Stimulation of rat erythrocyte P2X7 receptor induces the release of epoxyeicosatrienoic acids

Abstract

Background and purpose: Red blood cells (RBCs) are reservoirs of vasodilatory, antiaggregatory, and antiinflammatory lipid mediators-epoxyeicosatrienoic acids (EETs). This study addresses the formation and release of erythrocyte-derived EETs in response to ATP receptor stimulation that may represent an important mechanism regarding circulatory regulation.

Experimental approach: Erythrocyte EET formation and release were investigated by incubating rat RBCs in physiological salt solution with agents that effected ATP release via P2 receptor stimulation of phospholipase A2 and epoxygenase-like activities with activation of the ATP secretory mechanism. EETs were analyzed by gas and liquid chromatography-mass spectrometry.

Key results: EETs were released from rat RBCs: 14,15-, 11,12-, 8,9- and 5,6-EETs in a ratio of 1.2:1.0:0.9:0.8. EETs were produced by epoxidation of arachidonic acid catalyzed by hemoglobin. Spontaneous release of EETs, 0.66+/-0.14 ng per 10(9) RBCs, was dose-dependently increased by an ATP analog, BzATP, and inhibited by P2X(7) receptor antagonists. 5 microM ATP increased release of EETs over 20% to 0.83+/-0.15 ng per 10(9) RBCs; 10 microM BzATP tripled the amount of EET release to 1.87+/-0.20 ng per 10(9) RBCs. EET release by ATP or BzATP was not associated with hemolysis. Carbenoxolone, a gap junction inhibitor that inhibits ATP release, and glibenclamide, an inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR), which is required for ATP release, inhibited the spontaneous and stimulated EET release from RBCs.

Conclusions and implications: EETs are produced and released from RBCs via a mechanism that is mediated by ATP stimulation of P2X(7) receptors coupled to ATP transporters, pannexin-1 and CFTR.

Figure 1

Representative ESI LC-MS profiles (n=4–8, m/z 319) of the released HETEs and EETs from incubations of control (a), ATP 1 mM (b), BzATP 10 μM (c), AA 0.66 μM (d) and A23187 2 μM (e) with 4 × 10⁹ rat RBCs in 2 ml buffer for 30 min at 37°C. Eicosanoid products from RBCs include 15-, 13-, 12-, 11-, 10-, 9-, 7- and 5-HETEs as well as 14,15-, 11,12-, 8,9- and 5,6-EETs. EET, epoxyeicosatrienoic acid; HETE, hydroxyeicosatetraenoic acid.

Figure 2

Total EET release from rat erythrocytes in response to extracellular ATP. ATP from 1 μM to 1 mM was incubated with rat erythrocytes for 30 min at 37°C. n=8–12, ^*P<0.05 vs spontaneous EET release at 0.66±0.14 ng per 10⁹ RBCs. EET, epoxyeicosatrienoic acid.

Figure 3

Comparison of the potency of P2 receptor agonists on total EET release from rat erythrocytes. Rat RBCs were incubated with ATP, BzATP, 2MeSATP, ATPγS, ADP and UTP for 30 min at 37°C. EETs were analysed by GC-MS from the supernatent after centrifuging the RBCs. n=4–8, ^*P<0.05 vs control. BzATP, 2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate; EET, epoxyeicosatrienoic acid; 2MeSATP, 2-methylthio-ATP.

Figure 4

Dose-dependent response of the P2X₇ receptor agonist, BzATP, on EET release from rat erythrocytes. Incubations were carried out with BzATP from 0.1 to 100 μM. n= 6–10. BzATP, 2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate; EET, epoxyeicosatrienoic acid.

Figure 5

P2X₇ antagonists, BBG and Zn²⁺, inhibit EET release from rat erythrocytes. n=6–10, ^*P<0.05 vs basal in the same group. BzATP, 2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate; BBG, brilliant blue G; EET, epoxyeicosatrienoic acid.

Figure 6

Effects of ATP release inhibitors, glibenclamide, niflumic acid and carbenoxolone, on EET release from rat erythrocytes. n=6–12, ^*P<0.05 vs basal in the same group. BzATP, 2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate; EET, epoxyeicosatrienoic acid.

Figure 7

Effects of PLA₂ inhibitors, MAFP, BEL and S3319, on EET release from rat erythrocytes. MAFP, a Ca²⁺-dependent cytosolic PLA₂ inhibitor, BEL, a Ca²⁺-independent cytosolic PLA₂ inhibitor, and S3319, a secretory PLA₂ inhibitor were tested in the RBC incubations. n=6–8, ^*P<0.05 vs basal in the same group. BEL, bromoenol lactone; BzATP, 2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate; EET, epoxyeicosatrienoic acid; MAFP, methyl arachidonyl flurophosphonate.