Mechanisms Linking Ca2+ Homeostasis & Vascular Tone

这项研究的目的是了解整个动物的钙稳态和血管功能之间的联系机制。四个特定的目标将检验这一假说，即细胞外钙的升高，如发生在参与跨细胞钙离子运动的组织的间质中，激活血管周围神经钙感受器(CaSR)，该受体与产生和释放内源性大麻类血管扩张剂相结合，从而诱导局部血管松弛。支持这一假说的初步数据包括：我们证明十二指肠粘膜下层和肾皮质的间质钙离子浓度在生理范围内经历动态变化；我们发现背根神经节(DRG)和血管周围感觉神经表达功能性CaSR；我们证明钙离子诱导内源性大麻扩张剂从血管壁释放，并激活可能的anandamide受体。更多的研究表明，一氧化氮(NO)负向调节钙离子诱导的松弛，N18TG2神经母细胞瘤细胞表达CaSR。具体目标1将使用甲状旁腺缺乏的Gcm2-/-小鼠来挽救CaSR基因敲除表型，这将被用来检验神经元CaSR介导钙诱导的松弛的假设。特殊目的2将使用分离的动脉和培养的背根节神经元，结合内源性大麻素的GC-MS分析，了解这些化合物在介导钙离子诱导的松弛中的作用。具体目标3将使用药理学和分子遗传学方法来确定血管壁NO调节钙离子诱导的松弛的机制。具体目标4将使用分子、生理和药理学方法来继续我们的发现，即N18TG2细胞表达CaSR，并检验该细胞系可以作为研究神经元CaSR信号和递质产生的模型的假设。我们期待这些研究将使我们更全面地了解钙离子稳态与血管功能的分子机制，为感觉神经介导的松弛和血管内大麻素系统提供详细的机制信息，增加我们对神经元组织中CaSR传感功能的理解，并可能为新型血管扩张剂化合物的开发提供新的靶点。

The goal of the proposed studies is to understand the mechanisms linking whole animal Ca2+ homeostasis and vascular function. Four specific aims will test the hypothesis that elevation of extracellular Ca2+, as occurs in the interstitial compartment of tissues involved in transcellular Ca2+ movement, activates a perivascular nerve Ca2+ sensing receptor (CaSR) that is coupled with the production and release of a endocannabinoid vasodilator that induces local vasorelaxation. Preliminary data that support this hypothesis include our demonstration that the concentration of interstitial Ca2+ in the duodenal submucosa and renal cortex undergoes dynamic changes over a physiologic range; our finding that dorsal root ganglia (DRG) and perivascular sensory nerves express a functional CaSR; our demonstration that Ca2+ induces the release of an endocannabinoid dilator from the vessel wall with activity at the putative anandamide receptor. Additional work has revealed that nitric oxide (NO) negatively modulates Ca2+-induced relaxation and that N18TG2 neuroblastoma cells express a CaSR. Specific aim 1 will use the parathyroid deficient Gcm2 -/- mouse to rescue the CaSR knockout phenotype which will be used to test the hypothesis that a neuronal CaSR mediates Ca2+-induced relaxation. Specific aim 2 will use isolated arteries and cultured DRG neurons coupled with GC-mass spec analysis of endocannabinoids to understand the role of these compounds in mediating Ca2+-induced relaxation. Specific aim 3 will use pharmacologic and molecular genetic approaches to determine the mechanism by which vessel wall NO modulates Ca2+-induced relaxation. Specific aim 4 will use molecular, physiological and pharmacological approaches to pursue our finding that N18TG2 cells expresses a CaSR and to test the hypothesis that this cell line can serve as a model to study neuronal CaSR signaling and transmitter production. We anticipate that these studies will lead to a more complete understanding of the molecular mechanisms that link Ca2+ homeostasis with vascular function; provide detailed mechanistic information about sensory nerve mediated relaxation and the vascular endocannabinoid system, increase our understanding of CaSR sensing function in neuronal tissue, and may reveal new targets for the development of novel vasodilator compounds.