6 bnz camp

Endothelial cells (1.0 × 10⁵/cm²) were cultured on polycarbonate cell culture inserts (pore size 0.4 µm, porosity 0.9·10⁸/cm²; Nunc, ThermoFisher, Waltham, CA) coated with 0.1% gelatin for 48 h. When appropriate, cultures were serum starved for 24 h and cells were treated with fenoterol (1 µM; Boehringer Ingelheim, Germany), forskolin (10 µM; Tocris, UK), 6-Bnz-cAMP (300 µM) or 8-pCPT-2′-O-Me-cAMP (100 µM) (Biolog Life Science Institute, Germany). Parallel cultures were maintained under normoxic (21% oxygen tension) and hypoxic conditions (2% oxygen tension) for 48 h prior to experiments. Permeability was assessed by the addition of 10 µg/ml FITC-dextran in the upper compartments, and fluorescence in the lower compartments was assessed on a spectrofluorescence reader at Ex485/Em519 after 30 min.

RPMI 1640 culture medium and penicillin/streptomycin/amphotericin B solution were purchased from Invitrogen (Carlsbad, CA). PGE₂ and misoprostol were purchased from Cayman Chemical (Ann Arbor, MI); DMSO served as vehicle control. The protein kinase A (PKA)-specific cAMP analog 6-Bnz-cAMP was purchased from Biolog (Bremen, Germany). LPS was purchased from Sigma-Aldrich (St. Louis, MO).

H89, NKH 477, 8-Br-cAMP, and 8-Br-cGMP were from R&D Systems (Abingdon, Oxford, UK). Sp-cAMPS, 6-Bnz-cAMP, 8-pCPT-2′-O-Me-cAMP, Rp-cAMPS, Rp-8-CPT-cAMPS, ESI-09, and HJC0197 were from Biolog (Bremen, Germany). Ionomycin, SQ 22536, DDA and myristoylated-PKA inhibitor peptide (PKI) were from Merck-Millipore (Watford, UK). A membrane-permeant peptide inhibitor of A kinase-anchoring proteins (AKAPs) [stearated Ht31 AKAP inhibitor peptide (st-Ht31)] and its proline-modified inactive form (st-Ht31P) were from Promega (Southampton, UK). Thapsigargin was from Alomone Laboratories (Jerusalem, Israel). PAR1 peptide, histamine dihydrochloride, forskolin, IBMX, and PGE₂ were from Sigma-Aldrich (Welwyn Garden City, UK). Butaprost (free acid) and L902,688 were from Cayman Chemicals (Ann Arbor, MI). Membrane-permeant caged IP₃ (ci-IP₃PM) was from SiChem (Bremen, Germany). [2,8-³H] adenine ci-IP₃PM, d-2,3-O-isopropylidene-6-O-(2-nitro-4,5-dimethoxy)benzyl-myo-inositol 1,4,5-trisphosphate-hexakis(propionoxymethyl) ester was from Perkin Elmer (Seer Green, Bucks, UK). Fluo-8 was from Stratech Scientific Ltd (Newmarket, Suffolk, UK). Other reagents were from Sigma-Aldrich, sources specified previously (Pantazaka et al., 2013 (link)) or identified in this section.