FIGURE 2.

PGE₂ production, but not COX-2 up-regulation is dependent on cPLA₂-α enzyme activity. 80–90% confluent FLSs were stimulated with TNF (10 ng/ml), sPLA₂-IIA (4 μg/ml) either alone or in combination in the presence or absence of the cPLA₂-α-selective inhibitor (A) pyrrophenone or (B) pyrrolidine-1 at the concentrations shown for 16 h in Ham's/DMEM medium containing 0.1% BSA. Medium and cells were harvested, PGE₂ in medium was determined and the protein concentration in cell lysates determined as described under “Experimental Procedures.” Data are mean ± S.E. of triplicate determinations from cell cultures derived from each of four patients. **, p < 0.01; ***, p < 0.001 (Student's unpaired t test) relative to unstimulated cells unless indicated. C, cells were grown in 24-well plates and stimulated as described above. Cells were harvested, lysates were electrophoresed, transferred to nitrocellulose, probed with Abs, and labeled proteins were visualized on x-ray film by enhanced chemiluminescence, blots were scanned and densitometry performed and analyzed as described under “Experimental Procedures.” Representative blots from one cell culture are shown. Densitometry data are mean ± S.E. normalized relative to unstimulated cells from experiments performed on cell cultures derived from three separate patients.