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.
Fig 2.
IP receptor mediates the effect of 19(S)-HETE in MEG-01 cells.
(A) Effect of 1 μM 19(S)-HETE on cAMP levels in human monocytic cells (THP-1), human embryonic kidney cells (HEK-293T), human umbilical endothelial vein cells (HUVECs), human megakaryoblastic cells (MEG-01) and human liver hepatocellular carcinoma cells (Hep-G2). 19(S)-HETE response is shown as percentage of the forskolin (FSK)-induced cAMP increase. (B) Quantitative expression analysis of GPCRs in cells that respond (MEG-01) and in cells that do not respond (HUVECs) to 19(S)-HETE. (C) Ratio between effects induced by 19(S)-HETE (1 μM) in cells transfected with a pool of siRNA directed against a particular receptor and effects induced in cells treated with scrambled siRNA. Graph represents ranked averages of six independent experiments performed with 27 siRNA pools. (D) Effect of IP receptor antagonist, Cay10441 (3 μM), on 19(S)-HETE (1 μM) and cicaprost (1 μM)-induced cAMP accumulation in MEG-01 cells. Shown are mean values ± SD, n ≥ 3. *, P ≤ 0.05 (compared to buffer-treated controls).
Fig 3.
19(S)-HETE is an orthosteric prostacyclin receptor agonist.
(A) Effect of increasing concentrations of PGI2, 19(S)-HETE and 19(R)-HETE on cAMP levels in COS-1 transfected with IP receptor and an intracellular cAMP-sensitive bioluminescent probe (see Material and Methods). (B) Effect of different HETEs at 1 μM and of cicaprost (1 μM) on cAMP levels in COS-1 expressing IP receptor and the intracellular bioluminescent cAMP probe. (C) Effect of COX-1/2 blockers indomethacin (10 μM), NS-398 (10 μM) and FR122047 (1 μM) on formation of cAMP induced by 19(S)-HETE (1 μM), arachidonic acid (3 mM), cicaprost (1 μM) and forskolin (10 μM) in COS-1 cells expressing IP receptor. (D) Effect of iloprost and 19(S)-HETE on binding of 10 nM [3H]-iloprost to IP receptor expressed in COS-1 cells. Shown are mean values ± SEM, n ≥ 3. *, P ≤ 0.05; ns, not significant.
Fig 4.
19(S)-HETE is a selective IP receptor agonist.
(A) COS-1 cells expressing Gq/G11-coupled prostanoid receptors together with a Ca2+-sensitive bioluminescent fusion protein were exposed to 19(S)-HETE (3 μM) and their cognate prostanoid receptors ligands at 3 μM. EP1, PGE2 receptor subtype 1; FP, prostaglandin F2α receptor; TP, thromboxane A2 receptor. AUC, area under the curve of agonist-induced calcium transients recorded for 100 seconds. (B) Effect of 19(S)-HETE (3 μM) on various Gi-coupled prostanoid receptors expressed together with the promiscuous G-protein α-subunit Gα15 in COS-1 cells using a Ca2+-sensitive bioluminescent probe. Functionality of receptors was verified by recording responses after stimulation with the specific prostanoid receptors at 3 μM. EP3, PGE2 receptor, subtype 3; DP2, prostaglandin D2 receptor. (C) Gs-coupled prostanoid receptors were heterologously expressed together with a cAMP-sensitive bioluminescence probe and stimulated with 3 μM of 19(S)-HETE or their endogenous ligands. EP2 and EP4, prostaglandin E2 receptors, subtypes 2 and 4; DP1, prostaglandin D1 receptor; IP, prostacyclin receptor. Luminescence refers to the light generated 15 minutes after ligand stimulation and recorded with an integration time of 1250 ms.
Fig 5.
Vasorelaxant effects of 19(S)-HETE are mediated by the IP receptor.
(A-D) Effects of 19(S)-HETE (1 μM) or cicaprost (1 μM) on isolated mouse mesenteric arterial segments pre-contracted with 10 μM phenylephrine from wild-type (A, C) or from IP receptor deficient mice (B, D). (E-H) Effects of 19(S)-HETE (1 μM), cicaprost (1 μM) and isoproterenol (30 μM) on aortic segments pre-contracted with phenylephrine (10 μM). (I, J) Quantification of agonist effects shown in panels A-D and E-H expressed as difference between agonist-induced aortic segment tension changes and basal levels. (K-L) Effect of NS398 (10 μM) and FR122047 (1 μM), or of indomethacin (10 μM) on the ability of 19(S)-HETE (1 μM) and cicaprost (1 μM) to induce relaxation of isolated mesenteric arteries (K) and aortic segments (L) pre-contracted by 10 μM phenylephrine. Shown are mean values ± SEM, n ≥ 3. *,P ≤ 0.05 (compared to contraction induced by phenylephrine alone).
Fig 6.
Platelet-inhibitory effects of 19(S)-HETE are mediated by IP receptor.
(A-D) Effect of 19(S)-HETE (3 μM), cicaprost (1 μM) and sodium nitroprusside (SNP) (1 mM) pretreatment of platelets on aggregation of platelets isolated from wild-type (Ptgir+/+) and IP-receptor-deficient mice (Ptgir-/-) induced by 0.1 U/ml thrombin. Shown are representative aggregation traces of at least 3 independently performed experiments.