Lipoxygenase derived products of arachidonic acid contribute to endothelium-dependent hyperpolarization in rat middle cerebral arteries

Alister McNeish1, Kathryn Gauthier2, William Campbell2

1The University of Bath, Bath, UK, 2Medical College of Wisconsin, Milwaukee, USA

EDHF responses in the rat middle cerebral artery are reduced by inhibition of PLA2 indicating a metabolite of arachidonic acid (AA) contributes to the response; this metabolite is not an epoxyeicosatrienoic acid (EET) or an hydroxyeicosatetraenoic acid (HETE) (You, 2005, McNeish, 2006). Recently, products of 15-lipoxygenase, hydroxyepoxyeicosatrienoic acids (HEETAs) and their metabolites (generated by soluble epoxide hydrolase; sEH), trihydroxyeicosatrienoic acids (THETAs), have been characterised as hyperpolarizing factors in rabbit arteries where they stimulate SKCa channels (Chawengsub, 2008). We investigated if lipoxygenase products contribute to hyperpolarization in cerebral arteries.

Male Wistar rats (250-300g) were killed by CO2 inhalation. Segments of the middle cerebral artery were mounted in a wire myograph. Experiments were conducted in the presence of the lipoxygenase inhibitors ebselen (1μM) or NDGA (Nordihydroguaiaretic Acid; 1μM), or in the presence of the sEH inhibitor tAUCB (trans-4-[4-(3-Adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid; 10 μM). The Protease activated receptor 2 peptide agonist, SLIGRL (20μM) induced endothelium-dependant hyperpolarization and relaxation was assessed in the presence and absence of selective blockers of SKCa, (apamin, 50 nM), IKCa (TRAM-34, 1 μM) and BKCa (iberiotoxin, 100 nM). AA metabolites were measured by reverse phase HPLC of 14C labelled AA. Data are mean ± s.e.mean of 4 or more animals. Statistical comparisons were made using one way ANOVA with Bonferroni’s post test.

SLIGRL-induced hyperpolarizations (–27.2±2.8mV, n=10) were significantly inhibited by either ebselen or NDGA (15.6±3.0 mV and 10.7±2.3 mV, n=6 and 4, respectively, P<0.05). Inhibition of sEH (tAUCB 10 μM) did not increase overall hyperpolarization but did increase the component of hyperpolarization remaining after inhibition of IKCa and BKCa (15.8 ± 2.5 mV and 29.5 ± 5.2 mV, n=5 before and after tAUCB, respectively; P<0.05); this hyperpolarization was virtually abolished following addition of apamin (5.2 ± 2.4 mV, n=4). Analysis of metabolism of 14C AA confirmed that THETAs are produced by rat middle cerebral arteries.

The rat middle cerebral artery synthesises the lipoxygenase products THETAs which are derived from sEH dependant metabolism of HEETAs, both have been demonstrated to stimulate SKCa (Chewengsub, 2008). Inhibition of lipoxygenase reduced SLIGRL-induced hyperpolarization and inhibition of sEH increased SKCa mediated hyperpolarization. Therefore it is likely that the lipoxygenase metabolites that contribute to hyperpolarization in rat cerebral arteries are HEETAs. It is possible that sEH inhibitors may protect against endothelial dysfunction by up regulating SKCa function, in addition to their role in protecting EET signalling.

Supported by the British Heart Foundation

Chawengsub Y. et al., (2008) Am. J. Physiol. 294:H1348-56

McNeish A.J. et al., (2006) Stroke. 37: 1277-82

You Y. et al., (2005) Am. J. Physiol. 289: 1077-83