PURINOCEPTOR-MEDIATED ACTIVATION OF PAROTID CELLS

嘌呤受体介导的腮腺细胞激活

• 批准号: 3224137
• 负责人: Stephen Paul Soltoff
• 金额: $ 16.2万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-05-01 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3224137
• 关键词: G protein; acinar cell; adenosine triphosphate; biological signal transduction; calcium channel; crosslink; electrophysiology; guanine nucleotide binding protein; inhibitor /antagonist; ion transport; isothiocyanates; laboratory rat; membrane channels; neurotransmitter receptor; nucleotide analog; parotid gland; phospholipase C; protein structure function; purinergic receptor; radionuclides; receptor binding; salivation; stilbenes; voltage /patch clamp

The broad, long-term objective of this proposal is to understand on a molecular level the mechanism by which extracellular ATP (adenosine 5'- triphosphate) promotes receptor-mediated alterations of the ion transport events involved in saliva formation (fluid secretion) by the parotid acinar cell. the parotid cell secretes an isotonic primary fluid that is modified by the salivary ductal system, which adds and removes electrolytes. Secretory events are controlled by the release of neurotransmitters, and the parotid receives parasympathetic and sympathetic innervation. The former is believed to regulate fluid secretion, while the latter is involved in the stimulation of exocytosis and amylase release. ATP is costored and coreleased with neurotransmitters, and it may also act as a neurotransmitter. We found that ATP has a similar effect to muscarinic and other phospholipase C- linked agonists in activating the Ca2+-sensitive ion transport mechanisms involved in fluid secretion. However, it does this by a receptor- operated ion channel, and this is different from the mechanism of action of sympathetic and parasympathetic agonists. We found that a specific type of purinergic receptor, P2Z, is present on parotid acinar cells, and propose that when ATP binds to these receptors it activates fluid secretion by a pathway that is separate from other activation pathways in these cells. In this proposal, I will investigate the regulation of the ATP-activated ion current in parotid acinar cells, including determining whether a GTP-dependent protein is involved in its activation. We have identified a number of covalent (4,4'- diisothiocyanatostilbene- 2,2'-disulfonic acid [DIDS]) and noncovalent (Reactive Blue 2, Coomassie Blue) inhibitors of the physiological effects of ATP, including the ATP-stimulated entry of 45Ca2+. Using this assay, I will determine structural features that are important to the binding of these inhibitors to the P2Z receptor, and investigate their competition with ATP to determine if they are P2Z antagonists. In addition, I will use these inhibitors in binding studies using ATPalphaS (an effective P2Z agonist) to define pharmacological criteria for identifying the P2Z receptor. I will also attempt to covalently label the P2Z receptor by cross-linking ATPalpha[35S] to it. Together, these studies will provide a more complete understanding of the pharmacological and biochemical nature of this receptor and its involvement in fluid secretion and saliva formation.

该提议的广泛长期目标是了解 分子水平的细胞外ATP的机制（腺苷5'-- 三磷酸盐）促进离子转运的受体介导的改变 腮腺涉及唾液形成（流体分泌）的事件 腺泡细胞。 腮腺细胞分泌一种等渗的主要液体 由唾液导管系统修改，该系统添加和去除 电解质。 分泌事件由发布 神经递质，腮腺接受副交感神经和 同情神经。 据信前者可以调节液体 分泌，而后者参与刺激胞吐作用 和淀粉酶释放。 ATP被cosed和发行 神经递质，它也可以充当神经递质。 我们发现 该ATP的作用与毒蕈碱和其他磷脂酶C-的作用相似 激活Ca2+敏感的离子传输机制时相连的激动剂 参与流体分泌。 但是，它通过受体来做到这一点 操作的离子通道，这与作用机理不同 交感神经和副交感激动剂。 我们发现一个特定的 嘌呤能受体的类型存在于腮腺腺细胞上，并且 提出，当ATP与这些受体结合时，它会激活流体 与其他激活途径分开的途径分泌 在这些细胞中。 在此提案中，我将调查 腮腺腺泡细胞中的ATP激活离子电流，包括 确定GTP依赖性蛋白是否参与其 激活。 我们已经确定了许多共价（4,4'- 二硫代氨基苯甲酸 - 2,2'-二硫酸[DIDS]）和非共价 （反应性蓝2，Coomassie Blue）生理效应的抑制剂 ATP的of，包括45CA2+的ATP刺激入口。 使用此测定， 我将确定对绑定很重要的结构特征 这些对P2Z受体的抑制剂，并研究它们 与ATP竞争以确定它们是否是P2Z拮抗剂。 在 此外，我将使用ATPALLASE在结合研究中使用这些抑制剂 （有效的P2Z激动剂）定义药理学标准 识别P2Z受体。 我还将尝试共价标记 通过交联atpalpha [35s]的P2Z受体。 在一起，这些 研究将为药理学提供更完整的了解 该受体的生化性质及其参与流体 分泌和唾液形成。