Purinergic receptors in inflammation

性状（由申请方提供）：趋化性使多形核中性粒细胞（PMN）迅速到达感染和发炎部位。然而，过多的PMN流入会损害宿主组织。更好地了解控制中性粒细胞趋化性的机制，可能会导致改善炎症性疾病的治疗。基于我们最近的研究结果，ATP和腺苷参与中性粒细胞的趋化性，我们建议在这里研究如何调节这一嘌呤能信号传导过程，以防止组织损伤。嘌呤能信号传导具有三个基本组分：i）细胞外ATP和腺苷的来源; ii）响应于ATP和腺苷的嘌呤能受体，以及iii）通过将ATP水解为腺苷来调节细胞响应的外核苷酸酶。该提议基于以下工作假设：趋化剂从PMN释放ATP。ATP激活附近的P2 Y2受体，放大梯度感应。A3腺苷受体被募集到前沿，在那里腺苷由CD 39/E-NTPD酶1和碱性磷酸酶（ALP）产生。腺苷和通过A3受体的正反馈驱动细胞迁移，而通过A2 a受体的负反馈促进细胞后部的膜收缩。干扰这些嘌呤能信号传导过程抑制趋化性，从而改善脓毒症和创伤患者中PMN诱导的组织损伤和器官衰竭。具体目标如下：1。中性粒细胞释放ATP的机制：本节将重点讨论中性粒细胞释放细胞ATP以响应趋化刺激的机制。具体来说，我们将集中在hTTYH 3的tweety最大阴离子通道，连接蛋白半通道，和脱粒的参与。2.腺苷形成机制：实验的目的是检查主要的外核苷酸酶，负责释放的ATP转换为腺苷。主要重点将放在NTPDase 1和ALP的贡献。3.嘌呤能信号复合物：我们将探讨与ATP释放位点，嘌呤受体，和外核苷酸酶的趋化受体的共定位和研究，如果嘌呤信号簇，这些分子组成的提供“局部兴奋和全球抑制”的理论趋化模型中提出的。4.嘌呤能信号在体内的作用：我们将研究P2 Y2，A3，A2 a和NTPDase 1和ALP在小鼠模型中的作用，并测试靶向这些分子以防止宿主组织损伤的可行性。拟议的研究有望提高我们对控制趋化性机制的理解。这可能会导致新的治疗方法，以改善宿主组织损伤所造成的过度流入的活化中性粒细胞，例如，在创伤和感染性休克患者。公共卫生相关性：趋化性，中性粒细胞在健康和疾病中的关键功能反应仍然知之甚少。在这个项目中，我们建议确定细胞ATP和嘌呤能受体的释放如何控制趋化性，以及这种控制机制是否可以靶向预防创伤患者的炎症和宿主组织损伤。

DESCRIPTION (provided by applicant): Chemotaxis allows polymorphonuclear neutrophils (PMN) to rapidly reach infected and inflamed sites. However excessive influx of PMN damages host tissues. Better knowledge of the mechanisms that control PMN chemotaxis may lead to improved treatments of inflammatory diseases. Based on our recent findings that ATP and adenosine are involved in PMN chemotaxis, we propose to study here how to regulate this purinergic signaling process in order to prevent tissue damage. Purinergic signaling has three essential components: i) sources of the extracellular ATP and adenosine; ii) purinergic receptors that response to ATP and adenosine and, iii) ecto-nucleotidases that modulate cellular responses by hydrolyzing ATP to adenosine. This proposal is based on the following working hypothesis: Chemotactic agents release ATP from PMN. ATP activates nearby P2Y2 receptors, amplifying gradient sensing. A3 adenosine receptors are recruited to the leading edge where adenosine is generated by CD39/E- NTPDase1 and alkaline phosphatase (ALP). Adenosine and positive feedback through A3 receptors drives cell migration, while negative feedback through A2a receptors facilitates membrane retraction at the back of cells. Interfering with these purinergic signaling processes inhibits chemotaxis, which ameliorates PMN-induced tissue damage and organ failure in sepsis and trauma patients. The following specific aims will be addressed: 1. Mechanism of ATP release from PMN: This section will focus on the mechanisms by which PMN release cellular ATP in response to chemotactic stimulation. Specifically, we will focus on the involvement of hTTYH3 tweety maxi-anion channels, connexin hemi-channels, and degranulation. 2. Mechanism of adenosine formation: Experiments are designed to examine the major ecto-nucleotidases that are responsible for the conversion of released ATP to adenosine. Major emphasis will be placed on the contributions of NTPDase1 and ALP. 3. Purinergic signaling complexes: We will explore the co-localization of chemotactic receptors with ATP release sites, purinergic receptors, and ecto-nucleotidases and investigate if purinergic signaling clusters, comprised of these molecules provide "local excitation and global inhibition" as proposed in theoretical chemotaxis models. 4. Role of purinergic signaling in vivo: We will study the roles of P2Y2, A3, A2a, and NTPDase1 and ALP in mouse models and test the feasibility of targeting these molecules to prevent host tissue damage. The proposed studies are expected to improve our understanding of the mechanisms that control chemotaxis. This could lead to novel therapeutic approaches to ameliorate host tissue damage caused by excessive influx of activated PMN, for example, in trauma and septic shock patients. PUBLIC HEALTH RELEVANCE: Chemotaxis, a key functional response of neutrophils in health and disease is still poorly understood. In this project we propose to determine how release of cellular ATP and purinergic receptors control chemotaxis and whether this control mechanism can be pharmacologically targeted to prevent inflammation and host tissue damage in trauma patients.