Role of NTPDase1 and Extracellular Nucleotides in Smooth Muscle Cell Contraction

点击翻译按钮获取中文摘要

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.