KCC2 membrane diffusion tunes neuronal chloride homeostasis.

Neuronal Cl- homeostasis is regulated by the activity of two cation chloride co-transporters (CCCs), the K+-Cl- cotransporter KCC2 and the Na+-K+-Cl- cotransporter NKCC1, which are primarily extruding and importing chloride in neurons, respectively. Several neurological and psychiatric disorders including epilepsy, neuropathic pain, schizophrenia and autism are associated with altered neuronal chloride (Cl-) homeostasis. A current view is that the accumulation of intracellular Cl- in neurons as a result of KCC2 down-regulation and/or NKCC1 up-regulation may weaken inhibitory GABA signaling and thereby promote the development of pathological activities. CCC activity is determined mainly by their level of expression in the plasma membrane. Furthermore, CCCs undergo "diffusion-trapping" in the membrane, a mechanism that is rapidly adjusted by activity-dependent post-translational modifications i.e. phosphorylation/dephosphorylation of key serine and threonine residues. This represents probably the most rapid cellular mechanism for adapting CCC function to changes in neuronal activity. Therefore, interfering with these mechanisms may help restoring Cl- homeostasis and inhibition under pathological conditions. This article is part of the special issue entitled 'Mobility and trafficking of neuronal membrane proteins'.

A High-Throughput Screening Assay for NKCC1 Cotransporter Using Nonradioactive Rubidium Flux Technology.

A high-throughput screening (HTS) assay was developed for cotransporter, NKCC1, which is a potential target for the treatment of diverse disorders. This nonradioactive rubidium flux assay coupled with ion channel reader series provides a working screen for this target expressed in human embryonic kidney (HEK) cell line. An eightfold window of detection was achieved with the optimized assay. This new functional assay offered a robust working model for NKCC1 in determining reliable and concordant rank orders of the test compounds supporting its sensitivity and specificity. The robustness of manual assay indicated by Z' of 0.9 qualified its amenability to automation. The Z' of 0.7 was displayed by automated assay employed in high-throughput screening of compound libraries against this target. Being electrically neutral, the NKCC1 screening is difficult to achieve by both manual and automated electrophysiological techniques. These techniques, although considered gold standard, suffer from their inherent problems of being too slow to be in high-throughput format and with high running costs. In addition to being a functional assay for NKCC1, it is nontoxic as compared with thallium flux assay, which is prone to generate high number of false-positive/false-negative rates because of its innate fluorescence issues.

Peso y diabetes tipo 2: nuevas recomendaciones / Weight and type 2 diabetes: new recommendations

La gran mayoría de los pacientes con diabetes tipo 2 presenta un exceso de adiposidad. Existe un amplio consenso en que el adecuado tratamiento de la diabetes tipo 2 requiere un abordaje simultáneo del sobrepeso/ obesidad y del resto de los factores de riesgo cardiovascular. Las intervenciones no farmacológicas (dieta, ejercicio) representan la piedra angular del tratamiento del paciente con diabetes tipo 2. La pérdida de peso a través de la modificación de los estilos de vida ha demostrado claros beneficios en el paciente con diabetes tipo 2, requiriendo un enfoque individualizado y multidisciplinario, con programas estructurados dotados de recursos específicos. La ganancia de peso asociada al tratamiento con algunos fármacos antidiabéticos (secretagogos, glitazonas, insulina) puede dificultar el control glucémico, comprometer la adherencia al tratamiento, empeorar el perfil de riesgo vascular de los pacientes y limitar los beneficios del tratamiento. Por ello, la tendencia actual es abordar el tratamiento de la diabetes tipo 2 desde un punto de vista adipocéntrico, dando prioridad a los fármacos antidiabéticos que presenten un efecto ponderal neutro o que favorezcan la pérdida de peso (metformina, terapias incretínicas, inhibidores del cotransportador sodio-glucosa tipo 2). La cirugía metabólica representa una alternativa eficaz para los pacientes con diabetes tipo 2 y un índice de masa corporal ≥ 35 kg/m2 y permite la remisión de la diabetes en una alta proporción de casos, especialmente si la enfermedad no está muy evolucionada. Recientemente se ha publicado un consenso suscrito por diversas sociedades científicas españolas que realiza una serie de recomendaciones concretas sobre la actuación diagnóstica y terapéutica ante el paciente con diabetes y obesidad (AU)

Most patients with type 2 diabetes have excess adiposity. There is wide consensus that adequate treatment of type 2 diabetes requires a simultaneous approach to overweight/obesity and the remaining cardiovascular risk factors. Non-pharmacological interventions (diet, exercise) represent the cornerstone of the treatment of patients with type 2 diabetes. Weight loss through lifestyle modification has shown clear benefits in these patients, requiring an individualised and multidisciplinary approach with structured programmes endowed with specific resources. The weight gain associated with some antidiabetic drugs (secretagogues, glitazones, insulin) can hamper glycaemic control, compromising treatment adherence, worsening vascular risk profile, and limiting the benefits of treatment. Therefore, the current tendency is to adopt a weight-centred approach to the treatment of type 2 diabetes, giving priority to those antidiabetic drugs that have a neutral effect on weight or that favour weight loss (metformin, incretin therapies, sodium-glucose cotransporter-2 inhibitors). Metabolic surgery is an effective alternative for patients with type 2 diabetes and a BMI ≥35 kg/m2 and allows remission of diabetes in a large proportion of patients, especially if the disease is not very advanced. A consensus document supported by various Spanish scientific societies has recently been published. This document makes a series of specific recommendations on the diagnostic and therapeutic approach to patients with diabetes and obesity (AU)

Establishment of functional acinar-like cultures from human salivary glands.

Disorders of human salivary glands resulting from therapeutic radiation treatment for head and neck cancers or from the autoimmune disease Sjögren syndrome (SS) frequently result in the reduction or complete loss of saliva secretion. Such irreversible dysfunction of the salivary glands is due to the impairment of acinar cells, the major glandular cells of protein, salt secretion, and fluid movement. Availability of primary epithelial cells from human salivary gland tissue is critical for studying the underlying mechanisms of these irreversible disorders. We applied 2 culture system techniques on human minor salivary gland epithelial cells (phmSG) and optimized the growth conditions to achieve the maintenance of phmSG in an acinar-like phenotype. These phmSG cells exhibited progenitor cell markers (keratin 5 and nanog) as well as acinar-specific markers-namely, α-amylase, cystatin C, TMEM16A, and NKCC1. Importantly, with an increase of the calcium concentration in the growth medium, these phmSG cells were further promoted to acinar-like cells in vitro, as indicated by an increase in AQP5 expression. In addition, these phmSG cells also demonstrated functional calcium mobilization, formation of epithelial monolayer with high transepithelial electrical resistance (TER), and polarized secretion of α-amylase secretion after ß-adrenergic receptor stimulation. Taken together, suitable growth conditions have been established to isolate and support culture of acinar-like cells from the human salivary gland. These primary epithelial cells can be useful for study of molecular mechanisms involved in regulating the function of acinar cells and in the loss of salivary gland function in patients.