Kirschstein Timo, Sahre Theresa, Kernig Karoline, Protzel Chris, Porath Katrin, Köhling Rüdiger, Hakenberg Oliver W
Oscar Langendorff Institute of Physiology, University of Rostock, Gertrudenstrasse 9, 18057, Rostock, Germany.
Department of Urology, University of Rostock, Rostock, Germany.
BMC Urol. 2015 Oct 15;15:104. doi: 10.1186/s12894-015-0098-2.
Rho kinase (ROCK) and myosin-light chain kinase (MLCK) are key enzymes in smooth muscle contraction. Previous data have suggested that ROCK contribution to human detrusor contraction is increasing with age. Here, we have analyzed the transcriptional expression of Rho kinase isoforms (ROCK1 and ROCK2) as well as MLCK in the aging human detrusor smooth muscle obtained from resected tissue.
Small pieces of macroscopically healthy human detrusor smooth muscle (urothelium-free) were prepared for quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). Transcript expression (mRNA level) of the target genes ROCK1, ROCK2 and MLCK was normalized to three common reference genes (glyceraldehyde-3-phosphate dehydrogenase, β-actin, phosphoglycerate kinase 1).
We found that across all ages the expression level of ROCK (i.e. ROCK1 and ROCK2 together) was almost equal to that of MLCK in the human bladder. Further, ROCK2 showed a significantly higher expression level than ROCK1. Among all subjects, there was no significant correlation of any single target gene to age, but expression levels of ROCK and MLCK were inversely correlated. Moreover, the within-subject analysis revealed that the ROCK-to-MLCK ratio showed a significantly negative correlation to age. Thus, within a given subject, there is a relative ROCK down-regulation and concomitant MLCK up-regulation.
Together with previous data in human detrusor specimens showing increased ROCK contribution to detrusor contraction, we speculate that the drop of the ROCK-to-MLCK ratio may occur as an attempt to compensate for the increased Rho kinase activity.
Rho激酶（ROCK）和肌球蛋白轻链激酶（MLCK）是平滑肌收缩中的关键酶。先前的数据表明，ROCK对人类逼尿肌收缩的作用随年龄增长而增加。在此，我们分析了从切除组织中获取的衰老人类逼尿肌平滑肌中Rho激酶同工型（ROCK1和ROCK2）以及MLCK的转录表达。
制备肉眼可见健康的人类逼尿肌平滑肌小块（无尿路上皮）用于定量实时逆转录聚合酶链反应（RT-PCR）。将目标基因ROCK1、ROCK2和MLCK的转录表达（mRNA水平）标准化为三个常用参考基因（甘油醛-3-磷酸脱氢酶、β-肌动蛋白、磷酸甘油酸激酶1）。
我们发现，在所有年龄段中，人类膀胱中ROCK（即ROCK1和ROCK2之和）的表达水平几乎与MLCK相等。此外，ROCK2的表达水平显著高于ROCK1。在所有受试者中，任何单个目标基因与年龄均无显著相关性，但ROCK和MLCK的表达水平呈负相关。此外，受试者内分析显示，ROCK与MLCK的比值与年龄呈显著负相关。因此，在给定受试者中，存在相对的ROCK下调和伴随的MLCK上调。
结合先前人类逼尿肌标本中显示ROCK对逼尿肌收缩作用增加的数据，我们推测ROCK与MLCK比值的下降可能是为了补偿Rho激酶活性增加而出现的。
