Experimental work was performed using a clonal rat insulin-secreting cell line (BRIN-BD11) to measure the consumption or production of a range of important biochemical parameters  and Ca2+ levels. These measurements were then used to validate the theoretical model and fine-tune the parameters. Mathematical modelling was used to predict L-lactate and L-glutamate concentrations following D-glucose and/or L-alanine challenge and Ca2+ levels upon stimulation with a non metabolizable L-alanine analogue. Experimental data and mathematical model simulations combined suggest that L-alanine produces a potent insulinotropic effect via both a stimulatory impact on β-cell metabolism and as a direct result of the membrane depolarization due to Ca2+ influx triggered by L-alanine/Na+ co-transport. Our simulations indicate that both high intracellular ATP and Ca2+ concentrations are required in order to develop full insulin secretory responses. The model confirmed that K+ATP channel independent mechanisms of stimulation of intracellular Ca2+ levels, via generation of mitochondrial coupling messengers, are essential for promotion of the full and sustained insulin secretion response in β-cells.We integrated biological experimental data with mathematical modelling to gain insights into the role played by L-alanine in amino acid-stimulated insulin secretion (AASIS) and in D-glucose-stimulated insulin secretion (GSIS), details important to the understanding of complex β-cell metabolic coupling relationships. We present an ordinary differential equations (ODEs) based simplified kinetic model of core metabolic processes leading to ATP production  and Ca Pancreatic β-cells have been the subject of both experimental and theoretical interest for several decades as they play a key role in D-glucose homeostasis by adjusting insulin secretion according to blood D-glucose and other nutrients, endocrine and autocrine secretagogues 2) that are transferred to the mitochondrial electron transport chain (ETC), resulting in ATP generation. The rise in the ATP/ADP ratio closes the ATP-sensitive K+-channels (K+ATP) in the cell membrane leading to depolarization, influx of extracellular Ca2+ through the voltage-dependent Ca2+ (Ca2+ΔΨ) channels and mobilization of the insulin-containing vesicles causing their fusion with the plasma membrane and release of their cargo The current hypothesis of the mechanism of D-glucose-stimulated insulin secretion (GSIS) is that D-glucose enters the β-cell via a membrane-bound D-glucose transporter (GLUT1 or GLUT2) where it is metabolized in the pathway of glycolysis, resulting in pyruvate, which can then enter the mitochondria. Pyruvate is oxidized through the Tricarboxylic Acid (TCA) cycle producing reducing equivalents  and metabolites , can markedly affect the full development of insulin secretion Although the K+-dependent in vitro and in vivo+ co-transport  Amino acids represent a significant class of insulin secretion modulators as they are obtained from dietary proteins as well as being released by intestinal epithelial cells The amino acid L-alanine is one of the most abundant amino acids in plasma (0.5–0.7 mmol/l) A number of mathematical and computational models of the GSIS have been developed, although none of them has taken AASIS into account. In particular, modelling efforts have been focussed on the time dependent oscillatory behaviour of the insulin secretion, both of mitochondrial and electrical origin, which are lost in type two diabetes 2+ handling in β-cells focussed on describing the mechanism underlying oscillations rather than the regulatory properties of the network 2+ handling by the mitochondria with metabolism through ADP and membrane potential. It takes into account transport processes of the inner mitochondrial membrane , the Ca2+ uniporter and Na+/Ca2+ exchanger. A simplified model of GSIS, constituted by four ordinary differential equations for NADH, ADP, membrane potential and Ca2+ with pyruvate as input and ATP production as output, has been developed 2+ influx and glycolytic flux on the dynamics of the system and was able to capture the most noticeable features of the Magnus and Keizer model, using a simplified mathematical description. More recently, Fridlyand and co-workers 2+ and pyruvate. The model has been used to investigate the biochemical regulation and control of β-cells, such as alteration of the proton leak activity, NADH shuttles function and reactive oxygen species production.Magnus and Keizer built the first combined model of metabolism and CaSince the GSIS machinery is highly complex, mathematical models have also been developed separately for specific sections of the pathway such as metabolism and electrophysiology.Early stage models of mitochondrial energy metabolism include the work of Garfinkel 2+ to flow into the cell, alternating with a silent phase when Ca2+ is extruded and the membrane potential repolarizes. The cyclic alternation between depolarization and repolarization of the plasma membrane leads to oscillations in cytosolic Ca2+ concentrations Most current models of electrical activity in pancreatic β-cells focus on explaining the complex bursting behaviour. The bursting consists of an active phase when depolarization of the plasma membrane causes Ca2+ oscillations is based on the separation of timescales: a fast sub-system, constituted by the membrane potential and delayed rectifier potassium channels, generate the spiking during the active phase, whereas a slow variable provide the negative feedback responsible for the cyclical depolarization/repolarization The mechanism of the bursting of electrical activity and Ca2+ influx resulting in insulin granule exocytosis (model 2)  and the potent enhancing effect on D-glucose-stimulated insulin secretion (GSIS), as a first step to understanding the complex metabolic relationships in the β-cell. We consider the insulin secretion pathway divided in two main blocks: (i) core metabolic network leading to ATP production (model 1)  and combined  acute stimulus dose-response curves  minutes incubation with stimuli of interest.We are interested in modelling the mechanisms of possible metabolic stimulus-coupling effects rather than the time-dependent behaviour of the system. Thus, all simulations were allowed to run until the steady state was reached and all presented experimental results were recorded after 20h incubation in Krebs Ringer Bicarbonate Buffer (KRBB) supplemented with either various concentrations of D-glucose , which could reproduce experimental ATP data following D-glucose and/or L-alanine stimulation.An increase in ATP concentration is the main triggering event in the KIn order to estimate the input fluxes for model 1 corresponding to the range of D-glucose and L-alanine concentrations administered in the experiments  in the presence of a stimulatory concentration of D-glucose (16.7 mmol/l), L-alanine (10 mmol/l), their combination, 16.7 mmol/l D-glucose supplemented with a cell-permeable L-glutamate precursor  and a negative control  on intracellular L-glutamate content and corresponding insulin secretion levels were investigated. DMGLU was successfully utilized in inducing a dose-dependent increase in intracellular L-glutamate (results not shown) and the concentration of 5 mmol/l of DMGLU administered in combination with 16.7 mmol/l D-glucose were found to produce an “artificial” increase in intracellular L-glutamate of vsAs shown in + with a stoichiometric ratio 1∶1. This could lead to a substantial shift in the intracellular Na+ concentration, which could affect Ca2+ handling and ultimately account for the disparity in insulin secretion.L-alanine is taken up by β-cells at very high rate in BRIN-BD11 cells , L-alanine (10 mmol/l) and their combination are presented in 2+ concentration responses were found to positively correlate with insulin secretion.Intracellular Ca2+ concentration and insulin secretion respectively against The potent insulinotropic stimulus 16.7 mmol/l D-glucose plus 10 mmol/l L-alanine was associated with a relative fold increase of + co-transport in L-alanine-induced stimulation of insulin secretion, an experiment was designed whereby either 10 mmol/l of a non-metabolizable L-alanine analogue, AIB (α-aminoisobutyric acid), which shares the same co-transport mechanism of the amino acid, or the mitochondrial poison oligomycin (1.8 µg/ml) were employed.To determine the relative importance of oxidative metabolism and Na2+ concentration . Along with the Ca2+ pump current, the Na+/Ca2+ exchanger current  (We studied this using two separate models: a core metabolic model leading to ATP production (model 1)  with equimolar amounts of α-aminoisobutyric acid (AIB). AIB is an L-alanine non metabolizable analogue, thus it shares the same co-transport mechanism of the amino acids without the capability of being oxidised and hence producing ATP. The latter finding is consistent with a previous study + pump blocker, was used to demonstrate that L-alanine promotion of Na+ influx strongly affects insulin secretion. Moreover, it was demonstrated that replacing extracellular Na+ ions with equimolar concentration of N-methyl-D-glutamine (NMDG) in KRBB was effective in abolishing the effects of AIB and L-alanine electrical activity on elevation of intracellular Ca2+To gain further insights into what extent insulin secretory responses can be modulated by electrogenic Na2+ concentrations are necessary to develop full insulin secretory responses, in agreement with previously published findings + co-transport acts synergistically with membrane depolarization and thus K+ATP channel independent mechanisms of stimulation of Ca2+ levels in the β-cell that are essential for promotion of insulin secretion. Furthermore, both models combined suggest that the potent insulinotropic stimulus D-glucose plus L-alanine generates production of an, as yet, unidentified messenger involved in the amplifying pathway of insulin secretion.The analysis of our model simulations indicates that both high intracellular ATP and Ca+ATP dependent triggering pathway. Simulations carried out on model 1 suggest that L-alanine is also the source of metabolically derived putative stimulus-secretion messengers in insulin secretion, such as L-glutamate and citrate (latter result not shown), which could account for the full development of insulin secretion through the amplifying pathway. Moreover, L-alanine / Na+ co-transport mediated depolarization accounts for a substantial proportion of L-alanine stimulated insulin secretion.Collectively experimental data and simulation results suggest that L-alanine is particularly effective in inducing insulin secretion through three independent and complimentary mechanisms of action. Firstly, L-alanine is metabolized at high rates 2+ content is not the only player in insulin secretion when D-glucose and L-alanine are administered together  of powerful insulinotropic agents, as its action is robust, reliable, and controllable, contrarily from most commercially available drugs that lack coupling between metabolism and electrical activity.2+ effects on the mitochondrial dehydrogenases. However, the model is able to capture the essential behaviour of the experimental data, suggesting that the principal pathways involved are modelled with sufficient detail.The current model has multiple limitations. However, it is the only currently available model that accounts for amino acids-dependent insulin secretion mechanisms. Limitations of the current model includes the fact that model 1 in kinetic rates are either modelled using mass action or Michaelis-Menten functional expressions and do not take into account neither cooperativity nor other regulatory activities, like the Ca+ co-transport mechanism are still unknown and more comprehensive experimental data are needed to develop more informed expressions for the mathematical model 2 to be tested.Furthermore, details of Na2+ handling could enable testing of metabolic hypotheses and drug actions and constitute a valuable tool in planning targeted experiments. Furthermore, this study could constitute a starting point for a future refinement of individual steps generating mitochondrial derived coupling factors and Ca2+ handling, thus allowing for future integration of other biochemical pathways of interest.Our description of the β-cell’s core metabolic processes and CaRPMI-1640 culture media, foetal bovine serum (FBS) and plastic were purchased from Gibco . All other chemicals were obtained from Sigma-Aldrich Chemical  unless stated otherwise.2 and 95% air using a Forma Scientific incubator . Cells were cultured in 50–70 ml of RPMI-1640 tissue culture medium in T175cm2 sterile tissue culture flasks (Greiner) and passaged every 2–3 days by mechanical detachment with 0.025% (w/v) trypsin/EDTA solution. The passage number of cells used in this study was in the range 18–30.BRIN-BD11 cells were grown and maintained in RPMI-1640 tissue culture medium with 10% (v/v) foetal bovine serum, 0.1% antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin), 2 mmol/l L-glutamine and 11.1 mmol/l D-glucose, pH 7.4. BRIN-BD11 cells were grown and maintained at 37°C in a humidified atmosphere of 5% COThe general experimental workflow is illustrated in 2/T175cm2 flasks depending on the parameter to be assayed and were allowed to adhere over night before experiments. The following day, cells were washed with calcium and magnesium free phosphate buffer saline (PBS) and pre-incubated at 37°C for 40 min in Krebs Ringer bicarbonate buffer (KRBB)  supplemented with 1.1 mmol/l D-glucose. Supernatant was carefully removed and discarded before cells were washed with PBS.Briefly, BRIN-BD11 cells were seeded into either 24-well plates or T75cmThis was followed by an incubation period of 20 min in KRBB as described above but supplemented with either D-glucose only  or L-alanine only  or various combinations of D-glucose and L-alanine.g for 5 min at 4°C) and stored at −20°C for later determination of insulin and metabolites . Intracellular Ca2+ concentration was assessed by a cell-based experiment performed with flow cytometric techniques. Cells were also analysed for their respective ATP and L-glutamate content (lysates were stored at −80°C).After incubation, supernatant was removed, centrifuged (400 2+ concentrations) were normalized with respect to the cell protein content.All the results . Subsequently, cells were washed twice with PBS and the dye was extracted by disruption with acid ethanol . An aliquot (100 µl) of the resulting solutions was transferred to a 96-well plate and absorbance at 540 nm was recorded using a microplate spectrophotometer .BRIN-BD11 cells  according to instructions of the manufacturer and results were recorded using a luminometer . The assay is based on the ATP dependency of the light emitting luciferase-catalysed oxidation of luciferin for accurate measurement of extremely low concentration of ATP. An aliquot of cells lysates was stored at −80°C for later determination of proteins content.BRIN-BD11 cells were seeded in a T175cm2 flask (g for 5 min at 4°C), washed with PBS, briefly centrifuged again and incubated for further 20 minutes in KRBB supplemented with test stimuli. Intracellular Ca2+ concentrations were determined using an Accuri 6 flow cytometer . Fluo-4 AM dye was excited by a solid-state blue laser (488 nm), 50000 events per sample were acquired at a slow flow rate  and the fluorescent emissions were detected at 530/30 band pass filter (FL1). Data were acquired and analysed with BD Accuri CFlow® software and results are presented as percentage of control .BRIN-BD11 cells were seeded in a T75cmg for 5 min at 4°C.D-glucose consumption, L-alanine consumption and L-lactate production assays were carried out in 24 well plates  and , respectively and YSI 7100 Multiparameter Bioanalytical System . L-lactate production was measured using a lactate oxidase based assay kit supplied by BioVision.g for 15 min at 4°C. The supernatant fraction was transferred to a fresh tube and stored at −20°C for later analysis.Analogously to insulin secretion assays, cell proteins were incubated for 1 h with 150 µl of 1 X RIPA lysis reagent. Cells lysates were then removed to fresh ice-cold micro-centrifuge tubes and centrifuged at 14000 2 flask (2.0×107 cells), starved and stimulated as described previously. Subsequently, supernatant was removed and discarded, cells were washed twice with ice-cold PBS, lysed and total intracellular L-glutamate was determined according to the manufacturer’s instructions using a colorimetric method (BioVision). An aliquot of lysates was stored at −80°C for later protein determination.For L-glutamate determination, BRIN-BD11 cells were seeded in a T175cmCellular protein content was quantified using a BCA (bicinchoninic acid) protein assay kit , which utilizes a modification of the Biuret reaction.Cell size measurementBRIN-BD11 cell size was determined by using two independent approaches: (i) an indirect computer-based approach and (ii) direct measurement in suspension.BRIN-BD11 cells were washed with PBS twice, trypsinized, and removed in KRBB supplemented with 11.1 mmol/l D-glucose and 0.005% DMSO-dissolved neutral red. BRIN-BD11 cells were seeded at low density . Groups of data were compared using an unpaired Student’s t test or one-way ANOVA, where appropriate. The level of significance was set at P <0.05.We considered the following mathematical models focussing on:L-alanine trigger of insulin secretion and how it enhances GSIS through stimulation of oxidative metabolism;+-co-transport on triggering Ca2+ influx.The effect of direct membrane depolarization caused by L-alanine / Na2+ influx as output (We separated the insulin secretion pathway into two main modules: (i) core metabolic processes leading to ATP production with either D-glucose and/or L-alanine as input  can be found in Calcium handling model+ influx brought about by L-alanine co-transport can significantly increase the mean intracellular Ca2+ concentration at the steady state, possibly accounting for a substantial raise in insulin secretion.We are interested in computing whether an additional NaMembrane processes in β-cell are modelled according to the Hodgkin-Huxley theory of membrane electrical activity in which the rate of change of the plasma membrane potential 2+ sequestration events that have been reported in literature and are believed to modulate the burst mechanism. However, since electrophysiological events in β-cells are primarily driven by the plasma membrane oscillator and to date there are no data indicating that Na+ co-transport might affect ER Ca2+ stores or the sarco(endo)plasmic reticulum Ca2+ ATPase pumping rate, ER equations are not included in our model. Therefore, only one compartment was taken into account: we did not distinguish upon Ca2+ located in the cytosol or in the intracellular stores since our experimental data quantified only the total intracellular Ca2+ content. The model assumes that the extracellular space is constituted by a large volume, so that extracellular 2+, Na+ and K+ are taken from Our model is based on the work of Fridlyand + current (+ ATP-dependent current (+ Ca2+-activated current (2+ plasma membrane pump (2+ uniporter (voltage-dependent) current -insensitive channel + concentration is not the limiting factor for the transport as its level is one order of magnitude higher than the corresponding intracellular value. We model the Na+ current Click here for additional data file.Table S22+ handling in pancreatic β-cells: initial conditions and standard parameters list.Mathematical model of Ca(PDF)Click here for additional data file.


LA (20 µM for 2 min) stimulated a transient peak increase followed by a minor plateau increase in [Ca2+]i. Elongation of LA stimulation up to 10 min induced a strong and long-lasting elevation in [Ca2+]i. Activation of FFA receptors by the non-metabolic agonist GW9508 (40 µM for 10 min) resulted in an increase in [Ca2+]i similar to that of 2-min LA treatment. Inhibition of acyl-CoA synthetases by Triacsin C suppressed the strong and long-lasting increase in [Ca2+]i. The increase in [Ca2+]i induced by 2 min LA or GW9508 were fully eliminated by exhaustion of endoplasmic reticulum (ER) Ca2+ stores or by inhibition of phospholipase C (PLC). Removal of extracellular Ca2+ did not influence the transient peak increase in [Ca2+]i stimulated by 2 min LA or GW9508. The strong and long-lasting increase in [Ca2+]i induced by 10 min LA was only partially suppressed by extracellular Ca2+ removal or thapsigargin pretreatment, whereas remaining elevation in [Ca2+]i was eliminated after exhaustion of mitochondrial Ca2+ using triphenyltin. In conclusion, LA stimulates Ca2+ release from ER through activation of the FFA receptor coupled to PLC and mobilizes mitochondrial Ca2+ by intracellular metabolites in β-cells.The role of the free fatty acid (FFA) receptor and the intracellular metabolites of linoleic acid (LA) in LA-stimulated increase in cytosolic free calcium concentration ([Ca Long-chain free fatty acids (FFAs) diversely regulate pancreatic β-cell function under different conditions. FFAs acutely potentiate glucose-stimulated insulin secretion from both β-cell lines and β-cells in primary culture. On the other hand, they inhibit glucose-stimulated insulin secretion and induce β-cell apoptosis in a long term action on β-cells 2+ concentration ([Ca2+]i). The elevation of [Ca2+]i in β-cells, which may be achieved by either calcium influx through membrane channels or calcium release from intracellular calcium stores, triggers and amplifies the exocytosis of insulin granules. Both intracellular metabolites of FFAs and activation of GPR40 may regulate β-cell function via modulating levels of [Ca2+]i in β-cell. FFA metabolites, particularly long-chain acyl-CoA, have stimulatory effects on Ca2+ release from endoplasmic/sarcoplasmic reticulum in muscle cells 2+-mobilizing effects of long-chain acyl-CoA or other metabolites of FFAs in β-cells need to be clarified. On the other hand, GPR40 activation stimulated by FFAs leads to an acute increase in [Ca2+]i in β-cells. GPR40 activation by FFAs activates phospholipase C (PLC) to produce inositol triphosphate (InsP3) and the increase in [Ca2+]i through Ca2+ release from IP3-senstive intracellular Ca2+ pools 2+]i in β-cells is unknown.Insulin secretion is controlled by the levels of cytosolic Ca2+]i levels in primary cultured rat β-cells. The respective effects of the FFA receptor signaling pathway and the intracellular FFA metabolite signaling pathway on [Ca2+]i were carefully dissected and the multiple pathways for increase in [Ca2+]i were demonstrated.In the present study, we used linoleic acid to observe the effects of FFAs on i recording during day 3–6 in culture.Pancreatic islets were isolated from 10–12 week-old male Sprague-Dawley rats as previously described 2+]i was calculated according to the formula described by Grynkiewicz et al 2+]i(nM) =  Kd×(Fo/Fs)×(R-Rmin)/(Rmax-R), where Kd = the Fura-2 disassociation constant (225 nM), Fo = the 380 nm fluorescence in the absence of Ca2+, Fs = the 380 nm fluorescence with saturating Ca2+, R = the 340/380 nm fluorescence ratio, Rmax = the 340/380 nm ratio with saturating Ca2+, and Rmin =  the 340/380 nm ratio in the absence of Ca2+. Fo/Fs, Rmax and Rmin were determined in the recorded cells. Briefly, the cells were permeabilized by 20µM ionomycin for 10 min to allow sufficient extracellular Ca2+ entry, and the resulting 340/380 nm ratio is Rmax. After a steady value of Rmax had been obtained, the Rmin value was determined by chelating Ca2+ with 8 mM EGTA. In the experiments, cells were constantly perfused at a rate of 3 ml/min. Reagents were dissolved in the bath solution just before the recordings and delivered through the perfusion. The β-cells were identified by the response to 16 mM glucose stimulation with the increase in [Ca2+]i. The experiments of [Ca2+]i measurements were done after 15 min recovery from 16 mM glucose stimulation. In our preliminary experiment, the effects of LA, oleic acid, linolenic acid, and palmitic acid on increase in [Ca2+]i in beta-cells were observed and they showed same effects on increase in [Ca2+]i in beta-cells without difference. We choose LA as representative one for mechanism study in this study because LA has been shown clearly and convincingly to activate GPR40 and easy to use in experiment. It has been demonstrated that the EC50 of LA to activate GPR40 is 9.5µM 2+]i measurements was composed of (mM): 140 NaCl, 4.7 KCl, 2.6 CaCl2, 1.2 MgSO4, 1 NaHCO3, 1.2 Na2HPO4, 3 Glucose and 5 HEPES (pH = 7.4 with NaOH).Islet cells were loaded with 1 μM Fura-2/AM in RPMI-1640 medium for 30 minutes at 37°C. Cells were subsequently rinsed with bath solution and kept for 20 minutes in this solution to allow full deesterification of the dye. Fura-2 was alternately excited by ultraviolet light at 340 nm and 380 nm wavelength respectively (100 ms exposure and 2-seconds cycle) under the control of Dual-Wavelength Photometry Controller . The emission at 510 nm was detected by a photomultiplier tube . The signal was transmitted into DigiData and recorded by Axoscope 8.2 . [Ca2+]i levels. P<0.05 was taken as the minimum level of statistical significance.The data are represented as Mean ± SEM for each group. One-way ANOVA was used to analyze the statistical significance between different groups in the [Ca2+]i under 3 mM glucose in rat β-cells remained stable at 46±7 nM during 40 min recording  was not significantly different between 2 min LA stimulation and 10 min LA stimulation  and the second phase increase in [Ca2+]i (next 10 min). These parameters were used in the following results and discussion sections, when the increase in [Ca2+]i was analyzed in details.The mean [Camulation . The mea2+]i followed by a minor plateau increase in [Ca2+]i . Triacsin C, the ACS inhibitor, was used to study the effects of LA metabolites on the increase in [Ca2+]i in β-cells. Triacsin C (10 µM) itself did not influence [Ca2+]i in β-cells during 20 min incubation. After triacsin C treatment of β-cells for 5 min, 10 min LA treatment induced the transient peak increase in [Ca2+]i while the strong and long-lasting second phase increase in [Ca2+]i was significantly suppressed  was applied to cause activation of FFA receptors without metabolites in rat β-cells. GW9508  stimulated the transient peak increase in [Ca [Ca2+]i , n = 7,  [Ca2+]i . This reppressed , n = 7. ed group . The meareatment .2+]i, but did not influence the strong and long-lasting second phase increase in [Ca2+]i induced by 10 min LA stimulation  signaling pathways mulation . The meay U73122 ; whereasy U73122 . U73122 2+]i induced by 10 min LA stimulation  inhibitor and inhibits the transportation of long-chain acyl-CoA into mitochondria for β-oxidation mulation . Etomoxi2+-free bath solution, both LA and GW9508 induced the transient first phase increase in [Ca2+]i in β-cells. The following minor plateau increase in [Ca2+]i induced by 2 min LA and GW9508 was totally eliminated by removal of extracellular Ca2+  blocked the first and second phase increases in [Ca2+]i induced by 2 min LA and GW9508 in β-cells  stimulation of LA was probably mediated by calcium store-operated Ca2+ entry (SOCE). SOCE has been observed in many cell types, including β-cells 2+ release from endoplasmic reticulum 2+]i in β-cells, which was completely prevented by removal of extracellular calcium. It is suggested that Ca2+ influx is responsible for this component of [Ca2+]i increase. In addition, this plateau increase in [Ca2+]i was also blocked by elimination of Ca2+ release from InsP3-sensitive Ca2+ pools by thapsigargin treatment. This Ca2+ influx is therefore triggered by the calcium release from InsP3-sensitive Ca2+ pools and SOCE may be involved.The following increase in [Ca2+]i induced by 10 min LA stimulation is probably mitochondrial Ca2+ mobilization or leakage. In Ca2+-free solution and depletion of intracellular Ca2+ pool by thapsigargin treatment, the strong and long-lasting second phase increase in [Ca2+]i occurred in a similar pattern to that in normal Ca2+ solution by 10 min LA, excluding the contribution of Ca2+-influx and the thapsigargin-insensitive Ca2+ pool. Triphenyltin induces a formation of mitochondrial permeability transition pore (PTP) and triggers Ca2+ release from mitochondria 2+]i similar to that induced by 10 min LA under the condition of combination of thapsigargin treatment and removal of extracellular calcium. In addition, LA did not induce any further increase in [Ca2+]i after triphenyltin treatment of β-cells. It is therefore concluded that the strong and long-lasting second phase increase in [Ca2+]i in rat β-cells induced by 10 min LA may be due largely to the release of mitochondria calcium store. Mitochondria contain large amounts of Ca2+. The free matrix calcium concentration of mitochondria is in the range 0.5–2 µM 2+ binds mostly to phosphates to form a complex 2+ in the cell. Rapid reversibility of matrix calcium-phosphate complex can provide an increase in the free matrix Ca2+ concentration reaching 100-fold of the resting level 2+ efflux pathway from mitochondria 2+ efflux, probably through formation of PTP.The major component of the second phase increase in [Ca2+ mobilization. GW9508 did not mimic the strong and long-lasting second phase increase in [Ca2+]i and U73122 did not block it either. Therefore, metabolites of LA, rather than FFA receptor, may trigger the signaling pathway for the mitochondrial Ca2+ leakage. The slow increase and recovery of this second phase increase in [Ca2+]i also support that the metabolism pathway may be responsible for the mitochondrial Ca2+ mobilization as time is required for enzyme to form and remove metabolites of LA inside the β-cells. Long-chain Acyl-CoA may be responsible or at least partially responsible for mitochondrial Ca2+ mobilization. FFAs inside the cells are firstly acylated by ACS and then moved to the mitochondria for β-oxidation with the help of CPT1 or processed through other pathways such as protein acylation and triglyceride formation 2+]i by the inhibition of ACS with triacsin C, but not the inhibition of CPT1 with etomoxir, suggests that long-chain Acyl-CoA may be responsible for the metabolic effects of LA on [Ca2+]i changes. Long-chain acyl-CoA is an important intermediate linking fatty acid metabolism and regulation of cellular function 2+ release. Mitochondrial PTP induces apoptosis in various types of cells The intracellular metabolite of LA, probably Acyl-CoA, is responsible for the mitochondrial Ca2+]i in rat β-cells by LA stimulation. The patterns and Ca2+ sources of each pathway-linked increase in [Ca2+]i are different and may be associated to different β-cell functions.In summary, both FFA membrane receptor- and intracellular metabolite-mediated signaling pathways contribute to the increase in [Ca


The role of the inositol 1,4,5-trisphosphate receptor, the main intracellular Ca2+-release channel, in apoptosis is well recognized, but its role in autophagy only recently emerged and is therefore much less well understood. Positive as well as negative effects on autophagy have been reported for both the inositol 1,4,5-trisphosphate receptor and Ca2+. This review will critically present the evidence for a role of the inositol 1,4,5-trisphosphate receptor/Ca2+-release channel in autophagy and will demonstrate that depending on the cellular conditions it can either suppress or promote autophagy. Suppression occurs through Ca2+ signals directed to the mitochondria, fueling ATP production and decreasing AMP-activated kinase activity. In contrast, Ca2+-induced autophagy can be mediated by several pathways including calmodulin-dependent kinase kinase β, calmodulin-dependent kinase I, protein kinase C θ, and/or extracellular signal-regulated kinase.Autophagy is an important cell-biological process responsible for the disposal of long-lived proteins, protein aggregates, defective organelles and intracellular pathogens. It is activated in response to cellular stress and plays a role in development, cell differentiation, and ageing. Moreover, it has been shown to be involved in different pathologies, including cancer and neurodegenerative diseases. It is a long standing issue whether and how the Ca Autophagy is the name for a group of lysosomal degradation processes conserved throughout evolution ,2. It isThe latter is the best-studied form of autophagy and involves the formation of a typical double-membrane cistern, named phagophore, which surrounds and ultimately engulfs the cytoplasmic material to be degraded. The resulting vesicle is the autophagosome, which can fuse with late endosomes or lysosomes, leading to the breakdown of its content ,4. BecauAppropriate autophagy levels are not only needed for cellular homeostasis (removing of aggregated proteins and damaged organelles) ,6 but alwith autophagy rather than executed by autophagy [Depending on the type of stress faced by cells, autophagy can either provide the cell with building blocks and energy (e.g. during starvation) or help the cell to cope with potentially damaging elements  ,4,6,8,9.utophagy ,12.Impaired or altered autophagic flux has been implicated in several pathologies, including cancer and neurodegenerative disorders ,6,8.In order to avoid uncontrolled or excessive levels of autophagy, the process is tightly regulated. More than 30 autophagy genes (atg) have hereby been identified as essential regulators ,13. The 2-terminal kinase-1 (JNK1) [3) receptor (IP3R), stabilizing the Beclin 1/Bcl-2 interaction and inhibiting the induction of autophagy [Beclin 1 plays a central and critical role in these initial steps as a platform protein, recruiting other regulatory proteins to the PtdIns3K Complex III . It was 1 (JNK1) , and Bec1 (JNK1) . Either 1 (JNK1) . For ins1 (JNK1) . After s1 (JNK1) . Anotherutophagy . In addiutophagy . Furtherutophagy . In addiutophagy . Three aOnce the phagophore is formed, its further elongation depends on the formation of the Atg5-Atg12-Atg16L1 complex  and the 2+ is a well-known regulator of many intracellular processes, including apoptosis [3R, a Ca2+ channel mainly located in the ER, plays hereby a crucial role [3R may also play an important role in the control of autophagy, though the available data on the role of Ca2+ and the IP3R in autophagy are at least partially contradictory [Interestingly, the processes of apoptotic cell death and of pro-survival autophagy are interrelated in a complex way. They can have antagonistic, additive or even synergistic effects, depending on cell type and conditions . This inpoptosis ,37. Espeial role ,39. Moreadictory ,41.2+ store and in particular of the IP3R in the regulation of autophagy. While the role of the IP3R in controlling apoptosis is already well-established, its function in regulating autophagy only quite recently emerged. The available data however not only support a role for the IP3R and for IP3-induced Ca2+ release in the control of autophagy, but indicate that this role can depend on the exact conditions and so either has a preventing or an enhancing role with respect to autophagy.In this review, we will therefore critically review the available evidence on the role of the ER Ca2+ in the regulation of autophagy has been investigated since 1993 [2+ as both an increase and a decrease in the cytosolic [Ca2+ suppressed autophagy. This complexity was not directly resolved as further reports suggested that intracellular Ca2+ signaling and Ca2+-handling proteins inhibited autophagy, while other reports indicated a stimulatory role for Ca2+  has been shown to trigger Ca2+ release from these acidic compartments via the two-pore channels and so to induce autophagy [2+, possibly also activating Ca2+-induced Ca2+ release from the ER, and finally activating calmodulin-dependent kinase kinase β (CaMKKβ) and AMPK [The role of Cance 1993 , and theiewed in ). Althouutophagy . Interesand AMPK .2+ signals affect autophagy with hereby a prominent role for the ER as the main intracellular Ca2+ store and the IP3R as the most ubiquitously expressed intracellular Ca2+-release channel. Three genes encode an IP3R, leading to the expression of three IP3R isoforms . All IP3R isoforms are activated upon cell stimulation, phospholipase C activation and subsequent IP3 production. IP3 diffuses into the cytoplasm and binds and activates the IP3R, leading to IP3-induced Ca2+ release. The IP3R isoforms vary in a number of properties, including their affinity for IP3 and their regulation mechanisms. Main regulatory factors are the cytosolic and the luminal [Ca2+, ATP, their phosphorylation status and their interaction with regulatory proteins [2+ signals occurring in the cell regulate many intracellular processes, including cell death [In any case, there is no doubt that intracellular Caproteins -47. The ll death -39,48.3R in autophagy was based on the use of Li+[+ induced autophagy by inhibiting inositol monophosphatase, and subsequently decreasing IP3 levels. Autophagy was induced in an mTOR-independent manner, as no decrease in phosphorylation of mTOR substrates was observed, and it was proposed that the IP3R acted as an inhibitor of autophagy. This finding was confirmed in another study, demonstrating that in HeLa cells chemical inhibition of IP3Rs with xestospongin B (XeB), a potent and selective IP3R antagonist [3R expression using siRNA also induced autophagy [A first study implicating the role of the IPse of Li+. Li+ indtagonist  or supprutophagy .3R, several groups investigated the properties of the IP3R triple knock out chicken DT40 B lymphocytes (TKO cells) originally developed by T. Kurosaki [3R1 or IP3R3, but not of the type 2 ryanodine receptor, another ER Ca2+-release channel, suppressed autophagic levels [To further investigate the role of the IPKurosaki . These cKurosaki ,54, but Kurosaki . The varc levels ,54.3R depended on the binding of Beclin 1 to the IP3R [3R facilitates the binding of Beclin 1 to Bcl-2 by recruiting both proteins. This model is attractive, because Beclin 1 and Bcl-2 have been proposed to target distinct IP3R regions with Beclin 1 binding to the IP3-binding domain [3R/Bcl-2 complex and so induce autophagy [3R Ca2+-channel function would not be involved. Correlating with this, siRNA-mediated knock down of Beclin 1 did neither affect histamine-induced Ca2+ release nor the steady-state ER Ca2+ content [It was proposed that the control of autophagy by the IPthe IP3R . In thisg domain  and Bcl-g domain ,57. Moreutophagy . Accordi content .3R but independently of IP3-induced Ca2+ release was however not confirmed in other studies. TKO cells expressing channel-dead IP3R mutants  had, in contrast to those expressing wild-type IP3R1 or IP3R3, similar levels of autophagic markers as control TKO cells [2+ release through the IP3R is critical for the inhibition of autophagy by the IP3R, and it was proposed that this was related to a regulation of mTOR activity [Control of autophagy by the IPKO cells ,54. Thisactivity .3R on autophagy induction [3Rs are present in ER domains forming close associations with the mitochondria [2+ ions from the ER to the mitochondria [2 consumption, pyruvate dehydrogenase inhibition and AMPK activation. These observations suggest a mechanism in which constitutive Ca2+ release through IP3Rs fuels into the mitochondria, thereby augmenting mitochondrial bio-energetics and ATP production [2+ signals could be abolished via siRNA-mediated knock down or XeB-mediated inhibition of IP3Rs, leading to a decreased ATP production, an increased AMP/ATP ratio and subsequent AMPK activation and autophagy stimulation. As mTOR activation appeared unaffected, a non-canonical AMPK-dependent stimulation of autophagy was proposed [The group of K. Foskett performed a very detailed study to clarify the inhibitory effect of the IPnduction . It is wchondria . These dchondria ,59-61. Tchondria  showed ioduction . Also inproposed . A possiproposed .2+ store content, decreasing thereby IP3R-mediated Ca2+ transfer to the mitochondria, O2 consumption, and ATP production, and thus leading to AMPK stimulation and autophagy induction [3R.In follow-up on these results, recent data indicate that Bax inhibitor-1 (BI-1) overexpression promotes autophagy by decreasing ER Canduction . Moreove3R inhibition, it is clear that conditions suppressing IP3R activity cause autophagy induction via a mitochondrial pathway [2+- and IP3R-dependent mechanism, although an additional scaffold function for the IP3R cannot be excluded. Moreover, the relation between the IP3R and mTOR activity may form a feedback loop, as it was shown that the various IP3R isoforms can be phosphorylated and stimulated by mTOR [Although it is not yet clearly established by which pathway AMPK activation leads to autophagy induction in response to IP pathway . Under a by mTOR -65  [2+ stores [2+ originating from other compartments, the existing data support an important role for the ER Ca2+ stores in autophagy induction and in a number of studies a direct role for the IP3R herein was even presented.In contrast to the previous, other results indicated that intracellular Ca studies -69 were iewed in ). As theα, ATF6) , it can + stores . Althoug2+-induced autophagy was linked to Ca2+ mobilization via IP3Rs [3Rs was deduced from indirect evidence based on the use of the IP3R inhibitor 2-aminoethoxydiphenyl borate (2-APB) and the siRNA-mediated knock down of Ca2+/calmodulin-dependent phosphatase calcineurin putatively acting on the IP3R. However, 2-ABP is not specific for the IP3R [3R activity [In a first study, it was assumed that the Cdia IP3Rs . The invthe IP3R ,72, and activity ,73.3R in autophagy was performed in the slime mold Dictyostelium discoideum [3R gene was inactivated by random insertional mutagenesis or when IP3-induced Ca2+ signaling was blocked by BAPTA-AM [A second study indicating a role for the IPscoideum . Becausescoideum . ImportaBAPTA-AM .2+ homeostasis and dynamics, thereby sensitizing multiple Ca2+-handling mechanisms. During the initial phase of the autophagy response, the levels of the Ca2+-binding chaperones calreticulin and GRP78/BiP increased and ER Ca2+ leak decreased, resulting in an elevation of the steady state ER Ca2+ levels, while the sensitivity of the IP3R towards IP3 increased [2+-flux properties of the IP3R was not only indirectly due to the increased ER Ca2+-store content, but also involved direct effects through increased Beclin 1 binding to the IP3R. Functional experiments suggested that predominantly the N-terminal part of Beclin 1, containing the BH3 domain, was involved in this stimulation. Moreover, Beclin 1 appears to interact with the N-terminal part of the IP3R, especially in its suppressor domain (a.a. 1–225) [3R for IP3[2+-channel domain [3R is essential for the proper induction of autophagy upon nutrient starvation, as cells loaded with BAPTA-AM displayed an impaired autophagic flux [3R inhibitor XeB were more complex, because XeB induced autophagic flux in normal cells, but suppressed autophagic flux in starved cells [3R function in autophagy depending on the cellular condition. In normal cells, IP3Rs suppress autophagic flux by fueling Ca2+ into the mitochondria to sustain ATP production, thereby preventing AMPK activity [3Rs are required to promote Ca2+-signaling events that are critical for up-regulating autophagic flux [Finally, in mammalian cell lines, we recently determined that the induction of autophagy triggered by nutrient starvation occurred concomitantly with a remodeling of the proteins involved in ER Cancreased . This se. 1–225) . This doR for IP3 and at tl domain . Importagic flux . This isgic flux . Resultsed cells . This poactivity ,54. In ngic flux ,75.3-induced Ca2+ release [3R sensitization by Beclin 1 is an indirect effect, due to its effects on Bcl-2, e.g. by dissociating Bcl-2 from IP3Rs. However, a Beclin 1 mutant unable to bind Bcl-2 [3-induced Ca2+ release in vitro, indicating that these events were not due to a suppression of the inhibitory effect of Bcl-2. Nevertheless, in a cellular context, Bcl-2 seems to play an important role in tethering Beclin 1 at the ER membranes in the proximity of the IP3R channel [As Bcl-2 can suppress IP release ,82,83, ind Bcl-2  remained channel .3R in autophagy regulation, whereby depending on the state of the cells IP3-induced Ca2+ release can suppress or promote autophagy  may determine the specific outcome on autophagy. In addition, it can be expected that regulators of the IP3R may impinge on the cellular autophagy levels by modulating IP3-induced Ca2+ release. Main regulators in this context are obviously Beclin 1 and Bcl-2, though their exact regulation by associated proteins or phosphorylation events remains to be explored. Moreover, as these same proteins control apoptosis [Taken together these various results indicate a complex action of the IPy Figure . This copoptosis ,48, it i3-induced Ca2+ release can induce autophagy. However, the Ca2+-dependent mechanisms involved and the target(s) of the intracellular Ca2+ signal remain elusive, although different mechanisms have already been proposed  or of AMPK (compound C) as well as siRNA-mediated knock down of these enzymes, it was proposed that autophagy induced by thapsigargin or other Caget AMPK ,79,85. Tget AMPK , althougget AMPK . In partlmodulin , althouglmodulin .2+ is needed for PKCθ phosphorylation, though the mechanism remains elusive. Preliminary data indicate that the phospholipase C inhibitor U73122 partially inhibited ER stress-induced autophagy [3R. Interestingly, in amino acid starvation-induced autophagy no role for PKCθ was found, suggesting that this pathway is preferentially used during ER stress.In a study using hepatocytes and fibroblasts, thapsigargin induced autophagy through ER stress without inhibiting mTOR activity. Phosphorylation of protein kinase C θ (PKCθ) was hereby critical . Treatmeutophagy , underpi2+ treatment triggers Ca2+ release from the ER, putatively by activation of the IP3R, and induces both autophagy and apoptosis. Extracellular signal-regulated kinase (ERK) activation was observed, and inhibition of ERK selectively suppressed the autophagic response, but not the apoptotic cell death response [2+-induced autophagy, although the target of ERK was not elucidated. It is hereby interesting to note that ERK may be involved in the phosphorylation of Bcl-2, thereby leading to Beclin 1 release from Bcl-2 [In mesangial cells, Cdresponse . As ERK om Bcl-2 ,88. Moreom Bcl-2  or of thom Bcl-2 .2+ may therefore depend of one or more of these mechanisms, but the situation might also be more complex, as several other proteins regulating autophagy are known to be influenced by Ca2+. These proteins include DAPK, which is regulated by calmodulin and which can phosphorylate Beclin 1, thereby dissociating it from Bcl-2 and inducing autophagy [2+-binding protein family [The induction of autophagy by cytosolic Cautophagy . Finallyn family  as well n family  have als2+ is very plausible and that depending on the cell type or the cell state various mechanisms can be involved.Taken together, these data indicate that an induction or a regulation of autophagy by Ca2+- store content, the IP3R and IP3-induced Ca2+ release act on the autophagic process. Under normal conditions, a basal level of IP3-induced Ca2+ release from the ER to the mitochondria is responsible for a certain level of ATP production, sufficient for keeping AMPK inactive and therefore precluding induction of autophagy. If Ca2+ transfer to the mitochondria decreases below a certain threshold, ATP synthesis is not anymore guaranteed, AMP/ATP ratio increases and AMPK is activated, leading to autophagy. Under stress conditions however, Beclin 1 may interact with the IP3R, leading to an increased IP3-induced Ca2+ release. At least part of the released Ca2+ diffuses to the cytosol where it can stimulate the induction of autophagy via a not yet completely understood pathway. This pathway may either involve CaMKKβ and AMPK, or develop in an AMPK-independent way. Whatever the mechanism, this dual regulation of autophagy by the IP3R and Ca2+ is of paramount importance for the determination of cell fate. As autophagy is important in pathological situations as e.g. cancer and neurodegenerative diseases [2+ may lead to important therapeutical consequences.In conclusion, the available data indicate that ER Cadiseases ,8, the c2-APB, 2-aminoethoxydiphenyl borate; a.a., Amino acid; AMPK, AMP-activated kinase; ATG, Autophagy genes; BAPTA-AM, 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester); BI-1, Bax inhibitor-1; Bcl-2, B-cell lymphoma 2; CaMKI, Calmodulin-dependent kinase I; CaMKKβ, Calmodulin-dependent kinase kinase β; DAPK, Death-associated protein kinase; ER, Endoplasmic reticulum; ERK, Extracellular signal-regulated kinase; IP3, Inositol 1,4,5-trisphosphate; IP3R, Inositol 1,4,5-trisphosphate receptor; JNK1, c-Jun NH2-terminal kinase-1; LC3, Microtubule-associated protein 1 light chain 3; mTOR, Mammalian target of rapamycin; NAF-1, Nutrient-deprivation autophagy factor-1; PtdIns3K, Phosphatidylinositol 3-kinase; PKC, Protein kinase C; TKO Cells, IP3R triple knock out chicken DT40 B lymphocytes; ULK1, Unc-51-like kinase 1; XeB, Xestospongin B.The authors declare that they have no competing interests.JBP, JPD and GB wrote the manuscript. All authors approved the final version.


The effects of adrenomedullin in circulatory shock states are controversially discussed: while its exogenous supplementation improved organ function and survival  in expern = 11) or the adrenomedullin antibody HAM1101 . Fifteen hours later animals were anesthetized, mechanically ventilated and instrumented for a consecutive 6-hour observation period. Colloid fluid resuscitation and continuous i.v. noradrenaline were titrated to maintain normotensive (mean blood pressure >60 mmHg) and hyperdynamic hemodynamics. Creatinine blood levels and clearance were assessed as surrogate for glomerular filtration [Immediately after cecal ligation and puncture to induce peritonitis, mice randomly received vehicle (ltration . All datP < 0.001), increased total diuresis  vs. 0.6  ml, P = 0.028) resulting in improved fluid balance  vs. 0.26 , P = 0.011) and kidney function  vs. 2.0  μg/ml, P = 0.006; creatinine clearance: 400  vs. 197  μl/minute, P = 0.006).Adrenomedullin antagonism decreased the noradrenaline requirements needed to achieve target hemodynamics  vs. 0.02  μg/g/hour, In resuscitated murine septic shock, early modulation of excess adrenomedullin activity via antibody HAM1101 improves cardiovascular catecholamine responsiveness, ultimately associated with attenuation of acute kidney injury.


Compared with the control plants [cultivated in 0 mM Ca(NO3)2 medium], the maximal photochemical efficiency of PSII (Fv/Fm) in Ca2+-treated plants showed a slighter decrease after 5 h of stress, accompanied by higher non-photochemical quenching (NPQ), higher expression of antioxidative genes and less reactive oxygen species (ROS) accumulation. Meanwhile, higher content of D1 protein and higher ratio of (A+Z)/(V+A+Z) were also detected in Ca2+-treated plants under such stress. These results showed that Ca2+ could help protect the peanut photosynthetic system from severe photoinhibition under heat and HI stress by accelerating the repair of D1 protein and improving the de-epoxidation ratio of the xanthophyll cycle. Furthermore, EGTA (a chelant of Ca ion), LaCl3 (a blocker of Ca2+ channel in cytoplasmic membrane), and CPZ [a calmodulin (CaM) antagonist] were used to analyze the effects of Ca2+/CaM on the variation of (A+Z)/(V+A+Z) (%) and the expression of violaxanthin de-epoxidase (VDE). The results indicated that CaM, an important component of the Ca2+ signal transduction pathway, mediated the expression of the VDE gene in the presence of Ca to improve the xanthophyll cycle.Peanut is one of the calciphilous plants. Calcium (Ca) serves as a ubiquitous central hub in a large number of signaling pathways. The effect of exogenous calcium nitrate [Ca(NO D1 protein, encoded by the psbA gene, can be used to reflect the degree of photoinhibition of PSII de novo synthesis of this polypeptide in mature chloroplasts Plants are frequently subject to various environmental stresses. During summer, high temperature and high irradiance (HI) are the common stresses which plants are always faced with. Severe photo-oxidative damage to the photosynthetic apparatus is often attributed to the simultaneous occurrence of heat and HI and a decrease in photosynthesis often aggravates the amount of excess excitation energy During the long-term evolution, higher plants have developed many protective mechanisms to balance absorbed light energy with photosynthesis, thereby protecting the photosynthetic apparatus against photoinhibition 2+ acts as a regulator of many physiological and biochemical processes in response to abiotic stresses in plants 2+ in the cytoplast can be detected in plants in response to various stresses, such as high temperature 2+ improves plant resistance is related to maintaining a higher photosynthetic rate under stresses, and light-induced Ca2+ influx into chloroplasts not only influences the cytosolic concentration of free Ca2+ but also regulates the enzymatic processes inside the chloroplast 2+ improves the net photosynthetic rate (Pn), carboxylation efficiency, and apparent quantum yield (AQY) of tobacco leaves under high temperature stress 2+ could also improve the Pn and Rubisco activity of cucumber at suboptimal temperatures 2+ on photosynthesis are attributed to the improvement of the stability of PSII reaction centers by enhancing the activity of antioxidant enzymes to alleviate ROS accumulation 2, is dismuted by superoxide dismutase  to H2O2 and O22O2 is then converted into water by ascorbate peroxidase . Furthermore, exogenous Ca2+ could improve NPQ 2+ on the xanthophyll cycle are yet to be reported.Ca2+. The alteration in the level of Ca2+ triggers a full range of signal transduction pathways through high-affinity, Ca-binding proteins such as calmodulin (CaM) Environmental signals trigger rapid and transient increases in cytosolic CaArachis hypogaea L.) remains one of the most important oil-crops in the world, and Ca is by far the most critical nutrient for this crop to achieve high yields. Ca deficiency in soil can results in a decrease in yield, e.g. unfilled pods. Photosynthetic products are the sources of peanut seeds, and peanut seedlings are usually subject to high temperature and HI in the field during peanut pod development. However, the effects of Ca2+ on peanut photosynthesis are yet to be reported. To explore the effect of exogenous Ca2+ on photoinhibition and the relationship between the expression of Ca-binding protein and the de-epoxidation state of the xanthophyll cycle, 0 mM Ca(NO3)2 and 6 mM Ca(NO3)2-treated peanut plants were used in our experiments combined with some Ca2+ inhibitors. It seemed that Ca2+ and Ca2+-dependent signal transduction pathways were apparently involved in the xanthophyll cycle to dissipate excess energy when peanuts were exposed to high temperature and HI stress.Peanut (Arachis hypogaea L.) cultivar “Huayu 22” was used in this study. The peanut plants were incubated with two revised Hoagland solutions, respectively, in which an equal volume of 0 or 6 mM Ca(NO3)2 2 proved the best treatment in improving the heat and high irradiance stress of peanut plants) were presented. The seedlings incubated with 0 mM Ca(NO3)2 were marked as CK, and those incubated with 6 mM Ca(NO3)2 were marked as CA. The plants were grown at 25/20°C (day/night) under a 14 h photoperiod [300 µmol m−2 s−1 photon flux density (PFD)] for 20 d in a greenhouse. Functional leaves from plants were used in the experiments. To induce heat and high irradiance (HI) stress, with the adaxial side facing up, the detached leaves floating on the water were illuminated with 1 200 µmol m−2 s−1 PFD at high temperature (40°C).Peanut .Fresh leaf samples were dried in an oven at 105°C for 15 min, and then kept at 80°C to a constant weight. Approximately 150 mg of dried leaves was burned to ashes in an oven at 550°C. The ashes were dissolved in 65–68% HNO−2 s−1) which was low enough without inducing any significant variable fluorescence (Fv). The maximal fluorescence (Fm) was determined by 0.8 s saturating light of 8 000 µmol m−2 s−1 on a dark-adapted (adapted 15 min in darkness) leaf. The maximal photochemical efficiency (Fv/Fm) of PSII was calculated as Fv/Fm = (Fm-Fo)/Fm. NPQ was estimated as NPQ = Fm/Fm’-1 according to Chlorophyll fluorescence was measured with a portable fluorometer  according to the protocol described by 2 and filtered through three layers of cheesecloth. The filtrate was centrifuged at 5 000×g for 10 min. The thylakoid pellets were washed with isolation buffer, re-centrifuged, and finally suspended in isolation buffer. The chlorophyll contents were determined spectrophotometrically as described by 2 and stored at −70°C for use.Thylakoid membranes were prepared according to Thylakoid membrane proteins were denatured and separated using 12.5% polyacrylamide gradient gel that contained 6 M urea. 30 µg protein was applied to each well. The resolved protein was electroblotted to PVDF membrane and then probed with polyclonal antibodies raised in rabbits against the full-length D1 protein. The secondary antibody was peroxidase-conjugated goat anti-rabbit IgG. The D1 protein antibody was used at a dilution of 1∶500 and the secondary antibody was used at 1∶3 000.g for 25 min. Extracted solution (3 mL) was mixed with 1 mL of 0.1% titanium sulfate in 20% (v/v) H2SO4 and the mixture was then centrifuged at 6 000×g for 15 min. The intensity of the yellow supernatant was measured at 410 nm. H2O2 level was calculated using an extinction coefficient of 0.28 µmol−1 cm−1 according to the standard curve plotted with known H2O2 concentration.Hydrogen peroxide concentration was measured according to the method of 2•– was performed as described by g for 10 min at 4°C. The supernatant with phosphate buffer (pH 7.8) and 10 mM hydroxylammonium chloride was incubated at 25°C for 20 min, then 17 mM p-aminobenzene sulfonic acid and 7 mM α-naphthylamine were added, and the mixture was incubated at 25°C for 20 min. Finally, ethyl ether was added into the mixture that was centrifuged at 1 500×g for 5 min. The water phase was used to determine the absorbance at 530 nm. The O2•– generation was calculated per g fresh mass of leaves.The assay for O2 immediately after chlorophyll fluorescence measurement and stored at −70°C for use. Photosynthetic pigments were extracted from leaf disks with 80% ice-cold acetone. The extracts were centrifuged at 12 000×g for 5 min and supernatants were filtered through a 0.45 mm membrane filter before injection into reversed-phase high performance liquid chromatography (HPLC) using a Shimadzu Series model SCL-10AVP (Japan) equipped with an Elite Hypersil ODS2 4.6–250 mm cartridge column. Photosynthetic pigments were separated and quantified essentially following the method of Leaf disks were immersed in liquid NTotal RNA was extracted from the peanut leaves with the RNA simple kits  according to the manufacture’s protocol. The DNase-treated RNA was reverse-transcribed using M-MLV reverse transcriptase (TIANGEN). Real-time RT-PCR was performed on the Bio-Rad CFX96TM Real-time PCR System using SYBR Real Master Mix (TIANGEN). The PCR thermal cycle conditions were as following: denature at 95°C for 1 min and 40 cycles for 95°C 10 sec; 61°C 30 sec; 68°C 20 sec. Peanut TUA5 was used as internal reference gene for calculating relative transcriptional levels.AtpsbA, AhAPX, AhSOD, and AhCaM were acquired from GenBank database, the accession number were X79898, EF165068, DQ499511, and AY517930, respectively. The amino acid sequence of psbA gene showed high identities among different species, thus the two degenerate primers were designed according to the sequence from Arabidopsis thaliana. Primer sequences were as follows: TUA5-F, 5′-CTGATGTCGCTGTGCTCTTGG-3′; TUA5-R, 5′-CTGTTGAGGTTGGTGTAGGTAGG-3′; psbA-F, 5′-ATCTGCTAATGAAGGTTACAG-3′; psbA-R, 5′-ATACCTACTACCGGCCAAG-3′; APX-F, 5′-TGCTGGAACTTTTGATGTGG-3′; APX-R, 5′-AACTACACCGGCCAACTG-3′; SOD-F, 5′-CAGTTCTTAGCAGCAGTGAG-3′; SOD-R, 5′-GGAACCCATGAAGACCAG-3′; CaM-F, 5′-GGTGCTCGACAAGGATCAA-3′; CaM-R, 5′-ACTCCTCGTAGTTGATCTGC-3′; VDE-F, 5′-TGCCTATGAAATCAGATGTGG-3′; VDE-R, 5′-CAAGTTTGTTGTCTTCTGTGTG-3′.Gene sequence of CaM concentrations in peanut leaves were determined with the Plant calmodulin (CaM) ELISA Kit  according to the manufacture’s protocol.2+ and the de-epoxidation ratio of xanthophyll cycle pigments. EGTA was used as a chelant of calcium ion, LaCl3 was used as a blocker of Ca2+ channel in cytoplasmic membrane and CPZ was used as a CaM antagonist. CA seedlings were pretreated with a daily spray of 5 mM EGTA, 2 mM LaCl3, and 0.05 mM CPZ for 6 days, respectively.Some chemical agents were used to study the relationship between CaStatistical analyses were performed by analysis of variance (ANOVA) using SPSS version 13.0  and comparisons between the mean values were made by the least significant difference (LSD) at a 0.05 probability level.2+ culture media affected peanut seedling growth (3)2-treated peanut plants. Meanwhile, the contents of Ca2+ in CA peanut leaves and roots were respectively higher by 31.7% and 40.9% compared with that in CK  has been widely used as an indicator of photoinhibition. As shown in D1 protein, one of the major subunits of the PSII reaction center complex with rapid turnover, could be used to reflect the degree of photoinhibition. qRT-PCR and western blot analysis were performed to investigate the effects of the heat and HI stress on D1 protein at the levels of transcription and translation. Cross stress of heat and HI induced a decrease in both psbA expression  and D1 p2O2 and O2•–. As shown in 2O2 and O2•– in the chloroplasts of all tested plants increased after the heat and HI stress. CK seedlings accumulated more H2O2 and O2•– than those of CA seedlings after heat and HI stress for 5 h. Accordingly, higher expression of antioxidant enzymes APX , but the de-epoxidized ratio of the xanthophyll cycle pigments, (A+Z)/(V+A+Z), was higher in CA than in CK seedlings. At the end of the stress, the de-epoxidation level reached 32.1% and 40.8% in CK and CA seedlings, respectively . The accCaM gene and the protein level of CaM were analyzed in peanut leaves. After applying heat and HI stress for 5 h, either the transcriptional level /(V+A+Z) ratio in CA leaves /(V+A+Z) increased in CK and CA seedlings when exposed to the heat and HI stress, thus the energy dissipation and the xanthophyll cycle were both inhibited in Ca2+-deficit seedlings (2+ application seedlings suggested that Ca2+ could improve the xanthophyll cycle-dependent energy dissipation. To the best of our knowledge, the results of the present study are the first evidence of a relationship between Ca and the xanthophyll cycle in peanuts under the heat and HI stress.When exposed to photoinhibition, the xanthophyll cycle-dependent NPQ for higher plants is the most useful mechanism to dissipate excess energy. NPQ is induced in the presence of eedlings , 6. Amoneedlings  and higheedlings  in Ca2+ 2+ application for peanut seedlings could improve crop’s resistance to heat and HI stress by improving the xanthophyll cycle and ROS scavenging system. Moreover, the opposite effects of Ca2+-deficit cultivation may be related to lower membrane stability and integrity and may lower some enzymes’ activities because Ca2+ is believed to be involved in the formation of biological membrane and in the activation of some enzymes 2+-mediated signal transduction pathway on excess energy dissipation, especially the interconversions of the three pigment components of the xanthophyll cycle.Thus, it can be concluded that Ca2+ plays a pivotal role in signal transduction pathway under abiotic stress. Several families of Ca2+ sensors have been identified in higher plants. CaM and CaM-related proteins are the best known ones, which sense the “Ca signature” and participate in Ca2+-mediated signal transduction pathway and gene regulation during stress responses AhCaM gene in peanut leaves under Ca2+ application, and its expression was inhibited after the addition of EGTA and LaCl3 (AhCaM gene could be induced by the heat and HI stress in a Ca-dependent manner. Analysis of CaM at the protein level also verified that Ca2+ plays a role in resisting to heat and HI stress through Ca2+/CaM signal transduction pathway (2+ application, but was inhibited in CK, especially in EGTA, LaCl3, and CPZ-treated plants (VDE gene (2+ can improve the de-epoxidation state of the xanthophyll cycle through the Ca2+/CaM-mediated stimulation of the VDE gene. CaM is a small acidic protein that is primarily expressed in the cytoplasm. However, CaM has been found to be present in several organelles in plants, such as the nucleus and chloroplasts As a second messenger in plants, Cand LaCl3 , which i pathway . Additiod plants , which wVDE gene . That is


NaCl-induced calciuria may be associated with hypertension, urinary stone formation and osteoporosis, but its mechanism and long-term effects are not fully understood. This study examined alterations in the expressions of renal Ca2+ transporters, channels and claudins upon salt loading to better understand the mechanism of salt-induced urinary Ca2+ loss.Urinary Ca+/Ca2+ exchanger 1 (NCX1), Ca2+ pump (PCMA1b), Ca2+ channel (TRPV5), calbindin-D28k, and claudins  were analyzed by quantitative PCR, western blot and/or immunohistochemistry.Eight-week old Wistar-Kyoto rats were fed either 0.3% or 8% NaCl diet for 8 weeks. Renal cortical expressions of Na2+ increased 6.0 fold with high-salt diet. Renal cortical claudin-2 protein decreased by approximately 20% with decreased immunological staining on tissue sections. Claudin-16 and −19 expressions were not altered. Renal cortical TRPV5, calbindin-D28k and NCX1 expressions increased 1.6, 1.5 and 1.2 fold, respectively.Fractional excretion of Ca28k, and NCX1. Salt loading is known to reduce the proximal tubular reabsorption of both Na+ and Ca2+. The reduction in claudin-2 protein expression may be partly responsible for the reduced Ca2+ reabsorption in this segment. The concerted upregulation of more distal Ca2+-transporting molecules may be a physiological response to curtail the loss of Ca2+, although the magnitude of compensation does not seem adequate to bring the urinary Ca2+ excretion down to that of the normal-diet group.Chronic high-salt diet decreased claudin-2 protein and increased renal TRPV5, calbindin-D However2+-transporting molecule and claudin expressions. Chronic salt loading decreased the protein expression of claudin 2, a component of proximal, paracellular Ca2+ transport pathway. Concomitantly, dietary NaCl increased the expression of more distal, transcellular Ca2+ reabsorption machinery, TRPV5, calbindin-D28k and NCX1.This study, for the first time, examined the effects of long-term dietary sodium chloride on renal Ca2+ along with Na+[2+ excretion of salt-loaded rats increased approximately 6.0 fold. Generally, the cause for this phenomenon is attributed to an extracellular fluid volume expansion and/or to the reduced reabsorption of both Na+ and Ca2+ in the proximal tubule [Salt loading acutely increases urinary excretion of Ca with Na+,6. In thl tubule . Althougl tubule , from thl tubule , the use2+ in claudin-2 knockout mice is 3 times that of wild-type mice, further supporting a role of claudin-2 in proximal tubular paracellular Ca2+ reabsorption [+ and Ca2+ transport. Therefore, the decreased expression of claudin-2 with high-salt diet may, to some degree, account for the decrease in Ca2+ reabsorption, while limiting Na+ and water reabsorption, as Na+[2+ may pass through the pores formed by claudin-2. It has been reported that hyperosmolarity stress decreased claudin-2 expression in Madin-Darby canine kidney cells [2+ movement from the luminal to interstitial fluid in the proximal tubule, reduction in claudin-2 may underlie the increased urinary Ca2+ excretion observed under high-salt diet.Approximately 65% of calcium in the pro-urine is reabsorbed in the proximal tubule. In the proximal tubule, claudin-2 is postulated to form tight junction cation pores . Muto etsorption . In thisn, as Na+ and waten, as Na+ in additey cells , and hyp2+ reabsorption, mediating apical Na+ entry and consequently water reabsorption to produce osmotic gradient [2+ reabsorption in the proximal tubule. However, competition between Na+ and Ca2+ for paracellular transport binding site may occur in the proximal tubule. It has been reported that Ca2+ inhibits paracellular Na+ conductance by competitive binding on claudin-2 [+ and Ca2+ share a binding site, inversely, high Na+ may inhibit claudin-2 Ca2+ conductance. This competition between Na+ and Ca2+ may play a large role in the dietary NaCl-induced hypercalciuria.In the proximal tubule, NHE3 is shown to be important as a part of driving force for Cagradient . In our gradient . As regugradient , proteinlaudin-2 . If Na+ 2+ reabsorption. Claudin-16 and −19 are shown to be important for paracellular Mg2+ and Ca2+ in this segment. In our study, there was an increase in the fractional excretion of Mg, albeit smaller than that of Ca. However, there was no significant difference in renal claudin-16 or −19 mRNA in rats on high-salt diet. Extracellular volume expansion decreases transepithelial voltage and Mg2+ reabsorption in the TAL [2+ fractional excretion.Thick ascending limb of the loop of Henle is responsible for approximately 20% of Ca the TAL . Althoug2+ reabsorption [28k, and NCX1, was observed with salt loading in this study. Claudin-8, the distal tubular paracellular cation barrier, was not altered by salt loading. It may be that with salt loading, the proximal, paracellular Ca2+ reabsorption is reduced, and more distal, transcellular Ca2+ transport molecules are upregulated to facilitate Ca2+ reabsorption as a compensatory mechanism. However, salt loading may reduce the Ca2+ reabsorption via NCX1, as illustrated in Figure 2+ transport machinery with chronic salt-loading may partially compensate for the urinary Ca2+ loss, although with a limited effect.Distal nephron is the final and most-regulated site of urinary Casorption ,47. A co28k, and NCX1 upregulations by dietary NaCl, one possibility is the endocrine factors that regulate Ca2+-related molecules, such as parathyroid hormone [2D has been shown to increase the expressions of TRPV5, calbindinD28k, and NCX1 [2D was significantly lower in the high-salt group than the control group. Unless there is a significant difference between serum and intrarenal 1,25(OH)2D levels, it is likely that salt-induced transcellular Ca2+-transporter upregulation is mediated by pathway(s) other than 1,25(OH)2D.As for the mechanism of TRPV5, calbindin-D hormone  and vita hormone . For exaand NCX1 . HoweverThe weakness of the study includes a lack of regional expression data, as excised renal cortex was used in the study. Higher-resolution immunohistological staining experiments and qRT-PCR/Western blotting from micro-dissected tissue specimens are necessary in the future. However, this study aimed to lay the foundation for a more detailed mechanistic examination of the effects of chronically high dietary sodium on the expression of renal Ca transporters and on urinary calcium excretion.2+ in tubular fluid reaching the distal tubule, while the concerted upregulation of more distal Ca2+-handling molecules may curtail some of the Ca2+ loss in the urine. Findings of our study may have implications on further research on the pathophysiology of osteoporosis, urinary stone formation and hypertension associated with excessive salt intake.Our findings suggest that the decrease in renal claudin-2 protein by salt loading may increase the Ca+/Ca2+ exchanger 1; 1,25(OH)2D: 1,25-dihydroxyvitamin D3.NCX1: NaThe authors declare that they have no competing interests.MSY conceived the experiments in part with JY. KT, YM, RS and SA performed the experiments. HS, JK and TW gave advice on the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2369/13/160/prepub


In this study, we investigated the intracellular mechanism of RC-induced Ca2+ release, especially the role of Ca2+, in ferret ventricular muscle. Saponin-treated skinned trabeculae were placed in a glass capillary, and the amount of Ca2+ released from the SR by RC and caffeine (50 mM) was measured with fluo-3. It was estimated that in the presence of ATP about 45% of the Ca2+ content in the SR was released by RC. The amount of SR Ca2+ released by RC was unchanged by the replacement of ATP by AMP-PCP , suggesting that the suppression of the Ca2+ pump of SR at low temperature might not be a major mechanism in RC-induced Ca2+ release. The free Ca2+ concentration of the solution used for triggering RC-induced Ca2+ release was estimated to be only about 20 nM with fluo-3 or aequorin. When this solution was applied to the preparation at 3°C, only a small amount of Ca2+ was released from SR presumably by the Ca2+-induced Ca2+ release (CICR) mechanism. Thus, in mammalian cardiac muscles, RC releases a part of the (<50%) stored Ca2+ contained in the SR, and the mechanism of RC-induced Ca2+ release may differ from that of CICR, which is thought to play a role in frog skeletal muscle fibres that express ryanodine receptors of different types.Rapid lowering of the solution temperature  from 24 to 3°C within 3 s releases considerable amounts of Ca Thus, it was supposed that both caffeine and low temperature synergistically increase Ca2+ concentration around the SR and work to facilitate the CICR mechanism, leading to a massive Ca2+ release from SR ) by the following equation:tot was the total fluo-3 concentration in the assay solution and was 20 μM in the present study.After RC, Caboratory . At the 2+ released from the SR, defined as the change in total Ca2+ concentration in the capillary lumen (Δ[Ca]tot), could be expressed as the sum of changes in concentrations of free Ca2+ (Δ[Ca2+]), Ca2+ bound to fluo-3 (Δ[Fluo-3 · Ca]) and Ca2+ bound to other buffers  (Δ[B · Ca]):The amount of Ca2+ , Δ[Ca]tot could be approximated with Δ[Fluo-3 · Ca]. Figure 2+, Ca2+ release was induced by RC. The calibrated Δ[Fluo-3 · Ca] increased as the fluo-3 concentration was increased up to 200 μM, but was saturated at higher fluo-3 concentrations (200–400 μM). The result suggested that the fluo-3 concentration of 200 μM was high enough to predominantly bind released Ca2+ from the SR and that Δ[Fluo-3 · Ca] obtained at high fluo-3 concentrations (≥200 μM) could be an index of the amount of Ca2+ released from the SR . With 20 μM fluo-3 primarily used in the present study for economic reasons, on the other hand, Δ[Fluo-3 · Ca] had to be scaled to attain the value that would be obtained if the fluo-3 concentration was sufficiently high (≥200 μM). The scaling factor of 1.80 estimated from Fig. When the fluo-3 concentration is high enough to predominantly bind released Ca2+ in the SR. High concentrations of caffeine and AMP should maximise Ca2+ release, and the Ca2+ pump was expected to be inactive in the absence of ATP. Because caffeine significantly influences fluo-3 fluorescence and affinities of intracellular buffer sites for Ca2+, we carried out a separate set of calibrations of fluo-3 fluorescence signals; at the end of each experiment, Fmax and Fmin were estimated by the introduction of the G0RM0CafF assay solution plus 1 mM Ca2+ and the assay solution plus 1 mM EGTA, respectively. The relation between the fluo-3 concentration and Δ[Fluo-3 · Ca] was constructed in the G0RM0CafF assay solution  at 24°C for 1 min, fluo-3 was introduced (G0RM1.5F), and the bathing solution temperature was lowered from 24 to 3°C within 3 s , and the fluorescent Ca2+ signal was measured  were subtracted from the fluorescence signals obtained with SR Ca2+ loading . We counted an average of 5 s around the peak of the subtracted signal.For estimation of the amount of Ca2+ release by RC was calculated using the values obtained from protocol A and B.2.2+ release by RCEffect of adenine nucleotides on CaThe fractional SR Ca2+ release. For removal of ATP, the assay solution of protocol A  was simply substituted with 4.6 mM K2AMP-PCP.3.2+ released by the CICR mechanism at 3°CMeasurement of the amount of CaProtocol A was modified to study effects of ATP on RC-induced Ca2+ loading in SR at 24°C for 2 min, the preparation in a capillary was washed with the Ca2+-free solution , except for modifications of the solution temperatures. Following Cat test, and a significant difference of p < 0.05 was verified. Statistical values were given as mean ± SEM.We used two-tailed Student’s 2+ concentration in the solution used for RC containing 4.6 mM ATP and 20 μM fluo-3 with either fluo-3 or aequorin. The average free Ca2+ concentration was estimated to be 19 ± 2 nM (n = 11) measured with fluo-3  with aequorin at 3°C. There was no significant difference between the two values measured with fluo-3 and that with aequorin. Thus, the free Ca2+ concentration around SR was about 20 nM (pCa 7.7) before Ca2+ release was induced by RC.We measured the contaminated free Ca2+ loading of the SR, RC induced a large increase in the [Fluo-3-Ca] in the capillary space (a). The preparation was briefly washed with Ca2+-free solutions  was repeated without RC in the same preparation, an increase in [Fluo-3-Ca] was greater by about a factor of two than that induced by RC (c).Figure the Fluo--Ca in th2+ released by RC (a) was, on average, 3.16 ± 0.46 μM. On the other hand, the residual SR Ca2+ after RC (b) was, on average, 2.39 ± 0.61 μM. This value was significantly smaller (p < 0.01) than the 4.19 ± 0.49 μM predicted by the total amount of releasable Ca2+ (c = 7.34 ± 0.93 μM) minus the amount Ca2+ released by RC (a). This difference is likely due to Ca2+ leakage from the SR into solutions that contained EGTA during 1.5 min washing periods inserted between RC and caffeine applications  in the presence of ATP at 3°C  signal also showed a gradual rise that started about 10 s after the solution exchange  , which was higher than that estimated in the present study , unless an appropriate correction was made. Consequently, a fraction of Δ[Ca]tot induced by RC would have been overestimated in the previous study. In the present study, on the other hand, Δ[Ca]tot was deduced by scaling Δ[Fluo-3 · Ca]tot with factors separately estimated in the presence (2.78) and in the absence (1.80) of 50 mM caffeine (Fig. 2+ released by RC.In our previous study, we estimated the fraction of SR Ca ferrets . The int 0.90 μM . From th the rat . Furtherne heart . Therefo


Toward elucidating this, we used the isolated blood-perfused rat lung preparation. We loaded microvessels with the Ca2+ indicator, Fura 2 AM and then determined Ca2+ responses to infusions of lipopolysaccharide (LPS) into the microvessels. LPS induced a more than two-fold increase in the amplitude of cytosolic Ca2+ oscillations. Inhibiting inositol 1,4,5 trisphosphate receptors on endoplasmic reticulum (ER) Ca2+ stores with Xestospongin C (XeC), blocked the LPS-induced increase in the Ca2+ oscillation amplitude. However, XeC did not affect entry of external Ca2+ via plasma membrane Ca2+ channels in lung microvascular endothelial cells. This suggested that LPS augmented the oscillations via release of Ca2+ from ER stores. In addition, XeC also blocked LPS-mediated activation and nuclear translocation of nuclear factor-kappa B in lung microvessels. Further, inhibiting ER Ca2+ release blunted increases in intercellular adhesion molecule-1 expression and retention of naïve leukocytes in LPS-treated microvessels. Taken together, the data suggest that LPS-mediated Ca2+ release from ER stores underlies nuclear factor-kappa B activation and downstream inflammatory signaling in lung microvessels. Thus, we show for the first time a role for inositol 1,4,5 trisphosphate-mediated ER Ca2+ release in the induction of LPS responses in pulmonary microvascular endothelium. Mechanisms that blunt this signaling may mitigate endotoxin-induced morbidity.The pulmonary microvasculature plays a critical role in endotoxin-induced acute lung injury. However, the relevant signaling remain unclear. Specifically the role of endothelial Ca Sepsis is characterized by rapid retention of leukocytes in lung microvessels. Vascular administration of endotoxin , a model of sepsis, reveals a major role for the endothelium in this process. Using real-time fluorescence imaging, we recently reported that targeted infusion of LPS into lung capillaries and venules increased endothelial intercellular adhesion molecule-1 (ICAM-1) expression and augmented retention of LPS-untreated naïve leukocytes 2+, in tandem with calmodulin, can activate NF-κB and downstream gene transcription in multiple cell types 2+ underlies LPS-induced inflammatory responses 2+ plays a role in the induction of LPS-mediated responses.In endothelial cells, LPS acting via toll-like receptor 4 (TLR4) initiates transcriptional upregulation of proinflammatory and adhesion molecules 2+ plays a role in the induction of proinflammatory responses elicited by both receptor-mediated and -independent agonists 2+ from endoplasmic reticulum (ER) Ca2+ stores, via the second messenger inositol 1,4,5-trisphosphate (IP3) 2+ via plasma membrane Ca2+ channels 2+ from ER stores is associated with modulations in the amplitude and frequency of cytosolic Ca2+ oscillations 2+ response and its role in initiating downstream signaling remain undefined.In lung microvessels, cytosolic Ca2+ responses to infusions of LPS into lung microvessels using the isolated blood-perfused rat lung model. We then established the role of Ca2+ in the induction of downstream responses. Our data revealed that LPS initiates release of Ca2+ from ER stores, which then mediates the increase in endothelial ICAM-1 expression and microvascular leukocyte retention.Toward this, we determined endothelial cytosolic Caad libitum, and placed on a 12 h light-dark cycle. Surgery was only done on animals that were anesthetized and confirmed to be under a surgical plane of anesthesia.Animal use and studies were approved by the Institutional Animal Care and Use Committee of the University of Tennessee Health Science Center. Animals were housed and maintained by our Lab Animal Care Unit accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. Animals were given access to feed and water en bloc and placed on a microscope stage. The lungs were constantly inflated at an airway pressure of 5 cmH2O and continuously pump-perfused at 14 ml/min with autologous blood warmed to 37°C. The pulmonary artery and left atrial pressures were maintained at 10 and 3 cmH2O, respectively. The lung surface was kept moist with saline throughout the experiment.Adult male Sprague-Dawley rats weighing 250–350 g were anesthetized by intraperitoneal injection of Ketamine/Xylazine and exanguinated by cardiac puncture. The chest wall was opened and the pulmonary artery and left atrium were cannulated. Subsequently, the lungs and heart were excised Primary rat lung microvascular endothelial cells (RLMVEC) were purchased from VEC Tech  and cultured in medium 199 supplemented with growth factors. RLMVEC were used in passage numbers 3–5.sn-glycerol (OAG) were from Enzo Life Sciences . Alexa-Fluor 488-conjugated goat anti-mouse secondary Ab and Alexa-Fluor 488-conjugated goat anti-rabbit secondary Ab were from Invitrogen/Life Technologies . Mouse monoclonal anti-ICAM-1 antibody and anti-NF-κB p65 TRITC-conjugated Ab were from Santa Cruz Biotechnology . Anti-phosphoSer536 NF-κB p65 antibody was from Cell Signaling . The Ca2+ indicator Fura2-AM was from Invitrogen. All other chemicals were from Sigma.LPS from E. coli (serotype 0111:B4), nuclear stain Hoechst-33342, Ficoll gradient Histopaque-10771, and rhodamine 6G (R6G) were from Sigma Aldrich . Inhibitors Xestospongin C (XeC) and 2,5-Di-(t-butyl)-1,4-hydroquinone (t-BHQ) were from Calbiochem/EMD Millipore . KappaB-kinase NEMO-binding domain (IKK-NBD) peptide and 1-oleoyl;-2-acetyl-+, 5 mM K+, 1 mM Ca2+, 1 mM Mg2+, 10 mM glucose, 20 mM HEPES) with 4% dextran (40 kDa) and 1% fetal bovine serum. Ca2+-free HBS was prepared as similar to HBS, but without adding Ca2+. Prior to infusion into microvessels, 4% dextran and 0.05 mM EGTA were added to Ca2+-free HBS. Both HBS and Ca2+-free HBS were adjusted to a final pH of 7.4.Agents and leukocytes were infused into microvessels in a calcium rich HEPES-buffered Ringer’s solution  microcatheter was introduced through the left atrial cannula and blood cell-free conditions were established in microvessels by flushing with HBS in a small portion of the lung. We infused either HBS or LPS (100 µg/ml) for 30 min into microvessels, and then washed off luminal LPS with infusion of HBS for 60 min.Subsequently, R6G-labeled leukocytes were infused by microcathether into the LPS-treated microvessels and their transit through the vessel was imaged in real-time using a fluorescence microscope  fitted with the appropriate interference filters (excitation 545 nm and emission >560 nm). At the end of leukocyte infusion, we flushed the vessels with HBS. We then captured images of several microvessels within the LPS-treated region.Post-experiment, we analyzed the fluorescence images with Metamorph  and quantified the number of leukocytes retained in microvessels separately for all treatment groups.in situ immunofluorescence as previously reported Expression of ICAM-1, phosphorylation of NF-κB p65 subunit, and nuclear translocation of NF-κB p65 subunit were determined by 2+ release, we pretreated microvessels for 10 min before LPS infusion with XeC (25 µM) or t-BHQ (10 µM) with Ca2+ free HBS. To inhibit NF-κB, we infused IKK-NBD peptide (100 µM) that selectively inhibits NF-κB activation, for 30 min before and during LPS infusion.To determine the role of IP3-mediated ER Ca2+, we loaded microvessels with the cell-permeable ratiometric Ca2+ indicator Fura2-AM (10 µM) for 30 min. We then obtained images of the microvessels with our fluorescence microscope by exciting sequentially at 340 and 380 nm, and collecting the fluorescence emission at 515 nm. Images were obtained at 10 s intervals. The fluorescence emissions from both 340 and 380 excitations were quantified at a single region along a vessel wall. Changes in Ca2+ were indicated as changes in the ratio of the fluorescence emissions at 340 and 380 nm (F340/F380).To determine endothelial cytosolic Ca2+ oscillation. The results were confirmed by a blinded analysis.F340/F380 oscillation amplitude in lung microvessels were determined as reported previously To quantify baseline F340/F380 in a vessel, the value at a single region on the vessel wall was determined for 15 consecutive images, and then the mean F340/F380 quantified.RLMVECs were loaded with Fura2-AM for 40 min and washed with cell culture media. The cells were then imaged using parameters described above for microvessels. Images of cells were obtained from several distinct regions within a culture dish. After obtaining a series of images at baseline, either LPS (1 µg/ml) or OAG (100 µM) was added to the media and the imaging continued. To determine the role of IP3 receptor inhibition, RLMVECs were pretreated with XeC (10 µM) for 20 min before agonist treatment.To quantify F340/F380 in RLMVEC, an outline was drawn around cells in the image field and the F340/F380 calculated. For each RLMVEC, F340/F380 was calculated from three consecutive images and the mean taken as the F340/F380 for that cell. For each culture dish, a minimum of three cells per region and greater than three regions per dish were analyzed.All data are reported as mean±SEM. All multiple groups were compared with Kruskal-Wallis One Way ANOVA on Ranks followed by pair-wise multiple comparisons by Dunn’s method.2+ indicator Fura2-AM  6 on the plasma membrane 2+ . The images also indicated that pretreating microvessels with XeC blunted the LPS-induced p65 phosphorylation (2+ release mediated the phosphorylation of NF-κB p65 subunit in lung microvessels.To establish whether LPS activates NF-κB in an ER Cain situ immunofluorescence imaging. As evident from the confocal images, overlap between the NF-κB immunofluorescence and nuclear fluorescence was greater in LPS-treated microvessels compared to HBS-treated vessels (2+ release (Since the p65 subunit of NF-κB translocates to the nucleus upon activation 2+ from ER stores and attendant increase in the amplitude of cytosolic Ca2+ oscillations. In addition, our data also reveal that store Ca2+ release mediated the nuclear translocation of NF-κB. Thus, the current findings clearly attribute a major role for Ca2+ release from endothelial ER stores in the induction of LPS-induced inflammatory responses in lung microvessels.We show here for the first time that infusion of LPS into lung microvessels, induced release of Ca2+ release from ER stores mediated these responses. Further, the cell-permeable IKK-NBD peptide also blocked the translocation. IKK-NBD blocks association of the NF-κB essential modifier with the IKK complex and thus, NF-κB activation 2+ caused activation of NF-κB, leading to its nuclear translocation. While LPS-mediated NF-κB activation is well established 2+ in this process is less defined. In addition, while store Ca2+ release has been indicated to mediate LPS responses in inflammatory cells 2+ from ER stores may play a role in LPS-induced NF-κB activation. Calmodulin, a calcium sensor that is activated upon binding Ca2+, may be an intermediary in the Ca2+-dependent NF-κB activation 2+ are reported to elicit NF-κB-dependent responses LPS infusion into microvessels induced both the phosphorylation and nuclear translocation of the p65 subunit of NF-κB. However, pretreating microvessels with XeC limited both the phosphorylation and translocation, suggesting that Ca2+ via TRPC-permeable channels as the primary mechanism underlying cytosolic Ca2+ increase and downstream signaling 2+ oscillations were mediated by store Ca2+ release, as evinced by the inhibition of the LPS-induced response by the ER IP3 receptor blocker, XeC. This interpretation is supported by the finding that in RLMVEC, XeC did not inhibit cytosolic Ca2+ increases by OAG. As OAG induces entry of external Ca2+ via TRPC6 channels 2+ channels. In addition, using fluorescent probes that report store Ca2+ levels it has been shown previously that XeC inhibits store Ca2+ release, but not entry of external Ca2+ in lung microvessels 2+ increase by LPS is via IP3-dependent release of ER store Ca2+.Studies on LPS-induced responses, implicate influx of external Ca2+2+ release from ER stores. This possibility is supported by the observation that inhibiting ER Ca2+-ATPase pumps also blocked the LPS-induced ICAM-1 release. The difference in Ca2+ responses observed in this study from that recently reported in lung endothelial cells 2+ increase in primary RLMVEC by blocking ER store Ca2+ release. Thus in pulmonary microvessels, we interpret that LPS signals via release of Ca2+ from ER stores.Previous studies in lung microvessels suggest that receptor-mediated agonists initiate IP3-dependent release of store Ca2+ oscillations. Modulations in frequency and amplitude of cytosolic Ca2+ oscillations underlie several Ca2+-dependent signaling pathways in various cell types 2+ oscillation augmented reactive oxygen species production and P-selectin expression 2+ oscillations activate transcriptional events, possibly via the action of calcium/calmodulin kinases 2+ oscillations induced by LPS lead to NF-κB translocation to the nucleus and an increase in ICAM-1 expression.The effect of LPS on lung microvessels was to increase the amplitude of cytosolic CaIn the present study, ICAM-1 expression was elevated at 90 minutes post-LPS infusion. However, we reported that ICAM-1 expression was detectable in both venules and capillaries as early as 30 minutes after initiating LPS infusion 2+2+ signaling underlie differences in ICAM-1 expression in venules and capillaries. While the increase in the amplitude of Ca2+ oscillation was similar in both microvascular segments, the baseline Ca2+ was lower for capillaries compared to venules. Since, heterogeneity in baseline Ca2+ can lead to differences in the induction of Ca2+-dependent signaling 2+ at baseline contributed to their dissimilar ICAM-1 expression. Whether small differences in Ca2+ can significantly modulate endothelial responses and the specifics of the signaling mechanisms require further consideration.We reported that LPS-induced ICAM-1 expression in venules was higher than that in capillaries 2+ release and downstream responses in lung microvessels. Inhibiting IP3 receptors with XeC blocked induction of ICAM-1 and leukocyte retention, suggesting that blunting ER Ca2+ release may limit inflammatory responses in lung microvessels. In addition, reports indicate that inhibiting IP3-dependent store Ca2+ release also blunts LPS-induced cytokine production in macrophages 2+ from ER stores by blocking IP3 receptors may be effective in blunting LPS-induced inflammation and injury, and thus, facilitate survival.Our findings show that LPS induces IP3-dependent ER Ca2+ release leading to increase in cytosolic Ca2+ oscillations, activation followed by nuclear translocation of NF-κB, an increase in ICAM-1 expression and attendant leukocyte retention. These findings may lead to development of novel intervention strategies for endotoxin-induced lung injury.Thus, our data obtained using real-time fluorescence imaging reveal a new signaling pathway for LPS-induced responses in pulmonary microvessels that involves IP3-mediated Ca