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Aims: Vascular endothelial growth factor (VEGF) increases angiogenesis by stimulating endothelial cell (EC) migration. VEGF-induced nitric oxide (•NO) release from •NO synthase plays a critical role, but the proteins and signaling pathways that may be redox-regulated are poorly understood. The aim of this work was to define the role of •NO-mediated redox regulation of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) in VEGF-induced signaling and EC migration. Results: VEGF-induced EC migration was prevented by the •NO synthase inhibitor, N (G)-nitro-L-arginine methyl ester (LNAME). Either VEGF or •NO stimulated endoplasmic reticulum (ER) 45Ca2+ uptake, a measure of SERCA activity, and knockdown of SERCA2 prevented VEGF-induced EC migration and 45Ca2+ uptake. S-glutathione adducts on SERCA2b, identified immunochemically, were increased by VEGF, and were prevented by LNAME or overexpression of glutaredoxin-1 (Glrx-1). Furthermore, VEGF failed to stimulate migration of ECs overexpressing Glrx-1. VEGF or •NO increased SERCA S-glutathiolation and stimulated migration of ECs in which wild-type (WT) SERCA2b was overexpressed with an adenovirus, but did neither in those overexpressing a C674S SERCA2b mutant, in which the reactive cysteine-674 was mutated to a serine. Increased EC Ca2+ influx caused by VEGF or •NO was abrogated by overexpression of Glrx-1 or the C674S SERCA2b mutant. ER store-emptying through the ryanodine receptor (RyR) and Ca2+ entry through Orai1 were also required for VEGF- and •NO-induced EC Ca2+ influx. Innovation and Conclusions: These results demonstrate that •NO-mediated activation of SERCA2b via S-glutathiolation of cysteine-674 is required for VEGF-induced EC Ca2+ influx and migration, and establish redox regulation of SERCA2b as a key component in angiogenic signaling. Antioxid. Redox Signal. 00, 000–000.
Vascular endothelial growth factor (VEGF) is a potent stimulus of endothelial cell (EC) migration and angiogenesis. Although capable of directly promoting several kinase cascades, such as mitogen activated protein kinase (MAPK) and Src, through VEGF receptor phosphorylation, VEGF notably stimulates activity of the endothelial nitric oxide synthase (eNOS) to promote production of nitric oxide (•NO). The importance of •NO for angiogenesis is well established by studies showing decreased angiogenesis in response to hind-limb ischemia and decreased Matrigel plug angiogenesis (19, 15) in mice lacking eNOS (26, 29, 31). In addition, decreased •NO bioavailability due to disease may contribute to increased vascular permeability (22); decreased •NO-induced vessel relaxation (4, 21); and decreased angiogenesis (18). However, the mechanisms of •NO action in ECs and angiogenesis are not well understood.
Endothelial cell (EC) migration is required for both physiological and pathological angiogenesis. Nitric oxide (•NO)‐dependent signaling is required for vascular endothelial growth factor (VEGF)‐induced EC migration, but the protein targets that may be redox regulated are poorly understood. Here, we present novel evidence that S‐glutathiolation of SERCA2b cysteine‐674 is a novel specific redox‐regulated target of VEGF‐stimulated •NO production in ECs, which is required for VEGF‐induced Ca2+ influx and cell migration. These data represent a novel redox‐regulated mechanism of EC Ca2+ handling essential to angiogenic function, which constitutes a potential therapeutic target in diseases of altered angiogenesis.
In addition to classical signaling via cyclic guanine monophosphate-dependent mechanisms, •NO can elicit signaling through protein redox modifications. Reactive oxygen and nitrogen species (ROS/RNS), such as hydrogen peroxide, and the •NO metabolite, peroxynitrite, are potent mediators of cellular signaling and vascular function. Physiologically, low levels of ROS/RNS regulate cell signaling via reversible post-translational oxidative protein modifications, including S-nitrosation and S-glutathiolation, and these are important for transient changes in protein function (10). The sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) has been identified as an important target of both physiological and pathological redox modification with resulting changes in protein activity (4). S-glutathiolation of purified SERCA at cysteine-674 was demonstrated to cause a 50% increase in maximal Ca2+ uptake activity, and this protein modification has been associated with inhibition of smooth muscle cell (SMC) migration by •NO (28) and increased cardiac myocyte contractility caused by nitroxyl anion (14). ECs represent a distinct redox environment with regard to intracellular production of •NO by eNOS, and the redox regulation of SERCA within the endothelium has not been characterized.
SERCA is located on the endoplasmic reticulum (ER) and is responsible for uptake of Ca2+ into the ER required to maintain ER Ca2+ stores. In addition, changes in SERCA activity play an important role in intracellular signaling through regulation of extracellular Ca2+ influx (17). SERCA and its isoforms are the products of three genes: SERCA2b and SERCA3 are expressed in ECs (5, 13, 20), whereas SERCA2b is the principal isoform in smooth muscle (13). Unlike in SMCs, EC migration is enhanced by •NO (23), leading us to study here novel redox regulation mechanisms by which EC SERCA might participate in •NO-dependent, VEGF-induced cell migration.
Our findings show that VEGF induces an •NO-dependent S-glutathiolation of EC SERCA cysteine-674 and increase in SERCA activity that are required for stimulating EC migration into a scratch wound, as well as capillary tube formation. Additionally, knockdown of SERCA2 or overexpression of the C674S SERCA2b mutant prevents both VEGF- and •NO-induced EC migration. We found that VEGF and •NO stimulate Ca2+ influx from the extracellular space into the cytosol and that this is dependent upon both the plasma membrane Ca2+ influx channel, Orai1, and the ER ryanodine receptor (RyR) Ca2+-release channel, identifying a novel regulation of EC Ca2+ influx dependent on ER Ca2+-uptake and release mechanisms. These studies indicate that •NO-mediated S-glutathiolation of EC SERCA C674 induces a novel redox-regulated Ca2+ influx into ECs that is essential for angiogenic signaling.
VEGF (50ng/ml), the •NO donor, diethylenetriamine NONOate (DETA NONOate; 30μM), or vehicle was added to human aortic ECs (HAECs) for 1h before scratch wounding of the monolayer and were reapplied at the time of the scratch. Over 6h, both VEGF and DETA NONOate significantly increased the average distance of migration into the scratch (Fig. 1A). Co-treatment of cells with the •NO synthase inhibitor, N (G)-nitro-L-arginine methyl ester (LNAME, 30μM), prevented the VEGF-induced increase in migration without affecting the migration distance in the vehicle control, consistent with the role of eNOS and •NO in VEGF-induced EC migration. Measurement of SERCA activity as 45Ca2+ uptake showed that DETA NONOate or VEGF significantly increased SERCA activity (Fig. 1B). Additionally, VEGF-induced stimulation of SERCA activity was blocked by LNAME (Fig. 1B). VEGF and •NO also stimulated SERCA activity in bovine aortic endothelial cells (BAECs), and similar to HAECs, stimulation caused by VEGF was blocked by LNAME (Supplementary Fig. S1; Supplementary Data are available online at www.liebertonline.com/ars). These results indicate that VEGF via •NO stimulates both endothelial cell migration and SERCA activity.
Because ECs contain both SERCA3 and the SERCA2 splice isoform, SERCA2b (5, 13, 20), the specific contribution of SERCA2b to VEGF-induced signaling, was assessed by knockdown of SERCA2 in HAECs using selective siRNA. Knockdown was confirmed by both quantitative real time-polymerase chain reaction (qRT-PCR) for SERCA2 mRNA and western blot for SERCA2b with an isoform-specific antibody (Fig. 2A, B). Cell viability was confirmed by trypan blue exclusion (data not shown). In addition, the basal SERCA activity, assessed by 45Ca2+ uptake, was markedly inhibited by knockdown of SERCA2, indicating that it is the major isoform in these cells (Fig. 2C). Wound-healing migration assays over 6h showed no change in unstimulated migration between ECs treated with scrambled siRNA controls and siRNA against SERCA2, indicating that basal migration is not dependent on SERCA2 levels (Fig. 2D). However, knockdown of SERCA2 entirely prevented the VEGF-induced increase in HAEC migration, demonstrating an essential role for SERCA2 in VEGF-stimulated EC migratory signaling. Although SERCA3 was also present in these cells, as shown by Western blot and low levels of mRNA as measured by qRT-PCR, ECs treated with SERCA3 siRNA displayed 45Ca2+ uptake similar to that of those treated with scrambled siRNA and migrated similarly to controls in response to VEGF (Supplementary Fig. S2).
To assess S-glutathiolation of SERCA2b, HAECs were treated with VEGF (50ng/ml) for 15min before lysis. SERCA2b was immunoprecipitated using a polyclonal SERCA2 antibody and then western blotted for protein-bound glutathione and SERCA2b. Unstimulated SERCA2b was basally S-glutathiolated; however, addition of VEGF increased S-glutathiolation of SERCA2b, suggesting a role for redox regulation of SERCA in VEGF signaling (Fig. 3B). To confirm the specificity of the antibody probe, glutaredoxin-1 (Glrx-1), which specifically reduces S-glutathione adducts (11), was overexpressed by ~10-fold (Fig. 3A), and HAECs were treated with VEGF and assessed for SERCA2b S-glutathiolation. Glrx-1 inhibited VEGF-dependent glutathiolation of SERCA2b (Fig. 3B). Similarly, inhibition of •NO production by pretreatment with LNAME prevented VEGF-induced SERCA2b glutathiolation, confirming that •NO produced by VEGF signaling causes the S-glutathiolation of SERCA. To assess functional consequences of S-glutathiolation, HAECs overexpressing Glrx-1 were subjected to a wound-healing scratch assay over 6h or were assessed for formation of capillary tube-like structures over 24h. Compared to LacZ controls, Glrx-1 overexpression did not affect migration or tube formation of unstimulated HAECs (Fig. 3C, D and Supplementary Fig. S3A). In contrast, VEGF-stimulated migration and tube formation were prevented, unlike in the LacZ control. In addition, overexpression of Glrx-1 prevented •NO-induced increase in migration (Fig. 3C).
Wild-type (WT) SERCA2b or SERCA2b in which cysteine-674 was mutated to a serine (C674S) was overexpressed in HAECs using adenovirus (Fig. 4A). HAECs overexpressing WT SERCA2b demonstrated increased S-glutathiolation (Fig. 4B) in response to VEGF treatment. In contrast, VEGF-induced SERCA2b S-glutathiolation (Fig. 4B) was inhibited in cells overexpressing SERCA2b C674S. To assess the functional importance of SERCA2b C674, migration in response to either VEGF or •NO was measured over 6h. Overexpression of WT SERCA2b did not significantly influence the increased migration caused by DETA NONOate or VEGF compared with cells infected with an empty vector or LacZ (Fig. 4C, D). However, overexpression of SERCA2b C674S prevented increased migration due to either DETA NONOate (Fig. 4C) or VEGF (Fig. 4D). Similarly, expression of SERCA2b C674S, but not WT SERCA2b, prevented increased formation of EC capillary tube-like structures in response to VEGF (Fig. 4E and Supplementary Fig. S3B).
Ca2+ influx was essential for VEGF-induced migration, demonstrated by the Ca2+ entry blocker, nickel (100μM), which prevented both the increase in intracellular Ca2+ and HAEC migration caused by VEGF (Supplementary Fig. S4). To investigate the redox regulation of Ca2+ signaling in ECs, the effects of Glrx-1 overexpression and S-glutathiolation of SERCA2b on influx of extracellular Ca2+ into the cytosol after stimulation by VEGF were measured using Fura2. We first confirmed that Ca2+ influx was not stimulated by Ca2+ addition alone in dimethyl sulfoxide (DMSO) vehicle controls (Supplementary Fig. S4D). In the absence of extracellular Ca2+, VEGF had no significant effect on intracellular Ca2+ levels, but caused a robust increase in Ca2+ upon Ca2+ re-addition (Fig. 5) Demonstrating the importance of •NO production, we found that LNAME treatment inhibited VEGF-induced Ca2+ influx (Fig. 5A, B). In addition, we found that VEGF-induced increase in Ca2+ influx was significantly less in cells overexpressing Glrx-1 compared with cells expressing LacZ (Fig. 5C, D). This finding suggests that VEGF-induced influx of extracellular Ca2+ is regulated by a novel redox mechanism involving protein S-glutathiolation. To assess the specific role of SERCA2b in VEGF-induced Ca2+ influx, SERCA2 was knocked down using siRNA. VEGF-induced Ca2+ influx was significantly inhibited in cells with decreased SERCA2b expression compared to cells treated with nontargeting siRNA (Fig. 5E, F). Additionally, overexpression of SERCA2b C674S, but not WT SERCA2b, significantly decreased not only VEGF-induced Ca2+ influx but also •NO-induced Ca2+ influx (Fig. 5G, H), indicating a critical role for •NO-dependent redox regulation of SERCA2b C674 in VEGF- and •NO-induced Ca2+ signaling.
Orai1, a plasma membrane Ca2+ influx channel, was knocked down because of its reported importance to Ca2+ entry during VEGF stimulation (16). Forty-eight hours after EC transfection with siRNA specific to Orai1, Ca2+ influx in response to VEGF was significantly inhibited (Fig. 6A). In addition, we also found that •NO-induced Ca2+ influx was prevented when Orai-1 was knocked down (Fig. 6B).
Next, the potential mechanisms of VEGF-dependent Ca2+ store-emptying were assessed. It has previously been shown that HAECs have ER RyR Ca2+-release channels, and that their activation causes extracellular Ca2+ influx (7), but nothing is known about their involvement in VEGF signaling. First, we confirmed that caffeine caused Ca2+ influx in HAECs, and that this could be blocked by inhibiting RyR channel opening with ryanodine (100μM, data not shown). Ca2+ influx in response to either VEGF or •NO was then measured in the presence of the same ryanodine concentration. Inhibition of the RyR with ryanodine prevented both VEGF- and •NO-stimulated Ca2+ influx (Fig. 6C, D), suggesting that ER Ca2+ store-emptying, in addition to Ca2+ uptake by SERCA2b, is required for VEGF-induced Ca2+ influx.
Our studies reveal three novel findings regarding the mechanism of VEGF-induced EC migration. First, we identify that SERCA2b, and specifically the redox- active cysteine 674, is required for VEGF-induced EC migration. Second, VEGF-induced EC migration depends upon •NO-mediated S-glutathione adducts on SERCA2b cysteine-674, and both can be prevented by Glrx-1. Third, we demonstrate a novel redox-dependent regulation of VEGF-induced EC Ca2+ influx that depends on •NO-mediated redox regulation of SERCA2b. Although the role of •NO in VEGF-mediated EC migration is well recognized, its redox-regulated target proteins have not been fully established. Evidence that VEGF-induced •NO production regulates SERCA activity in ECs by S-glutathiolation, which controls Ca2+ influx, provides novel insights into the redox-regulation of VEGF-mediated angiogenic signaling.
Previous studies from our lab and others (2–4, 14, 28, 30) have demonstrated the importance of SERCA redox regulation in both physiology and disease, but its contribution to EC physiology has not been widely explored. In these studies, we determined that the ubiquitous SERCA isoform, SERCA2b, accounts for the majority of the SERCA activity and is required for VEGF- or •NO-induced migration. Although SERCA3 is also present in these cells, low mRNA levels, as measured by qRT-PCR (Supplementary Fig. S2), indicate that this isoform is poorly expressed in cultured HAECs. In addition, SERCA activity was unaffected, and ECs were still able to respond to VEGF in ECs treated with SERCA3 siRNA (Supplementary Fig. S2). In contrast, knockdown of SERCA2 eliminated the large majority of EC SERCA activity and accounted for VEGF-induced migration. A role for SERCA3 in other ECs cannot be excluded, because its expression varies markedly in culture (20). Although not addressed here directly, the arginine-rich environment of the amino acid sequence containing C675 of SERCA3 is similar to that which confers increased reactivity to C674 of SERCA2 (27), so that it too might be redox-sensitive.
Although redox regulation of SERCA2b has been explored previously in the context of SMCs, the presence of eNOS in ECs provides a unique redox environment that can affect SERCA function. We determined that VEGF stimulation in ECs required an eNOS activity, and that this led to S-glutathiolation of the SERCA2b-reactive cysteine-674. As a consequence of this redox modification, SERCA Ca2+ uptake activity was enhanced, and Ca2+ influx was stimulated. Interestingly, VEGF-induced Ca2+ influx was dependent on SERCA S-glutathiolation and could be prevented either by overexpression of Glrx-1 or SERCA2b C674S mutant or by knockdown of SERCA2, indicating that SERCA2b redox is critical for the VEGF-induced EC Ca2+ response. In addition, we determined that the RyR channel activity, which can mediate ER Ca2+ store-emptying, is required for VEGF-induced Ca2+ influx. RyRs have previously been found in HAECs, and their stimulation was shown to cause Ca2+ influx (32, 33), potentially by the way of store-operated Ca2+ entry. In agreement with previous reports, we found that VEGF-stimulated Ca2+ influx depended upon Orai1 channels (1, 16). Additionally, we found that •NO stimulates Orai1-dependent Ca2+ influx, supporting the involvement of these channels in a redox-sensitive pathway involving stimulation of SERCA-dependent ER Ca2+ uptake. We propose that the SERCA2b and RyR activity in response to VEGF plays an important role in Ca2+ cycling through the ER. Ca2+ influx is required for replenishment of that released from ER stores and is required for EC angiogenic signaling, including eNOS activation and redox-dependent Ca2+ reuptake by SERCA (as shown in Fig. 7). This model of •NO-induced cycling of Ca2+ through the ER is quite distinct from that proposed in SMCs, in which •NO-induced S-glutathiolation of SERCA refills SR stores and inhibits store-operated Ca2+ influx (4, 6). It is notable that the RyR activity is also known to be redox-regulated by •NO (8), and it is possible that in addition to SERCA, the RyR and potentially other redox-regulated proteins coordinate the Ca2+ response to VEGF and NO.
Our studies show that stimulation of EC SERCA2b activity by S-glutathiolation of cysteine-674 is essential for VEGF-induced Ca2+ influx and EC migration. We therefore propose that •NO-dependent stimulation of SERCA activity increases Ca2+ entry, and is required for driving early angiogenic events of migration and tube formation. These studies suggest that the redox status of SERCA may be important in inducing therapeutic angiogenesis or inhibiting pathological angiogenesis.
The Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and SERCA siRNAs were purchased from Invitrogen. TaqMan primers and real time-polymerase chain reaction (RT-PCR) reagents were purchased from Applied Biosystems. BAECs were purchased from Cell Systems. HAECs (19 years old, female) and the endothelial growth medium were purchased from Lonza. The SERCA3 antibody was from Affinity Bioreagents. The SERCA2 IID8 antibody was purchased from Santa Cruz Biochemicals. The rabbit polyclonal SERCA antibody was generated by Bethyl Laboratory. The glutathione antibody was from Virogen. Recombinant human VEGF-165 was purchased from R&D Biosciences. LNAME, thapsigargin, ryanodine, and DETA NONOate were purchased from Sigma.
Adenoviral WT SERCA and mutant SERCA (C674S) were designed as previously described (4). SERCA adenoviruses were screened for proper constructs by extraction of viral DNA using the RedExtract-N-Amp Tissue PCR kit (Sigma). Extracted DNA was subjected to polymerase chain reaction (PCR) using a forward primer specific to the viral promoter and a reverse primer located beyond the C674 codon: 5′-ACCGTCAGATCCGCTAGAGA-3′ and 5′-GCCACAATGGTGGAGAAGTT-3′. PCR products were cleaned using a Qiagen QiaQuick PCR Purification kit and then sequenced using the sequencing primer: 5′-GATCACTGGGGACAACAAGG-3′. SERCA adenovirus was purified using the double cesium chloride purification technique. Adenoviral E1A contamination of the purified SERCA adenovirus was excluded as described previously (12). ECs were infected to achieve equal expression of WT SERCA2b and SERCA2b C674S protein at a multiplicity of infection (MOI) of ≈10. Adenoviral Glrx-1 was previously reported (25) and was delivered at an MOI of ≈10. Cells were infected in FBS-free media and 10μg/ml Polybrene (Sigma) for 48h. Cells were quiesced for 24h in the medium with 0.1% serum before treatments.
Ca2+ uptake into the ER was measured using an oxalate-dependent ER 45Ca2+ uptake assay in cells in which the cell membrane was permeabilized. Cells were treated with VEGF (50ng/ml) with or without LNAME (30μM) or DETA NONOate (30μM) for times indicated. The medium was replaced with a Ca2+ uptake solution (in mM: 30 Tris–HCl, 100 KCl, 5 NaN3, 6 MgCl2, 0.15 EGTA, 0.12 CaCl2, and 10 oxalate), and ECs were permeablized with saponin (250μg/mL) before treatment with thapsigargin (TG) (10μM, 20min), such that the extravesicular Ca2+ concentration was controlled by buffer content. After treatment, cells were trypsinized and then incubated in a solution with 45Ca2+ (1μCi) and ATP (2mM). After 30min, cells were filtered through Whatman GF/C glass filters under vacuum and washed with physiological saline solution (PSS). Radioactivity was measured on a Beckman Coulter LS1801 scintillation counter. 45Ca2+ uptake was evaluated by counting radioactivity on the filters and normalized to protein concentration measured by the Bradford assay.
To knockdown SERCA expression, HAECs were cultured for 48h with the SERCA2-specific siRNA constructs 5′-ACCAGUAUGAUGGUCUGGUAGAAUU-3′ and 5′-AAUUCUACCAGACCAUCAUACUGGU-3′, the SERCA3-specific constructs 5′-CCAUCUACAGCAACAUGAAGCAAU-3′ and 5′-AAUUGCUUCAUGUUGCUGUAGAUGG-3′, or a scrambled siRNA control (Invitrogen) with Lipofectamine (Invitrogen) reagent in serum-free, antibiotic-free endothelial basal medium-2 (EBM-2).
BAECs or HAECs were grown to 80% confluency and quiesced overnight. Scratch wounds were applied to EC monolayers in low-serum media as previously described (28, 30). Inhibitors were given 1h before making a scratch wound with a pipette tip and reapplied at the time of the scratch. VEGF (50ng/ml) or DETA NONOate (30μM, released concentration ≈1μM (9) was given at the time of the scratch in a serum-free medium. Images were taken at 0 and 6h at three fixed locations along the scratch (Supplementary Fig. S1). Migration distances were averaged from the three measurements per condition using ImageJ software, and this was considered as n=1 (see Supplementary Methods for expanded details).
In vitro capillary tube-like formation on Growth Factor Reduced Matrigel (BD Biosciences) was performed as previously described (24). Briefly, 96-well plates were coated with Matrigel according to the manufacturer's instructions. Appropriately treated HAECs were seeded at a density of 1×104 cells/cm2 with or without 50ng/ml VEGF in low-serum endothelial growth media (Lonza) and incubated at 37°C overnight. Images were taken at 24h with the scale indicated in the images, and tube formation was quantified by scoring for the tube number by observers blinded to sample treatment.
Quantitative PCR was performed using gene-specific FAM-NFQ-conjugated TaqMan primers for human SERCA2 mRNA sequence 5′-GAGTTACCGGCTGAAGAAGGAAAAA-3′ or human SERCA3 mRNA sequence 5′-CTGGCTATCGGAGTGTACGTAGGCC-3′ (Applied Biosystems). A VIC-NFQ-conjugated human 18S primer was used to normalize mRNA expression levels. Expression was analyzed using the comparative CT (ΔΔCT) with StepOne™ Real Time PCR Software (Applied Biosystems).
HAECs were infected with Glrx-1 or LacZ for 48h and then quiesced in EBM-2 with 0.1% FBS. Cells were initially treated with VEGF over a time course of 0–60min to determine the time of peak S-glutathiolation. Cells were treated with VEGF for 5min or •NO gas solution (10μM) for 1min. Lysates were incubated with a custom polyclonal SERCA2 antibody and immunoblotted with a monoclonal GSH antibody (Virogen) and a monoclonal SERCA2 antibody (Santa Cruz).
HAECs plated on gelatin-coated glass coverslips were loaded with 2μM Fura2-AM (Invitrogen) in the presence of 0.02% pluronic F127 (Invitrogen) in serum-free endothelial growth media, and right before the experiment were transferred to nominally Ca2+-free PSS supplemented with 2.5mM probenecid (Alfa Aesar, Ward Hill, MA). Changes in intracellular Ca2+ (F340/F380) were monitored as previously described (7, 31). Briefly, cells were allowed to equilibrate in nominally Ca2+-free PSS for 1min before addition of VEGF, •NO, or TG. After 2.5min, Ca2+ (2mM) was added to the PSS. Ca2+ influx was recorded for 2min before addition of ionomycin (2μM) to permeablize the membrane and manganese (8mM) to quench the Fura2. A dual-excitation fluorescence-imaging system (Intracellular Imaging) was used for studies of individual cells. The changes in intracellular Ca2+ were expressed as ΔRatio, which was calculated for each cell as the difference between the maximal F340/F380 ratio after extracellular Ca2+ was added, and its level right before Ca2+ addition (see Supplementary Methods for expanded details).
Samples in the Laemmli buffer were run on 10% electrophoresis gels. Proteins were transferred onto supported nitrocellulose membranes and blocked with 5% milk. Primary antibodies were incubated overnight at 4°C in milk. Horseradish peroxidase (HRP)-conjugated or IR dye-conjugated secondary antibodies were used. Blots were imaged using film or the LICOR system.
Statistical analysis was performed using the Student's t-test or one-way analysis of variance with a Bonferroni multiple comparisons post-test. Results are expressed as means±SE. p<0.05 was considered significant.
This work was supported by the National Institute of Health Grants HL031607-29 (R.A.C. and X.Y.T.) and Grants RO1-HL54150 and RO1-HL071793 (V.B.), NIH Predoctoral Training Grant HL007501-26 (A.E.), and the Boston University Levinsky Fellowship (A.E.). These studies were supported by the Calcium Affinity Research Collaborative of the Evans Center, Department of Medicine, Boston University Medical Center.
No competing financial interests exist.