Calcium chelation independent effects of BAPTA on endogenous ANO6 channels in HEK293T cells.

Selective calcium chelator 1,2-Bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) is a common tool to investigate calcium signaling. However, BAPTA expresses various effects on intracellular calcium signaling, which are not related to its ability to bind Ca2+. In patch clamp experiments, we investigated calcium chelation independent effects of BAPTA on endogenous calcium-activated chloride channels ANO6 (TMEM16F) in HEK293T cells. We have found that application of BAPTA to intracellular solution led to two distinct effects on channels properties. On the one hand, application of BAPTA acutely reduced amplitude of endogenous ANO6 channels induced by 10 µM Ca2+ in single channel recordings. On the other hand, BAPTA application by itself induced ANO6 channel activity in the absence of the intracellular calcium elevation. Open channel probability was enhanced by increasing the intracellular BAPTA concentration from 0.1 to 1 and 10 mM. Another calcium chelator EGTA did not demonstrate chelation independent effects on the ANO6 activity in the same conditions. Due to off-target effects BAPTA should be used with caution when studying calcium-activated ANO6 channels.

Effect of EDTA with porous balloon on calcified lesion: An atherosclerotic cadaver study.

BACKGROUND: Ethylene diamine tetra-acetic acid (EDTA) is a chelating agent used to dissolve calcium deposits but evidence in decalcifying atherosclerotic lesions is limited. AIMS: We assessed the feasibility and efficacy of EDTA delivered via porous balloon to target calcified lesions in cadaveric below-the-knee (BTK) arteries. METHODS: Using porcine carotid arteries, EDTA concentration was measured in the arterial wall and outside the artery at the 0-, 0.5-, 4-, and 24-h circulation after the injection through a porous balloon. In cadaver BTK samples, the proximal and distal anterior tibial artery (ATA) and distal posterior tibial artery (PTA) were studied. EDTA-2Na/H2O or EDTA-3Na/H2O were administrated using a porous balloon, then circulated for 6 h for EDTA-3Na/H2O and 24 h for EDTA-2Na/H2O and EDTA-3Na/H2O. Micro-CT imaging of the artery segments before and after the circulation and cross-sectional analyses were performed to evaluate calcium burden. RESULTS: In the porcine carotid study, EDTA was delivered through a porous balloon present in the arterial wall and was retained there for 24 h. In BTK arteries, cross-sectional analyses of micro-CT revealed a significant decrease in the calcium area in the distal ATA segment under 24-h circulation with EDTA-2Na/H2O and in the distal ATA segment under 24-h circulation with EDTA-3Na/H2O. The proximal ATA segment under 6-h circulation with EDTA-3Na/H2O showed no significant change in any parameters of calcium CONCLUSION: EDTA-3Na/H2O or EDTA-2Na/H2O with longer circulation times resulted in greater calcium reduction in atherosclerotic lesion. EDTA may have a potential therapeutic option for the treatment of atherosclerotic calcified lesions.

Maintaining calcium homeostasis as a strategy to alleviate nephrotoxicity caused by evodiamine.

Evodiamine (EVO), the main active alkaloid in Evodia rutaecarpa, was shown to exert various pharmacological activities, especially anti-tumor. Currently, it is considered a potential anti-cancer drug due to its excellent anti-tumor activity, which unfortunately has adverse reactions, such as the risk of liver and kidney injury, when Evodia rutaecarpa containing EVO is used clinically. In the present study, we aim to clarify the potential toxic target organs and toxicity mechanism of EVO, an active monomer in Evodia rutaecarpa, and to develop mitigation strategies for its toxicity mechanism. Transcriptome analysis and related experiments showed that the PI3K/Akt pathway induced by calcium overload was an important step in EVO-induced apoptosis of renal cells. Specifically, intracellular calcium ions were increased, and mitochondrial calcium ions were decreased. In addition, EVO-induced calcium overload was associated with TRPV1 receptor activation. In vivo TRPV1 antagonist and calcium chelator effects were observed to significantly reduce body weight loss and renal damage in mice due to EVO toxicity. The potential nephrotoxicity of EVO was further confirmed by an in vivo test. In conclusion, TRPV1-mediated calcium overload-induced apoptosis is one of the mechanisms contributing to the nephrotoxicity of EVO due to its toxicity, whereas maintaining body calcium homeostasis is an effective measure to reduce toxicity. These studies suggest that the clinical use of EVO-containing herbal medicines should pay due attention to the changes in renal function of patients as well as the off-target effects of the drugs.

Fast recovery of disrupted tip links induced by mechanical displacement of hair bundles.

Hearing and balance rely on the capacity of mechanically sensitive hair bundles to transduce vibrations into electrical signals that are forwarded to the brain. Hair bundles possess tip links that interconnect the mechanosensitive stereocilia and convey force to the transduction channels. A dimer of dimers, each of these links comprises two molecules of protocadherin 15 (PCDH15) joined to two of cadherin 23 (CDH23). The "handshake" that conjoins the four molecules can be disrupted in vivo by intense stimulation and in vitro by exposure to Ca2+ chelators. Using hair bundles from the rat's cochlea and the bullfrog's sacculus, we observed that extensive recovery of mechanoelectrical transduction, hair bundle stiffness, and spontaneous bundle oscillation can occur within seconds after Ca2+ chelation, especially if hair bundles are deflected toward their short edges. Investigating the phenomenon in a two-compartment ionic environment that mimics natural conditions, we combined iontophoretic application of a Ca2+ chelator to selectively disrupt the tip links of individual frog hair bundles with displacement clamping to control hair bundle motion and measure forces. Our observations suggest that, after the normal Ca2+ concentration has been restored, mechanical stimulation facilitates the reconstitution of functional tip links.

Mitochondrial dysfunctions trigger the calcium signaling-dependent fungal multidrug resistance.

Drug resistance in fungal pathogens has risen steadily over the past decades due to long-term azole therapy or triazole usage in agriculture. Modification of the drug target protein to prevent drug binding is a major recognized route to induce drug resistance. However, mechanisms for nondrug target-induced resistance remain only loosely defined. Here, we explore the molecular mechanisms of multidrug resistance resulted from an efficient adaptation strategy for survival in drug environments in the human pathogen Aspergillus fumigatus We show that mutants conferring multidrug resistance are linked with mitochondrial dysfunction induced by defects in heme A biosynthesis. Comparison of the gene expression profiles between the drug-resistant mutants and the parental wild-type strain shows that multidrug-resistant transporters, chitin synthases, and calcium-signaling-related genes are significantly up-regulated, while scavenging mitochondrial reactive oxygen species (ROS)-related genes are significantly down-regulated. The up-regulated-expression genes share consensus calcium-dependent serine threonine phosphatase-dependent response elements (the binding sites of calcium-signaling transcription factor CrzA). Accordingly, drug-resistant mutants show enhanced cytosolic Ca2+ transients and persistent nuclear localization of CrzA. In comparison, calcium chelators significantly restore drug susceptibility and increase azole efficacy either in laboratory-derived or in clinic-isolated A. fumigatus strains. Thus, the mitochondrial dysfunction as a fitness cost can trigger calcium signaling and, therefore, globally up-regulate a series of embedding calcineurin-dependent-response-element genes, leading to antifungal resistance. These findings illuminate how fitness cost affects drug resistance and suggest that disruption of calcium signaling might be a promising therapeutic strategy to fight against nondrug target-induced drug resistance.

Calcium Ion Chelation Preserves Platelet Function During Cold Storage.

OBJECTIVE: Platelet transfusion is a life-saving therapy to prevent or treat bleeding in patients with thrombocytopenia or platelet dysfunction. However, for >6 decades, safe and effective strategies for platelet storage have been an impediment to widespread use of platelet transfusion. Refrigerated platelets are cleared rapidly from circulation, precluding cold storage of platelets for transfusion. Consequently, platelets are stored at room temperature with an upper limit of 5 days due to risks of bacterial contamination and loss of platelet function. This practice severely limits platelet availability for transfusion. This study is to identify the mechanism of platelet clearance after cold storage and develop a method for platelet cold storage. Approach and Results: We found that rapid clearance of cold-stored platelets was largely due to integrin activation and apoptosis. Deficiency of integrin ß3 or caspase-3 prolonged cold-stored platelets in circulation. Pretreatment of platelets with EGTA, a cell impermeable calcium ion chelator, reversely inhibited cold storage-induced platelet activation and consequently prolonged circulation of cold-stored platelets. Moreover, transfusion of EGTA-treated, cold-stored platelets, but not room temperature-stored platelets, into the mice deficient in glycoprotein Ibα significantly shortened tail-bleeding times and diminished blood loss. CONCLUSIONS: Integrin activation and apoptosis is the underlying mechanism of rapid clearance of platelets after cold storage. Addition of a cell impermeable calcium ion chelator to platelet products is potentially a simple and effective method to enable cold storage of platelets for transfusion.

Suppression of Presynaptic Glutamate Release by Postsynaptic Metabotropic NMDA Receptor Signalling to Pannexin-1.

The impact of pannexin-1 (Panx1) channels on synaptic transmission is poorly understood. Here, we show that selective block of Panx1 in single postsynaptic hippocampal CA1 neurons from male rat or mouse brain slices causes intermittent, seconds long increases in the frequency of sEPSC following Schaffer collateral stimulation. The increase in sEPSC frequency occurred without an effect on evoked neurotransmission. Consistent with a presynaptic origin of the augmented glutamate release, the increased sEPSC frequency was prevented by bath-applied EGTA-AM or TTX. Manipulation of a previously described metabotropic NMDAR pathway (i.e., by preventing ligand binding to NMDARs with competitive antagonists or blocking downstream Src kinase) also increased sEPSC frequency similar to that seen when Panx1 was blocked. This facilitated glutamate release was absent in transient receptor potential vanilloid 1 (TRPV1) KO mice and prevented by the TRPV1 antagonist, capsazepine, suggesting it required presynaptic TRPV1. We show presynaptic expression of TRPV1 by immunoelectron microscopy and link TRPV1 to Panx1 because Panx1 block increases tissue levels of the endovanilloid, anandamide. Together, these findings demonstrate an unexpected role for metabotropic NMDARs and postsynaptic Panx1 in suppression of facilitated glutamate neurotransmission.SIGNIFICANCE STATEMENT The postsynaptic ion and metabolite channel, pannexin-1, is regulated by metabotropic NMDAR signaling through Src kinase. This pathway suppresses facilitated release of presynaptic glutamate during synaptic activity by regulating tissue levels of the transient receptor potential vanilloid 1 agonist anandamide.

The interplay between α7 nicotinic acetylcholine receptors, pannexin-1 channels and P2X7 receptors elicit exocytosis in chromaffin cells.

Pannexin-1 (Panx1) forms plasma membrane channels that allow the exchange of small molecules between the intracellular and extracellular compartments, and are involved in diverse physiological and pathological responses in the nervous system. However, the signaling mechanisms that induce their opening still remain elusive. Here, we propose a new mechanism for Panx1 channel activation through a functional crosstalk with the highly Ca2+ permeable α7 nicotinic acetylcholine receptor (nAChR). Consistent with this hypothesis, we found that activation of α7 nAChRs induces Panx1-mediated dye uptake and ATP release in the neuroblastoma cell line SH-SY5Y-α7. Using membrane permeant Ca2+ chelators, total internal reflection fluorescence microscopy in SH-SY5Y-α7 cells expressing a membrane-tethered GCAMP3, and Src kinase inhibitors, we further demonstrated that Panx1 channel opening depends on Ca2+ signals localized in submembrane areas, as well as on Src kinases. In turn, Panx1 channels amplify cytosolic Ca2+ signals induced by the activation of α7 nAChRs, by a mechanism that seems to involve ATP release and P2X7 receptor activation, as hydrolysis of extracellular ATP with apyrase or blockage of P2X7 receptors with oxidized ATP significantly reduces the α7 nAChR-Ca2+ signal. The physiological relevance of this crosstalk was also demonstrated in neuroendocrine chromaffin cells, wherein Panx1 channels and P2X7 receptors contribute to the exocytotic release of catecholamines triggered by α7 nAChRs, as measured by amperometry. Together these findings point to a functional coupling between α7 nAChRs, Panx1 channels and P2X7 receptors with physiological relevance in neurosecretion.

In vivo optochemical control of cell contractility at single-cell resolution.

The spatial and temporal dynamics of cell contractility plays a key role in tissue morphogenesis, wound healing, and cancer invasion. Here, we report a simple optochemical method to induce cell contractions in vivo during Drosophila morphogenesis at single-cell resolution. We employed the photolabile Ca2+ chelator o-nitrophenyl EGTA to induce bursts of intracellular free Ca2+ by laser photolysis in the epithelial tissue. Ca2+ bursts appear within seconds and are restricted to individual target cells. Cell contraction reliably followed within a minute, causing an approximately 50% drop in the cross-sectional area. Increased Ca2+ levels are reversible, and the target cells further participated in tissue morphogenesis. Depending on Rho kinase (ROCK) activity but not RhoGEF2, cell contractions are paralleled with non-muscle myosin II accumulation in the apico-medial cortex, indicating that Ca2+ bursts trigger non-muscle myosin II activation. Our approach can be, in principle, adapted to many experimental systems and species, as no specific genetic elements are required.

Omega-3 and Omega-6 polyunsaturated fatty acids stimulate vascular differentiation of mouse embryonic stem cells.

Polyunsaturated fatty acids (PUFAs) and their metabolites may influence cell fate regulation. Herein, we investigated the effects of linoleic acid (LA) as ω-6 PUFA, eicosapentaenoic acid (EPA) as ω-3 PUFA and palmitic acid (PA) on vasculogenesis of embryonic stem (ES) cells. LA and EPA increased vascular structure formation and protein expression of the endothelial-specific markers fetal liver kinase-1, CD31 as well as VE-cadherin, whereas PA was without effect. LA and EPA increased reactive oxygen species (ROS) and nitric oxide (NO), activated endothelial NO synthase (eNOS) and raised intracellular calcium. The calcium response was inhibited by the intracellular calcium chelator BAPTA, sulfo-N-succinimidyl oleate which is an antagonist of CD36, the scavenger receptor for fatty acid uptake as well as by a CD36 blocking antibody. Prevention of ROS generation by radical scavengers or the NADPH oxidase inhibitor VAS2870 and inhibition of eNOS by L-NAME blunted vasculogenesis. PUFAs stimulated AMP activated protein kinase-α (AMPK-α) as well as peroxisome proliferator-activated receptor-α (PPAR-α). AMPK activation was abolished by calcium chelation as well as inhibition of ROS and NO generation. Moreover, PUFA-induced vasculogenesis was blunted by the PPAR-α inhibitor GW6471. In conclusion, ω-3 and ω-6 PUFAs stimulate vascular differentiation of ES cells via mechanisms involving calcium, ROS and NO, which regulate function of the energy sensors AMPK and PPAR-α and determine the metabolic signature of vascular cell differentiation.

Mechanical stretch induces myelin protein loss in oligodendrocytes by activating Erk1/2 in a calcium-dependent manner.

Myelin loss in the brain is a common occurrence in traumatic brain injury (TBI) that results from impact-induced acceleration forces to the head. Fast and abrupt head motions, either resulting from violent blows and/or jolts, cause rapid stretching of the brain tissue, and the long axons within the white matter tracts are especially vulnerable to such mechanical strain. Recent studies have shown that mechanotransduction plays an important role in regulating oligodendrocyte progenitors cell differentiation into oligodendrocytes. However, little is known about the impact of mechanical strain on mature oligodendrocytes and the stability of their associated myelin sheaths. We used an in vitro cellular stretch device to address these questions, as well as characterize a mechanotransduction mechanism that mediates oligodendrocyte responses. Mechanical stretch caused a transient and reversible myelin protein loss in oligodendrocytes. Cell death was not observed. Myelin protein loss was accompanied by an increase in intracellular Ca2+ and Erk1/2 activation. Chelating Ca2+ or inhibiting Erk1/2 activation was sufficient to block the stretch-induced loss of myelin protein. Further biochemical analyses revealed that the stretch-induced myelin protein loss was mediated by the release of Ca2+ from the endoplasmic reticulum (ER) and subsequent Ca2+ -dependent activation of Erk1/2. Altogether, our findings characterize an Erk1/2-dependent mechanotransduction mechanism in mature oligodendrocytes that de-stabilizes the myelination program.

A centrosome-localized calcium signal is essential for mammalian cell mitosis.

Mitosis defects can lead to premature ageing and cancer. Understanding mitosis regulation therefore has important implications for human disease. Early data suggested that calcium (Ca2+) signals could influence mitosis, but these have hitherto not been observed in mammalian cells. Here, we reveal a prolonged yet spatially restricted Ca2+ signal at the centrosomes of actively dividing cells. Local buffering of the centrosomal Ca2+ signals, by flash photolysis of the caged Ca2+ chelator diazo-2-acetoxymethyl ester, arrests mitosis. We also provide evidence that this Ca2+ signal emanates from the endoplasmic reticulum. In summary, we characterize a unique centrosomal Ca2+ signal as a functionally essential input into mitosis.-Helassa, N., Nugues, C., Rajamanoharan, D., Burgoyne, R. D., Haynes, L. P. A centrosome-localized calcium signal is essential for mammalian cell mitosis.

Modulation of calcium signaling depends on the oligosaccharide of GM1 in Neuro2a mouse neuroblastoma cells.

Recently, we demonstrated that the oligosaccharide portion of ganglioside GM1 is responsible, via direct interaction and activation of the TrkA pathway, for the ability of GM1 to promote neuritogenesis and to confer neuroprotection in Neuro2a mouse neuroblastoma cells. Recalling the knowledge that ganglioside GM1 modulates calcium channels activity, thus regulating the cytosolic calcium concentration necessary for neuronal functions, we investigated if the GM1-oligosaccharide would be able to overlap the GM1 properties in the regulation of calcium signaling, excluding a specific role played by the ceramide moiety inserted into the external layer of plasma membrane. We observed, by calcium imaging, that GM1-oligosaccharide administration to undifferentiated Neuro2a cells resulted in an increased calcium influx, which turned out to be mediated by the activation of TrkA receptor. The biochemical analysis demonstrated that PLCγ and PKC activation follows the TrkA stimulation by GM1-oligosaccharide, leading to the opening of calcium channels both on the plasma membrane and on intracellular storages, as confirmed by calcium imaging experiments performed with IP3 receptor inhibitor. Subsequently, we found that neurite elongation in Neuro2a cells was blocked by subtoxic administration of extracellular and intracellular calcium chelators, suggesting that the increase of intracellular calcium is responsible of GM1-oligosaccharide mediated differentiation. These results suggest that GM1-oligosaccharide is responsible for the regulation of calcium signaling and homeostasis at the base of the neuronal functions mediated by plasma membrane GM1.

Calcium chelation: a novel approach to reduce cryopreservation-induced damage to frozen platelets.

BACKGROUND: Cryopreserved platelets are phenotypically and functionally different to conventionally stored platelets. Calcium may be released from internal stores during the freeze-thaw process, initiating signaling events which lead to these alterations. It was hypothesized that the addition of a calcium chelator prior to cryopreservation may mitigate some of these changes. METHODS: Buffy coat-derived platelets that had been pooled and split were tested fresh and following cryopreservation (n = 8 per group). Platelets were cryopreserved using 5%-6% dimethylsulfoxide (DMSO) or were supplemented with increasing concentrations of the internal calcium chelator, BAPTA-AM (100 µM, 200 µM, or 400 µM), prior to storage at -80°C. RESULTS: Supplementation of platelets with BAPTA-AM prior to freezing improved platelet recovery in a dose response manner (400 µM: 84 ± 2%) compared to standard DMSO cryopreserved platelets (70 ± 4%). There was a loss of GPIbα, GPVI, and GPIIb/IIIa receptors on platelets following cryopreservation, which was rescued when platelets were supplemented with BAPTA-AM (400 µM: p < 0.0001 for all). Platelet activation markers, such as phosphatidylserine and P-selectin, were externalized on platelets following cryopreservation. However, the addition of BAPTA-AM significantly reduced the increase of these activation markers on cryopreserved platelets (400 µM: p < 0.0001 for both). Both cryopreserved platelet groups exhibited similar functionality as assessed by thromboelastography, forming clots at a faster rate than fresh platelets. CONCLUSIONS: This study demonstrates that calcium plays a crucial role in mediating cryopreservation-induced damage to frozen platelets. The addition of the calcium chelator, BAPTA-AM, prior to cryopreservation reduces this damage.

Long-term effects of citric acid-based bicarbonate haemodialysis on patient outcomes: a survival propensity score-matched study in western France.

BACKGROUND: Citric acid-based bicarbonate haemodialysis (CIT-HD) has gained more clinical acceptance over the last few years in France and is a substitute for other acidifiers [e.g. acetic acid (CH3COOH) and hydrochloric acid (HCl)]. This trend was justified by several clinical benefits compared with CH3COOH as well as the desire to avoid the consequences of the corrosive action of HCl, but a nationwide clinical report raised concerns about the long-term safety of CIT-HD. The aim of this study was to assess the long-term effects of CIT-HD exposure on patient outcomes in western France. METHODS: This is a population-based retrospective multicentre observational study performed in 1132 incident end-stage kidney disease patients in five sanitary territories in western France who started their renal replacement therapy after 1 January 2008 and followed up through 15 October 2018. Relevant data, collected prospectively with the same medical software, were anonymously aggregated for the purposes of the study. The primary goal of this study was to investigate the effects of citrate exposure on all-cause mortality. To provide a control group to CIT-HD one, propensity score matching (PSM) at 2:1 was performed in two steps: the first analysis was intended to be exploratory, comparing patients who received citrate ≤80% of the time (CIT-HD ≤80) versus those who received citrate >80% of the time (CIT-HD >80), while the second analysis was intended to be explanatory in comparing patients with 0% (CIT-HD0) versus 100% citrate time exposure (CIT-HD100). RESULTS: After PSM, in the exploratory part of the analysis, 432 CIT-HD ≤80 patients were compared with 216 CIT-HD >80 patients and no difference was found for all-cause mortality using the Kaplan-Meier model (log-rank 0.97), univariate Cox regression analysis {hazard ratio [HR] 1.01 [95% confidence interval (CI) 0.71-1.40]} and multivariate Cox regression analysis [HR 1.11 (95% CI 0.76-1.61)] when adjusted for nine variables with clinical pertinence and high statistical relevance in the univariate analysis. In the explanatory part of the analysis, 316 CIT-HD0 patients were then compared with 158 CIT-HD100 patients and no difference was found using the Kaplan-Meier model (log-rank 0.06), univariate Cox regression analysis [HR 0.69 (95% CI 0.47-1.03)] and multivariate Cox regression analysis [HR 0.87 (95% CI 0.57-1.33)] when adjusted for seven variables with clinical pertinence and high statistical relevance in the univariate analysis. CONCLUSIONS: Findings of this study support the notion that CIT-HD exposure ≤6 years has no significant effect on all-cause mortality in HD patients. This finding remains true for patients receiving high-volume online haemodiafiltration, a modality most frequently prescribed in this cohort.

Long-term mortality risk associated with citric acid- and acetic acid-based bicarbonate haemodialysis: a historical cohort propensity score-matched study in a large, multicentre, population-based study.

BACKGROUND: Citric acid-based bicarbonate dialysate (CiD) is increasingly used in haemodialysis (HD) to improve haemodynamic tolerance and haemocompatibility associated with acetic acid-based bicarbonate dialysate. Safety concerns over CiD have been raised recently after a French ecological study reported higher mortality hazard in HD clinics with high CiD consumption. Therefore, we evaluated the mortality risk associated with various acidifiers (AcD, CiD) of bicarbonate dialysate. METHODS: In this multicentre, historical cohort study, we included adult incident HD patients (European, Middle-East and Africa Fresenius Medical Care network; 1 January 2014 to 31 October 2018). We recorded acidifiers of bicarbonate dialysis and dialysate composition for each dialysis session. In the primary intention-to-treat analysis, patients were assigned to the exposed group if they received CiD in >70% of sessions during the first 3 months (CiD70%), whereas the non-exposed group received no CiD at all. In the secondary analysis, exposure was assessed on a monthly basis for the whole duration of the follow-up. RESULTS: We enrolled 10 121 incident patients during the study period. Of them, 371 met the criteria for inclusion in CiD70%. After propensity score matching, mortality was 11.43 [95% confidence interval (CI) 8.86-14.75] and 12.04 (95% CI 9.44-15.35) deaths/100 person-years in the CiD0% and CiD70% groups, respectively (P = 0.80). A similar association trend was observed in the secondary analysis. CONCLUSIONS: We did not observe evidence of increased mortality among patients exposed to CiD in a large European cohort of dialysis patients despite the fact that physicians were more inclined to prescribe CiD to subjects with worse medical conditions.

Impact of the dialysate acid component on haemodialysis mortality rates.

BACKGROUND: No prospective study has evaluated the long-term effect on mortality of the new acid concentrates added to bicarbonate dialysate. The aim of this pharmacoepidemiological study was to evaluate the association between hydrochloric or citric acid-based dialysate and mortality on haemodialysis (HD). METHODS: This study included 117 796 patients with 3 723 887 months on HD recorded in the national French Renal Epidemiology and Information Network registry. Dialysate acid components were retrospectively reconstructed for each facility. All patients on HD were associated each month with an exposure based on that at their facility of treatment. We took each patient's time-varying exposure into account to calculate the monthly mortality rates for each exposure. Incidence rate ratios (IRRs) for mortality were calculated with a Poisson regression, with acetic acid as the reference. Regressions were adjusted for initial clinical characteristics (age, gender, previous cardiovascular events, active malignancy, diabetes, pulmonary disease, mobility), dialysis technique and location (in-centre, outpatient centre, self-care unit) and ESRD vintage, updated monthly. RESULTS: The crude mortality rate per 1000 patient-months with citric acid {11.5 [95% confidence interval (CI) 11.1-12.0]} was lower than with either acetic acid [12.9 (95% CI 12.8-13.1)] or hydrochloric acid [12.8 (95% CI 12.2-13.5)]. For the 2014-17 period, the IRR for mortality with citric acid [adjusted IRR 0.94 (95% CI 0.90-0.99)] and with hydrochloric acid [adjusted IRR 0.86 (95% CI 0.79-0.94)] were significantly lower than with acetic acid. CONCLUSION: This post-marketing study of long-term exposure to dialysate acidifiers at the patient level found the use of citric and hydrochloric acid-based dialysates, compared with acetic acid, was associated with lower mortality.

Interaction between Calcium Chelators and the Activity of P2X7 Receptors in Mouse Motor Synapses.

The ability of P2X7 receptors to potentiate rhythmically evoked acetylcholine (ACh) release through Ca2+ entry via P2X7 receptors and via L-type voltage-dependent Ca2+ channels (VDCCs) was compared by loading Ca2+ chelators into motor nerve terminals. Neuromuscular preparations of the diaphragms of wild-type (WT) mice and pannexin-1 knockout (Panx1-/-) mice, in which ACh release is potentiated by the disinhibition of the L-type VDCCs upon the activation of P2X7 receptors, were used. Miniature end-plate potentials (MEPPs) and evoked end-plate potentials (EPPs) were recorded when the motor terminals were loaded with slow or fast Ca2+ chelators (EGTA-AM or BAPTA-AM, respectively, 50 µM). In WT and Panx1-/- mice, EGTA-AM did not change either spontaneous or evoked ACh release, while BAPTA-AM inhibited synaptic transmission by suppressing the quantal content of EPPs throughout the course of the short rhythmic train (50 Hz, 1 s). In the motor synapses of either WT or Panx1-/- mice in the presence of BAPTA-AM, the activation of P2X7 receptors by BzATP (30 µM) returned the EPP quantal content to the control level. In the neuromuscular junctions (NMJs) of Panx1-/- mice, EGTA-AM completely prevented the BzATP-induced increase in EPP quantal content. After Panx1-/- NMJs were treated with BAPTA-AM, BzATP lost its ability to enhance the EPP quantal content to above the control level. Nitrendipine (1 µM), an inhibitor of L-type VDCCs, was unable to prevent this BzATP-induced enhancement of EPP quantal content to the control level. We propose that the activation of P2X7 receptors may provide additional Ca2+ entry into motor nerve terminals, which, independent of the modulation of L-type VDCC activity, can partially reduce the buffering capacity of Ca2+ chelators, thereby providing sufficient Ca2+ signals for ACh secretion at the control level. However, the activity of both Ca2+ chelators was sufficient to eliminate Ca2+ entry via L-type VDCCs activated by P2X7 receptors and increase the EPP quantal content in the NMJs of Panx1-/- mice to above the control level.

Variations in Ca2+ Influx Can Alter Chelator-Based Estimates of Ca2+ Channel-Synaptic Vesicle Coupling Distance.

The timing and probability of synaptic vesicle fusion from presynaptic terminals is governed by the distance between voltage-gated Ca2+ channels (VGCCs) and Ca2+ sensors for exocytosis. This VGCC-sensor coupling distance can be determined from the fractional block of vesicular release by exogenous Ca2+ chelators, which depends on biophysical factors that have not been thoroughly explored. Using numerical simulations of Ca2+ reaction and diffusion, as well as vesicular release, we examined the contributions of conductance, density, and open duration of VGCCs, and the influence of endogenous Ca2+ buffers on the inhibition of exocytosis by EGTA. We found that estimates of coupling distance are critically influenced by the duration and amplitude of Ca2+ influx at active zones, but relatively insensitive to variations of mobile endogenous buffer. High concentrations of EGTA strongly inhibit vesicular release in close proximity (20-30 nm) to VGCCs if the flux duration is brief, but have little influence for longer flux durations that saturate the Ca2+ sensor. Therefore, the diversity in presynaptic action potential duration is sufficient to alter EGTA inhibition, resulting in errors potentially as large as 300% if Ca2+ entry durations are not considered when estimating VGCC-sensor coupling distances.SIGNIFICANT STATEMENT The coupling distance between voltage-gated Ca2+ channels and Ca2+ sensors for exocytosis critically determines the timing and probability of neurotransmitter release. Perfusion of presynaptic terminals with the exogenous Ca2+ chelator EGTA has been widely used for both qualitative and quantitative estimates of this distance. However, other presynaptic terminal parameters such as the amplitude and duration of Ca2+ entry can also influence EGTA inhibition of exocytosis, thus confounding conclusions based on EGTA alone. Here, we performed reaction-diffusion simulations of Ca2+-driven synaptic vesicle fusion, which delineate the critical parameters influencing an accurate prediction of coupling distance. Our study provides guidelines for characterizing and understanding how variability in coupling distance across chemical synapses could be estimated accurately.

Effects of intravesicular loading of a Ca2+ chelator and depolymerization of actin fibers on neurotransmitter release in frog motor nerve terminals.

Ca2+ -induced Ca2+ release (CICR) via type-3 ryanodine receptor enhances neurotransmitter release in frog motor nerve terminals. To test a possible role of synaptic vesicle in CICR, we examined the effects of loading of EGTA, a Ca2+ chelator, into synaptic vesicles and depolymerization of actin fibers. Intravesicular EGTA loading via endocytosis inhibited the ryanodine sensitive enhancement of transmitter release induced by tetanic stimulation and the associated rises in intracellular-free Ca2+ ([Ca2+ ]i : Ca2+ transients). Latrunculin A, a depolymerizer of actin fibers, enhanced both spontaneous and stimulation-induced transmitter release, but inhibited the enhancement of transmitter release elicited by successive tetanic stimulation. The results suggest a possibility that the activation of CICR from mobilized synaptic vesicles caused the enhancement of neurotransmitter release.