(2010) Extracellular 2,3-cyclic adenosine 5-monophosphate is a potent inhibitor of preglomerular vascular easy muscle cell and mesangial cell growth. crosstalk pathway between ENT2 and alveolar epithelial Adora2b in lung protection during ALI opens possibilities for combined therapies targeted to this protein set.Eckle, T., Hughes, K., Ehrentraut, H., Brodsky, K. S., Rosenberger, P., Choi, D.-S., Ravid, K., Weng, T., Xia, Y., Blackburn, M. R., Eltzschig, H. K. Crosstalk between the equilibrative nucleoside transporter ENT2 and alveolar Adora2b adenosine receptors dampens acute lung injury. or (mice with the GermlineCre mouse. To obtain Spc-Cre (alveolar epithelial specific) or VeCadherinCre (endothelial specific) mice, we bred mice with the Sftpc-Cre or VeCadherinCre mouse, respectively. for 5 min at 4C. Pulmonary tissue was flushed with 10 ml of saline the right ventricle, snap frozen in liquid nitrogen, and IL5R stored at ?80C (10, 14). RNA isolation and real-time polymerase chain reaction (PCR) Total RNA was extracted from tissue by TRIzol, followed by cDNA synthesis using an iScript cDNA Brompheniramine Synthesis Kit (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. Quantitative reverse transcriptase (RT)-PCR (ABI 7900HT; Applied Biosystems, Carlsbad, CA, USA) was performed to measure relative mRNA levels for various Brompheniramine transcripts, with Power SYBR Green PCR Grasp Mix (Applied Biosystems) made up of 1 M sense and 1 M antisense primers. Primers for real-time RT-PCR Real-time RT-PCR was performed with murine QuantiTect Primer Assay for -actin (QT01136772; Qiagen, Valencia, CA, USA) or the following primers (Invitrogen, Carlsbad, CA, USA): mouse ENT1 sense CTTGGGATTCAGGGTCAGAA or antisense ATCAGGTCACACGACACCAA; mouse ENT2 sense CATGGAAACTGAGGGGAAGA or antisense GTTCCAAAGGCCTCACAGAG; human ENT1 sense CTTGGGCTTGGAGAACAC or antisense AAGGCACCTGGTTTCTGTC; and human ENT2 sense CTTCCATACCCACTCTCTCACC or antisense GAGAGAGAGGGGATTGGGTC. Immunoblotting and immunohistochemistry experiments Rabbit polyclonal anti-ENT1 antibody (ab48607; Abcam Inc., Cambridge, MA, USA) and rabbit polyclonal anti-ENT2 antibody (ab48595; Abcam) were used to determine Ent1 or Ent2 protein content from whole lungs. For this purpose, we ventilated C57BL/6J mice (Charles River Laboratories, Wilmington, MA, USA) with the ventilator settings indicated in the physique legends. Animals were euthanized, and the remaining blood was removed from the pulmonary circulation by injection of 1 1 ml of phosphate-buffered saline (PBS) into the right ventricle. The lungs were excised and immediately frozen at ?80C until immunoblotting. For this purpose, tissues were homogenized and lysed for 10 min in ice-cold lysis buffer (107 polymorphonuclear neutrophils (PMNs)/500 l; 150 mM NaCl; 25 mM Tris, pH 8.0; 5 mM EDTA; 2% Triton X-100; and 10% mammalian tissue protease inhibitor cocktail; Sigma-Aldrich, St. Louis, MO, USA) and collected in microfuge tubes. After spinning at 14,000 to remove cell debris, the pellet was discarded. Proteins were solubilized in reducing Laemmli sample buffer and heated to 90C for 5 min. Samples were resolved on a 12% polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were blocked for 1 h at room temperature in PBS supplemented with 0.2% Tween 20 and 4% bovine serum albumin (BSA). The membranes were incubated in 10 g/ml Ent1 or Ent2 antibody for 1 h at room temperature, followed by 10-min washes in PBS. The membranes were then incubated in 1:3000 donkey anti-rabbit horseradish peroxidase. The wash was repeated, and proteins were detected by enhanced chemiluminescence. Measurement of BAL fluid albumin content The Brompheniramine albumin content of BAL fluid supernatants was measured with a mouse albumin enzyme-linked immunosorbent assay (ELISA) quantitation set according to the manufacturer’s instructions (Bethyl Laboratories, Montgomery, TX, USA). Samples were diluted 1:10,000 (10, 14). Blood gas analysis To assess pulmonary gas exchange, blood gas analyses were performed in subsets of experiments by obtaining arterial blood cardiac puncture. In short, a lateral thoracotomy was performed to access the left ventricle, and blood was obtained cardiac puncture. Analysis was performed immediately after collection with an i-Stat Analyzer (Abbott Laboratories, Abbott Park, IL, USA), and the arterial partial pressure of oxygen was measured, in addition to arterial partial carbon dioxide pressure and pH values (10, 14). Measurement of adenosine concentrations in BAL fluid Nucleosides were measured as described previously (44). In brief, to measure the nucleoside levels in BAL fluid, mice were anesthetized with 2.5% Avertin, and the lungs were lavaged 4 times with 0.3 ml of PBS containing 10 M dipyridamole (Sigma-Aldrich), 10 M deoxycoformycin (adenosine deaminase inhibitor; R&D Systems Inc., Minneapolis, MN, USA), and 10 M.
(2010) Extracellular 2,3-cyclic adenosine 5-monophosphate is a potent inhibitor of preglomerular vascular easy muscle cell and mesangial cell growth