REGULATION OF INTRACELLULAR CALCIUM IN PARATHYROID CELLS

甲状旁腺素的分泌受细胞外(EC)[Ca~(2+)]变化控制。 高钙对甲状旁腺激素释放有抑制作用，低钙对甲状旁腺素的释放有最大刺激作用。 EC[Ca~(2+)]也通过对Pre-Pre的影响来调节PTH的生物合成。 ProPTH mRNA水平和PTH基因转录水平。后一种影响是 可能在慢性适应血钙变化中起关键作用 活着。钙离子被认为与最近发现的一种膜钙离子相互作用 与磷脂酶C激活、1，4，5-InsP3形成偶联的传感器， [Ca~(2+)]i持续升高，最终抑制甲状旁腺素 分泌物。甲状旁腺细胞持续的细胞内钙反应 需要EC Ca~(2+)，我们推测，这是膜开放的结果 钙离子通道。关于药理方面的信息很少， 钙离子内流途径的生化或分子特性 甲状旁腺细胞。通过全细胞膜片钳技术，我们记录到了钙离子 电流对电压不敏感，对阳离子有选择性，并被 La3+和Gd3+。在微量荧光法研究中，Gd3+显著地还原 细胞内Ca~(2+)对高EC[Ca~(2+)]的反应 Gd3+-阻滞性电流在调节钙内流中的重要性。vt.在.的基础上 进一步分析，钙电流由2个组分组成。一 分量是其电导依赖于电压不敏感的电流 [Ca~(2+)]变化。这一电流被二氢吡啶阻断 钙通道拮抗剂，并受蛋白激酶A的负性调节。 其他电流成分是电压依赖的，由蛋白质调节 提出的研究有四个目的：(1)研究KC的作用。 钙离子浓度持续升高在甲状旁腺激素慢性抑制中的作用 通过测量甲状旁腺激素释放和前甲状旁腺激素mRNA的分泌和生物合成 与选择性诱导瞬变的药物孵育的细胞中的水平 或一过性加持续的[Ca~(2+)]i升高；(2)定义 甲状旁腺细胞内钙离子通道的特性及其评价 通过磷酸化和鸟苷酸的调节；(3)评估 钙电流对[Ca~(2+)]i持续升高的贡献及其意义 在高钙诱导的甲状旁腺素分泌/生物合成抑制中的作用 使用通道激动剂和拮抗剂；以及(4)从 甲状旁腺基因文库，编码二氢吡啶敏感的钙离子 通道，在非洲爪哇卵母细胞中表达，并确定这是否 通道可以耦合到钙离子传感器。甲状旁腺细胞的钙通道 可以作为关键机制，用于转换由 钙传感器与细胞内钙离子的相互作用。这些渠道可能会 有助于长期适应钙缺乏状态或慢性 体内的高钙血症条件。

PTH secretion is controlled by changes in the extracellular (EC) [Ca2+]. High EC Ca2+ inhibits, and low EC Ca2+ maximally stimulates PTH release. The EC [Ca2+] also modulates PTH biosynthesis through effects on pre- proPTH mRNA levels and PTH gene transcription. These latter effects are likely to be crucial in the chronic adaptation to changes in serum Ca2+ in vivo. Ca2+ is thought to interact with a recently identified membrane Ca2+ sensor which couples to phospholipase C activation, 1,4,5-InsP3 formation, sustained increases in [Ca2+]i, and eventually, to the inhibition of PTH secretion. Sustained intracellular Ca2+ responses in parathyroid cells require EC Ca2+ and, we hypothesize, result from the opening of membrane Ca2+ Channels. Little information is available on the pharmacologic, biochemical, or molecular properties of Ca2+ influx pathways in parathyroid cells. By whole-cell patch-clamping, we have recorded Ca2+ currents which are voltage-insensitive, cation-selective and blocked by La3+ and Gd3+. In microflurimetry studies, Gd3+ markedly reduces intracellular Ca2+ responses to high EC [Ca2+], underscoring the potential importance of Gd3+-blockable Currents in mediating Ca2+ influx. Upon further analysis, the Ca2+ currents are comprised of 2 components. One component is a voltage-insensitive current whose conductance is dependent on changes in the EC [Ca2+]. This current is blocked by dihydropyridine Ca2+ channel antagonists and negatively modulated by protein kinase A. The other current component is voltage-dependent and regulated by protein kinase C. The studies proposed have 4 aims: (1) to investigate the role of sustained increases in [Ca2+]i in mediating chronic suppression of PTH secretion and biosynthesis, by measuring PTH release and pre-proPTH mRNA levels in cells incubated with agents which selectively induce transient or transient plus sustained increases in [Ca2+]i; (2) to define the properties of Ca2+ channels in parathyroid cells and assess their regulation by phosphorylation and guanyl nucleotides; (3) to assess the contribution of Ca2+ currents to sustained increases in [Ca2+]i and their role in high EC Ca2+-induced suppression of PTH secretion/biosynthesis, using channel agonists and antagonists; and (4) to isolate a cDNA from a parathyroid cDNA library, which encodes a dihydropyridine-sensitive Ca2+ channel, as expressed in Xenopus oocytes, and to determine whether this channel can couple to the Ca2+ sensor. Ca2+ channels in parathyroid cells may serve as a key mechanism for transducing signals initiated by the interaction of EC Ca2+ with the Ca2+ sensor. These channels are likely to contribute to the longterm adaptation to Ca2+ deficiency states or chronic hypercalcemic conditions in vivo.