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

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 (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 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 Ca²⁺ sensitization via inactivation of myosin phosphatase, which links G_i and RhoA/Rho-kinase processes.