Endothelial mitochondria regulate the intracellular Ca²⁺ response to fluid shear stress

Abstract

Shear stress is known to stimulate an intracellular free calcium concentration ([Ca(2+)]i) response in vascular endothelial cells (ECs). [Ca(2+)]i is a key second messenger for signaling that leads to vasodilation and EC survival. Although it is accepted that the shear-induced [Ca(2+)]i response is, in part, due to Ca(2+) release from the endoplasmic reticulum (ER), the role of mitochondria (second largest Ca(2+) store) is unknown. We hypothesized that the mitochondria play a role in regulating [Ca(2+)]i in sheared ECs. Cultured ECs, loaded with a Ca(2+)-sensitive fluorophore, were exposed to physiological levels of shear stress. Shear stress elicited [Ca(2+)]i transients in a percentage of cells with a fraction of them displaying oscillations. Peak magnitudes, percentage of oscillating ECs, and oscillation frequencies depended on the shear level. [Ca(2+)]i transients/oscillations were present when experiments were conducted in Ca(2+)-free solution (plus lanthanum) but absent when ECs were treated with a phospholipase C inhibitor, suggesting that the ER inositol 1,4,5-trisphosphate receptor is responsible for the [Ca(2+)]i response. Either a mitochondrial uncoupler or an electron transport chain inhibitor, but not a mitochondrial ATP synthase inhibitor, prevented the occurrence of transients and especially inhibited the oscillations. Knockdown of the mitochondrial Ca(2+) uniporter also inhibited the shear-induced [Ca(2+)]i transients/oscillations compared with controls. Hence, EC mitochondria, through Ca(2+) uptake/release, regulate the temporal profile of shear-induced ER Ca(2+) release. [Ca(2+)]i oscillation frequencies detected were within the range for activation of mechanoresponsive kinases and transcription factors, suggesting that dysfunctional EC mitochondria may contribute to cardiovascular disease by deregulating the shear-induced [Ca(2+)]i response.

Thirty Years of Saying NO

“…[34][35][36] The G proteins are coupled to phospholipase C producing inositol trisphosphate (IP 3 ), which in turn binds to IP 3 receptors on the sarco/endoplasmic reticulum (SR) causing release of Ca 2+ from this internal store. 15,34,[37][38][39][40][41][42][43][44][45] This process is facilitated by the presence of Ca 2+ -binding protein S100A1, which is coupled to IP 3 receptors. 46 The depletion of Ca 2+ from the SR causes SR-transmembrane protein stromal interaction molecule 1 to oligomerize and redistribute to the interface between the SR and the cell membrane, where it activates store-operated calcium channels of the latter, [40][41][42][43][44][45][47][48][49] In addition, certain G-protein-coupled receptors (eg, B 2 -bradykinin receptors) are coupled to ADP-ribosylcyclase, the enzyme generating cyclic ADP ribose which then binds to ryanodine receptors of the SR; these receptors are also activated by Ca 2+ released upon IP 3 receptors stimulation, thereby further enhancing the release of intracellular Ca 2+ (calcium-induced calcium release).…”

“…The localized increased amounts of Ca 2+ are taken up by the SR calcium-ATPase and the mitochondrial calcium uniporter. 45 The uptake of Ca 2+ in the mitochondria then triggers the activation of the sodium/calcium exchanger in the mitochondria and hence rhythmic calcium release (oscillatory calcium signals) from these organelles, presumably causing a more harmonious calcium-dependent activation of eNOS; however, exaggerated (eg, in endothelial cells exposed to high cholesterol levels) oscillatory mitochondrial calcium signals may lead to reduced eNOS activity. 45,[47][48][49] Such mitochondrial Ca 2+ release in turn activates IP 3 and ryanodine receptors, and ) instead of NO; (B) even as homodimers, significant O 2 .…”

“…45 The uptake of Ca 2+ in the mitochondria then triggers the activation of the sodium/calcium exchanger in the mitochondria and hence rhythmic calcium release (oscillatory calcium signals) from these organelles, presumably causing a more harmonious calcium-dependent activation of eNOS; however, exaggerated (eg, in endothelial cells exposed to high cholesterol levels) oscillatory mitochondrial calcium signals may lead to reduced eNOS activity. 45,[47][48][49] Such mitochondrial Ca 2+ release in turn activates IP 3 and ryanodine receptors, and ) instead of NO; (B) even as homodimers, significant O 2 . production will occur when the effective concentrations of l-Arg is reduced below the levels required to saturate the enzyme; and (C) when sufficient l-Arg and BH 4 bind to coupled NOS, the electron (e -) flux provided by nicotinamide adenine dinucleotide phosphate (NADPH) passes through FAD, FMN, CaM, and Heme Fe and is eventually used in NO production.…”

“…Ca 2+ binds to calmodulin, together combining with the calmodulin-binding domain of eNOS; this facilitates the electron flux from the reductase to the oxygenase domains of the enzyme initiating NO production (Figure 1, top). [24][25][26]29,[37][38][39][40][41][42][43][44][45][46][47][48][49] In addition, the Ca…”

“…15,[81][82][83][84][85] Shear stress is signaled through the glycocalyx (a network of membrane-bound proteoglycans and glycoproteins, including syndecans and glypicans, at the luminal surface of the endothelium), membrane-associated proteins localized to the intercellular junctions (integrins and platelet endothelial cell adhesion molecules [CD31]), and proteins (G-protein-coupled receptors, ion channels, and PKs) of the plasma membrane and the caveolae. [81][82][83]85 Mechanisms responsible for the initial phase (lasting from seconds to ≈30 minutes) of eNOS activation by increases in shear stress, which are associated with an augmented intracellular calcium concentration include (1) opening of TRPV1 and TRPV4 channels for Ca 2+ influx and of calcium-activated potassium (KCa 2.3 and KCa 3.1 subtypes) channels causing hyperpolarization and increasing the electrochemical driving force for calcium entry 40,82,85 ; (2) increased interaction between the oligosaccharide components of the glycocalyx and lectinic moiety of G-protein-coupled receptors leading to the sensitization of the latter; (3) release of ATP activating purinergic P2Y 2 receptors that are coupled by G q proteins to activation of the phospholipase C/IP 3 pathway 45,86 ; (4) activation of the local production of bradykinin that stimulates the release of NO through a G q -dependent mechanism; and (5) increased production of angiotensin 1-7. 15,80,81,84 Shear stress also causes conformational change of caveolae promoting dissociation of eNOS from caveolin-1 and association of the enzyme with calmodulin.…”