Naturally Evolved Cellular Adaptations to Anoxia

巴克实验室正在研究一些脊椎动物物种，如淡水龟和金鱼，在没有氧气(缺氧)的情况下存活几天到几个月的细胞机制。我们对这种能力背后的基本机制很感兴趣，但我们的结果将在中风、心脏病发作和器官移植的治疗中具有明确的临床意义和应用。大脑和肝脏细胞模型都被用来研究组织耐缺氧的细胞机制，这些组织是电兴奋的和不是电兴奋的。在缺氧中存活的关键是关闭利用细胞活动的能量，例如合成新的蛋白质和保持跨细胞膜的离子梯度。我们之前已经证明，蛋白质合成减少了约90%，维持跨细胞膜离子梯度的泵减少了75%，离子通道活性(离子跨膜的途径)减少，在一种特殊情况下增加。降低这些能量消耗反应的速度是哺乳动物(人类)所缺乏的能力。然而，基本的生化途径对大多数物种来说都是常见的，尤其是爬行动物(海龟)、鱼、鸟类和哺乳动物。我们的目标是确定负责切断耐缺氧物种能量消耗过程的自然细胞途径。我们已经确定了几种可能的途径，并发现在缺氧期间，涉及谷氨酸的锥体神经元的兴奋信号减少，而涉及GABA(伽马氨基丁酸)和星状神经元的抑制活性增加。事实上，在缺氧期间，GABA电流的幅度增加了一倍，而且比在哺乳动物大脑中观察到的任何GABA电流都要大。这项拨款的一个主要焦点是测量缺氧期间释放GABA的星状神经元的电活动，以了解是什么触发了这种大量神经递质的释放。此外，使用高倍显微镜，我们将可视化星状神经元相对于锥体神经元的位置，以确定星状神经元是否专用于最近发现的一种神经传递形式--体积传递。大脑是一个可电兴奋的组织，我们也在研究非兴奋组织-肝脏中离子通道的缺氧调节。在这个资助周期中，我们将继续探索大脑和肝脏组织的电生理特性和第二信使途径，以更好地了解哪些离子通道是以减少能量需求和允许缺氧生存的方式进行调节的。更重要的是，我们的工作将证明，不同类型的细胞，即使在单个组织内，对缺氧挑战的反应也不同；因此，需要多种不同的策略来保护组织免受缺氧损伤。

The Buck lab is investigating the cellular mechanisms that permit some vertebrate species, such as freshwater turtles and goldfish to survive without oxygen (anoxia) for days to months. We are interested in the fundamental mechanisms underlying this ability but our results will have clear clinical significance and application in the treatment of stroke, heart attack and organ storage for transplant. Both brain and liver cell models are used to study the cellular mechanisms of anoxia tolerance in tissue that is electrically excitable and one that is not. Key to surviving anoxia is the shutting off of energy utilizing cellular activities, such as the synthesis of new proteins and maintaining ion gradients across cell membranes. We have previously shown that protein synthesis decreases by about 90%, the pump that maintains ion gradients across cell membranes decreases by 75%, and ion channel activity (pathways for ions to cross membranes) decreases and in one special case increases. The ability to reduce the rate of these energy consuming reactions is something that mammals (humans) lack. However, basic biochemical pathways are common to most species, certainly amongst reptiles (turtles), fish, birds and mammals. Our goal is to determine the natural cellular pathways responsible for shutting off energy consuming processes in anoxia-tolerant species. We have already identified several possible pathways and discovered that brain excitatory signaling by pyramidal neurons involving glutamate is decreased, while inhibitory activity involving GABA (gamma-aminobutyric acid) and stellate neurons is increased during anoxic periods. In fact, GABA currents double in magnitude during anoxia and are larger than any GABA current observed in mammal brain. A major focus of this grant is to measure the electrical activity of GABA releasing stellate neurons during anoxia to understand what triggers this large release of neurotransmitter. Furthermore, using high-powered microscopy we will visualize the position of the stellate neurons in relation to the pyramidal neurons to determine if the stellate neurons are specialized for a recently discovered form of neurotransmission - volume transmission. Brain is an electrically excitable tissue and we are also investigating anoxic regulation of ion channels in non-excitable tissue - liver. In this grant cycle we will continue to explore the electrophysiological properties and second messenger pathways of brain and liver tissue to better understand which ion channels are regulated in a way that reduces energetic demands and permits anoxic survival.  Importantly, our work will demonstrate that different cell types, even within a single tissue, respond differently to an anoxic challenge; therefore, multiple different strategies are needed to protect tissues from anoxic injury. **