Rolipram

Wild type HEK293T-vector and HEK293T–EPAC1 cells were plated at 10,000 cells/well in 30 μl growth media on a corning Epic 384 well cell assay microplate (PerkinElmer), and then incubated overnight at 37 °C. Cells were then equilibrated for 2 h at room temperature and then a baseline measurement was recorded using an Enspire plate reader. The cells were then treated with varying concentrations of the EPAC1 antagonist, ESI-09 (Sigma Aldrich) or the PKA inhibitors, H-89 (Tocris) or KT5720 (Tocris), in the presence or absence of forskolin (Tocris) and rolipram (Tocris) or 007-(8-pCPT-O′-Me-cAMP) (Biolog). Dynamic mass redistribution measurements (DMR) were then taken every minute for 60 min. All treatments were made up in DMSO (Fisher Scientific) and diluted in 1 × HBSS (Life Technologies)/20 mM HEPES (Sigma Aldrich) assay buffer. For data analysis DMR readings from HEK293T-vector cells were subtracted from HEK293T–EPAC1 cells.

cLTD was induced as previously described (Oh et al., 2006 (link)). Cultures were first incubated in ACSF for 30 min at room temperature: 125 mM NaCl, 2.5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 33 mM D-glucose, and 25 mM HEPES (pH 7.3), followed by stimulation with 50 μM NMDA in ACSF (no MgCl₂) for 5 min. After NMDA treatment, neurons were replaced in regular ACSF and then subjected to the corresponding procedure at indicated time points. Cultures were then washed with ice-cold PBS once and lysed in cold 1% NP-40 homogenization buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM PMSF, 1 mM Na₂VO₄, and 1× Sigma protease inhibitor and phosphatase inhibitor cocktails). When indicated, cultures were pre-treated or post-treated with BAPTA-AM (20 μM), FK506 (10 μM), MG132 (10 μM), or MK801 (10 μM; all from Tocris) and were present until cells were lysed. cLTP was induced as previously described (Otmakhov et al., 2004 (link)) by application of forskolin (50 μM) and rolipram (0.1 μM; both from Tocris), 60 min before or after cLTD. Cells were lysed 60 min after cLTP or cLTD treatment. Lysates were centrifuged at 12,000 × g for 10 min at 4°C and protein in the supernatant was quantified by Bradford method assay kit (Bio-Rad Laboratories). The intensity of the cLTD protocol may vary depending on the cultures.

Prostaglandin E1, MDL12330A, cilostamide, rolipram, and erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA) were obtained from Tocris Bioscience. MEM, penicillin/streptomycin, fetal bovine serum were purchased from Life Technologies. All other reagents were purchased from Sigma-Aldrich. Isoproterenol and IBMX solutions were prepared fresh daily.

D-glucose was purchased from Mallinckrodt Chemicals (Dublin, Ireland). IBMX (3-Isobutyl-1-methyl-1H-purine-2,6(3H,7H)-dione) was purchased from Sigma-Aldrich (St. Louis, MO). 8MM-IBMX (3-Isobutyl-8-(methoxymethyl)-1-methyl-1H-purine-2,6(3H,7H)-dione) was purchased from Santa Cruz Biotechnology (Dallas, TX). Cilostamide (N-cyclohexyl-N-methyl-4-(1,2- dihydro-2-oxo-6-quinolyloxy) butyramide), rolipram (4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidinone), and PF-04671536 (5-Methyl-3-[[(2R)-4-(2-thiazolylmethyl)-2-morpholinyl]methyl]-3H-1,2,3-triazolol[4 (link),5 (link)]pyrimidin-7-amine) were purchased from Tocris Bioscience (Bristol, UK). PF-04957325 (3-[[(2R)-4-(1,3-thiazol-2-ylmethyl)morpholin-2-yl]methyl]-5-(trifluoromethyl)triazolo[4,5-d]pyrimidin-7-amine) was purchased from MedChemExpress (Monmouth Junction, NJ). Unless otherwise indicated, all other reagents were purchased from Sigma-Aldrich.

ZD7288, (RS)-(±)-sulpiride, rolipram, forskolin and ivabradine hydrochloride were purchased from Tocris Bioscience (Ellisville, MO). DCZ dihydrochloride (water soluble) was purchased from Hello Bio Inc. (Princeton, NJ). Elacridar hydrochloride was purchased from Medchemexpress LLC (Monmouth Junction, NJ). Cocaine HCl was provided by the NIDA Drug Supply Program. All other common chemicals were obtained from Sigma-Aldrich (St. Louis, MO).