Department of Urology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
BJU Int. 2010 Aug;106(4):562-71. doi: 10.1111/j.1464-410X.2009.09156.x. Epub 2010 Jan 29.
To examine the effect of partial urethral obstruction (PUO) on the sphingosine-1-phosphate (S1P, a bioactive lipid shown to modulate smooth muscle, SM) pathway in the bladders of male rats, and to determine the effect of PUO on the RhoA/Rho-kinase (ROK) pathway, and whether there is a molecular cross-talk with the S1P pathways associated with bladder overactivity (S1P1-S1P3, where S1P1 is associated with nitric oxide-mediated SM relaxation, and S1P2 and S1P3 receptors are associated more with SM contraction via the ROK pathway).
In all, 20 male rats were divided into two groups and underwent PUO or a sham operation (control). After 2 weeks all rats were killed humanely and bladder specimens used for in vitro organ-bath physiological contractility studies, and for mRNA and protein analyses of major S1P/ROK pathway constituents via real-time polymerase chain reaction and Western blotting, respectively. In addition, early-passage SM cells were transfected with recombinant sphingosine kinase (SPHK, the enzyme that converts sphingosine to S1P).
Bladders from PUO rats had greater mRNA expression of the S1P2 and S1P3 receptors, as well as SPHK1, than the sham controls (4.78, 2.04 and 2.72 times, respectively). PUO rats also had significantly greater expression of RhoA and ROKalpha (1.76 and 2.19 times, respectively). Western blotting and organ-bath contractility studies showed similar changes at the protein and in vitro functional level, with an increased contractility of bladder strips from PUO rats to exogenous S1P. Transfection of SPHK into isolated SM cells increased ROK expression.
We show for the first time that the S1P signalling pathway is significantly upregulated in response to PUO in male rats at both the molecular and in vitro functional levels, correlating with an activation of the RhoA/ROK pathway. Further, we provide novel data that SPHK overexpression increases ROK expression in vitro, suggesting a novel hypothesis of S1P-induced bladder overactivity in the mechanism for PUO-induced bladder dysfunction and the S1P signalling pathway as a possible therapeutic target for bladder overactivity.
研究部分尿道梗阻（PUO）对雄性大鼠膀胱中鞘氨醇-1-磷酸（S1P，一种被证明可调节平滑肌[SM]的生物活性脂质）途径的影响，并确定 PUO 对 RhoA/ Rho-激酶（ROK）途径的影响，以及是否存在与膀胱过度活动相关的 S1P 途径的分子相互作用（S1P1-S1P3，其中 S1P1 与一氧化氮介导的 SM 松弛有关，而 S1P2 和 S1P3 受体更通过 ROK 途径与 SM 收缩有关）。
总共 20 只雄性大鼠被分为两组，分别进行 PUO 或假手术（对照组）。2 周后，所有大鼠均人道处死，用于离体器官浴生理收缩性研究，并通过实时聚合酶链反应和 Western 印迹分别对主要 S1P/ROK 途径成分的 mRNA 和蛋白质进行分析。此外，早期传代的 SM 细胞被重组鞘氨醇激酶（SPHK，将鞘氨醇转化为 S1P 的酶）转染。
与对照组相比，PUO 大鼠的 S1P2 和 S1P3 受体以及 SPHK1 的 mRNA 表达水平更高（分别为 4.78、2.04 和 2.72 倍）。PUO 大鼠 RhoA 和 ROKalpha 的表达也显著增加（分别为 1.76 和 2.19 倍）。Western 印迹和器官浴收缩性研究在蛋白质和体外功能水平上显示出相似的变化，PUO 大鼠膀胱条带对外源性 S1P 的收缩性增加。SPHK 转染到分离的 SM 细胞中增加了 ROK 的表达。
我们首次表明，在雄性大鼠中，PUO 会导致 S1P 信号通路在分子和体外功能水平上显著上调，与 RhoA/ROK 途径的激活相关。此外，我们提供了新的数据表明，SPHK 过表达可增加体外 ROK 的表达，这表明 S1P 诱导的膀胱过度活动的机制中存在 S1P 诱导的膀胱过度活动的新假说，以及 S1P 信号通路作为膀胱过度活动的可能治疗靶点。
