Injury-induced Dysregulation of Intracellular Ca2+ in Nociceptive Afferents

描述（由申请人提供）：与持续性炎症相关的疼痛和痛觉过敏至少部分是由于 伤害性传入的外周和中枢终末。我们以前曾报道，在这些感觉神经元中诱发的Ca 2+瞬变在持续性炎症的存在下更大，持续时间更长。虽然我们以前已经排除了一些机制，可能有助于炎症诱导的变化，在伤害性传入的Ca 2+的调节，我的初步结果表明，这些变化的基础上有两个不同的机制。一种是炎症诱导的Na+/Ca 2+交换剂（NCX）活性的降低，这是Ca 2+挤出的机制，并被认为是炎症诱导的诱发瞬时衰减率降低的基础。第二个是炎症诱导的上调的内部存储介导的Ca 2+内流途径，这似乎有助于增加的幅度诱发的Ca 2+瞬变。在这个建议中，我将集中在一系列实验中的第一个机制，这些实验旨在检验炎症超敏反应的维持至少部分是由于 皮肤伤害性传入神经中Na+/Ca 2+交换活性降低。我将首先确定哪种NCX亚型负责炎症诱导的NCX活性降低，因为这三种亚型都在皮肤伤害感受神经元中表达。然后，我将通过评估亚型的表达、密度和分布来确定炎症诱导的皮肤伤害性神经元中NCX活性降低的基础。最后，我将评估NCX活性降低对伤害性感受神经元兴奋性的功能影响。 神经元、神经源性炎症反应和伤害性行为。在所提出的实验中，我将采用一系列体外和体内试验，结合siRNA敲除和NCX的药理学抑制。因此，拟议的实验的成功完成将提供新的信息有关的蛋白质的功能的钙离子调节的关键，建议新的治疗方法治疗炎症性疼痛，并提供一个坚实的实验基础上，建立一个职业生涯作为一个独立的科学家。

DESCRIPTION (provided by applicant): The pain and hyperalgesia associated with persistent inflammation are due, at least in part, to an increase in excitability and transmitter release from both peripheral and central terminals of nociceptive afferents. We have previously reported that the evoked Ca2+ transients in these sensory neurons are bigger and longer lasting in the presence of persistent inflammation. While we have previously ruled out a number of mechanisms that may contribute to the inflammation-induced changes in the regulation of Ca2+ in nociceptive afferents, my preliminary results indicate two distinct mechanisms that underlie these changes. One is an inflammation- induced decrease in the activity of a Na+/Ca2+ exchanger (NCX), a mechanism of Ca2+ extrusion and suggested to be the basis for the inflammation-induced decrease in the evoked transient decay rate. The second is an inflammation-induced up-regulation of an internal store-mediated Ca2+ influx pathway, which appears to contribute to the increase in magnitude of the evoked Ca2+ transient. In this proposal, I will focus on the first mechanism in a series of experiments designed to test the hypothesis that the maintenance of the inflammatory hypersensitivity is due, at least in part, to a decrease in Na+/Ca2+ exchanger activity in cutaneous nociceptive afferents. I will first determine which NCX isoform(s) are responsible for the inflammation-induced decrease in NCX activity, as all three are expressed in cutaneous nociceptive neurons. I will then determine the basis for the inflammation-induced decrease in NCX activity in cutaneous nociceptive neurons through assessment of expression, density, and distribution of the isoform(s). Finally, I will assess the functional implications of a decrease in NCX activity on the excitability of nociceptive neurons, the neurogenic inflammatory response, and nociceptive behavior. In the proposed experiments, I will employ a complimentary array of in vitro and in vivo assays in combination with siRNA knockdown and pharmacological inhibition of NCX. Thus, the successful completion of the proposed experiments will provide novel information regarding the function of a protein critical for Ca2+ regulation, suggest novel therapeutic approaches for the treatment of inflammatory pain, and provide a solid experimental foundation upon which to build a career as an independent scientist.