Orai1 was discovered in T cells as a calcium-selective channel that is activated by store depletion. Recent studies suggest that it is expressed and functionally important also in blood vessels, not only because haematopoietic cells can incorporate in the vascular wall but also because Orai1 is expressed and functional in vascular smooth muscle cells and endothelial cells. This article summarises the arising observations in this new area of vascular research and debates underlying issues and challenges for future investigations. The primary focus is on vascular smooth muscle cells and endothelial cells. Specific topics include Orai1 expression; Orai1 roles in store-operated calcium entry and ionic currents of store-depleted cells; blockade of Orai1-related signals by Synta 66 and other pharmacology; activation or regulation of Orai1-related signals by physiological substances and compartments; stromal interaction molecules and the relationship of Orai1 to other ion channels, transporters and pumps; transient receptor potential canonical channels and their contribution to store-operated calcium entry; roles of Orai1 in vascular tone, remodelling, thrombus formation and inflammation; and Orai2 and Orai3. Overall, the observations suggest the existence of an additional, previously unrecognised, calcium channel of the vascular wall that is functionally important particularly in remodelling but probably also in certain vasoconstrictor contexts. It is nthanides .2+ selectivity of I-CRAC in endothelial cells has also been suggested to indicate a different molecular basis [The greater potency of Synta 66 on SOCE of vascular smooth muscle and endothelial cells (compared with blood cells) suggests a difference in the molecular mechanism of SOCE in these cells, even though the SOCE in all cases shows partial or strong dependence on Orai1. Different sensitivity to 2-APB  adds weiar basis . The ide2+ entry evoked by non-physiological stimuli. This is not to infer that they lack physiological relevance but it is necessary to consider if or when physiological stimuli can activate them. This is especially important because store depletion is a signal that leads to cell apoptosis and because physiological agonists can evoke Ca2+ release without causing significant store depletion, as demonstrated, for example, by simultaneous measurements of cytosolic and ER Ca2+ in endothelial cell lines [2+ and then used the Ca2+ add-back protocol to observe Ca2+ entry. While this protocol reduces confusion between Ca2+ release and Ca2+ entry, it is weakened by being a store depletion protocol (because the stores cannot refill after the Ca2+ release event). The experimental difficulty involved in avoiding inadvertent store depletion has been emphasised [2+ entry that depends on Orai1 in the continuous presence of extracellular Ca2+ and without store depletion. Two substances that activate the channels in this situation are the important growth factors PDGF and vascular endothelial growth factor (VEGF) [2+ entry in the continuous presence of extracellular Ca2+ but it was not reported if this effect was inhibited by Orai1 siRNA [2+ entry stimulated by lysophosphatidylcholine (0.3 μM) was suppressed by Orai1 siRNA even though the lysophosphatidylcholine did not evoke Ca2+ release, suggesting Ca2+-release-independent activation of Orai1 channels in vascular smooth muscle cells [The studies described above refer to Call lines , 65. Howphasised . Consequr (VEGF) , 59. ATPle cells .2+ entry by sphingosine-1-phosphate has been described in vascular smooth muscle cells [2+ release via G protein-coupled receptors, the SOCE-like signal occurred independently of sphingosine-1-phosphate receptors and was mimicked by intracellular sphingosine-1-phosphate [Intriguing stimulation of SOCE-like Cale cells . While shosphate . The SOC2+ signalling [2+-regulated adenylyl cyclase 6. A submembrane compartment for regulation of filamin A by Ca2+ and cyclic AMP was suggested to play a role in the control of endothelial cell shape [Localisation of Orai1 to membrane density fractions containing caveolin-1 was described in studies of pulmonary microvascular endothelial cells, suggesting compartmentalisation of Orai1-dependent Cagnalling . The frall shape .2+ signalling field as glycosylated phosphoproteins located to the cell surface. Although subsequent studies confirmed STIM1 localisation in the plasma membrane, its relevance to SOCE is now most commonly described in terms of STIM1 as a protein of the ER membrane [2+ content of the ER (or sarcoplasmic reticulum), detecting store depletion and relaying this information to Orai1 channels in the surface membrane through a complex process of conformational change, redistribution, clustering and physical association with Orai1 that clusters Orai1 proteins and activates them to enable Ca2+ entry [2+ sensitivity [A year before the discovery of Orai1 came the discovery of the relevance of stromal interaction molecules 1 and 2 (STIM1 and STIM2) to SOCE , 78. STImembrane . Prior tmembrane . STIM1 i2+ entry . STIM2 isitivity .STIM1 and STIM2 are expressed in vascular smooth muscle cells and endothelial cells. Knock-down of the expression of either type of STIM suppresses SOCE, with STIM1 knock-down usually showing the greater effect , 62. Det2+ channel subunit CaV1.2 [V1.2, leading to suppression of voltage-dependent Ca2+ entry in store-depleted cells and a reciprocal relationship with SOCE [+–Ca2+ exchanger, plasma membrane Ca2+ ATPase and SERCA is suggested from studies of other cell types, indicating that STIM1 has a broad coordinating role for Ca2+ transport mechanisms and emphasising that its function is not restricted to Orai1 interaction [+–Ca2+ exchanger (NCX1) was observed and Orai1 siRNA suppressed NCX1 expression as well as Orai1 expression [2+ ATPase (PMCA1) expression was also suppressed. Na+–Ca2+ exchange did not, however, influence SOCE [STIM1 does not only interact with Orai1 channels, it also binds ion channels that include TRPC1 and the voltage-gated Cat CaV1.2 , 106. Bit CaV1.2 . Studiest CaV1.2 , 64. Stuith SOCE . Clustereraction . Specifipression . Plasma nce SOCE . SERCA2ance SOCE . Becausence SOCE .2+ channels contribute to SOCE in vascular smooth muscle cells and endothelial cells. There are, however, also reports suggesting that TRPC channels contribute to SOCE in these cells [2+-selective and inwardly rectifying channels, whereas TRPC channels generate larger mixed cationic Ca2+- and Na+-permeable channels with complex rectification that includes substantial outward current. These two channel types are unlikely to participate in generating a common ion pore . Furthermore, while Orai1 channels have mostly been associated with activation by store depletion, there is plenty of evidence that TRPC channels do not require store depletion in order to be activated and may often be activated even without concomitant Ca2+ release [It is suggested by the above text that Orai1 Case cells , 101. Al release , 10, 1022+ transport proteins [It may be that both Orai1 and TRPC channels contribute to vascular SOCE. This would explain why some studies show partial suppression of SOCE by Orai1 or TRPC siRNA , 60. Difproteins . One stuproteins , 70. A sproteins .2+ entry phenomenon observed under non-physiological conditions (see above). From a biological perspective, however, many investigators have been using SOCE as a means to understand the physiological mechanism by which stores refill following IP3-evoked Ca2+ release. The refilling process is expected to be triggered by the SOCE protocol but the protocol also has other consequences that probably do not occur, or are less prominent, when a physiological agonist evokes Ca2+ release under physiological conditions at a physiological concentration. One of these consequences is ER stress. Given the emerging evidence of TRPC activation by stress factors [2+-independent phospholipase A2 [Important in this discussion is the definition of SOCE which, in practice, is a Ca factors , 28, 68,ipase A2 , 85, 103ipase A2 , Orai1 aipase A2  and the ipase A2 .2+ and Ca2+ sensor dependencies of TRPC channels [I–V in I-CRAC recording conditions and strong dependence on TRPC1 [Another method for investigating the physiological refilling process has been the I-CRAC protocol. In many studies, however, this too is non-physiological (see above). Furthermore, the protocol is designed to isolate and highlight I-CRAC. It is quite possible that the intricate Cachannels , 82, 83 2+-handling proteins such as Na+–Ca2+ exchanger; (4) Orai1 is the molecular basis of the I-CRAC Ca2+-selectivity filter and TRPCs do not contribute to it; (5) I-CRAC is not the only ionic current activated by store depletion; and (6) TRPCs or Orais can both be activated independently of store depletion or Ca2+ release.In summary, it is suggested that (1) Orai1 and TRPC form distinct ion channels that do not heteromultimerise with each other; (2) Orai1 and TRPC can both contribute to the SOCE phenomenon in vascular smooth muscle cells or endothelial cells; (3) Orai1 and TRPC interact physically with STIM1 and interplay with other Ca3-evoked Ca2+ release is one of the goals of the research. What we do know is that the Ca2+-ATPases of the stores, and especially SERCAs, are the refilling mechanism at the level of the stores and that they refill the stores using free Ca2+ from the cytosol. Therefore, in principle, any Ca2+ entry channel that contributes to the cytosolic free Ca2+ concentration near SERCA can contribute to store refilling; even Na+ entry acting indirectly via Na+–Ca2+ exchange can contribute. There is evidence that several types of Ca2+ entry channel can contribute in this way. The fascination in the field, however, has been that there might be a particular type of Ca2+ entry channel that is particularly specialised for providing Ca2+ to SERCA and in a restricted subcellular compartment. This specialised channel would seem to be the I-CRAC channel (i.e. the Orai1 channel). Evidence is pointing to the conclusion that such a specialised channel is a core feature across many cell types, including vascular smooth muscle cells and endothelial cells. Indeed, the original pioneering study of store refilling in vascular smooth muscle argued for a privileged Ca2+ entry mechanism that directly fills the stores from the extracellular medium with minimal impact on the global cytosolic Ca2+ concentration [2+ entry mechanism providing extracellular Ca2+ for store refilling or that it is the only Ca2+ entry channel activated by store depletion. It seems unlikely that cells would have evolved dependence on a single mechanism for store refilling when store depletion is a critical event leading to apoptosis.Elucidation of the physiological mechanism by which stores refill following IPntration . Neverth2+ stores in Ca2+-free extracellular medium, Ca2+ add-back was found to cause a contractile response in aorta that was larger in stroke-prone spontaneously hypertensive rats [2+ add-back response was not preceded by exposure to a SERCA inhibitor in normotensive animals [2+ elevation that is not well coupled to contraction [2+, and therefore, they suggest that Orai1 channels are important in physiological contractile responses of this artery. A note of caution, however, is that previous work on basilar artery suggested that SOCE had no effect on contraction of freshly isolated artery but strong effect on contraction after organ culture of the artery for 72 h [After a period of depletion of Caive rats . Deliverive rats . These dive rats . The Syn animals . Therefotraction . Howevertraction . The efffor 72 h , 12. Alt2+ evoked by PDGF in the continuous presence of extracellular Ca2+ [V1.3 potassium channel [Several studies have found that expression of Orai1 mRNA and protein are up-regulated when vascular smooth muscle cells undergo their switch from the contractile to the non-contractile (migrating and proliferating) phenotype (see above). It has also been observed that SOCE is larger in proliferating vascular smooth muscle cells , 42 and lar Ca2+ ; this filar Ca2+ . In vivolar Ca2+ , 107, silar Ca2+ . Similarlar Ca2+ . Collect channel , 25, 55.2+ evoked by VEGF [Endothelial cells also remodel using a phenotype that displays migrating and proliferating properties. Knock-down of Orai1 by siRNA inhibits the migration  and prol by VEGF , the pri by VEGF . In vitr by VEGF . Exogeno by VEGF . Synta 6 by VEGF . Similar by VEGF . A study by VEGF . A reduc by VEGF , similar by VEGF . OverallThis review focuses on two dominant cell types of the vascular wall but it should be borne in mind that Orai1 is also expressed in blood cells  which can interact with and integrate in the vascular wall as part of inflammatory and thrombotic events. Numerous studies suggest the importance of Orai1 channels in thrombus formation and inflammation , 32, 39.2+ in proliferating vascular smooth muscle cells [2+ (ARC) channel in HEK 293 cells have suggested that this channel is formed from a pentameric arrangement of Orai1 with Orai3 [Orai2 and Orai3 mRNAs are also detected in vascular smooth muscle cells and endothelial cells , 59, 80,le cells , 59, 77 le cells . There wle cells . Intriguth Orai3 . It is n2+ channel of blood vessels. The strongest evidence for expression and roles of Orai1 in the vasculature is in remodelling events that relate to neointimal hyperplasia and angiogenesis. Orai1 can play significant positive roles in migrating and proliferating behaviours of vascular smooth muscle and endothelial cells, all of which are important in events such as neointimal hyperplasia and angiogenesis. There is less evidence for the expression and roles of Orai1 in the contractile state of blood vessels but function is indicated and may be important in specific vessels under certain conditions. In both the remodelling and contractile contexts, there is need for more information on the expression and functional relevance of endogenous Orai1 channels especially in freshly isolated cells and tissues and, in vivo, in animals under physiological and pathological conditions.The evidence points to Orai1 as a novel Ca2+ stores. It would seem to be true that Orai1 provides such a mechanism, but strengths of the argument depend substantially on principles developed from studies of cell types other than vascular smooth muscle and endothelial cells or from over-expression approaches in cell lines. Reports on vascular smooth muscle cells and endothelial cells provide numerous indications that store depletion is associated with the activation or insertion not only of Orai1 channels but also additional types of Ca2+-permeable channel that impact on cytosolic Ca2+ concentrations directly or indirectly. The relationship between these channels and Orai1 requires further investigation and would benefit from the application of new technical approaches that provide better resolution in subcellular space, better information about associations between endogenous proteins in physiological cells and better information about activation of the channels in physiological and pathological contexts when Ca2+ signalling occurs in three-dimensional structures that are in slow turnover (quiescence) or actively remodelling.A fundamental implication from Orai1’s discovery is that it represents a long-sought, privileged and widespread mechanism for refilling of depleted Ca2+ release is not necessarily associated with store depletion and thus that a refilling process may be activated and maintained in the absence of store depletion. The reported activation of Orai1-dependent Ca2+ entry by PDGF or VEGF in the continuous presence of extracellular Ca2+ suggests the involvement of Orai1 in store refilling even when there is little or no store depletion. If there is indeed such efficient store refilling via Orai1, it raises questions about the physiological activation mechanism of Orai1 and the appropriateness of considering Orai1 only in terms of the store depletion-activated Orai1–STIM1 I-CRAC complex. Dependence of non-selective cationic current on Orai1 [An important step in the short term is to better address the relevance to physiological settings of experimentally induced store depletion events and the SOCE phenomenon. Several studies suggest that Caon Orai1  and the on Orai1  are suggOne of the most obvious problems in the field is the apparently conflicting published data sets on the molecular basis of SOCE. Put simply: Is SOCE mediated by Orai1, TRPC, other channels, etc., or all of them? How can different investigators use apparently similar experimental protocols and end up with such widely differing results and conclusions ? It would be helpful if experimental conditions were standardised. Another way forward would be to decrease emphasis on the SOCE phenomenon and focus attention instead on physiological activators of the channels and studies in physiological conditions. A further way forward is to accept that multiple channel types can contribute to SOCE in cells in vitro in planar culture or suspension but that the physiological relevance of these contributions depends on the exact cell type and the context. An intriguing study, for example, suggested the importance of the TRPC4 channel at the point in time when endothelial cells make contact . Such a Additional challenges ahead involve addressing (1) whether the vascular I-CRAC channel has a distinct molecular component compared with the I-CRAC channel in T cells, conferring a basis for distinction by pharmacology and, potentially, therapeutic drugs; (2) the roles of Orai2 and Orai3 in blood vessels (e.g. Is an ARC channel relevant?); and (3) the nature of the down-stream pathways of Orai1 channels and other channel types contributing to SOCE .2+ signalling and vascular fields. A previously unrecognised channel type of vascular smooth muscle cells and endothelial cells seems to have been identified and appears to have important functional consequences that could be relevant and significant for fundamental understanding and new therapeutic strategies. We are, however, at the beginning of this period of investigation and there is much still to learn and resolve. Application of new experimental methods and emphasis on other types of existing methods will be necessary as the field progresses.The discovery of Orai1 in T cells has led to an interesting and lively period of research in the Ca


Bitter tastants can activate bitter taste receptors on constricted smooth muscle cells to inhibit L-type calcium channels and induce bronchodilation. 2+ signals, as revealed in cultured ASM cells, to activate large-conductance Ca2+-activated K+ channels, which in turn hyperpolarize the membrane, leading to relaxation. Here we report that in mouse primary ASM cells bitter tastants neither evoke localized Ca2+ events nor alter spontaneous local Ca2+ transients. Interestingly, they increase global intracellular [Ca2+]i, although to a much lower level than bronchoconstrictors. We show that these Ca2+ changes in cells at rest are mediated via activation of the canonical bitter taste signaling cascade , and are not sufficient to impact airway contractility. But activation of TAS2Rs fully reverses the increase in [Ca2+]i induced by bronchoconstrictors, and this lowering of the [Ca2+]i is necessary for bitter tastant-induced ASM cell relaxation. We further show that bitter tastants inhibit L-type voltage-dependent Ca2+ channels (VDCCs), resulting in reversal in [Ca2+]i, and this inhibition can be prevented by pertussis toxin and G-protein βγ subunit inhibitors, but not by the blockers of PLCβ and IP3R. Together, we suggest that TAS2R stimulation activates two opposing Ca2+ signaling pathways via Gβγ to increase [Ca2+]i at rest while blocking activated L-type VDCCs to induce bronchodilation of contracted ASM. We propose that the large decrease in [Ca2+]i caused by effective tastant bronchodilators provides an efficient cell-based screening method for identifying potent dilators from among the many thousands of available bitter tastants.Bronchodilators are a standard medicine for treating airway obstructive diseases, and β2 adrenergic receptor agonists have been the most commonly used bronchodilators since their discovery. Strikingly, activation of G-protein-coupled bitter taste receptors (TAS2Rs) in airway smooth muscle (ASM) causes a stronger bronchodilation in vitro and in vivo than β2 agonists, implying that new and better bronchodilators could be developed. A critical step towards realizing this potential is to understand the mechanisms underlying this bronchodilation, which remain ill-defined. An influential hypothesis argues that bitter tastants generate localized Ca Bitter taste receptors (TAS2Rs), a G-protein-coupled receptor family long thought to be solely expressed in taste buds on the tongue, have recently been detected in airways. Bitter substances can activate TAS2Rs in airway smooth muscle to cause greater bronchodilation than β2 adrenergic receptor agonists, the most commonly used bronchodilators. However, the mechanisms underlying this bronchodilation remain elusive. Here we show that, in resting primary airway smooth muscle cells, bitter tastants activate a TAS2R-dependent signaling pathway that results in an increase in intracellular calcium levels, albeit to a level much lower than that produced by bronchoconstrictors. In bronchoconstricted cells, however, bitter tastants reverse the bronchoconstrictor-induced increase in calcium levels, which leads to the relaxation of smooth muscle cells. We find that this reversal is due to inhibition of L-type calcium channels. Our results suggest that under normal conditions, bitter tastants can activate TAS2Rs to modestly increase calcium levels, but that when smooth muscle cells are constricted, they can block L-type calcium channels to induce bronchodilation. We postulate that this novel mechanism could operate in other extraoral cells expressing TAS2Rs. Airway obstructive diseases (asthma and chronic obstructive pulmonary disease [COPD]) have become increasingly prevalent, currently affecting more than 300 million people worldwide. Dysfunction of airway smooth muscle (ASM) cells, a major cell type in the respiratory tree, plays a pivotal role in promoting progression of these diseases and in contributing to their symptoms of these diseases Bitter tastants represent a new class of compounds with potential as potent bronchodilators. Deshpande et al. recently found that cultured ASM cells express G-protein-coupled bitter taste receptors (TAS2Rs) 2+ concentration ([Ca2+]i) to a level comparable to that produced by potent bronchoconstrictors 2+ release from endoplasmic reticulum, which then activate large-conductance Ca2+-activated K+ channels thereby hyperpolarizing the membrane 2+-activated K+ channels but rather inhibit them 2+-activated K+ channel blockers did not affect the bronchodilation induced by bitter tastants Bitter tastant-induced bronchodilation was unexpected, because these agents appeared to increase intracellular Ca2+]i elevation leading to relaxation may be attributed to the fact that Ca2+ responses to bitter tastants were assessed in cultured human ASM cells, while the contractile responses to them were investigated in freshly dissected ASM tissues 2+ response is also modified. Therefore, to understand bitter tastant-induced bronchodilation, it is necessary to study the contraction and the underlying signaling in freshly isolated ASM tissues and cells. Using this approach in the present study, we found that bitter tastants activate the canonical bitter taste signaling cascade, slightly increasing global [Ca2+]i in resting cells, but not to a level sufficient to cause contraction. However, bitter tastants reverse the increase in [Ca2+]i evoked by bronchoconstrictors, leading to bronchodilation. This reversal is mediated by the suppression of L-type voltage-dependent Ca2+ channels (VDCCs) in a gustducin βγ subunit-dependent, yet PLCβ- and IP3R-independent manner. Hence, we propose that TAS2R activation in ASM stimulates two opposing Ca2+ signaling pathways, both mediated by Gβγ subunits, which increases [Ca2+]i at rest but blocks activated L-type VDCCs reversing the contraction they cause. These results provide the cellular and molecular basis of bitter tastant-induced bronchodilation that occurs in vitro and in vivo. They further reveal a Ca2+ signal that is well suited for screening and identifying potent bronchodilators from among the many thousands of available bitter tastants.The apparent conundrum of putative [Ca2+]i and ASM contraction in freshly isolated airway cells and tissues from mouse and human. Fluo-3 was used to assess the effect of bitter tastants on [Ca2+]i; chloroquine and denatonium, two substances commonly used to study bitter taste signaling, were used as bitter tastants.To uncover the mechanism underlying bitter tastant-induced bronchodilation as demonstrated in both in vitro and in vivo normal and asthmatic models of mice, and in vitro human airways  (0)  both in the presence of extracellular Ca2+  and in its absence , indicating that the source for this chloroquine response is from internal Ca2+ stores.We started our analysis by examining the Ca–100 µM) . Chloroq2+]i is sufficient to trigger contraction, we measured smooth muscle force formation in mouse airways. As shown in . To test whether spontaneous Ca2+ sparks mask the effect of bitter tastants on other forms of local Ca2+ releases, such as Ca2+ puffs due to the opening of IP3Rs 2+ responses to chloroquine in ASM cells pretreated with 100 µM ryanodine. In these cells, prior to chloroquine application, no spontaneous sparks were observed (n = 14). Chloroquine (10 µM) increased global [Ca2+] by 12%±4% (ΔF/F0 at its brightest location) in nine cells, and failed to cause any detectable Ca2+ increase in five cells. There were no detectable local Ca2+ events produced in any of the 14 cells. These results indicate that chloroquine at 10 µM does not increase local Ca2+ events (either Ca2+ puffs or Ca2+ sparks).Mouse ASM cells exhibit spontaneous Ca2+]i rise by bitter tastants. Since in taste cells, bitter tastants bind to TAS2R to activate the pertussis toxin (PTX) sensitive G-protein gustducin, which in turn induces a PLCβ2 and IP3 signaling cascade 2+]i to 21.1%±8.6% of the control cells  and −10.5%±7.3% of controls, respectively , a stable analogue of acetylcholine, which is the major neurotransmitter in parasympathetic nerves. As expected, Mch (100 µM) rapidly increased [Ca2+]i as fluo-3 fluorescence increased by 162%±26% (ΔF/F0), and concurrently caused cell shortening by 49%±8% . The reversal of the increase in [Ca2+]i was closely associated with relaxation in ASM cells from both mouse .Bitter tastants at µM levels can modestly increase I and the resulting cell length  in response to bitter tastants suggests that bitter tastants reduce [Ca2+]i, leading to bronchodilation. If this is the case, one would expect that bitter tastant-induced bronchodilation could be prevented if [Ca2+]i was clamped to a physiologically high level. To test this possibility, we used staphylococcal α-toxin  to make the ASM membrane permeable to ions such that the intracellular [Ca2+]i could be controlled at will. A major advantage of using this toxin is that it does not damage the cells; thus signaling processes such as the G-protein-coupled receptor mediated signaling remain intact 2+]i to 3 µM caused a robust increase in tension in mouse airway. More importantly, at this fixed [Ca2+]i level, denatonium, chloroquine and quinine  failed to relax ASM in the time frame they would have in Mch contracted airways without α-toxin treatment. Therefore, clamping [Ca2+]i at µM levels can prevent bitter tastant-induced bronchodilation, strongly arguing that reduction of [Ca2+]i by bitter tastants is necessary for their relaxation action. These results further imply that a decrease in Ca2+ sensitivity is probably not a major mechanism underlying bitter tastant-induced bronchodilation.The inverse relationship between changes in [Ca2+]i is necessary for bitter tastant-induced bronchodilation, we next addressed the molecule(s) that bitter tastants act on to reduce [Ca2+]i. Before addressing this critical question, it is appropriate to determine the Ca2+ pathways underlying Mch-induced contraction because a controversy persists 2+]i by releasing Ca2+ from internal stores and inducing Ca2+ influx from the extracellular space respectively 2+ release from the internal stores contributes to the early phase of Mch-induced contraction, and Ca2+ influx is required to sustain elevated [Ca2+]i and contraction. Indeed, we found that the sustained contraction by Mch in mouse ASM is largely dependent on Ca2+ influx . To further examine whether diltiazem inhibits muscarinic receptor-mediated contraction, we treated the airways with diltiazem at different concentrations before Mch administration. Since a unique feature of diltiazem in inhibiting L-type Ca2+ channels is its use-dependence of action , in this series of experiments we challenged the airways twice with KCl to activate the Ca2+ channels. As shown in n = 5). To directly demonstrate that diltiazem inhibits L-type Ca2+ channels, we studied the effect of diltiazem on L-type Ca2+ currents with patch-clamp recording. We found that diltiazem dose-dependently reduced depolarization-induced L-type Ca2+ currents at concentrations over the same range as that which blocked contraction  are another well-known class of L-type Ca% n = 5; . This re2+]i rise and contraction. At the single cell level, 10 µM FPL prevented chloroquine from reducing the [Ca2+]i increase caused by Mch  and mean peak currents at different depolarizing voltages (right panel) before and after 1 mM chloroquine. As is evident, chloroquine inhibited the L-type Ca2+ current when depolarizing voltages are between −30 mV and +40 mV.To directly examine the inhibitory role of bitter tastants on L-type VDCCs, we studied the effect of bitter tastants on L-type VDCC currents using patch clamp recording. 2+]i and contraction. KCl is a standard and common reagent used to study cellular processes mediated by depolarization. In airways, depolarization is expected to not only activate VDCCs in ASM but also those in cholinergic nerves as well (leading to release of Ach). Indeed, in ferret and pig, KCl activates both mechanisms to cause airway contraction + in these species To study whether this inhibition by chloroquine of L-type VDCCs could produce relaxation, we evaluated the effect of this bitter tastant on the depolarization-induced increase in i and contraction. In Ca2+ containing medium, KCl (60 mM) induced a marked increase in [Ca2+]i  Although L-type VDCCs are the major Ca [Ca2+]i  in isola [Ca2+]i . Yet in traction , consist2+ channels in the KCl-induced rise in [Ca2+]i and contraction, we investigated the influence of diltiazem on these two effects of KCl. We found that 100 µM diltiazem pretreatment reduced the KCl-induced increase in ΔF/F0 from 122%±19% to 16.8%±10% in isolated ASM cells . Moreover, FPL dose-dependently reversed chloroquine-induced relaxation in ASM pre-contracted by KCl , we reasoned that bitter tastants would be able to relax airways precontracted by KCl if bitter tastant's inhibition of L-type Cad by Mch . Indeed,d by Mch  and S2C.d by Mch , and (2)d by KCl . Similard by KCl , chloroqSM cells ; n = 7. l 60 mM; , and pred by KCl .2+]i increase in response to KCl in isolated single ASM cells. Pretreatment with PTX at 1 µg/ml for 6–8 h prevented chloroquine-induced reversal of the KCl-induced increase in [Ca2+]i, as did gallein (20 µM) and anti-βγ, a Gβγ blocking peptide (1 µM)  . Howeverng cells , failed ls  belong to seven transmembrane domain G-protein-coupled receptors. Long thought to only be expressed in the epithelium cells of the taste buds of the tongue, recent studies have revealed that these receptors also express in several extraoral tissues including brain, testis, immune cells, gastrointestinal tract, and respiratory system 2+ signaling compared to the study by Deshpande et al. 2+]i to a level comparable to bronchoconstrictors. In freshly isolated ASM, we found that bitter tastants only modestly increase [Ca2+]i to a level much lower than that produced by bronchoconstrictors. Second, Deshpande et al. 2+ events. However, in freshly isolated ASM, we found that bitter tastants do not increase local Ca2+ releases such as Ca2+ puffs and Ca2+ sparks. A reason for these two discrepancies may be that Deshpande et al.'s studies were conducted in cultured ASM cell lines; compared to freshly isolated ASM, these cells display a different phenotype by altering the expression of receptors, ion channels, and contractile proteins 2+-activated K+ channels strongly argue that bitter tastant-induced bronchodilation is highly unlikely to result from the generation of local Ca2+ events, which in turn activate large-conductance Ca2+-activated K+ channel and hyperpolarize the membrane as proposed This study revealed two major differences in Ca2+]i and cell shortening, we found that bitter tastant's ability to reverse the increase in [Ca2+]i caused by bronchoconstrictors is the underlying signal producing the bronchodilation. Three lines of evidence support this conclusion. First, in the presence of bronchoconstrictors, bitter tastants lowered [Ca2+]i while at the same time relaxing the precontracted cells, and this response was reversible. Second, clamping intracellular [Ca2+]i to levels produced by the bronchoconstrictors (low µM) prevented bitter tastants from relaxing airways. Third, enhancing and blocking Ca2+ influx via L-type Ca2+ channels can oppositely regulate the relaxation mediated by bitter tastants. These results reinforce the idea that [Ca2+]i is the critical signal governing ASM contractility.Since bitter tastants relax precontracted airways 2+ signals mediated by Gβγ upon activation of TAS2Rs revealed in this study are unique. It is expected that gustducin Gβγ activates PLCβ to generate IP3 and release Ca2+ from endo/sarcoplasmic reticulum to raise [Ca2+]i in ASM cells. But, unexpectedly, gustducin Gβγ also suppresses Ca2+ signaling mediated by Mch, which largely activates M3R, a Gq family receptor. In general, Gβγ from the Gi/Go family (to which TAS2Rs belong) tends to potentiate, rather than, inhibit the Ca2+ responses caused by the Gq family 2+ signaling by TAS2R activation is Gβγ isoform specific. Since Gβγ also mediates the ASM contractions induced by activation of M2R and γ-aminobutyric acid-B receptors 2+]i caused by bronchoconstrictors is isoform specific, and is likely via Gβ3γ13 dimers, which are released upon activation of TAS2Rs 2+ signaling mediated by Gβγ as revealed in the present study likely operates in these systems when they are stimulated by a combination of bitter tastants and other activators.The opposing Ca2+2+ channels can also be fully blocked by bitter tastants  would bind more weakly with these channels, thus decreasing the efficacy of these agents to inhibit these channels, should ASM cells from asthma patients have a more negative membrane potential. Bitter tastants, by their ability to inhibit L-type Ca2+ channels via activation of gustducin Gβγ, perhaps could circumvent the drawbacks of the currently available L-type Ca2+ channel blockers, and thus be a more effective asthma treatment. This is likely given that bitter tastants induce a stronger bronchodilation in both in vitro and in vivo asthmatic mouse models than do β2 agonists Gustducin βγ subunits inhibit L-type VDCCs to cause bronchodilation, highlighting the importance of these channels in mediating bronchoconstriction and their potential as a target for bronchodilators. Indeed, L-type VDCCs are expressed in ASM cells and their activation causes these cells to fully contract , S5, S6 50 of 430 nM and aristolochic acid activates human TAS2R43 with an EC50 of 81 nM 2+]i signal , as developed in the present study, are robust and quantitative and provide a powerful paradigm for identifying potential bronchodilators from among the many bitter tastants available.Although bitter tastants are promising candidates to be developed as a new class of bronchodilators, and the findings in the present study provide the cellular and molecular rationale for this line of inquiry, we would caution that chloroquine and denatonium examined in this study may not be ideal candidates because of the high concentration  needed to fully relax precontracted ASM. This caveat, however, should not dampen enthusiasm for this endeavor as there are many thousands of bitter tastants available from plants and animals, and numerous bitter small molecules synthesized by research laboratories and a variety of companies over the years. In fact, bitter tastants can stimulate bitter taste receptors at concentrations in the nanomolar range: strychnine activates human TAS2R46 with an ECExperimental protocols for animal research were approved by the Institutional Animal Care and Use Committees at the University of Massachusetts Medical School (protocol A-1473 to RZG).−1), and the trachea and mainstem bronchi were quickly removed and placed in a pre-chilled dissociation solution consisting of (in mM): 135 NaCl, 6 KCl, 5 MgCl2, 0.1 CaCl2, 0.2 EDTA, 10 HEPES, and 10 Glucose (pH 7.3). Tracheas and mainstem bronchi were dissected free from the surface of the connective tissue. The airway tissue was incubated in the dissociation medium containing papain 30 unit/ml, 1 mM DTT, and 0.5 mg/ml BSA, at 35°C for 30 min, and then transferred to a dissociation medium containing 3 unit/ml collagenase F and 0.5 mg/ml BSA, and incubated at 35°C for another 15 min to produce isolated ASM cells. Finally, the tissue was agitated with a fire polished wide-bore glass pipette to release the cells.C57BL/6 mice from 7 to 12 wk of age were anesthetized with intraperitoneally injected pentobarbitone . The rings were mounted on a wire myograph chamber (Danish Myo Technology), and a PowerLab recording device (AD Instruments) was used to record isometric tension. The ring preparations with zero tension were immersed in 5 ml of Krebs physiologic solution, bubbled with 95% O2 and 5% CO2 at 37°C. After 10 min equilibration, three stretches (each 2.5 mN) at 5 min intervals were applied to the rings. After these stretches the basal tones of the rings were usually settled at approximately 2 mN. To test the contractile response, each ring was stimulated twice with KCl (60 mM), separated by 20 min, before proceeding to other treatments. The order and treatment time of agonists and antagonists are indicated in the figure captions. In the experiments in n = 6) of that in their absence.C57BL/6 mice at 7–12 wk of age were sacrificed and the entire respiratory trees were rapidly removed and immersed in Krebs physiologic solution containing (in mM) 118.07 NaCl, 4.69 KCl, 2.52 CaCl2, 1.8 mM CaCl2, 10 mM HEPES, 5.6 mM glucose (pH 7.4). The rings were then transferred to and incubated in Ca2+-free H-T buffer for 5 min followed by another 5 min in buffer A . To skin ASM, the rings were incubated for 45 min with α-toxin  in buffer A at room temperature. After the permeabilization, the rings were treated with 10 µM ionomycin for 10 min to deplete intracellular Ca2+ stores.Mouse bronchi rings (4 mm in length) free of connective tissues were incubated for 5 min in HEPES-Tyrode (H-T) buffer which contained 137.0 mM NaCl, 2.7 mM KCl, 1.0 mM MgCl2EGTA, 5.83 mM MgCl2, 7.56 mM potassium propionate, 3.9 mM Na2ATP, 0.5 mM dithioerythritol, 16.2 mM phosphocreatine, 15 units/ml creatine kinase [pH 6.9]). The viability of the skinned muscle rings was examined by stimulation with pCa 4.5 solution  followed by the pCa 9.0 solution. The muscles that could generate sustained contraction in response to the pCa 4.5 solution and fully relax when exposed to the pCa 9.0 solution were used for subsequent experiments. To test the effect of bitter tastants, the viable muscle rings were induced to contract by exposure to pCa 5.5 solution followed by the administration of bitter tastants at the concentrations indicated in Skinned airway rings were mounted on the wire myograph chamber and washed two times with pCa 9 solution  for Ca2+ sparks.Fluorescence images using fluo-3 as a calcium indicator were obtained using a custom-built wide-field digital imaging system. The camera was interfaced to a custom made inverted microscope, and the cells were imaged using either a 20× Nikon 1.3 NA for global ) and then immunolabeled as described previously 3D fluorescence imaging was performed on an inverted wide field microscope (Nikon Diaphot 200) with excitation by a 100 W mercury lamp. Images were obtained through a 60× objective and digitally recorded on a cooled, back-thinned CDD camera (Photometrics), with an effective pixel size at the specimen of 83 nm in x-y and a z spacing of 100 nm. This resulted in a 3D stack of approximately 100 image planes for each cell.The fluorescence images were deconvolved with a constrained, iterative approach All chemicals, except fluo-3 (Invitrogen Co), gallein (Tocris Bioscience), anti-βγ blocking peptide (AnaSpec), anti-TAS2R107 (Santa Cruz Biotechnology), and purified IgG (Jackson ImmunoResearch Laboratories) were purchased from Sigma-Aldrich Co. For single cell studies, agonists and antagonists were applied locally to cells via a picospritzer at a constant pressure, so that the duration of its action and concentration could be controlled easily.n represents the number of cells or trachea and mainstem bronchi. Statistical analysis of differences was made with Student's paired or unpaired t-test and the significance level was set at p<0.05.Unless stated otherwise, data are reported as mean ± standard error of the mean (SEM) and Figure S12+]i in resting single cells (A) without a significant effect on the contractility (B) of relaxed mouse airways.Bitter tastant chloroquine dose-dependently increased i and contractile responses to bitter tastants and diltiazem in human ASM.Characteristics of [Ca (A) Bitter tastants reversed the [Ca2+]i rise and cell shortening induced by Mch. Measurements were taken at the steady state levels in response to Mch and chloroquine. The cell length before stimulation was considered as 100%. *p<0.05 paired Student's t-test; ***p<0.001; n = 6–12. (B) L-type VDCC blocker diltiazem dose-dependently reversed 10 µM Mch-induced contraction (n = 5 independent experiments). % relaxation = tension decrease due to diltiazem divided by tension increase due to Mch, times 100. The tension decrease at each concentration of diltiazem is measured once the tension stabilizes. The tension decrease at each increased concentration is always measured relative to the peak tension . (C) Chloroquine (1 mM) and diltiazem (100 µM) relaxed human intrapulmonary bronchi precontracted by 60 mM KCl (n = 3–5 independent experiments). % relaxation = tension decrease due to chloroquine divided by tension increase due to Mch, times 100. Bar charts are mean ± SEM. Human lung tissues were obtained (with informed consent) from patients undergoing surgery (lobectomy) for lung cancer at the Department of Surgery and the Department of Pathology at the University of Massachusetts Memorial Medical Center (Worcester). The tumors were identified as non–small cell carcinoma (adenocarcinoma or squamous cell carcinoma). Intrapulmonary airways were dissected out and cleaned free of the connective tissues. These airways were either cut into the rings (4 mM in length) for force measurements the same as for mouse airway tissues, or digested with the same enzymes, dissociation medium, and isolation procedure as for single mouse ASM cells. The experimental protocols on human tissues were approved by the Committee for Protection of Human Subjects in Research at the University of Massachusetts Medical School (protocol 13590 to RZG).(TIFF)Click here for additional data file.Figure S32+ influx plays a major role in producing and maintaining Mch-induced increases in [Ca2+]i and contraction in mouse ASM.Ca (A) In Ca2+ free medium, the tension generated by Mch was less than 20% of that in the presence of extracellular Ca2+. ***p<0.001, Student's paired t-test, n = 9 airway rings for the group with Ca2+, and n = 10 airway rings for the group without Ca2+. (B) Mch increased [Ca2+]i much less in Ca2+ free medium than in the presence of extracellular Ca2+. In the absence of extracellular Ca2+, Mch (10 µM) produced different patterns of changes in [Ca2+]i, so the area under each curve was calculated for 1 min of Mch stimulation and compared between the two conditions (right panel). ***p<0.001 with extracellular Ca2+ (n = 9 cells) versus without the Ca2+ (n = 12 cells); Student's unpaired t-test. (C) Ca2+ stores remained functional in the absence of extracellular Ca2+. The cells were placed in the absence of extracellular Ca2+ for ∼15 min, and then stimulated with two 10 µM Mch pulses 15 min apart. The chart on the right indicates that two Mch administrations produced comparable Ca2+ responses, i.e., Ca2+ stores are intact under experimental conditions in the present study. NS, p>0.05 for the response in the first pulse of Mch versus that in the second pulse, Student's paired t-test, n = 10. ΔF/F0 for (B) and (C) are the average over the entire cell.(TIFF)Click here for additional data file.Figure S42+ channel activation in Mch-induced contraction in mouse airway.Role of L-type Ca (A) Left panel: L-type VDCC blocker diltiazem dose-dependently reversed Mch-induced contraction (using tension as a proxy measure). Right panel: results for n = 6 airway rings. % relaxation was calculated the same as in 2+ channels. (v) Dose-response curve for diltiazem-mediated inhibition of contraction by KCl. Data are mean ± SEM (n = 5); % inhibition = (force by K0 − force by K2)/(force by K0)×100. (vi) Dose-response curve for diltiazem-mediated inhibition of contraction by Mch. Data are mean ± SEM (n = 5); % inhibition = (force by M0 – force by M1)/(force by M0)×100. Forces are measured at their peak, excluding the noise spikes due to the washing out of KCl or Mch. (C) Diltiazem inhibited L-type VDCC currents in a dose-dependent manner. Diltiazem was added to the bath solution cumulatively. Once at equilibrium at each level of diltiazem, cells were stimulated with a train of ten voltage pulses from −70 mV to 0 mV (inset) at 10 s intervals. Left panel displays patch clamp recordings of L-type Ca2+ currents in the control and in the presence of diltiazem at the given concentration (each in response to the tenth voltage pulse), and the right panel depicts the effect of diltiazem on the peak current at different concentrations. Ba2+ was used as the charge carrier. Data are mean ± SEM (n = 6); % inhibition = (peak current of the control − peak current at given diltiazem concentration)/peak current of the control ×100 (measured in the tenth voltage pulse). (D) Effect of nisoldipine on Mch-induced contraction. In light of the voltage-dependence of the inhibition of nisoldipine on L-type VDCCs, airway rings were modestly depolarized with 20 mM KCl (red bars) before Mch stimulation. Left panel displays a representative contractile response to 60 mM KCl and 1 µM Mch before and after 1 µM nisoldipine. Right panel shows the mean values (mean ± SEM) of inhibition by 1 µM nisoldipine of contraction evoked by KCl and Mch, respectively. % inhibition = /maximal force at the control ×100.(TIFF)Click here for additional data file.Figure S5KCl activates VDCCs in cholinergic nerves and ASM. (A) Atropine dose-dependently inhibited Mch-induced mouse airway contraction. The left panels display representative contractile responses to 3 µM Mch in the absence or the presence of atropine as marked near the traces, and the right panel shows the mean values  of inhibition by atropine of contraction evoked by 3 µM Mch. % inhibition = /maximal force from the control ×100. (B) Atropine inhibited KCl-induced mouse airway contraction. The left panel shows a representative tension recording in response to 60 mM KCl before and after 100 nM atropine, as marked beneath. The right panel shows the summarized results as the ratio of the force generated by the second KCl pulse over first KCl pulse .(TIFF)Click here for additional data file.Figure S62+]i and cause contraction in mouse ASM.KCl activates L-type VDCCs to increase [Ca (A) KCl failed to generate any global [Ca2+]i increase in the absence of extracellular Ca2+ in isolated single ASM cells. (i) A representative [Ca2+]i response to 60 mM KCl in the presence of extracellular Ca2+.  three examples showing that the same concentration of KCl did not increase Ca2+ in the zero Ca2+ medium. This failure was not due to the depletion of intracellular Ca2+ stores because 10 µM Mch still induced Ca2+ release either as a single peak or as oscillations. Eight cells gave rise to similar responses. ΔF/F0 is the average over the entire cell. (B) KCl (60 mM) caused virtually no increase in tension in the absence of extracellular Ca2+. The airways were placed in the Ca2+ free solution for 15 min before the measurement commenced. Left panel shows a pair of representative recordings and right panel the average results. **p<0.01, Student's paired t-test, n = 6 independent experiments. (C) KCl (60 mM)-induced increase in [Ca2+]i was markedly inhibited by prior application of L-type VDCC blocker diltiazem (100 µM). **p<0.01, Student's paired t-test, n = 9 for each conditions. (D) Diltiazem (100 µM) relaxed 60 mM KCl-induced contraction of mouse airways. Data are mean ± SEM (n = 6 independent experiments), and % relaxation definition and analysis are the same as in (TIFF)Click here for additional data file.Table S1Primers for Reverse Transcription-PCR.(TIFF)Click here for additional data file.Movie S12+]i and cell shortening; the first 120 images of this clip were analyzed and plotted in 0) because the cell changes its shape dramatically in response to stimuli (and changing thickness makes ΔF/F0 measures misleading).This clip shows 1 mM chloro reversed the 100 µM Mch-induced increase in [Ca(MOV)Click here for additional data file.


This review will focus on the regulation of myocardial contraction by peroxynitrite during health and disease, with a specific emphasis on cardiomyocyte Ca The heart plays a vital role in the cardiovascular system by providing the specialized tissues and organs of the body with a continual supply of oxygen and other essential nutrients. Modulation of myocardial contraction allows the heart to meet the demands of the body despite continual changes in metabolism. This modulation occurs at several different levels, including the level of the ventricular cardiomyocyte.2+ channels , and subsequently triggers the opening of the sarcoplasmic reticulum (SR) Ca2+-release channels or ryanodine receptors (RyRs). This results in an efflux of Ca2+ from the SR, which is the major Ca2+ store in the cardiomyocyte, in a process termed Ca2+-induced Ca2+-release. This process initiates the upstroke of the myocyte [Ca2+]i transient and this Ca2+ is now available for myocyte contraction. SR Ca2+ load, which represents the Ca2+ available for release from the SR, is a critical determinant of [Ca2+]i transient amplitude and thus myocyte contraction   and the Na+/Ca2+ exchanger (NCX) , thus allowing greater uptake of Ca2+ into the SR  Bers, , 2002. Tsα subunit. Gsα induces cAMP production via adenylate cyclase (AC). Increased cAMP then activates the cAMP-dependent protein kinase or PKA, which phosphorylates many different targets in the cardiomyocyte, including ICa, RyR, TnI, and PLB. The end result is increased Ca2+ influx, increased SR Ca2+ uptake, load, and release, and decreased myofilament Ca2+ sensitivity. PLB phosphorylation at Serine16 by PKA is one of the primary mechanisms responsible for the observed positive inotropic and lusitropic effects of β-AR stimulation  pathway results in positive inotropic and lusitropic effects via modulation of key excitation–contraction coupling proteins , on the other hand, is only expressed within cardiomyocytes during inflammatory responses which occur during many pathophysiological conditions of the myocardium. NOS2 produces much higher amounts of NO compared to constitutive isoforms and is [Ca2+]i independent   is a potent oxidant that is formed upon the reaction of NO and superoxide. This reaction occurs with an extremely high rate constant, making the formation of peroxynitrite quite favorable , to form additional reactive species   and via non-classical pathways (namely nitroxyl).NOS1 has been shown to co-immunoprecipitate with xanthine oxidoreductase  have been shown to produce positive inotropic effects on cardiomyocyte function. Conversely, high levels of peroxynitrite  are extremely detrimental to myocardial function by decreasing cardiomyocyte contraction.ex vivo myocardial preparations using SIN-1 . Interestingly, PP2a activity has been shown to be increased in heart failure  as well as the length of exposure . However, since peroxynitrite is highly reactive and rapidly decomposed, accurate measurements of concentration are difficult. In addition, determining intracellular concentrations of peroxynitrite with exogenously applied authentic peroxynitrite or the peroxynitrite donor SIN-1 is challenging, making direct comparisons to endogenously produced peroxynitrite formidable. In general, concentrations of 30 μM or less of authentic peroxynitrite or 100 μM or less of SIN-1 will result in positive inotropic effects as described. We have previously reported that 10 μM SIN-1 produces 3 nM/min peroxynitrite in our physiological saline solution  or long exposure times. These studies revealed irreversible effects of peroxynitrite that depressed myocyte contraction and inactivated SERCA (Ishida et al., In conclusion, peroxynitrite is emerging as a crucial modulator of myocardial function during health and disease. Under physiological conditions, low levels of peroxynitrite serve to maintain and/or increase basal and β-AR-stimulated contraction in the myocardium. This occurs via RyR and PLB phosphorylation through direct PKA activation and SERCA activation via S-nitrosylation Figure . ConversThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Voltage-gated Ca2+ channels (VGCC) and Ca2+-release activated channels (CRAC) enable Ca2+ flow into electrically excitable and non-excitable cells, respectively. VGCC are well characterized transducers of electrical activity that allow Ca2+ signaling into the cell in response to action potentials or subthreshold depolarizing stimuli. The identification of STromal Interaction Molecule (STIM) and Orai proteins has provided significant insights into the understanding of CRAC function and regulation. This review will summarize the current state of knowledge of STIM-Orai interaction and their contribution to cellular Ca2+ handling mechanisms. We will then discuss the bidirectional actions of STIM1 on VGCC and CRAC. In contrast to the stimulatory role of STIM1 on Orai channel activity that facilitates Ca2+ entry, recent reports indicated the ability of STIM1 to suppress VGCC activity. This new concept changes our traditional understanding of Ca2+ handling mechanisms and highlights the existence of dynamically regulated signaling complexes of surface expressed ion channels and intracellular store membrane-embedded Ca2+ sensors. Overall, STIM1 is emerging as a new class of regulatory proteins that fine-tunes Ca2+ entry in response to endoplasmic/sarcoplasmic reticulum stress.The spatial and temporal regulation of cellular calcium signals is modulated via two main Ca Regulation of its intracellular concentration ([Ca2+]i) represents a major determinant that controls signal transduction pathways such as secretion, excitation/contraction coupling, motility, transcription, growth, cell division or apoptosis  and out (efflux) of the cell through plasma membrane (PM) channels and transporters that facilitate Ca2+ movement between the extracellular milieu and cytoplasm across a Ca2+ concentration gradient  and reuptake (into the Ca2+ store). The main intracellular Ca2+ stores are the endoplasmic reticulum (ER) and sarcoplasmic reticulum (SR). A number of regulatory mechanisms have been proposed to mediate the cellular influx, efflux, release and reuptake of Ca2+, thus achieving Ca2+ homeostasis within the cell  is mediated by two principal means of Ca2+ entry. These routes are voltage-gated Ca2+ channels (VGCC) and ionotropic neurotransmitter receptors . The latter, SOCE, would in turn replenish the intracellular ER/SR stores  in a process referred to as SOCE  Orai, an integral PM protein being the pore-forming subunit of the CRAC channel , that regulate SOCE , and thus acts as a Ca2+ sensor. When the lumen of the ER/SR is full of Ca2+, STIM1 EF-hands are saturated with Ca2+ ions. In contrast, upon stores depletion, STIM1 molecules aggregate into oligomers  STIM1 overexpression attenuated CaV1.2 activity; while (2) CaV1.2-mediated responses were enhanced when STIM1 function or expression was impaired. Direct interaction between STIM1 and CaV1.2 proteins was ascertained and a set of experiments, using truncated forms of STIM1, documented that SOAR domain (STIM-Orai activating region, 344–442; Wang et al., V1.2 α1 subunit (1809–1908; Park et al., V1.2 interaction and functional studies revealed that the SOAR domain was necessary and sufficient to suppress CaV1.2 current (Figure V1.2 decreased as a consequence of long-term internalization of the channel from the PM (Figure V1.2 inhibitory interaction. Preventing STIM1 expression alone did not abolish CaV1.2 channel suppression while the simultaneous inhibition of both STIM1 and Orai1 was necessary to mask CaV1.2 inhibition by store depletion. In summary, the two groups reported for the first time that STIM1 effectively attenuates CaV1.2 activity.Using excitable cortical neurons and vascular smooth muscle cells, Park et al.  and Wangt Figure . A sloweM Figure . Despite2+ conductances inhibited upon store depletion has major implications for Ca2+ signaling in excitable and non-excitable cells. In excitable neuronal cells, VGCC are expressed at higher level than CRAC components and are the predominant Ca2+ influx route. In contrast, non-excitable cells display reduced VGCC expression and CRAC represents the main Ca2+ entry pathway due to the high expression of STIM1 and Orai (Liou et al., 2+ influx, a new modulatory role for STIM1 has emerged through which it inhibits Ca2+ entry mediated by VGCC. Noteworthy, the ability of STIM1 to reciprocally regulate Orai and CaV1.2 would imply that the mode of action of STIM1 is tissue specific. In other words, STIM1 would typically stimulate CRAC in non-excitable cells and inhibit VGCC in excitable cells (Park et al., 2+ entry by STIM1 seems to play a critical role in Ca2+ homeostasis by fine-tuning Ca2+ entry in cells that simultaneously express both channel types. By providing this missing piece of evidence, these studies resolved the predominant function of one channel over the other despite their co-expression in excitable and non-excitable tissues. That being said, whether other Ca2+ channels exhibit analogous or distinct modulation by STIM1 remains unknown.The description of CaV1.2 channel is not the only voltage-gated Ca2+ channel suppressed by STIM1. Interestingly, a study by Nguyen et al. (2+ channel. Using cardiomyocyte-derived HL-1 cells, an inhibitory association between STIM1 and CaV3.1 was reported and was suggested to limit excessive Ca2+ entry. Pathological Ca2+ influx in cardiac myocytes can trigger Ca2+ overload in the SR and cardiac arrhythmias (Sedej et al., V3.1 could, in theory, restrain such unwanted entry. Indeed, the authors showed that knocking down STIM1 increases T-type Ca2+ current density. They further suggested that STIM1 may regulate the expression of T-type α1 subunits as knocking down STIM1 augmented the surface expression of CaV3.1 channel. Whether CaV3.1 trafficking or stability at the PM is implicated remains unknown and requires further investigation. Intriguingly, these findings represent a novel regulatory pathway for Ca2+ handling in cardiac myocytes. This regulation may be implicated in the modulation of rhythmicity and excitability of native cardiac pacemaker cells where T-type Ca2+ channel expression and function is evident (Mangoni et al., Recent published experiments revealed that Can et al.  showed t2+ handling. Recent published work (Park et al., 2+ sensor to facilitate Ca2+ entry and replenish the stores, STIM1 suppresses the activity of voltage-gated Ca2+ channel in excitable cells. This inhibitory association prevents excessive cellular Ca2+ influx by mechanisms including direct protein-protein interaction and reduced VGCC surface expression (Park et al., 2+ in health and disease. Since STIM1 bi-directionally regulates VGCC and CRAC, SR/ER stress may lead to pathologies related to downstream Ca2+-dependent pathways. In fact, mutations in STIM1 elicit severe immunodeficiency syndromes associated with compromised SOCE (Feske et al., 2+ entry remain to be investigated; as it could be linked to diseases such as epilepsy, cardiac arrhythmia or hypertension. Finally, it will be fundamental to elucidate the cooperated interaction of PM Ca2+ channels and their associated subunits and how those signaling complexes respond to ER/SR stress through Ca2+ sensors such as STIM1 proteins.The interaction of STIM1 with CRAC or VGCC extends our understanding of the role of STIM1 in cellular CaOsama F. Harraz and Christophe Altier wrote the manuscript. Osama F. Harraz produced the figures.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


As the mechanisms underlying GO and calicheamicin-γ1 resistance are incompletely understood, we herein used flow cytometry-based single cell network profiling (SCNP) assays to study cellular responses of primary human AML cells to GO. Our data indicate that the extent of DNA damage is quantitatively impacted by CD33 expression and drug efflux activity. However, although DNA damage is required for GO-induced cytotoxicity, it is not sufficient for effective cell kill, suggesting that downstream anti-apoptotic pathways may function as relevant resistance mechanisms. Supporting this notion, we found activated PI3K/AKT signaling to be associated with GO resistance in vitro in primary AML cells. Consistently, the investigational AKT inhibitor MK-2206 significantly sensitized various human AML cells to GO or free calicheamicin-γ1 with particularly pronounced effects in otherwise GO or free calicheamicin-γ1 -resistant cells. Likewise, MK-2206 also sensitized primary AML cells to calicheamicin-γ1. Together, our findings illustrate the capacity of SCNP assays to discover chemotherapy-related biological pathways and signaling networks relevant to GO-induced genotoxic stress. The identification of AKT signaling as being associated with GO resistance in vitro may provide a novel approach to improve the in vivo efficacy of GO/calicheamicin-γ1 and, by extrapolation, other DNA damage-based therapeutics.Gemtuzumab ozogamicin (GO), an immunoconjugate between an anti-CD33 antibody and a calicheamicin-γ Initial phase 2 studies found an overall response rate of ∼30% with GO monotherapy in relapsed adult AML and led to accelerated marketing approval in the U.S. in 2000 1 derivative, which is then released intracellularly and causes single- and double-stranded DNA damage  and, to a lesser degree, multidrug resistance protein (MRP1), mediate resistance to GO With GO, the antibody primarily facilitates cellular uptake of the calicheamicin-γA damage  [5]. Conin vitro and, consequently, novel pharmacological approaches that can overcome GO resistance.Conversely, the impact of DNA repair and downstream signaling pathways on GO-mediated cytotoxicity has not been examined in detail. To this end, single cell network profiling (SCNP) assays using multiparametric flow cytometry have emerged as a versatile tool to simultaneously study several specific biological pathways and signaling networks at the single cell level within the context of complex tissues  without the need for isolation or purification of the cell populations of interest In accordance with the Declaration of Helsinki, all patients  provided written informed consent for the collection and use of their biospecimens for research purposes under studies approved by the Stanford University Research Compliance Office , the British Columbia Cancer Agency Research Ethics Board , and the Fred Hutchinson Cancer Research Center Institutional Review Board . Clinical data were de-identified in compliance with Health Insurance Portability and Accountability Act regulations.Pretreatment (“diagnostic”) peripheral blood or bone marrow specimens were obtained from patients with newly diagnosed AML treated at the British Columbia Cancer Agency, Lucile Packard Children’s Hospital (Stanford University), or FHCRC. Normal peripheral blood mononuclear cells (PBMCs) were commercially obtained from a healthy blood donor .U937 3), pelleted, and stained with cocktails of fluorochrome-conjugated antibodies. These cocktails included antibodies against cell surface markers for cell population gating  and up to 3 antibodies against intracellular signaling molecules for an 8-color flow cytometry assay. In these analyses, CD11b, CD34, and CD45 were consistently used in the SCNP assays while some specific conditions additionally included measurements of CD33 expression. Side-scatter properties and CD45 and/or CD33 expression were used to define the leukemic cell population. Antibodies against CD11b and CD34 were included to allow for further characterization of leukemic cell subsets but were not utilized for the analyses presented in this manuscript. Isotype controls or phosphopeptide blocking experiments were performed to characterize phospho-antibodies. Data was acquired on an LSR II and/or CANTO II flow cytometers using the FACS DIVA software (BD Biosciences). All flow cytometry data were analyzed with FlowJo  or WinList . Dead cells and debris were excluded by forward and side scatter properties combined with amine aqua viability dye exclusion. Leukemic cells were identified based side-scatter properties and expression of CD45 and/or CD33 as previously described SCNP assays were performed as described previously In pediatric specimens, CD33 was quantified using in-house biotin-conjugated CD33 followed by streptavidin-tagged Qdot605. In all adult specimens except AML-11, in which CD33-biotin-Qdot605 was not used because of limiting cell numbers, CD33 was quantified using PE-conjugated CD33, and CD33-biotin-Qdot605. To allow direct comparisons of CD33 expression levels between all specimens used in our studies, a linear regression model was construed between PE-conjugated and Qdot605-biotin-conjugated CD33 mean fluorescence intensities (MFIs), using AML samples and peripheral blood and bone marrow mononuclear cells from normal donors from our internal database with available data on both fluorophores, allowing calculation of equivalent Qdot605 MFI in specimen AML-11 using the equation: Qdot605 CD33 MFI = 0.6927×(CD33 PE MFI) +176.05  with or without the drug efflux inhibitor, PSC833 (2 µM), for 6–48 hours or incubation with staurosporine (2.33 µg/mL) for 6 hours, cells were restained with amine aqua viability dye prior to fixation and SCNP processing. Phosphorylation of histone H2AX at S139 (γH2AX) was used to quantify the extent of DNA damage in leukemic cells Untreated – LiveDrug treated]/[LiveUntreated], with “live” cells defined as cPARP−/aqua dye−. This metric ranges from values of 0 to 100% for each sample and normalizes for differences in sample quality or spontaneous apoptosis. The extent of GO-induced DNA damage was calculated as: % γH2AX+Drug treated – % γH2AX+Untreated.Following incubation with GO , IL-27 (50 ng/mL), PMA (400 nM), and SCF (20 ng/mL). Induced signaling responses were then measured using intracellular signaling molecules  and quantified with the LogFor kinetic assessment of GO induced DNA damage, human AML cell lines and normal PBMCs were incubated for 1–8 h with GO (30 ng/mL) with or without the drug efflux inhibitor, cyclosporin A (2.5 µg/mL), and processed as described above.2 and air. The cells were then lysed in RIPA lysis buffer with Complete EDTA-free protease inhibitor cocktail  on ice for 60 minutes and pre-cleared by centrifugation. 4× SDS sample buffer was added to RIPA soluble proteins to a final concentration of 1× SDS sample buffer. Samples were boiled, resolved by SDS-12%-PAGE, transblotted to nitrocellulose, and blots blocked with 5% non-fat dry milk in Tris-buffered saline. Blots were incubated with primary antibodies detecting p-AKT (S473) or total AKT (both CST) overnight at 4°C. After washing, incubation with HRP-conjugated secondary antibody for 1 hour at room temperature, and re-washing, immunoreactive signals were visualized with Amersham ECL Plus Western Blotting Detection System  and exposed to autoradiography film .Human AML cell lines were treated with MK-2206  or vehicle (DMSO) for 6 hours at 37°C in 5% CO1 (kindly provided by Wyeth-Ayerst [now Pfizer]) in the presence or absence of various doses of MK-2206. To partially reverse efflux-mediated drug resistance, parallel GO-treated cultures of KG-1 cells were treated with a low, subsaturating concentration (25 µM) of the drug efflux inhibitor, PK11195  1 in the presence or absence of MK-2206. After 3 days at 37°C in 5% CO2 and air, cell numbers and drug-induced cytotoxicity was determined by flow cytometry using 4',6-diamidino-2-phenylindole  to identify dead cells.In experiments with human AML cell lines, cells were incubated with increasing concentrations of GO or free calicheamicin-γ1 but with corresponding amounts of MK-2206. All statistical analyses were performed with Prism or InStat ; P<0.05 was considered statistically significant.Data on drug-induced cytotoxicity and DNA damage as well as SCNP are expressed as mean ± SEM. Data on GO-induced cytotoxicity and DNA damage were correlated with CD33 expression levels using Pearson’s linear correlation and expressed as correlation coefficient [95% confidence interval]. Two-way ANOVA for repeated measures was used to analyze cytotoxicity data in AML cell lines, which are shown as percent change in dead cells and cell numbers relative to parallel cultures treated without GO or free calicheamicin-γin vitro stimulation of diagnostic specimens from 6 pediatric and 6 adult patients containing >78% blast cells in all cases  or 48 hours . Importantly, GO failed to induce significant cytotoxicity after 24 or 48 hours in 2 cases (AML-07 and AML-08) with bright CD33 expression, indicating the presence of relevant resistance mechanisms that are unrelated to target antigen expression  and peripheral blood mononuclear cells from a healthy donor , with very marked increases noted in 2/6 specimens  but only minor effects seen in others . We then used SCNP assays to assess these signaling pathways in the pediatric AML samples  using established SCNP methodology for primary human AML 2-fold increase of 3.33±0.58 vs. 0.98±0.69, P = 0.060; for p-S6: mean log2-fold increase of 1.62±0.18 vs. 0.38±0.42, P = 0.054). PI3K pathway activity was similarly elevated in GO resistant samples vs. GO sensitive samples when modulated with FLT3 ligand (FLT3L) . Of note, no differences in basal levels p-S6 and p-Akt were observed between GO resistant vs. GO sensitive samples in the absence of SCF or FLT3L (data not shown).To identify downstream pathways that might affect GO cytotoxicity, we functionally assessed PI3K/AKT/mTOR, JAK/STAT, and Ras/Raf/MEK signaling pathways using SCNP assays; for details regarding modulators and assessed targets, please see Supporting 1-induced cytotoxicity, thus raising the possibility of using inhibitors of this signaling pathway as a novel means to increase the sensitivity of AML cells to calicheamicin-γ1-based therapies. To test this hypothesis, we used MK-2206, an investigational small molecule allosteric inhibitor of AKT currently in Phase 2 clinical development for the treatment of various human malignancies including AML. Consistent with previous investigations on solid tumor cancer as well as acute lymphoblastic leukemia cells 1-cytotoxicity in various human AML cell lines, which are relatively sensitive  or resistant  to single agent GO or calicheamicin-γ1. As shown in in vitro GO cytotoxicity in all 4 cell lines, with most marked effects noted in the GO-resistant TF-1 and KG-1 cell lines, suggesting that inhibition of PI3K/AKT survival signaling can sensitize otherwise resistant AML cells to GO-induced cytotoxicity. Of note, in KG-1 cells, which have active drug efflux, MK-2206 significantly sensitized the cells to GO cytotoxicity both in the absence as well as the presence of a drug efflux reversal agent, PK11195, supporting the relevance of PI3K/AKT pathway activation as a limiting factor for GO cytotoxicity even after inhibition of drug efflux. Consistent with the notion that GO-induced cytotoxic effects are primarily due to the calicheamicin-γ1 moiety rather than the anti-CD33 antibody, comparable results were obtained when the effect of MK-2206 on cytotoxicity induced by free calicheamicin-γ1 was assessed in these cell lines ; limited cell numbers precluded the simultaneous testing of GO in these assays. As shown in 1-induced cytotoxicity varied between these 3 specimens; nevertheless, in all 3 cases, MK-2206 enhanced the cytotoxic effects of calicheamicin-γ1.Taken together, these data suggest a potential role of PI3K/AKT signaling in the mechanism of resistance to GO or, by extension, calicheamicin-γll lines . Finallyin vitro.Clinical studies conducted over the last decade have unequivocally demonstrated that GO provides clinical benefit in a subset of patients with AML while being largely ineffective in others 1 derivative in vitro GO cytotoxicity on sufficient DNA damage, as determined by induced γH2AX levels, suggests that lack of γH2AX induction by GO could serve as an early biomarker of resistance to GO that could be available within hours after exposure to GO.Using SCNP assays, the data from the current study support three major conclusions. First, DNA damage is required but not sufficient for GO-induced cytotoxicity. This finding is consistent with genotoxic responses previously observed in AML samples treated with other agents such as etoposide or cytarabine/daunorubicin in vitro cellular resistance to GO in primary AML samples. This finding supports a model in which the toxicity of the calicheamicin-γ1 moiety is critically modulated by the ability of the cell to repair DNA damage as well as the activity of downstream pro- and anti-apoptotic pathways, and may offer a partial biological explanation for the observation that calicheamicin-γ1in vitro sensitivity varies over 100 000-fold among individual primary AML cell samples Second, our data are the first to indicate that anti-apoptotic pathways downstream of DNA damage contribute to 1. AKT is downstream of PI3K and the primary effector molecule of the PI3K signaling cascade in vitro data with MK-2206 indicate that AKT signaling modulates GO/calicheamicin-γ1 cytotoxicity and is associated with cellular resistance to these drugs. In turn, inhibition of AKT activation can greatly increase GO/calicheamicin-γ1 sensitivity, suggesting a role of AKT inhibitors such as MK-2206 as chemosensitizers for GO or, more generally, calicheamicin-γ1-based treatment strategies. Further studies will be required to fully understand the mechanistic details by which activation of this pathway interferes with GO/calicheamicin-γ1-induced cytotoxicity.Third, and most importantly, our studies identify activated PI3K/AKT signaling, measured after growth factor treatment, as a novel mechanism of resistance to GO and, by extension, calicheamicin-γin vitro GO and calicheamicin-γ1 resistance, our data illustrate the capacity of SCNP assays to functionally characterize the integrity of biological pathways and signaling networks relevant to cellular responses to genotoxins such as calicheamicin-γ1-based antibody-drug conjugates in vitro in primary samples from patients with AML. Although future studies with larger numbers of patient specimens will be required to validate our studies and quantify the contribution of AKT-mediated resistance to GO and calicheamicin-γ1 in detail, our data highlight the potential of SCNP assays to differentiate AML samples based on underlying biology which might be relevant to therapeutic interventions. Our data form the basis for future studies in which such assays are applied to AML samples from patients with documented clinical outcomes to validate findings from in vitro predictions. If validated, SCNP assays could serve to prospectively identify subsets of patients that are suitable for GO-based therapy – a critical advancement toward optimized drug use – and may help in the discovery of additional therapeutic targets,. By monitoring multiple cellular characteristics , SCNP assays may provide an ideal tool to prospectively inform about multiple GO-relevant resistance mechanisms simultaneously, and could help in the selection of the most appropriate chemosensitizing agents and drug combination strategies in patients under consideration for GO-containing therapy, thus further contributing to the individualization of AML treatment approaches.Together, by identifying an association between PI3K/AKT signaling activation and Figure S1Quantification of CD33 Qdot605 MFI in primary AML specimens. To compute the equivalent CD33 MFI value in the Qdot605 scale for sample AML-11 (which lacked this data point), a linear regression model was constructed comparing CD33 Qdot605 MFI with CD33 PE MFI and used to compute the equivalent Qdot605 MFI value for AML-11 based on its CD33 PE MFI value. AML samples and peripheral blood and bone marrow mononuclear cells from normal donors  with available data on both fluorophores (left table), was used to calculate a linear regression model between the CD33 Qdot605 and PE MFI scales (middle), allowing for computation of the equivalent CD33 Qdot605 MFI in specimen AML-11 using the equation: Qdot605-CD33 MFI  = 0.6927×(CD33-PE MFI) +176.05 (r = 0.94) (right).(TIFF)Click here for additional data file.Figure S2Effect of staurosporine on GO-resistant pediatric AML samples. Primary AML specimens were treated with staurosporine for 6 hours prior to flow cytometric assessment of apoptosis using cleaved caspase-3 (X-axis) and cleaved PARP (Y-axis). All GO-resistant samples except AML-10 showed robust apoptotic responses, as demonstrated by presence of >60–90% double positive cells upon treatment with staurosporine. Note: the apoptosis markers, cleaved caspase-3 and cleaved PARP, showed concordant results with an average of >90% of cells being either double positive or double negative for these markers.(TIFF)Click here for additional data file.Figure S3Kinetics of GO-induced DNA damage response in vitro. (A) Kinetics of GO-induced DNA damage measured by γH2AX levels. Treatment conditions are indicated by shape: GO alone (squares), drug efflux inhibitor  alone (circles), and GO in combination with cyclosporine A (crosses),.AML cell lines GDM-1 and KG-1 (left panels) were resistant to GO treatment alone with minimal induction of DNA damage at any time-points tested; however, U937 cells and primary myeloid PBMC (right panels) were sensitive to GO treatment alone with highest γH2AX levels observed at 6–8 hours. Co-treatment of GO with cyclosporine A sensitized MDR+ KG-1 and GDM-1 cells to GO-induced DNA damage.(TIFF)Click here for additional data file.Figure S4Inhibition of drug efflux activity by PSC833 increases GO sensitivity in some AML samples. Drug transporter activity was assessed by co-treatment of GO and the pump efflux inhibitor, PSC833. DNA damage  and apoptosis  responses are shown for AML-02 (left) and AML-05 (right) in the presence of GO alone, PSC833 alone, or GO in combination with PSC833. While PSC833 co-treatment substantially increased GO induced apoptosis in AML-02, PSC833 co-treatment did not sensitize AML-05 to GO induced apoptosis. Circles highlight γH2AX+ cells (red) and apoptotic cPARP+ cells (green).(TIFF)Click here for additional data file.Figure S51-induced cytotoxicity in human AML cell lines Effect of AKT inhibition on calicheamicin-γin vitro. Various doses of MK-2206 were incubated with increasing concentrations of calicheamicin-γ1 in (A) HL-60, (B) NB4, (C) TF-1, and (D) KG-1 cells. After 3 days, viability (left-side panel) and cell numbers (right-side panel) was determined by flow cytometry. *P<0.05 as compared to medium alone; **P<0.01 as compared to medium alone; ***P<0.001 as compared to medium alone; ****P<0.0001 as compared to medium alone.(TIFF)Click here for additional data file.Table S1List of reagents.(TIFF)Click here for additional data file.Table S2Conditions and staining cocktails used in SCNP signaling assays.(TIFF)Click here for additional data file.Table S32Fold signaling responses of AML samples.Log(TIFF)Click here for additional data file.


While an increase in [Ca2+]i excites neurons, [Ca2+]i overload elicits cytotoxicity, resulting in cell death. Intracellular Ca2+ is essential for many signal transduction mechanisms, and its level is precisely regulated by the Ca2+ extrusion system in the plasma membrane, which includes the Na+-Ca2+ exchanger (NCX). It has been demonstrated that Ca2+-ATPase is the primary mechanism for removing [Ca2+]i following excitatory activity in trigeminal ganglion (TG) neurons; however, the role of NCXs in this process has yet to be clarified. The goal of this study was to examine the expression/localization of NCXs in TG neurons and to evaluate their functional properties.Noxious stimulation and nerve injury induce an increase in intracellular Ca+ channels were prolonged by NCX inhibitors when [Ca2+]i in TG neurons was elevated beyond physiological levels.NCX isoforms  were expressed in primary cultured rat TG neurons. All the NCX isoforms were also expressed in A-, peptidergic C-, and non-peptidergic C-neurons, and located not only in the somata, dendrites, axons and perinuclear region, but also in axons innervating the dental pulp. Reverse NCX activity was clearly observed in TG neurons. The inactivation kinetics of voltage-dependent Na2+-homeostasis and somatosensory information processing by functionally coupling with voltage-dependent Na+ channels.Our results suggest that NCXs in TG neurons play an important role in regulating Ca Active Ca2+ efflux/extrusion via this system in plasma membrane involves either high-affinity, low-capacity Ca2+-ATPase (PMCA) or low-affinity, high-capacity Na+-Ca2+ exchangers (NCX) i) in both excitable and non-excitable cells and pump Ca2+ out of cells by means of the Na+ concentration gradient across the cell membrane i was measured. The changes in [Ca2+]i was expressed as F/F0 units, with RF340/F380 values (F) normalized with respect to the resting value (F0). To confirm that the primary cultured cells were indeed TG neurons, we assessed the cellular response to an extracellular solution containing high extracellular K+, which would induce depolarization, and hence Ca2+ influx in neurons  [+) solutions induced an increase in [Ca2+]i in 88.2% of tested primary cultured TG cells, while the remaining cells (11.8%) showed no response  -19. A seGene-specific primers for real-time RT-PCR analyses revealed the expression of NCX1, NCX2, and NCX3 mRNA in rat TG neurons , and then Na+ was replaced with equimolar Li+ (Li+-ECS) in order to reverse the Na+ gradient across the plasma membrane in the presence of extracellular Ca2+   Figure B. The int Figure B illustr2+ influx mediated by NCXs. In the presence of 1.5 mM [Ca2+]o, a series of applications of KB-R7943, SEA0400, or SN-6 significantly blocked Ca2+ influx by reverse NCX activity in a concentration-dependent manner  was obtained at a KB-R7943 concentration of 3.08 μM  was examined to investigate the contribution of NCX activity to excitability in TG neurons. Inward currents were elicited from TG neurons in 20-ms voltage clamp steps ranging from −80 to +80 mV. Voltage steps were applied in 10-mV increments from a holding potential (Vh) of −70 mV, in the presence of standard extracellular solution, and intracellular solution (ICS) containing either 44 nM, or 26 μM free Ca2+ . Current–voltage (I-V) relationships were determined by plotting the peak current amplitudes against the applied membrane potentials  and [Ca2+]i-overloaded conditions (26 μM [Ca2+]i). Inward currents were, however, significantly blocked by the voltage-dependent Na+ channel blocker tetrodotoxin (TTX) (1.0 μM) under both conditions  were carried mainly by TTX-sensitive voltage-dependent Na+ channels, with small component carried by TTX-resistant channels.The effect of NCX inhibitors on voltage-dependent NaINa without (open black) or with (filled black) a cocktail of NCX inhibitors . This was also the case in the [Ca2+]i-overloaded condition  of total INa between the physiological (open black circles) and [Ca2+]i-overloaded condition (open red circles), as shown in Figure 2+]i-overloaded condition and in the presence of NCX inhibitors, the values of τ  were significantly prolonged in a voltage-dependent manner . In addition, these cells, which were obtained from rat TG primary cultures, expressed total INa, indicating that the tested cells comprised mostly TG neurons rather than glial cells i-overload caused by Ca2+ influx via NCXs contributes to neuropathic pain. In contrast, the inactivation process of INa was significantly and voltage-dependently prolonged by the application of NCX inhibitors, only when the [Ca2+]i in TG neurons was overloaded   that i-overloaded conditions. In addition, a cocktail of these 3 NCX inhibitors  prolonged the inactivation process of INa under the [Ca2+]i-overloaded condition, but not under the physiological condition , but not SEA0400, has been shown to have an inhibitory effect on the peak amplitude of ocardium . In our + channels comprise a transmembrane glycoprotein consisting of a pore-forming α subunit, and an auxiliary β subunit which modulates the kinetics and voltage dependence of Na+ channel activation and inactivation -i was expressed as the fluorescence ratio (RF340/F380) at two excitation wavelengths of 380 nm and 340 nm.Primary cultured TG cells were incubated for 60 min at 37°C in Hank’s solution containing 10 μM fura-2 acetoxymethyl ester  and 0.1% (w/v) pluronic acid F-127 (Invitrogen), followed by rinsing with fresh Hank’s solution. A dish with fura-2-loaded TG neurons was mounted on the stage of a microscope  incorporated into the Aquacosmos system and software  equipped with an excitation wavelength selector and an intensified charge-coupled device camera (Hamamatsu Photonics). i increase in TG neurons o) in the Na+-ECS was substituted with equimolar extracellular Li+ concentration ([Li+]o) (Li+-ECS). To examine the K+-dependence of Na+-Ca2+ exchange in TG neurons, 5.0 mM K+ was added to Li+-ECS.Standard recording solution comprised Hank’s balanced salt solution containing the following: 137 mM NaCl, 5.0 mM KCl, 1.5 mM CaCl neurons : 91 mM N2, 5.0 mM EGTA (free [Ca2+] was 44 nM), and 10 mM HEPES (pH 7.2). For the [Ca2+]i-overloaded condition, the intracellular solution comprised following; 130 mM KCl, 2.0 mM Mg-ATP, 10 mM TEA-Cl, 10 mM CaCl2, 10 mM EGTA (free [Ca2+] was 26 μM) [2+]i measurement and patch-clamp recording, and applied at the appropriate concentration to primary cultured TG neurons via a gravity-fed perfusion system . For Na+ inward current recording, 1.0 μM tetrodotoxin was diluted with standard solution. Except for those noted above, all other reagents were obtained from Sigma-Aldrich .The intracellular solution (ICS) for patch-clamp recordings in the physiological condition contained the following: 140 mM KCl, 2.0 mM Mg-ATP, 10 mM TEA-Cl, 1.0 mM CaCls 26 μM) , and 20 2+-dependence of NCX activity and the dose–response relationship for the inhibitory effect of NCX inhibitors were obtained by fitting the data with following function:The Ca2+ concentration ([Ca2+]o) with a Hill coefficient (h) equal to 1; or where IC50 is the 50% inhibitory concentration for applied NCX inhibitors. Amax is maximal, and Amin is minimal response. The [X]o indicates applied concentration of extracellular Ca2+ or inhibitors. The kinetics of inactivation was determined by fitting the experimental data with a single exponential function using the pCLAMP software.where K is the equilibrium binding constant for applied extracellular Can observations, where n represents the number of separate experiments. The Wilcoxon t-test, Friedman test, or Kruskal-Wallis test and Dunn’s post hoc test were used to determine non-parametric statistical significance. A p value of less than 0.05 was considered significant. The statistical analysis was performed using Graph Pad Prism 5.0 .Data were expressed as the mean ± standard error (S.E.) or standard deviation (S.D.) of the mean of The authors declare no conflict of interest regarding the subject or materials discussed in this manuscript. In addition, the funders had no role in study design, data collection, analysis, decision to publish, or preparation of the manuscript.MZ, TI and YS were responsible for the conception and design of the experiments. HK, MS, US, MT, and YS were responsible for the acquisition, analysis and interpretation of the data. HK and YS were responsible for drafting and critically revising the article in terms of intellectual content. YS was responsible for final approval of the version to be submitted/published. All of the authors were involved in critically revising important intellectual content and giving final approval of the version of the manuscript to be published.