Fetal and adult cerebral and pulmonary artery BK channel adaptations to hypoxia

长期低氧(LTH)导致胎儿和成人脑血管的血管反应性发生深刻变化。尽管血管管腔内皮细胞和外周肾上腺素能神经在血管反应性中起主要作用，但血管平滑肌细胞(VSMCs)是血管张力的最终仲裁者。VSMC通常存在于部分收缩的 状态，产生肌源性张力，并能够进一步收缩或在暴露于可溶信号时放松。VSMC的反应性受许多因素的影响，包括发育和长期缺氧(LTH)，这两个因素都是拟议研究中的主要变量。VSMCs的发育变化包括收缩蛋白、膜成分、受体、信号通路和离子通道的变化。这些变化中的每一个都会影响血管的反应性。本实验室目前的研究表明，LTH对血管平滑肌的部分作用涉及钙激活、电压敏感的K+(BK或KCA)通道。BK通道可以说是血管树的脑和肺分支的VSMCs上最重要的单一通道。BK通道是VSMC反应性的关键，因为它们将细胞内游离钙水平与膜电位偶联。 此外，与其他已知的VSMC通道相比，它们受到更多的细胞外和细胞内因子的调节。事实上，BK通道存在于所有类型的平滑肌中，它们的激活通过膜的超极化间接地降低了收缩能力。在成熟动脉的血管平滑肌中，BK通道通过局部反应来调节膜电位 在ryanodine受体的控制下，由内库释放的“钙火花”引起的[Ca~(2+)]i升高。然而，在胎脑VSMCs中，BK通道主要对钙离子通过L型钙通道内流而引起的[Ca~(2+)]_i升高作出反应。由于单个SIO基因的选择性剪接，BK通道的功能和结构可能会发生变化，但很少 已知在发育或LTH过程中的通道功能和结构变化。 VSMCs和脑神经元的BK通道在功能上与某些蛋白激酶和磷酸酶结合在质膜上。我们发现，BK通道对各种调节因子的反应在发育过程中和对LTH的反应都不同，这表明BK通道对LTH的生理适应在胎儿和成人之间是不同的，其中一些对病理生理生存具有潜在的关键作用。结果表明：(1)LTH成人BK通道的钙调定点(3.3mM)与LTH和常氧胎儿(分别为3.0mM和4.7mM)相似，但仅为常氧成人(8.8mM；Lin等，2003)的一半。(2)LTH成人BK通道对细胞内氯化血红素抑制的敏感性(IC50=50 nM)是常氧成人或LTH和正常氧胎儿的4倍(IC50= 200海里)。(3)LTH成人BK通道在急性缺氧时被激活，其激活程度是LTH胎儿BK通道的两倍以上。(4)LTH成人BK通道的通道相关蛋白激酶A(PKA)活性几乎是LTH胎儿的两倍，但与LTH胎儿不同的是，BK通道几乎没有相关蛋白激酶G(PKG)活性。我们还发现，外源性激酶和磷酸酶对胎儿和成年绵羊基底动脉BK通道电压激活的作用相同。这表明相同的通道剪接变体在整个发育过程中都有表达。然而，我们的发现也表明，这些酶对LTH和常氧动物的BK通道产生不同的作用，无论 发展阶段。这表明，在LTH和常氧条件下，存在不同的剪接变体。在我们拟议的研究中，我们选择同时使用肺动脉和脑动脉，因为肺血管对急性缺氧的反应与脑动脉相反，而关于它们对LTH的反应、与发育年龄相关的差异或它们对氯化血红素的敏感性尚不清楚。

Long-term hypoxia (LTH) induces profound changes in vascular reactivity in both fetal and adult cerebral blood vessels. Although endothelial cells lining the blood vessel lumen and peripheral adrenergic nerves play major roles in vessel reactivity, the vascular smooth muscle cells (VSMCs) are the final arbiters of vascular tone. VSMCs normally exist in a partially contracted state, giving rise to myogenic tone, and are able to contract further or to relax upon exposure to soluble signals. VSMC responsiveness is influenced by many factors, including development and exposure to long-term hypoxia (LTH), both of which are principal variables in the proposed studies. Developmental changes in VSMCs include changes in contractile proteins, membrane composition, receptors, signal pathways, and ion channels. Each of these changes influences vascular reactivity. Current research from our laboratory indicates that some of the effects of LTH on vascular smooth muscle involve calcium-activated, voltage-sensitive K+ (BK or KCa) channels. BK channels are arguably the single most important channels on VSMCs of the cerebral and pulmonary branches of the vascular tree. BK channels are pivotal in terms of VSMC reactivity because they couple free intracellular calcium levels to membrane potential. In addition, they are modulated by more extra- and intracellular factors than any other known VSMC channels. Indeed, BK channels are found in all types of smooth muscle where their activation decreases contractility indirectly by hyperpolarizing the membrane. In vascular smooth muscle from mature arteries, BK channels regulate membrane potential by responding to local elevations of [Ca2+]i from "Ca 2+ sparks" released from internal stores under the control of ryanodine receptors. However, in fetal cerebral VSMCs, BK channels respond primarily to global elevations of [Ca2+]i due to Ca 2+ influxing through L-type calcium channels. BK channel function and structure can vary due to alternative splicing of a single SIo gene, but little is known about channel functional and structural changes during development or LTH. The BK channels of VSMCs and brain neurons associate functionally in the plasma membrane with certain protein kinases and phosphatases. We find that the responses of BK channels to various modulatory factors differ both during development and in response to LTH, indicating that the physiological adaptations of BK channels to LTH differ between the fetus and adult, some of which are potentially critical to pathophysiological survival. These include the following: (1) In LTH adults the Ca 2+ set point of BK channels (3.3 mu M) is similar to that of the LTH and normoxic fetus (3.0 and 4.7 mu M, respectively), but half that of normoxic adults (8.8 mu M; Lin et al., 2003). (2) In LTH adults the sensitivity of BK channels to inhibition by intracellular hemin (IC50 = 50 nM) is four times that of the normoxic adult or the LTH and normoxic fetus (IC50 = 200 nM). (3) In LTH adults the BK channels are activated by acute hypoxia to more than twice that of BK channels in the LTH fetus. (4) In LTH adults BK channels have almost twice the channel-associated protein kinase A (PKA) activity as that of the LTH fetus, but, unlike that of the LTH fetus, exhibit virtually no associated protein kinase G (PKG) activity. We also find identical actions of exogenous kinases and phosphatases on voltage activation of BK channels from fetal and adult ovine basilar arteries. This suggests that the same channel splice variant is expressed throughout development. However, our findings also indicate that these enzymes produce dissimilar actions on BK channels from LTH versus normoxic animals, regardless of developmental stage. This suggests that different splice variants occur under LTH and normoxia. In our proposed study we have opted to use both pulmonary, as well as cerebral, arteries because pulmonary vessels react oppositely to cerebral arteries in response to acute hypoxia, and nothing is yet known concerning their responses to LTH, the differences associated with developmental age, or their sensitivity to hemin.