Sorensen Dane W, Injeti Elisha R, Mejia-Aguilar Luisa, Williams James M, Pearce William J
Division of Physiology, Center for Perinatal Biology, Loma Linda University School of Medicine, Loma Linda, California.
Department of Pharmaceutical Sciences, Cedarville University School of Pharmacy, Cedarville, Ohio.
Am J Physiol Regul Integr Comp Physiol. 2021 Sep 1;321(3):R441-R453. doi: 10.1152/ajpregu.00293.2020. Epub 2021 Jul 28.
The rate-limiting enzyme for vascular contraction, myosin light chain kinase (MLCK), phosphorylates regulatory myosin light chain (MLC) at rates that appear faster despite lower MLCK abundance in fetal compared with adult arteries. This study explores the hypothesis that greater apparent tissue activity of MLCK in fetal arteries is due to age-dependent differences in intracellular distribution of MLCK in relation to MLC. Under optimal conditions, common carotid artery homogenates from nonpregnant adult female sheep and near-term fetuses exhibited similar values of and for MLCK. A custom-designed, computer-controlled apparatus enabled electrical stimulation and high-speed freezing of arterial segments at exactly 0, 1, 2, and 3 s, calculation of in situ rates of MLC phosphorylation, and measurement of time-dependent colocalization between MLCK and MLC. The in situ rate of MLC phosphorylation divided by total MLCK abundance averaged to values 147% greater in fetal (1.06 ± 0.28) than adult (0.43 ± 0.08) arteries, which corresponded, respectively, to 43 ± 10% and 31 ± 3% of the values measured in homogenates. Confocal colocalization analysis revealed in fetal and adult arteries that 33 ± 6% and 20 ± 5% of total MLCK colocalized with pMLC, and that MLCK activation was greater in periluminal than periadventitial regions over the time course of electrical stimulation in both age groups. Together, these results demonstrate that the catalytic activity of MLCK is similar in fetal and adult arteries, but that the fraction of total MLCK in the functional compartment involved in contraction is significantly greater in fetal than adult arteries.
血管收缩的限速酶，肌球蛋白轻链激酶（MLCK），尽管与成年动脉相比，胎儿动脉中MLCK的丰度较低，但它磷酸化调节性肌球蛋白轻链（MLC）的速率似乎更快。本研究探讨了这样一种假说，即胎儿动脉中MLCK更高的表观组织活性是由于MLCK与MLC在细胞内分布上的年龄依赖性差异所致。在最佳条件下，来自未怀孕成年雌性绵羊和近足月胎儿的颈总动脉匀浆显示出相似的MLCK的 和 值。一种定制设计的计算机控制设备能够在精确的0、1、2和3秒时对动脉段进行电刺激和高速冷冻，计算MLC磷酸化的原位速率，并测量MLCK和MLC之间随时间的共定位。MLC磷酸化的原位速率除以总MLCK丰度，胎儿动脉（1.06±0.28）的平均值比成年动脉（0.43±0.08）高147%，分别相当于匀浆中测量值的43±10%和31±3%。共聚焦共定位分析显示，在胎儿和成年动脉中分别有33±6%和20±5%的总MLCK与磷酸化MLC（pMLC）共定位，并且在两个年龄组的电刺激过程中，管腔周围区域的MLCK激活比外膜周围区域更大。总之，这些结果表明，胎儿和成年动脉中MLCK的催化活性相似，但参与收缩的功能区中总MLCK的比例在胎儿动脉中比成年动脉显著更高。
