Molecular Mechanisms Regulating Calcium Flux In Salivary

本项目旨在了解唾液腺细胞中钙信号的调节机制。神经递质对唾液腺液体分泌的刺激是通过胞浆[Ca-2+]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+的内流。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，我们的工作主要集中在唾液腺细胞内神经递质刺激的钙内流机制上。来自我们实验室和其他实验室的最新数据表明，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(由细胞内钙储存库中的钙的耗尽激活)和受体或第二信使操作的钙进入(通过第二信使直接由受体信号激活，如二酰甘油，响应神经递质刺激的磷脂酰肌醇二磷酸水解而产生)。这些钙进入途径普遍存在于可兴奋和不可兴奋的细胞中，并严重影响细胞的许多功能。这些钙进入途径的分子组成或调控机制(S)尚未在任何细胞类型中建立。 瞬时受体电位(TRPC)家族的离子通道蛋白被认为是神经递质激活的钙内流通道的分子成分。所有的TRPC都能被磷脂酰肌醇二磷酸的激动剂刺激激活，并促进这两种类型的钙内流。目前已鉴定的TRPC的生理功能(S)和调控尚未完全确定。此外，它们如何在商店经营和商店独立的钙进入途径中发挥作用尚不清楚。通过使用编码hTrp1的腺病毒(Adha-hTrp1)在大鼠SMG体内表达TRPC1，并通过检测天然和突变型TRPC1在人颌下腺细胞系HSG中的作用，我们先前报道了TRPC1参与调节唾液腺细胞内钙内流。此外，我们认为TRPC蛋白可以通过异构体相互作用产生不同的通道。 今年，我们已经完成了人类腮腺细胞系HSY中存在的一个由TRPC1+TRPC3形成的这样的通道的特征。我们已经报道，TRPC1+TRPC3异构体通道是由内钙库耗竭激活的。此外，我们绘制了这些蛋白质通过它们的N-末端结构域相互作用的图。TRPC1或TRPC3的N-端在HSY细胞中表达时，对钙离子内流和相关的阳离子电流产生显性-负性效应。我们今年还做了一些实验，以评估SOCE通路在激动剂和thapsigargin刺激的急性分散的小鼠颌下腺细胞钙离子内流中的作用。M受体激动剂卡巴胆碱和细胞内钙储备剂thapsigargin都能很好地激活细胞内钙离子的进入。在这两种情况下，钙离子进入的阻滞剂2-APB和低浓度的Gd都能阻止(90%)钙离子进入。对该制剂中电流的电生理测量显示出一种不同于HSG细胞(TRPC1通道)的线性非选择性电流，但更像HSY(TRPC1+TRPC3通道)细胞的电流。我们目前正在更详细地研究这一渠道活动。 我们还研究了Trp通道在调节唾液腺细胞体积中的作用，因为许多TRP与膜拉伸和渗透感觉有关。细胞容量调节是一个动态的过程，它与跨皮细胞渗透力和液体分泌的变化有关。AQP5是一种水通道，一直与液体分泌和调节量的变化有关，以响应非等渗条件。然而，渗透调节和调节细胞体积的机制还不是很清楚。TRPV4已被认为是一种渗透压和机械传感通道。我们研究了唾液腺细胞调节性体积减少(RVD)的机制，并报道了渗透感觉瞬时受体电位Val-loid 4(TRPV4)和水通道蛋白5(AQP5)之间的一种新的联系，AQP5参与调节水通透性和细胞体积。唾液腺细胞和腺泡细胞暴露于低渗环境可引起细胞体积增加和RVD的激活。低渗性也激活了随后的RVD所需的钙离子内流。这种钙内流与一种明显的非选择性阳离子电流有关，该电流被4-α-PDD激活，而被Ru红抑制，提示TRPV4参与其中。与此一致的是，内源性TRPV4和AQP5一起在细胞和腺泡顶区被检测到。重要的是，尽管细胞肿胀程度相似，但缺乏TRPV4或AQP5的小鼠的细胞在低张状态下表现出极大的减少钙内流和RVD丢失。此外，低渗增加了AQP5和TRPV4的结合和表面表达。肌动蛋白解聚可降低这两种作用和RVD。这些数据表明：(I)低张激活TRPV4依赖于AQP5，而不是细胞本身的肿胀；(Ii)TRPV4和AQP5共同控制调节性容量的减少。我们的数据表明，TRPV4和AQP5的耦合调控运输是由紧张性的变化触发的，这取决于细胞骨架的完整性。这些数据表明TRPV4在唾液腺功能中起着重要作用。

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.