Analyses of Ligand Binding to IP3 Receptors Using Fluorescence Polarization.

Fluorescence polarization (FP) can be used to measure binding of a small fluorescent ligand to a larger protein because the ligand rotates more rapidly in its free form than when bound. When excited with plane polarized light, the free fluorescent ligand emits depolarized light, which can be quantified. Upon binding, its rotation is reduced and more of the emitted light remains polarized. This allows FP to be used as a nondestructive assay of ligand binding. Here we describe a fast, high-throughput FP assay to quantify the binding of fluorescently labeled inositol 1,4,5-trisphosphate (IP3) to N-terminal fragments of the IP3 receptor. The assay is fast (1-6 h), it avoids use of radioactive materials and when measurements are performed at different temperatures, it can resolve Gibbs free energy (ΔG°), enthalpy (ΔH°), and entropy (ΔS°) changes of ligand binding

Disentangling astroglial physiology with a realistic cell model in silico

Electrically non-excitable astroglia take up neurotransmitters, buffer extracellular K+ and generate Ca2+ signals that release molecular regulators of neural circuitry. The underlying machinery remains enigmatic, mainly because the sponge-like astrocyte morphology has been difficult to access experimentally or explore theoretically. Here, we systematically incorporate multi-scale, tri-dimensional astroglial architecture into a realistic multi-compartmental cell model, which we constrain by empirical tests and integrate into the NEURON computational biophysical environment. This approach is implemented as a flexible astrocyte-model builder ASTRO. As a proof-of-concept, we explore an in silico astrocyte to evaluate basic cell physiology features inaccessible experimentally. Our simulations suggest that currents generated by glutamate transporters or K+ channels have negligible distant effects on membrane voltage and that individual astrocytes can successfully handle extracellular K+ hotspots. We show how intracellular Ca2+ buffers affect Ca2+ waves and why the classical Ca2+ sparks-and-puffs mechanism is theoretically compatible with common readouts of astroglial Ca2+ imaging

Spontaneous, pro-arrhythmic calcium signals disrupt electrical pacing in mouse pulmonary vein sleeve cells

The pulmonary vein, which returns oxygenated blood to the left atrium, is ensheathed by a population of unique, myocyte- like cells called pulmonary vein sleeve cells (PVCs). These cells autonomously generate action potentials that propagate into the left atrial chamber and cause arrhythmias resulting in atrial fibrillation; the most common, often sustained, form of cardiac arrhythmia. In mice, PVCs extend along the pulmonary vein into the lungs, and are accessible in a lung slice preparation. We exploited this model to study how aberrant Ca2+ signaling alters the ability of PVC networks to follow electrical pacing. Cellular responses were investigated using real-time 2-photon imaging of lung slices loaded with a Ca2+- sensitive fluorescent indicator (Ca2+ measurements) and phase contrast microscopy (contraction measurements). PVCs displayed global Ca2+ signals and coordinated contraction in response to electrical field stimulation (EFS). The effects of EFS relied on both Ca2+ influx and Ca2+ release, and could be inhibited by nifedipine, ryanodine or caffeine. Moreover, PVCs had a high propensity to show spontaneous Ca2+ signals that arose via stochastic activation of ryanodine receptors (RyRs). The ability of electrical pacing to entrain Ca2+ signals and contractile responses was dramatically influenced by inherent spontaneous Ca2+ activity. In PVCs with relatively low spontaneous Ca2+ activity (2+ activity (>1.5 Hz), electrical pacing was less effective; PVCs became unpaced, only partially-paced or displayed alternans. Because spontaneous Ca2+ activity varied between cells, neighboring PVCs often had different responses to electrical pacing. Our data indicate that the ability of PVCs to respond to electrical stimulation depends on their intrinsic Ca2+ cycling properties. Heterogeneous spontaneous Ca2+ activity arising from stochastic RyR opening can disengage them from sinus rhythm and lead to autonomous, pro-arrhythmic activity

Exposure to GSM RF fields does not affect calcium homeostasis in human endothelial cells, rat pheocromocytoma cells or rat hippocampal neurons

In the course of modern daily life, individuals are exposed to numerous sources of electromagnetic radiation that are not present in the natural environment. The strength of the electromagnetic fields from sources such as hairdryers, computer display units and other electrical devices is modest. However, in many home and office environments, individuals can experience perpetual exposure to an "electromagnetic smog", with occasional peaks of relatively high electromagnetic field intensity. This has led to concerns that such radiation can affect health. In particular, emissions from mobile phones or mobile phone masts have been invoked as a potential source of pathological electromagnetic radiation. Previous reports have suggested that cellular calcium (Ca2+) homeostasis is affected by the types of radiofrequency fields emitted by mobile phones. In the present study, we used a high-throughput imaging platform to monitor putative changes in cellular Ca2+ during exposure of cells to 900 MHz GSM fields of differing power (specific absorption rate 0.012-2 W/Kg), thus mimicking the type of radiation emitted by current mobile phone handsets. Data from cells experiencing the 900 Mhz GSM fields were compared with data obtained from paired experiments using continuous wave fields or no field. We employed three cell types (human endothelial cells, PC-12 neuroblastoma and primary hippocampal neurons) that have previously been suggested to be sensitive to radiofrequency fields. Experiments were designed to examine putative effects of radiofrequency fields on resting Ca2+, in addition to Ca2+ signals evoked by an InsP(3)-generating agonist. Furthermore, we examined putative effects of radiofrequency field exposure on Ca2+ store emptying and store-operated Ca2+ entry following application of the Ca2+ATPase inhibitor thapsigargin. Multiple parameters (e.g., peak amplitude, integrated Ca2+ signal, recovery rates) were analysed to explore potential impact of radiofrequency field exposure on Ca2+ signals. Our data indicate that 900 MHz GSM fields do not affect either basal Ca2+ homeostasis or provoked Ca2+ signals. Even at the highest field strengths applied, which exceed typical phone exposure levels, we did not observe any changes in cellular Ca2+ signals. We conclude that under the conditions employed in our experiments, and using a highly-sensitive assay, we could not detect any consequence of RF exposure

Robust Reproducible Resting State Networks in the Awake Rodent Brain

Resting state networks (RSNs) have been studied extensively with functional MRI in humans in health and disease to reflect brain function in the un-stimulated state as well as reveal how the brain is altered with disease. Rodent models of disease have been used comprehensively to understand the biology of the disease as well as in the development of new therapies. RSN reported studies in rodents, however, are few, and most studies are performed with anesthetized rodents that might alter networks and differ from their non-anesthetized state. Acquiring RSN data in the awake rodent avoids the issues of anesthesia effects on brain function. Using high field fMRI we determined RSNs in awake rats using an independent component analysis (ICA) approach, however, ICA analysis can produce a large number of components, some with biological relevance (networks). We further have applied a novel method to determine networks that are robust and reproducible among all the components found with ICA. This analysis indicates that 7 networks are robust and reproducible in the rat and their putative role is discussed

Weight Gain Is Associated with Medial Contact Site of Subthalamic Stimulation in Parkinson's Disease

The aim of our study was to assess changes in body-weight in relation to active electrode contact position in the subthalamic nucleus. Regular body weight measurements were done in 20 patients with advanced Parkinson's disease within a period of 18 months after implantation. T1-weighted (1.5T) magnetic resonance images were used to determine electrode position in the subthalamic nucleus and the Unified Parkinson's disease rating scale (UPDRS-III) was used for motor assessment. The distance of the contacts from the wall of the third ventricle in the mediolateral direction inversely correlated with weight gain (r = −0.55, p<0.01) and with neurostimulation-related motor condition expressed as the contralateral hemi-body UPDRS-III (r = −0.42, p<0.01). Patients with at least one contact within 9.3 mm of the wall experienced significantly greater weight gain (9.4±(SD)4.4 kg, N = 11) than those with both contacts located laterally (3.9±2.7 kg, N = 9) (p<0.001). The position of the active contact is critical not only for motor outcome but is also associated with weight gain, suggesting a regional effect of subthalamic stimulation on adjacent structures involved in the central regulation of energy balance, food intake or reward

Selective modulation of subtype III IP3R by Akt regulates ER Ca2+ release and apoptosis

Ca2+ transfer from endoplasmic reticulum (ER) to mitochondria can trigger apoptotic pathways by inducing release of mitochondrial pro-apoptotic factors. Three different types of inositol 1,4,5-trisphosphate receptor (IP3R) serve to discharge Ca2+ from ER, but possess some peculiarities, especially in apoptosis induction. The anti-apoptotic protein Akt can phosphorylate all IP3R isoforms and protect cells from apoptosis, reducing ER Ca2+ release. However, it has not been elucidated which IP3R subtypes mediate these effects. Here, we show that Akt activation in COS7 cells, which lack of IP3R I, strongly suppresses IP3-mediated Ca2+ release and apoptosis. Conversely, in SH-SY 5Y cells, which are type III-deficient, Akt is unable to modulate ER Ca2+ flux, losing its anti-apoptotic activity. In SH-SY 5Y-expressing subtype III, Akt recovers its protective function on cell death, by reduction of Ca2+ release. Moreover, regulating Ca2+ flux to mitochondria, Akt maintains the mitochondrial integrity and delays the trigger of apoptosis, in a type III-dependent mechanism. These results demonstrate a specific activity of Akt on IP3R III, leading to diminished Ca2+ transfer to mitochondria and protection from apoptosis, suggesting an additional level of cell death regulation mediated by Akt

INT reduction is a valid proxy for eukaryotic plankton respiration despite the inherent toxicity of INT and differences in cell wall structure

The reduction of 2-para (iodophenyl)-3(nitrophenyl)-5(phenyl) tetrazolium chloride (INT) is increasingly being used as an indirect method to measure plankton respiration. Its greater sensitivity and shorter incubation time compared to the standard method of measuring the decrease in dissolved oxygen concentration, allows the determination of total and size-fractionated plankton respiration with higher precision and temporal resolution. However, there are still concerns as to the method’s applicability due to the toxicity of INT and the potential differential effect of plankton cell wall composition on the diffusion of INT into the cell, and therefore on the rate of INT reduction. Working with cultures of 5 marine plankton (Thalassiosira pseudonana CCMP1080/5, Emiliania huxleyi RCC1217, Pleurochrysis carterae PLY-406, Scrippsiella sp. RCC1720 and Oxyrrhis marina CCMP1133/5) which have different cell wall compositions (silica frustule, presence/absence of calcite and cellulose plates), we demonstrate that INT does not have a toxic effect on oxygen consumption at short incubation times. There was no difference in the oxygen consumption of a culture to which INT had been added and that of a replicate culture without INT, for periods of time ranging from 1 to 7 hours. For four of the cultures (T. pseudonana CCMP1080/5, P. carterae PLY-406, E. huxleyi RCC1217, and O. marina CCMP1133/5) the log of the rates of dissolved oxygen consumption were linearly related to the log of the rates of INT reduction, and there was no significant difference between the regression lines for each culture (ANCOVA test, F = 1.696, df = 3, p = 0.18). Thus, INT reduction is not affected by the structure of the plankton cell wall and a single INT reduction to oxygen consumption conversion equation is appropriate for this range of eukaryotic plankton. These results further support the use of the INT technique as a valid proxy for marine plankton respiration

Serotonin Augments Gut Pacemaker Activity via 5-HT3 Receptors

Serotonin (5-hydroxytryptamine: 5-HT) affects numerous functions in the gut, such as secretion, muscle contraction, and enteric nervous activity, and therefore to clarify details of 5-HT's actions leads to good therapeutic strategies for gut functional disorders. The role of interstitial cells of Cajal (ICC), as pacemaker cells, has been recognised relatively recently. We thus investigated 5-HT actions on ICC pacemaker activity. Muscle preparations with myenteric plexus were isolated from the murine ileum. Spatio-temporal measurements of intracellular Ca2+ and electric activities in ICC were performed by employing fluorescent Ca2+ imaging and microelectrode array (MEA) systems, respectively. Dihydropyridine (DHP) Ca2+ antagonists and tetrodotoxin (TTX) were applied to suppress smooth muscle and nerve activities, respectively. 5-HT significantly enhanced spontaneous Ca2+ oscillations that are considered to underlie electric pacemaker activity in ICC. LY-278584, a 5-HT3 receptor antagonist suppressed spontaneous Ca2+ activity in ICC, while 2-methylserotonin (2-Me-5-HT), a 5-HT3 receptor agonist, restored it. GR113808, a selective antagonist for 5-HT4, and O-methyl-5-HT (O-Me-5-HT), a non-selective 5-HT receptor agonist lacking affinity for 5-HT3 receptors, had little effect on ICC Ca2+ activity. In MEA measurements of ICC electric activity, 5-HT and 2-Me-5-HT caused excitatory effects. RT-PCR and immunostaining confirmed expression of 5-HT3 receptors in ICC. The results indicate that 5-HT augments ICC pacemaker activity via 5-HT3 receptors. ICC appear to be a promising target for treatment of functional motility disorders of the gut, for example, irritable bowel syndrome