Journal: Cells

Article Title: Glial A 2B Adenosine Receptors Modulate Abnormal Tachykininergic Responses and Prevent Enteric Inflammation Associated with High Fat Diet-Induced Obesity

doi: 10.3390/cells9051245

Figure Lengend Snippet: Schematic representation of the design of experiments on cultured enteric glial cells (EGCs). EGCs were treated for three days with palmitate (PA, 400 μM). Then, cells were incubated for 2 h with lipopolysaccharide (LPS, 10 μg/mL) before the addition of adenosine deaminase (ADA, 3 U/mL), to minimize interferences by extracellular adenosine. Under these conditions, cells were treated for 19 h with MRS1754 (0.1 µM, A 2B R antagonist) or incubated for 1 h with MRS1754 before the addition of BAY60-6583 (0.05 µM, selective A 2B R agonist, 18 h). For the experiments with the inhibitor of glial cell-derived neurotrophic factor (GDNF) receptor signaling, cells were treated with RPI-1 (100 µM, 1,3-dihydro-5,6-dimethoxy-3-[(4-hydroxyphenyl)methylene]-H-indol-2-one) for 19 h. On the fourth day, cells were lysed for analysis of toll-like receptor 4 (TLR4) expression and the culture media were collected for analysis of substance P (SP), GDNF, and interleukin (IL)-1β release.

Article Snippet: The LMMP preparations were then incubated overnight at room temperature with guinea pig polyclonal anti-rat S100β (1:100; Synaptic Systems, Göttingen, Germany), rabbit polyclonal anti-human A 2B R (1:50; Merck Life Science, Milan, Italy), and mouse biotin-conjugated anti-human-HuC/D (1:100; Thermo Fisher Scientific, Milan, Italy) primary antibodies.

Techniques: Cell Culture, Incubation, Derivative Assay, Expressing

Journal: Cells

Article Title: Glial A 2B Adenosine Receptors Modulate Abnormal Tachykininergic Responses and Prevent Enteric Inflammation Associated with High Fat Diet-Induced Obesity

doi: 10.3390/cells9051245

Figure Lengend Snippet: ( A ) Representative confocal microphotographs showing the distribution of the neural marker HuC/D (yellow), the glial protein S100β (green), and A 2B Rs (magenta) in colonic longitudinal smooth muscle with attached myenteric plexus (LMMP) whole mount preparations from mice fed with standard diet (SD) or high-fat diet (HFD), in the absence or presence of 50 μM fluorocitrate (FC). White arrowheads indicate HuC/D + and A 2B R + neurons, while white stars indicate S100β + and A 2B R + glial cells. Scale bars = 22 μm; analysis of A 2B R ( B ) and S100B ( C ) density index in colonic LMMP whole mount preparations from mice fed with SD or HFD, in the absence or presence of 50 μM FC. * p < 0.05, ** p < 0.01 versus SD without FC; aa p < 0.01 versus HFD; n = 5 mice per group.

Article Snippet: The LMMP preparations were then incubated overnight at room temperature with guinea pig polyclonal anti-rat S100β (1:100; Synaptic Systems, Göttingen, Germany), rabbit polyclonal anti-human A 2B R (1:50; Merck Life Science, Milan, Italy), and mouse biotin-conjugated anti-human-HuC/D (1:100; Thermo Fisher Scientific, Milan, Italy) primary antibodies.

Techniques: Marker

Journal: Cells

Article Title: Glial A 2B Adenosine Receptors Modulate Abnormal Tachykininergic Responses and Prevent Enteric Inflammation Associated with High Fat Diet-Induced Obesity

doi: 10.3390/cells9051245

Figure Lengend Snippet: Schematic representation of the proposed role of A 2B Rs in the modulation of enteric glial cell (EGC) activity under experimental conditions mimicking obesity. In vitro incubation of EGCs with palmitate (PA, 400 μM) and lipopolysaccharide (LPS, 10 μg/mL) elicited an increase in TLR4 expression as well as substance P (SP), glial cell-derived neurotrophic factor (GDNF), and interleukin (IL)-1β release, suggesting an involvement of these cells in supporting the enteric inflammation and abnormal tachykininergic enteric motor responses associated with obesity. The pharmacological stimulation of A 2B Rs on EGCs, besides reducing TLR4 expression and IL-1β levels, can also counteract the abnormal increase in GDNF and SP release. A 2B R: adenosine A 2B receptor; AC: adenylyl cyclase; ATP: adenosine triphosphate; cAMP: cyclic adenosine monophosphate; NF-ҡB: nuclear factor kappa-light-chain-enhancer of activated B cells.

Article Snippet: The LMMP preparations were then incubated overnight at room temperature with guinea pig polyclonal anti-rat S100β (1:100; Synaptic Systems, Göttingen, Germany), rabbit polyclonal anti-human A 2B R (1:50; Merck Life Science, Milan, Italy), and mouse biotin-conjugated anti-human-HuC/D (1:100; Thermo Fisher Scientific, Milan, Italy) primary antibodies.

Techniques: Activity Assay, In Vitro, Incubation, Expressing, Derivative Assay