Journal: PLoS ONE

Article Title: Nucleolin Down-Regulation Is Involved in ADP-Induced Cell Cycle Arrest in S Phase and Cell Apoptosis in Vascular Endothelial Cells

doi: 10.1371/journal.pone.0110101

Figure Lengend Snippet: (A, B) The mRNA levels of P2Y1, 12, and 13 receptors in HUVEC (A) and HAEC (B). Cells were treated with the indicated concentrations of ADP for 24 h. The mRNA levels of P2Y1, 12 and 13 receptors were detected by RT-PCR. β-actin transcript was detected as a loading control. (C) The mRNA levels of P2Y1, P2Y12, and P2Y13 receptors in 5–8F cells. (D) The effect of P2Y1 receptor inhibitor MRS2179 (M2179) on ADP-induced down-regulation of nucleolin protein. HUVEC, pre-treated with the indicated concentrations of MRS2179 for 1 h, were re-treated with 100 µM ADP for 72 h. The nucleolin protein levels were detected by western blot. β-actin transcript was detected as a loading control. (E) The effect of P2Y12 receptor inhibitor PSB0739 (PSB) on ADP-induced down-regulation of nucleolin protein. (F) The effect of P2Y13 receptor antagonist MRS2211 (MRS) on ADP-induced down-regulation of nucleolin. (G, H) The effect of MRS2211 on the nucleolin protein levels in HUVEC (G) and HAEC (H). (I, J) The effect of P2Y13 receptor agonist 2-MeSADP on nucleolin protein levels in HUVEC (I) and HAEC (J). (K) 2-MeSADP did not regulate over-expressed nucleolin protein. Nucleolin-over-expressed HUVEC were treated with the indicated concentrations of ADP for 72 h. The GFP-nucleolin expression was detected by fluorescence microscope.

Article Snippet: P2Y1, 12, 13 agonist 2-MeSADP, P2Y1 selective inhibitor MRS2179, P2Y12 potential inhibitor PSB0739, P2Y13 competitive inhibitor MRS2211 were purchased from Tocris (Bristol, UK).

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Expressing, Fluorescence, Microscopy

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Adenosine A1and Prostaglandin E Receptor 3 Receptors Mediate Global Airway Contraction after Local Epithelial Injury

doi: 10.1165/rcmb.2012-0174oc

Figure Lengend Snippet: Figure 1. Adenosine released from a wounded single epithelial cell activates A1 receptors to induce airway contraction. (A) Bright-field images of a small airway embedded in a lung tissue slice before and z40 seconds after laser ablation demonstrate that damage of a single epithelial cell induces global airway contraction. Blue arrow points to the ablated epithelial cell, the apical membrane of which was ruptured by a pulsed femtosecond laser. Red dashed line outlines the lumen cross- sectional area before laser ablation. (B) Inhibition of A1 receptors with 50 mM PSB36 blocked local epithelial injury–induced airway contraction. (C) Local epithelial injury induced airway contraction, which leads to a roughly 30% reduction of the original cross-sectional area in control air- ways (n ¼ 33 airways from 12 rats). Statistical tests demonstrate that inhibition of adenosine receptors with 2 mM 9-chloro-2-(2-furanyl)- [1,2,4]triazolo[1,5-c]quinazolin-5-amine (CGS15943) (CGS, a nonselec- tive adenosine receptor antagonist; n ¼ 7 airways from 5 rats), inhibition of adenosine A1 receptors with 5–50 mM PSB36 (50 mM for 15 min incubation time, or 5 mM for 60 min; n ¼ 5 airways from 3 rats for 50 mM PSB36 treatment) or 4 mM SLV320 (n ¼ 9 airways from 4 rats), or deletion of adenosine with 5 units/ml adenosine deaminase (ADA; n ¼ 4 airways from 3 rats) significantly blocked the airway contraction induced by local epithelial injury, whereas inhibition of adenosine A2A receptors with 5 mM ZM241385 (n ¼ 4 airways from 2 rats), A2B receptors with 10 mM PSB603 (n ¼ 6 airways from 5 rats), or A3 receptors with 5 mM MRS1334 (n ¼ 8 airways from 6 rats) did not. (D) Statistical tests demonstrate that CGS15943 significantly blocked airway con- traction induced by 10 mM adenosine (ADO, a nonselective adenosine receptor agonist; n ¼ 4 airways from 4 rats for N6- cyclopentyladenosine [CPA] and n ¼ 4 airways from 4 rats for CPA 1 CGS treatment) or 10 mM ATP (n ¼ 5 airways from 5 rats for ATP and n ¼ 4 airways from 4 rats for ATP 1 CGS), but not 10 mM ATP-g-S (n ¼ 6 airways from 4 rats for ATP-g-S and n ¼ 5 airways from 4 rats for ATP-g-S 1 CGS). As a positive control, 25 mM KCl or 1 mM acetylcholine was used to verify the viability and contractility of airway smooth muscle cells after laser ablation experiments for each treatment. The ratio of lumen area was defined as the minimum cross- sectional area of airways after treatment divided by initial cross- sectional area. Statistical tests of significance of the ratio of lumen area between control airways and each treatment condition were per- formed with one-way ANOVA, and there are similar numbers of control airways for each treatment condition. *P , 0.05 was considered statis- tically significant.

Article Snippet: Adenosine, 9-chloro-2-(2-furanyl)-[1,2,4] triazolo[1,5-c]quinazolin-5-amine (CGS15943), 1-butyl-8-(hexahydro2,5-methanopentalen-3a(1H)-yl)-3,7-dihydro-3-(3-hydroxypropyl)-1Hpurine-2,6-dione (PSB36), 8-[4-[4-(4-chlorophenzyl)piperazide-1-sulfonyl) phenyl]]-1-propylxanthine (PSB603), SLV320, ZM241385, and MRS1334 were purchased from Tocris Bioscience (Ellisville, MO).

Techniques: Membrane, Inhibition, Control, Incubation, Positive Control