Karlene K. Gunter

Calcium and mitochondria.

The literature suggests that the physiological functions for which mitochondria sequester Ca2+ are (1) to stimulate and control the rate of oxidative phosphorylation, (2) to induce the mitochondrial permeability transition (MPT) and perhaps apoptotic cell death, and (3) to modify the shape of cytosolic Ca2+ pulses or transients. There is strong evidence that intramitochondrial Ca2+ controls both the rate of ATP production by oxidative phosphorylation and induction of the MPT. Since the results of these processes are so divergent, the signals inducing them must not be ambiguous. Furthermore, as pointed out by Balaban [J. Mol. Cell. Cardiol. 34 (2002 ) 11259–11271], for any repetitive physiological process dependent on intramitochondrial free Ca2+ concentration ([Ca2+]m), a kind of intramitochondrial homeostasis must exist so that Ca2+ influx during the pulse is matched by Ca2+ efflux during the period between pulses to avoid either Ca2+ buildup or depletion. In addition, mitochondrial Ca2+ transport modifies both spatial and temporal aspects of cytosolic Ca2+ signaling. Here, we look at the amounts of Ca2+ necessary to mediate the functions of mitochondrial Ca2+ transport and at the mechanisms of transport themselves in order to set up a hypothesis about how the mechanisms carry out their roles. The emphasis here is on isolated mitochondria and on general mitochondrial properties in order to focus on how mitochondria alone may function to fulfill their physiological roles even though the interactions of mitochondria with other organelles, particularly with endoplasmic and sarcoplasmic reticulum [Sci. STKE re1 (2004) 1–9], may also influence this story.

Neurons overexpressing heme oxygenase-1 resist oxidative stress-mediated cell death.

Abstract : This is the first report on the protective effect of heme oxygenase‐1 (HO‐1) overexpression against oxidative stress‐mediated neuronal cell death and demonstration of a decreased production of oxygen free radicals when HO‐1 levels are increased. HO‐1 is the heat shock/stress cognate of the heat shock protein 32 family of proteins. A known function of these proteins is α‐meso bridge‐specific cleavage of the heme molecule. For the present study, we used cerebellar granular neurons (CGNs) isolated from homozygous transgenic (Tg) mice that overexpress HO‐1 under neuron‐specific enolase control and nontransgenic (Ntg) littermates. The Tg mouse CGNs were characterized by increased levels of HO‐1 mRNA and protein, a lower resting intracellular calcium concentration, and a reduced HO‐1 transcriptional response to glutamate‐mediated oxidative stress. Compared with the Ntg neurons, when exposed to glutamate (30 μM or 3 mM), the magnitude of cell viability was increased and the number of cells exhibiting membrane permeability and chromatin condensation were significantly decreased in the Tg CGN cultures. The population of neurons surviving glutamate toxicity decreased when HO‐1 activity was inhibited by a peptide inhibitor. The neuroprotective effect by HO‐1 was extended to H2O2‐induced cell death. The mechanism of protection may involve in part a reduced production of reactive oxygen species upon exposure to glutamate. We suggest that induction of HO‐1 by pharmacological means may be a novel approach to amelioration of oxidative insults to neurons.

Transport of calcium by mitochondria

The identification of intramitochondrial free calcium ([Ca2+m) as a primary metabolic mediator [see Hansford (this volume) and Gunter, T. E., Gunter, K. K., Sheu, S.-S., and Gavin, C. E. (1994)Am. J. Physiol.267, C313–C339, for reviews] has emphasized the importance of understanding the characteristics of those mechanisms that control [Ca2+]m. In this review, we attempt to update the descriptions of the mechanisms that mediate the transport of Ca2+ across the mitochondrial inner membrane, emphasizing the energetics of each mechanism. New concepts within this field are reviewed and some older concepts are discussed more completely than in earlier reviews. The mathematical forms of the membrane potential dependence and concentration dependence of the uniporter are interpolated in such a way as to display the convenience of consideringVmax to be an explicit function of the membrane potential. Recent evidence for a transient rapid conductance state of the uniporter is discussed. New evidence concerning the energetics and stoichiometries of both Na+-dependent and Na+-independent efflux mechanisms is reviewed. Explicit mathematical expressions are used to describe the energetics of the system and the kinetics of transport via each Ca2+ transport mechanism.

Mn2+ sequestration by mitochondria and inhibition of oxidative phosphorylation

Manganese is known to accumulate in mitochondria and in mitochondria-rich tissues in vivo. Although Ca2+ enhances mitochondrial Mn2+ uptake, ATP-bound Mn2+ is not sequestered by suspended rat brain mitochondria, and ATP binds Mn2+ even more tightly than it binds Mg2+. Physiological levels of the polyamine spermine enhanced 54 Mn2+ uptake at the low [Ca2+]s characteristic of unstimulated cells (approximately 100 nM). With succinate as substrate, Mn2+ inhibited oxygen consumption by suspensions of rat liver mitochondria after the addition of ADP but not after the addition of uncoupler. With glutamate/malate as substrate, Mn2+ inhibited ADP-stimulated respiration and also slightly inhibited uncoupler-stimulated respiration. State 4 (resting) respiration was unchanged in all cases, indicating that the inner membrane retained its impermeability to protons. These results suggest that Mn2+ was not oxidized and that it can interfere directly with oxidative phosphorylation, most likely by binding to the F1 ATPase. Mn2+ may also bind to the NADH dehydrogenase complex, but not strongly enough to affect electron transport in vivo. It is suggested that accumulation of manganese within the mitochondria of globus pallidus may help explain the distinctive pathology of manganism.

The Ca2+ transport mechanisms of mitochondria and Ca2+ uptake from physiological-type Ca2+ transients

Mitochondria contain a sophisticated system for transporting Ca2+. The existence of a uniporter and of both Na+-dependent and -independent efflux mechanisms has been known for years. Recently, a new mechanism, called the RaM, which seems adapted for sequestering Ca2+ from physiological transients or pulses has been discovered. The RaM shows a conductivity at the beginning of a Ca2+ pulse that is much higher than the conductivity of the uniporter. This conductivity decreases very rapidly following the increase in [Ca2+] outside the mitochondria. This decrease in the Ca2+ conductivity of the RaM is associated with binding of Ca2+ to an external regulatory site. When liver mitochondria are exposed to a sequence of pulses, uptake of labeled Ca2+ via the RaM appears additive between pulses. Ruthenium red inhibits the RaM in liver mitochondria but much larger amounts are required than for inhibition of the mitochondrial Ca2+ uniporter. Spermine, ATP and GTP increase Ca2+ uptake via the RaM. Maximum uptake via the RaM from a single Ca2+ pulse in the physiological range has been observed to be approximately 7 nmole/mg protein, suggesting that Ca2+ uptake via the RaM and uniporter from physiological pulses may be sufficient to activate the Ca2+-sensitive metabolic reactions in the mitochondrial matrix which increase the rate of ATP production. RaM-mediated Ca2+ uptake has also been observed in heart mitochondria. Evidence for Ca2+ uptake into the mitochondria in a variety of tissues described in the literature is reviewed for evidence of participation of the RaM in this uptake. Possible ways in which the differences in transport via the RaM and the uniporter may be used to differentiate between metabolic and apoptotic signaling are discussed.

Uptake of Calcium by Mitochondria: Transport and Possible Function

Vertebrate mitochondria contain a complex system for transport of Ca 2+ and related ions, consisting of two saturable modes of Ca 2+ influx and two separate, saturable mechanisms of Ca 2+ efflux. The characteristics of the mechanisms of Ca 2+ uptake, the uniporter and the RaM, are discussed here and suggestions are made about how the mechanisms may work together and separately to mediate the two physiological roles with which they are most commonly associated ‐ control of the rate of cellular ATP production and induction of the permeability transition and apoptosis. It is argued that more subtlety of control of intramitochondrial free Ca 2+ concentration ([Ca 2+ ] m ) must be used by the uniporter and the RaM to fulfill their physiological roles than has been commonly recognized. This is because an increase in [Ca 2+ ] m is associated with both increased production of ATP which supports the continued life of the cell and with induction of the permeability transition and possibly apoptosis, which leads to cell death. The saturable mechanisms of mitochondrial Ca 2+ efflux and the Ca 2+ ‐induced mitochondrial permeability transition, which can transport Ca 2+ as well as other ions and molecules and is often considered as a Ca 2+ transport mechanism, are being reviewed separately.

Determination of the oxidation states of manganese in brain, liver, and heart mitochondria

Excess brain manganese can produce toxicity with symptoms that resemble those of Parkinsonism and causes that remain elusive. Manganese accumulates in mitochondria, a major source of superoxide, which can oxidize Mn2+ to the powerful oxidizing agent Mn3+. Oxidation of important cell components by Mn3+ has been suggested as a cause of the toxic effects of manganese. Determining the oxidation states of intramitochondrial manganese could help to identify the dominant mechanism of manganese toxicity. Using X‐ray absorbance near edge structure (XANES) spectroscopy, we have characterized the oxidation state of manganese in mitochondria isolated from brain, liver, and heart over concentrations ranging from physiological to pathological. Results showed that (i) spectra from different model manganese complexes of the same oxidation state were similar to each other and different from those of other oxidation states and that the position of the absorption edge increases with oxidation state; (ii) spectra from intramitochondrial manganese in isolated brain, heart and liver mitochondria were virtually identical; and (iii) under these conditions intramitochondrial manganese exists primarily as a combination of Mn2+ complexes. No evidence for Mn3+ was detected in samples containing more than endogenous manganese levels, even after incubation under conditions promoting reactive oxygen species (ROS) production. While the presence of Mn3+ complexes cannot be proven in the spectrum of endogenous mitochondrial manganese, the shape of this spectrum could suggest the presence of Mn3+ near the limit of detection, probably as MnSOD.

Release of Ca 2+ from Mitochondria via the Saturable Mechanisms and the Permeability Transition

The literature, reviewed in the previous article, supports three physiological roles for sequestration of calcium by mitochondria: 1) control of the rate of ATP production, 2) activation of the Ca 2+ ‐induced mitochondrial permeability transition (PT), and 3) modulation of cytosolic Ca 2+ transients. Removal of Ca 2+ from mitochondria permits rapid and efficient changes in the rate of ATP production to adapt to changing demands and can reverse the process of PT induction. Two separate, saturable mechanisms for facilitating Ca 2+ efflux from mitochondria exist. In addition, the permeability transition or PT, which may also remove Ca 2+ from the mitochondrial matrix, is intimately involved in other important functions such as apoptosis. Here we briefly review what is known about these important mitochondrial mechanisms and from their behavior speculate on their possible and probable functions.

Nanoparticle (NP) uptake by type I alveolar epithelial cells and their oxidant stress response

Mammalian cells take up nanoparticles (NPs) and some NPs increase ROS. We use imaging and measure ROS in parallel to evaluate NP-cell interactions with type I-like alveolar epithelial cells exposed to NPs at 1.2 µg/cm(2) . Titanium dioxide (Ti0(2)), gold (Au), silver (Ag), and manganese (Mn) were internalized by R3-1 cells; copper (Cu) NPs were observed at the cell surface only. TiO(2) and Au did not increase cell death but Mn and Cu did, with surviving cells recovering after initial Cu exposure. Ag NPs caused 80% of R3-1 cells to lift off the slides within one hour. Amplex Red was used to report H(2)O(2) production after exposure to 0.4 µg/cm(2) TiO(2), Au, Cu, Mn and Ag. TiO(2), Au, and Ag caused no significant increase in H(2)O(2) while Cu and Mn increased H(2)O(2). NPs that give up electrons, increase ROS production and cause cell death in R3-1 cells.

Manganese transport via the transferrin mechanism

Excessive manganese (Mn) uptake by brain cells, particularly in regions like the basal ganglia, can lead to toxicity. Mn(2+) is transported into cells via a number of mechanisms, while Mn(3+) is believed to be transported similarly to iron (Fe) via the transferrin (Tf) mechanism. Cellular Mn uptake is therefore determined by the activity of the mechanisms transporting Mn into each type of cell and by the amounts of Mn(2+), Mn(3+) and their complexes to which these cells are exposed; this complicates understanding the contributions of each transporter to Mn toxicity. While uptake of Fe(3+) via the Tf mechanism is well understood, uptake of Mn(3+) via this mechanism has not been systematically studied. The stability of the Mn(3+)Tf complex allowed us to form and purify this complex and label it with a fluorescent (Alexa green) tag. Using purified and labeled Mn(3+)Tf and biophysical tools, we have developed a novel approach to study Mn(3+)Tf transport independently of other Mn transport mechanisms. This approach was used to compare the uptake of Mn(3+)Tf into neuronal cell lines with published descriptions of Fe(3+) uptake via the Tf mechanism, and to obtain quantitative information on Mn uptake via the Tf mechanism. Results confirm that in these cell lines significant Mn(3+) is transported by the Tf mechanism similarly to Fe(3+)Tf transport; although Mn(3+)Tf transport is markedly slower than other Mn transport mechanisms. This novel approach may prove useful for studying Mn toxicity in other systems and cell types.