11,12-EET Stimulates the Association of BK Channel α and β1 Subunits in Mitochondria to Induce Pulmonary Vasoconstriction

Abstract

In the systemic circulation, 11,12-epoxyeicosatrienoic acid (11,12-EET) elicits nitric oxide (NO)- and prostacyclin-independent vascular relaxation, partially through the activation of large conductance Ca2+-activated potassium (BK) channels. However, in the lung 11,12-EET contributes to hypoxia-induced pulmonary vasoconstriction. Since pulmonary artery smooth muscle cells also express BK channels, we assessed the consequences of BKβ1 subunit deletion on pulmonary responsiveness to 11,12-EET as well as to acute hypoxia. In buffer-perfused mouse lungs, hypoxia increased pulmonary artery pressure and this was significantly enhanced in the presence of NO synthase (NOS) and cyclooxygenase (COX) inhibitors. Under these conditions the elevation of tissue EET levels using an inhibitor of the soluble epoxide hydrolase (sEH-I), further increased the hypoxic contraction. Direct administration of 11,12-EET also increased pulmonary artery pressure, and both the sEH-I and 11,12-EET effects were prevented by iberiotoxin and absent in BKβ1 −/− mice. In pulmonary artery smooth muscle cells treated with NOS and COX inhibitors and loaded with the potentiometric dye, di-8-ANEPPS, 11,12-EET induced depolarization while the BK channel opener NS1619 elicited hyperpolarization indicating there was no effect of the EET on classical plasma membrane BK channels. In pulmonary artery smooth muscle cells a subpopulation of BK channels is localized in mitochondria. In these cells, 11,12-EET elicited an iberiotoxin-sensitive loss of mitochondrial membrane potential (JC-1 fluorescence) leading to plasma membrane depolarization, an effect not observed in BKβ1 −/− cells. Mechanistically, stimulation with 11,12-EET time-dependently induced the association of the BK α and β1 subunits. Our data indicate that in the absence of NO and prostacyclin 11,12-EET contributes to pulmonary vasoconstriction by stimulating the association of the α and β1 subunits of mitochondrial BK channels. The 11,12-EET-induced activation of BK channels results in loss of the mitochondrial membrane potential and depolarization of the pulmonary artery smooth muscle cells.

The Pharmacology of the Cytochrome P450 Epoxygenase/Soluble Epoxide Hydrolase Axis in the Vasculature and Cardiovascular Disease

“…Again these effects of 11,12-EET relied on the activation of PKA. Mechanistically, it seems that 11,12-EET induces the physical association of the BKa and BKb1 subunits on the mitochondrial membrane (Loot et al, 2012). Whether this is a phenomenon exclusive to pulmonary VSMCs remains to be clarified but is of potential interest given that mitochondrial BK channels in the heart are thought to play a role in protection from ischemic injury (for review, see Singh et al, 2012) and both EETs and sEH inhibition protect mitochondrial function after stress (Katragadda et al, 2009;Batchu et al, 2012b).…”

“…However, a BKb1-dependent component to the EET-induced pulmonary vasoconstriction could be demonstrated after NO synthase inhibition. Rather than targeting BK channel activity on the plasma membrane, it seems that 11,12-EET contributes to pulmonary vasoconstriction by stimulating the association of the a and b1 subunits of mitochondrial BK channels to disrupt the mitochondrial membrane potential and depolarize pulmonary artery smooth muscle cells (Loot et al, 2012). Certainly, a substantial subpopulation of BK channels in pulmonary artery smooth muscle is localized to mitochondria (Roth et al, 2009).…”

“…In cancer cells and astrocytes, 11,12-EET attenuated reactive oxygen species generation, loss of mitochondrial function, and caspase activation induced by toxic stimuli the antileukemia drug arsenic trioxide Sarkar et al, 2014). On the other hand, 11,12-EET was found to acutely reduce the mitochondrial potential in pulmonary smooth muscle 1130 Fleming cells by promoting the association of the BKa and BKb1 subunits on the mitochondrial membrane, ultimately leading to the depolarization and contraction of pulmonary artery smooth muscle cells (Loot et al, 2012).…”

Endothelial Cell Regulation of Pulmonary Vascular Tone, Inflammation, and Coagulation

“…Mechanistically, much remains to be resolved regarding the effect of EETs on EC and PASMC in the lung, and the reasons underlying their differential effects in the pulmonary and systemic circulation. However, it is thought that in the systemic circulation, EETs activate Ca 2+ -dependent K + -channels, leading to EC hyperpolarization, which then spreads to the SMC (67,136). In the pulmonary vasculature, however, EET stimulation results in activation of K + channels in the mitochondrial membrane, depolarizing the mitochondria and subsequently, the SMC itself (136).…”

The Biological Actions of 11,12-Epoxyeicosatrienoic Acid in Endothelial Cells Are Specific to theR/S-Enantiomer and Require the GsProtein

“…3) It has been possible to design and generate a series of stable and specific EET agonists and antagonists (Gauthier et al, 2002;Falck et al, 2003a;Yang et al, 2008). 4) Many of the biologic actions of the EETs are dependent on the activation of PKA (Wong et al, 2000;Fukao et al, 2001;Popp et al, 2002;Fleming et al, 2007;Loot et al, 2012).…”