Receptor-operated Ca Entry Research Articles

The Rise in Tubular pH during Hypercalciuria Exacerbates Calcium Stone Formation.

In calcium nephrolithiasis (CaNL), most calcium kidney stones are identified as calcium oxalate (CaOx) with variable amounts of calcium phosphate (CaP), where CaP is found as the core component. The nucleation of CaP could be the first step of CaP+CaOx (mixed) stone formation. High urinary supersaturation of CaP due to hypercalciuria and an elevated urine pH have been described as the two main factors in the nucleation of CaP crystals. Our previous in vivo findings (in mice) show that transient receptor potential canonical type 3 (TRPC3)-mediated Ca2+ entry triggers a transepithelial Ca2+ flux to regulate proximal tubular (PT) luminal [Ca2+], and TRPC3-knockout (KO; -/-) mice exhibited moderate hypercalciuria and microcrystal formation at the loop of Henle (LOH). Therefore, we utilized TRPC3 KO mice and exposed them to both hypercalciuric [2% calcium gluconate (CaG) treatment] and alkalineuric conditions [0.08% acetazolamide (ACZ) treatment] to generate a CaNL phenotype. Our results revealed a significant CaP and mixed crystal formation in those treated KO mice (KOT) compared to their WT counterparts (WTT). Importantly, prolonged exposure to CaG and ACZ resulted in a further increase in crystal size for both treated groups (WTT and KOT), but the KOT mice crystal sizes were markedly larger. Moreover, kidney tissue sections of the KOT mice displayed a greater CaP and mixed microcrystal formation than the kidney sections of the WTT group, specifically in the outer and inner medullary and calyceal region; thus, a higher degree of calcifications and mixed calcium lithiasis in the kidneys of the KOT group was displayed. In our effort to find the Ca2+ signaling pathophysiology of PT cells, we found that PT cells from both treated groups (WTT and KOT) elicited a larger Ca2+ entry compared to the WT counterparts because of significant inhibition by the store-operated Ca2+ entry (SOCE) inhibitor, Pyr6. In the presence of both SOCE (Pyr6) and ROCE (receptor-operated Ca2+ entry) inhibitors (Pyr10), Ca2+ entry by WTT cells was moderately inhibited, suggesting that the Ca2+ and pH levels exerted sensitivity changes in response to ROCE and SOCE. An assessment of the gene expression profiles in the PT cells of WTT and KOT mice revealed a safeguarding effect of TRPC3 against detrimental processes (calcification, fibrosis, inflammation, and apoptosis) in the presence of higher pH and hypercalciuric conditions in mice. Together, these findings show that compromise in both the ROCE and SOCE mechanisms in the absence of TRPC3 under hypercalciuric plus higher tubular pH conditions results in higher CaP and mixed crystal formation and that TRPC3 is protective against those adverse effects.

Anti-inflammatory role of extracellular l-arginine through calcium sensing receptor in human renal proximal tubular epithelial (HK-2) cells.

Renal tubular epithelial cells are capable of synthesizing interleukins (IL) in response to a variety of proinflammatory cytokines. Moreover, elevated urinary levels of IL have been shown in patients with various forms of nephritic diseases. However, the underlying intracellular signaling mechanism is unclear. Here we show the immunological signaling role of l-Arginine (l-Arg) through Ca2+-sensing receptor (CaSR) in human kidney 2 (HK-2) renal proximal tubular epithelial cells, using Ca2+ imaging and patch clamp techniques and its mechanistic link to the downstream cellular function. Both pharmacological and siRNA inhibitors support the activation CaSR by extracellular l-Arg to induced Ca2+ entry via a Transient receptor potential canonical (TRPC) channel in HK-2 cells mainly through the receptor operated Ca2+ entry (ROCE). Activation of CaSR by l-Arg led to the rise in p-p38/p38 expression suggesting [Ca2+]i as a regulator for p38-signaling pathways. Notably, l-Arg activated CaSR-induced Ca2+ signaling reduced the expressions of key fibrotic, inflammatory, and apoptotic genes, suggesting its nephroprotective role via Ca2+ signaling through CaSR in HK-2 cells. Since we found that the IL-6 expressions were inversely proportional to the increasing concentrations of l-Arg in HK-2 cells, we measured the release of IL-6, which steadily decreased as the concentrations of l-Arg were elevated. Taken together, extracellular l-Arg is a negative regulator for IL-6-induced inflammatory process, through the activation of CaSR and TRPC channel by ROCE pathway and can have a potential to alleviate inflammatory renal diseases.

Reduction in SOCE and Associated Aggregation in Platelets from Mice with Platelet-Specific Deletion of Orai1.

Calcium signalling in platelets through store operated Ca2+ entry (SOCE) or receptor-operated Ca2+ entry (ROCE) mechanisms is crucial for platelet activation and function. Orai1 proteins have been implicated in platelet’s SOCE. In this study we evaluated the contribution of Orai1 proteins to these processes using washed platelets from adult mice from both genders with platelet-specific deletion of the Orai1 gene (Orai1flox/flox; Pf4-Cre termed as Orai1Plt-KO) since mice with ubiquitous Orai1 deficiency show early lethality. Platelet aggregation as well as Ca2+ entry and release were measured in vitro following stimulation with collagen, collagen related peptide (CRP), thromboxane A2 analogue U46619, thrombin, ADP and the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor thapsigargin, respectively. SOCE and aggregation induced by Thapsigargin up to a concentration of 0.3 µM was abrogated in Orai1-deficient platelets. Receptor-operated Ca2+-entry and/or platelet aggregation induced by CRP, U46619 or thrombin were partially affected by Orai1 deletion depending on the gender. In contrast, ADP-, collagen- and CRP-induced aggregation was comparable in Orai1Plt-KO platelets and control cells over the entire concentration range. Our results reinforce the indispensability of Orai1 proteins for SOCE in murine platelets, contribute to understand its role in agonist-dependent signalling and emphasize the importance to analyse platelets from both genders.

High magnesium mitigates the vasoconstriction mediated by different types of calcium influx from monocrotaline-induced pulmonary hypertensive rats.

What is the central question of this study? What is the involvement of Mg2+ in mitigating the vasoconstriction in pulmonary arteries and smaller pulmonary arteries in the monocrotaline-induced pulmonary arterial hypertension (MCT-PAH) rat model? What are the main finding and its importance? Both store-operated Ca2+ entry- and receptor-operated Ca2+ entry-mediated vasoconstriction were enhanced in the MCT-PAH model. High magnesium inhibited vasoconstriction by directly antagonizing Ca2+ and increasing NO release, and this was more notable in smaller pulmonary arteries. Increased extracellular magnesium concentration has been shown to attenuate the endothelin-1-induced contractile response via the release of nitric oxide (NO) from the endothelium in proximal pulmonary arteries (PAs) of chronic hypoxic mice. Here, we further examined the involvement of Mg2+ in the inhibition of vasoconstriction in PAs and distal smaller pulmonary arteries (sPAs) in a monocrotaline-induced pulmonary arterial hypertension (MCT-PAH) rat model. The data showed that in control rats vasoconstriction in sPAs is more intense than that in PAs. In MCT-PAH rats, store-operated Ca2+ entry (SOCE)- and receptor-operated Ca2+ entry (ROCE)-mediated contraction were significantly strengthened. However, there was no upregulation of the vasoconstriction mediated by voltage-dependent calcium entry (VDCE). Furthermore, high magnesium greatly inhibited VDCE-mediated contraction in PAs rather than sPAs, which was the opposite of the ROCE-mediated contraction. Moreover, monocrotaline pretreatment partly eliminated the endothelium-dependent vasodilatation in PAs, which in sPAs, however, was still promoted by magnesium due to the increased NO release in pulmonary microvascular endothelial cells (PMVECs). In conclusion, the findings suggest that both SOCE- and ROCE-mediated vasoconstriction in the MCT-PAH model are enhanced, especially in sPAs. The inhibitory effect of high magnesium on vasoconstriction can be achieved partly by its direct role as a Ca2+ antagonist and partly by increasing NO release in PMVECs.

TRPC3 promotes tumorigenesis of gastric cancer via the CNB2/GSK3β/NFATc2 signaling pathway.

The transient receptor potential canonical (TRPC) channels have been implicated in various types of malignancies including gastric cancer (GC). However, the detailed mechanisms of TRPC channels underlying cell proliferation and apoptosis of GC cells remain largely unknown. Here, we report that TRPC3 was highly expressed in clinical GC specimens and correlated with GC malignant progression and poor prognosis. Forced expression of TRPC3 in GC cells enhanced both receptor-operated Ca2+ entry (ROCE) and store-operated Ca2+ entry (SOCE) and promoted the nuclear factor of activated T cell 2 (NFATc2) nuclear translocation by AKT/GSK-3β and CNB2 signaling. Pharmacological inhibition of TRPC3 or CRISPR/Cas9-mediated TRPC3 knockout effectively inhibited the growth of GC cells both in vitro and in vivo. These effects were reversible by the rescue of TRPC3 expression. Furthermore, we confirmed the role of TRPC3 and the ROCE-AKT/GSK3β-CNB2/NFATc2 signaling cascade in regulating cell cycle checkpoint, apoptosis cascade, and intracellular ROS production in GC. Overall, our findings suggest an oncogenic role of TRPC3 in GC and may highlight a potential target of TRPC3 for therapeutic intervention of GC and its malignant progression.

Store-operated Ca2+ entry in primary murine lung fibroblasts is independent of classical transient receptor potential (TRPC) channels and contributes to cell migration

Stromal interaction molecules (STIM1, 2) are acting as sensors for Ca2+ in intracellular stores and activate Orai channels at the plasma membrane for store-operated Ca2+ entry (SOCE), while classical transient receptor potential (TRPC) channel mediate receptor-operated Ca2+ entry (ROCE). Several reports, however, indicate a role for TRPC in SOCE in certain cell types. Here, we analyzed Ca2+ influx and cell function in TRPC1/6-deficient (TRPC1/6−/−) and STIM1/2- deficient (STIM1/2ΔpmLF) primary murine lung fibroblasts (pmLF). As expected, SOCE was decreased in STIM1/2- deficient pmLF and ROCE was decreased in TRPC1/6−/− pmLF compared to control cells. By contrast, SOCE was not significantly different in TRPC1/6−/− pmLF and ROCE was similar in STIM1/2-deficient pmLF compared to Wt cells. Most interestingly, cell proliferation, migration and nuclear localization of nuclear factor of activated T-cells (NFATc1 and c3) were decreased after ablation of STIM1/2 proteins in pmLF. In conclusion, TRPC1/6 channels are not involved in SOCE and STIM1/2 deficiency resulted in decreased cell proliferation and migration in pmLF.

Upregulation of G protein‐couple receptor 68 (GPCR68) in pulmonary artery smooth muscle cells from animals and humans with pulmonary hypertension

IntroductionPulmonary arterial hypertension (PAH) is a progressive and fatal disease with no cure. It is characterized by elevated pulmonary vascular resistance (PVR) and pulmonary arterial pressure (PAP) in part due to pulmonary vascular remodeling and sustained pulmonary vasoconstriction. The increased pulmonary arterial smooth muscle cell (PASMC) proliferation contributes to excessive pulmonary vascular remodeling while a rise in cytosolic free Ca2+concentration ([Ca2+]cyt) in PASMC is a major trigger for pulmonary vasoconstriction and a key stimulus for PASMC proliferation. G‐protein coupled receptors (GPCR), which are attractive pharmacological targets, are important regulators of pulmonary vascular tone and PASMC phenotype. Stimulation of GPCR leads to Ca2+ influx and increased [Ca2+]cytdue to store‐operated Ca2+ entry (SOCE) via inositol 1,4,5‐trisphosphate (IP3) production and receptor‐operated Ca2+entry (ROCE) via diacylglycerol (DAG) production. GPCR68 (or OGR1) is a proton‐sensing GPCR that responds to extracellular acidity and regulates a variety of cellular functions. As a Gq/11‐coupled receptor, GPR68 activation is known to lead to Ca2+ release from the intracellular stores in IP3‐dependent manner but the role of GPCR68 in the development of PAH is still unknown.MethodsLung, heart, brain, liver, and kidney tissues were isolated from control rats. Pulmonary hypertension model was induced by a single dose of monocrotaline (MCT) in rats. PASMC isolated from control or MCT rats and healthy subjects or patients with idiopathic pulmonary arterial hypertension (IPAH) were cultured in 5% fetal bovine serum (FBS) smooth muscle growth media and incubated in a humidified 5% CO2 atmosphere at 37°C. Total RNA was isolated from tissue or cells using TRIZOL method. mRNA expression of GPCR68 was quantified by RT‐PCR and qRT‐PCR. The data was analyzed using ImageJ and statistical analysis was performed using paired or unpaired Student’s t‐test.ResultsRT‐PCR analysis revealed that GPCR68 is expressed in brain, lung, heart, liver, and kidney in normal rat tissues (n=5). GPCR68 mRNA level was significantly higher in lung tissue and PASMC isolated from MCT‐treated rats compared with controls. Moreover, our data shows that GPCR68 is expressed in both human PASMC and pulmonary arterial endothelial cells. qRT‐PCR showed that GPCR68 was upregulated in PASMC isolated from IPAH patients compared with cells isolated from healthy subjects.ConclusionOur finding suggest that GPR68 is involved in the development and progression of PAH and may play an important role in Ca2+ signaling and enhanced [Ca2+]cyt in PASMC from animals and humans with PAH.Support or Funding InformationThis work was supported, in part, by grants from the National Heart, Lung, and Blood Institute of the National Institutes of Health (HL125208, HL135807, and HL126609).

L-ornithine activates Ca2+ signaling to exert its protective function on human proximal tubular cells

Oxidative stress and reactive oxygen species (ROS) generation can be influenced by G-protein coupled receptor (GPCR)-mediated regulation of intracellular Ca2+ ([Ca2+]i) signaling. ROS production are much higher in proximal tubular (PT) cells; in addition, the lack of antioxidants enhances the vulnerability to oxidative damage. Despite such predispositions, PT cells show resiliency, and therefore must possess some inherent mechanism to protect from oxidative damage. While the mechanism in unknown, we tested the effect of l-ornithine, since it is abundantly present in PT luminal fluid and can activate Ca2+-sensing receptor (CaSR), a GPCR, expressed in the PT luminal membrane. We used human kidney 2 (HK2) cells, a PT cell line, and performed Ca2+ imaging and electrophysiological experiments to show that l-ornithine has a concentration-dependent effect on CaSR activation. We further demonstrate that the operation of CaSR activated Ca2+ signaling in HK-2 cells mediated by the transient receptor potential canonical (TRPC) dependent receptor-operated Ca2+ entry (ROCE) using pharmacological and siRNA inhibitors. Since PT cells are vulnerable to ROS, we simulated such deleterious effects using genetically encoded peroxide-induced ROS production (HyperRed indicator) to show that the l-ornithine-induced ROCE mediated [Ca2+]i signaling protects from ROS production. Furthermore, we performed cell viability, necrosis and apoptosis assays, and mitochondrial oxidative gene expression to establish that presence of l-ornithine rescued the ROS-induced damage in HK-2 cells. Moreover, l-ornithine-activation of CaSR can reverse ROS production and apoptosis via mitogen-activated protein kinase p38 activation. Such nephroprotective role of l-ornithine can be useful as the translational option for reversing kidney diseases involving PT cell damage due to oxidative stress or crystal nephropathies.

Abrogation of store-operated Ca2+ entry protects against crystal-induced ER stress in human proximal tubular cells

Calcium crystal internalization into proximal tubular (PT) cells results in acute kidney injury, nephrocalcinosis, chronic kidney disease (CKD), and kidney-stone formation. Ca2+ supersaturation in PT luminal fluid induces calcium crystal formation, leading to aberrant crystal internalization into PT cells. While such crystal internalization produces reactive oxygen species (ROS), cell membrane damage, and apoptosis; the upstream signaling events involving dysregulation of intracellular Ca2+ homeostasis and ER stress, remain largely unknown. We have recently described a transepithelial Ca2+ transport pathway regulated by receptor-operated Ca2+ entry (ROCE) in PT cells. Therefore, we examined the pathophysiological consequence of internalization of stone-forming calcium crystals such as calcium phosphate (CaP), calcium oxalate (CaOx), and CaP + CaOx (mixed) crystals on the regulation of intracellular Ca2+ signaling by measuring dynamic changes in Ca2+ transients in HK2, human PT cells, using pharmacological and siRNA inhibitors. The subsequent effect on ER stress was measured by changes in ER morphology, ER stress-related gene expression, endogenous ROS production, apoptosis, and necrosis. Interestingly, our data show that crystal internalization induced G-protein-coupled receptor-mediated sustained rise in intracellular Ca2+ concentration ([Ca2+]i) via store-operated Ca2+ entry (SOCE); suggesting that the mode of Ca2+ entry switches from ROCE to SOCE following crystal internalization. We found that SOCE components—stromal interacting molecules 1 and 2 (STIM1, STIM2) and ORAI3 (SOCE) channel were upregulated in these crystal-internalized cells, which induced ER stress, ROS production, and cell death. Finally, silencing those SOCE genes protected crystal-internalized cells from prolonged [Ca2+]i rise and ER stress. Our data provide insight into the molecular mechanism of crystal-induced Ca2+ dysregulation, ER stress, and PT cell death and thus could have a translational role in treating crystal nephropathies including kidney stones. Taken together, modulation of Ca2+ signaling can be used as a tool to reverse the pathological consequence of crystal-induced conditions including cardiovascular calcification.

Increased caveolin-1 expression enhances the receptor-operated Ca2+ entry in the aorta of two-kidney, one-clip hypertensive rats.

What is the central question of this study? The aim was to examine and compare the contributions of caveolin-1 to the contractile responses mediated by L-type voltage-dependent calcium channels, store-operated Ca2+ channels and receptor-operated Ca2+ channels in two different types of arteries from two-kidney, one-clip hypertensive rats. What is the main finding and its importance? We demonstrated that the density of caveolae and caveolin-1 expression were significantly upregulated in the aorta of two-kidney, one-clip hypertensive rats, but not in the third-order branches of mesenteric arteries. We highlight that caveolin-1 plays an important role in aortic constriction by enhancing receptor-operated Ca2+ entry in the hypertensive rat model. Calcium and its multiple regulatory mechanisms are crucial for the development of hypertension. Among these regulatory mechanisms, store-operated Ca2+ entry (SOCE) and receptor-operated Ca2+ entry (ROCE) mediate agonist-induced calcium influx, contributing to vascular contraction. The SOCE and ROCE are regulated by a variety of mechanisms involving caveolin-1 (Cav1), which has been found to be strongly associated with hypertension in gene polymorphism. In the present study, we investigated the role of Cav1 during the enhanced activity of calcium channels in hypertensive arteries. We demonstrated that the expression level of Cav1 was significantly increased in the aorta of two-kidney, one-clip (2K1C) hypertensive rats. The disruption of caveolae by methyl-β-cyclodextrin did not cause a marked difference in agonist-induced vasoconstriction in the third-order branches of the mesenteric arteries but strongly suppressed the aortic contractile response to endothelin-1 in the 2K1C group, which was not found in the control group. The increase in Cav1 by introduction of Cav1 scaffolding domain enhancing peptide promoted the 1-oleoyl-2-acetyl-glycerol-induced ROCE in hypertensive aortic smooth muscle cells but did not enhance the cyclopiazonic acid-induced SOCE. In the resistance arteries, similar changes were not observed, and no statistical changes of Cav1 expression were evident in the third-order branches of the mesenteric arteries. Our results indicate that increased Cav1 expression might promote the altered [Ca2+ ]i -induced aortic vasoreactivity by enhancing ROCE and be involved in the pathogenesis of hypertension.

Myosin Light Chain Kinase‐210 Induces ER‐PM Junctions and STIM1 Puncta Formation to Augment Store‐Operated Ca2+ Entry

Myosin light chain kinase‐Long (MYLK‐L or MYLK‐210), a Ca2+‐calmodulin dependent kinase containing a unique 922‐amino acid N‐terminal domain is a predominant isoform of MYLK expressed in endothelial cells (ECs). N‐terminus of MYLK‐210 has been shown to contain several single nucleotide polymorphisms (SNPs) in an actin binding region which is associated with severity of acute lung injury (ALI). However, significance of SNPs in regulating MYLK‐210 function remains unclear. The classical function of MYLK‐210 is to catalyze the phosphorylation of myosin light chain to induce EC contraction, leading to inter‐endothelial gap formation and increase permeability, a hall mark of ALI. Intriguingly, we showed that MYLK‐210, through its actin‐binding motif, regulates store‐operated Ca2+ entry (SOCE) but not receptor‐operated Ca2+ entry (ROCE). We showed that MYLK‐210 null endothelial cells (ECs) or MYLK‐210 depleted ECs displayed diminished Ca2+ entry in response to SOCE‐activator thapsigargin and thrombin. We generated MYLK‐210 constructs lacking C‐terminus (N‐MYLK) or N‐terminus (C‐MYLK) to investigate the role of specific domains of MYLK‐210 in inducing SOCE activity. We found that N‐MYLK but not C‐MYLK rescued SOCE activity in MYLK‐210 null ECs, indicating thereby that MYLK regulates SOCE in a kinase independent manner. Additionally, SOCE activation failed to increase lung vascular hyper‐permeability in MYLK‐210 null mice. We identified unique actin binding motif, DVRGLL within N terminus of MYLK‐L which enables MYLK‐210 to induce SOCE. Stromal‐interaction molecule 1 (STIM1), upon sensing the depletion of (Ca2+) from the endoplasmic reticulum (ER) store, organizes as puncta which translocate to plasma membrane that trigger SOCE via plasmalemmal Ca2+selective channels such as transient receptor potential channel 1 (TRPC1) and Orai1. Thus, we addressed if MYLK‐L controlled STIM1 function by regulating puncta formation. We showed that STIM1 formed puncta in control ECs but these puncta were markedly reduced in MYLK depleted ECs. Formation of endoplasmic reticulum (ER)‐plasma membrane (PM) junction is required for STIM1 activation of SOCE. Using MAPPER (membrane‐attached peripheral ER) that selectively labels ER–PM junctions, we found that MYLK induced ER‐PM junction, but these junctions were markedly reduced in the density in MYLK‐knockdown cells. Our study thereby defines for the first time the clinical validity of SNPs within actin binding region of MYLK in inducing ALI through organizing ER‐PM junctions, STIM1 activity, SOCE and lung vascular hyperpermeability.Support or Funding InformationNational Institute of Health (NIH)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Store-Operated Ca2+ Entry (SOCE) and Purinergic Receptor-Mediated Ca2+ Homeostasis in Murine bv2 Microglia Cells: Early Cellular Responses to ATP-Mediated Microglia Activation.

Microglia activation is a neuroinflammatory response to parenchymal damage with release of intracellular metabolites, e.g., purines, and signaling molecules from damaged cells. Extracellular purines can elicit Ca2+-mediated microglia activation involving P2X/P2Y receptors with metabotropic (P2Y) and ionotropic (P2X) cell signaling in target cells. Such microglia activation results in increased phagocytic activity, activation of their inflammasome and release of cytokines to sustain neuroinflammatory (so-called M1/M2 polarization). ATP-induced activation of ionotropic P2X4 and P2X7 receptors differentially induces receptor-operated Ca2+ entry (ROCE). Although store-operated Ca2+ entry (SOCE) was identified to modulate ROCE in primary microglia, its existence and role in one of the most common murine microglia cell line, BV2, is unknown. To dissect SOCE from ROCE in BV2 cells, we applied high-resolution multiphoton Ca2+ imaging. After depleting internal Ca2+ stores, SOCE was clearly detectable. High ATP concentrations (1 mM) elicited sustained increases in intracellular [Ca2+]i whereas lower concentrations (≤100 μM) also induced Ca2+ oscillations. These differential responses were assigned to P2X7 and P2X4 activation, respectively. Pharmacologically inhibiting P2Y and P2X responses did not affect SOCE, and in fact, P2Y-responses were barely detectable in BV2 cells. STIM1S content was significantly upregulated by 1 mM ATP. As P2X-mediated Ca2+ oscillations were rare events in single cells, we implemented a high-content screening approach that allows to record Ca2+ signal patterns from a large number of individual cells at lower optical resolution. Using automated classifier analysis, several drugs (minocycline, U73122, U73343, wortmannin, LY294002, AZ10606120) were tested on their profile to act on Ca2+ oscillations (P2X4) and sustained [Ca2+]i increases. We demonstrate specific drug effects on purinergic Ca2+ pathways and provide new pharmacological insights into Ca2+ oscillations in BV2 cells. For example, minocycline inhibits both P2X7- and P2X4-mediated Ca2+-responses, and this may explain its anti-inflammatory action in neuroinflammatory disease. As a technical result, our novel automated bio-screening approach provides a biomedical engineering platform to allow high-content drug library screens to study neuro-inflammation in vitro.

Increase in caveolae and caveolin-1 expression modulates agonist-induced contraction and store- and receptor-operated Ca2 + entry in pulmonary arteries of pulmonary hypertensive rats

Caveolin-1 (Cav-1) is a major component protein associated with caveolae in the plasma membrane and has been identified as a regulator of store-operated Ca2+ entry (SOCE) and receptor-operated Ca2+ entry (ROCE). However, the contributions of caveolae/Cav-1 of pulmonary arterial smooth muscle cells (PASMCs) to the altered Ca2+ signaling pathways in pulmonary arteries (PAs) during pulmonary hypertension (PH) have not been fully characterized. The present study quantified caveolae number and Cav-1 expression, and determined the effects of caveolae disruption on ET-1, cyclopiazonic acid (CPA) and 1-Oleoyl-2-acetyl-glycerol (OAG)-induced contraction in PAs and Ca2+ influx in PASMCs of chronic hypoxia (CH)- and monocrotaline (MCT)-induced PH rats. We found that the number of caveolae, and the Cav-1 mRNA and protein levels were increased significantly in PASMCs in both PH models. Disruption of caveolae by cholesterol depletion with methyl-β-cyclodextrin (MβCD) significantly inhibited the contractile response to ET-1, CPA and OAG in PAs of control rats. ET-1, SOCE and ROCE-mediated contractile responses were enhanced, and their susceptibility to MβCD suppression was potentiated in the two PH models. MβCD-induced inhibition was reversed by cholesterol repletion. Introduction of Cav-1 scaffolding domain peptide to mimic Cav-1 upregulation caused significant increase in CPA- and OAG-induced Ca2+ entry in PASMCs of control, CH and MCT-treated groups. Our results suggest that the increase in caveolae and Cav-1 expression in PH contributes to the enhanced agonist-induced contraction of PA via modulation of SOCE and ROCE; and targeting caveolae/Cav-1 in PASMCs may provide a novel therapeutic strategy for the treatment of PH.

A novel chimeric aequorin fused with caveolin-1 reveals a sphingosine kinase 1-regulated Ca2 + microdomain in the caveolar compartment

Caveolae are plasma membrane invaginations enriched in sterols and sphingolipids. Sphingosine kinase 1 (SK1) is an oncogenic protein that converts sphingosine to sphingosine 1-phosphate (S1P), which is a messenger molecule involved in calcium signaling. Caveolae contain calcium responsive proteins, but the effects of SK1 or S1P on caveolar calcium signaling have not been investigated. We generated a Caveolin-1–Aequorin fusion protein (Cav1–Aeq) that can be employed for monitoring the local calcium concentration at the caveolae ([Ca2+]cav). In HeLa cells, Cav1–Aeq reported different [Ca2+] as compared to the plasma membrane [Ca2+] in general (reported by SNAP25–Aeq) or as compared to the cytosolic [Ca2+] (reported by cyt-Aeq). The Ca2+ signals detected by Cav1–Aeq were significantly attenuated when the caveolar structures were disrupted by methyl-β-cyclodextrin, suggesting that the caveolae are specific targets for Ca2+ signaling. HeLa cells overexpressing SK1 showed increased [Ca2+]cav during histamine-induced Ca2+ mobilization in the absence of extracellular Ca2+ as well as during receptor-operated Ca2+ entry (ROCE). The SK1-induced increase in [Ca2+]cav during ROCE was reverted by S1P receptor antagonists. In accordance, pharmacologic inhibition of SK1 reduced the [Ca2+]cav during ROCE. S1P treatment stimulated the [Ca2+]cav upon ROCE. The Ca2+ responses at the plasma membrane in general were not affected by SK1 expression. In summary, our results show that SK1/S1P-signaling regulates Ca2+ signals at the caveolae. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

Chronic Hypoxia Increases Intracellular Ca(2+) Concentration via Enhanced Ca(2+) Entry Through Receptor-Operated Ca(2+) Channels in Pulmonary Venous Smooth Muscle Cells.

Hypoxic pulmonary hypertension (HPH) is characterized by pulmonary vascular remodeling. Intracellular Ca(2+)concentration ([Ca(2+)]i) is an essential signal for myocyte proliferation. Whether chronic hypoxia (CH) affects the basal [Ca(2+)]I and Ca(2+)entry through store- and/or receptor-operated calcium channels (SOCC, ROCC), and whether canonical transient receptor potential (TRPC) proteins are involved in CH-induced Ca(2+)influx and proliferation in pulmonary venous smooth muscle cells (PVSMCs) is examined. Rats were exposed to CH. PVSMCs were isolated from distal pulmonary veins. In freshly isolated PVSMCs, CH increased the basal [Ca(2+)]i; removal of Ca(2+)or application of SKF-96365 reversed the elevated [Ca(2+)]i, whereas nifedipine had no effect. Receptor-operated Ca(2+)entry (ROCE) was expressed in PVSMCs. In freshly isolated PVSMCs from CH rats, ROCE was enhanced, whereas store-operated Ca(2+)entry had no alteration. Furthermore, real-time polymerase chain reaction and western blotting showed that mRNA and protein expression level of TRPC6, but neither TRPC1 nor TRPC3, in pulmonary venous smooth muscle (PV) from CH rats and PVSMCs exposed to CH was greater than in normal PV and PVSMCs. The knockdown of TRPC6 in hypoxic PVSMCs with siRNA inhibited the enhanced ROCE and attenuated CH-induced PVSMCs proliferation. The enhanced Ca(2+)entry through ROCC, due to upregulated TRPC6, is a novel pathogenic mechanism contributing to the increased basal [Ca(2+)]iin PVSMCs and excessive PVSMC proliferation during the development of HPH.

Adenylate Cyclase/cAMP/Protein Kinase A Signaling Pathway Inhibits Endothelin Type A Receptor-Operated Ca2+ Entry Mediated via Transient Receptor Potential Canonical 6 Channels

Receptor-operated Ca²⁺ entry (ROCE) via transient receptor potential canonical channel 6 (TRPC6) is important machinery for an increase in intracellular Ca²⁺ concentration triggered by the activation of G(q) protein-coupled receptors. TRPC6 is phosphorylated by various protein kinases including protein kinase A (PKA). However, the regulation of TRPC6 activity by PKA is still controversial. The purpose of this study was to elucidate the role of adenylate cyclase/cAMP/PKA signaling pathway in the regulation of G(q) protein-coupled endothelin type A receptor (ET(A)R)-mediated ROCE via TRPC6. For this purpose, human embryonic kidney 293 (HEK293) cells stably coexpressing human ET(A)R and TRPC6 (wild type) or its mutants possessing a single point mutation of putative phosphorylation sites for PKA were used to analyze ROCE and amino acids responsible for PKA-mediated phosphorylation of TRPC6. Ca²⁺ measurements with thapsigargin-induced Ca²⁺-depletion/Ca²⁺-restoration protocol to estimate ROCE showed that the stimulation of ET(A)R induced marked ROCE in HEK293 cells expressing TRPC6 compared with control cells. The ROCE was inhibited by forskolin and papaverine to activate the cAMP/PKA pathway, whereas it was potentiated by Rp-8-bromoadenosine-cAMP sodium salt, a PKA inhibitor. The inhibitory effects of forskolin and papaverine were partially cancelled by replacing Ser28 (TRPC6(S28A)) but not Thr69 (TRPC6(T69A)) of TRPC6 with alanine. In vitro kinase assay with Phos-tag biotin to determine the phosphorylation level of TRPC6 revealed that wild-type and mutant (TRPC6(S28A) and TRPC6(T69A)) TRPC6 proteins were phosphorylated by PKA, but the phosphorylation level of these mutants was lower (approximately 50%) than that of wild type. These results suggest that TRPC6 is negatively regulated by the PKA-mediated phosphorylation of Ser28 but not Thr69.

Function and Regulation of Endothelin Type A Receptor–Operated Transient Receptor Potential Canonical Channels

The purpose of this study is to identify transient receptor potential canonical (TRPC) channels responsible for receptor-operated Ca(2+) entry (ROCE) triggered by activation of endothelin type A receptor (ET(A)R) and to clarify the importance of calmodulin (CaM) / inositol 1,4,5-trisphosphate (IP(3)) receptor binding (CIRB) domain at the C terminus of TRPC channels in ET(A)R-activated channel regulation. In HEK293 cells coexpressing ET(A)R and one of seven TRPC isoforms, ET(A)R stimulation induced ROCE through TRPC3, TRPC5, TRPC6, and TRPC7. The TRPC3- and TRPC6-mediated ROCE was inhibited by selective inhibitors of G(q) protein, phospholipase C (PLC), and CaM. The CIRB domain deletion mutants of TRPC3 and TRPC6 failed to induce ET(A)R-mediated ROCE. Either deletion of the CIRB domain or pharmacological inhibition of CaM did not inhibit the targeting of these channels to the plasma membrane. These results suggest that 1) TRPC3, TRPC5, TRPC6, and TRPC7 can function as ET(A)R-operated Ca(2+) channels; 2) G(q) protein, PLC, and CaM are involved in TRPC3- and TRPC6-mediated ROCE; 3) ET(A)R-mediated activation of TRPC3 and TRPC6 requires the CIRB domain; and 4) abolition of ET(A)R-induced ROCE by CIRB domain deletion and CaM inhibition is due to loss of CaM binding to the channels but not loss of cell surface TRPC3 and TRPC6.

Protein Kinase C-dependent Phosphorylation of Transient Receptor Potential Canonical 6 (TRPC6) on Serine 448 Causes Channel Inhibition

TRPC6 is a cation channel in the plasma membrane that plays a role in Ca(2+) entry following the stimulation of a G(q)-protein coupled or tyrosine kinase receptor. A dysregulation of TRPC6 activity causes abnormal proliferation of smooth muscle cells and glomerulosclerosis. In the present study, we investigated the regulation of TRPC6 activity by protein kinase C (PKC). We showed that inhibiting PKC with GF1 or activating it with phorbol 12-myristate 13-acetate potentiated and inhibited agonist-induced Ca(2+) entry, respectively, into cells expressing TRPC6. Similar results were obtained when TRPC6 was directly activated with 1-oleyl-2-acetyl-sn-glycerol. Activation of the cells with carbachol increased the phosphorylation of TRPC6, an effect that was prevented by the inhibition of PKC. The target residue of PKC was identified by an alanine screen of all canonical PKC sites on TRPC6. Unexpectedly, all the mutants, including TRPC6(S768A) (a residue previously proposed to be a target for PKC), displayed PKC-dependent inhibition of channel activity. Phosphorylation prediction software suggested that Ser(448), in a non-canonical PKC consensus sequence, was a potential target for PKCδ. Ba(2+) and Ca(2+) entry experiments revealed that GF1 did not potentiate TRPC6(S448A) activity. Moreover, activation of PKC did not enhance the phosphorylation state of TRPC6(S448A). Using A7r5 vascular smooth muscle cells, which endogenously express TRPC6, we observed that a novel PKC isoform is involved in the inhibition of the vasopressin-induced Ca(2+) entry. Furthermore, knocking down PKCδ in A7r5 cells potentiated vasopressin-induced Ca(2+) entry. In summary, we provide evidence that PKCδ exerts a negative feedback effect on TRPC6 through the phosphorylation of Ser(448).

Caveolin Dysfunction Impairs Pulmonary Endothelial Calcium Entry Following Chronic Hypoxia

Agonist‐induced Ca2+ entry into the pulmonary endothelium is dependent upon activation of both store‐operated Ca2+ entry (SOCE) and receptor operated Ca2+ entry (ROCE) pathways. In addition, there is recent evidence that trafficking of the relevant ion channels to the cell membrane by caveolin‐1 (cav‐1) is necessary for both forms of Ca2+ entry. We have previously reported that pulmonary endothelial cell (EC) SOCE and ROCE are reduced in the chronic hypoxia (CH) model of pulmonary hypertension. Therefore, we hypothesized that diminished endothelial Ca2+ entry following CH is due to impaired cav‐1 mediated trafficking of agonist‐induced Ca2+ entry pathways. To test this hypothesis we measured Ca2+ influx by fura‐2 fluorescence following the application of ATP (20 μM) in ECs pretreated with the caveolar disrupting agent methyl‐beta cyclodextrin (mβcd; 10 mM). We found that cholesterol depletion with mβcd attenuated agonist‐induced Ca2+ entry in control ECs to the level of ECs from CH rats. Furthermore, cholesterol repletion with a mβcd‐cholesterol (1:5) mixture or the introduction of the cav‐1 scaffolding peptide (AP‐CAV, 10 μM) rescued ATP‐induced Ca2+ entry in ECs from CH hypertensive arteries. These data suggest that CH‐induced pulmonary hypertension leads to impaired cav‐1 trafficking of ion channels necessary for receptor‐activated Ca2+ entry.

Ca2+ handling is altered when arterial myocytes progress from a contractile to a proliferative phenotype in culture

Phenotypic modulation of vascular myocytes is important for vascular development and adaptation. A characteristic feature of this process is alteration in intracellular Ca(2+) handling, which is not completely understood. We studied mechanisms involved in functional changes of inositol 1,4,5-trisphosphate (IP(3))- and ryanodine (Ry)-sensitive Ca(2+) stores, store-operated Ca(2+) entry (SOCE), and receptor-operated Ca(2+) entry (ROCE) associated with arterial myocyte modulation from a contractile to a proliferative phenotype in culture. Proliferating, cultured myocytes from rat mesenteric artery have elevated resting cytosolic Ca(2+) levels and increased IP(3)-sensitive Ca(2+) store content. ATP- and cyclopiazonic acid [CPA; a sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor]-induced Ca(2+) transients in Ca(2+)-free medium are significantly larger in proliferating arterial smooth muscle cells (ASMCs) than in freshly dissociated myocytes, whereas caffeine (Caf)-induced Ca(2+) release is much smaller. Moreover, the Caf/Ry-sensitive store gradually loses sensitivity to Caf activation during cell culture. These changes can be explained by increased expression of all three IP(3) receptors and a switch from Ry receptor type II to type III expression during proliferation. SOCE, activated by depletion of the IP(3)/CPA-sensitive store, is greatly increased in proliferating ASMCs. Augmented SOCE and ROCE (activated by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol) in proliferating myocytes can be attributed to upregulated expression of, respectively, transient receptor potential proteins TRPC1/4/5 and TRPC3/6. Moreover, stromal interacting molecule 1 (STIM1) and Orai proteins are upregulated in proliferating cells. Increased expression of IP(3) receptors, SERCA2b, TRPCs, Orai(s), and STIM1 in proliferating ASMCs suggests that these proteins play a critical role in an altered Ca(2+) handling that occurs during vascular growth and remodeling.