Arachidonic Acid Metabolite 19(S)-HETE Induces Vasorelaxation and Platelet Inhibition by Activating Prostacyclin (IP) Receptor.

19(S)-hydroxy-eicosatetraenoic acid (19(S)-HETE) belongs to a family of arachidonic acid metabolites produced by cytochrome P450 enzymes, which play critical roles in the regulation of cardiovascular, renal and pulmonary functions. Although it has been known for a long time that 19(S)-HETE has vascular effects, its mechanism of action has remained unclear. In this study we show that 19(S)-HETE induces cAMP accumulation in the human megakaryoblastic leukemia cell line MEG-01. This effect was concentration-dependent with an EC50 of 520 nM, insensitive to pharmacological inhibition of COX-1/2 and required the expression of the G-protein Gs. Systematic siRNA-mediated knock-down of each G-protein coupled receptor (GPCR) expressed in MEG-01 followed by functional analysis identified the prostacyclin receptor (IP) as the mediator of the effects of 19(S)-HETE, and the heterologously expressed IP receptor was also activated by 19(S)-HETE in a concentration-dependent manner with an EC50 of 567 nM. Pretreatment of isolated murine platelets with 19(S)-HETE blocked thrombin-induced platelets aggregation, an effect not seen in platelets from mice lacking the IP receptor. Furthermore, 19(S)-HETE was able to relax mouse mesenteric artery- and thoracic aorta-derived vessel segments. While pharmacological inhibition of COX-1/2 enzymes had no effect on the vasodilatory activity of 19(S)-HETE these effects were not observed in vessels from mice lacking the IP receptor. These results identify a novel mechanism of action for the CYP450-dependent arachidonic acid metabolite 19(S)-HETE and point to the existence of a broader spectrum of naturally occurring prostanoid receptor agonists

IP receptor mediates the effect of 19(S)-HETE in MEG-01 cells.

<p>(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. <i>*</i>, <i>P</i> ≤ 0.05 (compared to buffer-treated controls).</p

19(S)-HETE is an orthosteric prostacyclin receptor agonist.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163633#sec002" target="_blank">Material and Methods</a>). (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. *, <i>P</i> ≤ 0.05; ns, not significant.</p

Platelet-inhibitory effects of 19(S)-HETE are mediated by IP receptor.

<p>(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 (<i>Ptgir</i>^<i>+/+</i>) and IP-receptor-deficient mice (<i>Ptgir</i>^<i>-/-</i>) induced by 0.1 U/ml thrombin. Shown are representative aggregation traces of at least 3 independently performed experiments.</p

19(S)-HETE induces increase in cAMP levels in MEG-01 cells.

<p>(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 Gα_s 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 Gα_s 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. <i>*</i>, <i>P</i> ≤ 0.05; ns, not significant.</p

19(S)-HETE is a selective IP receptor agonist.

<p>(A) COS-1 cells expressing G_q/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 F_2α 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 G_i-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) G_s-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.</p

Myogenic vasoconstriction requires G12/G13 and LARG to maintain local and systemic vascular resistance

Myogenic vasoconstriction is an autoregulatory function of small arteries. Recently, G-protein-coupled receptors have been involved in myogenic vasoconstriction, but the downstream signalling mechanisms and the in-vivo-function of this myogenic autoregulation are poorly understood. Here, we show that small arteries from mice with smooth muscle-specific loss of G(12)/G(13) or the Rho guanine nucleotide exchange factor ARHGEF12 have lost myogenic vasoconstriction. This defect was accompanied by loss of RhoA activation, while vessels showed normal increases in intracellular [Ca2+]. In the absence of myogenic vasoconstriction, perfusion of peripheral organs was increased, systemic vascular resistance was reduced and cardiac output and left ventricular mass were increased. In addition, animals with defective myogenic vasoconstriction showed aggravated hypotension in response to endotoxin. We conclude that G(12)/G(13)- and Rho-mediated signaling plays a key role in myogenic vasoconstriction and that myogenic tone is required to maintain local and systemic vascular resistance under physiological and pathological condition