The culture medium was recovered for analysis of PGs, and cells were recovered for Northern or Western blot analysis. In experiment 3, cells were grown to confluency and the medium was replaced by fresh medium without serum. The cells were then incubated in the presence of PMA (100 nM) and in the presence or absence of increasing doses (0.01-100 nM) of in-domethacin to block all PGHS activity or with CGP 28238 to block PGHS-2. The culture supernatant was then recovered for measurement of PGI2. In experiment 4, cells were grown to confluency, the medium was replaced as described above, and cells were treated or not treated with CGP 28238 or indomethacin for 24 h. During the last 3 h, PMA (100 nM) was added to the cultures. The cells were then recovered for analysis of expression of PGHS-2 mRNA by Northern blot. ampicillin antibiotic
Accumulation of PGE2 and 6-keto-PGF1a (the stable metabolite of PGI2) in the incubation medium was determined by ELISA as described previously.
RNA Isolation and Northern Blot Analysis
Total RNA was prepared by the method of Chomczynski and Sacchi. In summary, after myocytes were homogenized in 4 M guanidium thiocyanate, RNA was extracted twice with phenol/chloroform-isoamyl alcohol (24:1), precipitated with isopropanol, and then washed with 70% ethanol.