Mechanisms Linking Ca2+ Homeostasis & Vascular Tone

这些研究的目的是了解整个动物体内钙稳态和血管功能的机制。四个具体的目标将测试的假设，即细胞外Ca2+的升高，发生在跨细胞Ca2+运动的组织的间质室，激活血管周围神经Ca2+感应受体（CaSR），这是耦合的生产和释放的内源性大麻素血管扩张剂，诱导局部血管舒张。支持这一假设的初步数据包括：我们证明了十二指肠粘膜下层和肾皮质间质Ca~（2+）浓度在生理范围内发生动态变化;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;我们证明了Ca2+诱导内源性大麻素扩张剂从血管壁释放，并对假定的大麻素受体具有活性。另外的研究表明，一氧化氮（NO）负调节钙离子诱导的舒张和N18 TG2神经母细胞瘤细胞表达CaSR。 具体目标1将使用甲状旁腺缺陷型Gcm 2-/-小鼠来拯救CaSR敲除表型，其将用于检验神经元CaSR介导Ca 2+诱导的松弛的假设。具体目标2将使用分离的动脉和培养的DRG神经元，结合内源性大麻素的GC-质谱分析，以了解这些化合物在介导Ca2+诱导的舒张中的作用。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Ca2+诱导的舒张的机制。具体目标4将使用分子，生理和药理学方法来追求我们的发现，即N18 TG2细胞表达CaSR，并测试该细胞系可以作为研究神经元CaSR信号传导和递质产生的模型的假设。我们预计，这些研究将导致一个更完整的理解的分子机制，连接Ca2+稳态与血管功能，提供详细的机械信息有关的感觉神经介导的放松和血管内源性大麻素系统，增加我们的理解在神经元组织中的CaSR传感功能，并可能揭示新的目标，为开发新的血管扩张剂化合物。

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.