The Ca2+ release-activated Ca2+ current (ICRAC) mediates store-operated Ca2+ entry in rat microglia

Ca²⁺ signaling plays a central role in microglial activation, and several studies have demonstrated a store-operated Ca²⁺ entry (SOCE) pathway to supply this ion. Due to the rapid pace of discovery of novel Ca²⁺ permeable channels, and limited electrophysiological analyses of Ca²⁺ currents in microglia, characterization of the SOCE channels remains incomplete. At present, the prime candidates are ‘transient receptor potential’ (TRP) channels and the recently cloned Orai1, which produces a Ca²⁺-release-activated Ca²⁺ (CRAC) current. We used cultured rat microglia and real-time RT-PCR to compare expression levels of Orai1, Orai2, Orai3, TRPM2, TRPM7, TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7 channel genes. Next, we used Fura-2 imaging to identify a store-operated Ca²⁺ entry (SOCE) pathway that was reduced by depolarization and blocked by Gd3+, SKF-96365, diethylstilbestrol (DES), and a high concentration of 2-aminoethoxydiphenyl borate (50 μM 2-APB). The Fura-2 signal was increased by hyperpolarization, and by a low concentration of 2-APB (5 μM), and exhibited Ca²⁺-dependent potentiation. These properties are entirely consistent with Orai1/CRAC, rather than any known TRP channel and this conclusion was supported by patch-clamp electrophysiological analysis. We identified a store-operated Ca²⁺ current with the same properties, including high selectivity for Ca²⁺ over monovalent cations, pronounced inward rectification and a very positive reversal potential, Ca²⁺-dependent current potentiation, and block by SKF-96365, DES and 50 μM 2-APB. Determining the contribution of Orai1/CRAC in different cell types is crucial to future mechanistic and therapeutic studies; this comprehensive multi-strategy analysis demonstrates that Orai1/CRAC channels are responsible for SOCE in primary microglia.     

A Reciprocal Shift in Transient Receptor Potential Channel 1 (TRPC1) and Stromal Interaction Molecule 2 (STIM2) Contributes to Ca2+ Remodeling and Cancer Hallmarks in Colorectal Carcinoma Cells

We have investigated the molecular basis of intracellular Ca²⁺ handling in human colon carcinoma cells (HT29) versus normal human mucosa cells (NCM460) and its contribution to cancer features. We found that Ca²⁺ stores in colon carcinoma cells are partially depleted relative to normal cells. However, resting Ca²⁺ levels, agonist-induced Ca²⁺ increases, store-operated Ca²⁺ entry (SOCE), and store-operated currents (I_SOC) are largely enhanced in tumor cells. Enhanced SOCE and depleted Ca²⁺ stores correlate with increased cell proliferation, invasion, and survival characteristic of tumor cells. Normal mucosa cells displayed small, inward Ca²⁺ release-activated Ca²⁺ currents (I_CRAC) mediated by ORAI1. In contrast, colon carcinoma cells showed mixed currents composed of enhanced I_CRAC plus a nonselective I_SOC mediated by TRPC1. Tumor cells display increased expression of TRPC1, ORAI1, ORAI2, ORAI3, and STIM1. In contrast, STIM2 protein was nearly depleted in tumor cells. Silencing data suggest that enhanced ORAI1 and TRPC1 contribute to enhanced SOCE and differential store-operated currents in tumor cells, whereas ORAI2 and -3 are seemingly less important. In addition, STIM2 knockdown decreases SOCE and Ca²⁺ store content in normal cells while promoting apoptosis resistance. These data suggest that loss of STIM2 may underlie Ca²⁺ store depletion and apoptosis resistance in tumor cells. We conclude that a reciprocal shift in TRPC1 and STIM2 contributes to Ca²⁺ remodeling and tumor features in colon cancer.     

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.     

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.     

TRPC4 forms store-operated Ca2+ channels in mouse mesangial cells

Studies were performed to identify the molecular component responsible for store-operated Ca²⁺ entry in murine mesangial cells (MMC). Because the canonical transient receptor potential (TRPC) family of proteins was previously shown to comprise Ca²⁺-selective and -nonselective cation channels in a variety of cells, we screened TRPC1–TRPC7 with the use of molecular methods and the fura 2 method to determine their participation as components of the mesangial store-operated Ca²⁺ (SOC) channel. Using TRPC-specific primers and RT-PCR, we found that cultured MMC contained mRNA for TRPC1 and TRPC4 but not for TRPC2, TRPC3, TRPC5, TRPC6, and TRPC7. Immunocytochemical staining of MMC revealed predominantly cytoplasmic expression of TRPC1 and plasmalemmal expression of TRPC4. The role of TRPC4 in SOC was determined with TRPC4 antisense and fura 2 ratiometric measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_i). SOC was measured as the increase in [Ca²⁺]_i after extracellular Ca²⁺ was increased from <10 nM to 1 mM in the continued presence of thapsigargin. We found that TRPC4 antisense, which reduced plasmalemmal expression of TRPC4, inhibited SOC by 83%. Incubation with scrambled TRPC4 oligonucleotides did not affect SOC. Immunohistochemical staining identified expressed TRPC4 in the glomeruli of mouse renal sections. The results of RT-PCR performed to distinguish between TRPC4- and TRPC4- were consistent with expression of both isoforms in brain but with only TRPC4- expression in MMC. These studies show that TRPC4- may form the homotetrameric SOC in mouse mesangial cells. canonical transient receptor potential; TRPC4-; TRPC4-; TRPC1; fura 2; glomerulus     

Heteromeric TRPV4-C1 channels contribute to store-operated Ca(2+) entry in vascular endothelial cells

There is controversy as to whether TRP channels participate in mediating store-operated current (I_SOC) and store-operated Ca²⁺ entry (SOCE). Our recent study has demonstrated that TRPC1 forms heteromeric channels with TRPV4 in vascular endothelial cells and that Ca²⁺ store depletion enhances the vesicle trafficking of heteromeric TRPV4-C1 channels, causing insertion of more channels into the plasma membrane in vascular endothelial cells. In the present study, we determined whether the enhanced TRPV4-C1 insertion to the plasma membrane could contribute to SOCE and I_SOC. We found that thapsigargin-induced SOCE was much lower in aortic endothelial cells derived from trpv4^−/− or trpc1^−/− knockout mice when compared to that of wild-type mice. In human umbilical vein endothelial cells (HUVECs), thapsigargin-induced SOCE was markedly reduced by knocking down the expression of TRPC1 and/or TRPV4 with respective siRNAs. Brefeldin A, a blocker of vesicular translocation, inhibited the SOCE. These results suggest that an enhanced vesicular trafficking of heteromeric TRPV4-C1 channels contributes to SOCE in vascular endothelial cells. Vascular tension studies suggest that such an enhanced trafficking of TRPV4-C1 channels may play a role in thapsigargin-induced vascular relaxation in rat small mesenteric arteries.     

Mechanisms of interleukin-1beta-induced Ca2+ signals in mouse cortical astrocytes: roles of store- and receptor-operated Ca2+ entry

Many neurodegenerative disorders are accompanied by chronic glial activation, which is characterized by the abundant production of proinflammatory cytokines, such as IL-1. IL-1 disrupts Ca²⁺ homeostasis and stimulates astrocyte reactivity. The mechanisms by which IL-1 induces Ca²⁺ dysregulation are not completely defined. Here, we examined how acute and chronic (24–48 h) treatment with IL-1 affect Ca²⁺ homeostasis in freshly dissociated and primary cultured mouse cortical astrocytes. Cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) was measured with fura-2 using digital imaging. An acute application of 10 ng/ml IL-1 induced Ca²⁺ mobilization from intracellular stores and activated store-operated Ca²⁺ entry (SOCE) and receptor-operated Ca²⁺ entry (ROCE) in both freshly dissociated and cultured actrocytes. Treatment of cultured astrocytes with IL-1 for 24 and 48 h elevated resting [Ca²⁺]_cyt, decreased Ca²⁺ store content [associated with sarco(endo)plasmic reticulum Ca²⁺-ATPase 2b downregulation], and augmented ROCE. Based on evidence that receptor-operated, but not store-operated Ca²⁺ channels are Ba²⁺ permeable, Ba²⁺ entry was used to distinguish receptor-operated Ca²⁺ channels from store-operated Ca²⁺ channels. ROCE was activated by the diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol (OAG). In the presence of extracellular Ba²⁺, OAG-induced elevations of cytosolic Ba²⁺ (fura-2 340-to-380-nm ratio) were significantly larger in astrocytes treated with IL-1. These changes in IL-1-treated astrocytes correlate with augmented expression of transient receptor potential cation channel (TRPC)6 protein, which likely mediates ROCE. Knockdown of the TRPC6 gene markedly reduced ROCE. The data suggest that IL-1-induced dysregulation of Ca²⁺ homeostasis is the result of enhanced ROCE and TRPC6 expression. The disruption of Ca²⁺ homeostasis appears to be an upstream component in the cascade of IL-1-activated pathways leading to neurodegeneration. transient receptor potential cation channel proteins     

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.     

TRPC1 inhibits apoptotic cell degeneration induced by dopaminergic neurotoxin MPTP/MPP

Disturbances in Ca²⁺ homeostasis have been implicated in a variety of neuropathological conditions including Parkinson's disease (PD). However, the importance of store-operated Ca²⁺ entry (SOCE) channels in PD remains to be investigated. In the present study, we have scrutinized the significance of TRPC1 in 1-methyl-4-phenyl-1,2,3,6-tetrahyrdro-pyridine (MPTP)-induced PD using C57BL/6 animal model and PC12 cell culture model. Both sub-acute and sub-chronic treatments of MPTP significantly reduced TRPC1, and tyrosine hydroxylase levels, but not TRPC3, along with increased neuronal death. Furthermore, MPTP induces mitochondrial dysfunction, which was associated with reduced mitochondrial membrane potential, decreased level of Bcl₂, Bcl-xl, and an altered Bcl-xl/Bax ratio thereby initiating apoptosis. Importantly, TRPC1 overexpression in PC12 cells showed significant protection against MPP⁺ induced neuronal apoptosis, which was attributed to the restoration of cytosolic Ca²⁺ and preventing loss of mitochondrial membrane potential. Silencing of TRPC1 or addition of TRPC1 channel blockers decreased mitochondrial membrane potential, whereas activation of TRPC1 restored mitochondrial membrane potential in cells overexpressing TRPC1. TRPC1 overexpression also inhibited Bax translocation to the mitochondria and thereby prevented cytochrome c release and mitochondrial-mediated apoptosis. Overall, these results provide compelling evidence for the role of TRPC1 in either onset/progression of PD and restoration of TRPC1 levels could limit neuronal degeneration in MPTP mediated PD.     

STIM1 long and STIM1 gate differently TRPC1 during store-operated calcium entry

During myogenesis, a long splice variant of STIM1, called STIM1L is getting expressed, while the level of STIM1 remains constant. Previous work demonstrated that STIM1L is more efficient in eliciting store-operated Ca²⁺ entry (SOCE), but no current analysis of the channel(s) activated by this new STIM1L isoform was performed until now. In this study, we investigate the ionic channel(s) activated by STIM1L and whether differences exist between the two STIM1 isoforms, using HEK-293 T cells as a model system. Our data show that STIM1 and STIM1L activate Orai1 channel but also the endogenously expressed TRPC1. The channel activation occurs in two steps, with first Orai1 activation followed, in a subset of cells, by TRPC1 opening. Remarkably, STIM1L more frequently activates TRPC1 and preferentially interacts with TRPC1. In low intracellular Ca²⁺ buffering condition, the frequency of TRPC1 opening increases significantly, strongly suggesting a Ca²⁺-dependent channel activation. The ability of STIM1L to open Orai1 appears decreased compared to STIM1, which might be explained by its stronger propensity towards TRPC1. Indeed, increasing the amount of STIM1L results in an enhanced Orai1 current. The role of endogenous TRPC1 in STIM1- and STIM1L-induced SOCE was confirmed by Ca²⁺ imaging experiments. Overall, our findings provide a detailed analysis of the channels activated by both STIM1 isoforms, revealing that STIM1L is more prone to open TRPC1, which might explain the larger SOCE elicited by this isoform.     

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.     

Canonical transient receptor potential TRPC7 can function as both a receptor- and store-operated channel in HEK-293 cells

Previous studies on the activation mechanism of canonical transient receptor potential (TRPC) channels have often produced conflicting conclusions. All seven have been shown to be activated by phospholipase C (PLC)-coupled receptors, but TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, and TRPC7 have also been proposed to function as store-operated channels.¹ In the case of TRPC3, the expression environment and the expression level appear to determine the mode of regulation. Evidence of a close structural relative of TRPC3, TRPC7, has been presented that this channel is activated by receptor activation or by store depletion. On the basis of previous findings for TRPC3, we reasoned that subtle differences in structure or expression conditions might account for the apparent distinct gating mechanisms of TRPC7. To reexamine the mode of activation of TRPC7, we stably and transiently transfected human embryonic kidney (HEK)-293 cells with cDNA encoding for human TRPC7. We examined the ability of a PLC-activating agonist and an intracellular Ca²⁺ store-depleting agent to activate these channels. Our findings demonstrate that when transiently expressed in HEK-293 cells, TRPC7 forms channels that are activated by PLC-stimulating agonists, but not by Ca²⁺ store depletion. However, when stably expressed in HEK-293 cells, TRPC7 can be activated by either Ca²⁺ store depletion or PLC activation. To our knowledge, this is the first demonstration of a channel protein that can be activated by both receptor- and store-operated modes in the same cell. In addition, the results reconcile the apparently conflicting findings of other laboratories regarding TRPC7 regulation. calcium signaling; nonselective cation channels     

Effects of organ culture on arterial gene expression and hypoxic relaxation: role of the ryanodine receptor

Organ culturespecifically inhibits vasorelaxation to acute hypoxia andpreferentially decreases specific voltage-dependent K⁺channel expression over other K⁺ and Ca²⁺channel subtypes. To isolate further potential oxygen-sensing mechanisms correlated with altered gene expression, we performed differential display analysis on RNA isolated from control and culturedcoronary arterial rings. We hypothesize that organ culture results inaltered gene expression important for vascular smooth musclecontractility important to the mechanism of hypoxia-induced relaxation.Our results indicate a milieu of changes suggesting both up- anddownregulation of several genes. The altered expression pattern of twopositive clones was verified by Northern analysis. Subsequent screeningof a porcine cDNA library indicated homology to the ryanodine receptor(RyR). RT-PCR using specific primers to the three subtypes of RyR showsan upregulation of RyR2 and RyR3 after organ culture. Additionally, thecaffeine- and/or ryanodine-sensitive intracellular Ca²⁺store was significantly more responsive to caffeine activation afterorgan culture. Our data indicate that organ culture increases expression of specific RyR subtypes and inhibits hypoxicvasorelaxation. Importantly, ryanodine blunted hypoxic relaxation incontrol coronary arteries, suggesting that upregulated RyR might play anovel role in altered intracellular Ca²⁺ handling during hypoxia.

     

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.     

Store-operated Ca2+ entry and Ca2+ responses to hypothalamic releasing hormones in anterior pituitary cells from Orai1−/− and heptaTRPC knockout mice

Store operated Ca²⁺ entry (SOCE) is the most important Ca²⁺ entry pathway in non-excitable cells. However, SOCE can also play a pivotal role in excitable cells such as anterior pituitary (AP) cells. The AP gland contains five different cell types that release six major AP hormones controlling most of the entire endocrine system. AP hormone release is modulated by Ca²⁺ signals induced by different hypothalamic releasing hormones (HRHs) acting on specific receptors in AP cells. TRH and LHRH both induce Ca²⁺ release and Ca²⁺ entry in responsive cells while GHRH and CRH only induce Ca²⁺ entry. SOCE has been shown to contribute to Ca²⁺ responses induced by TRH and LHRH but no molecular evidence has been provided. Accordingly, we used AP cells isolated from mice devoid of Orai1 channels (noted as Orai1−/− or Orai1 KO mice) and mice lacking expression of all seven canonical TRP channels (TRPC) from TRPC1 to TRPC7 (noted as heptaTRPC KO mice) to investigate contribution of these putative channel proteins to SOCE and intracellular Ca²⁺ responses induced by HRHs. We found that thapsigargin-evoked SOCE is lost in AP cells from Orai1−/− mice but unaffected in cells from heptaTRPC KO mice. Conversely, while spontaneous intracellular Ca²⁺-oscillations related to electrical activity were not affected in the Orai1−/− mice, these responses were significantly reduced in heptaTRPC KO mice. We also found that Ca²⁺ entry induced by TRH and LHRH is decreased in AP cells isolated from Orai1−/−. In addition, Ca²⁺ responses to several HRHs, particularly TRH and GHRH, are decreased in the heptaTRPC KO mice. These results indicate that expression of Orai1, and not TRPC channel proteins, is necessary for thapsigargin-evoked SOCE and is required to support Ca²⁺ entry induced by TRH and LHRH in mouse AP cells. In contrast, TRPC channel proteins appear to contribute to spontaneous Ca²⁺-oscillations and Ca²⁺ responses induced by TRH and GHRH. We conclude that expression of Orai1 and TRPC channels proteins may play differential and significant roles in AP physiology and endocrine control.     

Mechanisms of Cigarette Smoke Effects on Human Airway Smooth Muscle

Cigarette smoke contributes to or exacerbates airway diseases such as asthma and COPD, where airway hyperresponsiveness and airway smooth muscle (ASM) proliferation are key features. While factors such as inflammation contribute to asthma in part by enhancing agonist-induced intracellular Ca²⁺ ([Ca²⁺]i) responses of ASM, the mechanisms by which cigarette smoke affect ASM are still under investigation. In the present study, we tested the hypothesis that cigarette smoke enhances the expression and function of Ca²⁺ regulatory proteins leading to increased store operated Ca²⁺ entry (SOCE) and cell proliferation. Using isolated human ASM (hASM) cells, incubated in the presence and absence cigarette smoke extract (CSE) we determined ([Ca²⁺]i) responses and expression of relevant proteins as well as ASM proliferation, reactive oxidant species (ROS) and cytokine generation. CSE enhanced [Ca²⁺]i responses to agonist and SOCE: effects mediated by increased expression of TRPC3, CD38, STIM1, and/or Orai1, evident by attenuation of CSE effects when siRNAs against these proteins were used, particularly Orai1. CSE also increased hASM ROS generation and cytokine secretion. In addition, we found in the airways of patients with long-term smoking history, TRPC3 and CD38 expression were significantly increased compared to life-long never-smokers, supporting the role of these proteins in smoking effects. Finally, CSE enhanced hASM proliferation, an effect confirmed by upregulation of PCNA and Cyclin E. These results support a critical role for Ca²⁺ regulatory proteins and enhanced SOCE to alter airway structure and function in smoking-related airway disease.     

Expression of TRPC homologs in endothelial cells and smooth muscle layers of human arteries

TRPC channels are a group of Ca²⁺-permeable nonselective cation channels that mediate store-operated and/or agonist-stimulated Ca²⁺ influx in a variety of cell types. In this study, we extensively examined the expression patterns of TRPC homologs in human vascular tissues. RT-PCR amplified cDNA fragments of TRPC1 (505 bp), TRPC3 (372 bp), TRPC4 (499 bp), TRPC5 (325 bp), TRPC6 (509 bp), and TRPC7 (187 bp) from RNA isolated from cultured human coronary artery endothelial cells. In situ hybridization yielded strong labeling of TRPC1,3–6 in the endothelial and smooth muscle cells of human coronary and cerebral arteries. TRPC7 labeling was exclusively found in endothelial cells but not in smooth muscle cells. Results from immunohistochemical staining were consistent with those from in situ hybridization. Similar expression patterns of TRPC homologs were also observed in arterioles and vaso vasora. In conclusion, our study indicates that TRPC homologs are widely expressed in human vessels of all calibers, including medium-sized coronary arteries and cerebral arteries, smaller-sized resistance arteries, and vaso vasora. These results suggest a ubiquitous role of TRPC homologs in regulating blood supply to different regions and in controlling arterial blood pressure.     

Store-operated Ca2+ entry modulates sarcoplasmic reticulum Ca2+ loading in neonatal rabbit cardiac ventricular myocytes

Store-operated Ca²⁺ entry (SOCE), which is Ca²⁺ entry triggered by the depletion of intracellular Ca²⁺ stores, has been observed in many cell types, but only recently has it been suggested to occur in cardiomyocytes. In the present study, we have demonstrated SOCE-dependent sarcoplasmic reticulum (SR) Ca²⁺ loading (load_SR) that was not altered by inhibition of L-type Ca²⁺ channels, reverse mode Na⁺/Ca²⁺ exchange (NCX), or nonselective cation channels. In contrast, lowering the extracellular [Ca²⁺] to 0 mM or adding either 0.5 mM Zn²⁺ or the putative store-operated channel (SOC) inhibitor SKF-96365 (100 µM) inhibited load_SR at rest. Interestingly, inhibition of forward mode NCX with 30 µM KB-R7943 stimulated SOCE significantly and resulted in enhanced load_SR. In addition, manipulation of the extracellular and intracellular Na⁺ concentrations further demonstrated the modulatory role of NCX in SOCE-mediated SR Ca²⁺ loading. Although there is little knowledge of SOCE in cardiomyocytes, the present results suggest that this mechanism, together with NCX, may play an important role in SR Ca²⁺ homeostasis. The data reported herein also imply the presence of microdomains unique to the neonatal cardiomyocyte. These findings may be of particular importance during open heart surgery in neonates, in which uncontrolled SOCE could lead to SR Ca²⁺ overload and arrhythmogenesis. cardiac ontogeny; cardiac excitation-contraction coupling; calcium homeostasis     

Fine-tuning of store-operated calcium entry by fast and slow Ca2+-dependent inactivation: Involvement of SARAF

Store-operated Ca²⁺ entry (SOCE) is a functionally relevant mechanism for Ca²⁺ influx present in electrically excitable and non-excitable cells. Regulation of Ca²⁺ entry through store-operated channels is essential to maintain an appropriate intracellular Ca²⁺ homeostasis and prevent cell damage. Calcium-release activated channels exhibit Ca²⁺-dependent inactivation mediated by two temporally separated mechanisms: fast Ca²⁺-dependent inactivation takes effect in the order of milliseconds and involves the interaction of Ca²⁺ with residues in the channel pore while slow Ca²⁺-dependent inactivation (SCDI) develops over tens of seconds, requires a global rise in [Ca²⁺]_cyt and is a mechanism regulated by mitochondria. Recent studies have provided evidence that the protein SARAF (SOCE-associated regulatory factor) is involved in the mechanism underlying SCDI of Orai1. SARAF is an endoplasmic reticulum (ER) membrane protein that associates with STIM1 and translocate to plasma membrane-ER junctions in a STIM1-dependent manner upon store depletion to modulate SOCE. SCDI mediated by SARAF depends on the location of the STIM1-Orai1 complex within a PI(4,5)P₂-rich microdomain. SARAF also interacts with Orai1 and TRPC1 in cells endogenously expressing STIM1 and cells with a low STIM1 expression and modulates channel function. This review focuses on the modulation by SARAF of SOCE and other forms of Ca²⁺ influx mediated by Orai1 and TRPC1 in order to provide spatio-temporally regulated Ca²⁺ signals.     

Tau‐induced upregulation of C/EBPβ‐TRPC1‐SOCE signaling aggravates tauopathies: A vicious cycle in Alzheimer neurodegeneration

Intracellular accumulating of the hyperphosphorylated tau plays a pivotal role in neurodegeneration of Alzheimer disease (AD), but the mechanisms underlying the gradually aggravated tau hyperphosphorylation remain elusive. Here, we show that increasing intracellular tau could upregulate mRNA and protein levels of TRPC1 (transient receptor potential channel 1) with an activated store‐operated calcium entry (SOCE), an increased intraneuronal steady‐state [Ca²⁺]_i, an enhanced endoplasmic reticulum (ER) stress, an imbalanced protein kinases and phosphatase, and an aggravated tauopathy. Furthermore, overexpressing TRPC1 induced ER stress, kinases‐phosphatase imbalance, tau hyperphosphorylation and cognitive deficits in cultured neurons and mice, while pharmacological inhibiting or knockout TRPC1 attenuated the hTau‐induced deregulations in SOCE, ER homeostasis, kinases‐phosphatase balance, and tau phosphorylation level with improved synaptic and cognitive functions. Finally, an increased CCAAT‐enhancer‐binding protein (C/EBPβ) activity was observed in hTau‐overexpressing cells and the hippocampus of the AD patients, while downregulating C/EBPβ by siRNA abolished the hTau‐induced TRPC1 upregulation. These data reveal that increasing intracellular tau can upregulate C/EBPβ‐TRPC1‐SOCE signaling and thus disrupt phosphorylating system, which together aggravates tau pathologies leading to a chronic neurodegeneration.