Mitochondrial Redox Activity Research Articles

A polyphenol complex from Thymus vulgaris L. plants cultivated in the Campania Region (Italy): New perspectives against neuroblastoma

A thyme polyphenol extract (TPE) from Thymus vulgaris plants cultivated in the Campania Region (Italy) underwent qualitative and quantitative analyses using liquid chromatography–tandem mass spectrometry (LC–MS/MS) techniques and its effects were investigated on neuroblastoma cells. The extract, rich in rosmarinic acid and methoxylated flavones, inhibited mitochondrial redox activity and cell viability towards SH-SY5Y and SK-N-BE(2)-C cell lines at doses of 62.5 and 125 µg/mL. Cytotoxic TPE exposure resulted in an increase in intracellular reactive oxygen species (ROS) levels, and activation of caspases-8 and -3, consistent with our hypothesis that it possesses apoptosis-inducing properties. The occurrence of apoptosis was further established by attenuated total reflectance Fourier transform infrared (ATR FTIR) analysis: spectra from TPE-treated cells showed a significant decrease of nucleic acid/amide II ratio. The data obtained encourage further investigation and assessment of the efficacy of TPE in preclinical and clinical models to treat neuroblastoma, a major cause of mortality worldwide.

SiRNA knockdown of mitochondrial thymidine kinase 2 (TK2) sensitizes human tumor cells to gemcitabine.

Nucleoside metabolism enzymes are determinants of chemotherapeutic drug activity. The nucleoside salvage enzyme deoxycytidine kinase (dCK) activates gemcitabine (2', 2'-difluoro-2'-deoxycytidine) and is negatively regulated by deoxycytidine triphosphate (dCTP). Reduction of dCTP in tumor cells could, therefore, enhance gemcitabine activity. Mitochondrial thymidine kinase 2 (TK2) phosphorylates deoxycytidine to generate dCTP. We hypothesized that: (1) TK2 modulates human tumor cell sensitivity to gemcitabine, and (2) antisense knockdown of TK2 would decrease dCTP and increase dCK activity and gemcitabine activation. siRNA downregulation of TK2 sensitized MCF7 and HeLa cells (high and moderate TK2) but not A549 cells (low TK2) to gemcitabine. Combined treatment with TK2 siRNA and gemcitabine increased dCK. We also hypothesized that TK2 siRNA-induced drug sensitization results in mitochondrial damage that enhances gemcitabine effectiveness. TK2 siRNA and gemcitabine decreased mitochondrial redox status, DNA content, and activity. This is the first demonstration of a direct role for TK2 in gemcitabine resistance, or any independent role in cancer drug resistance, and further distinguishes TK2 function from that of other dTMP-producing enzymes [cytosolic TK1 and thymidylate synthase (TS)]. siRNA knockdown of TK1 and/or TS did not sensitize cancer cells to gemcitabine indicating that, among the 3 enzymes, only TK2 is a candidate therapeutic target for combination with gemcitabine.

A Novel Microfluidic-Based Array For High-Resolution High-Content Imaging of Islets Before Transplant.

Background: In order to facilitate in vitro study of pancreatic islets before the transplant, a microfluidic chip was designed to trap individual islet in an array format which allows us to study physiology and pathophysiology of islets in high-resolution fashion for high-content imaging. METHODS: (1) A microfluidic array was designed based on hydrodynamic principle for pressure drop in a micro channel. (2) Fluid flow simulation performed using COMSOL to verify design principle and minimal shear stress on islet cells. (3) A thin PDMS membrane was integrated as an oxygen controller. RESULTS: (1) Flow simulation verified the design principle, showing maximum velocity in trapping sites with minimum shear stress. (2) The device is capable of trapping up to 200 islets individually in an array format with 99% efficiency. (3)Both loading/trapping islets and fluid flow control were performed using gravity based flow control which eliminated the need for pumps. (4) The oxygen controller can provide fast oxygen microenvironment (less than 30 s) and varying and consistent oxygen concentrations and profiles. (5) As a result of having one layer design and using a very thin glass substrate,the device allowed multiparametric fluorescent and confocal imaging of islet cellular and subcellular metabolic activity and ion signaling such as mitochondrial energetics, ROS levels, calcium influx, and redox activity for high resolution high-content imaging purposes. CONCLUSION: We designed, fabricated, and validated a novel microfluidic platform that allows trapping individual islets with high efficiency. Device also allowed for co-culturing islets and hypoxia studies, showing an improvement over conventional hypoxia chambers. The device allowed high-resolution imaging of islet using fluorescence and confocal microscopy and high-content multiparametric imaging of key insulin stimulator-secretion coupling factors. This work demonstrates the feasibility of array-based cellomics analysis.

Cardioprotective effect of embelin on isoproterenol-induced myocardial injury in rats: Possible involvement of mitochondrial dysfunction and apoptosis

AimsPreventive and/or therapeutic interventions using natural products for ischemic heart disease have gained considerable attention worldwide. This study investigated the cardioprotective effect and possible mechanism of embelin, a major constituent of Embelia ribes Burm, using isoproterenol (ISO)-induced myocardial infarction model in rats. Materials and methodsRats were pretreated for three days with embelin (50mg/kg, p.o) before inducing myocardial injury by administration of ISO (85mg/kg) subcutaneously at an interval of 24h for 2 consecutive days. Serum was analyzed for cardiac specific injury biomarkers, lipids and lipoprotein content. Heart tissues were isolated and were used for histopathology, antioxidant and mitochondrial respiratory enzyme activity assays and western blot analysis. Key findingsResults showed that pretreatment with embelin significantly decreased the elevated levels of serum specific cardiac injury biomarkers (CK-MB, LDH and AST), serum levels of lipids and lipoproteins and histopathological changes when compared to ISO-induced controls. Exploration of the underlying mechanisms of embelin action revealed that embelin pretreatment restored the myocardial mitochondrial respiratory enzyme activities (NADH dehydrogenase, succinate dehydrogenase, cytochrome c oxidase and mitochondrial redox activity), strengthened antioxidant status and attenuated ISO-induced myocardial lipid peroxidation. Immunoblot analysis revealed that embelin interrupted mitochondria dependent apoptotic damage by increasing the myocardial expression of Bcl-2 and downregulating the expression of Bax, cytochrome c, cleaved-caspase-3 & 9 and PARP. Histopathology findings further strengthened the cardioprotective findings of embelin. SignificanceResult suggested that embelin may have a potential benefit in preventing ischemic heart disease like myocardial infarction.

Naringin ameliorates gentamicin-induced nephrotoxicity and associated mitochondrial dysfunction, apoptosis and inflammation in rats: Possible mechanism of nephroprotection

Gentamicin-induced nephrotoxicity has been well documented, although its underlying mechanisms and preventive strategies remain to be investigated. The present study was designed to investigate the protective effect of naringin, a bioflavonoid, on gentamicin-induced nephrotoxicity and to elucidate the potential mechanism. Serum specific renal function parameters (blood urea nitrogen and creatinine) and histopathology of kidney tissues were evaluated to assess the gentamicin-induced nephrotoxicity. Renal oxidative stress (lipid peroxidation, protein carbonylation, enzymatic and non-enzymatic antioxidants), inflammatory (NF-kB [p65], TNF-α, IL-6 and MPO) and apoptotic (caspase 3, caspase 9, Bax, Bcl-2, p53 and DNA fragmentation) markers were also evaluated. Significant decrease in mitochondrial NADH dehydrogenase, succinate dehydrogenase, cytochrome c oxidase and mitochondrial redox activity indicated the gentamicin-induced mitochondrial dysfunction. Naringin (100mg/kg) treatment along with gentamicin restored the mitochondrial function and increased the renal endogenous antioxidant status. Gentamicin induced increased renal inflammatory cytokines (TNF-α and IL-6), nuclear protein expression of NF-κB (p65) and NF-κB-DNA binding activity and myeloperoxidase (MPO) activity were significantly decreased upon naringin treatment. In addition, naringin treatment significantly decreased the amount of cleaved caspase 3, Bax, and p53 protein expression and increased the Bcl-2 protein expression. Naringin treatment also ameliorated the extent of histologic injury and reduced inflammatory infiltration in renal tubules. U-HPLS-MS data revealed that naringin co-administration along with gentamicin did not alter the renal uptake and/or accumulation of gentamicin in kidney tissues. These findings suggest that naringin treatment attenuates renal dysfunction and structural damage through the reduction of oxidative stress, mitochondrial dysfunction, inflammation and apoptosis in the kidney.

Myricitrin Alleviates Methylglyoxal-Induced Mitochondrial Dysfunction and AGEs/RAGE/NF-κB Pathway Activation in SH-SY5Y Cells

Advanced glycation end products (AGEs) have been identified in age-related intracellular protein deposits of neurodegenerative diseases. Methylglyoxal (MGO), a dicarbonyl metabolite, is a major precursor of AGEs which have been linked to the development of neurodegenerative diseases. Myricitrin, a flavanoid isolated from the root bark of Myrica cerifera, attenuated 6-OHDA-induced mitochondrial dysfunction and had a potential anti-Parkinson's disease in our previous investigation. The aims of this study were to investigate the protective effects of myricitrin against MGO-induced injury in SH-SY5Y cells and also to look for the possible mechanisms. The results showed that exposure of SH-SY5Y cells to MGO caused decreases of cell viability, intracellular ATP, mitochondrial redox activity, and mitochondrial membrane potential and an increase in reactive oxygen species generation. However, these mitochondrial dysfunctions were alleviated by co-treatment with myricitrin. Additionally, myricitrin was capable of inhibiting AGEs formation, blocking RAGE expression, and inhibiting NF-κB activation and translocation triggered by MGO in SH-SY5Y cells. Our results suggest that myricitrin alleviates MGO-induced mitochondrial dysfunction, and the possible mechanism is through modulating the AGEs/RAGE/NF-κB pathway. In summary, myricitrin might offer a promising therapeutic strategy to reduce the neurotoxicity of reactive dicarbonyl compounds, providing a potential benefit agent with age-related neurodegenerative diseases.

Baicalein Prevents 6-Hydroxydopamine-Induced Mitochondrial Dysfunction in SH-SY5Y Cells via Inhibition of Mitochondrial Oxidation and Up-Regulation of DJ-1 Protein Expression

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by progressive loss of dopaminergic (DA) neurons at the substantia nigra. Mitochondrial dysfunction is involved in the mechanism of cell damage in Parkinson’s disease (PD). 6-Hydroxydopamine (6-OHDA) is a dopamine analog which specifically damages dopaminergic neurons. Baicalein has been previously reported to have potential in the treatment of PD. The purpose of the present study was to investigate the mechanism of action of baicalein against 6-OHDA injury in SH-SY5Y cells. The results showed that baicalein significantly alleviated alterations of mitochondrial redox activity and mitochondrial membrane potential induced by 6-OHDA in a dose-dependent manner in SH-SY5Y cells compared with vehicle group. Futhermore, baicalein decreased the production of ROS and upregulated the DJ-1 protein expression in SH-SY5Y cells. In addition, baicalein also inhibited ROS production and lipid peroxidation (IC50 = 6.32 ± 0.03 μM) in rat brain mitochondia. In summary, the underlying mechanisms of baicalein against 6-OHDA-induced mitochondrial dysfunction may involve inhibition of mitochondrial oxidation and upregulation of DJ-1 protein expression.

Targeting oxidative stress, mitochondrial dysfunction and neuroinflammatory signaling by selective cyclooxygenase (COX)-2 inhibitors mitigates MPTP-induced neurotoxicity in mice

Several studies have pointed towards the role of oxidative stress, mitochondrial dysfunction and neuroinflammation in Parkinson's disease (PD). The present study was focused on the possible neuroprotective effect of selective cyclooxygenase (COX)-2-inhibitors: valdecoxib and NS-398 in 1-methyl-4-phenyl-1,2,3,6-tertahydropyridine (MPTP)-induced neurotoxicity in mice. MPTP administration in dose of 40 mg/kg, i.p (four injections of 10mg/kg, i.p. at an interval of 1h each) significantly induced the Parkinson-like symptoms in mice as indicated by change in locomotor activity, inability to correct posture (bar test), and oxidative stress (increased levels of lipid peroxidation, nitrite concentration, and depletion of antioxidant enzyme). MPTP administration significantly impaired mitochondrial complex-I activity and redox activity, upregulated the caspase-3 and NF-κB levels as compared to vehicle group. Treatment with valdecoxib (5 or 10 mg/kg, p.o.) or NS-398 (5 or 10mg/kg, p.o.) for 7 days significantly reversed behavioral, biochemical, mitochondrial complex alterations as well as attenuated the induction of proinflammatory mediators in MPTP-treated groups. The findings of the present study substantiate the neuroprotective role of selective COX-2 inhibitors in ameliorating MPTP-induced neurodegeneration in mice and suggest the possible therapeutic potential of these drugs in the management of PD.

Potential role of pioglitazone, caffeic acid and their combination against fatigue syndrome-induced behavioural, biochemical and mitochondrial alterations in mice

Chronic fatigue is an illness characterised by persistent and relapsing fatigue, often accompanied by numerous neuropsychiatric problems, such as anxiety and depression. The aetiology of chronic fatigue remains unclear so far. However, recent studies suggested the involvement of oxidative stress in this chronic debilitating disease. Alternatively, antioxidants have also been reported to have beneficial effect against chronic fatigue-like conditions. Therefore, present study has been designed to explore the potential role of pioglitazone, caffeic acid and their combination against chronic fatigue-like condition in mice. In the experimental protocol, the mice were put on the running wheel apparatus for 6 min test session daily for 21 days which produced fatigue-like condition. The locomotor activity and anxiety levels were measured on 0, 8th, 15th and 22nd days. The brains were isolated on 22nd day immediately after the behavioural assessments, oxidative damage and mitochondrial enzyme complexes were then estimated subsequently. Three weeks pioglitazone (5 and 10 mg/kg) and caffeic acid (5 and 10 mg/kg) pretreatment significantly attenuated the chronic fatigue-like condition (restored running wheel activity, locomotor activity and reduced anxiety-like behaviour) as compared to that in control (chronic fatigue) animals. Further, pioglitazone (5 and 10 mg/kg) and caffeic acid (5 and 10 mg/kg) drug treatments for 3 weeks significantly attenuated oxidative damage (decreased lipid peroxidation, nitrite concentration, restored reduction in glutathione and catalase levels), altered mitochondrial enzymes complex (I, II and IV) activities and mitochondrial redox activity (MTT assay) when compared with control. Further, combination of lower dose of pioglitazone (5 mg/kg) and caffeic acid (5 mg/kg) showed significant synergism in their protective effect which was significant as compared to their effect per se. The present study highlights the potential role of pioglitazone, caffeic acid and their combination in the pathophysiology of chronic fatigue-like condition in mice.

Possible nitric oxide modulation in protective effect of FK-506 against 3-nitropropionic acid-induced behavioral, oxidative, neurochemical, and mitochondrial alterations in rat brain

FK-506 is an immunosuppressant being widely used for allograft rejection cases in the present clinical scenario. Recently, the neuroprotective effect of FK-506 has also been reported against a number of neurodegenerative diseases in rodents. This study was designed to explore the possible protective effect of FK-506 and its interaction with nitric-oxide modulators against 3-nitropropionic acid (3-NP)-induced behavioural, biochemical, neurochemical, and mitochondrial alterations in striatum, cortex, and hippocampus regions of the brain. Systemic administration of 3-nitropropionic acid produces Huntington-like symptoms in rats. 3-NP (10 mg/kg) treatment for 14 days impaired locomotor activity, grip strength, and body weight. 3-NP treatment significantly raised malondialdehyde, nitrite concentration, depleted antioxidant enzymes (SOD and catalase), and levels of bioamines (dopamine and norepinephrine) in striatum, cortex, and hippocampus areas of rat brain. Significant alterations in mitochondrial enzyme complexes (I, II, and IV) activities and mitochondrial redox activity have also been altered significantly by 3-NP. Pretreatment with FK-506 (0.5, 1, and 2 mg/kg) significantly reversed these behavioral, biochemical, and cellular alterations. L-arginine treatment with a subeffective dose FK-506 (1 mg/kg) reversed the protective effect of FK-506. However, L-NAME pretreatment with FK-506 (1 mg/kg) potentiated the protective effect of FK-506. The present study shows that FK-506 attenuates 3-NP-induced neurotoxicity and nitric-oxide modulation might be involved in its protective action.

Mediator-assisted simultaneous probing of cytosolic and mitochondrial redox activity in living cells

This work describes an electron transfer mediator-assisted amperometric flow injection method for assessing redox enzyme activity in different subcellular compartments of the phosphoglucose isomerase deletion mutant strain of Saccharomyces cerevisiae, EBY44. The method is demonstrated using the ferricyanide–menadione double mediator system to study the effect of dicoumarol, an inhibitor of cytosolic and mitochondrial oxidoreductases and an uncoupler of the electron transport chain. Evaluation of the role of NAD(P)H-producing pathways in mediating biological effects is facilitated by introducing either fructose or glucose as the carbon source, yielding either NADH or NADPH through the glycolytic or pentose phosphate pathway, respectively. Respiratory noncompetent cells show greater inhibition of cytosolic menadione-reducing enzymes when NADH rather than NADPH is produced. Spectrophotometric in vitro assays show no difference between the cofactors. Respiratory competent cells show cytosolic inhibition only when NADPH is produced, whereas production of NADH reveals uncoupling at low dicoumarol concentrations and inhibition of complexes III and IV at higher concentrations. Spectrophotometric assays only indicate the presence of cytosolic inhibition regardless of the reduced cofactor used. This article shows the applicability of the amperometric method and emphasizes the significance of determining biological effects of chemicals in living cells.

Acute systemic inflammation induces central mitochondrial damage and mnesic deficit in adult Swiss mice

The aim of this study was to investigate how the brain is affected during systemic inflammation. For this purpose, Swiss mice were challenged with a single intraperitoneal dose of lipopolysaccharide (LPS; 250microg/mouse) to mimic aspects of systemic infection. Spatial learning in Y-maze test demonstrated a differential learning profile during the training test between control and LPS-treated mice, with an alteration in the latter group. We show that systemic LPS-induced inflammation and oxidative injury as assessed by reactive oxygen species (ROS) and nitrites/nitrates (NOx) production associated with reduced glutathione (GSH) depletion, cyclooxygenase-2 (COX-2) expression, and lipid peroxidation. LPS also induced a loss in mitochondrial integrity as shown by a significant decrease in membrane potential and impairment in mitochondrial redox activity. Thus, peripheral inflammation by producing brain inflammation and oxidative injury causes mnesic deficits. It remains to determine whether such events can induce neuronal dysfunction/degeneration and, with time, lead to cholinergic deficiency, amyloid deposits and cognitive impairments as they occur in Alzheimer's disease.

α-Glycerylphosphorylethanolamine rescues astrocytes from mitochondrial impairment and oxidative stress induced by amyloid β-peptides

The present work shows that α-glycerylphosphorylethanolamine (α-GPE) is effective in recovering astrocytes from mitochondrial membrane integrity and potential derangement and cellular oxidative stress that occur under amyloid β-peptides-induced reactive gliosis. α-Glycerylphosphorylethanolamine (α-GPE), a new compound with nootropic properties, known to improve in vivo the learning and memory processes, has been tested for its protective properties on an in vitro model of degeneration. Rat primary astrocytic cultures treated with two amyloid-derived peptides, Aβ (1–40) and Aβ 3(pE)–42, showed a marked reduction of the mitochondrial redox activity and membrane potential, together with an increase of oxidative species production. Plasma membrane lipid peroxidation (LPO) as well as generation of peroxides is greatly increased under Aβ-peptides toxicity. These features, typical of the reactive gliosis that accompanies neuronal degeneration, were readily recovered by pretreatment with α-GPE. α-GPE, likely improving the fluidity of cell membrane, has the potential to recover astrocytes from the general redox derangement induced by different amyloid fragments and possibly to protect from inflammation, gliosis and neurodegeneration. This is the first evidence of an antioxidant effect of the ethanolamine derivative on a rat model of chronic gliosis.

1-Methyl-4-phenylpyridinium (MPP +) Decreases Mitochondrial Oxidation-Reduction (REDOX) Activity and Membrane Potential (Δψ m) in Rat Striatum

Mitochondrial dysfunction has long been implicated in the death of nigrostriatal dopaminergic neurons in Parkinson's disease (PD) and its experimental models. Here we further analyzed changes in the mitochondrial oxidation-reduction (REDOX) activity and membrane potential (Δψ m) of striatal synaptosomes after the infusion of 1-methyl-4-phenylpyridinium (MPP +) into rat striatum. MPP + (40 nmol) treatment produced decreases in mitochondrial REDOX activity and Δψ m at 18 h, as measured by fluorometric analysis with both Alamar blue and JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide) dyes. At this time point, tyrosine hydroxylase (TH) and dopamine transporter (DAT) protein levels were not altered, but both decreased at 7 days after MPP + (40 nmol) infusion. Both measures of mitochondrial dysfunction induced by MPP + (40 nmol) at 18 h were attenuated, at least in part, by pretreatment with a selective dopamine uptake inhibitor GBR-12909 (1-(2-(bis(4-fluorophenyl)methoxy)ethyl)-4-(3-phenylpropyl) piperazine). In addition, GBR-12909 partially attenuated MPP + (40 nmol)-caused a loss of striatal nerve terminal as indicated by decreases in TH and DAT immunoreactivities as well as dopamine and its metabolites levels. The present study indicates that decreases in mitochondrial REDOX activity and Δψ m may play a role in MPP +-induced dopaminergic neurotoxicity, and further provides that improvement of mitochondrial dysfunction may be a better way to slow progressive dopaminergic neurodegeneration commonly associated with PD.

Acetaminophen protects hippocampal neurons and PC12 cultures from amyloid β-peptides induced oxidative stress and reduces NF-κB activation

The present findings show that an atypical non-steroidal anti-inflammatory drug, such as acetaminophen, retains the ability to recover amyloid β-peptides driven neuronal apoptosis through the impairment of oxidative stress. Moreover, this compound reduces the increased NF-κB binding activity, which occurs in these degenerative conditions. Therapeutic interventions aimed at reducing the inflammatory response in Alzheimer’s disease (AD) recently suggested the application of non-steroidal anti-inflammatory drugs. Although the anti-inflammatory properties of acetaminophen are controversial, it emerged that in an amyloid-driven astrocytoma cell degeneration model acetaminophen proved to be effective. On these bases, we analyzed the role of acetaminophen against the toxicity exerted by different Aβ-peptides on rat primary hippocampal neurons and on a rat pheochromocytoma cell line. We found a consistent protection from amyloid β-fragments 1-40 and 1-42-induced impairment of mitochondrial redox activity on both cell cultures, associated with a marked reduction of apoptotic nuclear fragmentation. An antioxidant component of the protective activity emerged from the analysis of the reduction of phospholipid peroxidation, and also from a significant reduction of cytoplasmic accumulation of peroxides in the pheochromocytoma cell line. Moreover, activation of NF-κB by amyloid-derived peptides was greatly impaired by acetaminophen pre-treatment in hippocampal cells. This evidence points out antioxidant and anti-transcriptional properties of acetaminophen besides the known capability to interfere with inflammation within the central nervous system, and suggests that it can be exploited as a possible therapeutic approach against AD.

Inhibition of glycolysis alters potassium ion transport and mitochondrial redox activity in rat brain.

To examine the relationships between brain glycolysis, ion transport, and mitochondrial reduction/oxidation (redox) activity, extracellular potassium ion activity (K+0) and redox shifts of cytochrome oxidase (cytochrome a,a3) were recorded previous to and during superfusion of rat cerebral cortex with the glycolytic inhibitor iodoacetic acid (IAA). IAA produced oxidation of cytochrome a,a3, increased local oxygenation, increased K+0, and, in response to neuronal activation, slowed rates of K+0 reaccumulation. Rates of rereduction of cytochrome a,a3, after the oxidation of this cytochrome by stimulation, were also slowed by IAA. These effects of IAA demonstrate the dependence of K+0 reaccumulation on the integrity of glycolysis, support the concept that active processes are involved in brain ion transport, and suggest a link between ATP supplied by glycolysis and ion transport activity. These data are also compatible with the suggestion that residual dysfunctions after brain ischemia result from derangements in glycolytic functioning rather than from limitations in oxygen availability or oxidative metabolic activity.

Potassium Ion Homeostasis and Mitochondrial Redox Activity in Brain: Relative Changes as Indicators of Hypoxia

This study was directed at relating ion transport and mitochondrial redox activity during hypoxia, as a step toward definition of brain oxygen sufficiency. To accomplish this, extracellular potassium ion activity (K+o) was recorded by ion-selective microelectrodes while reduction/oxidation (redox) ratios of cytochrome oxidase (cytochrome a,a3) were monitored by reflection spectrophotometry in cerebral cortex of rats anesthetized with pentobarbital. In normoxia, neuronal activation by direct cortical stimulation produced transient oxidation of cytochrome a,a3 and elevation of K+o. Moderate hypoxia (PaO2 above 50 mm Hg) resulted in reduction of cytochrome a,a3 but only slight elevation of K+o. At this level of hypoxia, cytochrome a,a3 continued to respond to neuronal activation with transient shifts toward oxidation and rates of K+o reaccumulation were unchanged from control. When PaO2 was further decreased below a critical threshold, stimulus-provoked oxidative responses of mitochondrial reactants were replaced by shifts toward reduction, but rates of reaccumulation of K+, spilled into the extracellular space by neuronal activation, remained unchanged. Only during severe hypoxia (PaO2 less than 20 mm Hg) was it possible in some animals to record a slowing in the reaccumulation of K+o without provocation of spreading cortical depression. These data indicate that ion transport activity in cerebral cortex is more refractory to hypoxia than is mitochondrial redox functioning. They suggest an in vivo parallel to the "cushioning" effect of mitochondria in vitro, in which oxygen consumption remains constant despite fluctuations in oxygenation and redox ratios, and also that there may be a greater anaerobic capacity to provide energy for ion transport in mammalian brain than has previously been appreciated.

Pyruvate dehydrogenase activity in hamster small intestine during development.

Total pyruvate dehydrogenase activities in hamster intestine increase from 40 nmol/min (munits) per g of intestine in the foetal animals to 460 munits/g in the adult, whereas the fraction of the enzyme in the active form increases from 34 to 42% of the total activity over the same period. However, a complete conversion of the enzyme into the active form is observed in the neonatal animal immediately after birth. Results from experiments in vitro suggested that the activation of pyruvate dehydrogenase is controlled, in part, by the [NAD+]/[NADH] ratio. This proposal was tested in vivo by examining the proportion of the enzyme in the active form during conditions when the [NAD+]/[NADH] ratio was markedly altered, and the data show a direct relationship between the mitochondrial redox state and activity of the active form.

Intracellular redox changes in functioning cerebral cortex. II. Effects of direct cortical stimulation.

Intracellular redox changes in functioning cerebral cortex. II. Effects of direct cortical stimulation.