Free Calcium Concentration Research Articles

Evodiamine Inhibits Angiotensin II-Induced Rat Cardiomyocyte Hypertrophy.

To investigate the effects of evodiamine (Evo), a component of Evodiaminedia rutaecarpa (Juss.) Benth, on cardiomyocyte hypertrophy induced by angiotensin II (Ang II) and further explore the potential mechanisms. Cardiomyocytes from neonatal Sprague Dawley rats were isolated and characterized, and then the cadiomyocyte cultures were randomly divided into control, model (Ang II 0.1 μmol/L), and Evo (0.03, 0.3, 3 μmol/L) groups. The cardiomyocyte surface area, protein level, intracellular free calcium ([Ca2+]i) concentration, activity of nitric oxide synthase (NOS) and content of nitric oxide (NO) were measured, respectively. The mRNA expressions of atrial natriuretic factor (ANF), calcineurin (CaN), extracellular signal-regulated kinase-2 (ERK-2), and endothelial nitric oxide synthase (eNOS) of cardiomyocytes were analyzed by real-time reverse transcriptionpolymerase chain reaction. The protein expressions of calcineurin catalytic subunit (CnA) and mitogen-activated protein kinase phosphatase-1 (MKP-1) were detected by Western blot analysis. Compared with the control group, Ang II induced cardiomyocytes hypertrophy, as evidenced by increased cardiomyocyte surface area, protein content, and ANF mRNA expression; increased intracellular free calcium ([Ca2+]i) concentration and expressions of CaN mRNA, CnA protein, and ERK-2 mRNA, but decreased MKP-1 protein expression (P<0.05 or P<0.01). Compared with Ang II, Evo (0.3, 3 μmol/L) significantly attenuated Ang II-induced cardiomyocyte hypertrophy, decreased the [Ca2+]i concentration and expressions of CaN mRNA, CnA protein, and ERK-2 mRNA, but increased MKP-1 protein expression (P<0.05 or P<0.01). Most interestingly, Evo increased the NOS activity and NO production, and upregulated the eNOS mRNA expression (P<0.05). Evo signifificantly attenuated Ang II-induced cardiomyocyte hypertrophy, and this effect was partly due to promotion of NO production, reduction of [Ca2+]i concentration, and inhibition of CaN and ERK-2 signal transduction pathways.

5-Fluorouracil-induced mitochondrial oxidative cytotoxicity and apoptosis are increased in MCF-7 human breast cancer cells by TRPV1 channel activation but not Hypericum perforatum treatment.

5-Fluorouracil (5-FU) is a widely used chemotherapy agent for breast cancer, although drug resistance is a critical issue regarding the use of this agent in the disease. Calcium signaling is a well-known main cause of proliferation and apoptosis in breast cancer cells. Although previous studies have implicated TRPV1 inhibitor, anticancer, and apoptotic roles of Hypericum perforatum (HPer) in several cells, the synergistic inhibition effects of HPer and 5-FU in cancer and the stimulation of ongoing apoptosis have not yet been clarified in MCF-7 cells. Therefore, we investigated the apoptotic and antioxidant properties of 5-FU with/without HPer through activation of TRPV1 in MCF-7 cells. The MCF-7 cells were divided into four groups: the control group, the HPer-treated group (0.3mM), the 5-FU-treated group (25μM), and the 5-FU+HPer-treated group. The intracellular free calcium ion concentration ([Ca2+]i) increased with 5-FU treatments, but they decreased with the HPer and HPer+5-FU treatments. The [Ca2+]i is further decreased in the four groups by TRPV1 channel antagonist (capsazepine and 0.01mM) treatments. However, mitochondrial membrane depolarization and apoptosis levels, and the PARP1, caspase 3, and caspase 9 expression levels were increased by 5-FU treatment, although the values were decreased by the HPer and 5-FU+HPer treatments. Cell viability level was also decreased by 5-FU treatment. In conclusion, antitumor and apoptosis effects of 5-FU are up-regulated by activation of TRPV1 channels, but its action was down-regulated by HPer treatment. It seems that HPer cannot be used for increasing the antitumor effect of 5-FU through modulation of the TRPV1.

Feeding wet distillers grains plus solubles contributes to sarcoplasmic reticulum membrane instability

Feeding wet distillers grains plus solubles (WDGS) increases polyunsaturated fatty acid (PUFA) levels in beef. It was hypothesised that WDGS in feedlot diets increases PUFA concentration in the sarcoplasmic reticulum (SR) membrane, thereby altering membrane integrity, resulting in more rapid intracellular calcium leakage and improved tenderness. The objective of this study was to evaluate this hypothesis. Ninety-six crossbred steers were fed either a corn-based diet with 0% WDGS or 50% WDGS. Fifteen strip loins per treatment were collected, fabricated into steaks, aged and placed under retail display conditions. Steaks were used to measure tenderness, proteolysis, free calcium concentrations, lipid oxidation, sarcomere length and SR membrane fatty acid, phospholipid lipid, neutral lipid and total lipid profiles. Compared with steaks from steers fed 0% WDGS, steaks from steers fed 50% WDGS were more tender (P &lt; 0.05) and had greater (P &lt; 0.05) free calcium concentrations early post-mortem. Feeding 50% WDGS also tended to increase (P &lt; 0.10) total PUFA concentrations, decrease (P &lt; 0.10) total phospholipid concentration and increase (P &lt; 0.10) total neutral lipid concentration for SR membrane. Steaks from steers fed 0% WDGS had greater (P &lt; 0.05) lipid oxidation (TBARS values) than steaks from steers fed 50% WDGS after extended aging. Although differences in tenderness between the two treatments were detected, there were no corresponding differences (P &gt; 0.10) in sarcomere length or proteolysis. This study showed that feeding WDGS may increase tenderness, possibly by increasing free calcium in muscle early post-mortem. However, the true mechanism that contributes to these differences is still unclear.

Chitosan signaling in guard cells requires endogenous salicylic acid.

An elicitor chitosan (CHT) induces stomatal closure but the mechanism remains to be clarified. A phytohormone salicylic acid (SA) is crucial for elicitor-induced defense signaling in plants. Here we investigated whether endogenous SA is required for CHT signaling in guard cells. In the SA-deficient nahG mutant, treatment of CHT did not induce either apoplastic reactive oxygen species (ROS) production or stomatal closure but co-treatment of CHT and SA induced both apoplastic ROS production and stomatal closure, indicating the involvement of endogenous SA in CHT-induced apoplastic ROS production and CHT-induced stomatal closure. Furthermore, CHT induced transient cytosolic free calcium concentration increments in the nahG mutant in the presence of exogenous SA but not in the absence of exogenous SA. These results provide evidence that endogenous SA is a crucial element in CHT-induced stomatal closure.

Silence of inositol 1,4,5-trisphosphate receptor expression decreases cyantraniliprole susceptibility in Bemisia tabaci

Cyantraniliprole is the second active ingredient of anthranilic diamide insecticide, and the first to control a cross-spectrum of chewing and sucking pests such as sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). The inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR) are two families of Ca2+ release channels to raise the cytoplasmic free calcium concentration when it is activated by various extracellular stimuli. Previous study proved the over-expression of ryanodine receptor (RyR) was associated with the resistance to diamide insecticides, while the roles of IP3R in diamide resistance remain unknown. In this study, a full-length cDNA sequence of IP3R was cloned from B. tabaci through RT-PCR and rapid amplification of cDNA ends (RACE). The gene (named BtIP3R) is 9922bps long, with an open reading frame (ORF) of 8202bps, encoding a predicted IP3R of 2733 amino acids. The BtIP3R shares 47–78% identity with other insect IP3Rs. Quantitative real-time PCR (qRT-PCR) analysis showed that the BtIP3R was highly expressed in larva, pseudopupa, and female adult, while lowly expressed in egg and male adult. RNA interference (RNAi) by dietary introduction of double-stranded RNA (dsRNA) of BtIP3R significantly reduced the mRNA levels of the target gene in the adult, and dramatically decreased the susceptibility of adult B. tabaci to cyantraniliprole. The results shed light on further understanding of cyantraniliprole resistance mechanisms in B. tabaci as well as in other insects.

The Lectin Chaperone Calnexin Is Involved in the Endoplasmic Reticulum Stress Response by Regulating Ca2+ Homeostasis in Aspergillus nidulans.

The Ca2+-mediated signaling pathway is crucial for environmental adaptation in fungi. Here we show that calnexin, a molecular chaperone located in the endoplasmic reticulum (ER), plays an important role in regulating the cytosolic free calcium concentration ([Ca2+]c) in Aspergillus nidulans Inactivation of calnexin (ClxA) in A. nidulans caused severe defects in hyphal growth and conidiation under ER stress caused by the ER stress-inducing agent dithiothreitol (DTT) or high temperature. Importantly, defects in the ΔclxA mutant were restored by the addition of extracellular calcium. Furthermore, the CchA/MidA complex (the high-affinity Ca2+ channels), calcineurin (calcium/calmodulin-dependent protein phosphatase), and PmrA (secretory pathway Ca2+ ATPase) were required for extracellular calcium-based restoration of the DTT/thermal stress sensitivity in the ΔclxA mutant. Interestingly, the ΔclxA mutant exhibited markedly reduced conidium formation and hyphal growth defects under the low-calcium condition, which is similar to defects caused by mutations in MidA/CchA. Moreover, the phenotypic defects were further exacerbated in the ΔclxA ΔmidA ΔcchA mutant, which suggested that ClxA and MidA/CchA are both required under the calcium-limiting condition. Using the calcium-sensitive photoprotein aequorin to monitor [Ca2+]c in living cells, we found that ClxA and MidA/CchA complex synergistically coordinate transient increase in [Ca2+]c in response to extracellular calcium. Moreover, ClxA, in particular its luminal domain, plays a role in mediating the transient [Ca2+]c in response to DTT-induced ER stress in the absence of extracellular calcium, indicating ClxA may mediate calcium release from internal calcium stores. Our findings provide new insights into the role of calnexin in the regulation of calcium-mediated response in fungal ER stress adaptation.IMPORTANCE Calnexin is a well-known molecular chaperone conserved from yeast to humans. Although it contains calcium binding domains, little is known about the role of calnexin in Ca2+ regulation. In this study, we demonstrate that calnexin (ClxA) in the filamentous fungus Aspergillus nidulans, similar to the high-affinity calcium uptake system (HACS), is required for normal growth and conidiation under the calcium-limiting condition. The ClxA dysfunction decreases the transient cytosolic free calcium concentration ([Ca2+]c) induced by a high extracellular calcium or DTT-induced ER stress. Our findings provide the direct evidence that calnexin plays important roles in regulating Ca2+ homeostasis in addition to its role as a molecular chaperone in fungi. These results provide new insights into the roles of calnexin and expand knowledge of fungal stress adaptation.

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using calcium-sensitive fluorescent dyes that change their emission intensity or wavelength depending on the concentration of free calcium in the cell. There are several methods used to stain cells with calcium dyes. Most common are the processes of loading the dyes through a micropipette or pre-incubating with the acetoxymethyl ester forms of the dyes. However, these methods are not quite applicable to neuromuscular junctions (NMJs) due to methodological issues that arise. In this article, we present a method for loading a calcium-sensitive dye through the frog nerve stump of the frog nerve into the nerve endings. Since entry of external calcium into nerve terminals and the subsequent binding to the calcium dye occur within the millisecond time-scale, it is necessary to use a fast imaging system to record these interactions. Here, we describe a protocol for recording the calcium transient with a fast CCD camera.

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction.

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using calcium-sensitive fluorescent dyes that change their emission intensity or wavelength depending on the concentration of free calcium in the cell. There are several methods used to stain cells with calcium dyes. Most common are the processes of loading the dyes through a micropipette or pre-incubating with the acetoxymethyl ester forms of the dyes. However, these methods are not quite applicable to neuromuscular junctions (NMJs) due to methodological issues that arise. In this article, we present a method for loading a calcium-sensitive dye through the frog nerve stump of the frog nerve into the nerve endings. Since entry of external calcium into nerve terminals and the subsequent binding to the calcium dye occur within the millisecond time-scale, it is necessary to use a fast imaging system to record these interactions. Here, we describe a protocol for recording the calcium transient with a fast CCD camera.

Dermal exposure to weathered MC252 crude oil results in echocardiographically identifiable systolic myocardial dysfunction in double-crested cormorants (Phalacrocorax auritus)

During the Deepwater Horizon Natural Resource Damage Assessment, gross morphologic cardiac abnormalities, including softer, more distensible musculature, were noted upon gross necropsy in hearts from laughing gulls and double-crested cormorants exposed to weathered MC252 crude oil. A species specific, echocardiographic technique was developed for antemortem evaluation of function that was used to evaluate and better characterize cardiac dysfunction. Control (n=12) and treated (n=13) cormorant groups of similar sex-ratio and ages were dermally treated with approximately 13ml of water or weathered MC252 crude oil, respectively, every 3 days for 6 dosages. This resulted in a low to moderate external exposure. Upon visualization and clinical assessment of the hearts of all test subjects, comprehensive diagnostic cardiographic measurements were taken twice, prior to oil application and after a 21day dermal oil exposure. Oil-treated birds showed a decrease in cardiac systolic function, as characterized by an increased left ventricular internal dimension-systole and left ventricular stroke volume as well as concurrent decreased left ventricular ejection fraction and left ventricular fractional shortening when compared to both control birds’ and the treated birds’ time zero values. These changes are indicative of a possible dilative cardiomyopathy induced by oil exposure, although further elucidation of possible collagen damage is recommended. Arrhythmias including tachycardia in two treated birds and bradycardia in all treated birds were documented, indicating further clinically significant abnormalities induced by MC252 oil that warrant further investigation. A statistically significant increase in free calcium concentration, important to muscular and neurologic function in treated birds was also noted. This study documents that weathered MC252 oil caused clinically significant cardiac dysfunction that could result in mortality and decrease recruitment.

Biophysical characterization of nanostructured TiO2 as a good substrate for hBM-MSC adhesion, growth and differentiation

Mesenchymal stem cells from human bone marrow (hBM-MSC) are widely utilized for clinical applications involving bone healing. In this context, their use has been often optimized in association to variously designed titanium substrates, being this material of great use in orthopaedic implants.According to recent findings, the ability of hBM-MSC to differentiate towards a specific lineage is not only driven by biochemical signals, but physical stimuli, such as rigidity or roughness of the substrate, can also support a commitment towards osteogenic differentiation. Moreover, the presence of features with defined dimensional scales, in particular nanometer-size, also proved to elicit specific biological effects. Here we evaluated the effectiveness of a nano-patterned titanium surface in sustaining hBM-MSC adhesion, growth and differentiation by means of a panel of biophysical tools: morphometry, electrophysiology, intracellular calcium measurements and immunocytochemistry. The results substantiate the idea that this micro-textured titanium dioxide is a good surface for growth and differentiation of hBM-MSC and it exhibits a stimulating action mainly in the initial period of differentiation. Moreover, the basal concentration of free cytosolic Calcium [Ca2+]i is confirmed to be a good hallmark of the hBM-MSC maturation stage. The study could provide relevant hints to help improving the biocompatibility and osteointegration potential of clinical titanium implants.

PCramoll and rCramoll lectins induce cell death in human prostate adenocarcinoma (PC-3) cells by impairment of mitochondrial homeostasis

Lectins from Cratylia mollis seed have shown potential in vivo antitumor actions, however the mechanism have not yet been addressed. Here we evaluated the antitumor effects of native (pCramoll) and recombinant (rCramoll) lectins from C. mollis against human prostate adenocarcinoma (PC-3) cells. The viability of PC-3 cells was analyzed with the MTT assay and ANNEXIN V/propidium iodide staining. The actions of pCramoll or rCramoll on mitochondrial superoxide production, free cytosolic calcium concentration and mitochondrial membrane potential were evaluated using fluorescent probes (MitoSox Red, Fura 2-AM and safranin O, respectively). pCramoll and rCramoll reduced the viability of PC-3 cells in a dose-dependent manner. Both lectins increased the generation of mitochondrial superoxide as well as the concentration of cytosolic calcium. These changes led to a decrease in oxidative phosphorylation, which impaired the formation of ATP. The resulting cell death was not blocked by MPT (mitochondrial permeability transition) inhibitors (Debio 025 or bongkrekic acid). Thus pCramoll and rCramoll promote PC-3 cell death through calcium signaling, leading to mitochondrial collapse. This work provides more insights into the action of pCramoll and rCramoll against cancer cells. These lectins represent valuable tools for biomedical research.

Self-organized mechano-chemical dynamics in amoeboid locomotion of Physarum fragments

The aim of this work is to quantify the spatio-temporal dynamics of flow-driven amoeboid locomotion in small (∼100 μm) fragments of the true slime mold Physarum polycephalum. In this model organism, cellular contraction drives intracellular flows, and these flows transport the chemical signals that regulate contraction in the first place. As a consequence of these non-linear interactions, a diversity of migratory behaviors can be observed in migrating Physarum fragments. To study these dynamics, we measure the spatio-temporal distributions of the velocities of the endoplasm and ectoplasm of each migrating fragment, the traction stresses it generates on the substratum, and the concentration of free intracellular calcium. Using these unprecedented experimental data, we classify migrating Physarum fragments according to their dynamics, finding that they often exhibit spontaneously coordinated waves of flow, contractility and chemical signaling. We show that Physarum fragments exhibiting symmetric spatio-temporal patterns of endoplasmic flow migrate significantly slower than fragments with asymmetric patterns. In addition, our joint measurements of ectoplasm velocity and traction stress at the substratum suggest that forward motion of the ectoplasm is enabled by a succession of stick-slip transitions, which we conjecture are also organized in the form of waves. Combining our experiments with a simplified convection-diffusion model, we show that the convective transport of calcium ions may be key for establishing and maintaining the spatio-temporal patterns of calcium concentration that regulate the generation of contractile forces.

Structural features of the lipid bilayer of the red blood cell membrane in people with isolated hypercholesterolemia without coronary artery disease and in patients with coronary artery disease

To identify the specific features of modifying the lipid composition of the red blood cell membrane in people with isolated hypercholesterolemia without coronary heart disease (CHD) and in CHD patients with different functional classes of angina pectoris. 92 men with Functional Class II-IV stable angina, 22 men with isolated hypercholesterolemia (HC) without CHD, and 18 healthy men with normal cholesterol levels were examined. Blood plasma cholesterol levels were determined in all the examinees. The erythrocyte membrane was examined for the levels of lipid peroxidation (LPO) products, free cholesterol, calcium, and phospholipid fractions and for the activity of Na+/K+-, Mg2+- and Ca2+-ATPases; a bicycle exercise test was carried out. As compared with persons with normal blood cholesterol levels, the examinees with HC without CHD were found to have higher levels of LPO products, lower levels of readily oxidizable phospholipid fractions, decreased activity of membrane-bound Na+/K+-, Mg2+-, and Ca2+-ATPases, and elevated concentrations of free cholesterol and calcium in the red blood cell membrane. Having the pronounced activity of free radical oxidation, the patients with CHD were detected to have deeper similar structural changes in the cell membrane, which became more severe with a larger number of affected coronary arteries. The findings suggest that the lipid structure of the cell membrane undergoes changes associated with its functional depression just at the preclinical stage of atherogenesis. Biomembrane structural modification in patients with angina pectoris progresses in proportion to the number of affected coronary arteries, by limiting the functional reserve of the cell and whole body.

Release of calcium into the myofibrillar space in response to active shortening of striated muscle.

The study was undertaken to explore whether shortening of striated muscle during activity is associated with release of bound Ca2+ into the myofibrillar space as has previously been proposed in order to explain the depressant effect of active shortening. The experiments were carried out on single muscle fibres isolated from the anterior tibialis muscle of Rana temporaria. The fibres were loaded with the calcium sensitive indicator Fluo-3. The fibres, stimulated to produce a partially fused isometric tetanus, were subjected to a shortening ramp or, alternatively, to a stretch ramp during activity while force, fibre length, sarcomere length and the Fluo-3 signal were recorded. A shortening ramp performed during a partially fused tetanus caused an increase in the myofibrillar free calcium concentration and produced, simultaneously, a decrease in active force. The isometric force recovered gradually after the shortening ramp, while the intracellular Ca2+ concentration stayed above the control level during the remainder of the stimulation period. A stretch ramp applied during a partially fused tetanus caused a considerably smaller change in the myofibrillar Ca2+ concentration. The results provide evidence that the myosin cross-bridges interact with the calcium binding sites on the thin filaments during active shortening, causing sustained release of calcium and reduced contractile strength.

Rapid frequency-dependent changes in free mitochondrial calcium concentration in rat cardiac myocytes.

Calcium ions regulate mitochondrial ATP production and contractile activity and thus play a pivotal role in matching energy supply and demand in cardiac muscle. The magnitude and kinetics of the changes in free mitochondrial calcium concentration in cardiac myocytes are largely unknown. Rapid stimulation frequency-dependent increases but relatively slow decreases in free mitochondrial calcium concentration were observed in rat cardiac myocytes. This asymmetry caused a rise in the mitochondrial calcium concentration with stimulation frequency. These results provide insight into the mechanisms of mitochondrial calcium uptake and release that are important in healthy and diseased myocardium. Calcium ions regulate mitochondrial ATP production and contractile activity and thus play a pivotal role in matching energy supply and demand in cardiac muscle. Little is known about the magnitude and kinetics of the changes in free mitochondrial calcium concentration in cardiomyocytes. Using adenoviral infection, a ratiometric mitochondrially targeted Förster resonance energy transfer (FRET)-based calcium indicator (4mtD3cpv, MitoCam) was expressed in cultured adult rat cardiomyocytes and the free mitochondrial calcium concentration ([Ca2+ ]m ) was measured at different stimulation frequencies (0.1-4Hz) and external calcium concentrations (1.8-3.6mm) at 37°C. Cytosolic calcium concentrations were assessed under the same experimental conditions in separate experiments using Fura-4AM. The increases in [Ca2+ ]m during electrical stimulation at 0.1Hz were rapid (rise time=49±2ms), while the decreases in [Ca2+ ]m occurred more slowly (decay half time=1.17±0.07s). Model calculations confirmed that this asymmetry caused the rise in [Ca2+ ]m during diastole observed at elevated stimulation frequencies. Inhibition of the mitochondrial sodium-calcium exchanger (mNCE) resulted in a rise in [Ca2+ ]m at baseline and, paradoxically, in an acceleration of Ca2+ release. rapid increases in [Ca2+ ]m allow for fast adjustment of mitochondrial ATP production to increases in myocardial demand on a beat-to-beat basis and mitochondrial calcium release depends on mNCE activity and mitochondrial calcium buffering.

Dynamics of volume-averaged intracellular Ca2+ in a rat CNS nerve terminal during single and repetitive voltage-clamp depolarizations.

The intracellular concentration of free calcium ions ([Ca2+ ]i ) in a nerve terminal controls both transmitter release and synaptic plasticity. The rapid triggering of transmitter release depends on the local micro- or nanodomain of highly elevated [Ca2+ ]i in the vicinity of open voltage-gated Ca2+ channels, whereas short-term synaptic plasticity is often controlled by global changes in residual [Ca2+ ]i , averaged over the whole nerve terminal volume. Here we describe dynamic changes of such global [Ca2+ ]i in the calyx of Held - a giant mammalian glutamatergic nerve terminal, which is particularly suited for biophysical studies. We provide quantitative data on Ca2+ inflow, Ca2+ buffering and Ca2+ clearance. These data allow us to predict changes in [Ca2+ ]i in the nerve terminal in response to a wide range of stimulus protocols at high temporal resolution and provide a basis for the modelling of short-term plasticity of glutamatergic synapses. Many aspects of short-term synaptic plasticity (STP) are controlled by relatively slow changes in the presynaptic intracellular concentration of free calcium ions ([Ca2+ ]i ) that occur in the time range of a few milliseconds to several seconds. In nerve terminals, [Ca2+ ]i equilibrates diffusionally during such slow changes, such that the globally measured, residual [Ca2+ ]i that persists after the collapse of local domains is often the appropriate parameter governing STP. Here, we study activity-dependent dynamic changes in global [Ca2+ ]i at the rat calyx of Held nerve terminal in acute brainstem slices using patch-clamp and microfluorimetry. We use low concentrations of a low-affinity Ca2+ indicator dye (100μm Fura-6F) in order not to overwhelm endogenous Ca2+ buffers. We first study voltage-clamped terminals, dialysed with pipette solutions containing minimal amounts of Ca2+ buffers, to determine Ca2+ binding properties of endogenous fixed buffers as well as the mechanisms of Ca2+ clearance. Subsequently, we use pipette solutions including 500μm EGTA to determine the Ca2+ binding kinetics of this chelator. We provide a formalism and parameters that allow us to predict [Ca2+ ]i changes in calyx nerve terminals in response to a wide range of stimulus protocols. Unexpectedly, the Ca2+ affinity of EGTA under the conditions of our measurements was substantially lower (KD =543±51nm) than measured in vitro, mainly as a consequence of a higher than previously assumed dissociation rate constant (2.38±0.20 s-1 ), which we need to postulate in order to model the measured presynaptic [Ca2+ ]i transients.

Contribution of Mitochondrial Calcium Uptake to Intracellular Calcium Homeostasis Studied with Organelle-Targetted Probes in Skeletal Muscle Fibers

Organelle-targetted Cameleons provide a useful tool to quantify variations in local free calcium concentration. We studied murine FDB single fibers with a probe targetted to sarcoplasmic reticulum (SR) and a probe targetted to mitochondria and assuming organelle volumes available in the literature (Luff & Atwood, J Cell Biol 1971). In wild type C57 mice, SR free calcium concentration was found to be 570 uM and, assuming a bound/free calcium ratio of 60/1 (Murphy et al, J Physiol 2009) and a volume of 5%, this corresponded to 1.7 mmole/fiber liter. Following similar approach the amount released was 0.4 and 0.6 mmole/fiber liter in a 2 s - 60Hz tetanus and, respectively, after 20 mM caffeine administration. Resting mitochondrial calcium concentration was 140 nM and, assuming a ratio of bound to free of 100/1 and a volume of 5%, this corresponded to a 1.2 umol/fiber liter. According to this line of reasoning, the increase in mitochondrial calcium content was 1.32 and 1.4 umole/fiber liter in a 2 s - 60Hz tetanus and, respectively, after 20 mM caffeine administration. The results showed that the fraction of calcium released by SR and taken up by mitochondria is below 1 % of the calcium release, with minimal impact on cytosolic calcium level, but likely sufficient to activate metabolic mitochondrial response. Supported by Telethon Grant GGP 13 213.

Role of Antioxidants in Neonatal Hypoxic-Ischemic Brain Injury: New Therapeutic Approaches.

Hypoxic–ischemic brain damage is an alarming health and economic problem in spite of the advances in neonatal care. It can cause mortality or detrimental neurological disorders such as cerebral palsy, motor impairment and cognitive deficits in neonates. When hypoxia–ischemia occurs, a multi-faceted cascade of events starts out, which can eventually cause cell death. Lower levels of oxygen due to reduced blood supply increase the production of reactive oxygen species, which leads to oxidative stress, a higher concentration of free cytosolic calcium and impaired mitochondrial function, triggering the activation of apoptotic pathways, DNA fragmentation and cell death. The high incidence of this type of lesion in newborns can be partly attributed to the fact that the developing brain is particularly vulnerable to oxidative stress. Since antioxidants can safely interact with free radicals and terminate that chain reaction before vital molecules are damaged, exogenous antioxidant therapy may have the potential to diminish cellular damage caused by hypoxia–ischemia. In this review, we focus on the neuroprotective effects of antioxidant treatments against perinatal hypoxic–ischemic brain injury, in the light of the most recent advances.

Structure-specific effects of lipidated oxytocin analogs on intracellular calcium levels, parental behavior, and oxytocin concentrations in the plasma and cerebrospinal fluid in mice.

Oxytocin (OT) is a neuroendocrine nonapeptide that plays an important role in social memory and behavior. Nasal administration of OT has been shown to improve trust in healthy humans and social interaction in autistic subjects in some clinical trials. As a central nervous system (CNS) drug, however, OT has two unfavorable characteristics: OT is short‐acting and shows poor permeability across the blood–brain barrier, because it exists in charged form in the plasma and has short half‐life. To overcome these drawbacks, an analog with long‐lasting effects is required. We previously synthesized the analog, lipo‐oxytocin‐1 (LOT‐1), in which two palmitoyl groups are conjugated to the cysteine and tyrosine residues. In this study, we synthesized and evaluated the analogs lipo‐oxytocin‐2 (LOT‐2) and lipo‐oxytocin‐3 (LOT‐3), which feature the conjugation of one palmitoyl group at the cysteine and tyrosine residues, respectively. In human embryonic kidney‐293 cells overexpressing human OT receptors, these three LOTs demonstrated comparably weak effects on the elevation of intracellular free calcium concentrations after OT receptor activation, compared to the effects of OT. The three LOTs and OT exhibited different time‐dependent effects on recovery from impaired pup retrieval behavior in sires of CD38‐knockout mice. Sires treated with LOT‐1 showed the strongest effect, whereas others had no or little effects at 24 h after injection. These results indicated that LOTs have structure‐specific agonistic effects, and suggest that lipidation of OT might have therapeutic benefits for social impairment.

Basic principles of confocal microscopy of cаlcium signals

Background: The study of fast processes occurring in living cells, for example, the dynamics of calcium ions or other ions, is actual for modern biophysics, physiology and medicine and can be carried out by laser scanning confocal microscopy. Confocal microscopy as a method of studying biological objects has a number of features requiring an understanding of the delicate mechanisms underlying it. Objectives: The aim of the work is the description of principles and details of measurements of calcium signals in the living cells using laser scanning confocal microscopy and fluorescent indicators. Materials and methods: The analysis of the methodological and practical aspects of studies of calcium signals was carried out by confocal microscopy in the work. Results: Confocal microscopy allows to study the changes in the concentration of free calcium within the cell and even a small part of it by a fluorescent dye, which increases the contrast in comparison with conventional fluorescence microscopy through an additional confocal aperture located in the front of the detector and the use of laser light source and scanning the object. To register calcium signals it is necessary to make a selection of a number of adequate parameters: the registration frequency, the size of a single scanning element (pixel), the sensitivity of the detector, the intensity of laser irradiation, the diameter of the confocal gap, the corresponding filters for exciting and emitting the fluorescent dye used and the corresponding dichroic mirror. An important stage of a setup of a confocal system is the determination of the value of the scattering function of a point. Compensation of the process of bleaching of the fluorescence dye and reduction of phototoxicity, minimization of the scattering process of the image allow to increase a reproducibility of the experiments. Conclusions: Using modern laser scanning confocal microscopes for registration of calcium signals from a small group of channels (for example, rianodine receptors) or the cell part, it is necessary to carefully select the parameters of the hardware setup, which are determined by the nature of the object of study and the peculiarities of the experimental conditions. This will allow to receive the reliable information about the functioning of both individual channels and the mechanisms of Ca2+ signaling of the whole cell or its part.