Journal: Cell Communication and Signaling : CCS

Article Title: Downregulation of connexin 43-based gap junctions underlies propofol-induced excessive relaxation in hypertensive vascular smooth muscle cells

doi: 10.1186/s12964-023-01176-3

Figure Lengend Snippet: Propofol attenuated Ca 2+ transfer between neighboring HUASMCs by inhibiting the function of Cx43-GJs. A , B Western blot ( A ) and cell immunofluorescence ( B ) were used to test Cx43 expression. Cx43 protein in ( B ) was stained in green and cell nuclei were stained in blue. GAPDH served as a loading control. n = 3 per group. C “Parachute” dye coupling assay was applied to measure the function of GJs. In merged images, donor cells were stained in yellow by Calcein (green) and Dil (red); receiver cells were stained in green with Calcein. Each dot represents the ratio of “recipient cells” (green)/ “donor cells” (yellow) in one field. Three different fields were captured in every experiment. The experiment was repeated 3 times ( n = 3 per group). D Detection of [Ca 2+ ] i with fluo3-AM by flow cytometry on HUASMCs. * p < 0.05, ** p < 0.01, *** p < 0.001. All values expressed as mean ± SEM

Article Snippet: In addition, cells were treated with an intracellular calcium chelator (BAPTA-AM, 10 μM, APExBIO, Houston, USA), the specific Cx43 gap junction agonist retinoic acid (RA, 1 μM, MedChemExpress, New Jersey, USA) [ ], a specific Cx43 gap junction blocker (Gap26, 0.25 mg/ml, APExBIO, Houston, USA), and a selective RhoA inhibitor (50 μM, Rhosin hydrochloride, APExBIO).

Techniques: Western Blot, Immunofluorescence, Expressing, Staining, Flow Cytometry

Journal: Cell Communication and Signaling : CCS

Article Title: Downregulation of connexin 43-based gap junctions underlies propofol-induced excessive relaxation in hypertensive vascular smooth muscle cells

doi: 10.1186/s12964-023-01176-3

Figure Lengend Snippet: Enhancing Cx43-GJs function with retinoic acid reversed propofol-induced excessive relaxation in Ang II-pretreated HUASMCs. A F-actin was stained with FITC-phalloidin (green) and cell nuclei was stained with DAPI (blue) in HUASMCs. B p-MLC2 and MLC2 were labeled by cellular immunofluorescence (red) and cell nuclei were stained by DAPI (blue) in HUASMCs. C Bar plots showed the statistical results of F-actin level, p-MLC2 level and MLC2 level. Each dot represents the fluorescence intensity of one field. Three different fields were captured in every experiment. The experiment was repeated 3–5 times ( n = 3–5 per group). ** p < 0.01, *** p < 0.001. All values expressed as mean ± SEM

Article Snippet: In addition, cells were treated with an intracellular calcium chelator (BAPTA-AM, 10 μM, APExBIO, Houston, USA), the specific Cx43 gap junction agonist retinoic acid (RA, 1 μM, MedChemExpress, New Jersey, USA) [ ], a specific Cx43 gap junction blocker (Gap26, 0.25 mg/ml, APExBIO, Houston, USA), and a selective RhoA inhibitor (50 μM, Rhosin hydrochloride, APExBIO).

Techniques: Staining, Labeling, Immunofluorescence, Fluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: Downregulation of connexin 43-based gap junctions underlies propofol-induced excessive relaxation in hypertensive vascular smooth muscle cells

doi: 10.1186/s12964-023-01176-3

Figure Lengend Snippet: Gap26 and Cx43-siRNA significantly inhibited Cx43-GJs function in Ang II-pretreated HUASMCs. A Cell immunofluorescence was used to test Cx43 expression. Cx43 protein in was stained in green and cell nuclei were stained in blue. n = 3–5 per group. B , C “Parachute” dye coupling assay was applied to measure the function of GJs. In merged images, donor cells were stained in yellow by Calcein (green) and Dil (red); receiver cells were stained in green with Calcein. Each dot represents the ratio of “recipient cells” (green)/ “donor cells” (yellow) in one field. Three different fields were captured in every experiment. The experiment was repeated 3 times ( n = 3 per group). * p < 0.05, *** p < 0.001. All values expressed as mean ± SEM

Article Snippet: In addition, cells were treated with an intracellular calcium chelator (BAPTA-AM, 10 μM, APExBIO, Houston, USA), the specific Cx43 gap junction agonist retinoic acid (RA, 1 μM, MedChemExpress, New Jersey, USA) [ ], a specific Cx43 gap junction blocker (Gap26, 0.25 mg/ml, APExBIO, Houston, USA), and a selective RhoA inhibitor (50 μM, Rhosin hydrochloride, APExBIO).

Techniques: Immunofluorescence, Expressing, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Downregulation of connexin 43-based gap junctions underlies propofol-induced excessive relaxation in hypertensive vascular smooth muscle cells

doi: 10.1186/s12964-023-01176-3

Figure Lengend Snippet: Inhibition of Cx43-GJs function attenuated Ang II-increased [Ca 2+ ] i , F-actin polymerization and MLC2 phosphorylation. A Detection of [Ca 2+ ] i with fluo3-AM by flow cytometry on HUASMCs. B F-actin was stained with FITC-phalloidin (green) and cell nuclei was stained with DAPI (blue) in HUASMCs. Each dot represents the fluorescence intensity of one field. Three different fields were captured in every experiment. The experiment was repeated 3 times ( n = 3 per group). C p-MLC2 and MLC2 were labeled by cellular immunofluorescence (red) and cell nuclei were stained by DAPI (blue) in HUASMCs. Each dot represents the fluorescence intensity of one field. Three different fields were captured in every experiment. The experiment was repeated 3–4 times ( n = 3–4 per group). D Quantitation of fluorescence intensity ( B , C ) in each group. * p < 0.05, ** p < 0.01, *** p < 0.001. All values expressed as mean ± SEM

Article Snippet: In addition, cells were treated with an intracellular calcium chelator (BAPTA-AM, 10 μM, APExBIO, Houston, USA), the specific Cx43 gap junction agonist retinoic acid (RA, 1 μM, MedChemExpress, New Jersey, USA) [ ], a specific Cx43 gap junction blocker (Gap26, 0.25 mg/ml, APExBIO, Houston, USA), and a selective RhoA inhibitor (50 μM, Rhosin hydrochloride, APExBIO).

Techniques: Inhibition, Flow Cytometry, Staining, Fluorescence, Labeling, Immunofluorescence, Quantitation Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Downregulation of connexin 43-based gap junctions underlies propofol-induced excessive relaxation in hypertensive vascular smooth muscle cells

doi: 10.1186/s12964-023-01176-3

Figure Lengend Snippet: Propofol inhibited the activation of RhoA/LIMK2/cofilin and RhoA/MLCK signaling pathways. A Levels of P-LIMK2, LIMK2, P-cofilin and cofilin on HUASMCs were tested by western blot. GAPDH served as a loading control. n = 4 per group. B Levels of MLCK on HUASMCs were tested by western blot. GAPDH served as a loading control. n = 3 per group. C Levels of p-LIMK2, LIMK2, p-cofilin and cofilin on HUASMCs were tested by western blot. GAPDH served as a loading control. n = 3 per group. D Levels of MLCK on HUASMCs were tested by western blot. GAPDH served as a loading control. n = 3 per group. * p < 0.05, ** p < 0.01, *** p < 0.001. All values expressed as mean ± SEM. E The scheme of the mechanism that propofol induces excessive relaxation in Ang II-HUASMCs. (1) Comparison of the number and function of Cx43-GJs and [Ca 2+ ] i in normal and Ang II-HUASMCs. (2) ① When HUASMCs are treated with propofol, propofol causes a prominent decrease in the function of Cx43-GJs in Ang II-HUASMCs compared with normal HUASMCs; ② This results in a more substantial decrease of intracellular Ca 2+ mobility in Ang II-HUASMCs; ③-④ Subsequently, propofol also leads to a stronger inhibition on the activation of RhoA protein and its downstream- RhoA/LIMK2/cofilin and RhoA/MLCK signaling pathways in Ang II-HUASMCs; ⑤ Ultimately, compared to normal HUASMCs, the activation of F-actin polymerization and MLC2 phosphorylation were both mitigated more dramatically in Ang II-HUASMCs, which caused excessive relaxation

Article Snippet: In addition, cells were treated with an intracellular calcium chelator (BAPTA-AM, 10 μM, APExBIO, Houston, USA), the specific Cx43 gap junction agonist retinoic acid (RA, 1 μM, MedChemExpress, New Jersey, USA) [ ], a specific Cx43 gap junction blocker (Gap26, 0.25 mg/ml, APExBIO, Houston, USA), and a selective RhoA inhibitor (50 μM, Rhosin hydrochloride, APExBIO).

Techniques: Activation Assay, Western Blot, Inhibition

Journal: Frontiers in Molecular Neuroscience

Article Title: NF-κB and AP-1 are required for the lipopolysaccharide-induced expression of MCP-1, CXCL1, and Cx43 in cultured rat dorsal spinal cord astrocytes

doi: 10.3389/fnmol.2022.859558

Figure Lengend Snippet: The treatment with TAK-242, CBX, Gap26 or Gap19 inhibited astrocyte hemichannel activity and the release of MCP-1 and CXCL1 from cultured astrocytes. (A) Representative photomicrographs showed EtBr uptake in astrocytes after LPS treatment and the inhibition promoted by TAK-242, CBX, Gap26, or Gap19. Scale bar: 100 μm. (B) Astrocytes were incubated in the presence of LY before and after LPS application. The fluorescence diffusion distance of LY is representative as the gap junction function in astrocytes. Scale bar: 200 μm. (C) Quantification of EtBr uptake intensity in different groups (at least 50 cells were counted per condition). * p < 0.05 vs. Veh-treated group, + p < 0.05 vs. Veh + LPS-treated group. (D) The histogram showed an analysis of the distance between the dye transfer front and the scrape line of each group. * p < 0.05 vs. Veh-treated group, + p < 0.05 vs. Veh + LPS-treated group, n = 5 per group. (E) The effect of LPS-induced MCP-1 release was suppressed by TAK-242, CBX, Gap26, or Gap19. * p < 0.05 vs. Veh-treated group, + p < 0.05 vs. Veh + LPS-treated group, n = 4 per group. (F) The effect of LPS-induced CXCL1 release was suppressed by TAK-242, CBX, Gap26 or Gap19. * p < 0.05 vs. Veh-treated group, + p < 0.05 vs. Veh + LPS-treated group, all data were expressed as mean ± standard deviation of at least four independent experiments ( n = 4 per group).

Article Snippet: Gap26 (a specific gap junction and hemichannel blocker corresponding to residues 63–75 of Cx43; AS-62644) ( ) was purchased from AnaSpec.

Techniques: Activity Assay, Cell Culture, Inhibition, Incubation, Fluorescence, Diffusion-based Assay, Standard Deviation