Time course of TNF-α–induced increase of PDE2 activity and mRNA. HUVECs were stimulated with TNF-α (10 ng/mL) and assayed for PDE2 activity (A) and PDE2A3 mRNA (B). PDE2 activity was measured in HUVEC lysates of cells stimulated up to 24 hours, normalized to total cellular protein, and expressed as pmol cAMP (or cGMP) × minute^-1 × mg protein^-1. Untreated controls were run in parallel for 3 time points (6, 12, and 24 hours). Data presented are mean ± SEM of 4 independent experiments. (B) Semiquantitative RT-PCR analysis of PDE2 mRNA in HUVECs. Cells were stimulated up to 48 hours with TNF-α (10 ng/mL). After RT reaction, PCR was run with 32 cycles (PDE2A3) and 28 cycles (glyceraldehyde-3-phosphate dehydrogenase, GAPDH). Representative ethidium bromide–stained agarose gels are shown. RT-PCR product sizes for PDE2A3 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were 583 base pair (bp) and 598 bp, respectively. PCR for GAPDH served as internal control. The gels are representative of 4 independent experiments. ^*P < .05 versus t = 0; ^**P< .01 versus controls.