Thorpe Richard B, Hubbell Margaret C, Silpanisong Jinjutha, Williams James M, Pearce William J
Center for Perinatal Biology and Divisions of Physiology, Pharmacology, and Biochemistry, Loma Linda University School of Medicine, Loma Linda, California.
Center for Perinatal Biology and Divisions of Physiology, Pharmacology, and Biochemistry, Loma Linda University School of Medicine, Loma Linda, California
Am J Physiol Heart Circ Physiol. 2017 Jul 1;313(1):H207-H219. doi: 10.1152/ajpheart.00480.2016. Epub 2017 May 26.
Long-term hypoxia (LTH) attenuates nitric oxide-induced vasorelaxation in ovine middle cerebral arteries. Because cGMP-dependent protein kinase (PKG) is an important mediator of NO signaling in vascular smooth muscle, we tested the hypothesis that LTH diminishes the ability of PKG to interact with target proteins and cause vasorelaxation. Prominent among proteins that regulate vascular tone is the large-conductance Ca-sensitive K (BK) channel, which is a substrate for PKG and is responsive to phosphorylation on multiple serine/threonine residues. Given the influence of these proteins, we also examined whether LTH attenuates PKG and BK channel protein abundances and PKG activity. Middle cerebral arteries were harvested from normoxic and hypoxic (altitude of 3,820 m for 110 days) fetal and adult sheep. These arteries were denuded and equilibrated with 95% O-5% CO in the presence of -nitro-l-arginine methyl ester (l-NAME) to inhibit potential confounding influences of events upstream from PKG. Expression and activity of PKG-I were not significantly affected by chronic hypoxia in either fetal or adult arteries. Pretreatment with the BK inhibitor iberiotoxin attenuated vasorelaxation induced by 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate in normoxic but not LTH arteries. The spatial proximities of PKG with BK channel α- and β1-proteins were examined using confocal microscopy, which revealed a strong dissociation of PKG with these proteins after LTH. These results support our hypothesis that hypoxia reduces the ability of PKG to attenuate vasoconstriction in part through suppression of the ability of PKG to associate with and thereby activate BK channels in arterial smooth muscle. Using measurements of contractility, protein abundance, kinase activity, and confocal colocalization in fetal and adult ovine cerebral arteries, the present study demonstrates that long-term hypoxia diminishes the ability of cGMP-dependent protein kinase (PKG) to cause vasorelaxation through suppression of its colocalization and interaction with large-conductance Ca-sensitive K (BK) channel proteins in cerebrovascular smooth muscle. These experiments are among the first to demonstrate hypoxic changes in BK subunit abundances in fetal cerebral arteries and also introduce the use of advanced methods of confocal colocalization to study interaction between PKG and its targets.
长期缺氧（LTH）会减弱一氧化氮诱导的绵羊大脑中动脉血管舒张。由于环磷酸鸟苷依赖性蛋白激酶（PKG）是血管平滑肌中NO信号的重要介质，我们检验了以下假设：LTH会降低PKG与靶蛋白相互作用并引起血管舒张的能力。在调节血管张力的蛋白质中，大电导钙敏感钾（BK）通道尤为突出，它是PKG的底物，对多个丝氨酸/苏氨酸残基的磷酸化有反应。鉴于这些蛋白质的影响，我们还研究了LTH是否会降低PKG和BK通道蛋白丰度以及PKG活性。从常氧和缺氧（海拔3820米，持续110天）的胎儿和成年绵羊中采集大脑中动脉。去除这些动脉的内皮，并在存在L-硝基-L-精氨酸甲酯（L-NAME）的情况下用95%O₂-5%CO₂平衡，以抑制PKG上游事件的潜在混杂影响。PKG-I的表达和活性在胎儿或成年动脉中均未受到慢性缺氧的显著影响。用BK抑制剂iberiotoxin预处理可减弱常氧但非LTH动脉中8-（4-氯苯硫基）鸟苷3',5'-环一磷酸诱导的血管舒张。使用共聚焦显微镜检查PKG与BK通道α-和β1-蛋白的空间接近性，结果显示LTH后PKG与这些蛋白强烈解离。这些结果支持了我们的假设，即缺氧部分通过抑制PKG与动脉平滑肌中BK通道结合并激活的能力来降低PKG减弱血管收缩的能力。通过测量胎儿和成年绵羊脑动脉的收缩性、蛋白丰度、激酶活性以及共聚焦共定位，本研究表明长期缺氧会降低环磷酸鸟苷依赖性蛋白激酶（PKG）通过抑制其与脑血管平滑肌中大电导钙敏感钾（BK）通道蛋白的共定位和相互作用来引起血管舒张的能力。这些实验是最早证明胎儿脑动脉中BK亚基丰度的缺氧变化的实验之一，并且还引入了先进的共聚焦共定位方法来研究PKG与其靶标的相互作用。
