| Summary: | <p>Abstract</p> <p>Background</p> <p>In peripheral airways, acetylcholine induces contraction via activation of muscarinic M2-and M3-receptor subtypes (M<sub>2</sub>R and M<sub>3</sub>R). 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 [Ca<sup>2+</sup>]<sub>i </sub>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.</p> <p>Methods</p> <p>To address this issue, we studied precision cut lung slices derived from FVB and M<sub>2</sub>R-KO and M<sub>3</sub>R-KO mice.</p> <p>Results</p> <p>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 M<sub>3</sub>R-KO mice when SPHK was inhibited by DHS and DMS but was unaffected in M<sub>2</sub>R-KO mice. Quantitative RT-PCR revealed that the disruption of the M<sub>2</sub>R and M<sub>3</sub>R genes had no significant effect on the expression levels of the SPHK1-isoform in peripheral airways.</p> <p>Conclusion</p> <p>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 M<sub>2</sub>R. Given the important role of muscarinic mechanisms in pulmonary disease, these findings should be of considerable therapeutic relevance.</p>
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