Modulation of endothelial cell KCa3.1 Channels during endothelium-derived hyperpolarizing factor signaling in mesenteric resistance arteries

Arterial hyperpolarization to acetylcholine (ACh) reflects coactivation of KCa3.1 (IKCa) channels and KCa2.3 (SKCa) channels in the endothelium that transfers through myoendothelial gap junctions and diffusible factor(s) to affect smooth muscle relaxation (endothelium-derived hyperpolarizing factor [EDHF] response). However, ACh can differentially activate KCa3.1 and KCa2.3 channels, and we investigated the mechanisms responsible in rat mesenteric arteries. KCa3.1 channel input to EDHF hyperpolarization was enhanced by reducing external [Ca2+]o but blocked either with forskolin to activate protein kinase A or by limiting smooth muscle [Ca2+]i increases stimulated by phenylephrine depolarization. Imaging [Ca2+]i within the endothelial cell projections forming myoendothelial gap junctions revealed increases in cytoplasmic [Ca2+]i during endothelial stimulation with ACh that were unaffected by simultaneous increases in muscle [Ca2+]i evoked by phenylephrine. If gap junctions were uncoupled, KCa3.1 channels became the predominant input to EDHF hyperpolarization, and relaxation was inhibited with ouabain, implicating a crucial link through Na+/K+-ATPase. There was no evidence for an equivalent link through KCa2.3 channels nor between these channels and the putative EDHF pathway involving natriuretic peptide receptor-C. Reconstruction of confocal z-stack images from pressurized arteries revealed KCa2.3 immunostain at endothelial cell borders, including endothelial cell projections, whereas KCa3.1 channels and Na+/K+-ATPase {alpha}2/{alpha}3 subunits were highly concentrated in endothelial cell projections and adjacent to myoendothelial gap junctions. Thus, extracellular [Ca2+]o appears to modify KCa3.1 channel activity through a protein kinase A-dependent mechanism independent of changes in endothelial [Ca2+]i. The resulting hyperpolarization links to arterial relaxation largely through Na+/K+-ATPase, possibly reflecting K+ acting as an EDHF. In contrast, KCa2.3 hyperpolarization appears mainly to affect relaxation through myoendothelial gap junctions. Overall, these data suggest that K+ and myoendothelial coupling evoke EDHF-mediated relaxation through distinct, definable pathways

Visualising urban sustainability

Developing sustainable urban environments is complex and requires a number of factors – including social, economic and environmental sustainability - to be taken into account. This project is prototype software that produces a 3D virtual model of urban developments allowing viewers to see the short and long-term implications of courses of action

Outreach work : child sexual exploitation : a rapid evidence assessment

This briefing is based on a rapid review of the available literature on outreach work with children and young people. It is intended to provide the ReachOut project with an overview of different approaches to outreach; what it generally aims to achieve; what distinguishes it from centre--‐based work and how it is applicable to children and young people involved in, or at risk of, child sexual exploitation. We highlight what is known about ‘detached’ and other approaches that aim to reach vulnerable populations who are not accessing mainstream services. We hope it will be useful in informing ReachOut’s thinking about the role and value of its own outreach activities

Characterisation of the vasodilation effects of DHA and EPA, n-3 PUFAs (fish oils), in rat aorta and mesenteric resistance arteries

Background and Purpose Increasing evidence suggests that the omega-3 polyunsaturated acids (n-3 PUFA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), are beneficial to cardiovascular health, promoting relaxation of vascular smooth muscle cells and vasodilation. Numerous studies have attempted to study these responses, but to date there has not been a systematic characterisation of both DHA and EPA mediated vasodilation in conduit and resistance arteries. Therefore, we aimed to fully characterise the n-3 PUFA-induced vasodilation pathways in rat aorta and mesenteric artery. Methods Wire myography was used to measure the vasomotor responses of freshly dissected rat mesenteric artery and aorta. Arteries were pre-constricted with U46619 and cumulative concentrations of either DHA or EPA (10 nM-30 μM) were added. The mechanisms by which n-3 PUFA relaxed arteries were investigated using inhibitors of vasodilator pathways, which include: nitric oxide synthase (NOS; L-NAME), cycloxygenase (COX; indomethacin), cytochrome P450 epoxygenase (CYP450; clotrimazole); and calcium-activated potassium channels (KCa), SKCa (apamin), IKCa (TRAM-34) and BKCa (paxilline). Results Both DHA- and EPA-induced relaxations were partially inhibited following endothelium removal in rat mesenteric arteries. Similarly, in aorta EPA-induced relaxation was partially suppressed due to endothelium removal. CYP450 also contributed to EPA-induced relaxation in mesenteric artery. Inhibition of IKCa partially attenuated DHA-induced relaxation in aorta and mesenteric artery along with EPA-induced relaxation in mesenteric artery. Furthermore, this inhibition of DHA- and EPA-induced relaxation was increased following the additional blockade of BKCa in these arteries. Conclusions This study provides evidence of heterogeneity in the vasodilation mechanisms of DHA and EPA in different vascular beds. Our data also demonstrates that endothelium removal has little effect on relaxations produced by either PUFA. We demonstrate IKCa and BKCa are involved in DHA-induced relaxation in rat aorta and mesenteric artery; and EPA-induced relaxation in rat mesenteric artery only. CYP450 derived metabolites of EPA may also be involved in BKCa dependent relaxation. To our knowledge this is the first study indicating the involvement of IKCa in n-3 PUFA mediated relaxation

Inhibition of endothelium-derived hyperpolarizing factor by ascorbate in the bovine eye

The aim of this study was to characterise vasodilator responses in the perfused ciliary vascular bed of the bovine eye. When bovine eyes were perfused at a constant rate of 2.5 ml min-1, infusion of the powerful vasodilator, papaverine (150 muM), produced a very small reduction in perfusion pressure. Under the same conditions, the nitric oxide synthase inhibitor, L- NAME (100 muM), had no effect but the inhibitor of soluble guanylate cyclase, ODQ (10 muM), produced a small vasoconstrictor response. These results indicate that there is a small component of intrinsic (myogenic) tone that may be suppressed by a basal release of nitric oxide. In the bovine eye, vasodilatation to acetylcholine or bradykinin was unaffected by L- NAME (100 muM), or the cyclo-oxygenase inhibitor, flurbiprofen (30 muM), but was significantly attenuated following treatment with a high concentration of KC1 (30 muM), or by damaging the endothelium with the detergent, CHAPS (0.3%, 2 min). Thus agonist-induced vasodilatation is not mediated by nitric oxide or prostacyclin but involves a K+ conductance and is endothelium-dependent. Acetylcholine-induced vasodilatation in the bovine eye was unaffected by glibenclamide (10 muM), an inhibitor of ATP-sensitive K+ channels (KATP), but was significantly attenuated by TEA (10 mM), a non-selective inhibitor of K+ channels. The blockade of vasodilatation by TEA but not glibenclamide could indicate that a calcium- sensitive K+ channel is involved in the response. The small conductance calcium-sensitive K+ channel (SKCa) inhibitor, apamin (100 nM), and the large conductance calcium-sensitive K+ channel (BKCa) inhibitor, iberiotoxin (50 nM), had no significant effect on acetylcholine-induced vasodilatation. In contrast, the intermediate (IKCa)/large conductance calcium-sensitive K+ channel inhibitor, charybdotoxin (50 nM), powerfully blocked these vasodilator responses, and uncovered a vasoconstrictor response. Thus, vasodilator responses appear to involve the opening of IKCa channels. The combination of apamin (100 nM) with a sub-threshold concentration of charybdotoxin (10 nM) significantly attenuated acetylcholine-induced vasodilatation, but the combination of apamin (100 nM) with iberiotoxin (50 nM) had no effect. This profile of blockade is consistent with the vasodilator responses being mediated by endothelium-derived hyperpolarizing factor (EDHF). Ascorbate is known to protect nitric oxide dependent vasodilatation under conditions of oxidant stress, however, EDHF-mediated vasodilator responses induced by acetylcholine or bradykinin were powerfully blocked when ascorbate (50 muM, 120 min) was included in the perfusion medium; with acetylcholine a normally masked muscarinic vasoconstrictor response was also uncovered. These results indicate that, ascorbate at a physiologically relevant concentration, can inhibit EDHF-mediated vasodilatations. The blockade of EDHF-mediated vasodilatation by ascorbate was time-dependent (maximum blockade at 120 min) and concentration-dependent (10-150 muM). Thus, the blocking action of ascorbate has a slow onset and occurs concentrations across the normal plasma concentration range (10 -150 muM). The ability of ascorbate to block EDHF-mediated vasodilatation in the bovine eye is likely to result from its reducing properties, since this action was mimicked by two other reducing agents, namely, N-acetyl-L-cysteine (1 mM) and dithiothreitol (100 muM), but not by the redox-inactive analogue, dehydroascorbate (50 muM). In the bovine eye, vasodilatations induced by the KATP opener, levcromakalim (100 pmol-30 nmol), or the nitric oxide donor, glyceryl trinitrate (10 nmol), were completely unaffected by the infusion of ascorbate (50 muM). Furthermore, the L-NAME induced vasoconstrictor response in the presence of U46619 (~200 nM) was unaffected by infusion of ascorbate (50 muM). Thus, the blockade of EDHF-mediated vasodilatation by ascorbate is highly selective and does not result from non-selective damage of the endothelium as basal release of nitric oxide is unaffected. (Abstract shortened by ProQuest.)

Regulation of KCa2.3 andendothelium-dependent hyperpolarization (EDH) in the rat middle cerebral artery: the role of lipoxygenase metabolites and isoprostanes

Background and Purpose. In rat middle cerebral arteries, endothelium-dependent hyperpolarization (EDH) is mediated by activation of calcium-activated potassium(KCa) channels specifically KCa2.3 and KCa3.1. Lipoxygenase (LOX) products function as endothelium-derived hyperpolarizing factors (EDHFs) in rabbit arteries by stimulating KCa2.3. We investigated if LOX products contribute to EDH in rat cerebral arteries. Methods. Arachidonic acid (AA) metabolites produced in middle cerebral arteries were measured using HPLC and LC/MS. Vascular tension and membrane potential responses to SLIGRL were simultaneously recorded using wire myography and intracellular microelectrodes. Results. SLIGRL, an agonist at PAR2 receptors, caused EDH that was inhibited by a combination of KCa2.3 and KCa3.1 blockade. Non-selective LOX-inhibition reduced EDH, whereas inhibition of 12-LOX had no effect. Soluble epoxide hydrolase (sEH) inhibition enhanced the KCa2.3 component of EDH. Following NO synthase (NOS) inhibition, the KCa2.3 component of EDH was absent. Using HPLC, middle cerebral arteries metabolized 14C-AA to 15- and 12-LOX products under control conditions. With NOS inhibition, there was little change in LOX metabolites, but increased F-type isoprostanes. 8-iso-PGF2α inhibited the KCa2.3 component of EDH. Conclusions. LOX metabolites mediate EDH in rat middle cerebral arteries. Inhibition of sEH increases the KCa2.3 component of EDH. Following NOS inhibition,loss of KCa2.3 function is independent of changes in LOX production or sEH inhibition but due to increased isoprostane production and subsequent stimulation of TP receptors. These findings have important implications in diseases associated with loss of NO signaling such as stroke; where inhibition of sEH and/or isoprostane formation may of benefit

Thromboxane A2 inhibition of SKCa after NO synthase block in rat middle cerebral artery

NO/prostanoid independent, EDHF-mediated hyperpolarization and dilation in rat middle cerebral arteries is mediated solely by endothelial cell IK(Ca). However, when the NO-pathway is also active, both SK(Ca) and IK(Ca) contribute to EDHF responses. As the SK(Ca) component can be inhibited by stimulation of thromboxane A(2) (TxA(2)) TP receptors and NO has the potential ability to inhibit thromboxane synthesis, we investigated whether TxA(2) might explain loss of functional input from SK(Ca) during NOS inhibition in cerebral arteries. EXPERIMENTAL APPROACH: Rat middle cerebral arteries were mounted in a wire myograph. Endothelium-dependent responses to the PAR2 agonist, SLIGRL were assessed as simultaneous changes in smooth muscle membrane potential and tension. KEY RESULTS: Responses were obtained in the presence of L-NAME as appropriate. Inhibition of TP receptors with either ICI 192,605 or SQ 29,548, did not affect EDHF mediated hyperpolarization and relaxation, but in their presence neither TRAM-34 nor apamin (to block IK(Ca) and SK(Ca) respectively) individually affected the EDHF response. However, in combination they virtually abolished it. Similar effects were obtained in the presence of the thromboxane synthase inhibitor, furegrelate, which additionally revealed an iberiotoxin-sensitive residual EDHF hyperpolarization and relaxation in the combined presence of TRAM-34 and apamin. CONCLUSIONS AND IMPLICATIONS: In the rat middle cerebral artery, inhibition of NOS leads to a loss of the SK(Ca) component of EDHF responses. Either antagonism of TP receptors or block of thromboxane synthase restores an input through SK(Ca). These data indicate that NO normally enables SK(Ca) activity in rat middle cerebral arteries

Barnardo’s ReachOut: final evaluation report March 2019

ReachOut is a preventative child sexual exploitation (CSE) project established in 2016 under a partnership funding agreement between Barnardo’s, the KPMG Foundation, Department for Education, Communities and Local Government and Rotherham Metropolitan Borough Council (RMBC). An independent evaluation was commissioned from the University of Bedfordshire with DMSS Research both to evaluate the impact of the project and to provide ongoing learning and feedback. A diverse staff team was recruited from a range of professional backgrounds including criminal justice, social work and youth work. There have been three main strands of work undertaken by ReachOut in order to achieve its aims: •Outreach work to raise awareness and provide support to children and young people in their communities  •Healthy relationship education in schools and other settings •Direct support for children and young people identified as at risk of CSE. These have operated at three levels of prevention: universal, including outreach at community events across Rotherham, helping to convey the message  that CSE is relevant to everyone; primary prevention, including education work in schools reaching over 2000 children and young people; targeted prevention with groups and communities identified as potentially more vulnerable to CSE as well as direct work with around 300 individual children and young people. Over the course of the three years, evaluators have carried out interviews with ReachOut staff and managers and representatives from external agencies; observed sessions of delivery; interviewed samples of young people and parents; analysed feedback questionnaires from school students and staff; reviewed project monitoring and samples of case records

From Dish to Bedside: Lessons Learned While Translating Findings from a Stem Cell Model of Disease to a Clinical Trial

While iPSCs have created unprecedented opportunities for drug discovery, there remains uncertainty concerning the path to the clinic for candidate therapeutics discovered with their use. Here we share lessons that we learned, and believe are generalizable to similar efforts, while taking a discovery made using iPSCs into a clinical trial

Comparative Reflections on Community-Oriented Policing (COP) in Post-Conflict Central America

In this article we discuss the comparative impact and significance of Community-Oriented Policing (COP) in Central America (Guatemala, El Salvador and Nicaragua). We emphasize in particular the formal role of COP as a means to re-establish trust between the state and community, demonstrate professionalism and to evidence the democratic accountability of the police to the population. Although these formal goals remain the goal of community oriented policing, we demonstrate in this article that there has been an increased emphasis on more kinetic or militarized forms of policing in recent years. Hard handed, heavily armed and interventionist police policies have spread from El Salvador to Guatemala, and more recently Nicaragua. Moves towards more aggressive policing are explained by governments and police forces as a necessary response to the rising threat of gangs and drug cartels and horrifying levels of homicide statistics. However, as we highlight there is also evidence of these changes reflecting undemocratic shifts within national administrations and the repositioning of people within government and national institutions with links to these countries' earlier military governments.The net effect of these changes we argue is to erode the intentions of COP initiatives, and severely reduce levels of trust and accountability between people and the democratic state