Regulation of the mitochondrial proton gradient by cytosolic Ca2+ signals

Mitochondria convert the energy stored in carbohydrate and fat into ATP molecules that power enzymatic reactions within cells, and this process influences cellular calcium signals in several ways. By providing ATP to calcium pumps at the plasma and intracellular membranes, mitochondria power the calcium gradients that drive the release of Ca2+ from stores and the entry of Ca2+ across plasma membrane channels. By taking up and subsequently releasing calcium ions, mitochondria determine the spatiotemporal profile of cellular Ca2+ signals and the activity of Ca2+-regulated proteins, including Ca2+ entry channels that are themselves part of the Ca2+ circuitry. Ca2+ elevations in the mitochondrial matrix, in turn, activate Ca2+-dependent enzymes that boost the respiratory chain, increasing the ability of mitochondria to buffer calcium ions. Mitochondria are able to encode and decode Ca2+ signals because the respiratory chain generates an electrochemical gradient for protons across the inner mitochondrial membrane. This proton motive force (Δp) drives the activity of the ATP synthase and has both an electrical component, the mitochondrial membrane potential (ΔΨ m ), and a chemical component, the mitochondrial proton gradient (ΔpH m ). ΔΨ m contributes about 190mV to Δp and drives the entry of Ca2+ across a recently identified Ca2+-selective channel known as the mitochondrial Ca2+ uniporter. ΔpH m contributes ~30mV to Δp and is usually ignored or considered a minor component of mitochondria respiratory state. However, the mitochondrial proton gradient is an essential component of the chemiosmotic theory formulated by Peter Mitchell in 1961 as ΔpH m sustains the entry of substrates and metabolites required for the activity of the respiratory chain and drives the activity of electroneutral ion exchangers that allow mitochondria to maintain their osmolarity and volume. In this review, we summarize the mechanisms that regulate the mitochondrial proton gradient and discuss how thermodynamic concepts derived from measurements in purified mitochondria can be reconciled with our recent findings that mitochondria have high proton permeability in situ and that ΔpH m decreases during mitochondrial Ca2+ elevation

Droplet Digital PCR Shows the D-Loop to be an Error Prone Locus for Mitochondrial DNA Copy Number Determination

Absolute quantification of mitochondrial DNA copy number (mCN) provides important insights in many fields of research including cancer, cardiovascular and reproductive health. Droplet digital PCR (ddPCR) natively reports absolute copy number, and we have developed a single-dye, multiplex assay to measure rat mCN that is accurate, precise and affordable. We demonstrate simple methods to optimize this assay and to determine nuclear reference pseudogene copy number to extend the range of mCN that can be measured with this assay. We evaluated two commonly used mitochondrial DNA reference loci to determine mCN, the ND1 gene and the D-Loop. Harnessing the absolute measures of ddPCR, we found that the D-Loop amplifies with a copy number of ~1.0–1.5 relative to other sites on the mitochondrial genome. This anomalous copy number varied significantly between rats and tissues (aorta, brain, heart, liver, soleus muscle). We advocate for avoiding the D-Loop as a mitochondrial reference in future studies of mCN. Further, we report a novel approach to quantifying immunolabelled mitochondrial DNA that provides single-cell estimates of mCN that closely agree with the population analyses by ddPCR. The combination of these assays represents a cost-effective and powerful suite of tools to study mCN

Mitochondria and microdomains in vascular smooth muscle cell Ca²⁺ signalling

Contraction of vascular smooth muscle (VSM) is regulated by fluctuations in the intracellular concentration of free ionic calcium ([Ca²⁺][sub i]). The spatio-temporal regulation of [Ca²⁺][sub i] relies on the sub-cellular architecture of the smooth muscle cell and the juxtaposition of opposing plasmalemma (PM), sarcoplasmic reticulum (SR) and mitochondria. This thesis addresses two related aspects of Ca²⁺-signaling in VSM: 1) basal Ca²⁺-entry across the PM and 2) mitochondrial Ca²⁺-uptake during agonist mediated stimulation in cultured rat aorta smooth muscle cells. Basal Ca²⁺-entry into resting cells, measured with radio-labeled ⁴⁵Ca²⁺, was blocked (~80%) by organic inhibitors of L-type voltage-gated Ca²⁺-channels (nifedipine), store-/receptoroperated Ca²⁺-channels (SKF-96365) and inositol-1,4,5-trisphosphate receptors (IP₃R) (2-APB). At increasing concentrations, gadolium (Gd³⁺) biphasically inhibited Ca²⁺-uptake. The maximal effect of the first phase (100μM Gd³⁺) was equally as effective as combined treatment with 2-APB and SKF-96365. At 0.2-10mM, Gd³⁺ inhibited Ca²⁺ influx to a greater extent than the organic inhibitors. We concluded that basal Ca²⁺ entry primarily occurred via basal activity of excitable channels, and PCR analysis suggests this influx to involve L-type voltage-gated Ca²⁺-channels, and TRPC1, TRPC4 and TRPC6. Next, we used Ca²⁺-sensitive proteins (aequorins and pericams) and dyes (fura-2) to measure parallel [Ca²⁺] changes in the mitochondrial matrix, subplasmalemmal cytosol and bulk cytosol. Replacing extracellular Na⁺ with n-methyl-d-glucamine (NMDG) caused Ca²⁺-entry by reversal of the Na⁺/Ca²⁺-exchanger (NCX), which was selectively blocked by KB-R7943. NCX-reversal increased mitochondrial and subplasmalemmal but not bulk cytosolic [Ca²⁺] revealing a local interaction of the SR, NCX and mitochondria. Furthermore, NCX-reversal and mitochondrial Ca²⁺-uptake appear to occur during the [Ca²⁺][sub i] plateau phase of the response to purinergic stimulation (ATP). However, this mitochondrial Ca²⁺-uptake does not increase [Ca²⁺][sub MT] because of a compensatory stimulation of mitochondrial Ca²⁺-extrusion (blocked by CGP-37157). Finally, we dissected the [Ca²⁺][sub MT] response to SR Ca²⁺-release in response to agonist-mediated stimulation. ATP and [ARG⁸]-vasopressin transiently increased [Ca²⁺][sub MT] by activating both IP₃R and ryanodine receptors (RyR) (selectively inhibited by 2-APB and procaine). Image analysis of fluorescently labeled mitochondria, IP₃R and RyR corroborated functional evidence that IP₃R and RyR release Ca²⁺ from separate sub-compartments of the SR and that physiological [Ca²⁺][sub MT] elevations rely on IP₃R-RyR cross-talk.Medicine, Faculty ofAnesthesiology, Pharmacology and Therapeutics, Department ofGraduat

Regulation of the mitochondrial proton gradient by cytosolic Ca(2+) signals

Mitochondria convert the energy stored in carbohydrate and fat into ATP molecules that power enzymatic reactions within cells, and this process influences cellular calcium signals in several ways. By providing ATP to calcium pumps at the plasma and intracellular membranes, mitochondria power the calcium gradients that drive the release of Ca(2+) from stores and the entry of Ca(2+) across plasma membrane channels. By taking up and subsequently releasing calcium ions, mitochondria determine the spatiotemporal profile of cellular Ca(2+) signals and the activity of Ca(2+)-regulated proteins, including Ca(2+) entry channels that are themselves part of the Ca(2+) circuitry. Ca(2+) elevations in the mitochondrial matrix, in turn, activate Ca(2+)-dependent enzymes that boost the respiratory chain, increasing the ability of mitochondria to buffer calcium ions. Mitochondria are able to encode and decode Ca(2+) signals because the respiratory chain generates an electrochemical gradient for protons across the inner mitochondrial membrane. This proton motive force (Δp) drives the activity of the ATP synthase and has both an electrical component, the mitochondrial membrane potential (ΔΨ( m )), and a chemical component, the mitochondrial proton gradient (ΔpH( m )). ΔΨ( m ) contributes about 190 mV to Δp and drives the entry of Ca(2+) across a recently identified Ca(2+)-selective channel known as the mitochondrial Ca(2+) uniporter. ΔpH( m ) contributes ~30 mV to Δp and is usually ignored or considered a minor component of mitochondria respiratory state. However, the mitochondrial proton gradient is an essential component of the chemiosmotic theory formulated by Peter Mitchell in 1961 as ΔpH( m ) sustains the entry of substrates and metabolites required for the activity of the respiratory chain and drives the activity of electroneutral ion exchangers that allow mitochondria to maintain their osmolarity and volume. In this review, we summarize the mechanisms that regulate the mitochondrial proton gradient and discuss how thermodynamic concepts derived from measurements in purified mitochondria can be reconciled with our recent findings that mitochondria have high proton permeability in situ and that ΔpH( m ) decreases during mitochondrial Ca(2+) elevations

Regulation of plasma membrane calcium fluxes by mitochondria

The role of mitochondria in cell signaling is becoming increasingly apparent, to an extent that the signaling role of mitochondria appears to have stolen the spotlight from their primary function as energy producers. In this chapter, we will review the ionic basis of calcium handling by mitochondria and discuss the mechanisms that these organelles use to regulate the activity of plasma membrane calcium channels and transporters

Dynamic regulation of the mitochondrial proton gradient during cytosolic calcium elevations

Mitochondria extrude protons across their inner membrane to generate the mitochondrial membrane potential (ΔΨ(m)) and pH gradient (ΔpH(m)) that both power ATP synthesis. Mitochondrial uptake and efflux of many ions and metabolites are driven exclusively by ΔpH(m), whose in situ regulation is poorly characterized. Here, we report the first dynamic measurements of ΔpH(m) in living cells, using a mitochondrially targeted, pH-sensitive YFP (SypHer) combined with a cytosolic pH indicator (5-(and 6)-carboxy-SNARF-1). The resting matrix pH (∼7.6) and ΔpH(m) (∼0.45) of HeLa cells at 37 °C were lower than previously reported. Unexpectedly, mitochondrial pH and ΔpH(m) decreased during cytosolic Ca(2+) elevations. The drop in matrix pH was due to cytosolic acid generated by plasma membrane Ca(2+)-ATPases and transmitted to mitochondria by P(i)/H(+) symport and K(+)/H(+) exchange, whereas the decrease in ΔpH(m) reflected the low H(+)-buffering power of mitochondria (∼5 mm, pH 7.8) compared with the cytosol (∼20 mm, pH 7.4). Upon agonist washout and restoration of cytosolic Ca(2+) and pH, mitochondria alkalinized and ΔpH(m) increased. In permeabilized cells, a decrease in bath pH from 7.4 to 7.2 rapidly decreased mitochondrial pH, whereas the addition of 10 μm Ca(2+) caused a delayed and smaller alkalinization. These findings indicate that the mitochondrial matrix pH and ΔpH(m) are regulated by opposing Ca(2+)-dependent processes of stimulated mitochondrial respiration and cytosolic acidification

Mitochondrial regulation of sarcoplasmic reticulum Ca2+ content in vascular smooth muscle cells

Subplasmalemmal ion fluxes have global effects on Ca(2+) signaling in vascular smooth muscle. Measuring cytoplasmic and mitochondrial [Ca(2+)]and [Na(+)], we previously showed that mitochondria buffer both subplasmalemmal cytosolic [Ca(2+)] and [Na(+)] in vascular smooth muscle cells. We have now directly measured sarcoplasmic reticulum [Ca(2+)] in aortic smooth muscle cells, revealing that mitochondrial Na(+)/Ca(2+) exchanger inhibition with CGP-37157 impairs sarcoplasmic reticulum Ca(2+) refilling during purinergic stimulation. By overexpressing hFis1 to remove mitochondria from the subplasmalemmal space, we show that the rate and extent of sarcoplasmic reticulum refilling is augmented by a subpopulation of peripheral mitochondria. In ATP-stimulated cells, hFis-1-mediated relocalization of mitochondria impaired the sarcoplasmic reticulum refilling process and reduced mitochondrial [Ca(2+)] elevations, despite increased cytosolic [Ca(2+)] elevations. Reversal of plasmalemmal Na(+)/Ca(2+) exchange was the primary Ca(2+) entry mechanism following ATP stimulation, based on the effects of KB-R7943. We propose that subplasmalemmal mitochondria ensure efficient sarcoplasmic reticulum refilling by cooperating with the plasmalemmal Na(+)/Ca(2+) exchanger to funnel Ca(2+) into the sarcoplasmic reticulum and minimize cytosolic [Ca(2+)] elevations that might otherwise contribute to hypertensive or proliferative vasculopathies

OPA1 promotes pH flashes that spread between contiguous mitochondria without matrix protein exchange

The chemical nature and functional significance of mitochondrial flashes associated with fluctuations in mitochondrial membrane potential is unclear. Using a ratiometric pH probe insensitive to superoxide, we show that flashes reflect matrix alkalinization transients of ∼0.4 pH units that persist in cells permeabilized in ion-free solutions and can be evoked by imposed mitochondrial depolarization. Ablation of the pro-fusion protein Optic atrophy 1 specifically abrogated pH flashes and reduced the propagation of matrix photoactivated GFP (paGFP). Ablation or invalidation of the pro-fission Dynamin-related protein 1 greatly enhanced flash propagation between contiguous mitochondria but marginally increased paGFP matrix diffusion, indicating that flashes propagate without matrix content exchange. The pH flashes were associated with synchronous depolarization and hyperpolarization events that promoted the membrane potential equilibration of juxtaposed mitochondria. We propose that flashes are energy conservation events triggered by the opening of a fusion pore between two contiguous mitochondria of different membrane potentials, propagating without matrix fusion to equilibrate the energetic state of connected mitochondria

Estrogen modulation of endothelium-derived relaxing factors by human endothelial cells

We report the modulatory effects of estrogen on release of endothelium-derived relaxing factors (EDRFs) in a human endothelial cell line, EA.hy926. Using bioassay, we showed that EA.hy926 released EDRF including nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) measured by relaxation of pre-contracted endothelium-denuded rabbit aortic rings. This EDRF production was significantly higher in cells treated for 24 h with 17-β-estradiol (10 mol/L) than control cells. Addition of l-NAME to the perfusate of cells caused the relaxation induced by the endothelial cell perfusate to become transient and abolished the enhancement of relaxation due to estrogen treatment. Addition of K channel blockers to the perfusate abolished the l-NAME-resistant relaxation of the bioassay ring. Using real-time PCR, we demonstrated that eNOS expression in estrogen-treated cells was significantly higher than controls. These results show that estrogen exerts a potentially important vasculo-protective effect by stimulating NO but not EDHF production. © 2004 Elsevier Inc. All rights reserved. -6 C