Heterocellular regulation of surfactant secretion

最近，I型细胞已被牵连作为潜在的mechanosensors刺激表面活性剂分泌的II型细胞通过细胞间传输的瞬时增加胞质钙（钙波）。有两个主要途径：缝隙连接细胞间通讯和旁分泌刺激嘌呤受体。我们将使用多种方法的组合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，我们将确定是否破坏不同的细胞间通讯途径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是否增加的表面活性剂脂质在通气参数的增加是由于通过间隙连接，嘌呤能受体，或两者的信号传播。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讯途径。用于这些研究的分泌表面活性剂产生细胞包括成体原代II型细胞。虽然成人II型细胞表达P2 Y2嘌呤能受体，但新生儿细胞不表达，从而提供了该受体的“敲除”模型的等价物。作为II型细胞的伴侣，我们将使用培养6天的II型细胞（第6天细胞）作为培养物中I型样细胞的最佳可用模型。我们还将使用其他细胞作为II型细胞的伴侣，包括具有不同连接蛋白表达的Hela转染子。初步数据表明，成人II型细胞与第6天细胞的共培养刺激表面活性剂分泌。将在共培养物中检查抑制剂对钙波传输和表面活性剂分泌的影响。用编码显性阴性Cx43构建体的腺载体转导的第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。将在共培养物中检查抑制剂对钙波传输和表面活性剂分泌的影响。用编码显性负性Cx43构建体的腺载体转导的第6天细胞将有助于阐明Cx43在刺激表面活性剂分泌中的作用。在目标3中，我们将确定在培养和原位的具有不同表型的原代肺泡上皮细胞的嘌呤能受体表达谱。最后，在目标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物来确定机械扩张是否诱导细胞间信号传递到II型细胞。通过定义分子--调节表面活性物质分泌的细胞间通讯的分子成分，我们希望确定在急性肺损伤或呼吸机诱导的损伤期间被破坏的可能的靶点，这些靶点可能通过引起表面活性物质分泌的失调而加重肺部疾病的程度。

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.