Control of Membrane Excitability by Potassium and Chloride Leak Conductances

The permeability of the neuronal membrane to different ions determines both resting membrane potential (RMP) and input conductance. These parameters determine the cells response to synaptic input. In this thesis I have examined how the molecular properties of potassium and chloride ion channels can influence neuronal excitability in ways that have not previously been considered. For example, two‐pore domain potassium (K2P) channels open at rest to generate a persistent potassium ion efflux. In addition to its accepted role in setting the RMP, I have tested the hypothesis that this conductance is sufficient to repolarise the membrane during an action potential (AP) in the absence of voltage‐dependent potassium channels (Kv). We tested this prediction using heterologous expression of TASK3 or TREK1 K2P channels combined with conductance injection to simulate the presence of a voltage‐gated sodium conductance. These experiments demonstrated that K2P channels are sufficient to support APs during short and prolonged depolarising current pulses. The membranes permeability to chloride ions can also be affected by extrasynaptic GABAA receptors containing the delta subunit (δ‐GABAARs) that produce a tonic conductance due to their high apparent affinity for GABA. The anaesthetics Propofol and THIP are both believed to alter neuronal excitability by enhancing this persistent chloride flux. We have examined how this anaesthetic action is affected by the steady‐state ambient GABA concentrations that are believed to exist in vivo. Surprisingly, the anaesthetic enhancement of δ‐GABAARs is lost at low ambient GABA concentrations. Therefore, I would suggest that the anaesthetic potency of these drugs is affected by the resting ambient GABA concentration in a manner that has not previously been appreciated. In the current Thesis I have examined the molecular and pharmacological properties of two very different ion channel families that both generate a leak conductance, and I will present models that link the behaviour of these ion channels to their ability to modulate neuronal excitability

Linking Induction and Transrepression of PPARβ/δ with Cellular Function

The copyrights of all papers published in this journal are retained by the respective authors as per the 'Creative Commons Attribution License' (http://creativecommons.org/licenses/by/3.0/).Peroxisome proliferator activated receptors (PPARs) are ligand-activated transcription factors and members of the nuclear hormone receptor superfamily. PPARβ/δ is ubiquitously expressed and has a central role in homeostasis, and has been suggested as a therapeutic target for a number of metabolic and cardiovascular disorders. This important nuclear receptor controls transcription under different modes of molecular activity which directly control the cellular function and fate of tissues. This complex activity of induction and transrepression of gene expression (with and without exogenous ligands) is poorly understood and yet understanding this molecular control through novel drug development would led to control over a key molecular switch in all cells. This review outlines the main molecular mechanisms of PPARβ/δ, and links the modes of activity to the signalling pathways in inflammation, proliferation and senescence, with the goal to understand how this will translate into novel drug design to control the PPARβ/δ molecular switch.Peer reviewe

Evolution of the Subject:Synthetic Biology in Fine Art Practice

I will discuss how art practice within a genetics laboratory can provide a situated account of scientific knowledge through a performative exploration of subjectivity. The focus of this paper is my doctoral research on my relationship with the microbial organism through its use as synthetic biology resource. Drawing source material from synthetic biology and art practices that employ biotechnology, I undertake a slow, performative practice tracing my subjective experience in the laboratory through devising a method of converting my thought into DNA and then physically inserting this DNA into the body of the common laboratory micro-organism, Escherichia coli (E. coli). Diffracted through readings of post-humanist and vital materialist works, I (re)consider my intuitive engagement with the materials, methods and tools of synthetic biology both as an imposition upon the constructed space of the laboratory and upon the constructed body of the organism. Through the ongoing art project, Pithos, a practical engagement with the organism as vessel, and Transformation, a participatory performance workshop that reconsiders genetic modification as an act of assembling lively material, I construct narratives that reframe our relation to biological material through intra-action, kinship and responsibility

Effects of Apamin and Charybdtoxin on Endothelium Independent Vasodilatation : Implications in the study of EDHF

Endothelial derived hyperpolarizing factor (EDHF), together with endothelial derived NO and prostacyclin represent the major endogenous vasodilator hormone pathways (Coleman et al, 2004). Since the chemical nature of EDHF is uncertain, many studies have relied on pharmacological tools. Often EDHF described as the endothelial dependent dilation which is resistant to a combination of drugs that inhibit NO synthase (usually L-NAME) and cyclo-oxygenase (usually indomethacin) but abolished by those that block large and small Ca2+-activated K+ channels (BKCa and SKCa; with charybdotoxin plus apamin respectively). This approach relies on each of these drugs being selective for endothelial dependent responses. In the current study we have taken a different approach to most and performed experiments where inhibitors were added to endothelium denuded vessels after dilators induced stable vasodilatation. Male Wistar rats (200 ± 15.4g) were killed by lethal exposure to CO2. Second order mesenteric arteries were mounted in isometric wire myographs and vasomotor responses recorded as described previously (Harrington and Mitchell, 2004). In some experiments vessels were contracted with EC80 concentration of U46619, dilation was then induced by the addition of 3x10-6M acetylcholine or 3x10-6M SNP. When either L-NAME (10-4M) or charybdotoxin (10-7M) plus apamin (5x10-7M) were added after acetylcholine the vasodilatation was immediately and completely reversed (Figure A and B respectively). In other experiments, endothelium independent vasodilation induced by SNP, were also reversed by charybdotoxin plus apamin, but not L-NAME. Figure A. Example trace of an artery re-contracting following the addition of L-NAME in pre-contracted arteries dilated with acetylcholine. Bar Graphs show re-contraction of arteries in response to L-NAME or charybdotoxin plus apamin (C+A) in arteries with endothelium, dilated with acetylcholine (Figure B) or without endothelium dilated with SNP (Figure C). This data demonstrates that charybdotoxin and apamin have pharmacological effects independent of the endothelium, at the level of smooth muscle cell function. These observations suggest results obtained with these drugs in relation to EDHF studies, should be treated with caution. Harrington L and Mitchell JA (2004) Br J Pharmacol 143: 611-617 Coleman et al (2004) Clin Ex Pharm Phys 31; 641-649Peer reviewedFinal Accepted Versio

The ‘EDHF’ Antagonist 14, 15 Epoxyeicosa-5(Z)-Enoic Acid has Vasodilator Properties in Mesenteric Vessels

There is now overwhelming evidence for Epoxyeicosatrienoic acids (EETs) as endothelial derived hyperpolarising factor (EDHF). Most recently, a number of pharmacological tools have been developed for the study of EETs in relation to EDHF responses. EETs have been shown to cause relaxation by activating smooth muscle large conductance Ca2+ sensitive K+ (BKCa) (Archer et al, 2003). This dilatory response has been shown to be specifically inhibited by its analogue 14, 15-epoxyeicosa-5 (Z) enoic acid (14, 15 EEZE) in both human internal mammary artery and bovine coronary artery (Archer et al, 2003). Here we have investigated the antagonist effects of 14, 15 EEZE in murine arteries. Male Black 6 mice (12-18 weeks) were killed by lethal exposure to CO2. First order arteries were isolated and mounted in wire myographs immersed in physiological salt solution (PSS). Arteries were equilibrated (30 mins) and tensions normalised as described previously (Mulvany and Halpern, 1977). Arteries incubated for 30 minutes with or without 3µg/ml 14, 15 EEZE. A concentration response curve to 11, 12 EET was performed cumulatively on arteries pre-contracted with EC80 U46619. In some experiments, arteries were pre-contracted with EC80 U46619, and concentration response to 14, 15 EEZE performed cumulatively.Non peer reviewe

Nitric oxide-dependent vasodilation is compromised in isolated pulmonary arteries from COX knockout mice

Cyclooxygenase (COX) has two isoforms and is essential for prostanoid synthesis. COX-1 is constitutive whilst COX-2 is induced in inflammation. Two COX products, prostacyclin (PGI2) and thromboxane (TxA2), regulate vessel tone; PGI2 mediates vasodilation and platelet inhibition, and TxA2 opposes this. PGI2 therapies are used in pulmonary arterial hypertension (PAH). Endogenous TxA2/PGI2 has been linked to PAH in animal models, but the mechanism and isoform involved is debated. We hypothesized that pulmonary artery (PA) from COX-1–/– and COX-2–/– mice would have altered vasodilatory function compared with wild-type (WT; C57Bl6) mice. Vasomotor responses to contractile and relaxant agents were measured by myography. PA from all mice responded similarly to contraction by high potassium or the TxA2 mimetic, U46619. Relaxation to PGI2 receptor or PPARβ/ agonists was also similar in all PAs. However, COX-1–/– and, to a lesser extent, COX-2–/– PA had impaired vasodilation to acetylcholine (ACh), which stimulates endothelial nitric oxide (NO) release, and COX-1–/– PA also dilated less to sodium nitroprusside (SNP); an NO donor that works on smooth muscle (Fig 1). These data indicate an interaction between COX and NO sensing pathways in pulmonary vessels, and have implications for our understanding of PAH.Non peer reviewedFinal Accepted Versio

BioDwelling:A participatory approach to living with living material

BioDwelling is an arts-led research project that brings ethical concerns of culture, gender and multispecies relationality from the feminist technosciences into direct conversation with the emerging field of biotechnological architecture (bio-architecture). Working within a multi-disciplinary bio-architecture research group, we develop a practice-led methodology to facilitate the exploration of questions that arise when we begin to engineer more-than-human dwelling spaces. In this article we give a brief overview of the work of the Hub for Biotechnology in the Built Environment (HBBE) and the Responsible Interactions research theme, which outlines the project context. We go on to describe the BioDwelling methodology and introduce three arts-led interventions that draw on feminist science and cultural theory to invite reflections on the ethical implications of working with living materials to build (or grow?) spaces in which humans intend to live. We conclude with a summary of the project to date, reflections on methods and possible next steps.</p

Role of the endothelium and COX-1 in prostacyclin generation by whole vessels stimulated with different agonists

Prostacyclin is an important cardioprotective hormone produced by the vascular wall, whose synthesis is dependent on cyclo-oxygenase (COX) enzymes. In healthy vessels the endothelium is thought to be the main site of prostacyclin release (Moncada et al 1977). Two isoforms of COX exist, and we have recently published data demonstrating that it is COX-1 rather than COX-2 that drives the production of prostacyclin in mouse aorta (Kirkby et al 2012). In this study we aimed to extend these observations by investigating what proportion of the COX-1 driven aortic prostacyclin production that comes from the endothelium versus the rest of the vessel wall (smooth muscle layers and adventitia). To do this, we explored how removal of the endothelium would influence the ability of aortic tissue to release prostacyclin in response to a range of agonists that are known to activate the endothelium and the vessel wallNon peer reviewe

Alchemical Sensing: Creating an Embodied Experience of the Unseen Organism

This paper presents the research surrounding the audiovisual installation, Stars Beneath our Feet (2015) by Louise Mackenzie. It introduces the concept of alchemical sensing to describe the layered use of scientific technology in the context of an audio-visual art installation as an alternative frame of reference that attempts an embodied understanding of the unseen organism. The process of translation through layers of technology is considered as alchemical in reference to the ancient Greek and Egyptian origins of the tradition. Not alchemical in the sense of seeking immortality or turning metal into gold, but alchemical in the anima mundi sense of seeking out the ‘essence’ of matter. Referencing the development of the field of sonification, the acoustic artwork of Joe Davis and Katie Egan and of Anne Niemetz and Andrew Pelling, the use of Atomic Force Microscopy, Python, Photosounder and MAX MSP were employed to construct an embodied audio sense of the micro-organism, Dunaliella salina. Movements detected were translated using both sonification and audification techniques into sound files that were used to form the audio component of Stars Beneath Our Feet: an installation as part of Lumiere Durham 2015, a four- day international light festival produced in the UK by Artichoke. The video component of the installation was made using a combination of dark field microscopy and DSLR camera to produce moving images that focus on a perspective of micro-organisms that is other to that commonly used within scientific research. The objective of ‘looking at’ the organisms in this expanded manner and ‘listening to’ the sounds of data obtained via technological interpretation of the movement of micro- organisms in the context of an art installation adds a broader sensory dimension to our understanding of the unseen organism, one which encompasses their being in the world without consideration of their use as resources. https://vimeo.com/14712064

Role of COX-1 and COX-2 in the release of prostanoids in murine lung and isolated lung fibroblasts

Cyclooxygenase (COX) is the first enzyme in the conversion of arachidonic acid to prostanoids. There are two isoforms of COX; COX-1, which is constitutively expressed with a homeostatic role in most tissues, and COX-2, which while constitutively expressed in some discreet sites is generally inducible by growth factors and during inflammation. In the current study, we have used tissues and cells from knock-out mice to investigate the relative contributions of COX-1 and COX-2 to PGE2 production by lung tissue ex vivo and by proliferating lung fibroblasts in vitro. Lung tissues from WT (C57Bl6), COX-1-/- and COX-2-/- mice were immediately dissected (<15 min after death) and incubated (37 °C) for 30 min in DMEM containing 50 µM calcium ionophore (A23187). Release of PGE2 was determined by competitive immunoassay. In parallel studies, murine lung fibroblasts from COX-1-/- and COX-2-/- mice were explanted and cultured before being seeded in 96-well plates at sub-confluence (5000-8000/well) and incubated for 24-48 hours in the presence of 10% FCS. Accumulated release of PGE2 was then measured as above. Over 30 min PGE2 was released by lung pieces from wild type (1117 ± 55 pg/ml) and COX-2-/- (2013 ± 255 pg/ml) but not from COX-1-/- (<61pg/ml) mice (n=4). In contrast, proliferating lung fibroblasts from COX-1-/- (4978.9 ± 1392 pg/ml) mice released higher levels of PGE2 than cells from COX-2-/- (1194 ± 617 ng/ml) mice (n=4 using cells from 2-3 separate mice for each genotype). These results show that COX-1 activity underpins the stimulated release of PGE2 in healthy mouse lung tissue. Conversely, COX-2 activity predominates in proliferating lung fibroblasts, which may be important as COX-derived PGE2 mediates proliferation of lung fibroblasts (Trends Immunol.2004;25(1):40-6). Our results suggest a switch in COX isoform in lung cells during proliferation which could be relevant to our understanding of conditions such as idiopathic pulmonary fibrosis.Non peer reviewedFinal Accepted Versio