Saed G M, Carretero O A, MacDonald R J, Scicli A G
Henry Ford Hospital, Hypertension Research Division, Detroit, Mich 48202.
Circ Res. 1990 Aug;67(2):510-6. doi: 10.1161/01.res.67.2.510.
Glandular kallikrein (EC 3.4.21.8) belongs to a subgroup of serine proteases coded by a multigene family. A kininogenase resembling glandular kallikrein has been identified in vascular tissue; however, it is not clear whether it is synthesized by vascular tissue or taken up from plasma. To determine the potential for kallikrein synthesis in vascular tissues, we tested whether messenger RNA (mRNA) for glandular kallikrein is present in rat arteries and veins. Poly(A+) RNA was isolated from pools of arteries or veins (n = 3, 30 rats each). Poly(A+) RNA from the kidney and liver was used as a positive and negative control, respectively. As a probe, we used rat pancreatic kallikrein 32P-labeled complementary DNA, which recognizes mRNA of the entire rat kallikrein family. Slot-blot analysis indicated that kallikrein mRNA was present in mRNA from the arteries, veins, and kidney but not from the liver. Poly(A+) RNA from arteries and veins contained approximately 1% as much kallikrein mRNA as that from the kidney. To confirm the slot-blot results and determine whether the mRNA for true glandular kallikrein was present in vascular tissue, we employed a polymerase chain reaction assay, first using primers specific for the entire kallikrein family (which amplify a 430-bp fragment) and then using primers specific for true glandular kallikrein mRNA (which amplify a 370-bp fragment). After the polymerase chain reaction assay, both arteries and veins showed fragments of these sizes when tested with rat kallikrein complementary DNA probe, thus confirming the presence of glandular kallikrein mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)
腺激肽释放酶(EC 3.4.21.8)属于由多基因家族编码的丝氨酸蛋白酶亚组。在血管组织中已鉴定出一种类似于腺激肽释放酶的激肽原酶;然而,尚不清楚它是由血管组织合成的还是从血浆中摄取的。为了确定血管组织中激肽释放酶合成的可能性,我们检测了大鼠动脉和静脉中是否存在腺激肽释放酶的信使核糖核酸(mRNA)。从动脉或静脉池(n = 3,每组30只大鼠)中分离出聚腺苷酸加尾(Poly(A+))RNA。分别以肾脏和肝脏的Poly(A+) RNA作为阳性和阴性对照。作为探针,我们使用了大鼠胰腺激肽释放酶32P标记的互补DNA,它能识别整个大鼠激肽释放酶家族的mRNA。狭缝印迹分析表明,激肽释放酶mRNA存在于动脉、静脉和肾脏的mRNA中,但不存在于肝脏中。动脉和静脉的Poly(A+) RNA中激肽释放酶mRNA的含量约为肾脏的1%。为了证实狭缝印迹结果并确定血管组织中是否存在真正的腺激肽释放酶mRNA,我们采用了聚合酶链反应检测法,首先使用针对整个激肽释放酶家族的引物(扩增出一个430碱基对的片段),然后使用针对真正的腺激肽释放酶mRNA的引物(扩增出一个370碱基对的片段)。在聚合酶链反应检测后,当用大鼠激肽释放酶互补DNA探针检测时,动脉和静脉均显示出这些大小的片段,从而证实了腺激肽释放酶mRNA的存在。(摘要截短于250字)