Free Calcium Concentration Research Articles

Basophil Stimulation and Signaling Pathways.

Despite the growing use of flow cytometry to analyze the functional characteristics of basophils, the intracellular signaling cascades that control their ability to elaborate various pro-allergic and inflammatory mediators and cytokines remain comparatively obscure. Additionally, some studies require the analysis of pro-allergic and inflammatory mediators, such as histamine, LTC4, and various basophil-derived cytokines (e.g., IL-4 and IL-13). Elucidation of intracellular signaling proteins by Western blotting, cytosolic free calcium concentration by spectrofluorophotometry, and detection of mediator releases, as well as analysis of gene expressions by RT-PCR, generally requires relatively large numbers of purified basophils. In selected assays, flow cytometry enables the analysis of relatively low cell numbers and purity for the expression of intracellular signaling proteins or measurement of cytosolic free calcium concentrations by basophil-specific gating strategies. Unfortunately, many aspects of signal transduction relevant to human basophils cannot be readily extrapolated from the use of basophil or mast cell lines. This chapter therefore focuses on how to employ primary human basophils for studying mediator releases and signaling characteristics.

Parallel Acceleration on Removal of Optical Mapping Baseline Wandering.

Optical mapping measurements on hearts stained with fluorescent dyes is imagining method widely accepted and recognized as a tool to study complex spatial-temporal dynamics of cardiac electro-physiology. One shortcoming of the method is baseline wandering in obtained fluorescence signals as signals relevant to transmembrane potential (Vm) change and free intracellular calcium concentration ([Ca]i +2), the two most used dyes, are calculated as a relative signal change in respect to the fluorescence baseline. These changes are small fractional changes often smaller than 10 %. Baseline fluorescence drifts due to dye photo-bleaching, heart contraction/movement artifacts, and stability of the excitation light source over time. Depending on experimental instrumentation, recording duration, signal to noise levels and study aims of the optical imagining, many research groups adopted their own techniques tailored to a specific experimental data. Here we present a technique based on finite impulse response (FIR) filters with paralleled acceleration implemented on GPUs and multi-core CPU, in MATLAB.

Inorganic polyphosphate is required for sustained free mitochondrial calcium elevation, following calcium uptake

Mitochondrial free calcium is critically linked to the regulation of cellular metabolism. Free ionic calcium concentration within these organelles is determined by the interplay between two processes: exchange across the mitochondrial inner membrane and calcium-buffering within the matrix. During stimulated calcium uptake, calcium is primarily buffered by orthophosphate, preventing calcium toxicity while allowing for well-regulated yet elevated calcium loads. However, if limited to orthophosphates only, this buffering system is expected to lead to the irreversible formation of insoluble precipitates, which are not observed in living cells, under physiological conditions. Here, we demonstrate that the regulation of free mitochondrial calcium requires the presence of free inorganic polyphosphate (polyP) within the organelle. We found that the overexpression of a mitochondrial-targeted enzyme hydrolyzing polyP leads to the loss of the cellular ability to maintain elevated calcium concentrations within the organelle, following stimulated cytoplasmic signal. We hypothesize that the presence of polyP prevents the formation of calcium-phosphate insoluble clusters, allowing for the maintenance of elevated free calcium levels, during stimulated calcium uptake.

PH Variability and its Relationship with Sarcomere Length and Free Calcium in Beef from Commercial Cattle in Puerto Rico

ObjectivesResearch conducted at the University of Puerto Rico noted that beef with elevated pH values (> 5.86) resulted in more tender meat (P ≤ 0.05). It has been established that proteolytic degradation mechanisms can be influenced by pH and calcium concentration in muscle. Beef with pH values ≥ 5.86 is classified as Dark Firm and Dry (DFD) but there are negative implications associated with greater pH values. However, observations indicating increased tenderness with increased pH raise the question: can variations in pH be associated with differences in sarcomere length (SL) and free calcium concentration (FCC)? Therefore, the objectives of this project were to: (1) document pH distribution; (2) determine the incidence of DFD; and (3) evaluate the relationship between pH, SL, and FCC in commercial cattle harvested in Puerto Rico.Materials and MethodsLongissimus lumborum samples (n = 51) were obtained and background information was noted including number of permanent incisors (PI), type (Dairy or Beef), and gender. The pH values were used to categorize beef into the following groups: Low (≤ 5.40), Normal (5.41 to 5.59), High (5.60 to 5.85) and DFD (≥ 5.86). Meat was flash frozen, powdered, and placed on a microscope slide and a Helium-Neon laser was used to determine SL. A subset of samples was sent off and prepared at the University of Nebraska-Lincoln for FCC quantification (Ward Laboratories; Kearney, NE) with an inductively coupled plasma emission spectrometer (iCAP 6500 Radial; Thermo Electron, Cambridge, UK). All statistical analyses were conducted in SAS (9.4). The Proc FREQ was used to determine pH category distributions and incidence of DFD. The Proc GLIMMIX and Tukey adjustment (α = 0.05) were used to determine the effects of number of PI, type, and gender on pH category, SL and FCC. The Proc CORR was used to evaluate the relationship between pH category, SL and FCC.ResultsThe pH category distribution for the current samples was as follows: 3.92% Low, 41.18% Normal, 35.29% High and 19.61% DFD. The SL ranged from 1.69 to 1.46 mm with an average of 1.53 mm. The FCC ranged from 132.19 to 31.39 mM with an average of 64.23 mM. Longer sarcomeres were detected in cattle with eight and zero PI (1.57 and 1.56 mm, respectively); cattle with two and four PI had intermediate SL (1.53 and 1.52 mm, respectively), and cattle with six PI had the shortest sarcomeres (1.51 mm; P = 0.03). Dairy cattle had longer sarcomeres relative to beef cattle (1.56 vs. 1.52 mm; P = 0.02). Dairy cattle tended to have increased FCC relative to beef cattle (70.72 vs. 58.38 mM; P = 0.08). Also, FCC tended to be greater within the Normal and Low pH categories relative to the High and DFD categories (72.36 vs. 57.31 mM; P = 0.06). The SL and FCC had no relationship (P > 0.05) within the Low, Normal and High pH categories. However, DFD beef had longer SL (0.78; P = 0.01), while having decreased FCC (–0.66; P = 0.04).ConclusionOver half (54.90%) of the beef samples analyzed fell into the High and DFD pH categories, with nearly 20% being classified as DFD. Although, a clear relationship was not established between SL and FCC within the Low, Normal or High pH categories, the results indicate that the increased pH in samples surpassing the DFD threshold correspond to longer sarcomeres and decreased free calcium.

A Proposed Mechanism for Texture Property of Woody Breast in Broilers

ObjectivesWoody breast is a myopathy observed in chicken breast meat (Pectoralis major) characterized by its tough and rubbery texture. However, the exact causation of woody breast texture is still unknown. We hypothesize that sarcoplasmic reticulum (SR) dysfunctionality early postmortem results in rapid leakage of intracellular calcium may partially contribute to the abnormal meat texture observed in woody breast meat. The objective of this preliminary study was to investigate this hypothesis.Materials and MethodsFourteen Ross line broiler breast fillets (7 severe woody breast and 7 normal) were collected at 3 h postmortem from a commercial processing plant located in the southeast United States. The 7 woody breast samples also exhibited moderate to severe white striping. The 7 normal samples did not exhibit any signs of white striping or woody breast. Each sample was trimmed, weighed, vacuum packaged and frozen at –20°C at approximately 8 h postmortem. One 1.9 cm strip across the cranial end of each fillet was fabricated and pulverized in liquid nitrogen to measure sarcomere length (Laser Scan Confocal Microscope with a 100x/NA 1.4 objective), calpain activity (immunoblotting for µ-calpain autolysis), proteolysis (immunoblotting for troponin-T degradation) and collagen content (hydroxyproline content). Purge was also collected from each sample to evaluate protein (bicinchoninic acid assay) and free calcium concentration (atomic absorption).ResultsWoody breast fillets were heavier than normal chicken breast fillets (522.9 vs. 446.9g; P < 0.05). Woody breast samples tended to have shorter sarcomeres (1.70 vs. 2.02 µm; P = 0.0543) and less intact troponin-T compared to normal breast samples (relative intact troponin-T band density: 49.98 vs. 56.97%; P = 0.0515) at 8 h postmortem. It was interesting to note that no µ-calpain band was detected through immunoblotting for both the woody breast and normal samples at 8 h postmortem. Other studies have found similar results as poultry µ-calpain autolyzed at a much rapid rate than µ-calpain in mammalian species. In addition, the purge from woody breast samples also had higher levels of free calcium compared to normal samples (6.2 vs. 4.2 nmol calcium/mg protein; P < 0.05). Lastly, there was more collagen present in the woody breast samples compared to normal chicken breast samples (3.89 vs. 2.08 mg collagen/g muscle tissue; P < 0.05).ConclusionThe results indicated that the cause of texture abnormality of woody breast may be the combined effects of more calcium being released from the SR early postmortem resulting in shorter sarcomere length and more collagen being deposited in the chicken breast meat. Additional research with the focus on SR integrity and functionality as well as collagen crosslinks are needed to further elucidate the basic mechanism of woody breast texture formation.

Immunosuppressive effects of a novel potassium channel toxin Ktx-Sp2 from Scorpiops Pocoki

BackgroundThe cDNA Library of venomous animals could provide abundant bioactive peptides coding information and is an important resource for screening bioactive peptides that target and regulate disease-related ion channels. To further explore the potential medicinal usage of the transcriptome database of Scorpiops Pocoki’s venom gland, this research identified the function of a new potassium channel toxin Ktx-Sp2, whose gene was screened from the database by sequence alignment.ResultsThe mature peptide of Ktx-Sp2 was obtained by genetic engineering. Whole-cell patch-clamp experiment showed that Ktx-Sp2 peptide could effectively block three types of exogenous voltage-gated potassium channels—Kv1.1, Kv1.2 and Kv1.3, among which, the blocking activity for Kv1.3 was relatively high, showing selectivity to some extent. Taking Jurkat T cells as the cell model, this study found that Ktx-Sp2 peptide could also effectively block endogenous Kv1.3, significantly reduce the free calcium concentration in Jurkat T cells, inhibit the activation of Jurkat T cells and reduce the release of inflammatory cytokines IL-2, showing a strong immunosuppressant effect.ConclusionsThis study further proves that the transcriptome database of the Scorpiops Pocoki venom gland is an important resource for discovery of novel bioactive polypeptide coding genes. The newly screened Kv1.3 channel blocker Ktx-Sp2 expanded the range of leading compounds for the treatment of autoimmune diseases and promoted the development and application of scorpion toxin peptides in the field of biomedicine.

The Mechanism, Regulatory Effect of Wnt5α/Ca2+ Signaling Pathway on hMSCs Osteogenic Differentiation Under Infection Conditions

Background: The paper aimed to reveal the role and possible regulatory mechanisms of Wnt5 gene and its non-canonical Beta-catenin signaling pathway in osteogenic differentiation and bone repairment of hMSCs under infection environment. Material and Methods : CCK-8 method was employed to detect the effect of SPA on the proliferation of different hMSCs cells. The histological staining of alizarin red was used to observe the differentiation of cells into osteoblasts. The semi-quantitative detection of osteoblast alkaline phosphatase (ALP) was performed. Fluo-2 assay was employed to measure the intracellular calcium concentration of different hMSCs before and after osteogenic differentiation stimulated by SPA. RT-PCR was applied to detect the specific gene fluorescence of osteoblasts differentiation. Western Blotting was employed to determine the differentiation of hMSCs into osteoblasts. Results: Different hMSCs were able to proliferate in vitro under the stimulation of 100 ng/mL SPA. CCK-8 kit was used to detect lentivirus-infected hMSCs and non-transduced hMSCs in vitro. The results showed that all the cells gradually proliferate with time and an increasing rate. The hMSCs treated with different Wnt5 genes were stained with alizarin red after osteogenic differentiation, and the activities of AMSCs ALP increased. The intracellular free calcium concentration increased with the induction time and peaked in the Wnt5 overexpression group. PCR results showed that ALP, Runx-2, Collagen I, OC, Osterix and BSP genes were all increased in contrast with the non-induction Control group. Conclusion: Under the inflammatory condition of SPA virulence factors, non-canonical Wnt signaling pathway is associated with the classical Wnt/ β-catenin signaling pathway in the process of osteogenic differentiation of hMSCs in vitro, which has a certain synergistic effect.

Photoreversible stretching of a BAPTA chelator marshalling Ca2+-binding in aqueous media.

Free calcium ion concentration is known to govern numerous biological processes and indeed calcium acts as an important biological secondary messenger for muscle contraction, neurotransmitter release, ion-channel gating, and exocytosis. As such, the development of molecules with the ability to instantaneously increase or diminish free calcium concentrations potentially allows greater control over certain biological functions. In order to permit remote regulation of Ca2+, a selective BAPTA-type synthetic receptor / host was integrated with a photoswitchable azobenzene motif, which upon photoirradiation would enhance (or diminish) the capacity to bind calcium upon acting on the conformation of the adjacent binding site, rendering it a stronger or weaker binder. Photoswitching was studied in pseudo-physiological conditions (pH 7.2, [KCl] = 100 mM) and dissociation constants for azobenzene cis- and trans-isomers have been determined (0.230 μM and 0.102 μM, respectively). Reversible photoliberation/uptake leading to a variation of free calcium concentration in solution was detected using a fluorescent Ca2+ chemosensor.

The Synergy of Ciprofloxacin and Carvedilol against Staphylococcus aureus-Prospects of a New Treatment Strategy?

Staphylococcus aureus infections are common and difficult to treat. The increasing number of drug-resistant staphylococcal infections has created the need to develop new strategies for the treatment of these infections. The synergistic antimicrobial activity of different pharmaceuticals seems to be an interesting alternative. The aim of this study was to assess the synergistic activity of ciprofloxacin and carvedilol against S. aureus strains. The antibacterial potential of ciprofloxacin and carvedilol was evaluated according to the CLSI guidelines. The calcium content in S. aureus cells was measured using flow cytometry and atomic absorption spectroscopy. Moreover, confocal and scanning electron microscopy were used to determine the mechanism of antibacterial synergy of ciprofloxacin and carvedilol. The antibacterial effect of ciprofloxacin was higher in the presence of carvedilol than in S. aureus cultures containing the antibiotic only. A significant increase in S. aureus membrane permeability was also observed. The simultaneous administration of the tested compounds caused damage to S. aureus cells visualized by SEM. Enhancement of the antimicrobial action of ciprofloxacin by carvedilol was correlated with an increase in free calcium content in S. aureus cells, morphological changes to the cells, and a reduction in the ability to form bacterial aggregates.

Effect of External Calcium and other Divalent Cations on K+ Contractures in Denervated Slow Skeletal Muscle Fibers of the Frog

The influence of Ca2+ and other divalent cations on contractile responses of slow skeletal muscle fibers of the frog (Rana pipiens) under conditions of chronic denervation was investigated.Isometric tension was recorded from slow bundles of normal and denervated cruralis muscle in normal solution and in solutions with free calcium concentration solution or in solutions where other divalent cations (Sr2+, Ni2+, Co2+ or Mn2+) substituted for calcium. In the second week after nerve section, in Ca2+-free solutions, we observed that contractures (evoked from 40 to 80 mM-K+) of non-denervated muscles showed significantly higher tensions (p<0.05), than those from denervated bundles. Likewise, in solutions where calcium was substituted by all divalent cations tested, with exception of Mn2+, the denervated bundles displayed lower tension than non-denervated, also in the second week of denervation. In this case, the Ca2+ substitution by Sr2+ caused the higher decrease in tension, followed by Co2+ and Ni2+, which were different to non-denervated bundles, as the lowest tension was developed by Mn2+, followed by Co2+, and then Ni2+ and Sr2+. After the third week, we observed a recovery in tension. These results suggest that denervation altering the binding capacity to divalent cations of the voltage sensor.

Vibration of osteoblastic cells using a novel motion-control platform does not acutely alter cytosolic calcium, but desensitizes subsequent responses to extracellular ATP.

Low-magnitude high-frequency mechanical vibration induces biological responses in many tissues. Like many cell types, osteoblasts respond rapidly to certain forms of mechanostimulation, such as fluid shear, with transient elevation in the concentration of cytosolic free calcium ([Ca2+ ]i ). However, it is not known whether vibration of osteoblastic cells also induces acute elevation in [Ca2+ ]i . To address this question, we built a platform for vibrating live cells that is compatible with microscopy and microspectrofluorometry, enabling us to observe immediate responses of cells to low-magnitude high-frequency vibrations. The horizontal vibration system was mounted on an inverted microscope, and its mechanical performance was evaluated using optical tracking and accelerometry. The platform was driven by a sinusoidal signal at 20-500 Hz, producing peak accelerations from 0.1 to 1 g. Accelerometer-derived displacements matched those observed optically within 10%. We then used this system to investigate the effect of acceleration on [Ca2+ ]i in rodent osteoblastic cells. Cells were loaded with fura-2, and [Ca2+ ]i was monitored using microspectrofluorometry and fluorescence ratio imaging. No acute changes in [Ca2+ ]i or cell morphology were detected in response to vibration over the range of frequencies and accelerations studied. However, vibration did attenuate Ca2+ transients generated subsequently by extracellular ATP, which activates P2 purinoceptors and has been implicated in mechanical signaling in bone. In summary, we developed and validated a motion-control system capable of precisely delivering vibrations to live cells during real-time microscopy. Vibration did not elicit acute elevation of [Ca2+ ]i , but did desensitize responses to later stimulation with ATP.

RyRs mediate lead-induced neurodegenerative disorders through calcium signaling pathways

Heavy metal lead (Pb) is widely distributed in the environment and can induce neurodegeneration. Accumulating evidence has shown that ryanodine receptors (RyRs) play vital roles in neurodegenerative brain. However, whether aberrant RyRs levels contribute to Pb-induced neurodegeneration has largely remained unknown. In the present study, we report the important role of elevated levels of RyRs in Pb-induced neurodegeneration. Pb was found to upregulate the levels of RyRs in the rat hippocampal tissues and rat pheochromocytoma (PC12) cells. Furthermore, exposure to Pb induced neurodegenerative cognitive impairment in rats, depressed the long-term potentiation (LTP) in the rat brain slices, increased the neuronal intracellular free calcium concentration ([Ca2+]i), inhibited the phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and cyclic adenosine 3′,5′-monophosphate (cAMP) response element binding protein (CREB) as well as the expression of anti-apoptotic protein B-cell lymphoma 2 (Bcl2), and activated the phosphorylation of extracellular regulated protein kinases (Erk) protein both in vitro and in vivo. In addition, the knockdown of RyR3 in PC12 cells significantly decreased the [Ca2+]i levels, increased the CaMKIIα and CREB phosphorylation, decrease the phosphorylation of Erk, and elongated the cognitive function-related neurite outgrowth after exposure to Pb. Moreover, treatment with a RyRs agonist showed the involvement of RyRs in Pb-induced depression in LTP in the rat brain slices. In summary, we determined that Pb-mediated upregulation of RyRs led to neurodegeneration via high levels of free calcium, depression of the calcium-dependent CaMKIIα/CREB mnemonic signaling pathway, and activation of the calcium-dependent Erk/Bcl2 apoptotic signaling pathway. These findings on the impact of Pb on the levels of RyRs could further improve our understanding of Pb-induced neurotoxicity and provide a promising molecular target to antagonize Pb-induced neurodegenerative diseases.

Intracellular Calcium Recording After Purinoceptor Activation Using a Video-Microscopy Equipment.

Calcium is one of the most important intracellular messengers, triggering a wide range of cellular responses. Changes in intracellular free calcium concentration can be measured using calcium sensitive fluorescent dyes, which are either EGTA- or BAPTA-based organic molecules that change their spectral properties in response to Ca2+ binding. One of the most common calcium indicators is the ratiometric dye Fura-2. The main advantage of using ratiometric dyes is that the ratio signal is independent of the illumination intensity, dye concentration, photobleaching, and focus changes among others, allowing for the concentration of intracellular calcium to be determined independently of these artifacts. In this protocol, we describe the use of Fura-2 to measure intracellular calcium elevations in single cultured cells after purinoceptor activation using a video-microscopy equipment. This method, usually known as calcium imaging, allows for real-time quantification of intracellular calcium dynamics and can be adapted to measure agonist mediated intracellular calcium responses due to the activation of different purinergic receptors in several cellular models using the appropriate growth conditions.

Self-Correcting Method for the Measurement of Free Calcium and Magnesium Concentrations by 1H NMR.

A method for the direct measurement of free Ca2+ and Mg2+ concentrations in the range of 1-100 mM by NMR spectroscopy is demonstrated. The method automatically corrects for the effect of ionic strength on the activity of the species in solution and works satisfactorily even when significant concentrations of competitive ions are present. The method requires only the measurement of the 1H chemical shifts of our reporter ligands, glycolate and sulfoacetate, and is easily implemented using NMR imaging techniques. As a proof of concept, we extract the thermodynamic binding constants and conformer distributions of analyte ligands using an in situ ion gradient. Existing approaches for the measurement of free Ca2+ or Mg2+ concentrations by NMR operate only at very low ion concentrations or else require careful recalibration for different sample conditions. By providing the free Ca2+ or Mg2+ concentrations, the proposed methodology significantly enhances the information obtainable via NMR investigations of ion-responsive systems.

Disturbed cardiac mitochondrial and cytosolic calcium handling in a metabolic risk-related rat model of heart failure with preserved ejection fraction.

AimCalcium ions play a pivotal role in matching energy supply and demand in cardiac muscle. Mitochondrial calcium concentration is lower in animal models of heart failure with reduced ejection fraction (HFrEF), but limited information is available about mitochondrial calcium handling in heart failure with preserved ejection fraction (HFpEF).MethodsWe assessed mitochondrial Ca2+ handling in intact cardiomyocytes from Zucker/fatty Spontaneously hypertensive F1 hybrid (ZSF1)‐lean (control) and ZSF1‐obese rats, a metabolic risk‐related model of HFpEF. A mitochondrially targeted Ca2+ indicator (MitoCam) was expressed in cultured adult rat cardiomyocytes. Cytosolic and mitochondrial Ca2+ transients were measured at different stimulation frequencies. Mitochondrial respiration and swelling, and expression of key proteins were determined ex vivo.ResultsAt rest, mitochondrial Ca2+ concentration in ZSF1‐obese was larger than in ZSF1‐lean. The diastolic and systolic mitochondrial Ca2+ concentrations increased with stimulation frequency, but the steady‐state levels were larger in ZSF1‐obese. The half‐widths of the contractile responses, the resting cytosolic Ca2+ concentration and the decay half‐times of the cytosolic Ca2+ transients were higher in ZSF1‐obese, likely because of a lower SERCA2a/phospholamban ratio. Mitochondrial respiration was lower, particularly with nicotinamide adenine dinucleotide (NADH) (complex I) substrates, and mitochondrial swelling was larger in ZSF1‐obese.ConclusionThe free mitochondrial calcium concentration is higher in HFpEF owing to alterations in mitochondrial and cytosolic Ca2+ handling. This coupling between cytosolic and mitochondrial Ca2+ levels may compensate for myocardial ATP supply in vivo under conditions of mild mitochondrial dysfunction. However, if mitochondrial Ca2+ concentration is sustainedly increased, it might trigger mitochondrial permeability transition pore opening.

The Role of Two-Pore Channels in Norepinephrine-Induced [Ca2+]i Rise in Rat Aortic Smooth Muscle Cells and Aorta Contraction.

Second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) triggers Ca2+ release via two-pore channels (TPCs) localized in endolysosomal vesicles. The aim of the present work is to evaluate the role of TPCs in the action of norepinephrine (NE), angiotensin II (AngII), vasopressin (AVP), and 5-hydroxytriptamine (5-HT) on free cytoplasmic calcium concentration ([Ca2+]i) in smooth muscle cells (SMCs) isolated from rat aorta and on aorta contraction. To address this issue, the NAADP structural analogue and inhibitor of TPCs, NED 19, was applied. We have demonstrated a high degree of colocalization of the fluorescent signals of cis-NED 19 and endolysosmal probe LysoTracker in SMCs. Both cis- or trans-NED 19 inhibited the rise of [Ca2+]i in SMCs induced by 100 μM NE by 50–60%. IC50 for cis- and trans-NED 19 were 2.7 and 8.9 μM, respectively. The inhibition by NED 19 stereoisomers of the effects of AngII, AVP, and 5-HT was much weaker. Both forms of NED 19 caused relaxation of aortic rings preconstricted by NE, with relative potency of cis-NED 19 several times higher than that of trans-NED 19. Inhibition by cis-NED 19 of NE-induced contraction was maintained after intensive washing and slowly reversed within an hour of incubation. Cis- and trans-NED 19 did not cause decrease in the force of aorta contraction in response to Ang II and AVP, and only slightly relaxed aorta preconstricted by 5-HT and by KCl. Suppression of TPC1 in SMCs with siRNA caused a 40% decrease in [Ca2+]i in response to NE, whereas siRNA against TPC2 did not change NE calcium signaling. These data suggest that TPC1 is involved in the NE-stimulated [Ca2+]i rise in SMCs. Inhibition of TPC1 activity by NED 19 could be the reason for partial inhibition of aortic rings contraction in response to NE.

Multiparametric real-time sensing of cytosolic physiology links hypoxia responses to mitochondrial electron transport.

Hypoxia regularly occurs during plant development and can be induced by the environment through, for example, flooding. To understand how plant tissue physiology responds to progressing oxygen restriction, we aimed to monitor subcellular physiology in real time and in vivo. We establish a fluorescent protein sensor-based system for multiparametric monitoring of dynamic changes in subcellular physiology of living Arabidopsis thaliana leaves and exemplify its applicability for hypoxia stress. By monitoring cytosolic dynamics of magnesium adenosine 5'-triphosphate, free calcium ion concentration, pH, NAD redox status, and glutathione redox status in parallel, linked to transcriptional and metabolic responses, we generate an integrated picture of the physiological response to progressing hypoxia. We show that the physiological changes are surprisingly robust, even when plant carbon status is modified, as achieved by sucrose feeding or extended night. Inhibition of the mitochondrial respiratory chain causes dynamics of cytosolic physiology that are remarkably similar to those under oxygen depletion, highlighting mitochondrial electron transport as a key determinant of the cellular consequences of hypoxia beyond the organelle. A broadly applicable system for parallel in vivo sensing of plant stress physiology is established to map out the physiological context under which both mitochondrial retrograde signalling and low oxygen signalling occur, indicating shared upstream stimuli.

A Ca2+-Binding Protein that Regulates the Lifetime of the Transducin Active State

Abstract—It has been previously shown in the study of the kinetic behavior of cGMP-specific phosphodiesterase in preparations of the outer segments of bovine retinal rods, which was temporarily activated by 2 μM GTP at high and low concentrations of free ions of Ca2+ (100 μM and <10 nm, respectively), that the lifetime of cGMP-specific phosphodiesterase in the active state significantly decreased (by ≈2 times) when low concentrations of free calcium ions were used. This suggested that there is a Ca2+-binding protein in the outer segments of bovine retinal rods that can interact with the so-called free transducin and regulate the rate of hydrolysis of GTP, which is bound in its active site, depending on the concentration of free Ca2+ ions. This paper is devoted to a discussion of the known Ca2+-binding proteins that occur in the outer segments of bovine retinal rods that could play the role of a potential regulator.

NMDA-induced increases in intracellular Ca2+ concentrations in osteoblasts

Objective To study the effect of N-Methyl-D-asparticacid (NMDA) on the intracellular free calcium concentration ([Ca2+ ]i) in primary cultured rat calvaria osteoblasts. Methods A calcium imaging technique was applied to observe [Ca2+ ]i changes in primary cultured rat calvaria osteoblasts after stimulating by NMDA with various concentrations or pretreated with NMDA receptor noncompetitive antagonism MK801 (Dizocilpin). Results Different concentrations of NMDA caused [Ca2+ ]i increases in varying degrees and by different ways. NMDA could evoke transient increase and secondary change in [Ca2+ ]i including calcium oscillation or steady increase. MK801 inhibited NMDA-induced [Ca2+ ]i increase in varying degrees. Conclusion These results indicated that there are abundant functional NMDA receptors expressed in primary cultured rat calvaria osteoblasts, showing different forms and varying degrees of [Ca2+ ]i increases in response to different concentrations of NMDA. The characters of the blocking effect of MK801 to NMDA-induced [Ca2+ ]i increasing, indicated that the NMDA receptors expressed in primary cultured rat calvaria osteoblasts differ in channel properties from those in central nervous system. Key words: Osteoblasts; NMDA receptors; Calcium oscillation

Pseudo-plateau bursting and mixed-mode oscillations in a model of developing inner hair cells

Inner hair cells (IHCs) are excitable sensory cells in the inner ear that encode acoustic information. Before the onset of hearing IHCs fire calcium-based action potentials that trigger transmitter release onto developing spiral ganglion neurones. There is accumulating experimental evidence that these spontaneous firing patterns are associated with maturation of the IHC synapses and hence involved in the development of hearing. The dynamics organising the IHCs’ electrical activity are therefore of interest.Building on our previous modelling work we propose a three-dimensional, reduced IHC model and carry out non-dimensionalisation. We show that there is a significant range of parameter values for which the dynamics of the reduced (three-dimensional) model map well onto the dynamics observed in the original biophysical (four-dimensional) IHC model. By estimating the typical time scales of the variables in the reduced IHC model we demonstrate that this model could be characterised by two fast and one slow or one fast and two slow variables depending on biophysically relevant parameters that control the dynamics. Specifically, we investigate how changes in the conductance of the voltage-gated calcium channels as well as the parameter corresponding to the fraction of free cytosolic calcium concentration in the model affect the oscillatory model bahaviour leading to transition from pseudo-plateau bursting to mixed-mode oscillations. Hence, using fast-slow analysis we are able to further our understanding of this model and reveal a path in the parameter space connecting pseudo-plateau bursting and mixed-mode oscillations by varying a single parameter in the model.