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 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 (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.
Kume
Hiroaki
H
Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, 65 Tsrumai-cho, Showa-ku, Nagoya 466-8550, Japan. hkume@med.nagoya-u.ac.jp.
Takeda
Naoya
N
Oguma
Tetsuya
T
Ito
Satoru
S
Kondo
Masashi
M
Ito
Yasushi
Y
Shimokata
Kaoru
K
D000818:Animals; D016535:Bronchial Hyperreactivity / Q000139:chemically induced*; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D006168:Guinea Pigs; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000502:physiology*; D008246:Lysophospholipids / Q000494:pharmacology*; D008297:Male; D043263:Myosin-Light-Chain Phosphatase / Q000502:physiology*; D037342:Pertussis Toxin / Q000494:pharmacology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D015398:Signal Transduction; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D054460:rho-Associated Kinases
D047908:Intracellular Signaling Peptides and Proteins; D008246:Lysophospholipids; C060506:sphingosine 1-phosphate; D037342:Pertussis Toxin; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D043263:Myosin-Light-Chain Phosphatase; D013110:Sphingosine; D002118:Calcium