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


METHODS
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.


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
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.
Jiang
H
H
Department of Pharmacology, New York Medical College, Valhalla, NY, USA. Houli_jiang@nymc.edu
Zhu
A G
AG
Mamczur
M
M
Falck
J R
JR
Lerea
K M
KM
McGiff
J C
JC
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001095:Arachidonic Acids / Q000032:analysis / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D002229:Carbenoxolone / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D005905:Glyburide / Q000494:pharmacology; D054513:Group IV Phospholipases A2; D006461:Hemolysis / Q000187:drug effects; D007476:Ionophores / Q000494:pharmacology; D008297:Male; D009544:Niflumic Acid / Q000494:pharmacology; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D051381:Rats; D017207:Rats, Sprague-Dawley; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058486:Receptors, Purinergic P2X7; D021241:Spectrometry, Mass, Electrospray Ionization
D001095:Arachidonic Acids; D007476:Ionophores; C546470:P2rx7 protein, rat; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D016718:Arachidonic Acid; D000001:Calcimycin; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D009544:Niflumic Acid; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D000255:Adenosine Triphosphate; D010741:Phospholipases A; D054513:Group IV Phospholipases A2; D054467:Phospholipases A2; D002229:Carbenoxolone; D005905:Glyburide