However, its role in the context of mitochondrial Ca2+ homeostasis is not yet clear. Several studies that were based on PPIF inhibition or knock out suggested that mPTP is involved in the Ca2+ efflux mechanism, while other observations have revealed the opposite result.The mitochondrial permeability transition pore (mPTP) has long been known to have a role in mitochondrial calcium (Ca1/FO ATP synthase has been recently found to be a fundamental component of the mPTP. In this work, we focused on the contribution of the mPTP in the Ca2+ efflux mechanism by modulating the expression of the c subunit. We observed that forcing mPTP opening or closing did not impair mitochondrial Ca2+ efflux. Therefore, our results strongly suggest that the mPTP does not participate in mitochondrial Ca2+ homeostasis in a physiological context in HeLa cells.The c subunit of the mitochondrial F Mitochondria are intracellular organelles involved in several cellular functions such as ATP production, fatty acid oxidation, Ca2+2+ uptake and release mechanisms are based on the utilization of gated channels for Ca2+ uptake and exchangers for release that are dependent upon the negative mitochondrial membrane potential, which represents the driving force for Ca2+ accumulation in the mitochondrial matrix 2+ uniporter (MCU), which is encoded by the recently discovered gene ccdc109a 2+ influx, while the mitochondrial Na+/Ca2+ antiporter (NCLX) 2+ efflux. In spite of this information, the mitochondrial Ca2+ efflux mechanism has not been completely elucidated. It is mostly accepted that Na+/Ca2+ exchange activity is relevant for excitable cells 2+ efflux 2+ efflux, one based on the H+/Ca2+ antiporter A key signaling messenger that is able to transduce life or death signals to mitochondria is intracellular CaThe mPTP is a high-conductance channel that is located at the contact sites between the inner and outer mitochondrial membranes. This channel is responsible for the non-selective permeability state of the mitochondrial inner membrane. The transition to this state for small molecules is referred to as the mitochondrial permeability transition (MPT). The molecular composition of the mPTP is not yet clear, but several proteins have been shown to be components that participate in mPTP activity, including voltage-dependent anion channels (VDAC) 2+ ions, prooxidant and proapoptotic proteins, a decrease in the mitochondrial membrane potential, pH variations and adenine nucleotides all sensitize the opening of the pore Ca2+ or oxidative signal and different type of cell death 2+ efflux mechanism 2+ homeostasis under non-pathological conditions. Recently, we suggested that, similar to PPIF, the c subunit of the FO ATP synthase constitutes a critical component of the mPTP and that it is required for the MPT, mitochondrial fragmentation and cell death induced by oxidative stress or mitochondrial Ca2+ overload MPT resulting from mPTP opening is usually considered a transducer event in between Ca2+ release; therefore, it does not participate in mitochondrial Ca2+ homeostasis in a non-pathological context, at least in HeLa cells.By modulating c subunit expression and using pharmacological approaches, we revealed the important finding that the mPTP is not necessary for mitochondrial Ca22.12+ homeostasis, we have investigated the kinetics of the mitochondrial and cytosolic Ca2+ influx and efflux. We have recently suggested that the c subunit of the F1/FO ATP synthase is a component of the mPTP that is required for the MPT-driven mitochondrial fragmentation that is induced by cytosolic Ca2+ overload and oxidative stress 2+ assay during c subunit silencing  or a mix of siRNAs that targeted ATP5G1, ATP5G2 and ATP5G3 (siATP5G) for 48 h  and in the Ca2+ uptake rate, but no significant variations in the rate of Ca2+ release from the mitochondria were observed  (2+ influx and efflux (By taking advantage of aequorin technology for 48 h C. Silencobserved D. Furthe > 0.05) E or in td efflux F were de2+ dynamics were monitored in the presence of a well-established blocker of the mitochondrial Ca2+ efflux system, CGP37157, which is an inhibitor of the mitochondrial Na+/Ca2+ exchanger To confirm the experimental method, mitochondrial Ca2+ uptake was induced with histamine (100 μM) in the continuous presence of the inhibitor.HeLa cells were exposed to CGP37157 (10 μM) for 2 min, and Ca2+ uptake by the mitochondria   and a significant decrease of 31% in the rate of Ca2+ release from the mitochondria due to inhibition of the mitochondrial Na+/Ca2+ exchanger . The absence of significant differences between the cytosolic Ca2+ levels   and Ca2+ influx and efflux kinetics  confirmed the specificity of CGP37157.CGP37157 caused an expected increase in Ca2+ homeostasis.In our experimental settings, HeLa cells appeared to display higher RNA levels of ATP5G1 compared to its homologs. We thus decided to overexpress a Myc-tagged ATP5G1, which was under the control of the cytomegalovirus immediate early promoter, to verify its effect on mitochondrial Ca2+ assay , without significantly affect the kinetics of Ca2+ influx and efflux   E and F.2.22+ efflux, we used a pharmacological approach; specifically, Cyclosporine A (CsA) and hydrogen peroxide (H2O2) were used as an inhibitor of the mPTP To confirm the absence of an effect of mPTP modulation on Ca2+]m. As previously reported 2+ uptake   was added to HeLa cells 30 min before stimulation with histamine (100 μM) and measurements of m, but does not affect efflux rates in living cells.Overall, the data show that the mPTP pharmacological opening inversely correlate with the amplitude and influx rate of [Ca2.32+ efflux only during saturation of the Ca2+ export system +/Ca2+ exchanger.Multiple authors have proposed that the mPTP exerts a significant effect on mitochondrial Ca2+]m during agonist stimulation with a concomitant reduction of the efflux rate. If mPTP opening extrudes Ca2+ only during the saturation of other efflux systems, then c subunit depletion should result in a strengthened inhibition of the efflux rate. However, silencing of ATP5G in HeLa cells exposed to CGP37157 did not display any significant differences in peak amplitude (p > 0.05), uptake, or release speed , exposure to CGP37157 induced a significant increase in the  of 1 μM. The cells were finally perfused with IB that was supplemented with Ruthenium Red (RuR), which is an inhibitor of the mitochondrial Ca2+ uniporter, and deprived of free Ca2+ to induce a maximal rate of Ca2+ release from the mitochondria. The same experiment was performed during modulation of the expression of the c subunit or in the presence of CsA (1 μM), which was added 30 min before the beginning of the measurements. The exposure of permeabilized cells transfected with scrambled siRNA to external [Ca2+] (1 μM) induced a rapid increase in the [Ca2+]m that was stabilized at a value of 3.09 ± 0.24, which was analogous to the [Ca2+]m that was reached in cells transfected with siATP5G  or during exposure to CsA (p > 0.05). This finding confirmed that the mPTP does not affect the maximal mitochondrial Ca2+ efflux rate at the standard [Ca2+]m.The efficiency of the mitochondrial Caor ATP5G B. Neglig2+/H+ antiporter. Therefore, the Ca2+ efflux rates were monitored in the presence of the mitochondrial Na+/Ca2+ exchanger CGP37157 and were elicited by 2 μM RuR and in the presence of external Ca2+. CGP37157 dramatically increased mitochondrial Ca2+ uptake, causing fast aequorin consumption when external Ca2+ 1 μM was added ([Ca2+]m > 14 μM data not show). In this second group of permeabilized cells, Ca2+ uptake was elicited by adding external Ca2+ (0.5 μM). Administration of RuR induced slow (but not absent) Ca2+ release from the mitochondrial matrix in scrambled, siRNA-transfected HeLa cells  and efflux (through the Na+/Ca2+ or H+/Ca2+ exchanger) systems Correct Ca2+ efflux system due to the non-specific channel properties that were observed during its opening 2+ homeostasis that could be associated with alterations in the mitochondrial Ca2+ efflux . On the other hand, several other reports have indicated the opposite 2+ homeostasis.Some authors have proposed that the mPTP is a component of the Ca1/FO ATP synthase is a component of the mPTP, we have focused our attention on the involvement of the mPTP in mitochondrial Ca2+ homeostasis under non-pathological conditions. The novelty of our study lies in the regulation of mPTP activity by manipulating the c subunit of the F1/FO ATP synthase and measuring the [Ca2+]m and [Ca2+]c using the pH-independent, luminescent, Ca2+-sensitive probe, aequorin Driven by our recent work suggesting that the c subunit of the F2+]m revealed a minimum reduction in mitochondrial Ca2+ loading after c subunit silencing, but no variations in the rate of Ca2+ release from the mitochondria were observed. Furthermore, no changes in the cytosolic Ca2+ levels were observed. As previously shown in this condition, we forced the closing of the mPTP, and the cells were protected from damage.Measurements of the m and uptake rate that were observed in this model. To validate these results, we took advantage of the well-known PPIF inhibitor CsA that was reported to be capable of increasing the mitochondrial membrane potential (MMP) and [Ca2+]m2+ uptake and the peak amplitude occurred, but, again, no differences in the rate of mitochondrial Ca2+ efflux were observed (which was in accordance with the observation after c subunit silencing).We previously reported that, in this cell line, c subunit silencing generates a moderate reduction of the MMP, which is the opposite of the significant depolarization that occurs upon c subunit overexpression. Variations in the MMP, which is the driving force for Ca2+ efflux. Most of these reports were based on PPIF inhibition or knock out. It could then be speculated that PPIF could somehow affect other participants of the Ca2+ efflux system, as proposed by Wei and co-workers However, we cannot completely ignore the several respectable reports proposing that there is an effect of the mPTP on mitochondrial Ca2+ efflux. To test this hypothesis, we induced mPTP opening through the use of the prototypic, pro-oxidant hydrogen peroxide. Exposure to this stress agent results in mPTP opening with a consequent reduction in the maximal [Ca2+]m after agonist exposure and an increase in calcein quenching by cobalt. In this condition, a decrease in the mitochondrial Ca2+ influx and efflux were observed, which was in agreement with the reduced driving force, while an opened, non-specific Ca2+ channel (such as the mPTP) should produce a significant increase in the Ca2+ efflux rate.It could be argued that c subunit overexpression did not lead to an mPTP opening event sufficient to generate a detectable Ca2+]m was partially recovered in cells silenced for ATP5G or pretreated with CsA, protection from the mPTP did not modify the kinetics of Ca2+ efflux. In contrast, overexpression of ATP5G1 promoted H2O2-induced mPTP opening, causing a further reduction in the maximal [Ca2+]m under agonist stimulation. In spite of this reduction, the efflux rate did not display a significant stimulation, but rather a small inhibition. These observations provide strong confirmation that, even following potent induction of its opening, the mPTP does not participate in Ca2+ efflux. We previously confirmed that induction of the mPTP by H2O2 caused slow MMP dissipation (probably due to the induction of the low conductance mode of mPTP) 2+]m recovering phase seems to correlate with the levels of MMP previously observed in such conditions 2+]m by the mPTP should indirectly occur due to regulation of the MMP.Nonetheless, although the m levels are not sufficient to induce a calcium efflux through the mPTP. Furthermore, an effect of on the [Ca2+]m was still observable as an increase in the [Ca2+]m and influx rate when blocking mNCX (characters dependent on the MMP); nonetheless, the efflux rate was still not affected.It was proposed that the effects of the mPTP on the kinetics of Ca2+ overload approach was proposed. Recently, several components of the mitochondrial Ca2+ uptake machinery have been revealed 2+ influx rate and [Ca2+]m. Such an increase was not further stimulated by inhibition of the mPTP opening through treatment with CsA, which suggested that the mitochondrial network was near its maximal uptake capacity. Interestingly, MCUa overexpression also resulted in a reproducible tendency to increase the Ca2+ efflux rate. Treatment with CsA did not generate any significant effects on the calcium efflux rates of MCUa-overexpressing cells. Overall, these results clearly indicate that overload of the [Ca2+]m in our cell model is not sufficient to induce a mPTP-dependent Ca2+ efflux.An alternative Ca2+ efflux system is dependent upon [Ca2+]m, which is exposed; and modulation of the expression of the c subunit as well as exposure to CsA results in alterations of the [Ca2+]m.The Ca2+]m, Ca2+ efflux measurements were performed in permeabilized cells that had been stimulated with external Ca2+ (1 μM) to obtain a stable level of [Ca2+]m in the control mitochondria. After that plateau was reached, the external Ca2+ was removed, and the MCU inhibitor Ruthenium Red was added to avoid Ca2+ reuptake. The rate of Ca2+ release from the mitochondria under these conditions was considered the maximum efflux speed that could be reached, and no significant differences in the rate after mPTP inhibition with CsA or during ATP5G silencing or its overexpression were observed. It is reasonable to assume that because mitochondria are energized by the addition of ADP, pyruvate, and succinate in permeabilized cells, the efflux rate is driven mostly by the mNCX and the H+/Ca2+ exchangers.To avoid misinterpretation of the data due to different values for the  values using a computer algorithm based on the Ca2+ response curve of wild-type and mutant aequorins.All aequorin measurements were performed in KRB supplemented with 1 mM CaCl2HPO4, 5 mM succinic acid, 5 mM malic acid, 1 mM MgCl2, 20 mM HEPES, 1 mM pyruvate, 0.5 mM ATP (pH 7 at 37 °C). Intracellular buffer was supplemented with 2 mM EGTA and 2 mM HEDTA-buffered [Ca2+] 1 μM or 0.5 μM (intracellular buffer/Ca2+), calculated with the Chelator software 2+ uptake rate was calculated as the first derivative by using Microsoft Excel (Microsoft Co.); all the slopes were calculated at the same [Ca2+]m to ensure comparable activity of efflux system.In the experiments with permeabilized cells, a buffer mimicking the cytosolic ionic composition, (intracellular buffer) was used: 130 mM KCl, 10 mM NaCl, 2 mM K5.22+ phosphate procedure as previously described, a plasmid coding for mtDsRed was added to the transfection for all procedures. After 36 h of expression HeLa were loaded with 1 mM Calcein acetoxymethyl ester and Co2+ as instructed by the Image-IT® LIVE Mitochondrial Transition Pore Assay Kit (Molecular Probes-Life Technologies). Cells were then imaged by means of 490 ± 20 nm excitation and 525 nm long pass emission filters on a Axiovert 200 M fluorescence microscope equipped with a 40× water immersion objective . Finally, images were analyzed with a custom Cell Profiler pipeline (http://www.cellprofiler.org/), briefly, after a background normalization Calcein average intensity was calculated over the mtDsRed mask.At day one HeLa Cells were seeded on 25 mm glass coverslip. After 24 h were transfected with Hyperfect or standard Ca5.3t-tests. p values < 0.05 were considered statistically significant.Data were expressed as the mean ± SEM and were analyzed with Microsoft Excel (Microsoft Co.). Statistical significance was determined by means of ANOVA followed by two-tailed unpaired Student's


Cymbopogon citratus (a plant with antihypertensive properties).β-Citronellol can act against pathogenic microorganisms that affect airways and, invirtue of the popular use of β-citronellol-enriched essential oils in aromatherapy,we assessed its pharmacologic effects on the contractility of rat trachea.Contractions of isolated tracheal rings were recorded isometrically through a forcetransducer connected to a data-acquisition device. β-Citronellol relaxed sustainedcontractions induced by acetylcholine or high extracellular potassium, buthalf-maximal inhibitory concentrations (IC50) for K+-elicitedstimuli were smaller than those for cholinergic contractions. It also inhibitedcontractions induced by electrical field stimulation or sodium orthovanadate withpharmacologic potency equivalent to that seen against acetylcholine-inducedcontractions. When contractions were evoked by selective recruitment ofCa2+ from the extracellular medium, β-citronellol preferentiallyinhibited contractions that involved voltage-operated (but not receptor-operated)pathways. β-Citronellol (but not verapamil) inhibited contractions induced byrestoration of external Ca2+ levels after depleting internalCa2+ stores with the concomitant presence of thapsigargin and recurrentchallenge with acetylcholine. Treatment of tracheal rings with L-NAME, indomethacinor tetraethylammonium did not change the relaxing effects of β-citronellol.Inhibition of transient receptor potential vanilloid subtype 1 (TRPV1) or transientreceptor potential ankyrin 1 (TRPA1) receptors with selective antagonists caused nochange in the effects of β-citronellol. In conclusion, β-citronellol exertedinhibitory effects on rat tracheal rings, with predominant effects on contractionsthat recruit Ca2+ inflow towards the cytosol by voltage-gated pathways,whereas it appears less active against contractions elicited by receptor-operatedCa2+ channels.β-Citronellol is an alcoholic monoterpene found in essential oils such The acyclic monoterpenoid β-citronellol has odor qualities that make it useful in the perfume industry  Stapf. (Poaceae) and Lippiaalba (Mill.) N.E. Brown. (Verbenaceae), aromatic plants that haveantihypertensive properties (2+) influx from the extracellular medium as well as inhibition ofrelease of intracellular Ca2+ from Ca2+ stores appear to mediateits inhibitory effects on vascular smooth muscle  improved upper respiratorysymptoms in volunteers diagnosed with pharyngotonsillitis, viral laryngitis, or viraltracheitis  areuseful for the treatment of respiratory diseases, but knowledge regarding the mode ofaction is restricted to folk medicine. However, a prospective randomized double-blindcontrolled trial revealed that a spray application containing the essential oil ofacheitis . Mulyaniacheitis  showed trculosis . This firculosis .Inhalatmplaints , but eviviarecruitment of Ca2+ channels on smooth muscle cells (SMCs).The present study was designed to determine the pharmacologic profile of β-citronellolon the contractility of isolated tracheal rings from rats. The emphasis was on theability of β-citronellol to inhibit the contractile events mediated Male Wistar rats (200-250 g) were obtained from populations maintained at thevivarium of the Departamento de Fisiologia e Farmacologia, Universidade Federal doCeará . Rats were maintained under conditions of constanttemperature (22±2°C) with a 12-h light-dark cycle and free access to food and water.All animals were cared for in compliance with regulations set by the BrazilianNational Council for Control of Experimentation with Animals. All proceduresdescribed herein were approved by the Animal Ethics Committee of the UniversidadeFederal do Ceará (protocol CEPA #28/12).ip). Tracheal rings were obtained by cutting (in atransverse direction) isolated trachea after careful dissection in a dish containingphysiologic salt solution to remove adjacent tissues. From each trachea, three tofour rings were prepared for maintenance in a 5-mL organ bath filled with physiologicsalt solution at 37°C under continuous bubbling with 5% CO2 inO2 and pH 7.4. Each tracheal ring was suspended by two parallelstainless-steel rods passed through its lumen, as described previously  concentration (60 mM) or acetylcholine). In the steady state of a given sustained contraction,concentration-effect curves were obtained by exposing preparations to increasingconcentrations of β-citronellol, which was added cumulatively to the organ bath (12min for each concentration). Control preparations received only the vehicle at anidentical experimental time. In preparations contracted with KCl or ACh,concentration-effect curves to β-citronellol were constructed in the absence orpresence of antagonists, as indicated below. In another set of experiments, a singleconcentration of β-citronellol was chosen and contractions were evoked to recruit adesired smooth muscle contractile pathway under certain circumstances. Othercontractile agents were used and more experimental details for each protocol areprovided in the Results and Discussion section.Tracheal rings were challenged to contract in response to contractile stimuli . Solutions with high KClcontent involved addition of appropriate amounts of a 3-M KCl solution (in distilledwater) directly to the organ bath to achieve the desired concentration. For someexperiments, barium ions (Ba2+) substituted for Ca2+ in thephysiologic salt solution.The physiologic salt solution used was Krebs-Henseleit, which had the followingcomposition: 118.0 mmol/L NaCl, 4.7 mmol/L KCl, 2.5 mmol/L CaClG-nitroarginine methylester , tetraethylammonium , sodiumorthovanadate (ID 24899708), capsazepine (ID 24277967), indomethacin , A-967079 (CAS Number 1170613-55-4), HC-030031 (CAS Number 349085-38-7),thapsigargin (ID 24278762) and verapamil (ID 24277881) were purchased fromSigma-Aldrich (USA).(±)-β-Citronellol , ACh (PubChem ID 24891113), atropine (ID24890401), 5-hydroxytryptamine (ID 24278124), L-Nv/v).Salts  were purchased from Sigma-Aldrich or Merck(Germany).In general, stock solutions were prepared in distilled water and stored at -20°C.β-Citronellol was dissolved directly in physiologic solution containing 2% Tween 80and sonicated immediately before addition in the bath chamber. The maximumconcentration of the vehicle in the organ bath was 0.01% (50) and effective concentration (EC50) values were calculated byinterpolation from semi-logarithmic plots, reported as geometric means , and compared using the Mann-Whitney U-test. Contractileresponses of tracheal tissues were quantified and normalized as a percentage of thefinal contractile response to K+ (60 mM) obtained after the equilibrationperiod or as a percentage of the ACh-induced sustained contraction as indicated.Significance of results was determined using ANOVA and, if significant, followed by amultiple comparison test. P<0.05 was considered significant.Data are reported as means±SE. Half-maximal inhibitory concentration µM; n=9] significantly lower than those needed to reverse contractionselicited by ACh   µM (n=6). Thisvalue was higher than the IC50 needed to inhibit K+-inducedcontractions , but not significantly different in comparisonwith the IC50 estimated for ACh-elicited contractions. Such results under EFSare consistent with the cholinergic nature of intramural neurons involved with theexcitatory parasympathetic input towards tracheal smooth muscle  but part of its action is secondary to the opening of chloride ion(Cl-) channels that can mediate membrane depolarization with furtheropening of Ca2+ channels gated by voltage (2+ channels) (One explanation for the higher pharmacologic potency of β-citronellol againstcontractions evoked by Koupling" . It has  voltage . Neverthhannels) ,22, whic50 of 243.0(190.2-310.5) µM (n=6), which did not differ significantly from the IC50required to relax ACh-induced contractions . Sodiumorthovanadate is a well-known tyrosine (Tyr) phosphatase inhibitor that indirectlyshifts the kinase-phosphatase balance towards phosphorylation of Tyr kinases. Suchfindings are in accordance with the lower potency of β-citronellol against contractionsevoked by metabotropic cascades if we consider that Tyr kinases have been reported to bedownstream pathways in the contractile responses evoked by cholinergic agonists actingthrough G protein-coupled muscarinic receptors  with ICeceptors .2+ channels in SMCs). First, a concentration-effect curve wasconstructed by adding verapamil to the steady-state contraction induced by 60 mMK+. +, whereas50 of ACh from 1.9(1.4-2.7) in control (n=8) to 17.8 (4.9-64.7) µM in the presence of verapamil , but did not interfere significantly with the maximal effectachieved in the concentration-effect of ACh  µM. Just like verapamil, β-citronellol could not reduce themaximal contractile effect reached upon use of higher concentrations of ACh. The profileof the inhibitory action of β-citronellol against cholinergic contractions clearlydiffered from experiments in which increasing concentrations of K+ (10-120mM) were used as contractile stimuli . Under Ca2+-free conditions, addition of 60 mMK+ did not produce sustained contraction, and the contractile tonus oftracheal preparations remained at levels recorded under resting conditions. Still in thepresence of high K+, cumulative addition of Ca2+  (2+ (0.1-20 mM) substituted for Ca2+ in suchprocedures   andwas hannels) . When Baocedures , similarocedures . It has  in SMCs -26.2+-free conditions (medium containing EGTA and 10 µM verapamil). Thispharmacologic approach aimed to diminish the influence of Ca2+ influx throughvoltage-operated channels when Ca2+ (0.1-20 mM) was added cumulatively to theextracellular medium. In contrast, the development of contractile force was seenprobably because SMCs can also enable Ca2+ influx through metabotropicpathways such as the receptor-operated channels activated by phospholipase C-linked Gproteins in response to ACh occupancy in muscarinic receptors (2+ addition reached a magnitude comparableto that seen with β-citronellol-untreated control preparations (eceptors . Under sarations . Such fi2+ through store-operated Ca2+ channels("capacitative Ca2+ entry") (2+-free medium after treatment with thapsigargin(1 µM), a non-competitive inhibitor of sarcoplasmic/endoplasmic reticulumCa2+ ATPase (2+-free conditions can be appliedvia activation of M3/M2 receptors by ACh (60µM) and its downstream signal-transduction molecule inositol 1,4,5-trisphosphate, whichelicits Ca2+ mobilization from the sarcoplasmic reticulum to the cytosol promotes activation of capacitative Ca2+inflow (though the exact sequence of cell events in this pathway is not known).Irrespective of the amount of Ca2+ restored in the extracellular medium, SMCscan produce force even after removal of the cholinergic agent from the extracellularsolution, a condition that resembles constitutive activity in pharmacologic receptors(Transmembrane influx of Ca entry") ,29 can b+ ATPase . Recurre cytosol. Ca2+ reeceptors.2+ stores could be confirmed by observation of unmeasurable responses,ACh was removed from the Ca2+-free extracellular milieu and2.5 mM Ca2+ was added. 2+. Interestingly, verapamil (1µM) did not change smooth-muscle contraction under these circumstances, a featurealready reported for D-600    . Thus, ohannels) .2+ channels in SMCs, a non-selective cation channel activatedby capsaicin . Indeed, in SMCs. However in SMCs. However5 µMACh . Similar50 values requiredto reverse K+- or ACh-induced contractions were not altered significantly , INDO (10 µM) or TEA (5 mM) but the ICWhitney) . The resWhitney) ,39. In a effects .2+ influx from the extracellularmilieu to produce myorelaxant actions. β-Citronellol can alsoinhibit contractions mediated by metabotropic pathways, but with lower pharmacologicpotency. It is unlikely that β-citronellol acts as a direct blocking agent on L-typeCa2+-channels on rat tracheal SMCs.In conclusion, the present study showed that β-citronellol has inhibitory properties onairway SMCs, a feature that should be considered in inhalatory therapies withβ-citronellol-rich essential oils. β-Citronellol has higher potency to inhibitvoltage-gated pathways. Our findings are in accordance with the notion thatβ-citronellol can antagonize transmembrane Ca


A detailed analysis of the temporal and spatial organization of Ca2+ spikes in IPS-CM clusters revealed that the disaggregation of EBs between 0.5 and 2 weeks produced IPS-CM characterized by spontaneous beating and high levels of regional Ca2+ synchronization. These phenomena were typically absent in IPS-CM obtained from older EBs (>2 weeks). The maintenance of all spontaneously active IPS-CM clusters under “on plate” culture conditions promoted the progressive reduction in regional Ca2+ synchronization and the loss of spontaneous Ca2+ spiking. Raising the extracellular [Ca2+] surrounding these quiescent IPS-CM clusters from ~0.4 to 1.8 mM unmasked discrete behaviors typified by either (a) long-lasting Ca2+ elevation that returned to baseline or (b) persistent, large-amplitude Ca2+ oscillations around an increased cytoplasmic [Ca2+]. The different responses of IPS-CM to elevated extracellular [Ca2+] could be traced back to their routes of derivation. The data point to the possibility of predictably influencing IPS-CM phenotype and response to external activation via defined interventions at early stages in their maturation.Induced pluripotent stem cell-derived cardiomyocytes (IPS-CM) are considered by many to be the cornerstone of future approaches to repair the diseased heart. However, current methods for producing IPS-CM typically yield highly variable populations with low batch-to-batch reproducibility. The underlying reasons for this are not fully understood. Here we report on a systematized approach to investigate the effect of maturation in embryoid bodies (EB) vs. “on plate” culture on spontaneous activity and regional Ca The ability to reprogramme adult human somatic cells into an embryonic-like pluripotent state  vs. “on plate” culture] with the extent of regional Ca2 culture surfaces that had been pre-coated with GelTrex (1:400 (v/v) in incomplete Dulbecco's Modified Eagle Medium (Life Technologies) for 45 min at 37°C). iPS were grown on a 3-day cycle and adherent cells were detached in small clumps to preserve the structural integrity of iPS populations , NaH2PO4 (2), pH7.4)] for 5–10 min at room temperature, rehydrated in PBS (30 min) and component cells were stained using propidium iodide  in PBS which was allowed to equilibrate through fixed EBs. PI labels all post-fixation cellular material  were acquired. Z-axis image stacks were reconstructed into 360° rotational views of EB structure using Imaris 6.0 (Bitplane). Three-dimensional reconstructions of EBs at 0, 1, 2, and 3 weeks are given in Supplementary Movies EBs were fixed using 4% (v/v) formaldehyde in phosphate buffered saline  at ~0.4 mM. Imaging of user-defined areas containing IPS-CM clusters was performed using CLSM and a 63X oil-immersion objective  1.4) . Fluorescence and the corresponding brightfield images in B-areas  were acquired at 512 × 512 pixel resolution at 14.7 frames per second as described  conditions for 330 s, a bolus of CaCl2 was added to raise extracellular [Ca2+] to ~1.8 mM and imaging was continued for 210 s  in PBS] and IPS-CM nuclei were counter-stained with DAPI . Subsequently, five random A-areas of each plate were imaged using a 10X objective .At half-week intervals throughout the 4-week protocol  were subdivided into square grids comprised of 49 identical regions of interest . For each component C-area, the signal intensity traces, corresponding to changes in Ca2+-dependent fluo4 fluorescence with time, were exported using LAS-AF Report function (Leica Microsystems) and transferred into Matlab . Data were de-trended and super-threshold Ca2+ signal events (“spikes”) were detected using a custom-made algorithm that calculates local maximum of the first derivative  in the signal where F = fluorescence signal intensity and t = time).Image series obtained from B-areas . The perfect temporal co-incidence of Ca2+ spikes in one C-area with those in another C-area generates a straight line with a correlation co-efficient  of 1. We considered an R-value of ≥ 0.85 as a stringent threshold for robust correlation. For each C-area, the number of the other 48 C-areas that exhibited temporally co-incident Ca2+ spikes that met this criterion of an R-value ≥ 0.85 was calculated. This process was iterated through all 49 C-areas in the 7 × 7 grid. The extent of regional Ca2+ synchronization was thus defined as the proportion of a B-area that exhibited co-incident (synchronized) Ca2+ spikes and was expressed as a physical area (μm2). Regional Ca2+ synchronization in B-areas was visualized by linear interpolation and then assigned a pseudo-color look-up table that spanned the range of values determined.To compute the extent of Ca2+ spikes in C-areas was also used to calculate the mean inter-spike interval (ISI) and the standard deviation of ISI . The numerical value of THI increase as the Ca2+ spikes become more temporally disordered . Cell densities in B-areas  were calculated from the same IPS-CM clusters that had been user-selected for Ca2+ imaging. Plating heterogeneity was defined as the standard deviation of regional cell density in A- and B-areas normalized to the corresponding value obtained immediately following the disaggregation of 0-week EBs, which was assigned 1.The density and plating heterogeneity of IPS-CM were calculated from data obtained from five random A-areas on each plate  as described  (Supplementary Movie n = 9 EBs), the physical dimensions of which did not change over the study. Although there was evidence of a progressive time-dependent change in cellular arrangement within EBs . In three-dimensional reconstructions of EBs we did not observe cavitation or the formation of acellular hypoxic cores (which would not be stained by PI) over the course of the study . Likewise, mitotic capacity in post-EB IPS-CM was extremely low (0.01%) and we recorded just one incidence of cell division during the entire study  to analyse IPS-CM density and distribution following EB disaggregation Figure , we dete2+ spike synchronization in IPS-CM clusters and focussed our analysis to the study of IPS-CM clusters in B-areas ext was raised from ~0.4 mM to 1.8 mM, unmasked strikingly different responses in these quiescent IPS-CM. In some IPS-CM the increased [Ca2+]ext triggered a large elevation in intracellular [Ca2+] which lasted for several minutes  gives insights into some of the culture-dependent determinants that modulate IPS-CM Ca2+ handling behavior.We investigated the extent of regional Cas Figure . Intercein vivo is modulated by interactions with neighboring cells and many approaches for directing cardiomyocyte maturation via the control of cell-to-cell communication have been developed  cardiomyocytes. Importantly, maintenance of these IPS-CM under “on plate” conditions was associated with the progressive elimination of regional Ca2+ synchronization  was used throughout this study and the spontaneous contractility observed in EBs across the 4-week protocol reveals that 0.4 mM [Ca2+]ext is fully compatible with the development and persistence of spontaneous Ca2+ oscillations. It is plausible however that the comparatively low extracellular Ca2+ environment used in Burridge's protocol  does not represent a “physiologically” high Ca2+ environment .IPS-CM clusters derived from EBs at 0.5–1.5 weeks exhibited the highest levels of regional Can Figure  and spony Figure . The latn Figure  and the s Figure . There af, funny current) and T-type Ca2+ channels  underscored by different modes of derivation  can result in very different response to external cues . Spontaneous and Ca2+-triggered behavior in IPS-CM clusters can be traced back to their route of derivation and therefore a major finding of this study is that adaptive changes in Ca2+-signal organization under different maturation conditions are determinants of IPS-CM behavior. This finding is entirely in agreement with reports that cell function and fate may be imprinted via early-stage genetic and epigenetic events  and IPS-CM tolerance to massive scale culturing required to generate the vast numbers of cells required for heterologous and autologous cellular transplantation in new heart repair approaches , Wellcome Trust (094219/Z/10/Z), and the Wales Deanery .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The initial Ca2+ influx mediated by plasma membrane ion channels has been suggested to be crucial for the adaptive signaling. NADPH oxidase (Nox)-mediated production of reactive oxygen species (ROS) has also been suggested to play crucial roles in regulating adaptation to salinity stress in several plant species including halophytes. Respiratory burst oxidase homolog (Rboh) proteins show the ROS-producing Nox activity, which are synergistically activated by the binding of Ca2+ to EF-hand motifs as well as Ca2+-dependent phosphorylation. We herein review molecular identity, structural features and roles of the Ca2+-permeable channels involved in early salinity and osmotic signaling, and comparatively discuss the interrelationships among spatiotemporal dynamic changes in cytosolic concentrations of free Ca2+, Rboh-mediated ROS production, and downstream signaling events during salinity adaptation in planta.Salinity stress, which induces both ionic and osmotic damage, impairs plant growth and causes severe reductions in crop yield. Plants are equipped with defense responses against salinity stress such as regulation of ion transport including Na Therefore, plants have developed various systems to scavenge ROS   . Nox-mednscripts . Growth ts  and K+-permeable NSCCs, resulting in K+ leakage from roots , putative PM Ca2+-permeable MS channel components are suggested to play a role in the regulation of mechanical responses via signal transduction pathways dependent on Ca2+ and ROS : Salinity stress causes osmotic shock to trigger the activation of PM MS Ca2+-permeable channels and Ca2+ influx. Elevated cytosolic Ca2+ leads to the activation of various Ca2+ sensor proteins including CaMs, CMLs, CBLs, and CDPKs. Rbohs are synergistically activated by Ca2+-binding to the EF-hand motifs and phosphorylation to produce ROS. Elevated cytosolic Ca2+ and apoplastic ROS may also activate not only ROS-activated PM Ca2+-permeable channels including annexins but also as-yet-unidentified Ca2+-permeable channels/transporters localized at the endomembranes to sustain further Ca2+ mobilization. The signaling network dependent on Ca2+ and ROS may play a crucial role in regulating downstream events such as the Ca2+-dependent activation of Na+/H+ exchangers, SOS1 and NHX1 , while it was unaffected in a halophyte (S. portulacastrum)  . These f+ exclusion from the cytosol may not require a high level of antioxidant activity. Such exclusion is most probably achieved by the orchestrated action of several complementary mechanisms including SOS1-mediated Na+ exclusion from the cell (+ sequestration by tonoplast NHXs (+ back-leak into the cytosol (Halophytes do not always need to possess high antioxidant activity because of their higher capacity to prevent ROS generation . Salt-tothe cell , vacuolaast NHXs  and effi cytosol ,b.2+]cyt increase mediated by PM Ca2+-permeable MS channels and their interacting molecules is a hallmark response to osmotic stress (2+-independent osmotic sensory mechanisms may also play a role. Effectors of ROS and signaling cascades downstream of Rbohs need to be elucidated to better understand their roles. The crosstalk of Rboh/Nox-mediated Ca2+-dependent ROS production with other signaling pathways involving ABA, MAPK, and NO as well as jasmonic acid may be important in transcriptional regulation.Although a rapid [Cac stress , Ca2+-inArabidopsis (Rboh in halophytes may have characteristic consensus sequences involved in chromatin modification, and may, thus, enhance plant salinity tolerance.In addition, mild salinity stress in the seedling stage induces osmotic priming in bidopsis . Since sbidopsis , the proThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Importantly, we demonstrate that the reduction of extracellular calcium content by the chelating agent ethylenediaminetetraacetic acid can decrease intra-mitochondrial ROS production and enhance cell viability. Antioxidant administration also reduces mitochondrial ROS production and cell loss, but this treatment does not synergize with Ca2+ chelation. Our results disclose novel intracellular pathways involved in PSY-induced death that may be exploited for therapeutic purposes to delay GLD onset and/or slow down its progression.Globoid cell leukodystrophy (GLD) is a metabolic disease caused by mutations in the galactocerebrosidase (GALC) gene. GALC is a lysosomal enzyme whose function is to degrade galacto-lipids, including galactosyl-ceramide and galactosyl-sphingosine . GALC loss of function causes progressive intracellular accumulation of PSY. It is widely held that PSY is the main trigger for the degeneration of myelinating cells and progressive white-matter loss. However, still little is known about the molecular mechanisms by which PSY imparts toxicity. Here, we address the role of calcium dynamics during PSY-induced cell death. Using the human oligodendrocyte cell line MO3.13, we report that cell death by PSY is accompanied by robust cytosolic and mitochondrial calcium (Ca GALC loss of function causes progressive accumulation of PSY, a cytotoxic metabolite that has been assumed as the main cause for GLD pathogenesis.2 PSY leads to Schwann cell and oligodendrocyte death, but still little is known about the molecular mechanisms by which PSY imparts toxicity. It has been demonstrated that PSY accumulates in cell membrane raft micro-domains, disrupting their architecture3 and inhibiting protein kinase C translocation to the plasma membrane.4 Recently, increased raft clustering was also reported in cultured dorsal root ganglion neurons prepared from the GLD murine model , and this was associated with the dysregulation of tyrosine kinase receptor A membrane recruitment and ligand-tyrosine kinase receptor A activated endocytosis.5 PSY induces p53-mediated apoptotic cell death,6 tumor necrosis factor-related apoptosis,7 activation of secretory phospholipase A2,8 cytochrome C release from mitochondria and apoptosis activation via the caspase-9 pathway.9 Moreover, several authors found that peroxisomal β-oxidation was significantly inhibited and very long-chain fatty acid levels and reactive oxygen species (ROS) production were increased in PSY-treated cells.11Globoid cell leukodystrophy (GLD), also known as Krabbe disease, is a childhood leukodystrophy triggered by mutations in the galactocerebrosidase (GALC) gene; the physio-pathological hallmarks of GLD are progressive demyelination, reactive astrocytosis and microgliosis.2+) is an essential ion for cell life, acting as a key second messenger in almost all cellular functions. It is well established that Ca2+ is one of the main second messengers involved in apoptotic cell death in neurons and in other cell types; sustained cytosolic Ca2+ increase can activate apoptosis.12 This can originate from extracellular influx or by release from intracellular stores like the endoplasmic reticulum.13 Importantly, mitochondria are also involved in Ca2+ homeostasis.14 Mitochondrial Ca2+ in basal conditions is maintained at low concentrations, but mitochondria are organelles that can take up high Ca2+ concentrations; indeed, different stimuli, such as nutrients, hormones or neurotransmitters that increase the cytoplasmic Ca2+ content also induce intra-mitochondrial Ca2+ increase.15 If this increase is relevant, ROS production increases and this is associated with mitochondrial membrane destruction, release of cytochrome C and apoptosis induction.16 During this process, pro-apoptotic Bcl2 family of proteins plays a crucial role by regulating the intracellular/mitochondrial Ca2+ content, and by inducing mitochondrial permeabilization, the essential step for cytochrome C release and caspase activation.17Calcium  in serum-free condition to exclude the sphingolipid content present in serum (as sphingosine 1-phosphate (S1P)),19 which could have hindered the PSY effect. After treatment, cells were harvested and stained with Annexin V-FITC conjugate and propidium iodide (PI), and analyzed by flow cytometry , 48±7% (P<0.001) and 67±7% (P<0.001) for 3 μM, 5 μM and 10 μM PSY concentration, respectively  to also monitor mitochondrial potential loss during the last phases of apoptosis.20 The stained cells were treated for 6–8 h with PSY 10 μM and imaged by time-lapse confocal microscopy to follow the cytoplasmic Ca2+ kinetics from PSY administration (t=0) until cell death occurred.Because Ca2+ peak after treatment (t≈5–8 min), followed by a second, slower Ca2+ increase that preceded cell death (t≤8 h from PSY administration) showed two peaks  and the cells failed to show any significant change in calcium or mitochondrial potential with respect to the baseline, demonstrating the specificity of PSY effects (μM) treatment . We first verified the correct probe localization inside mitochondria: to this end, MTCD2CPV-transfected cells were stained with TMRM and analyzed by high-resolution confocal microscopy. High-magnification images were acquired along the z axis by sequential acquisitions of the FRET signal and the TMRM fluorescence to avoid cross talking between TMRM and YFP excitation. Experiments confirmed the co-localization of the MTCD2CPV probe with TMRM , followed by recovery to baseline levels and by a second more protracted Ca2+ elevation. This last event started before the onset of mitochondrial potential decrease, and was maintained until cell death occurred .2XRos to monitor both cytoplasmic calcium and ROS production. PSY induced an early peak in CM-H2XRos fluorescence soon after administration; this peak corresponded with the early calcium peak detected by Fluo-3 fluorescence. After this peak, the signal gradually increased reaching its maximum before a second cytoplasmic Ca2+ increase and indicating robust ROS elevation for 50 min<t<200 min  and the cells failed to show any important change in CM-H2XRos fluorescence or mitochondrial potential with respect to the baseline, demonstrating the specificity of PSY effects . Initially, we performed preliminary dose–response experiments to determine the maximal EDTA concentration not affecting cell viability or ROS production. On the basis of the data reported in 2+ reduction in our cell-culture medium . MO3.13 cells were treated with PSY 3–10 μM in presence of EDTA for 24 h, then flow cytometry was carried out to assess viability as previously described . Our data demonstrate that EDTA can protect MO3.13 from PSY-induced cell death. Indeed, EDTA increased cell viability 1.5-fold in case of PSY 5 μM, and twofold in case of PSY 10 μM with respect to cells treated with same concentrations of PSY but without EDTA , respectively. More recently, two papers by Lloyd-Evans et al.31 reported Ca2+ release in rat brain microsomes upon PSY treatment, with PSY acting as an agonist of ryanodine receptors. Interestingly, these references documented relevant Ca2+ alterations  within ≈10 min from PSY (up to 30 μM) administration, but Ca2+ kinetics on longer time-scales was not investigated. Moreover, in contrast with other lysosomal storage disorders, no clear evidence has been still reported that Ca2+ dysregulation is a pathogenic factor in GLD.32 Our data are full in agreement with the above-presented literature, extending the characterization of Ca2+ dynamics until PSY-induced cell death occurs; PSY accumulation is indeed believed to be the main cause of oligodendrocyte loss in GLD. Moreover, in order to make our analysis more relevant for shedding light on GLD pathogenesis, we used a differentiated human oligodendrocyte cell line (starved MO3.13 cells) for all the experiments. MO3.13 cells are indeed widely used for GLD research  because of their very active sphingolipid metabolism similar to that of primary oligodendrocytes.34 Our experiments of Ca2+ chelation directly support the hypothesis that Ca2+ dysregulation by PSY can trigger oligodendrocyte loss.It is well known that several sphingolipid messengers such as ceramide, glucosyl-ceramide, ceramide-1-P, sphingosine and sphingosine-1-phosphate can regulate intracellular Ca21 In the apoptotic population, after the mentioned early Ca2+ peak occurring few minutes after treatment, the cytoplasmic Ca2+ presented a second robust elevation during the last phases of apoptosis execution , but mitochondria can sense cytoplasmic Ca2+ variations and transiently sustain high Ca2+ concentrations (hundreds of μM),42 thus contributing to Ca2+ homeostasis in the cell. Slight mitochondrial Ca2+ increases can regulate the mitochondrial activity by activating the Krebs cycle dehydrogenases, by promoting the supply of oxidable substrates and by regulating the activity of ATP synthase.16 This is one of the mechanisms used by cells to reduce the cytoplasmic Ca2+, because ATP production can stimulate the sarco endoplasmic calcium ATPase that transfers Ca2+ from the cytoplasm to the endoplasmic reticulum.43 We reported a progressive increase of the mitochondrial potential during the first phases following PSY administration, indicating stimulation of mitochondrial activity and suggesting the activation of the endoplasmic reticulum for storing the Ca2+ excess. Conversely, robust mitochondrial Ca+ elevations lead to ROS production that runs out with mitochondrial membranes destruction, release of cytochrome C and apoptosis induction.16 Following the first Ca2+ peak, we indeed measured a second, sustained Ca2+ increase, and enhancement of mitochondrial ROSs (t<200 min) before cell death. These data fit with the paradigm of cell death mediated by mitochondrial Ca2+ increase and ROS production, as previously hypothesized by Formichi and co-workers.7 Interestingly, mitochondrial abnormalities, such as loss of mitochondrial Ca2+ buffering and malfunctioning in the lysosomal-mitochondrial axis, have been also reported in other lysosomal storage disorders.44Our data demonstrate that the cytoplasmic Ca2+ elevation and mitochondria-mediated cell death, we hypothesized that protection from PSY cytotoxicity could result from reducing the extracellular calcium content and mitochondrial oxidative stress, possibly in a synergic way. We found that Ca2+ chelation from the extracellular medium by EDTA (1 mM) improves cell survival following PSY administration . Moreover, given the important role of mitochondrial ROS production, antioxidants that specifically target mitochondria might be tested. The MitoQ, for example, is a mitochondria-targeted antioxidant designed to accumulate within mitochondria 2+ that are coupled to mitochondrial ROS production. Chelation of extracellular calcium decreases intra-mitochondrial ROS production and enhances cell viability. Antioxidant treatment also reduces mitochondrial ROS production and cell loss, but this therapy does not synergize with Ca2+ chelation. Altogether, these data provide novel information on the intracellular pathways activated during PSY-induced toxicity.In this study, we have followed longitudinally calcium dynamics and mitochondrial ROS during PSY-induced oligodendrocyte cell death. We report that PSY treatment causes elevations of cytosolic and mitochondrial CaHuman oligodendrocyte MO3.13 cells  were maintained in DMEM medium supplemented with 2 mM L-glutamine, 1% penicillin/streptomicyn and 10% heat-inactivated fetal bovine serum , at 37 °C in humidified atmosphere containing 5% CO2.2; 24 h after plating, the medium was removed and cells were washed two times with phosphate-buffered saline (PBS) 1 × . Then, cells were cultured in serum-free medium  or serum-free medium+PSY 1–10 μM. For EDTA and NAC experiments, cells were pre-treated for 30 min with EDTA 1 mM or/and NAC 5 mM, then treated with PSY 3–10 μM for 24 h. PSY was diluted in DMSO and prepared as a 10 mM stock solution, EDTA was diluted in water and prepared as 1 M stock solution and NAC was diluted in water as 1 M stock solution; control cultures received the same quantity of vehicle (DMSO or/and water). Given the concentrations of Ca2+ and Mg2+ in the culture medium , the binding constants for EDTA with Ca2+ and Mg2+ (log Ka=10.65 for Ca2+ and 8.79 for Mg2+) and the final concentration od EDTA (1 mM), the theoretical amount of free Ca2+ and free Mg2+ in the medium supplemented with EDTA is calculated to be approximately 0.8 mM for both ions , that is, ≈60% reduction in Ca2+ content. We also measured the pH in DMEM and DMEM+EDTA (1 mM) and we did not find important medium acidification (pH from 7.4 to 7.25).MO3.13 cells were seeded at 25000 cells/cm2) containing Annexin V-FITC conjugate 1 μM  and PI 100 nM  and incubated in ice for 30 min. Flow cytometry was performed by using a S3 flow cytometer  equipped with 488 and 561 nm diode-pumped solid-state lasers. Annexin V-FITC was excited using the 488 nm laser, and fluorescence was collected through a 552/50 nm band-pass filter and 505 nm long-pass filter; PI was excited with the 561 nm laser and the fluorescence was collected through a 605/40 nm band-pass filter and a 570 nm long-pass fiter. At least 20 000 gated events were acquired for each sample. Data were analysed using the Bio-Rad ProSort software.After treatments, MO3.13 cells were harvested and spun for 5 min at 1400 rpm; the obtained pellets were washed with PBS 1 × and spun again for 5 min at 1400 rpm. Cells were re-suspended in binding buffer  for 30 min. Fluorescence and phase contrast images were acquired by using an Eclipse Ti inverted microscope  equipped with a × 20 air Nikon objective, N.A. 0.75, ApoFluor and a CCD ORCA R2 . Hoechst 33342 was excited by a mercury arc lamp using a 450/50 nm band-pass filter, and emission was collected using an UV-2E/C filter (Nikon). Cell death was qualitatively assessed on the basis of cellular and nuclear morphology evaluating the presence of condensed or fragmented nuclei.Cells were fixed with paraformaldeyde 2% for 30 min at room temperature; after fixation, cells were washed once with PBS and stained with Hoechst 33342 . Preparation of cell lysates and western blotting were carried out as described in Reimertz et al.A total of 1 × 102) into 24-well plates for 2 days and then treated with serum-free medium. After 24 h of treatment, cells were fixed for 30 min in 2% paraformaldehyde, permeabilised with 0.1% Tween-20 and blocked with 5% horse serum for 15 min. Cells were then incubated overnight with anti-myelin basic protein antibody . Cover slips were washed with PBS (10 min for three times), incubated with anti-rabbit biotinylated secondary antibody  for 90 min, washed with PBS and then stained with Alexa 488 Streptavidin  for 30 min. Finally, the cover slips were mounted onto glass microscope slides in presence of Vectashield mounting medium with DAPI . Confocal images were acquired as z-stacks by using a scanned on z axis with a SP2 TCS-NT Leica  laser scanning confocal microscope, equipped with 40 × 1.2 NA Plan Apo oil objective. The optical slice was set to 1 μM (FWHM).MO3.13 cells were plated on glass cover slips (25000 cells/cm2) and treated as described before; after treatments, the cells were stained with MitoTracker Red CM-H2XRos  for 30 min at 37 °C in humidified atmosphere containing 5% CO2. MitoTracker Red CM-H2XRos was diluted with anhydrous DMSO to obtain 1 mM of stock solution and used 1 μM as final concentration. After staining, cells were harvested and spun for 5 min at 1400 rpm; the obtained pellets were washed with PBS and spun again for 5 min at 1400 rpm. Flow cytometry was performed by using a S3 flow cytometer (Bio-Rad) equipped with a 488 nm and 561 diode-pumped solid-state lasers. MitoTracker Red CM-H2XRos was excited with a 561 nm laser and fluorescence emission was collected through a 605/40 nm band-pass filter and 570 nm long-pass fiter. At least 20 000 gated events were acquired for each sample. Data were analyzed using the Bio-Rad ProSort Software.MO3.13 cells were seeded in 24 well plates (25000 cells/cm2) were plated in WillCo  dishes 24 h before experiments to reach 50–60% of confluence and to obtain a stable substrate adhesion. One hour before the experiment, MO3.13 cells were deprived by serum, stained with Fluo-3 AM 1 μM for 30 min, and then incubated in serum-free medium with TMRM (50 nM). TMRM concentration was chosen to avoid quenching of the dye. One hour later, the WillCo dish was mounted into SP2 TCS-NT Leica laser scanning confocal microscope, equipped with 40 × 1.2 NA Plan Apo oil objective and with an incubating chamber (T=37 °C and 5% CO2). Fluo-3 was excited using the 488-nm laser line, and the emitted fluorescence was collected through a 530/30-nm band-pass filter; TMRM was excited using the 568-nm laser line, and the emitted fluorescence was collected through a 590-nm long-pass filter. Excitatory lights were kept to the minimum power to minimize photodamage and photobleaching. The pinhole of the microscope was set to obtain an optical slice of 0.5 μm and samples were scanned two times at 512 × 512 pixel resolution with a time interval of 15 s. After at least 30 min of baseline recording, PSY 10 μM was added to the medium and cellular response was recorded for 6–8 h.MO3.13 cells .47 In this construct, a Ca2+-responsive element  is present that, case of Ca2+ variations, alters the efficiency of FRET between two fluorescent proteins. The transfection solution was prepared diluting 1 μg of plasmid and 1 μl of Lipofectamine 2000  reagent in OPTIMEM medium (Life Technologies) following the manufacturer's instructions, and incubated at room temperature for 30 min. After liposome reaction, solutions were used to transfect the cells. After 90 min incubation, the transfection medium was removed and replaced with fresh medium. Experiments were performed 72 h after the transfection to have ≈80% of the cells with the correct MTCD2CPV localization into the mitochondria. Ninety minutes before experiments, MO3.13 cells were deprived of serum and after 1 h stained for 1 h with TMRM 50 nM and mounted into the incubator chamber for the acquisition in time-lapse at 37 °C in a humidified atmosphere containing 5% CO2. Images were collected with a time interval of 15 s, with a resolution of 512 × 512 pixels. The MTCD2CPV calcium probe was excited with the 458 laser line of a SP2 TCS-NT Leica confocal microscope and the emissions of CFP and YFP were recorded at 485 and 535 nm, respectively. TMRM was excited with 568-nm laser line in sequential acquisition after cameleon recording to exclude YFP excitation and cross-talking. For each time point, both fluorescent and transmitted light images were acquired. The quantification was performed by using the MetaMorph 5.0 software  expressing the calcium concentration as ratio of emissions FRET/CFP (535/485). The images were elaborated with the plug-in ‘FRET analyzer' of the Image J free software (http://imagej.nih.gov/ij/).For mitochondrial calcium measurements, 1 × 102) were plated into WillCo dishes 24 h before experiments to reach 50–60% of confluence and to obtain a stable substrate adhesion. After Fluo-3 AM loading (as described in 'Cytoplasmic Ca2+ imaging'), medium was removed and replaced with serum-free medium with MitoTracker Red CM-H2XRos 1 μM. Cells were stained for 30 min at 37 °C in a humidified atmosphere containing 5% CO2 and then imaged by using a SP2 TCS-NT Leica confocal microscope, equipped with 40 × 1.2 NA Plan Apo oil objective and with an incubating chamber . Fluo-3 signal was recorded as described in 'Cytoplasmic Ca2+ imaging'. MitoTracker Red CMH2XRos was excited using the 568-nm laser line, and the emitted fluorescence was collected through a 590-nm long-pass filter. Excitatory lights were kept to the minimum power to minimize photodamage and photobleaching. The pinhole of the microscope was set to obtain an optical slice of 0.5 μm and samples were scanned two times at 512 × 512 pixel resolution with a time interval of 15 s. After at least of 30 min of baseline recording, PSY was added to the medium and cells responses was recorded for 6–8 h.MO3.13 cells (50000 cells/cmn' indicates the number of the performed experiments). Data are reported as the averaged value of the means of single experiments +/− the standard error of the mean (mean +/− S.E.M.). Each distribution of the means or, when required, of full datasets has been tested for normality . For parametric data, Student's t-test  or one-way ANOVA (Tukey's or Dunnett's post tests) were used; for non-parametric data, Mann-Whitney test (two-tailed) or Kruskal-Wallis test was used. Minimum statistical significance will be set for P values<0.05 . NS stands for ‘not significant'.Inferential statistics was used to compare datasets from different experimental groups. All the experiments were repeated at least three times independently (in figure legends, ‘


The purpose of this study was to test the hypothesis that Ca2+ release is significantly impaired in the skeletal muscle of HF patients in whom exercise capacity is severely diminished compared to age-matched healthy volunteers.Exercise intolerance in chronic heart failure (HF) has been attributed to abnormalities of the skeletal muscles. Muscle function depends on intact excitation-contraction coupling (ECC), but ECC studies in HF models have been inconclusive, due to deficiencies in the animal models and tools used to measure calcium -evoked Ca2+ release in 4 HF patients and 4 age-matched healthy controls. The mean peak Ca2+ release flux in fibers obtained from HF patients (10±1.2 µM/ms) was markedly (2.6-fold) and significantly (p<0.05) smaller than in fibers from healthy volunteers (28±3.3 µM/ms). This impairment in AP-evoked Ca2+ release was ubiquitous and was not explained by differences in the excitability mechanisms since single APs were indistinguishable between HF patients and healthy volunteers.Using state-of-the-art electrophysiological and optical techniques in single muscle fibers from biopsies of the locomotive 2+ release, is impaired in single muscle fibers in HF patients.These findings prove the feasibility of performing electrophysiological experiments in single fibers from human skeletal muscle, and offer a new approach for investigations of myopathies due to HF and other diseases. Importantly, we have demonstrated that one step in the ECC process, AP-evoked Ca Chronic heart failure (HF) patients with systolic dysfunction have benefited from advances in pharmacological and device therapies that have reduced mortality 2+) cycling in humans with HF. The integrity of important steps in the ECC chain of events, such as the Ca2+ release from the sarcoplasmic reticulum (SR), is a decisive factor for normal exercise ability; ECC impairments are expected to critically contribute to muscle weakness and poor exercise tolerance Skeletal muscle function depends critically on both intact energy metabolism and on the robustness of the sequence of events linking the electrical with the mechanical activation of the muscle fibers . The role of metabolic deficiencies in skeletal muscle function in HF patients on optimal medical therapy has recently been questioned 2+ release in skeletal muscle fibers have not been utilized in animal models. Altogether, the findings from animal studies are inconclusive and contradictory vastus lateralis (VL) muscles directly from HF patients and healthy age-matched volunteers, and used state-of-the-art electrophysiological and optical techniques to test our hypothesis that the ECC process is significantly impaired in the locomotive skeletal muscle of HF patients compared to healthy volunteers. Our approach puts special emphasis on the fibers’ ability to generate Ca2+ transients in response to action potential (AP) stimulation and, by using low affinity Ca2+ indicators in conjunction with high internal EGTA concentrations Animal models of HF have been used to investigate ECC abnormalities that may recapitulate the skeletal myopathy seen in HF patients The study was approved by the University of California Los Angeles (UCLA) Human Subjects Protection Committee, and the participants gave their written informed consent.Advanced HF patients, New York Heart Association Class II–III meeting the following eligibility criteria were recruited from the Ahmanson-UCLA Cardiomyopathy Center: 1) age 21–65 years, 2) Left ventricular ejection fraction <35%, 3) HF duration > 1 year, 4) not involved in a formal exercise training program, 5) on stable, guideline appropriate HF medications, and not taking warfarin or non-warfarin oral anticoagulants. Age and gender-matched healthy, non-smoking controls, without chronic illnesses and taking no daily medications, who drank <2 drinks/day, and who did not participate in regular exercise (30 minutes most days), served as controls.The procedure was performed at the UCLA Out-Patient Surgery Center under local anesthesia. An incision was made in the lateral aspect of the thigh, at about 20 cm proximal to the patella, and a sample of vastus lateralis (VL) muscle measuring 40 mm in length, 8 mm in width and 5 mm in depth was obtained. The biopsy was transported to the lab in ice-cold oxygenated Tyrode solution, where it was pinned in a Sylgard bottomed Petri dish containing oxygenated Tyrode solution and divided into 10–12 bundles (∼2 mm in diameter), as delimited by the perimysium. Bundle pulling was kept at a minimum at all times during dissection. When kept in oxygenated Tyrode solution at room temperature, bundles remained viable for >10 hours. Although not studied systematically, viability was found to be longer the greater the biopsy length. In many cases, bundles 40 mm in length contracted upon stimulation with 10 Hz trains after 1.5 hours from biopsy.A bundle was pinned down at slack length in a dish containing Tyrode, which was subsequently slowly exchanged by relaxing solution (see Solutions). In this solution, fibers became electrically and mechanically inactivated, thus preventing contractures during dissection and increasing the rate of success of intact fiber isolation. A peripheral smaller bundle comprised of ∼20 fibers was dissected aside using small Vannas scissors and #5 tweezers. The tissue was divided progressively into smaller bundles until a single fiber of about 20 mm in length was obtained. For this finer dissection, #5 tweezers and retina surgical scissors  were used. Fiber stretching was avoided at all times. Only fiber segments crystalline in appearance, devoid of contractured regions, freely flexible, and exhibiting a sharp banding pattern were used for electrophysiological experiments. The degree of difficulty of dissection of fibers from HF biopsies was not significantly different from that of control biopsies.2+ sensor was added to the lateral compartments. Finally, the chamber was placed on the stage of the microscope and connected to the perfusion system and the electrical amplifier. Fibers requiring more than −70 nA to be repolarized to −90 mV were discarded. Recordings were started 15–20 min after adding the Ca2+ sensor to the lateral pools. The circuit used to stimulate the fibers and to record the membrane potential and currents has been previously described [25]. One cut end of the fiber was connected to a current source, the other was connected to a high impedance amplifier, and the central segment of the fiber was held at virtual ground. APs were elicited by current pulses  applied at one cut end. Membrane potential was measured as the difference between the potential at the other cut end (i.e. the myoplasm) and the central pool (i.e. the extracellular milieu).Fibers were mounted in an inverted double-gap grease chamber, under stereoscopic microscope observation, as previously described 2+ concentration changes were measured using the low affinity dye OregonGreenBapta488-5N (OGB-5N) 2+ were added to the internal solution with two purposes in mind: a) to prevent fiber movements that would distort optical recordings and potentially dislodge the grease seals; and, b) to allow for the calculation of peak Ca2+ flux from OGB-5N Ca2+ transients using a single compartment model that is extensively described elsewhere 2+ dependent fluorescence transients (referred to simply as Ca2+ transients) are reported as ΔF/F transients (dimensionless). These transients are used to calculate Ca2+ flux transients (μM/ms).AP evoked Ca2+ transient before calculating ΔF/F transients.The optical setup was similar to that described elsewhere The composition of solutions (mM) was:2, 2CaCl2, 10 glucose, 10 MOPS.Tyrode: 150 NaCl, 4KCl, 1MgCl2SO4, 10 MOPS, 1 MgCl2, 1 CaCl2, 10 glucose.Relaxing solution: 120 K2, 5 reduced Glutathione, 5 ATP-TRIS, 5 Creatine Phosphate-Na2, 20 EGTA, 10 Ca(OH)2.Internal solution: 92 K-Aspartate, 20 MOPS, 1 MgCl2O.All solutions were adjusted to ph = 7.4 and had 300±5 mOsmol/kg HVoltage, current and optical signals were filtered at 10, 5 and 2 kHz , respectively, and acquired at 10µs/point. Optical and electrical data from control and HF fibers were compared using the Student’s t test. Significance was set at p<0.05.Four HF patients at an advanced stage, and four healthy age-matched human volunteers participated in these studies. The major features of the cohorts are presented in In contrast to previous reports in rat muscles r changed only slightly throughout the typical 30 min of electrophysiological experimentation. Interestingly, these values are comparable to those previously measured in frog fibers mounted in a similar experimental chamber Additional direct evidence for the fibers’ intactness was obtained by measuring their spontaneous repolarization after exchanging the high K relaxing solution with Tyrode in the central compartment of the experimental chamber. Isolated fibers from both healthy volunteers and HF patients repolarized spontaneously to a similar membrane potential (−13.6±3.42 mV [n = 8] and −16.5±5.1 mV [n = 10] respectively), and required almost identical holding currents in order to maintain them at a resting potential of −90 mV (−59.2±18.3 nA [n = 8] and −60.8±28.5 nA [n = 10] respectively), and V2+ in response to AP stimulation. A smaller population of fibers was used for this purpose . The fibers were allowed to equilibrate with internal solutions containing high [EGTA] and free Ca2+ concentration [Ca2+] close to the physiological value ∼0.1 µM 2+ transient recorded in a fiber from a healthy volunteer and equilibrated with internal solution containing 30∶15 EGTA:Ca (see Solutions). In response to the AP (2+ transient) was elicited that reached a peak ΔF/F of ∼0.24. Interestingly, knowing the concentration and Ca2+ binding properties of the Ca2+ indicator (OGB-5N) and EGTA, we were able to estimate with accuracy the Ca2+ release flux (in μM/ms) responsible for this fluorescence transient 2+ release, was ∼29 µM/ms. In comparison, Ca2+ transients recorded from a muscle fiber dissected from a HF patient’s VL biopsy under the same conditions as those used for the fiber from the healthy volunteer were ∼3-fold smaller amplitude  conditions. As demonstrated in 2+ fluxes determined in fibers from healthy volunteers equilibrated with 30∶15 (n = 2) and 20∶10 (n = 3) EGTA mixtures were comparable to each other. Since the mean flux from fibers was independent of the EGTA:Ca ratio, the data from healthy volunteers was pooled. The results demonstrate that the mean peak Ca2+ release flux in fibers obtained from HF patients equilibrated with a 30∶15 EGTA:Ca mixture  is substantially (2.6-fold) and significantly (p<0.05) smaller than that in fibers from healthy volunteers . We then compared the frequency distribution of peak Ca2+ fluxes from healthy volunteers and HF patients , time to peak (TTP), and maximal rate of depolarization ([dV/dt]max) of APs recorded in fibers isolated from the skeletal muscle biopsies of HF patients and healthy volunteers  they circumvent the inherent limitations of studying ECC in models of HF by utilizing actual tissue obtained directly from patients afflicted with the HF condition; 2) they confirm the feasibility of performing electrophysiological experiments characterizing individual steps of the ECC process in single fibers from human skeletal muscle according to fiber type; and critically, 3) they offer new approaches to investigate other myopathic conditions that may also involve impairments in the ECC process, such as chronic obstructive pulmonary disease and chronic renal disease Using state-of-the-art electrophysiological and optical techniques, we have performed studies in single fibers procured from the 2A/α2C adrenoceptor knockout mouse that leads to sympathetic hyperactivity-induced HF and quantified exercise dysfunction 2A/α2C adrenoceptors are absent in all tissues, including the skeletal muscle, it is difficult to know whether muscle changes are due to HF, or to the genetic modification. In fact, in this model, the sympathetic hyperactivity leads to early skeletal muscle changes attributable to relatively increased β2-adrenoceptor activity 2+ cycling proteins and muscle fiber type composition, generally confound data interpretation and detract from these models. As noted earlier, some Animal models of HF, including the infarct model and the sympathetic hyperactivity-induced model, are critical to our further investigations and improved understanding of human diseases, but have inherent limitations 2+ release evoked by AP stimulation in human skeletal muscle fibers in health or disease. Two reports of the Ca2+ release flux in healthy human fibers performed under voltage-clamp conditions have been published 2+ fluxes would be recorded, the values calculated in these reports were significantly smaller than ours, by a factor of at least 2-fold. Differences in the fibers’ integrity, the use of Ca2+ sensors, and calculation methodologies may partially explain these differences. In one study, percutaneous needle biopsies were obtained, yielding only short muscle segments 2+ release fluxes suggests that our fibers were possibly in better physiological condition throughout the experimentation.To our knowledge there are no previous studies of the Ca2+ release, is impaired in fibers from the VL muscle of HF patients compared to those of healthy volunteers. We have previously reported a reduced expression of sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) and dihydropyridine receptor (DHPR) in homogenates of VL muscle from HF patients compared to healthy volunteers, whereas SERCA1a and ryanodine receptor (RyR1) levels were not different 2+ content is reduced in fibers from HF patients compared to those from healthy volunteers, and/or that there are orphaned RyR1s. Either alteration could result in a significant reduction of Ca2+ fluxes potentially leading to reduced exercise performance and increased fatigability characteristic of HF patients. Diminished peak Ca2+ release was present in response to single APs; a finding that would be expected to result in smaller peak twitch and tetanic tension (i.e. muscle weakness and premature fatigability). Further investigations are necessary to identify the exact step(s) in the ECC process that lead to this marked impairment in AP-evoked Ca2+ release in HF patients. Comparisons of the physiological trigger for Ca2+ release (the AP which initiates the ECC process) in healthy volunteers and HF patients were performed and essentially eliminated this as a potential cause .The amplitude and duration of the transverse tubular system depolarization are key factors in determining the features of Ca+ channels (NaV1.4) is significantly increased in soleus muscles of rat models of muscle disuse, a condition to which HF patients may be prone The maximum depolarization rates of the AP in control and HF fibers are similar to that found in frog fibers using a similar method 2+ transients have been reported to differ between fast and slow muscle fibers, and to correlate with the myosin heavy chain (MHC) type expressed; specifically, Ca2+ release rates are larger in type II than in type I fibers Both the kinetics and magnitude of the Ca2+ fluxes in HF patients, without overlap with the peak Ca2+ fluxes in healthy volunteers, consistent with a ubiquitous abnormality affecting the ECC process in every fiber type (I and II) of VL muscles. In other words, the level of Ca2+ release from HF fibers with the greatest Ca2+ flux, , was even lower than the level of Ca2+ release from healthy fibers with the lowest Ca2+ flux, presumably type I fibers. A simple fiber-type shift (i.e. from type I to type II) should have resulted in an overall right-shifted distribution of HF fibers, not seen in 2+ release.Interestingly, we found a narrow, left-shifted distribution of peak Ca2+ release that affects all the fibers, independent of their type, in VL muscles.In summary, our results strongly support the hypothesis that fibers from HF patients have marked and ubiquitous impairments in ECC, specifically AP-evoked Ca2+ release fluxes reported here are possibly smaller than those that could be obtained at normal body temperatures. Though future experimentation will take this into account, the observed impairments in Ca2+ release of fibers from HF patients are likely to represent a general limitation rather than an experimental peculiarity linked to the choice of temperature.The use of humans, in place of animals, for investigation, has its own limitations, which we acknowledge here: First, patients are on medications including statins, which, for ethical reasons, cannot be interrupted, and these medications may have systemic effects. Nonetheless, it is well established that despite optimal medical therapy HF patients have severe exercise limitations. We would argue that it is precisely these individuals who need to be studied, since despite the best medical therapy, their exercise capacity continues to be impaired. It is worth noting that no patient was diagnosed with a statin myopathy. Secondly, patients have diverse etiologies of HF, however, the severity of exercise dysfunction, and the characteristics of the skeletal myopathy, including the fiber type transformation, are known to be independent of the specific etiology of the HF 2+ release is significantly diminished in fibers obtained from the locomotive VL muscle in HF patients. This diminished Ca2+ release is not attributable to significant alterations in the AP of fibers from HF patients compared to fibers from healthy volunteers, since the electrical activity in both groups is virtually identical. Our previous findings of diminished DHPR and SERCA2 expression are suggestive of decreased SR Ca2+ content, and/or orphaned RyR1; the relative contributions of these abnormalities and others in the ECC process await further investigation. This decrease in Ca2+ flux was present in every fiber tested, consistent with a ubiquitous abnormality involving the ECC process in every fiber type in the VL muscle of HF patients, and unlikely to be attributable to a simple fiber type shift. Finally, using this approach, Ca2+ fluxes in individual fibers can be compared between muscles of locomotion, vulnerable to disuse, and compared to fibers in non-locomotive muscles, such as the deltoid, to assess if abnormalities of the ECC process are a systemic problem, attributable to systemic neurohumoral activation and inflammation, or a localized problem, more easily explained by disuse. The impact of exercise training on the impairment of ECC and Ca2+ release has widespread clinical relevance, both in understanding the role of disuse in this impairment, as well as an important therapeutic approach, and warrants further study.We report for the first time in single fibers from HF patients compared to healthy volunteers, that AP-evoked CaFigure S1The transverse tubular system in live fibers from control and HF individuals. Muscle bundles dissected from biopsies obtained from control individuals (A) and HF patients (B) were stretched to a sarcomere length similar to that in the electrophysiological experiments and pinned down on a Sylgard bottomed dish. The fibers were stained with di-8-ANEPPS and imaged using a two-photon laser scanning microscope (Biorad Radiance 2000). The dye was excited with a 920 nm laser beam, and the emission was collected using a dichroic/band-pass combination of 506 nm/620–650 nm. It can be seen in the optical section in A that, as previously described for other mammalian fibers, di-8-ANEPPS clearly demonstrate the regular double row pattern of the transverse tubular system. The surface membrane can also be seen at the right upper part of the image. The image in B demonstrate that the transverse tubular system of fibers from HF patients is indistinguishable from that of control fibers. The scale represent 15 µm in both images.(EPS)Click here for additional data file.


NKCC1 is a secondary active transporter, mainly localized in astrocytes, driven by transmembrane Na+/K+ gradients generated by the Na+,K+-ATPase. The astrocytic Na+,K+-ATPase is stimulated by small increases in extracellular K+ concentration and by the β-adrenergic agonist isoproterenol. Larger K+ increases, as occurring during ischemia, also stimulate NKCC1, creating cell swelling. This study showed no edema after 3 hr medial cerebral artery occlusion but pronounced edema after 8 hr reperfusion. The edema was abolished by inhibitors of specifically β1-adrenergic pathways, indicating failure of K+-mediated, but not β1-adrenoceptor-mediated, stimulation of Na+,K+-ATPase/NKCC1 transport during reoxygenation. Ninety percent reduction of extracellular Ca2+ concentration occurs in ischemia. Ca2+ omission abolished K+ uptake in normoxic cultures of astrocytes after addition of 5 mM KCl. A large decrease in ouabain potency on K+ uptake in cultured astrocytes was also demonstrated in Ca2+-depleted media, and endogenous ouabains are needed for astrocytic K+ uptake. Thus, among the ionic changes induced by ischemia, the decrease in extracellular Ca2+ causes failure of the high-K+-stimulated Na+,K+-ATPase/NKCC1 ion/water uptake, making β1-adrenergic activation the only stimulus and its inhibition effective against edema.Infarct size and brain edema following ischemia/reperfusion are reduced by inhibitors of the Na The EGFR tyrosine kinase inhibitor, Tyrphostin AG 1478 (N-[(2R)-2-(hydroxamidocarbonymethyl)-4-methylpentanoyl]-L-tryptophan methylamide); the metalloproteinase inhibitor, GM 6001 ; the MEK inhibitor, U0126, ; and the Src inhibitor, PP1 (4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolopyrimidine) were obtained from Calbiochem . Horse serum was either drawn from selected animals . 42K was obtained from New England Nuclear, Lachine, Quebec (no longer available). Pentobarbital sodium was obtained from Sinopharm Chemical Reagent Co., Ltd. .Most chemicals, including isoproterenol, ethacrynic acid, ouabain hydrochloride, the  animals  or boughμL physiological saline, at the following concentrations: ethacrynic 50 mM; betaxolol 3 mM; ICI118551 10 μM; H89 500 μM; PTX 20 μM; PP1 1 mM; AG1478 100 μM; GM6001 1 mM; U0126 1 mM. With a brain weight in the rat of 1-2 g the intracerebral concentrations are probably at least 100 times smaller but dependent upon the distribution, which may vary between the individual drugs.All experiments were carried out in accordance with the USA National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications number 80-23) revised 1978, and all experimental protocols were approved by the Institutional Animal Care and Use Committee of China Medical University. The experiments were carried out using rats, because it would have been very difficult to effectively perform MCAO in mice, used routinely for our cultures. In general, rats and mice are believed to react in rather similar manners to brain ischemia. Male Sprague-Dawley rats, weighing 280–320 g, were housed in cages on a 12 h light/dark cycle in a temperature-controlled (23–25°C) colony room with free access to food and water. Focal brain ischemia was induced unilaterally by occlusion of the right middle cerebral artery (MCAO) as previously described by Longa et al.  in animaWet weights (WW) were separately measured in right hemispheres (ischemia side) and left hemispheres (control side) after removal of cerebellum, pons, and olfactory bulbs. Dry weights (DW) were determined after 24 hr heating at 120°C as described by Bigdeli et al. , and per2, 7.5 mM glucose, and 20% horse serum. The cultures were incubated at 37°C in a humidified atmosphere of CO2/air (5 : 95%). The medium was exchanged with fresh medium of similar composition on day 3 and subsequently every 3-4 days. At day 3, the serum concentration was reduced to 10%, and after the age of 2 weeks, 0.25 mM dibutyryl cyclic AMP (dBcAMP) was included in the medium. This compound increases intracellular cyclic AMP and promotes differentiation in astrocyte cultures derived from newborn brain. Recently the usefulness of astrocyte cultures has been authoritatively reviewed by Lange et al. i, which in astrocytes leads to metalloproteinase-mediated release of a growth factor that transactivates the epidermal growth factor receptor (EGFR). Both the metalloproteinase inhibitor GM6001 and the EGFR inhibitor AG1478 inhibit ERK1/2 phosphorylation. So does U0126, an inhibitor of ERK1/2 phosphorylation. As can be seen from β1-adrenergic edema formation in vivo after ischemia/reperfusion. In contrast, the Src inhibitor PP1 that is active in the β2- but not β1-adrenergic signaling [ in vivo results further supports the validity of the used cultures as models of their in situ counterparts. This is important for the studies described below.The signaling pathways for both hibitors . As showignaling , has a signaling  had no e2+ in the medium addition of 5 mM KCl causes a relatively fast increase in intracellular K+ concentration similar in magnitude to that previously observed [2+ no such increase occurs. In contrast the effect of the β1-adrenergic agonist dobutamine is independent of the presence of Ca2+.observed . In the 42K uptake by μM concentrations of ouabain, expressed as percentage of the uninhibited rate in cultures incubated either in medium with a normal Ca2+ concentration (1.8 mM CaCl2) or in medium without added Ca2+. Since no Ca2+ chelator was added, this medium is likely to contain traces of Ca2+. The K+ concentration in the medium was 5.4 mM with 42K adding ~0.3 mM. Under control conditions (no ouabain present) the uptake rates were identical in the presence and absence of Ca2+ . However, the potency of ouabain was drastically reduced by ouabain as seen from 2+ is obvious at 100 μM ouabain. The potency of ouabain on the fraction with lower affinity was always inhibited by Ca2+ deficiency. However, at the lowest ouabain concentrations there was a tendency towards a negative inhibition, that is, a stimulation, mimicking the stimulatory effect on Na+,K+-ATPase activity normally only observable at low nanomolar ouabain concentrations [trations .+ concentration, decrease in extracellular Ca2+ concentration) have profound effects on edema formation and its inhibition by β1-adrenergic antagonists. The finding that edema does not develop during the ischemic period but only after reoxygenation is consistent with the energy dependence of the Na+,K+-ATPase/NKCC1 transport system and indicates that the MCAO effectively prevented blood supply. It is in agreement with the observation by Goyagi et al. [β1-adrenergic antagonist 30 min after the onset of a 2-hour-long ischemic period drastically reduces infarct size and improves neurological deficit score after 7 days. Iwata et al. [β1-adrenoceptor beginning 60 min after 8 min bilateral carotid artery occlusion combined with hypotension reduced neuronal injury after forebrain ischemia, although motor activity was not improved. Goyagi et al. [β1-adrenergic antagonist beginning before the insult, and motor deficit index scores were significantly lower and neuronal survival was better in rats treated with β1-adrenoceptor antagonists beginning 30 min before 10 min of spinal cord ischemia [β1-adrenergic antagonists provided long-term improvement of histological outcome, they had no effect on neurological outcome and spatial memory retention 14 days later, tested with administration beginning 30 min before the onset of ischemia and continuing for 24 hrs [β1-adrenergic antagonists to improve long-term functional recovery in brain ischemia. This is because β1-adrenergic stimulation exerts beneficial effects, such as the stimulation of glycogenolysis, which is essential for formation of memory [β1-adrenergic antagonist as soon as extracellular K+ concentration has normalized. This will in the presence of β1-adrenergic antagonist probably mainly be by neuronal uptake. It is unlikely to require ouabain-mediated signaling, since it in astrocytes serves to promote Na+ uptake for stimulation of the intracellular site of the Na+,K+-ATPase [+ during excitation.This study has demonstrated that the ionic alterations evoked by ischemia  are consistent with previously observed rates of K+ uptake in similar cultures [+,K+-ATPase subunits [2+ in the medium  [+-stimulated K+ uptake. In the present context it is important that the K+-induced metabolic stimulation is abolished when Ca2+ is excluded from the medium [The rates for active Kcultures  and the subunits . The drae medium  is in agtrations  confirmstrations , 54. The (20 mM) , indicat2+ depletion plays a major role by abolishing K+-induced, but not β1-adrenoceptor-mediated stimulation of the Na+-K+-ATPase/NKCC1 transport system, additional factors may also be involved in edema formation. Thus, the cellular increase in Ca2+, which is the reason for its extracellular decrease, promotes NADH hyperoxidation and electrical dysfunction after anoxia in hippocampal slices [2+ overload, since cultured astrocytes rapidly die following anoxia and reperfusion when the gaseous and interstitial ionic changes of transient brain ischemia are simulated, and their death required external Ca2+ [β1-adrenergic agonists (or of ethacrynic acid) used in the present study is unknown. Finally, it should be noted that prestroke use of β1-blockers in a thorough study was found not to affect stroke severity or outcome clinically [β1-blocker treatment might receive doses providing lower intracerebral concentrations than those employed in this study and needed for protection against edema. To counteract edema formation it may be essential to use high doses of β1-adrenergic antagonists until extracellular K+ has normalized. It is unknown how long that takes, but an at least hour-long increase in extracellular glucose level after seizures [+ level, might suggest that this could take a long time, especially when astrocytic K+ reaccumulation is inhibited.Although the present observations together with literature data thus strongly suggest that extracellular Cal slices . Not onlnal Ca2+ . Importanal Ca2+ . To whatinically . This coseizures , which aβ1-adrenergic inhibition on infarct size and edema in experimental ischemia/reperfusion are convincing, even when added after the onset of ischemia. This is a great advantage for potential clinical use, and it is consistent with the present observation in this study that most or all of the swelling occurs after reperfusion. The study also confirmed an antiedema effect of NKKC1 inhibitors and of β1-adrenergic antagonists and signaling inhibitors, and it presented evidence that abnormal Ca2+ homeostasis resulting from ischemia is the pathophysiological basis for the mechanism by which β1-adrenergic inhibitors can inhibit brain edema after anoxia/reperfusion. This seems to be because Ca2+ deficiency counteracts stimulation of the Na+,K+-ATPase/NKCC1 transport system by elevated extracellular K+, which requires signaling by endogenous ouabains, and is thus dependent on extracellular Ca2+. Accordingly β1-adrenoceptor activity is left as its only astrocytic stimulus, and it was shown to be independent of the presence of extracellular Ca2+. Even in the presence of a β1-adrenergic antagonist extracellular K+ will eventually be reabsorbed by the neuronal Na+,K+-ATPase, which is unlikely to require extracellular Ca2+, but this will not lead to swelling in adult brain, where NKCC1 is highly enriched in astrocytes.The beneficial effects of


At a large model synapse, the calyx of Held, transmitter release is controlled by several Ca2+ channels in a "domain overlap" mode, at least in young animals. To study the geometrical constraints of Ca2+ channel placement in domain overlap control of release, we used stochastic MCell modelling, at active zones for which the position of docked vesicles was derived from electron microscopy (EM). We found that random placement of Ca2+ channels was unable to produce high slope values between release and presynaptic Ca2+ entry, a hallmark of domain overlap, and yielded excessively large release probabilities. The simple assumption that Ca2+ channels can be located anywhere at active zones, except below a critical distance of ~ 30 nm away from docked vesicles ("exclusion zone"), rescued high slope values and low release probabilities. Alternatively, high slope values can also be obtained by placing all Ca2+ channels into a single supercluster, which however results in significantly higher heterogeneity of release probabilities. We also show experimentally that high slope values, and the sensitivity to the slow Ca2+ chelator EGTA-AM, are maintained with developmental maturation of the calyx synapse. Taken together, domain overlap control of release represents a highly organized active zone architecture in which Ca2+ channels must obey a certain distance to docked vesicles. Furthermore, domain overlap can be employed by near-mature, fast-releasing synapses.The spatial arrangement of Ca 2+ channels provide the rise in intracellular Ca2+ concentration necessary to initiate the membrane fusion of transmitter—filled vesicles at synapses. Because Ca2+ diffuses away from Ca2+ channels, the distance between Ca2+ channels and vesicles on the range of tens of nanometers is a crucial determinant of the vesicle fusion probability. However, there is still little experimental evidence on how Ca2+ channels and vesicles co-localize in the nanospace of a single synapse. We show by computational modelling that the channels should be located at some distance to vesicles (~ 30 nm), to allow for release control by several channels, a release mechanism found at many synapses. In realistic synapses with a high density of docked vesicles, this translates into a likely localization of Ca2+ channels at membrane sites not occupied by docked vesicles. Thus, we present a computational model of how Ca2+ channels can be localized in an active zone with several docked vesicles, to enable control of release by several Ca2+ channels.Ca Furthermore, docked vesicles likely create a hindrance for Ca2+ diffusion towards the more distant vesicles . We el; ref. –47. We c2+ channels at active zones of CNS synapses. At hippocampal synapses, freeze-fracture replica labeling has shown several loosely organized, stripe-like clusters of particles detected by an antibody against CaV2.1 (P/Q-type Ca2+ channel)  within an active zone, as predicted by a previous modelling study i. In addition, the lowermost data points at very low release probabilities were often inaccurate despite the number of individual repetitions ; in this case, the lowermost data points were not included in the fit range. In order to evaluate the statistical robustness of the slope estimates, the entire simulation was repeated 3–5 times, using different random seeds for the spatial arrangements of Ca2+ channels; these repetitions are referred to as "independent spatial seeds" in the main text.We used the Monte Carlo simulator MCell  to tracklux e.g. , bottom.2+ was reflected off the walls; the size of the compartment was chosen based on published nearest-neighbor distances of active zones at the immature rat calyx of Held [2+ channels, endogenous Ca2+ buffers and Ca2+ sensors for release. Docked vesicles were present as spheres with 45 nm diameter inaccessible to Ca2+ ions. Ca2+ channels opened and closed stochastically during the AP according to a two-state model  buffer were present in the simulations, with parameters as given in 2+ sensor was a kinetic model of Ca2+ binding and vesicle fusion, with parameters which reflect extensive previous Ca2+ uncaging studies at the calyx of Held synapse. The specific kinetic model was a 5-site model  were in the range of 3–10 MΩ and 5–25 MΩ for post- and presynaptic recordings, and were compensated by up to 80 and 50% for post- and presynaptic recordings respectively, using the patch-clamp amplifier . For postsynaptic recordings, the remaining Rs error was compensated offline. Brief steps to +60 mV of various lengths were applied from a holding potential of -70 mV, resulting in the recruitment of an increasing number of Ca2+ channels giving rise to Ca2+ "tail" currents during the repolarization phase . Mice were killed by decapitation, sometimes following a brief isoflurane anesthesia in the oldest age group, in a protocol approved by the Veterinary office of the Canton of Vaud, Switzerland. Transverse brain slices of 200 μm thickness were made with a vibratome , and stored in a submerged keeping chamber containing a standard bicarbonate buffered solution , bubbled with 95% Ocordings , the exton phase . In fibeon phase , afferen3, 1.25 NaH2PO4, 25 glucose, 1 MgCl2, 2 CaCl2, 0.4 ascorbic acid, 3 myo-inositol and 2 Na-pyruvate, continuously bubbled with 95% O2/5% CO2 (pH 7.4). The intracellular (pipette) solution for pre- and postsynaptic recordings contained (in mM): 130 Cs-gluconate, 20 TEA-Cl, 10 HEPES, 5 Na2-phosphocreatine, 4 MgATP, 0.3 Na2GTP (pH 7.2 adjusted with CsOH). For pre- or postsynaptic recordings, this solution was supplemented with 0.1 or 5 mM EGTA, respectively. All chemicals were from Sigma Aldrich/Fluka  unless indicated.The extracellular solution contained (in mM): 125 NaCl, 2.5 KCl, 25 NaHCOS1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.