REGULATION OF INTRACELLULAR CALCIUM IN PARATHYROID CELLS

PTH分泌受细胞外（EC）[Ca2 +]的变化控制。 高EC Ca 2+抑制，低EC Ca 2+最大限度地刺激PTH释放。 EC [Ca2 +]还通过影响PTH的前代谢来调节PTH的生物合成。 proPTH mRNA水平和PTH基因转录。后一种效应是 可能是至关重要的慢性适应血清钙的变化， vivo. Ca2+被认为与最近发现的膜Ca2+相互作用 该传感器与磷脂酶C激活、1，4，5-InsP3形成 持续增加[Ca2 +] i，并最终抑制PTH 分泌物甲状旁腺细胞内持续的钙反应 需要EC Ca2+，我们推测，这是由于膜的开放 Ca2+通道。关于药理学的信息很少， 生物化学或分子特性的Ca2+内流途径， 副甲状腺细胞通过全细胞膜片钳技术，我们记录了Ca 2 + 电流是电压不敏感的，阳离子选择性的，被 La~（3+）和Gd~（3+）。 在微量荧光法研究中，Gd 3+显著降低 细胞内Ca 2+对高EC [Ca 2 +]的反应，强调了 Gd~（3+）可阻断电流在介导Ca~（2+）内流中的重要性后 进一步分析，Ca~（2+）电流由2个分量组成。一 分量是电压不敏感电流，其电导取决于 EC [Ca2 +]的变化。 这种电流被二氢吡啶阻断 Ca 2+通道拮抗剂和负调节蛋白激酶A。的 另一种电流成分是电压依赖性的，受蛋白质调节 激酶C本研究有4个目的：（1）探讨 [Ca2 +] i持续升高介导PTH慢性抑制 分泌和生物合成，通过测量PTH释放和pre-proPTH mRNA 与选择性诱导瞬时 或短暂加持续增加[Ca2 +] i;（2）定义 甲状旁腺细胞中钙通道的特性，并评估其 通过磷酸化和鸟苷酸调节;（3）评估 Ca~（2+）电流对[Ca~（2+）] i持续增加的贡献及其 在高EC Ca 2+诱导的PTH分泌/生物合成抑制中的作用， 使用通道激动剂和拮抗剂;和（4）从一种或多种细胞中分离cDNA， 甲状旁腺cDNA文库，其编码二氢吡啶敏感的Ca 2 + 通道，表达在非洲爪蟾卵母细胞，并确定是否这 通道可以耦合到Ca2+传感器。甲状旁腺细胞的钙通道 可以作为一个关键机制，用于转换由 EC Ca 2+与Ca 2+传感器的相互作用。这些渠道可能 有助于长期适应钙缺乏状态或慢性 体内高钙条件。

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.