Mitochondrial ATP production provides long-range control of endothelial inositol trisphosphate–evoked calcium signaling

Abstract

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 Ca2+ signaling or modify reactive oxygen species (ROS). To control Ca2+ signaling, these organelles are often observed close to influx and release sites and may be tethered near Ca2+ transporters. In this study, we used high-resolution, wide-field fluorescence imaging to investigate the regulation of Ca2+ 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 (IP3)-mediated Ca2+ signaling predominated near these contact sites and required polarized mitochondria. Of note, mitochondrial control of Ca2+ signals occurred even when mitochondria were far from Ca2+ release sites. Indeed, the endothelial mitochondria were mobile and moved throughout the cytoplasm. Mitochondrial control of Ca2+ signaling was mediated by ATP production, which, when reduced by mitochondrial depolarization or ATP synthase inhibition, eliminated local IP3-mediated Ca2+ release events. ROS buffering did not significantly alter local Ca2+ release events. These results highlight the importance of mitochondrial ATP production in providing long-range control of endothelial signaling via IP3-evoked local Ca2+ release in intact endothelium.

Endothelial TRPV4 channels modulate vascular tone by Ca2+‐induced Ca2+ release at inositol 1,4,5‐trisphosphate receptors

“…To crosscheck the contribution of IP 3 Rs in the propagating Ca 2+ waves, we used caffeine—a potent inhibitor of IP 3 Rs (Ehrlich, Kaftan, Bezprozvannaya, & Bezprozvanny, ; Parker & Ivorra, ; Saleem, Tovey, Molinski, & Taylor, ). Caffeine, which does not evoke Ca 2+ release in the endothelial cells under study and inhibits Ca 2+ release evoked by IP 3 (Wilson et al, ), also blocked GSK‐evoked propagating Ca 2+ waves (Figure S8). Interestingly, caffeine also reduced the slow global Ca 2+ rise suggesting an effect of caffeine also on TRPV4 channels.…”

“…Caffeine, while often used as a RyR activator, is a potent inhibitor of IP 3 Rs in the same concentration range used to activate RyR (Ehrlich et al, ; Parker & Ivorra, ; Saleem et al, ). Caffeine does not evoke Ca 2+ release in endothelial cells (Wilson, Saunter, Girkin, & McCarron, ) and inhibits Ca 2+ release induced by photolysis of caged‐IP 3 in the endothelium (Wilson et al, ). That caffeine was effective in inhibiting propagating Ca 2+ waves supports the conclusion that IP 3 Rs contribute to TRPV4‐mediated Ca 2+ responses.…”

“…In some experiments, the endothelium was loaded with Cal‐520 (5 μM) and a membrane permeant, caged IP 3 (5 μM), and the Ca 2+ response to local photolysis of caged IP 3 examined. In these experiments, the endothelium was imaged as above, and a xenon flash lamp (Rapp Optoelecktronic, Germany) was used to uncage IP 3 (Olson, Chalmers, & McCarron, ; Olson, Sandison, Chalmers, & McCarron, ; Wilson et al, ).…”

“…While RyRs may be expressed in endothelial cells (Moccia, Berra‐Romani, & Tanzi, ; Mumtaz, Burdyga, Borisova, Wray, & Burdyga, ; Rusko, Wang, & Vanbreemen, ), the functional role of the channels in the endothelium is not clear given that RyR activators such as caffeine do not increase cytoplasmic Ca 2+ in endothelial cells (Borisova, Wray, Eisner, & Burdyga, ; Wilson et al, ; Wilson, Lee, & McCarron, ). Ca 2+ release from the internal store in endothelial cells may be mediated mainly by IP 3 Rs (Mumtaz et al, ; Wilson et al, ). Ca 2+ release via IP 3 R occurs in response to physical forces and circulating vasoactive substances and contributes to the control of several vascular activities such as vasorelaxation, endothelial permeability, and production of anti‐thrombotic factors (Sun, Geyer, & Komarova, ).…”

FK506 regulates Ca2+ release evoked by inositol 1,4,5‐trisphosphate independently of FK‐binding protein in endothelial cells

“…The higher percentage of cells showing basal activity is likely to be a consequence of the smaller number of cells in the endothelial patches (when compared to the intact artery), and the use of sodium pyruvate in the PSS which leads to a greater basal Ca 2+ response to shear stress (Wilson, Lee, & McCarron, 2016). The lower number of cells showing baseline spontaneous Ca 2+ signals in caffeine probably reflects a caffeine-induced inhibition of IP 3 R activity that generates the spontaneous activity (Brown, Sayers, Kirk, Michell, & Michelangeli, 1992;Missiaen, Taylor, & Berridge, 1992;Parker & Ivorra, 1991;Wilson et al, 2019). These results show that ACh evokes a substantial Ca 2+ increase while caffeine does not.…”