Moreover, it was shown that an adenosine receptor\related increase in cAMP enhanced the membrane current and intracellular Ca2+ concentration in endothelial cells and that these effects were both blocked by CNG channel inhibitors (Cheng em et?al /em . RT\PCR. Scrape loading/dye transfer was used to evaluate the impact of the A2A and A2B adenosine receptor subtype agonist 7-Epi 10-Desacetyl Paclitaxel 2\phenylaminoadenosine (2\PAA) on the gap junction coupling. We found that 2\PAA stimulated cAMP synthesis and enhanced gap junction coupling in a concentration\dependent manner. This enhancement was accompanied by an increase Rabbit Polyclonal to Cytochrome P450 4F2 in gap junction plaques formed by Cx43. Inhibition of protein kinase A did not affect the 2\PAA\related enhancement of gap junction coupling. In contrast, the cyclic nucleotide\gated (CNG) channel inhibitor l\model for BBB endothelial cells (Weksler +?(represents the relative dye diffusion distance measured at the time point 0?h and represents the asymptotic value of 7-Epi 10-Desacetyl Paclitaxel the dye diffusion distance that would be achieved by 2\PAA treatment for an infinite time. From the asymptote at 4 C. The cell pellet was resuspended in 15?l RIPA buffer (25?mm Tris HCl, pH?7.6, 150?mm NaCl, 1% nonidet P\40, 1% sodium desoxycholate, 0.1% SDS, freshly added 1% phosphatase inhibitor mix II (Serva, Heidelberg, Germany), 0.5% protease inhibitor cocktail (Roche, Waiblingen, Germany), 1.5?mm PMSF) and kept for 15?min on ice before centrifugation for 15?min at 14,000??at 4 C. The protein concentration in the supernatant was determined with a Bradford assay (Sigma\Aldrich) using bovine serum albumin (BSA) as standard. The protein solution was mixed with 1??Laemmli buffer (13?mm Tris HCl, 2% glycerol, 0.4% SDS, 0.002% Bromophenol Blue, 10?mm DTT, pH 6.8) and heated at 70 C for 10?min. Aliquots of 30?g of protein per lane were separated in a 5% SDS\polyacrylamide stacking gel and a 8% or 12% separation gel. The proteins were transferred onto a nitrocellulose membrane using a semi\dry blotting system (transfer buffer: 25?mm Tris HCl, pH?8.3, 192?mm glycine, 0.1% SDS, 20% methanol). Afterwards, the membranes were blocked in 5% non\fat dry 7-Epi 10-Desacetyl Paclitaxel milk powder in TBS (50?mm Tris HCl, 75?mm NaCl, pH 7.4) containing 0.1% Tween?20 (TBS\T) for 2?h at room temperature. Anti\\tubulin antibody for the loading control (Sigma\Aldrich, T4026) was diluted 1:7500, anti\CNGA2 antibody (Alomone Labs, Jerusalem, Israel, APC\045) was diluted 1:750 and anti\Cx37 antibody (Abcam, ab58918) was diluted 1:700 in TBS\T and applied to the membranes at 4 C overnight. After washing, the secondary anti\rabbit and the secondary anti\mouse antibody (each diluted 1:10,000 in TBS\T, Sigma\Aldrich, A9169 and A9044) were each applied for 1?h at room temperature. The detection was carried out with SuperSignal West chemiluminescent substrate (Thermo Fisher Scientific) and imaged with a CCD camera imaging system (Intas Science Imaging, G?ttingen, Germany). The presence of CNGA2 and Cx37 protein was confirmed in at least five different cell passages. Measurement of intracellular cAMP concentration Approximately 4.5??105 hCMEC/D3 cells per well were seeded in a 24 multiwell plate and grown for 48?h until confluent. Measurement of cAMP levels was performed using the cAMP\Screen Chemiluminescent Immunoassay System (Thermo Fisher Scientific) according to the manufacturer’s instructions with slight modifications as described below. 100?l of lysis buffer were added per well to the cells and 7-Epi 10-Desacetyl Paclitaxel incubated for 30?min at 37 C with gentle agitation. 90?l of lysed cell suspension were added to each well of the supplied ELISA 96 multiwell plate. 30?l of the diluted cAMP\AP conjugate and 60?l of the anti\cAMP antibody were added per well, followed by an incubation for 1?h at 37 C with gentle agitation. Afterwards the wells were washed three times with 200?l wash buffer before addition of 100?l chemiluminescent substrate and incubation for 30?min at room temperature. Luminometric measurement was 7-Epi 10-Desacetyl Paclitaxel performed with a Varioskan Flash plate reader (Thermo Fisher Scientific) with a measurement time of 1 1?s per well. Defined cAMP concentrations served as standard. Chemiluminescence values of treated cell samples were normalized to those obtained from vehicle\treated.