MOLECULAR MECHANISMS REGULATING CALCIUM FLUX IN SALIVARY GLANDS

神经递质对唾液腺液体分泌的刺激是通过胞浆[Ca]([Ca+]i)的双相升高来调节的；最初的一过性增加是由于内部释放，而后者的持续增加是由于Ca~(2+)内流。持续的液体分泌直接依赖于[Ca~(2+)]的持续升高，即Ca~(2+)内流。本研究旨在了解唾液腺细胞内钙离子内流的调节机制。唾液细胞中的钙离子内流是许多其他非兴奋性细胞中存在的储存操作的钙离子内流机制的典型。然而，在任何类型的细胞中，这种离子通量的机制还没有确定。在我们以前的研究中，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侧质膜钙内流的蛋白(S)。在本报告所述期间，我们继续朝着这一方向努力。利用我们以前开发的方法，我们从大鼠颌下腺质膜中纯化了一条钙内流途径，其转运和电生理特性与从大鼠腮腺质膜中纯化的钙内流途径相似。此外，通过凝胶过滤层析，颌下腺和腮腺活性部分的表观分子量分别为135kD和160kD。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研究。我们建立了膜片钳系统，并利用全细胞构型研究了人颌下腺细胞系(HSG)钙依赖性钾通道的活动。这种离子通道对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，我们首次证实了TRP1和TRP3基因在大鼠腮腺和颌下腺以及HSG细胞中的存在。Trp基因被认为编码一条钙离子内流通道，这可能参与了钙库操作的钙离子内流机制。

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.