Free Calcium Concentration Research Articles

Regucalcin Transgenic Rat: A Novel Animal Model for Metabolic Disorder Associated with Osteoporosis

Regucalcin transgenic (TG) rat has been generated to determine a role of endogenous regucalcin in metabolic disorders. Regucalcin TG rats induced a prominent increase in regucalcin protein in the various tissues. Bone loss and hyperlipidemia were found to induce in regucalcin TG rats with growing and aging. Bone loss induced in regucalcin TG rats was resulted from enhanced osteoclastogenesis and suppressed osteoblastic mineralization in rat marrow cells. Moreover, hyperlipidemia induced in regucalcin TG rats was seen with increasing age; serum triglyceride, high-density lipoprotein (HDL)-cholesterol, free fatty acid, albumin and calcium concentrations were markedly increased in regucalcin TG. Decrease in lipid and glycogen contents in the liver tissues was also induced in regucalcin TG rats. The gene expression of leptin and adiponectin was suppressed in the adipose and liver tissues of TG rats. Regucalcin possessed insulin resistance in liver cells. Regucalcin revealed stimulatory effects on adipogenesis that is differentiated from messenchymal stem cells to adipocytes in bone marrow cells. Regucalcin may be implicated to obesity and diabetes associated with osteoporosis. Regucalcin TG rat model may be a useful tool in the development of therapeutic tools for metabolic disorder associated with osteoporosis in vivo.

The expression of the truncated isoform of somatostatin receptor subtype 5 associates with aggressiveness in medullary thyroid carcinoma cells

The truncated somatostatin receptor variant sst5TMD4 associates with increased invasiveness and aggressiveness in breast cancer. We previously found that sst5 activation may counteract sst2 selective agonist effects in a medullary thyroid carcinoma (MTC) cell line, the TT cells, and that sst5TMD4 is overexpressed in poorly differentiated thyroid cancers. The purpose of this study is to evaluate sst5TMD4 expression in a series of human MTC and to explore the functional role of sst5TMD4 in TT cells. We evaluated sst5TMD4 and sst5 expression in 36 MTC samples. Moreover, we investigated the role of sst5TMD4 in TT cells evaluating cell number, DNA synthesis, free cytosolic calcium concentration ([Ca(2+)]i), calcitonin and vascular endothelial growth factor levels, cell morphology, protein expression, and invasion. We found that in MTC the balance between sst5TMD4 and sst5 expression influences disease stage. sst5TMD4 overexpression in TT cells confers a greater growth capacity, blocks sst2 agonist-induced antiproliferative effects, modifies the cell phenotype, decreases E-cadherin and phosphorylated β-catenin levels, increases vimentin, total β-catenin and phosphorylated GSK3B levels (in keeping with the development of epithelial to mesenchymal transition), and confers a greater invasion capacity. This is the first evidence indicating that sst5TMD4 is expressed in human MTC cells, where it associates with more aggressive behavior, suggesting that sst5TMD4 might play a functionally relevant role.

Renal autoregulation in health and disease.

Intrarenal autoregulatory mechanisms maintain renal blood flow (RBF) and glomerular filtration rate (GFR) independent of renal perfusion pressure (RPP) over a defined range (80-180 mmHg). Such autoregulation is mediated largely by the myogenic and the macula densa-tubuloglomerular feedback (MD-TGF) responses that regulate preglomerular vasomotor tone primarily of the afferent arteriole. Differences in response times allow separation of these mechanisms in the time and frequency domains. Mechanotransduction initiating the myogenic response requires a sensing mechanism activated by stretch of vascular smooth muscle cells (VSMCs) and coupled to intracellular signaling pathways eliciting plasma membrane depolarization and a rise in cytosolic free calcium concentration ([Ca(2+)]i). Proposed mechanosensors include epithelial sodium channels (ENaC), integrins, and/or transient receptor potential (TRP) channels. Increased [Ca(2+)]i occurs predominantly by Ca(2+) influx through L-type voltage-operated Ca(2+) channels (VOCC). Increased [Ca(2+)]i activates inositol trisphosphate receptors (IP3R) and ryanodine receptors (RyR) to mobilize Ca(2+) from sarcoplasmic reticular stores. Myogenic vasoconstriction is sustained by increased Ca(2+) sensitivity, mediated by protein kinase C and Rho/Rho-kinase that favors a positive balance between myosin light-chain kinase and phosphatase. Increased RPP activates MD-TGF by transducing a signal of epithelial MD salt reabsorption to adjust afferent arteriolar vasoconstriction. A combination of vascular and tubular mechanisms, novel to the kidney, provides for high autoregulatory efficiency that maintains RBF and GFR, stabilizes sodium excretion, and buffers transmission of RPP to sensitive glomerular capillaries, thereby protecting against hypertensive barotrauma. A unique aspect of the myogenic response in the renal vasculature is modulation of its strength and speed by the MD-TGF and by a connecting tubule glomerular feedback (CT-GF) mechanism. Reactive oxygen species and nitric oxide are modulators of myogenic and MD-TGF mechanisms. Attenuated renal autoregulation contributes to renal damage in many, but not all, models of renal, diabetic, and hypertensive diseases. This review provides a summary of our current knowledge regarding underlying mechanisms enabling renal autoregulation in health and disease and methods used for its study.

Protective effects of diltiazem against vascular endothelial cell injury induced by angiotensin-II and hypoxia

To provide pharmacological data for future clinical studies, this study investigated the protective effects of diltiazem on vascular endothelial cell (VEC) injury induced by angiotensin-II (AngII), hypoxia, and a combination of both treatments. The concentration of intracellular free calcium and the mitochondrial membrane potential in VEC were assessed as indicators of cell injury. An in vivo hypoxic animal model was used to test the protective effect of diltiazem on vascular endothelial tissues. Our study showed that AngII and hypoxia decreased the mitochondrial membrane potential in VEC, which was significantly inhibited by diltiazem. Diltiazem protected against VEC injury induced by the increased concentration of intracellular free calcium, which was associated with AngII and hypoxia. Diltiazem reduced the apoptosis of rat VEC under a sustained hypoxic condition. In addition, it reduced AngII and endothelin I levels in rat vascular endothelial tissues. Our study confirmed that AngII and hypoxia induced VEC injury by regulating the levels of mitochondrial membrane potential and intracellular free calcium. Diltiazem, a calcium channel blocker, protected VEC from AngII- and hypoxia-induced injury.

Distinct mPTP activation mechanisms in ischaemia-reperfusion: contributions of Ca2+, ROS, pH, and inorganic polyphosphate.

The mitochondrial permeability transition pore (mPTP) plays a central role for tissue damage and cell death during ischaemia-reperfusion (I/R). We investigated the contribution of mitochondrial inorganic polyphosphate (polyP), a potent activator of Ca(2+)-induced mPTP opening, towards mPTP activation and cardiac cell death in I/R. A significant increase in mitochondrial free calcium concentration ([Ca(2+)]m), reactive oxygen species (ROS) generation, mitochondrial membrane potential depolarization (ΔΨm), and mPTP activity, but no cell death, was observed after 20 min of ischaemia. The [Ca(2+)]m increase during ischaemia was partially prevented by the mitochondrial Ca(2+) uniporter (MCU) inhibitor Ru360 and completely abolished by the combination of Ru360 and the ryanodine receptor type 1 blocker dantrolene, suggesting two complimentary Ca(2+) uptake mechanisms. In the absence of Ru360 and dantrolene, mPTP closing by polyP depletion or CSA decreased mitochondrial Ca(2+) uptake, suggesting that during ischaemia Ca(2+) can enter mitochondria through mPTP. During reperfusion, a burst of endogenous polyP production coincided with a decrease in [Ca(2+)]m, a decline in superoxide generation, and an acceleration of hydrogen peroxide (H2O2) production. An increase in H2O2 correlated with restoration of mitochondrial pHm and an increase in cell death. mPTP opening and cell death on reperfusion were prevented by antioxidants Trolox and MnTBAP [Mn (III) tetrakis (4-benzoic acid) porphyrin chloride]. Enzymatic polyP depletion did not affect mPTP opening during reperfusion, but increased ROS generation and cell death, suggesting that polyP plays a protective role in cellular stress response. Transient Ca(2+)/polyP-mediated mPTP opening during ischaemia may serve to protect cells against cytosolic Ca(2+) overload, whereas ROS/pH-mediated sustained mPTP opening on reperfusion induces cell death.

Two Dimensional Finite Element Model to Study Calcium Distribution in Oocytes

Cytosolic free calcium concentration is a key regulatory factor and perhaps the most widely used means of controlling cellular function. Calcium can enter cells through different pathways which are activated by specific stimuli including membrane depolarization, chemical signals and calcium depletion of intracellular stores. One of the important components of oocyte maturation is differentiation of the Ca 2+ signaling machinery which is essential for egg activation after fertilization. Eggs acquire the ability to produce the fertilization-specific calcium signal during oocyte maturation. The calcium concentration patterns required during different stages of oocyte maturation are still not completely known. Also the mechanisms involved in calcium dynamics in oocyte cell are still not well understood. In view of above a two dimensional FEM model has been proposed to study calcium distribution in an oocyte cell. The parameters such as buffers, ryanodine receptor, SERCA pump and voltage gated calcium channel are incorporated in the model. Based on the biophysical conditions the initial and boundary conditions have been framed. The model is transformed into variational form and Ritz finite element method has been employed to obtain the solution. A program has been developed in MATLAB 7.10 for the entire problem and executed to obtain numerical results. The numerical results have been used to study the effect of buffers, RyR, SERCA pump and VGCC on calcium distribution in an oocyte cell.

Different NaCl-induced calcium signatures in the Arabidopsis thaliana ecotypes Col-0 and C24.

A common feature of stress signalling pathways are alterations in the concentration of cytosolic free calcium ([Ca2+]cyt), which allow the specific and rapid transmission of stress signals through a plant after exposure to a stress, such as salinity. Here, we used an aequorin based bioluminescence assay to compare the NaCl-induced changes in [Ca2+]cyt of the Arabidopsis ecotypes Col-0 and C24. We show that C24 lacks the NaCl specific component of the [Ca2+]cyt signature compared to Col-0. This phenotypic variation could be exploited as a screening methodology for the identification of yet unknown components in the early stages of the salt signalling pathway.

Fluoride Affects Calcium Homeostasis by Regulating Parathyroid Hormone, PTH-Related Peptide, and Calcium-Sensing Receptor Expression.

Parathyroid hormone (PTH), PTH-related peptide (PTHrP), and calcium-sensing receptor (CaSR) play important roles in maintaining calcium homeostasis. Here, we study the effect of fluoride on expression of PTH, PTHrP, and CaSR both in vitro and in vivo. MC3T3-E1 cells and Sprague-Dawley rats were treated with different concentrations of fluoride. Then, the free calcium ion concentration in cell culture supernatant and serum were measured by biochemical analyzer. The expression of PTH, PTHrP, and CaSR was analyzed by qRT-PCR and Western blot. We found that the low dose of fluoride increased ionized calcium (i[Ca(2+)]) and the high dose of fluoride decreased i[Ca(2+)] in cell culture supernatant. The low dose of fluoride inhibited the PTH and PTHrP expression in MC3T3-E1 cells. The high dose of fluoride improved the PTHrP expression in MC3T3-E1 cells. Interestingly, we found that NaF decreased serum i[Ca(2+)] in rats. Fluoride increased CaSR expression at both messenger RNA (mRNA) and protein levels in MC3T3-E1 cells and rats. The expression of PTHrP protein was inhibited by fluoride in rats fed regular diet and was increased by fluoride in rats fed low-calcium diet. Fluoride also increased the expression of PTH, NF-kappaB ligand (RANKL), and osteoprotegerin (OPG) in rats. The ratio of RANKL/OPG in rats fed low-calcium food in presence or absence of fluoride was significantly increased. These results indicated that fluoride might be able to affect calcium homeostasis by regulating PTH, PTHrP, and CaSR.

A single-compartment model of calcium dynamics in nerve terminals and dendrites.

This introduction describes a single-compartment model of calcium dynamics that has been applied to fluorescence measurements of intracellular free calcium concentration ([Ca(2+)]i) changes in neurons. The model describes intracellular calcium handling under simplified conditions, for which analytical expressions for the amplitude and the time constants of [Ca(2+)]i changes can be explicitly derived. In particular, it reveals the dependence of the measured [Ca(2+)]i changes on the calcium indicator concentration. Applied to experimental data from small cells or subcellular compartments, the model equations have been extremely useful for obtaining quantitative information about essential parameters of Ca(2+) influx, buffering, and clearance. We illustrate also several changes that occur when the basic assumptions do not hold (e.g., when calcium diffusion, dye saturation, or kinetic effects become significant). Finally, we discuss how the changes in calcium dynamics, which are explained by the model, have been exploited for measuring properties of calcium-driven reactions, such as those regulating short-term synaptic enhancement, vesicle recycling, and adaptation.

Calcium signals inhibition sensitizes ovarian carcinoma cells to anti-Bcl-xL strategies through Mcl-1 down-regulation.

Ovarian carcinoma is the leading cause of death from gynecologic cancer in the developed world and is characterized by acquired chemoresistance leading to an overall 5-year survival rate of about 30 %. We previously showed that Bcl-xL and Mcl-1 cooperatively protect platinum-resistant ovarian cancer cells from apoptosis. Despite BH3-mimetics represent promising drugs to target Bcl-xL, anti-Mcl-1 strategies are still in pre-clinical studies and required new investigations. Calcium is a universal second messenger and dysregulation of calcium signal is often observed during carcinogenesis. As change in cytosolic free calcium concentration [Ca2+]i is known to control the fate of the cell by regulating Bcl-2 family members, we wonder if calcium signal could impact on Mcl-1 expression and if its pharmacological inhibition could be useful to sensitize ovarian carcinoma cells to anti-Bcl-xL strategies. We therefore studied the effect of different calcium signals inhibitors in ovarian carcinoma cell lines SKOV3 and IGROV1-R10 and analysed their effects on proliferation and Mcl-1 expression. We also exposed these cells to these inhibitors in combination with anti-Bcl-xL strategies (siRNA or BH3-mimetic: ABT-737). We found that calcium signaling regulates Mcl-1 through translational events and a calmodulin-mediated pathway. BAPTA-AM and calmodulin inhibitor combination with ABT-737 leads to apoptosis, a process that is reversed by Mcl-1 enforced expression. As Mcl-1 represents a crucial hurdle to the success of chemotherapy, these results could open to new area of investigation using calcium modulators to directly or indirectly target Mcl-1 and thus efficiently sensitize ovarian carcinoma cells to anti-Bcl-xL strategies.Electronic supplementary materialThe online version of this article (doi:10.1007/s10495-015-1095-3) contains supplementary material, which is available to authorized users.

Modulation of calmodulin lobes by different targets: an allosteric model with hemiconcerted conformational transitions.

Calmodulin is a calcium-binding protein ubiquitous in eukaryotic cells, involved in numerous calcium-regulated biological phenomena, such as synaptic plasticity, muscle contraction, cell cycle, and circadian rhythms. It exibits a characteristic dumbell shape, with two globular domains (N- and C-terminal lobe) joined by a linker region. Each lobe can take alternative conformations, affected by the binding of calcium and target proteins.Calmodulin displays considerable functional flexibility due to its capability to bind different targets, often in a tissue-specific fashion. In various specific physiological environments (e.g. skeletal muscle, neuron dendritic spines) several targets compete for the same calmodulin pool, regulating its availability and affinity for calcium. In this work, we sought to understand the general principles underlying calmodulin modulation by different target proteins, and to account for simultaneous effects of multiple competing targets, thus enabling a more realistic simulation of calmodulin-dependent pathways. We built a mechanistic allosteric model of calmodulin, based on an hemiconcerted framework: each calmodulin lobe can exist in two conformations in thermodynamic equilibrium, with different affinities for calcium and different affinities for each target. Each lobe was allowed to switch conformation on its own. The model was parameterised and validated against experimental data from the literature. In spite of its simplicity, a two-state allosteric model was able to satisfactorily represent several sets of experiments, in particular the binding of calcium on intact and truncated calmodulin and the effect of different skMLCK peptides on calmodulin’s saturation curve. The model can also be readily extended to include multiple targets. We show that some targets stabilise the low calcium affinity T state while others stabilise the high affinity R state. Most of the effects produced by calmodulin targets can be explained as modulation of a pre-existing dynamic equilibrium between different conformations of calmodulin’s lobes, in agreement with linkage theory and MWC-type models.

Cardiac tissue slices: preparation, handling, and successful optical mapping.

Cardiac tissue slices are becoming increasingly popular as a model system for cardiac electrophysiology and pharmacology research and development. Here, we describe in detail the preparation, handling, and optical mapping of transmembrane potential and intracellular free calcium concentration transients (CaT) in ventricular tissue slices from guinea pigs and rabbits. Slices cut in the epicardium-tangential plane contained well-aligned in-slice myocardial cell strands (“fibers”) in subepicardial and midmyocardial sections. Cut with a high-precision slow-advancing microtome at a thickness of 350 to 400 μm, tissue slices preserved essential action potential (AP) properties of the precutting Langendorff-perfused heart. We identified the need for a postcutting recovery period of 36 min (guinea pig) and 63 min (rabbit) to reach 97.5% of final steady-state values for AP duration (APD) (identified by exponential fitting). There was no significant difference between the postcutting recovery dynamics in slices obtained using 2,3-butanedione 2-monoxime or blebistatin as electromechanical uncouplers during the cutting process. A rapid increase in APD, seen after cutting, was caused by exposure to ice-cold solution during the slicing procedure, not by tissue injury, differences in uncouplers, or pH-buffers (bicarbonate; HEPES). To characterize intrinsic patterns of CaT, AP, and conduction, a combination of multipoint and field stimulation should be used to avoid misinterpretation based on source-sink effects. In summary, we describe in detail the preparation, mapping, and data analysis approaches for reproducible cardiac tissue slice-based investigations into AP and CaT dynamics.

Activation of the bovine retinal rod outer segment cGMP-specific phosphodiesterase by the transducin-GTP[S] complex in a physiologically significant range of free calcium ion concentrations

The basal- and GTP[S]-stimulated activities of cGMP-specific phosphodiesterase were measured in totally bleached bovine retinal rod outer segments suspensions at high and low free calcium ions concentration (≈100 μM and <10 nM, respectively). The obtained results suggest that the activity of free phosphodiesterase in rod outer segment membranes and all following processes (transducin activation, interaction transducin-GTP[S] complex with phosphodiesterase, and activity of the enzyme stimulated by transducin-GTP[S] complex) do not depend on the concentration of free calcium ions in a physiologically significant range of its concentration changes in rod outer segments.

Calcium Transport across Plasma Membrane in Early Stages of Chronic Kidney Disease – Impact of Vitamin D3 Supplementation

Mini review summarizes the results of studies focused on elucidating pathophysiological mechanisms of altered intracellular calcium homeostasis in the peripheral blood mononuclear cells of patients with early stages of chronic kidney disease. The basic mechanisms of calcium entry as well as those of its removal are impaired by the disease. These disturbances cause an increase concentration of free cytosolic calcium which can result in a change of broad range of cellular processes and expression patterns of various signaling molecules. Vitamin D3 supplementation is the standard treatment of frequent vitamin D3 insufficiency or deficiency in these patients. It can be assumed that vitamin D3 through the pleiotropic effects may participate in modulation of intracellular calcium homeostasis. Vitamin D3 supplementation resulted in a reduction of cytosolic free calcium affecting some of the transport systems involved in cell calcium entry as well as calcium exit. Normalization of the cytosolic free calcium concentration can have a beneficial effect on intracellular signaling. The mechanisms for regulating and controlling intracellular calcium homeostasis in chronic kidney disease patients are currently still under investigation.

Calcium Sensitive Ring-Like Oligomers of Synaptotagmin: Implications for Regulation of Neurotransmitter Release

The synaptic vesicle protein synaptotagmin-1 (SYT) is required to couple calcium influx to the membrane fusion machinery. However, the structural mechanism underlying this process is unclear. Using electron microscopy we find an unexpected circular arrangement (ring) of SYT's cytosolic domain (C2AB) formed on lipid monolayers in the absence of free calcium. Rings vary in diameter from 18 nm to 43 nm, corresponding to 11 to 26 molecules of SYT. Continuous stacking of the SYT rings occasionally converts both lipid monolayers and liposomal bilayers into protein-coated tubes. Helical reconstruction of the SYT tubes shows that the C2B interacts with the membrane and is involved in ring formation, while the C2A domain points radially outwards. SYT rings are rapidly disrupted by physiological concentrations of free calcium but not by magnesium. Assuming that calcium-free SYT rings are physiologically relevant, these results suggest a simple and novel mechanism by which SYT regulates neurotransmitter release: the ring acts as a spacer to prevent the completion of SNARE complex assembly thereby clamping fusion (in the absence of calcium). When the ring disassembles in the presence of calcium, then fusion proceeds unimpeded.

Alpha-lipoic acid protects against cadmium-induced hepatotoxicity via calcium signalling and gap junctional intercellular communication in rat hepatocytes.

This study investigated the protective effect of alpha-lipoic acid (LA) on cadmium (Cd)-induced hepatotoxicity in BRL 3A rat liver cells. We demonstrated that LA ameliorated Cd-induced cellular injury in cell viability and nuclear fragmentation in BRL 3A cells. Furthermore, LA markedly ameliorated Cd-induced gap junctional intercellular communication (GJIC) inhibition and Cx43 mRNA expression decrease, as well as disassembly of gap junctions. The gap junction blocker carbenoxolone disodium (CBX) as well as LA protected healthy cells from Cd-exposed cells in Transwell co-culture system. LA also protected BRL 3A cells against Cd-induced elevation of the intracellular concentration of free calcium ([Ca(2+)]i). Pretreatment with a chelater of intracellular Ca(2+) BAPTA-AM or chelater of extracellular Ca(2+) EGTA attenuated Cd-induced cytotoxicity and GJIC inhibition. CBX exacerbated the decrease in cell viability and further elevated the increase in [Ca(2+)]i induced by Cd, whereas BAPTA-AM partly attenuated these phenomena, while EGTA had little effects. These results suggested that Cd-induced hepatotoxicity via GJIC inhibition and [Ca(2+)]i elevation, which originates mainly from intracellular stores. GJIC inhibition has dual effects: (i) it restricts release of Ca(2+) from the cell, which exacerbates the [Ca(2+)]i elevation and cytotoxicity induced by Cd; and (ii) it protects healthy cells from their dangerous neighbors by blocking intercellular communication. Above all, our results indicated that LA partly prevented Cd-induced cytotoxicity via GJIC and calcium signaling in BRL 3A rat liver cells.

Depolarization-induced Intracellular Free Calcium Concentration Increases Show No Desensitizing Effect in Rat Odontoblasts.

Odontoblasts play an important role in the transduction of the sensory signals underlying dentinal pain. Transmembrane voltage-independent Ca(2+) influx in odontoblasts has been well described. Voltage-dependent Ca(2+) influx has also been reported, but its biophysical properties remain unclear. The aim of the present study was to investigate the desensitizing effect of voltage-dependent Ca(2+) influx in rat odontoblasts by measuring depolarization-induced intracellular free Ca(2+) concentrations ([Ca(2+) ]i ). Odontoblasts on dental pulp slices from newborn rats were acutely isolated and [Ca(2+) ]i measured by using fura-2 fluorescence. Repeated application of extracellular high-K(+) solution (50 mM), which induces membrane depolarization-elicited repeated and transient increases in [Ca(2+) ]i in the presence of extracellular Ca(2+). Increases in depolarization-induced [Ca(2+) ]i showed no significant desensitizing effect (p >0.05; Friedman test). These results suggest that odontoblasts express a voltage-dependent Ca(2+) influx pathway with no desensitizing properties.

Purification, characterization and neuroprotective effects of a polysaccharide from Gynostemma pentaphyllum

In the present study, we investigated the neuroprotective effects of GPP1, a purified polysaccharide from Gynostemma pentaphyllum, on Aβ (25–35)-induced neuronal toxicity and explore its potential mechanisms in PC12 cells. The results showed pretreatment with GPP1 prior to Aβ (25–35) exposure significantly protected PC12 cells from Aβ (25–35)-induced cell death, lactate dehydrogenase (LDH) release, DNA damage, intracellular free calcium concentration ([Ca2+]i) influx, mitochondrial dysfunction and mitochondrial cytochrome c (Cyt-C) release. In addition, pretreatment with GPP1 also protected PC12 cells against Aβ (25–35)-induced the accumulation of reactive oxygen species (ROS) and lipid peroxidation product MDA, the reduction in glutathione (GSH) level and superoxide dismutase (SOD) activity, the increased Bax/Bcl-2 protein expression ratio and caspase-3 activation. Our findings suggest that GPP1 exerts a neuroprotective effect against Aβ (25–35)-induced neurotoxicity in PC12 cells, at least in part, via inhibiting oxidative stress and suppressing the mitochondrial apoptotic pathway.

Ca2+-selective electrode: A simple method to measure the phytase-aided release of bound calcium in soymilk

Soymilk is a nutritious beverage widely consumed worldwide. However, the presence of phytic acid hinders the bioavailability of minerals, such as calcium, underscoring the necessity for dephosphorylation. The aim of the study was to develop a quick and simple method to measure the phytase-aided release of bound calcium in soymilk. Freed calcium ion (Ca2+) by the phytase treatment (maximum activity of 48U/L) at different temperatures (45–65°C) and pH (4.8–5.6) was analyzed using a calcium ion-selective electrode (ISE) that produced almost a perfect linear relationship between the enzyme activity and the concentration of free calcium (r=0.98–0.99). The free calcium concentration in untreated soymilk (31mmol/L) increased up to 77mmol/L (p<0.05) with phytase treatment. The entire range of Ca2+ liberation was sensitively quantified with the ISE indicating the feasibility of this simple method for the assessment of the efficacy of phytase to improve the bioavailability of calcium, hence, nutritional value, of soymilk.

Acidic environment activates inflammatory programs in fibroblasts via a cAMP–MAPK pathway

The tissue micromilieu in disorders (inflammation, ischemia, tumor) often shows pronounced metabolic acidosis that may alter signaling and transcriptional activity in resident cells which can be of special importance for omnipresent fibroblasts. In the present study we investigated the impact of metabolic acidosis on rat fibroblasts with special emphasis on their role in inflammation by regulation of TNF-α, MCP-1, COX-2 and iNOS expression and the signaling pathways involved.Extracellular acidosis led to an enhanced expression of TNF-α, COX-2 and iNOS in parallel to an activation of p38 and ERK1/2 kinases that was not observed by sole intracellular acidosis. Accordingly, the protein amounts of TNF-α and COX-2 as well as the production of nitrate and nitrite were elevated. Acidosis-induced expression of COX-2 and iNOS depended on p38 kinase, but not on ERK1/2. In contrast acidosis-induced TNF-α expression was independent of both kinases. Although GPR4, GPR68 and GPR132 are expressed in fibroblasts, the involvement of these potential candidate pH sensors could be ruled out since no acidosis-induced elevation in intracellular cAMP or free calcium content was observed. Furthermore our data show that MAPK activation by an acidic micromilieu depends on Ser/Thr phosphatase activity, but not on the production of reactive oxygen species and is sensitive to cAMP antagonism by Rp-cAMPS. In conclusion, our results show that an acidic microenvironment induces a differential transcriptional program of pathological relevant genes in fibroblasts via the cAMP-phosphatase–MAPK pathway and thereby generates a parainflammatory situation that can result in tissue remodeling.