Molecular Mechanisms Regulating Calcium Flux In Salivary

本项目旨在了解唾液腺细胞钙信号的介导和调控机制。唾液腺液体分泌的神经递质刺激是通过细胞质[Ca-2+]的双期升高介导的；最初是由于内部释放引起的短暂性增加，后来是由于Ca-2+内流引起的持续增加。持续的液体分泌直接依赖于[Ca-2+]的持续升高，从而依赖于神经递质刺激的钙内流。在过去的8-10年里，我们一直致力于研究神经递质刺激的唾液腺细胞钙内流机制。最近来自我们实验室和其他实验室的数据表明，两种类型的钙进入机制可以促进这种钙信号；储存操作的钙进入（由细胞内钙储存中的钙耗尽激活）和受体或第二信使操作的钙进入（由受体通过第二信使信号直接激活，如二酰基甘油，这是在神经递质刺激的磷脂酰肌醇二磷酸水解反应中产生的）。这些钙进入途径普遍存在于可兴奋性和不可兴奋性细胞中，并严重影响许多细胞功能。这些钙进入途径的分子成分或调节机制尚未在任何细胞类型中建立。

This project is aimed towards understanding the mechanisms which mediate and regulate calcium signaling in salivary gland cells. Neurotransmitter stimulation of fluid secretion in salivary glands is mediated via a biphasic elevation in cytosolic [Ca-2+]; an initial transient increase due to internal release and a latter sustained increase due to Ca-2+ influx. Sustained fluid secretion is directly dependent upon the sustained elevation of [Ca-2+] and thus on neurotransmitter-stimulated calcium influx. In the past 8-10 years, our efforts have been focused on the neurotransmitter-stimulated calcium influx mechanism in salivary gland cells. Recent data from our laboratory and others demonstrate that two types of calcium entry mechanisms can contribute to this calcium signal; store-operated calcium entry (which is activated by the depletion of calcium in the intracellular calcium store) and receptor-or second messenger-operated calcium entry (which is activated directly by receptor signaling via second messengers such as diacylglycerol that are generated in response to neurotransmitter-stimulated phosphatidyl inositol bisphosphate hydrolysis). These calcium entry pathways are ubiquitously present in excitable and non-excitable cells and critically affect a number cellular functions. The molecular components or regulatory mechanism(s) of these calcium entry pathways have not yet been established in any cell type. Members of the transient receptor potential (TRPC) family of ion channel proteins have been proposed as molecular components of the neurotransmitter-activated calcium influx channels. All TRPCs have the ability to be activated by agonist-stimulation of phosphatidyl inositol bisphosphate hydrolysis and contribute to both types of calcium entry. The physiological function(s) and regulation of the presently identified TRPCs have not yet been fully established. Further, how they contribute to both store-operated and store-independent calcium entry pathways is not known. By expressing TRPC1 in vivo in rat SMG by using an adenovirus encoding hTrp1 (AdHA-hTrp1) and by examining the role of native and mutant TRPC1 in the human submandibular gland cell line, HSG, we had previously reported that TRPC1 is involved in the regulation of store-operated calcium influx in salivary gland cells. Further, we had suggested that TRPC proteins can generate distinct channels via heteromeric interactions. This year we have completed characterization of one such channel formed of TRPC1+TRPC3 that is present in the human parotid gland cell line, HSY. We have reported that TRPC1+TRPC3 heteromeric channel is activated by internal calcium store depletion. Further, we mapped out that the proteins interact via their N-terminal domains. The N-terminus of either TRPC1 or TRPC3 when expressed in HSY cells exerts dominant-negative effects on store-operated calcium entry and the associated cation current. We have also done experiments this year to assess the contribution of the SOCE pathway to agonist and thapsigargin-stimulated Ca2+ entry in acutely dispersed mouse submandibular gland cell preparations. Ca2+ entry is robustly activated by both the muscarinic agonist, carbachol, as well as the intracellular calcium store depleting agent, thapsigargin. Entry under both conditions was blocked (>90%) by the store-operated calcium entry inhibitors, 2-APB and low concentrations of gadolinium. Electrophysiological measurements of current in this preparation demonstrated a linear non-selective current that is different from the current seen in HSG cells (TRPC1 channels), but more like the one in HSY (TRPC1+TRPC3 channel) cells. We are currently studying this channel activity in greater detail. We have also examined the role of TRP channels in regulation of cell volume in salivary gland cells since a number of TRPs have been associated with membrane stretch and osmosensing. Cell volume regulation is a dynamic process which is correlated with changes in transepithelial osmotic forces and fluid secretion. AQP5 is a water channel that has been has been associated with fluid secretion and regulatory volume changes in response to anisosmotic conditions. However, the mechanisms involved in osmosensation and regulation of cell volume are not clearly understood. TRPV4 has been proposed as an osmo- and mechanosensor channel. We examined the mechanism of regulatory volume decrease (RVD) in salivary gland cells and report a novel association between osmosensing transient receptor potential vanalloid 4 (TRPV4) and aquaporin 5 (AQP5) which is involved in regulating water permeability and cell volume. Exposure of salivary gland cells and acini to hypotonicity elicited increase in cell volume and activation of RVD. Hypotonicity also activated calcium entry which was required for subsequent RVD. This calcium entry was associated with a distinct non-selective cation current that was activated by 4-alpha-PDD and inhibited by ruthenium red, suggesting involvement of TRPV4. Consistent with this, endogenous TRPV4 was detected in cells and in the apical region of acini along with AQP5. Importantly, cells from mice lacking either TRPV4 or AQP5 displayed greatly reduced calcium entry and loss of RVD in response to hypotonicity although the extent of cell swelling was similar. Furthermore, hypotonicity increased the association and surface expression of AQP5 and TRPV4. Both effects, and RVD, were reduced by actin depolymerization. These data suggest that (i) activation of TRPV4 by hypotonicity depends on AQP5, not on cell swelling per se, and (ii) TRPV4 and AQP5 concertedly control regulatory volume decrease. Our data show that coupled regulated trafficking of TRPV4 and AQP5 is triggered by changes in tonicity and that this depends on cytoskeletal intactness. These data suggest an important role for TRPV4 in salivary gland function.