REGULATION OF PULMONARY ENDOTHELIAL PERMEABILITY

在患有慢性心力衰竭的人和动物中，肺经历了重要的保护性适应，这些患者能够经常耐受伴随的肺动脉高压，而不会发生肺泡水肿和呼吸窘迫。为了最佳地维持心力衰竭患者的肺功能，至关重要的是，旨在改善心功能的治疗不会对这些保护性肺适应产生不利干扰。这意味着首先我们必须了解心衰导致肺高压耐受性和急性肺损伤抵抗力增强的机制。由于肺内皮屏障在调节液体进入肺实质的运动中起着关键作用，因此我们的研究旨在了解该屏障在心力衰竭中的功能适应。我们的工作假设是，心力衰竭导致肺内皮对损伤敏感性的功能性“下调”，这是由于细胞内Ca2+储存耗尽后p450介导的调节Ca2+进入的信号的改变，以及内皮细胞cAMP含量和周转量的改变。具体目的是确定1)p450介导的存储损耗和肺内皮Ca2+进入之间的信号在心力衰竭发生后是否存在缺陷；2)心力衰竭后，肺内皮通透性增加的阻力是由于质膜离子通道调节的改变和Ca2+进入的减少；3)在心力衰竭时，肺内皮中cAMP的升高是维持内皮屏障完整的关键因素。这一建议涉及到几位具有互补专业知识的研究人员的合作努力，以便以多学科的方式调查这个问题。为了验证这一假设，我们将比较正常狗和心室快速起搏引起心力衰竭狗的肺分离和原代分离的宏观和微血管内皮细胞的肺内皮通透性。生化方法将包括微粒体P450代谢和cAMP周转的测量。免疫印迹法将用于评估肺和肺内皮中特异性P450环氧合酶、腺苷酸环化酶和磷酸二酯酶异构体的蛋白表达。Fura PE3，一种比例染料，将用于测量Ca2+瞬态，而膜片钳技术将允许直接测量存储操作的Ca2+通道活性。

In humans and animals with chronic heart failure, the lung undergoes important protective adaptations as evidenced by the ability of these patients to frequently tolerate the accompanying pulmonary hypertension without the development of alveolar edema and respiratory distress. To optimally maintain pulmonary function in heart failure, it is critical that therapies which are directed at improving cardiac function do not adversely interfere with these protective lung adaptations. This means that first we must understand the mechanisms by which heart failure results in the tolerance to pulmonary hypertension and the increased resistance to acute lung injury. Since the pulmonary endothelial barrier plays a key role in regulating movement of fluid into lung parenchyma, our inquiry is directed at understanding adaptations in function of this barrier in heart failure. Our working HYPOTHESIS is that heart failure results in a the functional "down-regulation" of pulmonary endothelial sensitivity to injury, due to alterations in P450-mediated signaling regulating Ca2+ entry subsequent to depletion of intracellular Ca2+ stores and to alterations in endothelial cell cAMP content and turnover. The Specific Aims are to determine if 1) P450-mediated signaling between store depletion and Ca2+ entry in pulmonary endothelium is deficient following the development of heart failure; 2) after heart failure, resistance to increases in lung endothelial permeability is due to altered regulation of plasma membrane ion channels and resultant decreases in Ca2+ entry; and 3) an elevation in cAMP in lung endothelium is a critical element which contributes to maintenance of an intact endothelial barrier in heart failure. This proposal involves the collaborative efforts of several investigators with complementary expertise in order to investigate this problem in a multidisciplinary fashion. To address this hypothesis, pulmonary endothelial permeability will be compared in isolated lung and in primary isolates of macro- and microvascular endothelial cells from normal dogs and dogs with heart failure induced by rapid ventricular pacing. Biochemical approaches will include measurement of microsomal P450 metabolism and turnover of cAMP. Immunoblotting will be used to evaluate protein expression for specific P450 epoxygenase, adenylyl cyclase and phosphodiesterase isoforms in lung and lung endothelium. Fura PE3, a ratiometric dye, will be used to measure Ca2+ transients, while patch clamp techniques will allow direct measurement of store operated Ca2+ channel activity.