MOLECULAR MECHANISMS REGULATING CALCIUM FLUX IN SALIVARY GLANDS

唾液腺液体分泌的神经递质刺激是通过细胞质[Ca] （[Ca2+]i）的双期升高介导的；最初的短暂增加是由于内部释放，后持续的增加是由于Ca2+内流。持续的液体分泌直接依赖于[Ca2+]的持续升高，即Ca2+内流。本项目旨在了解介导和调节唾液腺细胞Ca2+内流的机制。唾液细胞中的Ca2+内流是许多其他不可兴奋细胞中存储操作的Ca2+内流机制的原型。然而，在任何类型的细胞中，这种离子通量的机制尚未确定。在我们之前的研究中，我们已经开始努力纯化和鉴定参与介导大鼠腮腺腺泡细胞基底外侧质膜Ca2+内流的蛋白质。在本报告所述期间，我们继续朝着这个方向努力。利用我们之前开发的方法，我们从大鼠颌下腺质膜中纯化了一条钙内流通道，该通道与从大鼠腮腺质膜中纯化的通道具有相似的转运和电生理特性。此外，通过凝胶过滤层析，下颌骨和腮腺活性组分的表观分子量分别为135kD和160 kD。为了补充我们正在进行的研究，今年我们启动了分子生物学和电生理学的研究。我们建立了膜片钳系统，利用全细胞结构研究了人颌下腺细胞系（HSG）中钙依赖性K通道的活性。这种离子通道对于维持唾液腺细胞的膜电位至关重要，从而调节钙稳态和液体分泌等过程。利用分子生物学技术，我们首次证明了TRP1和TRP3基因在大鼠腮腺和下颌骨腺以及HSG细胞中的存在。TRP基因被认为编码钙内流通道，这可能与储存操作的钙进入机制有关。

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.