Calcium Channels in Neonatal Pulmonary Hypertension

描述(申请人提供)：该试点项目的目标是明确电压门控L型钙通道在新生儿肺高压发病机制中的作用。利用已建立的新生仔猪缺氧性肺高压模型，体内血流动力学显示，暴露于慢性缺氧(CH)21天的新生仔猪的静息肺张力增加。对同一CH仔猪的分离、灌流肺的研究表明，硝苯地平敏感的Ca1通道是引起血管张力异常的原因之一，在来自小肺动脉的膜片钳血管平滑肌细胞上观察到高密度的Cal电流。随后的研究发现，在CH仔猪的肺血管系统中，Cal通道的成孔α1C亚基显著上调，这与促进Cal通道转运的辅助P2A亚基的表达增加相对应。有趣的是，血栓素A2和内皮素-1这两种与新生儿PH有关的血管收缩物质在培养的小肺动脉中也诱导了同样的钙通道异常。最后，降低21日龄CH仔猪血栓素A2利用率的预防性治疗可阻止肺血管中钙通道的上调，从而减轻PH的发生。根据这些关键发现，血管收缩物质上调钙离子通道有助于新生猪肺高压的发生，我们将致力于以下特定目标的研究：(A)确定新生仔猪肺小动脉钙通道的分子组成；(B)明确血栓素A2和内皮素-1上调新生仔猪肺小动脉钙通道的机制；以及(C)确定血栓素A2和内皮素-1是否在体内上调肺血管钙通道以促进肺高压的发生。这些研究将首次提供关于促进新生儿PH期间肺血管中异常钙通道表达的刺激和机制的详细信息，并将有助于确定缓解婴幼儿PH的治疗靶点。

DESCRIPTION (provided by applicant): The goal of this pilot project is to define the contribution of voltage-gated, L-type Ca2+ (CaL) channels to the pathogenesis of pulmonary hypertension (PH) in the neonate. Using a well established model of hypoxia-induced PH in neonatal piglets, in-vivo hemodynamics revealed an increased resting pulmonary tone in neonatal piglets exposed to chronic hypoxia (CH) for 21 days. Studies in isolated, perfused lungs from the same CH piglets demonstrated that nifedipine-sensitive CaL, channels contributed to the anomalous vascular tone, and an elevated density of CaL current was observed in patch-clamped vascular smooth muscle cells from small pulmonary arteries. Subsequent studies revealed a striking upregulation of the pore-forming alpha1C subunit of the CaL channel in the pulmonary vasculature of CH piglets, which corresponded to an increased expression of the ancillary p2a subunit that promotes CaL channel trafficking. Interestingly, this same pattern of CaL channel abnormalities was induced in cultured small pulmonary arteries by thromboxane A2 and endothelin-1, two vasoconstrictor substances implicated in neonatal PH. Finally, prophylactic therapy to lower thromboxane A2 availability in 21-day CH piglets prevented the upregulation of CaL channels in the pulmonary vasculature and mitigated the development of PH. Based on these key findings suggesting that the upregulation of CaL channels by vasoconstrictor substances contributes to neonatal PH, we will pursue specific aims designed to: (a) identify the molecular composition of CaL channels in small pulmonary arteries of neonatal piglets, (b) define the mechanistic basis by which thromboxane A2 and endothelin-1 upregulate CaL channels in small pulmonary arteries of CH piglets, and (c) determine if thromboxane A2 and endothelin-1 upregulate CaL channels in the pulmonary vasculature in vivo to contribute to the development of PH. These studies will provide the first detailed information on the stimuli and mechanisms that promote abnormal CaL, channel expression in the pulmonary vasculature during neonatal PH, and will assist in identifying therapeutic targets to mitigate PH in infants and young children.