Phosphate-activated glutaminase activity is enhanced in brain, intestine and kidneys of rats following portacaval anastomosis

AIM: To assess whether portacaval anastomosis (PCA) in rats affects the protein expression and/or activity of glutaminase in kidneys, intestines and in three brain areas of cortex, basal ganglia and cerebellum and to explain the neurological alterations found in hepatic encephalopathy (HE). METHODS: Sixteen male Wistar rats weighing 250-350 gwere grouped into sham-operation control (n = 8) or portacaval shunt (n = 8). Twenty-eight days after the procedure, the animals were sacrificed. The duodenum, kidney and brain were removed, homogenised and mitochondria were isolated. Ammonia was measured in brain and blood. Phosphate-activated glutaminase (PAG) activity was determined by measuring ammonia production following incubation for one hour at 37 with O-phthalaldehyde (OPA) and specific activity expressed in units per gram of protein (μkat/g of protein). Protein expression was measured by immunoblotting. RESULTS: Duodenal and kidney PAG activities together with protein content were significantly higher in PCA group than in control or sham-operated rats (duodenum PAG activity was 976.95±268.87 μkat/g of protein in PCA rats vs 429.19±126.92 μkat/g of protein in shamoperated rats; kidneys PAG activity was 1259.18 ± 228.79 μkat/g protein in PCA rats vs 669.67± 400.8 μkat/g of protein in controls, P < 0.05; duodenal protein content: 173% in PCA vs sham-operated rats; in kidneys the content of protein was 152% in PCA vs sham-operated rats). PAG activity and protein expression in PCA rats were higher in cortex and basal ganglia than those in shamoperated rats (cortex: 6646.6 ± 1870.4 μkat/g of protein vs 3573.8 ± 2037.4 μkat/g of protein in control rats, P < 0.01; basal ganglia, PAG activity was 3657.3 ± 1469.6 μkat/g of protein in PCA rats vs 2271.2 ± 384 μkat/g of protein in sham operated rats, P < 0.05; In the cerebellum, the PAG activity was 2471.6 ± 701.4 μkat/g of protein vs 1452.9 ± 567.8 μkat/g of protein in the PCA and sham rats, respectively, P < 0.05; content of protein:cerebral cortex: 162% ± 40% vs 100% ± 26%, P < 0.009;and basal gangl ia: 140% ± 39% vs 100% ± 14%,P < 0.05; but not in cerebel lum: 100% ± 25% vs 100% ± 16%, P = ns). CONCLUSION: Increased PAG activity in kidney and duodenum could contribute significantly to the hyperammonaemia in PCA rats, animal model of encephalopathy. PAG is increased in non-synaptic mitochondria from the cortex and basal ganglia and could be implicated in the pathogenesis of hepatic encephalopathy. Therefore, PAG could be a possible target for the treatment of HE orliver dysfunction

Addressing acute stress among professionals caring for COVID-19 patients: lessons learned during the first outbreak in Spain (March–April 2020)

To describe lessons learned during the first COVID-19 outbreak in developing urgent interventions to strengthen healthcare workers’ capacity to cope with acute stress caused by health care pressure, concern about becoming infected, despair of witnessing patients’ suffering, and critical decision-making requirements of the SARS-CoV-2 pandemic during the first outbreak in Spain. Methods: A task force integrated by healthcare professionals and academics was activated following the first observations of acute stress reactions starting to compromise the professionals’ capacity for caring COVID-19 patients. Literature review and qualitative approach (consensus techniques) were applied. The target population included health professionals in primary care, hospitals, emergencies, and nursing homes. Interventions designed for addressing acute stress were agreed and disseminated. Findings: There are similarities in stressors to previous outbreaks, and the solutions devised then may work now. A set of issues, interventions to cope with, and their levels of evidence were defined. Issues and interventions were classified as: adequate communication initiative to strengthen work morale (avoiding information blackouts, uniformity of criteria, access to updated information, mentoring new professionals); resilience and recovery from physical and mental fatigue (briefings, protecting the family, regulated recovery time during the day, psychological first aid, humanizing care); reinforce leadership of intermediate commands (informative leadership, transparency, realism, and positive messages, the current state of emergency has not allowed for an empirical analysis of the effectiveness of proposed interventions. Sharing information to gauge expectations, listening to what professionals need, feeling protected from threats, organizational flexibility, encouraging teamwork, and leadership that promotes psychological safety have led to more positive responses. Attention to the needs of individuals must be combined with caring for the teams responsible for patient care. Conclusions: Although the COVID-19 pandemic has a more devastating effect than other recent outbreaks, there are common stressors and lessons learned in all of them that we must draw on to increase our capacity to respond to future healthcare crises

Papel de la hiperamoniemia sobre la actividad glutaminasa en diferentes tejidos y estrés oxidativo en ratas sometidas a derivación porto-cava

Como se sabe a partir de los datos publicados, el amonio es el principal causante de la Encefalopatía Hepática (EH). La hipótesis de partida del presente estudio es que la principal fuente de amonio plasmático es el amonio esplénico, y que este se forma debido a una mayor actividad de la glutaminasa activada por fosfato (GAP) en el intestino y el riñón. Como segunda hipótesis de trabajo se parte de que los altos niveles de amonio plasmático y la elevada actividad de la enzima glutaminasa (GAP) en astrocitos, en determinadas áreas del cerebro, podrían ser factores contribuyentes a la elevada concentración extracelular del glutamato, observada en modelos animales de EH y enfermos con EH causada por fallo hepático. Como tercera hipótesis se plantea el posible papel de la hiperamonemia y la elevada concentración de glutamato en los fluidos cerebrales extracelulares, como agentes desencadenantes del estrés oxidativo. Por ello los objetivos del presente trabajo son estudiar, en un modelo animal de ratas con DPC, los siguiente puntos: 1. Establecer los niveles de amonio en plasma y en tejidos extrahepáticos y cerebro. 2. Establecer la actividad glutaminasa en tejidos extrahepáticos y cerebro. 3. Establecer el grado de oxidación de las proteínas del cerebro, mediante la cuantificación de grupos carbonilos, como índice de oxidación. 4. Estudiar el papel de determinados productos: histidina, glutamato, lactitol, neomicina IGF-1 y flumazenilo, sobre la actividad glutaminasa, con el fin de iniciar estudios encaminados a reducir su actividad (inhibición) como posible vía terapéutica o de tratamiento de la EH. CONCLUSIONES: 1. La derivación porto-cava en ratas es un buen modelo de encefalopatía hepática mínima, que mimetiza un estado de hiperamonemia crónica y disfunción hepática, caracterizado por elevación del amonio en sangre, disminución del peso corporal, variación del perfil de aminoácidos en sangre, incrementándose los aminoácidos aromáticos y disminuyendo los aminoácidos ramificados, así como una elevación de los niveles de óxido nítrico. 2. La actividad GAP está aumentada en riñón y duodeno de ratas sometidas a DPC y este incremento contribuye de manera significativa a la hiperamonemia sistemática. 3. La actividad GAP está incrementada en el córtex y ganglios basales, y correlaciona con la formación de grupos carbonilos. El aumento de la actividad GAP en el astrocito provoca hiperamoniemia y estrés oxidativo, así como oxidación proteica excesiva destacando la oxidación del factor regulador de la hipoxia 3-alfa (HIF-3-α). 4. El tratamiento con IGF-1 evita la pérdida de peso y disminuye la concentración de óxido nítrico en las ratas sometidas a DPC; pero no disminuye la producción de amonio. 5. La inhibición in vitro de la glutaminasa podría ayudar a la elección de una batería de compuestos, en la búsqueda de fármacos inhibidores de la glutaminasa para el tratamiento de la encefalopatía hepática, basada en una aproximación de estructura-actividad