Epoxyeicosatrienoic and dihydroxyeicosatrienoic acids dilate human coronary arterioles via BK(Ca) channels: implications for soluble epoxide hydrolase inhibition

Abstract

Epoxyeicosatrienoic acids (EETs) are metabolized by soluble epoxide hydrolase (sEH) to form dihydroxyeicosatrienoic acids (DHETs) and are putative endothelium-derived hyperpolarizing factors (EDHFs). EDHFs modulate microvascular tone; however, the chemical identity of EDHF in the human coronary microcirculation is not known. We examined the capacity of EETs, DHETs, and sEH inhibition to affect vasomotor tone in isolated human coronary arterioles (HCAs). HCAs from right atrial appendages were prepared for videomicroscopy and immunohistochemistry. In vessels preconstricted with endothelin-1, three EET regioisomers (8,9-, 11,12-, and 14,15-EET) each induced a concentration-dependent dilation that was sensitive to blockade of large-conductance Ca2+-activated K+ (BK(Ca)) channels by iberiotoxin. EET-induced dilation was not altered by endothelial denudation. 8,9-, 11,12-, and 14,15-DHET also dilated HCA via activation of BK(Ca) channels. Dilation was less with 8,9- and 14,15-DHET but was similar with 11,12-DHET, compared with the corresponding EETs. Immunohistochemistry revealed prominent expression of cytochrome P-450 (CYP450) 2C8, 2C9, and 2J2, enzymes that may produce EETs, as well as sEH, in HCA. Inhibition of sEH by 1-cyclohexyl-3-dodecylurea (CDU) enhanced dilation caused by 14,15-EET but reduced dilation observed with 11,12-EET. DHET production from exogenous EETs was reduced in vessels pretreated with CDU compared with control, as measured by liquid chromatography electrospray-ionization mass spectrometry. In conclusion, EETs and DHETs dilate HCA by activating BK(Ca) channels, supporting a role for EETs/DHETs as EDHFs in the human heart. CYP450s and sEH may be endogenous sources of these compounds, and sEH inhibition has the potential to alter myocardial perfusion, depending on which EETs are produced endogenously.

Fig. 1

Epoxyeicosatrienoic acid (EET)-induced dilation of human coronary arterioles (HCAs). A: 8,9-, 11,12-, and 14,15-EET each induces a dose-dependent relaxation of HCAs (n = 9, 18, and 25, respectively). 14,15-EET is a less potent dilator than 8,9- and 11,12-EET (†P < 0.05 vs. 8,9-EET curve, ‡P < 0.05 vs. 11,12-EET curve). B: endothelial denudation does not diminish or enhance vasodilation to 14,15-EET [n = 5, P = not significant (NS)]. n, No. of vessels.

Fig. 2

Effect of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel blockade on EET- and dihydroxyeicosatrienoic acids (DHET)-induced dilation of HCAs. A–C: dilation to 8,9-, 11,12-, and 14,15-EET is inhibited by iberiotoxin (100 nmol/l, n = 6, 7, and 6, respectively). D–F: 8,9-, 11,12-, and 14,15-DHET each induces a dose-dependent relaxation of HCAs that is sensitive to iberiotoxin (100 nmol/l, n = 6, 6, and 6, respectively). 8,9- and 14,15-DHET are less potent dilators than their respective EETs, whereas 11,12-DHET is similar to 11,12-EET. n, No. of vessels. †P < 0.05 vs. control curve, *P < 0.05 vs. control at specific dose, ‡P < 0.05 vs. respective EET curve.

Fig. 3

Expression of cytochrome P-450 (CYP450) epoxygenases and soluble epoxide hydrolase (sEH) in the microvasculature of human right atrial appendage. Representative images of tissue sections (5 μm thick) following immunohistochemical staining. A positive reaction is indicated by the production of a brown pigment. A–C: CYP2C8, -2C9, and -2J2 are expressed in the endothelium of HCA, as well as in the vascular smooth muscle and surrounding tissues (n = 5, 6, and 4, respectively). D: sEH is expressed in HCA endothelium and in surrounding tissues (n = 4). E and F: staining was absent when each primary antibody was omitted or replaced by preimmune serum, ruling out nonspecific binding of the secondary antibody (n = 5, 6, 4, and 4, respectively). n, No. of patients. Bar, 100 μm.

Fig. 4

Effect of sEH inhibition on EET-mediated dilation of HCA. A: 11,12-EET-mediated dilation is reduced in the presence of 1-cyclohexyl-3-dodecylurea (CDU; 1 μmol/l, n = 6). B: 14,15-EET-induced dilation is enhanced in the presence of CDU (n = 6). C: 11,12-DHET-induced dilation is not altered in the presence of CDU (n = 5, P = NS). n, No. of vessels. †P < 0.05 vs. control curve, *P < 0.05 vs. control at specific dose.

Fig. 5

Effect of sEH inhibition on the metabolism of EETs to DHETs by HCA. After incubations using 11,12- and 14,15-EET, EETs and DHETs were quantified by using liquid chromatography electrospray-ionization mass spectrometry. A greater percentage of EETs were hydrolyzed to their corresponding DHET derivatives in the presence of HCAs than in buffer alone (n = 3, *P < 0.05). However, production of DHETs was reduced when separate vessels from the same patient were pretreated with CDU (†P < 0.05). n, No. of patients.