cAMP accumulation in intact cells was assayed as described previously.25–27 Briefly, for measuring intracellular cAMP, HEK293 cells or rat cardiac myocytes in 24-well plates prelabeled with [3H]adenine (0.5–2 μCi/well for 24 hours) were washed once with HEPES-buffered DMEM and incubated (37°C for 30 minutes) in the same medium containing 1 mmol/L 3-isobutyl 1-metylxanthine with or without ISO and WNNOs. The reaction was terminated by aspiration and by the immediate addition of ice-cold 5% trichloroacetic acid with 1 mmol/L cAMP/ATP (0.5 mL/well). [3H]cAMP and [3H]ATP were separated on AG 40W-X4 Dowex and alumina columns, and the data are presented as the ratio of [3H]cAMP to [3H]cAMP plus [3H]ATP, as described previously.
cAMP accumulation in intact cells was assayed as described previously.25–27 Briefly, for measuring intracellular cAMP, HEK293 cells or rat cardiac myocytes in 24-well plates prelabeled with [3H]adenine (0.5–2 μCi/well for 24 hours) were washed once with HEPES-buffered DMEM and incubated (37°C for 30 minutes) in the same medium containing 1 mmol/L 3-isobutyl 1-metylxanthine with or without ISO and WNNOs. The reaction was terminated by aspiration and by the immediate addition of ice-cold 5% trichloroacetic acid with 1 mmol/L cAMP/ATP (0.5 mL/well). [3H]cAMP and [3H]ATP were separated on AG 40W-X4 Dowex and alumina columns, and the data are presented as the ratio of [3H]cAMP to [3H]cAMP plus [3H]ATP, as described previously.