REGULATION OF INTRACELLULAR CALCIUM IN PARATHYROID CELLS

甲状旁腺激素（PTH）的释放是通过改变 细胞外[Ca ~（2+）]。高钙抑制，低钙刺激 甲状旁腺激素分泌。升高EC Ca 2+可迅速、持续地增加 细胞内游离Ca ~（2+）（[Ca ~（2+）]i）与Ca ~（2+）动员 第二信使三磷酸肌醇（β 3）-1，4，5-β 3释放钙 2+从各种细胞的细胞内储存，在T细胞中， 还通过开放膜Ca 2+通道刺激Ca 2+内流。 海 海胆卵和泪腺细胞，1，3，4，5 InsP 4，产生自 1，4，5-InsP 3 3-激酶和ATP共同作用， 1，4，5-β-D-3介导膜Ca 2+内流。 1，4，5-丙三醇的作用 和1，3，4，5-双胍4对EC Ca ~（2+）诱导的[Ca ~（2+）] 2+]i尚未得到解决，也没有Ca 2+流入机制， 用膜片钳技术直接研究了甲状旁腺细胞。 我们 假设甲状旁腺细胞表达钙受体或传感器， 2+，当激活时诱导[Ca 2 +]的快速和持续增加， 这是由于细胞内Ca 2+动员和膜Ca 2+内流。 我们 提出1，4，5-β 3介导细胞内的初始释放， Ca 2+和膜Ca 2+通道--由1，4，5-三羟甲基丙烷门控 和/或1，3，4，5-InsP 4，通过GTP结合蛋白，或通过Ca 2+受体 分子本身-负责[Ca 2 +]i的持续增加。 本提案的目标是确定和描述 甲状旁腺细胞中钙离子动员的机制， 电生理学和生物化学方法。 具体来说，我们将 确定高EC Ca 2+是否诱导1，3，4，5-InsP 3和1，4，5-InsP 4 积累; 1，3，4，5-β 4是否调节Ca 2+的再摄取 1，4，5 - 2013年释放;以及1，4，5 - 2013年生产是否 对Ca 2+的动员和吸收至关重要。 我们将实现 后者通过阻断磷脂酰肌醇4，5-双PO 4（PIP 2）水解 和1，4，5-lnsp 3代与磷脂酶C抑制剂和 与PIP 2和1，4，5-β 3类似物结合的单克隆抗体 1，4，5-β 3S，其动员1，4，5-β 3-敏感的Ca 2+池， 抵抗磷酸化到1，3，4，5 - 14-通过膜片钳，我们将 评估这些细胞是否表达电压不敏感的Ca 2+通道 受第二信使或鸟苷酸调控， 直接通过Ca 2+受体分子。 这些方法应提供 甲状旁腺细胞[Ca 2 +]i调节的重要见解 并确定可能参与的Ca 2+内流机制， 甲状旁腺素分泌过多状态的发病机制。

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.