To examine the intermolecular aspects of CICR during hypertrophy, we utilized loose-patch confocal imaging to visualize the signaling between a single L-type Ca2+ channel (LCC) and ryanodine receptors (RyRs) in aortic stenosis rat models of compensated (CHT) and decompensated (DHT) hypertrophy. We found that the LCC-RyR intermolecular coupling showed a 49% prolongation in coupling latency, a 47% decrease in chance of hit, and a 72% increase in chance of miss in DHT, demonstrating a state of “intermolecular failure.” Unexpectedly, these modifications also occurred robustly in CHT due at least partially to decreased expression of junctophilin, indicating that intermolecular failure occurs prior to cellular manifestations. As a result, cell-wide Ca2+ release, visualized as “Ca2+ spikes,” became desynchronized, which contrasted sharply with unaltered spike integrals and whole-cell Ca2+ transients in CHT. These data suggested that, within a certain limit, termed the “stability margin,” mild intermolecular failure does not damage the cellular integrity of excitation-contraction coupling. Only when the modification steps beyond the stability margin does global failure occur. The discovery of “hidden” intermolecular failure in CHT has important clinical implications.Pressure overload–induced hypertrophy is a key step leading to heart failure. The Ca 2+ channels) and calcium release channels on a specialized calcium-regulating organelle called the sarcoplasmic reticulum. This signaling process is defective in heart failure. We have found that the signaling efficiency between a single L-type channel and its controlled Ca2+ release channels decreases during the transition from hypertrophy to heart failure. Moreover, we find unexpectedly that the signaling failure between channels occurs even before any obvious defect in the cardiac cell's ability to contract is seen. In normal cells, the timing between calcium influx and release is rapid; but in hypertrophy before heart failure manifests, there is a delay in this signaling process. In seeking the underlying mechanisms of this intermolecular failure, we find that a protein known as junctophilin, which anchors the sarcoplasmic reticulum to the cell membrane system, is expressed at a lower level. These results reveal early molecular events associated with the progression of hypertrophy, and may provide new insights for developing methods of early diagnosis and treatment to prevent heart failure.High blood pressure induces hypertrophy, a thickening of the cardiac muscle that eventually leads to heart failure, a leading cause of morbidity and mortality. The contractile power of the heart depends in part on signaling between calcium channels on the cell membrane , intermolecular coupling at a molecular level is already slipping. In response to pressure overload, the heart produces an adaptive response in the form of cardiac hypertrophy to maintain adequate cardiac output and tissue perfusion –3. In th2+-induced Ca2+ release (CICR) [2+ influx through L-type Ca2+ channels (LCCs) on the cell surface membrane (including T-tubules) activates ryanodine receptor (RyR) Ca2+ release from the sarcoplasmic reticulum (SR) to generate cell-wide Ca2+ transients [2+ cycling proteins, e.g., SR Ca2+ pumps (SERCA), Na+-Ca2+ exchangers, and their regulatory mechanisms, are also important in determining the amplitude and kinetics of Ca2+ transients [2+ transients triggered by comparable LCC currents are decreased in amplitude and/or slowed in kinetics in most models of decompensated hypertrophy (DHT) and heart failure [2+ influx through LCCs becomes less effective in triggering RyR Ca2+ release [At the cellular level, the contractile power during excitation-contraction coupling (E-C coupling) is governed by a mechanism known as Cae (CICR) ,6. In thansients –9. Besidansients . All theansients ,10–14. Mansients . However failure ,13. Thes release . It is t2+ imaging protocol in conjunction with a loose-seal patch clamp technique to investigate LCC-RyR intermolecular coupling [2+ signaling during the pathogenesis leading to heart failure.During past years, we have developed a local Cacoupling ,16. In t2+ current (ICa) and intracellular Ca2+ transients when the cell membrane was depolarized to 0 mV  (2+ release (The unique advantage of Ca control A. In acc-tubules B. This ps (CRUs) B and a d release A.(R) on the sensitivity (S) of RyRs to LCC Ca2+ influx (L). Based on the stochasticity of LCC-RyR coupling [R per unit time (dR/dt) relates positively to L, S, and RyR availability. Due to the refractoriness of Ca2+ release [R). Therefore, by the first order of approximation,2+ release then produces a Ca2+ transient C(t), which can be described byk represents the apparent rate constant for Ca2+ removal from the cytosol. Assuming that both activation and inactivation of LCCs follow exponential kinetics  of ICa, which could also be reproduced by the simulation in which slowed ICa inactivation  decreased chance of hit, (2) increased chance of miss, (3) the prolonged latency of LCC-RyR coupling, and (4) the prolonged delay and progressive temporal dispersion of cell-wide Ca2+ spikes  becomes lower in peak, it lasts longer in time ((R) relatively unchanged. However, with further decrease of LCC-RyR coupling efficiency, an increasing fraction of CRUs tends not to be activated before ICa inactivation  as described previously . Briefly2, 1.2 MgCl2, 1.2 NaH2PO4, 10 glucose, 0.02 tetrodotoxin, and 10 HEPES, (pH 7.35) adjusted with NaOH. An EPC7 amplifier  was used for the whole-cell patch clamp technique. For Ca2+ transient measurement, the patch pipette filling solution contained (in mM): 127 CsCl, 10 NaCl, 1 MgCl2, 5 MgATP, 15 tetraethylammonium chloride, 10 HEPES, and 0.2 fluo-4 pentapotassium , (pH 7.2) adjusted with CsOH. When Ca2+ spikes occurring at T-tubules were measured, 10 mM EGTA and 4 mM CaCl2 were included in the pipette solution with the CsCl decreased to 115 mM. ICa was activated by 200-ms depolarization pulses at 10-s intervals.Single ventricular myocytes were isolated using an enzymatic method described previously . The celp) of 3–5 MΩ (∼1 μm at the tip) was gently pressed onto the cell surface to form a low-resistance seal (Rs = 20–30 M). The patch membrane voltage (VP) was determined based on resting potential (RP) and command voltage (Vcom) by VP = RP − VcomRs/(Rs+Rp). The pipettes were filled with extracellular solution except that 20 mM Ca2+ and 10 μM FPL64176 were included to facilitate the visualization of Ca2+ sparklets from LCCs.The loose-seal patch clamp technique, as described previously , was per2+ indicator fluo-4 was already in the pipette solution. To load fluo-4 in loose-patch experiments, cells were incubated in 2.5 μM fluo-4 AM (Molecular Probes) in extracellular solution for about 5 min in the dark at 37 °C. Ca2+ transients, Ca2+ spikes from individual T-tubules, and Ca2+ sparks were measured using a Zeiss LSM-510 inverted confocal microscope . All image data were taken in the line-scanning mode along the long axis of the myocyte excited at 488 nm. The Ca2+ level was reported as F/F0, where F0 is the resting or diastolic fluo-4 fluorescence.For whole-cell experiments, the CaTotal RNA from isolated ventricular myocytes was extracted using Trizol reagent, and first-strand cDNA was generated using the ImProm-II Transcription System . Quantitative real-time RT-PCR was performed using the primers of junctophilin type-2  and GAPDH . Amplifications were performed in 35 cycles using an Opticon continuous fluorescence detection system  with SYBR Green fluorescence (Molecular Probes). Each cycle consisted of 30 s at 94 °C, 30 s at 56 °C, and 30 s at 72 °C. All samples were quantified using the comparative Ct method for relative quantitation of gene expression, normalized to GAPDH .t-test for unpaired samples; a value of p < 0.05 was considered significant. When fitted data were compared, their difference was divided by the standard error and compared with the standard normal distribution. During the experiments, we considered the time difference between CHT (7–9 wk after surgery) and DHT (9–11 wk). Sham-operated controls were set for each group. As the data from these two control groups did not differ significantly, they were combined together as the “control” group in this manuscript.Results are expressed as mean plus or minus the standard error of the mean (SEM). Statistical analysis was performed using the Student Figure S1ICa (C–E) among control, CHT, and DHT groups (n ≥ 16 cells from four or more rats for each group). The kinetics of ICa is characterized by the fast (C) and slow (D) time constants. A single asterisk (*) indicates p < 0.05.Comparison of cell capacitance (A), contraction amplitude (B), and kinetics of (19 KB PDF)Click here for additional data file.Figure S22+ transient, and then repolarized to various potentials (−20 to +50 mV), which activated ICa with different amplitudes. The resulted Ca2+ transient was recorded by confocal microscopy.(A) Representative records of the experiment. The cell membrane was first depolarized beyond reversal potential (to +80 mV), which could not activate Ca2+ transient amplitude and membrane potential. Note the leftward shift of the curves when ICa duration shortens.(B) The sigmoid relationship between Ca(64 KB PDF)Click here for additional data file.Figure S32+ indicator, fluo-5F was used to avoid indicator saturation. No significant difference (p > 0.05) could be identified among the control, CHT, and DHT groups (n ≥ 15 cells from three or more rats for each group).The low-affinity Ca(17 KB PDF)Click here for additional data file.Figure S4(A) Representative immunostaining images of LCC (red) and RyR (green) distribution in cardiomyocytes.n ≥ 10 cells from three rats in each group).(B) Percentage of LCC fluorescence co-localized with that of RyRs ((31 KB PDF)Click here for additional data file.Table S1(73 KB PDF)Click here for additional data file.


Therefore, we hypothesized that the relaxation of airway SMCs induced by agents that increase cAMP results from the down-regulation or slowing of the frequency of the Ca2+ oscillations.It has been shown that the contractile state of airway smooth muscle cells (SMCs) in response to agonists is determined by the frequency of Ca2+ signaling of airway SMCs contracted with methacholine (MCh) was investigated in murine lung slices with phase-contrast and laser scanning microscopy.The effects of isoproterenol (ISO), forskolin (FSK) and 8-bromo-cAMP on the relaxation and Ca2+ oscillations within the SMCs and the relaxation of contracted airways. The decrease in the Ca2+ oscillation frequency correlated with the extent of airway relaxation and was concentration-dependent. The mechanism by which cAMP reduced the frequency of the Ca2+ oscillations was investigated. Elevated cAMP did not affect the re-filling rate of the internal Ca2+ stores after emptying by repetitive exposure to 20 mM caffeine. Neither did elevated cAMP limit the Ca2+ available to stimulate contraction because an elevation of intracellular Ca2+ concentration induced by exposure to a Ca2+ ionophore (ionomycin) or by photolysis of caged-Ca2+ did not reverse the effect of cAMP. Similar results were obtained with iberiotoxin, a blocker of Ca2+-activated K+ channels, which would be expected to increase Ca2+ influx and contraction. By contrast, the photolysis of caged-IP3 in the presence of agonist, to further elevate the intracellular IP3 concentration, reversed the slowing of the frequency of the Ca2+ oscillations and relaxation of the airway induced by FSK. This result implied that the sensitivity of the IP3R to IP3 was reduced by FSK and this was supported by the reduced ability of IP3 to release Ca2+ in SMCs in the presence of FSK.All three cAMP-elevating agents simultaneously induced a reduction in the frequency of Ca2+ oscillation frequency by reducing internal Ca2+ release through IP3 receptors.These results indicate that the relaxant effect of cAMP-elevating agents on airway SMCs is achieved by decreasing the Ca A major symptom of asthma is an excessive contraction of airway smooth muscle cells (SMCs) which results in airway hyper-reactivity. To alleviate this acute and chronic airway constriction, β-adrenergic agonists, that relax SMCs, are commonly administered . Yet, dei), such as forskolin (FSK) that directly activates AC, theophylline that inhibits phosphodiesterase and 8-bromo-cAMP that is an analog of cAMP i). However, it is unknown how any of these effects would influence the frequency of Ca2+ oscillations of SMCs.In previous studies with isolated tracheal SMCs, cAMP was found to modulate the frequency of Callations ,16. Howele sites . These i+ influx ,15,16,18membrane -21, an i+ efflux  or uptak+ efflux ,24, or aormation . All of 2+ oscillations, we examined the effect of isoproterenol (ISO) and other cAMP elevating agents (FSK and 8-bromo-cAMP) on methacholine (MCh)-induced contractility and Ca2+ signaling of airway SMCs in murine lung slices. The importance of using lung slices, instead of cultured or isolated cells, is that Ca2+ signaling and airway contractility can be measured simultaneously. We found that cAMP induced relaxation of MCh-contracted airways and reduced the frequency of the Ca2+ oscillations. In addition, we confirmed that cAMP did not affect Ca2+ influx or the refilling of depleted Ca2+ stores. By contrast, we found that an elevation of intracellular IP3 concentration ([IP3]i) reversed the effect of FSK by increasing the frequency of the Ca2+ oscillations to induce contraction. In addition, we also show that cAMP inhibits the Ca2+ release via the IP3 receptor. From these data, we suggest that airway relaxation induced by cAMP is achieved by decreasing the Ca2+ oscillation frequency of airway SMCs by inhibiting IP3-induced Ca2+ mobilization.To explore the hypothesis that cAMP induces intrapulmonary airway relaxation by down-regulating Ca3/PM was obtained from Alexis Biochemicals  and dissolved in DMSO, aliquoted and frozen at -80°C. Other reagents were obtained from Sigma-Aldrich or Calbiochem. Hanks' balanced salt solution was supplemented with 20 mM HEPES buffer (sHBSS) and adjusted to pH 7.4.Cell culture reagents were obtained from GIBCO/Invitrogen Corp. Oregon Green 488 BAPTA-1 AM and O-nitrophenyl EGTA AM (NP-EGTA AM) were obtained from Molecular Probes . Caged-iso-InsP3. Slices were maintained in culture media at 37°C and 10% CO2 for up to 3 days.The preparation of lung slices has been previously described in detail . BrieflyLung slices were observed in a custom-made perfusion chamber constructed from 2 cover-glasses sealed with thin strips of silicone grease  on a Plexiglas support. The slices were held in place with a small sheet of nylon mesh  with a small hole to view the selected airway and avoid the mesh from influencing the contraction of the airway. A gravity-driven perfusion system with a multi-tube manifold with a single output  was used, in conjunction with valves  under TTL control, to exchange solutions in the chamber. The chamber volume was about 100 μl. The perfusion rate was 800 μl/min.The lung slices were observed with an inverted microscope  with a 10× objective and a zoom adapter. Phase-contrast images were recorded using a CCD camera , a camera interface  and image acquisition software . Digital images were recorded in time-lapse (0.5 Hz) and analyzed with "NIH /Scion Image" software (Scion Corp.). The lumen area of the airway was measured by summing the number of pixels below a selected threshold grey level with respect to time. All experiments were performed at room temperature .Slices were loaded for 30 minutes at 30°C with 20 μM Oregon Green 488 BAPTA-1 AM in sHBSS containing 0.1% pluronic  and 100 μM sulfobromophthalein. Lung slices were de-esterified for another 30 minutes at 30°C in sHBSS containing 100 μM sulfobromophthalein before being placed in the perfusion chamber as described above.Fluorescence imaging was performed using a custom-built video-rate confocal or 2-photon scanning laser microscope. Either a 488 nm beam from a diode laser or an 800 nm beam from a Ti-sapphire laser  pumped with a 5 W 525 nm diode laser  was scanned across the specimen with two oscillating mirrors (X- and Y-scan) through an inverted microscope . For confocal microscopy, the emitted fluorescence (>510 nm) was separated from the excitation light by a dichroic mirror, a long pass filter and a confocal aperture . For 2-p2+ (NP-EGTA) or caged-IP3 was used to experimentally increase the [Ca2+]i or [IP3]i. Slices were initially loaded with Oregon Green 488 BAPTA-1 AM as described above. Subsequently, slices were incubated, at RT, with either 1 μM NP-EGTA AM (for 15 min) or 2 μM Caged-iso-InsP3/PM (for 1 hr) in sHBSS containing 0.1% pluronic and 100 μM sulfobromophthalein followed by de-esterification for 30 min in the sHBSS containing 100 μM sulfobromophthalein. The details of the flash photolysis setup have been previously described i  from the SR or the subsequent refilling of the depleted internal Ca2+ store.To explore the mechanisms by which cAMP reduced the frequency of the Cares Fig. . In viewres Fig. . This trion Fig. . The ration Fig. . The ration Fig. . The sim2+]i, brought about by decreasing Ca2+ influx and/or increasing Ca2+ efflux or reuptake, has been proposed as a mechanism by which cAMP relaxed airway SMCs, we examined the effect of elevating [Ca2+]i during the response of SMCs to cAMP. The [Ca2+]i was increased with 3 different agents: 1) NP-EGTA, a caged-Ca2+ compound, which upon UV photolysis releases Ca2+, 2) ionomycin, a Ca2+ selective ionophore, and 3) iberiotoxin (IbTX), a specific inhibitor of BKCa channels, which enhances Ca2+ influx by blocking K+ efflux to induce hyper-polarization.Because a reduction of i Fig  and 9B (2+]i of airway SMCs was observed when the lung slices treated with MCh (200 nM) and FSK (10 μM) were subsequently exposed to IbTX (50 nM). The airway responded with a quick and full relaxation within 3 mins . When the intensity and exposure time of UV light was optimized, repetitive UV flashes could induce similar repetitive Ca2+ waves in the same SMC.Because Ca2+ oscillations and the accompanying relaxation of SMCs were observed when the slices were exposed to MCh (200 nM) and FSK (10 μM). But, in response to UV illumination  and photolysis of caged IP3, the frequency of the Ca2+ oscillations increased significantly for a short period  induced a single spike of elevated [Ca2+]i in untreated airway SMCs  in a variety of cell types, we examined the effect of CICR via RyR in airway SMCs. We found that exposure to ryanodine  induced only a small and slow decrease in the MCh-induced airway contraction  is involved in Ca2+ conditions required for the induction of CICR, we released Ca2+ by flash photolysis of caged Ca2+. When a restricted area within a single SMC was exposed to UV light , AC can also be inhibited by receptor-coupled G-inhibitory proteins (Gi). Therefore, a potential mechanism for the down-regulation of the ISO response is the inhibition of AC by activation of M2 muscarinic receptors and their associated Gi by MCh i within the SMCs initially increases but subsequently declines. The idea that increases in [cAMP]i correlate with relaxation was addressed by circumventing the β-adrenergic receptor by either activating adenylyl cyclase with FSK or by increasing the [cAMP]i directly with 8-bromo-cAMP. A major characteristic of FSK or 8-bromo-cAMP-induced relaxation was that the relaxation was sustained and that re-contraction never occurred. This response is consistent with the idea that the [cAMP]i must be maintained or increased if relaxation is to be sustained and that some form of desensitization of β-adrenoceptor occurs allowing re-contraction.Because β-adrenoceptor agonists primarily relax SMCs by increasing [cAMP]i with airway relaxation has been reported in mice i of the Ca2+ oscillations is substantially high, that increases in [cAMP]i induce airway relaxation by reducing the frequency of Ca2+ oscillations in SMCs rather than by lowering the baseline of global [Ca2+]i.The correlation of increases in [cAMP] in mice , rats i did not significantly affect the Ca2+ signaling or contractile response induced by repetitive exposures to caffeine. These results demonstrate that the Ca2+ capacity of the internal store was fully restored to normal in the presence of cAMP and imply that Ca2+ loading of the SR by Ca2+ pumps was not significantly altered.To test the first hypothesis, that the internal Ca2+ influx to limit the Ca2+ available to replenish internal stores and maintain the ongoing Ca2+ oscillation, we used flash photolysis of caged-Ca2+ or exposure to ionomycin, a Ca2+ ionophore, to directly increase the available Ca2+. We found that while a transient global increase in [Ca2+]i resulted from the photolysis of caged-Ca2+, this had little effect on the slow frequency of the Ca2+ oscillations in the presence of FSK. Furthermore, the application of ionomycin initially inhibited the FSK-reduced Ca2+ oscillation to relax the airway. However, the gradual increase in [Ca2+]i induced by continued exposure to ionomycin subsequently induced re-contraction of the airway. Because in both experiments, an increase in the frequency of the Ca2+ oscillations was not observed, these results indicate that a lowered Ca2+ influx is not a limiting factor. It is possible that ionomycin might have emptied the Ca2+ of SR to inhibit the Ca2+ oscillations; however considering the higher concentration of Ca2+ in the extracellular environment it is unlikely that the internal store would have been emptied before an influx of Ca2+ had occurred.To investigate the second possibility that cAMP decreased Ca2+ influx appears to involve membrane hyper-polarization resulting from cAMP-activation of BKCa channels to down-regulate voltage-dependent Ca2+ influx i is not the primary cause of the modulation of the frequency of Ca2+ oscillations.Another mechanism inhibiting SMC Ca+ influx -21. Specnea-pigs , horse i was increased by flash photolysis. This response was accompanied by the re-contraction of airway SMCs. The idea that the sensitivity of the IP3R to IP3 is down-regulated by FSK is further supported by the finding that photolysis of caged IP3 induced a propagating Ca2+ wave in the absence, but not in the presence of FSK. A similar effect of cAMP on Ca2+ release has also been reported in other types of SMCs or non-excitable cells i; the receptor is closed by high Ca2+ concentrations at a constant IP3 concentration [3]i, the bell-shaped response curve is shifted to the left, with the result that the IP3R closes at a lower [Ca2+]i i ,52. In v3R is a reduction in the production of IP3. This idea is based on the evidence that G protein-activated PLCβ activity can be inhibited by PKA-induced phosphorylation [An additional mechanism that would augment the inhibitory action of cAMP on the IPrylation ,5,53,54.2+ [2+/calmodulin while phosphorylation of Rho-kinase or protein kinase C can increase the activity of MLCP. We have also found that cAMP can down-regulate the Ca2+-sensitivity of agonist-induced contraction in lung slices [2+ oscillation frequency.An alternative mechanism for cAMP-induced SMC relaxation is the desensitization of contractile apparatus to Ca2+ ,24,40,55g slices . However2+ oscillations controls the contractile state of airway SMCs and that compounds that elevate cAMP slow the frequency of Ca2+ oscillations within SMCs to proportionally induce airway relaxation. Furthermore, cAMP appears to regulate the Ca2+ oscillation frequency by inhibiting Ca2+ release through IP3R rather than influencing Ca2+ influx or the refilling of internal stores. In addition, we find that, under normal conditions, the RyR is not essential for Ca2+ oscillations of airway SMCs.In summary, we conclude that the frequency of Ca


We compared the effect of extracellular ATP on rat trachea and extrapulmonary bronchi (EPB) and both human and rat intrapulmonary bronchi (IPB), and investigated the implicated signalling pathways.Extracellular ATP may modulate airway responsiveness. Studies on ATP-induced contraction and [Ca2+]i was monitored fluorimetrically using indo 1 in freshly isolated and cultured tracheal myocytes. Statistical comparisons were done with ANOVA or Student's t tests for quantitative variables and χ2 tests for qualitative variables. Results were considered significant at P < 0.05.Isometric contraction was measured on rat trachea, EPB and IPB isolated rings and human IPB isolated rings. i response. The contractile response was not reduced by thapsigargin and RB2, a P2Y receptor inhibitor, except in rat and human IPB. By contrast, removal of external Ca2+, external Na+ and treatment with D600 decreased the ATP-induced response. The contraction induced by α-β-methylene ATP, a P2X agonist, was similar to that induced by ATP, except in IPB where it was lower. Indomethacin and H-89, a PKA inhibitor, delayed the return to baseline in extrapulmonary airways.In rat airways, extracellular ATP (102+ influx in addition with, in IPB, P2Y receptors stimulation and Ca2+ release from intracellular Ca2+ stores. Extracellular Ca2+ influx occurs through L-type voltage-dependent channels activated by external Na+ entrance through P2X receptors. The transience of the response cannot be attributed to ATP degradation but to purinoceptor desensitization and, in extrapulmonary airways, prostaglandin-dependent PKA activation.Extracellular ATP induces a transient contractile response in human and rat airways, mainly due to P2X receptors and extracellular Ca Bergner and co-workers reported, in mouse freshly ASM cells, that ATP induced an oscillating [Ca2+]i response [2+]i response [2+]i response to intracellular Ca2+, whereas in pig cultured ASM cells, Sawai and co-workers showed that the ATP-induced [Ca2+]i response was decreased in the presence of extracellular Ca2+ i responses of isolated tracheal myocytes were monitored fluorimetrically using the Ca2+-sensitive probe indo-1 as previously described [2+]i was estimated from the 405/480 ratio using a calibration for indo-1 determined within cells.[Caescribed . Briefly2+]i was observed during test ejections of PSS (data not shown). Generally, each record of [Ca2+]i response was obtained from a different cell. Each type of experiment was repeated for the number of cells indicated in the text.ATP or ACh was applied to the tested cell by a pressure ejection from a glass pipette located close to the cell. No change in i (data not shown). DMEM, ITS, penicillin, streptomycin, amphotericin B and foetal bovine serum were from GIBCO-BRL .Collagenase (type CLS1) was from Worthington Biochemical Corp. . Bovine serum albumin, acetylcholine, carbachol, ATP, ATP-γ-S, α-β-methylene ATP, D600, RB2, H-89, caffeine and thapsigargin were purchased from Sigma . Indo-1 AM was from Calbiochem . Indo-1 AM and thapsigargin were dissolved in dimethyl sulphoxide which maximal concentration used in our experiments was < 0.1% and had no effect on the resting value of the i response of isolated cells were carried out with Student's t tests for quantitative variables and χ2 tests for qualitative variables. Results were considered significant at P < 0.05Data are given as mean ± SEM. The maximal contraction R10, is shown in figure ATP induced a fast and transient contraction of rat isolated airway rings which amplitude depended on the concentration of agonist and the location along the airway tree. Original trace obtained in IPB is presented in figure -3 M) on epithelium-denuded rings. Measurements were repeated on 6 to 8 specimens. The response pattern was similar to that obtained in intact rings , 10-5 M (n = 65), 10-4 M (n = 97), and 10-3 M (n = 82) on myocytes freshly isolated from rat trachea. Original representative [Ca2+]i responses are shown in figure 2+]i rise followed, in some cases, by several subsequent [Ca2+]i oscillations. The percentage of responding cells, the amplitude of the [Ca2+]i peak, and the percentage of oscillating responses were concentration-dependent. Similar experiments were performed with 10-5ACh (n = 61), a concentration that induces the maximal [Ca2+]i response [2+]i peak was 627 ± 30.2 nM, the percentage of oscillating response was 39.3%, and the frequency of oscillations was 7.83 ± 0.69 oscillations/min. Compared to the cholinergic response, the percentage of responding cells to 10-3 M ATP and the frequency of oscillations were significantly lower, but not the amplitude of the peak nor the percentage of oscillating responses.In a first set of experiments, ATP was applied at 10response . The per2+]i response to ATP only in cultured cells [2+]i response to 10-3 M ATP in cells cultured for 3 days (n = 27) in non-proliferating medium and 10 days in proliferating medium(n = 35)  with that obtained in freshly isolated cells. After 2 days of culture in non-proliferating medium, the percentage of responding cells as well as the amplitude of the [Ca2+]i peak in responding cells were significantly reduced  figure . Culturee figure . To see d figure  and 4D, 2+ stores in the response to ATP, we performed the following experiments: in the absence of extracellular Ca2+, rings from rats airways (n = 6 to 8) were exposed to 10-6 M thapsigargin, an irreversible SERCA blocker. Ca2+ release from the SR was triggered by 5 mM caffeine application for 30 min, followed by wash up. Such a protocol ensures the emptiness of the SR, which was verified by the fact that in these conditions, the contractile response to ACh, which has been shown to act via intracellular Ca2+ release from the SR i response in the responding cells, as shown in figure 2+]i oscillations.Experiments in the absence of external Ca2+, we tested the effect of 10-5 M D600, an inhibitor of the L-type voltage-dependent Ca2+ channels on the contractile response to 10-3 M ATP (n = 7 to 10). As shown in figure max was significantly reduced in the presence of D600. In a following series of experiments, 10-3 M ATP was applied to the rings in the absence of extracellular Na+. In these conditions, the ATP-induced response was significantly reduced in each type of rings, as shown in figure + did not modify the contractile response to the depolarizing agent KCl (30 mM) (n = 5 to 7), as shown in figure Since ATP-induced response appeared to be dependent on extracellular Ca-4 M. As with ATP at the same concentration, the α-β-methylene ATP-induced contraction was transient. The amplitude of the contractile response was not different from experiments with ATP in similar conditions in extrapulmonary airways, but was significantly reduced in IPB . RB2 also significantly reduced the contractile response of human IPB (n = 8). Results are shown in figure B figure . TR10 wa-7 to 10-4 M. Results are shown in figure R10 (n = 5 to 10).In order to evaluate a possible role of ATP degradation in the transience of the response, we assessed the effect of the non-hydrolysable ATP analogous, ATP-γ-S, from 10-5 M indomethacin. Rat tissues were incubated in the presence of indomethacin 30 min before ATP stimulation. The maximal contractile response was not significantly modified  declare that they have no competing interests.2+]i recording on isolated cells, participated in the analysis of the data, and helped the draft of the manuscript. RM participated in the design of the study and helped the draft of the manuscript. ER conceived the study, participated in its design, helped in [Ca2+]i recordings, performed statistical analysis and drafted the manuscript.BM carried out the contractile experiments and [Ca


See also Nurden AT. Does ATP act through P2X1 receptors to regulate platelet activation and thrombus formation? This issue, pp 907–9.2+ mobilization; however, the extent to which secondary activation of adenosine 5′-triphosphate (ATP)-gated P2X1 receptors contributes to intracellular Ca2+ responses remains unclear. We now show that selective inhibition of P2X1 receptors substantially reduces the [Ca2+]i increase evoked by several important agonists in human platelets; for collagen, thromboxane A2, thrombin, and adenosine 5′-diphoshate (ADP) the maximal effect was a reduction to 18%, 34%, 52%, and 69% of control, respectively. The direct contribution of P2X1 to the secondary Ca2+ response was far greater than that of either P2Y receptors activated by co-released ADP, or via synergistic P2X1:P2Y interactions. The relative contribution of P2X1 to the peak Ca2+ increase varied with the strength of the initial stimulus, being greater at low compared to high levels of stimulation for both glycoprotein VI and PAR-1, whereas P2X1 contributed equally at both low and high levels of stimulation of thromboxane A2 receptors. In contrast, only strong stimulation of P2Y receptors resulted in significant P2X1 receptor activation. ATP release was detected by soluble luciferin:luciferase in response to all agonists that stimulated secondary P2X1 receptor activation. However, P2X1 receptors were stimulated earlier and to a greater extent than predicted from the average ATP release, which can be accounted for by a predominantly autocrine mechanism of activation. Given the central role of [Ca2+]i increases in platelet activation, these studies indicate that ATP should be considered alongside ADP and thromboxane A2 as a significant secondary platelet agonist.In the platelet, it is well established that many G-protein- and tyrosine kinase-coupled receptors stimulate phospholipase-C-dependent Ca Althougectively . The impablished ,6. In coensitize  and theiregation . Howeverregation ,9. One ethrombin ,10,11. Wm) and type VII apyrase (0.32 U mL−1) as described elsewhere i under closely matched conditions. 600 nmα,β-meATP did not interfere with the luciferin-luciferase assay for ATP .ATP secretion from washed platelet suspensions was measured in a Model 400 lumi-aggregometer as above, following the kit manufacturer’s guidelines. The luminescence channel output was amplified tenfold before acquisition to computer. Platelets used in luminescence studies were loaded with fura-2 to provide parallel measurements of ATP secretion and [Ca2+]i are representative of experiments from ≥ four separate donors. The magnitude of Ca2+ responses was assessed from the peak increase above prestimulus level. Average values represent the mean ± SEM, with statistical analysis performed using Student’s paired t-test. Significance is indicated at levels of 0.05 (*), 0.01 (**), 0.005 (***), and 0.001 (****).Sample records of [Ca2+]i responses, we first derived conditions that provide maximal block, but with strict limits on selectivity. Thus, whilst 3 μm was the minimum concentration of the suramin derivative NF449 i increases over only a narrow range of concentrations; consequently, small and large responses could be achieved with 0.5 and 1.0 μm U46619, respectively. Inhibition of P2X1 receptors caused a marked decrease in the peak [Ca2+]i increase at both these concentrations; peak responses to 1 and 0.5 μm U46619 were reduced to 42 ± 11% and 38 ± 4%, respectively, by 1 μm NF449 and 34 ± 7% and 35 ± 3%, respectively, after α,β-meATP  to the effect of P2X1 desensitization alone.We also examined the role of P2X,β-meATP . Block o1 receptors in ADP-evoked responses is difficult to study because of contamination of commercial samples with ATP i independently of ADP-activated P2Y receptors. This enhancement of the initial Ca2+ responses may explain the previously reported ability of P2X1 receptors to potentiate aggregation at low levels of collagen and thrombin [1 receptors as a secondary Ca2+ influx pathway could also help explain the resistance to thrombosis displayed by P2X1−/− mice [1 receptors [An increase in [Caoduction ,27. The oduction –30. The thrombin ,10,11. T−/− mice  and the eceptors .2+ by a combination of IP3-dependent store release and influx across the plasma membrane [2+ store depletion (store-operated Ca2+ influx) or the products of PLC such as diacylglycerol (termed store-independent Ca2+ influx) [1 receptors should be considered as a significant additional contributor to early Ca2+ influx in the platelet following stimulation by agonists acting via G-protein-coupled receptors and tyrosine-kinase-coupled receptors.Platelet receptors coupled to phospholipase-C activation mobilize Camembrane . The cur influx) ,23,24. W1 is able to potentiate the non-selective cation channel coupled to P2Y receptors in murine megakaryocytes and to accelerate and amplify the P2Y-evoked Ca2+ mobilization in suspensions of human platelets [1 and P2Y receptors are active, the present study shows that P2X1 does not rely upon interactions with co-activated P2Y receptors to contribute as a secondary Ca2+-elevating pathway in the platelet. Serotonin release from dense granules also has little or no role in the Ca2+ responses in these experiments as a maximal concentration of serotonin (1 μm) evoked a Ca2+ increase of < 15 nm, and 100 nm methiothepin (a concentration that blocked responses to 1 μm serotonin) had no significant effect on the Ca2+ increase evoked by 1 μm U46619 . A likely explanation for the important independent role of P2X1 is that the primary agonist already stimulates the phospholipase-C-dependent Ca2+ fluxes subsequently targeted by released ADP via P2Y receptors, whereas P2X1 receptor-cation channels represent a separate route for elevating Ca2+. In addition, we show that P2X1 receptors can be activated only when small total amounts of ATP are released, thus allowing this pathway to contribute at low levels of dense granule secretion. Moreover, our experiments were conducted in the presence of apyrase, indicating the ability of ATP to activate P2X1 receptors even in the presence of significant levels of ectonucleotidase activity. The rapid kinetics of P2X1 activation by its ligand [2. In particular, because P2X1 causes elevation of only Ca2+ (and Na+), stimulation of complementary signaling pathways by P2Y12 receptors remains crucial for collagen and thromboxaneA2 receptor-evoked aggregation [We have previously shown that P2Xlatelets . Althoug1 receptors occurs via an autocrine rather than a paracrine manner. First, the bulk phase ATP level when P2X1 maximally contributes is sufficient to activate less than 10% of P2X1 receptors (1 receptors contribute to the collagen-evoked Ca2+ increase earlier than the average extracellular ATP increases (1 receptors at sites of secretion may also explain how ATP release can so efficiently activate P2X1 receptors. Although there is no direct evidence for such localization at present, lipid rafts may play an important role as these microdomains in platelets contain both P2X1 receptors and SNARE proteins, and raft disruption leads to inhibition of P2X1 responses and exocytosis [Two pieces of evidence suggest that secondary activation of P2Xeceptors . Secondlncreases . These oocytosis ,32.1 receptors can represent a significant pathway for early Ca2+-mobilization following activation of a variety of major receptors linked through both G-proteins and tyrosine kinases in the platelet. Thus, P2X1 receptors should be considered alongside store-operated and store-independent channels as an important route for Ca2+ influx, and therefore to be a more significant potential antithrombotic target than previously recognized.In conclusion, we show that P2X


We therefore tested several Rho-kinase inhibitors and a cell-permeable analogue of cAMP (dbcAMP) for their ability to reverse ET-1-induced constriction and Ca2+-sensitization.Vasospasm of the spiral modiolar artery (SMA) may cause ischemic stroke of the inner ear. Endothelin-1 (ET-1) induces a strong, long-lasting constriction of the SMA by increasing contractile apparatus Ca2+sensitivity was evaluated by correlating vascular diameter and smooth muscle cell [Ca2+]i, measured by fluo-4-microfluorometry and videomicroscopy.The present study employed SMA isolated from gerbil temporal bones. Ca50 ICof 3, 15, and 111 μmol/L, respectively. DbcAMP stimulated a dose-dependent vasodilation (50 Ec= 1 mmol/L) and a reduction of [Ca2+]i (50 EC= 0.3 μmol/L) of ET-1-preconstricted vessels (1 nmol/L). Fasudil and dbcAMP both reversed the ET-1-induced increase in Ca2+ sensitivity.The Rho-kinase inhibitors Y-27632, fasudil, and hydroxy-fasudil reversed ET-1-induced vasoconstriction with an 2+-sensitization. Therefore, Rho-kinase inhibitors or cAMP modulators could possess promise as pharmacological tools for the treatment of ET-1-induced constriction, ischemic stroke and sudden hearing loss.Rho-kinase inhibition and dbcAMP reversed ET-1-induced vasoconstriction and Ca The inner ear's blood supply depends solely on the spiral modiolar artery (SMA), a functional end artery. Vasospasm/constriction of the SMA can cause an ischemic stroke of the inner ear, leading to sudden sensorineural hearing loss (SSHL). Thus, investigating the mechanisms controlling the inner ear microcirculation is a prerequisite for the development of new strategies to treat SSHL.2+ ([Ca2+]i) and/or by an increase in the Ca2+ sensitivity of the contractile apparatus ex) was raised to 3 and 10 mmol/L by addition of CaCl2. A maximal vasodilation was induced by the removal of extracellular Ca2+. The nominally Ca2+-free solution contained (in mmol/L) 150 NaCl, 3.6 KC1, 1.0 MgCl2, 1.0 EGTA, 5.0 HEPES, and 5.0 glucose, pH = 7.4. Fluo-4-AM (Molecular Probes) was dissolved in anhydrous DMSO and stored in 1 mmol/L aliquots. Y-27632 was kindly provided by Welfide. Fasudil was obtained from Calbiochem. Fasudil (obtained from Tocris Cookson) was modified to hydroxyfasudil by Dr. Duy Hua, Dept of Chemistry, Kansas State University. All other chemicals were obtained from Sigma.The physiologic salt solution (PSS) contained (in mmol/L) 150 NaCl, 3.6 KC1, 1.0 MgClExperiments were conducted on tissues isolated from gerbils under a protocol that was approved by the Institutional Animal Care and Use Committee at Kansas State University. Gerbils were anesthetized with sodium pentobarbital (100 mg/kg i.p.) and decapitated. Temporal bones were removed, opened and placed into a micro-dissection chamber containing PSS at 4°C. The SMA was isolated from the cochlea by micro-dissection as described previously . Briefly2+]i has been described previously ex. The [Ca2+]i was monitored as fluorescence intensity and was normalized to the basal fluorescent emission prior to the beginning of each experiment. The fluorescence and diameter values taken for statistical analysis represent averages of the [Ca2+]i-fluorescence and the vascular diameter over 30 sec beginning 30 sec after the onset of stimulation. We carefully validated the [Ca2+]i -measurements with the non-ratiometric Ca2+-dye fluo-4 by (i) excluding artefacts due to vessel diameter changes , (ii) excluding significant differences in the magnitude of [Ca2+]i -changes between preparations, and (iii) assessing the same optimal dye-loading conditions in each experiment i, a strong and long-lasting vasoconstriction and a robust increase in the vasomotion of the gerbil spiral modiolar artery . Note that ET-1-induced a transient [Ca2+]i increase and a sustained vasoconstriction while exposure to 10 mmol/l Ca2+ induced an increase in [Ca2+]i and a parallel vasoconstriction.ET-1 (10 nmol/L) induced a transient increase in [Caery Fig. . The [Ca2+]i-levels. The Rho-kinase inhibitors Y-27632, fasudil and hydroxy-fasudil reversed ET-1-induced constriction (10 nmol/L) in a dose-dependent manner , 15 μmol/L  and 111 μmol/L , respectively. The Ca2+ sensitivity of the contractile apparatus was assessed as linear slopes obtained from correlations of [Ca2+]i and vascular diameter. ET-1 (100 pmol/L) increased the Ca2+ sensitivity . This possibly indicates that basal Rho-kinase activity is rather low under normal conditions i and Ca2+ sensitivity. Although cAMP reduces intracellular Ca2+ levels, the present data demonstrate that the main pathway targets the Ca2+ sensitivity of the contractile apparatus. This interpretation is consistent with findings obtained in other preparations such as permeabilized intestinal and bronchial smooth muscle. In these studies the authors also could show that cAMP mediates a Ca2+ desensitization of the contractile apparatus [2+ desensitization and vasodilation in the SMA [We show that cAMP is a potent vasodilating second messenger in the SMA, acting by mechanisms which decrease [Capparatus ,31. cAMP the SMA . The datAlthough the sudden loss of hearing causes substantial distress and pronounced long-term effects in affected individuals, adequate strategies to clinically treat the disorder are lacking. One subgroup of SSHL is believed to arise from SMA vasospasm(s) which ultimately lead to ischemic stroke of the inner ear . The tarThe author(s) declare that they have no competing interests.EQS conducted all of the experiments and was involved in all aspects of data analysis and manuscript preparation. PW assisted with all facets of this study, assisting with data analysis and manuscript preparation. PW also provided laboratory space, equipment and financial support. WA provided financial support and made substantial contributions to conception and design of the present study.All authors have been involved in drafting the manuscript and revising it critically for important intellectual content and given final approval of the version to be published.The pre-publication history for this paper can be accessed here:


Described here is a genetic approach for non-invasive detection of localized Ca2+ concentration ([Ca2+]) rises in live animals using bioluminescence imaging (BLI). Transgenic mice conditionally expressing the Ca2+-sensitive bioluminescent reporter GFP-aequorin targeted to the mitochondrial matrix were studied in several experimental paradigms. Rapid [Ca2+] rises inside the mitochondrial matrix could be readily detected during single-twitch muscle contractions. Whole body patterns of [Ca2+] were monitored in freely moving mice and during epileptic seizures. Furthermore, variations in mitochondrial [Ca2+] correlated to behavioral components of the sleep/wake cycle were observed during prolonged whole body recordings of newborn mice. This non-invasive imaging technique opens new avenues for the analysis of Ca2+ signaling whenever whole body information in freely moving animals is desired, in particular during behavioral and developmental studies.Rapid and transient elevations of Ca Abnormalities in the homeostatic regulation of Ca2+ signaling have pathological consequences relevant to many common diseases  Calcium (Ca2+ concentration ([Ca2+]) rises that are restricted in space (e.g. nano- & micro-domains), amplitude (100 nM–100's µM) and time (microseconds to seconds). The propagation of Ca2+ waves or other second messengers associated with Ca2+ signaling may also affect remote cellular regions, tissues, or other parts of an organism. In addition, Ca2+ oscillations of varying frequencies are important for gene expression and other rhythmic activities The selective regulation of many signal transduction pathways is partly facilitated by intracellular Ca2+ signals , optical imaging methods can provide the high degree of spatio-temporal resolution necessary for their characterization. Recently, these methods have been extended to in vivo approaches allowing [Ca2+] within the intact animal to be investigated under more physiological conditions in vivo imaging of the neonatal brain by fiber-optic based detection of Ca2+ sensitive dyes, led to the identification of early network Ca2+ oscillations (ENOs) occurring in the cortex of newborn mice during sleep 2+ sensitive probe was expressed in the muscles of live animals and gave accurate information about [Ca2+] in the mitochondrial matrix ([Ca2+]m) during relaxation/contraction cycles ca. 1 mm2), preventing longitudinal analyses or studies on Ca2+ signals over long distances and simultaneously across multiple systems.In keeping with the versatile nature of Cai.e. background noise is limited to that of the light detector). In recent years, whole animal bioluminescence imaging (BLI) has emerged as a sensitive method for localizing gene expression or cell migration in live animals 2+-reporter, which is based on the light emitting system of the jellyfish, Aequorea victoria2+ binding, aequorin undergoes a conformational change that oxidizes its substrate coelenterazine (CLZN) and chemiluminescence resonance energy transfer (CRET) to the GFP moiety occurs, with an emission maximum in the green (λ = 510 nm). GA has a low Ca2+ binding affinity, large dynamic range of light emission, is stable and has little, if any, toxicity, making it a potentially useful reporter for application in BLI studies Bioluminescent probes in which light is produced by enzymatic breakdown of a substrate have an excellent signal-to-noise ratio  transgene. The transcription unit was introduced by knock-in to the Hypoxanthine Phosphorylated Ribosyl Transferase (hprt) locus on the X chromosome mtGA contains the targeting sequence of subunit VIII of cytochrome c oxidase for localization in the mitochondrial matrix lox stop lox sequence 3′ to the strong ubiquitous promoter, CAG . Direct visualization of GFP fluorescence in fresh or fixed tissue shows expression of the reporter protein in muscle fibers of the tibialis anterior muscle (n = 52) (Transgenic mice carrying the r muscle . Electro(n = 52) . Finally(n = 52) . The res(n = 52) , thus co2+ oscillation waves (ENOs) in the developing neocortex 2+ oscillations in the cytosol have been reported to occur in synchrony with [Ca2+] changes in the mitochondrial matrix 2+]m oscillations could be detected in acute slices prepared from the neonatal mtGA mouse brain. For the Ca2+ dependent CRET reaction to occur, the genetically expressed mtGA must be reconstituted with the aequorin substrate, CLZN (mtGA-CLZN). As described in previous studies 2+]m were detected in both the cortex and hippocampus of acute brain slices prepared from 0 to 2 day old newborns (n = 3).Two-photon imaging of acute brain slices from newborn rats previously revealed large-scale, highly synchronized early network Ca2+ events decreased in frequency or were absent in brain slices from older animals (P4–P12) (data not shown). However, highly synchronized large-scale oscillations could be induced in brain slices from P4–P12 mice by lowering the external concentration of Mg2+ (n = 25) or by addition of bicuculline (10 µM) (data not shown). Blockade of Na+-channels with tetrodotoxin  (n = 3), completely abolished the [Ca2+]m oscillations, suggesting that they depend on neuronal activity. In addition, oscillations induced by low Mg2+ were reversibly blocked by the NMDA receptor antagonist, D-APV (50 µM), while those induced by bicuculline were reduced by both D-APV and the antagonist for AMPA receptors, CNQX (2 µM) (data not shown). Oscillatory rises in Ca2+ were absent or significantly reduced after incubation with the NADH-ubiquinone oxidoreductase (complex I) inhibitor, piericidin A (2 µM) (n = 2) or FCCP (2 µM) (n = 3), confirming the mitochondrial origin of these responses.These CaA (2 µM) , n = 2 oP (2 µM) , n = 3, 2+ from the sarcoplasmic reticulum (SR), which is rapidly sequestered by mitochondria 2+-responses associated with contraction of the hindlimb muscles could be detected within intact tissues of adult mice. In these studies, CLZN was introduced via a tail-vein injection of the substrate. We found that tetanic stimulation of the sciatic nerve in mice bearing mtGA-CLZN, lead to a rapid increase in light emission in the hindlimb muscles   . In contRenilla luciferase, indicate that CLZN has limited access to some tissues and that it can be unstable in biological media n = 4 mice, see 2+]m transients corresponding to tetanic contraction of the hindlimb muscle could be detected for up to 100 stimulations over a 1–2 hour period (n = 104). Similar optical signals were recorded when the same mouse was re-injected with CLZN on different days. Intramuscular injection of Ru360, a specific inhibitor of mitochondrial Ca2+ uptake, attenuated Ca2+ rises evoked by tetanic stimulation (70 Hz) of the sciatic nerve  2+-responses with good spatial resolution in intact tibialis anterior muscle fibers, in situ. Applying the same protocols for stimulation of the sciatic nerve as Rudolf et al., we obtained comparable results with BLI. However, in our studies we monitored light responses from the entire hindlimb muscle (or at least a large number of fibers from that muscle) using a larger field of view and a lower spatial resolution. 10 s trains of stimuli were applied every 60 s to the sciatic nerve at different frequencies (1–50 Hz) (n = 4 mice). Results show that mitochondrial Ca2+ uptake/release during single twitch muscle contractions (1 Hz) can be recorded in vivo, with a time resolution down to 40 ms . The intensity of the muscle contraction, i.e. the number of muscle fibres recruited, also increased with an increased stimulation voltage and there was a strong correlation between the mean light intensity and the peak amplitude of the electromyogram ) using an imaging system that allows co-registration of the video and bioluminescent images. Three major behavioral states, corresponding to the known components of sleep/wake cycles, were identified: (i) whole body startles and myoclonic twitches (see 2+-transients having short durations (<1 s) . Variations in [Ca2+]m detected from whole body recordings of newborn mice had distinct patterns (2+-response patterns were observed to be made up of two major forms (a) fast Ca2+ transients having short durations of 100's of ms to 1 s, which were predominant (2+ responses (>1 s) (n = 9) and another group of P1–3 (n = 8). Sustained Ca2+ responses had highly variable durations in both groups (>1–200 s) (2+ transients were often observed to precede sustained responses (see n = 4). The mean interval between fast Ca2+ transients and sustained responses was not significantly different between the two groups  compared to older P1–P3 animals  ). Overall, the percentage of time recorded in the absence of sustained Ca2+ responses was 88.5±3.5 % (n = 9) and 76.9±3.5 % (n = 8) for P0–1 and P1–3, respectively , nor when the substrate was directly injected into the brain (n = 3).In further studies, bioluminescence was monitored in newborn animals over longer recording durations (1–1.5 hours). Similarly to studies on the hindlimb muscle, signal was detectable within 15 minutes, and for up to 10 hours after i.p. injection in pups that had been returned to their litter (patterns . Whole bo groups . However events) . Sustainectively . Recent GA mouse . However2+-transients could be detected during kainate-induced seizure in non-anaesthetised 6–7 day old mice. Imaging was started immediately after i.p. injection of kainic acid (25 mg/Kg) and was continued for up to 1 hour. In the sequence of images shown  and We next investigated if Caes shown , the dynstration . Elevatebona fide physiological context, i.e. in live, un-restrained and non-anaesthetised animals. Our objective here was to develop an approach allowing non-invasive in vivo detection of local Ca2+-signaling in freely moving animals. A genetic approach was chosen because it allows specific in vivo targeting, endowing optical signals with a precise knowledge of their origin even when detection takes place from outside of the living animal. In previous studies we showed that the Ca2+-sensitive genetically encoded bioluminescent protein GFP-aequorin, could be expressed in several organelles such as the mitochondrial matrix for detection of localized [Ca2+] in cells 2+] will enable the role of this compartment to be examined in physiological processes  and in pathological conditions (e.g. excitotoxicity and metabolic disorders).A challenge in biology is to monitor signal transduction in a 2+ concentration of the mitochondrial matrix to be non-invasively investigated. Whole animal recordings of light emission during induced Ca2+ concentration changes in hindlimb muscle mitochondria during contraction/relaxation cycles are well correlated with previously reported data obtained by fluorescence microscopy in both in vivoin vitroin vivo imaging of calcium signaling has remained more or less invasive, precluding its use in freely moving, unrestrained and behaving animals. In contrast to methods using fluorescence imaging, bioluminescence does not require light excitation and the high contrast-to-noise ratio afforded by mtGA-CLZN is based on the absence of background bioluminescence in tissues. Furthermore, the acquisition of signals is undertaken with a photon counting system based on a cooled GaAs intensified charge-coupled device having no readout noise. The importance of this approach is that the imaging parameters  do not need to be predefined. This opens up the possibility to detect Ca2+ signals covering a broad temporal range (from 100's milliseconds to 100's seconds), which can be identified in post-processing of the data according to signal intensities and kinetic properties.When transgenically expressed mtGA is reconstituted with coelenterazine (mtGA-CLZN), it provides a high signal over background, allowing elevations in the Caa posteriori reconstruction of the image set in one or several optimized sequence combinations, without loss of quantitation accuracy. In particular, the smoothing algorithms use unitary gain filters that have no effect on output values as long as measurements are made in a region of interest larger than the smoothing kernel  and in the absence of light absorption and scattering by tissues, a spatial resolution of 100 µm or 200 µm depending on the field of view chosen by the operator . Schematically, the photon fluxes reaching the detection system are a combination of (i) the photon emission rate, which depends on the biological construction and the biological activity, and (ii) the absorption and scattering of photons by the living tissues, which depend on their depth of emission and their wavelength. This latter component cannot be easily measured and is unaccounted for in the estimation of the “true” spatial resolution of the images. In practice, either the temporal or the spatial resolution, or both, need to be decreased in order to allow sufficient statistics of photon counting. In the different experiments reported here, which represent a panel of different levels of light emission in different volumes of biological tissue, either the intrinsic temporal resolution limit (40 ms) was reached at the extent of a relatively large degradation (i.e. smoothing) of the spatial resolution . The preparation was then centrifuged (×2), at 800×g for 10 min. Each time the supernatant was then collected and centrifuged again at 9,000×g for 10 min to pellet the mitochondrial population, which was resuspended in , and centrifuged at 8,000×g for 10 min. Further mitochondrial purification was performed by Ficoll gradient as previously described Purified fractions of mitochondria were obtained at 4°C from adult transgenic mice using a similar method as previously reported SDS-PAGE was performed according to the method of Laemmli (1970), using 12% gels. Immunoblotting was undertaken on Immobilon-P membranes . Incubations were undertaken first with rabbit polyclonal antibodies  in PBS containing 5 % milk supplemented with 0.3% Tween-20, and immunoreactive proteins were then observed by incubation with HRP-conjugated goat anti-rabbit IgG antibodies followed by enhanced chemiluminescence (ECL) western blotting detection reagents .The complete anterior tibialis muscle was removed, washed in PBS 1X solution then fixed for 1h in PFA 4% solution . After washing in PBS 1X solution, the muscle was teased apart in PBS to obtain isolated fibers. GFP fluorescence was directly visualized in fibers after washing in PBS and mounting in FluorSave reagent . For the immunofluorescence studies, non-specific antibody binding sites were blocked by incubating fixed muscular fibers for 30 min in PBS containing 10% normal goat serum (NGS)/0.25% Triton X-100. After incubation, the fibers were immunostained with a 1∶500 dilution of anti-GFP polyclonal antibody  and a 1∶250 dilution of anti-cytochrome C monoclonal antibody (BD Biosciences) in PBS containing 2% NGS/0.25% Triton X-100/0.2% Bovine Serum Albumin (BSA) washed with PBS containing 0.25% Triton X-100/0.2% BSA several times, incubated with a 1∶1000 dilution of a Cy™3 conjugated anti-mouse antibody (Jackson ImmunoResearch) and a 1∶1000 dilution of a Alexa®Fluor 488 goat anti-rabbit antibody . The stained preparation was then mounted in FluorSave reagent. Confocal analysis was performed using an Axiovert 200M laser scanning confocal microscope  through a 63×/1.4 NA, oil-immersion objective using LP560 and BP505-550 filters. The pinhole aperture was set at 98 µm and images were digitized at 8-bit resolution into a 512×512 array.Anterior tibialis muscle was dissected out and fixed for 1 h with 4% PFA  in 0.1 M phosphate buffer . Muscle blocks were post-fixed for 30 min with PBS containing 4% PFA, 0.1% glutaraldehyde  and 0.2% picric acid (Sigma-Aldrich), followed by 0.5% osmium tetroxide (Electron Microscopy Sciences) for 30 minutes in the same buffer and embedded in LR-White resin (Electron Microscopy Sciences). The post-embedding inmunogold GFP labeling was followed as previously reported 2 solution was injected into the well and recording was continued for a further 30 sec. Light emitted through short band-pass filters ‘blue’ (460/30 nm) and ‘green’ (515/20 nm) was detected in independent experiments using a luminometer . The ratio of light detected  through the ‘green’ to ‘blue’ filters was then determined. All experiments were carried out at room temperature.Cells were prepared as previously described i.p) injection for neonates. Coelenterazine was injected at 2–4 mg/kg mouse, in a volume of 100–150 µl for adults and 20–40 µl for neonates.Coelenterazine  was dissolved in ethanol to a stock concentration of 10 mM and stored at −20°C. Care was taken to protect the substrate from light and oxygen. The stock solution was then diluted in sodium phosphate buffer immediately before tail-vein (i.v.) for adults or intra-peritoneal (2/5 % CO2) artificial cerebrospinal fluid (ACSF) without added CaCl2, containing 124 mM NaCl, 3 mM KCl, 1.3 mM MgCl2, 25 mM NaHCO3, 1.25 mM NaHPO4 and 10 mM glucose. Horizontal or coronal slices (400 µm thick) were rapidly cut using a vibratome  and then transferred to another smaller chamber kept at room temperature and containing ACSF (as above but with 2 mM CaCl2 added) and also 5–10 µM coelenterazine. The chambers containing the slices were then maintained in the dark for at least 1 hour before being transferred to a slice chamber (Warner Instruments Inc. USA) for imaging and this was then placed onto an inverted microscope, where perfusion (1 ml/min) with oxygenated ACSF was continued throughout the remainder of the experiment. Slices were imaged through a 10X Plan-NEOFLUAR objective on a combined bioluminescence/fluorescence wide field microscope system as previously described 2 (256×256 pixels), was analysed for each experiment. Stock solutions of TTX , D-APV , CNQX , FCCP , Piericidin A , were stored at −20°C and diluted in ACSF before addition to the perfusate.0–2 day old mouse pups were decapitated and brains were rapidly removed. After removal of the cerebellum, the brain was placed on a small platform and immersed into ice-cold oxygenated  was injected intramuscularly, in small aliquots (<20 µl) in 5 or 6 different locations across the hindlimb muscles.8–12 week old male mice with ubiquitous expression of mtGA in all cells of the muscles were used in experiments. Animals were anaesthetised by isoflurane and the fur covering the area of the hindlimb was shaved to maximize light transmission. The sciatic nerve was then isolated and a custom made platinum bipolar electrode was attached for stimulation of muscle contraction in the hindlimb. The anaesthetised mouse was then transferred to the imaging chamber and anaesthetic was continuously administered by nose cones throughout the imaging procedure. An isolated stimulator  was used to apply a monopolar voltage pulse (polarity was chosen to have the lowest threshold voltage), which was usually in the range of 0.5–1.5 V/0.1-5 ms. The DS2A was driven by a pulse generator  or a 4030 timer generator (Digitimer Ltd). Variable stimulation frequencies were applied as described in the text, with each pulse having 5 ms duration. This protocol was based on similar work previously described in vivo BLI images were acquired using either one of two whole-body small animal imaging systems based on a highly sensitive photon counting technique: the “Photon Imager” or the “Video Imager” . Both systems consist of a light tight chamber housing a third generation cooled GaAs intensified charge-coupled device (ICCD) camera (1080×1440 pixels) operating in a photon counting mode and an F 1.4 objective lens. The field of view (FOV) is either 16×12 cm or 8×6 cm depending on the platform position, yielding an “intrinsic” resolution of the camera of 100 µm  or 200 µm (largest FOV). This true spatial resolution in the final image depends on light absorption and scattering in tissues and also on image smoothing, which is user-dependant. Post acquisition, spatial smoothing of the BLI data is available (i) during reconstruction of the BLI image, by a Gaussian filter with a FWHM  size of 3, 5, or 9 times the pixel size; or (ii) during reconstruction of the composite (BLI plus video) image, by a Gaussian filter with a FWHM expressed in mm. Information regarding the processing of images is given in the legend of each figure. Color scales represent photons/pixel, unless otherwise stated.All With the “Photon Imager”, video images can be taken before or after recording the bioluminescence images, for superimposition. The “Video Imager” is very similar to the “Photon Imager”, for the exception that it enables simultaneous registration of bioluminescence images and video images using infrared LEDs. The light collected by the objective lens is split by a 45° angle mirror into two beams. One of these beams is then recorded as the video signal on a CCD camera and the other beam is acquired as the BLI signal (as described above) after filtering with a short-pass filter. Both systems operate at up to 25 frames/s (40 ms exposure time) but longer integration times can be selected after acquisition for data analysis and replay.An average of 10 electromyograms (EMG) were recorded by a ball shaped silver electrode  covered with AgCl and positioned under the leg skin in the vicinity of the tibialis muscle while stimulating the sciatic nerve. The induced contractions were of the isotonic type, the leg being free to move. EMG signals were recorded through a NPI  amplifier system  onto a “D.A.T.” recorder  for further measurement and analysis. Measurements were performed using an Axon Instruments  TL1 acquisition system operated with Pclamp-6 software ; analysis was made with the Origin-7 graph package . The same stimulation system as described above was used. The light intensity during muscle contractions was recorded as a function of time using 40 ms frames. Light and EMG records were synchronised by generating a dim pulse of light from a small LED located near by the animal under study in the imager, in synchrony with the trigger applied to the isolated stimulator.2+-induced bioluminescence was co-registered with the video recording using an acquisition rate of 25 Hz . Motor twitches and coordinated movements were also visually recorded by placing a scoring of 1 for each frame (1 frame/s) when movements were present and 0 when they were not. The resulting plot was then generated using Microsoft Excel Software. For comparison to Ca2+-responses, coordinated movements are defined as sustained motor activity of the limbs or head. Motor twitches are defined as phasic, rapid, and independent movements of one limb or the tail. Startles are sudden phasic contractions of the body muscles, with the simultaneous involvement of all extremities 1–2 day old mice expressing mtGA were injected (i.p.) with native coelenterazine . After 1–2 hours, non-anaesthetised mice were imaged at room temperature (25°C) un-restrained and freely moving. Ca2+-responses (<1 s duration) and sustained Ca2+-responses (>1 s) was determined. A threshold was set for Ca2+-responses at 30 photons/s above the mean of the baseline in subsections of the trace. The interval between sustained Ca2+-responses was determined by taking the time point at the start of one sustained response to the time point at the start of the next sustained response. The interval between fast Ca2+-transients was determined between each sleep/wake cycle (i.e. between sustained responses). The duration of sustained Ca2+-responses was determined as the time point at the start of the rising phase until the last visible peak or shoulder above the threshold, in order to avoid including the decay phase.In additional experiments, freely moving animals were also monitored with the Photon Imager. 0–3 day old mice expressing mtGA were injected (as above) with coelenterazine and imaged 1–2 hours later at room temperature (25°C). Mouse pups were placed inside a circular cardboard barrier that was 4 cm in diameter for 30 min prior to the beginning of recordings. Recordings of mice were then undertaken for up to 1 hour. For experimental analysis, a single region of interest was drawn over the entire area within the barrier and plots with 120 ms of time integration over 1500 s were analysed. The interval between fast Ca2+ fluxes were monitored in the whole animal during seizure. Kainic acid (Sigma-Aldrich) was prepared by dissolving the lyophilized drug first in a drop of NaOH and then making a further dilution in PBS to the required concentration.Mice (P6–P18) were injected (i.p.) with coelenterazine (4 µg/g). After 2 hours, kainic acid (25 mg/kg) was injected (i.p.) and the distribution of mitochondrial CaData was analyzed using Microsoft®Excel 2002. Results are given as the mean±s.e.m.2+], calcium concentration; [Ca2+]m, [Ca2+] in the mitochondrial matrix; BLI, bioluminescence imaging; ENO, early network oscillations; GFP, green fluorescent protein; GA, GFP-aequorin; CLZN, coelenterazine; CRET, chemiluminescence resonance energy transfer; hprt, Hypoxanthine Phosphorylated Ribosyl Transferase; mtGA, mitochondrially targeted GFP-aequorin; TTX, tetrodotoxin; D-APV, D-2-amino-5-phosphonovaleric acid; CNQX, 6-cyano-7-nitro-quinoxaline-2-3-dione; FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; SR, sarcoplasmic reticulum; YC2, second generation yellow cameleon; FOV, field of view; BRET, bioluminescence resonance energy transfer; ES, embryonic stem cells; CAG, pCMV-chicken β-actin; RLU, Relative light units; FWHM, Full-Width Half-Maximum.Abbreviations:  (bioluminescence) were co-registered together with the video image in a newborn mouse (P1). The movie was recorded with the “Video Imager”. The animation represents consecutive frames recorded over approximately 1.5 s (see the corresponding light emission profile in (0.89 MB MOV)Click here for additional data file.Movie S3An example of coordinated movement. As for (2.45 MB MOV)Click here for additional data file.Movie S4Visualization of mitochondrial Ca2+-fluxes during kainic acid-induced seizure. A 7 day old mouse pup was injected (i.p.) with coelenterazine. After 2 hours, kainic acid (25 mg/kg) was injected (i.p.) and the distribution of mitochondrial Ca2+ fluxes were monitored in the whole animal during seizure. The sequence shows whole animal Ca2+-induced bioluminescence during kainic acid induced seizure. Frames have a resolution of 300 µm for each pixel. Color scale is 0.02–1.0 photons/pixel. The integration time for each image is 30 s (6 frames/s).(0.75 MB MOV)Click here for additional data file.Movie S5Whole body patterns of Ca2+- induced bioluminescence approximately 50 mins after kainate-induced seizure in a 7 day old mouse pup. A video image was recorded at the beginning of the acquisition and is merged with the bioluminescence recording, which consists of 2 s frames. Smoothing has been applied to the bioluminescent image overlay to a resolution of 1 mm and the color scale is 0.01–0.1 photons/pixel. There are 50 frames in total (6 frames/s).(1.18 MB MOV)Click here for additional data file.


Tracheal epithelial cells are anchored to a dynamic basement membrane that contains a variety of extracellular matrix proteins including collagens and laminins. During development, wound repair and disease of the airway epithelium, significant changes in extracellular matrix proteins may directly affect cell migration, differentiation and events mediated by intercellular communication. We hypothesized that alterations in cell matrix, specifically type I collagen and laminin α3β3γ2 (LM-332) proteins within the matrix, directly affect intercellular communication in ciliated rabbit tracheal epithelial cells (RTEC).2+ waves. Expression of connexin (Cx) mRNA and proteins were assayed by reverse transcriptase – polymerase chain reaction and immunocytochemistry, respectively.Functional coupling of RTEC was monitored by microinjection of the negatively charged fluorescent dyes, Lucifer Yellow and Alexa 350, into ciliated RTEC grown on either a LM-332/collagen or collagen matrix. Coupling of physiologically significant molecules was evaluated by the mechanism and extent of propagated intercellular Ca2+ waves, the mechanism of transfer was dependent on matrix: RTEC grown on LM-332/collagen propagated Ca2+waves via extracellular purinergic signaling whereas RTEC grown on collagen used gap junctions. Comparison of RTEC grown on collagen or LM-332/collagen matrices revealed a reorganization of Cx26, Cx43 and Cx46 proteins.When compared to RTEC grown on collagen alone, RTEC grown on LM-332/collagen displayed a significant increase in dye transfer. Although mechanical stimulation of RTEC grown on either LM-332/collagen or collagen alone resulted in intercellular CaAlterations in airway basement membrane proteins such as LM-332 can induce connexin reorganizations and result in altered cellular communication mechanisms that could contribute to airway tissue function. In order to determine if purine release was a component of intercellular Ca2+ wave propagation, mechanical stimulation was repeated in the presence of the nucleotidase, apyrase. The addition of 50 U/ml apyrase significantly reduced the number of cells participating in a mechanically-induced Ca2+ wave in RTEC grown on LM-332/collagen matrices to 3.0 ± 0.7 cells  and structural  proteins. Laminins are one of many basement membrane extracellular matrix molecules that can contribute to cell support and signalling of the developing airway ,5,7,9,12Direct cellular coupling through gap junctions has been traditionally monitored by transfer of low molecular weight fluorescent dyes or by measurement of electrical conductance. Both ciliated and aciliated RTEC have been shown to be electrically coupled . Initial2+ waves between RTEC grown on collagen or LM-332/collagen matrices and used specific inhibitors to identify intercellular signalling pathways. A role for gap junctions in mechanically induced Ca2+ waves in RTEC grown on collagen matrices has been firmly established i of the stimulated cell through release of Ca2+ from intracellular stores. The changes in [Ca2+]i in adjacent cells is through a gap junctional mediated, IP3-dependent Ca2+ release [2+]i.Although dye coupling techniques are recognized as valuable experimental tools to identity functional gap junctions, it has become increasingly clear that gap junctions made of different connexin isoforms can also allow the differential transfer of physiologically relevant molecules ,47,48. Tablished ,42,49-51ted cell ,51 that  release ,35,42,51 release ,52,53. I2+ waves are conserved. However, inhibitor studies are consistent with a shift in the mechanism of coordination of Ca2+ changes to a paracrine/purinergic signalling pathway. Although cultured RTEC cells grown on collagen [2+]i, it is only the RTEC grown on LM-332/collagen that utilize purinergic signalling in response to mechanical stimulation to coordinate [Ca2+]i changes. This pronounced switch in communication mechanisms in RTEC cultures in response to LM-332 suggests that differences in mechanically-induced Ca2+ communication between rabbit i. It should not be discounted that a subtle change in the pattern of Ca2+ signalling can have a significant change on the cellular physiology of the signal i. All authors contributed to design of experiments, drafting of the manuscript and approved of the final manuscript.BEI contributed cell culture, initial microinjection experiments and was responsible for all immunocytochemistry experiments. CEO contributed to cell culture, microinjection experiments and was responsible for RT-PCR. SB contributed to cell culture and was responsible for digital imaging of [Ca


Influx of Ca2+ in non excitable smooth muscle is believed to be predominantly through store operated channels (SOC) or receptor operated channels (ROC). Whereas agonists can activate both SOC and ROC in a range of smooth muscle types, the specific trigger for SOC activation is depletion of the sarcoplasmic reticulum Ca2+ stores. The mechanism underlying SOC activation following depletion of intracellular Ca2+ stores in smooth muscle has not been identified.Control of cytosolic calcium plays a key role in airway myocyte function. Changes in intracellular CaTo investigate the roles of the STIM homologues in SOC activation in airway myocytes, specific siRNA sequences were utilised to target and selectively suppress both STIM1 and STIM2. Quantitative real time PCR was employed to assess the efficiency and the specificity of the siRNA mediated knockdown of mRNA. Activation of SOC was investigated by both whole cell patch clamp electrophysiology and a fluorescence based calcium assay.2+ influx in response to store depletion by cyclopiazonic acid (60%) or histamine but not bradykinin. siRNA to STIM2 had no effect on these responses. In addition STIM1 suppression resulted in a more or less complete abrogation of SOC associated inward currents assessed by whole cell patch clamp.Transfection of 20 nM siRNA specific for STIM1 or 2 resulted in robust decreases (>70%) of the relevant mRNA. siRNA targeted at STIM1 resulted in a reduction of SOC associated Ca2+ store depletion or following agonist stimulation with histamine in human airway myocytes. These are the first data demonstrating a role for STIM1 in a physiologically relevant, non-transformed endogenous expression cell model.Here we show that STIM1 acts as a key signal for SOC activation following intracellular Ca The contractile/relaxant state of the airway myocyte is a key determinant of airway calibre thus contributing to bronchoconstriction in diseases such as asthma. Previous studies have demonstrated that the contractile response of airway myocytes is dependent initially upon release of intracellular Ca2+ from the sarcoplasmic reticulum  but tha2+ entry ) in cont2+ entry .2+ stores. Contractile agonists such as acetylcholine, histamine and bradykinin may vary in their ability to differentially activate ROC or SOC although all agonists are known to induce activation of phospholipase C with consequent IP3 mediated Ca2+ release from the intracellular stores. The mechanism underlying signalling for subsequent Ca2+ influx in response to store depletion, and hence refilling of the sarcoplasmic reticulum Ca2+ stores, remains unknown. In the current study we have set out to define the signals for SOC activation in human airway myocytes following both store depletion and agonist activation by spasmogens.SOC activation in many cell types including smooth muscle is known to involve depletion of the intracellular sarcoplasmic reticulum CaCRAC) in T lymphocytes via a mechanism which has been proposed to involve translocation of STIM1 from endoplasmic reticulum like sites to the cell membrane i.HASMs (passage 4–5) were plated in black walled, clear bottom 96 well plates and loaded with Fluo-4AM (Molecular probes) for 1 hour at room temperature in culture medium (DMEM) supplemented with 10% FCS and 2.5 mM probenecid . Cells were then washed with Hanks' balanced saline solution containing 10 mM Hepes, 2.5 mM probenecid, 0.1 mM CaCl2, 10 mM EGTA, 30 mM HEPES, 3.62 mM CaCl2. External Solution: 140 mM NaCl, 5 mM CsCl, 1 mM MgCl2, 10 mM D-Glucose, 10 mM HEPES, CaCl2 (as indicated). K+ was replaced by Cs+ in both external and internal solutions to block K+ currents and Cl- was replaced by an equal molar concentration of methanesulfonate to minimize Cl- currents. Nifedipine (5 μM) was included in the external solution. Pipettes were drawn from borosilicate glass and had resistances of 5–8 MΩ when filled with internal solution. HASM cells were placed directly into the cell chamber, allowed to settle and then were continuously perfused with external solution at a constant speed of 6 ml/min. Experimental drugs were delivered through a puffer pipette positioned 50 μm around the cells. Cells were held at a membrane potential of -60 mV and current-voltage relationships were analysed every 5s from voltage ramps from -100 to +100 mV at a rate of 0.5 Vs-1. Currents were filtered at 1 kHz and sampled at 4 KHz. Individual cell current densities were calculated by dividing peak current amplitude at maximum activation of inward current (at -100 mV) by cell capacitance.The conventional whole-cell patch-clamp technique  was emplHASMs grown on coverslips, transfected with either 20 nM scrambled siRNA or 20 nM STIM1 siRNA were fixed with 4% formaldehyde. Cells were permeabilized (0.5% TritonX-100) and blocked with 20% goat serum in PBS for 20 min. Cells were incubated with primary antibody   overnight at 4°C followed by labeling with Alexa fluor 488 (Molecular probes). Cells were visualized on a Zeiss LS 510 confocal microscope .Averaged data are presented as mean ± sem. Where appropriate, statistical significance was assessed by unpaired Students T tests or one-way ANOVA followed by the Dunnets test for multiple group comparisons. Data were considered significant at *P < 0.05 or **P < 0.01.Initially we assessed the expression of the two known human homologues STIM1 and STIM2 in cells relevant to airway function figure . Both ST2+]i) in monolayers of cultured human airway myocytes. SOC mediated Ca2+ influx was induced by depletion of the sarcoplasmic reticulum Ca2+ store using a combination of low external Ca2+(0.1 mM) and incubation with the SERCA inhibitor cyclopiazonic acid . Incubation of airway myocytes in low Ca2+ in the presence of CPA resulted in an initial small rise in cytosolic Ca2+ indicative of store depletion. Re-addition of extracellular Ca2+ resulted in rapid influx into the cell through SOCs. siRNA targeted at STIM1 resulted in a dose-dependent reduction of SOC associated Ca2+ influx .We next evaluated the ability of siRNA targeted to STIM1 and STIM2 to inhibit SOC using a fluorescence assay utilising Fluo-4AM, designed to measure changes in intracellular free calcium concentration . SOC currents were induced by voltage ramps at a rate of 0.5 mV·ms-1 from a holding potential of -60 mV. Inward currents (SOC currents) measured at -100 mV were then compared. Changes in current density of SOC are illustrated in Figure 2+ free buffer). The STIM1 suppressed cells showed a reduced SOC inward current compared with cells treated with scrambled or STIM2 siRNA sequences. We also noticed a change in the reversal potential in the STIM1 suppressed cells towards a more negative potential: the significance of this is uncertain but similar findings on ICRAC currents have been reported using EF-hand mutants of STIM1 in RBL cells [We next used whole cell patch clamp electrophysiology approaches to confirm that siRNA targeted at STIM1 but not STIM2 is able to inhibit SOC activation. SOC currents were activated by reducing external CaBL cells .2+ entry in human airway myocytes are shown in figure 2+ responses to be H1 receptor mediated in these cells. Under conditions of low extracellular Ca2+ the sustained rise in intracellular Ca2+ seen following agonist stimulation is reduced, an effect mimicked by a range of di and tri-valent cations including Ni2+, La3+ and Gd3+ and also by the putative ROC inhibitor SKF96365 [2+ response to agonist. Following pre-incubation with siRNA targeted at STIM1 we observed a significant reduction in the magnitude of Ca2+ influx induced by histamine  declare that they have no competing interests.Samantha Peel performed the mRNA expression and calcium studies, Bo Liu performed the electrophysiology and Ian P Hall wrote the paper. All authors were involved in the design of the studies, discussion of the results and preparation of the final manuscript.