Sympathetic Neurotransmitters and Ouabain Hypertension

哇巴因诱导的高血压的特征是交感神经系统过度活跃和血管平滑肌收缩增加。它还可能涉及小动脉中交感神经肌肉传递的变化。这项拟议的研究旨在首先确定交感神经递质在小动脉中释放的某些基本机制，然后确定哇巴因对这些机制的影响。这项研究的基本前提是：1)交感神经肌肉传递和动脉收缩重要地涉及两种共同递质，ATP和去甲肾上腺素(NE)；2)这两种神经递质是由尚未完全了解的钙依赖机制差异释放的，可能涉及不同的突触小泡、“残留[钙]”和储存的钙(除钙进入外)。关于哇巴因作用机制的一个总体假设是，抑制神经末梢Na泵增加了‘残留’[Ca2]和/或储存的[Ca2](从而增加了NE和ATP的释放)，并且神经末梢[Ca2]的增加是由Na/Ca交换介导的。具体目标是：1)在单个交感神经静脉曲张时确定ATP和NE释放的概率，2)测量ATP和NE递质包的大小(量子)，3)检验NE和ATP的不同释放是由不同类型的突触小泡释放的假设，4)检验急性、低剂量哇巴因抑制交感神经静脉曲张中Na/K-ATPase的α3亚型并改变概率的假设 5)决定哇巴因高血压大鼠的释放概率或量子大小是否发生改变。大鼠和小鼠肠系膜小动脉将被加载荧光钙指示剂，并在肌图仪中进行研究，该仪器允许同时进行共聚焦荧光成像、电刺激/记录和记录等长力的发展。具有基因改变的钠/钙交换器或三磷酸腺苷受体的小鼠将被使用。交界性钙瞬变(JCATS)将被用来测量神经释放的ATP。碳纤维微电极和安培法将被用来测量NE的释放，即‘NE氧化电流’(NEOCs)。这项研究将首次同时测量去甲肾上腺素和三磷酸腺苷的释放，从而确定控制神经性动脉收缩的一些基本机制。 将阐明交感神经参与哇巴因诱发高血压的机制。

Ouabain-induced hypertension is characterized by hyperactivity of the sympathetic nervous system and increased contraction of vascular smooth muscle. It may also involve changes in sympathetic neuromuscular transmission in small arteries. The proposed research aims first to determine certain basic mechanisms of sympathetic transmitter release in small arteries, and then, to determine how these are affected by ouabain. Basic premises of the research are i) that sympathetic neuromuscular transmission and arterial contraction importantly involve the two co-transmitters, ATP and nor-epinephrine (NE) and, ii) that these two neurotransmitters are differentially released by Ca 2+ dependent mechanisms that are not yet completely known, possibly involving different synaptic vesicles, 'residual [Ca2+] ' and stored Ca 2+ (in addition to Ca 2+ entry). An overall hypothesis on mechanisms of ouabain actions is that inhibition of nerve terminal Na + pumps increases 'residual' [Ca 2+] and/or stored Ca 2+(thereby increasing NE and ATP release) and that the increases in terminal [Ca 2+] are mediated by Na/Ca exchange. Specific Aims are: 1) Determine the probabilities, at individual sympathetic nerve varicosities, of ATP and NE release, 2) Measure the sizes of ATP and NE transmitter packets ('quanta'), 3) Test the hypothesis that differential release of NE and ATP results from the release of different types of synaptic vesicles, 4) Test the hypothesis that acute, low-dose, ouabain inhibits the alpha3-isoform of the Na/K-ATPase in sympathetic varicosities and changes the probability of release, but not quantal size, 5) Determine whether release probability or quantal size is altered in ouabain hypertensive rats. Rat and mouse mesenteric small arteries will be loaded with fluorescent Ca 2+ indicators and studied in a myograph that permits simultaneous confocal fluorescence imaging, electrical stimulation/recording, and recording of isometric force development. Mice with genetically altered Na/Ca exchangers or ATP receptors will be used. Junctional Ca 2+ transients (jCaTs) will be used to measure neurally released ATP. Carbon fiber microelectrodes and amperometry will be used to measure NE release as 'NE oxidation currents' (NEOCs). The research will measure NE and ATP release together for the first time and thereby determine some of the basic mechanisms that control neurogenic contractions of arteries. will elucidate the mechanisms by which sympathetic nerves contribute to ouabain-induced hypertension.