Xenopus oocyte is a useful cell model to study Ca2+ homeostasis and cell cycle regulation, two highly interrelated processes. Here, we used antisense oligonucleotides to investigate the role in the oocyte of stromal interaction molecule (STIM) proteins that are fundamental elements of the store-operated calcium-entry (SOCE) phenomenon, as they are both sensors for Ca2+ concentration in the intracellular reservoirs as well as activators of the membrane channels that allow Ca2+ influx.The 2+ release, and the process of maturation stimulated by progesterone. Activation of SOC channels was monitored electrically by measuring the Tin response, a Ca2+-influx-dependent Cl− current, while maturation was assessed by germinal vesicle breakdown (GVBD) scoring and electrophysiology.Endogenous STIM1 and STIM2 expression was demonstrated, and their synthesis was knocked down 48–72 h after injecting oocytes with specific antisense sequences. Selective elimination of their mRNA and protein expression was confirmed by PCR and Western blot analysis, and we then evaluated the effect of their absence on two endogenous responses: the opening of SOC channels elicited by G protein-coupled receptor (GPCR)-activated CaTin currents and GVBD were strongly reduced or eliminated in cells devoid of STIM2; STIM1 knockdown had no effect on the maturation process, but it reduced the Tin response by 15 to 70%. Thus, the endogenous SOCE response in Xenopus oocytes depended mainly on STIM2, and its expression was necessary for entry into meiosis induced by progesterone.It was found that STIM2, but not STIM1, was essential in both responses, and The online version of this article (doi:10.1186/s12899-014-0009-x) contains supplementary material, which is available to authorized users. Xenopus oocyte has been a useful cell model to determine the underlying mechanisms responsible for the increase of the cytoplasmic Ca2+ concentration through its release from intracellular reservoirs , and the P2Y2 Xenopus tropicalis  subtypes were used, and for muscarinic receptors, the M1 subtype [human cDNA Clone ID IOH56940 ]. Another group was injected with 25–50 nl of H2O for control experiments.In order to express the desired membrane receptors, cDNA coding for P2Y2, P2Y8, or M1 receptors were cloned into the plasmid pEXENEX1 and linearized with SalI or HindIII, then purified and transcribed to capped RNA with T7 polymerase using the mMESSAGE mMACHINE kit . Oocytes were injected with 25–50 ng of the respective cRNA (1 ng/nl). For purinergic receptors the P2Y8 The antisense sequences were designed to target the initiation translation region, a strategy that has been successfully used in several experimental protocols; antisense oligonucleotide strongly inhibits mRNA expression via an RNAse-H-dependent mechanism .c current in Ca2+-free Ringer solution  and for stim2 (90%) from Xenopus tropicalis [GenBank: XM_004916759.1] ; this GPCR expression did not affect the level of The results obtained for all conditions described above are summarized in Figure Tin response generation. Prior to this analysis we tested whether or not as-STIM injection affected the Ca2+-dependent Cl− oscillatory current (Iosc) generated by the first application of one of the agonists , reached a peak after 400–480 s, and then slowly returned to basal levels after 680–800 s . Consistent with previous studies i increase produced by both mechanisms in the oocyte, here we used the Ca2+-dependent Cl− current as an endogenous sensor whose amplitude accurately reflects the concentration of Ca2+ beneath the plasmatic membrane. This is especially true for Ca2+ influx, since this occurs in the plasma membrane where the Ca2+-dependent Cl− channels are co-expressed with the SOC channels responsible for the influx. Thus, monitoring an endogenous Ca2+ sensor such as the Cl− channel offers not only spatial and temporal advantages, but also amplifies the normally small Ca2+ current through SOC channels and avoids altering the Ca2+ dynamics with further pharmacological manipulations. STIM protein expression and function was then studied in the Xenopus oocyte using this tool.Calcium release and influx are two phenomena well studied in the  systems . Followiresponse . The Ca2 studies ,42,43, t2+ is released from the ER, STIM proteins are activated, rapidly translocated, and oligomerized into junctions formed between the ER and the plasma membrane, where they bind to and activate highly selective Ca2+ channels formed by Orai proteins that allow Ca2+ influx , in which case as-STIM2 might affect the activation of Ca2+-dependent processes required prior to meiosis entry.Significant inhibition of the maturation process was observed in oocytes devoid of STIM2 protein. Here, we provided clear-cut evidence of STIM2 involvement during or in preparation for maturation, since its absence eliminated the process of GVBD. Once again, this result contrasted with the lack of effect in STIM1-knockdown oocytes, whose maturation was similar to that of control oocytes. Indeed, it has been shown that the function of STIM1 is downregulated during the maturation process, which contributes to elimination of the SOC response in  oocytes -24; a si oocytes , althoug oocytes . There i oocytes . In frog+-influx . FurtherXenopus oocyte and in other cell types as well.In this study, STIM2 is fundamental for the endogenous SOC response in the oocyte, although an association with STIM1 seemed to be necessary for its full generation. The mechanism responsible for the clear dependence of meiosis entry on STIM2 expression is a fundamental question that remains open, and its elucidation might help to understand the function of STIM proteins in the


Consequently, the recently proposed rapid upward migration of brine and HF fluid, predicted to occur as a result of increased HF activity, does not appear to be physically plausible. Unrealistically high estimates of upward flow are the result of invalid assumptions about HF and the hydrogeology of sedimentary basins.Recent increases in the use of hydraulic fracturing (HF) to aid extraction of oil and gas from black shales have raised concerns regarding potential environmental effects associated with predictions of upward migration of HF fluid and brine. Some recent studies have suggested that such upward migration can be large and that timescales for migration can be as short as a few years. In this article, we discuss the physical constraints on upward fluid migration from black shales  to shallow aquifers, taking into account the potential changes to the subsurface brought about by HF. Our review of the literature indicates that HF affects a very limited portion of the entire thickness of the overlying bedrock and therefore, is unable to create direct hydraulic communication between black shales and shallow aquifers via induced fractures. As a result, upward migration of HF fluid and brine is controlled by preexisting hydraulic gradients and bedrock permeability. We show that in cases where there is an upward gradient, permeability is low, upward flow rates are low, and mean travel times are long (often >10 A few recent studies have considered this possibility , howeveras well;  did not 6 years). Hydraulic fracturing increases permeability at depth, however, it affects a much smaller thickness than that of the overlying bedrock and occurs over too short a timescale to affect natural vertical head gradients. After an HF stimulation, hydrocarbon extraction creates a low pressure zone that draws fluids toward the target formation, thereby eliminating any potential for upward flow. In sum, rapid upward migration of HF fluid or brine via bedrock would require the co-occurrence of upward head gradients and high bedrock permeabilities. As we discuss in this article, these two conditions are mutually exclusive, indicating that widespread and rapid upward migration of HF fluid and brine through bedrock is not physically plausible.In this article, we discuss the constraints on upward fluid migration from black shales to shallow aquifers. Our analysis applies to a number of black shales that are currently being targeted for oil and gas development, such as the Marcellus, the Barnett, the Bakken, the Niobrara, and the Eagle Ford. Not surprisingly, our discussion focuses on permeabilities, head gradients, and the relationships between the two, as these variables control the direction and magnitude of vertical fluxes. We show that in cases where upward head gradients exist, permeability is low, and therefore, vertical fluxes are low. Additionally, timescales for transport are long , fluids are present in the sedimentary column, including fresh water, brine, oil, and natural gas . Fluids All sedimentary basins have layered structures, although sediment thickness and stratigraphy vary within and between basins . This lakeff) for flow perpendicular to bedding is approximated as a harmonic mean  and sandstone (10−15 m2)  and therefore, multiple, sometimes thick, low-k layers may limit vertical flow rates  over timescales of tens to hundreds of millions of years   and calcow rates , 2012. TThe grain-size distribution is the dominant control on permeability, however, other factors are also important at depth, including effective stress, partial saturation, and cementation, often reducing permeability by orders of magnitude.k = k0[1 − (Pe/P1)m]3, where k0 is on the order of 10−17 m2, P1 is 19.3 (±1.6) MPa, m is 0.159 (±0.007), and Pe is the effective stress  is given below, where S is saturation and n is a fitted parameter  in porous media also reduces permeability. One common relationship for relative permeability  is another important process that reduces permeability. Both types of cementation reduce permeability, although diagenetic cement has generally a larger effect  and the layered structure of sedimentary basins will constrain the vertical permeability of bedrock above black shales toward the low end of measured values. Low permeability layers at depth in sedimentary basins are common, due to the effects of effective stress, cementation, and partial saturation. Only a thin low-k shales . Therefok rocks . Under either driving mechanism, in order for upward flow to occur, the head gradient must be large enough to overcome density gradients associated with increasing salinity with depth. There are certainly instances of upward head gradients in the vicinity of some black shales, however, high upward head gradients would need to be sustained over thick sequences  of highly permeable bedrock to drive a significant amount of brine into shallow fresh groundwater. As discussed below, these two conditions are mutually exclusive, suggesting that high upward fluxes of brine are not physically plausible.The necessary ingredient for upward flow is, of course, an upward head gradient. There are areas in sedimentary basins in which natural conditions create upward head gradients, and these are generated by one of two mechanisms—topography or overpressure. Topographic gradients create focused discharge areas , whereas overpressured zones may be more wide spread but are inherently associated with low-Topographic gradients can establish long flow paths that penetrate to great depths and traverse entire basins . The first is an upper limit imposed by the mechanical properties of rock, that is, if dh/dz is high enough it will fracture the rock and relieve built-up pressure. The second is a lower limit needed to overcome density stratification, due to the tendency for dense brine to form a stable fluid layer at depth, with less dense fresh water floating on top.When upward flow occurs, there are two fundamental controls on the upward head gradient  when pore pressure exceeds the least compressive stress, σmin, which holds fractures closed. The principal direction of σmin varies with depth; it is typically vertical in shallow bedrock and horizontal at depth , fractures propagate vertically. In either case, the upper bound for σmin is approximately the overburden stress . With ρr at approximately 2300 kg m−3 and ρw at approximately 1230 kg m−3 for brine , The lower limit to d,000 ppm , with dequations . This coρb = 1230 kg m−3 and ρw = 1000 kg m−3, Equation For The black shales targeted for HF are predominantly in basins where burial and rapid gas generation are no longer occurring. Consequently, in most cases where overpressure is present, it was likely generated in the past. Therefore, the question that must be answered is what permeabilities would allow overpressure to persist over the time since sediment deposition and rapid gas generation have ceased to be important. These timescales are generally on the order of tens to hundreds of millions of years for basins in the United States .z is overburden thickness, α is the compressibility of bedrock, t is the timescale for diffusion of pressure, and μ is the viscosity of water. Note that this relationship applies for homogeneous or heterogeneous rock, as well as for permeability dominated by fractures or the matrix. For timescales of 10 million to 100 million years, overburden thicknesses of 1000 to 5000 m , α = 10−9 Pa−1  and, therefore, upward fluid fluxes will be extremely small.There are clearly mechanisms that can drive upward flow in sedimentary basins, however, regardless of the driving mechanism, flow rates are low and flow paths are long. For topographically driven flow, water that penetrates deep enough to drive basin-scale brine migration must travel large horizontal distances  before reemerging at the surface. Over the past 200 million years, maximum cross basin  flow rates at depth in many U.S. basins have been on the order of cm yearkeff and transient elevated pressure to create upward head gradients. A large amount of monitoring data on fracture height growth has been recently published and shows that fractures have remained well below potable groundwater, as discussed in the next section. Conversely, there are no data on pressure propagation away from the fracture network, however, there are well known scaling relationships that can be readily used to bound the extent of pressure propagation. In any event, water introduced is most likely to be trapped by capillary tension, which causes gas-filled bedrock to soak up water like a dry sponge  have recently been published for the Barnett, Eagle Ford, Marcellus, Woodford, and Niobrara Shales . These ds) from the fracture network at which a change in pore pressure would occur in response to HF, yielding, keff of 10−20 to 10−16 m2 , changes in pore pressure depend on rock and fluid properties that control pressure propagation. Equation The short duration and localized pressure pulse associated with HF stimulations is in sharp contrast with the long duration and large-scale depressurization brought about by hydrocarbon production. For example, Equation Modeling fluid flow is unfortunately complex at the depths where black shales occur, but is nevertheless necessary for modeling chemical transport. Some complicating issues arise in modeling analyses, such as the limited amount of data for these deep formations , the potThe difficulties associated with flow modeling are not insurmountable, as long as they are dealt with in a physically sound manner. For example, the flow of oil and gas may not need to be specifically modeled if the flow of water is of greater interest; it may be reasonable in some instances to model hydrocarbon-bearing formations with an appropriately reduced permeability due to the effects of partial saturation. Modeling frameworks have been developed to handle variable density flow and heat transport , both ofThe key issue that is difficult to overcome in any case is the lack of data on deep formations. Such data limitations are always a problem, and therefore, model assumptions and results should always be evaluated with simple scaling analyses to provide reality checks. We have provided some scaling relationships in this article that may be helpful in some circumstances. In the following subsections, we discuss some simple approaches for bounding the magnitude of potential upward fluid fluxes and travel times—both of which are topics discussed in recent studies  that hav−1 , however, it is also difficult to conceive of a monitoring program that would provide meaningful information, given the constraints on pressure wave propagation and fluid flow at depth. For example, low permeability rocks prevent pressure waves from propagating in a short enough period to be observed over human timescales , and a pt = Ln/q, are constrained by physically plausible combinations of upward head gradients, permeability, effective porosity (n), and the depth interval between the fracture zone and shallow groundwater (L). For most basins in the United States that are targeted for HF, upward head gradients, if present, are likely to be relicts of past overpressure generating mechanisms . With this permeability, a unit head gradient , and effective porosity values ranging from 10−2 (for the matrix) to 10−5  are not physically possible over thick sedimentary intervals above black shales.A variable related to the upward flux is the timescale for transport. The timescales for vertical transport, chanisms  and assoat depth , althougat depth . Short tVertical permeabilities are dominated by the least permeable layer. The stratigraphy above black shales is typically dominated by shales, siltstones, and mudstones, and many of these layers have inherently low permeability, which is further reduced by high effective stress at depth, cementation, and partial saturation.Hydraulic fracturing affects a much smaller thickness of rock than that of the overburden. Similarly, the elevated pressures associated with HF are both short lived and localized to the fracture network, due to bedrock properties that limit pressure propagation at depth. Therefore, upward migration of HF fluid or brine would be controlled by natural vertical head gradients and would have to traverse a thick interval of low permeability bedrock in order to reach shallow groundwater.6 years). In older overpressured basins, permeabilities required to maintain elevated subsurface pressure over geologic time are on the order of 10−20 m2 or lower, resulting in negligible vertical flow rates.Natural upward head gradients do occur, and are driven by either topography or relict overpressure at depth. In either case, flow rates are low and timescales for transport are long (often >10Much of the groundwork for understanding and modeling fluid flow and chemical transport within sedimentary basins has been previously established. Bulk limits and scaling functions are already available to provide reality checks on flow and transport analyses, some of which we have summarized in this paper. The characteristics of sedimentary basins in which black shales are located do not allow for rapid upward migration of HF fluid or brine over short timescales for the following reasons: Our analysis and literature review indicate that where upward flow occurs, both permeability and flow rates are low, and therefore, timescales for transport are long. Overall, the rapid upward migration scenarios that have been recently suggested  are not


We measured changes in myoplasmic [Ca2+] ([Ca2+]i) using two KATP-channel-deficient models: (1) a pharmacological approach exposing fibers to glibenclamide, a channel blocker, and (2) a genetic approach using fibers from null mice for the Kir6.2 gene. Fatigue was elicited with one tetanic contraction every sec for 3 min. For all conditions, large differences in fatigue kinetics were observed from fibers which had greater tetanic [Ca2+]i at the end than at the beginning of fatigue to fibers which eventually completely failed to release Ca2+ upon stimulation. Compared to control conditions, KATP-channel-deficient fibers had a greater proportion of fiber with large decreases in tetanic [Ca2+]i, fade and complete failure to release Ca2+ upon stimulation. There was, however, a group of KATP-channel-deficient fibers that had similar fatigue kinetics to those of the most fatigue-resistant control fibers. For the first time, differences in fatigue kinetics were observed between Kir6.2-/- and glibenclamide-exposed muscle fibers. Verapamil significantly reduced unstimulated and tetanic [Ca2+]i. It is concluded that not all fibers are dependent on the myoprotection of KATP channels and that the decrease in unstimulated force by verapamil reported in a previous studies in glibenclamide-exposed fibers is due to a reduction in Ca2+ load by reducing Ca2+ influx through CaV1.1 channels between and during contractions.One objective of this study was to document how individual FDB muscle fibers depend on the myoprotection of KATP channels during fatigue. Verapamil, a CaV1.1 channel blocker, prevents large increases in unstimulated force during fatigue in KATP-channel-deficient muscles. A second objective was to determine if verapamil reduces unstimulated [Ca The ATP-sensitive K+ (KATP) channel is a component of these mechanisms. The channel links energy metabolism to the electrical activity of the cell membrane as it is activated during metabolic stress when intracellular ATP and pH decrease and intracellular ADP and extracellular adenosine increase  that the slower fatigue rate and better recovery in the presence of verapamil was due to less Ca2+-induced fiber damage. [Ca2+]i was, however, not measured in the Cifelli et al. study. So, it is possible that the lower unstimulated [Ca2+]i is not just the results of lower Ca2+ influx through CaV1.1, but also to lower Ca2+ release during contractions since the maintenance of the plateau phase during a tetanus requires a Ca2+ influx through CaV1.1 channels  exposing fibers from CD-1 mice  to glibenclamide, a KATP channel inhibitor  and (2) using Kir6.2-/- fibers , a genetic model that does not express functional KATP channels in skeletal muscle  muscles. K2+ measurements. All measurements were carried out the day the fibers were prepared because the stimulation threshold and the number of contracting fibers remained constant for at least 3 h after being transferred to the experimental chamber used to measure [Ca2+]i, while after an overnight incubation at 37°C some fibers completely lose their capacity to contract, whereas others have an increased stimulation threshold over a three hr period in the experimental chamber . Fibers were continuously perfused in physiological solution with a flow rate of 5 mL/min. Experimental temperature of 37°C was controlled for by simultaneously heating the plate in which the chamber was mounted, and the physiological solution running through the chamber, using a dual channel heater controller . The fiber temperature was about 22°C after positioning the RC-25 chamber on the microscope, and that temperature had to be increased to a the physiological 37°C. As discussed by Selvin et al. , the tem2, 3.1 MgCl2, 25 NaHCO3, 2 NaH2PO4, 5.5 D-glucose and 0.2% FBS. All solutions were continuously bubbled with 95% O2–5% CO2 and had a pH of 7.4. FBS was added in all solutions because it prevents (1) a complete loss of contractility and (2) increases in stimulation threshold over time especially when fibers are stressed with a fatigue bout : 118.5 NaCl, 4.7 KCl, 2.4 CaClFibers were stimulated using field stimulation, generated by two platinum electrodes running along each side of the chamber containing the fibers. The electrodes were connected to a Grass S88 stimulator and SIU5 isolation unit  which were used to generate 200 msec trains of 0.3 msec 10 V pulses at a frequency of 140 Hz.2+]i was then calculated from the ratio as previously described , similar to the protocol described by Westerblad and Allen . Fura-2 2+  our mean tetanic [Ca2+]i values reported in this study are within the range reported in previous studies in which fura-2 or indo-1 was used .For most of the experiments, ANOVAs were used to determine significant differences. Split plot designs were used because fibers were tested at all-time levels. ANOVA calculations were made using the Version 9.3 GLM  procedures of the Statistical Analysis Software . When a main effect or an interaction was significant, the least square difference (L.S.D.) was used to locate the significant differences , complete fade was significantly greater in the presence of glibenclamide (58%), of glibenclamide/verapamil (92%), and in Kir6.2-/- fibers (50%). Complete failure to release Ca2+ was observed in 22% of control fibers, a value significantly different from the 53% for glibenclamide-exposed fibers and 0% in glibenclamide/verapamil-exposed fibers. The proportion of Kir6.2-/- fibers (25%) with complete Ca2+ failure was similar to control.Partial fade was observed in 44% of the control fibers Fig.A. This p-/- and glibenclamide-exposed fibers, with values of 108 and 103 sec, respectively. Partial fade eventually became complete fade by a mean time of 170 sec for control fibers, a value that was significantly longer than the 137 and 155 sec for Kir6.2-/- and glibenclamide-exposed fibers, respectively. Complete failure to release Ca2+ occurred at 167, 153, and 174 sec for control, glibenclamide-exposed and Kir6.2-/- fibers, respectively. Glibenclamide- and verapamil-exposed fibers had the shortest time period before partial fade occurred (18 sec). However, of the total of 29 fibers exposed for 30 min to glibenclamide prior to fatigue, only three (10%) had some partial fade compared to 9 of 14 (64%) fibers concomitantly exposed to glibenclamide and verapamil. That is, a large portion of glibenclamide- and verapamil-exposed fibers already had partial fade prior to the fatigue stimulation. Notably, complete fade started within 69 sec of fatiguing stimulation, but complete failure to release Ca2+ did not occur for the glibenclamide- and verapamil-exposed fibers.Next, we measured how long fibers were able to maintain a plateau phase Fig.B. Contro2+]i data points are averaged to calculate [Ca2+]i values because of the signal noises associated with light measurement. During contractions, the averaging is easy when there is a [Ca2+]i plateau phase as shown in Figure2+]i during contractions changed during fatigue, we measured two parameters. The first parameter was the plateau tetanic [Ca2+]i to represent the average [Ca2+]i during a tetanic contraction. In unfatigued fibers, [Ca2+]i reached its maximum in <20 msec following the first stimulation . So, plateau tetanic [Ca2+]i was calculated by averaging all [Ca2+]i data points from the 20th to the 200th msec (the time of the last stimulation). The second parameter was the peak tetanic [Ca2+]i. When there was no fade, peak tetanic [Ca2+]i was calculated from the 20th to the 200th msec as shown in Figure2+]i had the same value. When there was a fade, such as the 98th contractions in Figure2+]i value was calculated from at least 10 data points; in other cases, such as the 114th contractions in Figure2+]i could only be calculated by averaging three data points. It is important to note that for the latter case, peak [Ca2+]i was underestimated as the Ca2+ fura-2 interaction is too slow for fura-2 to fully follow rapid changes in [Ca2+]i  those which had a higher plateau tetanic [Ca2+]i at the end of fatigue; (2) fibers with decreases ranging between 0.09 and 0.47 μmol/L; and (3) fibers with decreases exceeding 0.5 μmol/L. For the first group, plateau tetanic [Ca2+]i significantly increased from 1.2 to 1.7 μmol/L and then returned to values close to prefatigue values within 90 sec . The largest decreases in unstimulated [Ca2+]i after the first 60 sec were observed in fibers (Group C) that had the largest reductions in plateau tetanic [Ca2+]i . The highest mean value for unstimulated [Ca2+]i among the three groups of control fibers was 174 mmol/L compared to 208 and 203 mmol/L for Kir6.2-/- and glibenclamide-exposed fibers, respectively. Finally, contrary to the study of Cifelli et al. . Second, the highest unstimulated [Ca2+]i for glibenclamide–verapamil-exposed fibers was 95 nmol/L  than the 203 nmol/L in glibenclamide-exposed fibers  the higher was the unstimulated [Ca2+]i.Regardless of the experimental conditions, unstimulated [Ca.848 Fig.. It sugg2+]i failed to fully recover after 15 min of recovery  that not all fibers were adversely affected by a lack of KATP channel deficiency; and (4) that verapamil affected both unstimulated and tetanic [Ca2+]i.This study reports for the first time at 37°C: (1) large differences in fatigue kinetics among skeletal muscle fibers; (2) differences in fatigue kinetics between Kir6.22+]i .Many studies on muscle fatigue have been carried out at 22°C using single FDB fibers by the Allen/Westerblad groups , a Na+ channel inhibitor, to prevent action potentials because long pulses eventually depolarized t-tubular membrane activating CaV1.1 channels  decreases in tetanic [Ca2+]i exceeding 0.5 μmol/L, (2) fade, and (3) complete failure to release Ca2+ upon stimulation (the latter observed with glibenclamide-exposed fibers). So, as previously suggested, KATP channels play an important myoprotective role during muscle activity, especially during fatigue.Studies using KATP channel agonist have provided evidence that the channel contributes to a decrease in force by reducing action potential amplitude and the amount of Ca2+]i that remained above prefatigue level while plateau tetanic [Ca2+]i decreased slightly but not significantly  protein family (Higgins + channels in lung tissue (Mall et al. 2+ channels (Shibasaki et al. -/- fibers.There is no evidence that glibenclamide affects other channels than the KATP channel in unfatigued skeletal muscle (Light and French  Higgins . When ac-/- and glibenclamide-exposed fibers is the fact that Kir6.2-/- represents a chronic loss of KATP channel activity that allows for compensatory mechanisms to develop. In fact, daily O2 uptake is greater in Kir6.2-/- than in wild-type mice (Alekseev et al. -/- FDB bundles and only during recovery in wild-type bundles (Scott -/- fibers on the myoprotection of KATP channels.Another possibility to explain the differences between Kir6.2es Scott . So, one-/- muscles. However, this study is reporting greater differences between glibenclamide and Kir6.2-/- single FDB single fibers than the study of Cifelli et al. (-/- fibers to prevent large contractile dysfunctions. This notion is further supported by the fact that Cifelli et al. reported that 65% of the KATP-channel-deficient fibers supercontracted, an effect not observed in this study.The remaining question is why no difference between the two KATP-channel-deficient models had not been reported in previous studies? The lack of difference with EDL and soleus is most likely because of the presence of a significant anoxic core during fatigue as per Barclays’ model Barclay  preventii et al. . In thisi et al. . It is t2+-fura-2 fluorescence may come from cytosolic Ca2+, an important point to consider for unstimulated [Ca2+]i. Unstimulated [Ca2+]i of unfatigued fiber was still above resting level one sec after a contraction (e.g., 1st contraction, Fig.2+]i increased to values approaching 180 nmol/L (Fig.2+]i.In this study, fibers were loaded with fura-2 by exposing them to fura-2 AM, which can permeate membrane organelles before esterases cleave the AM. Consequently, not all of the Caol/L Fig.. It is t2+]i significantly increased in control, glibenclamide-exposed and Kir6.2-/- fibers, mostly between the 60th and 90th sec. The subsequent changes in unstimulated [Ca2+]i appeared to depend on the fate of the plateau [Ca2+]i. That is, decreases in unstimulated [Ca2+]i occurred only in fibers with decreases in plateau [Ca2+]i; the decreases in unstimulated [Ca2+]i being the largest in fibers with the largest decreases in plateau [Ca2+]i giving rise to a linear relationship between the two parameters (Fig.2+ release during contraction diminishes, Ca2+ pumps are more capable of lowering [Ca2+]i between contractions.Unstimulated i in glibenclamide-exposed fibers, but the effect appears not limited to a reduction in Ca2+ influx between contractions. This is because verapamil also caused significant reductions in tetanic [Ca2+]i primarily because of substantial fade, an effect also observed for tetanic force (Cifelli et al. 2+]i from the glibenclamide- and verapamil-exposed fibers fell closed to the relationship from the other experimental conditions (Fig.2+ load due to lower Ca2+ influx through CaV1.1 channels between and during contractions. Another verapamil effect was a significant improvement to recover tetanic [Ca2+]i in glibenclamide-exposed fibers (Fig.2+]i in KATP-channel-deficient fibers causes fiber damage resulting in lower capacity to recover.Cifelli et al.  reportedions Fig.. Thus, tions Fig. is the rbers Fig.B and D. bers Fig.. Thus, t-/- fibers develop less contractile dysfunctions than glibenclamide-exposed wild-type fibers and it is suggested that the greater the physiological or metabolic stress the smaller the differences between the two KATP channel deficiency models. Finally, this study provides further evidence that a large Ca2+ influx through CaV1.1 channels between and during contractions in KATP-channel-deficient fibers causes a Ca2+ load resulting in large increases in unstimulated [Ca2+]i and force resulting in lower capacity to recover, possibly due to some fiber damage.In conclusion, this study demonstrated large differences in fatigue kinetics among FDB fibers during fatigue at 37°C, differences that are much greater than those reported in previous studies carried out at 22°C. Abolishing KATP channel activity using pharmacological or genetic approach causes contractile dysfunctions in most but not all FDB fibers; it is proposed that oxidative fibers are the least dependent and glycolytic the most dependent on the myoprotection from KATP channels. This study shows for the first time that Kir6.2None declared.


Glucagon from the pancreatic α-cells is a major blood glucose-regulating hormone whose most important role is to prevent hypoglycaemia that can be life-threatening due to the brain’s strong dependence on glucose as energy source. Lack of blood glucose-lowering insulin after malfunction or autoimmune destruction of the pancreatic β-cells is the recognized cause of diabetes, but recent evidence indicates that diabetic hyperglycaemia would not develop unless lack of insulin was accompanied by hypersecretion of glucagon. Glucagon release has therefore become an increasingly important target in diabetes management. Despite decades of research, an understanding of how glucagon secretion is regulated remains elusive, and fundamentally different mechanisms continue to be proposed. The autonomous nervous system is an important determinant of glucagon release, but it is clear that secretion is also directly regulated within the pancreatic islets. The present review focuses on pancreatic islet mechanisms involved in glucose regulation of glucagon release. It will be argued that α-cell-intrinsic processes are most important for regulation of glucagon release during recovery from hypoglycaemia and that paracrine inhibition by somatostatin from the δ-cells shapes pulsatile glucagon release in hyperglycaemia. The electrically coupled β-cells ultimately determine islet hormone pulsatility by releasing synchronizing factors that affect the α- and δ-cells. They also provided indirect evidence that glucagon originates from the A2 cells by showing that they undergo pronounced nuclear atrophy after glucagon injections  channels to depolarize the β-cell and open voltage-dependent L-type Ca2+ channels. Subsequent influx of Ca2+ raises the cytoplasmic Ca2+ concentration ([Ca2+]i), which is the most important trigger of insulin release. This model consequently implies that the β-cells have an intrinsic capacity to sense glucose and release insulin as required, but [Ca2+]i-triggered secretion can also be enhanced or reduced by increase or decrease of cAMP, as well as by modulation of protein kinase C and the protein phosphatase calcineurin, mediated by paracrine effects of α-cell glucagon, δ-cell somatostatin and neurotransmitters like acetylcholine and noradrenaline  channels to hyperpolarize the α-cells and close voltage-dependent Ca2+ channels  and hyperglycaemic (>8 mM) concentrations of glucose. However, maximal inhibition of glucagon release from mouse and human islets is reached already at 5–7 mM glucose , which c2+ is generally assumed to be the final trigger of both insulin and glucagon secretion, and pulsatile insulin release is indeed paralleled by synchronous β-cell oscillations of [Ca2+]i ..i subunihe SSTR2 , perturbtin gene  enhancestin gene , but in tin gene . In humatin gene . On the tin gene ,65,74.ATP channels to depolarize islet cells, stimulates the release of both insulin (ATP channel closure in α-cells is stimulatory (see below) and that this stimulation is counteracted by the simultaneously enhanced release of inhibitory somatostatin. Further evidence for a role of somatostatin in the regulation of glucagon secretion in hyperglycaemia is obtained from the kinetics of secretion. Pulsatile hormone release from human and mouse islets is generated by hyperglycaemia with coinciding pulses of insulin and somatostatin that are synchronized in opposite phase to the glucagon pulses (2+]i (discussed above), such a role requires that somatostatin inhibits glucagon release when [Ca2+]i is peaking. This may well be the case since SSTR2 blockade potently amplifies glucagon release from mouse islets exposed to 20 mM glucose without marked effects on the [Ca2+]i oscillations in α-cells , metabolism of glucose is essential for β-cell recognition of the sugar as stimulus. Considering that hormone secretion is glucose-regulated also in islet α- and δ-cells and that these cell types originate from a common progenitor , it seemKm GLUT2 , which snsporter ,85. HoweKm GLUT1 , and gluKm GLUT1 ,85. The  β-cells  and may  glucose , but glu glucose ,88 and t glucose ,47,89, F glucose , and NAD glucose  in α- th glucose . If gluc glucose .2+]i oscillations at high glucose, whereas the α-cells are active in the absence of the sugar. Glucose-induced closure of the KATP channels depolarizes the β-cells to open L-type Ca2+ channels that show half-maximal activation at –19 mV, and this Ca2+ permeability dominates the upstroke of the action potentials in the β-cell -sensitive Na+ channels that open at potentials more positive than –30 mV  and mediate most Ca2+ influx, but their blockade has little effect on secretion. Conversely, blocking the non-L-type channels (20%) has modest effects on [Ca2+]i but inhibits secretion to a similar extent as glucose elevation from 1 mM to 6 or 7 mM (2+ from the endoplasmic reticulum (ER)  and account for most of the exocytosis, although they open very briefly and only mediate a fraction of the Ca2+ entry  ,94, and lum (ER) , entry ochannels ,95. In h2+ entry . The facgnalling ,91,97,98 release .ATP channels to depolarize the β-cell (see above). The most cited model for α-cell intrinsic regulation assumes the same sequence of events, meaning that glucose inhibits glucagon release by depolarizing the α-cells . Reduced Na+/K+ pumping may consequently underlie a more positive equilibrium potential for K+ in α- than in β-cells as speculated above. Moreover, if the energy utilization for ouabain-sensitive Na+/K+ pumping decreases with extracellular K+ elevation up to 14 mM like in squid axon , hyperpolarization seems to be the most common observation plasmic reticulum Ca2+ ATPase (SERCA-pump) to sequester Ca2+ in the endoplasmic reticulum (ER). It is not until the depolarization is sufficient to open voltage-dependent Ca2+ channels that [Ca2+]i increases (2+]i by sequestration in the ER was early observed in guinea pig α-cells (1- and β-adrenergic receptors to generate inositol (3) and cAMP, respectively   . Accordie (AMPK) . Howevere (AMPK) . Current2+]i activity with oscillations at 1–3 mM glucose  that act in the narrow intercellular space of islets but whose effect is lost by dilution when studying purified or isolated α-cells. Indeed, increasing glucose in the 1–20 mM range concentration-dependently inhibits [Ca2+]i signalling in isolated α-cells, provided that they are already depolarized . Ev. Ev2+]i  glucose . The quegnalling ,91,97,98 release , but ava2+ as the trigger of secretion. However, one should also consider the possibility that Ca2+ has a more permissive role and that α-cell-intrinsic glucose regulation in terms of inhibition and stimulation is mediated by modulating factors like cAMP and perhaps diacylglycerol. This alternative is particularly attractive considering that glucose only modestly decreases α-cell [Ca2+]i signalling, which likely explains why even maximally inhibited glucagon secretion is only reduced to 25%–45% of maximal release  and the Swedish Diabetes Association .


Calsequestrin1 (CSQ1), a sarcoplasmic reticulum Ca2+ buffering protein, inhibits SOCE, but the mechanism of action is unknown. We identified an interaction between CSQ1 and STIM1 in HEK293 cells. An increase in monomeric CSQ1 induced by depleted Ca2+ stores, or trifluoperazine (TFP), a blocker of CSQ folding and aggregation, enhanced the CSQ1-STIM1 interaction. In cells with Ca2+ stores depleted, TFP further increased CSQ1 monomerization and CSQ1-STIM1 interaction, but reduced the association of STIM1 with Orai1 and SOCE. Over-expression of CSQ1 or a C-terminal (amino acid 388–396) deletion mutant significantly promoted the association of CSQ1 with STIM1, but suppressed both STIM1-Orai1 interaction and SOCE, while over-expression of the C-terminal (amino acid 362–396) deletion mutant had no effect. The physical interaction between low polymeric forms of CSQ1 and STIM1 likely acts by interfering with STIM1 oligimerization and inhibits STIM1-Orai1 interaction, providing a brake to SOCE under physiological conditions. This novel regulatory mechanism for SOCE may also contribute to the pathological Ca2+ overload in calsequestrin deficient diseases, such as malignant hyperthermia and ventricular tachycardia.Interaction between the endoplasmic reticulum (ER)-located stromal interaction molecue1 (STIM1) and the plasma membrane-located Ca It is essential for the mediation of diverse cell functions such as cell activation, proliferation, differentiation, growth and cytokine production and release12+ release-activated Ca2+ channel pore forming subunit, Orai132+ stores, STIM1 senses the lowered ER Ca2+content through its amino terminus in the ER lumen and then oligomerizes and redistributes to an ER membrane compartment closely juxtaposed to the adjacent plasma membrane, forming STIM1 clusters52+ influx from the extracellular space78Store-operated Ca2+ ATPase 2 (SERCA2), the microtubule plus-end binding protein (EB1), Ca2+ release activated Ca2+ channel regulator2A, surfeit locus protein4 and septins have been reported to interact with STIM11011Although the basic mechanism by which STIM1 activates Orai1 channels is fairly well understood, the cellular machinery that regulates and modulates STIM1-Orai1 interaction and SOCE is not so well known. Recent studies have revealed several modulators of the STIM1-Orai1 complex, located either in the plasma or ER lumen. For examples, STIM2, ER Ca2+ buffering protein in the sarcoplasmic reticulum (SR). It has two isoforms, CSQ1  and CSQ2 (cardiac)132+ buffer. However, more recent studies have demonstrated that this SR protein also plays a key role in regulating the ryanodine receptor (RyR)-mediated Ca2+ release in skeletal muscle cells. It acts as a luminal Ca2+ sensor in regulating RyR activities via its interactions with triadin and junctin15162+ concentration is lower than 10 μM. As luminal Ca2+ concentration increases, it undergoes molecule compaction, dimerization and polymerization182+ concentration of 1 mM through interaction of its carboxyl-terminal aspartate-rich region, a main Ca2+ binding motif, with triadin or junctin151720CSQ gene expression may result in cardiac arrhythmias and malignant hyperthermia222+ release in skeletal muscle cells, CSQ1 can also function as an inhibitor of SOCE24Calsequestrin (CSQ) is a major Ca2+ release as in muscle2+ store depletion induced by thapsigargin (TG) in HEK293 cells, while over-expression of the C-terminal deletion mutant C35 (deletion of amino acids 362–396) has no effect. Furthermore, we report, for the first time, that a direct physical interaction between CSQ1 and STIM1 prevents STIM1 interaction with Orai1 and thus down-regulates SOCE.Unlike skeletal muscle, the function of CSQ in non-muscle cells is rather unclear. In a previous study, we reported that CSQ1 is expressed in non-muscle cells and functions as a regulator of Ca2+ stores by 1 μM TG (2+ entry through the gating of SOC channels was observed (data not shown). CSQ1 also co-immunoprecipitated with STIM1, and this association was enhanced by TG stimulation , a CSQ inhibitor that binds to CSQ and inhibits its folding, compaction and the subsequent aggregation21It is well known that the polymeric status of CSQ is regulated by the level of SR Ca2+-free medium , referred as C9, and HA-tagged CSQ1 lacking residues 371–405 (wild-type residues 362–396), referred to as C35  was reduced by over-expression of HA-CSQ1 or C9-CSQ1 compared with vector control, but not by the C35-CSQ1 mutant  depletion of ER Ca2+ stores, addition of Mn2+ resulted in a more rapid fluorescence decrease, due to SOCE302+ quench, the fluorescence decrease induced by TG in HA-CSQ1 group appeared to be the smallest followed by C9 and then C35 groups when compared with control group  compared with those in control siRNA-treated cells (2+]i was found to be sensitive to 2-APB (30 μM) addition (data not shown), a compound commonly used to inhibit SOCE.Finally, the Casfection  were deted cells . FurtherPrevious studies have shown that CSQ1 regulates SOCE, and that the aspartate-rich segment is responsible for its interference with the SOCE pathway in skeletal myocytes24212+ as well as triadin/junctin and to act as a suppressor of SOCE17181920212+ binding causing more monomer CSQ1 formation18192033The aspartate-rich carboxyl-terminus of CSQ1 has been reported to have multiple functions to bind Ca2+ stores are full in the resting state, and CSQ1 linear polymers binding large amounts of Ca2+ are anchored in the vicinity of Ca2+ release channels213 receptor channels in the case of non-excitable cells. There is a dynamic equilibrium of dissociation and compaction between CSQ1 polymers and monomers, in which polymers should predominate due to the abundant ER Ca2+. In this condition, a small fraction of monomers bind to undefined luminal sites on STIM1. Upon ER store depletion, CSQ1 polymers dissociate into low-polymeric forms, such as dimers and monomers, which in turn increases binding of CSQ1 to STIM1. Meanwhile, depletion of Ca2+ also results in Ca2+ dissociation from the STIM1 EF-hand motif, which causes STIM1 oligomerization, and then migration, aggregation and accumulation in pre-existing ER-PM junctions. Finally, the direct binding of STIM1 to Orai1 recruits Orai1 into puncta resulting in localized Ca2+ influx. However, this Ca2+ influx is somewhat limited by CSQ1 sequestration of STIM1 that may somehow inhibit clustered STIM1 movement to the cell periphery . Formaldehydrate solution, thapsigargin, trifluoroperazine dihydrochloride, (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride, protease inhibitor cocktail were from Sigma-Aldrich . HA-tag mouse monoclonal antibody (Ab24779) was from Abcam. All of the other primary and secondary antibodies including mouse monoclonal anti-CSQ1 (SC-137080), goat polyclonal anti-CSQ2 (SC-16576), rabbit polyclonal anti-CSQ1 (SC-2005), mouse monoclonal anti-STIM1 (SC-166840), mouse monoclonal anti-Orai1 (SC-377281), rabbit polyclonal anti-HA tag (SC-805), mouse monoclonal anti-actin (SC-2005), mouse monoclonal anti-GAPDH (SC-2005), goat anti-mouse IgG-HRP (SC-2005), goat anti-rabbit IgG-HRP (SC-2004) were from Santa Cruz Biotechnology, Inc. .2. Plasmids were transiently transfected into HEK293 cells by Lipofectamine 2000 reagent following the manufacturer’s protocol using 2 μg/ml in DMEM of each construct.HEK293 cells were purchased from ATTC and cultured in DMEM  supplemented with 10% fetal bovine serum  at 37 °C under 95% air and 5% COCSQ1 between Val49 and IIe50. The deletion mutants were C9 (deletion of wild-type amino acids 388–396) and C35 (deletion of wild-type amino acids 362–396). Nucleotide sequences of these constructs were verified by sequencing. These constructs (20 μg each) were transfected into HEK293 cells for 36 h and protein expression was confirmed by Western blot analysis. Briefly, cells were seeded 24 h ahead into 10 cm dishes with antibiotics-free, 10% FBS-containing DMEM and allowed growing to 80% confluence. We diluted 20 μg plasmid DNA and 20 μl Lipofectamine 2000 reagent with FBS-free DMEM to 500 μl in two separated tubes and incubated for 5 min at room temperature, and then added the diluted DNA to diluted Lipofectamine 2000 reagent and mixed by gently inverting the tubes 3–5 times and incubated the DNA-lipid complex for 15 min at room temperature. We replaced the growth medium with 9 ml FBS-free DMEM in the culture dish and added 1 ml DNA-lipid complex to cell dishes. After 4 h incubation at 37 °C under 95% air and 5% CO2, we changed them with 10% FBS-containing DMEM and cultured the cells for 36 h before harvest. Through the whole process, the DMEM medium was antibiotic-free.Plasmids were constructed by Life Technologies CO.LTD (AB & Invitrogen). HA-tagged human wild-type CSQ1 and its mutant cDNA were generated by PCR. Each cDNA were cloned into the pIRES2-EGFP expression vector. HA tag (YPYDVPDYA) was inserted downstream of the predicted signal sequence of the wild-type CSQ1 knockdown siRNA (CSQ1-KD) and non-specific control siRNA (Con-siRNA) plasmids were constructed by Life Technologies CO.LTD (AB & Invitrogen). They were transfected into HEK293 cells as previously reportedCell protein extracts were prepared by washing the cells with PBS and then lysing the cells with lysis buffer, supplemented with freshly added 1 mM PMSF, protease inhibitor cocktail. Protein samples were denaturized by incubation in loading buffer at 95 °C for 5 min. Proteins were resolved on 10% SDS-PAGE and electroblotted onto nitrocellulose membranes. The membranes were subsequently blocked for 1 h at room temperature in 5% non-fat milk with Tris-buffered saline/Tween-20 (TBST), pH 7.4. Then the membranes were incubated overnight at 4 °C with anti-CSQ1 (1:1000), anti-STIM1 (1:1000), anti-Orai1 (1:1000), anti-HA (1:5000) primary antibodies for detection of CSQ1, STIM1, Orai1, HA-tag, respectively. After washing with TBST, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000) at room temperature for 1 h and then exposed to Super Signal West Pico enhanced chemiluminescence reagents for 2 min. Blots were then exposed to Kodak photographic films. The gray scale intensity of bands was measured using densitometry software Quantity One from Bio-Rad.Non-denaturing were treated in the sample buffer containing 150 mM sodium chloride, 0.2% Triton X-100 and 50 mM Tris, pH 8.0. All the samples were then subjected to non-denaturing electrophoresis using a resolving gel containing 10% (w/v) acrylamide, 0.33% bisacrylamide, 10% ammonium persulphate, 0.1% N,N,N’, N’-tetramethylethylenediamine (TEMED), 1.5 M Tris, pH 8.8. The stacking gel contained 5% (w/v) acrylamide, 0.15% bisacrylamide, 0.07% ammonium persulphate, 0.1%TEMED, and 1 M Tris, pH 6.8. The electrode buffer contained 25 mM Tris/glycine buffer, pH 8.5. The gel was run at 4 °C for 4 h at 80 V. Gels were elctrolotted on nitrocellulose membranes. Then the membrane was treated as described above.Cells were harvested, washed with PBS and then lysed with lysis buffer: supplemented with freshly added 1 mM PMSF, protease inhibitor cocktail. Then the lysates were centrifuged at 4 °C, 15000 g for 15 min. The supernatants were precleared with rProtein G  or Protein A  beads and protein concentration was determined, using the Pierce BCA Protein Reagent Kit. The cell extracts were then adjusted to approximately 1–2 mg/ml total protein with lysis buffer and mixed with corresponding primary antibodies (0.2 μg/100 μg proteins) as indicated and incubated at 4 °C overnight under constant agitation. Equal amounts of samples were mixed with normal IgG as negative controls. Beads were subsequently added to the mixtures (≥5 μl per 1 μg IgG) and agitation was continued for 4 h at 4 °C. The beads were then washed with lysis buffer and proteins were eluted by incubation in loading buffer at 95 °C for 5 min. The eluted proteins were resolved on 10% SDS-PAGE and analyzed by Western blotting as described above.4 cells per well). After 24 h, various treatments were applied to the cells, and they were then fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde for 15 min. The cells were then permeabilized by 0.5% Triton X-100 in PBS for 30 min and blocked in 5% bovine serum albumin for 30 min at room temperature. The primary antibodies were diluted (1:400) in PBS and added to the permeabilized cells, which were incubated overnight at 4 °C. Dye-conjugated secondary antibodies  were added for 1 h at room temperature. For nuclear staining, cells were incubated with Hochest33342 for 10 min at room temperature. After mounting in mounting medium, a 3D Z-stack was acquired at 0.5 μm intervals by Leica SP8 microscopy equipped with a 63× oil immersion objective (NA 1.4), pinhole = 1 μm. For the measurement of STIM1 aggregation, HEK293 cells expressing empty vectors or exogenous HA-CSQ1, C9 or C35 truncations were suspended in Ca2+-free HBSS and then stimulated with 1 μM TG plus 5 μM ionomycin for 5 min. Then the cells were immuno-labeled with mouse anti-STIM1  mAb at a dilution of 1:400. Alexa Fluor 594 goat-anti-mouse secondary antibody was used at 1:400. The bottom of each cell was firstly determined, and then the x-y image of the plane 3 μm above the bottom was taken by Leica SP8 microscopy, the pinhole = 1.5 μm. The selection of STIM1 particles in the cell periphery was performed by Leica Qwin Standard V2.8, and the number in each cell was normalized to the cell circumference, and then compared between groups.Cells were plated on 10 × 10 mm cover slides in 6 well plates  formaldehyde solution for 5 min. The cross-linking reaction was terminated by the addition of a 1 M glycine stock solution, pH 8.0, to a final concentration of 0.1 M and incubation was continued for 5 min on ice. Cells were collected by centrifugation at 300 g for 1 min and washed 3 times with ice-cold PBS. Then cells were lysed with lysis buffer supplemented with 1% SDS, 0.1% Triton-X100, 1 mM PMSF, 10 μl protease inhibitor cocktail under constant agitation for 5 min at 4 °C. The lysates were then centrifuged at 4 °C, 15000 g for 15 min and the supernatants were collected for protein concentration assay and Western blotting without boiling before loading in the comb hole.As previously described382+([Ca2+]i) was measured using the fluorescent probe Fura2. HEK293 cells suspensions were loaded with Fura2 by incubation in culture medium supplemented with 1 μM Fura2 acetoxy methyl ester at 37 °C for 30 min. After washing by centrifugation with 1.8 mM Ca2+-containing Hepes-buffered saline solution (HBSS) and then Ca2+-free HBSS, cells were resuspended in Ca2+ free HBSS for subsequent use in experiments. The fluorescence was monitored in a stirred cuvette with fluorescence spectrophotometer HITACHI F-7000 at 37 °C. The excitation wavelengths were alternated between 340 and 380 nm and emission was measured at 510 nm. At the end of each incubation, a final concentration of 0.2% Triton X-100 and 5 mM EGTA were added to obtain maximal (Fmax) and minimal (Fmin) fluorescence, respectively. Values of [Ca2+]i were calculated from the ratios of fluorescence with excitation at 340 nM to that at 380 nM by the FL Solutions Intracellular Cation Scan software based on the formula described by Grynkiewicz2+ (0.2 mM) in 1 μM TG-treated cells expressing HA-CSQ1 or either of the two truncation mutants was measured at the Ca2+-independent excitation wavelength of Fura2 (360 nm) in Ca2+-free HBSS. Cells were treated with 1 μM TG for 250 s to deplete ER Ca2+ stores, and were pre-incubated with 20 μM TFP for 10 min or not as indicated. The initial rate (50 s) of decline of Fura2 fluorescence upon Mn2+ addition was calculated and expressed as percent of the rate for the vector control (-TFP). The maximally quenched fluorescence signal was achieved at the end of the experiment by lysing the cells with 0.5% Triton X-100 (as 0%). Basal rate (no TG) was subtracting before calculating each rate, and the vector rate was then set as 100%.Cytosolic free Ca2+ pumps was assessed by monitoring the rate of quenching of intracellular Fura2 by Mn2+ entering the cells. Transfected HEK293 cells were loaded with 2 μM Fura2-AM in DMEM for 30 min at 37 oC and then washed in 1.8 mM Ca2+-containing HBSS and then Ca2+-free HBSS as for [Ca2+]i measurements. The fluorescence was monitored in a stirred cuvette with fluorescence spectrophotometer HITACHI F-7000 at 37 °C. The excitation wavelength was set at the isosbestic point 360 nm and emission was measured at 510 nm. Following a 60 s stabilization period, 1 μM TG was added for 250 s, followed by 0.2 mM Mn2+ for 450 s. Subsequent addition of 0.5% Triton X-100 allows complete quench of Fura2 to obtain a background value.The rate of divalent cation influx unaffected by CaData are presented as the means ± SD of three to twelve independent measurements. Statistical comparisons between groups were carried out with a two-tailed unpaired Student’s t-test followed by one-way analysis of variance (ANOVA) as appropriate. P < 0.05 was considered statistically significant.How to cite this article: Wang, L. et al. Retrograde regulation of STIM1-Orai1 interaction and store-operated Ca2+ entry by calsequestrin. Sci. Rep.5, 11349; doi: 10.1038/srep11349 (2015).


Human adipose mesenchymal stem cells (haMSCs) are multipotent adult stem cells of great interest in regenerative medicine or oncology. They present spontaneous calcium oscillations related to cell cycle progression or differentiation but the correlation between these events is still unclear. Indeed, it is difficult to mimic haMSCs spontaneous calcium oscillations with chemical means. Pulsed electric fields (PEFs) can permeabilise plasma and/or organelles membranes depending on the applied pulses and therefore generate cytosolic calcium peaks by recruiting calcium from the external medium or from internal stores. We show that it is possible to mimic haMSCs spontaneous calcium oscillations  using 100 μs PEFs or 10 ns PEFs. We propose a model that explains the experimental situations reported. PEFs can therefore be a flexible tool to manipulate cytosolic calcium concentrations. This tool, that can be switched on and off instantaneously, contrary to chemicals agents, can be very useful to investigate the role of calcium oscillations in cell physiology and/or to manipulate cell fate. Human mesenchymal stem cells (hMSCs) have the ability to differentiate into different cell types including adipocytes, chondroblasts and osteoblasts12Even if the differentiating protocols using chemical agents to differentiate haMSC into adipocytes456791012131152+ oscillations implicating (i) endoplasmic reticulum (ER) Ca2+ channels like the inositol 1,4,5-trisphosphate receptor (InsP3R) and plasma membrane (PM) Ca2+ channels as well as (ii) store-operated Ca2+ channels (SOCCs) and (iii) voltage-operated Ca2+ channels (VOCCs)2+ release-recapture ER mechanisms amplified by the entry of external Ca2+ through PM Ca2+ channelset al. reported that differentiated hMSCs present less Ca2+ oscillations than undifferentiated hMSCs and that blocking these oscillations by using a 10 V/m continuous electric field (EF) facilitates differentiation into osteogenic lineage2+ for stem cells and differentiation2+ HMSCs present spontaneous Ca2+ peaks by permeabilising not only PM but also internal membranes such as the ER membranes, allowing the release of the Ca2+ stored in the ERMicrosecond pulsed electric field (μsPEF) of about 100 kV/m are commonly used to induce PM permeabilisation to different types of molecules . Even if for each cell the rhythm of the Ca2+ oscillations was rather stable, there was a large intercellular variability in the interval between two oscillations which is in agreement with the idea of asynchronous oscillations . From 17 MV/m to 36 MV/m cells presented Ca2+ peaks starting with sharp rises  were applied with 200 s of interval, in order to compare the electrically induced Ca2+ peaks to the spontaneous ones. Most of the cells responded to each nsPEF by an increase in the cytosolic Ca2+ concentration. With external Ca2+, nsPEF of 11 MV/m allowed reproducing Ca2+ peaks with comparable amplitude and duration as the spontaneous ones without stopping the spontaneous oscillations  without stopping the spontaneous oscillations.In llations . If nsPEntration . But, thllations . On Fig.2+ and they also presented Ca2+ peaks with sharp rises in response to nsPEFs due to the release of Ca2+ from the internal stores and mostly the ER .Reproduction of spontaneous oscillations (same amplitude and duration) was achieved using one μsPEF of 100 μs and 15–31 kV/m. However, not all the cells responded. The higher the μsPEF amplitude, the higher the percentage of cells presenting a Cantration . The low2+ peaks . The ampSpontaneous oscillations were not interrupted by the application of six μsPEFs . The add2+ (S-MEM) there was no increase in Ca2+ fluorescence when applying one single μsPEF of 31 kV/m (data not shown) proving that external Ca2+ was involved in the Finally, in the absence of external Ca2+ ions, can cross the electropermeabilized membranes. We used PEFs to generate artificial Ca2+ peaks: we show that one nsPEFs or one μsPEFs can provoke a Ca2+ peak similar to the spontaneous oscillations . The presence or absence of serum in the media did not change the effect of the pulse, (data not shown).PEFs provoke the transient permeabilization of the cells membranes. The extent of this transient electropermeabilisation  depends on the PEF amplitude, duration, number and repetition frequency. As a consequence, hydrophilic molecules, including Ca2+ release induced by nsPEFs in non-excitable cells. It was shown that 10 nsPEFs of 30 ns at 2.5 MV/m2+ release from the ER. In the former case, nsPEF affected organelles without affecting PM Ca2+ channels2+ oscillations (the haMSC) a single nsPEF as short as 10 ns can cause a Ca2+ release and a Ca2+ peak that can be almost identical to one Ca2+ oscillation as discussed below. The shape of the haMSC spontaneous oscillations is in agreement with the hypothesis described by Kawano et al.2+ stored in the ER and amplified by external Ca2+ entry through PM Ca2+ channels). Actually, some haMSCs were not presenting spontaneous Ca2+ oscillations during the observation time  pathway involving the release of calcium from the ER and activation of VOCCS and SOCCS is also involved in the amplification of the electrically induced Ca2+ peaks if the initially μsPEF-induced Ca2+ permeabilisation is large enough  seems to be interesting to synchronously cause a larger proportion of cells reproducing the same shape of CaIt is known that nsPEFs can permeabilise the ER membrane2931332+ peak and the higher the nsPEF strength, the higher the Ca2+ peak amplitude as well as the sharper the beginning of the peak  which is the only source of Ca2+ in this case  could enhance the internal stores depletion.nsPEF amplitudes of more than 9.5 MV/m were needed to induce a Cathe peak : Ca2+ pehis case . Conside 24 MV/m  or to th2+ peaks amplitudes were either lower than or as high as the ones of the spontaneous oscillations (2+ peaks of a lower amplitude than the spontaneous oscillations (4 times shorter than the μsPEF). For nsPEFs of less than 9,5 MV/m, only the external Ca2+ was involved. For higher nsPEF amplitudes, both the PM and the organelles membranes were permeabilised. When nsPEFs of 11 MV/m to 13 MV/m were applied, the Ca2+ increases were more important and the shape of the beginning of the peak was similar to that of spontaneous oscillations , explaining why there is no additive effect and why the amplitude of the induced Ca2+ peaks is similar to the Ca2+ oscillations amplitude.Callations . These dllations d 1 couldl effect . This ob2+ ions as permeabilization marker, the amplitude of the electric pulses necessary to permeabilise the PM is lower than the PEF amplitude necessary to permeabilize the organelles membrane. Interestingly, the comparison is made here using the same marker (Ca2+) and the same system to visualise it . Our conclusions are supported by Semenov et al.2+).A decade ago, it was thought that nsPEFs would only permeabilise organelles, but it has been shown that nsPEFs also permeabilise PM2+ could no longer be mobilised across the membranes. The data also suggest that induced PM permeabilisation or organelles membrane permeabilisation was fully reversible.The characteristics of the permeabilisation marker and the sensitivity of the techniques used to detect it are important parameters that must be taken into account. Indeed, we showed elsewhere2+ concentration, the Ca2+ should be pumped back to the ER through SERCA pumps (sarco/ER Ca2+ ATPase) and/or released to the external medium through the NCX channel (Na+/Ca2+ exchanger) and PMCA (PM Ca2+ ATPase) as demonstrated by Kawano et al.Whatever the mechanism for the rise in cytosolic Ca2+ concentrations on demand and to reproduce spontaneous Ca2+ oscillations in haMSCs using nsPEFs and μsPEFs. The important parameters  in the regulation of cell physiology by Ca2+ peaks are not known but with these new tools, different shapes of Ca2+ peaks can be produced. Interestingly, the perturbation of cell membranes is minimal and fully reversible when the electric pulses are delivered at repetition frequencies compatible with the spontaneous oscillations rates. This way to manipulate the cytosolic Ca2+ concentration presents the great advantage that it can be instantaneously switched on and off, which is not possible with chemical agents. Thus it is possible to manipulate cytosolic Ca2+ concentrations on demand by electrical manipulation and to activate or not SOCCs and VOCCs. This approach can help to understand the implication of Ca2+ oscillations in biological processes like cell differentiation and to determine the important parameters of the Ca2+ peaks in cell physiology.In conclusion, we developed new flexible tools to control internal Ca2 atmosphere by passing them every 3–4 days (one passage corresponding to one doubling time of the population). HaMSCs were isolated and characterised as reported in Liew et al.2 and 10% foetal bovine serum) and S-MEM . Serum was not used in the buffer without calcium (SMEM) since it contains some calcium.HaMSCs were isolated from surgical waste of individuals undergoing elective lipoaspiration. Samples were obtained after written informed consent from all the donors, in accordance with France and European legislations6et al.A commercial generator purchased from FID  was used to treat the cells. It generates trapezoidal monopolar pulses of 10 ns. The exposure devices were microchambers with parallel gold electrodes, 25 μm thick, with a gap between the electrodes of 300 μm or 150 μm. This device allows a high spatial homogeneity of the electric field (variation lower than 1.6% in z and lower than 2–10% maximum in x) as described in Dalmay TM  connected to two parallel stainless steel rods of 1 mm diameter, 4 mm apart that were shaped to enter a 24 plate well and used as electrodes. The whole system was installed on an inverted microscope (see below).Electric pulses of 100 μs were generated using a Cliniporator2+ oscillations, the cells were incubated with 5 μM of the Ca2+ fluorescent marker, Fluo-4 AM  for 30 min in a humidified atmosphere with 5% CO2 at 37 °C in complete DMEM. To easily localise the cells, the incubation buffer also contained 375 nM of the nuclear fluorescent dye Hoechst 33342 . There is no interference between Fluo-4 and Hoechst 33342 fluorescences because their emission wavelengths are separated enough. The slides were washed three times with PBS , turned upside down and placed on the top of the microchamber filled with complete DMEM or S-MEM (the cells being thus located inside the channel).Cells were cultured on glass cover slides for at least 24 hours to obtain well spread out cells. In order to visualise the Ca2. Indeed, the microscope had a heating stage controlled by a Tempcontrol 37-2 digital  and the CO2 was maintained to 5% with a CTI-Controller 3700 digital (Pecon GmbH) into a Plexiglas chamber placed over the heating stage.All the observations done with microsecond pulses exposure were done at 37 °C with 5% of COImages of haMSCs were taken every 10 s for 10 to 40 min with a Zeiss AxioCam Hrc controlled by the Axio Vision 4.6 software  on a Zeiss Axiovert S100 epifluorescence inverted microscope. The pulses were always delivered after at least 5 min of observation and 2 s before the next image. Bright field images and Fluo-4 and Hoechst 33342 fluorescence images were sequentially taken using shutters controlled by the AxioVision 4.6 software. The minimum opening time of the shutters for the fluorescent light was about 500 ms. In order to decrease the light energy applied on the cells, a 90% density Filter Thorlabs  was used.The Hoechst 33342 was used to track the cells during videomicroscopy. Indeed, nuclei were recognised and the individual cells were tracked using the Cell Profiler software , allowing the automatic measurement of the Fluo-4 fluorescence of each cell on every image. Curves were plotted with a program in Matlab.Different increasing field amplitudes were applied on the same cells of a coverslide as it was difficult to compare the responses on totally different cells because of the intercellular variability. On each group of cells, 7 to 8 PEFs of 10 ns were applied with an interval of 300 s. The range of field amplitudes was from 2.5 MV/m up to 40 MV/m.On the box plot, minima and maxima are represented at the ends of the whiskers. The rectangle is defined by the median and by the first and the third quartile of the sample.How to cite this article: de Menorval, M.-A. et al. Electric pulses: a flexible tool to manipulate cytosolic calcium concentrations and generate spontaneous-like calcium oscillations in mesenchymal stem cells. Sci. Rep.6, 32331; doi: 10.1038/srep32331 (2016).


Activation of the UPRmt protects against stresses imposed by reactive oxygen species, respiratory chain deficits, and pathologic bacteria. Consistent with the UPRmt’s role in adaption, we found that either its pharmacological or genetic activation by ethidium bromide (EtBr) or RNAi of the mitochondrial AAA-protease spg-7 was sufficient to reduce death in an anoxia-based Caenorhabditis elegans model of ischemia-reperfusion injury. The UPRmt-specific transcription factor atfs-1 was necessary for protection and atfs-1 gain-of-function (gf) mutants were endogenously protected from both death and dysfunction. Neurons exhibited less axonal degeneration following non-lethal anoxia-reperfusion (A-R) when the UPRmt was pre-activated, and consistent with the concept of mitochondrial stress leading to cell non-autonomous (ie. “remote”) effects, we found that restricted activation of the UPRmt in neurons decreased A-R death. However, expression of the atfs-1(gf) mutant in neurons, which resulted in a robust activation of a neuronal UPRmt, did not upregulate the UPRmt in distal tissues, nor did it protect the worms from A-R toxicity. These findings suggest that remote signaling requires additional component(s) acting downstream of de facto mitochondrial stress.The mitochondrial unfolded protein response (UPR We. We14]. mt reflects a balance between mitochondrial and nuclear ATFS-1 targeting, which is likely to be prone to overexpression artifacts. Moreover, cell-specific promoters exhibit a range of temporal and quantitative differences in expression levels. To circumvent these issues, we first generated a genetic model where FLP recombinase is used to activate gene expression [atfs-1 transgene was cloned where the promoter is interrupted by a wCherry insert that is itself flanked by FLP recombinase sites  transgene was placed into a genomic atfs-1(lf) background, the worms were unable to activate the UPRmt Phsp-60::GFP reporter in response to spg-7(RNAi)  , suggestreporter , suggestIn an attempt to discern regulated trafficking of the ATFS-1 transcription factor, a translational GFP fusion was expressed in the MosSCI system, but there was no detectable fluorescent signal even under conditions where we expected to observe increased nuclear trafficking . We hypoatfs-1(et15) gf missense mutation was incorporated into the MosSCI transgene, to generate a cell-specific compensatory response in the absence of stress. When FLP recombinase was expressed using the pan-neural promoter rab-3  mutant background as a presumed negative control. However, we found that regardless of whether the MosSCI atfs-1(gf) transgene was in a genomic atfs-1(lf) or atfs-1(+) background, the UPRmt was not activated in distal tissues  background: flp null 73 ± 11%, Prab-3:flp 69 ± 13%, error is SEM, p = 0.69; mt reporter  A-R toxicity and 2.) axonal degeneration in neurons subject to sub-lethal A-R. We also find that mitochondrial dysfunction elicited through RNAi targeting of the ETC complex IV subunit cco-1 in neurons can coordinate stress responses remotely to decrease A-R toxicity. Somewhat surprisingly however, neuronal expression of an atfs-1(gf) mutant did not reduce death, although it did function intrinsically to protect neurons themselves from axonal damage. Our results suggest that neuronal survival is not the major determinant of A-R survival and that de facto mitochondrial stress is required to coordinate remote stress responses through an unknown signal(s). However, our results do not discount a role for atfs-1 acting in parallel to this unidentified signal, or being required in the target tissues independent of the signaling tissue. It’s interesting that the homeodomain-containing transcription factor DVE-1 and the small ubiquitin-like protein UBL-5 are both encoded by genes required for signaling the UPRmt [atfs-1. In addition, mitochondrial function has been shown to regulate mTORC1 via ROS signaling , which mt has been suggested to be a surveillance response, whereby interruptions in core physiologic processes such as ETC function coordinate adaptation throughout the organism [mt activation in the germline (or stem cells?) may act as a sentinel for mitochondrial damage. Ethidium bromide, which causes mitochondrial stress and induces the UPRmt, has been shown to preferentially affect germline maintenance in C. elegans. There are multiple precedents for germline-soma signaling. For example, translation  is monitored as a surveillance mechanism [mt mediated metabolic checkpoint [glp-1 mutants which do not generate oocytes have altered fat stores and are greatly protected from A-R [The UPRorganism . It seemechanism , and lipechanism . The gerechanism –48. Witheckpoint . Finallyfrom A-R .C. elegans also alter lifespan [atfs-1 is dispensable for cco-1 RNAi mediated longevity [mt requires atfs-1 and, at least cell autonomously, that an ATFS-1(gf) that favors nuclear targeting is sufficient to reduce A-R death. Undoubtedly there are multiple signaling pathways that respond to mitochondrial stress, and the genetic requirements for protection from stress may overlap, but are likely to have insult specific components.Genetic manipulations that impair mitochondrial function in lifespan . Similarlifespan . One comongevity . In contongevity  indicatemt like other compartment specific unfolded protein responses is aimed at restoring proteostasis in the face of stress. A general concept that emerges from multiple studies of stress is that adaptive signaling is generally beneficial in the short term, but chronic activation of these pathways is maladaptive. However, it’s still unclear whether this holds true for the UPRmt. For example, it’s been established that pre-activation of the UPRmt protects against statin toxicity [Pseudomonas infection [C. elegans mutants in electron transport chain (ETC) subunits require atfs-1 for survival [mt is strictly beneficial?The UPRtoxicity  and Pseunfection . In addisurvival , suggestspg-7(RNAi) can cause larval maturation to stall, as can high concentrations of EtBr, both of which are means of activating the UPRmt. One might argue that these phenotypes result from excess mitochondrial dysfunction that cannot be compensated for by activation of the UPRmt. However, it is worth noting that the atfs-1(et15) gf mutant is generally smaller and matures more slowly than wildtype worms and displays reduced fecundity [mt may be primarily beneficial, further work is required to delineate the downside of chronic activation.Unfortunately, there are several observations suggesting that this is not as straightforward as would appear, and probably depends upon the degree of activation. Treatment with ecundity . In addiecundity . This ma atfs-1et gf mutanatfs-1 and the UPRmt can influence A-R damage and survival. We further hypothesize that mitochondrial stress activates an as-of-yet unidentified signaling pathway(s) to coordinate adaptive responses throughout the organism. While the mammalian ATFS-1 ortholog has yet to be identified, the similarities between the UPRmt in mammals and worms  B.) atfs-1(tm4525) mutants fail to exhibit EtBr mediated protection . Grey diamonds are means with the error shown as standard deviations.A.) Box-and-whisker plot of A-R toxicity following treatment with 30 μg/ml EtBr to activate the UPR(PDF)Click here for additional data file.S2 Figatfs-1(gf) transgenic worms grown on  control plates and  plates containing 30μg/mL EtBr. The transgenic marker for FLP (Pmyo-2::mCherry) is shown in panels A and B, while activation of the UPRmt reporter Phsp-60::GFP is shown in panels C and D. The genomic background is atfs-1(+). These data indicate that failure to activate distal tissue stress responses in this strain does not result from an intrinsic dysfunction.Representative fluorescent photomicrographs of MosSCI FLP-out (PDF)Click here for additional data file.S3 Figatfs-1(gf) transgenic worms grown on  control plates and  plates containing 30μg/mL EtBr. Transmitted light images are shown in panels A-E, while fluorescent images are shown in panels F-J. Relevant information is labeled at the top of the figure. Note that the strain in panels E and J lacks the UPRmt reporter Phsp-60::GFP and that the MosSCI FLP-out atfs-1(gf) is fused to GFP. These panels have been overexposed. The failure to detect either mitochondrial or nuclear GFP (the green is autofluorescence) exemplifies the low expression level of a single copy transgene and its likely degradation under basal conditions.Representative photomicrographs of MosSCI FLP-out (PDF)Click here for additional data file.S1 TableThis is an Excel spreadsheet with the raw data organized by figure. Technical and experimental replicates are indicated.(XLSX)Click here for additional data file.


Obesity results from imbalance between food intake and energy expenditure. The energy homeostasis is regulated by hypothalamic neurons that receive different neural, hormonal and metabolic signals. Insulin is one of the main hormones that regulate energy homeostasis and acts through a cascade of intracellular signaling that depends on the activation of several proteins, such as Akt. Our hypothesis is that PH domain and Leucine rich repeat Protein Phosphatase 1 (PHLPP1) inhibits Akt activity by dephosphorylating serine 473 residues, impairing the insulin action in the hypothalamus. However, PHLPP1 expression and the role played by it on the hypothalamus of diet induced obesity (DIO) animals are not fully understood.To investigate PHLPP1 protein expression in the hypothalamus of DIO rats and to assess whether the PHLPP1 silencing improves insulin action and decreases body adiposity.Eight-week-old male Wistar rats were distributed into two groups. One group was fed with standard diet and the other group with high-fat diet, during eight weeks. After this period, the hypothalamus of Chow and DIO animals were dissected for analysis of PHLPP1 protein expression. In another experiment, DIO animals were cannulated in the lateral ventricle and received intracerebroventricular (ICV) treatment to silence PHLPP1 expression, or its control, scramble (siRNA-SCR), during 7 days. Body weight and food intake were measured daily. On day 8, fasted animals received an insulin or saline ICV injection and their hypothalamus was extracted after 15 min to evaluate the PHLPP1 expression and insulin pathway proteins. Retroperitoneal and epididymal adipose depots were dissected and weighted.Initially, DIO animals showed increased protein expression of PHLPP1 in hypothalamus compared to the Chow group. After 7 days of ICV treatment with siRNA-PHLPP1, DIO rats showed reduced expression of this phosphatase and led to greater weight loss and reduction in adiposity, epipydimal and retroperitoneal. Interestingly, hypothalamic PHLPP1 inhibition restored the phosphorylation of Akt in the hypothalamus. Conversely, it was not observed significant changes in food intake and fasting glucose.Hypothalamic PHLPP1 silencing in DIO animals restored, at least in part, the insulin signaling, promoting reduction in body weight and adiposity.