Intracellular Buffering Research Articles

Modulation of Dopaminergic Neuronal Excitability by Zinc through the Regulation of Calcium-related Channels.

Depending on the intracellular buffering of calcium by chelation, zinc has the following two apparent effects on neuronal excitability: enhancement or reduction. Zinc increased tonic activity in the depolarized state when neurons were intracellularly dialyzed with EGTA but attenuated the neuronal activity when BAPTA was used as an intracellular calcium buffer. This suggests that neuronal excitability can be modulated by zinc, depending on the internal calcium buffering capacity. In this study, we elucidated the mechanisms of zinc-mediated alterations in neuronal excitability and determined the effect of calcium-related channels on zinc-mediated alterations in excitability. The zinc-induced augmentation of firing activity was mediated via the inhibition of small-conductance calcium-activated potassium (SK) channels with not only the contribution of voltage-gated L-type calcium channels (VGCCs) and ryanodine receptors (RyRs), but also through the activation of VGCCs via melastatin-like transient receptor potential channels. We suggest that zinc modulates the dopaminergic neuronal activity by regulating not only SK channels as calcium sensors, but also VGCCs or RyRs as calcium sources. Our results suggest that the cytosolic calcium-buffering capacity can tightly regulate zinc-induced neuronal firing patterns and that local calcium-signaling domains can determine the physiological and pathological state of synaptic activity in the dopaminergic system.

Chromaffin Cells of the Adrenal Medulla: Physiology, Pharmacology, and Disease.

Chromaffin cells (CCs) of the adrenal gland and the sympathetic nervous system produce the catecholamines (epinephrine and norepinephrine; EPI and NE) needed to coordinate the bodily "fight-or-flight" response to fear, stress, exercise, or conflict. EPI and NE release from CCs is regulated both neurogenically by splanchnic nerve fibers and nonneurogenically by hormones (histamine, corticosteroids, angiotensin, and others) and paracrine messengers [EPI, NE, adenosine triphosphate, opioids, γ-aminobutyric acid (GABA), etc.]. The "stimulus-secretion" coupling of CCs is a Ca2+ -dependent process regulated by Ca2+ entry through voltage-gated Ca2+ channels, Ca2+ pumps, and exchangers and intracellular organelles (RE and mitochondria) and diffusible buffers that provide both Ca2+ -homeostasis and Ca2+ -signaling that ultimately trigger exocytosis. CCs also express Na+ and K+ channels and ionotropic (nAChR and GABAA ) and metabotropic receptors (mACh, PACAP, β-AR, 5-HT, histamine, angiotensin, and others) that make CCs excitable and responsive to autocrine and paracrine stimuli. To maintain high rates of E/NE secretion during stressful conditions, CCs possess a large number of secretory chromaffin granules (CGs) and members of the soluble NSF-attachment receptor complex protein family that allow docking, fusion, and exocytosis of CGs at the cell membrane, and their recycling. This article attempts to provide an updated account of well-established features of the molecular processes regulating CC function, and a survey of the as-yet-unsolved but important questions relating to CC function and dysfunction that have been the subject of intense research over the past 15 years. Examples of CCs as a model system to understand the molecular mechanisms associated with neurodegenerative diseases are also provided. Published 2019. Compr Physiol 9:1443-1502, 2019.

Relationship between skeletal muscle carnosine content and cycling sprint performance

Background: High-intensity exercise is limited by muscle acidosis, among other factors. Supplementation with buffering agents can delay the onset of muscular fatigue and improve performance ( Saunders et al., 2017 , Heibel et al., 2018 ). These include supplements that can increase the extracellular buffering capacity (e.g., sodium bicarbonate, which increases blood bicarbonate) and the intracellular buffering capacity (e.g., beta-alanine, which increases muscle carnosine). Since high-intensity performance has been associated with hydrogen ion buffering capacity ( Rampinini et al., 2009 ), we sought to examine whether pre-exercise blood bicarbonate concentration and muscle carnosine content are associated with repeated-sprint cycling performance. Purpose: To determine the relationship between blood bicarbonate and muscle carnosine of trained cyclists and high-intensity performance during the 4-bout Wingate cycling test. Methods: Forty-one trained male cyclists (age: 35 ± 7 y; weight: 74.7 ± 10.5 kg; height: 1.78 ± 0.08 m) that had been training for at least 2 years (experience: 9 ± 8 years; training hours: 12 ± 6 hours∙week -1 ; training distance: 261 ± 165 km∙week -1 ) participated in the study. Participants were required not to have used supplements containing creatine in the 3 months, or beta-alanine in the 6 months prior to the study. A 4-bout Wingate cycling test (30 s all-out with 3-min passive recovery between bouts) protocol was performed on a mechanically braked cycle-ergometer. Total mechanical work (TMW) was determined for each single bout and for the overall protocol (i.e., the four bouts altogether). Blood samples were taken immediately before and after exercise for the determination of blood pH and bicarbonate using a blood gas analyser (Rapid Point 350, Siemens, Germany). Sample muscles were obtained from the m. vastus lateralis using the biopsy technique; carnosine was determined using tandem liquid chromatography-mass spectrometry (HPLC-ESI + -MS/MS) as described by Carvalho et al. (2018) . Data are presented as mean ± 1 standard deviation. The relationship between blood bicarbonate and muscle carnosine variables and exercise performance was determined using Pearson’s correlation and a paired t-test was used to compare blood variables before and after the exercise test. All data were analysed using GraphPad 6.0 and statistical signii¬cance was accepted at p 0.05; Figure 2). A significant decrease in blood bicarbonate (rest 29.6 ± 2.1 vs post 14.6 ± 3.7 mmol∙L -1 ; p < 0.0001) and blood pH (rest 7.353 ± 0.035 vs post 7.048 ± 0.094; p < 0.0001; Figure 3) was shown following the cycling protocol. Discussion: This study showed no association between repeated sprint cycling performance and blood bicarbonate or muscle carnosine, suggesting that neither initial blood bicarbonate concentration nor muscle carnosine content influences performance during this type of exercise. Interestingly, sodium bicarbonate and beta-alanine supplementation have previously been shown to improve repeated Wingate performance, particularly in the final bouts ( Tobias et al., 2013 , Artioli et al., 2007 , Painelli et al., 2014 ). Since fatigue during high-intensity activity is multifactorial, this may indicate that small baseline variations in blood bicarbonate and muscle carnosine are not large enough to translate into performance benefits. It is likely that supplementation with sodium bicarbonate and beta-alanine, on the other hand, result in changes of sufficient magnitude to delay fatigue and improve performance. Conclusions: The current study showed that initial levels of blood bicarbonate and muscle carnosine were not associated with repeated Wingate performance, suggesting that these variables are not good predictors of cycling sprint performance.

Metal ligands in micronutrient acquisition and homeostasis.

Acquisition and homeostasis of micronutrients such as iron (Fe) and zinc (Zn) pose specific challenges. Poor solubility and high reactivity require controlled synthesis and supply of ligands to complex these metals extracellularly and intracellularly. Cytosolic labile pools represent only a minute fraction of the total cellular content. Several low-molecular-weight ligands are known in plants, including sulfur ligands (cysteine and peptides), nitrogen/oxygen ligands (S-adenosyl-l-methionine-derived molecules and histidine), and oxygen ligands (phenolics and organic acids). Some ligands are secreted into the extracellular space and influence the phytoavailability of metal ions. A second principal function is the intracellular buffering of micronutrients as well as the facilitation of long-distance transport in xylem and phloem. Furthermore, low-molecular-weight ligands are involved in the storage of metals, predominantly in vacuoles. A detailed molecular understanding is hampered by technical limitations, in particular the difficulty to detect and quantify cellular metal-ligand complexes. More, but still too little, is known about ligand synthesis and the transport across membranes, either with or without a complexed metal. Metal ligands have an immediate impact on human well-being. Engineering metal ligand synthesis and distribution in crops has tremendous potential to improve the nutritional quality of food and to tackle major human health risks.

Unraveling the Potential Role of Glutathione in Multiple Forms of Cell Death in Cancer Therapy.

Glutathione is the principal intracellular antioxidant buffer against oxidative stress and mainly exists in the forms of reduced glutathione (GSH) and oxidized glutathione (GSSG). The processes of glutathione synthesis, transport, utilization, and metabolism are tightly controlled to maintain intracellular glutathione homeostasis and redox balance. As for cancer cells, they exhibit a greater ROS level than normal cells in order to meet the enhanced metabolism and vicious proliferation; meanwhile, they also have to develop an increased antioxidant defense system to cope with the higher oxidant state. Growing numbers of studies have implicated that altering the glutathione antioxidant system is associated with multiple forms of programmed cell death in cancer cells. In this review, we firstly focus on glutathione homeostasis from the perspectives of glutathione synthesis, distribution, transportation, and metabolism. Then, we discuss the function of glutathione in the antioxidant process. Afterwards, we also summarize the recent advance in the understanding of the mechanism by which glutathione plays a key role in multiple forms of programmed cell death, including apoptosis, necroptosis, ferroptosis, and autophagy. Finally, we highlight the glutathione-targeting therapeutic approaches toward cancers. A comprehensive review on the glutathione homeostasis and the role of glutathione depletion in programmed cell death provide insight into the redox-based research concerning cancer therapeutics.

The Physiological Roles of Carnosine and β-Alanine in Exercising Human Skeletal Muscle.

Carnosine (β-alanyl-L-histidine) plays an important role in exercise performance and skeletal muscle homeostasis. Dietary supplementation with the rate-limiting precursor β-alanine leads to an increase in skeletal muscle carnosine content, which further potentiates its effects. There is significant interest in carnosine and β-alanine across athletic and clinical populations. Traditionally, attention has been given to performance outcomes with less focus on the underlying mechanism(s). Putative physiological roles in human skeletal muscle include acting as an intracellular pH buffer, modulating energy metabolism, regulating Ca handling and myofilament sensitivity, and scavenging of reactive species. Emerging evidence shows that carnosine could also act as a cytoplasmic Ca-H exchanger and form stable conjugates with exercise-induced reactive aldehydes. The enigmatic nature of carnosine means there is still much to learn regarding its actions and applications in exercise, health, and disease. In this review, we examine the research relating to each physiological role attributed to carnosine, and its precursor β-alanine, in exercising human skeletal muscle.

Paying the Toll for Inflammation

Paying the Toll for Inflammation

Presynaptic loss of dynamin-related protein 1 impairs synaptic vesicle release and recycling at the mouse calyx of Held.

This study characterizes the mechanisms underlying defects in synaptic transmission when dynamin-related protein 1 (DRP1) is genetically eliminated. Viral-mediated knockout of DRP1 from the presynaptic terminal at the mouse calyx of Held increased initial release probability, reduced the size of the synaptic vesicle recycling pool and impaired synaptic vesicle recycling. Transmission defects could be partially restored by increasing the intracellular calcium buffering capacity with EGTA-AM, implying close coupling of Ca2+ channels to synaptic vesicles was compromised. Acute restoration of ATP to physiological levels in the presynaptic terminal did not reverse the synaptic defects. Loss of DRP1 impairs mitochondrial morphology in the presynaptic terminal, which in turn seems to arrest synaptic maturation. Impaired mitochondrial biogenesis and function is implicated in many neurodegenerative diseases, and likely affects synaptic neurotransmission prior to cellular loss. Dynamin-related protein 1 (DRP1) is essential for mitochondrial fission and is disrupted in neurodegenerative disease. In this study, we used the mouse calyx of Held synapse as a model to investigate the impact of presynaptic DRP1 loss on synaptic vesicle (SV) recycling and sustained neurotransmission. In vivo viral expression of Cre recombinase in ventral cochlear neurons of floxed-DRP1 mice generated a presynaptic-specific DRP1 knockout (DRP1-preKO), where the innervated postsynaptic cell was unperturbed. Confocal reconstruction of the calyx terminal suggested SV clusters and mitochondrial content were disrupted, and presynaptic terminal volume was decreased. Using postsynaptic voltage-clamp recordings, we found that DRP1-preKO synapses had larger evoked responses at low frequency stimulation. DRP1-preKO synapses also had profoundly altered short-term plasticity, due to defects in SV recycling. Readily releasable pool size, estimated with high-frequency trains, was dramatically reduced in DRP1-preKO synapses, suggesting an important role for DRP1 in maintenance of release-competent SVs at the presynaptic terminal. Presynaptic Ca2+ accumulation in the terminal was also enhanced in DRP1-preKO synapses. Synaptic transmission defects could be partially rescued with EGTA-AM, indicating close coupling of Ca2+ channels to SV distance normally found in mature terminals may be compromised by DRP1-preKO. Using paired recordings of the presynaptic and postsynaptic compartments, recycling defects could not be reversed by acute dialysis of ATP into the calyx terminals. Taken together, our results implicate a requirement for mitochondrial fission to coordinate postnatal synapse maturation.

Apparent calcium dependence of vesicle recruitment.

Synaptic transmission relies on the recruitment of neurotransmitter-filled vesicles to presynaptic release sites. Increased intracellular calcium buffering slows the recovery from synaptic depression, suggesting that vesicle recruitment is a calcium-dependent process. However, the molecular mechanisms of vesicle recruitment have only been investigated at some synapses. We investigate the role of calcium in vesicle recruitment at the cerebellar mossy fibre to granule cell synapse. We find that increased intracellular calcium buffering slows the recovery from depression following physiological stimulation. However, the recovery is largely resistant to perturbation of the molecular pathways previously shown to mediate calcium-dependent vesicle recruitment. Furthermore, we find two pools of vesicles with different recruitment speeds and show that models incorporating two pools of vesicles with different calcium-independent recruitment rates can explain our data. In this framework, increased calcium buffering prevents the release of intrinsically fast-recruited vesicles but does not change the vesicle recruitment rates themselves. During sustained synaptic transmission, recruitment of new transmitter-filled vesicles to the release site counteracts vesicle depletion and thus synaptic depression. An elevated intracellular Ca2+ concentration has been proposed to accelerate the rate of vesicle recruitment at many synapses. This conclusion is often based on the finding that increased intracellular Ca2+ buffering slows the recovery from synaptic depression. However, the molecular mechanisms of the activity-dependent acceleration of vesicle recruitment have only been analysed at some synapses. Using physiological stimulation patterns in postsynaptic recordings and step depolarizations in presynaptic bouton recordings, we investigate vesicle recruitment at cerebellar mossy fibre boutons. We show that increased intracellular Ca2+ buffering slows recovery from depression dramatically. However, pharmacological and genetic interference with calmodulin or the calmodulin-Munc13 pathway, which has been proposed to mediate Ca2+ -dependence of vesicle recruitment, barely affects vesicle recovery from depression. Furthermore, we show that cerebellar mossy fibre boutons have two pools of vesicles: rapidly fusing vesicles that recover slowly and slowly fusing vesicles that recover rapidly. Finally, models adopting such two pools of vesicles with Ca2+ -independent recruitment rates can explain the slowed recovery from depression upon increased Ca2+ buffering. Our data do not rule out the involvement of the calmodulin-Munc13 pathway during stronger stimuli or other molecular pathways mediating Ca2+ -dependent vesicle recruitment at cerebellar mossy fibre boutons. However, we show that well-established two-pool models predict an apparent Ca2+ -dependence of vesicle recruitment. Thus, previous conclusions of Ca2+ -dependent vesicle recruitment based solely on increased intracellular Ca2+ buffering should be considered with caution.

Comparison of sustained-release and rapid-release \u03b2-alanine formulations on changes in skeletal muscle carnosine and histidine content and isometric performance following a muscle-damaging protocol

β-alanine supplementation increases muscle carnosine content and improves anaerobic exercise performance by enhancing intracellular buffering capacity. β-alanine ingestion in its traditional rapid-release formulation (RR) is associated with the symptoms of paresthesia. A sustained-release formulation (SR) of β-alanine has been shown to circumvent paresthesia and extend the period of supply to muscle for carnosine synthesis. The purpose of this investigation was to compare 28 days of SR and RR formulations of β-alanine (6 g day−1) on changes in carnosine content of the vastus lateralis and muscle fatigue. Thirty-nine recreationally active men and women were assigned to one of the three groups: SR, RR, or placebo (PLA). Participants supplementing with SR and RR formulations increased muscle carnosine content by 50.1% (3.87 mmol kg−1ww) and 37.9% (2.62 mmol kg−1ww), respectively. The change in muscle carnosine content in participants consuming SR was significantly different (p = 0.010) from those consuming PLA, but no significant difference was noted between RR and PLA (p = 0.077). Although participants ingesting SR experienced a 16.4% greater increase in muscle carnosine than RR, fatigue during maximal voluntary isometric contractions was significantly attenuated in both SR and RR compared to PLA (p = 0.002 and 0.024, respectively). Symptoms of paresthesia were significantly more frequent in RR compared to SR, the latter of which did not differ from PLA. Results of this study demonstrated that only participants consuming the SR formulation experienced a significant increase in muscle carnosine. Differences in the muscle carnosine response between these formulations may have practical significance for athletic populations in which small changes may have important implications on performance.

Abstract 3486: Metabolomics in estrogen receptor or triple-negative breast cancer

Abstract Breast cancer is the most frequently diagnosed cancer in women. Metabolic syndrome and obesity may contribute to triple-negative breast cancer (TNBC) and there are obesity-related alterations in metabolites. Therefore, we pilot-tested metabolomics in ER-negative or TNBC using urine samples from 60 breast cancer cases. In total, 475 metabolites were quantified in different super-pathways: 147 in amino acid, 38 in carbohydrate, 27 in cofactors and vitamins, 12 in energy, 50 in lipid, 31 in nucleotide, 17 in peptide, and 153 in xenobiotics. Our data in 13 ER- and 47 ER+ showed that urinary beta-alanine is higher (3.35+3.73 vs. 1.82+2.26) in ER- breast cancer patients. Our data also show, the first time, that the urinary beta-alanine was higher in TNBC (3.60+4.33; N=8) compared to other subtypes (1.93+2.32; N=52). Beta-alanine is a nonessential amino acid that has previously been shown to be metabolized into carnosine, which functions as an intracellular buffer. In breast cancer cells, beta-alanine reduces both tumor cell migration and proliferation without acting in a cytotoxic fashion. It also significantly increased breast cancer cell sensitivity to doxorubicin. Our data on xanthine are also consistent with the previous study on breast tumor that slightly higher level was observed in ER- patient (1.41+1.35 vs. 1.16+0.88). The level of xanthine was significantly higher in TNBC compared to other subtypes (1.67+1.69 vs. 1.14+0.84). More importantly, our pilot data also showed that urinary mannose levels were significantly higher in ER- tumor (2.21+1.90 vs. 0.90+1.21 p=0.032) and TNBC (2.19+1.77 vs. 1.03+1.38, p=0.037), which is consistent with a previous study that reported that serum mannose levels can differentiate patients with metastatic and localized breast cancer. Metabolic drugs for cancer, such as glutaminase inhibitors, are currently under development and being tested in clinical trials. Therefore, if our data are validated, metabolomics of ER- or TNBC will contribute to innovative discoveries that will impact breast cancer prevention and precision medicine targeting metabolomic pathways. Citation Format: Jennifer J. Hu, Cristiane Takita, Eunkyung Lee, Jean Wright. Metabolomics in estrogen receptor or triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3486.

PKA and PKC Modulators Affect Ion Channel Function and Internalization of Recombinant Alpha1 and Alpha1-Beta Glycine Receptors.

Glycine receptors (GlyRs) are important mediators of fast inhibitory neurotransmission in the mammalian central nervous system. Their function is controlled by multiple cellular mechanisms, including intracellular regulatory processes. Modulation of GlyR function by protein kinases has been reported for many cell types, involving different techniques, and often yielding contradictory results. Here, we studied the effects of protein kinase C (PKC) and cAMP-dependent protein kinase A (PKA) on glycine induced currents in HEK293 cells expressing human homomeric α1 and heteromeric α1-β GlyRs using whole-cell patch clamp techniques as well as internalization assays. In whole-cell patch-clamp measurements, modulators were applied in the intracellular buffer at concentrations between 0.1 μM and 0.5 μM. EC50 of glycine increased upon application of the protein kinase activators Forskolin and phorbol-12-myristate-13-acetate (PMA) but decreased in the presence of the PKC inhibitor Staurosporine aglycon and the PKA inhibitor H-89. Desensitization of recombinant α1 receptors was significantly increased in the presence of Forskolin. Staurosporine aglycon, on the other hand decreased desensitization of heteromeric α1-β GlyRs. The time course of receptor activation was determined for homomeric α1 receptors and revealed two simultaneous effects: cells showed a decrease of EC50 after 3–6 min of establishing whole-cell configuration. This effect was independent of protein kinase modulators. All modulators of PKA and PKC, however, produced an additional shift of EC50, which overlay and eventually exceeded the cells intrinsic variation of EC50. The effect of kinase activators was abolished if the corresponding inhibitors were co-applied, consistent with PKA and PKC directly mediating the modulation of GlyR function. Direct effects of PKA- and PKC-modulators on receptor expression on transfected HEK cells were monitored within 15 min of drug application, showing a significant increase of receptor internalization with PKA and PKC activators, while the corresponding inhibitors had no significant effect on receptor surface expression or internalization. Our results confirm the observation that phosphorylation via PKA and PKC has a direct effect on the GlyR ion channel complex and plays an important role in the fine-tuning of glycinergic signaling.

Effect of Skeletal Muscle Carnosine Content on Apoptotic Signaling

Carnosine (Carn) has a pluripotent role in skeletal muscle (SkM) physiology, enhancing intracellular buffering, acting as an antioxidant, antiglycating and ion‐chelating agent, and also acts as a diffusible Ca2+/H+ exchanger. Carn role as an antioxidant may reduce apoptosis, but has not been fully investigated in human SkM. Therefore, the purpose of this study was to investigate if SkM Carn influences apoptotic signaling in response resistance exercise. Fourteen recreational active men completed a lower‐body resistance exercise protocol consisting of the squat, leg press, and leg extension exercises. Blood samples were obtained before (PRE), immediately‐(IP), one‐(1H), five‐(5H), 24‐(24H) &amp; 48‐(48H) hours post‐resistance exercise. SkM biopsies were obtained from the vastus lateralis at BL, 1H, 5H, &amp; 48H post‐resistance exercise. SkM carnosine content was assessed via HPLC. Participants were placed into either a high Carn (HI: n = 7; Carn = 12.25 ± 2.40 mmol·kg−1WW) or a low Carn (LO: n =7; Carn = 5.60 ± 1.27 mmol·kg−1WW) group for analysis. Multiplex signaling assay kits were used to quantify phosphorylation status of apoptotic markers (JNK, FADD, p53, BAD, Bcl‐2). Circulating measures of muscle damage [myoglobin (Mb)] and oxidative stress [8‐hydroxy‐2′ – deoxyguanosine (8OHdG)] were assessed via ELISA. To control for BL differences a repeated measures analysis of covariance was used to analyze SkM data. Area under the curve (AUC) analysis was determined by the trapezoidal method. Although no significant interactions between the groups were observed for FADD, BAD, p53, Mb, or 8‐OHdG (p &gt; 0.05)in response to resistance exercise, significant interactions were observed for JNK (p = 0.019) and Bcl‐2 (p = 0.032). Post‐hoc analysis indicated a significantly greater response at 1H for HI compared to LO in JNK (p = 0.035), while a trend towards an increase (p = 0.065) was observed for Bcl‐2. In addition, changes in Bcl‐2 and JNK following exercise were only significant in HI (p = 0.009 and p = 0.007, respectively), but not LO (p = 0.370 and p = 0.369, respectively). AUC analysis for 8OHdG indicated that circulating concentrations for HI (6,107.5 ± 2,144.6 ng·mL−1·min−1) was significantly (p = 0.037, D = 1.25) less than LO (8,838.7 ± 2,222.3 ng·mL−1·min−1). In conclusion, our results indicated that participants with higher Carn have a reduction in pro‐apoptotic proteins, with an attenuated circulating oxidative stress response to exercise.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inducible glutathione S-transferase (IrGST1) from the tick Ixodes ricinus is a haem-binding protein

Blood-feeding parasites are inadvertently exposed to high doses of potentially cytotoxic haem liberated upon host blood digestion. Detoxification of free haem is a special challenge for ticks, which digest haemoglobin intracellularly. Ticks lack a haem catabolic mechanism, mediated by haem oxygenase, and need to dispose of vast majority of acquired haem via its accumulation in haemosomes. The knowledge of individual molecules involved in the maintenance of haem homeostasis in ticks is still rather limited. RNA-seq analyses of the Ixodes ricinus midguts from blood- and serum-fed females identified an abundant transcript of glutathione S-transferase (gst) to be substantially up-regulated in the presence of red blood cells in the diet. Here, we have determined the full sequence of this encoding gene, ir-gst1, and found that it is homologous to the delta-/epsilon-class of GSTs. Phylogenetic analyses across related chelicerates revealed that only one clear IrGST1 orthologue could be found in each available transcriptome from hard and soft ticks. These orthologues create a well-supported clade clearly separated from other ticks' or mites’ delta-/epsilon-class GSTs and most likely evolved as an adaptation to tick blood-feeding life style. We have confirmed that IrGST1 expression is induced by dietary haem(oglobin), and not by iron or other components of host blood. Kinetic properties of recombinant IrGST1 were evaluated by model and natural GST substrates. The enzyme was also shown to bind haemin in vitro as evidenced by inhibition assay, VIS spectrophotometry, gel filtration, and affinity chromatography. In the native state, IrGST1 forms a dimer which further polymerises upon binding of excessive amount of haemin molecules. Due to susceptibility of ticks to haem as a signalling molecule, we speculate that the expression of IrGST1 in tick midgut functions as intracellular buffer of labile haem pool to ameliorate its cytotoxic effects upon haemoglobin intracellular hydrolysis.

Intracellular fixation buffer inactivates Newcastle disease virus in chicken allantoic fluid, macrophages and splenocytes

Inactivation of Newcastle disease virus (NDV) has been routinely achieved with heat, β-propiolactone, binary ethylenimine, ultraviolet light and formalin. However, these strategies have not been tested for cell surface ligand or receptor phenotype in viral-infected chicken immune cells. To study the capacity of fixation buffers to preserve surface markers while inactivating NDV, a primary splenocyte culture was infected with NDV and incubated with a commercial intracellular fixation buffer (ICB), formulated with 4% formaldehyde. Splenocytes were fixed with a 1:2 dilution of ICB in phosphate buffered saline (PBS) for 45min at 23°C or 4°C and inactivation of NDV was tested in addition to recognition of antigens by antibodies in fixed and non-fixed splenocytes via flow cytometric analysis. The binding and percentage of splenic CD4+ and CD8+ cells were not affected. In addition, NDV titers as high as 109.5 and 107.6 EID50 in allantoic fluid (AF) and macrophages, respectively, were successfully inactivated after 45min at 23°C and 4°C, confirming the ICB’s effectiveness in inactivating high concentrations of NDV. In conclusion, high concentrations of NDV in AF, chicken splenocytes, and macrophages can be inactivated using ICB. Additionally, this method did not compromise cell phenotyping of enriched chicken splenocytes.

Increased Ca buffering underpins remodelling of Ca2+ handling in old sheep atrial myocytes.

Key points Ageing is associated with an increased risk of cardiovascular disease and arrhythmias, with the most common arrhythmia being found in the atria of the heart.Little is known about how the normal atria of the heart remodel with age and thus why dysfunction might occur.We report alterations to the atrial systolic Ca2+ transient that have implications for the function of the atrial in the elderly.We describe a novel mechanism by which increased Ca buffering can account for changes to systolic Ca2+ in the old atria.The present study helps us to understand how the processes regulating atrial contraction are remodelled during ageing and provides a basis for future work aiming to understand why dysfunction develops. Many cardiovascular diseases, including those affecting the atria, are associated with advancing age. Arrhythmias, including those in the atria, can arise as a result of electrical remodelling or alterations in Ca2+ homeostasis. In the atria, age‐associated changes in the action potential have been documented. However, little is known about remodelling of intracellular Ca2+ homeostasis in the healthy aged atria. Using single atrial myocytes from young and old Welsh Mountain sheep, we show the free Ca2+ transient amplitude and rate of decay of systolic Ca2+ decrease with age, whereas sarcoplasmic reticulum (SR) Ca content increases. An increase in intracellular Ca buffering explains both the decrease in Ca2+ transient amplitude and decay kinetics in the absence of any change in sarcoendoplasmic reticulum calcium transport ATPase function. Ageing maintained the integrated Ca2+ influx via I Ca‐L but decreased peak I Ca‐L. Decreased peak I Ca‐L was found to be responsible for the age‐associated increase in SR Ca content but not the decrease in Ca2+ transient amplitude. Instead, decreased peak I Ca‐L offsets increased SR load such that Ca2+ release from the SR was maintained during ageing. The results of the present study highlight a novel mechanism by which increased Ca buffering decreases systolic Ca2+ in old atria. Furthermore, for the first time, we have shown that SR Ca content is increased in old atrial myocytes.

The intracellular angiotensin system buffers deleterious effects of the extracellular paracrine system

The ‘classical’ renin–angiotensin system (RAS) is a circulating system that controls blood pressure. Local/paracrine RAS, identified in a variety of tissues, including the brain, is involved in different functions and diseases, and RAS blockers are commonly used in clinical practice. A third type of RAS (intracellular/intracrine RAS) has been observed in some types of cells, including neurons. However, its role is still unknown. The present results indicate that in brain cells the intracellular RAS counteracts the intracellular superoxide/H2O2 and oxidative stress induced by the extracellular/paracrine angiotensin II acting on plasma membrane receptors. Activation of nuclear receptors by intracellular or internalized angiotensin triggers a number of mechanisms that protect the cell, such as an increase in the levels of protective angiotensin type 2 receptors, intracellular angiotensin, PGC-1α and IGF-1/SIRT1. Interestingly, this protective mechanism is altered in isolated nuclei from brains of aged animals. The present results indicate that at least in the brain, AT1 receptor blockers acting only on the extracellular or paracrine RAS may offer better protection of cells.

Metabolic inhibition reduces cardiac L-type Ca2+ channel current due to acidification caused by ATP hydrolysis.

Metabolic stress evoked by myocardial ischemia leads to impairment of cardiac excitation and contractility. We studied the mechanisms by which metabolic inhibition affects the activity of L-type Ca2+ channels (LTCCs) in frog ventricular myocytes. Metabolic inhibition induced by the protonophore FCCP (as well as by 2,4- dinitrophenol, sodium azide or antimycin A) resulted in a dose-dependent reduction of LTCC current (ICa,L) which was more pronounced during β-adrenergic stimulation with isoprenaline. ICa,L was still reduced by metabolic inhibition even in the presence of 3 mM intracellular ATP, or when the cell was dialysed with cAMP or ATP-γ-S to induce irreversible thiophosphorylation of LTCCs, indicating that reduction in ICa,L is not due to ATP depletion and/or reduced phosphorylation of the channels. However, the effect of metabolic inhibition on ICa,L was strongly attenuated when the mitochondrial F1F0-ATP-synthase was blocked by oligomycin or when the cells were dialysed with the non-hydrolysable ATP analogue AMP-PCP. Moreover, increasing the intracellular pH buffering capacity or intracellular dialysis of the myocytes with an alkaline solution strongly attenuated the inhibitory effect of FCCP on ICa,L. Thus, our data demonstrate that metabolic inhibition leads to excessive ATP hydrolysis by the mitochondrial F1F0-ATP-synthase operating in the reverse mode and this results in intracellular acidosis causing the suppression of ICa,L. Limiting ATP break-down by F1F0-ATP-synthase and the consecutive development of intracellular acidosis might thus represent a potential therapeutic approach for maintaining a normal cardiac function during ischemia.

FCS experiments to quantify Ca2+ diffusion and its interaction with buffers.

Ca2+ signals are ubiquitous. One of the key factors for their versatility is the variety of spatio-temporal distributions that the cytosolic Ca2+ can display. In most cell types Ca2+ signals not only depend on Ca2+ entry from the extracellular medium but also on Ca2+ release from internal stores, a process which is in turn regulated by cytosolic Ca2+ itself. The rate at which Ca2+ is transported, the fraction that is trapped by intracellular buffers, and with what kinetics are thus key features that affect the time and spatial range of action of Ca2+ signals. The quantification of Ca2+ diffusion in intact cells is quite challenging because the transport rates that can be inferred using optical techniques are intricately related to the interaction of Ca2+ with the dye that is used for its observation and with the cellular buffers. In this paper, we introduce an approach that uses Fluorescence Correlation Spectroscopy (FCS) experiments performed at different conditions that in principle allows the quantification of Ca2+ diffusion and of its reaction rates with unobservable (non-fluorescent) Ca2+ buffers. To this end, we develop the necessary theory to interpret the experimental results and then apply it to FCS experiments performed in a set of solutions containing Ca2+, a single wavelength Ca2+ dye, and a non-fluorescent Ca2+ buffer. We show that a judicious choice of the experimental conditions and an adequate interpretation of the fitting parameters can be combined to extract information on the free diffusion coefficient of Ca2+ and of some of the properties of the unobservable buffer. We think that this approach can be applied to other situations, particularly to experiments performed in intact cells.

Non-linear impact of glutathione depletion on C. elegans life span and stress resistance

The redox environment in cells and organisms is set by low-molecular mass and protein-bound thiols, with glutathione (GSH) representing a major intracellular redox buffer. Subtle thiol oxidation elicits signal transduction processes and adaptive responses to cope with stressors, whereas highly oxidizing conditions may provoke cell death. We here tested how thiol depletion affects life span, stress resistance and stress signaling in the model organism Caenorhabditis elegans. Diethyl maleate (DEM), an α,β-unsaturated carbonyl compound that conjugates to GSH and other thiols, decreased C. elegans life span at a concentration of 1mM. In contrast, low and moderate doses of DEM (10–100µM) increased mean and maximum life span and improved resistance against oxidative stress. DEM-induced life span extension was not detectable in worms deficient in either the FoxO orthologue, DAF-16, or the Nrf2 orthologue, SKN-1, pointing to a collaborative role of the two transcription factors in life span extension induced by thiol depletion. Cytoprotective target genes of DAF-16 and SKN-1 were upregulated after at least 3 days of exposure to 100µM DEM, but not 1mM DEM, whereas only 1mM DEM caused upregulation of egl-1, a gene controlled by a p53-orthologue, CEP-1. In order to test whether depletion of GSH may elicit effects similar to DEM, we suppressed GSH biosynthesis in worms by attenuating γ-glutamylcysteine synthetase (gcs-1) expression through RNAi. The decline in GSH levels elicited by gcs-1 knockdown starting at young adult stage did not impair viability, but increased both stress resistance and life expectancy of the worms. In contrast, gcs-1 knockdown commencing right after hatching impaired nematode stress resistance and rendered young adult worms prone to vulval ruptures during egg-laying. Thus, modest decrease in GSH levels in young adult worms may promote stress resistance and life span, whereas depletion of GSH is detrimental to freshly hatched and developing worms.