Activation of the SPHK/S1P signalling pathway is coupled to muscarinic receptor-dependent regulation of peripheral airways.
BACKGROUND
In peripheral airways, acetylcholine induces contraction via activation of muscarinic M2-and M3-receptor subtypes (M2R and M3R). Cholinergic hypersensitivity is associated with chronic obstructive pulmonary disease and asthma, and therefore the identification of muscarinic signaling pathways are of great therapeutic interest. A pathway that has been shown to be activated via MR and to increase [Ca2+]i includes the activation of sphingosine kinases (SPHK) and the generation of the bioactive sphingolipid sphingosine 1-phosphate (S1P). Whether the SPHK/S1P signaling pathway is integrated in the muscarinic control of peripheral airways is not known.


METHODS
To address this issue, we studied precision cut lung slices derived from FVB and M2R-KO and M3R-KO mice.


RESULTS
In peripheral airways of FVB, wild-type, and MR-deficient mice, SPHK1 was mainly localized to smooth muscle. Muscarine induced a constriction in all investigated mouse strains which was reduced by inhibition of SPHK using D, L-threo-dihydrosphingosine (DHS) and N, N-dimethyl-sphingosine (DMS) but not by N-acetylsphingosine (N-AcS), a structurally related agent that does not affect SPHK function. The initial phase of constriction was nearly absent in peripheral airways of M3R-KO mice when SPHK was inhibited by DHS and DMS but was unaffected in M2R-KO mice. Quantitative RT-PCR revealed that the disruption of the M2R and M3R genes had no significant effect on the expression levels of the SPHK1-isoform in peripheral airways.


CONCLUSIONS
These results demonstrate that the SPHK/S1P signaling pathway contributes to cholinergic constriction of murine peripheral airways. In addition, our data strongly suggest that SPHK is activated via the M2R. Given the important role of muscarinic mechanisms in pulmonary disease, these findings should be of considerable therapeutic relevance.
Pfaff
Melanie
M
Institute for Anatomy and Cell Biology, Justus-Liebig-University Giessen, Germany. Mel.pfaff@gmx.de
Powaga
Norbert
N
Akinci
Sibel
S
Schütz
Werner
W
Banno
Yoshiko
Y
Wiegand
Silke
S
Kummer
Wolfgang
W
Wess
Jürgen
J
Haberberger
Rainer Viktor
RV
D000818:Animals; D002478:Cells, Cultured; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D008168:Lung / Q000378:metabolism*; D008246:Lysophospholipids / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D043585:Receptor, Muscarinic M2 / Q000378:metabolism*; D043587:Receptor, Muscarinic M3 / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism; D014018:Tissue Distribution; D000099157:Sphingosine Kinase
D008246:Lysophospholipids; D017853:Phosphotransferases (Alcohol Group Acceptor); D043585:Receptor, Muscarinic M2; D043587:Receptor, Muscarinic M3; D013110:Sphingosine; D000099157:Sphingosine Kinase; C060506:sphingosine 1-phosphate