Sphingosine 1-phosphate causes airway hyper-reactivity by Rho-mediated myosin phosphatase inactivation

Hiroaki Kume, Naoya Takeda, Tetsuya Oguma, Satoru Ito, Masashi Kondo, Yasushi Ito, Kaoru Shimokata

Research output: Contribution to journalArticle

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Abstract

In the present study, we investigated whether extracellular sphingosine 1-phosphate (S1P) is involved in airway hyperreactivity in bronchial asthma. The effects of S1P on the response to methacholine was examined in the fura-2-loaded strips of guinea pig tracheal smooth muscle using simultaneous recording of the isometric tension and the ratio of fluorescence intensities at 340 and 380 nm (F 340/F 380). A 15-min pretreatment with S1P (>100 nM) markedly enhanced methacholine-induced contraction without elevating F 340/F 380. This effect of S1P was suppressed in the presence of Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)- cyclohexane-carboxamide], a selective inhibitor of Rho-kinase, in a concentration-dependent manner. Moreover, pretreatment with pertussis toxin caused an inhibition in S1P-induced hyper-reactivity to methacholine in a time- and concentration-dependent manner. In contrast, although S1P-induced Ca 2+ mobilization was attenuated by SKF96365 and verapamil, the subsequent response to methacholine was unaffected. A 15-min pretreatment with lower concentrations of S1P (<100 nM), which is clinically attainable, did not increase methacholine-induced contraction. However, when the incubation was lengthened to 6 h, S1P (<100 nM) enhanced the subsequent response to methacholine. Next, application of S1P to cultured human bronchial smooth muscle cells increased the proportion of active RhoA (GTP-RhoA) and phosphorylation of myosin phosphatase target subunit 1 (MYPT1). This phosphorylation of MYPT1 was significantly inhibited by application of Y-27632 and by pretreatment with pertussis toxin. Our findings demonstrate that exposure of airway smooth muscle to S1P results in airway hyper-reactivity mediated by Ca 2+ sensitization via inactivation of myosin phosphatase, which links G i and RhoA/Rho-kinase processes.

Original languageEnglish
Pages (from-to)766-773
Number of pages8
JournalJournal of Pharmacology and Experimental Therapeutics
Volume320
Issue number2
DOIs
Publication statusPublished - 01-02-2007

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Myosin-Light-Chain Phosphatase
Methacholine Chloride
rho-Associated Kinases
1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole
Pertussis Toxin
Smooth Muscle
Phosphorylation
sphingosine 1-phosphate
Fura-2
Verapamil
Guanosine Triphosphate
Smooth Muscle Myocytes
Guinea Pigs
Asthma
Fluorescence

All Science Journal Classification (ASJC) codes

  • Pharmacology

Cite this

Kume, Hiroaki ; Takeda, Naoya ; Oguma, Tetsuya ; Ito, Satoru ; Kondo, Masashi ; Ito, Yasushi ; Shimokata, Kaoru. / Sphingosine 1-phosphate causes airway hyper-reactivity by Rho-mediated myosin phosphatase inactivation. In: Journal of Pharmacology and Experimental Therapeutics. 2007 ; Vol. 320, No. 2. pp. 766-773.
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abstract = "In the present study, we investigated whether extracellular sphingosine 1-phosphate (S1P) is involved in airway hyperreactivity in bronchial asthma. The effects of S1P on the response to methacholine was examined in the fura-2-loaded strips of guinea pig tracheal smooth muscle using simultaneous recording of the isometric tension and the ratio of fluorescence intensities at 340 and 380 nm (F 340/F 380). A 15-min pretreatment with S1P (>100 nM) markedly enhanced methacholine-induced contraction without elevating F 340/F 380. This effect of S1P was suppressed in the presence of Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)- cyclohexane-carboxamide], a selective inhibitor of Rho-kinase, in a concentration-dependent manner. Moreover, pretreatment with pertussis toxin caused an inhibition in S1P-induced hyper-reactivity to methacholine in a time- and concentration-dependent manner. In contrast, although S1P-induced Ca 2+ mobilization was attenuated by SKF96365 and verapamil, the subsequent response to methacholine was unaffected. A 15-min pretreatment with lower concentrations of S1P (<100 nM), which is clinically attainable, did not increase methacholine-induced contraction. However, when the incubation was lengthened to 6 h, S1P (<100 nM) enhanced the subsequent response to methacholine. Next, application of S1P to cultured human bronchial smooth muscle cells increased the proportion of active RhoA (GTP-RhoA) and phosphorylation of myosin phosphatase target subunit 1 (MYPT1). This phosphorylation of MYPT1 was significantly inhibited by application of Y-27632 and by pretreatment with pertussis toxin. Our findings demonstrate that exposure of airway smooth muscle to S1P results in airway hyper-reactivity mediated by Ca 2+ sensitization via inactivation of myosin phosphatase, which links G i and RhoA/Rho-kinase processes.",
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Sphingosine 1-phosphate causes airway hyper-reactivity by Rho-mediated myosin phosphatase inactivation. / Kume, Hiroaki; Takeda, Naoya; Oguma, Tetsuya; Ito, Satoru; Kondo, Masashi; Ito, Yasushi; Shimokata, Kaoru.

In: Journal of Pharmacology and Experimental Therapeutics, Vol. 320, No. 2, 01.02.2007, p. 766-773.

Research output: Contribution to journalArticle

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T1 - Sphingosine 1-phosphate causes airway hyper-reactivity by Rho-mediated myosin phosphatase inactivation

AU - Kume, Hiroaki

AU - Takeda, Naoya

AU - Oguma, Tetsuya

AU - Ito, Satoru

AU - Kondo, Masashi

AU - Ito, Yasushi

AU - Shimokata, Kaoru

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N2 - In the present study, we investigated whether extracellular sphingosine 1-phosphate (S1P) is involved in airway hyperreactivity in bronchial asthma. The effects of S1P on the response to methacholine was examined in the fura-2-loaded strips of guinea pig tracheal smooth muscle using simultaneous recording of the isometric tension and the ratio of fluorescence intensities at 340 and 380 nm (F 340/F 380). A 15-min pretreatment with S1P (>100 nM) markedly enhanced methacholine-induced contraction without elevating F 340/F 380. This effect of S1P was suppressed in the presence of Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)- cyclohexane-carboxamide], a selective inhibitor of Rho-kinase, in a concentration-dependent manner. Moreover, pretreatment with pertussis toxin caused an inhibition in S1P-induced hyper-reactivity to methacholine in a time- and concentration-dependent manner. In contrast, although S1P-induced Ca 2+ mobilization was attenuated by SKF96365 and verapamil, the subsequent response to methacholine was unaffected. A 15-min pretreatment with lower concentrations of S1P (<100 nM), which is clinically attainable, did not increase methacholine-induced contraction. However, when the incubation was lengthened to 6 h, S1P (<100 nM) enhanced the subsequent response to methacholine. Next, application of S1P to cultured human bronchial smooth muscle cells increased the proportion of active RhoA (GTP-RhoA) and phosphorylation of myosin phosphatase target subunit 1 (MYPT1). This phosphorylation of MYPT1 was significantly inhibited by application of Y-27632 and by pretreatment with pertussis toxin. Our findings demonstrate that exposure of airway smooth muscle to S1P results in airway hyper-reactivity mediated by Ca 2+ sensitization via inactivation of myosin phosphatase, which links G i and RhoA/Rho-kinase processes.

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