Lubomirov Lubomir T, Weber Greta, Schroeter Mechthild, Metzler Doris, Bust Maria, Korotkova Tatiana, Hescheler Jürgen, Todorov Vladimir T, Pfitzer Gabriele, Grisk Olaf
Institute of Physiology, Brandenburg Medical School Theodor Fontane, Germany; Institute of Vegetative Physiology, Center of Physiology, University of Cologne, Germany; Vascular Biology Research Group (RenEVA), Research Institute, Medical University-Varna, Varna, Bulgaria; Institute of Physiology and Pathophysiology, Faculty of Health - School of Medicine, Biomedical Center for Education and Research (ZBAF), Witten/Herdecke University, Witten, Germany; Research Cluster, Molecular Mechanisms of Cardiovascular Diseases, Brandenburg Medical School Theodor Fontane, Neuruppin, Germany.
Institute of Physiology, Brandenburg Medical School Theodor Fontane, Germany.
Eur J Pharmacol. 2025 Jan 5;986:177133. doi: 10.1016/j.ejphar.2024.177133. Epub 2024 Nov 17.
The femoral artery (FA) is the largest vessel in the hindlimb circulation and its proper tone regulation ensures adequate blood supply to muscle tissue. We investigated whether an alanine mutation of the targeting subunit of myosin-light-chain-phosphatase (MLCP), MYPT1, at threonine 696 (MYPT1-T696A/+), decisive for enzyme acivity, affects the responsiveness of young and old FAs (y-FAs and o-FAs) to activation of nitric-oxide/soluble-guanylate-cyclase/protein-kinase-G cascade (NO/sGC/PKG). Contractile responses of the vessels were measured by wire myography. Phosphorylation of the regulatory myosin-light-chain at serine 19 (MLC-S19), the myosin-light-chain-phosphatase targeting subunit, MYPT1-T696, the PKG-sensitive site of MYPT1 at S695 (MYPT1-S695) and S668 (MYPT1-S668), and the regulatory phosphorylation of eNOS at S1177 (eNOS-S1177) were determined in arterial homogenates by Western blot. In FAs of all ages, the MYPT1-T696A-mutation did not alter vessel diameter and the contractile reactivity to the thromboxaneA-analogue, U46619 and the RhoA kinase inhibitor, Y27632. In contrast, the mutation T696 into alanine attenuated the relaxing effect of exogenous NO (DEA-NONOate) in y-FAs. The effect of a direct sGC activation by cinaciguat was also attenuated in both age groups of MYPT1-T696A/+, but strongly in o-FA. The MYPT1-T696A-mutation also attenuated acetylcholine-induced relaxation, but only in o-FAs. Similary, the alanine mutation attenuated the acetylcholine effect on MLC-S19- and MYPT1-T696 only in WT o-FAs. Interestingly, neither eNOS-S1177 nor the phosphorylation of the PKG phosphospecific sites, MYPT1-S695 and MYPT1-S668 were altered by MYPT1-T696A-mutation or aging. These findings suggest that the alanine mutation of MYPT1-T696 reduces the ability of the NO/cGMP/PKG-system to relax FAs in aging.
股动脉（FA）是后肢循环中最大的血管，其适当的张力调节可确保向肌肉组织提供充足的血液供应。我们研究了肌球蛋白轻链磷酸酶（MLCP）的靶向亚基MYPT1在苏氨酸696处的丙氨酸突变（MYPT1-T696A/+）（这对酶活性起决定性作用）是否会影响年轻和老年FA（y-FA和o-FA）对一氧化氮/可溶性鸟苷酸环化酶/蛋白激酶G级联反应（NO/sGC/PKG）激活的反应性。通过线肌张力描记法测量血管的收缩反应。通过蛋白质免疫印迹法在动脉匀浆中测定丝氨酸19（MLC-S19）处调节性肌球蛋白轻链、肌球蛋白轻链磷酸酶靶向亚基MYPT1-T696、MYPT1在S695（MYPT1-S695）和S668（MYPT1-S668）处的PKG敏感位点以及内皮型一氧化氮合酶在S1177（eNOS-S1177）处的调节性磷酸化。在所有年龄段的FA中，MYPT1-T696A突变均未改变血管直径以及对血栓素A类似物U46619和RhoA激酶抑制剂Y27632的收缩反应性。相反，将T696突变为丙氨酸减弱了外源性NO（DEA- NONOate）对y-FA的舒张作用。在MYPT1-T696A/+的两个年龄组中，西那卡瓜直接激活sGC的作用也减弱了，但在o-FA中减弱得更明显。MYPT1-T696A突变也减弱了乙酰胆碱诱导的舒张作用，但仅在o-FA中。同样，丙氨酸突变仅在野生型o-FA中减弱了乙酰胆碱对MLC-S19和MYPT1-T696的作用。有趣的是，MYPT1-T696A突变或衰老均未改变eNOS-S1177以及PKG磷酸特异性位点MYPT1-S695和MYPT1-S668的磷酸化。这些发现表明，MYPT1-T696的丙氨酸突变降低了衰老过程中NO/cGMP/PKG系统舒张FA的能力。
