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

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

The extracellular calcium-sensing receptor promotes porcine egg activation via calcium/calmodulin-dependent protein kinase II.

Extracellular calcium is required for intracellular Ca2+ oscillations needed for egg activation, but the regulatory mechanism is still poorly understood. The present study was designed to demonstrate the function of calcium-sensing receptor (CASR), which could recognize extracellular calcium as first messenger, during porcine egg activation. CASR expression was markedly upregulated following egg activation. Functionally, the addition of CASR agonist NPS R-568 significantly enhanced pronuclear formation rate, while supplementation of CASR antagonist NPS2390 compromised egg activation. There was no change in NPS R-568 group compared with control group when the egg activation was performed without extracellular calcium addition. The addition of NPS2390 precluded the activation-dependent [Ca2+ ]i rise. When egg activation was conducted in intracellular Ca2+ chelator BAPTA-AM and NPS R-568 containing medium, CASR function was abolished. Meanwhile, CASR activation increased the level of the [Ca2+ ]i effector p-CAMKII, and the presence of KN-93, an inhibitor of CAMKII, significantly reduced the CASR-mediated increasement of pronuclear formation rate. Furthermore, the increase of CASR expression following activation was reversed by inhibiting CAMKII activity, supporting a positive feedback loop between CAMKII and CASR. Altogether, these findings provide a new pathway of egg activation about CASR, as the extracellular Ca2+ effector, promotes egg activation via its downstream effector and upstream regulator CAMKII.

Maladaptive cortical hyperactivity upon recovery from experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) patients exhibit neuropsychological symptoms in early disease despite the immune attack occurring predominantly in white matter and spinal cord. It is unclear why neurodegeneration may start early in the disease and is prominent in later stages. We assessed cortical microcircuit activity by employing spiking-specific two-photon Ca2+ imaging in proteolipid protein-immunized relapsing-remitting SJL/J mice in vivo. We identified the emergence of hyperactive cortical neurons in remission only, independent of direct immune-mediated damage and paralleled by elevated anxiety. High levels of neuronal activity were accompanied by increased caspase-3 expression. Cortical TNFα expression was mainly increased by excitatory neurons in remission; blockade with intraventricular infliximab restored AMPA spontaneous excitatory postsynaptic current frequencies, completely recovered normal neuronal network activity patterns and alleviated elevated anxiety. This suggests a dysregulation of cortical networks attempting to achieve functional compensation by synaptic plasticity mechanisms, indicating a link between immune attack and early start of neurodegeneration.

Intracellular calcium chelating agent (BAPTA-AM) aids stallion semen cooling and freezing-thawing.

This study aimed to investigate the effects of different concentrations of 1,2-bis-(o-aminophenoxy)-ethane-N,N,N0 N0-tetraacetic acid, tetra-acetoxymethyl ester (BAPTA-AM), an intracellular calcium chelating agent, on stallion semen cooling and freezing-thawing. After collection, semen was extended (1:1 v/v) on a skim milk-based extender, centrifuged and resuspended at 400 million/ml into cooling or freezing extenders containing 0, 5, 25, 50, 100 and 200 µΜ BAPTA-AM. Motility parameters were assessed after cooling in Equitainer at 5°C for 12, 24, 48, 72 and 120 hr and after freezing-thawing. In addition, mitochondrial membrane potential, intracellular ATP, reactive oxygen species and malondialdehyde concentrations were measured in cryopreserved-thawed semen. Cooled stored (48 hr) semen containing 50 µΜ BAPTA-AM and control extender (0 µΜ BAPTA-AM) was used to assess fertility. Inclusion of 50 µΜ BAPTA-AM resulted in superior sperm motility parameters during cooled storage when compared to other groups (p < 0.05). Furthermore, semen cryopreserved in extender containing 50 µΜ BAPTA-AM showed increased intracellular ATP and mitochondrial membrane potential, whereas reactive oxygen species and malondialdehyde were increased after thawing for all groups (p < 0.05). Addition of 50 µΜ BAPTA-AM to cooling extender resulted in similar pregnancy rates to the control group (75% vs. 73.6%, respectively; p > 0.05). In conclusion, the addition of BAPTA-AM to semen extenders aided stallion semen cryopreservation in a dose-dependent manner. Furthermore, the cooling extender supplemented with 50 µΜ BAPTA-AM could be used to prolong the sperm motility during cooling without apparently compromising fertility. Field trials should be conducted to assess fertility of cryopreserved stallion semen with BAPTA-AM.

Seihai-to (TJ-90)-Induced Activation of Airway Ciliary Beatings of Mice: Ca2+ Modulation of cAMP-Stimulated Ciliary Beatings via PDE1.

Sei-hai-to (TJ-90, Qing Fei Tang), a Chinese traditional medicine, increases ciliary beat frequency (CBF) and ciliary bend angle (CBA) mediated via cAMP (3',5'-cyclic adenosine monophosphate) accumulation modulated by Ca2+-activated phosphodiesterase 1 (PDE1A). A high concentration of TJ-90 (≥40 µg/mL) induced two types of CBF increases, a transient increase (an initial increase, followed by a decrease) and a sustained increase without any decline, while it only sustained the CBA increase. Upon inhibiting increases in intracellular Ca2+ concentration ([Ca2+]i) by 10 µM BAPTA-AM (Ca2+-chelator, 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) or Ca2+/calmodulin-dependent PDE1 by 8MmIBMX (a selective PDE1 inhibitor), TJ-90 (400 µg/mL) induced only the sustained CBF increase without any transient CBF increase. The two types of the CBF increase (the transient increase and the sustained increase) induced by TJ-90 (≥40 µg/mL) were mimicked by the stimulation with both procaterol (100 pM) and ionomycin (500 nM). Thus, TJ-90 stimulates small increases in the intracellular cAMP concentration ([cAMP]i) and [Ca2+]i in airway ciliary cells of mice. These small increases in [cAMP]i and [Ca2+]i cause inducing a transient CBF increase or a sustained CBF increase in an airway ciliary cells, depending on the dominant signal, Ca2+-signal, or cAMP-signal.

Calcium ion regulation by BAPTA-AM and ruthenium red improved the fertilisation capacity and developmental ability of vitrified bovine oocytes.

Vitrification reduces the fertilisation capacity and developmental ability of mammalian oocytes; this effect is closely associated with an abnormal increase of cytoplasmic free calcium ions ([Ca2+]i). However, little information about the mechanism by which vitrification increases [Ca2+]i levels or a procedure to regulate [Ca2+]i levels in these oocytes is available. Vitrified bovine oocytes were used to analyse the effect of vitrification on [Ca2+]i, endoplasmic reticulum Ca2+ (ER Ca2+), and mitochondrial Ca2+ (mCa2+) levels. Our results showed that vitrification, especially with dimethyl sulfoxide (DMSO), can induce ER Ca2+ release into the cytoplasm, consequently increasing the [Ca2+]i and mCa2+ levels. Supplementing the cells with 10 µM 1,2-bis (o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM or BAPTA) significantly decreased the [Ca2+]i level and maintained the normal distribution of cortical granules in the vitrified bovine oocytes, increasing their fertilisation ability and cleavage rate after in vitro fertilisation (IVF). Treating vitrified bovine oocytes with 1 µM ruthenium red (RR) significantly inhibited the Ca2+ flux from the cytoplasm into mitochondria; maintained normal mCa2+ levels, mitochondrial membrane potential, and ATP content; and inhibited apoptosis. Treating vitrified oocytes with a combination of BAPTA and RR significantly improved embryo development and quality after IVF.

Aspirin-induced heat stress resistance in chicken myocardial cells can be suppressed by BAPTA-AM in vitro.

Our recent studies have displayed the protective functions of aspirin against heat stress (HS) in chicken myocardial cells, and it may be associated with heat shock proteins (HSPs). In this study, we further investigated the potential role of HSPs in the aspirin-induced heat stress resistance. Four of the most important HSPs including HspB1 (Hsp27), Hsp60, Hsp70, and Hsp90 were induced by aspirin pretreatment and were suppressed by BAPTA-AM. When HSPs were induced by aspirin, much slighter HS injury was detected. But more serious damages were observed when HSPs were suppressed by BAPTA-AM than those cells exposed to HS without BAPTA-AM, even the myocardial cells have been treated with aspirin in prior. Comparing to other HSPs, HspB1 presented the largest increase after aspirin treatments, 86-fold higher than the baseline (the level before HS). These findings suggested that multiple HSPs participated in aspirin's anti-heat stress function but HspB1 may contribute the most. Interestingly, during the experiments, we also found that apoptosis rate as well as the oxidative stress indicators (T-SOD and MDA) was not consistently responding to heat stress injury as expected. By selecting from a series of candidates, myocardial cell damage-related enzymes (CK-MB and LDH), cytopathological tests, and necrosis rate (measured by flow cytometry assays) are believed to be reliable indicators to evaluate heat stress injury in chicken's myocardial cells and they will be used in our further investigations.

Calcium-Mediated Induction of Paradoxical Growth following Caspofungin Treatment Is Associated with Calcineurin Activation and Phosphorylation in Aspergillus fumigatus.

The echinocandin antifungal drug caspofungin at high concentrations reverses the growth inhibition of Aspergillus fumigatus, a phenomenon known as the "paradoxical effect," which is not consistently observed with other echinocandins (micafungin and anidulafungin). Previous studies of A. fumigatus revealed the loss of the paradoxical effect following pharmacological or genetic inhibition of calcineurin, yet the underlying mechanism is poorly understood. Here, we utilized a codon-optimized bioluminescent Ca(2+) reporter aequorin expression system in A. fumigatus and showed that caspofungin elicits a transient increase in cytosolic free Ca(2+) ([Ca(2+)]c) in the fungus that acts as the initial trigger of the paradoxical effect by activating calmodulin-calcineurin signaling. While the increase in [Ca(2+)]c was also observed upon treatment with micafungin, another echinocandin without the paradoxical effect, a higher [Ca(2+)]c increase was noted with the paradoxical-growth concentration of caspofungin. Treatments with a Ca(2+)-selective chelator, BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid], or the L-type Ca(2+) channel blocker verapamil abolished caspofungin-mediated paradoxical growth in both the wild-type and the echinocandin-resistant (EMFR-S678P) strains. Concomitant with increased [Ca(2+)]c levels at higher concentrations of caspofungin, calmodulin and calcineurin gene expression was enhanced. Phosphoproteomic analysis revealed that calcineurin is activated through phosphorylation at its serine-proline-rich region (SPRR), a domain previously shown to be essential for regulation of hyphal growth, only at a paradoxical-growth concentration of caspofungin. Our results indicate that as opposed to micafungin, the increased [Ca(2+)]c at high concentrations of caspofungin activates calmodulin-calcineurin signaling at both a transcriptional and a posttranslational level and ultimately leads to paradoxical fungal growth.

Age-associated potency decline in bovine oocytes is delayed by blocking extracellular Ca(2+) influx.

Oocyte aging due to delayed fertilization is associated with declining quality and developmental potential. Intracellular calcium (Ca(2+)) concentration ([Ca(2+)]i) regulates oocyte growth, maturation, and fertilization and has also been implicated in aging. Using bovine oocytes, we tested the hypothesis that oocyte aging could be delayed by reducing [Ca(2+)]ivia blocking the influx of extracellular Ca(2+) or chelating ooplasmic free Ca(2+). After IVM, cumulus-oocyte complexes or denuded oocytes were cultured in medium supplemented with 1-octanol, phorbol 12-myristate 13-acetate, or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis-acetoxymethyl ester (BAPTA-AM) to manipulate [Ca(2+)]i. Addition of 1-mM 1-octanol increased blastocyst development rates in the cumulus-oocyte complexes aged for 6 hours by IVF and for 6, 12, and 24 hours by parthenoactivation, and this effect was independent of the presence of cumulus cells. The intracellular levels of ATP, Glutathione, and Glutathione disulfide were not affected by 1-octanol, but [Ca(2+)]i was significantly decreased. When oocytes were cultured in Ca(2+)-free medium for 12 hours, the blastocyst development rate was greater and the beneficial effects of 1-octanol on oocyte aging were abolished. However, when the medium was supplemented with phorbol 12-myristate 13-acetate, [Ca(2+)]i increased and the blastocyst development rate decreased. Moreover, BAPTA-AM reduced [Ca(2+)]i and increased blastocyst development rates after IVF or parthenoactivation. We conclude that the age-associated developmental potency decline was delayed by blocking the influx of extracellular Ca(2+) or reducing ooplasmic free Ca(2+). 1-Octanol, BAPTA-AM, or Ca(2+)-free medium could be used to lengthen the fertilization windows of aged bovine oocytes.

A novel explanation for observed CaMKII dynamics in dendritic spines with added EGTA or BAPTA.

We present a simplified reaction network in a single well-mixed volume that captures the general features of CaMKII dynamics observed during both synaptic input and spine depolarization. Our model can also account for the greater-than-control CaMKII activation observed with added EGTA during depolarization. Calcium input currents are modeled after experimental observations, and existing models of calmodulin and CaMKII autophosphorylation are used. After calibration against CaMKII activation data in the absence of chelators, CaMKII activation dynamics due to synaptic input via n-methyl-d-aspartate receptors are qualitatively accounted for in the presence of the chelators EGTA and BAPTA without additional adjustments to the model. To account for CaMKII activation dynamics during spine depolarization with added EGTA or BAPTA, the model invokes the modulation of CaV2.3 (R-type) voltage-dependent calcium channel (VDCC) currents observed in the presence of EGTA or BAPTA. To our knowledge, this is a novel explanation for the increased CaMKII activation seen in dendritic spines with added EGTA, and suggests that differential modulation of VDCCs by EGTA and BAPTA offers an alternative or complementary explanation for other experimental results in which addition of EGTA or BAPTA produces different effects. Our results also show that a simplified reaction network in a single, well-mixed compartment is sufficient to account for the general features of observed CaMKII dynamics.

Ischemia-induced endothelial cell swelling and mitochondrial dysfunction are attenuated by cinnamtannin D1, green tea extract, and resveratrol in vitro.

Polyphenols possess antioxidant and anti-inflammatory properties. Oxidative stress (OS) and inflammation have been implicated in the pathogenesis of cytotoxic brain edema in cerebral ischemia. In addition, OS and pro-inflammatory cytokines also damage the endothelial cells and the neurovascular unit. Endothelial cell swelling may contribute to a leaky blood-brain barrier which may result in vasogenic edema in the continued presence of the existing cytotoxic edema. We investigated the protective effects of polyphenols on cytotoxic cell swelling in bEND3 endothelial cultures subjected to 5 hours oxygen-glucose deprivation (OGD). A polyphenol trimer from cinnamon (cinnamtannin D1), a polyphenol-rich extract from green tea, and resveratrol prevented the OGD-induced rise in mitochondrial free radicals, cell swelling, and the dissipation of the inner mitochondrial membrane potential. Monocyte chemoattractant protein (also called CCL2), a chemokine, but not tumor necrosis factor-α or interleukin-6, augmented the cell swelling. This effect of monochemoattractant protein 1-1 was attenuated by the polyphenols. Cyclosporin A, a blocker of the mitochondrial permeability transition pore, did not attenuate cell swelling but BAPTA-AM, an intracellular calcium chelator did, indicating a role of [Ca(2+)]i but not the mPT in cell swelling. These results indicate that the polyphenols reduce mitochondrial reactive oxygen species and subsequent cell swelling in endothelial cells following ischemic injury and thus may reduce brain edema and associated neural damage in ischemia. One possible mechanism by which the polyphenols may attenuate endothelial cell swelling is through the reduction in [Ca(2+)]i.

Hyperthermia induces apoptosis through endoplasmic reticulum and reactive oxygen species in human osteosarcoma cells.

Osteosarcoma (OS) is a relatively rare form of cancer, but OS is the most commonly diagnosed bone cancer in children and adolescents. Chemotherapy has side effects and induces drug resistance in OS. Since an effective adjuvant therapy was insufficient for treating OS, researching novel and adequate remedies is critical. Hyperthermia can induce cell death in various cancer cells, and thus, in this study, we investigated the anticancer method of hyperthermia in human OS (U-2 OS) cells. Treatment at 43 °C for 60 min induced apoptosis in human OS cell lines, but not in primary bone cells. Furthermore, hyperthermia was associated with increases of intracellular reactive oxygen species (ROS) and caspase-3 activation in U-2 OS cells. Mitochondrial dysfunction was followed by the release of cytochrome c from the mitochondria, and was accompanied by decreased anti-apoptotic Bcl-2 and Bcl-xL, and increased pro-apoptotic proteins Bak and Bax. Hyperthermia triggered endoplasmic reticulum (ER) stress, which was characterized by changes in cytosolic calcium levels, as well as increased calpain expression and activity. In addition, cells treated with calcium chelator (BAPTA-AM) blocked hyperthermia-induced cell apoptosis in U-2 OS cells. In conclusion, hyperthermia induced cell apoptosis substantially via the ROS, ER stress, mitochondria, and caspase pathways. Thus, hyperthermia may be a novel anticancer method for treating OS.

Transduction without tip links in cochlear hair cells is mediated by ion channels with permeation properties distinct from those of the mechano-electrical transducer channel.

Tip links between adjacent stereocilia are believed to gate mechano-electrical transducer (MET) channels and mediate the electrical responses of sensory hair cells. We found that mouse auditory hair cells that lack tip links due to genetic mutations or exposure to the Ca(2+) chelator BAPTA can, however, still respond to mechanical stimuli. These MET currents have unusual properties and are predominantly of the opposite polarity relative to those measured when tip links are present. There are other striking differences, for example, the channels are usually all closed when the hair cell is not stimulated and the currents in response to strong stimuli can be substantially larger than normal. These anomalous MET currents can also be elicited early in development, before the onset of mechano-electrical transduction with normal response polarity. Current-voltage curves of the anomalous MET currents are linear and do not show the rectification characteristic of normal MET currents. The permeant MET channel blocker dihydrostreptomycin is two orders of magnitude less effective in blocking the anomalous MET currents. The findings suggest the presence of a large population of MET channels with pore properties that are distinct from those of normal MET channels. These channels are not gated by hair-bundle links and can be activated under a variety of conditions in which normal tip-link-mediated transduction is not operational.

Paclitaxel induces apoptosis in breast cancer cells through different calcium--regulating mechanisms depending on external calcium conditions.

Previously, we reported that endoplasmic reticulum calcium stores were a direct target for paclitaxel initiation of apoptosis. Furthermore, the actions of paclitaxel attenuated Bcl-2 resistance to apoptosis through endoplasmic reticulum-mediated calcium release. To better understand the calcium-regulated mechanisms of paclitaxel-induced apoptosis in breast cancer cells, we investigated the role of extracellular calcium, specifically; whether influx of extracellular calcium contributed to and/or was necessary for paclitaxel-induced apoptosis. Our results demonstrated that paclitaxel induced extracellular calcium influx. This mobilization of extracellular calcium contributed to subsequent cytosolic calcium elevation differently, depending on dosage. Under normal extracellular calcium conditions, high dose paclitaxel induced apoptosis-promoting calcium influx, which did not occur in calcium-free conditions. In the absence of extracellular calcium an "Enhanced Calcium Efflux" mechanism in which high dose paclitaxel stimulated calcium efflux immediately, leading to dramatic cytosolic calcium decrease, was observed. In the absence of extracellular calcium, high dose paclitaxel's stimulatory effects on capacitative calcium entry and apoptosis could not be completely restored. Thus, normal extracellular calcium concentrations are critical for high dose paclitaxel-induced apoptosis. In contrast, low dose paclitaxel mirrored controls, indicating that it occurs independent of extracellular calcium. Thus, extracellular calcium conditions only affect efficacy of high dose paclitaxel-induced apoptosis.

Interplay of reactive oxygen species, intracellular Ca2+ and mitochondrial homeostasis in the apoptosis of prostate cancer cells by deoxypodophyllotoxin.

The limited treatment option for recurrent prostate cancer and the eventual resistance to conventional chemotherapy drugs has fueled continued interest in finding new anti-neoplastic agents of natural product origin. We previously reported anti-proliferative activity of deoxypodophyllotoxin (DPT) on human prostate cancer cells. Using the PC-3 cell model of human prostate cancer, the present study reveals that DPT induced apoptosis via a caspase-3-dependent pathway that is activated due to dysregulated mitochondrial function. DPT-treated cells showed accumulation of the reactive oxygen species (ROS), intracellular Ca (i)(2+) surge, increased mitochondrial membrane potential (MMP, ΔΨ(m)), Bax protein translocation to mitochondria and cytochrome c release to the cytoplasm. This resulted in caspase-3 activation, which in turn induced apoptosis. The antioxidant N-acetylcysteine (NAC) reduced ROS accumulation, MMP and Ca (i)(2+) surge, on the other hand the Ca(2+) chelator BAPTA inhibited the Ca( i)(2+) overload and MMP without affecting the increase of ROS, indicating that the generation of ROS occurred prior to Ca(2+) flux. This suggested that both ROS and Ca( i)(2+) signaling play roles in the increased MMP via Ca (i)(2+)-dependent and/or -independent mechanisms, since ΔΨ(m) elevation was reversed by NAC and BAPTA. This study provides the first evidence for the involvement of both ROS- and Ca( i)(2+)-activated signals in the disruption of mitochondrial homeostasis and the precedence of ROS production over the failure of Ca(2+) flux homeostasis.

Screening quality for Ca2+-activated potassium channel in IonWorks Quattro is greatly improved by using BAPTA-AM and ionomycin.

INTRODUCTION: IonWorks automated patch clamp systems are being widely used for ion channel drug discovery, but the perforated patch mode of these systems makes it difficult to obtain a steady intracellular Ca(2+) concentration ([Ca(2+)](i)). This difficulty prevents obtaining high-quality data regarding Ca(2+)-activated channels such as BK and SK channels. We examined the methods for stabilizing [Ca(2+)](i) in the IonWorks Quattro automated patch clamp system to evaluate BK channels. METHODS: Electrophysiological recordings were performed using the single-hole or population patch clamp mode of IonWorks Quattro. To increase [Ca(2+)](i), ionomycin was used. The variation in the BK current and the effect of BK channel modulators were examined in the presence and absence of an intracellular Ca(2+) chelator, BAPTA-AM (20µM). RESULTS: BK current activated by step pulses to +100mV in the presence of ionomycin exhibited large variation (ranging from 0.086 to 11nA). In individual cells, oscillation of the current amplitude was observed when five repetitive pulses were applied at 0.1Hz. Approximately 30% of cells exhibited current variation exceeding 20% when the variation was calculated using the first and third pulses. However, BAPTA-AM treatment before current measurement decreased the number of cells displaying large variation (>20%) to 5%. In the presence of BAPTA-AM, the BK channel modulators NS1619 and 12,14-dichlorodehydroabietic acid increased the BK current at concentrations of 10µM or more showing clear concentration dependency, whereas in its absence, the effect of both compounds was detected only at 30µM. DISCUSSION: The main finding of this study is that the [Ca(2+)](i) variation in the basal condition is very large and hinders the accurate evaluation of compounds in Ca(2+)-activated ion channels. The application of BAPTA-AM and ionomycin greatly improved the precision of BK channel screening, and this method should be applicable to other Ca(2+)-activated ion channels such as SK channels.

Discrete neocortical dynamics predict behavioral categorization of sounds.

The ability to group stimuli into perceptual categories is essential for efficient interaction with the environment. Discrete dynamics that emerge in brain networks are believed to be the neuronal correlate of category formation. Observations of such dynamics have recently been made; however, it is still unresolved if they actually match perceptual categories. Using in vivo two-photon calcium imaging in the auditory cortex of mice, we show that local network activity evoked by sounds is constrained to few response modes. Transitions between response modes are characterized by an abrupt switch, indicating attractor-like, discrete dynamics. Moreover, we show that local cortical responses quantitatively predict discrimination performance and spontaneous categorization of sounds in behaving mice. Our results therefore demonstrate that local nonlinear dynamics in the auditory cortex generate spontaneous sound categories which can be selected for behavioral or perceptual decisions.

Sleep oscillations in the thalamocortical system induce long-term neuronal plasticity.

Long-term plasticity contributes to memory formation and sleep plays a critical role in memory consolidation. However, it is unclear whether sleep slow oscillation by itself induces long-term plasticity that contributes to memory retention. Using in vivo prethalamic electrical stimulation at 1 Hz, which itself does not induce immediate potentiation of evoked responses, we investigated how the cortical evoked response was modulated by different states of vigilance. We found that somatosensory evoked potentials during wake were enhanced after a slow-wave sleep episode (with or without stimulation during sleep) as compared to a previous wake episode. In vitro, we determined that this enhancement has a postsynaptic mechanism that is calcium dependent, requires hyperpolarization periods (slow waves), and requires a coactivation of both AMPA and NMDA receptors. Our results suggest that long-term potentiation occurs during slow-wave sleep, supporting its contribution to memory.

[Mesenchymal stromal cells synchronize the rhythm of protein synthesis under the effect of an exogenous signal].

A comparative study was performed of dense 5-hour cultures of rat hepatocytes and equal-density cultures of mesenchymal stromal cells (MSC) isolated from human adipose tissue of rat bone marrow. The cells were grown on collagen-coated class slides in serum-free medium. Unlike in hepatocytes, no rhythm of protein synthesis was initially revealed in MSC, but such a rhythm manifested itself when the culture medium was supplemented with melatonin (2 nM, 5 min). The results of experiments with cytoplasmic calcium chelator BAPTA-AM and protein kinase inhibitor H7 indicate that the mechanism of protein synthesis synchronization in MSC consists in calcium-dependent phosphorylation of cell proteins.

Gintonin, a ginseng-derived lysophosphatidic acid receptor ligand, attenuates Alzheimer's disease-related neuropathies: involvement of non-amyloidogenic processing.

Ginseng extracts show cognition-enhancing effects in Alzheimer's disease (AD) patients. However, little is known about the active components and molecular mechanisms of how ginseng exerts its effects. Recently, we isolated a novel lysophosphatidic acid (LPA) receptor-activating ligand from ginseng, gintonin. AD is caused by amyloid-ß protein (Aß) accumulation. Aß is derived from amyloid-ß protein precursors (AßPPs) through the amyloidogenic pathway. In contrast, non-amyloidogenic pathways produce beneficial, soluble AßPPα (sAßPPα). Here, we describe our investigations of the effect of gintonin on sAßPPα release, Aß formation, Swedish-AßPP transfection-mediated neurotoxicity in SH-SY5Y neuroblastoma cells, and Aß-induced neuropathy in mice. Gintonin promoted sAßPPα release in a concentration- and time-dependent manner. Gintonin action was also blocked by the Ca2+ chelator BAPTA, α-secretase inhibitor TAPI-2, and protein-trafficking inhibitor brefeldin. Gintonin decreased Aß1-42 release and attenuated Aß1-40-induced cytotoxicity in SH-SY5Y cells. Gintonin also rescued Aß1-40-induced cognitive dysfunction in mice. Moreover, in a transgenic mouse AD model, long-term oral administration of gintonin attenuated amyloid plaque deposition as well as short- and long-term memory impairment. In the present study, we demonstrated that gintonin mediated the promotion of non-amyloidogenic processing to stimulate sAßPPα release to restore brain function in mice with AD. Gintonin could be a useful agent for AD prevention or therapy.