Cyclosporin A inhibits inositol 1,4,5-trisphosphate-dependent Ca2+ signals by enhancing Ca2+ uptake into the endoplasmic reticulum and mitochondria.

Cytosolic Ca(2+) ([Ca(2+)](i)) oscillations may be generated by the inositol 1,4,5-trisphosphate receptor (IP(3)R) driven through cycles of activation/inactivation by local Ca(2+) feedback. Consequently, modulation of the local Ca(2+) gradients influences IP(3)R excitability as well as the duration and amplitude of the [Ca(2+)](i) oscillations. In the present work, we demonstrate that the immunosuppressant cyclosporin A (CSA) reduces the frequency of IP(3)-dependent [Ca(2+)](i) oscillations in intact hepatocytes, apparently by altering the local Ca(2+) gradients. Permeabilized cell experiments demonstrated that CSA lowers the apparent IP(3) sensitivity for Ca(2+) release from intracellular stores. These effects on IP(3)-dependent [Ca(2+)](i) signals could not be attributed to changes in calcineurin activity, altered ryanodine receptor function, or impaired Ca(2+) fluxes across the plasma membrane. However, CSA enhanced the removal of cytosolic Ca(2+) by sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA), lowering basal and inter-spike [Ca(2+)](i). In addition, CSA stimulated a stable rise in the mitochondrial membrane potential (DeltaPsi(m)), presumably by inhibiting the mitochondrial permeability transition pore, and this was associated with increased Ca(2+) uptake and retention by the mitochondria during a rise in [Ca(2+)](i). We suggest that CSA suppresses local Ca(2+) feedback by enhancing mitochondrial and endoplasmic reticulum Ca(2+) uptake, these actions of CSA underlie the lower IP(3) sensitivity found in permeabilized cells and the impaired IP(3)-dependent [Ca(2+)](i) signals in intact cells. Thus, CSA binding proteins (cyclophilins) appear to fine tune agonist-induced [Ca(2+)](i) signals, which, in turn, may adjust the output of downstream Ca(2+)-sensitive pathways.

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.

Influence of nitric oxide on cellular and mitochondrial integrity in oxidatively stressed astrocytes.

Astrocytes provide protection and trophic support to neurons, but like neurons are vulnerable to oxidative stress. Decreased function of astrocytes resulting from oxidative stress could contribute to neurodegeneration. Our goal is to understand the intracellular events associated with oxidative stress in astrocytes. Because nitric oxide (NO) has been implicated as a contributor to oxidative stress in the brain, we examined in this study whether NO contributed to oxidative stress in astrocytes. Stimulation of NO decreases superoxide levels, preserves mitochondrial membrane potential, and decreases mitochondrial swelling in astrocytes treated with peroxide. Chelation of NO is associated with increased cell death, mitochondrial swelling, and loss of mitochondrial membrane potential, in response to peroxide treatment. Peroxide treatment increased intracellular calcium and the peroxide-induced changes in intracellular calcium were not altered in response to NO. Iron-loading increases peroxide-induced oxidative stress in astrocytes, but induction of NO limited the iron effect, suggesting an interaction between iron and NO. These data suggest endogenously produced NO protects astrocytes from oxidative stress, perhaps by preserving mitochondrial function.

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.

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.

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 physiological significance of this behavior in nonexcitable cells, in which the primary mechanism of Ca2+ mobilization is through (1,4,5)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+ mobilization. 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 (1,4,5)IP3-sensitive intracellular Ca2+ channels. Sinusoidal [Ca2+]i oscillations represent a mechanistically distinct type of temporal organization, 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 (1,4,5)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 frequency-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.

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.

Coordination of Ca2+ signaling by intercellular propagation of Ca2+ waves in the intact liver.

Activation of the inositol lipid signaling system results in cytosolic Ca2+ oscillations and intra- and intercellular Ca2+ waves in many isolated cell preparations. However, this form of temporal and spatial organization of signaling has not been demonstrated in intact tissues. Digital imaging fluorescence microscopy was used to monitor Ca2+ at the cellular and subcellular level in intact perfused rat liver loaded with fluorescent Ca2+ indicators. Perfusion with low doses of vasopressin induced oscillations of hepatocyte Ca2+ that were coordinated across entire lobules of the liver by propagation of Ca2+ waves along the hepatic plates. At the subcellular level these periodic Ca2+ waves initiated from the sinusoidal domain of cells within the periportal region and propagated radially across cell-cell contacts into the pericentral region, or until terminated by annihilation collision with other Ca2+ wave fronts. With increasing agonist dose, the frequency but not the amplitude of the Ca2+ waves increased. Intracellular Ca2+ wave rates were constant, but transcellular signal propagation was determined by agonist dose, giving rise to a dose-dependent increase in the rate at which Ca2+ waves spread through the liver. At high vasopressin doses, a single Ca2+ wave was observed and the direction of Ca2+ wave propagation was reversed, initiating in the pericentral region and spreading to the periportal region. It is concluded that intercellular Ca2+ waves may provide a mechanism to coordinate responses across the functional units of the liver.

Subcellular organization of calcium signalling in hepatocytes and the intact liver.

Hepatocytes respond to inositol 1,4,5-trisphosphate (InsP3)-linked agonists with frequency-modulated oscillations in the intracellular free calcium concentration ([Ca2+]i), that occur as waves propagating from a specific origin within each cell. The subcellular distribution and functional organization of InsP3-sensitive Ca2+ pools has been investigated, in both intact and permeabilized cells, by fluorescence imaging of dyes which can be used to monitor luminal Ca2+ content and InsP3-activated ion permeability in a spatially resolved manner. The Ca2+ stores behave as a luminally continuous system distributed throughout the cytoplasm. The structure of the stores, an important determinant of their function, is controlled by the cytoskeleton and can be modulated in a guanine nucleotide-dependent manner. The nuclear matrix is devoid of Ca2+ stores, but Ca2+ waves in the intact cell propagate through this compartment. The organization of [Ca2+]i signals has also been investigated in the perfused liver. Frequency-modulated [Ca2+]i oscillations are still observed at the single cell level, with similar properties to those in the isolated hepatocyte. The [Ca2+]i oscillations propagate between cells in the intact liver, leading to the synchronization of [Ca2+]i signals across part or all of each hepatic lobule.

Continuous measurement of mitochondrial pH gradients in isolated hepatocytes by difference ratio spectroscopy.

The pH gradient, delta pH, present across the inner mitochondrial membrane in isolated rat hepatocytes was continuously monitored with a novel spectroscopic technique that utilizes the weak acid fluorescein. Unlike most cytosolic pH indicators, such as 2',7'-bis(carboxyethyl)-5,(6)-carboxyfluorescein (BCECF), fluorescein freely distributes between the cytosolic and mitochondrial compartments. As is typical for weak acids, the distribution between these two compartments is governed by the magnitude of the pH gradient. Since fluorescein has two ionizable groups, the fluorescein dianion is concentrated in the mitochondrial compartment 100-fold per delta pH unit. In this compartment, fluorescein absorbance (or excitation) spectra are red-shifted about 6-8 nm in the matrix environment, as compared to the cytosolic dye at equivalent pH values. The combination of favorable mitochondrial accumulation and red-shifted spectra enables mitochondrial pH to be continuously monitored qualitatively in whole cells by dual wavelength spectroscopy (510 minus 540 nm). When the cytosolic pH is determined by independent means, the mitochondrial pH can be quantitated, based on the theoretical dependence of the fluorescein distribution ratio on delta pH, the ratio of cytosolic to mitochondrial volumes, and the known extinction coefficients for the dye in the cytosolic and mitochondrial compartments. The sensitivity of the method for following kinetic responses in mitochondrial pH is especially noteworthy; a 0.1-unit change in delta pH is easily distinguished, with a time resolution of less than a second.