Biomedical subjects

Gary Fiskum

Publications and source records attributed to Gary Fiskum.

At least 19 recordsLinked to original sources

PubMed2025

Acute Changes in Rat Tissue Gene Expression Following Exposure to Flight Relevant Hypobaria.

Aeromedical evacuation (AE) is an invaluable tool for the transport of critically injured patients to care facilities. There is increasing evidence obtained from animal models and human patients that exposure to AE-relevant hypobaria within a few days of injury can worsen outcomes. The cause of this secondary injury is not well understood but it may be related to changes in gene expression induced by exposure to evacuation-relevant conditions. In order to explore the causes of secondary injury, gene expression induced by AE-relevant flight conditions was analyzed. Adult male rats were exposed to flight cabin-relevant hypobaria (8000 or 4000 ft equivalents) and/or different oxygen concentrations (21% or 100%) for 5 or 10 hr. At the end of the exposures, RNA was isolated from lung, blood, heart, and brain (hippocampus), levels of gene expression were measured via microarray analysis, and canonical pathway analysis identified the primary gene pathways enriched by the exposures. This information should be useful to not only optimize the health status of trauma patients undergoing aeromedical evacuation but also help determine which gene expression pathways could be modulated to optimize the therapeutic efficacy of the body's endogenous protection and repair mechanisms.

Animals↗

PubMed2007

Hypotensive hemorrhage increases calcium uptake capacity and Bcl-XL content of liver mitochondria.

We tested the hypothesis that the response of mitochondrial uptake of calcium and content of Bcl proteins to reversible hemorrhagic shock increases the vulnerability for hepatocellular death. Pentobarbital-anesthetized rats were bled to a mean arterial pressure of 30 to 40 mmHg for 1 h. A subset was then resuscitated (isotonic sodium chloride solution, three times shed volume). Liver mitochondria were isolated at the end of hemorrhage and 1.5 h after the onset of resuscitation. Resuscitation accelerated mitochondrial respiration in the presence of adenosine diphosphate (state 3) above control (P<0.01). The respiratory control ratio ([RCR] state 3/state 4) was calculated using the respiratory rate in the presence of carboxyatractyloside (state 4). The RCR was depressed at the end of hemorrhage and recovered completely in response to resuscitation (P<0.05). The mitochondrial capacity for calcium uptake increased at the end of hemorrhage and remained greater than control (P<0.01) after resuscitation when plasma ornithine carbamoyltransferase (an index of hepatocellular injury) was greater than control (P<0.05). At this time, the capacity for calcium uptake was correlated with plasma ornithine carbamoyltransferase (r=0.819, P<0.01). Mitochondrial content of Bcl-xL, an antiapoptotic protein, was increased at the end of hemorrhage (P<0.03) with no further change after resuscitation and no change in mitochondrial Bak, a proapoptotic protein. Thus, mitochondrial mechanisms are triggered early during reversible hypovolemia that may limit the intensity of intracellular calcium signaling and its potential to cause cellular injury and death.

Animals↗

PubMed2006

Anoxia-induced changes in pyridine nucleotide redox state in cortical neurons and astrocytes.

NAD(P)H autofluorescence was used to verify establishment of metabolic anoxia using primary cultures of cortical neurons and astrocytes. Cells on cover slips were placed in a chamber and O(2) was displaced by continuous infusion of argon. Perfusion with medium at PO(2) < 0.4 mm Hg caused an increase in NAD(P)H fluorescence, albeit to levels lower than that obtained with cyanide. Addition of the nitric oxide-generating agent DETA-NO to the hypoxic medium further increased fluorescence to the level with cyanide. Fluorescence under anoxia remained high in the presence of glucose, but declined in neurons and not in astrocytes when glucose was substituted with 2-deoxyglucose. Reoxygenation of neurons resulted in a decline in fluorescence and a loss in fluorescent gradient between fully reduced and fully oxidized (plus respiratory uncoupler). We conclude that (1) DETA-NO is useful for generating metabolic anoxia in the presence of argon (2) Exogenous glucose is necessary to maintain NAD(P)H in a reduced state during metabolic anoxia in neurons but not astrocytes (3) Neurons undergo a partially irreversible decline in NAD(P)H fluorescence during metabolic anoxia and reoxygenation that could contribute to prolonged metabolic failure.

Animals↗

PubMed2006

Neuron-specific conditional expression of a mitochondrially targeted fluorescent protein in mice.

Mitochondrial dysfunction contributes to the pathophysiology of both acute and chronic neurodegenerative disorders. Quantification of mitochondrial bioenergetic properties generally requires the use of isolated brain mitochondria. However, the involvement of neuronal mitochondrial dysfunction in these disorders is limited by the lack of markers, and therefore isolation procedures, that distinguish neuronal compared with astrocyte mitochondria. To address this and other issues concerning neuronal mitochondria in the CNS, transgenic mice were generated that express a fluorescent protein targeted specifically to neurons. A neuron-specific promoter, CaMKIIalpha (calcium/calmodulin-dependent kinase IIalpha) driven tTA (tetracycline transactivator) mice were crossed with TRE (tetracycline responsive element) driven mitochondrial targeted enhanced yellow fluorescent protein (eYFP) mice. Expression of eYFP in the bigenic mouse brain was observed only in neuronal mitochondria of striatum, forebrain, and hippocampus and was enhanced by the removal of the tetracycline analog doxycycline (Dox) in the diet. The respiratory control ratio of synaptic and nonsynaptic mitochondria isolated from eYFP-expressing mice was the same as control mice, suggesting that neuronal mitochondria expressing eYFP maintain normal bioenergetic functions. More importantly, the development of Dox-inducible, neuron targeted mito/eYFP transgenic mice offer a unique in vivo model for delineating the participation of neuronal mitochondria in neuronal survival and death.

Animals↗

PubMed2006

Oximetry-guided reoxygenation improves neurological outcome after experimental cardiac arrest.

BACKGROUND AND PURPOSE: Current guidelines suggest that cardiac arrest (CA) survivors should be ventilated with 100% O(2) after resuscitation. Breathing 100% O(2) may worsen neurological outcome after experimental CA. This study tested the hypothesis that graded reoxygenation, with oximetry guidance, can safely reduce FiO(2) after resuscitation, avoiding hypoxia while promoting neurological recovery. METHODS: Mature dogs underwent 10 minutes of CA and restoration of spontaneous circulation with 100% O(2.) Animals were randomized to 1-hour additional ventilation on 100% FiO(2) or to rapid lowering of arterial O(2) saturation to <96% but >94% with pulse oximeter guidance. Animals were awakened at hour 23, and the neurological deficit score (0=normal; 100=brain-dead) was measured. Reanesthetized animals were perfusion-fixed and the brains removed for histopathology. RESULTS: The neurological deficit score was significantly better in oximetry (O) dogs. O dogs appeared aware of their surroundings, whereas most hyperoxic (H) animals were stuporous (neurological deficit score=43.0+/-5.9 [O] versus 61.0+/-4.2 [H]; n=8, P<0.05). Stereological analysis revealed fewer injured CA1 neurons in O animals (cresyl violet: 35.5+/-4.3% [O] versus 60.5+/-3.3% [H]; P<0.05). There were also fewer fluoro-Jade B-stained degenerating CA1 neurons in O animals (3320+/-267 [O] versus 6633+/-356 [H] per 0.1 mm(3); P<0.001). CONCLUSIONS: A clinically applicable protocol designed to reduce postresuscitative hyperoxia after CA results in significant neuroprotection. Clinical trials of controlled normoxia after CA/restoration of spontaneous circulation should strongly be considered.

Animals↗

PubMed2006

Postischemic hyperoxia reduces hippocampal pyruvate dehydrogenase activity.

The pyruvate dehydrogenase complex (PDHC) is a mitochondrial matrix enzyme that catalyzes the oxidative decarboxylation of pyruvate and represents the sole bridge between anaerobic and aerobic cerebral energy metabolism. Previous studies demonstrating loss of PDHC enzyme activity and immunoreactivity during reperfusion after cerebral ischemia suggest that oxidative modifications are involved. This study tested the hypothesis that hyperoxic reperfusion exacerbates loss of PDHC enzyme activity, possibly due to tyrosine nitration or S-nitrosation. We used a clinically relevant canine ventricular fibrillation cardiac arrest model in which, after resuscitation and ventilation on either 100% O2 (hyperoxic) or 21-30% O2 (normoxic), animals were sacrificed at 2 h reperfusion and the brains removed for enzyme activity and immunoreactivity measurements. Animals resuscitated under hyperoxic conditions exhibited decreased PDHC activity and elevated 3-nitrotyrosine immunoreactivity in the hippocampus but not the cortex, compared to nonischemic controls. These measures were unchanged in normoxic animals. In vitro exposure of purified PDHC to peroxynitrite resulted in a dose-dependent loss of activity and increased nitrotyrosine immunoreactivity. These results support the hypothesis that oxidative stress contributes to loss of hippocampal PDHC activity during cerebral ischemia and reperfusion and suggest that PDHC is a target of peroxynitrite.

Animals↗

PubMed2006

Cyclosporin A increases mitochondrial calcium uptake capacity in cortical astrocytes but not cerebellar granule neurons.

Isolated brain mitochondria are a heterogeneous mixture from different cell types and these subsets may have differing sensitivities to Ca2+-induced membrane permeability transition (MPT) and to inhibition of the MPT by cyclosporin A (CsA). This study tested the hypothesis that mitochondria within primary cultures of astrocytes and neurons exhibit different energy-dependent Ca2+ uptake capacities and different degrees to which CsA increases their uptake capacity. Astrocytes and neurons were suspended in a cytosol-like medium containing respiratory substrates, ATP, and Mg2+ in the presence of digitonin to selectively permeabilize the plasma membrane. Uptake of added Ca2+ by mitochondria within the cells was measured by Calcium Green 5N fluorescent monitoring of the medium [Ca2+]. Permeabilized astrocytes had a fourfold higher Ca2+ uptake capacity, relative to neurons and a twofold higher content based on relative contents of mitochondria assessed by measurements of mitochondrial DNA and cytochrome oxidase subunit 1 protein. In astrocytes the Ca2+ uptake capacity was increased twofold by preincubation with 2-5 microM CsA, while in neurons CsA had no effect. Similar results were obtained using measurements of the effects of added Ca2+ on mitochondrial membrane potential. FK506, a drug similar to CsA but without MPT inhibitory activity, had no effect on either cell type. These results are consistent with the presence of a calcium-induced MPT in astrocytes, even in the presence of ATP, and indicate that the MPT in cerebellar granule neurons is resistant to CsA inhibition. Some of the protective effects of CsA in vivo may therefore be mediated by preservation of mitochondrial functional integrity within astrocytes.

Animals↗

PubMed2006

Normoxic resuscitation after cardiac arrest protects against hippocampal oxidative stress, metabolic dysfunction, and neuronal death.

Resuscitation and prolonged ventilation using 100% oxygen after cardiac arrest is standard clinical practice despite evidence from animal models indicating that neurologic outcome is improved using normoxic compared with hyperoxic resuscitation. This study tested the hypothesis that normoxic ventilation during the first hour after cardiac arrest in dogs protects against prelethal oxidative stress to proteins, loss of the critical metabolic enzyme pyruvate dehydrogenase complex (PDHC), and minimizes subsequent neuronal death in the hippocampus. Anesthetized beagles underwent 10 mins ventricular fibrillation cardiac arrest, followed by defibrillation and ventilation with either 21% or 100% O2. At 1 h after resuscitation, the ventilator was adjusted to maintain normal blood gas levels in both groups. Brains were perfusion-fixed at 2 h reperfusion and used for immunohistochemical measurements of hippocampal nitrotyrosine, a product of protein oxidation, and the E1alpha subunit of PDHC. In hyperoxic dogs, PDHC immunostaining diminished by approximately 90% compared with sham-operated dogs, while staining in normoxic animals was not significantly different from nonischemic dogs. Protein nitration in the hippocampal neurons of hyperoxic animals was 2-3 times greater than either sham-operated or normoxic resuscitated animals at 2 h reperfusion. Stereologic quantification of neuronal death at 24 h reperfusion showed a 40% reduction using normoxic compared with hyperoxic resuscitation. These results indicate that postischemic hyperoxic ventilation promotes oxidative stress that exacerbates prelethal loss of pyruvate dehydrogenase and delayed hippocampal neuronal cell death. Moreover, these findings indicate the need for clinical trials comparing the effects of different ventilatory oxygen levels on neurologic outcome after cardiac arrest.

Animals↗

PubMed2006

The potential role of mitochondria in pediatric traumatic brain injury.

Mitochondria play a central role in cerebral energy metabolism, intracellular calcium homeostasis and reactive oxygen species generation and detoxification. Following traumatic brain injury (TBI), the degree of mitochondrial injury or dysfunction can be an important determinant of cell survival or death. Literature would suggest that brain mitochondria from the developing brain are very different from those from mature animals. Therefore, aspects of developmental differences in the mitochondrial response to TBI can make the immature brain more vulnerable to traumatic injury. This review will focus on four main areas of secondary injury after pediatric TBI, including excitotoxicity, oxidative stress, alterations in energy metabolism and cell death pathways. Specifically, we will describe what is known about developmental differences in mitochondrial function in these areas, in both the normal, physiologic state and the pathologic state after pediatric TBI. The ability to identify and target aspects of mitochondrial dysfunction could lead to novel neuroprotective therapies for infants and children after severe TBI.

Aging↗

PubMed2005

Physiologic progesterone reduces mitochondrial dysfunction and hippocampal cell loss after traumatic brain injury in female rats.

Growing literature suggests important sex-based differences in outcome following traumatic brain injury (TBI) in animals and humans. Progesterone has emerged as a key hormone involved in many potential neuroprotective pathways after acute brain injury and may be responsible for some of these differences. Many studies have utilized supraphysiologic levels of post-traumatic progesterone to reverse pathologic processes after TBI, but few studies have focused on the role of endogenous physiologic levels of progesterone in neuroprotection. We hypothesized that progesterone at physiologic serum levels would be neuroprotective in female rats after TBI and that progesterone would reverse early mitochondrial dysfunction seen in this model. Female, Sprague-Dawley rats were ovariectomized and implanted with silastic capsules containing either low or high physiologic range progesterone at 7 days prior to TBI. Control rats received ovariectomy with implants containing no hormone. Rats underwent controlled cortical impact to the left parietotemporal cortex and were evaluated for evidence of early mitochondrial dysfunction (1 h) and delayed hippocampal neuronal injury and cortical tissue loss (7 days) after injury. Progesterone in the low physiologic range reversed the early postinjury alterations seen in mitochondrial respiration and reduced hippocampal neuronal loss in both the CA1 and CA3 subfields. Progesterone in the high physiologic range had a more limited pattern of hippocampal neuronal preservation in the CA3 region only. Neither progesterone dose significantly reduced cortical tissue loss. These findings have implications in understanding the sex-based differences in outcome following acute brain injury.

Animals↗

PubMed2005

Isolation of mitochondria with high respiratory control from primary cultures of neurons and astrocytes using nitrogen cavitation.

To study neurons or glia-specific mitochondria one needs to isolate these organelles from primary neuronal or astrocytic cell culture. This work provides novel method for isolation of functional and morphologically intact mitochondria from neurons and astrocytes in cell cultures. In the first step, mitochondria are released from cells by disruption of cell membranes using a nitrogen cavitation technique. This technique is based on rapid decompression of a cell suspension from within a pressure vessel. Mitochondria released from cell bodies are then separated from the rest of cell homogenate by Percoll gradient centrifugation. This is a relatively rapid technique that yields to very well coupled mitochondria that exhibited functional and morphological characteristics comparable to mitochondria isolated from brain tissue using common techniques. This technique thus will allow examination of mitochondria that are exclusively cell specific in origin.

Animals↗

PubMed2005

Methoxychlor inhibits brain mitochondrial respiration and increases hydrogen peroxide production and CREB phosphorylation.

The organochlorine insecticide methoxychlor (mxc) is an established reproductive toxicant that affects other systems including the central nervous system (CNS), possibly by mechanisms involving oxidative stress. This study tested the hypothesis that mxc inhibits brain mitochondrial respiration, resulting in increased production of reactive oxygen species (ROS). Oxygen electrode measurements of mitochondrial respiration and Amplex Red measurements of H(2)O(2) production were performed with rat brain mitochondria exposed in vitro to mxc (0-10 microg/ml) and with brain mitochondria from mice chronically exposed in vivo to mxc (0-64 mg/kg/day) for 20 days by intraperitoneal injection. In vitro mxc exposure inhibited ADP-dependent respiration (state 3) using both complex I- and II-supported substrates. Similarly, state 3 respiration was inhibited following in vivo mxc exposure using complex I substrates. H(2)O(2) production was stimulated after in vitro mxc treatment in the presence of complex I substrates, but not in mitochondria isolated from in vivo mxc-treated mice. Because previous studies demonstrated a relationship between oxidative stress and CREB phosphorylation, we also tested the hypothesis that mxc elevates phosphorylated CREB (pCREB) in mitochondria. Enzyme-linked immunosorbent assay (ELISA) measurements demonstrated that pCREB immunoreactivity was elevated by in vitro mxc exposure in the presence or absence of respiratory substrates, indicating that stimulation of H(2)O(2) production is not necessary for this effect. These multiple effects of mxc on mitochondria may play an important role in its toxicity, particularly in the CNS.

Animals↗

PubMed2005

Delayed therapy of experimental global cerebral ischemia with acetyl-L-carnitine in dogs.

Acetyl-L-carnitine (ALCAR), when administered immediately following restoration of spontaneous circulation (ROSC) from experimental cardiac arrest (CA) has previously been demonstrated to promote normalization of brain energy metabolism and neurologic recovery following 10 min CA. In order to determine ultimate efficacy for this or other drugs, clinical trials must be performed in human subjects. In several human clinical trials, though, drug administration has been significantly delayed following resuscitation from CA. These experiments test the hypothesis that post-resuscitative delay in ALCAR administration will impair the ability of this drug to promote neurologic recovery. Neurological deficit scoring (23 h) as well as frontal cortex lactate levels (2 and 24 h) were compared following resuscitation from 10 min CA in dogs receiving either ALCAR or drug vehicle 30 min following ROSC. Dogs treated with ALCAR 30 min following ROSC from 10 min CA exhibited more normal cerebral cortex lactate levels than did vehicle control animals. There was no difference, however, in neurologic deficit scores between groups, with all animals demonstrating moderate to severe clinical neurologic impairment at 23 h following ROSC. A 30-min delay in ALCAR administration following ROSC from 10 min CA impairs the ability of this drug to promote neurologic recovery despite apparent normalization of brain lactate levels.

Acetylcarnitine↗

PubMed2005

Inhibition of mitochondrial neural cell death pathways by protein transduction of Bcl-2 family proteins.

Bcl-2 and other closely related members of the Bcl-2 family of proteins inhibit the death of neurons and many other cells in response to a wide variety of pathogenic stimuli. Bcl-2 inhibition of apoptosis is mediated by its binding to pro-apoptotic proteins, e.g., Bax and tBid, inhibition of their oligomerization, and thus inhibition of mitochondrial outer membrane pore formation, through which other pro-apoptotic proteins, e.g., cytochrome c, are released to the cytosol. Bcl-2 also exhibits an indirect antioxidant activity caused by a sub-toxic elevation of mitochondrial production of reactive oxygen species and a compensatory increase in expression of antioxidant gene products. While classic approaches to cytoprotection based on Bcl-2 family gene delivery have significant limitations, cellular protein transduction represents a new and exciting approach utilizing peptides and proteins as drugs with intracellular targets. The mechanism by which proteins with transduction domains are taken up by cells and delivered to their targets is controversial but usually involves endocytosis. The effectiveness of transduced proteins may therefore be limited by their release from endosomes into the cytosol.

Animals↗

PubMed2005

Diacylglycerols activate mitochondrial cationic channel(s) and release sequestered Ca(2+).

Mitochondria contribute to cytosolic Ca(2+) homeostasis through several uptake and release pathways. Here we report that 1,2-sn-diacylglycerols (DAG's) induce Ca(2+) release from Ca(2+)-loaded mammalian mitochondria. Release is not mediated by the uni-porter or the Na(+)/Ca(2+) exchanger, nor is it attributed to putative catabolites. DAG's-induced Ca(2+) efflux is biphasic. Initial release is rapid and transient, insensitive to permeability transition inhibitors, and not accompanied by mitochondrial swelling. Following initial rapid release of Ca(2+) and relatively slow reuptake, a secondary progressive release of Ca(2+) occurs, associated with swelling, and mitigated by permeability transition inhibitors. The initial peak of DAG's-induced Ca(2+) efflux is abolished by La(3+) (1 mM) and potentiated by protein kinase C inhibitors. Phorbol esters, 1,3-diacylglycerols and 1-monoacylglycerols do not induce mitochondrial Ca(2+) efflux. Ca(2+)-loaded mitoplasts devoid of outer mitochondrial membrane also exhibit DAG's-induced Ca(2+) release, indicating that this mechanism resides at the inner mitochondrial membrane. Patch clamping brain mitoplasts reveal DAG's-induced slightly cation-selective channel activity that is insensitive to bongkrekic acid and abolished by La(3+). The presence of a second messenger-sensitive Ca(2+) release mechanism in mitochondria could have an important impact on intracellular Ca(2+) homeostasis.

Animals↗

PubMed2005

Expression of cellular FLICE inhibitory proteins (cFLIP) in normal and traumatic murine and human cerebral cortex.

Cellular Fas-associated death domain-like interleukin-1-beta converting enzyme (FLICE) inhibitory proteins (cFLIPs) are endogenous caspase homologues that inhibit programmed cell death. We hypothesized that cFLIPs are differentially expressed in response to traumatic brain injury (TBI). cFLIP-alpha and cFLIP-delta mRNA were expressed in normal mouse brain-specifically cFLIP-delta (but not cFLIP-alpha) protein was robustly expressed. After controlled cortical impact (CCI), cFLIP-alpha expression increased initially then decreased to control levels at 12 h, increasing again at 24-72 h (P<0.05). cFLIP-delta expression was decreased in brain homogenates by 12 h after CCI, then increased again at 24 to 72 h (P<0.05). cFLIP-delta immunostaining was markedly reduced in injured cortex, but not hippocampus, at 3 to 72 h after CCI. In cortex, reduced cFLIP-delta staining was found in TUNEL-positive cells, but in hippocampus TUNEL-positive cells expressed cFLIP-delta immunoreactivity. cFLIP-delta was increased in a subset of reactive astrocytes in pericontusional cortex and hippocampus at 48 to 72 h. Low levels of both cFLIP isoforms were detected in human cortical tissue with no TBI, from four patients undergoing brain surgery for epilepsy and <24 h post mortem from three patients without CNS pathologic assessment. In cortical tissue surgically removed <18 h after severe TBI (n=3), cFLIP-alpha expression was increased relative to epilepsy controls (P<0.05) but not relative to post-mortem controls. The data suggest differential spatial and temporal regulation of cFLIP-alpha and cFLIP-delta expression that may influence the magnitude of cell death and further implicate programmed mechanisms of cell death after TBI.

Adolescent↗

PubMed2005

Calcium-dependent dephosphorylation of brain mitochondrial calcium/cAMP response element binding protein (CREB).

Calcium-mediated signaling regulates nuclear gene transcription by calcium/cAMP response element binding protein (CREB) via calcium-dependent kinases and phosphatases. This study tested the hypothesis that CREB is also present in mitochondria and subject to dynamic calcium-dependent modulation of its phosphorylation state. Antibodies to CREB and phosphorylated CREB (pCREB) were used to demonstrate the presence of both forms in isolated mitochondria and mitoplasts from rat brain. When energized mitochondria were exposed to increasing concentrations of Ca2+ in the physiological range, pCREB was lost while total CREB remained constant. In the presence of Ru360, an inhibitor of the mitochondrial Ca2+ uptake uniporter, calcium-dependent loss of pCREB levels was attenuated, suggesting that intramitochondrial calcium plays an important role in pCREB dephosphorylation. pCREB dephosphorylation was not, however, inhibited by the phosphatase inhibitors okadaic acid and Tacrolimus. In the absence of Ca2+, CREB phosphorylation was elevated by the addition of ATP to the mitochondrial suspension. Exposure of mitochondria to the pore-forming molecule alamethicin that causes osmotic swelling and release of intermembrane proteins enriched mitochondrial pCREB immunoreactivity. These results further suggest that mitochondrial CREB is located in the matrix or inner membrane and that a kinase and a calcium-dependent phosphatase regulate its phosphorylation state.

Adenosine Triphosphate↗

PubMed2005

Regulation of mitochondrial gene expression by energy demand in neural cells.

Mitochondrial DNA (mtDNA) encodes critical subunit proteins of the oxidative phosphorylation (OXPHOS) complex that generates ATP. This study tested the hypothesis that mitochondrial gene expression in neural cells is regulated by energy demand, as modified via stimulation of cellular sodium transport. Exposure of PC12S cells to the sodium ionophore monensin (250 nm) for 1-6 h caused a 13-60% decrease in cellular ATP (from 15 to 5 nmol per mg protein at 6 h). Levels of mitochondrial DNA-encoded mRNAs (mt-mRNAs) increased significantly (150%) within the first hour of exposure to monensin, and then decreased significantly (50%) at 3-4 h. Levels of mtDNA-encoded 12S rRNA and nuclear DNA-encoded OXPHOS subunit mRNAs were not significantly affected. Exposure of primary cerebellar neuronal cultures to the excitatory amino acid glutamate caused a similar rapid and significant increase followed by a significant decrease in cell mt-mRNA levels. The monensin-induced initial increase in mt-mRNA levels was abolished by pretreatment with actinomycin D or by reducing extracellular sodium ion concentration. The monensin-induced delayed reduction in mt-mRNA levels was accelerated in the presence of actinomycin D, and was accompanied by a 67% reduction in the half-life (from 3.6 to 1.2 h). Exposure of PC12S cells to 2-deoxy-d-glucose significantly decreased cellular ATP levels (from 14.2 to 7.1 nmol per mg protein at 8 h), and increased mt-mRNA levels. These results suggest a physiological transcriptional mechanism of regulation of mitochondrial gene expression by energy demand and a post-transcriptional regulation that is independent of energy status of the cell.

Actins↗