Signaling from blood vessels to CNS axons through nitric oxide

Brain function is usually perceived as being performed by neurons with the support of glial cells, the network of blood vessels situated nearby serving simply to provide nutrient and to dispose of metabolic waste. Revising this view, we find from experiments on a rodent central white matter tract (the optic nerve) in vitro that microvascular endothelial cells signal persistently to axons using nitric oxide (NO) derived from the endothelial NO synthase (eNOS). The endogenous NO acts to stimulate guanylyl cyclase-coupled NO receptors in the axons, leading to a raised cGMP level which then causes membrane depolarization, apparently by directly engaging hyperpolarization-activated cyclic nucleotide-gated ion channels. The tonic depolarization and associated endogenous NO-dependent cGMP generation was absent in optic nerves from mice lacking eNOS, although such nerves responded to exogenous NO, with raised cGMP generation in the axons and associated depolarization. In addition to the tonic activity, exposure of optic nerves to bradykinin, a classical stimulator of eNOS in endothelial cells, elicited reversible NO- and cGMP-dependent depolarization through activation of bradykinin B-2 receptors, to which eNOS is physically complexed. No contribution of other NO synthase isoforms to either the action of bradykinin or the continuous ambient NO level could be detected. The results suggest that microvascular endothelial cells participate in signal processing in the brain and can do so by generating both tonic and phasic NO signals

All things being equal: Does it matter for equity how you organise and pay for health care? A review of the International Evidence

Over the last 25 years, the health care systems of most high-income countries have experienced extensive—usually market-based—organizational and financial reforms. The impact of these system changes on health equity has been hotly debated. Examining evidence from systematic reviews of the effects of health care system organizational and financial reforms will add empirical information to this debate, identify any evidence gaps, and help policy development. Systematic review methodology was used to locate and evaluate published systematic reviews of quantitative intervention studies (experimental and observational) of the effects on equity in health care access and/or health status of health care system organizational and financial reforms (system financing, funding allocations, direct purchasing arrangements, organization of service provision, and health and social care system integration) in high-income countries. Nine systematic reviews were identified. Private insurance and out-of-pocket payments as well as the marketization and privatization of services have either negative or inconclusive equity effects. The evidence base on the health equity effects of managed care programs or integrated partnerships between health and social services is inconclusive. There were no relevant studies located that related to resource allocation reforms. The systematic review-level evidence base suggests that financial and organizational health care system reforms have had either inconclusive or negative impacts on health equity both in terms of access relative to need and in terms of health outcomes

Building Community: A Tool Kit for Youth and Adults in Charting Assets and Creating Change

Extension workers around the country are discovering a new way to help communities become more of what they want to be, and link them to extension resources, through the use of Building Community: A Tool Kit for Youth and Adults In Charting Assets and Creating Change. Developed by the Innovation Center for Community and Youth Development with Extension partners, the tool kit focuses on the gifts that a community brings to their desire for change--gifts of the past, place, people, and relationships--and has proven itself to be a powerful tool for sustainable community development

After Atos Healthcare: is the Employment and Support Allowance fit for purpose and does the Work Capability Assessment have a future?

In June 2014 the BBC reported that it had seen a number of UK Department for Work and Pensions documents relating to the Employment and Support Allowance (ESA). These documents, which included six memos written by civil servants and government advisers, tell us very little that we did not know already; namely that the ESA is in crisis, and that waiting lists for assessment and appeals are unacceptable. However, what is significant is that these concerns are being raised within the Department for Work and Pensions itself. The underlying drift is that the question of whether the current model of ESA and Work Capability Assessment is sustainable is now firmly on the UK government’s radar

Desperately seeking reductions in health inequalities: perspectives of UK researchers on past, present and future directions in health inequalities research

Following government commitments to reducing health inequalities from 1997 onwards, the UK has been recognised as a global leader in health inequalities research and policy. Yet health inequalities have continued to widen by most measures, prompting calls for new research agendas and advocacy to facilitate greater public support for the upstream policies that evidence suggests are required. However, there is currently no agreement as to what new research might involve or precisely what public health egalitarians ought to be advocating. This article presents an analysis of discussions among 52 researchers to consider the feasibility that research-informed advocacy around particular solutions to health inequalities may emerge in the UK. The data indicate there is a consensus that more should be been done to learn from post-1997 efforts to reduce health inequalities, and an obvious desire to provide clearer policy guidance in future. However, discussions as to where researchers should now focus their efforts and with whom researchers ought to be engaging reveal three distinct ways of approaching health inequalities, each of which has its own epistemological foundations. Such differences imply that a consensus on reducing health inequalities is unlikely to materialise. Instead, progress seems most likely if all three approaches are simultaneously enabled

Orthogonalization of vectors with minimal adjustment

Two transformations are proposed that give orthogonal components with a one-to-one correspondence between the original vectors and the components. The aim is that each component should be close to the vector with which it is paired, orthogonality imposing a constraint. The transformations lead to a variety of new statistical methods, including a unified approach to the identification and diagnosis of collinearities, a method of setting prior weights for Bayesian model averaging, and a means of calculating an upper bound for a multivariate Chebychev inequality. One transformation has the property that duplicating a vector has no effect on the orthogonal components that correspond to nonduplicated vectors, and is determined using a new algorithm that also provides the decomposition of a positive-definite matrix in terms of a diagonal matrix and a correlation matrix. The algorithm is shown to converge to a global optimum

Surface plasmon resonance using the catalytic domain of soluble guanylate cyclase allows the detection of enzyme activators.

Soluble Guanylate Cyclase (sGC) is the receptor for the signalling agent nitric oxide (NO) and catalyses the production of the second messenger cyclic guanosine monophosphate (cGMP) from guanosine triphosphate (GTP). The enzyme is an attractive drug target for small molecules that act in the cardiovascular and pulmonary systems, and has also shown to be a potential target in neurological disorders. We have discovered that 5-(indazol-3-yl)-1,2,4-oxadiazoles activate the enzyme in the absence of added NO and shown they bind to the catalytic domain of the enzyme after development of a surface plasmon resonance assay that allows the biophysical detection of intrinsic binding of ligands to the full length sGC and to a construct of the catalytic domain

A new small molecule inhibitor of soluble guanylate cyclase

Soluble guanylate cyclase (sGC) is a haem containing enzyme that regulates cardiovascular homeostasis and multiple mechanisms in the central and peripheral nervous system. Commonly used inhibitors of sGC activity act through oxidation of the haem moiety, however they also bind haemoglobin and this limits their bioavailability for in vivo studies. We have discovered a new class of small molecule inhibitors of sGC and have characterised a compound designated D12 (compound 10) which binds to the catalytic domain of the enzyme with a KD of 11 μM in a SPR assay

Improved genetically-encoded, FlincG-type fluorescent biosensors for neural cGMP imaging.

Genetically-encoded biosensors are powerful tools for understanding cellular signal transduction mechanisms. In aiming to investigate cGMP signaling in neurones using the EGFP-based fluorescent biosensor, FlincG (fluorescent indicator for cGMP), we encountered weak or non-existent fluorescence after attempted transfection with plasmid DNA, even in HEK293T cells. Adenoviral infection of HEK293T cells with FlincG, however, had previously proved successful. Both constructs were found to harbor a mutation in the EGFP domain and had a tail of 17 amino acids at the C-terminus that differed from the published sequence. These discrepancies were systematically examined, together with mutations found beneficial for the related GCaMP family of Ca(2+) biosensors, in a HEK293T cell line stably expressing both nitric oxide (NO)-activated guanylyl cyclase and phosphodiesterase-5. Restoring the mutated amino acid improved basal fluorescence whereas additional restoration of the correct C-terminal tail resulted in poor cGMP sensing as assessed by superfusion of either 8-bromo-cGMP or NO. Ultimately, two improved FlincGs were identified: one (FlincG2) had the divergent tail and gave moderate basal fluorescence and cGMP response amplitude and the other (FlincG3) had the correct tail, a GCaMP-like mutation in the EGFP region and an N-terminal tag, and was superior in both respects. All variants tested were strongly influenced by pH over the physiological range, in common with other EGFP-based biosensors. Purified FlincG3 protein exhibited a lower cGMP affinity (0.89 μM) than reported for the original FlincG (0.17 μM) but retained rapid kinetics and a 230-fold selectivity over cAMP. Successful expression of FlincG2 or FlincG3 in differentiated N1E-115 neuroblastoma cells and in primary cultures of hippocampal and dorsal root ganglion cells commends them for real-time imaging of cGMP dynamics in neural (and other) cells, and in their subcellular specializations

Cellular targets of nitric oxide in the hippocampus.

In the hippocampus, as in many other CNS areas, nitric oxide (NO) participates in synaptic plasticity, manifested as changes in pre- and/or postsynaptic function. While it is known that these changes are brought about by cGMP following activation of guanylyl cyclase-coupled NO receptors attempts to locate cGMP by immunocytochemistry in hippocampal slices in response to NO have failed to detect the cGMP elevation where expected, i.e. in the pyramidal neurones. Instead, astrocytes, unidentified varicose fibres and GABA-ergic nerve terminals are reported to be the prominent NO targets, raising the possibility that NO acts indirectly via other cells. We have re-investigated the distribution of cGMP generated in response to endogenous and exogenous NO in hippocampal slices using immunohistochemistry and new conditions designed to optimise cGMP accumulation and, hence, its detectability. The conditions included use of tissue from the developing rat hippocampus, a potent inhibitor of phosphodiesterase-2, and an allosteric enhancer of the NO-receptive guanylyl cyclase. Under these conditions, cGMP was formed in response to endogenous NO and was found in a population of pyramidal cell somata in area CA3 and subiculum as well as in structures described previously. The additional presence of exogenous NO resulted in hippocampal cGMP reaching the highest level recorded for brain tissue (1700 pmol/mg protein) and in cGMP immunolabelling throughout the pyramidal cell layer. Populations of axons and interneurones were also stained. According with these results, immunohistochemistry for the common NO receptor β1-subunit indicated widespread expression. A similar staining pattern for the α1-subunit with an antibody used previously in the hippocampus and elsewhere, however, proved to be artefactual. The results indicate that the targets of NO in the hippocampus are more varied and extensive than previous evidence had suggested and, in particular, that the pyramidal neurones participating in NO-dependent synaptic plasticity are direct NO targets