Fig 1. 19(S)-HETE induces increase in cAMP levels in MEG-01 cells.
(A) Effect of 19(S)-HETE and related compounds on intracellular cAMP levels in MEG-01 cells. cAMP concentration was measured 15 minutes after the addition of 1 μM of the indicated compounds and 10 μM of the positive control, forskolin. (B) Effects of COX-1/2 inhibition on 19(S)-HETE-induced cAMP increase in MEG-01 cells. Cells were pretreated for 30 minutes with indomethacin (10 μM), with NS398 (10 μM) and FR122047 (1 μM) or buffer (solvent) and were then stimulated with 19(S)-HETE (1 μM), arachidonic acid (3 mM) and forskolin (10 μM) for 15 minutes. (C) Effect of increasing concentration of 19(S)-HETE and its regiomer 19(R)-HETE on cAMP levels in MEG-01 cells. (D) Role of Gαs in 19(S)-HETE- and forskolin-induced cAMP accumulation in MEG-01 cells. MEG-01 cells were reverse-transfected with scrambled siRNA (ctrl. siRNA) or Gαs siRNA pools and 72h later were assayed for their responsiveness to 19(S)-HETE (1 μM) and forskolin (10 μM). Intracellular cAMP concentration was determined as described in Material and Methods. Shown are mean values ± SD, n ≥ 3. *, P ≤ 0.05; ns, not significant.