Calcium signaling defects underlying salivary gland dysfunction

Salivary glands secrete saliva, a mixture of proteins and fluids, which plays an extremely important role in the maintenance of oral health. Loss of salivary secretion causes a dry mouth condition, xerostomia, which has numerous deleterious consequences including opportunistic infections within the oral cavity, difficulties in eating and swallowing food, and problems with speech. Saliva secretion is regulated by stimulation of specific signaling mechanisms within the acinar cells of the gland. Neurotransmitter-stimulated increase in cytosolic [Ca²⁺] ([Ca²⁺]_i) in acinar cells is the primary trigger for salivary fluid secretion from salivary glands, the loss of which is a critical factor underlying dry mouth conditions in patients. The increase in [Ca²⁺]_i regulates multiple ion channel and transport activities that together generate the osmotic gradient which drives fluid secretion across the apical membrane. Ca²⁺ entry mediated by the Store-Operated Ca²⁺ Entry (SOCE) mechanism provides the essential [Ca²⁺]_i signals to trigger salivary gland fluid secretion. Under physiological conditions depletion of ER-Ca²⁺ stores is caused by activation of IP3R by IP3 and this provides the stimulus for SOCE. Core components of SOCE in salivary gland acinar cells are the plasma membrane Ca²⁺ channels, Orai1 and TRPC1, and STIM1, a Ca²⁺-sensor protein in the ER, which regulates both channels. In addition, STIM2 likely enhances the sensitivity of cells to ER-Ca²⁺ depletion thereby tuning the cellular response to agonist stimulation. Two major, clinically relevant, conditions which cause irreversible salivary gland dysfunction are radiation treatment for head-and-neck cancers and the autoimmune exocrinopathy, Sjögren's syndrome (pSS). However, the exact mechanism(s) that causes the loss of fluid secretion, in either condition, is not clearly understood. A number of recent studies have identified that defects in critical Ca²⁺ signaling mechanisms underlie salivary gland dysfunction caused by radiation treatment or Sjögren's syndrome (pSS). This chapter will discuss these very interesting and important studies.     

Dissection of Calcium Signaling Events in Exocrine Secretion

The secretion of fluid and electrolytes by salivary gland acinar cells requires the coordinated regulation of multiple ion channel and transporter proteins, signaling components, and water transport. Importantly, neurotransmitter stimulated increase in the cytosolic free [Ca²⁺] ([Ca²⁺]_i) is critical for the regulation of salivary gland secretion as it regulates several major ion fluxes that together establish the sustained osmotic gradient to drive fluid secretion. The mechanisms that act to modulate these increases in [Ca²⁺]_i are therefore central to the process of salivary fluid secretion. Such modulation involves membrane receptors for neurotransmitters, as well as mechanisms that mediate intracellular Ca²⁺ release, and Ca²⁺ entry, as well as those that maintain cellular Ca²⁺ homeostasis. Together, these mechanisms determine the spatial and temporal aspects of the [Ca²⁺]_i signals that regulate fluid secretion. Molecular cloning of these transporters and channels as well as development of mice lacking these proteins has established the physiological significance of key components that are involved in regulating [Ca²⁺]_i in salivary glands. This review will discuss these important studies and the findings which have led to resolution of the Ca²⁺ signaling mechanisms that determine salivary gland fluid secretion.     

TRPC1, Orai1, and STIM1 in SOCE: Friends in tight spaces

Store-operated calcium entry (SOCE) is a ubiquitous Ca²⁺ entry pathway that is activated in response to depletion of ER-Ca²⁺ stores and critically controls the regulation of physiological functions in miscellaneous cell types. The transient receptor potential canonical 1 (TRPC1) is the first member of the TRPC channel subfamily to be identified as a molecular component of SOCE. While TRPC1 has been shown to contribute to SOCE and regulate various functions in many cells, none of the reported TRPC1-mediated currents resembled I_CRAC, the highly Ca²⁺-selective store-dependent current first identified in lymphocytes and mast cells. Almost a decade after the cloning of TRPC1 two proteins were identified as the primary components of the CRAC channel. The first, STIM1, is an ER-Ca²⁺ sensor protein involved in activating SOCE. The second, Orai1 is the pore-forming component of the CRAC channel. Co-expression of STIM1 and Orai1 generated robust I_CRAC. Importantly, STIM1 was shown to also activate TRPC1 via its C-terminal polybasic domain, which is distinct from its Orai1-activating domain, SOAR. In addition, TRPC1 function critically depends on Orai1-mediated Ca²⁺ entry which triggers recruitment of TRPC1 into the plasma membrane where it is then activated by STIM1. TRPC1 and Orai1 form discrete STIM1-gated channels that generate distinct Ca²⁺ signals and regulate specific cellular functions. Surface expression of TRPC1 can be modulated by trafficking of the channel to and from the plasma membrane, resulting in changes to the phenotype of TRPC1-mediated current and [Ca²⁺]_i signals. Thus, TRPC1 is activated downstream of Orai1 and modifies the initial [Ca²⁺]_i signal generated by Orai1 following store depletion. This review will summarize the important findings that underlie the current concepts for activation and regulation of TRPC1, as well as its impact on cell function.     

IP3 receptors and Ca2+ entry

Inositol 1,4,5-trisphosphate receptors (IP₃R) are the most widely expressed intracellular Ca²⁺ release channels. Their activation by IP₃ and Ca²⁺ allows Ca²⁺ to pass rapidly from the ER lumen to the cytosol. The resulting increase in cytosolic [Ca²⁺] may directly regulate cytosolic effectors or fuel Ca²⁺ uptake by other organelles, while the decrease in ER luminal [Ca²⁺] stimulates store-operated Ca²⁺ entry (SOCE). We are close to understanding the structural basis of both IP₃R activation, and the interactions between the ER Ca²⁺-sensor, STIM, and the plasma membrane Ca²⁺ channel, Orai, that lead to SOCE. IP₃Rs are the usual means through which extracellular stimuli, through ER Ca²⁺ release, stimulate SOCE. Here, we review evidence that the IP₃Rs most likely to respond to IP₃ are optimally placed to allow regulation of SOCE. We also consider evidence that IP₃Rs may regulate SOCE downstream of their ability to deplete ER Ca²⁺ stores. Finally, we review evidence that IP₃Rs in the plasma membrane can also directly mediate Ca²⁺ entry in some cells.     

The dynamic complexity of the TRPC1 channelosome

A rise in cytoplasmic [Ca²⁺] due to store-operated Ca²⁺ entry (SOCE) triggers a plethora of responses, both acute and long term. This leads to the important question of how this initial signal is decoded to regulate specific cellular functions. It is now clearly established that local [Ca²⁺] at the site of SOCE can vary significantly from the global [Ca²⁺] in the cytosol. Such Ca²⁺ microdomains are generated by the assembly of key Ca²⁺ signaling proteins within the domains. For example, GPCR, IP₃ receptors, TRPC3 channels, the plasma membrane Ca²⁺ pump and the endoplasmic reticulum (ER) Ca²⁺ pump have all been found to be assembled in a complex and all of them contribute to the Ca²⁺ signal. Recent studies have revealed that two other critical components of SOCE, STIM1 and Orai1, are also recruited to these regions. Thus, the entire machinery for activation and regulation of SOCE is compartmentalized in specific cellular domains which facilitates the specificity and rate of protein-protein interactions that are required for activation of the channels. In the case of TRPC1-SOC channels, it appears that specific lipid domains, lipid raft domains (LRDs), in the plasma membrane, as well as cholesterol-binding scaffolding proteins such as caveolin-1 (Cav-1), are involved in assembly of the TRPC channel complexes. Thus, plasma membrane proteins and lipid domains as well as ER proteins contribute to the SOCE-Ca²⁺ signaling microdomain and modulation of the Ca²⁺ signals per se. Of further interest is that modulation of Ca²⁺ signals, i.e. amplitude and/or frequency, can result in regulation of specific cellular functions. The emerging data reveal a dynamic Ca²⁺ signaling complex composed of TRPC1/Orai1/STIM1 that is physiologically consistent with the dynamic nature of the Ca²⁺ signal that is generated. This review will focus on the recent studies which demonstrate critical aspects of the TRPC1 channelosome that are involved in the regulation of TRPC1 function and TRPC1-SOC-generated Ca²⁺ signals.     

TRPC1 regulates calcium‐activated chloride channels in salivary gland cells

Calcium‐activated chloride channel (CaCC) plays an important role in modulating epithelial secretion. It has been suggested that in salivary tissues, sustained fluid secretion is dependent on Ca²⁺ influx that activates ion channels such as CaCC to initiate Cl^? efflux. However direct evidence as well as the molecular identity of the Ca²⁺ channel responsible for activating CaCC in salivary tissues is not yet identified. Here we provide evidence that in human salivary cells, an outward rectifying Cl^? current was activated by increasing [Ca²⁺]_i, which was inhibited by the addition of pharmacological agents niflumic acid (NFA), an antagonist of CaCC, or T16Ainh‐A01, a specific TMEM16a inhibitor. Addition of thapsigargin (Tg), that induces store‐depletion and activates TRPC1‐mediated Ca²⁺ entry, potentiated the Cl^? current, which was inhibited by the addition of a non‐specific TRPC channel blocker SKF96365 or removal of external Ca²⁺. Stimulation with Tg also increased plasma membrane expression of TMEM16a protein, which was also dependent on Ca²⁺ entry. Importantly, in salivary cells, TRPC1 silencing, but not that of TRPC3, inhibited CaCC especially upon store depletion. Moreover, primary acinar cells isolated from submandibular gland also showed outward rectifying Cl^? currents upon increasing [Ca²⁺]_i. These Cl^? currents were again potentiated with the addition of Tg, but inhibited in the presence of T16Ainh‐A01. Finally, acinar cells isolated from the submandibular glands of TRPC1 knockout mice showed significant inhibition of the outward Cl^? currents without decreasing TMEM16a expression. Together the data suggests that Ca²⁺ entry via the TRPC1 channels is essential for the activation of CaCC. J. Cell. Physiol. 9999: 2848–2856, 2015. © 2015 Wiley Periodicals, Inc.

     

Effects of intratracheal administration of nuclear factor-kappaB decoy oligodeoxynucleotides on long-term cigarette smoke-induced lung inflammation and pathology in mice

Background

Sildenafil, a potent phosphodiesterase type 5 (PDE5) inhibitor, has been proposed as a treatment for pulmonary arterial hypertension (PAH). The mechanism of its anti-proliferative effect on pulmonary artery smooth muscle cells (PASMC) is unclear. Nuclear translocation of nuclear factor of activated T-cells (NFAT) is thought to be involved in PASMC proliferation and PAH. Increase in cytosolic free [Ca²⁺] ([Ca²⁺]_i) is a prerequisite for NFAT nuclear translocation. Elevated [Ca²⁺]_i in PASMC of PAH patients has been demonstrated through up-regulation of store-operated Ca²⁺ channels (SOC) which is encoded by the transient receptor potential (TRP) channel protein. Thus we investigated if: 1) up-regulation of TRPC1 channel expression which induces enhancement of SOC-mediated Ca²⁺ influx and increase in [Ca²⁺]_i is involved in hypoxia-induced PASMC proliferation; 2) hypoxia-induced promotion of [Ca²⁺]_i leads to nuclear translocation of NFAT and regulates PASMC proliferation and TRPC1 expression; 3) the anti-proliferative effect of sildenafil is mediated by inhibition of this SOC/Ca²⁺/NFAT pathway.

Methods

Human PASMC were cultured under hypoxia (3% O₂) with or without sildenafil treatment for 72 h. Cell number and cell viability were determined with a hemocytometer and MTT assay respectively. [Ca²⁺]_i was measured with a dynamic digital Ca²⁺ imaging system by loading PASMC with fura 2-AM. TRPC1 mRNA and protein level were detected by RT-PCR and Western blotting respectively. Nuclear translocation of NFAT was determined by immunofluoresence microscopy.

Results

Hypoxia induced PASMC proliferation with increases in basal [Ca²⁺]_i and Ca²⁺ entry via SOC (SOCE). These were accompanied by up-regulation of TRPC1 gene and protein expression in PASMC. NFAT nuclear translocation was significantly enhanced by hypoxia, which was dependent on SOCE and sensitive to SOC inhibitor SKF96365 (SKF), as well as cGMP analogue, 8-brom-cGMP. Hypoxia-induced PASMC proliferation and TRPC1 up-regulation were inhibited by SKF and NFAT blocker (VIVIT and Cyclosporin A). Sildenafil treatment ameliorated hypoxia-induced PASMC proliferation and attenuated hypoxia-induced enhancement of basal [Ca²⁺]_i, SOCE, up-regulation of TRPC1 expression, and NFAT nuclear translocation.

Conclusion

The SOC/Ca²⁺/NFAT pathway is, at least in part, a downstream mediator for the anti-proliferative effect of sildenafil, and may have therapeutic potential for PAH treatment.     

Distinct Mechanisms of [Ca2+]i Oscillations in HSY and HSG Cells: Role of Ca2+ Influx and Internal Ca2+ Store Recycling

This study examined [Ca²⁺]_i oscillations in the human salivary gland cell lines, HSY and HSG. Relatively low concentrations of carbachol (CCh) induced oscillatory, and higher [CCh] induced sustained, steady-state increases in [Ca²⁺]_i and K _Ca currents in both cell types. Low IP₃, but not thapsigargin (Tg), induced [Ca²⁺]_i oscillations, whereas Tg blocked CCh-stimulated [Ca²⁺]_i oscillations in both cell types. Unlike in HSG cells, removal of extracellular Ca²⁺ from HSY cells (i) did not affect CCh-stimulated [Ca²⁺]_i oscillations or internal Ca²⁺ store refill, and (ii) converted high [CCh]-induced steady-state increase in [Ca²⁺]_i into oscillations. CCh- or thapsigargin-induced Ca²⁺ influx was higher in HSY, than in HSG, cells. Importantly, HSY cells displayed relatively higher levels of sarcoendoplasmic reticulum Ca²⁺ pump (SERCA) and inositoltrisphosphate receptors (IP₃Rs) than HSG cells. These data demonstrate that [Ca²⁺]_i oscillations in both HSY and HSG cells are primarily determined by the uptake of Ca²⁺ from, and release of Ca²⁺ into, the cytosol by the SERCA and IP₃R activities, respectively. In HSY cells, Ca²⁺ influx does not acutely contribute to this process, although it determines the steady-state increase in [Ca²⁺]_i. In HSG cells, [Ca²⁺]_i oscillations directly depend on Ca²⁺ influx; Ca²⁺ coming into the cell is rapidly taken up into the store and then released into the cytosol. We suggest that the differences in the mechanism of [Ca²⁺]_i oscillations HSY and HSG cells is related to their respective abilities to recycle internal Ca²⁺ stores. Received: 30 October 2000/Revised: 26 February 2001     

TRPC1 and ORAI1 channels in colon cancer

Colon cancer cells, like other types of cancer cells, undergo the remodeling of the intracellular Ca²⁺ homeostasis that contributes to cancer cell hallmarks including enhanced cell proliferation, migration, and survival. Colon cancer cells display enhanced store-operated Ca²⁺ entry (SOCE) compared with their non-cancer counterparts. Colon cancer cells display an abnormal expression of SOCE molecular players including Orai1 and TRPC1 channels, and the stromal interacting molecule (STIM) 1 and 2. Interestingly, upregulation of Orai1 and TRPC1 channels and their contribution to SOCE are associated with cancer malignancy in colon cancer cells. In a specific cellular model of colon cancer, whereas in non-cancer colon cells SOCE is composed of the Ca²⁺ release activated (CRAC) currents, in colon cancer cells SOCE is composed of CRAC- and cationic, non-selective store operated (SOC) currents. Former SOCs are mediated by TRPC1 channels. Moreover, colon cancer cells also display dysregulation of the expression of 1,4,5-triphosphate receptors (IP₃R) that could contribute to the enhanced SOCE. Another important factor underlying the enhanced SOCE is the differential mitochondrial modulation of the CRAC and SOC currents in non-cancer and colon cancer cells. In colon cancer cells, mitochondria take up more Ca²⁺ that prevent the Ca²⁺-dependent inactivation of the SOCs, leading to sustained Ca²⁺ entry. Notably, the inhibition of SOCE in cancer colon cells abolishes their cancer hallmarks. Robust evidence has shown the efficiency of non-steroidal anti-inflammatory drugs (NSAIDs) and difluoromethylornithine (DFMO) to reverse the enhanced cell proliferation, migration, and apoptosis resistance of cancer cells. In colon cancer cells, both NSAIDs and DFMO decrease SOCE, but they target different molecular components of SOCE. NSAIDs decrease the Ca²⁺ uptake by mitochondria, limiting their ability to prevent the Ca²⁺-dependent inactivation of the SOCs that underlie SOCE. On the other hand, DFMO inhibits the expression of TRPC1 channels in colon cancer cells, eliminating their contribution to SOCE. The identification of players of SOCE in colon cancer cells may help to better understand the remodeling of the Ca²⁺ homeostasis in cancer. Importantly, the use of different pharmacological tools that target different SOCE molecular players in colon cancer cells may play a pivotal role in designing better chemoprevention strategies.     

Functional organization of TRPC-Ca channels and regulation of calcium microdomains

TRP family of proteins are components of unique cation channels that are activated in response to diverse stimuli ranging from growth factor and neurotransmitter stimulation of plasma membrane receptors to a variety of chemical and sensory signals. This review will focus on members of the TRPC sub-family (TRPC1–TRPC7) which currently appear to be the strongest candidates for the enigmatic Ca²⁺ influx channels that are activated in response to stimulation of plasma membrane receptors which result in phosphatidyl inositol-(4,5)-bisphosphate (PIP₂) hydrolysis, generation of IP₃ and DAG, and IP₃-induced Ca²⁺ release from the intracellular Ca²⁺ store via inositol trisphosphate receptor (IP₃R). Homomeric or selective heteromeric interactions between TRPC monomers generate distinct channels that contribute to store-operated as well as store-independent Ca²⁺ entry mechanisms. The former is regulated by the emptying/refilling of internal Ca²⁺ store(s) while the latter depends on PIP₂ hydrolysis (due to changes in PIP₂ per se or an increase in diacylglycerol, DAG). Although the exact physiological function of TRPC channels and how they are regulated has not yet been conclusively established, it is clear that a variety of cellular functions are controlled by Ca²⁺ entry via these channels. Thus, it is critical to understand how cells coordinate the regulation of diverse TRPC channels to elicit specific physiological functions. It is now well established that segregation of TRPC channels mediated by interactions with signaling and scaffolding proteins, determines their localization and regulation in functionally distinct cellular domains. Furthermore, both protein and lipid components of intracellular and plasma membranes contribute to the organization of these microdomains. Such organization serves as a platform for the generation of spatially and temporally dictated [Ca²⁺]_i signals which are critical for precise control of downstream cellular functions.     

Distinct Contributions of Orai1 and TRPC1 to Agonist-Induced [Ca2+]i Signals Determine Specificity of Ca2+-Dependent Gene Expression

Regulation of critical cellular functions, including Ca²⁺-dependent gene expression, is determined by the temporal and spatial aspects of agonist-induced Ca²⁺ signals. Stimulation of cells with physiological concentrations of agonists trigger increases [Ca²⁺]_i due to intracellular Ca²⁺ release and Ca²⁺ influx. While Orai1-STIM1 channels account for agonist-stimulated [Ca²⁺]_i increase as well as activation of NFAT in cells such as lymphocytes, RBL and mast cells, both Orai1-STIM1 and TRPC1-STIM1 channels contribute to [Ca²⁺]_i increases in human submandibular gland (HSG) cells. However, only Orai1-mediated Ca²⁺ entry regulates the activation of NFAT in HSG cells. Since both TRPC1 and Orai1 are activated following internal Ca²⁺ store depletion in these cells, it is not clear how the cells decode individual Ca²⁺ signals generated by the two channels for the regulation of specific cellular functions. Here we have examined the contributions of Orai1 and TRPC1 to carbachol (CCh)-induced [Ca²⁺]_i signals and activation of NFAT in single cells. We report that Orai1-mediated Ca²⁺ entry generates [Ca²⁺]_i oscillations at different [CCh], ranging from very low to high. In contrast, TRPC1-mediated Ca²⁺ entry generates sustained [Ca²⁺]_i elevation at high [CCh] and contributes to frequency of [Ca²⁺]_i oscillations at lower [agonist]. More importantly, the two channels are coupled to activation of distinct Ca²⁺ dependent gene expression pathways, consistent with the different patterns of [Ca²⁺]_i signals mediated by them. Nuclear translocation of NFAT and NFAT-dependent gene expression display “all-or-none” activation that is exclusively driven by local [Ca²⁺]_i generated by Orai1, independent of global [Ca²⁺]_i changes or TRPC1-mediated Ca²⁺ entry. In contrast, Ca²⁺ entry via TRPC1 primarily regulates NFκB-mediated gene expression. Together, these findings reveal that Orai1 and TRPC1 mediate distinct local and global Ca²⁺ signals following agonist stimulation of cells, which determine the functional specificity of the channels in activating different Ca²⁺-dependent gene expression pathways.     

Tuning store-operated calcium entry to modulate Ca2+-dependent physiological processes

The intracellular calcium signaling processes are tightly regulated to ensure the generation of calcium signals with the specific spatiotemporal characteristics required for regulating various cell functions. Compartmentalization of the molecular components involved in the generation of these signals at discrete intracellular sites ensures the signaling specificity and transduction fidelity of the signal for regulating downstream effector processes. Store-operated calcium entry (SOCE) is ubiquitously present in cells and is critical for essential cell functions in a variety of tissues. SOCE is mediated via plasma membrane Ca²⁺ channels that are activated when luminal [Ca²⁺] of the endoplasmic reticulum ([Ca²⁺]_ER) is decreased. The ER-resident stromal interaction molecules, STIM1 and STIM2, respond to decreases in [Ca²⁺]_ER by undergoing conformational changes that cause them to aggregate at the cell periphery in ER-plasma membrane (ER-PM) junctions. At these sites, STIM proteins recruit Orai1 channels and trigger their activation. Importantly, the two STIM proteins concertedly modulate Orai1 function as well as the sensitivity of SOCE to ER-Ca²⁺ store depletion. Another family of plasma membrane Ca²⁺ channels, known as the Transient Receptor Potential Canonical (TRPC) channels (TRPC1-7) also contribute to sustained [Ca²⁺]_i elevation. Although Ca²⁺ signals generated by these channels overlap with those of Orai1, they regulate distinct functions in the cells. Importantly, STIM1 is also required for plasma membrane localization and activation of some TRPCs. In this review, we will discuss various molecular components and factors that govern the activation, regulation and modulation of the Ca²⁺ signal generated by Ca²⁺ entry pathways in response to depletion of ER-Ca²⁺ stores. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.     

Interleukin-13 enhanced Ca2+ oscillations in airway smooth muscle cells

Physiological mechanisms associated with interleukin-13 (IL-13), a key cytokine in asthma, in intracellular Ca²⁺ signaling in airway smooth muscle cells (ASMCs) remain unclear. The aim of this study was to assess effects of IL-13 on Ca²⁺ oscillations in response to leukotriene D4 (LTD4) in human cultured ASMCs.LTD4-induced Ca²⁺ oscillations in ASMCs pretreated with IL-13 were imaged by confocal microscopy. mRNA expressions of cysteinyl leukotriene 1 receptors (CysLT1R), CD38, involved with the ryanodine receptors (RyR) system, and transient receptor potential canonical (TRPC), involved with store-operated Ca²⁺ entry (SOCE), were determined by real-time PCR. In IL-13-pretreated ASMCs, frequency of LTD4-induced Ca²⁺ oscillations and number of oscillating cells were significantly increased compared with untreated ASMCs. Both xestospongin C, a specific inhibitor of inositol 1,4,5-triphosphate receptors (IP₃R), and ryanodine or ruthenium red, inhibitors of RyR, partially blocked LTD4-induced Ca²⁺ oscillations. Ca²⁺ oscillations were almost completely inhibited by 50 μM of 2-aminoethoxydiphenyl borate (2-APB), which dominantly blocks SOCE but not IP₃R at this concentration. Pretreatment with IL-13 increased the mRNA expressions of CysLT1R and CD38, but not of TRPC1 and TRPC3.We conclude that IL-13 enhances frequency of LTD4-induced Ca²⁺ oscillations in human ASMCs, which may be cooperatively modulated by IP₃R, RyR systems and possibly by SOCE.     

Chronic Hypoxia Increases TRPC6 Expression and Basal Intracellular Ca2+ Concentration in Rat Distal Pulmonary Venous Smooth Muscle

Background

Hypoxia causes remodeling and contractile responses in both pulmonary artery (PA) and pulmonary vein (PV). Here we explore the effect of hypoxia on PV and pulmonary venous smooth muscle cells (PVSMCs).

Methods

Chronic hypoxic pulmonary hypertension (CHPH) model was established by exposing rats to 10% O₂ for 21 days. Rat distal PVSMCs were isolated and cultured for in vitro experiments. The fura-2 based fluorescence calcium imaging was used to measure the basal intracellular Ca²⁺ concentration ([Ca²⁺]_i) and store-operated Ca²⁺ entry (SOCE). Quantitative RT-PCR and western blotting were performed to measure the expression of mRNA and levels of canonical transient receptor potential (TRPC) protein respectively.

Results

Hypoxia increased the basal [Ca²⁺]_i and SOCE in both freshly dissociated and serum cultured distal PVSMCs. Moreover, hypoxia increased TRPC6 expression at mRNA and protein levels in both cultured PVSMCs exposed to prolonged hypoxia (4% O₂, 60 h) and distal PV isolated from CHPH rats. Hypoxia also enhanced proliferation and migration of rat distal PVSMCs.

Conclusions

Hypoxia induces elevation of SOCE in distal PVSMCs, leading to enhancement of basal [Ca²⁺]_i in PVSMCs. This enhancement is potentially correlated with the increased expression of TRPC6. Hypoxia triggered intracellular calcium contributes to promoted proliferation and migration of PVSMCs.     

Flufenamic acid is a tool for investigating TRPC6-mediated calcium signalling in human conditionally immortalised podocytes and HEK293 cells

Mutations in the cation channel TRPC6 result in a renal-specific phenotype of familial nephrotic syndrome, affecting intracellular calcium ([Ca²⁺]_i) signalling in the glomerular podocyte. Tools to study native TRPC6 activity are scarce, although there has been recent success with flufenamic acid (FFA). We confirm the specificity of FFA for TRPC6 both in an artificial expression system and in a human conditionally immortalised podocyte cell line (ciPod).Cells were loaded with fura-2AM and changes in intracellular calcium ([Ca²⁺]_i) were calculated. 200 μM FFA induced an increase in [Ca²⁺]_i in HEK293 cells with native TRPC6 expression, which was enhanced by overexpression of TRPC6 and completely blocked in the absence of extracellular calcium. Expressed TRPC7 did not significantly affect the response to FFA whereas expressed TRPC3 reduced it. FFA also induced an increase ciPod in [Ca²⁺]_i, which was inhibited using SKF96365 and 2-APB, but not indomethacin. In ciPod, adenovirus (Ad-v) wild type (WT) TRPC6 increased [Ca²⁺]_i activity to FFA compared to native TRPC6, whereas activity was significantly reduced with Ad-v dominant negative (DN) TRPC6. The niflumic acid (NFA) induced increase in [Ca²⁺]_i in ciPod was not affected by Ad-v TRPC6 DN, and in HEK293 cells was not affected by WT TRPC6.In conclusion, FFA activates TRPC6 [Ca²⁺]_i signalling in both ciPod and HEK293 cells independently of TRPC3 and TRPC7, and independently of properties of the fenamate family.     

Ammonium Increases Ca<Superscript>2+</Superscript> Signalling and Up-Regulates Expression of TRPC1 Gene in Astrocytes in Primary Cultures and in the In Vivo Brain

Rapid rise in ammonium concentration in the brain is the major pathogenic factor in hepatic encephalopathy that is manifested by state of confusion, forgetfulness and irritability, psychotic symptoms, delusions, lethargy, somnolence and, in the terminal stages, coma. Primary cultures of mouse astrocytes were used to investigate effects of chronic treatment (3 days) with ammonium chloride (ammonium) at 3 mM, this being a relevant concentration for hepatic encephalopathy condition, on metabotropic receptor agonist-induced increases in free cytosolic Ca²⁺ concentration [(Ca²⁺)_i], measured with fura-2 based microfluorimetry and on store-operated Ca²⁺ entry (SOCE) activated following treatment with the SERCA inhibitor thapsigargin. The agonists used were the β-adrenergic agonist isoproterenol, the α₂-adrenergic agonist dexmedetomidine, the InsP₃ receptor (InsP₃R) agonist adenophostin A and ryanodine receptor agonist 4-Chloro-m-cresol (4-CMC). Agonist-induced [Ca²⁺]_i responses were significantly increased in astrocytes chronically exposed to ammonium. Similarly, the SOCE, meditated by the transient receptor potential channel 1 (TRPC1), was significantly augmented. The ammonium-induced increase in SOCE was a result of an up-regulation of mRNA and protein expression of TRPC1 in astrocytes. Increase in TRPC1 expression and in SOCE were both prevented by ouabain antagonist canrenone. Similar up-regulation of TRPC1 gene expression was found in the brain of adult mice subjected to intraperitoneal injection of urease for 3 days. In transgenic mice tagged with an astrocyte-specific or a neurone-specific markers and treated with intraperitoneal injections of urease for 3 days, the fluorescence-activated cell sorting of neurones and astrocytes demonstrated that TRPC1 mRNA expression was up-regulated in astrocytes, but not in neurones.     

Effects of chronic treatment with fluoxetine on receptor-stimulated increase of [Ca2+]i in astrocytes mimic those of acute inhibition of TRPC1 channel activity

Primary cultures of mouse astrocytes were used to investigate effects by chronic treatment (3-21 days) with fluoxetine (0.5-10 μM) on capacitative Ca²⁺ influx after treatment with the SERCA inhibitor thapsigargin and on receptor agonist-induced increases in free cytosolic Ca²⁺ concentration [Ca²⁺]_i, determined with Fura-2. The agonists were the 5-HT_2B agonist fluoxetine, the α₂-adrenergic agonist dexmedetomidine, and ryanodine receptor (RyR) and IP₃ receptor (IP₃R) agonists. In untreated sister cultures each agonist distinctly increased [Ca²⁺]_i, but in cultures treated for sufficient length of time or with sufficiently high doses of fluoxetine, acute administration of fluoxetine, dexmedetomidine, or RyR or IP₃R agonists elicited reduced, in some cases abolished, effects. Capacitative Ca²⁺ entry, meditated by TRPC1 channels, was sufficiently inhibited to cause a depletion of Ca²⁺ stores, which could explain the reduced agonist effects. All effects of chronic fluoxetine administration could be replicated by TRPC1 channel antibody or siRNA. Since increases in astrocytic [Ca²⁺]_i regulate release of gliotransmitters, these effects may have profound effects on brain function. They may be important for therapeutic effects of all 5 conventional ‘serotonin-specific reuptake inhibitors’ (SSRIs), which at concentrations used therapeutically (∼1 μM) share other of fluoxetine's chronic effects (Zhang et al., Neuron Glia Biol. 16 (2010) 1-13).     

Inhibition of SOC/Ca2+/NFAT pathway is involved in the anti-proliferative effect of sildenafil on pulmonary artery smooth muscle cells

Background

Sildenafil, a potent phosphodiesterase type 5 (PDE5) inhibitor, has been proposed as a treatment for pulmonary arterial hypertension (PAH). The mechanism of its anti-proliferative effect on pulmonary artery smooth muscle cells (PASMC) is unclear. Nuclear translocation of nuclear factor of activated T-cells (NFAT) is thought to be involved in PASMC proliferation and PAH. Increase in cytosolic free [Ca²⁺] ([Ca²⁺]_i) is a prerequisite for NFAT nuclear translocation. Elevated [Ca²⁺]_i in PASMC of PAH patients has been demonstrated through up-regulation of store-operated Ca²⁺ channels (SOC) which is encoded by the transient receptor potential (TRP) channel protein. Thus we investigated if: 1) up-regulation of TRPC1 channel expression which induces enhancement of SOC-mediated Ca²⁺ influx and increase in [Ca²⁺]_i is involved in hypoxia-induced PASMC proliferation; 2) hypoxia-induced promotion of [Ca²⁺]_i leads to nuclear translocation of NFAT and regulates PASMC proliferation and TRPC1 expression; 3) the anti-proliferative effect of sildenafil is mediated by inhibition of this SOC/Ca²⁺/NFAT pathway.

Methods

Human PASMC were cultured under hypoxia (3% O₂) with or without sildenafil treatment for 72 h. Cell number and cell viability were determined with a hemocytometer and MTT assay respectively. [Ca²⁺]_i was measured with a dynamic digital Ca²⁺ imaging system by loading PASMC with fura 2-AM. TRPC1 mRNA and protein level were detected by RT-PCR and Western blotting respectively. Nuclear translocation of NFAT was determined by immunofluoresence microscopy.

Results

Hypoxia induced PASMC proliferation with increases in basal [Ca²⁺]_i and Ca²⁺ entry via SOC (SOCE). These were accompanied by up-regulation of TRPC1 gene and protein expression in PASMC. NFAT nuclear translocation was significantly enhanced by hypoxia, which was dependent on SOCE and sensitive to SOC inhibitor {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400"}}SKF96365 (SKF), as well as cGMP analogue, 8-brom-cGMP. Hypoxia-induced PASMC proliferation and TRPC1 up-regulation were inhibited by SKF and NFAT blocker (VIVIT and Cyclosporin A). Sildenafil treatment ameliorated hypoxia-induced PASMC proliferation and attenuated hypoxia-induced enhancement of basal [Ca²⁺]_i, SOCE, up-regulation of TRPC1 expression, and NFAT nuclear translocation.

Conclusion

The SOC/Ca²⁺/NFAT pathway is, at least in part, a downstream mediator for the anti-proliferative effect of sildenafil, and may have therapeutic potential for PAH treatment.