MOLECULAR MECHANISMS REGULATING CALCIUM FLUX IN SALIVARY GLANDS

调节唾液腺钙通量的分子机制

• 批准号: 6161792
• 负责人: INDU S. AMBUDKAR
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6161792
• 关键词: basolateral membrane; calcium channel; calcium flux; laboratory rat; parotid gland; potassium channel; salivary glands; submandibular gland; tissue /cell culture; voltage /patch clamp

Neurotransmitter stimulation of fluid secretion in salivary glands is mediated via a biphasic elevation in cytosolic [Ca] ([Ca2+]i); an initial transient increase due to internal release and a latter sustained increase due to Ca2+ influx. Sustained fluid secretion is directly dependent upon the sustained elevation of [Ca2+], i.e. on Ca2+ influx. This project is aimed towards understanding the mechanisms which mediate and regulate Ca2+ influx in salivary gland cells. Ca2+ influx in salivary cells is prototypical of the store-operated Ca2+ influx mechanisms present in many other non-excitable cells. However, the mechanism of this ion flux has not yet been determined in any cells type. In our previous studies we had initiated efforts to purify and identify the protein(s) involved in mediating Ca2+ influx across the basolateral plasma membrane of rat parotid acinar cells. In this reporting period we have continued our efforts in this direction. By using the methods developed by us previously, we have purified a calcium influx pathway from rat submandibular gland plasma membranes which displays similar transport and electrophysiological properties as that purified from rat parotid gland plasma membranes. Further, by using gel filtration chromatography, the apparent molecular weights are 135kD and 160 kD, respectively, for the active fractions from submandibular and parotid glands. To complement our ongoing studies, this year we initiated molecular biological and electrophysiological studies. We have set up a patch clamp system and by using the whole cell configuration we have studied the Ca-dependent K- channel activity in a human submandibular gland cell line (HSG). This ion channel is critical for maintaining the membrane potential in salivary gland cells thus regulating processes such as calcium homeostasis and fluid secretion. Using molecular biology techniques we have demonstrated for the first time the presence of TRP1 and TRP3 genes in rat parotid and submandibular glands and in HSG cells. The TRP genes have been suggested to encode a calcium influx channel, which might be involved in the store- operated calcium entry mechanism.

神经递质刺激唾液腺液体分泌， 通过胞质[Ca]（[Ca 2 +]i）的双相升高介导;初始 由于内部释放而引起的短暂增加和随后的持续增加 由于Ca 2+的涌入。 持续的液体分泌直接依赖于 [Ca ~（2+）]的持续升高，即对Ca ~（2+）内流的影响。 这个项目是 旨在了解介导和调节Ca 2+的机制 唾液腺细胞内流。 唾液细胞中的Ca 2+内流是 钙池操作的Ca 2+内流机制的原型存在于许多 其他非兴奋性细胞。 然而，这种离子流的机制 尚未在任何细胞类型中确定。 在我们之前的研究中， 开始努力纯化和鉴定参与的蛋白质， 介导大鼠基底外侧质膜Ca ~（2+）内流 腮腺腺泡细胞 在本报告所述期间，我们继续 朝着这个方向努力。 通过使用我们开发的方法 之前，我们已经从大鼠中纯化了钙内流途径， 下颌下腺质膜显示类似的运输和 电生理特性与从大鼠腮腺纯化的电生理特性相同 质膜 此外，通过使用凝胶过滤色谱， 表观分子量分别为135 kD和160 kD， 下颌下腺和腮腺的活性成分。 以补充 我们正在进行的研究，今年我们启动了分子生物学， 电生理学研究。 我们建立了膜片钳系统， 使用全细胞构型，我们研究了Ca依赖的K- 人下颌下腺细胞系（HSG）中的通道活性。 该离子 通道是维持唾液中膜电位的关键 腺细胞从而调节诸如钙稳态的过程， 液体分泌 利用分子生物学技术， 首次在大鼠腮腺中发现TRP 1和TRP 3基因， 下颌下腺和HSG细胞。 TRP基因被认为是 编码钙内流通道，这可能与储存有关- 操作钙进入机制。