Abstract
In the present study, we investigated whether extracellular sphingosine 1-phosphate (S1P) is involved in airway hyper-reactivity 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 (F340/F380). A 15-min pretreatment with S1P (>100 nM) markedly enhanced methacholine-induced contraction without elevating F340/F380. 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 Ca2+ 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 Ca2+ sensitization via inactivation of myosin phosphatase, which links Gi and RhoA/Rho-kinase processes.
Footnotes
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This work was supported by the Ministry of Education, Science, Sports, and Culture of Japan (Grants-in-Aid 17590785 to H.K. and 17790531 to S.I.).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.106.110718.
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ABBREVIATIONS: S1P, sphingosine 1-phosphate; BAL, bronchoalveolar lavage; ASM, airway smooth muscle; PTX, pertussis toxin; MCh, methacholine; BSM, bronchial smooth muscle; PBS, phosphate-buffered saline; MBS, myosin-binding subunit; MYPT1, myosin phosphatase target subunit 1; SKF96365, 1-{β-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride; Y-27632, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane-carboxamide; PD98059, 2′-amino-3′-methoxyflavone; MP, myosin phosphatase.
- Received July 18, 2006.
- Accepted November 10, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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