P271	Cytochrome P450 2C-derived epoxyeicosatrienoic acids regulate Ca2+ signalling and hyperpolarization in endothelial cells.
1A.Rueben, 1R.Popp, 1B.Keserü, 2B.D.Hammock, 1R.Busse, 1I.Fleming
1Institut für kardiovaskuläre Physiologie, JWG-Universität, Frankfurt/Main, DE; 2Department of Entomology and UC Davis Cancer Center, University of California, Davis, US.

Epoxyeicosatrienoic acids (EETs) have been suggested to act as calcium-influx factors, controlling the entry of Ca2+ into non-excitable cells. The aim of the present study was to investigate the role of EETs generated by cytochrome P450 (CYP) 2C9 on Ca2+ signalling and the activation of Ca2+-dependent K+ channels in human endothelial cells (ECs). Stimulation of cultured ECs, which no longer express CYP 2C protein, with bradykinin (BK) elicited both a Ca2+ transient and membrane hyperpolarization. These responses were not affected by sulfaphenazole (sulfa), a selective inhibitor of CYP 2C9 or by 1-adamantyl-3-cyclohexylurea (ACU), an inhibitor of the soluble epoxide hydrolase (sEH) that metabolizes EETs. Incubation of ECs expressing CYP 2C9 resulted in a significant increase in the plateau phase of the BK-induced Ca2+ transient. Sulfa abrogated the effects of enhanced CYP expression on intracellular Ca2+, whereas ACU enhanced the Ca2+ influx during the plateau phase. Increased CYP expression was also associated with a small change in the EC membrane potential and in the magnitude and duration of the BK-induced hyperpolarization, effects that were sensitive to sulfa. ACU significantly prolonged the BK-induced hyperpolarization of these cells from 83+/-30 to 205+/-80 seconds, an effect prevented by sulfa. The potentiating effects of ACU on Ca2+ signalling and hyperpolarization were also abrogated by the EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid, by ruthenium red, an inhibitor of the transient receptor potential (TRP) V4 ion channel and by the PK A inhibitor, adenosine 3',5'-cyclic monophosphothioate. We assessed the effects of ACU on the EDHF-mediated relaxation of isolated porcine coronary arteries in the presence of diclofenac and Nω-nitro-L-arginine. Preincubation of arterial rings with ACU resulted in an enhanced EDHF-mediated relaxation to BK, which was abolished in the presence of sulfa. These data indicate that EETs generated intracellularly by CYP 2C9 act as Ca2+ influx factors and modulate endothelial cell Ca2+ as well as agonist-induced membrane hyperpolarization via a PKA- and possibly TRPV4-dependent pathway. Moreover, our data highlight the role of the sEH in the control of EET signalling.

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