Role of NTPDase1 and Extracellular Nucleotides in Smooth Muscle Cell Contraction

我实验室的研究旨在阐明细胞外核苷酸信号的作用，并确定其在哺乳动物细胞中的作用模式。ATP和ADP等核苷酸由平滑肌细胞（SMC）和支配SMC的神经等细胞以受调节的方式释放。一旦离开细胞，这些分子就会激活特定的p2受体。外核苷酶将ATP和ADP水解为腺苷，终止后者的作用，并引起腺苷（P1）受体的激活，这通常导致与p2受体激活不同的生理效应。我们最近证明了NTPDase1是血管SMC中主要的外核苷酸酶，并且NTPDase1缺乏的小鼠通过P2Y6揭示了核苷酸的主要血管收缩作用。最近，我们观察到ntppdase在输精管的非血管性SMC中表达，其活性对于阻止P2X1功能是必要的。输精管中ntpase的缺失导致精子蠕动受损，精液中精子浓度降低，生育能力降低。因此，我们假设ntpase是一般非血管性SMC表面的主要外核苷酶，其活性通过调节P2受体激动剂水平影响收缩。本研究计划的长期目标是阐明细胞外核苷酸信号在非血管平滑肌（SM）收缩中的作用和机制。为了解决这个问题，我们设计了以下目标。******目的1：确定非血管SMC是否表达外核苷酸酶活性并鉴定酶。***目的2：评价目的1鉴定的外核苷酶在SM收缩中的作用。目的3：确定参与SM收缩调控的P2受体。******目标1将通过含SM的组织，如呼吸道、生殖道和胃肠道组织的酶组织化学来完成。这些SM中存在的外核苷酶将通过定量RT-PCR、免疫组织化学和Western blot进行鉴定。目的2将通过比较WT组织与第一个目标中发现的外核苷酶缺乏的小鼠组织的等长收缩，以及在不同刺激（包括外源性ATP和ADP）下，评估SMC原代培养物中的钙动员来完成。在第2个目标中观察到的P2受体参与SM收缩的鉴定将在第3个目标中进行评估，使用拮抗剂、sirna和前2个实验提出的P2受体候选物缺失的KO小鼠。******在这项工作中，我们期望定义一个涉及细胞外核苷酸信号的非血管SM收缩的基本机制。此外，我们将确定参与这一生物过程的外核苷酸酶和P2受体。**

The research of my laboratory aims to elucidate the roles of extracellular nucleotide signaling and to define their mode of action in mammalian cells. Nucleotides such as ATP and ADP are released in a regulated manner by cells such as smooth muscle cells (SMC) and nerves innervating SMC. Once outside the cell, these molecules activate specific P2-receptors. The hydrolysis of ATP and ADP to adenosine by ectonucleotidases terminates the latter's effects and causes the activation of adenosine (P1) receptors which often results in different physiological effects to those elicited by P2-receptor activation. We recently demonstrated that NTPDase1 was the dominant ectonucleotidase in vascular SMC and that mice deficient in NTPDase1 unmasked a major vasoconstrictor effect of nucleotides via P2Y6. More recently we observed that NTPDase1 was expressed in non-vascular SMC of the vas deferens and that its activity was necessary to prevent P2X1 functionality. The absence of NTPDase1 in vas deferens led to impaired peristalsis, reduced spermatozoa concentration in the semen, and reduced fertility. We therefore hypothesize that NTPDase1 is the major ectonucleotidase at the surface of non-vascular SMC in general and that its activity affects contraction via the regulation of the P2 receptor agonist levels. The long term objective of this research program is to elucidate the role and mechanisms of extracellular nucleotide signaling in non-vascular smooth muscle (SM) contraction. To address this problem, we have designed the following objectives. ******Obj 1: Determine whether non-vascular SMC express ectonucleotidase activity and identify the enzyme(s).***Obj 2: Evaluate the role of the ectonucleotidase(s) identified in Obj 1 in SM contraction.***Obj 3: Identify the P2 receptors involved in the regulation of SM contraction.******Obj 1 will be done by enzyme histochemistry of SM containing tissues such as those from the respiratory tract, the reproductive tract and the gastrointestinal tract. The identity of the ectonucleotidase present in these SM will be carried out by quantitative RT-PCR, immunohistochemistry and Western blot. Obj 2 will be done by the comparison of isometric contraction of WT tissues with tissues from mice deficient for the ectonucleotidase identified in the first objective and also by assessing calcium mobilization in primary SMC cultures under different stimuli including exogenous ATP and ADP. The identification of the P2 receptors involved in SM contraction observed in the 2nd objective will be evaluated in the 3rd objective with antagonists, SiRNAs and KO mice deficient for the P2 receptor candidate suggested by the former experiments.******In this work we expect to define a basic mechanism of non-vascular SM contraction involving extracellular nucleotide signaling. In addition we will identify the ectonucleotidase and the P2 receptors involved in this biological process. **