Vera Pedro L, Iczkowski Kenneth A, Leng Lin, Bucala Richard, Meyer-Siegler Katherine L
Research and Development Service, Bay Pines Veterans Affairs Medical Center, Bay Pines, Florida, USA.
J Urol. 2005 Jul;174(1):338-43. doi: 10.1097/01.ju.0000161606.15696.79.
Macrophage migration inhibitory factor (MIF) is released into intraluminal fluid (ILF) during bladder inflammation in the rat. We investigated the forms of MIF that are released. We examined MIF release after subcutaneous substance P (SP) or intravesical capsaicin and studied proteins associated with excreted MIF in ILF.
Anesthetized male rats with the bladder isolated from the kidneys were injected with SP subcutaneously (saline vehicle) or with intravesical capsaicin (vehicle, 0.1 mM and 1 mM). After 1 hour the ILF was removed and MIF levels were determined using enzyme-linked immunosorbent assay or Western blotting procedures under native, nonreducing and reducing conditions. Mass spectrometry was used to identify proteins associated with MIF in ILF and results were verified by immunoprecipitation.
SP and intravesical capsaicin increased the total amount of MIF in ILF. MIF was found in high molecular weight complexes that resolved into 2 bands under nonreducing conditions. SP and capsaicin differentially increased the MIF bands. Mass spectrometry determined that MIF was complexed with acute phase proteins. MIF immunoprecipitation followed by Western blotting confirmed that MIF was complexed to alpha1-inhibitor-3.
MIF is complexed with alpha1-inhibitor-3, a member of the alpha-2-macroglobulin proteinase inhibitor family, in the rat. Although SP and capsaicin increased the total amount of MIF detected by enzyme-linked immunosorbent assay in ILF, the patterns of MIF complexes elicited by these 2 treatments were different. These findings suggest that in association with other proteins MIF forms part of a complex elicited by bladder inflammation.
在大鼠膀胱炎症期间，巨噬细胞移动抑制因子（MIF）会释放到管腔内液（ILF）中。我们研究了释放的MIF的形式。我们检测了皮下注射P物质（SP）或膀胱内注射辣椒素后MIF的释放情况，并研究了与ILF中排出的MIF相关的蛋白质。
将膀胱与肾脏分离的麻醉雄性大鼠皮下注射SP（生理盐水载体）或膀胱内注射辣椒素（载体，0.1 mM和1 mM）。1小时后取出ILF，在天然、非还原和还原条件下，使用酶联免疫吸附测定或蛋白质印迹法测定MIF水平。采用质谱法鉴定与ILF中MIF相关的蛋白质，并通过免疫沉淀验证结果。
SP和膀胱内注射辣椒素增加了ILF中MIF的总量。在高分子量复合物中发现了MIF，在非还原条件下该复合物可分解为两条带。SP和辣椒素对MIF条带的增加存在差异。质谱分析确定MIF与急性期蛋白形成复合物。MIF免疫沉淀后进行蛋白质印迹证实MIF与α1-抑制因子-3形成复合物。
在大鼠中，MIF与α2-巨球蛋白蛋白酶抑制剂家族的成员α1-抑制因子-3形成复合物。尽管SP和辣椒素增加了通过酶联免疫吸附测定在ILF中检测到的MIF总量，但这两种处理引起的MIF复合物模式不同。这些发现表明，与其他蛋白质相关联，MIF构成了由膀胱炎症引发的复合物的一部分。
