Redox State Of Cytochrome Research Articles

Influence of beta-galactose-specific mitochondrial lectins from prostate hyperplasic tissue on mitochondrial properties

Galactose-specific lectins (Gal-lectins) were isolated from the mitochondrial fraction of prostate post-operational hyperplasic tissue of two diagnoses: benign prostate hyperplasic tissue with low-grade intraepithelial neoplasia (LGPIN) and benign prostate hyperplasic tissue with atypical adenomatous hyperplasia (AAH). They had similar molecular weight and other properties. Effects of these lectins were investigated in vitro model experiments on bovine liver cells mitochondrial properties. Time-dependent changes: (i) in the amount of H2O2; (ii) redox state of Cu in cytochrome oxidase and (iii) redox state of heme in cytochrome a+a3 (cyt a+a3) of cytochrome c oxidase complex were studied. Gal-lectins from both sources increase the amount of H2O2 and decrease the redox state of Cu in cytochrome oxidase and heme in cyt a+a3. However the Gallectin from tissue with more severed transformation (AAH) expresses significantly more strong and long-lasting influence. These effects are mediated by galactose binding domain of the lectins as are completely abolished by the inclusion of galactose in reaction medium. Accumulation of H2O2 and long-lasting decrease in the redox state of key enzymes of mitochondrial respiration chain could induce defective functioning of these organelles and whole cells. Obtained data point the possible way, which enhances further transformation of prostate tissue by release of Gallectins from damaged mitochondria.

Light scattering change precedes loss of cerebral adenosine triphosphate in a rat global ischemic brain model

Measurement of intrinsic optical signals (IOSs) is an attractive technique for monitoring tissue viability in brains since it enables noninvasive, real-time monitoring of morphological characteristics as well as physiological and biochemical characteristics of tissue. We previously showed that light scattering signals reflecting cellular morphological characteristics were closely related to the IOSs associated with the redox states of cytochrome c oxidase in the mitochondrial respiratory chain. In the present study, we examined the relationship between light scattering and energy metabolism. Light scattering signals were transcranially measured in rat brains after oxygen and glucose deprivation, and the results were compared with concentrations of cerebral adenosine triphosphate (ATP) measured by luciferin–luciferase bioluminescence assay. Electrophysiological signal was also recorded simultaneously. After starting saline infusion, EEG activity ceased at 108 ± 17 s, even after which both the light scattering signal and ATP concentration remained at initial levels. However, light scattering started to change in three phases at 236 ± 15 s and then cerebral ATP concentration started to decrease at about 260 s. ATP concentration significantly decreased during the triphasic scattering change, indicating that the start of scattering change preceded the loss of cerebral ATP. The mean time difference between the start of triphasic scattering change and the onset of ATP loss was about 24 s in the present model. DC potential measurement showed that the triphasic scattering change was associated with anoxic depolarization. These findings suggest that light scattering signal can be used as an indicator of loss of tissue viability in brains.

Ultrafast Proteinquake Dynamics in Cytochromec

We report here our systematic studies of the heme dynamics and induced protein conformational relaxations in two redox states of ferric and ferrous cytochrome c upon femtosecond excitation. With a wide range of probing wavelengths from the visible to the UV and a site-directed mutation we unambiguously determined that the protein dynamics in the two states are drastically different. For the ferrous state the heme transforms from 6-fold to 5-fold coordination with ultrafast ligand dissociation in less than 100 fs, followed by vibrational cooling within several picoseconds, but then recombining back to its original 6-fold coordination in 7 ps. Such impulsive bond breaking and late rebinding generate proteinquakes and strongly perturb the local heme site and shake global protein conformation, which were found to completely recover in 13 and 42 ps, respectively. For the ferric state the heme however maintains its 6-fold coordination. The dynamics mainly occur at the local site, including ultrafast internal conversion in hundreds of femtoseconds, vibrational cooling on the similar picosecond time scale, and complete ground-state recovery in 10 ps, and no global conformation relaxation was observed.

Absorption Measurements of Cell Monolayers Relevant to Mechanisms of Laser Phototherapy: Reduction or Oxidation of Cytochrome c Oxidase Under Laser Radiation at 632.8 nm

The objective of this work was a further investigation of redox mechanisms of laser phototherapy on the cellular level. Cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, is believed to work as the photoacceptor to modulate cellular metabolism in laser phototherapy. The changes in the absorption spectra of HeLa-cell monolayers before and after irradiation at 632.8 nm using fast multi-channel recording were evaluated by the intensity ratio between the peaks at 770 and 670 nm (intensity ratio criterion). By the intensity ratio criterion, the irradiation effects (reduction or oxidation of the photoacceptor) depended on the initial redox status of cytochrome c oxidase. The irradiation (three times at 632.8 nm, dose = 6.3 x 103 J/m(2), tau(irrad.) = 10 sec, tau(record.) = 600 msec) of cells initially characterized by relatively oxidized cytochrome c oxidase caused first a reduction of the photoacceptor, and then its oxidation (a bell-shaped curve). The irradiation by the same scheme of the cells with initially relatively reduced cytochrome c oxidase caused first oxidation and then a slight reduction of the enzyme (a curve opposite to the bell-shaped curve). The experimental results of our work demonstrate that irradiation at 632.8 nm causes either a (transient) relative reduction of the photoacceptor, putatively cytochrome c oxidase, or its (transient) relative oxidation, depending on the initial redox status of the photoacceptor. The maximum in the bell-shaped dose-dependence curve or the minimum of the reverse curve is the turning point between the prevailing of oxidation or reduction processes. Our results are evidence that the bell-shaped dose dependences recorded for various cellular responses are characteristic also for redox changes in the photoacceptor, cytochrome c oxidase.

S12.35 Mitochondria as regulators of apoptosis through the redox state of cytochrome c

S12.35 Mitochondria as regulators of apoptosis through the redox state of cytochrome c

Redox State of Cytochrome c Regulates Cellular ROS and Caspase Cascade in Permeablized Cell Model

In a permeablized cell system, oxidized cyt c is able to induce caspase cascade whereas reduced cyt c cannot. In in vitro experiments, oxidized cyt c can promote H(2)O(2) generation. It is suggested that the redox state of cyt c might regulate the initiation of apoptosis via regulation of cellular ROS level.

Mitochondrial Regulation of Caspase Activation by Cytochrome Oxidase and Tetramethylphenylenediamine via Cytosolic Cytochrome c Redox State

Cytochrome c release from mitochondria induces caspase activation in cytosols; however, it is unclear whether the redox state of cytosolic cytochrome c can regulate caspase activation. By using cytosol isolated from mammalian cells, we find that oxidation of cytochrome c by added cytochrome oxidase stimulates caspase activation, whereas reduction of cytochrome c by added tetramethylphenylenediamine (TMPD) or yeast lactate dehydrogenase/cytochrome c reductase blocks caspase activation. Scrape-loading of cells with this reductase inhibited caspase activation induced by staurosporine. Similarly, incubating intact cells with ascorbate plus TMPD to reduce intracellular cytochrome c strongly inhibited staurosporine-induced cell death, apoptosis, and caspase activation but not cytochrome c release, indicating that cytochrome c redox state can regulate caspase activation. In homogenates from healthy cells cytochrome c was rapidly reduced, whereas in homogenates from apoptotic cells added cytochrome c was rapidly oxidized by some endogenous process. This oxidation was prevented if mitochondria were removed from the homogenate or if cytochrome oxidase was inhibited by azide. This suggests that permeabilization of the outer mitochondrial membrane during apoptosis functions not just to release cytochrome c but also to maintain it oxidized via cytochrome oxidase, thus maximizing caspase activation. However, this activation can be blocked by adding TMPD, which may have some therapeutic potential.

Metabolic stress as a contributor to physiological opening of TRP channels in blowfly (Calliphora vicina)

Abstract Purpose: TRP channels in blowflies can be reversibly activated by metabolic stress. Activation can be caused by hypoxia or mitochondrial uncoupling and results in membrane depolarization. The changes where traced as changes in extracellular ionic composition, photoreceptor membrane currents and changes in membrane potential. Idea of specific signaling pathway opening TRP channels was rejected with findings that impairment of PIP2 under lack of ATP, results in activation of TRP channels. Even though the mechanism of metabolic stress TRP channel activation is elucidated, it remained unanswered what rate of metabolic stress is needed to open the channels and whether under physiological conditions photoreceptor cells encounter the rate of metabolic stress that can significantly contribute to opening of TRP channels. Methods: Extracellular concentrations of [K+]o, [Na+]o and [Ca2+]o were measured using the ion‐selective microelectrodes. The redox states of respiratory pigments (flavoproteins and cytochromes) in the eyes of blowflies (Calliphora vicina) were measured with time‐resolved absorption spectroscopy and use of principal components analysis. Anoxia was reached in under 2s time. Results: Changes in [K+]o, [Na+]o and [Ca2+]o appeared with latency of 8.06 ± 1.38 s, 10.38 ± 2.48 s and 13.11 ± 3.09 s, respectively (mean ± s.e.m., n=7). First detectable changes in redox state of cytochrome c respiratory pigment started at 1.79 ± 0.19 s and reached 39.4% ± 5.4 % of the maximal reduction by the time changes in ionic composition were detectable. Conclusions: Our conclusion was that high degree of maximal redox state change is needed (39.4%) to open TRP channels, what is unlikely to happen under physiological conditions.

Modification by Homocysteine Thiolactone Affects Redox Status of Cytochrome c

Homocysteine (Hcy)-thiolactone mediates a post-translational incorporation of Hcy into protein in humans. Protein N-homocysteinylation is detrimental to protein structure and function and is linked to pathophysiology of hyperhomocysteinemia observed in humans and experimental animals. The modification by Hcy-thiolactone can be detrimental directly by affecting the function of an essential lysine residue or indirectly by interfering with the function of other essential residues or cofactors. Previous work has shown that cytochrome c is very sensitive to Hcy-thiolactone, which causes formation of N-Hcy-cytochrome c multimers. However, it was unclear what sites in cytochrome c were prone to Hcy attachment and whether N-linked Hcy can affect the structure and redox function of cytochrome c. Here we show that 4 lysine residues (Lys8 or -13, Lys86 or -87, Lys99, and Lys100) of cytochrome c are susceptible to N-homocysteinylation. We also show that N-homocysteinylation of 1 mol of lysine/mol of protein affects the redox state of the heme ligand of cytochrome c by rendering it reduced. The modification causes subtle structural changes, manifested as increased resistance of the N-Hcy-cytochrome c to proteolysis by trypsin, chymotrypsin, and Pronase. However, no major secondary structure perturbations were observed as shown by circular dichroism spectroscopy. Our data illustrate how N-homocysteinylation can interfere with the function of heme-containing proteins.

Protective activities of Vaccinium antioxidants with potential relevance to mitochondrial dysfunction and neurotoxicity

Both the neurotransmitter dopamine (DA) and a neurotoxic metabolite, 6-hydroxy DA, can be oxidized to generate hydrogen peroxide and other reactive species (ROS). ROS promote oxidative stress and have been implicated in dopaminergic neurodegeneration, e.g., Parkinson's disease (PD). There is also evidence for a relation between catecholamine-mediated oxidative damage in dopaminergic neurons and the effects of these neurotransmitters on the redox state of cytochrome c (Cytc). In neurons and other cells, oxidative stress may be enhanced by abnormal release of Cytc and other mitochondrial proteins into the cytoplasm. Cytc release can result in apoptosis; but sub-apoptogenic-threshold release can also occur, and may be highly damaging in the presence of DA metabolites. Loss of mitochondrial membrane integrity, a pathological situation of relevance to several aging-related neurodegenerative disorders including PD, contributes to release of Cytc; and the level of such release is known to be indicative of the extent of mitochondrial dysfunction. In this context, we have used a Cytc-enhanced 6-hydroxy DA oxidation reaction to gauge dietary antioxidant activities. Anthocyanin-rich preparations of Vaccinium species ( Vaccinium myrtillus, Vaccinium corymbosum, and Vaccinium oxycoccus) as well as a purified glycosylated anthocyanidin were compared. The most potent inhibition of oxidation was observed with V. myrtillus preparation: 50% inhibition with 7 μM of total anthocyanins. This activity was 1.5–4 times higher than that for the other preparations or for the purified anthocyanin. Ascorbate (Vitamin C), at up to 4-fold higher concentrations, did not result in significant inhibition in this assay. Antioxidant activity in the assay correlated strongly ( r 2 > 0.91, P < 0.01) with reported Vaccinium content of anthocyanins and total cyanidins, but not quercetin or myricetin. The results provide evidence for the high potency of anthocyanins towards a potentially neurotoxic reaction, and provide a basis for in vivo testing of these flavonoids and their physiological metabolites in the context of neuro- and mitochondrio-protective effects.

Fertilization induced changes in sea urchin sperm: Mitochondrial deformation and phosphatidylserine exposure

This study demonstrates that the single mitochondrion of the sea urchin sperm undergoes a shape change at fertilization that is linked to respiration. The mitochondrion swells and shifts to the lateral side of the sperm head on contact with the homologous egg jelly or egg surface; Mg(2+)- or Na(+)-free seawater or respiratory inhibitors also induce this change. During the mitochondrial deformation, the sperm decreases the rate of oxygen consumption and their redox-state of cytochromes is disrupted b-c(1)/c. Simultaneously, the adenine nucleotides content changes precipitously. This suggests that mitochondrial morphology is strongly associated with respiratory activities in the sea urchin sperm. These changes in mitochondrial morphology and function are similar to the mitochondrial changes in apoptotic cells such as swelling, decrease in its membrane potential, and release of cytochrome c. In apoptotic cells, the exposure of phosphatidylserine from the inner to outer leaflet of the plasma membrane is one of prominence phenomena. This change was visualized by staining the sea urchin sperm with Annexin V-Fluorescein. It is possible that mitochondrial deformation is an initial sign of sperm destruction, which like as apoptotic cells.

Evaluation of Cerebral Oxygenation During Electrophysiological Studies

To assess cerebral cortical oxygenation and cellular homeostasis during EPS and RF ablation, using near infrared spectroscopy. Standard EPS was performed in 11 sedated patients (female = 6, mean age = 45 ± 11 years) with symptomatic SVT documented on ECG and no pre-existing cardiovascular or cerebrovascular disease. A NIRO-300 spectrophotometer was used to continuously measure absolute changes in the concentrations of chromophores which reflect intravascular and mitochondrial levels of oxygenation, namely oxygenated haemoglobin (hbo2), deoxygenated haemoglobin (Hb) and the net redox status of cytochrome c oxidase (cyt-a,a3). MABP was monitored simultaneously. RF ablations were performed on 10 patients [AV nodal reentrant tachycardia=6; AV reentrant tachycardia= 4; cycle length = 348 ± 69 msec (range: 225-425 msec)]. No significant changes occurred in hbo2, Hb and cyt-a,a3 during atrial and ventricular EPS pacing protocols, induced SVT, or RF ablation. MABP decreased during pacing at cycle lengths < 400 msec (p<0.005). Cellular energy dynamics were stable during atrial and ventricular pacing, SVT, and RF ablation. This data suggests that overall cerebral tissue oxygenation is preserved during EPS.

Absorption measurements of a cell monolayer relevant to phototherapy: Reduction of cytochrome c oxidase under near IR radiation

Phototherapy uses monochromatic light in the optical region of 600–1000 nm to treat in a non-destructive and non-thermal fashion various soft-tissue and neurological conditions. This kind of treatment is based on the ability of light red-to-near IR to alter cellular metabolism as a result of its being absorbed by cytochrome c oxidase. To further investigate the involvement of cytochrome c oxidase as a photoacceptor in the alteration of the cellular metabolism, we have aimed our study at, first, recording the absorption spectra of HeLa-cell monolayers in various oxygenation conditions (using fast multichannel recording), secondly, investigating the changes caused in these absorption spectra by radiation at 830 nm (the radiation wavelength often used in phototherapy), and thirdly, comparing between the absorption and action spectra recorded. The absorption measurements have revealed that the 710- to 790-nm spectral region is characteristic of a relatively reduced photoacceptor, while the 650- to 680-nm one characterizes a relatively oxidized photoacceptor. The ratio between the peak intensities at 760 and 665 nm is used to characterize the redox status of cytochrome c oxidase. By this criterion, the irradiation of the cellular monolayers with light at λ = 830 nm ( D = 6.3 × 10 3 J/m 2) causes the reduction of the photoacceptor. A similarity is established between the peak positions at 616, 665, 760, 813, and 830 nm in the absorption spectra of the cellular monolayers and the action spectra of the long-term cellular responses (increase in the DNA synthesis rate and cell adhesion to a matrix).

Identification of a Cd2+- and Zn2+-Binding Site in CytochromecUsing FTIR Coupled to an ATR Microdialysis Setup and NMR Spectroscopy

Fourier transform infrared (FTIR) difference spectroscopy allows the study of molecular changes occurring at active sites in proteins with high sensitivity. Reactions are triggered by light, potential, or temperature steps and more recently by the diffusion of buffers containing effectors above membrane proteins deposited as films on ATR crystals. We have adapted a microdialysis system to an ATR, to study metal sites in soluble proteins. In this study, we identified a Cd(2+)- or Zn(2+)-binding site in cytochrome c with dissociation constants of 17 and 42 microM, respectively, which affects the oxidation rate of ferrocytochrome c by hydrogen peroxide. Using the microdialysis ATR-FTIR setup, we determined that a histidine and the carboxylate group of a glutamate are involved in Zn(2+) binding. The implication of His 33 and Glu 104 in the binding site was deduced from the comparison of FTIR data recorded with horse heart and the variant tuna cytochrome c lacking these two amino acids. A two-dimensional NMR analysis of the Zn(2+)-binding site in horse heart cytochrome c confirmed that His 33 and residues close to the C terminus are sensitive to Zn(2+) binding. This study demonstrates that the microdialysis ATR-FTIR setup is promising for the analysis of metal sites in proteins. From H(2)O/(2)H(2)O exchange experiments, we concluded that the impact of Zn(2+) and Cd(2+) binding on the oxidation kinetics of ferrocytochrome c by H(2)O(2) is associated to the perturbation of a hydrogen-bonding network involving His 33 that is sensitive to the redox state of cytochrome c.

Plastocyanin redox kinetics in spinach chloroplasts: evidence for disequilibrium in the high potential chain

Reduction kinetics of cytochrome f, plastocyanin (PC) and P 700 (‘high-potential chain’) in thylakoids from spinach were followed after pre-oxidation by a saturating light pulse. We describe a novel approach to follow PC redox kinetics from deconvolution of 810–860 nm absorption changes. The equilibration between the redox-components was analyzed by plotting the redox state of cytochrome f and PC against that of P 700. In thylakoids with (1) diminished electron transport rate (adjusted with a cytochrome bf inhibitor) or (2) de-stacked grana, cytochrome f and PC relaxed close to their thermodynamic equilibriums with P 700. In stacked thylakoids with non-inhibited electron transport, the equilibration plots were complex and non-hyperbolic, suggesting that during fast electron flux, the ‘high-potential chain’ does not homogeneously equilibrate throughout the membrane. Apparent equilibrium constants <5 were calculated, which are below the thermodynamic equilibrium known for the ‘high potential chain’. The disequilibrium found in stacked thylakoids with high electron fluxes is explained by restricted long-range PC diffusion. We develop a model assuming that about 30% of Photosystem I mainly located in grana end-membranes and margins rapidly equilibrate with cytochrome f via short-distance transluminal PC diffusion, while long-range lateral PC migration between grana cores and distant stroma lamellae is restricted. Implications for the electron flux control are discussed.

Endogenous NO regulates superoxide production at low oxygen concentrations by modifying the redox state of cytochrome c oxidase.

We have investigated in whole cells whether, at low oxygen concentrations ([O(2)]), endogenous nitric oxide (NO) modulates the redox state of the mitochondrial electron transport chain (ETC), and whether such an action has any signaling consequences. Using a polarographic-and-spectroscopic-coupled system, we monitored redox changes in the ETC cytochromes b(H), cc(1), and aa(3) during cellular respiration. The rate of O(2) consumption (VO(2)) remained constant until [O(2)] fell below 15 microM, whereas the onset of reduction of cytochromes aa(3), part of the terminal ETC enzyme cytochrome c oxidase, occurred at approximately 50 microM O(2). Incubation of the cells with an inhibitor of NO synthase lowered significantly (P < 0.05) the [O(2)] at which reduction of the cytochromes occurred. We also measured intracellular superoxide (O(2)(-)) production at different [O(2)] and found there was no increase in O(2)(-) generation in control cells, or those treated with the NO synthase inhibitor, when incubated at 21% O(2). However, after 30-min exposure of control cells to 3% O(2), an increase in O(2)(-) generation was observed, accompanied by translocation to the nucleus of the transcription factor NF-kappa B. Both of these responses were diminished by NO synthase inhibition. Our results suggest that endogenous NO, by enhancing the reduction of ETC cytochromes, contributes to a mechanism by which cells maintain their VO(2) at low [O(2)]. This, in turn, favors the release of O(2)(-), which initiates the transcriptional activation of NF-kappa B as an early signaling stress response.

Two Sites of Interaction of Anions with Cytochrome a in Oxidized Bovine Cytochrome c Oxidase

An interaction between cytochrome a in oxidized cytochrome c oxidase (CcO) and anions has been characterized by EPR spectroscopy. Those anions that affect the EPR g = 3 signal of cytochrome a can be divided into two groups. One group consists of halides (Cl-, Br-, and I-) and induces an upfield shift of the g = 3 signal. Nitrogen-containing anions (CN-, NO2-, N3-, NO3-) are in the second group and shift the g = 3 signal downfield. The shifts in the EPR spectrum of CcO are unrelated to ligand binding to the binuclear center. The binding properties of one representative from each group, azide and chloride, were characterized in detail. The dependence of the shift on chloride concentration is consistent with a single binding site in the isolated oxidized enzyme with a Kd of approximately 3 mm. In mitochondria, the apparent Kd was found to be about four times larger than that of the isolated enzyme. The data indicate it is the chloride anion that is bound to CcO, and there is a hydrophilic size-selective access channel to this site from the cytosolic side of the mitochondrial membrane. An observed competition between azide and chloride is interpreted by azide binding to three sites: two that are apparent in the x-ray structure plus the chloride-binding site. It is suggested that either Mg2+ or Arg-438/Arg-439 is the chloride-binding site, and a mechanism for the ligand-induced shift of the g = 3 signal is proposed.

Monitoring cytochrome redox changes in the mitochondria of intact cells using multi-wavelength visible light spectroscopy

We have developed an optical system based on visible light spectroscopy for the continuous study of changes in the redox states of mitochondrial cytochromes in intact mammalian cells. Cells are suspended in a closed incubation chamber in which oxygen and nitric oxide (NO) concentrations can be monitored during respiration. Simultaneously the cells are illuminated with a broad-band tungsten–halogen light source. Emergent light in the visible region (from 490–650 nm) is detected using a spectrophotometer and charge-coupled device camera system. Intensity spectra are then converted into changes in optical attenuation from a ‘steady-state’ baseline. The oxidised-minus-reduced absorption spectra of the mitochondrial cytochromes are fitted to the attenuation spectra using a multi-wavelength least-squares algorithm. Thus, the system can measure changes in the redox states of the cytochromes during cellular respiration. Here we describe this novel methodology and demonstrate its validity by monitoring the action of known respiratory chain inhibitors, including the endogenous signalling molecule NO, on cytochrome redox states in human leukocytes.

Light Scattering Changes in Isolated Brain Mitochondria during Anoxia - Magnesium Effect on Morphological Changes and Respiration -

Under pathological conditions, morphological changes of the cells and tissues may differ from those of normal conditions, which can be reflected by changes in light scattering (LS). Consequently, LS has been recognized as a potential non-invasive tool for optical diagnosis of living tissue. This paper aimed to identify the basic properties of LS of isolated brain mitochondria in vitro under normoxic and anoxic conditions in the presence and absence of Mg2+. An increase in LS was observed during anoxia in both the presence and absence of Mg2+. In both cases, the changes in LS initiated by anoxia and reoxygenation started concomitantly with the reduction of heme aa3. The rates of LS changes were slower than those of heme aa3, particularly in the presence of Mg2+. Mg2+ inhibited the morphological responses of mitochondria caused by the addition of ADP and ATP, and significantly reduced the oxygen consumption rate in state 4. These results are due to modulation of the K+/H+ antiporter affected by Mg2+. In addition, the mitochondria were well coupled, although the basal level of LS fell after addition of Mg2+. Therefore, the observed responses of mitochondria at anoxia were physiological and independent of the presence or absence of Mg2+. The relationship between LS and redox state of cytochrome c oxidase, an anoxic indicator, provides a basis to assess the tissue conditions in vivo.

Do common anaesthetic protocols influence cerebral oxygenation?

Near-infrared-spectroscopy (NIRS; Jobsis 1977) has been used in humans to measure regional cerebral oxygen saturation (rsO2), decreases of which can be indicative of a critical cerebral O2 supply (Kirkpatrick et al. 1998). This study investigated changes in rsO2, the redox status of cytochrome a/a3, and the cerebral total hemoglobin content using NIRS during different sedative and anaesthetic techniques in dogs. Eighty four foxhounds and 32 beagles aged 12.9 ± 13.4 (6.3–94.4) (±SD (range)) months and weighing 24.8 ± 6.6 (2.7–43) kg were divided randomly into four groups. Group AM received 0.1 mg kg−1 acepromazine and 0.5 mg kg−1 l-methadone/fenpipramid (l-Polamivet) IV, group DM 0.5 mg kg−1 diazepam and 0.5 mg kg−1 l-Polamivet IV, and group MM 40 µg kg−1 medetomidine and 0.5 mg kg−1 l-Polamivet IV. Group P was anaesthetized (7 mg kg−1) and maintained (0.3 mg kg−1 minute−1) with propofol. Thirty minutes after sedation groups AM, DM and MM received 8 µg kg−1 naloxone IV, and at 35 minutes group MM received 200 µg kg−1 atipamezole IV. NRIS was monitored continuously (recording interval, 1 second). Blood gases were analysed before and every 5 minutes after sedation or induction, and at 2 and 7 minutes post-naloxone injection or end of propofol infusion. Descriptive and comparative statistics (global: Kruskal–Wallis test, level of significance α = 0.05, group comparison: Mann–Whitney test, α = 0.0083), and Spearman's correlation were determined using the statistical software SPSS. Before drug administration rsO2 was 65 ± 7% (n = 109). One minute after drug administration significant differences between groups were found (Table 1). There was a significant increase of about 8% in rsO2 over time in the p group, whereas groups AM, DM and MM showed a significant decrease with the greatest reduction of 20% in group MM. One minute after naloxone IV pre-induction values were rapidly attained in groups AM (65.2 ± 6%, n = 31) and DM (65.4 ± 5.4%, n = 24) while in group MM (52.1 ± 10.8%, n = 27) pre-induction values of rsO2 were not reached until 1 minute after atipamezole IV (62.9 ± 7.4%). Periodic breathing (MM) was mirrored by variations of rsO2. There were no significant changes or intergroup differences in the redox state of cytochrome a/a3 or of total cerebral hemoglobin content indicating cerebral blood volume (Wyatt et al. 1990). The 20% decrease in rsO2 in group MM is in the range regarded as indicative of a critical cerebral O2 supply in humans (Kirkpatrick et al. 1998). The effects of naloxone and atipamezole and the correlation with blood gas analysis demonstrate an influence of respiratory depression on the vascular cerebral O2 status whereas the lack of cytochrome a/a3 reduction indicates an undisturbed cellular O2 supply (Jobsis 1977). In spite of some limitations in terms of applicability (Alef 2002). NIRS seems to enable the influence of clinical routines on cerebral oxygenation to be monitored although further validation of NIRS in the dog and the cat is required.