Role of NTPDase1 and Extracellular Nucleotides in Smooth Muscle Cell Contraction

NTPDase1 和细胞外核苷酸在平滑肌细胞收缩中的作用

• 批准号: RGPIN-2016-05867
• 负责人: Sévigny, Jean
• 金额: $ 3.21万
• 依托单位: Université Laval
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=688810
• 关键词: Role; NTPDase1; Extracellular; Nucleotides; Smooth

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. **

本实验室的研究旨在阐明细胞外核苷酸信号的作用，并确定其在哺乳动物细胞中的作用模式。诸如ATP和ADP的核苷酸由诸如平滑肌细胞（SMC）和支配SMC的神经的细胞以调节的方式释放。 一旦在细胞外，这些分子激活特定的P2受体。 ATP和ADP通过外核苷酸酶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受体激活引起的生理效应不同的生理效应。 我们最近证明，NTPDase 1是占主导地位的外核苷酸酶在血管平滑肌细胞和小鼠NTPDase 1缺陷揭示了一个主要的血管收缩作用的核苷酸通过P2 Y 6。 最近，我们观察到NTPDase 1在输精管的非血管SMC中表达，并且其活性对于阻止P2 X1功能是必要的。 输精管中缺乏NTPD酶1导致精子增殖受损，精液中精子浓度降低，生育力降低。 因此，我们假设NTPDase 1是一般非血管SMC表面的主要外核苷酸酶，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，我们制定了以下目标。** 目的1：确定非血管SMC是否表达外核苷酸酶活性并鉴定酶。*目标2：评价目标1中鉴定的外核苷酸酶在SM收缩中的作用。*目的3：鉴定参与SM收缩调节的P2受体。目标1将通过含SM组织（如呼吸道、生殖道和胃肠道组织）的酶组织化学进行。 将通过定量RT-PCR、免疫组织化学和Western印迹法对这些SM中存在的外核苷酸酶进行鉴定。 目的2将通过比较WT组织与第一个目的中鉴定的外核苷酸酶缺陷小鼠组织的等长收缩，以及通过评估不同刺激（包括外源性ATP和ADP）下原代SMC培养物中的钙动员来完成。 将在第3个目标中使用拮抗剂、siRNA和先前实验提出的P2受体候选物缺陷的KO小鼠评价在第2个目标中观察到的参与SM收缩的P2受体的鉴定。**在这项工作中，我们希望确定一个基本的机制，非血管SM收缩涉及细胞外核苷酸信号。 此外，我们将确定外核苷酸酶和P2受体参与这一生物过程。**