Endothelial Expression of Neuronal Nitric Oxide Synthase

 描述(申请人提供)：脑微血管功能障碍与中风后的脑损伤有关，然而，其潜在的机制尚不清楚。一氧化氮合酶(NOS)有内皮型(ENOS)和神经型(NNOS)两种亚型，分别以它们首次被发现的位置命名。我们的初步研究首次利用PCR和免疫印迹技术在新鲜分离的大鼠脑微血管和大鼠、小鼠和人的脑微血管内皮细胞(BMECs)中发现了nNOS。我们发现内皮型nNOS在结构和功能上都不同于eNOS，nNOS在神经元中表达。因此，我们将内皮型nNOS命名为Ennos。我们的初步研究表明，抑制BMECs中的eNOS或神经元中的nNOS会增加超氧化物歧化水平，降低NO水平。此外，eNOS抑制会导致线粒体储备呼吸能力降低。同样，抑制神经元中的nNOS会增加超氧化物歧化水平，降低NO水平。相反，抑制内皮型一氧化氮合酶导致超氧化物歧化水平降低，一氧化氮水平升高，并增强线粒体储备呼吸能力。因此，与神经元来源的nNOS和eNOS不同，eNOS以非偶联状态存在。初步研究还表明，内皮型一氧化氮合酶对基础状态和血管紧张素II诱导的BMECs中超氧化物歧化水平有显着贡献，其水平与NADPH氧化酶相当，但独立于NADPH氧化酶。最后，抑制缺氧-葡萄糖剥夺和复氧过程中的所有一氧化氮合酶亚型(OGD-R)可减少胞浆和线粒体来源的超氧化物歧化产物，从而提高BMECs的存活率，这表明了Ennos在BMECs中的生理学意义。实验性中风引起的脑损伤在eNOS组更大，但在nNOS基因敲除组中减少，然而，nNOS抑制提供神经保护的确切机制从未被检验过。我们推测，Ennos在功能上不同于eNOS和神经源性的nNOS。我们进一步假设，Ennos是OGD-R对BMECs损伤的主要介质，是缺血后血脑屏障破坏的重要调节器。目的1在体外培养的骨髓微血管内皮细胞和神经元中，内皮型一氧化氮合酶与内皮型一氧化氮合酶和n一氧化氮合酶在功能上不同于eNOS和nNOS，它们在产生超氧化物歧化酶和一氧化氮，以及在OGD-R后对线粒体功能的调节上具有不同的作用。目的2确定内皮型一氧化氮合酶和内皮型一氧化氮合酶对OGD-R后BMECs活性和结构完整性的功能意义。目的3确定内皮型一氧化氮合酶和内皮型一氧化氮合酶在脑缺血后血脑屏障完整性和微血管功能障碍中的不同作用。这些研究将从根本上促进我们对神经血管单位最重要的单一调节因子--一氧化氮合酶的机制认识，并将为靶向一氧化氮合酶治疗卒中微血管功能障碍提供突破性的发现。

 DESCRIPTION (provided by applicant): Cerebral microvascular dysfunction has been implicated in the brain injury following stroke, however, the underlying mechanisms are unclear. Nitric oxide synthase (NOS) has endothelial (eNOS) and neuronal (nNOS) isoforms that were named after the locations where they were first identified. Our preliminary studies, for the first time, identified nNOS in freshly isolated rat brain microvessels and brain microvascular endothelial cells (BMECs) from rat, mouse, and humans utilizing PCR and immunoblot techniques. We found that endothelial nNOS is structurally and functionally distinct from eNOS and the nNOS expressed in the neurons. Therefore, we named the endothelial nNOS as enNOS. Our preliminary studies revealed that inhibition of eNOS in BMECs or nNOS in neurons increased the levels of superoxide and decreased NO levels. Furthermore, eNOS inhibition results in diminished mitochondrial reserve respiratory capacity. Similarly, inhibition of nNOS in neurons increased superoxide levels and decreased NO levels. In contrast, enNOS inhibition led to diminished superoxide levels, increased NO levels, and enhanced mitochondrial reserve respiratory capacity. Thus, unlike nNOS of neuronal origin and eNOS, enNOS exists in the uncoupled state. Preliminary studies also showed that enNOS significantly contributes to baseline as well as angiotensin II- induced superoxide levels in BMECs that is comparable to but independent of NADPH oxidase. Finally, inhibition of all NOS isoforms during oxygen-glucose deprivation and reoxygenation (OGD-R) decreased superoxide generation from cytosol and mitochondrial sources resulting in increased survival of BMECs which indicates the physiological significance of enNOS in BMECs. Experimental stroke-induced brain damage is greater in eNOS but diminished in nNOS knockouts, however, the exact mechanisms underlying the nNOS inhibition afforded neuroprotection have never been examined. We hypothesize that enNOS is functionally distinct from eNOS and nNOS of neuronal origin. We further hypothesize that enNOS is the primary mediator of OGD-R injury to BMECs and is an important modulator of post-ischemic BBB disruption. Aim 1 will demonstrate that enNOS is functionally distinct from eNOS and nNOS in generating superoxide versus NO and in modulating mitochondrial function after OGD-R in cultured BMECs and neurons. Aim 2 will determine the functional significance of enNOS and eNOS on post-OGD-R viability and structural integrity of BMECs. Aim 3 will determine the differential role of enNOS and eNOS on the post-ischemic BBB integrity and microvascular dysfunction. The proposed studies will fundamentally advance our mechanistic understanding of NOS, the single most important regulator of neurovascular unit, and will provide breakthrough findings to target enNOS for treating microvascular dysfunction in stroke.