RESUMEN
BACKGROUND: Paediatric palliative and hospice care aims to improve the quality of life of children with life-limiting and life-threatening conditions and their families. The number of these patients has risen significantly in recent years, resulting in an increased need for palliative care for this population. Although the need for paediatric palliative and hospice care is growing, meaningful outcome evaluation to demonstrate its effectiveness as a complex healthcare intervention is in its early stages. For complex interventions (programmes), theory-based evaluations have grown in prominence in recent years. They seek to understand how and why an intervention works by uncovering its underlying mechanisms by means of programme theory. To support both outcome evaluation in paediatric palliative care and a reflective practice of programme theorizing, we aimed to describe the construction of a programme theory for a specialist paediatric palliative and hospice care programme in Austria and to offer a reflective account of its development process. METHODS: We drew on a combination of theory-based evaluation frameworks to construct a programme theory consisting of an action and a change component. Through multiple iterations, incorporating different stakeholders' perspectives and drawing on different sources of knowledge and theory, we theorized how and why the programme likely achieves its intended outcomes. RESULTS: The programme theory outlines the proposed chains of events, causal mechanisms and outcomes of a specialist paediatric palliative and hospice care programme for children and families in several areas corresponding to its main conceptual tenets. Through a range of activities and interventions, the programme triggers coping and adaptation mechanisms that ultimately contribute to family and child wellbeing in physical, psychological, social, and spiritual dimensions. Established trust and partnership between children/families and healthcare professionals as well as a person-centered and family-centered approach were identified as enabling factors. CONCLUSIONS: Our findings provide insights into how a specialized paediatric palliative and hospice care programme works to achieve its intended outcomes for children and families. This helps demonstrate its impact, contributing to meaningful outcome evaluation and service improvement.
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Cuidados Paliativos al Final de la Vida , Cuidados Paliativos , Pediatría , Humanos , Cuidados Paliativos/métodos , Cuidados Paliativos/normas , Cuidados Paliativos al Final de la Vida/métodos , Cuidados Paliativos al Final de la Vida/organización & administración , Pediatría/métodos , Desarrollo de Programa/métodos , Niño , Evaluación de Programas y Proyectos de Salud/métodos , Calidad de Vida/psicologíaRESUMEN
Impairment of excretion and enzymatic processing of nitrogen, for example, because of liver or kidney failure, or with urea cycle and creatine synthesis enzyme defects, surprisingly leads to primarily neurologic symptoms, yet the exact mechanisms remain largely mysterious. In guanidinoacetate N-methyltransferase (GAMT) deficiency, the guanidino compound guanidinoacetate (GAA) increases dramatically, including in the cerebrospinal fluid (CSF), and has been implicated in mediating the neurological symptoms in GAMT-deficient patients. GAA is synthesized by arginine-glycine amidinotransferase (AGAT), a promiscuous enzyme that not only transfers the amidino group from arginine to glycine, but also to primary amines in, for example, GABA and taurine to generate γ-guanidinobutyric acid (γ-GBA) and guanidinoethanesulfonic acid (GES), respectively. We show that GAA, γ-GBA, and GES share structural similarities with GABA, evoke GABAA receptor (GABAA R) mediated currents (whereas creatine [methylated GAA] and arginine failed to evoke discernible currents) in cerebellar granule cells in mouse brain slices and displace the high-affinity GABA-site radioligand [3 H]muscimol in total brain homogenate GABAA Rs. While γ-GBA and GES are GABA agonists and displace [3 H]muscimol (EC50 /IC50 between 10 and 40 µM), GAA stands out as particularly potent in both activating GABAA Rs (EC50 ~6 µM) and also displacing the GABAA R ligand [3 H]muscimol (IC50 ~3 µM) at pathophysiologically relevant concentrations. These findings stress the role of substantially elevated GAA as a primary neurotoxic agent in GAMT deficiency and we discuss the potential role of GAA in arginase (and creatine transporter) deficiency which show a much more modest increase in GAA concentrations yet share the unique hyperexcitability neuropathology with GAMT deficiency. We conclude that orthosteric activation of GABAA Rs by GAA, and potentially other GABAA R mimetic guanidino compounds (GCs) like γ-GBA and GES, interferes with normal inhibitory GABAergic neurotransmission which could mediate, and contribute to, neurotoxicity.
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Creatina , Receptores de GABA-A , Ratones , Animales , Creatina/farmacología , Muscimol , Glicina/farmacología , Ácido gamma-Aminobutírico , ArgininaRESUMEN
Previous studies indicate that moderate-to-high ethanol (EtOH) concentrations enhance dopamine (DA) neurotransmission in the mesolimbic DA system from the ventral tegmental area (VTA) and projecting to the nucleus accumbens core (NAc). However, voltammetry studies demonstrate that moderate-to-high EtOH concentrations decrease evoked DA release at NAc terminals. The involvement of γ-aminobutyric acid (GABA) receptors (GABAA Rs), glycine (GLY) receptors (GLYRs) and cholinergic interneurons (CINs) in mediating EtOH inhibition of evoked NAc DA release were examined. Fast scan cyclic voltammetry, electrophysiology, optogenetics and immunohistochemistry techniques were used to evaluate the effects of acute and chronic EtOH exposure on DA release and CIN activity in C57/BL6, CD-1, transgenic mice and δ-subunit knockout (KO) mice (δ-/-). Ethanol decreased DA release in mice with an IC50 of 80 mM ex vivo and 2.0 g/kg in vivo. GABA and GLY decreased evoked DA release at 1-10 mM. Typical GABAA R agonists inhibited DA release at high concentrations. Typical GABAA R antagonists had minimal effects on EtOH inhibition of evoked DA release. However, EtOH inhibition of DA release was blocked by the α4 ß3 δ GABAA R antagonist Ro15-4513, the GLYR antagonist strychnine and by the GABA ρ1 (Rho-1) antagonist TPMPA (10 µM) and reduced significantly in GABAA R δ-/- mice. Rho-1 expression was observed in CINs. Ethanol inhibited GABAergic synaptic input to CINs from the VTA and enhanced firing rate, both of which were blocked by TPMPA. Results herein suggest that EtOH inhibition of DA release in the NAc is modulated by GLYRs and atypical GABAA Rs on CINs containing δ- and Rho-subunits.
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Dopamina/metabolismo , Etanol/farmacología , Núcleo Accumbens/efectos de los fármacos , Receptores de GABA/efectos de los fármacos , Animales , Agonistas del GABA/farmacología , Antagonistas del GABA/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones TransgénicosRESUMEN
Muscimol, the major psychoactive ingredient in the mushroom Amanita muscaria, has been regarded as a universal non-selective GABA-site agonist. Deletion of the GABAA receptor (GABAA R) δ subunit in mice (δKO) leads to a drastic reduction in high-affinity muscimol binding in brain sections and to a lower behavioral sensitivity to muscimol than their wild type counterparts. Here, we use forebrain and cerebellar brain homogenates from WT and δKO mice to show that deletion of the δ subunit leads to a > 50% loss of high-affinity 5 nM [3 H]muscimol-binding sites despite the relatively low abundance of δ-containing GABAA Rs (δ-GABAA R) in the brain. By subtracting residual high-affinity binding in δKO mice and measuring the slow association and dissociation rates we show that native δ-GABAA Rs in WT mice exhibit high-affinity [3 H]muscimol-binding sites (KD ~1.6 nM on α4ßδ receptors in the forebrain and ~1 nM on α6ßδ receptors in the cerebellum at 22°C). Co-expression of the δ subunit with α6 and ß2 or ß3 in recombinant (HEK 293) expression leads to the appearance of a slowly dissociating [3 H]muscimol component. In addition, we compared muscimol currents in recombinant α4ß3δ and α4ß3 receptors and show that δ subunit co-expression leads to highly muscimol-sensitive currents with an estimated EC50 of around 1-2 nM and slow deactivation kinetics. These data indicate that δ subunit incorporation leads to a dramatic increase in GABAA R muscimol sensitivity. We conclude that biochemical and behavioral low-dose muscimol selectivity for δ-subunit-containing receptors is a result of low nanomolar-binding affinity on δ-GABAA Rs.
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Encéfalo/metabolismo , Muscimol/metabolismo , Receptores de GABA-A/metabolismo , Animales , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Unión ProteicaRESUMEN
The role of GABAA receptor (GABAAR)-mediated tonic inhibition in interneurons remains unclear and may vary among subgroups. Somatostatin (SOM) interneurons in the hilus of the dentate gyrus show negligible expression of nonsynaptic GABAAR subunits and very low tonic inhibition. To determine the effects of ectopic expression of tonic GABAAR subtypes in these neurons, Cre-dependent viral vectors were used to express GFP-tagged GABAAR subunits (α6 and δ) selectively in hilar SOM neurons in SOM-Cre mice. In single-transfected animals, immunohistochemistry demonstrated strong expression of either the α6 or δ subunit; in cotransfected animals, both subunits were consistently expressed in the same neurons. Electrophysiology revealed a robust increase of tonic current, with progressively larger increases following transfection of δ, α6, and α6/δ subunits, respectively, indicating formation of functional receptors in all conditions and likely coassembly of the subunits in the same receptor following cotransfection. An in vitro model of repetitive bursting was used to determine the effects of increased tonic inhibition in hilar SOM interneurons on circuit activity in the dentate gyrus. Upon cotransfection, the frequency of GABAAR-mediated bursting in granule cells was reduced, consistent with a reduction in synchronous firing among hilar SOM interneurons. Moreover, in vivo studies of Fos expression demonstrated reduced activation of α6/δ-cotransfected neurons following acute seizure induction by pentylenetetrazole. The findings demonstrate that increasing tonic inhibition in hilar SOM interneurons can alter dentate gyrus circuit activity during strong stimulation and suggest that tonic inhibition of interneurons could play a role in regulating excessive synchrony within the network. SIGNIFICANCE STATEMENT: In contrast to many hippocampal interneurons, somatostatin (SOM) neurons in the hilus of the dentate gyrus have very low levels of nonsynaptic GABAARs and exhibit very little tonic inhibition. In an effort to increase tonic inhibition selectively in these interneurons, we used Cre-dependent viral vectors in SOM-Cre mice to achieve interneuron-specific expression of the nonsynaptic GABAAR subunits (α6 and δ) in vivo. We show, for the first time, that such recombinant GFP-tagged GABAAR subunits are expressed robustly, assemble to form functional receptors, substantially increase tonic inhibition in SOM interneurons, and alter circuit activity within the dentate gyrus.
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Giro Dentado/citología , Red Nerviosa/metabolismo , Neuronas/metabolismo , Receptores de GABA-A/metabolismo , Somatostatina/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/genética , Animales , Giro Dentado/efectos de los fármacos , Agonistas del GABA/farmacología , Antagonistas del GABA/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Vectores Genéticos/metabolismo , Humanos , Isoxazoles/farmacología , Masculino , Ratones , Ratones Transgénicos , Inhibición Neural/efectos de los fármacos , Inhibición Neural/genética , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Pentilenotetrazol/farmacología , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Pirimidinas/farmacología , Receptores de GABA-A/genética , Somatostatina/genéticaRESUMEN
The molecular mechanism(s) of action of anesthetic, and especially, intoxicating doses of alcohol (ethanol [EtOH]) have been of interest even before the advent of the Research Society on Alcoholism. Recent physiological, genetic, and biochemical studies have pin-pointed molecular targets for anesthetics and EtOH in the brain as ligand-gated ion channel (LGIC) membrane proteins, especially the pentameric (5 subunit) Cys-loop superfamily of neurotransmitter receptors including nicotinic acetylcholine (nAChRs), GABAA (GABAA Rs), and glycine receptors (GlyRs). The ability to demonstrate molecular and structural elements of these proteins critical for the behavioral effects of these drugs on animals and humans provides convincing evidence for their role in the drugs' actions. Amino acid residues necessary for pharmacologically relevant allosteric modulation of LGIC function by anesthetics and EtOH have been identified in these channel proteins. Site-directed mutagenesis revealed potential allosteric modulatory sites in both the trans-membrane domain (TMD) and extracellular domain (ECD). Potential sites of action and binding have been deduced from homology modeling of other LGICs with structures known from crystallography and cryo-electron microscopy studies. Direct information about ligand binding in the TMD has been obtained by photoaffinity labeling, especially in GABAA Rs. Recent structural information from crystallized procaryotic (ELIC and GLIC) and eukaryotic (GluCl) LGICs allows refinement of the structural models including evaluation of possible sites of EtOH action.
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Anestésicos/farmacología , Depresores del Sistema Nervioso Central/farmacología , Receptores de Canales Iónicos con Asa de Cisteína Activados por Ligando/efectos de los fármacos , Etanol/farmacología , Modelos Moleculares , Secuencia de Aminoácidos , Anestésicos/metabolismo , Animales , Depresores del Sistema Nervioso Central/metabolismo , Receptores de Canales Iónicos con Asa de Cisteína Activados por Ligando/metabolismo , Etanol/metabolismo , Humanos , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Estructura MolecularRESUMEN
Dementia and autoimmune diseases are prevalent conditions with limited treatment options. Taurine and homotaurine (HT) are naturally occurring sulfonate amino acids, with taurine being highly abundant in animal tissues, but declining with age in the blood. HT is a blood-brain barrier permeable drug under investigation for Alzheimer's disease. HT also has beneficial effects in a mouse model of multiple sclerosis likely through an anti-inflammatory mechanism mediated by GABAA receptor (GABAAR) agonism in immune cells. While both taurine and HT are structural GABA analogs and thought to be GABA mimetics at GABAARs, there is uncertainty concerning their potency as GABA mimetics on native GABAARs. We show that HT is a very potent GABA mimetic, as it evokes GABAAR-mediated currents with an EC50 of 0.4 µM (vs. 3.7 µM for GABA and 116 µM for taurine) in murine cerebellar granule cells in brain slices, with both taurine and HT having similar efficacy in activating native GABAARs. Furthermore, HT displaces the high affinity GABAAR ligand [3H]muscimol at similarly low concentrations (HT IC50 of 0.16 µM vs. 125 µM for taurine) in mouse brain homogenates. The potency of taurine and HT as GABAAR agonists aligns with endogenous concentrations of taurine in the blood and with HT concentrations achieved in the brain following oral administration of HT or the HT pro-drug ALZ-801. Consequently, we discuss that GABAARs subtypes, similar to the ones we studied here in neurons, are plausible targets for mediating the potential beneficial effects of taurine in health and life-span extension and the beneficial HT effects in dementia and autoimmune conditions.
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There are increasing discussions on theory dynamics in nursing research. We aimed at mapping the theoretical publication output by nursing researchers from the European German-speaking area. We conducted a focused mapping review and synthesis, focusing on nursing journals articles with a theory-related aim. We identified 32 eligible publications, reflecting 2% of the nursing journal articles affiliated with researchers from our target region. Twenty-one articles involved an inductive approach. Eleven articles intended to test or revise a theory. The theoretical publication output with a theory-related aim was low. Theory-building efforts were fragmented and mostly without reference to a meta-theoretical level.
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Developing and evaluating health interventions for the benefit of patients is notoriously difficult. This also applies to the discipline of nursing, owing to the complexity of nursing interventions. Following significant revision, the updated guidance of the Medical Research Council (MRC) adopts a pluralistic view to intervention development and evaluation, including a theory-based perspective. This perspective promotes the use of program theory, aiming to understand how and under what circumstances interventions lead to change. In this discussion paper, we reflect the recommended use of program theory in the context of evaluation studies addressing complex nursing interventions. First, we review the literature by investigating the question whether and how evaluation studies targeting complex interventions used theory and to what extent program theories may contribute to enhance the theoretical foundations of intervention studies in nursing. Second, we illustrate the nature of theory-based evaluation and program theories. Third, we argue how this may impact theory building in nursing in general. We finish by discussing which resources, skills and competencies are necessary to fulfill the demanding task of undertaking theory-based evaluations. We caution against an oversimplified interpretation of the updated MRC guidance regarding the theory-based perspective, e.g. by using simple linear logic models, rather than articulating program theories. Instead, we encourage researchers to embrace the corresponding methodology, i.e. theory-based evaluation. With the prevailing perspective of knowledge production in crisis, we might be on the verge of a paradigm shift in health intervention research. Viewed through this lens, the updated MRC guidance could lead to a renewed understanding of what constitutes useful knowledge in nursing. This may facilitate knowledge production and, thereby, contribute to improve nursing practice for the benefit of the patient. TWEETABLE ABSTRACT: The latest iteration of the MRC Framework for developing and evaluating complex healthcare interventions could lead to a renewed understanding of what constitutes useful knowledge for nursing.
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Investigación Biomédica , Atención a la Salud , HumanosRESUMEN
Decreased expression of the δ subunit of the GABAA receptor (GABAAR) has been found in the dentate gyrus in several animal models of epilepsy and other disorders with increased excitability and is associated with altered modulation of tonic inhibition in dentate granule cells (GCs). In contrast, other GABAAR subunits, including α4 and γ2 subunits, are increased, but the relationship between these changes is unclear. The goals of this study were to determine if viral transfection of δ subunits in dentate GCs could increase δ subunit expression, alter expression of potentially-related GABAAR subunits, and restore more normal network excitability in the dentate gyrus in a mouse model of epilepsy. Pilocarpine-induced seizures were elicited in DOCK10-Cre mice that express Cre selectively in dentate GCs, and two weeks later the mice were injected unilaterally with a Cre-dependent δ-GABAAR viral vector. At 4-6 weeks following transfection, δ subunit immunolabeling was substantially increased in dentate GCs on the transfected side compared to the nontransfected side. Importantly, α4 and γ2 subunit labeling was downregulated on the transfected side. Electrophysiological studies revealed enhanced tonic inhibition, decreased network excitability, and increased neurosteroid sensitivity in slices from the δ subunit-transfected side compared to those from the nontransfected side of the same pilocarpine-treated animal, consistent with the formation of δ subunit-containing GABAARs. No differences were observed between sides of eYFP-transfected animals. These findings are consistent with the idea that altering expression of key subunits, such as the δ subunit, regulates GABAAR subunit assemblies, resulting in substantial effects on network excitability.
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Epilepsia , Neuroesteroides , Animales , Giro Dentado/metabolismo , Epilepsia/inducido químicamente , Epilepsia/metabolismo , Ratones , Ratones Endogámicos C57BL , Pilocarpina/metabolismo , Pilocarpina/farmacología , Receptores de GABA-A/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Extrasynaptic GABA(A) receptors (eGABARs) allow ambient GABA to tonically regulate neuronal excitability and are implicated as targets for ethanol and anesthetics. These receptors are thought to be heteropentameric proteins made up of two α subunits-either α4 or α6-two ß2 or ß3 subunits, and one δ subunit. The GABA analog 4,5,6,7-tetrahydroisoxazolo (5,4-c)pyridin-3(-ol) (THIP) has been proposed as a selective ligand for eGABARs. Behavioral and in vitro studies suggest that eGABARs have nanomolar affinity for THIP; however, all published studies on recombinant versions of eGABARs report micromolar affinities. Here, we examine THIP sensitivity of native eGABARs on cerebellar neurons and on reconstituted GABARs in heterologous systems. Concentration-response data for THIP, obtained from cerebellar granule cells and molecular layer interneurons in wild-type and δ subunit knockout slices, confirm that submicromolar THIP sensitivity requires δ subunits. In recombinant experiments, we find that δ subunit coexpression leads to receptors activated by nanomolar THIP concentrations (EC(50) of 30-50 nM for α4ß3δ and α6ß3δ), a sensitivity almost 1,000-fold higher than receptors formed by α4/6 and ß3 subunits. In contrast, γ2 subunit expression significantly reduces THIP sensitivity. Even when δ subunit cDNA or cRNA was supplied in excess, high- and low-sensitivity THIP responses were often apparent, indicative of variable mixtures of low-affinity αß and high-affinity αßδ receptors. We conclude that δ subunit incorporation into GABARs leads to a dramatic increase in THIP sensitivity, a defining feature that accounts for the unique behavioral and neurophysiological properties of THIP.
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Agonistas del GABA/farmacología , Neuronas GABAérgicas/efectos de los fármacos , Isoxazoles/farmacología , Receptores de GABA-A/efectos de los fármacos , Receptores de GABA-A/fisiología , Animales , Corteza Cerebelosa/citología , Relación Dosis-Respuesta a Droga , Agonistas del GABA/administración & dosificación , Agonistas del GABA/farmacocinética , Neuronas GABAérgicas/fisiología , Células HEK293 , Humanos , Isoxazoles/administración & dosificación , Isoxazoles/farmacocinética , Ratones , Ratones Noqueados , Oocitos , Técnicas de Placa-Clamp , Isoformas de Proteínas/fisiología , Multimerización de Proteína , Subunidades de Proteína , Receptores de GABA-A/química , Receptores de GABA-A/deficiencia , Receptores de GABA-A/genética , Proteínas Recombinantes de Fusión/fisiología , Solubilidad , Xenopus laevisRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Previously, the phytochemical constituents of Biebersteinia heterostemon Maxim (BHM) and Arenaria kansuensis Maxim (AKM) were studied and the evaluation of anxiolytic effect based on their extracts was also investigated. The two traditional Tibetan herbs, BHM and AKM, have been widely used in Qinghai-Tibet Plateau for cardiopulmonary disorders and neuropsychiatric diseases. The anxiolytic activities of a number of agents mediated by α2/3-containing GABAA receptors (GABAARs) have been demonstrated through the genetic and pharmacological studies. Flavonoids, such as flavones and flavanols, are a class of ligands that act at GABAARs and exhibit anxiolytic effects in vivo. Here, the flavonoids are the predominant constituents isolated from BHM and AKM. And our purpose is to investigate structure-activity relationships of the flavonoid compounds with binding to BZ-S of GABAAR complexes, and to search for anxiolytic constituents that lack undesirable-effects such as sedation and myorelaxation. MATERIALS AND METHODS: The flavonoid constituents were separated and purified through the repeatedly silica gel or/and C18 column chromatography. The affinities of the compounds for BZ-S of GABAARs were detected by the radioreceptor binding assay with bovine cerebellum membranes, in which the different recombinant subunits-containing GABAARs were expressed in HEK 293T cells. The behavior tests, including elevated plus maze, locomotor activity, holeboard, rotarod and horizontal wire, were used to determine and evaluate the anxiolytic, sedative, and myorelaxant effects of these flavonoids. RESULTS: Eleven total flavonoid compounds were obtained from the Tibetan herbs (BHM and AKM). The flavones with 6-and/or 8-OMe possessed the most potent binding affinity to GABAARs, which were based on the result of structure-activity relationships analysis. Demethoxysudachitin (DMS, Ki = 0.59 µM), a flavone that binds to recombinant α1-3/5 subunit-containing GABAARs, was isolated from BHM, and exhibited high anxiolytic activity, without inducing sedation and myorelaxation. Moreover, the anxiolytic effect of DMS was antagonized by flumazenil, suggesting that a mode of action was mediated via the BZ-S of GABAARs. CONCLUSIONS: This present study indicated that the flavones, especially DMS, are novel GABAAR ligands and therapeutic potential candidates for anxiety.
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Ansiolíticos/farmacología , Arenaria , Conducta Animal/efectos de los fármacos , Flavonoides/farmacología , Geraniaceae , Extractos Vegetales/farmacología , Receptores de GABA-A/efectos de los fármacos , Animales , Ansiolíticos/química , Ansiolíticos/aislamiento & purificación , Ansiolíticos/toxicidad , Arenaria/química , Arenaria/toxicidad , Conducta Exploratoria/efectos de los fármacos , Flavonoides/química , Flavonoides/aislamiento & purificación , Flavonoides/toxicidad , Geraniaceae/química , Geraniaceae/toxicidad , Células HEK293 , Humanos , Ligandos , Medicina Tradicional Tibetana , Ratones Endogámicos C57BL , Estructura Molecular , Actividad Motora/efectos de los fármacos , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/toxicidad , Unión Proteica , Ratas , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Relación Estructura-ActividadRESUMEN
GABA(A) receptors (GABA(A)Rs) have long been a focus as targets for alcohol actions. Recent work suggests that tonic GABAergic inhibition mediated by extrasynaptic δ subunit-containing GABA(A)Rs is uniquely sensitive to ethanol and enhanced at concentrations relevant for human alcohol consumption. Ethanol enhancement of recombinant α4ß3δ receptors is blocked by the behavioral alcohol antagonist 8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester (Ro15-4513), suggesting that EtOH/Ro15-4513-sensitive receptors mediate important behavioral alcohol actions. Here we confirm alcohol/alcohol antagonist sensitivity of α4ß3δ receptors using human clones expressed in a human cell line and test the hypothesis that discrepant findings concerning the high alcohol sensitivity of these receptors are due to difficulties incorporating δ subunits into functional receptors. To track δ subunit incorporation, we used a functional tag, a single amino acid change (H68A) in a benzodiazepine binding residue in which a histidine in the δ subunit is replaced by an alanine residue found at the homologous position in γ subunits. We demonstrate that the δH68A substitution confers diazepam sensitivity to otherwise diazepam-insensitive α4ß3δ receptors. The extent of enhancement of α4ß3δH68A receptors by 1 µM diazepam, 30 mM EtOH, and 1 µM ß-carboline-3-carboxy ethyl ester (but not 1 µM Zn(2+) block) is correlated in individual recordings, suggesting that δ subunit incorporation into recombinant GABA(A)Rs varies from cell to cell and that this variation accounts for the variable pharmacological profile. These data are consistent with the notion that δ subunit-incorporation is often incomplete in recombinant systems yet is necessary for high ethanol sensitivity, one of the features of native δ subunit-containing GABA(A)Rs.
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Azidas/farmacología , Benzodiazepinas/farmacología , Etanol/antagonistas & inhibidores , Etanol/farmacología , Receptores de GABA-A/biosíntesis , Regulación Alostérica , Animales , Carbolinas/farmacología , Cationes Bivalentes , Línea Celular , Diazepam/farmacología , Humanos , Mutación , Técnicas de Placa-Clamp , Subunidades de Proteína/biosíntesis , Subunidades de Proteína/genética , Ratas , Receptores de GABA-A/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Transfección , Zinc/farmacologíaRESUMEN
Aim: The aim of this study was to translate and culturally adapt the PCPI-S into German and to eventually test its psychometric properties in long-term care settings. Background: Person-centred practice has been widely adopted internationally as a best-practice model in nursing and health care. To ensure a sustainable implementation of this practice and to successively promote it, person-centred practice should be evaluated on a regular basis. The Person-centred Practice Inventory-Staff (PCPI-S), which is based on McCormack & McCance's Person-centred Practice Framework, is a new instrument for this purpose by assessing perceptions of person-centredness among healthcare staff. Design: A two-phase research design was used involving the translation and cultural adaption of the PCPI-S from English to German (PCPI-S-G; Phase 1) and a quantitative cross-sectional survey (Phase 2). Methods: Construct validity was evaluated using confirmatory factor analysis (CFA), and internal consistency was calculated using Cronbach's α. Results: Phase 1 was conducted using an internationally recommended checklist for translations and cultural adaptations. In Phase 2, the PCPI-S-G was tested in 15 residential care homes in Austria with a sample of 255 staff members. The CFA showed good construct validity and supported the theoretical framework. The internal consistency for the three constructs of the PCPI-S was excellent, revealing Cronbach's α-scores from 0.902-0.941.
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Actitud del Personal de Salud , Atención Dirigida al Paciente , Austria , Estudios Transversales , Humanos , Reproducibilidad de los Resultados , Encuestas y CuestionariosRESUMEN
General anesthetics, once thought to exert their effects through non-specific membrane effects, have highly specific ion channel targets that can silence neuronal populations in the nervous system, thereby causing unconsciousness and immobility, characteristic of general anesthesia. Inhibitory GABA(A) receptors (GABA(A)Rs), particularly highly GABA-sensitive extrasynaptic receptor subtypes that give rise to sustained inhibitory currents, are uniquely sensitive to GABA(A)R-active anesthetics. A prominent population of extrasynaptic GABA(A)Rs is made up of alpha4, beta2 or beta3, and delta subunits. Considering the demonstrated importance of GABA receptor beta3 subunits for in vivo anesthetic effects of etomidate and propofol, we decided to investigate the effects of GABA anesthetics on "extrasynaptic" alpha4beta3delta and also binary alpha4beta3 receptors expressed in human embryonic kidney (HEK) cells. Consistent with previous work on similar receptor subtypes we show that maximal GABA currents through "extrasynaptic" alpha4beta3delta receptors, receptors defined by sensitivity to EtOH (30mM) and the beta-carboline beta-CCE (1microM), are enhanced by the GABA(A)R-active anesthetics etomidate, propofol, and the neurosteroid anesthetic THDOC. Furthermore, we show that receptors formed by alpha4beta3 subunits alone also show high GABA sensitivity and that saturating GABA responses of alpha4beta3 receptors are increased to the same extent by etomidate, propofol, and THDOC as are alpha4beta3delta receptors. Therefore, both alpha4beta3 and alpha4beta3delta receptors show low GABA efficacy, and GABA is also a partial agonist on certain binary alphabeta receptor subtypes. Increasing GABA efficacy on alpha4/6beta3delta and alpha4beta3 receptors is likely to make an important contribution to the anesthetic effects of etomidate, propofol and the neurosteroid THDOC.
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Expresión Génica/efectos de los fármacos , Hipnóticos y Sedantes/farmacología , Receptores de GABA-A/biosíntesis , Proteínas Recombinantes/biosíntesis , Ácido gamma-Aminobutírico/farmacología , Línea Celular Transformada , Depresores del Sistema Nervioso Central/farmacología , Desoxicorticosterona/análogos & derivados , Desoxicorticosterona/farmacología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Estimulación Eléctrica/métodos , Etanol/farmacología , Etomidato/farmacología , Humanos , Técnicas de Placa-Clamp/métodos , Propofol/farmacología , Transfección/métodosRESUMEN
Neuronal mechanisms underlying alcohol intoxication are unclear. We find that alcohol impairs motor coordination by enhancing tonic inhibition mediated by a specific subtype of extrasynaptic GABA(A) receptor (GABAR), alpha6beta3delta, expressed exclusively in cerebellar granule cells. In recombinant studies, we characterize a naturally occurring single-nucleotide polymorphism that causes a single amino acid change (R100Q) in alpha6 (encoded in rats by the Gabra6 gene). We show that this change selectively increases alcohol sensitivity of alpha6beta3delta GABARs. Behavioral and electrophysiological comparisons of Gabra6(100R/100R) and Gabra6(100Q/100Q) rats strongly suggest that alcohol impairs motor coordination by enhancing granule cell tonic inhibition. These findings identify extrasynaptic GABARs as critical targets underlying low-dose alcohol intoxication and demonstrate that subtle changes in tonic inhibition in one class of neurons can alter behavior.
Asunto(s)
Depresores del Sistema Nervioso Central/farmacología , Etanol/farmacología , Inhibición Neural/fisiología , Neuronas/fisiología , Desempeño Psicomotor/fisiología , Receptores de GABA-A/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Animales Recién Nacidos , Conducta Animal , Cerebelo/citología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Genotipo , Técnicas In Vitro , Inhibición Neural/efectos de los fármacos , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp/métodos , Polimorfismo de Nucleótido Simple/fisiología , Subunidades de Proteína/fisiología , Desempeño Psicomotor/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/genética , Transfección/métodos , Xenopus laevis , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
There is a need for treatments that can safely promote regulatory lymphocyte responses. T cells express GABA receptors (GABAA-Rs) and GABA administration can inhibit Th1-mediated processes such as type 1 diabetes and rheumatoid arthritis in mouse models. Whether GABAA-R agonists can also inhibit Th17-driven processes such as experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS), is an open question. GABA does not pass through the blood-brain barrier (BBB) making it ill-suited to inhibit the spreading of autoreactivity within the CNS. Homotaurine is a BBB-permeable amino acid that antagonizes amyloid fibril formation and was found to be safe but ineffective in long-term Alzheimer's disease clinical trials. Homotaurine also acts as GABAA-R agonist with better pharmacokinetics than that of GABA. Working with both monophasic and relapsing-remitting mouse models of EAE, we show that oral administration of homotaurine can (1) enhance CD8+CD122+PD-1+ and CD4+Foxp3+ Treg, but not Breg, responses, (2) inhibit autoreactive Th17 and Th1 responses, and (3) effectively ameliorate ongoing disease. These observations demonstrate the potential of BBB-permeable GABAA-R agonists as a new class of treatment to enhance CD8+ and CD4+ Treg responses and limit Th17 and Th1-medaited inflammation in the CNS.
Asunto(s)
Agonistas del GABA/administración & dosificación , Esclerosis Múltiple/tratamiento farmacológico , Taurina/análogos & derivados , Administración Oral , Animales , Barrera Hematoencefálica/metabolismo , Modelos Animales de Enfermedad , Agonistas del GABA/farmacología , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Taurina/administración & dosificación , Taurina/farmacología , Células TH1/efectos de los fármacos , Células TH1/inmunología , Células Th17/efectos de los fármacos , Células Th17/inmunologíaRESUMEN
BACKGROUND: Our previous studies on Asterothamnus centrali-asiaticus Novopokr. (ACN) and Arenaria kansuensis Maxim. (AKM) had led to the isolation of some phytochemical constituents and evaluation of anticonvulsant effect based on their extracts. ACN and AKM have been widely used in traditional Tibetan herbs for neuropsychiatric diseases and cardiopulmonary disorders. PURPOSE: The purpose is to investigate structure-activity relationships of flavonoids isolated from ACN and AKM, for binding to the benzodiazepine site (BZ-S) of γ-aminobutyric acid type A (GABAA) receptor complex, and to search for anticonvulsant compounds without undesirable effects such as myorelaxation and sedation. STUDY DESIGN AND METHODS: The affinities of these flavonoids for the BZ-S of GABAA receptors were determined by [3H]flunitrazepam binding to mouse cerebellum membranes in vitro. And the anticonvulsant, myorelaxant and sedative effects were determined by pentylenetetrazol (PTZ)-induced seizure and electrogenic seizure protection, rotarod test and locomotor activity test, respectively. RESULTS: Fifteen and thirteen flavonoids were isolated from ACN and AKM, respectively. Structure-activity relationships analysis indicated that 6-and/or 8-OMe flavones exhibited the most potent binding affinity to GABAA receptors. Furthermore, 2',4',5,7-tetrahydroxy-5',6-dimethoxyflavone (DMF, IC50 value of 0.10⯵M), a flavone isolated from ACN, presented high anticonvulsant activity against chemical-induced seizures and electrogenic seizures, without myorelaxation and sedation. CONCLUSION: This study suggested that these flavones, especially DMF, are new BZ receptor ligands and prospective therapeutic candidates for seizures.
Asunto(s)
Anticonvulsivantes/farmacología , Arenaria/química , Asteraceae/química , Flavonoides/farmacología , Antagonistas de Receptores de GABA-A/farmacología , Receptores de GABA-A/metabolismo , Convulsiones/tratamiento farmacológico , Animales , Anticonvulsivantes/aislamiento & purificación , China , Flavonoides/aislamiento & purificación , Flunitrazepam , Masculino , Medicina Tradicional Tibetana , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Pentilenotetrazol/efectos adversos , Extractos Vegetales/farmacología , Estudios Prospectivos , Convulsiones/inducido químicamenteRESUMEN
GABAA receptors (GABARs) are heteromultimeric proteins composed of five subunits. The specific subunit composition determines critical properties of a GABAR such as pharmacological sensitivities and whether the receptor contributes to synaptic or extrasynaptic forms of inhibition. Classically, synaptic but not extrasynaptic GABARs are thought to respond to benzodiazepines, whereas the reverse has been suggested for ethanol. To examine the effects of subunit composition on GABAR function in situ, we took advantage of two naturally occurring alleles of the rat gene for GABAR subunit alpha6 (Gabra6(100R) and Gabra6(100Q)). Depending on their subunit partners, these two variants of alpha6 can lead to differential sensitivities to benzodiazepines and ethanol. An examination of synaptic and extrasynaptic GABA-mediated currents in cerebellar granule cells from Gabra6(100R/100R) and Gabra6(100Q/100Q) rats uncovered marked allele-dependent differences in benzodiazepine sensitivity. Unexpectedly, we found that the benzodiazepines flunitrazepam and diazepam enhanced extrasynaptic inhibition mediated by delta subunit-containing GABARs in Gabra6(100Q/100Q) rats. Complementary experiments on recombinant GABARs confirmed that, at subsaturating [GABA], flunitrazepam potentiates alpha6/delta subunit-containing GABARs. Based on data and a simple theoretical analysis, we estimate that the average extrasynaptic [GABA] is approximately 160 nm in perfused slices. These results (1) demonstrate contributions of alpha6 subunits to both synaptic and extrasynaptic GABA responses, (2) establish that delta subunit-containing GABARs are benzodiazepine sensitive at subsaturating [GABA] and, (3) provide an empirical estimate of extrasynaptic [GABA] in slices.
Asunto(s)
Inhibición Neural/genética , Neuronas/metabolismo , Polimorfismo Genético/genética , Receptores de GABA-A/genética , Sinapsis/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Benzodiazepinas/farmacología , Corteza Cerebelosa/fisiología , Resistencia a Medicamentos/genética , Etanol/farmacología , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/genética , Femenino , Inhibición Neural/efectos de los fármacos , Neuronas/efectos de los fármacos , Oocitos , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Sinapsis/efectos de los fármacos , Sinapsis/genética , Membranas Sinápticas/efectos de los fármacos , Membranas Sinápticas/genética , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/genética , Xenopus laevisRESUMEN
GABA(A) receptors (GABA(A)Rs) are the main inhibitory neurotransmitter receptors and have long been implicated in mediating at least part of the acute actions of ethanol. For example, ethanol and GABAergic drugs including barbiturates and benzodiazepines share many pharmacological properties. Besides the prototypical synaptic GABA(A)R subtypes, nonsynaptic GABA(A)Rs have recently emerged as important regulators of neuronal excitability. While high doses (> or =100 mM) of ethanol have been reported to enhance activity of most GABA(A)R subtypes, most abundant synaptic GABA(A)Rs are essentially insensitive to ethanol concentrations that occur during social ethanol consumption (< 30 mM). However, extrasynaptic delta and beta3 subunit-containing GABA(A)Rs, associated in the brain with alpha4 or alpha6 subunits, are sensitive to low millimolar ethanol concentrations, as produced by drinking half a glass of wine. Additionally, we found that a mutation in the cerebellar alpha6 subunit (alpha6R100Q), initially reported in rats selectively bred for increased alcohol sensitivity, is sufficient to produce increased alcohol-induced motor impairment and further increases of alcohol sensitivity in recombinant alpha6beta3delta receptors. Furthermore, the behavioral alcohol antagonist Ro15-4513 blocks the low dose alcohol enhancement on alpha4/6/beta3delta receptors, without reducing GABA-induced currents. In binding assays alpha4beta3delta GABA(A)Rs bind [(3)H]Ro15-4513 with high affinity, and this binding is inhibited, in an apparently competitive fashion, by low ethanol concentrations, as well as analogs of Ro15-4513 that are active to antagonize ethanol or Ro15-4513's block of ethanol. We conclude that most low to moderate dose alcohol effects are mediated by alcohol actions on alcohol/Ro15-4513 binding sites on GABA(A)R subtypes.