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Arteries and veins are lined by nonproliferating endothelial cells that play a critical role in regulating blood flow. Endothelial cells also regulate tissue perfusion, metabolite exchange, and thrombosis. It is thought that endothelial cells rely on ATP generated via glycolysis, rather than mitochondrial oxidative phosphorylation, to fuel each of these energy-demanding processes. However, endothelial metabolism has mainly been studied in the context of proliferative cells, and little is known about energy production in endothelial cells within the fully formed vascular wall. Using intact arteries isolated from rats and mice, we show that inhibiting mitochondrial respiration disrupts endothelial control of vascular tone. Basal, mechanically activated, and agonist-evoked calcium activity in intact artery endothelial cells are each prevented by inhibiting mitochondrial ATP synthesis. Agonist-evoked calcium activity was also inhibited by blocking the transport of pyruvate, the master fuel for mitochondrial energy production, through the mitochondrial pyruvate carrier. The role for mitochondria in endothelial cell energy production is independent of species, sex, or vascular bed. These data show that a mitochondrial ATP supply is necessary for calcium-dependent, nitric oxide-mediated endothelial control of vascular tone, and identifies the critical role of endothelial mitochondrial energy production in fueling perfused blood vessel function., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Physiological Society.)

Hydrogen peroxide (H 2 O 2 ) is a mitochondrial-derived reactive oxygen species (ROS) that regulates vascular signalling transduction, vasocontraction and vasodilation. Although the physiological role of ROS in endothelial cells is acknowledged, the mechanisms underlying H 2 O 2 regulation of signalling in native, fully-differentiated endothelial cells is unresolved. In the present study, the effects of H 2 O 2 on Ca 2+ signalling were investigated in the endothelium of intact rat mesenteric arteries. Spontaneous local Ca 2+ signals and acetylcholine evoked Ca 2+ increases were inhibited by H 2 O 2 . H 2 O 2 inhibition of acetylcholine-evoked Ca 2+ signals was reversed by catalase. H 2 O 2 exerts its inhibition on the IP 3 receptor as Ca 2+ release evoked by photolysis of caged IP 3 was supressed by H 2 O 2 . H 2 O 2 suppression of IP 3 -evoked Ca 2+ signalling may be mediated by mitochondria. H 2 O 2 depolarized mitochondria membrane potential. Acetylcholine-evoked Ca 2+ release was inhibited by depolarisation of the mitochondrial membrane potential by the uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP) or complex 1 inhibitor, rotenone. We propose that the suppression of IP 3 -evoked Ca 2+ release by H 2 O 2 arises from the decrease in mitochondrial membrane potential. These results suggest that mitochondria may protect themselves against Ca 2+ overload during IP 3 -linked Ca 2+ signals by a H 2 O 2 mediated negative feedback depolarization of the organelle and inhibition of IP 3 -evoked Ca 2+ release., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Endothelial cells are reported to be glycolytic and to minimally rely on mitochondria for ATP generation. Rather than providing energy, mitochondria in endothelial cells may act as signaling organelles that control cytosolic Ca 2+ signaling or modify reactive oxygen species (ROS). To control Ca 2+ signaling, these organelles are often observed close to influx and release sites and may be tethered near Ca 2+ transporters. In this study, we used high-resolution, wide-field fluorescence imaging to investigate the regulation of Ca 2+ signaling by mitochondria in large numbers of endothelial cells (∼50 per field) in intact arteries from rats. We observed that mitochondria were mostly spherical or short-rod structures and were distributed widely throughout the cytoplasm. The density of these organelles did not increase near contact sites with smooth muscle cells. However, local inositol trisphosphate (IP 3 )-mediated Ca 2+ signaling predominated near these contact sites and required polarized mitochondria. Of note, mitochondrial control of Ca 2+ signals occurred even when mitochondria were far from Ca 2+ release sites. Indeed, the endothelial mitochondria were mobile and moved throughout the cytoplasm. Mitochondrial control of Ca 2+ signaling was mediated by ATP production, which, when reduced by mitochondrial depolarization or ATP synthase inhibition, eliminated local IP 3 -mediated Ca 2+ release events. ROS buffering did not significantly alter local Ca 2+ release events. These results highlight the importance of mitochondrial ATP production in providing long-range control of endothelial signaling via IP 3 -evoked local Ca 2+ release in intact endothelium., (© 2019 Wilson et al.)

Arteries and veins are lined by nonproliferating endothelial cells that play a critical role in regulating blood flow. Endothelial cells also regulate tissue perfusion, metabolite exchange, and thrombosis. It is thought that endothelial cells rely on ATP generated via glycolysis, rather than mitochondrial oxidative phosphorylation, to fuel each of these energy-demanding processes. However, endothelial metabolism has mainly been studied in the context of proliferative cells, and little is known about energy production in endothelial cells within the fully formed vascular wall. Using intact arteries isolated from rats and mice, we show that inhibiting mitochondrial respiration disrupts endothelial control of vascular tone. Basal, mechanically activated, and agonist-evoked calcium activity in intact artery endothelial cells are each prevented by inhibiting mitochondrial ATP synthesis. Agonist-evoked calcium activity was also inhibited by blocking the transport of pyruvate, the master fuel for mitochondrial energy production, through the mitochondrial pyruvate carrier. The role for mitochondria in endothelial cell energy production is independent of species, sex, or vascular bed. These data show that a mitochondrial ATP supply is necessary for calcium-dependent, nitric oxide-mediated endothelial control of vascular tone, and identifies the critical role of endothelial mitochondrial energy production in fueling perfused blood vessel function. [ABSTRACT FROM AUTHOR]

Background and Purpose: Ca2+ influx via TRPV4 channels triggers Ca2+ release from the IP3‐sensitive internal store to generate repetitive oscillations. Although mitochondria are acknowledged regulators of IP3‐mediated Ca2+ release, how TRPV4‐mediated Ca2+ signals are regulated by mitochondria is unknown. We show that depolarised mitochondria switch TRPV4 signalling from relying on Ca2+‐induced Ca2+ release at IP3 receptors to being independent of Ca2+ influx and instead mediated by ATP release via pannexins. Experimental Approach TRPV4‐evoked Ca2+ signals were individually examined in hundreds of cells in the endothelium of rat mesenteric resistance arteries using the indicator Cal520. Key Results: TRPV4 activation with GSK1016790A (GSK) generated repetitive Ca2+ oscillations that required Ca2+ influx. However, when the mitochondrial membrane potential was depolarised, by the uncoupler CCCP or complex I inhibitor rotenone, TRPV4 activation generated large propagating, multicellular, Ca2+ waves in the absence of external Ca2+. The ATP synthase inhibitor oligomycin did not potentiate TRPV4‐mediated Ca2+ signals. GSK‐evoked Ca2+ waves, when mitochondria were depolarised, were blocked by the TRPV4 channel blocker HC067047, the SERCA inhibitor cyclopiazonic acid, the PLC blocker U73122 and the inositol trisphosphate receptor blocker caffeine. The Ca2+ waves were also inhibited by the extracellular ATP blockers suramin and apyrase and the pannexin blocker probenecid. Conclusion and Implications: These results highlight a previously unknown role of mitochondria in shaping TRPV4‐mediated Ca2+ signalling by facilitating ATP release. When mitochondria are depolarised, TRPV4‐mediated release of ATP via pannexin channels activates plasma membrane purinergic receptors to trigger IP3‐evoked Ca2+ release. [ABSTRACT FROM AUTHOR]