Alpha-synuclein modulation of Ca2+ signaling in human neuroblastoma (SH-SY5Y) cells

Parkinson's disease (PD) is characterized in part by the presence of alpha-synuclein (alpha-syn) rich intracellular inclusions (Lewy bodies). Mutations and multiplication of the alpha-synuclein gene (SNCA) are associated with familial PD. Since Ca2+ dyshomeostasis may play an important role in the pathogenesis of PD, we used fluorimetry in fura-2 loaded SH-SY5Y cells to monitor Ca2+ homeostasis in cells stably transfected with either wild-type alpha-syn, the A53T mutant form, the S129D phosphomimetic mutant or with empty vector (which served as control). Voltage-gated Ca2+ influx evoked by exposure of cells to 50 mM K+ was enhanced in cells expressing all three forms of alpha-syn, an effect which was due specifically to increased Ca2+ entry via L-type Ca2+ channels. Mobilization of Ca2+ by muscarine was not strikingly modified by any of the alpha-syn forms, but they all reduced capacitative Ca2+ entry following store depletion caused either by muscarine or thapsigargin. Emptying of stores with cyclopiazonic acid caused similar rises of [Ca2+](i) in all cells tested (with the exception of the S129D mutant), and mitochondrial Ca2+ content was unaffected by any form of alpha-synuclein. However, only WT alpha-syn transfected cells displayed significantly impaired viability. Our findings suggest that alpha-syn regulates Ca2+ entry pathways and, consequently, that abnormal alpha-syn levels may promote neuronal damage through dysregulation of Ca2+ homeostasis.

Hypoxic modulation of ca(2+) signaling in human venous and arterial endothelial cells

Our understanding of vascular endothelial cell physiology is based on studies of endothelial cells cultured from various vascular beds of different species for varying periods of time. Systematic analysis of the properties of endothelial cells from different parts of the vasculature is lacking. Here, we compare Ca(2+) homeostasis in primary cultures of endothelial cells from human internal mammary artery and saphenous vein and how this is modified by hypoxia, an inevitable consequence of bypass grafting (2.5% O(2), 24 h). Basal [Ca(2+)]( i ) and store depletion-mediated Ca(2+) entry were significantly different between the two cell types, yet agonist (ATP)-mediated mobilization from endoplasmic reticulum stores was similar. Hypoxia potentiated agonist-evoked responses in arterial, but not venous, cells but augmented store depletion-mediated Ca(2+) entry only in venous cells. Clearly, Ca(2+) signaling and its remodeling by hypoxia are strikingly different in arterial vs. venous endothelial cells. Our data have important implications for the interpretation of data obtained from endothelial cells of varying sources.

Modulation of potassium ion channel proteins utilising antibodies

The application of antibodies to living cells has the potential to modulate the function of specific proteins by virtue of their high specificity. This specificity has proven effective in determining the involvement of many proteins in neuronal function where specific agonists and antagonists do not exist, e.g. ion channel subunits. We discuss a way to utilise subunit specific antibodies to target individual channel subunits in electrophysiological experiments to determine functional roles within native neurones. Utilising this approach, we have investigated the role of the voltage-gated potassium channel Kv3.1b subunit within a region of the brainstem important in the regulation of autonomic function. We provide some useful control experiments in order to help validate this method. We conclude that antibodies can be extremely valuable in determining the functions of specific proteins in living neurones in neuroscience research.

Carbon monoxide inhibits L-type Ca2+ channels via redox modulation of key cysteine residues by mitochondrial reactive oxygen species

Conditions of stress, such as myocardial infarction, stimulate up-regulation of heme oxygenase (HO-1) to provide cardioprotection. Here, we show that CO, a product of heme catabolism by HO-1, directly inhibits native rat cardiomyocyte L-type Ca2+ currents and the recombinant alpha1C subunit of the human cardiac L-type Ca2+ channel. CO (applied via a recognized CO donor molecule or as the dissolved gas) caused reversible, voltage-independent channel inhibition, which was dependent on the presence of a spliced insert in the cytoplasmic C-terminal region of the channel. Sequential molecular dissection and point mutagenesis identified three key cysteine residues within the proximal 31 amino acids of the splice insert required for CO sensitivity. CO-mediated inhibition was independent of nitric oxide and protein kinase G but was prevented by antioxidants and the reducing agent, dithiothreitol. Inhibition of NADPH oxidase and xanthine oxidase did not affect the inhibitory actions of CO. Instead, inhibitors of complex III (but not complex I) of the mitochondrial electron transport chain and a mitochondrially targeted antioxidant (Mito Q) fully prevented the effects of CO. Our data indicate that the cardioprotective effects of HO-1 activity may be attributable to an inhibitory action of CO on cardiac L-type Ca2+ channels. Inhibition arises from the ability of CO to promote generation of reactive oxygen species from complex III of mitochondria. This in turn leads to redox modulation of any or all of three critical cysteine residues in the channel's cytoplasmic C-terminal tail, resulting in channel inhibition.

Modulation of hTREK-1 by carbon monoxide

TREK-1 is a background K channel important in the regulation of neuronal excitability. Here, we demonstrate that recombinant human TREK-1 is activated by low concentrations of carbon monoxide (CO) and nitric oxide (NO), applied via their respective donor molecules. Related channels hTASK-1 and hTASK-3 were unaffected by CO. Effects of both CO and NO were prevented by preincubation of cells with the protein kinase G inhibitor, Rp-8-Br-PET-cGMPS. The effects of CO were independent of NO formation. At higher concentrations, both NO and CO were inhibitory. As both NO and CO are important neuronal gasotransmitters and TREK is crucial in regulating neuronal excitability, our results provide a novel means by which these gases may modulate neuronal activity.

Voltage-gated potassium currents within the dorsal vagal nucleus: inhibition by BDS toxin

Voltage-gated potassium (Kv) channels are essential components of neuronal excitability. The Kv3.4 channel protein is widely distributed throughout the central nervous system (CNS), where it can form heteromeric or homomeric Kv3 channels. Electrophysiological studies reported here highlight a functional role for this channel protein within neurons of the dorsal vagal nucleus (DVN). Current clamp experiments revealed that blood depressing substance (BDS) and intracellular dialysis of an anti-Kv3.4 antibody prolonged the action potential duration. In addition, a BDS sensitive, voltage-dependent, slowly inactivating outward current was observed in voltage clamp recordings from DVN neurons. Electrical stimulation of the solitary tract evoked EPSPs and IPSPs in DVN neurons and BDS increased the average amplitude and decreased the paired pulse ratio, consistent with a presynaptic site of action. This presynaptic modulation was action potential dependent as revealed by ongoing synaptic activity. Given the role of the Kv3 proteins in shaping neuronal excitability, these data highlight a role for homomeric Kv3.4 channels in spike timing and neurotransmitter release in low frequency firing neurons of the DVN.

Immunopharmacology - antibodies for specific modulation of proteins involved in neuronal function

The application of antibodies to living neurones has the potential to modulate function of specific proteins by virtue of their high specificity. This specificity has proven effective in determining the involvement of many proteins in neuronal function where specific agonists and antagonists do not exist, e.g. ion channel subunits. We discuss studies where antibodies modulate functions of voltage gated sodium, voltage gated potassium, voltage gated calcium hyperpolarisation activated cyclic nucleotide (HCN gated) and transient receptor potential (TRP) channels. Ligand gated channels studied in this way include nicotinic acetylcholine receptors, purinoceptors and GABA receptors. Antibodies have also helped reveal the involvement of different intracellular proteins in neuronal functions including G-proteins as well as other proteins involved in trafficking, phosphoinositide signalling and neurotransmitter release. Some suggestions for control experiments are made to help validate the method. We conclude that antibodies can be extremely valuable in determining the functions of specific proteins in living neurones in neuroscience research.

Cosmic ray modulation of infra-red radiation in the atmosphere

Cosmic rays produce molecular cluster ions as they pass through the lower atmosphere. Neutral molecular clusters such as dimers and complexes are expected to make a small contribution to the radiative balance, but atmospheric absorption by charged clusters has not hitherto been observed. In an atmospheric experiment, a narrowband thermopile filter radiometer centred on 9.15 {\mu}m, an absorption band previously associated with infra-red absorption of molecular cluster ions, was used to monitor changes following events identified by a cosmic ray telescope sensitive to high-energy (>400 MeV) particles, principally muons. The average change in longwave radiation in this absorption band due to molecular cluster ions is 7 mWm sup{-2}. The integrated atmospheric energy density for each event is 2 Jm sup{-2}, representing an amplification factor of 10 sup{12} compared to the estimated energy density of a typical air shower. This absorption is expected to occur continuously and globally, but calculations suggest that it has only a small effect on climate.

Wave activity for large-amplitude disturbances described by the primitive equations on the sphere

Pseudomomentum and pseudoenergy are both measures of wave activity for disturbances in a fluid, relative to a notional background state. Together they give information on the propagation, growth, and decay of disturbances. Wave activity conservation laws are most readily derived for the primitive equations on the sphere by using isentropic coordinates. However, the intersection of isentropic surfaces with the ground (and associated potential temperature anomalies) is a crucial aspect of baroclinic wave evolution. A new expression is derived for pseudoenergy that is valid for large-amplitude disturbances spanning isentropic layers that may intersect the ground. The pseudoenergy of small-amplitude disturbances is also obtained by linearizing about a zonally symmetric background state. The new expression generalizes previous pseudoenergy results for quasigeostrophic disturbances on the β plane and complements existing large-amplitude results for pseudomomentum. The pseudomomentum and pseudoenergy diagnostics are applied to an extended winter from the European Centre for Medium-Range Weather Forecasts Interim Re-Analysis data. The time series identify distinct phenomena such as a baroclinic wave life cycle where the wave activity in boundary potential temperature saturates nonlinearly almost two days before the peak in wave activity near the tropopause. The coherent zonal propagation speed of disturbances at tropopause level, including distinct eastward, westward, and stationary phases, is shown to be dictated by the ratio of total hemispheric pseudoenergy to pseudomomentum. Variations in the lower-boundary contribution to pseudoenergy dominate changes in propagation speed; phases of westward progression are associated with stronger boundary potential temperature perturbations.

Carbon monoxide inhibition of Cav3.2 T-type Ca2+ channels reveals tonic modulation by thioredoxin

T-type Ca2+ channels play diverse roles in tissues such as sensory neurons, vascular smooth muscle, and cancers, where increased expression of the cytoprotective enzyme, heme oxygenase-1 (HO-1) is often found. Here, we report regulation of T-type Ca2+ channels by carbon monoxide (CO) a HO-1 by-product. CO (applied as CORM-2) caused a concentration-dependent, poorly reversible inhibition of all T-type channel isoforms (Cav3.1-3.3, IC50 ∼3 μM) expressed in HEK293 cells, and native T-type channels in NG108-15 cells and primary rat sensory neurons. No recognized CO-sensitive signaling pathway could account for the CO inhibition of Cav3.2. Instead, CO sensitivity was mediated by an extracellular redox-sensitive site, which was also highly sensitive to thioredoxin (Trx). Trx depletion (using auranofin, 2-5 μM) reduced Cav3.2 currents and their CO sensitivity by >50% but increased sensitivity to dithiothreitol ∼3-fold. By contrast, Cav3.1 and Cav3.3 channels, and their sensitivity to CO, were unaffected in identical experiments. Our data propose a novel signaling pathway in which Trx acts as a tonic, endogenous regulator of Cav3.2 channels, while HO-1-derived CO disrupts this regulation, causing channel inhibition. CO modulation of T-type channels has widespread implications for diverse physiological and pathophysiological mechanisms, such as excitability, contractility, and proliferation

An exact local conservation theorem for finite-amplitude disturbances to non-parallel shear flows, with remarks on Hamiltonian structure and on Arnol'd's stability theorems

Disturbances of arbitrary amplitude are superposed on a basic flow which is assumed to be steady and either (a) two-dimensional, homogeneous, and incompressible (rotating or non-rotating) or (b) stably stratified and quasi-geostrophic. Flow over shallow topography is allowed in either case. The basic flow, as well as the disturbance, is assumed to be subject neither to external forcing nor to dissipative processes like viscosity. An exact, local ‘wave-activity conservation theorem’ is derived in which the density A and flux F are second-order ‘wave properties’ or ‘disturbance properties’, meaning that they are O(a2) in magnitude as disturbance amplitude a [rightward arrow] 0, and that they are evaluable correct to O(a2) from linear theory, to O(a3) from second-order theory, and so on to higher orders in a. For a disturbance in the form of a single, slowly varying, non-stationary Rossby wavetrain, $\overline{F}/\overline{A}$ reduces approximately to the Rossby-wave group velocity, where (${}^{-}$) is an appropriate averaging operator. F and A have the formal appearance of Eulerian quantities, but generally involve a multivalued function the correct branch of which requires a certain amount of Lagrangian information for its determination. It is shown that, in a certain sense, the construction of conservable, quasi-Eulerian wave properties like A is unique and that the multivaluedness is inescapable in general. The connection with the concepts of pseudoenergy (quasi-energy), pseudomomentum (quasi-momentum), and ‘Eliassen-Palm wave activity’ is noted. The relationship of this and similar conservation theorems to dynamical fundamentals and to Arnol'd's nonlinear stability theorems is discussed in the light of recent advances in Hamiltonian dynamics. These show where such conservation theorems come from and how to construct them in other cases. An elementary proof of the Hamiltonian structure of two-dimensional Eulerian vortex dynamics is put on record, with explicit attention to the boundary conditions. The connection between Arnol'd's second stability theorem and the suppression of shear and self-tuning resonant instabilities by boundary constraints is discussed, and a finite-amplitude counterpart to Rayleigh's inflection-point theorem noted

Modulation of stretch-induced myocyte remodeling and gene expression by nitric oxide: a novel role for lipoma preferred partner in myofibrillogenesis

Prolonged hemodynamic load as a result of hypertension eventually leads to maladaptive cardiac adaptation and heart failure. The signalling pathways that underlie these changes are still poorly understood. The adaptive response to mechanical load is mediated by mechanosensors which convert the mechanical stimuli into a biological response. We examined the effect of cyclic mechanical stretch on myocyte adaptation using neonatal rat ventricular myocytes with 10% (adaptive) or 20% (maladaptive) maximum strain, 1Hz for 48 hours to mimic in vivo mechanical stress. Cells were also treated with and without L-NAME, a general nitric oxide synthase (NOS) inhibitor to suppress NO production. Maladaptive 20% mechanical stretch led to a significant loss of intact sarcomeres which was rescued by LNAME (P<0.05, n≥5 cultures). We hypothesized that the mechanism was through NOinduced alteration of myocyte gene expression. L-NAME up-regulated the mechanosensing proteins Muscle LIM protein (MLP (by 100%, p<0.05, n=4 cultures)) and lipoma preferred partner, a novel cardiac protein (LPP (by 80%, p<0.05, n=4 cultures)). L-NAME also significantly altered the subcellular localisation of LPP and MLP in a manner that favoured growth and adaptation. These findings suggest that NO participates in stretch-mediated adaptation. The use of isoform selective NOS inhibitors indicated a complex interaction between iNOS and nNOS isoforms regulate gene expression. LPP knockdown by siRNA led to formation of α-actinin aggregates and Z-bodies showing that myofibrillogenesis was impaired. There was an up-regulation of E3 ubiquitin ligase (MUL1) by 75% (P<0.05, n=5 cultures). This indicates that NO contributes to stretch-mediated adaptation via the upregulation of proteins associated mechansensing and myofibrillogenesis, thereby presenting potential therapeutic targets during the progression of heart failure. Keywords: Mechanotransduction, heart failure, stretch, heart, hypertrophy

Phenolic acid intake, delivered via moderate champagne wine consumption, improves spatial working memory via the modulation of hippocampal and cortical protein expression/activation.

Aims: While much data exist for the effects of flavonoid-rich foods on spatial memory in rodents, there are no such data for foods/beverages predominantly containing hydroxycinnamates and phenolic acids. To address this, we investigated the effects of moderate Champagne wine intake, which is rich in these components, on spatial memory and related mechanisms relative to the alcohol- and energy-matched controls. Results: In contrast to the isocaloric and alcohol-matched controls, supplementation with Champagne wine (1.78 ml/kg BW, alcohol 12.5% vol.) for 6 weeks led to an improvement in spatial working memory in aged rodents. Targeted protein arrays indicated that these behavioral effects were paralleled by the differential expression of a number of hippocampal and cortical proteins (relative to the isocaloric control group), including those involved in signal transduction, neuroplasticity, apoptosis, and cell cycle regulation. Western immunoblotting confirmed the differential modulation of brain-derived neurotrophic factor, cAMP response-element-binding protein (CREB), p38, dystrophin, 2',3'-cyclic-nucleotide 3'-phosphodiesterase, mammalian target of rapamycin (mTOR), and Bcl-xL in response to Champagne supplementation compared to the control drink, and the modulation of mTOR, Bcl-xL, and CREB in response to alcohol supplementation. Innovation: Our data suggest that smaller phenolics such as gallic acid, protocatechuic acid, tyrosol, caftaric acid, and caffeic acid, in addition to flavonoids, are capable of exerting improvements in spatial memory via the modulation in hippocampal signaling and protein expression. Conclusion: Changes in spatial working memory induced by the Champagne supplementation are linked to the effects of absorbed phenolics on cytoskeletal proteins, neurotrophin expression, and the effects of alcohol on the regulation of apoptotic events in the hippocampus and cortex. Antioxid. Redox Signal. 00, 000-000.

Utilizing electronic effects in the modulation of BTPhen ligands with respect to the partitioning of minor actinides from lanthanides

Effects of bromine substitution at the 5 and 5,6-positions of the 1,10-phenanthroline nucleus of BTPhen ligand on their extraction properties for Ln(III) andAn(III) cations have been studied. Compared to C5-BTPhen, electronic modulation in BrC5-BTPhen and Br2C5-BTPhen enabled these ligands to be fine-tuned in order to enhance the separation selectivity of Am(III) from Eu(III)