“
“Reliable neuronal communication depends on accurate temporal correlation between the action potential and neurotransmitter release. Although a requirement for Ca2+ in neurotransmitter release is amply documented, recent studies have shown that voltage-sensitive G protein coupled receptors (GPCRs) are also involved in this process. However, how slow-acting GPCRs control fast neurotransmitter release is an unsolved question. Here we examine whether the

recently discovered fast depolarization-induced charge movement in the M-2-muscarinic receptor (M2R) is responsible for M2R-mediated control of acetylcholine release. We show that inhibition of the M2R charge mTOR inhibitor cancer movement in Xenopus oocytes correlated well with inhibition of acetylcholine release at the mouse neuromuscular junction. Our results suggest that, in addition to Ca2+ influx, charge movement in GPCRs is also necessary

for release control.”
“While epithelial cell culture models (e.g., Caco-2 cell line) are widely used to assess the absorption of drug molecules across healthy intestinal mucosa, there are no suitable in vitro models of the intestinal barrier in the state of inflammation. Thus development of novel drugs and formulations for the treatment of inflammatory bowel disease is largely bound to animal models. We here report on the development of a complex in vitro model of the inflamed intestinal mucosa, starting with the selection Selleck Dorsomorphin of suitable enterocyte cell line and proinflammatory stimulus and progressing to the setup and characterization of a three-dimensional coculture of human intestinal epithelial cells and immunocompetent macrophages and dendritic cells. In the 3D setup, controlled inflammation Screening Library concentration can be induced allowing the mimicking of pathophysiological changes occurring in vivo in the inflamed intestine. Different combinations of proinflammatory stimuli (lipopolysaccharides from Escherichia coil and Salmonella

typhimurium, interleukin-1 beta, interferon-gamma) and intestinal epithelial cell lines (Caco-2, HT-29, T84) were evaluated, and only Caco-2 cells were responsive to stimulation, with interleukin-1 beta being the strongest stimulator. Caco-2 cells responded to the proinflammatory stimulus with a moderate upregulation of proinflammatory markers and a slight, but significant, decrease (20%) of transepithelial electrical resistance (TEER) indicating changes in the epithelial barrier properties. Setting up the coculture model, macrophages and dendritic cells derived from periphery blood monocytes were embedded in a collagen layer on a Transwell filter insert and Caco-2 cells were seeded atop. Even in the presence of immunocompetent cells Caco-2 cells formed a tight monolayer. Addition of IL-1 beta increased inflammatory cytokine response more strongly compared to Caco-2 single culture and stimulated immunocompetent cells proved to be highly active in sampling apically applied nanoparticles.