Alkalinization-Induced Changes in Intracellular Calcium in Rat Spinal Cord Neurons

It is well-known that pH changes can influence a lot of cellular processes. In this work, we have specifically studied the influence of alkalinization, which can be developed in spinal cord neurons during hyperventilation (respiratory alkalosis) and chronic renal failure (metabolic alkalosis) on calcium homeostasis. Application of Tyrode solution with increased pH (pH = 8.8) to secondary sensory neurons isolated from rat spinal dorsal horn induced elevation of intracellular free calcium concentration in the cytosol ([Ca2+]i) if applied after membrane depolarization. Repetitive application of alkaline solution led to disappearance of such elevations. Depletion of endoplasmic reticulum (ER) calcium stores by 30 mM caffeine almost completely blocked the effect of elevated extracellular pH. If caffeine-induced [Ca2+]i transients were evoked during alkalinization, their amplitudes were decreased by 41%. Preapplication of 500 nM ionomycin resulted in disappearance of alkalinization-induced [Ca2+]i transients, whereas prolonged applications (for 20 min) of 200 nM thapsigargin, a blocker of Ca2+ ATPase of the endoplasmic reticulum, resulted in disappearance of the rapid phase of the [Ca2+]i transients induced by alkalinization. Preapplication of the mitochondrial protonophore CCCP (10 microM) also induced changes in the alkalinization-induced calcium response--it lost its peak and was transformed into an irregular wave terminating in several seconds. The data obtained indicate that alkalinization induces an increase of [Ca2+]i level in the investigated neurons via a combined action of both intracellular Ca2+-accumulating structures--the endoplasmic reticulum and mitochondria. This suggestion was supported by morphological data that both structures in these neurons are tightly connected and may interact during release of accumulated calcium ions.     

Age-dependent changes in Ca2+ homeostasis in peripheral neurones: implications for changes in function

Calcium ions represent universal second messengers within neuronal cells integrating multiple cellular functions, such as release of neurotransmitters, gene expression, proliferation, excitability, and regulation of cell death or apoptotic pathways. The magnitude, duration and shape of stimulation-evoked intracellular calcium ([Ca2+]i) transients are determined by a complex interplay of mechanisms that modulate stimulation-evoked rises in [Ca2+]i that occur with normal neuronal function. Disruption of any of these mechanisms may have implications for the function and health of peripheral neurones during the aging process. This review focuses on the impact of advancing age on the overall function of peripheral adrenergic neurones and how these changes in function may be linked to age-related changes in modulation of [Ca2+]i regulation. The data in this review suggest that normal aging in peripheral autonomic neurones is a subtle process and does not always result in dramatic deterioration in their function. We present studies that support the idea that in order to maintain cell viability peripheral neurones are able to compensate for an age-related decline in the function of at least one of the neuronal calcium-buffering systems, smooth endoplasmic reticulum calcium ATPases, by increased function of other calcium-buffering systems, namely, the mitochondria and plasmalemma calcium extrusion. Increased mitochondrial calcium uptake may represent a 'weak point' in cellular compensation as this over time may contribute to cell death. In addition, we present more recent studies on [Ca2+]i regulation in the form of the modulation of release of calcium from smooth endoplasmic reticulum calcium stores. These studies suggest that the contribution of the release of calcium from smooth endoplasmic reticulum calcium stores is altered with age through a combination of altered ryanodine receptor levels and modulation of these receptors by neuronal nitric oxide containing neurones.     

Neuronal calcium stores

Neuronal calcium stores associated with specialized intracellular organelles, such as endoplasmic reticulum and mitochondria, dynamically participate in generation of cytoplasmic calcium signals which accompany neuronal activity. They fulfil a dual role in neuronal Ca2+ homeostasis being involved in both buffering the excess of Ca2+ entering the cytoplasm through plasmalemmal channels and providing an intracellular source for Ca2+. Increase of Ca2+ content within the stores regulates the availability and magnitude of intracellular calcium release, thereby providing a mechanism which couples the neuronal activity with functional state of intracellular Ca2+ stores. Apart of 'classical' calcium stores (endoplasmic reticulum and mitochondria) other organelles (e.g. nuclear envelope and neurotransmitter vesicles) may potentially act as a functional Ca2+ storage compartments. Calcium ions released from internal stores participate in many neuronal functions, and might be primarily involved in regulation of various aspects of neuronal plasticity.     

Involvement of the Endoplasmic Reticulum of Chromaffin Cells of the Rat Adrenal Gland in Calcium Signaling

We studied the involvement of the endoplasmic reticulum (ER) in calcium signaling in rat chromaffin cells. For this purpose, the following agents influencing the activity of the ER were used: (i) Caffeine that activates the release of Ca²⁺ from the endoplasmic store and (ii) thapsigargin that suppresses accumulation of calcium in the ER. The intracellular Ca²⁺ concentration was measured with the help of a calcium-sensitive dye, Fura-2AM, using the microfluorescent technique. Applications of caffeine led to a rise in the level of free Ca²⁺ in the cell cytosol and also to a decrease in the amplitude of calcium transients induced by depolarization of the plasma membrane under the action of a hyperpotassium solution. Under conditions of repeated caffeine applications, the amplitude of transients decreased to 9% of its initial value, which is explained by exhaustion of the calcium stores. The action of caffeine was restored when the calcium stores were re-filled under the action of depolarization of the plasma membrane. Thapsigargin completely removed the effect of caffeine and did not influence KCl-induced transients. Therefore, our experiments are indicative of a significant importance of the ER calcium stores for calcium signaling in chromaffin cells, which allows us to hypothesize that these stores play an important role in the control of secretion of catecholamines.     

Calcium Release From the Internal Stores as a Possible Target for Antinociceptive Treatment in Rats with Experimentally-Induced Diabetes

Distal neuropathy is the most common complication of diabetes mellitus, and it is highly important to reveal the cellular mechanisms leading to its development. In our experiments, neurons of control and streptozotocin-treated diabetic rats were examined. Changes in the intracellular free calcium concentrations ([Ca²⁺] _i ) were fluorometrically measured in primary and secondary nociceptive (dorsal root ganglion, DRG, and dorsal horn, DH, respectively) neurons. The [Ca²⁺] _i elevation was induced by different agents, which can release calcium from the endoplasmic reticulum (ER) calcium stores. The amplitudes of calcium elevation induced by application of caffeine and ionomicine in DRG and DH neurons of diabetic rats were significantly lower, as compared with the control. Application of ATP and glutamate to a Ca-free extracellular solution induced calcium release from the IP₃-sensitive store in DH neurons. Release of calcium from the IP₃-sensitive ER calcium stores became significantly smaller in neurons from diabetic rats. Taken together, these data indicate that significant changes in the calcium regulating mechanisms of the ER develop under diabetes conditions.     

Endoplasmic reticulum calcium release is modulated by actin polymerization

Intracellular calcium ions regulate the structure and functions of cytoskeletal proteins. On the other hand, recent studies have shown that the cytoskeleton, and actin filaments in particular, can modulate calcium influx through plasma membrane ligand- and voltage-gated channels. We now report that calcium release from inositol trisphosphate (IP3) and ryanodine-sensitive endoplasmic reticulum (ER) stores is modulated by polymerization and depolymerization of actin filaments in cultured hippocampal neurons. Depolymerization of actin filaments with cytochalasin D attenuates calcium release induced by carbamylcholine (CCh; a muscarinic agonist for IP3 pathway), caffeine (a ryanodine receptor agonist) and thapsigargin (an inhibitor of the ER calcium- ATPase) in both the presence and absence of extracellular calcium. Conversely, the actin polymerizing agent jasplakinolide potentiates calcium release induced by CCh, caffeine and thapsigargin. Cytochalasin D attenuated, while jasplakinolide augmented, thapsigargin-induced JNK activation and neuronal cell death. Our data show that the actin cytoskeleton regulates ER calcium release, suggesting roles for actin in the various physiological and pathological processes that involve calcium release.     

Elevation of intracellular glucosylceramide levels results in an increase in endoplasmic reticulum density and in functional calcium stores in cultured neurons.

Gaucher disease is a glycosphingolipid storage disease caused by defects in the activity of the lysosomal hydrolase, glucocerebrosidase (GlcCerase), resulting in accumulation of glucocerebroside (glucosylceramide, GlcCer) in lysosomes. The acute neuronopathic type of the disease is characterized by severe loss of neurons in the central nervous system, suggesting that a neurotoxic agent might be responsible for cellular disruption and neuronal death. We now demonstrate that upon incubation with a chemical inhibitor of GlcCerase, conduritol-B-epoxide (CBE), cultured hippocampal neurons accumulate GlcCer. Surprisingly, increased levels of tubular endoplasmic reticulum elements, an increase in [Ca(2+)](i) response to glutamate, and a large increase in [Ca(2+)](i) release from the endoplasmic reticulum in response to caffeine were detected in these cells. There was a direct relationship between these effects and GlcCer accumulation since co-incubation with CBE and an inhibitor of glycosphingolipid synthesis, fumonisin B(1), completely antagonized the effects of CBE. Similar effects on endoplasmic reticulum morphology and [Ca(2+)](i) stores were observed upon incubation with a short-acyl chain, nonhydrolyzable analogue of GlcCer, C(8)-glucosylthioceramide. Finally, neurons with elevated GlcCer levels were much more sensitive to the neurotoxic effects of high concentrations of glutamate than control cells; moreover, this enhanced toxicity was blocked by pre-incubation with ryanodine, suggesting that [Ca(2+)](i) release from ryanodine-sensitive intracellular stores can induce neuronal cell death, at least in neurons with elevated GlcCer levels. These results may provide a molecular mechanism to explain neuronal dysfunction and cell death in neuronopathic forms of Gaucher disease.     

Caffeine-sensitive calcium stores in presynaptic nerve endings: a physiological role?

Ca2+-sensitive minielectrodes and the fluorescent cytosolic calcium probes, quin2 and fura2, were used to study some aspects of calcium homeostasis in intact and permeabilized synaptosomes from whole rat brain. Experiments in permeabilized synaptosomes revealed the existence of a vesicular, non-mitochondrial, ATP-dependent calcium uptake system with a vanadate sensitivity similar to that of brain microsomes, or endoplasmic reticulum-type calcium sequestering organelles. By using the fluorescent probes it was possible to show that caffeine mobilizes calcium from these internal stores in intact synaptosomes. Our results indicate a role of the caffeine sensitive calcium stores in the buffering of calcium loads elicited by plasma membrane depolarization.     

Advancing age alters rapid and spontaneous refilling of caffeine-sensitive calcium stores in sympathetic superior cervical ganglion cells.

Intracellular calcium concentration ([Ca2+]i) release from smooth endoplasmic reticulum (SER) stores plays an important role in cell signaling. These stores are rapidly refilled via influx through voltage-gated calcium channels or spontaneously via store-operated calcium channels and subsequent pumping by SER Ca2+-ATPases. We measured [Ca2+]i transients in isolated fura 2-loaded superior cervical ganglion cells from 6-, 12-, 20-, and 24-mo-old Fischer 344 rats. For rapid refilling, [Ca2+]i transients were elicited by a 1) 5-s exposure to K+, 2) caffeine to release Ca2+ from SER stores, 3) K+ to refill SER Ca2+ stores, and 4) caffeine. The percent difference between the peak and rate of rise of the first and second caffeine-evoked [Ca2+]i transient significantly declined over the age range of 12-24 mo. To estimate spontaneous refilling, cells were depolarized for 5 s with 68 mM K+ (control), followed by a 10-s exposure to 10 mM caffeine "conditioning stimulus" to deplete [Ca2+]i stores. Caffeine was then rapidly applied for 5 s at defined intervals from 60 to 300 s. Integrated caffeine-evoked [Ca2+]i transients were measured and plotted as a percentage of the K+ response vs. time. The derivative of the refilling time curves significantly declined over the age range from 12-24 mo. Overall, these data suggest that the ability of superior cervical ganglion cells to sustain release of [Ca2+]i following rapid or spontaneous refilling declines with advancing age. Compromised ability to sustain calcium signaling may possibly alter the overall function of adrenergic neurons innervating the cerebrovasculature.     

Vanilloid receptor 1 regulates multiple calcium compartments and contributes to Ca2+-induced Ca2+ release in sensory neurons

Vanilloid receptor 1 belongs to the transient receptor potential ion channel family and transduces sensations of noxious heat and inflammatory hyperalgesia in nociceptive neurons. These neurons contain two vanilloid receptor pools, one in the plasma membrane and the other in the endoplasmic reticulum. The present experiments characterize these two pools and their functional significance using calcium imaging and 45Ca uptake in stably transfected cells or dorsal root ganglion neurons. The plasma membrane localized receptor is directly activated by vanilloids. The endoplasmic reticulum pool was demonstrated to be independently activated with 20 microm capsaicin or 1.6 microm resiniferatoxin using a bathing solution containing 10 microm Ruthenium Red (to selectively block plasma membrane-localized receptors) and 100 microm EGTA. We also demonstrate an overlap between the endoplasmic reticulum-localized vanilloid receptor regulated stores and thapsigargin-sensitive stores. Direct depletion of calcium via activation of endoplasmic reticulum-localized vanilloid receptor 1 triggered store operated calcium entry. Furthermore, we found that, in the presence of low extracellular calcium (10(-5) m), either 2 microm capsaicin or 0.1 nm-1.6 microm resiniferatoxin caused a pronounced calcium-induced calcium release in either vanilloid receptor-expressing neurons or heterologous expression systems. This phenomenon may allow new insight into how nociceptive neuron function in response to a variety of nociceptive stimuli both acutely and during prolonged nociceptive signaling.     

Characteristics of store-operated calcium channels activated by depletion of the endoplasmic reticulum ryanodine-sensitive calcium stores in rat dorsal root ganglion neurons

In neurons of the rat dorsal root ganglia (DRG), using a patch-clamp technique in the whole-cell configuration, we studied the characteristics of calcium channels activated by depletion of the ryanodine-sensitive calcium stores of the endoplasmic reticulum. Current-voltage (I-V) relationships of these store-operated calcium channels were obtained by subtraction of the integral I-V characteristics after application of caffeine from the integral I-V characteristics of calcium channels in the control. Currents through store-operated calcium channels could be induced by application of a series of hyperpolarization current pulses to the cell under conditions of replacement of a calcium-free solution containing caffeine by a caffeine-free solution containing 2 mM Ca²⁺. In this case, the following two main conditions were abserved: Voltage-operated calcium channels were inactivated, while a gradient of the electrochemical potential for calcium ions was increased, which made easier passing of these currents through store-operated calcium channels. Therefore, we found that in DRG neurons, despite the presence of great numbers of both voltage-operated and receptor-dependent calcium channels, one more mechanism underlying the entry of calcium through store-operated channels does exist. Neirofiziologiya/Neurophysiology, Vol. 39, No. 3, pp. 195–200, May–June, 2007.     

Mitochondrial Enzymes and Endoplasmic Reticulum Calcium Stores as Targets of Oxidative Stress in Neurodegenerative Diseases

Considerable evidence indicates that oxidative stress accompanies age-related neurodegenerative diseases. Specific mechanisms by which oxidative stress leads to neurodegeneration are unknown. Two targets of oxidative stress that are known to change in neurodegenerative diseases are the mitochondrial enzyme alpha-ketoglutarate dehydrogenase complex (KGDHC) and endoplasmic reticulum calcium stores. KGDHC activities are diminished in all common neurodegenerative diseases and the changes are particularly well documented in Alzheimer's disease (AD). A second change that occurs in cells from AD patients is an exaggerated endoplasmic reticulum calcium store [i.e., bombesin-releasable calcium stores (BRCS)]. H(2)O(2), a general oxidant, changes both variables in the same direction as occurs in disease. Other oxidants selectively alter these variables. Various antioxidants were used to help define the critical oxidant species that modifies these responses. All of the antioxidants diminish the oxidant-induced carboxy-dichlorofluorescein (cDCF) detectable reactive oxygen species (ROS), but have diverse actions on these cellular processes. For example, alpha-keto-beta-methyl-n-valeric acid (KMV) diminishes the H(2)O(2) effects on BRCS, while trolox and DMSO exaggerate the response. Acute trolox treatment does not alter H(2)O(2)-induced changes in KGDHC, whereas chronic treatment with trolox increases KGDHC almost threefold. The results suggest that KGDHC and BRCS provide targets by which oxidative stress may induce neurodegeneration and a useful tool for selecting antioxidants for reversing age-related neurodegeneration.     

Properties of the caffeine-sensitive intracellular calcium stores in mammalian neurons

Using indo-1- and fura-2-based microfluorometry for measuring the cytoplasmic free calcium concentration ([Ca²⁺] _in ), the properties of caffeine-induced Ca²⁺ release from internal stores were studied in rat cultured central and peripheral neurons, including dorsal root ganglion (DRG) neurons, neurons from then. cuneatus, CA1 and CA3 hippocampal regions, and pyramidal neocortical neurons. Under resting conditions, the Ca²⁺ content of internal stores in DRG neurons was high enough to produce caffeine-triggered [Ca²⁺] _in transients. Prolonged exposure of caffeine depleted the caffeine-sensitive stores of releasable Ca²⁺; the degree of this depletion depended on caffeine concentration. The depletion of the caffeine-sensitive internal stores to some extent was linked to calcium extrusion via La³⁺-sensitive plasmalemmal Ca²⁺-ATPases. Caffeine-induced Ca²⁺ release deprived internal stores in DRG neurons, but they refilled themselves spontaneously within 10 min. Pharmacological manipulation with caffeine-sensitive stores interferred with the depolarization-induced [Ca²⁺] _in transients. In the presence of low caffeine concentration (0.5–1.0 mM) in the extracellular solution, the rate of rise of the depolarization-triggered [Ca²⁺] _in transients significantly increased (by a factor of 2.15 ± 0.29) suggesting the occurrence of Ca²⁺-induced Ca²⁺ release. When the caffeine-sensitive stores were emptied by prolonged application of caffeine, the amplitude and rate of rise of the depolarization-induced [Ca²⁺] _in transients decreased. These findings suggest the involvement of internal caffeine-sensitive calcium stores in generation of calcium signal in sensory neurons. In contrast, in all types of central neurons tested the resting Ca²⁺ content of internal stores was low, but the stores could be charged by transmembrane Ca²⁺ entry through voltage-operated calcium channels. After charging, the stores in central neurons spontaneously lost releasable calcium content and within 10 min they became completely empty again. We suggest that internal Ca²⁺ stores in peripheral and central neurons, although having similar pharmacological characteristics, handle Ca²⁺ ions in a different manner. Calcium stores in sensory neurons are continuously filled by releasable calcium and after discharging they can be spontaneously refilled, whereas in central neurons internal calcium stores can be charged by releasable calcium only transiently. Caffeine-evoked [Ca²⁺] _in transients in all types of neurons were effectively blocked by 10 mM ryanodine, 5 mM procaine, 10 mM dantrolene, or 0.5 mM Ba²⁺, thus sharing the basic properties of the Ca²⁺-induced Ca²⁺ release from endoplasmic reticulum.Neirofiziologiya/Neurophysiology, Vol. 26, No. 1, pp. 16–25, January–February, 1994.     

Apoptosis and calcium: new roles for cytochrome c and inositol 1,4,5-trisphosphate

Mounting evidence suggests that calcium released from internal stores plays a critical role in the progression of apoptosis. The primary calcium release channel on endoplasmic reticulum membranes is the inositol 1,4,5-trisphosphate receptor (IP3R). Deletion of the gene for IP3R results in defects in apoptosis in response to multiple stimuli. Conversely, augmented IP3R levels are associated with increased cell death. A mechanistic basis for altered IP3R function during apoptosis was revealed with the discovery that cytochrome c binds to IP3R early in apoptosis. This interaction blocks the calcium-dependent inhibition of IP3R function, resulting in increased calcium release from internal stores. The resultant cytoplasmic and mitochondrial calcium overload culminates in cell-wide cytochrome c release and maximal caspase activation. These findings highlight the importance of intracellular calcium stores in apoptosis, and the multi-functional role of cytochrome c released from mitochondria in cell death.     

Calcium-induced calcium release contributes to somatic secretion of serotonin in leech Retzius neurons

We analyzed the contribution of calcium (Ca2+)-induced Ca2+ release to somatic secretion in serotonergic Retzius neurons of the leech. Somatic secretion was studied by the incorporation of fluorescent dye FM1-43 upon electrical stimulation with trains of 10 impulses and by electron microscopy. Quantification of secretion with FM1-43 was made in cultured neurons to improve optical resolution. Stimulation in the presence of FM1-43 produced a frequency-dependent number of fluorescent spots. While a 1-Hz train produced 19.5+/-5.0 spots/soma, a 10-Hz train produced 146.7+/-20.2 spots/soma. Incubation with caffeine (10 mM) to induce Ca2+ release from intracellular stores without electrical stimulation and external Ca2+, produced 168+/-21.7 spots/soma. This staining was reduced by 49% if neurons were preincubated with the Ca2+- ATPase inhibitor thapsigargin (200 nM). Moreover, in neurons stimulated at 10 Hz in the presence of ryanodine (100 microM) to block Ca2+-induced Ca2+ release, FM1-43 staining was reduced by 42%. In electron micrographs of neurons at rest or stimulated at 1 Hz in the ganglion, endoplasmic reticulum lay between clusters of dense core vesicles and the plasma membrane. In contrast, in neurons stimulated at 20 Hz, the vesicle clusters were apposed to the plasma membrane and flanked by the endoplasmic reticulum. These results suggest that Ca2+-induced Ca2+ release produces vesicle mobilization and fusion in the soma of Retzius neurons, and supports the idea that neuronal somatic secretion shares common mechanisms with secretion by excitable endocrine cells.     

Caffeine-induced modulation of network oscillation in a molluscan olfactory center

Calcium release from intracellular stores has various actions in neurons, but its effects on network oscillation have not been well understood. The olfactory center (procerebrum, PC) of the terrestrial slug Limax valentianus shows a regular oscillation in the local field potential (LFP). Here we report that caffeine, which is an agonist for ryanodine receptors and triggers calcium release from intra-cellular stores, has strong modulatory effects on the PC. In isolated PC neurons, caffeine enhanced the cytoplasmic calcium concentration, and this was blocked by ryanodine. Caffeine elevated the frequency and amplitude of the LFP oscillation, which was also blocked by ryanodine. The time lag between the frequency and amplitude effects suggests distinct mechanisms for the modulation of these two parameters. These results suggest that calcium release from intracellular stores through ryanodine receptors activates network activity in the PC.     

Calcium-induced release of calcium regulates differentiation of cultured spinal neurons.

Voltage-dependent calcium influx has been shown to regulate the differentiation of cultured amphibian spinal neurons. We have examined the transient elevation of intracellular calcium induced by depolarization, using calcium indicators and confocal microscopy with high temporal and spatial resolution. Rapid calcium elevations in both the nucleus and the cytosol are primarily due to calcium-dependent release of calcium from intracellular stores. Depletion of stores associated with the endoplasmic reticulum reduces all transients. Elevations diminish with neuronal maturation. Depletion of stores of intracellular calcium at early times affects neuronal differentiation in a manner similar to the prevention of influx. The results indicate that both influx and release are necessary to promote neuronal differentiation.     

The role of the presenilin-1 homologue gene sel-12 of Caenorhabditis elegans in apoptotic activities

Many cases of autosomal dominant early onset familial Alzheimer's disease result from mutations in presenilin-1 (PS1). In this study, we examined the role of the PS1 homologue gene sel-12 of Caenorhabditis elegans under oxidative stress and clarified the sel-12-induced apoptosis. A genetic null allele mutant, sel-12(ar171), showed resistance to oxidative stress and prevented mitochondrial dysfunction-induced apoptosis. On the other hand, another allele mutant, sel-12(ar131), that carries a missense mutation showed a proapoptotic activity, which may be the result of a gain of function property. Also, sel-12(ar131)-induced apoptosis was ced-3- and ced-4-dependent. Dantrolene, which specifically inhibits Ca(2+) release from endoplasmic reticulum stores, prevents sel-12(ar131)-induced apoptosis. SEL-12, which is localized in the endoplasmic reticulum, may induce apoptosis through abnormal calcium release from the endoplasmic reticulum. Together, with the previous finding that human PS1 could substitute for SEL-12, these results suggest the similar involvement of PS1-inducing apoptosis under oxidative stress and mitochondrial dysfunction in the Alzheimer's Disease brain.     

Ceramide increases mitochondrial free calcium levels via caspase 8 and Bid: role in initiation of cell death

We investigated how the mitochondrial phase of ceramide-mediated cell death is initiated in nerve growth factor (NGF)-differentiated PC12 cells. We distinguished three independent effects of ceramide: free radical production; a transient increase in cytosolic free calcium; and a long-lasting increase in mitochondrial free calcium. Only the latter led to cell death, which could be prevented by buffering of mitochondrial calcium with the calcium binding protein calbindin D-28K ectopically expressed in mitochondria. We showed that mitochondrial calcium did not increase as a result of the increase in cytosolic free calcium levels. Rather, it appears to derive from the endoplasmic reticulum (ER) since dantrolene, which inhibits release of calcium from ER into cytosol through ryanodine receptors, prevented the increase in cytosolic free calcium but potentiated the increase in mitochondrial free calcium. This suggests that a transfer of calcium occurs directly, or very locally, between the two organelles. This transfer implicated activation of caspase 8 and cleavage of its substrate Bid, a previously unknown function of these cell death intermediaries. The increase in mitochondrial free calcium was also responsible for the release of cytochrome c into the cytosol, underlining the critical role it plays in ceramide-mediated cell death.     

Contribution of intracellular calcium stores to an increase in cytosolic calcium concentration induced by Mannheimia haemolytica leukotoxin

The contribution of intracellular calcium stores to Mannheimia haemolytica leukotoxin (LKT)-induced increase in cytosolic calcium concentration was studied by pharmacologically inhibiting transport of calcium across the plasma and endoplasmic reticulum membranes of bovine neutrophils exposed to LKT. Active intracellular storage of calcium by sarcoplasmic/endoplasmic reticulum calcium ATPase, influx of extracellular calcium across the plasma membrane, and release of stored calcium via inositol triphosphate receptors and ryanodine-sensitive calcium channels were inhibited using thapsigargin, lanthanum chloride, xestospongin C, and magnesium chloride, respectively. Pre-incubation with thapsigargin attenuated the increase in cytosolic calcium concentration produced by LKT, thus confirming the involvement of intracellular calcium stores. Inhibitory effects of lanthanum chloride, xestospongin C, and magnesium chloride indicated that the increase in cytosolic calcium concentration induced by LKT resulted from both influx of calcium across the plasma membrane and release of calcium from intracellular stores.