Department of Biomedicine, Aarhus University, Aarhus C, Denmark.
Department of Molecular Medicine and Surgery, Integrative Physiology, Karolinska Institutet, Stockholm, Sweden.
Acta Physiol (Oxf). 2018 Sep;224(1):e13059. doi: 10.1111/apha.13059. Epub 2018 Mar 25.
The Na,K-ATPase is involved in a large number of regulatory activities including cSrc-dependent signalling. Upon inhibition of the Na,K-ATPase with ouabain, cSrc activation is shown to occur in many cell types. This study tests the hypothesis that acute potentiation of agonist-induced contraction by ouabain is mediated through Na,K-ATPase-cSrc signalling-dependent sensitization of vascular smooth muscle cells to Ca .
Agonist-induced rat mesenteric small artery contraction was examined in vitro under isometric conditions and in vivo in anaesthetized rats. Arterial wall tension and [Ca ] in vascular smooth muscle cells were measured simultaneously. Changes in cSrc and myosin phosphatase targeting protein 1 (MYPT1) phosphorylation were analysed by Western blot. Protein expression was examined with immunohistochemistry. The α1 and α2 isoforms of the Na,K-ATPase were transiently downregulated by siRNA transfection in vivo.
Ten micromolar ouabain, but not digoxin, potentiated contraction to noradrenaline. This effect was not endothelium-dependent. Ouabain sensitized smooth muscle cells to Ca , and this was associated with increased phosphorylation of cSrc and MYPT1. Inhibition of tyrosine kinase by genistein, PP2 or pNaKtide abolished the potentiating effect of ouabain on arterial contraction and Ca sensitization. Downregulation of the Na,K-ATPase α2 isoform made arterial contraction insensitive to ouabain and tyrosine kinase inhibition.
Data suggest that micromolar ouabain potentiates agonist-induced contraction of rat mesenteric small artery via Na,K-ATPase-dependent cSrc activation, which increases Ca sensitization of vascular smooth muscle cells by MYPT1 phosphorylation. This mechanism may be critical for acute control of vascular tone.
钠钾-ATP 酶参与了许多调节活动，包括 cSrc 依赖性信号转导。在哇巴因抑制钠钾-ATP 酶后，许多细胞类型中 cSrc 的激活被证明发生。本研究检验了这样一个假设，即哇巴因急性增强激动剂诱导的收缩是通过钠钾-ATP 酶-cSrc 信号依赖性血管平滑肌细胞对 Ca 的敏感性来介导的。
在等长条件下体外检测激动剂诱导的大鼠肠系膜小动脉收缩，并在麻醉大鼠体内检测。同时测量血管平滑肌细胞的动脉壁张力和[Ca]。通过 Western blot 分析 cSrc 和肌球蛋白磷酸酶靶向蛋白 1（MYPT1）的磷酸化变化。用免疫组织化学法检测蛋白表达。用 siRNA 转染体内瞬时下调钠钾-ATP 酶的α1和α2 同工型。
10μM 哇巴因，但不是地高辛，增强去甲肾上腺素的收缩作用。这种作用与内皮无关。哇巴因使平滑肌细胞对 Ca 敏感，这与 cSrc 和 MYPT1 的磷酸化增加有关。用 genistein、PP2 或 pNaKtide 抑制酪氨酸激酶，消除了哇巴因对动脉收缩和 Ca 敏感性的增强作用。钠钾-ATP 酶α2 同工型的下调使动脉收缩对哇巴因和酪氨酸激酶抑制不敏感。
数据表明，微摩尔哇巴因通过钠钾-ATP 酶依赖性 cSrc 激活增强大鼠肠系膜小动脉激动剂诱导的收缩，通过 MYPT1 磷酸化增加血管平滑肌细胞对 Ca 的敏感性。这种机制可能对急性血管张力控制至关重要。
