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

Rory Edward Morty,Annemarieke E Loot,Matthias Oelze,Benjamin Keserü,Ingrid Fleming,Isabelle Moneke,Andreas Daiber,Tetyana Syzonenko

doi:10.1371/journal.pone.0046065

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.

Highlights

Acute hypoxic pulmonary vasoconstriction is an adaptive response of the pulmonary circulation that directs blood flow from poorly oxygenated to better ventilated areas thereby maintaining pulmonary gas exchange [1]
Among the intracellular mediators that are generated during acute hypoxia are the cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) [6,7], and preventing their metabolism by inhibiting the soluble epoxide hydrolase can markedly potentiate hypoxic pulmonary vasoconstriction [8]
As vascular responses that are dependent on the activation of BK channels are frequently only detectable in the absence of nitric oxide (NO) and PGI2 [24], we assessed the sensitivity of acute hypoxic vasoconstriction to soluble epoxide hydrolase (sEH) inhibition in the presence of L-NA (300 mmol/L) and diclofenac (10 mmol/L)

Summary

Introduction

Acute hypoxic pulmonary vasoconstriction is an adaptive response of the pulmonary circulation that directs blood flow from poorly oxygenated to better ventilated areas thereby maintaining pulmonary gas exchange [1]. To-date, the pulmonary oxygen sensor and the signaling cascade leading to hypoxic pulmonary vasoconstriction have not been fully elucidated. The most likely candidates for pulmonary O2 sensing are the mitochondria of the pulmonary resistance artery smooth muscle cells. It is unclear whether mitochondria increase or decrease reactive oxygen species (ROS) output in response to hypoxia, inhibition of the electron transport chain, as occurs under moderate hypoxia, does seem to be a prerequisite for hypoxic pulmonary vasoconstriction [2,3]. Among the intracellular mediators that are generated during acute hypoxia are the cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) [6,7], and preventing their metabolism by inhibiting the soluble epoxide hydrolase (sEH) can markedly potentiate hypoxic pulmonary vasoconstriction [8]

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Results

Conclusion