5-Oxo-ETE regulates tone of guinea pig airway smooth muscle via activation of Ca2+ pools and Rho-kinase pathway

2004 ◽  
Vol 287 (4) ◽  
pp. L631-L640 ◽  
Author(s):  
Frederic Mercier ◽  
Caroline Morin ◽  
Martin Cloutier ◽  
Sonia Proteau ◽  
Joshua Rokach ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Airway Smooth Muscle ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Eicosatetraenoic Acid ◽  
Chlorobenzoic Acid ◽  
Tp Receptor ◽  
Lower Amplitude ◽  
Inotropic Responses

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a proinflammatory mediator, but its effects on airway smooth muscle (ASM) have never been assessed. Tension measurements performed on guinea pig ASM showed that 5-oxo-ETE induced sustained concentration-dependent positive inotropic responses (EC50 = 0.89 μM) of somewhat lower amplitude than those induced by carbamylcholine and the thromboxane A2 (TXA2) agonist U-46619. Transient inotropic responses to 5-oxo-ETE were recorded in Ca2+-free medium, suggesting mobilization of intracellular Ca2+. Meanwhile, the sustained contraction, which required Ca2+ entry, was partially blocked by 1 μM nifedipine (an L-type Ca2+ channel blocker) but relatively insensitive to 100 μM Gd3+. The 5-oxo-ETE responses were also inhibited by indomethacin and SC-560 [a cyclooxygenase (COX-1) inhibitor] pretreatments but not by NS-398 (a selective COX-2 inhibitor). The contractile effects of 5-oxo-ETE on ASM were inhibited by the selective TXA2 receptor (TP receptor) antagonist SQ-29548 (−75%) and by 2-(p-amylcinnamoyl) amino-4-chlorobenzoic acid pretreatment, a phospholipase A2 inhibitor (−66%), suggesting that the major part of its effect is mediated by the release of TXA2. ASM responses to 5-oxo-ETE were also blocked by the Rho-kinase inhibitor Y-27632, which also partially inhibited the response to the TP receptor agonist U-46619, suggesting that the contractile response is due in part to Ca2+ sensitization of ASM cell myofilaments.

Capsazepine, a vanilloid antagonist, abolishes tonic responses induced by 20-HETE on guinea pig airway smooth muscle

2005 ◽  
Vol 288 (3) ◽  
pp. L460-L470 ◽  
Author(s):  
É Rousseau ◽  
Martin Cloutier ◽  
Caroline Morin ◽  
Sonia Proteau
Keyword(s):  
Arachidonic Acid ◽  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Eicosatetraenoic Acid ◽  
Dependent Manner ◽  
Release Process ◽  
Concentration Dependent Manner ◽  
Intracellular Ca ◽  
Inotropic Responses ◽  
Enzymatic Pathways

The aim of this study was to delineate the mode of action of 20-hydroxy-eicosatetraenoic acid (20-HETE) in airway smooth muscle (ASM) cells. ASM metabolizes arachidonic acid by various enzymatic pathways, including the cytochrome P-450 (CYP-450) ω-hydroxylase, which leads to the production of 20-HETE, a bronchoconstrictive eicosanoid. The present study demonstrated that 20-HETE induced concentration-dependent tonic responses in ASM, whereas transient responses were recorded in Ca2+-free solution, suggesting an intracellular Ca2+ release process. 20-HETE inotropic responses were abolished by 36 μM 2-aminoethoxydiphenyl borate or 1 μM thapsigargin but were insensitive to 10 μM ryanodine, indicating that inositol triphosphate receptors likely control the release of intracellular Ca2+. Sustained tension, which required Ca2+ entry, was partially blocked by 1 μM nifedipine (an L-type) and 100 μM Gd3+ (a nonselective cationic channel blocker). Moreover, in the absence of selective 20-HETE receptor antagonists, 20-HETE tonic responses were inhibited in a concentration-dependent manner (0.1–10 μM) by capsazepine, a well-characterized vanilloid receptor antagonist. Capsazepine was also observed to reverse cumulative responses to 20-HETE and capsaicin, a TRPV1 agonist. In addition, capsazepine pretreatment largely modified the sustained inotropic responses to 20-HETE, suggesting that 20-HETE cross-reacted with TRPV1 receptors with a low affinity (μM) or that its specific receptor was inhibited by the vanilloid antagonist. Data obtained using RHC-80267, ONO-RS-082, and eicosatetraynoic acid, respective inhibitors of diacylglycerol-lipase, phospholipase A2, and CYP-450 ω-hydroxylase, reveal that intracellular arachidonic acid production and its 20-HETE metabolite may be responsible for the activation of nonselective cationic channels and tonic responses.

Hyperoxic conditions inhibit airway smooth muscle myosin phosphatase in rat pups

2007 ◽  
Vol 292 (1) ◽  
pp. L68-L73 ◽  
Author(s):  
Paul G. Smith ◽  
Albana Dreshaj ◽  
Subhendu Chaudhuri ◽  
Baran M. Onder ◽  
Maroun J. Mhanna ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Electrical Field Stimulation ◽  
Smooth Muscle Myosin ◽  
Postnatal Life ◽  
Myosin Phosphatase ◽  
Rat Pups ◽  
Muscle Myosin

Exposure of rat pups to 100% oxygen is a model for studying neonatal lung injury. Airway reactivity is increased in this model, in part due to impaired airway smooth muscle (ASM) relaxation. We compared biochemical determinants of ASM contractility in rat pups exposed to 100% oxygen for 7 days vs. littermates raised in room air. The baseline quantities of ASM contractile proteins, extent of phosphorylation of the 20-kDa myosin regulatory light chain (LC20), and amount of the myosin-binding subunit of smooth muscle myosin phosphatase (MYPT) were all comparable between the two groups. Bethanechol-induced contraction increased the extent of phosphorylation of both LC20 and MYPT in the hyperoxic group (45% and 70% over control, respectively). Relaxation after electrical field stimulation demonstrated greater phosphorylation of both LC20 and MYPT in the hyperoxic group compared with controls (67% and 84%, respectively). To determine if hyperoxia induced changes in the isoforms of MYPT, isoform expression was also compared but differences were not found. To determine potential mechanisms whereby MYPT phosphorylation was increased by hyperoxia, separate tracheas were treated with the Rho kinase inhibitor Y-27632. This treatment completely eliminated differences in MYPT phosphorylation between the groups. Because phosphorylation of MYPT impairs the phosphatase activity of myosin phosphatase, these data suggest that hyperoxic conditioning during early postnatal life impairs relaxation through prolonging LC20 phosphorylation. This mechanism might contribute to increased ASM reactivity seen in bronchopulmonary dysplasia.

Increase in passive stiffness at reduced airway smooth muscle length: potential impact on airway responsiveness

2010 ◽  
Vol 298 (3) ◽  
pp. L277-L287 ◽  
Author(s):  
Ynuk Bossé ◽  
Dennis Solomon ◽  
Leslie Y. M. Chin ◽  
Kevin Lian ◽  
Peter D. Paré ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Muscle Length ◽  
Passive Stiffness ◽  
Tidal Breathing ◽  
Reference Length ◽  
Length Reduction

The amplitude of strain in airway smooth muscle (ASM) produced by oscillatory perturbations such as tidal breathing or deep inspiration (DI) influences the force loss in the muscle and is therefore a key determinant of the bronchoprotective and bronchodilatory effects of these breathing maneuvers. The stiffness of unstimulated ASM (passive stiffness) directly influences the amplitude of strain. The nature of the passive stiffness is, however, not clear. In this study, we measured the passive stiffness of ovine ASM at different muscle lengths (relative to in situ length, which was used as a reference length, Lref) and states of adaptation to gain insights into the origin of this muscle property. The results showed that the passive stiffness was relatively independent of muscle length, possessing a constant plateau value over a length range from 0.62 to 1.25 Lref. Following a halving of ASM length, passive stiffness decreased substantially (by 71%) but redeveloped over time (∼30 min) at the shorter length to reach 65% of the stiffness value at Lref, provided that the muscle was stimulated to contract at least once over a ∼30-min period. The redevelopment and maintenance of passive stiffness were dependent on the presence of Ca2+ but unaffected by latrunculin B, an inhibitor of actin filament polymerization. The maintenance of passive stiffness was also not affected by blocking myosin cross-bridge cycling using a myosin light chain kinase inhibitor or by blocking the Rho-Rho kinase (RhoK) pathway using a RhoK inhibitor. Our results suggest that the passive stiffness of ASM is labile and capable of redevelopment following length reduction. Redevelopment and maintenance of passive stiffness following muscle shortening could contribute to airway hyperresponsiveness by attenuating the airway wall strain induced by tidal breathing and DI.

Regulatable stiffness in relaxed airway smooth muscle: a target for asthma treatment?

2012 ◽  
Vol 112 (3) ◽  
pp. 337-346 ◽  
Author(s):  
Abdul Raqeeb ◽  
Yuekan Jiao ◽  
Harley T. Syyong ◽  
Peter D. Paré ◽  
Chun Y. Seow
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Muscle Layer ◽  
Radial Force ◽  
Calcium Entry Blocker ◽  
Passive Stiffness

The airway smooth muscle (ASM) layer within the airway wall modulates airway diameter and distensibility. Even in the relaxed state, the ASM layer possesses finite stiffness and limits the extent of airway distension by the radial force generated by parenchymal tethers and transmural pressure. Airway stiffness has often been attributed to passive elements, such as the extracellular matrix in the lamina reticularis, adventitia, and the smooth muscle layer that cannot be rapidly modulated by drug intervention such as ASM relaxation by β-agonists. In this study, we describe a calcium-sensitive component of ASM stiffness mediated through the Rho-kinase signaling pathway. The stiffness of ovine tracheal smooth muscle was assessed in the relaxed state under the following conditions: 1) in physiological saline solution (Krebs solution) with normal calcium concentration; 2) in calcium-free Krebs with 2 mM EGTA; 3) in Krebs with calcium entry blocker (SKF-96365); 4) in Krebs with myosin light chain kinase inhibitor (ML-7); and 5) in Krebs with Rho-kinase inhibitor (Y-27632). It was found that a substantial portion of the passive stiffness could be abolished when intracellular calcium was removed; this calcium-sensitive stiffness appeared to stem from intracellular source and was not sensitive to ML-7 inhibition of myosin light chain phosphorylation, but was sensitive to Y-27632 inhibition of Rho kinase. The results suggest that airway stiffness can be readily modulated by targeting the calcium-sensitive component of the passive stiffness within the muscle layer.

Insulin increases the expression of contractile phenotypic markers in airway smooth muscle

2007 ◽  
Vol 293 (1) ◽  
pp. C429-C439 ◽  
Author(s):  
Dedmer Schaafsma ◽  
Karol D. McNeill ◽  
Gerald L. Stelmack ◽  
Reinoud Gosens ◽  
Hoeke A. Baarsma ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Cell Morphology ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Protein Accumulation ◽  
Mrna Levels ◽  
Phenotypic Markers ◽  
Serum Free ◽  
Contractile Phenotype

We have previously demonstrated that long-term exposure of bovine tracheal smooth muscle (BTSM) strips to insulin induces a functional hypercontractile phenotype. To elucidate molecular mechanisms by which insulin might induce maturation of contractile phenotype airway smooth muscle (ASM) cells, we investigated effects of insulin stimulation in serum-free primary BTSM cell cultures on protein accumulation of specific contractile phenotypic markers and on the abundance and stability of mRNA encoding these markers. In addition, we used microscopy to assess insulin effects on ASM cell morphology, phenotype, and induction of phosphatidylinositol (PI) 3-kinase signaling. It was demonstrated that protein and mRNA levels of smooth muscle-specific contractile phenotypic markers, including sm-myosin, are significantly increased after stimulation of cultured BTSM cells with insulin (1 μM) for 8 days compared with cells treated with serum-free media, whereas mRNA stability was unaffected. In addition, insulin treatment promoted the formation of large, elongate ASM cells, characterized by dramatic accumulation of contractile phenotype marker proteins and phosphorylated p70S6K (downstream target of PI 3-kinase associated with ASM maturation). Insulin effects on protein accumulation and cell morphology were abrogated by combined pretreatment with the Rho kinase inhibitor Y-27632 (1 μM) or the PI 3-kinase inhibitor LY-294002 (10 μM), indicating that insulin increases the expression of contractile phenotypic markers in BTSM in a Rho kinase- and PI 3-kinase-dependent fashion. In conclusion, insulin increases transcription and protein expression of contractile phenotypic markers in ASM. This could have important implications for the use of recently approved aerosolized insulin formulations in diabetes mellitus.

scholarly journals The contribution of Ca2+ signaling and Ca2+ sensitivity to the regulation of airway smooth muscle contraction is different in rats and mice

2009 ◽  
Vol 296 (6) ◽  
pp. L947-L958 ◽  
Author(s):  
Yan Bai ◽  
Michael J. Sanderson
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Laser Scanning ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Smooth Muscle Contraction ◽  
Laser Scanning Microscopy ◽  
Airway Smooth Muscle Cells ◽  
Similar Frequency ◽  
Sustained Contraction

To determine the relative contributions of Ca2+ signaling and Ca2+ sensitivity to the contractility of airway smooth muscle cells (SMCs), we compared the contractile responses of mouse and rat airways with the lung slice technique. Airway contraction was measured by monitoring changes in airway lumen area with phase-contrast microscopy, whereas changes in intracellular calcium concentration ([Ca2+]i) of the SMCs were recorded with laser scanning microscopy. In mice and rats, methacholine (MCh) or serotonin induced concentration-dependent airway contraction and Ca2+ oscillations in the SMCs. However, rat airways demonstrated greater contraction compared with mice, in response to agonist-induced Ca2+ oscillations of a similar frequency. Because this indicates that rat airway SMCs have a higher Ca2+ sensitivity compared with mice, we examined Ca2+ sensitivity with Ca2+-permeabilized airway SMCs in which the [Ca2+]i was experimentally controlled. In the absence of agonists, high [Ca2+]i induced a sustained contraction in rat airways but only a transient contraction in mouse airways. This sustained contraction of rat airways was relaxed by Y-23672, a Rho kinase inhibitor, but not affected by GF-109203X, a PKC inhibitor. The subsequent exposure of Ca2+-permeabilized airway SMCs, with high [Ca2+]i, to MCh elicited a further contraction of rat airways and initiated a sustained contraction of mouse airways, without changing the [Ca2+]i of the SMCs. Collectively, these results indicate that airway SMCs of rats have a substantially higher innate Ca2+ sensitivity than mice and that this strongly influences the transduction of the frequency of Ca2+ oscillations into the contractility of airway SMCs.

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

2004 ◽  
Vol 287 (4) ◽  
pp. L641-L648 ◽  
Author(s):  
Katsuaki Endou ◽  
Kunihiko Iizuka ◽  
Akihiro Yoshii ◽  
Hideo Tsukagoshi ◽  
Tamotsu Ishizuka ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Muscle Contraction ◽  
Airway Smooth Muscle ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Smooth Muscle Contraction ◽  
Common Mechanism ◽  
Human Bronchus ◽  
Rho Kinase Inhibitor ◽  
Dependent Protein

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

Urokinase potentiates PDGF-induced chemotaxis of human airway smooth muscle cells

2003 ◽  
Vol 284 (6) ◽  
pp. L1020-L1026 ◽  
Author(s):  
Stephen M. Carlin ◽  
Michael Roth ◽  
Judith L. Black
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Airway Smooth Muscle ◽  
Kinase Inhibitor ◽  
Airway Remodeling ◽  
Muscle Cells ◽  
Mek Inhibitor ◽  
Airway Smooth Muscle Cells ◽  
Human Airway Smooth Muscle ◽  
Human Airway

We investigated the chemotactic action of PDGF and urokinase on human airway smooth muscle (HASM) cells in culture. Cells were put in collagen-coated transwells with 8-μm perforations, incubated for 4 h with test compounds, then fixed, stained, and counted as migrated nuclei by microscopy. Cells from all culture conditions showed some basal migration (migration in the absence of stimuli during the assay), but cells preincubated for 24 h in 10% FBS or 20 ng/ml PDGF showed higher basal migration than cells quiesced in 1% FBS. PDGFBB, PDGFAA, and PDGFABwere all chemotactic when added during the assay. PDGF chemotaxis was blocked by the phosphatidyl 3′-kinase inhibitor LY-294002, the MEK inhibitor U-0126, PGE2, formoterol, pertussis toxin, and the Rho kinase inhibitor Y-27632. Urokinase alone had no stimulatory effect on migration of quiescent cells but caused a dose-dependent potentiation of chemotaxis toward PDGF. Urokinase also potentiated the elevated basal migration of cells pretreated in 10% FBS or PDGF. This potentiating effect of urokinase appears to be novel. We conclude that PDGF and similar cytokines may be important factors in airway remodeling by redistribution of smooth muscle cells during inflammation and that urokinase may be important in potentiating the response.

Abstract 641: Expression Of GLUT4 In Vascular Smooth Muscle Affects Endothelial Function In Hypertension.

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Kevin B Atkins ◽  
Jharna Saha ◽  
Frank C Brosius
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Interesting Phenomenon ◽  
Glut4 Expression ◽  
Endothelial Denudation ◽  
Rho Kinase Inhibitor ◽  
Total Body ◽  
Rats And Mice

Expression of GLUT4 is decreased in arterial smooth muscle of hypertensive rats and mice, and total body overexpression of GLUT4 in mice prevents enhanced arterial reactivity. To demonstrate that the effect on vascular response to GLUT4 overexpression is vascular rather than systemic in origin we utilized smooth muscle-specific GLUT4 transgenic mice (SMG4). GLUT4 expression in aortae of SMG4 compared to WT mice was increased 2-3 fold. Adult wild-type (WT) and SMG4 mice were made hypertensive or not through implantation of angiotensin II (AngII; 1.4mg/kg/d for 2 wks) or vehicle containing osmotic mini-pumps. Both WT and SMG4 mice AngII-treated mice exhibited significantly increased systolic blood pressure. In AngII-treated WT mice (WT-AngII) aortic GLUT4 expression was significantly decreased, whereas GLUT4 expression in aortae of AngII-treated SMG4 mice (SMG4-AngII) was maintained. The phosphorylation of ERM and MYPT1(Thr850) were significantly increased in aortae of WT-AngII compared to WT-Sham and SMG4-AngII mice. Responsiveness to the contractile agonists, phenylephrine, 5-HT, and PGF 2 was significantly increased in endothelium-intact aortic rings from WT-AngII mice, but remained normal in aortae of SMG4-AngII mice. Following pretreatment with Rho-kinase inhibitor Y-27632, relative inhibition of contractility to 5-HT was equal in aortae from WT-AngII and SMG4-AngII-treated mice. With endothelial denudation, contractility to 5-HT was equally enhanced in aortae of WT-AngII and SMG4-AngII-treated mice. Interestingly, whereas acetylcholine stimulated relaxation was significantly decreased in aortic rings of WT-AngII mice, relaxation in rings from SMG4-AngII mice was not significantly different from WT or SMG4. These results demonstrate an interesting phenomenon whereby decreased expression of GLUT4 in vascular smooth muscle leads to an endothelial dysfunction that not only impairs relaxation, but also enhances contractility.

Sign in / Sign up

Export Citation Format

Share Document