Temperature-dependent release of ATP from human erythrocytes: Mechanism for the control of local tissue perfusion

Copyright @ 2012 The AuthorsThis article has been made available through the Brunel Open Access Publishing Fund.Human limb muscle and skin blood flow increases significantly with elevations in temperature, possibly through physiological processes that involve temperature-sensitive regulatory mechanisms. Here we tested the hypothesis that the release of the vasodilator ATP from human erythrocytes is sensitive to physiological increases in temperature both in vitro and in vivo, and examined potential channel/transporters involved. To investigate the source of ATP release, whole blood, red blood cells (RBCs), plasma and serum were heated in vitro to 33, 36, 39 and 42°C. In vitro heating augmented plasma or ‘bathing solution’ ATP in whole blood and RBC samples, but not in either isolated plasma or serum samples. Heat-induced ATP release was blocked by niflumic acid and glibenclamide, but was not affected by inhibitors of nucleoside transport or anion exchange. Heating blood to 42°C enhanced (P < 0.05) membrane protein abundance of cystic fibrosis transmembrane conductance regulator (CFTR) in RBCs. In a parallel in vivo study in humans exposed to whole-body heating at rest and during exercise, increases in muscle temperature from 35 to 40°C correlated strongly with elevations in arterial plasma ATP (r2 = 0.91; P = 0.0001), but not with femoral venous plasma ATP (r2 = 0.61; P = 0.14). In vitro, however, the increase in ATP release from RBCs was similar in arterial and venous samples heated to 39°C. Our findings demonstrate that erythrocyte ATP release is sensitive to physiological increases in temperature, possibly via activation of CFTR-like channels, and suggest that temperature-dependent release of ATP from erythrocytes might be an important mechanism regulating human limb muscle and skin perfusion in conditions that alter blood and tissue temperature.This article is made available through the Brunel Open Access Publishing Fund

Extracellular Ca2+ modulates ADP-evoked aggregation through altered agonist degradation: implications for conditions used to study P2Y receptor activation

ADP is considered a weak platelet agonist due to the limited aggregation responses it induces in vitro at physiological concentrations of extracellular Ca2+ [(Ca2+)o]. Lowering [Ca2+]o paradoxically enhances ADP-evoked aggregation, an effect that has been attributed to enhanced thromboxane A2 production. This study examined the role of ectonucleotidases in the [Ca2+]o-dependence of platelet activation. Reducing [Ca2+]o from millimolar to micromolar levels converted ADP (10 μmol/l)-evoked platelet aggregation from a transient to a sustained response in both platelet-rich plasma and washed suspensions. Blocking thromboxane A2 production with aspirin had no effect on this [Ca2+]o-dependence. Prevention of ADP degradation abolished the differences between low and physiological [Ca2+]o resulting in a robust and sustained aggregation in both conditions. Measurements of extracellular ADP revealed reduced degradation in both plasma and apyrase-containing saline at micromolar compared to millimolar [Ca2+]o. As reported previously, thromboxane A2 generation was enhanced at low [Ca2+]o, however this was independent of ectonucleotidase activity. P2Y receptor antagonists cangrelor and MRS2179 demonstrated the necessity of P2Y12 receptors for sustained ADP-evoked aggregation, with a minor role for P2Y1. In conclusion, Ca2+-dependent ectonucleotidase activity is a major factor determining the extent of platelet aggregation to ADP and must be controlled for in studies of P2Y receptor activation

Cmr is a redox-responsive regulator of DosR that contributes to M. tuberculosis virulence.

Mycobacterium tuberculosis (MTb) is the causative agent of pulmonary tuberculosis (TB). MTb colonizes the human lung, often entering a non-replicating state before progressing to life-threatening active infections. Transcriptional reprogramming is essential for TB pathogenesis. In vitro, Cmr (a member of the CRP/FNR super-family of transcription regulators) bound at a single DNA site to act as a dual regulator of cmr transcription and an activator of the divergent rv1676 gene. Transcriptional profiling and DNA-binding assays suggested that Cmr directly represses dosR expression. The DosR regulon is thought to be involved in establishing latent tuberculosis infections in response to hypoxia and nitric oxide. Accordingly, DNA-binding by Cmr was severely impaired by nitrosation. A cmr mutant was better able to survive a nitrosative stress challenge but was attenuated in a mouse aerosol infection model. The complemented mutant exhibited a ∼2-fold increase in cmr expression, which led to increased sensitivity to nitrosative stress. This, and the inability to restore wild-type behaviour in the infection model, suggests that precise regulation of the cmr locus, which is associated with Region of Difference 150 in hypervirulent Beijing strains of Mtb, is important for TB pathogenesis

Intravascular ADP and soluble nucleotidases contribute to acute prothrombotic state during vigorous exercise in humans

This article has been made available through the Brunel Open Access Publishing Fund and is available from the specified link - Copyright @ 2007 The Physiological Society .Extracellular ATP and ADP trigger vasodilatatory and prothrombotic signalling events in the vasculature. Here, we tested the hypothesis that nucleotide turnover is activated in the bloodstream of exercising humans thus contributing to the enhanced platelet reactivity and haemostasis. Right atrial, arterial and venous blood samples were collected from endurance-trained athletes at rest, during submaximal and maximal cycle ergometer exercise, and after early recovery. ATP-specific bioluminescent assay, together with high-performance liquid chromatographic analysis, revealed that plasma ATP and ADP concentrations increased up to 2.5-fold during maximal exercise. Subsequent flow cytometric analysis showed that plasma from exercising subjects significantly up-regulated the surface expression of P-selectin in human platelets and these prothrombotic effects were diminished after scavenging plasma nucleotides with exogenous apyrase. Next, using thin layer chromatographic assays with [gamma-P-32]ATP and H-3/C-14-labelled nucleotides, we showed that two soluble nucleotide-inactivating enzymes, nucleotide pyrophosphatase/phosphodiesterase and nucleoside triphosphate diphosphohydrolase, constitutively circulate in human bloodstream. Strikingly, serum nucleotide pyrophosphatase and hydrolase activities rose during maximal exercise by 20-25 and 80-100%, respectively, and then declined after 30 min recovery. Likewise, soluble nucleotidases were transiently up-regulated in the venous blood of sedentary subjects during exhaustive exercise. Human serum also contains 5'-nucleotidase, adenylate kinase and nucleoside diphosphate (NDP) kinase; however, these activities remain unchanged during exercise. In conclusion, intravascular ADP significantly augments platelet activity during strenuous exercise and these prothrombotic responses are counteracted by concurrent release of soluble nucleotide-inactivating enzymes. These findings provide a novel insight into the mechanisms underlying the enhanced risk of occlusive thrombus formation under exercising conditions

Comparative investigation of the pathogenicity of three Mycobacterium tuberculosis mutants defective in the synthesis of p-hydroxybenzoic acid derivatives.

p-Hydroxybenzoic acid derivatives (p-HBADs) are glycoconjugates secreted by all Mycobacterium tuberculosis isolates whose contribution to pathogenicity remains to be determined. The pathogenicity of three transposon mutants of M. tuberculosis deficient in the biosynthesis of some or all forms of p-HBADs was studied. Whilst the mutants grew similarly to the wild-type strain in macrophages and C57BL/6 mice, two of the mutants induced a more severe and diffuse inflammation in the lungs. The lack of production of some or all forms of p-HBADs in these two mutants also correlated with an increased secretion of the pro-inflammatory cytokines tumour-necrosis factor α, interleukin 6 and interleukin 12 in vivo. We propose that the loss of production of p-HBADs by tubercle bacilli results in their diminished ability to suppress the pro-inflammatory response to infection and that this ultimately provokes extensive pulmonary lesions in the C57BL/6 model of tuberculosis infection

P2Y receptors as regulators of lung endothelial barrier integrity

Endothelial cells (ECs), forming a semi-permeable barrier between the interior space of blood vessels and underlying tissues, control such diverse processes as vascular tone, homeostasis, adhesion of platelets, and leukocytes to the vascular wall and permeability of vascular wall for cells and fluids. Mechanisms which govern the highly clinically relevant process of increased EC permeability are under intense investigation. It is well known that loss of this barrier (permeability increase) results in tissue inflammation, the hall mark of inflammatory diseases such as acute lung injury and its severe form, acute respiratory distress syndrome. Little is known about processes which determine the endothelial barrier enhancement or protection against permeability increase. It is now well accepted that extracellular purines and pyrimidines are promising and physiologically relevant barrier-protective agents and their effects are mediated by interaction with cell surface P2Y receptors which belong to the superfamily of G-protein-coupled receptors. The therapeutic potential of P2Y receptors is rapidly expanding field in pharmacology and some selective agonists became recently available. Here, we present an overview of recently identified P2Y receptor agonists that enhance the pulmonary endothelial barrier and inhibit and/or reverse endothelial barrier disruption

Mechanical modeling of initiation of localized yielding under plane stress conditions in rigid-rigid polymer alloys

Two-dimensional Finite Element Method simulations, which involve consideration of the nonlinearity of a material, have been conducted to gain understanding about the rigid-rigid polymer toughening concept we proposed. The simulation results for the plane stress condition indicate that as long as the inclusion phase possesses (i) a 60% difference in the tangent modulus from that of the matrix at any given strain level prior to failure or (ii) smaller yield or craze stain than the yield strain of the matrix, then, localized shear yielding will occur around the inclusion. A toughened rigid-rigid polymer alloy system can then be obtained. The plain strain case is also discussed with an implementation of the rigid-rigid polymer toughening concept.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38110/1/760311106_ftp.pd

The Feasibility of Offshore Wind Power off the coast of County Waterford, Ireland

Current European Union regulations state that by 2020, 16% of all of Ireland’s energy will be required to come from renewable energy. Due to Ireland’s large maritime territory added to some of the strongest offshore wind speeds in all of Europe, offshore wind power would seem likely to play a very important role in future Irish energy developments. This study examines the feasibility of an offshore wind farm off the coast of County Waterford, Ireland. As of yet no detailed energy assessment study has been carried out in this area of the Celtic Sea. A literature review was conducted to provide a background into current offshore policy in Ireland, and to investigate the various components of undertaking a feasibility study. Hydrographic and oceanographic data were provided by the Marine Institute of Ireland in order to evaluate the feasibility of offshore power in this area. Maps were produced to analyse the hydrographic data, and three potential areas for development were formulated. Wind, tidal and wave data were studied to provide information on the seascape of the area. Following the collection of results, site assessments were undertaken to evaluate each site. From analysis of the site assessments, it was found that a small area off the coast of Dungarvan Harbour would be the most practical location for an offshore wind farm. Nine 4.1 MW-rated turbines constructed on drilled monopile foundations were found to be capable of providing 15 MW under average wind conditions. The study found large differences between the three sites, in both seabed conditions and energy outputs. It was concluded that certain areas of the south coast of Ireland would be possible for the developments of future offshore wind farms, providing adequate environmental and visual impact studies are carried out. Other conclusions that were reached included similar energy outputs to the Arklow Bank Wind Farm (Ireland’s only current offshore farm), and possible benefits to tourism in Waterford due to the underground cables from offshore turbines replacing planned overhead transmission lines

Bust of John Wesley

S/d ‘Coade, London, 1793’. This ‘artificial stone’ was invented by Elizabeth Coade, and was immensely durable and often used for outdoor sculptures