Lawrence D. Robb-Gaspers

Decoding of cytosolic calcium oscillations in the mitochondria

Frequency-modulated oscillations of cytosolic Ca2+ ([Ca2+]c) are believed to be important in signal transduction, but it has been difficult to correlate [Ca2+]c oscillations directly with the activity of Ca(2+)-regulated targets. We have studied the control of Ca(2+)-sensitive mitochondrial dehydrogenases (CSMDHs) by monitoring mitochondrial Ca2+ ([Ca2+]m) and the redox state of flavoproteins and pyridine nucleotides simultaneously with [Ca2+]c in single hepatocytes. Oscillations of [Ca2+]c induced by IP3-dependent hormones were efficiently transmitted to the mitochondria as [Ca2+]m oscillations. Each [Ca2+]m spike was sufficient to cause a maximal transient activation of the CSMDHs and [Ca2+]m oscillations at frequencies above 0.5 per minute caused a sustained activation of mitochondrial metabolism. By contrast, sustained [Ca2+]c increases yielded only transient CSMDH activation, and slow or partial [Ca2+]c elevations were ineffective in increasing [Ca2+]m or stimulating CSMDHs. We conclude that the mitochondria are tuned to oscillating [Ca2+]c signals, the frequency of which can control the CSMDHs over the full range of potential activities.

Spatial and temporal aspects of cellular calcium signaling.

Cytosolic Ca2+ signals are often organized in complex temporal and spatial patterns, even under conditions of sustained stimulation. In this review we discuss the mechanisms and physi‐ological significance of this behavior in nonexcitable cells, in which the primary mechanism of Ca2+ mobilization is through (l,4,5)IP3‐dependent Ca2+ release from intracellular stores. Oscillations of cytosolic free Ca2+ ([Ca2+]i) are a common form of temporal organization; in the spatial domain, these [Ca2+]i oscillations may take the form of [Ca2+]i waves that propagate throughout the cell or they may be restricted to specific subcellular regions. These patterns of Ca2+ signaling result from the limited range of cytoplasmic Ca2+ diffusion and the feedback regulation of the pathways responsible for Ca2+ mo‐bilization. In addition, the spatial organization of [Ca2+]i changes appears to depend on the strategic distribution of Ca2+ stores within the cell. One type of [Ca2+]i oscillation is baseline spiking, in which discrete [Ca2+]i spikes occur with a frequency, but not amplitude, that is determined by agonist dose. Most current evidence favors a model in which baseline [Ca2+]i spiking results from the complex interplay between [Ca2+]i and (1,4,5)IP3 in regulating the gating of (l,4,S)IP3‐sensitive intracellular Ca2+ channels. Sinusoidal [Ca2+]i oscillations represent a mechanistically distinct type of temporal organiza‐tion, in which agonist dose regulates the amplitude but has no effect on oscillation frequency. Sinusoidal [Ca2+]i oscillations can be explained by a negative feedback effect of protein kinase C on the generation of (l,4,S)IP3 at the level of phospholipase C or its activating G‐protein. The physiological significance of [Ca2+]i oscillations and waves is becoming more established with the observation of this behavior in intact tissues and by the recognition of Ca2+‐dependent processes that are adapted to respond to fre‐ quency‐modulated oscillatory [Ca2+]i signals. In some cells, these [Ca2+]i signals are targeted to control processes in limited cytoplasmic domains, and in other systems [Ca2+]i waves can be propagated through gap junctions to coordinate the function of multicellular systems.—Thomas, A. P., Bird, G. S. J., Hajnóczky, G., Robb‐Gaspers, L. D., Putney, J. W., Jr. Spatial and temporal aspects of cellular calcium signaling. FASEB J. 10, 1505‐1517 (1996)

Integrating cytosolic calcium signals into mitochondrial metabolic responses

Stimulation of hepatocytes with vasopressin evokes increases in cytosolic free Ca2+ ([Ca2+]c) that are relayed into the mitochondria, where the resulting mitochondrial Ca2+ ([Ca2+]m) increase regulates intramitochondrial Ca2+‐sensitive targets. To understand how mitochondria integrate the [Ca2+]c signals into a final metabolic response, we stimulated hepatocytes with high vasopressin doses that generate a sustained increase in [Ca2+]c. This elicited a synchronous, single spike of [Ca2+]m and consequent NAD(P)H formation, which could be related to changes in the activity state of pyruvate dehydrogenase (PDH) measured in parallel. The vasopressin‐induced [Ca2+]m spike evoked a transient increase in NAD(P)H that persisted longer than the [Ca2+]m increase. In contrast, PDH activity increased biphasically, with an initial rapid phase accompanying the rise in [Ca2+]m, followed by a sustained secondary activation phase associated with a decline in cellular ATP. The decline of NAD(P)H in the face of elevated PDH activity occurred as a result of respiratory chain activation, which was also manifest in a calcium‐dependent increase in the membrane potential and pH gradient components of the proton motive force (PMF). This is the first direct demonstration that Ca2+‐mobilizing hormones increase the PMF in intact cells. Thus, Ca2+ plays an important role in signal transduction from cytosol to mitochondria, with a single [Ca2+]m spike evoking a complex series of changes to activate mitochondrial oxidative metabolism.

Coupling between cytosolic and mitochondrial calcium oscillations: role in the regulation of hepatic metabolism

Mitochondria are strategically localized at sites of Ca2+ release, such that increases in cytosolic free Ca2+ ([Ca2+]c) from either internal Ca2+ stores or Ca2+ influx across the plasma membrane can be rapidly transported into the mitochondrial matrix. The consequent elevation in mitochondrial Ca2+ ([Ca2+]m) stimulates the Ca2+-sensitive intramitochondrial dehydrogenases, resulting in elevation of NAD(P)H. The preferential coupling between increases in [Ca2+]c and [Ca2+]m is one proposed mechanism to coordinate mitochondrial ATP production with cellular energy demand. In liver cells, hormones that act through the second messenger inositol 1,4, 5-trisphosphate (IP3) generate oscillatory [Ca2+]c signals, which result from a periodic Ca2+- and IP3-mediated activation/deactivation of intracellular Ca2+ release channels. The [Ca2+]c spiking frequency increases with agonist dose, whereas the amplitude of each [Ca2+]c spike is constant. This frequency modulation of [Ca2+]c spiking encodes the signal from the extracellular agonist, which is then decoded by the internal Ca2+-sensitive proteins such as the Ca2+-sensitive intramitochondrial dehydrogenases. Our studies have investigated the relationship between IP3-dependent [Ca2+]c signals and [Ca2+]m in primary cultured hepatocytes. In addition, the changes in cellular [Ca2+] levels have been correlated with the regulation of intramitochondrial NAD(P)H levels, pyruvate dehydrogenase activity and the magnitude of the mitochondrial proton motive force.

Influence of calcium and iron on cell death and mitochondrial function in oxidatively stressed astrocytes.

Astrocytes protect neurons and oligodendrocytes by buffering ions, neurotransmitters, and providing metabolic support. However, astrocytes are also vulnerable to oxidative stress, which may affect their protective and supportive functions. This paper examines the influence of calcium and iron on astrocytes and determines if cell death could be mediated by mitochondrial dysfunction. We provide evidence that the events associated with peroxide‐induced death of astrocytes involves generation of superoxide at the site of mitochondria, loss of mitochondrial membrane potential, and depletion of ATP. These events are iron‐mediated, with iron loading exacerbating and iron chelation reducing oxidative stress. Iron chelation maintained the mitochondrial membrane potential, prevented peroxide‐induced elevations in superoxide levels, and preserved ATP levels. Although increased intracellular calcium occurred after oxidative stress to astrocytes, the calcium increase was not necessary for collapse of mitochondrial membrane potential. Indeed, when astrocytes were oxidatively stressed in the absence of extracellular calcium, cell death was enhanced, mitochondrial membrane potential collapsed at an earlier time point, and superoxide levels increased. Additionally, our data do not support opening of the mitochondrial permeability transition pore as part of the mechanism of peroxide‐induced oxidative stress of astrocytes. We conclude that the increase in intracellular calcium following peroxide exposure does not mediate astrocytic death and may even provide a protective function. Finally, the vulnerability of astrocytes and their mitochondria to oxidative stress correlates more closely with iron availability than with increased intracellular calcium. J. Neurosci. Res. 55:674–686, 1999. 

Coordination of calcium signalling by endothelial-derived nitric oxide in the intact liver.

Calcium ions (Ca2+) and nitric oxide (NO) are key signalling molecules that are implicated in the regulation of numerous cellular processes. Here we show that, in the intact liver, stimulation of endothelial cells by bradykinin coordinates the propagation of vasopressin-dependent intercellular Ca2+ waves across hepatic plates, and markedly increases the frequency of Ca2+ oscillations in individual hepatocytes. Modulation of Ca2+ oscillations by bradykinin is lost following isolation of hepatocytes, but restored in co-cultures of hepatocytes and endothelial cells. The sensitizing effects of bradykinin are mimicked by NO donors and abrogated by NO inhibitors. Thus, crosstalk between NO and Ca2+ signalling pathways through the microvasculature is probably an important mechanism for the coordination of liver function and may have a function in other organs.

Imaging Whole Organs — Single cell [Ca2+]i measurements in the perfused liver

Cytosolic free calcium ([Ca2+]i) signaling mediates many of the cellular responses to extracellular stimuli, including hormones, growth factors and neurotransmitters. Over a decade ago, Woods et al.1 first demonstrated that hormones coupled to the activation of inositol lipid-specific phospholipase C cause discrete, periodic [Ca2+]i spikes or transients in isolated hepatocytes. Since then the basic control mechanisms governing the phosphoinositide-dependent Ca2+ signaling pathway, in nonexcitable cells, has been the focus of intense scrutiny. This work was recently reviewed by our laboratory and by others.2–4 Much less is known about the spatial and temporal regulation of single cell [Ca2+]i in the intact liver or any other intact organ. Single cell [Ca2+]i oscillations have been measured in some intact organs including: hepatocytes in isolated perfused liver,5–7 endothelial cells lining the microvessels of a perfused lung,8 omega neuron in a conscious cricket brain,9 and pyramidal neurons in anaesthetized rat brain.10 Additional insight into in vivo [Ca2+]i regulatory mechanisms have been uncovered in tissue preparations which maintain the functional unit of the intact organ such as, pyramidal neurons and gray matter astrocytes from hippocampal slices,11 vascular smooth muscle cells from rat tail artery segments,12 tracheal epithelial cells13–14 and pancreatic acini clusters.15–16

Mitochondrial Ca2+ Signalling

Historically, the role played by mitochondria in cellular Ca2+ homeostasis has been the subject of considerable controversy (Fiskum & Lehninger, 1982; Akerman & Nicholls, 1983). The view that mitochondria, by virtue of possessing a huge capacity for Ca2+ uptake (Lehninger, 1967) act in the cell as mobilizable stores of the ion (Exton, 1980) capable of regulating cytosolic Ca2+ (Nicholls, 1978) has seen a considerable revision of the past 10–15 years (Denton & McCormack, 1980; Hansford, 1994; Crompton, 1985; McCormack et al., 1990; Denton & McCormack, 1986). This resulted firstly from the recognition that Ca2+ cycles constantly across the inner mitochondrial inner membrane (Nicholls & Crompton, 1980), so that the concentration of Ca2+ within the mitochondrial matrix ([Ca2+]m) is related to cytosolic Ca2+ by the kinetic properties of the transporters, rather than purely thermodynamic considerations. Thus, intramitochondrial Ca2+ concentrations in the low micromolar range have become generally accepted, rather than earlier assumptions of [Ca2+]m levels in the millimolar range or higher. Secondly, mitochondria from all vertebrate sources studied so far, as well as certain plants, have been shown to possess three important intramitochondrial oxidative enzymes whose activity is controlled in the micromolar range of Ca2+ ions (Denton et al., 1972; Denton et al., 1978; McCormack & Denton, 1979; Rutter, 1990). These findings led to the model first proposed by Denton and McCormack (Denton & McCormack, 1980) that increases in intramitochondrial Ca2+ concentration ([Ca2+]m), triggered after stimulus-induced increases in cytosolic free calcium concentration ([Ca2+]c), could activate oxidative metabolism, generating NADH or the respiratory chain, and enhanced ATP synthesis. This would then fuel the Ca2+-activated ATP-requiring events in the cytosol, such as muscle contraction, secretion or gene expression, without requiring a drop in intracellular ATP levels. Considerable evidence supporting this model has accrued since its proposal. For reviews see (McCormack et al., 1990; Denton & McCormack, 1990). However, support for the model has been considered to be at odds with the view that mitochondria could play any significant role in controlling cytosolic [Ca2+].