Regulation of K_Ca2.3 and endothelium-dependent hyperpolarization (EDH) in the rat middle cerebral artery: the role of lipoxygenase metabolites and isoprostanes

Abstract

Background and Purpose. In rat middle cerebral arteries, endothelium-dependent hyperpolarization (EDH) is mediated by activation of calcium-activated potassium (KCa) channels specifically KCa2.3 and KCa3.1. Lipoxygenase (LOX) products function as endothelium-derived hyperpolarizing factors (EDHFs) in rabbit arteries by stimulating KCa2.3. We investigated if LOX products contribute to EDH in rat cerebral arteries.Methods. Arachidonic acid (AA) metabolites produced in middle cerebral arteries were measured using HPLC and LC/MS. Vascular tension and membrane potential responses to SLIGRL were simultaneously recorded using wire myography and intracellular microelectrodes.Results. SLIGRL, an agonist at PAR2 receptors, caused EDH that was inhibited by a combination of KCa2.3 and KCa3.1 blockade. Non-selective LOX-inhibition reduced EDH, whereas inhibition of 12-LOX had no effect. Soluble epoxide hydrolase (sEH) inhibition enhanced the KCa2.3 component of EDH. Following NO synthase (NOS) inhibition, the KCa2.3 component of EDH was absent. Using HPLC, middle cerebral arteries metabolized 14C-AA to 15- and 12-LOX products under control conditions. With NOS inhibition, there was little change in LOX metabolites, but increased F-type isoprostanes. 8-iso-PGF2α inhibited the KCa2.3 component of EDH.Conclusions. LOX metabolites mediate EDH in rat middle cerebral arteries. Inhibition of sEH increases the KCa2.3 component of EDH. Following NOS inhibition, loss of KCa2.3 function is independent of changes in LOX production or sEH inhibition but due to increased isoprostane production and subsequent stimulation of TP receptors. These findings have important implications in diseases associated with loss of NO signaling such as stroke; where inhibition of sEH and/or isoprostane formation may of benefit.

Actions and Mechanisms of Polyunsaturated Fatty Acids on Voltage-Gated Ion Channels

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Peroxynitrite formed during a transient episode of brain ischaemia increases endothelium‐derived hyperpolarization‐type dilations in thromboxane/prostaglandin receptor‐stimulated rat cerebral arteries

“…Concentrations of most inhibitors were chosen based on previous studies (McNeish & Garland 2007, McNeish et al 2012, Gauthier et al 2014. Concentrations of most inhibitors were chosen based on previous studies (McNeish & Garland 2007, McNeish et al 2012, Gauthier et al 2014.…”

“…We submitted HMVEC to H/R, a cell culture model of I/R (Koo et al 2001). Rho activation is an important regulator of the activity of some ion channels (Ghisdal et al 2003, Luykenaar et al 2009), which may play a role on TP-dependent inhibition of SK Ca channel function in the endothelium (McNeish et al 2012, Gauthier et al 2014. 2a and Figure S2, expression of hypoxia-inducible factor 1a (HIF-1a) was higher in cells that have undergone H/R than in those kept in normoxia.…”

“…Thromboxane A 2 (TxA 2 ) is one of the multiple factors released by activated platelets that can mediate further platelet recruitment, activation and smooth muscle cell constriction, contributing to the prothrombotic state in I/R (Chamorro 2009). In addition, previous studies suggest a role of endothelial thromboxane/prostaglandin receptor (TP) stimulation in suppressing the small-conductance calcium-activated potassium (SK Ca ) channel component of EDH (Plane & Garland 1996, Crane & Garland 2004, McNeish & Garland 2007, McNeish et al 2012, Gauthier et al 2014. Thus, TxA 2 -induced attenuation of EDHmediated relaxation may represent an additional mechanism by which TxA 2 could impair tissue perfusion, especially in disease states where NO is defective (Ellinsworth et al 2014).…”

“…TxA 2 binds to TP in both smooth muscle and endothelial cells and triggers the RhoA/Rho kinase pathway, which is hyperactivated in the ischaemic brain (Koumura et al 2011), regulating many cellular functions including ion channel activity (Ghisdal et al 2003, Luykenaar et al 2009). Rho kinase activation is linked to inhibition of endothelial cell SK Ca channel function in rat middle cerebral artery (MCA;McNeish et al 2012, Gauthier et al 2014, although the precise mechanism is still unknown. One possibility is that inhibition of SK Ca channel function is dependent upon actin cytoskeleton remodelling (Gorovoy et al 2005, Prasain & Stevens 2009).…”