Mechanical ventilation preserves diaphragm mitochondrial function in a rat sepsis model.

BACKGROUND: To describe the effect of mechanical ventilation on diaphragm mitochondrial oxygen consumption, ATP production, reactive oxygen species (ROS) generation, and cytochrome c oxidase activity and content, and their relationship to diaphragm strength in an experimental model of sepsis. METHODS: A cecal ligation and puncture (CLP) protocol was performed in 12 rats while 12 controls underwent sham operation. Half of the rats in each group were paralyzed and mechanically ventilated. We performed blood gas analysis and lactic acid assays 6 h after surgery. Afterwards, we measured diaphragm strength and mitochondrial oxygen consumption, ATP and ROS generation, and cytochrome c oxidase activity. We also measured malondialdehyde (MDA) content as an index of lipid peroxidation, and mRNA expression of the proinflammatory interleukin-1ß (IL-1ß) in diaphragms. RESULTS: CLP rats showed severe hypotension, metabolic acidosis, and upregulation of diaphragm IL-1ß mRNA expression. Compared to sham controls, spontaneously breathing CLP rats showed lower diaphragm force and increased susceptibility to fatigue, along with depressed mitochondrial oxygen consumption and ATP production and cytochrome c oxidase activity. These rats also showed increased mitochondrial ROS generation and MDA content. Mechanical ventilation markedly restored mitochondrial oxygen consumption and ATP production in CLP rats; lowered mitochondrial ROS production by the complex 3; and preserved cytochrome c oxidase activity. CONCLUSION: In an experimental model of sepsis, early initiation of mechanical ventilation restores diaphragm mitochondrial function.

Electrophysiological properties of the mitochondrial permeability transition pores: Channel diversity and disease implication.

The mitochondrial permeability transition pore (mPTP) is a channel that, when open, is responsible for a dramatic increase in the permeability of the mitochondrial inner membrane, a process known as the mitochondrial permeability transition (mPT). mPTP activation during Ca2+ dyshomeostasis and oxidative stress disrupts normal mitochondrial function and induces cell death. mPTP opening has been implicated as a critical event in many diseases, including hypoxic injuries, neurodegeneration, and diabetes. Discoveries of recent years indicate that mPTP demonstrates very complicated behavior and regulation, and depending on specific induction or stress conditions, it can function as a high-conductance pore, a small channel, or a non-specific membrane leak. The focus of this review is to summarize the literature on the electrophysiological properties of the mPTP and to evaluate the evidence that it has multiple molecular identities. This review also provides perspective on how an electrophysiological approach can be used to quantitatively investigate the biophysical properties of the mPTP under physiological, pharmacological, pathophysiological, and disease conditions.

The SR/ER-mitochondria calcium crosstalk is regulated by GSK3ß during reperfusion injury.

Glycogen synthase kinase-3ß (GSK3ß) is a multifunctional kinase whose inhibition is known to limit myocardial ischemia-reperfusion injury. However, the mechanism mediating this beneficial effect still remains unclear. Mitochondria and sarco/endoplasmic reticulum (SR/ER) are key players in cell death signaling. Their involvement in myocardial ischemia-reperfusion injury has gained recognition recently, but the underlying mechanisms are not yet well understood. We questioned here whether GSK3ß might have a role in the Ca(2+) transfer from SR/ER to mitochondria at reperfusion. We showed that a fraction of GSK3ß protein is localized to the SR/ER and mitochondria-associated ER membranes (MAMs) in the heart, and that GSK3ß specifically interacted with the inositol 1,4,5-trisphosphate receptors (IP3Rs) Ca(2+) channeling complex in MAMs. We demonstrated that both pharmacological and genetic inhibition of GSK3ß decreased protein interaction of IP3R with the Ca(2+) channeling complex, impaired SR/ER Ca(2+) release and reduced the histamine-stimulated Ca(2+) exchange between SR/ER and mitochondria in cardiomyocytes. During hypoxia reoxygenation, cell death is associated with an increase of GSK3ß activity and IP3R phosphorylation, which leads to enhanced transfer of Ca(2+) from SR/ER to mitochondria. Inhibition of GSK3ß at reperfusion reduced both IP3R phosphorylation and SR/ER Ca(2+) release, which consequently diminished both cytosolic and mitochondrial Ca(2+) concentrations, as well as sensitivity to apoptosis. We conclude that inhibition of GSK3ß at reperfusion diminishes Ca(2+) leak from IP3R at MAMs in the heart, which limits both cytosolic and mitochondrial Ca(2+) overload and subsequent cell death.

The voltage-dependent anion channel (VDAC): function in intracellular signalling, cell life and cell death.

Research over the last decade has extended the prevailing view of mitochondria to include functions well beyond the critical bioenergetics role in supplying ATP. It is now recognized that mitochondria play a crucial role in cell signaling events, inter-organelle communication, aging, many diseases, cell proliferation and cell death. Apoptotic signal transmission to the mitochondria results in the efflux of a number of potential apoptotic regulators to the cytosol that trigger caspase activation and lead to cell destruction. Accumulating evidence indicates that the voltage-dependent anion channel (VDAC) is involved in this release of proteins via the outer mitochondrial membrane. VDAC in the outer mitochondrial membrane is in a crucial position in the cell, forming the main interface between the mitochondrial and the cellular metabolisms. VDAC has been recognized as a key protein in mitochondria-mediated apoptosis since it is the proposed target for the pro- and anti-apoptotic Bcl2-family of proteins and due to its function in the release of apoptotic proteins located in the inter-membranal space. The diameter of the VDAC pore is only about 2.6-3 nm, which is insufficient for passage of a folded protein like cytochrome c. New work suggests pore formation by homo-oligomers of VDAC or hetero-oligomers composed of VDAC and pro-apoptotic proteins such as Bax or Bak. This review provides insights into the central role of VDAC in cell life and death and emphasizes its function in the regulation of mitochondria-mediated apoptosis and, thereby, its potential as a rational target for new therapeutics.

Mitochondria regulate inactivation of L-type Ca2+ channels in rat heart.

1. L-type Ca2+ channels play an important role in vital cell functions such as muscle contraction and hormone secretion. Both a voltage-dependent and a Ca2+-dependent process inactivate these channels. Here we present evidence that inhibition of the mitochondrial Ca2+ import mechanism in rat (Sprague-Dawley) ventricular myocytes by ruthenium red (RR), by Ru360 or by carbonyl cyanide m-chlorophenylhydrazone (CCCP) decreases the magnitude of electrically evoked transient elevations of cytosolic Ca2+ concentration ([Ca2+]c). These agents were most effective at stimulus rates greater than 1 Hz. 2. RR and CCCP also caused a significant delay in the recovery from inactivation of L-type Ca2+ currents (I(Ca)). This suggests that sequestration of cytosolic Ca2+, probably near the mouth of L-type Ca2+ channels, into mitochondria during cardiac contractile cycles, helps to remove the Ca2+-dependent inactivation of L-type Ca2+ channels. 3. We conclude that impairment of mitochondrial Ca2+ transport has no impact on either L-type Ca2+ currents or SR Ca2+ release at low stimulation frequencies (e.g. 0.1 Hz); however, it causes a depression of cytosolic Ca2+ transients attributable to an impaired recovery of L-type Ca2+ currents from inactivation at high stimulation frequencies (e.g. 3 Hz). The impairment of mitochondrial Ca2+ uptake and subsequent effects on Ca2+ transients at high frequencies at room temperature could be physiologically relevant since the normal heart rate of rat is around 5 Hz at body temperature. The role of mitochondria in clearing Ca2+ in the micro-domain near L-type Ca2+ channels could be impaired during high frequencies of heart beats such as in ventricular tachycardia, explaining, at least in part, the reduction of muscle contractility.

Efflux of glutathione conjugate of monochlorobimane from striatal and cortical neurons.

Evidence for the presence of a novel transporter in primary cultures of rat striatal neurons and mouse cortical neurons similar in function to the multidrug resistance-associated protein (MRP1) is presented. Functional activity was assessed by efflux studies with the glutathione conjugate of monochlorobimane (B-SG). The glutathione transferase-catalyzed formation of B-SG in rat striatal neurons and mouse cortical neurons was inhibited by ethacrynic acid. The efflux of B-SG from rat striatal neurons and mouse cortical neurons was lower at 20 degrees C than at 37 degrees C and was lower in cells with reduced ATP concentrations compared with cells with constitutive ATP concentrations. In addition, the efflux of B-SG was inhibited by MK-571 in both rat striatal and mouse cortical neurons and by probenecid in rat striatal neurons, but not in mouse cortical neurons. Verapamil did not inhibit B-SG efflux in either rat striatal or mouse cortical neurons. Although functionally similar to MRP1, Western blot analysis with commercially available antibodies directed against human and mouse MRP1 failed to show MRP1-like protein in either whole-cell homogenates of rat striatal neurons or mouse cortical neurons, indicating that the described neuronal transporter differs in structure from human or mouse MRP1 or lacks epitopes in common with MRP1.

Identification of a ryanodine receptor in rat heart mitochondria.

Recent studies have shown that, in a wide variety of cells, mitochondria respond dynamically to physiological changes in cytosolic Ca(2+) concentrations ([Ca(2+)](c)). Mitochondrial Ca(2+) uptake occurs via a ruthenium red-sensitive calcium uniporter and a rapid mode of Ca(2+) uptake. Surprisingly, the molecular identity of these Ca(2+) transport proteins is still unknown. Using electron microscopy and Western blotting, we identified a ryanodine receptor in the inner mitochondrial membrane with a molecular mass of approximately 600 kDa in mitochondria isolated from the rat heart. [(3)H]Ryanodine binds to this mitochondrial ryanodine receptor with high affinity. This binding is modulated by Ca(2+) but not caffeine and is inhibited by Mg(2+) and ruthenium red in the assay medium. In the presence of ryanodine, Ca(2+) uptake into isolated heart mitochondria is suppressed. In addition, ryanodine inhibited mitochondrial swelling induced by Ca(2+) overload. This swelling effect was not observed when Ca(2+) was applied to the cytosolic fraction containing sarcoplasmic reticulum. These results are the first to identify a mitochondrial Ca(2+) transport protein that has characteristics similar to the ryanodine receptor. This mitochondrial ryanodine receptor is likely to play an essential role in the dynamic uptake of Ca(2+) into mitochondria during Ca(2+) oscillations.

Mitochondrial calcium in heart cells: beat-to-beat oscillations or slow integration of cytosolic transients?

Mitochondria have been implicated in intracellular Ca2+ signaling in many cell types. The inner mitochondrial membrane contains Ca2+-transporting proteins, which catalyze Ca2+ uptake and extrusion. Intramitochondrial (matrix) Ca2+, in turn, regulates the activity of Krebs cycle dehydrogenases and, ultimately, the rate of ATP synthesis. In the myocardium, controversy remains whether the fast cytosolic Ca2+ transients underlying excitation-contraction coupling in beating cells are rapidly transmitted into the matrix compartment or slowly integrated by the mitochondrial Ca2+ transporters. This mini-review critically summarizes the recent experimental work in this field.

Transport of Ca2+ from sarcoplasmic reticulum to mitochondria in rat ventricular myocytes.

Studies with electron microscopy have shown that sarcoplasmic reticulum (SR) and mitochondria locate close to each other in cardiac muscle cells. We investigated the hypothesis that this proximity results in a transient exposure of mitochondrial Ca2+ uniporter (CaUP) to high concentrations of Ca2+ following Ca2+ release from the SR and thus an influx of Ca2+ into mitochondria. Single ventricular myocytes of rat were skinned by exposing them to a physiological solution containing saponin (0.2 mg/ml). Cytosolic Ca2+ concentration ([Ca2+]c) and mitochondrial Ca2+ concentration ([Ca2+]m) were measured with fura-2 and rhod2, respectively. Application of caffeine (10 mM) induced a concomitant increase in [Ca2+]c and [Ca2+]m. Ruthenium red, at concentrations that block CaUP but not SR release, diminished the caffeine-induced increase in [Ca2+]m but not [Ca2+]c. In the presence of 1 mM BAPTA, a Ca2+ chelator, the caffeine-induced increase in [Ca2+]m was reduced substantially less than [Ca2+]c. Moreover, inhibition of SR Ca2+ pump with two different concentrations of thapsigargin caused an increase in [Ca2+]m, which was related to the rate of [Ca2+]c increase. Finally, electron microscopy showed that sites of junctions between SR and T tubules from which Ca2+ is released, or Ca2+ release units, CRUs, are preferentially located in close proximity to mitochondria. The distance between individual SR Ca2+ release channels (feet or ryanodine receptors) is very short, ranging between approximately 37 and 270 nm. These results are consistent with the idea that there is a preferential coupling of Ca2+ transport from SR to mitochondria in cardiac muscle cells, because of their structural proximity.

Rapid report: a novel technique for quantitative measurement of free Ca2+ concentration in rat heart mitochondria.

1. The free mitochondrial Ca2+ concentration ([Ca2+]m) in rat heart mitochondria was measured quantitatively by loading the mitochondria with fura-2 and then injecting them into Xenopus laevis oocytes. 2. When oocytes were incubated with a physiological solution, the free cytosolic Ca2+ concentration ([Ca2+]c) in the oocytes was 82 +/- 11 nM (n = 20, mean +/- s.e.m.) and the [Ca2+]m of the injected rat heart mitochondria was 116 +/- 10 nM (n = 18, mean +/- s.e.m.). 3. Inhibition of the oocyte endoplasmic reticular Ca2+-ATPase with thapsigargin produced a transient increase in averaged [Ca2+]c at sub-micromolar concentrations. 4. Injection of cardiac mitochondria blunted the peak and prolonged the duration of thapsigargin-induced [Ca2+]c transients as a result of Ca2+ sequestration by the cardiac mitochondria. 5. These results demonstrate that the present technique provides a new approach for studying [Ca2+]m regulation quantitatively under physiological environments. Furthermore, it clearly shows that cardiac mitochondria can modify the shape of thapsigargin-induced cytosolic Ca2+ pulses in Xenopus oocytes.

Signaling mechanisms underlying muscarinic receptor-mediated increase in contraction rate in cultured heart cells.

We have investigated the mechanisms by which stimulation of cardiac muscarinic receptors result in paradoxical stimulatory effects on cardiac function, using cultured neonatal rat ventricular myocytes as a model system. Application of low concentrations of carbachol (CCh) (EC50 = 35 nM) produced an atropine-sensitive decrease in spontaneous contraction rate, while, in cells pretreated with pertussis toxin, higher concentrations of CCh (EC50 = 26 microM) elicited an atropine-sensitive increase in contraction rate. Oxotremorine, an m2 muscarinic acetylcholine receptor (mAChR) agonist, mimicked the negative but not the positive chronotropic response to CCh. Reverse transcription followed by polymerase chain reaction carried out on mRNA obtained from single cells indicated that ventricular myocytes express mRNA for the m1, m2, and, possibly, m4 mAChRs. The presence of m1 and m2 mAChR protein on the surface membranes of the cultured ventricular myocytes was confirmed by immunofluorescence. The CCh-induced positive chronotropic response was significantly inhibited by fluorescein-tagged antisense oligonucleotides directed against the m1, but not the m2 and m4, mAChR subtypes. The response was also inhibited by antisense oligonucleotides against Gqalpha protein. Finally, inhibition of CCh-induced phosphoinositide hydrolysis with 500 microM neomycin or 5 microM U73122 completely abolished the CCh-induced positive chronotropic response. These results are consistent with the stimulatory effects of mAChR activation on the rate of contractions in cultured ventricular myocytes being mediated through the m1 mAChR coupled through Gq to phospholipase C-induced phosphoinositide hydrolysis.

Identification of a novel point mutation (S65T) in alpha-galactosidase A gene in Chinese patients with Fabry disease. Mutations in brief no. 169. Online.

Fabry disease is an X-linked inborn error of sphingolipid catabolism resulting from deficient enzyme activity of alpha-galactosidase A. The molecular defects of human alpha-galactosidase A gene causing Fabry disease have been characterized, including gene rearrangement and point mutations, which show the genetic heterogeneity in Fabry disease. To characterize the molecular defects of these patients, each exon of alpha-galactosidase A gene including intron-exon junctions were PCR amplified using biotin-labelled primer and sequenced using magnetic beads solid-phase sequencing. A G to C transversion was identified in the last nucleotide of exon 1 in two unrelated Chinese patients. This mutation obliterates an EcoN1 restriction site. Family studies show close linkage with the affected family members. Screening of 100 alleles (22 males, 39 females) of unrelated normal Chinese can not find this mutation. This mutation not only changes the amino acid from serine to threonine, but also likely cause splicing defects. To our knowledge, this is the first report of mutation in Chinese patients with Fabry disease, and a novel mutation causing Fabry disease not reported in literature previously.

3-Nitropropionic acid exacerbates N-methyl-D-aspartate toxicity in striatal culture by multiple mechanisms.

We examined the effects of 3-nitropropionic acid-induced succinate dehydrogenase inhibition on neuronal ATP content, N-methyl-D-aspartate-induced neuronal death, resting membrane potential, and N-methyl-D-aspartate-induced changes in cytosolic calcium concentration ([Ca2+]c) in cultured rat striatal neurons. Exposure of cultures to 3 mM 3-nitropropionic acid for 3 h did not cause overt toxicity, but reduced ATP content by 35%. Treatment with 3-nitropropionic, or removal of Mg2+ from the medium, enhanced subsequent N-methyl-D-aspartate toxicity, reducing the LC50 from 250 microM to 12 microM or 30 microM, respectively. Even after Mg2+ removal, enhancement of N-methyl-D-aspartate toxicity by 3-nitropropionic acid remained pronounced, with the LC50 further decreasing to 3 microM. The mean resting membrane potential of neurons treated with 3-nitropropionic acid was -37 mV, while that in control neurons was -61 mV. Treatment with 3-nitropropionic did not affect baseline [Ca2+]c as determined by fura-2 microfluorimetry. N-methyl-D-aspartate (30 microM) caused a rapid rise in [Ca2+]c, the initial magnitude of which was not affected by 3-nitropropionic acid. However, after a 1-h treatment, [Ca2+]c was dramatically higher in 3-nitropropionic acid-treated neurons. This increased calcium load was washed out slowly and only partially, although calcium in control neurons washed out rapidly and almost completely. These results suggest that in striatal neurons, the enhancement of N-methyl-D-aspartate toxicity caused by succinate dehydrogenase inhibition may be due to synergism between partial relief of the Mg2+ blockade of the N-methyl-D-aspartate receptor and other mechanisms, including disruption of neuronal calcium regulation. This synergism may be relevant to the neuronal death observed in neurodegenerative disorders.

Visualization of NMDA receptor-induced mitochondrial calcium accumulation in striatal neurons.

Ca2+ influx through NMDA receptor-gated channels and the subsequent rise in intracellular Ca2+ concentration ([Ca2+]i) have been implicated in cytotoxic processes that lead to irreversible neuronal injury. While many studies have focused on cytosolic Ca2+ homeostasis, much less is known about Ca2+ fluxes in subcellular organelles, such as mitochondria. The mitochondria play an important role in Ca2+ homeostasis by sequestering cytosolic Ca2+ loads. However, mitochondrial Ca2+ overload can impair ATP synthesis, induce free radical formation, and lead to lipid peroxidation. Thus, it is also important to understand the mitochondrial Ca2+ fluxes induced by NMDA. In this study, changes in mitochondrial Ca2+ concentration ([Ca2+]m) in cultured striatal neurons were monitored with a Ca(2+)-binding fluorescent probe, rhod-2, and laser scanning confocal microscopy. The rhod-2 fluorescence signal was highly localized in mitochondrial areas of confocal images. A rapid increase of [Ca2+]m was observed when neurons were treated with 100 microM NMDA. The increased [Ca2+]m induced by NMDA could not be observed in the presence of ruthenium red, an inhibitor of the mitochondrial Ca2+ uniporter, or CCCP, a protonophore that breaks down the mitochondrial membrane potential necessary for Ca2+ uptake. The magnitude and reversibility of changes in [Ca2+]m induced by NMDA were variable. In neurons receiving multiple pulses of NMDA, [Ca2+]m did not return to baseline. The elevated [Ca2+]m may persist indefinitely and may rise further after successive NMDA exposures. These data demonstrate that Ca2+ accumulates in mitochondria in response to NMDA receptor activation. This Ca2+ accumulation may play a role in the excitotoxic mitochondrial dysfunction induced by NMDA.

Does mammalian heart contain only the M2 muscarinic receptor subtype?

Five muscarinic acetylcholine receptor (mAChR) subtypes, m1-m5, have been cloned and sequenced to date. The question as to which mAChR subtypes exist in mammalian heart has been studied extensively and is still under considerable debate. We used the reverse transcriptase-polymerase chain reaction to amplify mRNA from adult rat ventricular myocytes, and found that these cells express mRNA for m1 and m2 mAChRs. Immunocytochemical analysis confirmed that m1 and m2, but not m3, mAChR proteins are present on the surface of these cells. Finally, the functional significance of these receptors was examined. Administration of the m1 mAChR antagonist pirenzepine inhibited the stimulatory effect of the muscarinic agonist carbachol on Ca transients. These findings are consistent with the presence of at least two mAChR subtypes in mammalian heart, m1 and m2, and suggest that activation of m1 mAChRs is involved in the stimulatory effects of muscarinic agonists in mammalian heart.

[3H]benzylpempidine, a new radioligand for probing a putative channel site on nicotinic cholinergic receptors.

A study was undertaken to assess the receptor binding characteristics of [3H]4-benzylpempidine to an allosteric site on calf brain membranes associated with nicotinic cholinergic receptors and to compare the binding affinity of novel arylpempidine analogs with their ability to antagonize the behavioral effects of nicotine in mice. Scatchard analysis of the binding yielded a K(d) of 20 nM and a B(max) of 330 fmols/mg membrane protein. [3H]4-benzylpempidine appears to be a more satisfactory ligand than [3H]mecamylamine, since it possessed a 50-fold greater affinity and its binding was far less sensitive to inorganic ions and Tris. Among the arylpempidine analogs 4-m-chlorobenzylidenepempidine and 4-benzylidenepempidine had the lowest K(i) values (1.4 nM and 5.0 nM, respectively) and were the most potent in antagonizing nicotine-induced seizures in mice. Although the K(i) values for pempidine and mecamylamine were 1-2 orders of magnitude greater than any of the arylpempidines, the dose required to antagonize nicotine-induced seizures in mice was comparable to the arylpempidines. One explanation for this apparent discrepancy in the correlation of binding affinity and nicotine antagonism is the lower brain penetration of arylpempidines compared to mecamylamine, following their systemic administration to mice.

Phosphatidic acid: a potential signal transducer for cardiac hypertrophy.

Phosphatidic acid (PA) is mainly formed by the hydrolysis of phosphatidylcholine due to the activation of phospholipase D (PLD). PA is also generated by phosphorylation of diacylglycerol (DAG) due to the action of DAG kinase and is converted to DAG under the action of PA phosphohydrolase. Most of the positive inotropic agents which are known to stimulate cardiac hypertrophy, have been shown to increase the level of PA in cardiac sarcolemma. Although the growth factor-like effect of PA has been recognized in a wide variety of tissues, there is a lack of similar information in adult cardiomyocytes. By using single cardiomyocytes, we have now shown that PA increased the basal [Ca2+]i level without significant effect on the amplitude of Ca2+ transients. PA (10-50 mu M) also increased the [Ca2+]i in cardiac cell suspension. PA has also been shown to stimulate protein synthesis in cardiomyocytes, which is inhibited by a PKC inhibitor as well as a Ca2+ chelator. PA at the concentration of 1-50 mu M was observed to stimulate the activity of PLC in cardiac sarcolemma; this effect was attenuated by a PLC inhibitor. Since DAG, formed due to the activation of PLC, is considered to play a crucial role in regulating the activity of protein kinase C (PKC), the positive feedback effect of PA on this pathway may be essential for maintaining the sustained elevation in the activity of PKC during the development of cardiac hypertrophy. In view of these observations and other facts available in the literature, it is suggested that PA may be a potential signal transducer for the development of cardiac hypertrophy.

Histamine induces oscillations of mitochondrial free Ca2+ concentration in single cultured rat brain astrocytes.

1. The free Ca2+ concentration of mitochondria ([Ca2+]m) in cultured rat brain astrocytes was measured with a fluorescent Ca2+ indicator, rhod-2, and laser confocal microscopy. 2. Confocal images revealed a rhod-2 distribution that matched mitochondrial localization. 3. Using a Ca2+ ionophore, ionomycin, to clamp the [Ca2+]m from 0 to 100 microM in order to obtain the minimal and maximal fluorescence of rhod-2 in situ, a 3.5 +/- 0.4-fold increase in fluorescence intensity was observed, suggesting that the fluorescence of intramitochondrial rhod-2 was responding in a Ca(2+)-sensitive manner, thereby allowing measurements of [Ca2+]m in single astrocytes. 4. Exposure of fura-2-loaded astrocytes to 100 microM histamine produced a rapid and transient increase in cytosolic Ca2+ concentration ([Ca2+]c) that lasted for several tens of seconds. The spike in [Ca2+]c was frequently followed by variable numbers of repetitive oscillations of Ca2+, which appeared to dampen in amplitude with time. 5. This pattern of histamine-induced [Ca2+]c oscillations was also observed in rhod-2-loaded cells suggesting that [Ca2+]m fluctuated with a similar frequency. 6. The oscillations of [Ca2+]m, but not of [Ca2+]c, were abolished by a proton ionophore, carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), and by Ruthenium Red, a mitochondrial Ca(2+)-uniporter inhibitor. 7. These results suggest that the mitochondrial Ca2+ transport systems in cultured rat brain astrocytes are able to relay receptor-mediated [Ca2+]m oscillations into mitochondria.