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.
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