Role of NTPDase1 and Extracellular Nucleotides in Smooth Muscle Cell Contraction

本实验室的研究旨在阐明细胞外核苷酸信号的作用，并确定它们在哺乳动物细胞中的作用模式。ATP和ADP等核苷酸是由SMC和支配SMC的神经等细胞以受调控的方式释放的。一旦在细胞外，这些分子就会激活特定的P2受体。胞外核糖核酸酶将ATP和ADP水解为腺苷，终止后者的作用，引起腺苷(P1)受体的激活，从而导致不同于P2受体激活的生理效应。我们最近证明了NTPDase1是血管SMC中的主要外切核苷酸酶，并且NTPDase1基因缺陷的小鼠通过P2Y6揭示了核苷酸的主要血管收缩作用。最近，我们观察到NTPDase1在输精管的非血管SMC中表达，它的活性是阻止P2X1功能所必需的。输精管中缺乏NTPDase1导致蠕动功能受损，精子在精液中的浓度降低，生育能力下降。因此，我们假设NTPDase1是非血管SMC表面的主要胞外核苷酸酶，其活性通过调节P2受体激动剂水平而影响收缩。本研究的长期目标是阐明细胞外核苷酸信号在非血管平滑肌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。*目标1：确定非血管SMC是否表达胞外核苷酸酶活性并鉴定其活性(S)。*目标2：评价目标1中鉴定的胞外核苷酸酶(S)在SM收缩中的作用。*目标3：识别参与SM收缩调节的P2受体。*目标1将通过含有呼吸道、生殖道和胃肠道组织的SM的酶组织化学来完成。我们将通过定量RT-PCR、免疫组织化学和Western印迹来鉴定这些SM中存在的外源核酸酶。OBJ-2将通过比较WT组织与第一个目标中确定的缺乏ECA核苷酸酶的小鼠组织的等长收缩，以及评估不同刺激条件下原代SMC培养中的钙动员来完成OBJ-2。在第二个目标中观察到的与SM收缩有关的P2受体的鉴定将在第三个目标中用先前实验建议的缺乏P2受体候选的拮抗剂、siRNAs和KO小鼠进行评估。*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，我们还将确定参与这一生物过程的胞外核苷酸酶和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. **