Heterocellular regulation of surfactant secretion

最近，I型细胞被认为是潜在的机械传感器，通过胞质钙（钙波）的短暂增加在细胞间传递刺激II型细胞分泌表面活性剂。这主要有两种途径：间隙连接细胞间通讯和旁分泌刺激嘌呤能受体。我们将使用多种方法来确定哪些细胞间信号通路调节表面活性剂的分泌。使用具有重叠作用模式的化学上不同的抑制剂，我们将确定破坏不同的细胞间通讯途径是否会抑制代谢标记的内源性表面活性剂脂质的分泌（目的1）。这将使我们能够确定随着通气参数的增加而增加的表面活性剂脂质是由于信号通过间隙连接、嘌呤能受体或两者传播所致。在目标2中，共培养系统将用于识别用于传递异细胞钙波和刺激表面活性剂分泌的细胞间通信途径。用于这些研究的分泌表面活性剂的细胞包括成人原代II型细胞。虽然成年II型细胞表达P2Y2嘌呤能受体，但新生细胞不表达，为该受体提供了相当于“敲除”的模型。作为II型细胞的伙伴，我们将使用培养6天的II型细胞（第6天细胞）作为培养i型样细胞的最佳模型。我们还将使用其他细胞作为II型细胞的合作伙伴，包括具有不同连接蛋白表达的Hela转染。初步数据表明，成人II型细胞与第6天细胞共培养可刺激表面活性剂的分泌。抑制剂对钙波传输和表面活性剂分泌的影响将在共培养中进行检验。用腺载体编码显性阴性Cx43构建体转导的第6天细胞也将有助于阐明Cx43在刺激表面活性剂分泌中的作用。抑制剂对钙波传输和表面活性剂分泌的影响将在共培养中进行检验。第6天的细胞用腺载体编码显性阴性Cx43构建体将有助于阐明Cx43在刺激表面活性剂分泌中的作用。在Aim 3中，我们将定义培养和原位不同表型的原代肺泡上皮细胞的嘌呤能受体表达谱。最后，在Aim 4中，我们将使用在可变形膜基质上培养的肺泡上皮细胞的异细胞共培养来确定机械膨胀是否诱导细胞间信号传递给II型细胞。通过定义细胞-细胞通讯中对调节表面活性剂分泌重要的分子成分，我们希望确定在急性肺损伤或呼吸机诱导损伤期间被破坏的可能靶标，这些靶标可能通过引起表面活性剂分泌的失调而加剧肺部疾病的程度。

Recently, type I cells have been implicated as potential mechanosensors for stimulating surfactant secretion by type II cells by intercellular transmission of transient increases in cytosolic calcium (calcium waves). There are two major pathways for this: gap junctional intercellular communication and paracrine stimulation of purinergic receptors. We will use a combination of approaches to determine which intercellular signaling pathways regulate surfactant secretion. Using chemically distinct inhibitors with overlapping modes of action, we will determine whether disrupting different intercellular communication pathways inhibits secretion of metabolically labeled endogenous surfactant lipid (Aim 1). This will enable us to determine whether increased surfactant lipid in response to increases in ventilation parameters are due to signals propagated through gap junctions, purinergic receptors, or both. In Aim 2, co-cultures systems will be used to identify intercellular communication pathways used to transmit heterocellular calcium waves and to stimulate surfactant secretion. Secretion Surfactant producing cells used for these studies include adult primary type II cells. While adult type II cells express P2Y2 purinergic receptors, neonatal cells do not, providing the equivalent of a "knock-out" model for this receptor As partners for type II cells, we will use type II cells cultured for 6 days (Day 6 cells) as the best available model for type I-like cell in culture. We will also use other cells as partners for type II cells, including Hela transfectants with differing connexin expression. Preliminary data indicates that co-culture of adult type II cells with Day 6 cells stimulates surfactant secretion. The effect of inhibitors on calcium wave transmission and surfactant secretion will be examined in co-cultures. Day 6 cells transduced with adenovectors encoding dominant negative Cx43 constructs will also help elucidate roles for Cx43 in stimulating surfactant secretion. The effect of inhibitors on calcium wave transmission and surfactant secretion will be examined in co-cultures. Day 6 cells transduced with adenovectors encoding dominant negative Cx43 constructs will help elucidate roles for Cx43 in stimulating surfactant secretion. In Aim 3, we will define the purinergic receptor expression profile for primary alveolar epithelial cells with different phenotypes in culture and in situ. Finally, in Aim 4, we will use heterocellular co-cultures of alveolar epithelial cells cultured on deformable membrane substrate to determine whether mechanical distension induces intercellular signals transmitted to type II cells. By defining the molecular the molecular constituents of cell-cell communication important for regulating surfactant secretion, we hope to identify possible targets that are disrupted during acute lung injury or ventilator induced injury and that may exacerbate the extent of lung disease by causing misregulation of surfactant secretion.