Upon sensing, they transduce electrical and Ca2+ signals via their cation channel activities. These functional features of TRP channels allow the body to react and adapt to different forms of environmental changes. Indeed, members of one class of TRP channels have emerged as sensors of gaseous messenger molecules that control various cellular processes. Nitric oxide (NO), a vasoactive gaseous molecule, regulates TRP channels directly via cysteine (Cys) S-nitrosylation or indirectly via cyclic GMP (cGMP)/protein kinase G (PKG)-dependent phosphorylation. Recent studies have revealed that changes in the availability of molecular oxygen (O2) also control the activation of TRP channels. Anoxia induced by O2-glucose deprivation and severe hypoxia (1% O2) activates TRPM7 and TRPC6, respectively, whereas TRPA1 has recently been identified as a novel sensor of hyperoxia and mild hypoxia (15% O2) in vagal and sensory neurons. TRPA1 also detects other gaseous molecules such as hydrogen sulfide (H2S) and carbon dioxide (CO2). In this review, we focus on how signaling by gaseous molecules is sensed and integrated by TRP channels.The transient receptor potential ( These gene products form cation channels that detect and transduce cellular stimuli into electrical signals  or chemical signals [via changes in intracellular Ca2+ concentration ([Ca2+]i)]  that, when activated by receptor stimulation, induce phospholipase C (PLC) to hydrolyze phosphatidylinositol-4,5-biphosphate (PIP2) into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG)  Ca2+ stores, can also be categorized as RACCs. In contrast, TRPV Ca2+-permeable channels can be functionally defined as thermosensors  concentration (pH < 5.6); the intrinsic ligand, anandamide; and receptor-driven PLC activity  proteins are the product of Clapham, . The TRPClapham, , and conClapham, . The solClapham, . PungentClapham, . TRPA1 hClapham, . Thus, TClapham, , all of 2), nitric oxide (NO), carbon monoxide (CO), hydrogen sulfide (H2S), and carbon dioxide (CO2), have been shown to play important roles in biological signal transduction. These molecules share several unique physicochemical properties and exert their biological activities through mechanisms distinct from those of other signaling molecules as summarized by Suematsu  receptors activates neuronal NOS (nNOS)  both positively  revealed that cGMP has a dual effect on Ca2+ entry. Increasing cGMP levels by up to 10-fold above that in control cells treated with N-nitro-L-arginine, a specific inhibitor of NOS, is associated with activation of Ca2+ entry. Further increases in cGMP to levels up to 80-fold above control inhibit Ca2+ entry in a concentration-dependent manner. The biphasic effect of cGMP provides the cells with a negative feedback mechanism and inhibits Ca2+ entry during periods of high [Ca2+]i, allowing oscillatory behavior in Ca2+ entry. In vascular endothelial cells isolated from bovine aorta, exogenous NO gas potentiates Ca2+ influx, which markedly increases the sustained phase of [Ca2+]i elevation and possibly NO production 1,2,3,4-oxatriazolium] (25 μM), or the alkylator, N-ethylmaleimide (10 μM), is strongly activated by transient external Ca2+ removal that depletes internal Ca2+ stores, closely resembling activation of SOC activity in the same cells -dependent mechanism. These results suggest a role of cGMP and PKG in the regulation of Ca2+ entry in vascular endothelial cells ] to SNAP, in control cells and cells heterologously expressing either TRPC1 or TRPC4β (a splice isoform of TRPC4) are not statistically significant, a larger fraction (7–9%) of the TRP-expressing cells shows a Δ ratio (340/380) >0.5 when compared to control cells (2–5%). Cells heterologously co-expressing TRPC4β and TRPC5 give responses to SNAP comparable to those in cells heterologously expressing TRPC5 alone, whereas cells heterologously co-expressing TRPC1 and TRPC5 give slightly suppressed yet robust responses. Co-immunoprecipitation of TRPC5 with TRPC1 or TRPC4β suggests that heteromultimeric TRPC5/TRPC1 and TRPC5/TRPC4β channels also have NO sensitivity. The thermosensor TRP channels TRPV1, TRPV3, and TRPV4 also show SNAP (300 μM)-induced activation, as predicted from conserved Cys residues in the corresponding regions of these homologs. Indeed, substitutions of two conserved Cys residues in TRPV1 lead to significantly suppressed responses to SNAP and to Cys S-nitrosylation. Notably, the sensitivity of TRPV1 to H+ and heat is enhanced by SNAP but abolished by the mutations, despite normal surface expression and intact control H+ and heat responses. Thus, channel activation regulated by nitrosylation is conserved among a number of TRP channels belonging to different subfamilies.Some TRP channels are potently regulated by Cys modifications, including Cys S-nitrosylation by NO in heterologous systems and bovine aortic endothelial cells , which reduces both S-nitrosothiols and disulfides to thiols, fully suppressed SNAP-activated TRPC5 channel activity, suggesting that both nitrosylation and disulfide bond formation are involved in SNAP-induced TRPC5 activation. In an S-nitrosylation assay . Among numerous caveolin-associated proteins linked to signaling cascades  and another NO donor (6)-(E)-Ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide (NOR3) (300 μM) activate human TRPA1 in heterologous systems and mouse TRPA1 in dissociated sensory neurons . For capsaicin, injections of capsaicin at >1000-times the in vitro EC50 are typically used to observe acute pain behavior. It is therefore not surprising that significant acute nocifensive behavior was not observed in terms of NO donors. Another potential explanation for the lack of a phenotype in individual knockout mice is due to functional compensation by each other. Indeed, TRPV1 and TRPA1 have overlapping expression in a subset of DRG neurons  in the hind paw after PLC and PKA pathway activation , causes nociceptive behavior  through voltage-dependent Ca2+ channels (VDCCs). Phosphorylation of the inositol 1,4,5-trisphosphate receptor (IP3R)-associated protein, IRAG (IP3R-associated PKG substrate), is thought to inhibit agonist-induced Ca2+ release from internal stores. The possibility has also been suggested that phosphorylation of phospholamban by PKG increases the activity of sarcoplasmic Ca2+-ATPase, facilitating the active transport of Ca2+ into internal stores and thereby decreasing [Ca2+]i. In many different types of blood vessel, activation of PKG has been found to inhibit vasoconstrictor-induced Ca2+ influx through plasmalemmal pathways other than those employing VDCCs . The TRPC6 protein is abundantly expressed in various types of VSMCs and has been shown to be a constituent of vasoconstrictor-activated cation channels, which increase Ca2+ entry into VSMCs via direct Ca2+ permeation or secondary activation of a VDCC and/or Na+-Ca2+ exchanger , PKG-mediated mechanisms may work as a universal negative feedback to regulate neurohormonal Ca2+ mobilization across the VSMC membrane  and gaseous molecules such as NO and CO  environment. Because of the ambivalent physiological nature of O2, aerobic life forms must adapt to hyperoxia and low O2 environment (hypoxia) by sensing O2 availability and transmitting this information to effector systems.Of the gaseous molecules, On Csete, . Thus, O2 availability can be acute or chronic  in arterial blood  in humans. The failure of AET in the face of a clear role for excitotoxicity in acute neurological disorders is paradoxical -induced cation influx and currents in smooth muscle cells are largely absent in precapillary pulmonary arteries of Trpc6 knockout mice, although recombinant TRPC6 expressed heterologously cannot be activated by hypoxia   hydroxylation by Pro hydroxylases (PHDs), and direct oxidation of Cys residues. In normoxia, PHDs hydroxylate conserved Pro394 within the tenth AnkR domain of TRPA1, inhibiting its activity. In hypoxia, the decrease in O2 concentration diminishes PHD activity, relieving TRPA1 from the inhibitory action of Pro hydroxylation and leading to its activation. This recovery of TRPA1 activity can be achieved by insertion of fresh, unmodified TRPA1 proteins into the plasma membrane or by dehydroxylation of modified proteins through an unidentified molecular mechanism. In hyperoxia, O2 activates TPRA1 by oxidizing Cys633, Cys856 or both. Cys633 and Cys856 are located within the seventeenth AnkR domain and the intracellular linker region between S4 and S5, respectively. TRPA1 can assume at least two oxidized forms during hyperoxia: a relatively unstable oxidized state (state 1) readily reversed by glutathione, and a relatively stable oxidized state (state 2). SH groups on the key Cys residues (Cys633 and Cys856) may be modified to sulfenic acid (S-OH) in state 1, and form disulfide bonds (S-S) in state 2. This oxidation mechanism over-rides the inhibition by Pro hydroxylation to activate TRPA1. In mice, disruption of the Trpa1 gene abolishes hyperoxia- and mild hypoxia (15% O2)-induced cationic currents in vagal and sensory neurons and thereby impedes enhancement of in vivo vagal discharges induced by hyperoxia and hypoxia. The results suggest a new O2-sensing mechanism mediated by TRPA1   is comparable to atmospheric PO2  produces pain responses through potentiation of T-type Ca2+ channels probably in the primary afferents  causes activation of human and mouse TRPA1 in heterologous expression systems, suggesting that TRPA1 is indeed a molecular target for H2S in the bladder  activates capsaicin-sensitive sensory neurons in isolated rat urinary bladder , in addition to the primary activation trigger immediately upstream, the TRP channel plays an important role in forming positive feedback or feed-forward loops in the cascade. For example, in vascular endothelial cells, Ca2+ influx via TRPV1 and TRPA1 in controlling Ca2+ signaling pathways in neurons remain elusive. Considering that the PHD-HIF pathway is central to chronic hypoxia responses that increase red blood cell mass and stimulate new blood vessel growth (Semenza and Wang, 2+ influx through O2-sensitive TRPA1 channels on the functional regulation of PHD and HIF. Studies of TRP channels have been extended dramatically from the simple functional description of single molecules to the holistic analysis and integration of the molecular systems controlled by TRP channels.The accumulating evidence summarized above strongly suggests that TRPV1 and TRPA1 are sensors that transduce gaseous signals into electrical signals in sensory and vagal neurons. The chemosensory inputs to these neurons are propagated toward the central nervous system to induce pain sensation or to change ventilatory patterns. However, the roles of CaThe 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.


Background: Cellular transducers of Wnt signaling, β-catenin and Ca2+, are considered to act via independent canonical and non-canonical pathways.Results: Six Wnt ligands activated cytosolic and intranuclear Ca2+ and nuclear translocation of β-catenin.Conclusion: Wnt β-catenin and Ca2+ signaling are coupled in mammalian cells.Significance: A coupled model of Wnt/Ca2+ and β-catenin signaling that challenges the canonical and non-canonical categorization is proposed. 2+ and β-catenin, a 92-kDa negatively charged transcription factor, transduce Wnt signaling via the non-canonical, Wnt/Ca2+ and canonical, Wnt/β-catenin pathways independently. The nuclear envelope is a barrier to large protein entry, and this process is regulated by intracellular calcium [Ca2+]i and trans-nuclear potential. How β-catenin traverses the nuclear envelope is not well known. We hypothesized that Wnt/Ca2+ and Wnt/β-catenin pathways act in a coordinated manner and that [Ca2+]i release facilitates β-catenin entry into the nucleus in mammalian cells. In a live assay using calcium dyes in PC3 prostate cancer cells, six Wnt peptides  mobilized [Ca2+]i but Wnt11 did not. Based upon dwell time (range = 15–30 s) of the calcium waveform, these Wnts could be classified into three classes: short, 3A and 5A; long, 7A and 10B; and very long, 4 and 9B. Wnt-activated [Ca2+]i release was followed by an increase in intranuclear calcium and the depolarization of both the cell and nuclear membranes, determined by using FM4-64. In cells treated with Wnts 5A, 9B, and 10B, paradigm substrates for each Wnt class, increased [Ca2+]i was followed by β-catenin translocation into the nucleus in PC3, MCF7, and 253J, prostate, breast, and bladder cancer cell lines; both the increase in Wnt 5A, 9B, and 10B induced [Ca2+]i release and β-catenin translocation are suppressed by thapsigargin in PC3 cell line. We propose a convergent model of Wnt signaling network where Ca2+ and β-catenin pathways may act in a coordinated, interdependent, rather than independent, manner.Ca Macromopathways . The 92-pathways ) is at tpathways –28, sugg2+ and K+ concentrations  across the NE . Cytosontration ; an incrs the NE , 30, 31,ransport , 32–34. ransport  consistiransport . In HeLaport  whether Wnts other than Wnt5A also increase in [Ca2+]i; (ii) that increase in [Ca2+]i due to Wnt/Ca2+ signaling activation results in depolarization of nuclear membranes; (iii) that in cells, activated by multiple Wnt ligands, β-catenin is translocated into the nucleus. By using a live fluorochrome assay and subsequent assessment of β-catenin translocation to nucleoplasm in Wnt ligand-activated mammalian cancer cell lines , we demonstrate that a number of Wnt ligands increase intracellular and intranuclear Ca2+, with distinct kinetics, depolarization of the NE and translocation of β-catenin to the nucleus; both the increase in Wnt induced [Ca2+]i release and β-catenin translocation are suppressed by thapsigargin, an inhibitor of microsomal Ca2+-ATPase  were obtained from ATCC via John R. W. Masters, University College London and cultured in RPMI 1640 (Invitrogen) medium containing 5 mRecombinant Wnt  peptides were purchased from R&D Systems . Wnt-peptide stock solutions were prepared in PBS.2+]i stores, in some cases PC3 cells, loaded with calcium dyes, were incubated with 1–5 μm of thapsigargin (Sigma), resuspended in dimethyl sulfoxide, for 20 min at 37 °C, prior to addition of Wnt ligand and live calcium imaging (as described above).The imaging protocol was developed in our laboratory and has been described in detail elsewhere . Brieflyi.e. excitation with 488 nm for both indicators and emission detection at 500–560 nm for Fluo4 and 650–800 for FM4-64. All other imaging/data acquisition conditions were the same as above.Experiments with the membrane potential sensitive dye FM4 64 (Invitrogen) and Fluo4 were performed in exactly the same manner as for the Fluo4/FuraRed but with different emission spectra detection for the FM4 64 indicator, −1 . The calcium wave , in response to Wnt ligands, traveled across the FluoroDish.The experiments were performed using a 35-mm, glass-bottomed FluoroDish containing 1 ml of PBS at 37 °C to which ligands were added at desired concentration. In a static, non-superfusing assay system, there is a lag time before the start of a response, in this case, an increase in the intensity of the Fluo4 signal due to the release of free calcium. To measure the diffusion constant under these conditions, we used pontamine sky blue (Sigma), which fluoresces in the red spectrum with an excitation of 488 nm, using the standard practice for adding ligands; the diffusion constant was measured to be 23.3 ± 2.1 μm·s2+]i release in PC3 cells in response to different Wnt ligands. The time constants  were calculated by first fitting the Fluo4/FuraRed ratio to a single Gaussian function (OriginLab Corp.); a lower (10%) and upper (90%) amplitude thresholding method was then used to calculate the time constants (s). Statistical significance was calculated using Mann-Whitney U test.After image acquisition, regions of interest were drawn over individual PC3 cells (up to 25) in a single frame, and calcium intensity measurements were made using FluoView 1000 software (Olympus). The measurements were exported as a tab-delimited file into a spreadsheet for further analysis, and Fluo4/Fura Red ratios were calculated. The waveform, a product of the ratio of Fluo4 and FuraRed over the period of measurement was used to measure the kinetics of the activation of [Ca2+]i experiments using dyes (see above), or grown in eight-well chamber glass slides  were used. For Wnt ligand treatment, the culture medium replaced with PBS  at 37 °C. The experiments were replicated for Wnt ligand or vehicle control as described for [Ca2+]i measurements. Briefly, Wnt ligands were added and chamber slides incubated at 37 °C for up to 20 min. Chamber wells were washed 3× with 0.5 ml of PBS, and cells were fixed with 0.5 ml of 4% formalin for 30 min at 4 °C. Cells were washed 3× with ice-cold PBS and stored in 0.5 ml of PBS at 4 °C for immunostaining. In some experiments, PC3 cells were treated with thapsigargin as described above.Immunostaining was performed in two different laboratories, independently, with the experimenter blind to the identity of the treatment. Either cells used in FluoroDish, post i . The ratio of Fluo4/FuraRed was plotted as a function of time  were calculated by first fitting the Fluo4/FuraRed ratio to a single Gaussian function. We then used a lower (10%) and upper (90%) amplitude thresholding method to calculate the timing parameters. The amplitude for [Ca2+]i release in response to Wnt ligands represents the peak of the calcium waveform. The amplitude of calcium release was Wnt ligand concentration-dependent . A number of Wnt ligands  also activated [Ca2+]i release in MCF7 and 253J human breast and urinary tract cell lines, respectively , indicating that this phenomenon was not specific to PC3 cell line. Furthermore, Wnt5A also activated [Ca2+]i in Cos7 simian kidney cell line .Addition of all Wnt ligands tested, with the exception of Wnt11, caused a change in ratio of Fluo4/Fura Red in PC 3 cells  was abolished after treatment of PC3 cells with thapsigargin (5 μm) (2+]i abolishes Wnt induced [Ca2+]i release.Thapsigargin is an inhibitor of the endoplasmic reticulum Cai stores . Thapsig release . The incn (5 μm) . These r2+]i release in response to Wnt peptides (100 ng/ml) were calculated as described under “Experimental Procedures” and shown in A and C). DTs reflect the time the [Ca2+]i signal was sustained at peak amplitude after application of Wnt peptides. The DT analysis (B) indicates that of the six Wnts activating [Ca2+]i release could be classified into three classes: short, 3A and 5A (DT < 15 s); long, 7A and 10B (DT > 25 s); and very long, 4 and 9B (DT > 30 s).Time constants for the rise, dwell, and fall time of [Caanalysis B indicat2+]i release was further analyzed by using three different concentrations for each Wnt ligand. Box-whisker plots for the rise, dwell, and fall time as a function of low, medium, and high concentrations of each Wnt ligand is given in supplemental Figs. S2–S7). Time constants were significantly different between at least two different concentrations for Wnts 3A, 4, 5A, 9B, and 10B , but no difference in these time constants was observed for Wnt7A . We compared the DT constants for Wnts 3A, 4, 5A, 9B, and 10B in more detail to elucidate the mechanisms of [Ca2+]i release by fitting the DT data to calculate the reaction-rate expression for a first-order reaction  to their receptor and show, also for the first time, that the molecular dynamics of [Ca2+]i release, via receptor/ligand interaction by different Wnts, could provide critical regulatory mechanisms of Wnt/Ca2+ signaling.The kinetics of [Careaction . Strikin2+ follows increase in cytosolic Ca2+ (2+]i (Fluo4) signal in the green channel (500–560 nm) upon addition of the Wnt ligands in these cells . After ∼300 s, there was an increase in the intensity of the FM4-64 signal in the red channel (650–800 nm); there was depolarization of the cell membrane, as expected .Calcium is a key regulator of the electrical activity in the nucleus . Intranulic Ca2+ . We therexpected . There w2+ signaling is a decrease in cell motility . The rate of wound closure in wounded PC3 cells in the presence of Wnt 5A or 9B was similar to that of control untreated cells . However, there was a significant decrease in the rate of wound closure in cells treated with thapsigargin even in the presence of Wnt ligands. These results indicate that downstream mechanisms of Wnt/Ca2+ signaling are similar for the classical non-canonical (Wnt 5A) or canonical (Wnt 9B) Wnt ligands. Similar results were obtained for Wnts 3A and 10B when [Ca2+]i was inhibited by incubation with thapsigargin .One functional consequence of inhibition of downstream Wnt/Camotility . To test2+]i release using immunofluorescence. Compared with vehicle controls, there was an increased in expression of β-catenin in PC3 cells treated with Wnts5A, 9B, and 10B (A); the expression of β-catenin is visibly increased in PC3 cells treated with Wnt5A (B), Wnt9B (C), and Wnt 10B (D) and is visible in the nucleus. Similar results were obtained for Wnt 3A and Wnt4 in PC3 cells . Co-localization of β-catenin and DAPI signals was quantified using Fiji software . Furthermore, inhibition of Wnt-induced [Ca2+]i release, by incubation of cells with thapsigargin, reduced the translocation of β-catenin into the nucleus , indicating a link between β-catenin translocation, store-operated, thapsigargin-inhibitable [Ca2+]i release and perhaps alterations in nuclear envelope potential.β-Catenin expression was determined subsequent to Wnt ligand activation and i release was followed by translocation of β-catenin to the nucleus. The data for MCF7 and 253J cells, with β-catenin expression visible in the cell nucleus after treatment with Wnt ligands, are shown in supplemental Fig. S13. These results indicate that increase in [Ca2+]i in response to a variety of Wnt ligands leads to a depolarization of the nuclear envelope and β-catenin translocation from the cytosol to the nucleus.We also used the paradigm ligands of the Wnt classes  . A discharge of [Ca2+]i from the endoplasmic reticulum and inhibition of microsomal Ca2+-ATPase by incubation of cells with thapsigargin abolished the increase in Wnt-induced [Ca2+]i release . Thapsigargin is resuspended in dimethyl sulfoxide. Dimethyl sulfoxide induces Ca2+ spikes in a variety of cells . Thapsigargin also induced Ca2+ transient, which declined to reach the base line within 120 s , unlike thapsigargin treatment, which abolished Wnt-induced [Ca2+]i release . The Wnpolarity –47. The polarity , 22; how release  and reduof cells ; our resin 120 s . However release , indicat2+, depolarize the NE, and increase the translocation of β-catenin to the nucleoplasm, suggesting a coupled rather than independent Wnt/Ca2+ and β-catenin pathway.The results presented here provide first evidence that key transducers of canonical and non-canonical pathways converge to activate Wnt signaling in mammalian cells. A new convergent model of Wnt signaling network is proposed , in whicXenopus embryos  and inde.g. Wnt/β-catenin or Wnt/Ca2+ or both) could determine, for example, cell fate of a stem cell or whether cancer cells metastasize (e.g. by acquiring increased capacity for motility). Wnt 11 is the only ligand tested in our study that did not mobilize [Ca2+]i . A possible explanation for this may reside in primary amino acids that deviates from the consensus Wnt sequence in five amino acid residues, in close proximity to RWNC motif .However, these distinctions have been blurred when it was shown that Wnts associated with the so-called non-canonical pathway are capable of transforming cells in culture , 53, 54.in vitro, biochemical experiments, Sharma and colleagues  but also small (<10 kDa) macromolecules across NE  and reduction in β-catenin translocation into the nucleus in the presence of thapsigargin  suggest a putative role of membrane potential regulating ions as regulators of Wnt signaling in PC3 cells.Other mechanisms involve silencing of NPC channel activity for transport of medium-sized (∼40 kDa) transcription factors (such as NFκB or SP1) . Ca2+ ioolecules , and it cross NE . Translo2+]i release (2+]i results in the increased intranuclear Ca2+ within 300 s of Wnt ligand activation (2+ increase and depolarization of the NE, there is also an evident increase in the nuclear expression of β-catenin (In the convergent model of Wnt signaling in mammalian cells proposed here , binding release . The shativation . Subsequ-catenin  and 7. T-catenin .


To confirm direct interaction and locate the calmodulin-binding domain(s), we designed a series of FRET biosensors that consist of enhanced cyan and yellow fluorescent proteins flanking each of GPER’s submembrane domains (SMDs). Responses of these biosensors showed that all four submembrane domains directly bind calmodulin. Modifications of biosensor linker identified domains that display the strongest calmodulin-binding affinities and largest biosensor dynamics, including a.a. 83–93, 150–175, 242–259, 330–351, corresponding respectively to SMDs 1, 2, 3, and the juxta-membranous section of SMD4. These biosensors bind calmodulin in a strictly Ca2+-dependent fashion and with disparate affinities in the order SMD2>SMD4>SMD3>SMD1, apparent Kd values being 0.44±0.03, 1.40±0.16, 8.01±0.29, and 136.62±6.56 µM, respectively. Interestingly, simultaneous determinations of biosensor responses and suitable Ca2+ indicators identified separate Ca2+ sensitivities for their interactions with calmodulin. SMD1-CaM complexes display a biphasic Ca2+ response, representing two distinct species (SMD1 sp1 and SMD1 sp2) with drastically different Ca2+ sensitivities. The Ca2+ sensitivities of CaM-SMDs interactions follow the order SMD1sp1>SMD4>SMD2>SMD1sp2>SMD3, EC50(Ca2+) values being 0.13±0.02, 0.75±0.05, 2.38±0.13, 3.71±0.13, and 5.15±0.25 µM, respectively. These data indicate that calmodulin may regulate GPER-dependent signaling at the receptor level through multiple interaction sites. FRET biosensors represent a simple method to identify unknown calmodulin-binding domains in G protein-coupled receptors and to quantitatively assess binding properties.The G protein-coupled estrogen receptor 1 (GPER) has been demonstrated to participate in many cellular functions, but its regulatory inputs are not clearly understood. Here we describe a new approach that identifies GPER as a calmodulin-binding protein, locates interaction sites, and characterizes their binding properties. GPER coimmunoprecipitates with calmodulin in primary vascular smooth muscle cells under resting conditions, which is enhanced upon acute treatment with either specific ligands or a Ca Along with menopause comes a substantial increase in the risk of cardiovascular diseases 2+-binding motifs. Ca2+-bound CaM has a dumbbell conformation with two EF-hand motifs on either end connected by a central helix. This central helix functions as a tether that is bent upon target interaction while the two lobes exert concerted effects 2+ binding exposes hydrophobic patches, promoting CaM’s interaction with its target proteins. In this fashion, CaM is the ubiquitous transducer of cellular Ca2+ signals and is involved in virtually all aspects of cellular functions due to its interaction with and requirement for the activities of hundreds of target proteins Calmodulin (CaM) is a highly conserved 148-a.a. protein that contains four EF-hand Ca2 dopamine receptor 2+-dependent, information regarding the specific Ca2+ sensitivities of these interactions is lacking. Knowledge of the Ca2+ sensitivities of GPCR-CaM interactions will be of value in determining the roles of CaM interaction with each submembrane domain at different physiological scenarios in cells.G protein-coupled receptors (GPCRs) represent a superfamily of cell surface proteins that convey extracellular inputs to vast changes in cellular functions via dynamic associations with heterotrimeric G proteins and numerous other partners at their submembrane domains. Recently, CaM has been demonstrated to interact with a number of G protein-coupled receptors (GPCRs), such as the metabotropic glutamate receptors mGluR1 and mGluR5 d and Ca2+ sensitivities. Using FRET-based biosensors designed to scan all submembrane domains of GPER, we identified up to four binding sites located each in submembrane domains 1, 2, 3 and 4 of GPER that have distinct binding affinities for CaM. In addition, we simultaneously incorporated these biosensors and suitable Ca2+ indicators in single assays to precisely determine the Ca2+ sensitivities of the interactions between CaM and these different domains in GPER. This approach in turn revealed quite distinct sensitivities to Ca2+ of the interactions between CaM and GPER’s submembrane domains. Interestingly, submembrane domain 1 interacts with CaM with biphasic Ca2+ dependency, representing two distinct populations of complexes formed at drastically different free Ca2+ values that range from cytoplasmic resting levels to those that occur during Ca2+ entry. The results demonstrate that FRET biosensor-based screening is an effective and efficient approach to identify unknown CaM-binding domains in GPCRs that allows for highly quantitative assessment of their binding properties.With respect to estrogen biology, it is completely unknown if CaM plays a role GPER-dependent signaling. We have begun to test the idea that CaM is directly involved in GPER-dependent signaling in the vasculature at both the receptor level and downstream effectors. In this paper, we describe a new approach to identify GPER as a novel CaM-binding protein, locate all of its CaM-binding domains, and determine their binding characteristics including KThe protocol to isolate primary vascular smooth muscle cells from porcine aortas  was approved by the Des Moines University Institutional Biosafety Committee.2 at 37°C for one week. This procedure consistently yields highly homologous populations VSMCs, verified by both morphological properties and smooth muscle α-actin immunofluorescence staining. This protocol was approved by Des Moines University Institutional Biosafety Committee.Descending segments of porcine thoracic aortas were freshly collected from a local slaughter house. Primary vascular smooth muscle cells (VSMCs) were isolated from these aortas using a modification of the methods published by Bolzon g for 5 min at 4°C. Following pre-clearing, the protein content of the eluant was determined using the BCA assay (Pierce). One milligram of total cellular proteins was then rocked with the resin-antibody conjugates for 3 hours in 1-mL columns at 4°C prior to eluting. Following electrophoresis and transfer, membranes were cut between the levels of the bait and the prey proteins prior to incubation with separate primary antibodies against CaM  and GPER .Co-immunoprecipitation was performed using a Pierce immunoprecipitation kit  as per manufacturer’s instructions. The use of an amino-reactive resin in this approach prevents the co-elution of the antibody heavy and light chains that may co-migrate with the bands of the proteins of interest. Typically, 10 µL of resin was conjugated with appropriate amounts of primary antibody. After appropriate treatment, cell lysis was performed on monolayer cells without trypsinization to avoid complications having to do with shear during the process. Cells were lysed on ice for 15 minutes and the lysate was centrifuged at 21,000×x, where x denotes the amino acid numbering of the GPER insert between the fluorophores. In total, the BSGPERx made were BSGPER83–93, BSGPERGGG83–93GGG, BSGPER150–170, BSGPER150–175, BSGPER242–259, BSGPER330–345, BSGPER330–351, BSGPER330–361, BSGPER330–375, and BSGPER362–375 . Biosensors were introduced into a bacterial expression pET vector with a 6-histidine tag at the C terminus downstream of ECFP .Biosensors to screen for CaM-binding domains in GPER were constructed based on the principle of fluorescent resonance energy transfer (FRET). Sub-membrane domains (SMDs) 1–4 from human GPER , correspR362–375 . The BSGx were expressed in BL21(DE3) competent cells. An overnight culture of 5 mL was inoculated into 300 mL of 2XYT culture medium and shaken at 37°C until OD 0.6–0.8. Expression was then induced with 0.5 mM isopropyl 1-β thiogalacto-pyranoside (IPTG) and the culture was grown at 22°C for an additional 24 hrs. Expressed biosensors were then purified using standard affinity chromatography with Cb2+ affinity resin (Pierce). Eluant was dialyzed twice against large volumes of 50 mM Tris, 100 mM KCl, pH 7.5 at 4°C. Purified BSGPERx were stored at –80°C.BSGPERg for 30 min, 10 mM CaCl2 was added to the supernatant and heated to 65°C for 15 minutes. Supernatant was brought to room temperature and centrifuged again at 100,000× g for 30 min in the presence of 4 µL/mL DNAse and 3 mM MgCl2. Supernatant was loaded onto a CL-4B Phenyl Sepharose column. Columns were subjected to three long successive washes with buffer A , B , followed by a brief wash with buffer A. CaM was eluted with 50 mM Tris, 100 mM NaCl, and 5 mM EGTA. Eluant was dialyzed twice against 50 mM Tris, 100 mM KCl, pH 7.5 and concentrated using Centriprep (Millipore) prior to use. This protocol consistently produces high yields of pure CaM.CaM was purified with a slight modification of previously published approach. A bacterial expression pET vector encoding the rat CaMI gene was transformed into BL21(DE3) E. Coli. An overnight culture of 10 mL was added to 1L of culture media and grown at 37°C until O.D. reached 0.6–0.8. Expression was induced by 1 mM IPTG. The culture was then grown for 4 hrs at 37°C. After centrifugation, pellets were resuspended in 50 mM Tris, 100 mM NaCl, pH 7.5 at room temperature. Resuspension was treated with 200 µg/mL lysozyme, protease inhibitor cocktail (0.06 units/mL) and 0.01 µL/mL PMSF for 1 hr. Cells were lysed by sonication. After centrifugation at 100,000× x were tested in vitro for direct binding with purified CaM. BSGPERx were mixed in titration buffer  containing 0.1 mg/mL bovine serum albumin and 1 mM CaCl2 in a quartz cuvette . Small aliquots of purified CaM were added to the mixture as biosensor responses were monitored in a QuantaMaster™-40 spectrofluometer . All titrations were performed at 22°C. Direct interactions between the specified GPER fragments and CaM were identified by disruption of FRET between donor ECFP and acceptor EYFP. Biosensor fractional responses were determined by the formulamin and Rmax are the ratios between emission intensities at λ475 and λ535 nm (F475/F535) when the biosensor is in unbound and maximally bound state, respectively. Fractional biosensor responses could also be determined using the emission intensity at λ475 nm (F475) or λ535 nm (F535), as follow.max and Fmin represent the observed, maximal and minimal intensities at λ535 nm, as described previously fract; however, we determined that the use of ratiometric responses (formula (1)) negated this necessity. Precise fractional biosensor responses were plotted against titrated CaM concentrations. Apparent Kd values for BSGPERx-CaM interactions were obtained by fitting biosensor’s fractional responses as a function of CaM concentration to hyperbolic or quadratic binding equations BSGPERfract, [BS] and [CaM] are fractional response of BSGPERx ( BSGPERx ) and themax and Rmin, reflects the conformational change that occurs upon CaM binding to the linker. This parameter was used as additional indicator of the location of the CaM-binding domain.Dynamic range (DR) in biosensor responses to CaM, operationally defined as the fold difference between R2+ sensitivities of the interactions between CaM and the different submembrane domains of GPER were determined by simultaneous spectrofluorometric measurements of the responses of a suitable Ca2+ indicator and BSGPERx in the presence of saturating CaM concentration and incremental increases in free Ca2+. Saturating CaM concentrations were obtained from determinations of apparent Kd values of interactions between BSGPERx and Ca2+-CaM described above. Free Ca2+ concentrations were determined by the responses of X-Rhod5F emission intensity at λ600 nm with excitation at λ580 nm, which were simultaneously monitored with biosensor response. Biosensor FRET emission ratio (F475/F535) following excitation at λ430 nm were concurrently monitored in a QuantaMaster-40 spectrofluorometer. Typically, starting reaction mixture contained 0.5 µM GPER biosensor, 2 µM X-Rhod5F, 0.1 mg/mL BSA, 0.25 mM Br2BAPTA and saturating CaM concentrations (obtained from Kd titrations) in 25 mM Tris, 100 mM KCl, pH 7.5 at 22°C. In some assays with high saturating CaM concentrations, the concentration of Br2BAPTA was increased to guarantee chelation of Ca2+ prior to the start of Ca2+ titrations (see Results). In this nominally Ca2+-free condition, there was no interaction between CaM and BSGPERx. Incremental Ca2+ was then titrated into the reaction mix. Free Ca2+ values were calculated using the formulain vitro Kd value (µM) of X-Rhod5F for Ca2+; Fmin and Fmax are its fluorescence intensities measured at λ600 nm under nominally Ca2+-free and Ca2+-saturating conditions, respectively.Ca2+ sensitivities of biosensor-CaM interactions were then determined as the EC50(Ca2+) values, which were derived from fits of biosensor fractional responses (BSfract) against corresponding free Ca2+ concentration using the equation:fract is obtained from n is the Hill coefficient.The Cat-test, assuming unequal variances between control and treated groups. Statistical significance was determined as P<0.05.Data are expressed as means ± SD of at least three independent experiments. Statistical analysis was performed using Student’s 2, 5 nM), the GPER agonist G-1 (100 nM), or the SERCA pump inhibitor thapsigargin (2 µM) for 3 minutes in the presence of 1 mM extracellular Ca2+. Both E2 and G-1 have been demonstrated to trigger intracellular Ca2+ signals 2+ entry. GPER was immunoprecipitated from the lysate and the pull-down fractions were probed for CaM  of their responses, operationally defined as the fold difference between the observed Rmin and Rmax . This v2+-saturated CaM (Ca2+-CaM). BSGPER83–93, whose linker corresponds to GPER’s SMD1, showed reciprocal responses in its emission at λ475 nm and λ535 nm only to fairly high CaM concentrations . After maximal responses were obtained for BSGPER83–93, BSGPER150–175, BSGPER242–259, BSGPER330–375  was added to chelate Ca2+ in the mixture. This addition completely reversed the responses for these biosensors  of GPER as linkers between the FRET donor ECFP and acceptor EYFPc and screened for direct interactions with purified CaM. trations , and wasCa2+-CaM , albeit Ca2+-CaM . The low represents a fairy short sequence (11 residues) that does not follow any known CaM-binding motifs and yet apparently binds CaM in a strictly Ca2+-dependent fashion, albeit with relatively low affinity. BSGPER150–175 (SMD2) contains a 20-a.a. segment (a.a. 150–170) of hydrophobic residues interspersed among positively charged residues, while the remaining part (a.a. 170–175) is a patch of dense basic, positively charged residues  in CaM binding to SMD2. d value for CaM-BSGPERx interaction using the example of BSGPER150–170. Incremental purified CaM was added to a mixture of biosensor and saturating Ca2+ concentration, and the fluorescence emission intensities of donor ECFP  and are summarized in The 18-residue SMD3 (a.a. 242–259) contains several hydrophobic residues interspersed among a very dense group of basic residues. The hydrophobic residues here again do not appear to follow any of the common motifs of CaM-binding sequences. Given the short length of this submembrane domain, it is likely that the entire domain interacts with CaM . As for sequence . The SMD0 µM CaM ; this se2+-dependent nature of these interactions  and emission (λ605 nm) peaks are far to the right to the emission peak of the EYFPc moiety of the biosensors. 2+ was titrated to a mixture of biosensor, saturating concentration of CaM. Starting mixture contained 0.25–3 mM BAPTA to guarantee nominally Ca2+-free condition at the beginning of the titration.The disparate affinities for CaM binding of the four domains identified above suggest that they may interact with CaM under different physiological scenarios. Given the complete Caractions , knowledtermined , saturat83–93, BSGPER150–175, BSGPER242–259, and BSGPER330–351 to saturating CaM concentrations as a function of free Ca2+. The four CaM-binding domains display significantly different Ca2+ sensitivities in their interaction with CaM. BSGPER150–175, which has the strongest affinity among the four , despite a slightly increased dynamic range due to a longer linker . Fits of this species formation against free Ca2+  value of 0.13±0.02 µM. Analyses of BSGPERGGG83–93GGG–CaM species 1  values are well in the range of resting free cytoplasmic Ca2+ concentrations in cells. BSGPER83–93–CaM species 2 are complexes formed at drastically higher free Ca2+ concentrations than species 1. Fits of the entire population formed as a function of free Ca2+ values follow the behavior of this more abundant species  value of 3.71±0.13 µM. This value is well within cytoplasmic Ca2+ values measured within the sustained phase of vasopressin-induced Ca2+ entry in smooth muscle cells 50(Ca2+) values and the Hill coefficients for the four CaM binding domains in their Ca2+-dependent interactions with CaM.Interestingly, BSGPERentified , displayse curve , the majr linker  vs 10A. behavior  identicar  values determined, SMD2, while apparently being the most avid binder of CaM among the four SMDs, only possesses an intermediate Ca2+ sensitivity (EC50(Ca2+) ∼2.3 µM) for its interaction with CaM. Knowledge of the free Ca2+ concentrations produced in different physiological scenarios then can be used together with these values in predicting what interactions will take place in cells. Indeed, from our co-immunoprecipitation data, it is obvious that GPER and CaM are in the same complexes even at resting conditions. Based on the condition of the co-IP reaction, this would at first sight seem to be a Ca2+-independent interaction; however, based on the Ca2+ sensitivity data presented in 2+ concentration (species 1), with an EC50(Ca2+) value of ∼ 130 nM, a value in the range of resting cytoplasmic Ca2+ concentrations in many cell types. So from the standpoint of free Ca2+ value, this interaction can certainly occur in unstimulated cells. From the standpoint of available CaM, it is noteworthy that the interaction between CaM and SMD1 is of low affinity. So the question arises as to whether this interaction can occur in cells. It has been demonstrated that CaM is a limiting factor in cells and CaM targets compete for Ca2+-dependent interactions with CaM 2+ levels, the competition among targets for Ca2+-dependent interaction with CaM must be much reduced, as only those interactions that are highly sensitive to Ca2+, such as that of SMD1-CaM species 1, can take place. A similar case was recently demonstrated, where the interaction between endothelial nitric oxide synthase and CaM, and hence nitric oxide production, can occur at fairly low free Ca2+ concentrations (EC50(Ca2+) ∼ 150 nM) and that phosphorylation of the synthase substantially reduces, albeit by no means negate, the Ca2+ dependency of the interactions (EC50(Ca2+) ∼ 50 nM) 2+-independent. In the case of GPER83–93-CaM complex species 1, the biochemical results obtained here lend a nice plausible interpretation of the co-IP data obtained in control cells. In addition, it is noteworthy that GPER has been found in a number of different locations in cells in addition to the plasma membrane 2+ values in the Golgi network have been shown to be in the range of 100–250 µM, while that in the ER can reach above 450 µM 2+) values for these interactions are all in the sub- or low micromolar range (2+ sensitivity or in a Ca2+-independent fashion, and showed up in the GPER pull-down fractions probed for CaM and vice-versa, under resting conditions. Nevertheless, our in-vitro data clearly demonstrate absolute Ca2+ dependence of the interactions between CaM and GPER and provide parameters that can sufficiently explain the co-IP data under resting condition.As all of these interactions are Caar range . Of cour2+ release from VSMCs can raise cytoplasmic Ca2+ level above 1 µM, while sub-plasma membrane Ca2+ can reach 45 µM and remain above 5 µM for a sustained period during subsequent Ca2+ entry 2+ signal, while SMD3 and 2 can interact with Ca2+-CaM during the peak and sustained phase of agonist-induced Ca2+ entry in VSMCs. In this regard, it is interesting to consider the observation that thapsigargin, which stimulates store-operated Ca2+ entry independently of estrogen activity, enhanced the association between CaM and GPER independently of specific ligand binding in VSMCs (2+ signal can activate GPER or alter its functions as a G protein-coupled receptor by promoting its multi-level interactions with CaM. Consistent with this idea, it has been demonstrated that experimental shear stress, which triggers Ca2+ signals in vascular endothelial cells, causes conformational changes in the B2 bradykinin receptor independently of ligand binding Under stimulated conditions, it has been shown that vasopressin-induced SR Cain VSMCs . An inte2+ response curve of SMD1-CaM complex formation. It is possible that sequential binding of the C-terminal lobe and N-terminal lobes of CaM to SMD1 is responsible for this behavior and that the short nature of GPER’s submembrane domain 1 sequence sets the stage such property to be discerned. In a speculative scheme, at low Ca2+ concentrations, such as at rest in cells, CaM’s C-terminal lobe may interact with SMD1, causing a small conformational change in this domain; as free Ca2+ increases, such as during SR Ca2+ release or Ca2+ entry, the N-terminal lobe may now contribute to the interaction, causing a much larger conformational change. Consistent with this possibility, Ca2+ binding to separate N- and C-terminal halves of CaM in the presence of a peptide from the CaM-binding domain of the plasma membrane Ca2+-ATPase has been shown to be of biphasic nature At the moment we do not have a complete explanation for the biphasic nature of the Ca2+-dependent CaM-binding protein, whose interaction with CaM stabilizes its dimerization, modulates the interaction with estrogen responsive element and activates transcription 2+ sensitivity, the effects of CaM binding to each of the sub-membrane domains on GPER’s functions present an interesting and necessary area of investigations.In summary, we have used a novel approach to identify four CaM-binding domains in GPER and characterize properties of their interactions with CaM. Our data demonstrate that this approach can be used with ease to locate CaM binding sequences in GPCRs in particular and perhaps in other proteins in general. The results of our study suggest that CaM may play a regulatory role in actions of estrogen mediated by GPER. It is interesting to note that the nuclear estrogen receptor ERα has been demonstrated to be a Ca


However, the exact role of Ca2+ in spheroid formation remains unclear. The mechanism of action of natural anti-oxidants such as lipoic acid, which provide neuroprotection during oxidative stress in MS model, also remains unclear.Axonal severing is critical to the irreversible disability that occurs over the course of multiple sclerosis (MS). Reactive oxygen species (ROS) are implicated in neurodegenerative aspects of MS: axonal spheroid formation, severing, and axoplasmic Ca2O2. Ca2+ dynamics and its sources were examined during spheroids formation using real time imaging, ratiometric Ca2+ indicators and immunocytochemistry.Primary cortical neurons from adult mice were subjected to physiologically-relevant levels of H2+ by 30 min. Onset of axonal spheroid formation began at 15 min when Ca2+ increase was 2.2 fold. Axonal severing occurred at sites of spheroids around 90-120 min. Analysis of small axonal segments revealed an uneven distribution of Ca2+ during exposure to H2O2. Micrometers apart, focal Ca2+ increases in small axonal domains ranged from 2.8 to 4.4 fold. Domains with a 3.8 to 4.4-fold increase correlated with the sites of spheroids, suggesting high focal extracellular Ca2+ influx at these sites. Several treatments significantly attenuated Ca2+ increase and completely abolished spheroid formation under ROS: removal of extracellular Ca2+; N-type Ca2+ channel blocker omega-conotoxin GVIA; L-type Ca2+ channel blocker amlodipine; and reverse Na+/ Ca2+ exchanger (NCX1) blocker KB-R7943. Aggregation of reverse NCX1 and N-type voltage-gated Ca2+ channel was detected at spheroids.Exposure to ROS led to a 3.5 fold increase in axoplasmic Ca2+ and spheroid formation and suggest that focal aggregation of the reverse NCX1 and N-type Ca2+ channel plays central role in high focal Ca2+ increase during oxidative stress. These findings provide a basis for investigating the neuroprotective mechanism of the natural anti-oxidant lipoic acid during oxidative stress.Our results reveal a correlation between focal axoplasmic Ca


Previous studies employing cytoplasmic Ca2+ indicators revealed that these Ca2+ waves were stimulated by a combination of inositol 1,4,5-trisphosphate- and Ca2+-induced Ca2+ release from the endo/sarcoplasmic reticulum. Herein, we present the first report of endothelin-1 stimulated waves of Ca2+ depletion from the sarcoplasmic reticulum of vascular smooth muscle cells using a calsequestrin-targeted Ca2+ indicator. Our findings confirm that these waves are due to regenerative Ca2+-induced Ca2+ release by the receptors for inositol 1,4,5-trisphosphate. Our main new finding is a transient elevation in SR luminal Ca2+ concentration ([Ca2+]SR) both at the site of wave initiation, just before regenerative Ca2+ release commences, and at the advancing wave front, during propagation. This strongly suggests a role for [Ca2+]SR in the activation of inositol 1,4,5-trisphosphate receptors during agonist-induced calcium waves. In addition, quantitative analysis of the gradual decrease in the velocity of the depletion wave, observed in the absence of external Ca2+, indicates continuity of the lumen of the sarcoplasmic reticulum network. Finally, our observation that the depletion wave was arrested by the nuclear envelope may have implications for selective Ca2+ signalling.Agonist-stimulated smooth muscle Ca The unexpected aspect of this discovery was that the tonic increases in both the bulk smooth muscle [Ca2+]i and force were based on wave-like [Ca2+]i oscillations in individual VSMCs. Because of the asynchronous nature of these cellular Ca2+ waves, summation over thousands of cells in the vascular media results in maintained average [Ca2+]i elevation and vascular tone. Nevertheless, the repetitive Ca2+ waves would still confer the advantage of added informational content due to frequency encoding and prevention of potential harm due to prolonged elevated [Ca2+]i.In vascular smooth muscle cells (VSMCs), fluctuations in cytoplasmic Ca2+ waves, previous studies were limited, to some degree, by the fact that they reported changes in cytoplasmic Ca2+, which represent the result of Ca2+ release from the sarcoplasmic reticulum (SR) rather than the process itself. In this study, in order to obtain more direct insight into regenerative Ca2+ release in VSMCs, we have used a specific Ca2+ indicator targeted to calsequestrin in the SR lumen that we refer to as D1SR. The VSMCs used in this study constituted a primary culture, which remained highly contractile during the study. Here, we report a wave of Ca2+ depletion from the SR itself, which in this case is activated by applying the vascular autocoid endothelin-1 (ET-1). The observed agonist-induced SR Ca2+ depletion wave provides valuable new information about the role of SR luminal Ca2+ concentration ([Ca2+]SR) in the initiation and propagation of Ca2+ waves, which was not attainable by recording fluctuations in [Ca2+]i.Although a great deal of valuable information has subsequently accumulated on the mechanism of Ca−1) NaCl 140, glucose 10, KCl 5, HEPES 5, CaCl2 1.5 and MgCl2 1 (pH 7.4) was used for all calcium measurements and confocal microscopy. The nominal zero-Ca2+ PSS was prepared in the same way as normal PSS without the addition of calcium. Endothelin-1 (ET-1) was obtained from Sigma-Aldrich . Thapsigargin (Tg), a cell permeable inhibitor of sarcoplasmic reticular Ca2+-ATPase (SERCA), and xestospongin C (Xes-C), a potent membrane-permeable blocker of IP3-mediated Ca2+ release, were purchased from EMD Chemicals . Sodium orthovanadate, an inhibitor of secretory pathway Ca2+ ATPase (SPCA) pumps, was purchased from Sigma-Aldrich. Stock solutions of ET-1 and Tg were prepared in dimethyl sulfoxide (DMSO). For all experiments, vehicle-treated (0 µM) groups were incubated with equal volume of DMSO, respectively (the maximum volume of solvents used with the highest concentration of drugs). Further dilutions of reagents were made in zero-Ca2+ PSS buffer. The calcium dye Fluo-4 AM, ER-Tracker, Mito-Tracker Green FM, BODIPY® TR-X thapsigargin, and CellLight™ Golgi-GFP BacMam 2.0 were all purchased from Invitrogen .HEPES-PSS containing (in mmol·L−1), and streptomycin (100 µg.mL−1)  at 37°C, in a humidified incubator in 5% CO2. See following sections for a detailed description of the transfection protocol and buffer/reagents used in the study.Rat aortic SMCs were originated from the laboratories of Drs. Urs Ruegg and Nicolas Demaurex , and prepared from aorta of male Wistar Kyoto rats (200–300 g) as previously described 2+, conformational changes in the CaM-M13 domain increase the fluorescence resonance energy transfer (FRET) between the flanking CFP and YFP yielding a Ca2+ response. SMCs were transfected with adenoviral D1SR constructs at a multiplicity of infection of 100 (MOI = 100). Following overnight incubation at 37°C, cells were replenished with fresh medium. Fluorescence microscopy was used to assess transfection efficiency and cellular morphology at 48 h post transfection. For all experiments, a transfection efficiency of 80–90% was achieved.D1SR indicator, a modified variant of the D1ER cameleon Ratiometric FRET images were acquired with a 63× oil-immersion objective (Leica DM16000 inverted microscope) and a cooled Hamamatsu 9100-02 electron multiplier CCD camera. Cells were excited at 440 nm, and 513 nm; and imaged using 488 nm/535 nm (for donor and FRET channels) and 535 nm (for acceptor channel) band-pass filters (three images/time point). The acceptor channel was simultaneously recorded to monitor photobleaching. We set the intensity of light at 15% transmitted light, and excitation exposure times at 150 ms with 10 s intervals.in vitro measurement of cytoplasmic Ca2+ signals, all parameters  were maintained constant during the experiment. The cell culture was illuminated using an Argon-Krypton laser (488 nm) and a high-gain photomultiplier tube collected the emission (505–550 nm). The customized Hamamatsu 9100-02 electron multiplier CCD camera delivers 1000×1000 pixels, imaged to Shannon-Nyquist specifications for the 63× objectives, providing a larger field of view. The representative fluorescence traces shown reflect the averaged fluorescence signals from 15 regions of interest (ROIs) in each cell. The measured changes in Fluo-4 AM fluorescence level are proportional to the relative changes in cytoplasmic Ca2+ ([Ca2+]i). The confocal images were analyzed off-line with the Improvision Volocity software (Perkin-Elmer). Fluorescence traces were extracted from the movies and were normalized to initial fluorescence values.During 2+ traces were analyzed by Improvision Volocity software, using built-in regions-of-interest (ROI) function to select the areas of interest that is at least 30 pixels long in length and width. For traces involving specific sites, shape and size of ROI were adjusted to avoid artifacts and saturated areas. For distance and velocity analysis, one ROI was selected per frame (time point) based on the movement of the Ca2+ wave. The resulting data were formatted on Microsoft Excel 2007, and analyzed by GraphPad Prism 5.0. Pseudo-colour visualization was performed by ImageJ, using customized lookup tables to assign colour for each pixel intensity values. Line scan was performed first by analyzing pixel intensity of a series of small ROIs (1–3 pixels in length per ROI) along a line using a customized Python script to output change in intensity over time along the line. The script was confirmed to output same values when analyzing same ROI on Volocity. The resulting values were then graphed using Gnuplot 4.4 with custom lookup table.All data used for Cat-test with two-tailed distribution using GraphPad Prism 5 .Data are presented as means ± SEM of at least four independent experiments. Significance was determined using Student’s 2+]SR, we transfected rat aortic SMCs with an adenoviral vector expressing the specific FRET-based SR Ca2+ indicator, D1SR. To monitor fluctuations in [Cain situ in semi-permeablilized rat aortic SMCs in intracellular HEPES solution  with 5 mM HEDTA as described before 2 to prepare buffers with free Ca2+ concentrations ([Ca2+]free) ranging from 1 µM to 10 mM . D1SR fluorescent ratio (0R/R) was plotted against [Ca2+]free in 12 cells from four independent cultures and an exponential fit was applied to the data to determine the apparent dissociation constant Kd (197±47 µM) and the Hill slope (n = 0.87±0.19)  and 10 mM free calcium (15 mM CaHEDTA), respectively.The D1SR indicator was calibrated 87±0.19) . [Ca2+]S2+]SR in the peripheral (marked with red circle) and peri-nuclear (marked with black circle) areas of SMCs. In the cell periphery, ET-1 causes a drop in [Ca2+]SR (from 1.08±0.12 mM to 450±50 µM), and subsequent SERCA inhibition results in an additional decline (to 55±11 µM). In the peri-nuclear areas, however, the ET-stimulated changes in [Ca2+]SR are moderate (from 1.05±0.07 mM to 700±50 µM). Although a clear loss of [Ca2+]SR is evident following Tg application, a considerable amount of D1SR signal remains in the peri-nuclear areas (427±23 µM), indicating the presence of organelles containing calsequestrin and Ca2+ that are not sensitive to SERCA blockade. A plausible explanation for this residual [Ca2+]SR is that in these cells the SR is contiguous with membranous organelles or endosomes, which accumulate Ca2+ via the Tg-insensitive “secretory pathway Ca2+ ATPase” (SPCA). An alternative explanation is provided by a recent report from Ledeen’s group 2+ after SERCA blockade. Furthermore, since the nuclear envelope and the SR are confluent, this Ca2+, taken up by the NCX is capable of diffusing into the peri-nuclear SR.The traces in 2+]SR with fluctuations in [Ca2+]i, we used the cytoplasmic Ca2+ indicator fluo-4 AM in the same VSMC preparation (2+]i as opposed to uridine-5′-triphosphate (UTP), which stimulates a large maintained increase in [Ca2+]i. Subsequent inhibition of SERCA with Tg (2 µM) elicits another large [Ca2+]i transient due to depletion of SR luminal Ca2+. Therefore, at least in the case of ET-1, it is clear that the luminal SR Ca2+ indicator shows dynamics, which are different from those observed with the cytoplasmic Ca2+ sensitive dye, fluo-4 AM.In order to compare the changes in SR  is added. In the presence of ET-1, focal fluctuations in [Ca2+]SR can be observed, and just before the onset of the Ca2+ depletion wave, [Ca2+]SR is elevated at the site of wave initiation , from which a wave of Ca2+ depletion radiates out into the surrounding SR network , the transiently refilled neighbouring SR locus (marked as B), and the next SR locus further ahead of the depletion wave (marked as C). After passage of the ET-induced Ca2+ depletion wave, the SR was capable of refilling upon subsequent re-perfusion with normal (1.5 mM) Ca2+ solution, confirming the reversibility of the observed SR depletion wave  prior to the application of ET-1 (2+ waves 3R. This notion was further corroborated by the observation that our cultured SMCs failed to respond to caffeine (data not shown), confirming the lack of functional RyRs in these cells, which is in agreement with a previous report by Vallot et al A plausible explanation for the initiation of SR Ca of ET-1 . Additio2+]SR depletion wave is to identify a line intersecting the site of wave initiation and recording the changes in 0R/R values along this line vs. time (line scan). The resulting “heat map” illustrates that initially the area of [Ca2+]SR depletion expands rapidly, but as time proceeds, it asymptotically approaches a final limit  reported previously for intact smooth muscle Ca2+ waves in the presence of extracellular Ca2+2+ waves increase with the level of activation and the concentration of IP32+]SR at the rim as the wave progresses , and Ca2+ sources (SERCA) (3Rs (which yield cytoplasmic Ca2+ blips) 2+]SR. Whenever an increase in local [Ca2+]SR exceeds the threshold for regenerative opening of an IP3R cluster, sufficient Ca2+ would be released to initiate a regenerative Ca2+ wave.The observed transient increase in SR at the rim of the depletion wave  the appearance of functionally segregated compartments in the SR of SMCs, and 3) the existence of a luminal Ca2+ binding site of the IP3R To analyze this proposed mechanism, we developed a preliminary and simplified two-dimensional quantitative model for the propagation of the observed depletion waves, which is based on previous observations and suggested models arguing for 1) a continuous SR lumen in regards to Ca2+ depletion wave (CDW) event, the [Ca2+]SR is at a normal resting level (CSR) and that it needs to reach a critical level (C*SR) for release. Then, for example, following the depletion of a given SR compartment, for a nearest neighbouring compartment to release, its [Ca2+]SR needs to change by C*SR−CSR = 200 µM, which translates to about 1500 Ca2+. For the sake of order-of-magnitude calculations, let us say that SERCA pumps operating at 300 s−1 would take a time interval Δ1t = 5 s to cause a 200 µM [Ca2+]SR change. We calculate the depletion wave velocity, vCDW, as the inter-IP3R-cluster distance, d, divided by the time, Δt, taken to raise the [Ca2+]SR in the new compartment from normal to critical: vCDW = d/Δt. For the sake of completeness, the wave velocity should also include a component for the Ca2+ diffusion time from cluster to cluster. This component is however of the order of milliseconds and therefore negligible when compared to SR compartment refill times.Using the symbols and data in vCDW,1 = d/Δ1t = 0.1 µm/s. If we now assume that SR compartments 2, 3, etc lose part of their Ca2+ toward the depleted neighboring SR compartments by the percentages indicated in 2+]SR will be C2,SR, C3,SR, etc., where these values are lower than CSR. The same line of reasoning then suggests that, for compartment 2 to release, its [Ca2+]SR needs to change by C*SR− C2,SR>200 µM, which will take a time Δ2t>Δ1t and, in turn, yields a velocity for the second step as vCDW,2 = d/Δ2t<vCDW,1. To consider what would happen in a portion of SR with Ca2+ tight compartments, to a first degree of approximation, we can re-use the model above with 0% Ca2+ communication between compartments. It should be evident then that the interval needed to refill each compartment before release is the same as the putative wave progresses. Since the average inter-cluster distance is the same, the resulting wave velocity would be a constant value vCDW = d/Δt at each step. The results reported in 2+ flux into the model indeed caused incremental decrease of the simulated Ca2+ depletion wave velocity  shows that the depleted areas of colliding waves coalesce (white arrow) without a dividing line of Ca2+ loaded SR as seen at the periphery of both depletion waves (red arrows).Close inspection of the SR depletion waves also provides novel insight into sub-cellular differential Ca2+ depletion waves in the smooth muscle sarcoplasmic reticulum, we describe a crucial role for luminal [Ca2+]SR in the initiation and propagation of cytoplasmic Ca2+ waves. This additional insight was gained because the dynamics of SR Ca2+ depletion do not simply mirror [Ca2+]i elevations, but display their own unique characteristics. The disparity between the [Ca2+]i and [Ca2+]SR transients is most likely due to significant contributions by plasma membrane Ca2+ fluxes to smooth muscle Ca2+ homeostasis and excitation.In this first report of Ca2+ waves in VSM are propagated by regenerative CICR at the IP3Rs 2+ free medium 2+ raises the local [Ca2+]i in the neighbouring nano-domain occupied by IP3Rs, which are about to be activated. Although such spatio-temporal resolution of cytoplasmic Ca2+ waves has not yet been reported, our data conclusively show that this is indeed the case. The only possible explanation for the increased [Ca2+]SR at the wave rim is that the SERCA pumps respond to the local [Ca2+]i elevations before activation of IP3R ahead of the Ca2+ wave. Our observation of a diffuse distribution of both SERCA and IP3R over the entire SR  and sinks (IP3Rs) in the walls of the SR lumen Click here for additional data file.Movie S2ET-induced SR calcium depletion wave.(WMV)Click here for additional data file.Movie S3Arrest of depletion wave at the nuclear envelope.(WMV)Click here for additional data file.


Regucalcin was shown to play a multifunctional role in cell regulation, such as maintainance of intracellular Ca2+ homeostasis and suppression of signal transduction, protein synthesis, nuclear function, cell proliferation and apoptosis in various types of cells and tissues. Cardiac excitation-contraction coupling is based on the regulation of intracellular Ca2+ concentration by the Ca2+ pump in the sarcoplasmic reticulum of heart muscle cells. Regucalcin, which is expressed in the heart, was found to increase rat heart sarcoplasmic reticulum Ca2+-ATPase activity and ATP-dependent Ca2+ uptake and mitochondrial Ca2+-ATPase activity. Regucalcin was also shown to suppress Ca2+-dependent protein tyrosine phosphatase, Ca2+/calmodulin-dependent protein phosphatase  and nitric oxide (NO) synthase activity in the heart cytoplasm. Moreover, regucalcin was found to activate superoxide dismutase (SOD), which plays a significant role in the prevention of cell death and apoptosis in the heart. Regucalcin may be a key molecule in heart muscle cell regulation through Ca2+ signaling. Regucalcin may also play a pathophysiological role in heart failure. The aim of this study was to review the recent findings regarding the role of regucalcin in Ca2+ signaling in the heart.Regucalcin was first identified in 1978 as a regulatory protein of Ca Regucalcin was also shown to suppress protein synthesis and activate proteolysis, suggesting a role in protein turnover. The overexpression of endogenous regucalcin was demonstrated to suppress cell proliferation  activity and ATP-dependent Ca2+ uptake and mitochondrial Ca2+-ATPase activity  of rat heart muscle was previously investigated (−10-10−8 M) at physiological levels in the enzyme reaction mixture (−5 M), a specific inhibitor of the microsomal Ca2+-ATPase  was shown to stimulate ATP-dependent 45Ca2+ uptake by the microsomes , a modifying reagent of sulfhydryl (SH) groups (2+-ATPase activity. In the presence of DTT (5 mmol/l), regucalcin was not able to enhance SERCA2a activity , an inhibitor of enzyme phosphorylation (2+-ATPase activity was not modulated in the presence of dibutyryl cyclic AMP (cAMP), inositol 1,4,5-trisphosphate, or calmodulin, which is an intracellular signaling factor  groups . Dithiotactivity . The abo+-ATPase . The stirylation . In addig factor . Thus, r2+-ATPase is activated through cAMP-dependent phosphorylation of phospholamban following hormonal stimulation. The function of the endogenous activator protein of SERCA2a has not been clearly determined. Regucalcin, which is present in the cytoplasm of heart muscle cells, may play an important role as an endogenous activator in the regulation of SERCA2a activity in rat heart muscle  in the enzyme reaction mixture   to the enzyme reaction mixture led to an increase in Ca2+-ATPase activity in heart mitochondria in the presence of 50 μM CaCl2  . An incrμM CaCl2 . Regucals lipids . The stiniporter . The stiactivity . Of note TG rats . The abo2+ transients in regucalcin-transfected COS-7 cells and increase their Ca2+ storage capacity -induced Cacapacity . These ocapacity . Therefoa levels .2+/calmodulin-dependent protein phosphatase , a calmodulin-binding protein, was shown to possess a Ca2+-dependent and calmodulin-stimulated protein phosphatase activity  to the enzyme reaction mixture led to an increase in protein phosphatase activity towards three phosphoaminoacids (−9 and 10−8 M) was found to inhibit protein phosphatase activity towards three phosphoaminoacids in the presence of ethylene glycol-bis(2-amino-ethyl)-N,N,N′,N′-tetraacetic acid (EGTA) (2 (10 μM). Thus, regucalcin was found to inhibit the activity of various protein phosphatasees, dependently or independently of Ca2+.The role of regucalcin in the regulation of protein phosphatase activity in the heart muscle cytosol was demonstrated using regucalcin TG rats . Proteininoacids . This ind (EGTA) . The inh2+ addition on increasing protein phosphatase activity towards three phosphoaminoacids was not observed in the heart cytoplasm of TG rats  to the enzyme reaction mixture containing the heart cytosolic protein led to an increase in NO synthase activity (2+ effect was inhibited by trifluoperazine (TFP), an antagonist of calmodulin, indicating the presence of Ca2+/calmodulin-dependent NO synthase activity in rat heart muscle cytosol (−9 or 10−8 M)  synthase activity in the cytosol of rat heart muscle . The addactivity . The Ca2 cytosol . The act 10−8 M) . This efe enzyme . The pre2+ (10 μM) addition on NO synthase activity was weakened in the heart muscle cytoplasm obtained from regucalcin TG rats  plays a role in the prevention of cell death and apoptosis in the heart . A decre in rats . Regucal in rats .−10-10−8 M) at a physiological concentration to the enzyme reaction mixture containing the heart cytoplasm obtained from wild-type rats led to an increase in SOD activity, indicating that regucalcin directly activates this enzyme (The addition of regucalcin (10s enzyme . The stis enzyme . The adds enzyme . The acts enzyme . Moreoves enzyme . The aboRegucalcin was found to increase SOD activity in rat heart cytoplasm. Regucalcin exerts an inhibitory effect on NO synthase activity in the heart cytosol . The proAgeing is an important risk factor of cardiovascular diseases, including heart failure. The role of regucalcin in cardiac remodelling was previously reported . Regucal2+-related signal transduction in various types of cells and tissues, including the liver and kidney (2+ homeostasis due to activating the Ca2+-ATPase in the sarcoplasmic reticulum and mitochondria of rat heart cells. Regucalcin suppresses Ca2+/calmodulin-dependent enzymes, including protein phosphatase and NO synthase, which are associated with Ca2+ signaling. Ca2+ signaling is one of the most important components in cardiac excitation-contraction coupling. This coupling system may be regulated through regucalcin. Regucalcin may play a physiological role in the regulation of Ca2+-related heart cell functions. Whether regucalcin is associated with other protein molecules that are involved in cardiac excitation-contraction coupling in heart cells, remains to be elucidated.Regucalcin plays a pivotal role as a suppressor protein in Cad kidney –12. MoreMoreover, regucalcin was found to play a pivotal role as a suppressor of NO synthase and an activator of SOD in the heart cytoplasm. The overproduction of NO may lead to heart cell damage. SOD plays a pivotal role in the suppression of free radical production that leads to heart failure. Regucalcin may play a physiological role by exerting protective effects against heart failure, through the activation of SOD or the suppression of NO overproduction in heart cells. The pathophysiological role of regucalcin in heart dysfunction remains to be fully elucidated. However, the currently available evidence indicate that regucalcin may be a target molecule in heart disease.


In nucleated cells, fluoxetine could either inhibit or stimulate suicidal death or apoptosis. The present study tested whether fluoxetine influences eryptosis. To this end cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V binding, hemolysis from hemoglobin release and [Ca2+]i from Fluo-3 fluorescence intensity. As a result, a 48 h exposure of erythrocytes to fluoxetine (≥25 µM) significantly decreased forward scatter, increased annexin V binding and enhanced [Ca2+]i. The effect on annexin V binding was significantly blunted, but not abolished, in the absence of extracellular Ca2+. In conclusion, fluoxetine stimulates eryptosis, an effect at least in part due to increase of cytosolic Ca2+ activity. The antidepressant fluoxetine inhibits ceramide producing acid sphingomyelinase. Ceramide is in turn known to trigger eryptosis the suicidal death of erythrocytes characterized by cell shrinkage and exposure of phosphatidylserine at the erythrocyte surface. Ceramide is effective through sensitizing the erythrocytes to the pro-eryptotic effect of increased cytosolic Ca Fluoxetine is a widely used antidepressant ,2, which2+ activity ([Ca2+]i) [2+]i may increase due to Ca2+ entry through Ca2+ permeable cation channels [2+]i is followed by cell shrinkage due to activation of Ca2+ sensitive K+ channels i further triggers translocation of phosphatidylserine to the erythrocyte surface thus disrupting the phosphatidylserine asymmetry of the erythrocyte cell membrane [Eryptosis is characterized by erythrocyte shrinkage and translocation of phosphatidylserine to the erythrocyte cell membrane surface . Ceramid[Ca2+]i) . [Ca2+]ichannels ,14. The channels , cellulaed water . Increasmembrane . Eryptosmembrane  and caspmembrane ,21,22,23membrane , Janus amembrane , casein membrane ,27, p38 membrane , PAK2 kimembrane  as well membrane  and sunimembrane  sensitiv2+]i, cell volume and phosphatidylserine exposure at the cell surface were determined prior to and following fluoxetine exposure. As a result, fluoxetine increases [Ca2+]i, decreases erythrocyte volume and enhances the phosphatidylserine abundance at the erythrocyte surface. The present study explored whether fluoxetine impacts on eryptosis. To this end, i). Thus, Fluo-3 fluorescence intensity was utilized to estimate [Ca2+]i. To this end, the erythrocytes were bathed for 48 h in Ringer solution without or with fluoxetine (1–50 µM), loaded with Fluo-3-AM and Fluo-3 fluorescence intensity (arbitrary units) quantified by FACS analysis. As illustrated in 2+]i in human erythrocytes.Cell membrane scrambling could be triggered by increase of cytosolic Ca2+ in the stimulation of cell membrane scrambling following fluoxetine treatment, experiments were performed either in the presence of 1 mM extracellular Ca2+ or in the absence of extracellular Ca2+ and presence of the Ca2+ chelator EGTA (1 mM). As illustrated in 2+. Accordingly, Ca2+ removal significantly blunted the effect of fluoxetine (50 µM) on annexin V binding. However, even in the absence of extracellular Ca2+ the percentage of annexin V binding erythrocytes was significantly increased by fluoxetine treatment  is the main stimulator of cell membrane scrambling and subsequent phosphatidylserine translocation from the inner leaflet of the cell membrane to the outer leaflet of the cell membrane [2+ entry is accomplished by Ca2+ permeable non selective cation channels involving the transient receptor potential channel TRPC6 [2+ permeable cation channels in the erythrocyte cell membrane are activated by oxidative stress [The fluoxetine induced cell membrane scrambling with breakdown of phosphatidylserine asymmetry of the erythrocyte cell membrane was significantly blunted in the absence of extracellular Camembrane . The Ca2el TRPC6 . The Ca2e stress . 2+]i, which activates Ca2+ sensitive K+ channels [− exit and the cellular loss of KCl with osmotically obliged water eventually leads to the observed cell shrinkage [The erythrocyte shrinkage following fluoxetine treatment again presumably results from increase of [Cachannels ,83. The hrinkage .2+ activity, an effect, however, reaching statistical significance only at high fluoxetine concentrations. The possibility must be considered that the sensitivity of Fluo-3 fluorescence for the measurement of cytosolic Ca2+ activity may be limited and thus higher concentrations of cytosolic Ca2+ activity are required to significantly detect alterations of Fluo-3 fluorescence intensity than the cytosolic Ca2+ concentrations required to trigger cell membrane scrambling resulting in statistically significant increases of annexin V binding. Nevertheless, even in the nominal absence of extracellular Ca2+ fluoxetine still significantly increases annexin V binding, i.e., cell membrane scrambling. Thus, additional mechanisms are presumably involved in the stimulation of erythrocyte membrane scrambling by fluoxetine. Eryptosis could be triggered by oxidative stress or weakened antioxidant defence [According to Fluo-3 fluorescence intensity, fluoxetine does increase cytosolic Ca defence ,82,84. M defence . Eryptos defence ,21,22,23 defence , casein  defence ,27, p38  defence  and PAK2 defence  or by in defence  and cGMP defence . A stimulation of eryptosis may result in anemia. Eryptotic erythrocytes are rapidly removed from circulating blood . As longExcessive eryptosis may further lead to adhesion of phosphatidylserine exposing erythrocytes to endothelial CXCL16/SR PSO . At leasToxic concentrations of the antidepressant fluoxetine stimulates eryptosis, the suicidal erythrocyte death.


In this study, oxidative stress was produced by exposure to 2+]i: an initial, inositol trisphosphate (IP3)-mediated release of Ca2+ from endoplasmic reticulum (ER) stores followed by a sustained phase of Ca2+ entry . Under non-cytotoxic conditions, tBHP increased resting [Ca2+]i; a 90 minute exposure to tBHP (0.5-10 mM ) increased [Ca2+]i from 26 to up to 127 nM and decreased [Ca2+]L by 55%. The initial response to 10 μM carbamylcholine was depressed by tBHP in the absence, but not the presence, of extracellular calcium. SOCE, however, was depressed in both the presence and absence of extracellular calcium. Acute exposure to tBHP did not block calcium influx through open SOCE channels. Activation of SOCE following thapsigargin-induced depletion of ER calcium was depressed by tBHP exposure. In calcium-free media, tBHP depressed both SOCE and the extent of thapsigargin-induced release of Ca2+ from the ER. M3 receptor binding parameters  were not affected by exposure to tBHP.Activation of M3 muscarinic receptors induced a biphasic increase in [Ca2+]i, [Ca2+]L, IP3 receptor mediated release of calcium from the ER, and calcium entry through the SOCE. tBHP had little effect on M3 receptor binding or G protein coupling. Thus, oxidative stress affects multiple aspects of calcium homeostasis and calcium dependent signaling.Oxidative stress induced by tBHP affected several aspects of M3 receptor signaling pathway in CHO cells, including resting [Ca An aliquot (15–30 μg protein) of CHO-M3 cells homogenized in 50 mM Tris–HCl (pH 7.4), was incubated with [3H]MS in 50 mM Tris–HCl (pH 7.4) for 60 minutes at room temperature in a final volume of 1 ml. In some experiments, the cells were exposed to 2 mM tBHP for 90 min before harvesting. Binding was determined by filtering the suspension through glass fiber filters (1 μm pore size) and measuring the amount of radioactivity bound to membranes trapped on the filters by liquid scintillation counting. Non-specific binding was determined in the presence of 10 μM atropine.Muscarinic receptors were labeled using [3H]MS binding parameters  were determined by nonlinear regression (DeltaGraph) using a mass action expression for a single population of receptor binding sites. Binding parameters were averaged from 3 independent experiments. Agonist binding was determined by the ability of carbamylcholine to inhibit the binding of 0.3 nM [3H]MS. 5′-Guanylyl-imidodiphosphate (Gpp(NH)p) was included in some experiments to assess receptor – G protein interactions.[2+]i) was monitored using the calcium-sensitive fluorescent dye, fura-2 (Molecular Probes), in a ratiometric assay, as previously described i.. The fluorescence from 18–25 cells was measured in each plate. Cells that did not maintain a constant resting [Ca2+]i. (1–2 cells/plate), or that completely failed to respond to carbamylcholine (< 1 cell/plate), were excluded from the analysis. Carbamylcholine  was added after a baseline level was recorded for 30–40 sec; the carbamylcholine remained in the medium throughout the measurement period (3–5 min).[Ca2+ in the lumen of the ER ([Ca2+]L) was measured using the low-affinity calcium indicator, Mag-Fura 2 (Molecular Probes). Fluorescence at 512 nm was measured following excitation at 340 nm and 380 nm using the InCyt imaging system. CHO cells  were seeded onto 29 mm glass bottom plates and incubated at 37°C for approximately 18 hours. The media was removed and the cells rinsed with BSS and then incubated with Mag-Fura 2 (50 μM) for 60 minutes at room temperature. Mag-Fura 2 was removed and the cells incubated for an additional 30 minutes. Prior to imaging, the BSS was removed and replaced with Ca2+- and Mg2+-free BSS. During imaging, the cells were permeabilized by exposure to 0.005% saponin for 70 seconds to release cytosolic calcium. This caused a rapid decrease in total fluorescence intensity following excitation at both 340 and 380 nm, but elimination of the pervasive cytosolic signal allowed detection of ER lumen calcium levels 30 seconds after saponin addition. Following stabilization of the signal (≈100 sec), R340/380 was averaged for 10 seconds in approximately 20 cells on each plate.The concentration of CaParameters  from two populations were compared as the means from 3–20 independent experiments  using Student’s t-test. Values from experiments with multiple independent variables were compared by ANOVA and Tukey’s test using GraphPad Prism software. Significant differences were indicated by P values of < 0.05.The effects of oxidative stress on signal transduction in CHO cells expressing the human M3 muscarinic receptor were studied.2+]i) of CHO-M3 cells in BSS in this series of experiments was 25.6 ± 8.8 nM . Stimulation with the muscarinic agonist carbamylcholine (10 μM) led to a rapid and sustained increase in cytosolic calcium to a concentration that generally ranged from 500 to 700 nM , did not decrease over the time courses of these experimental protocols.CHO-M3 cell viability and the production of reactive oxidative species were revealed by the ability of the cells to reduce the fluorescent reagents MTS and CM-HThe production of ROS in response to a 90 min exposure to tBHP is also illustrated in Figure 2+]i was compromised in a concentration-dependent manner, suggesting a disruption of capacitive calcium entry from the extracellular medium .M3 receptor-mediated responses to carbamylcholine were measured following exposure of CHO-M3 cells to tBHP for 90 min  were increased following exposure to tBHP  are summarized in Figure 2+]i was increased following exposure to 10 and 20 mM tBHP; following exposure to 20 mM tBHP, [Ca2+]i was increased from 26 to 127 nM.Resting intracellular calcium concentrations . However, the time course for the return to baseline [Ca2+]i was consistently slower following exposure to tBHP , suggesting impairment of the calcium transporters that normally restore [Ca2+]i to baseline levels [2+]i in response to reintroduction of calcium was 24 ± 8 nM/sec in control cells, significantly greater  than the initial rates following treatment with any tBHP concentration from 1–10 mM (range: 5.6 – 10.5 nM/sec). However, the rates of calcium entry following reintroduction of calcium in cells exposed to the different concentrations of tBHP (1–10 mM) were not different.M3 receptor-mediated responses to carbamylcholine were also measured in the absence of extracellular calcium Figure . CHO-M3 M Figure . In cont2+]i were measured in cells exposed to various concentrations of tBHP for 90 min , and this rate was not affected by exposure to 0.5 – 20 mM tBHP (range: 1.9 - 4.1 nM/sec).Thapsigargin (1 μM)-induced changes in [Can Figure . ThapsigP Figure a. The abP Figure a. The ex2+]i returned to baseline levels (about 6 min), calcium was added to the extracellular medium. SOCE visualized in this manner was depressed by 30-70% in a dose-dependent manner. The initial rate of rise [Ca2+]I in response to reintroduction of calcium was 40.0 ± 5.1 nM/sec in control cells, more than twice the rates observed in cells exposed to 0.5 – 5 mM tBHP .IP3 receptor-mediated calcium release and SOCE were monitored by reintroducing extracellular calcium (2 mM) to CHO-M3 cells following exposure to thapsigargin (1 μM) in a calcium-free medium Figure b. While 2+]i during the initial response to IP3 receptor- or thapsigargin-mediated release from the ER could reflect changes in the luminal concentration of calcium ([Ca2+]L). Accordingly, [Ca2+]L was measured using an alternate calcium-sensitive dye (Mag-Fura2) and depletion of cytosolic [Ca2+]I by permeabilization of the plasma membrane  reflected ER Ca2+ levels insofar as: 1) R340/380 was immediately diminished following exposure to 10 μM carbamylcholine, and 2) pretreatment with the ER Ca2+-ATPase inhibitor, thapsigargin, prevented the increase in R340/380 observed upon permeabilization. Pretreatment with tBHP (1–20 mM) for 90 min caused a decrease in [Ca2+]L to ≈ 55% of control levels.The influences of tBHP on [Cae Figure . The inc3H]MS. [3H]MS binding was well described by a binding isotherm for ligand binding to a single population of independent receptors . The GTP analog, Gpp(NH)p, shifted the inhibition curves to the right, reflecting a conversion of receptors in a state characterized by a high affinity for agonists to a low agonist affinity conformation. These affinity states reflect the state of receptor coupling to transducer G proteins, with receptor-G protein complexes having a higher affinity for agonists than uncoupled receptors. The ability of carbamylcholine to inhibit [3H]MS binding was not affected by a 90 min exposure to 2 mM tBHP (IC50’s = 1.8 ± 0.3 μM in control and 2.4 ± 0.6 μM in exposed cells). Carbamylcholine inhibition of [3H]MS binding was depressed in the presence of 5’-Gpp(NH)p (IC50′s = 5.0 ± 1.2 μM compared to 1.8 ± .03 mM in control cells). Exposure of the cells to tBHP did not affect guanine nucleotide depression of agonist binding affinity . These results indicate that receptor-G protein uncoupling is not compromised by exposure to tBHP.Agonist binding to muscarinic receptors reveals multiple aspects of receptor organization. Agonist binding was inferred from the ability of carbamylcholine to inhibit the binding of 0.3 nM [r Figure b. Carbam3H]MS dissociation in the presence of the allosteric ligand . Accordn Figure . M2 muscn Figure b. Again,The influence of oxidative stress on muscarinic signaling pathways was investigated in CHO cells, which are widely used in the study of muscarinic signaling pathways. Wild type CHO cells do not express muscarinic receptors or acetylcholinesterase, but express the basic components of both the phospholipase Cβ  and aden2+]i. and selectively inhibited extracellular calcium entry in endothelial cells in response to bradykinin, while having a lesser effect on calcium release from the ER i and extracellular calcium entry (SOCE) were consistently depressed by oxidative stress. The present study extends these observations to the CHO cell model, and include concurrent measurements of oxidant effects on muscarinic receptor systems, immediate SOCE entry, ER calcium concentrations, and cytotoxic effects.Oxidative stress from endogenous or environmental stress affects multiple aspects of calcium metabolism ,10. For m the ER . Bogeskitivation . The spetivation . The preq proteins. Gαq, in turn, stimulates phospholipase Cβ which hydrolyzes the membrane phosphatidylinositol 4.5-bisphosphate releasing diacylglycerol and inositol trisphosphate (IP3). IP3 interacts with its cognate receptor on the endoplasmic reticulum to release stored calcium. This depletion of ER calcium induces an interaction between STIM proteins in the ER and Orai channels in the plasma membranes that allows calcium to enter the cell i.; 2) a decrease in [Ca2+]L; 3) a decrease in agonist-stimulated release of calcium from the ER mediated by IP3 receptors ; and 4) a pronounced decrease in SOCE . These effects are consistent with other accounts of oxidative stress increasing the cytosolic content of calcium [Four major effects of induction of acute oxidative stress were observed in the present study: 1) an increase in resting [Ca calcium .2+]i. and to reduce MTS. However, cytotoxicity was observed when the cells were exposed to these same concentrations of tBHP for longer periods of time . In contrast, acute exposure to tBHP following the activation of SOCE did not block calcium entry , produced a greater increase in resting [Ca2+]i, and had a greater effect on SOCE (completely eliminating SOCE at certain nontoxic concentrations). It seems likely that the effects of ZnO do not reflect a simple increase in oxidative stress produced by an hydroxyl radical generating agent.Exposure to metal oxide nanoparticles creates oxidative stress in a variety of cells, and one impetus for the present study was the inhibitory effects of metal oxide nanoparticles on signaling mediated by CHO-M3 cells . ZnO nan2+]i, [Ca2+]L, IP3 receptor mediated release of calcium from the ER, and calcium entry through the SOCE. In contrast, tBHP had little effect on M3 receptor binding or G protein coupling. Thus, oxidative stress affects multiple aspects of calcium homeostasis and calcium dependent signaling.Stress caused by a potent oxidant affected several aspects of M3 receptor signaling pathway in CHO cells, including resting [Ca3H] N-methylscopolamine; MTS: 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PLCβ: Phospholipase Cβ; SOCE: Store operated calcium entry; tBHP: tert-butyl hydroperoxide.BSS: Basal salt solution; [Ca2+]i: Intracellular calcium concentration; [Ca2+]L: Calcium concentration in the lumen of the endoplasmic reticulum; CHO: Chinese hamster ovary; ER: Endoplasmic reticulum; IP3: Inositol trisphosphate; DCFDA: 2′,7′-dichlorodihydrofluorescein; Gpp(NH0)p: 5′-guanylyl-imidodiphosphate; [3H]MS: [The authors declare that they have no competing interests.T-HT performed the ligand binding measurements and performed data analysis and reviewed the manuscript H-JW measured calcium responses and cell viability and revised the manuscript JDE, performed data analysis and reviewed the manuscript RAR and measured ER calcium concentration, C-TC, AGM, EKS and ALM measured calcium responses, performed data analysis and reviewed the manuscript Y-WH was involved in study design and preparing the manuscript RSA designed the experiments, analyzed the data and prepared the manuscript. All authors read and approved the final manuscript.