Neurogenic Calcium Signals in Small Arteries

描述（由申请人提供）：本研究将集中于神经源性Ca 2+信号在自主神经系统控制动脉直径中的作用和机制，特别是平滑肌细胞（SMC）中通过交感神经“三联体"共递质ATP、去甲肾上腺素（NE）和神经肽Y（NPY）的协同作用产生的Ca 2+信号。虽然神经源性血管收缩的成分可归因于ATP、NE和NPY，但SMC中潜在的Ca 2+信号尚不清楚。在低频神经刺激过程中，大鼠肠系膜小动脉平滑肌细胞内Ca 2+的初步共聚焦成像显示了新的Ca 2+瞬变，与兴奋性连接电位（EJPs）相关，暂时称为“连接Ca 2+瞬变”（jCaTs）。高频电刺激时，可产生Ca 2+“闪烁”（可能是平滑肌细胞动作电位所致）和Ca 2+波。刺激血管周围感觉神经可使血管舒张;初步结果表明Ca 2+火花频率增加。具体的实验目标包括：（1）验证神经元释放的ATP引起钙内流，而不引起钙波的假说;（2）验证神经元释放的ATP引起钙内流，而不引起钙波的假说; 3）检验神经释放的NE激发Ca 2+波激活收缩的假设，（4）验证了神经元释放的NPY通过激活SMCS上的Y1受体，改变Ca 2+波的频率，从而调节NE的作用，（5）检验感觉神经释放的降钙素基因相关肽（CGRP）降低Ca 2+放电频率，从而引起血管舒张的假说。将来自大鼠和小鼠（包括P2 Xz受体缺陷型转基因小鼠）的肠系膜小动脉加载fluo-4，并固定在共聚焦肌电描记器上，用于同时进行Ca 2+成像、等长力记录和膜电位测量。实时共聚焦显微镜将用于提供足够快的2D图像，以解决jCaTs，闪光和波。该研究将测试交感神经肌肉传递和自主神经系统控制平滑肌收缩的综合假设：神经释放的ATP激活P2 X1受体。由此产生的Ca 2+内流激活了一个小的收缩，产生EJPs，并被可视化为jCaTs。EJPs可以累加以触发动作电位，可视化为Ca 2+“闪光”神经元释放的NE和NPY（作为代谢型或G蛋白衬里的受体）增加Ca 2+波的频率，其激活强烈收缩。通过可视化新的生理神经源性Ca 2+信号和完整动脉的收缩，该研究旨在提供有关神经递质在控制动脉功能中的协调作用的新信息。

DESCRIPTION (provided by applicant): The research will focus on the roles and mechanisms of neurogenic Ca 2+ signals in autonomic nervous system control of arterial diameter, particularly the Ca2+ signals generated in smooth muscle cells (SMC) by the concerted action of the sympathetic 'triad' of co-transmitters, ATP, norepinephfine (NE) and neuropeptide Y (NPY). Although components of neurogenic vasoconstriction are attributable to ATP, NE and NPY, the underlying Ca 2+ signals in SMC are not known. Preliminary confocal imaging of Ca 2+ within the SMC of intact pressurized rat mesenteric small arteries during low frequency nerve stimulation has revealed novel Ca2+ transients, putatively associated with excitatory junction potentials (EJPs) and provisionally called "junctional Ca 2+ transients" (jCaTs). With higher frequency stimulation, Ca 2+ 'flashes' (may be due to SMC action potentials) and Ca 2+waves are generated. Stimulation of perivascular sensory nerves elicits vasodilation; preliminary results implicate an increase in the frequency of Ca 2+ sparks. Specific Experimental goals include: 1) Test the hypothesis that jCaTs arise from Ca 2+entering SMCs through P2X1 receptors, which are activated by the ATP contained in one or a few quanta of transmitter, 2) Test the hypothesis that neurally released ATP elicits Ca 2+ influx, but not Ca 2+ waves, 3) Test the hypothesis that neurally released NE elicits Ca 2+ waves that function to activate contraction, 4) Test the hypothesis that neurally released NPY activates the Y1 receptor on SMCS to modulate the effects of NE by changing the frequency of Ca 2+ waves, and 5) Test the hypothesis that calcitonin gene related peptide (CGRP) released from sensory nerves decrease Ca 2+ spark frequency; thus causing vasodilation. Mesenteric small arteries from rats and mice (including a P2Xz-receptor deficient transgenic mouse) will be loaded with fluo-4 and mounted on a confocal myograph for simultaneous Ca2+ imaging, recording of isometric force, and measurement of membrane potential. A real-time confocal microscope will be used to provide 2D images fast enough to resolve jCaTs, flashes and waves. The research will test a comprehensive hypothesis on sympathetic neuromuscular transmission and the control of smooth muscle contraction by the autonomic nervous system: Neurally released ATP activates P2X1 receptors. The resulting Ca 2+ influx activates a small contraction, generates EJPs and is visualized as jCaTs. EJPs may summate to trigger action potentials, visualized as Ca 2+ 'flashes' Neurally released NE and NPY (as metabotropic or G protein-lined receptors) increase the frequency of Ca 2+ waves, which activate strong contraction. By visualizing novel, physiological neurogenic Ca 2+ signals and contraction of intact arteries, the research seeks to provide new information on the concerted action of neurotransmitters in controlling arterial function.