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兴奋性增加和递质释放。 伤害性传入的外周终末和中枢终末。我们之前已经报道过，在存在持续性炎症的情况下，这些感觉神经元中诱发的钙瞬变更大、持续时间更长。虽然我们之前已经排除了一些机制，这些机制可能有助于炎症诱导的伤害性传入细胞内钙离子调节的变化，但我的初步结果表明，这些变化背后有两个不同的机制。一种是炎症导致Na+/Ca~(2+)交换器(NCX)活性降低，这是Ca~(2+)排出的一种机制，并被认为是炎症导致诱发的瞬时衰减率下降的基础。第二个是炎症诱导的内部钙离子内流途径的上调，这似乎是诱发的钙瞬变幅度增加的原因之一。在这项提案中，我将重点介绍一系列实验中的第一个机制，这些实验旨在测试炎性超敏反应的维持至少部分归因于 皮肤伤害性传入Na~+/Ca~(2+)交换活性降低。我将首先确定哪个Ncx亚型(S)负责炎症诱导的Ncx活性降低，因为这三个亚型都在皮肤伤害性神经元中表达。然后，我将通过评估异构体的表达、密度和分布来确定炎症导致皮肤伤害性神经元Ncx活性降低的基础(S)。最后，我将评估Ncx活性降低对伤害性感受器兴奋性的功能影响。 神经元、神经源性炎症反应和伤害性行为。在拟议的实验中，我将采用一系列补充的体外和体内测试，结合siRNA敲除和NCX的药理抑制。因此，拟议实验的成功完成将提供有关对钙调节至关重要的蛋白质功能的新信息，为治疗炎症性疼痛提供新的治疗方法，并为建立独立科学家的职业生涯提供坚实的实验基础。

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.