversible modification at M3AChRs may act as a feedback mechanism to terminate the cGMP dependent muscarinic signal transduction cascades at the sarcolema of BTSM.

Sarcoplasmic reticulum Ca2+ refilling is determined by L-type Ca2+ and store operated Ca2+ channels in guinea pig airway smooth muscle

Sarcoplasmic reticulum Ca2+ refilling (SRREF) is crucial to sustain the agonists induced airway smooth muscle contraction. Nevertheless, its mechanisms have not been clearly described yet, although L-type voltage dependent, store operated, receptor operated channels and the Na+/Ca2+ exchanger in its reverse mode (NCXREV) have been proposed as Ca2+ handling proteins participating in this process. We found that in guinea pig and bovine tracheal smooth muscle, SRREF induced by caffeine was completely abolished by thapsigargin, even in the presence of Bay K8644, an activator of the L-type Ca2+ channel. Activation of NCXREV in guinea pig tracheal myocytes increased SRREF in ~70%, while opening of the L-type Ca2+ channels with Bay K8644 and favoring the capacitative Ca2+ entry with 2-APB (32μM) also augmented the SRREF by ~170% and ~71%, respectively. Methoxyverapamil (D-600, an L-type Ca2+ channel blocker), 2-APB (100µM, antagonist of the capacitative Ca2+ entry) and PPADS (NCXREV blocker) diminished the SRREF by ~63%, ~72% and ~31%, respectively. The simultaneous addition of D-600 and 2-APB annulled SRREF. These last results were also seen when carbachol was used instead of caffeine. In tracheal rings, 2-APB and nifedipine abolished the carbachol-induced contraction. We concluded that the sarcoplasmic reticulum Ca2+ pump is the only mechanism involved in the SRREF and that L-type Ca2+ voltage dependent and store operated Ca2+ channels are the principal membranal Ca2+ handling proteins that provide extracellular Ca2+ for SRREF and carbachol-induced contraction in the guinea pig airway smooth muscle; NCXREV seems to play a minor role.

Gz- and not Gi-proteins are coupled to pre-junctional μ-opioid receptors in bovine airways

We investigated the signal transmission pathway by which activation of μ-opioid receptors attenuates acetylcholine (ACh) release in bovine trachealis. Electrical stimulation (ES)-induced [3H]-ACh release was determined in bovine tracheal smooth muscle strips pre-incubated with either the Gi-protein inhibitor pertussis toxin (PTX, 500ng/ml and 1μg/ml) or the Gz-protein specific inhibitor arachidonic acid (AA, 10−6M and 10−5M) and then treated with DAMGO (D-Ala2,N-MePhe4,Gly-ol5-enkephalin) 10−5M. Indomethacin 10−5M was used to block AA cascade. The inhibitory effect of DAMGO on ES-induced [3H]-ACh release was PTX-insensitive, but, by contrast, ablated by AA in a concentration-dependent manner. AA 10−5M alone reduced [3H]-ACh release, an effect that was prevented by iberiotoxin 10−7M, suggesting an involvement of Ca2+-activated K+-channels. Western blot analysis consistently showed immunoreactive bands against a specific antibody anti-Gz-α subunit at ∼40kDa, consistent with the presence of Gz-protein. The present findings suggest that in isolated bovine trachealis, activation of μ-opioid receptors inhibits ACh-release through a signal transmission pathway involving Gz-protein rather than Gi-protein.

Focal Adhesion Kinase Regulates Collagen I–Induced Airway Smooth Muscle Phenotype Switching

Increased extracellular matrix (ECM) deposition and airway smooth muscle (ASM) mass are major contributors to airway remodeling in asthma. Recently, we demonstrated that the ECM protein collagen I, which is increased surrounding asthmatic ASM, induces a proliferative, hypocontractile ASM phenotype. Little is known, however, about the signaling pathways involved. Using bovine tracheal smooth muscle, we investigated the role of focal adhesion kinase (FAK) and downstream signaling pathways in collagen I-induced ASM phenotype modulation. Phosphorylation of FAK was increased during adhesion to both uncoated and collagen I-coated culture dishes, without differences between these matrices. Nor were any differences found in cellular adhesion. Inhibition of FAK activity by overexpression of the FAK deletion mutants FAT (focal adhesion targeting domain) and FRNK (FAK-related nonkinase) attenuated adhesion. After attachment, FAK phosphorylation increased in a time-dependent manner in cells cultured on collagen I, whereas no activation was found on an uncoated plastic matrix. In addition, collagen I increased in a time- and concentration-dependent manner the cell proliferation, which was fully inhibited by FAT and FRNK. Similarly, the specific pharmacologic FAK inhibitor PF-573228 [6-((4-((3-(methanesulfonyl)benzyl)amino)-5-trifluoromethylpyrimidin-2-yl) amino)-3,4-dihydro-1H-quinolin-2-one] as well as specific inhibitors of p38 mitogen-activated protein kinase (MAPK) and Src also fully inhibited collagen I-induced proliferation, whereas partial inhibition was observed by inhibition of phosphatidylinositol-3-kinase (PI3-kinase) and mitogen-activated protein kinase kinase (MEK). The inhibition of cell proliferation by these inhibitors was associated with attenuation of the collagen I-induced hypocontractility. Collectively, the results indicate that induction of a proliferative, hypocontractile ASM phenotype by collagen I is mediated by FAK and downstream signaling pathways.

Effects of selenoprotein P on the contraction and relaxation of the airway smooth muscle

Selenoprotein P (SeP) not only represents the major selenoprotein in plasma, but also provides more than 50% of the total plasma selenium. However, there is no report concerning the direct action of selenium or selenium-containing compounds on the contraction and relaxation of the airway smooth muscle. Therefore, we investigated the effects of SeP and sodium selenite (SS) on the indirectly induced contraction and relaxation of the cat bronchi, and gel contraction of cultured bovine tracheal smooth muscle cells (BTSMC) induced by ATP. In the present results, SeP or SS suppressed the amplitude of twitch-like contractions of cat bronchiole without affecting the non-adrenergic and non-cholinergic (NANC) relaxations evoked by electrical field stimulation. SeP also suppressed the ATP-induced gel contraction of BTSMC. These results suggest that SeP suppresses the amplitude of twitch-like contraction of cat bronchiole by acting directly on the bronchiolar smooth muscle.

Glucocorticosteroids and β2-Adrenoceptor Agonists Synergize to Inhibit Airway Smooth Muscle Remodeling

Airway remodeling, including increased airway smooth muscle (ASM) mass and contractility, contributes to increased airway narrowing in asthma. Increased ASM mass may be caused by exposure to mitogens, including platelet-derived growth factor (PDGF) and collagen type I, which induce a proliferative, hypocontractile ASM phenotype. In contrast, prolonged exposure to insulin induces a hypercontractile phenotype. Glucocorticosteroids and β₂-adrenoceptor agonists synergize to increase glucocorticosteroid receptor translocation in ASM cells; however, the impact of this synergism on phenotype modulation is unknown. Using bovine tracheal smooth muscle, we investigated the effects of the glucocorticosteroids fluticasone (10 nM), budesonide (30 nM), and dexamethasone (0.1-1 μM) and the combination of low concentrations of fluticasone (3-100 pM) and fenoterol (10 nM) on ASM phenotype switching in response to PDGF (10 ng/ml), collagen type I (50 μg/ml), and insulin (1 μM). All glucocorticosteroids inhibited PDGF- and collagen I-induced proliferation and hypocontractility, with the effects of collagen I being less susceptible to glucocorticosteroid action. At 100-fold lower concentrations, fluticasone (100 pM) synergized with fenoterol to prevent PDGF- and collagen I-induced phenotype switching. This inhibition of ASM phenotype switching was associated with a normalization of the PDGF-induced decrease in the cell cycle inhibitors p21(WAF1/CIP1) and p57(KIP2). At this concentration, fluticasone also prevented the insulin-induced hypercontractile phenotype. At even lower concentrations, fluticasone (3 pM) synergized with fenoterol to inhibit this phenotype switch. Collectively, these findings indicate that glucocorticosteroids and β₂-agonists synergistically inhibit ASM phenotype switching, which may contribute to the increased effectiveness of combined treatment with glucocorticosteroids and β₂-agonists in asthma.

The Relaxant Action of Nicorandil in Bovine Tracheal Smooth Muscle

The relaxant mechanisms of nicorandil were examined by comparing its effects with those of sodium nitroprusside and cromakalim in bovine tracheal smooth muscle. In preparations contracted with methacholine (0.3 µmol/l) or high K⁺ (40 mmol/l), nicorandil and sodium nitroprusside caused concentration-dependent relaxations. Their relaxant effects on high K⁺-contracted preparations were smaller than those on methacholine-contracted muscle. Cromakalim relaxed methacholine-contracted preparations, whereas it had no effect on high K⁺-contracted muscle. The inhibitor of soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 5 µmol/l) completely prevented the relaxation induced by lower concentrations (<30 µmol/l) of nicorandil, whereas it partially attenuated relaxation caused by higher concentrations. The ATP-sensitive K⁺ (K_ATP) channel blocker glibenclamide only partially attenuated the relaxant responses to nicorandil (at 100 and 300 µmol/l). Combination treatment with ODQ and glibenclamide almost completely prevented nicorandil-induced relaxations. The large-conductance Ca²⁺-activated K⁺ channel (Maxi K⁺ channel) inhibitor iberiotoxin significantly prevented the relaxations induced by lower concentrations (3 and 10 µmol/l) of nicorandil. The preventive effect of iberiotoxin was markedly enhanced under the blockade of K_ATP channels with glibenclamide. These results suggest that nicorandil relaxes bovine tracheal smooth muscle through 2 mechanisms: opening of K_ATP channels and activation of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway. Nicorandil may also activate Maxi K⁺ channels, possibly through the NO-cGMP pathway, and the interaction of K_ATP channels and Maxi K⁺ channels may affect the relaxant effect of nicorandil in bovine tracheal smooth muscle.

Functional consequences of human airway smooth muscle phenotype plasticity

Airway smooth muscle (ASM) phenotype plasticity, characterized by reversible switching between contractile and proliferative phenotypes, is considered to contribute to increased ASM mass and airway hyper-responsiveness in asthma. Further, increased expression of collagen I has been observed within the ASM bundle of asthmatics. Previously, we showed that exposure of intact bovine tracheal smooth muscle (BTSM) to collagen I induces a switch from a contractile to a hypocontractile, proliferative phenotype. However, the functional relevance of this finding for intact human ASM has not been established. We investigated the effects of exposure of human tracheal smooth muscle (HTSM) strips to monomeric collagen I and PDGF on contractile responses to methacholine and KCl. Expression of contractile proteins sm-α-actin and sm-MHC was assessed by Western blot analysis. The proliferation of HTSM cells was assessed by cell counting, measuring mitochondrial activity (Alamarblue conversion) and [(3) H]-thymidine incorporation. Proliferation of intact tissue slices was assessed by [(3) H]-thymidine incorporation. Culturing HTSM strips in the presence of collagen I or PDGF for 4 days reduced maximal contractile responses to methacholine or KCl and the expression of contractile proteins. Conversely, collagen I and PDGF increased proliferation of HTSM cells and proliferative responses in tissue slices. PDGF additively increased the proliferation of HTSM cells cultured on collagen I; this additive effect was not observed on contractility, contractile protein expression or proliferation of intact tissue. These findings indicate that collagen I and PDGF induce a functionally hypocontractile, proliferative phenotype of human ASM, which may contribute to airway remodelling in asthma.

Cross-bridge apparent rate constants of human gallbladder smooth muscle

This paper studies human gallbladder (GB) smooth muscle contractions. A two-state cross-bridge model was used to estimate the apparent attachment and detachment rate constants, as well as increased Ca2+ concentration from the peak active stress during the isometric contraction. The active stress was computed from a mechanical model based entirely on non-invasive routine ultrasound scans. In the two-state cross-bridge model, the two apparent rate constants, representing the total attached/detached cross-bridges, respectively, were estimated using active stress prediction for 51 subjects undergoing cholecystokinin-provocation test, together with estimates from the four-state cross-bridge model for a swine carotid, bovine tracheal and guinea pig GB smooth muscles. The study suggests that the apparent rate constants should be patient-specific, i.e. patients with a lower stress level are characterized by smaller apparent rate constants. In other words, the diseased GB may need to develop fast cycling cross-bridges to compensate in the emptying process. This is a first step towards more quantitative and non-invasive measures of GB pain, and may provide useful insight in understanding GB motility and developing effective drug treatments.

TAK1 plays a major role in growth factor-induced phenotypic modulation of airway smooth muscle

Increased airway smooth muscle (ASM) mass is a major feature of airway remodeling in asthma and chronic obstructive pulmonary disease. Growth factors induce a proliferative ASM phenotype, characterized by an increased proliferative state and a decreased contractile protein expression, reducing contractility of the muscle. Transforming growth factor-β-activated kinase 1 (TAK1), a mitogen-activated protein kinase kinase kinase, is a key enzyme in proinflammatory signaling in various cell types; however, its function in ASM is unknown. The aim of this study was to investigate the role of TAK1 in growth factor-induced phenotypic modulation of ASM. Using bovine tracheal smooth muscle (BTSM) strips and cells, as well as human tracheal smooth muscle cells, we investigated the role of TAK1 in growth factor-induced proliferation and hypocontractility. Platelet-derived growth factor- (PDGF; 10 ng/ml) and fetal bovine serum (5%)-induced increases in DNA synthesis and cell number in bovine and human cells were significantly inhibited by pretreatment with the specific TAK1 inhibitor LL-Z-1640-2 (5Z-7-oxozeaenol; 100 nM). PDGF-induced DNA synthesis and extracellular signal-regulated kinase-1/2 phosphorylation in BTSM cells were strongly inhibited by both LL-Z-1640-2 pretreatment and transfection of dominant-negative TAK1. In addition, LL-Z-1640-2 inhibited PDGF-induced reduction of BTSM contractility and smooth muscle α-actin expression. The data indicate that TAK1 plays a major role in growth factor-induced phenotypic modulation of ASM.

Testosterone Inhibits cAMP-Phosphodiesterases in Heart Extracts from Rats and Increases cAMP Levels in Isolated Left Atria

Several mechanisms have been proposed to explain the acute cardiovascular effects elicited by androgens, such as vasodilation and positive inotropism. Phosphodiesterases (PDEs) are important modulators of cardiac contractility. However, an effect on PDEs by androgens in cardiac tissues has not previously been reported. In this study, extracts from rat ventricles and isolated left atria were assayed for cAMP-dependent PDE activity. To study the tissue selectivity, the enzymatic activity was also assayed in extracts from bovine tracheal smooth muscle and Chinese hamster ovary (CHO) cells. Functional assays were also performed with isolated atria. Testosterone, but not 5α- and 5β-dihydrotestosterone, inhibited cAMP-PDE activity in extracts from left ventricles and atria. In atria, the inhibition of cAMP-PDE activity was associated with an increase in intracellular levels of cAMP and a cardiotonic response. This effect was not elicited in tracheal muscle strips or CHO cell extracts, suggesting the possibility of tissue and cAMP-PDE selectivity. The results of these studies suggest a new mechanism of action of testosterone in the rat heart, which might contribute to the reported cardiotonic effect.