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

长期缺氧（LTH）诱导胎儿和成人脑血管血管反应性的深刻变化。虽然内皮细胞内衬的血管腔和外周肾上腺素能神经在血管反应性中起主要作用，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，引起肌原性紧张，并且能够在暴露于可溶性信号时进一步收缩或舒张。VSMC的反应性受到许多因素的影响，包括发育和暴露于长期缺氧（LTH），这两者都是拟议研究中的主要变量。VSMC的发育变化包括收缩蛋白、膜组成、受体、信号通路和离子通道的变化。这些变化中的每一个都影响血管反应性。我们实验室的研究表明，LTH对血管平滑肌的一些作用涉及钙激活的电压敏感性K+（BK或KCa）通道。BK通道可以说是血管树的脑和肺分支的VSMC上最重要的通道。BK通道在VSMC反应性方面是关键的，因为它们将细胞内游离钙水平与膜电位偶联。 此外，它们比任何其他已知的VSMC通道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，它们的激活通过使膜超极化而间接降低收缩性。在来自成熟动脉的血管平滑肌中，BK通道通过响应局部的 [Ca 2 +]i升高是由兰尼碱受体控制的内贮释放的“Ca 2+火花”引起的。而在胎脑VSMCs中，BK通道主要对[Ca 2 +]i的整体升高起反应，[Ca 2 +]i的升高是由于Ca 2+通过L-型钙通道流入所致。BK通道的功能和结构可以由于单个SIo基因的选择性剪接而变化，但很少 已知在发育或LTH期间通道功能和结构的变化。 VSMC和脑神经元的BK通道在质膜中与某些蛋白激酶和磷酸酶功能相关。我们发现，BK通道的各种调制因子的反应不同的发育过程中，并在响应LTH，表明BK通道的生理适应LTH胎儿和成人之间的不同，其中一些是潜在的病理生理生存的关键。其中包括：（1）在LTH成人中，BK通道的Ca 2+设定点（3.3 μ M）与LTH和正常含氧量胎儿的Ca 2+设定点（分别为3.0和4.7 μ M）相似，但只有正常含氧量成人的一半（8.8 μ M; Lin等，2003年）。(2)在LTH成人中，BK通道对细胞内氯化血红素抑制的敏感性（IC 50 = 50 nM）是含氧量正常的成人或LTH和含氧量正常的胎儿的4倍（IC 50 = 50 nM）。 200 nM）。(3)在LTH成人中，BK通道被急性缺氧激活，其激活程度是LTH胎儿中BK通道的两倍以上。(4)在LTH成人BK通道有几乎两倍的通道相关蛋白激酶A（PKA）活性的LTH胎儿，但不像LTH胎儿，表现出几乎没有相关的蛋白激酶G（PKG）活性。我们还发现相同的行动外源性激酶和磷酸酶的电压激活的BK通道从胎儿和成年绵羊基底动脉。这表明在整个发育过程中表达相同的通道剪接变体。然而，我们的研究结果也表明，这些酶产生不同的行动对BK通道从LTH与常氧动物，无论 发育阶段这表明在LTH和常氧下发生不同的剪接变体。在我们提出的研究中，我们选择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，而且还不知道它们对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。

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.