REGULATION OF INTRACELLULAR CALCIUM IN PARATHYROID CELLS

甲状旁腺激素(PTH)的释放受 胞外(EC)[Ca2+]。高钙抑制，低钙刺激 甲状旁腺素分泌。升高EC Ca~(2+)可迅速、持续地增加 细胞内游离钙与钙动员 第二信使三磷酸肌醇-1，4，5-Insp3释放钙 2+来自多种细胞的胞内存储，在T细胞中，可以 也可通过开放膜钙通道来刺激钙内流。在海里 海胆卵和泪腺细胞，1，3，4，5InsP4，产生自 1，4，5-Insp3由1，4，5-InsP3 3-激酶和ATP共同作用 1，4，5-Insp3介导膜内钙内流。1，4，5-Insp3的作用 和1，3，4，5-Insp4对EC Ca~(2+)诱导的[Ca~(2+)]i升高的调节 2+]i还没有被解决，也没有钙离子内流机制 用膜片钳技术直接研究甲状旁腺细胞。我们的 假说甲状旁腺细胞表达钙离子受体或感受器 2+被激活后可引起[Ca~(2+)]快速持续升高， 由于细胞内钙离子的动员和膜上钙离子的内流。我们 1，4，5-Insp3介导细胞内初始释放 钙离子和膜钙通道--被1，4，5-Insp3门控 和/或1，3，4，5-InsP4，通过GTP结合蛋白，或通过钙离子受体 分子本身--负责持续增加[Ca~(2+)]i。 当前提案的目标是确定和描述 甲状旁腺细胞钙离子动员机制的研究 电生理学和生化方法。具体来说，我们将 确定高钙是否诱导1，4，5-InsP3和1，3，4，5Insp4 1，3，4，5-Insp4是否调节钙再摄取 由1，4，5-Insp3释放；以及1，4，5-Insp3的生产是否将 对钙的动员和吸收是必不可少的。我们将完成 后者通过阻断磷脂酰肌醇4，5-双P04(PIP2)的水解 和1，4，5-lnsp3的生成与磷脂酶C抑制剂和 与PIP2和1，4，5-Insp3类似物结合的单抗 1，4，5-Insp3S，它动员1，4，5-Insp3敏感的钙池和 抵抗1，3，4，5Insp4的磷酸化-通过膜片钳，我们将 评估这些细胞是否表达电压不敏感的钙通道 它们受第二信使或鸟苷或 直接通过钙离子受体分子。这些方法应该提供 甲状旁腺细胞[Ca~(2+)]_i调节的重要启示 并确定可能涉及的钙离子内流机制 甲状旁腺激素高分泌状态的发病机制。

Parathyroid hormone (PTH) release is regulated by changes in the extracellular (EC) [Ca2+]. High Ca2+ inhibits, and low Ca2+ stimulates PTH secretion. Raising EC Ca2+ produces prompt, sustained increases in intracellular free Ca2+ ([Ca 2+]i) and the Ca2+- mobilizing second-messenger inositol trisphosphate (Insp3)- 1,4,5-Insp3 releases Ca 2+ from intracellular stores in a variety of cells and, in T-cells, can also stimulate Ca2+ influx by opening membrane Ca2+ channels. In sea urchin eggs and lacrimal cells, 1,3,4,5 InsP4, generated from 1,4,5-Insp3 by 1,4,5-InsP3 3-kinase and ATP, acts together with 1,4,5-Insp3 to mediate membrane Ca2+ influx. The role of 1,4,5-Insp3 and 1,3,4,5-Insp4 in the regulation of EC Ca2+- induced increases in [Ca 2+]i has not been addressed nor have the Ca2+ influx mechanisms in parathyroid cells been studied directly by patch-clamp techniques. Our hypothesis is that parathyroid cells express receptors or sensors for Ca 2+ which when activated induce rapid and sustained increases in [Ca2+], due to intracellular Ca2+ mobilization and membrane Ca2+ influx. We propose that 1,4,5-Insp3 mediates the initial release of intracellular Ca2+ and that membrane Ca2+ channels-- gated either by 1,4,5-Insp3 and/or 1,3,4,5-InsP4, by a GTP-binding protein, or by the Ca2+ receptor molecule itself -- are responsible for sustained increases in [Ca2+]i . The goal of the current proposal is to identify and characterize the mechanisms for Ca2+ mobilization in parathyroid cells using electrophysiologic and biochemical approaches. Specifically, we will determine whether high EC Ca2+ induces 1 4,5-InsP3 and 1,3,4,5Insp4 accumulation; whether 1,3,4,5-Insp4 regulates the reuptake of Ca2+ released by 1,4,5-Insp3; and whether 1,4,5-Insp3 production is to essential to Ca2+ mobilization and uptake. We will accomplish the latter by blocking phosphatidyl inositol 4,5-bis P04 (PIP2) hydrolysis and 1,4,5-lnsp3 generation with a phospholipase C inhibitor and a monoclonal antibody which binds to PIP2 and by the 1,4,5-Insp3 analogue 1,4,5-Insp3S, which mobilizes 1,4,5-Insp3-sensitive Ca2+ pools and resists phosphorylation to 1,3,4,5Insp4- By patch-clamping, we will assess whether these cells express voltage-insensitive Ca2+ channels which are regulated by second-messengers or guanyl nucleotides or directly by the Ca2+ receptor molecule. These approaches should provide important insights into the regulation of [Ca2+]i in parathyroid cells and identify Ca2+ influx mechanisms that may be involved in the pathogenesis of PTH hypersecretory states.