Pfaff Melanie, Powaga Norbert, Akinci Sibel, Schütz Werner, Banno Yoshiko, Wiegand Silke, Kummer Wolfgang, Wess Jürgen, Haberberger Rainer Viktor
Institute for Anatomy and Cell Biology, Justus-Liebig-University Giessen, Germany.
Respir Res. 2005 May 31;6(1):48. doi: 10.1186/1465-9921-6-48.
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.
To address this issue, we studied precision cut lung slices derived from FVB and M2R-KO and M3R-KO mice.
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.
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.
在外周气道中，乙酰胆碱通过激活毒蕈碱M2和M3受体亚型（M2R和M3R）诱导收缩。胆碱能超敏反应与慢性阻塞性肺疾病和哮喘相关，因此确定毒蕈碱信号通路具有重要的治疗意义。一条已被证明通过MR激活并增加[Ca2+]i的信号通路包括鞘氨醇激酶（SPHK）的激活和生物活性鞘脂鞘氨醇-1-磷酸（S1P）的生成。SPHK/S1P信号通路是否整合到外周气道的毒蕈碱控制中尚不清楚。
为了解决这个问题，我们研究了来自FVB、M2R基因敲除（M2R-KO）和M3R基因敲除（M3R-KO）小鼠的精密肺切片。
在FVB、野生型和MR缺陷小鼠的外周气道中，SPHK1主要定位于平滑肌。毒蕈碱在所有研究的小鼠品系中均诱导收缩，使用D，L-苏式-二氢鞘氨醇（DHS）和N，N-二甲基鞘氨醇（DMS）抑制SPHK可减少收缩，但不影响SPHK功能的结构相关试剂N-乙酰鞘氨醇（N-AcS）则无此作用。当SPHK被DHS和DMS抑制时，M3R-KO小鼠外周气道几乎没有收缩的初始阶段，但M2R-KO小鼠不受影响。定量逆转录聚合酶链反应（RT-PCR）显示，M2R和M3R基因的破坏对外周气道中SPHK1异构体的表达水平没有显著影响。
这些结果表明，SPHK/S1P信号通路参与了小鼠外周气道的胆碱能收缩。此外，我们的数据强烈表明SPHK是通过M2R激活的。鉴于毒蕈碱机制在肺部疾病中的重要作用，这些发现应具有相当大的治疗相关性。
