Damon Poburko

Dynamic Regulation of the Mitochondrial Proton Gradient during Cytosolic Calcium Elevations

Mitochondria extrude protons across their inner membrane to generate the mitochondrial membrane potential (ΔΨm) and pH gradient (ΔpHm) that both power ATP synthesis. Mitochondrial uptake and efflux of many ions and metabolites are driven exclusively by ΔpHm, whose in situ regulation is poorly characterized. Here, we report the first dynamic measurements of ΔpHm 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 ΔpHm (∼0.45) of HeLa cells at 37 °C were lower than previously reported. Unexpectedly, mitochondrial pH and ΔpHm decreased during cytosolic Ca2+ elevations. The drop in matrix pH was due to cytosolic acid generated by plasma membrane Ca2+-ATPases and transmitted to mitochondria by Pi/H+ symport and K+/H+ exchange, whereas the decrease in ΔpHm 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 Ca2+ and pH, mitochondria alkalinized and ΔpHm 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 Ca2+ caused a delayed and smaller alkalinization. These findings indicate that the mitochondrial matrix pH and ΔpHm are regulated by opposing Ca2+-dependent processes of stimulated mitochondrial respiration and cytosolic acidification.

The mechanism of phenylephrine-mediated [Ca2+]i oscillations underlying tonic contraction in the rabbit inferior vena cava

1 We characterized the mechanisms in vascular smooth muscle cells (VSMCs) that produce asynchronous, wave‐like Ca2+ oscillations in response to phenylephrine (PE). Confocal imaging was used to observe [Ca2+]i in individual VSMCs of intact inferior vena cava (IVC) from rabbits. 2 It was found that the Ca2+ waves were initiated by Ca2+ release from the sarcoplasmic reticulum (SR) via inositol 1,4,5‐trisphosphate‐sensitive SR Ca2+ release channels (IP3R channels) and that refilling of the SR Ca2+ store through the sarcoplasmic‐endoplasmic reticulum Ca2+‐ATPase (SERCA) was required for maintained generation of the repetitive Ca2+ waves. 3 Blockade of L‐type voltage‐gated Ca2+ channels (L‐type VGCCs) with nifedipine reduced the frequency of PE‐stimulated [Ca2+]i oscillations, while additional blockade of receptor‐operated channels/store‐operated channels (ROCs/SOCs) with SKF96365 abolished the remaining oscillations. Parallel force measurements showed that nifedipine inhibited PE‐induced tonic contraction by 27% while SKF96365 abolished it. This indicates that stimulated Ca2+ entry refills the SR to support the recurrent waves of SR Ca2+ release and that both L‐type VGCCs and ROCs/SOCs contribute to this process. 4 Application of the Na+‐Ca2+ exchanger (NCX) inhibitors 2′,4′‐dichlorobenzamil (forward‐ and reverse‐mode inhibitor) and KB‐R7943 (reverse‐mode inhibitor) completely abolished the nifedipine‐resistant component of [Ca2+]i oscillations and markedly reduced PE‐induced tone. 5 Thus, we conclude that each Ca2+ wave depends on initial SR Ca2+ release via IP3R channels followed by SR Ca2+ refilling through SERCA. Na+ entry through ROCs/SOCs facilitates Ca2+ entry through the NCX operating in the reverse mode, which refills the SR and maintains PE‐induced [Ca2+]i oscillations. In addition some Ca2+ entry through L‐type VGCCs and ROCs/SOCs serves to modulate the frequency of the oscillations and the magnitude of force development.

Transient Receptor Potential Channel 6–Mediated, Localized Cytosolic [Na+] Transients Drive Na+/Ca2+ Exchanger–Mediated Ca2+ Entry in Purinergically Stimulated Aorta Smooth Muscle Cells

The Na+/Ca2+ exchanger (NCX) is increasingly recognized as a physiological mediator of Ca2+ influx and significantly contributes to salt-sensitive hypertension. We recently reported that Ca2+ influx by the NCX (1) is the primary mechanism of Ca2+ entry in purinergically stimulated rat aorta smooth muscle cells and (2) requires functional coupling with transient receptor potential channel 6 nonselective cation channels. Using the Na+ indicator CoroNa Green, we now directly observed and characterized the localized cytosolic [Na+] ([Na+]i) elevations that have long been hypothesized to underlie physiological NCX reversal but that have never been directly shown. Stimulation of rat aorta smooth muscle cells caused both global and monotonic [Na+]i elevations and localized [Na+]i transients (LNats) at the cell periphery. Inhibition of nonselective cation channels with SKF-96365 (50 &mgr;mol/L) and 2-amino-4-phosphonobutyrate (75 &mgr;mol/L) reduced both global and localized [Na+]i elevations in response to ATP (1 mmol/L). This effect was mimicked by expression of a dominant negative construct of transient receptor potential channel 6. Selective inhibition of NCX-mediated Ca2+ entry with KB-R7943 (10 &mgr;mol/L) enhanced the LNats, whereas the global cytosolic [Na+] signal was unaffected. Inhibition of mitochondrial Na+ uptake with CGP-37157 (10 &mgr;mol/L) increased both LNats and global cytosolic [Na+] elevations. These findings directly demonstrate NCX regulation by LNats, which are restricted to subsarcolemmal, cytoplasmic microdomains. Analysis of the LNats, which facilitate Ca2+ entry via NCX, suggests that mitochondria limit the cytosolic diffusion of LNats generated by agonist-mediated activation of transient receptor potential channel 6–containing channels.

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.

Glutamate Transport Decreases Mitochondrial pH and Modulates Oxidative Metabolism in Astrocytes

During synaptic activity, the clearance of neuronally released glutamate leads to an intracellular sodium concentration increase in astrocytes that is associated with significant metabolic cost. The proximity of mitochondria at glutamate uptake sites in astrocytes raises the question of the ability of mitochondria to respond to these energy demands. We used dynamic fluorescence imaging to investigate the impact of glutamatergic transmission on mitochondria in intact astrocytes. Neuronal release of glutamate induced an intracellular acidification in astrocytes, via glutamate transporters, that spread over the mitochondrial matrix. The glutamate-induced mitochondrial matrix acidification exceeded cytosolic acidification and abrogated cytosol-to-mitochondrial matrix pH gradient. By decoupling glutamate uptake from cellular acidification, we found that glutamate induced a pH-mediated decrease in mitochondrial metabolism that surpasses the Ca2+-mediated stimulatory effects. These findings suggest a model in which excitatory neurotransmission dynamically regulates astrocyte energy metabolism by limiting the contribution of mitochondria to the metabolic response, thereby increasing the local oxygen availability and preventing excessive mitochondrial reactive oxygen species production.

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

Subplasmalemmal ion fluxes have global effects on Ca2+ signaling in vascular smooth muscle. Measuring cytoplasmic and mitochondrial [Ca2+]and [Na+], we previously showed that mitochondria buffer both subplasmalemmal cytosolic [Ca2+] and [Na+] in vascular smooth muscle cells. We have now directly measured sarcoplasmic reticulum [Ca2+] in aortic smooth muscle cells, revealing that mitochondrial Na+/Ca2+ exchanger inhibition with CGP-37157 impairs sarcoplasmic reticulum Ca2+ 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 [Ca2+] elevations, despite increased cytosolic [Ca2+] elevations. Reversal of plasmalemmal Na+/Ca2+ exchange was the primary Ca2+ 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+/Ca2+ exchanger to funnel Ca2+ into the sarcoplasmic reticulum and minimize cytosolic [Ca2+] elevations that might otherwise contribute to hypertensive or proliferative vasculopathies.

Agonist-induced mitochondrial Ca2+ transients in smooth muscle

We investigated the role of mitochondria (MT) in calcium signaling in a culture of rat aortic smooth muscle cells. We used targeted aequorin to selectively measure [Ca2+] in this organelle. Our results reveal that smooth muscle cell stimulation with agonists causes a large, transient increase in mitochondrial [Ca2+] ([Ca2+]m). This large transient can be blocked with inhibitors of the sarco‐endoplasmic reticulum Ca2+‐ATPase, suggesting a close relationship between the sarcoplasmic reticulum (SR) and the mitochondria. FCCP completely abolished the response to agonists, and targeted mitochondrial GFP revealed a vast tubular network of MT in these cells. When added before stimulation with ATP, IP3 inhibitors partially blocked the ATP‐induced rise in mitochondrial Ca2+ release. The role of the Na+/Ca2+ exchanger (NCX) was examined by removing extracellular Na+. This procedure prevented the decrease in the [Ca2+]m transient normally seen on removal of extracellular Ca2+. We propose a functional linkage of MT and SR dependent on a narrow junctional space between the two organelles in which Ca2+ diffusion is restricted. Approximately half of the mitochondria appear to be associated with the superficial SR, which communicates with the extracellular space via NCX.—Szado, T., Kuo, K.‐H., Bernard‐Helary, K., Poburko, D., Lee, C. H., Seow, C., Ruegg, U. T., van Breemen, C. Agonist‐induced mitochondrial Ca2+ transients in smooth muscle. FASEB J. 17, 28–37 (2003)

Postsynaptic GluA1 enables acute retrograde enhancement of presynaptic function to coordinate adaptation to synaptic inactivity

Prolonged blockade of AMPA-type glutamate receptors in hippocampal neuron cultures leads to homeostatic enhancements of pre- and postsynaptic function that appear correlated at individual synapses, suggesting some form of transsynaptic coordination. The respective modifications are important for overall synaptic strength but their interrelationship, dynamics, and molecular underpinnings are unclear. Here we demonstrate that adaptation begins postsynaptically but is ultimately communicated to presynaptic terminals and expressed as an accelerated turnover of synaptic vesicles. Critical postsynaptic modifications occur over hours, but enable retrograde communication within minutes once AMPA receptor (AMPAR) blockade is removed, causing elevation of both spontaneous and evoked vesicle fusion. The retrograde signaling does not require spiking activity and can be interrupted by NBQX, philanthotoxin, postsynaptic BAPTA, or external sequestration of BDNF, consistent with the acute release of retrograde messenger, triggered by postsynaptic Ca2+ elevation via Ca2+-permeable AMPARs.

Ca2+ signaling in smooth muscle: TRPC6, NCX, and LNats in nanodomains

Following the recent observation of localized cytosolic subplasmalemmal [Na+] elevations (LNats) in rat aortic smooth muscle cells, we discuss here the current evidence for the structural and molecular roles of cytosolic nanodomains at close junctions of the plasma membrane (PM) and sarcoplasmic reticulum (SR) in the generation of LNats. These junctions, the loss of which might contribute to vascular aging and disease, provide a platform for ion metabolism signalplexes and the interaction of localized Na+ and Ca2+ gradients. We moreover suggest the existence in the junctions of a Na+ diffusional barrier as a necessary condition for the generation of LNats. LNats are likely a fundamental feature of near membrane ion signaling in many cell types, and their discovery offers new possibilities for elucidating the mechanism, function and pathogenesis of Na+ and Ca2+ signaling nanodomains.

Amiloride derivatives induce apoptosis by depleting ER Ca2+ stores in vascular endothelial cells

Background and purpose:  Amiloride derivatives are blockers of the Na+/H+ exchanger (NHE) and at micromolar concentrations have protective effects on cardiac and brain ischaemia/reperfusion injury but at higher concentrations also induce apoptosis. Here, we aimed to elucidate the mechanism related to this cytotoxic action.