Reverse mode Na+/Ca2+ exchange mediated by STIM1 contributes to Ca2+ influx in airway smooth muscle following agonist stimulation
© The Author(s) 2010
Received: 19 December 2009
Accepted: 2 December 2010
Published: 2 December 2010
Agonist stimulation of airway smooth muscle (ASM) results in IP3 mediated Ca2+ release from the sarcoplasmic reticulum followed by the activation of store operated and receptor operated non-selective cation channels. Activation of these non-selective channels also results in a Na+ influx. This localised increase in Na+ levels can potentially switch the Na+/Ca2+ exchanger into reverse mode and so result in a further influx of Ca2+. The aim of this study was to characterise the expression and physiological function of the Na+/Ca2+ exchanger in cultured human bronchial smooth muscle cells and determine its contribution to agonist induced Ca2+ influx into these cells.
The expression profile of NCX (which encodes the Na+/Ca2+ exchanger) homologues in cultured human bronchial smooth muscle cells was determined by reverse transcriptase PCR. The functional activity of reverse mode NCX was investigated using a combination of whole cell patch clamp, intracellular Ca2+ measurements and porcine airway contractile analyses. KB-R7943 (an antagonist for reverse mode NCX) and target specific siRNA were utilised as tools to inhibit NCX function.
NCX1 protein was detected in cultured human bronchial smooth muscle cells (HBSMC) cells and NCX1.3 was the only mRNA transcript variant detected. A combination of intracellular Na+ loading and addition of extracellular Ca2+ induced an outwardly rectifying current which was augmented following stimulation with histamine. This outwardly rectifying current was inhibited by 10 μM KB-R7943 (an antagonist of reverse mode NCX1) and was reduced in cells incubated with siRNA against NCX1. Interestingly, this outwardly rectifying current was also inhibited following knockdown of STIM1, suggesting for the first time a link between store operated cation entry and NCX1 activation. In addition, 10 μM KB-R7943 inhibited agonist induced changes in cytosolic Ca2+ and induced relaxation of porcine peripheral airways.
Taken together, these data demonstrate a potentially important role for NCX1 in control of Ca2+ homeostasis and link store depletion via STIM1 directly with NCX activation.
The bronchoconstrictor response of the asthmatic airway depends on airway narrowing caused by inappropriate ASM contraction. The ASM layer is also a potential source of pro-inflammatory mediators and so plays a key role in the pathogenesis of this disease. Control of intracellular Ca2+ is critical to regulation of ASM function and mediates many processes including contraction, proliferation and gene expression .
Cytoplasmic Ca2+ levels contribute significantly to the contractile/relaxant state of an airway myocyte. Initiation of contraction is dependent upon cytosolic inositol 1, 4, 5-trisphosphate (IP3) mediated release of intracellular Ca2+ from the sarcoplasmic reticulum (SR). Sustained contraction is thought to be dependent upon Ca2+ influx from extracellular sources through receptor operated cation (ROC) or store operated cation (SOC) channels on the plasmalemma. The latter are activated following depletion of SR Ca2+ via a mechanism involving STIM1 and ORAI homologues [2, 3]. In addition, we have previously shown expression of a range of TRPC homologues in HBSMC . Plasma membrane Na+/Ca2+ exchange acting in reverse mode is well documented in cardiac muscle, but only recently has become hypothesised as an additional mechanism of Ca2+ influx in smooth muscle [1, 5, 6]. Under physiological conditions, Na+/Ca2+ exchange is the principle mechanism of Ca2+ extrusion, maintaining low intracellular Ca2+ levels by transporting one Ca2+ in exchange for three Na+ . Under experimental conditions including membrane depolarization and localised increases in intracellular Na+, the direction of ion exchange can be reversed resulting in Na+ extrusion and Ca2+ influx. Certain pathophysiological conditions can replicate such conditions including ischemia and exposure to endogenous ouabain . Inhibition of the Na+/K+ pump by ouabain, resulting in intracellular Na+ accumulation has been shown to contract human bronchial smooth muscle . In addition, KB-R7943 (a selective antagonist for reverse mode Na+/Ca2+ exchange) reduces agonist induced contraction of ASM [1, 5, 10].
It is clear that reverse mode NCX can be activated following localised intracellular Na+ accumulation, via inhibition of the Na+/K+ pump. An hypothesis where Na+ influx through non-selective ROC or SOC channels is sufficient to trigger Ca2+ influx though NCX has recently been proposed [11–13]. NCX1 has also been demonstrated to interact both physically and functionally with certain TRPC homologues .
The focus of this study was to define the functional role of NCX in cultured HBSMC. This included investigating the presence of transcript variants, exploring electrophysiological properties of reverse mode NCX currents, its contribution to agonist evoked Ca2+ signals and airway contraction. In addition we show evidence of a link between SOC entry mediated by STIM1 and the activation of NCX1.
Human bronchial tissue was obtained from consenting patients with no history of asthma. Ethical approval was obtained from the Nottingham local ethical research committee.
Human bronchial smooth muscle cells
Primary human bronchial smooth muscle cells (HBSMCs) were isolated from bronchial tissue by enzyme (collagenase) digestion and expanded through culture in Dulbecco's Modified Eagle Medium (DMEM) (containing 2 mM glutamine and 10% foetal calf serum (FCS)) using published methods . Briefly, bronchial tissue was surgically removed from patients and placed in sterile, ice cold DMEM (without calcium or magnesium). The smooth muscle was removed from the surrounding epithelial and connective tissue and washed with DMEM. The smooth muscle was then finely chopped with a scalpel and incubated at 37°C with collagenase H (2 mg/ml) for 2-3 hours. Every hour, the cell mixture was filtered through a 100 μM cell strainer, pelleted by centrifugation and seeded into a 25 cm3 culture flask. Cells were allowed to reach confluency before being passaged further. Cells between passage 3-6 were used in the experiments described in this study.
Transfection of siRNAs
NCX1 siRNA (AAGGAGAAGGAAATGAAACTG) and STIM1 siRNA (AAGGGAAGACCTCAATTACCA) were designed by and purchased from Ambion (Huntingdon, Cambridge, UK). A non-sense, scrambled siRNA was used as a negative control for transfection. Cells were transfected with 20 nM siRNA in FCS free medium over a period of 6 hours, the medium was then replaced with serum containing medium and cultured for a further 42 hours at 37°C, 5% CO2. The transfection reagent used was Lipofectamine 2000 (Invitrogen, Paisley, UK) at a final concentration of 1 μl/ml as per manufacturers' instructions.
Reverse transcriptase PCR
For reverse transcriptase (RT) PCR, primers against NCX2 and 3 were designed to span an intron-exon boundary (to eliminate genomic DNA contamination) using Primer 3 software . Primer sequences are as follows; NCX2 forward:CATTCATGGAGGGAGCAGTT, NCX2 reverse: ATGACCAGGATGGAGACACC, NCX3 forward: AAGGTGCTGTTTGCCTGTGT, NCX3 reverse:TTTGCTGGCAAACGTATCTG. Primers for NCX1 (forward: TGTGAGTGAGAGCATTGGC, reverse: CTCTTTGCTGGTCAGTGGCT) were designed by Pitt et al  to span the alternatively spliced region and distinguish between transcript variants. Cycling was performed 35 times; 94°C, followed by 55°C (annealing temperature), then 72°C (all for 90 seconds) followed by 10 mins at 72°C. PCR products were visualized by ethidium bromide staining and confirmed by direct sequencing.
Real-Time PCR (Taqman)
siRNA targeted mRNA knockdown was measured using real time, quantitative PCR (Taqman). Gene specific primers and probes against NCX1 were designed to span an intron-exon boundary using Beacon designer 7 (Stratagene): Forward primer; GCCTACTGACAGCTTTCATTGG, Reverse primer; TGTGTCTGGCACTGATGTTCC, probe; TGGCTTCCCACTTTGGCTGCACCA. 18 s RNA was used as a housekeeping gene to correct for equal cDNA input. The NCX1 probe was dual labelled with 5' FAM and 3'TAMRA dyes and was purchased from Applied Biosystems (Foster City, CA) along with the universal mastermix and the 18 s pre-designed assay. Each sample was run in duplicate and mRNA knockdown was measured from mRNA obtained from 3 separate experiments. The relative expression of the target gene was calculated using the comparative method (2-ΔΔCt) .
HBSMC grown on cover slips were fixed with 4% formaldehyde and semi-permeabilized with 0.1% saponin as described . Cells were incubated with mouse NCX1 antibody (1:200) (Abcam, Cambridge, UK) followed by labeling with anti-mouse Alexa fluor 488 (1:400) (Molecular probes). Cells were visualized on a spinning disk confocal microscope using a Zeiss Axio Observer D1, Hamamatsu electron multiplier CCD camera C0100-13 with a Yokogawa spinning disk system.
Measurement of Intracellular Calcium
Intracellular Ca2+ levels ([Ca2+]i) in confluent, monolayers of HBSMC was estimated using Fluo-4AM. Cells were plated in 48 well plates and loaded with Fluo-4AM (Molecular probes) for 1 hour at room temperature in DMEM supplemented with 10% FCS, 2 mM glutamine and 2.5 mM probenecid (Sigma Chemical Co, Poole, UK). After loading, cells were washed with hanks balanced saline solution (HBSS) containing 10 mM HEPES, 2.5 mM probenecid, 2 mM CaCl2 and 1 mM MgCl2. After a 10 second baseline, cells were stimulated with either bradykinin (BK,1 μM) or histamine (HA,10 μM) for a total time period of 2 minutes. 10 μM KB-R7943 or equivalent DMSO vehicle control was added to the cells shortly before assay. Ca2+ imaging was performed on the spinning disk confocal microscope system. Confocal images (1 image/second) were collected at 525 nm emission after excitation at 491 nm. Volocity analysis software (Improvision) was used to analyze the images. Each experiment incorporated at least 5 repeat wells per condition and each experiment was repeated at least 4 times. Data are presented as changes in mean fluorescence intensity (FI) compared with the baseline and peak fluorescence values was used as an estimation of changes in [Ca2+]i.
Porcine lung slices
Whole porcine lungs were obtained from a local abattoir (Nottingham). An isolated, distal lobe was inflated to ~90% capacity with 2% agarose (Type VII, Sigma) solution (in HBSS) by inserting a tube via the trachea; the airways were cleared of agarose by injecting a pulse of air. Slices were cut ~130 μM thick using a vibrotome tissue slicer (EMS-4000, Electron Microscope Sciences). Lung slices were transferred to DMEM, supplemented with 2 mM L-glutamine and penicillin-streptomycin and incubated for 2~3 days at 37°C/5% CO2. Phase contrast images were recorded at a rate of 1 image per second. The difference in brightness between the airway lumen and surrounding alveolar tissue was used to detect the airway edge and calculate the area of the airway. Airway narrowing was expressed as the percentage decrease in airway area compared to the area of the fully relaxed airway (prior to agonist stimulation).
Unless otherwise stated, statistical significance between groups was assessed by unpaired T tests where *p < 0.05, **p < 0.01 were considered significant.
NCX1 is expressed in HBSMCs
RT-PCR revealed that only NCX1 was present in HBSMCs (Figure 1A). Human brain cDNA was used as a positive control  to validate primers designed for NCX2 and NCX3. The NCX1 primers span the variable region of NCX1 and so distinguish between splice variants (designed by Pitt and colleagues ) (Figure 1C). RT-PCR gave a single PCR product (Figure 1A) and sequence analysis revealed that within the variable region, only exons B and D were present. This was repeated for three different donors. Therefore the isoform that we found in HBSMCs was NCX1.3 and is the same variant that has previously been found in this tissue type . In addition, positive staining of NCX1 protein was found in permeabilised HBSMCs (Figure 1B). A Negative control sample where only secondary antibody was applied revealed no positive staining (data not shown).
Reverse mode NCX currents are induced in HBSMCs using ion replacement experimental conditions and are augmented by stimulation with histamine
The cells were initially perfused with a Ca2+-free extracellular solution containing 140 mM Na+. Extracellular Ca2+ was applied to the Na+ loaded cell along with physiological (140 mM) or reduced (5 mM) Na+ to induce an outwardly rectifying current mediated by reverse mode NCX1 i.e. 3Na+ efflux and 1Ca2+ influx according to the stoichiometry of this exchanger (3Na+:1Ca2+). The intracellular solution was buffered to low Ca2+ (~14 nM), and high Na+ (20 mM) which favors reverse mode NCX1. Under these conditions, perfusion with Ca2+ free extracellular solution with 140 mM Na+ induced a current, with a linear current-voltage relationship and a reversing potential of ~ 0 mV (Figure 2D). The characteristics of this current resemble that of SOC channels. Re-addition of 2 mM extracellular Ca2+ reduced these currents and subsequently activated a small transient outwardly rectifying current with a reversal potential of -22.56 ± 4.67 mV (n = 14). A typical example of the current activated by extracellular application of 2 mM Ca2+ is illustrated in Figure 2A. This outwardly rectifying current was abolished by 10 μM KB-R7943 (Figure 2B-D). Furthermore, when extracellular Na+ was reduced from 140 mM to 5 mM (along with re-addition of 2 mM extracellular Ca2+), the outwardly rectifying current was significantly augmented. The peak current density following extracellular 2 mM Ca2+ re-addition with 5 mM Na+ was increased by 243% compared to that with 140 mM Na+. This increase in outwardly rectifying current was also abolished by 10 μM KB-R7943 (Figure 2A). These results suggest that NCX1 is present in cultured HBSMCs and is activated upon these specific changes in experimental conditions.
Knockdown of NCX1 and STIM1 by siRNA results in reduction of histamine induced outwardly rectifying currents
KB-R7943 partially inhibits agonist evoked Ca2+signals in HBSMCs
KB-R7943 relaxes pre-contracted porcine airways
This present study provides, for the first time, molecular and electrophysiological data as well as cellular and tissue pharmacological evidence supporting the presence of a functional NCX1 protein that can operate in reverse mode and thus presents an additional mechanism of Ca2+ influx in HBSMCs. We also provide additional evidence in support of an existing hypothesis whereby Na+ influx through SOC channels activates reverse mode NCX; to our knowledge we are the first to show a link between STIM1 mediated Na+ influx and the activation of NCX1.
We initially confirmed expression of NCX1 mRNA and protein in HBSMCs. The splice variant we identified was NCX1.3 and is consistent with previous findings . We could not detect the presence of other NCX1 splice variants or the expression of NCX2 or NCX3 in this cell type. NCX1 (weak band) NCX2 and NCX3 expression was detected in human brain and served as a positive control for these primer pairs. Expression of NCX1 protein was also confirmed by immunohistochemistry using specific NCX1 antibodies. We found that NCX1 protein was expressed both on the membrane and in the cytoplasm of the cells. There is some evidence of membrane bound NCX1 protein being trafficked into the cytoplasm which could explain this result .
Following confirmation of the expression of NCX1.3 in cultured HBSMCs, we then studied the functional role of this protein and its contribution to agonist evoked Ca2+ signals in these cells. NCX1.1 is the splice variant expressed in cardiomyocytes and has been extensively studied and its electrophysiological properties are well characterized . Few studies have been performed in cells expressing NCX1.3; therefore we firstly aimed to characterize the currents carried by NCX1.3 in HBSMCs using whole cell patch clamp.
Experimental conditions were adjusted to favour the activation of reverse mode NCX1 currents. Cells were first loaded with Na+ through perfusion with a Ca2+ free extracellular buffer and the addition of ouabain. Reverse mode NCX1 was subsequently activated via re-addition of 2 mM extracellular Ca2+. The outwardly rectifying current displayed typical characteristics to that of Na+ efflux through NCX1 and were inhibited by KB-R7943 (a relatively selective inhibitor of reverse mode NCX1). These currents were greatly enhanced by stimulation with the contractile agonist HA and these HA enhanced currents were inhibited by KB-R7943. However, because at high concentrations KB-R7943 can have effects on other channels, we also used knockdown of NCX1 by siRNA, with similar results. Perfusion with Ca2+ free extracellular buffer activates a SOC like current prior to the activation of the NCX1 mediated outwardly rectifying current. Interestingly, inhibition of this SOC current by prior knockdown of STIM1 also inhibited HA induced NCX1 currents. As far as we are aware, these are the first data that provide evidence of a functional link between STIM1 mediated SOC activation and reverse mode NCX1 activation.
The electrophysiology data provided evidence of a functionally active NCX1 that is activated following HA induced SOC activation. We then further tested its physiological relevance through measurement of agonist induced Ca2+ signals and contraction of ASM. The addition of 10 μM KB-R7943 almost completely abolished NCX1 sensitive currents. However, in comparison the equivalent concentration of inhibitor only marginally inhibited HA induced Ca2+ signals. The experimental conditions used to yield the electrophysiology results were designed to optimise and isolate NCX1 currents. Experimental conditions implemented in Figure 7 were under more physiological conditions and therefore a range of other Ca2+ influx pathways (insensitive to NCX1 inhibition) would have been activated. Inhibition of reverse mode NCX1 by KB-R7943 resulted in dose dependent relaxation of pre-contracted small airways, illustrating the functional importance of NCX1 in maintaining contraction. Addition of KB-R7943 also inhibited both BK and HA induced intracellular Ca2+ signals in HBSMCs.
Abnormal Ca2+ handling within ASM is likely to contribute to inappropriate contraction, a major symptom of asthma. The understanding of how intracellular Ca2+ is regulated under physiological and pathophysiological situations forms an important aspect in the search for new therapeutic targets for the treatment of asthma. In recent years, there have been major advances in the understanding of Ca2+ homeostasis driven in part by the identification of STIM1 and ORAI1 as critical regulators of Ca2+ influx in many tissue types. In HBSMCs, we have shown that the interaction between STIM1 and ORAI1 plays an important role in the activation of SOC channels [2, 3]. The precise molecular identity of these SOC channels is still under debate with strong evidence for both ORAI and TRPC homologues. Our data supports the hypothesis whereby histamine (HA) stimulation results in IP3 mediated Ca2+ store release, resulting in store depletion of Ca2+. This in turn activates SOC channels via a STIM1 dependent mechanism. Activation of SOC channels permits influx of Na+ (plus a small amount of Ca2+). This localized increase of intracellular Na+ results in membrane depolarization that activates reverse mode NCX1 to further promote Ca2+ influx.
In summary, we provide evidence demonstrating that reverse mode NCX1 plays a role in the control of Ca2+ homeostasis in ASM. This role is dependent on STIM1 activation of SOC following intracellular SOC depletion. Whilst other Ca2+ entry mechanisms may also play a part in Ca2+ influx, these data suggest targeting NCX may provide a novel therapeutic strategy in diseases such as asthma.
airway smooth muscle
foetal calf serum
human bronchial smooth muscle cells
receptor operated cation
store operated cation
The work presented in this manuscript is funded by the Medical Research Council
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