Physicochemical Investigation of Taste

描述(由申请人提供)：生物体已经进化出将其细胞内和细胞外液体隔间的酸度控制在狭窄的生理范围内的机制。在系统层面上，在大脑、心脏、肠道和舌头中充当氢离子传感器的细胞会激发反射性反应，从而缓解并最终消除酸性挑战。酸味感受器检测到细胞内pH的下降，但在强酸的情况下，如胃酸(HCI)，质子进入味觉细胞的机制尚不清楚。来自NADPH氧化酶基因敲除小鼠的初步数据表明，质子通过两个氢离子通道穿过味觉细胞的顶膜。1)与对照组相比，在NADPH氧化酶基因敲除小鼠中，大约60%的味觉神经反应缺失，这表明一个味觉质子通道与NADPH氧化酶活性有关。2)环磷酸腺苷可增强NADPH氧化酶基因敲除小鼠对盐酸的剩余反应。我们将通过在电压钳条件下记录麻醉野生型和NADPH氧化酶基因敲除小鼠的味觉神经，并使用极化的单个味蕾制备的荧光成像方法测量味觉细胞顶端和基底侧膜上的氢离子通量，来详细研究这两种通道的功能。酸味反应的相部分的转导不依赖于钙，而补酸部分依赖于钙。新的数据表明，这种阶段性转导涉及到细胞内pH降低引起的味觉细胞体积的减少。PH引起的体积减少依赖于细胞骨架肌动蛋白。细胞骨架的化学破坏或细胞的渗透预收缩会阻止时相反应，但不会阻止主药。我们将详细探讨时相酸度反应和细胞体积变化之间的联系。味觉细胞中的pH恢复机制塑造了酸味反应。我们有证据表明，其中一种是Pendrin，一种氯化氢-碳酸氢盐交换剂。我们将调查它的作用以及相关转运蛋白在二氧化碳酸化反应中的作用。由于垂蛋白也运输甲酸，我们将研究它在甲酸味觉反应中的作用。该提案研究了酸味的三个关键过程：1)酸进入机制，2)分离的时相和紧张性转导机制，以及3)pH恢复和适应机制。这项研究将揭示酸味防御机制如何防止酸的摄取。

DESCRIPTION (provided by applicant): Organisms have evolved mechanisms for controlling the acidity of their intracellular and extracellular fluid compartments to within narrow physiological limits. On the systemic level, cells that function as hydrogen ion sensors in the brain, the heart, the gut, and the tongue evoke reflexive responses that serve to mitigate and ultimately eliminate the acidic challenge. Sour taste receptors detect a decrease in intracellular pH, but in the case of strong acids such as gastric acid (HCI) the mechanism by which protons enter taste cells remains unknown. Preliminary data from NADPH oxidase knockout mice indicate that protons cross the apical membranes of taste cells through two hydrogen ion channels. 1) About 60% of the taste neural response to HCI is absent in NADPH oxidase knockout mice relative to controls, suggesting that one taste proton channel is linked to NADPH oxidase activity. 2) The remaining response to HCI in NADPH oxidase knockout mice is enhanced by cyclic AMP. We will investigate the function of both channels in detail by recording from taste nerves in anesthetized wild type and NADPH oxidase knockout mice under voltage clamp conditions, and by making hydrogen ion flux measurements across the apical and basolateral membranes of taste cells using a polarized single taste bud preparation with fluorescence imaging methods. Transduction for the phasic part of the sour response is calcium-independent while the tonic part depends on calcium. New data indicate that the phasic transduction involves a decrease in taste cell volume induced by a decrease in intracellular pH. The pH-induced volume decrease depends on cytoskeletal actin. Chemical disruption of the cytoskeleton or osmotically pre-shrinking the cells blocks the phasic response, but not the tonic. We shall explore the link between the phasic sour response and cell volume changes in detail. pH recovery mechanisms in taste cells shape the sour taste response. We have evidence that one of these is pendrin, a chloride-bicarbonate exchanger. We will investigate its role and the role of related transporters in sour responses to carbon dioxide. Since pendrin also transports formate, we will investigate its role in formic acid taste responses. The proposal investigates the three key processes in sour taste: 1) acid entry mechanisms, 2) separate phasic and tonic transduction mechanisms, and 3) pH recovery and adaptation mechanisms. The research will reveal how the sour taste defense mechanism functions to prevent the ingestion of acids.