Calcium-induced Release Research Articles

Modelling and experimental analysis of the role of interacting cytosolic and vacuolar pools in shaping low temperature calcium signatures in plant cells

A major challenge to understanding low temperature calcium signatures in plants is defining how these signatures emerge from the interactions of different molecular components that are stored in different subcellular pools of a plant cell. Here we develop an integrative model that incorporates the interactions of Ca²⁺, H⁺, K⁺, Cl⁻ and ATP in both cytosolic and vacuolar pools. Our analysis reveals how these four major ions along with ATP forms a complex network to relate the emergence of calcium signatures to other responses (e.g. pH response). Modelling results are in agreement with experimental observations for both cytosolic free calcium concentration ([Ca²⁺](c)) and pH. The model is further validated by experimentally measuring the response of [Ca²⁺](c) to six fluctuating (rather than constant) temperature profiles. We found that modelling results are in reasonable agreement with experimental observations, in particular, if the rate of reducing temperature is relatively high. In addition, we show that both calcium-induced calcium release (CICR) at the vacuolar membrane and transport of ions from the cytosolic pool to the vacuolar membrane play important roles in the interaction between cytosolic and vacuolar pools. In combination they control the amount and timing of calcium release from the vacuolar to cytosolic pool, shaping the specific calcium signature. The methodology and principles developed here establish an integrative view on the role of cytosolic and vacuolar pools in shaping calcium signatures in general, and they are universally applicable to study of the interactions of multiple subcellular pools.

Sarcoplasmic Reticulum Calcium Depletion Waves in Vascular Smooth Muscle Cells: An Inside View of Spatiotemporal Calcium Regulation

We present results from a novel study of Ca2+ waves in vascular smooth muscle cells using a sarcoplasmic reticulum (SR)-targeted Ca2+ indicator that specifically binds to the luminal protein calsequestrin.Agonist-stimulated waves of elevated cytoplasmic calcium concentration regulate blood vessel tone, and vasomotion in vascular smooth muscle. Previous studies employing cytoplasmic calcium indicators revealed that these calcium waves are generated by a combination of inositol 1,4,5-trisphosphate (IP3) and calcium-induced calcium release (CICR) from the SR. Our findings confirm that these waves are due to regenerative CICR by the receptors for IP3 (IP3R).The main new finding from our bservations is a transient elevation in luminal SR Ca2+ concentration ([Ca2+]_SR) both at the site of wave initiation, just before regenerative Ca2+ release commences, and at the advancing wave front, during wave propagation. This strongly suggests a role of [Ca2+]_SR in activation of IP3R.In addition, we find that these depletion waves are characterized by a decreasing velocity as they progress. We developed a quantitative diffusional model to analyze this finding and conclude that the gradual decrease in the velocity of the SR Ca2+ depletion wave, observed in the absence of external calcium, indicates continuity of the lumen of the sarcoplasmicreticulum network.Finally, our observation that the depletion wave was arrested by the nuclear envelope may have implications for selective Ca2+ signalling.

Humanin Peptides Regulate Calcium Flux in the Mammalian Neuronal, Glial and Endothelial Cells under Stress Conditions

Humanin (HN) and humanin-like substances are short polypeptides that prevent neuronal cell death and dysfunction related to progression of Alzheimer?s disease. HN activates oligomeric receptor (CNFTR/WSX-1/gp130 complex) in the extracellular environment, and it is believed to interact with intracellular proteins, such as Bcl2 family. HN stimulates JAK2/STAT3 pathway that regulates apoptosis and cell death, however exact molecular mechanism of HN mediated cytoprotection remains unknown. The goal of our study was to evaluate effect HN (HNM) and HN-like peptides, which include HNG, HN10d and HN 10dV, on the intracellular calcium release in the cultured brain cells (LN18, C8D1A) and endothelial HUVECs under stress conditions. We incubated the cells with low amounts of HN (4 ?M) for 24 hours, generated cellular stress by addition of either 25 ?M β-amyloid (neuronal and glial cells) or 5 ng/mL of TNF-α (endothelial cells); and induced calcium release with 10 ?M ATP. We demonstrated that HN regulated calcium flux in all three cell types, although it was more pronounced in the endothelial than brain cells. HNM led to decreased calcium flux in C8D1A, HUVECs and LN18; HN10d ? in HUVECs, and 10dV ? in the glial cells. We hypothesized that a site of HN activity and calcium release was located in the endoplasmic reticulum (ER), in which there was slightly altered expression of selected ER-stress associated proteins upon incubation with HNs. There was no effect of the exogenous HNs on the calcium release from the isolated mitochondria. We demonstrated mRNA expression of HNM, HN10d and HN10dV in the neuronal LN18 cells; however their levels were not affected by β-amyloid treatment. It might indicate that stress conditions force HN intracellular translocation instead of change in HN gene expression. Thus it is possible that localization of HN in the ER may regulate intracellular calcium amounts, which ultimately determine fate of the cell.

TRPC-Mediated Current Is Not Involved in Endocannabinoid-Induced Short-Term Depression in Cerebellum

It has been reported that activation of metabotropic glutamate receptor 1 (mGluR1) can mediate endocannabinoid-induced short-term depression of synaptic transmission in cerebellar parallel fiber (PF)-Purkinje cell (PC) synapse. mGluR1 has signaling pathways involved in intracellular calcium increase which may contribute to endocannabinoid release. Two major mGluR1-evoked calcium signaling pathways are known: (1) slow-kinetic inward current carried by transient receptor potential canonical (TRPC) channel which is permeable to Ca2+; (2) IP3-induced calcium release from intracellular calcium store. However, it is unclear how much each calcium source contributes to endocannabinoid signaling. Here, we investigated whether calcium influx through mGluR1-evoked TRPC channel contributes to endocannabinoid signaling in cerebellar Purkinje cells. At first, we applied SKF96365 to inhibit TRPC, which blocked endocannabinoid-induced short-term depression completely. However, an alternative TRP channel inhibitor, BTP2 did not affect endocannabinoid-induced short-term depression although it blocked mGluR1-evoked TRPC currents. Endocannabinoid signaling occurred normally even though the TRPC current was mostly blocked by BTP2. Our data imply that TRPC current does not play an important role in endocannabinoid signaling. We also suggest precaution in applying SKF96365 to inhibit TRP channels and propose BTP2 as an alternative TRPC inhibitor.

Pixel-Wise Fitting: Noise-Free Analytical 3D Visualisation of Calcium Handling in Cardiac Myocytes

Realtime imaging of fast signaling events has yielded important insight and has greatly fostered our understanding of fundamental processes such as subcellular calcium signalling in cardiac and neuronal cells. Scanning technology has pushed the edge of acquisition speeds into dimensions in which the level of single pixel noise has become limiting for the interpretation of the data. Here, we demonstrate that for excitable cells, such as cardiac myocytes, for which signaling (i.e. excitation-contraction coupling, ECC) occurs on the millisecond timescale, such technical limitations can be overcome by a pixel-wise fitting approach using high-speed 2-dimensional confocal calcium data (280 nm lateral resolution and 146 frames/s) and a mathematical approach designed to approximate local calcium transients. Such an approach not only produces virtually noise-free single pixel fluorescence data originating from subcellular volumes as small as 0.08 fl. It also allows extraction of 2D information on signalling events such as calcium induced calcium release in a very robust manner. Using such an analytical approach enabled us to tackle important questions of cardiac ECC under physiological conditions but also helped us to reveal novel information about the pro-arrhythmogenic calcium alternans by demonstrating that microscopic alternans preceeds macroscopic alternans. The applicability of such an analytical, pixel-wise fitting approach can be transferred to other biological systems and may thus help to overcome present technical limitations such as diminished signal to noise levels in high-speed live cell imaging.This work was supported by the DFG, BMBF and the Medical Faculty.

Transverse Tubule Structure is Related to Contractile Function in Human Heart Failure

The transverse tubules (t-tubules) of cardiac myocytes facilitate a rapid and synchronous contraction. Loss of t-tubule structure has been reported in several animal models of cardiac failure and also in human heart failure. The loss of t-tubule structure is thought to disrupt calcium induced calcium release and contribute to impaired cardiac contraction. We sought to determine if a relationship between contractile performance and t-tubule structure may contribute to human heart failure. To test this idea we exploited the regional heterogeneity in contractility found within the failing human heart. MRI analysis was performed on patients with non-ischemic dilated cardiomyopathy awaiting a cardiac transplant. Tagged MRI was used to locate regions of different fractional shortening (circumferential strain) within the same heart. A model of the diseased heart was then created using MRI images from which a sampling map was created and used to sample tissue regions from the patient's heart (at transplantation). Using this methodology we were able obtain 14 tissue samples from 5 diseased hearts with corresponding contractility data ranging from poor (%Sc 2) to near normal function (%Sc 15). Tissue sections from these regions were labelled with fluorescent wheat germ agglutinin (WGA) and imaged using confocal microscopy. Fourier analysis of WGA labelled t-tubule images was used to assess t-tubule integrity of the cardiac myocytes using a modification of an established technique. Briefly, the height of the peak in the power spectrum corresponding to sarcomere spacing of t-tubules provided a metric of integrity (TT power). The mean TT power showed a strong positive relationship with fractional shortening data (R2=0.61 p<0.001) suggesting that loss of t-tubule structure plays a role in reduced contractile function in human heart failure.

How to stop the fire? Control of Ca2+-induced Ca2+release in cardiac muscle

Cardiac excitation–contraction coupling in higher vertebrates is driven by the process of calcium-induced calcium release (CICR). According to the original hypothesis, an action potential initiates a small Ca2+ influx through plasmalemmal Ca2+ channels which indirectly activates contraction by triggering a much larger Ca2+ release from ryanodine receptors (RyR2s), Ca2+ channels on the sarcoplasmic reticulum (SR). It is believed that upon relaxation, SR Ca2+ release ceases via cytosolic Ca2+-dependent inactivation of RyR2s, which allows resequestration of Ca2+ to the SR by SR Ca2+-ATPase. Thus CICR, where Ca2+ serves as trigger, output and negative regulator, is an inherently unstable process. Accordingly, uncontrollable Ca2+ release is implicated in many hallmark features of cardiac disease; diastolic pro-arrhythmic aftercontractions and/or loss of SR Ca2+ resulting in reduced systolic Ca2+ transients and thereby impaired contractility.

Vulnerability of the Retinal Microvasculature to Hypoxia: Role of Polyamine-Regulated KATPChannels

It is uncertain why retinal capillaries are particularly vulnerable to hypoxia. In this study, it was hypothesized that their specialized physiology, which includes being the predominant microvascular location of functional adenosine triphosphate-sensitive potassium (K(ATP)) channels, boosts their susceptibility to hypoxia-induced cell death. Cell viability, ionic currents, intracellular calcium, and pericyte contractility in microvascular complexes freshly isolated from the rat retina were assessed using trypan blue dye exclusion, perforated-patch recordings, fura-2 fluorescence, and time-lapse videos. Chemical hypoxia was induced by antimycin, an oxidative phosphorylation inhibitor. In freshly isolated retinal microvascular complexes, chemical hypoxia caused more cell death in capillaries than in arterioles. Indicative of the role of polyamine-dependent K(ATP) channels, antimycin-induced capillary cell death was markedly decreased in microvessels treated with the polyamine synthesis inhibitor, difluoromethylornithine, or the K(ATP) channel inhibitor, glibenclamide. These inhibitors also diminished the antimycin-induced hyperpolarization, as well as the antimycin-induced intracellular calcium increase, which was significantly dependent on extracellular calcium and was diminished by the inhibitor of calcium-induced calcium release (CICR), dantrolene. Consistent with the importance of the CICR-dependent increase in capillary cell calcium, dantrolene significantly decreased hypoxia-induced capillary cell death. We also found that activation of the polyamine/K(ATP) channel/Ca(2+) influx/CICR pathway not only boosted the vulnerability of retinal capillaries to hypoxia, but also caused the contraction of capillary pericytes, whose vasoconstrictive effect may exacerbate hypoxia. The vulnerability of retinal capillaries to hypoxia is boosted by a mechanism involving the polyamine/K(ATP) channel/Ca(2+) influx/CICR pathway. Discovery of this pathway should provide new targets for pharmacological interventions to minimize hypoxia-induced damage in retinal capillaries.

Deciphering the structure and function of FcεRI/mast cell axis in the regulation of allergy and anaphylaxis: a functional genomics paradigm.

Allergy and anaphylaxis are inflammatory disorders caused by immune reactions mainly induced by immunoglobulin-E that signal through the high-affinity FcεRI receptor to release the inflammatory mediators from innate immune cells. The FcεRI/mast cell axis is potently involved in triggering various intracellular signaling molecules to induce calcium release from the internal stores, induction of transcription factors such as NF-kB, secretion of various cytokines as well as lipid mediators, and degranulation, resulting in the induction of allergy and anaphylaxis. In this review, we discuss various cellular and molecular mechanisms triggered through FcεRI/mast cell axis in allergy and anaphylaxis with a special emphasis on the functional genomics paradigm.

Synaptically evoked Ca2+release from intracellular stores is not influenced by vesicular zinc in CA3 hippocampal pyramidal neurones

The co-release of neuromodulatory substances in combination with classic neurotransmitters such as glutamate and GABA from individual presynaptic nerve terminals has the capacity to dramatically influence synaptic efficacy and plasticity. At hippocampal mossy fibre synapses vesicular zinc is suggested to serve as a cotransmitter capable of regulating calcium release from internal stores in postsynaptic CA3 pyramidal cells. Here we investigated this possibility using combined intracellular ratiometric calcium imaging and patch-clamp recording techniques. In acute hippocampal slices a brief train of mossy fibre stimulation produced a large, delayed postsynaptic Ca(2+) wave that was spatially restricted to the proximal apical dendrites of CA3 pyramidal cells within stratum lucidum. This calcium increase was sensitive to intracellularly applied heparin indicating reliance upon release from internal stores and was triggered by activation of both group I metabotropic glutamate and NMDA receptors. Importantly, treatment of slices with the membrane-impermeant zinc chelator CaEDTA did not influence the synaptically evoked postsynaptic Ca(2+) waves. Moreover, mossy fibre stimulus evoked postsynaptic Ca(2+) signals were not significantly different between wild-type and zinc transporter 3 (ZnT3) knock-out animals. Considered together our data do not support a role for vesicular zinc in regulating mossy fibre evoked Ca(2+) release from CA3 pyramidal cell internal stores.

Abstract 18375: A Luminal Calcium Sensing Deficient Mutation of the Cardiac Ryanodine Receptor Diminishes Stress-Induced Ventricular Tachycardias in Calsequestrin Null Mice

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease associated with syncope and sudden cardiac death. Naturally occurring mutations in the cardiac ryanodine receptor (RyR2) and calsequestrin (CASQ2) have been linked to CPVT. The sarcoplasmic reticulum (SR) calcium content and the sensitivity of RyR2 to activation by luminal calcium are some of the key factors determining the propensity for Store Overload Induced Calcium Release (SOICR) and SOICR-evoked VTs. We have recently found that the residue E4782 located in the helix bundle crossing (the ion gate) of the RyR2 calcium release channel acts as a SR store calcium sensor responsible for luminal calcium regulation of RyR2 and SOICR. Interestingly, we found that E4782Q mutation completely protected against SOCR-evoked VTs in a CPVT mouse model harboring the disease-causing RyR2 mutation R4496C. In this present study, we determined whether the E4782Q mutation is capable of rescuing the VT phenotype of the CASQ2 null mice, another mouse model of CPVT. We bred E4872Q+/- mice with the CASQ2 null mice to produce CASQ2-/- or CASQ2+/- with or without the heterozygous E4872Q mutation. After the injection of epinephrine (1.6mg/kg) and caffeine (120mg/kg), CASQ2-/- / E4872Q-/- and CASQ2+/- / E4872Q -/- mice showed long lasting VTs during 30-min of ECG recording (83% and 33%, respectively). On the other hand, CASQ2-/- / E4872Q+/- and CASQ2+/- / E4872Q -/- displayed little or no VTs (6.5% and 0.8%, respectively, p &lt; 0.001, when compared with CASQ2-/- / E4872Q -/-, and CASQ2+/- /E4872Q -/-, n=7~9, for each group). Thus, the E4872Q mutation nearly completely rescued the CPVT phenotype in CASQ2 null mice. These observations indicate that targeting the luminal calcium sensor of the RyR2 calcium release channel represents a new and promising therapeutic strategy for treating CPVT and other calcium mediated cardiac arrhythmias.

Spaceflight regulates ryanodine receptor subtype 1 in portal vein myocytes in the opposite way of hypertension

Gravity has a structural role for living systems. Tissue development, architecture, and organization are modified when the gravity vector is changed. In particular, microgravity induces a redistribution of blood volume and thus pressure in the astronaut body, abolishing an upright blood pressure gradient, inducing orthostatic hypotension. The present study was designed to investigate whether isolated vascular smooth muscle cells are directly sensitive to altered gravitational forces and, second, whether sustained blood pressure changes act on the same molecular target. Exposure to microgravity during 8 days in the International Space Station induced the decrease of ryanodine receptor subtype 1 expression in primary cultured myocytes from rat hepatic portal vein. Identical results were found in portal vein from mice exposed to microgravity during an 8-day shuttle spaceflight. To evaluate the functional consequences of this physiological adaptation, we have compared evoked calcium signals obtained in myocytes from hindlimb unloaded rats, in which the shift of blood pressure mimics the one produced by the microgravity, with those obtained in myocytes from rats injected with antisense oligonucleotide directed against ryanodine receptor subtype 1. In both conditions, calcium signals implicating calcium-induced calcium release were significantly decreased. In contrast, in spontaneous hypertensive rat, an increase in ryanodine receptor subtype 1 expression was observed as well as the calcium-induced calcium release mechanism. Taken together, our results shown that myocytes were directly sensitive to gravity level and that they adapt their calcium signaling pathways to pressure by the regulation of the ryanodine receptor subtype 1 expression.

Calcium Waves in the Xenopus Oocytes Trigger Store Operated Calcium Entry

The oscillation of cytoplasmic calcium concentration exists in a large number of non-excitable cells such as astrocytes in the brain, intestinal cells and oocytes (Fig.1). This rhythmic activity carries information into the cell, regulating various processes such as gene expression or transmitter release. In some cells, such as Xenopus oocytes, the oscillations can rely only on calcium being released in the cytoplasm from intracellular stores after stimulation of inositol triphosphate (IP3) receptors located on the endoplasmic reticulum (ER). To refill the intracellular stores in the ER, the cell has to import extracellular calcium. This is achieved through the STIM/Orai pathway where an ER calcium sensor (STIM) is activated by store depletion and in turn interacts and opens a calcium channel in the plasma membrane (Orai) leading to calcium influx, a process known as Store Operated Calcium Entry (SOCE). In the present work we evaluated the interactions between SOC and intracellular calcium oscillations in Xenopus oocytes. Intracellular calcium levels were monitored using the amplitude of the endogenous calcium-activated chloride currents. Following injection into the cells of a non-hydrolysable analog of IP3 (IP3-DF), long lasting calcium oscillations could be triggered as well as SOCE ( Fig. 1 ). The calcium oscillations were more efficient in promoting SOC than store depletion by ionomycin (2.34 ± 0.55 nA, n=13 vs 0.05 ± 0.02 nA, n=8), the mechanisms involved are currently under study. We also observed that activation of SOC could trigger a calcium wave in a dose-dependent manner following a “Calcium Induced Calcium Release” pattern. This suggests that SOC might not only be involved in replenishing the calcium stores but also in the regulation of the amplitude and timing of calcium oscillations. The well known down-regulation of SOC during oocyte maturation should therefore have important functional consequences on intracellular calcium oscillations.

Calcium-induced calcium release from the sarcoplasmic reticulum can be evaluated with a half-logistic function model in aequorin-injected cardiac muscles

Release of calcium (Ca(2+)) from the sarcoplasmic reticulum (SR) induced by Ca(2+) influx through voltage-dependent sarcolemmal L-type Ca(2+) channels (CICR) in cardiac muscle cells has been implicated as a potential target contributing to anesthetic-induced myocardial depression. In an earlier study, we found that (1) a half-logistic (h-L) function, which represents a half-curve of a sigmoid logistic function with a boundary at the inflection point, curve-fits the first half of the ascending phases of the isometric myocardial tension and isovolumic left ventricular (LV) pressure waveforms better than a mono-exponential (m-E) function and (2) the h-L time constants are useful as inotropic indices. We report here our investigation of the potential application of an h-L function to the analysis of the first half of the ascending phase of the Ca(2+) transient curve (faCaT) that precedes and initiates myocardial contraction and the increase in LV pressure. Ca(2+) transients (CaT) were measured using the Ca(2+)-sensitive photoprotein aequorin, which was microinjected into seven isolated rabbit right ventricular and 15 isolated mouse LV papillary muscles. The faCaT data from the beginning of twitch stimulation to the maximum of the first-order time derivative of Ca(2+) concentration (dCa/dt(max)) was curve-fitted by the least-squares method using h-L and m-E function equations. The mean correlation coefficient (r) values of the h-L and m-E curve-fits for the faCaTs were 0.9740 and 0.9654 (P < 0.05) in the rabbit and 0.9895 and 0.9812 (P < 0.0001) in the mouse. The h-L curves tracked the amplitudes and time courses of the faCaTs in cardiac muscles more accurately than m-E functions. Based on this result, we suggest that the h-L time constant may be a more reliable index than the m-E time constant for evaluating the rate of CICR from the SR in myocardial Ca(2+) handling. The h-L approach may provide a more useful model for the study of CICR during the contraction process induced by anesthetic agents.

Apoptosis induced clustering of IP3R1 in nuclei of non-differentiated PC12 cells

Inositol 1,4,5-trisphosphate (IP(3)) receptors are emerging as key sites for regulation by pro- and anti-apoptotic factors. Induction of apoptosis for 3 h increased mRNA and protein levels of type 1 IP(3) receptors in non-differentiated (ND), but not in differentiated (D) PC12 cells. Inhibitors of the IP(3) R's calcium release-2-aminoethoxydiphenyl borate (2-APB) and xestospongin-completely prevented Bax and caspase-3 mRNA increase after treatment with the apoptosis inducer set (AIK), and this reinforces the importance of IP(3) R1 in the apoptosis of ND PC12 cells. Apoptosis induction not only increases the IP(3) R1 protein, but it also causes formation of IP(3) R1 clusters in the nucleus which most likely result from fusion of the nucleoplasmic reticulum and/or IP(3) R1 translocation to the nucleus. This is quite similar to the observations noted after overexpression of IP(3) R1 in PC12 cells. The amount of IP(3) induced calcium release was higher in control than in AIK-treated cells. From our results we propose that after the apoptosis induction the amount of intranuclear calcium decreased dramatically due to the increase of calcium permeability of the nuclear calcium store vesicles. Therefore, increase of the calcium permeability may result from IP(3) receptors translocation to nuclei that can boost the calcium transport through IP(3) receptors.

Control of intracellular calcium bursting oscillations using method of self-organization

The contribution of this paper is to show a method for controlling intracellular calcium bursting oscillations including frequency and magnitude that have not been controllable before. The method is based on an understanding of system level behaviors as a phenomenon of self-organization. In these systems, the overall function is a consequence of the interaction between the system’s elements. Therefore, if the aim is to control the system’s signals and overall function, it seems reasonable to interact with its elements. In this paper the method is investigated by means of the calcium-induced calcium release (CICR) model proposed by Borghans et al. and a number of representative simulations of intracellular calcium bursting oscillations are used to illustrate the control of these oscillations.

N-Acylhomoserine lactones are potent neutrophil chemoattractants that act via calcium mobilization and actin remodeling

In gram-negative bacteria, cell-cell communication based on HSL QS molecules is known to coordinate the production of virulence factors and biofilms. These bacterial signals can also modulate human immune cell behavior. Using a Transwell migration assay, we found that human primary neutrophils are strongly stimulated by 3O-C(12)-HSL and -C(10)-HSL but not C(4)-HSL in a concentration-dependent manner. Moreover, 3O-C(12)-HSL and -C(10)-HSL activate PLCγ1 but not -γ2, mobilize intracellular calcium, and up-regulate IP(3)R. These changes were paralleled by F-actin accumulation, primarily in the leading edge of neutrophils, as evidenced by phalloidin staining and confocal microscopy. F- and G-actin isolation and quantification by immunoblotting revealed that the F/G-actin ratio was increased significantly after treatment with all three HSLs. Furthemore, 3O-C(12)-HSL- and 3O-C(10)-HSL treatment resulted in phosphorylation of Rac1 and Cdc42. In contrast, C(4)-HSL had negligible influence on the phosphorylation status of PLC and Rac1/Cdc42 and failed to attract neutrophils and induce calcium release. The calcium inhibitor thapsigargin, which blocks ER calcium uptake, strongly prevented neutrophil migration toward 3O-C(12)-HSL and -C(10)-HSL. These findings show that the bacterial QS molecules 3O-C(12)-HSL and -C(10)-HSL may attract human neutrophils to the sites of bacterial infection and developing biofilms. Indeed, recognition of HSL QS signals by neutrophils may play a critical role in their recruitment during infections.

&amp;#x3B3;-Tocotrienol Induces Apoptosis in Human T Cell Lymphoma through Activation of Both Intrinsic and Extrinsic Pathways

Tocotrienols are members of vitamin E family and possess broad biological activities including antioxidant, anti-inflammatory and antitumor effects. In the present study, we examine the potential of α-tocotrienol (AT) and γ-tocotrienol (GT) in inhibiting the proliferation of human T cell lymphoma Jurkat cells and elucidate the pathways involved in anti tumor effects of GT. GT but not AT inhibited proliferation and induced apoptosis in Jurkat cells in a dose dependent manner. GT treatment resulted in elevated mitochondrial ROS production, activation of JNK and suppression of ERK and p38 MAPK. GT also induced calcium release, loss of mitochondrial membrane potential and cytochrome c release from the mitochondria. These changes were accompanied by increase in Bax expression with a concomitant decrease in Bcl-xl expression suggesting activation of mitochondrial apoptotic pathway. GT induced increase in mitochondrial ROS was abrogated by catalase. Besides, GT also up-regulated surface expression of Fas and FasL on Jurkat cells. Further, caspase activation and PARP degradation were also seen in cells treated with GT. Inhibitors of caspase-8 and caspase-9 significantly abrogated GT mediated apoptosis. In contrast GT was not toxic to normal human peripheral blood mononuclear cells suggesting differential cytotoxicity towards normal lymphocytes and transformed lymphoma cells. Cellular uptake studies with tocotrienols showed higher intracellular accumulation of GT as compared to AT which may be responsible for its better antitumor activity. Our results show antitumor effects of GT in human lymphoma cells via increased mitochondrial ROS generation and activation of both intrinsic and extrinsic apoptotic pathways.

Timing Is Everything, Even for Cholinergic Control

Synaptic plasticity is widely considered to be a cellular mechanism underlying learning and memory. In this issue of Neuron, Gu and Yakel show that the precise timing of a single cholinergic pulse of activity can determine whether plasticity will occur at a glutamatergic synapse and confer long-term potentiation versus depression.

Inositol 1,4,5-Trisphosphate Receptor-Mediated Calcium Release in Purkinje Cells: From Molecular Mechanism to Behavior

The inositol 1,4,5-trisphosphate (IP(3)) receptor is highly expressed in cerebellar Purkinje cells and mediates conspicuous calcium release from intracellular calcium stores. Receptor stimulation, such as through mGluR1, activates the G(q)-PLC pathway, which leads to IP(3)-induced calcium release and subsequent cellular responses, including cerebellar long-term depression in Purkinje cells. Recent studies have demonstrated the regulatory mechanisms of IP(3) receptor, revealing activation via IP(3) and Ca(2+), inactivation via high concentrations of Ca(2+), and modulation by various proteins that bind to the IP(3) receptor. Novel calcium imaging techniques and caged compounds provide analysis of calcium signals at the single spine level in relation to the induction of long-term depression. Genetically encoded indicators for calcium or IP(3) could provide alternate Ca(2+) or IP(3) imaging, in particular, for in vivo observations. IP(3)-induced calcium release participates in early development of dendritic branch formation, and loss-of-function mutations or hyper-activation could result various diseases. The IP(3) receptor plays a central role in calcium signaling in Purkinje cells, affecting a wide variety of cellular functions, including development, plasticity, maintenance of synaptic functions, and cerebellar motor control.