NPY Induced Regulation of Sympathetic Neurotransmission

描述(申请人提供)：神经肽Y(NPY)已知与去甲肾上腺素(NE)和三磷酸腺苷(ATP)共同定位于血管交感神经元，在那里它被认为起到协同递质/协同调节剂的作用。最近的证据表明，NPY通过作用于连接后的Y1受体，在交感神经介导的血管收缩中发挥生理作用。NPY还可通过Y2受体抑制去甲肾上腺素和很可能的ATP释放，增加NPY-ir的释放，并通过Y3和Y5受体抑制NE的合成。这些结果表明，交感神经递质的NE合成和释放过程可能受NPY的不同调节，提示交感神经传递的节前调节具有额外的控制水平。然而，这一作用的生理作用还没有完全确定。我们获得的证据表明，NPY的节前作用的一个重要机制是通过抑制钙通道。也有证据表明，SNARE蛋白在自身和异种受体优先释放和调节递质释放中发挥作用。本研究的目的是进一步研究NPY在交感神经递质释放和去甲肾上腺素释放的调节中的生理作用(目标1和目标2)，以及NPY和其他介质参与节前调节的机制(目标3)。目标1提出的研究的基本原理如下：如果NPY对递质释放有内源性调节，那么针对结合前NPY自身受体的激动剂和拮抗剂应该以与内源性激动剂(如释放的NPY)激活的受体一致的方式改变诱发的递质释放。换句话说，外源性给予激动剂和拮抗剂的效果应该随着内源性NPY的生物相浓度的不同而变化。效应细胞的反应应与抑制或刺激NPY释放一致。在活体内展示功能性受体似乎也是必要的。在目标2中，我们研究了一系列选择性Y1和Y2激动剂和拮抗剂对神经刺激引起的NE、NPY-ir和ATP的释放以及肠系膜动脉床灌流压的影响。这将在内源性NPY浓度升高之前和之后完成：1)增加神经刺激的频率或2)降低灌注率；或3)通过急性或慢性组织水平的耗尽而降低NPY浓度。我们还将检查激动剂和拮抗剂在穿孔大鼠制剂中的体内效应。在Aim 2中，类似的实验将评估NPY药物对神经刺激引起的NE合成增加的影响，这是通过抑制脱羧酶后DOPA的积累来衡量的。在目标3中，我们将确定肉毒杆菌神经毒素(BoNTs)阻断SNARE蛋白是否抑制NE、NPY-ir和ATP的诱导释放，作为优先释放或分化调制的机制。我们还将研究通过SNARE蛋白的信号是否有助于NPY的节前调制。这些研究将为NPY在交感神经传递的节前调节中的功能作用和作用机制提供有用的新信息。

DESCRIPTION (provided by applicant): Neuropeptide Y (NPY) is known to be co-localized, with norepinephrine (NE) and adenosine triphosphate (ATP) in vascular sympathetic neurons where it is thought to play a role as a co-transmitter/co-modulator. Recent evidence has established that NPYplays a physiological role in sympathetically mediated vasoconstriction by acting on postjunctional Y1 receptors. NPY is also known to exert prejunctional effects leading to inhibition of NE and most likely ATP release and an increase in NPY-ir release via Y2 receptors as well as inhibition of NE synthesis via Y3 and Y5 receptors. These results suggest that the process of NE synthesis and release of sympathetic co-transmitters may be differentially modulated by NPY suggesting an additional level of control in the prejunctional regulation of sympathetic neurotransmission. However the physiological role of this action has not been completely established. We have obtained evidence that an important mechanism for the prejunctional effects of NPY is through inhibition of Ca 2+channels. There is also evidence for a role of SNARE proteins in the preferential release and modulation of transmitter releaseby auto and heteroreceptors. The purpose of the present proposal is to further investigate the physiological role (Aims 1 and 2) on the NPY induced modulation of sympathetic co-transmitter release and NE release and the mechanisms involved (Aim 3) in the prejunctional modulation by NPY and other mediators. The rationale for studies proposed in Aim 1 is as follows: If there is endogenous modulation of transmitter release by NPY, then agonists and antagonists specific for the prejunctional NPY autoreceptor should alter evoked transmitter release in a manner consistent with the receptors being activated by endogenous agonist (e.g. released NPY). In other words, the effect of exogenously administered agonists and antagonists should vary with the biophase concentration of endogenous NPY. The response of the effector cell should be consistent with inhibition or stimulation ofNPY release. It would also seem necessary to demonstrate functional receptors in vivo. A similar rationale exists for Aim 2. In Aim 1 we examine the effect of a series of selective Y1 and Y2 agonists and antagonists on the nerve stimulation evoked release of NE, NPY-ir and ATP as well as perfusion pressure in the mesenteric arterial bed. This will be done before and after the endogenous NPY concentration is elevated by: 1) increasing the frequency of nerve stimulation or 2) decreasing the perfusion rate; or 3) after the NPY concentration has been reduced by depletion of tissue levels acutely or chronically. We will also examine the in vivo effect of agonists and antagonists in the pithed rat preparation. In Aim 2 similar experiments will evaluate the effect of NPY drugs on the nerve stimulation evoked increase in NE synthesis as measured by DOPA accumulations after decarboxylase inhibition. In Aim 3 we will determine if interruption of SNARE proteins by Botulinum neurotoxins (BoNTs) inhibit the evoked release of NE, NPY-ir and ATP as a mechanism for preferential release or differentiated modulation. We will also examine if signaling through SNARE proteins contributes to the prejunctional modulation by NPY. These studies will provide useful new information on the functional role and mechanism of action of NPY in the prejunctional regulation of sympathetic neurotransmission.