Endogenous Substrates Utilized by Rat Brain in Severe Insulin-Induced Hypoglycemia

Abstract: Several previous studies have demonstrated that severe hypoglycemia is accompanied by consumption of endogenous brain substrates (glycolytic and citric acid cycle metabolites and free amino acids) and some have shown a loss of structural components as well, notably phospholipids. In the present study, on paralysed and artificially ventilated rats, we measured cerebral oxygen and glucose consumption during 30 min of hypoglycemic coma (defined as hypoglycemia of sufficient severity to cause cessation of spontaneous EEG activity) and calculated the non-glucose oxygen consumption. In an attempt to estimate the missing substrate we measured tissue concentrations of phospholipids and RNA. After 5 min of hypoglycemic coma, tissue phospholipid content decreased by about 8% with no further change during the subsequent 55 min. A similar reduction remained after 90 min of recovery, induced by glucose administration following 30 min of coma. Since no preferential loss of polyenoic fatty acids or of ethanolamine phosphoglycerides occurred, it is concluded that loss of phospholipids was due to phospholipase activity rather than to peroxidative degradation. The free fatty acid concentration increased sixfold after 5 min of coma and remained elevated during the course of hypoglycemia. A 9% reduction in tissue RNA content was observed after 30 min of hypoglycemia. Calculations indicated that available endogenous carbohydrate and amino acid substrates were essentially consumed after 5 min of coma, and that other non-glucose substrates must have accounted for approximately 50μmol·g^?1 of oxygen (8.3 μmol·g^?1 in terms of glucose equivalents) within the 5–30 min period. The 10% reduction in phospholipid-bound fatty acids was more than sufficient (in four- to fivefold excess) to account for this oxygen consumption. However, since no further degradation occurred in the 5–30 min period, there is no simple, direct, quantitative relationship between oxygen consumption and cortical fatty acid oxidation during this interval. The possibility thus remains that unmeasured exogenous or endogenous substrates were utilized.     

Increased Potassium Conductance of Brain Mitochondria Induces Resistance to Permeability Transition by Enhancing Matrix Volume

Modulation of K⁺ conductance of the inner mitochondrial membrane has been proposed to mediate preconditioning in ischemia-reperfusion injury. The mechanism is not entirely understood, but it has been linked to a decreased activation of mitochondrial permeability transition (mPT). In the present study K⁺ channel activity was mimicked by picomolar concentrations of valinomycin. Isolated brain mitochondria were exposed to continuous infusions of calcium. Monitoring of extramitochondrial Ca²⁺ and mitochondrial respiration provided a quantitative assay for mPT sensitivity by determining calcium retention capacity (CRC). Valinomycin and cyclophilin D inhibition separately and additively increased CRC. Comparable degrees of respiratory uncoupling induced by increased K⁺ or H⁺ conductance had opposite effects on mPT sensitivity. Protonophores dose-dependently decreased CRC, demonstrating that so-called mild uncoupling was not beneficial per se. The putative mitoK_ATP channel opener diazoxide did not mimic the effect of valinomycin. An alkaline matrix pH was required for mitochondria to retain calcium, but increased K⁺ conductance did not result in augmented ΔpH. The beneficial effect of valinomycin on CRC was not mediated by H₂O₂-induced protein kinase Cϵ activation. Rather, increased K⁺ conductance reduced H₂O₂ generation during calcium infusion. Lowering the osmolarity of the buffer induced an increase in mitochondrial volume and improved CRC similar to valinomycin without inducing uncoupling or otherwise affecting respiration. We propose that increased potassium conductance in brain mitochondria may cause a direct physiological effect on matrix volume inducing resistance to pathological calcium challenges.     

Ionophoric actions of avenaciolide on mitochondrial cations

Avenaciolide produces an initial stimulation of mitochondrial respiration followed at high doses of the drug by a decline in respiration to less than the unstimulated rate; under these conditions the mitochondria are insensitive to ADP and to uncoupler. At lower avenaciolide concentrations followed by ADP there is a sustained acceleration of respiration which is sensitive to EDTA and oligomycin, pointing to the existence of a Mg-requiring ATPase.Spectrophotometric tests with bromthymol blue and fluorimetry show a similarity between the responses to avenaciolide and divalent cations.Mitochondrial contents of substrate anions and cations are altered by avenaciolide; the extent of the changes depend on the level of the drug used and also on the composition of the medium. If K⁺ is present with an energy source, the uptake of K⁺ at the start of an incubation is enhanced by avenaciolide when supplied at less than 25–30 nmole/mg protein, and the K⁺ gain is accompanied by an uptake of substrate anion; at higher concentrations of avenaciolide the direction of flow is reversed with loss of K⁺, divalent cations, and substrate anions. In potassium-free media, or in the absence of energy only losses of ions are found.Addition of avenaciolide to mitochondria onto which [¹⁴C]octyl malonate had previously been adsorbed results in a discharge of the labeled compound.     

Adenosine diphosphate and calcium stimulation of respiration in mitochondria from alloxan diabetic rats

Liver mitochondria from normal and alloxan diabetic rats, isolated in 0.25 M sucrose, were assayed with an oxygen electrode for ADP/O and Ca⁺²/O ratios, respiratory ratio, and respiratory control index. Mitochondria were incubated with two substrates, succinate and β-hydroxybutyrate; two types of ionic media, Na⁺ medium (Na⁺ the major monovalent cation) and K⁺ medium (K⁺ the major monovalent cation); and two respiratory stimulants, ADP (352 μM) and Ca⁺² (187 μM). Significant differences between respiratory rates and ADP/O ratios were dependent upon the substrate and ionic medium employed. The results confirm previous studies which showed no alteration in ADP/O ratio but decreased State 3 respiratory rates under similar conditions of K⁺ medium with ADP stimulation in the diabetic. Furthermore, the State 3 respiration was prolonged compared to normal. Ca⁺² stimulation was the same in normal and diabetic mitochondria in K⁺ medium. Studies in Na⁺ media revealed more significant differences in RCI's, respiratory rates, and ADP/O ratios that were substrate dependent as well as ion dependent. The results from these various studies can be accounted for by an hypothesis linking mitochondrial K⁺ interaction with alterations in the diabetic mitochondria.     

Effects of Tl<Superscript>+</Superscript> on ion permeability,membrane potential and respiration of isolated rat liver mitochondria

It is known that permeability of the inner mitochondrial membrane is low to most univalent cations (K⁺, Na⁺, H⁺) but high to Tl⁺. Swelling, state 4, state 3, and 2,4-dinitrophenol (DNP)-stimulated respiration as well as the membrane potential (ΔΨ_mito) of rat liver mitochondria were studied in media containing 0–75 mM TlNO₃ either with 250 mM sucrose or with 125 mM nitrate salts of other monovalent cations (KNO₃, or NaNO₃, or NH₄NO₃). Tl⁺ increased permeability of the inner mitochondrial membrane to K⁺, Na⁺, and H⁺, that was manifested as stimulation of the swelling of nonenergized and energized mitochondria as well as via an increase of state 4 and dissipation of ΔΨ_mito. These effects of Tl⁺ increased in the order of sucrose <K⁺ <Na⁺ ≤ NH₄⁺. They were stimulated by inorganic phosphate and decreased by ADP, Mg²⁺, and cyclosporine A. Contraction of energized mitochondria, swollen in the nitrate media, was markedly inhibited by quinine. It suggests participation of the mitochondrial K⁺/H⁺ exchanger in extruding of Tl⁺-induced excess of univalent cations from the mitochondrial matrix. It is discussed that Tl⁺ (like Cd²⁺ and other heavy metals) increases the ion permeability of the inner membrane of mitochondria regardless of their energization and stimulates the mitochondrial permeability transition pore in low conductance state. The observed decrease of state 3 and DNP-stimulated respiration in the nitrate media resulted from the mitochondrial swelling rather than from an inhibition of respiratory enzymes as is the case with the bivalent heavy metals.     

CEREBRAL METABOLIC CHANGES IN PROFOUND, INSULIN-INDUCED HYPOGLYCEMIA, AND IN THE RECOVERY PERIOD FOLLOWING GLUCOSE ADMINISTRATION

Severe hypoglycemia was induced by insulin in lightly anaesthetized (70°_o N₂O) and artificially ventilated rats. Brain tissue was frozen in situ after spontaneous EEG potentials had disappeared for 5. 10. 15 or 30 min and cerebral cortex concentrations of labile organic phosphates, glycolytic metabolites, ammonia and amino acids were determined. In other experiments, recovery was induced by glucose injection at the end of the period of EEG silence. All animals with an isoelectric EEG showed extensive deterioration of the cerebral energy state. and gross perturbation of amino acid concentrations. The latter included a 4-fold rise in aspartate concentration and reductions in glutamate and glutamine concentrations to 20 and 5^o_o of control levels respectively. There was an associated rise in ammonia concentration to about 3μmol-g^-1. Administration of glucose brought about extensive recovery of cerebral energy metabolism. For example, after an isoelectric period of 30 min tissue concentrations of phosphocreatine returned to or above normal, the accumulation of ADP and AMP was reversed, there was extensive resynthesis of glycogen and glutamine and full normalisation of tissue concentrations of pyruvate. α-ketoglutarate. GABA and ammonia. However, even after 3 h of recovery there was a reduction in the ATP concentration and thereby in adenine nucleotide pool, moderate elevations of lactate content and the lactate pyruvate ratio, and less than complete restoration of the amino acid pool. It is concluded that some cells may have been irreversibly damaged by the hypoglycemia.     

Adenine nucleotide translocase mediates the K(ATP)-channel-openers-induced proton and potassium flux to the mitochondrial matrix

K_ATP channel openers have been shown to protect ischemic-reperfused myocardium by mimicking ischemic preconditioning, although their mechanisms of action have not been fully clarified. In this study we investigated the influence of the adenine nucleotide translocase (ANT) inhibitors–carboxyatractyloside (CAT) and bongkrekic acid (BA)–on the diazoxide- and pinacidil-induced uncoupling of isolated rat heart mitochondria respiring on pyruvate and malate (6 + 6 mM). We found that both CAT (1.3 M) and BA (20 M) markedly reduced the uncoupling of mitochondrial oxidative phosphorylation induced by the K_ATP channel openers. Thus, the uncoupling effect of diazoxide and pinacidil is evident only when ANT is not fixed by inhibitors in neither the C- nor the M-conformation. Moreover, the uncoupling effect of diazoxide and pinacidil was diminished in the presence of ADP or ATP, indicating a competition of K_ATP channel openers with adenine nucleotides. CAT also abolished K⁺-dependent mitochondrial respiratory changes. Thus ANT could also be involved in the regulation of K_ATP-channel-openers-induced K⁺ flux through the inner mitochondrial membrane.     

Ischemia-Induced Changes in Cerebral Mitochondrial Free Fatty Acids, Phospholipids, and Respiration in the Rat

Abstract: Changes in the free fatty acid pool size and fatty acyl chain composition of mitochondrial membrane phospholipids and their relation to disruption of mitochondrial function were examined in rat brains after 30 min of cerebral ischemia (Pulsinelli-Brierley model) and 60 min of normoxic reoxygenation. During ischemia, significant hydrolysis of polyunsaturated molecular species from diacyl phosphatidylcholine, particularly fatty acyl 20:4 (arachidonic acid; 20% decrease) and 22:6 (docosahexaenoic acid; 15% decrease), was observed. Thirty minutes of ischemia caused a 16% loss of 18:2 (linoleic acid) from phosphatidylethanolamine. Recirculation for 60 min did not return the polyunsaturated fatty acid content of phospholipids to normal. Total content of free fatty acids increased during ischemia, particularly 18:2 and 22:6, which exhibited the most dramatic rise. The free fatty acid pool size continued to increase during 60 min of recirculation. The respiratory control ratio decreased significantly during 30 min of ischemia with no apparent recovery following 60 min of reoxygenation. The degree of free radical-mediated lipid peroxidation in mitochondria was significantly increased during ischemia and reperfusion. It was concluded that (a) 30 min of cerebral ischemia caused differential degradation in each of the phospholipid classes and preferential hydrolysis of the polyunsaturated molecular species and (b) 60 min of normoxic reperfusion failed to promote reacylation of the mitochondrial phospholipids and restoration of normal respiration.     

Hypoxic preconditioning induces neuroprotection against transient global ischemia in adult rats via preserving the activity of Na(+)/K(+)-ATPase

We demonstrated previously that 30 min of hypoxic preconditioning (HPC) applied 1 day before 10 min of transient global cerebral ischemia (tGCI) reduced neuronal loss in the hippocampal CA1 subregion in adult rats. The aim of the present study was to investigate the role of Na⁺/K⁺-ATPase and protein kinase Mζ (PKMζ) in the protective effect of HPC against tGCI in adult rats. We found that the activity of Na⁺/K⁺-ATPase decreased in the hippocampal CA1 subregion after 10 min of tGCI. This effect was not seen after 30 min of HPC in adult rats. Corresponding to the changes in Na⁺/K⁺-ATPase activity, the surface expression of Na⁺/K⁺-ATPase α1 subunit increased after HPC. Furthermore, HPC dramatically reduced the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells in the hippocampal CA1 subregion after tGCI. However, neither PKMζ nor phosphorylation of PKMζ was changed after tGCI or HPC. The results of the present study are consistent with the hypothesis that both enhanced recovery of Na⁺/K⁺-ATPase activity due to preserved the protein levels of Na⁺/K⁺-ATPase α1 subunit and reduced DNA fragmentation after tGCI contribute to the protection afforded by HPC. However, PKMζ activation does not appear to play a role in this neuroprotection.     

Influence of ATP-dependent K<Superscript>+</Superscript>-channel opener on K<Superscript>+</Superscript>-cycle and oxygen consumption in rat liver mitochondria

The influence of the K_ATP⁺-channel opener diazoxide on the K⁺ cycle and oxygen consumption has been studied in rat liver mitochondria. It was found that diazoxide activates the K_ATP⁺-channel in the range of nanomolar concentrations (50–300 nM, K _1/2 ∼ 140 nM), which results in activation of K⁺/H⁺ exchange in mitochondria. The latter, in turn, accelerates mitochondrial respiration in respiratory state 2. The contribution of K_ATP⁺-channel to the mitochondrial potassium cycle was estimated using the selective K_ATP⁺-channel blocker glibenclamide. The data show that the relative contribution of K_ATP⁺-channel in the potassium cycle of mitochondria is variable and increases only with the decrease in the ATP-independent component of K⁺ uptake. Possible mechanisms underlying the observed phenomena are discussed. The experimental results more fully elucidate the role of K_ATP⁺-channel in the regulation of mitochondrial functions, especially under pathological conditions accompanied by impairment of the mitochondrial energy state.     

THE MECHANISM OF THE EXCLUSION OF SODIUM IONS AND THE CONCENTRATION OF POTASSIUM IONS BY GUINEA-PIG CEREBRAL CORTEX SLICES

—Guinea pig cerebral slices were incubated in oxygenated Krebs-Ringer bicarbonate glucose saline for periods of 1 s to 60 min, and their swelling and Na⁺ and K⁺ cone were measured. The swelling was at the rate of 8 per cent for the 1st min, and 0·8 per cent for the next 29 min; it fell significantly during the subsequent 30 min (P= 0·05). The Na⁺ and K⁺ concn in the tissue fluctuated during the 1st min of incubation, but the Na⁺ concn had risen to a mean of 108 mm after 1 min incubation and the K⁺ concn had fallen to a mean of 52 mm by 3 min. The concentrations of these cations did not change significantly after these times. Cerebral slices were also incubated for 30 min in isotonic media modified such that Na⁺, + K⁺, Na⁺+ choline⁺, or K⁺+ choline⁺ always added up to 150 mm . It was found that about half of the swelling (20-25 per cent) was independent of the Na⁺ or K⁺ concn and a further 20-25 per cent of the swelling varied with the cations only if Na⁺ and K⁺ were both present and was a function of the K⁺ concn in the medium (0·15 per cent m-mol). The Na⁺ concn in the tissue was a mean 8·4 mm after incubation in a Na⁺-free medium and 7·1 mm in K⁺ after incubation in a K⁺-free medium. Cerebral slices in the presence of Na⁺+ K⁺ excluded one molecule of Na⁺ for every four molecules in the incubating medium; they accumulated K⁺ from the medium until the concn in the medium exceeded 130 mm .     

Effects of a high fat diet on liver mitochondria: increased ATP-sensitive K<Superscript>+</Superscript> channel activity and reactive oxygen species generation

High fat diets are extensively associated with health complications within the spectrum of the metabolic syndrome. Some of the most prevalent of these pathologies, often observed early in the development of high-fat dietary complications, are non-alcoholic fatty liver diseases. Mitochondrial bioenergetics and redox state changes are also widely associated with alterations within the metabolic syndrome. We investigated the mitochondrial effects of a high fat diet leading to non-alcoholic fatty liver disease in mice. We found that the diet does not substantially alter respiratory rates, ADP/O ratios or membrane potentials of isolated liver mitochondria. However, H₂O₂ release using different substrates and ATP-sensitive K⁺ transport activities are increased in mitochondria from animals on high fat diets. The increase in H₂O₂ release rates was observed with different respiratory substrates and was not altered by modulators of mitochondrial ATP-sensitive K⁺ channels, indicating it was not related to an observed increase in K⁺ transport. Altogether, we demonstrate that mitochondria from animals with diet-induced steatosis do not present significant bioenergetic changes, but display altered ion transport and increased oxidant generation. This is the first evidence, to our knowledge, that ATP-sensitive K⁺ transport in mitochondria can be modulated by diet.     

Temperature response of oxygen uptake in brain mitochondria from normal and ischemic rats

Mitochondria isolated from rat brains following 30 min of complete (decapitation) ischemia showed a 3-fold increase in free fatty acid content, but no significant decreases in the total fatty acid or phospholipid content.This free fatty acid increase was associated with an altered mitochondrial function: a 50% inhibition of state 3 (+ ADP) respiration and a decrease in the respiratory control ratio from 5.5 to 3 to 24°C. The P:O ratio remained unchanged at 3, and there was no increase in stage 4 respiration. When glutamate and malate supported respiration was determined as a function of temperature in control mitochondria, the resulting Arrhenius plot of the state 3 respiration was biphasic with a transition temperature around 30°C, while ischemic mitochondria exhibited a linear Arrhenius plot with energy of activation (approximately 10 kcal/mol) similar to that of control mitochondria below the transition temperature.The difference in temperature response between control and ischemic mitochondria reflects a change in mitochondrial lipid composition, and is therefore a functional manifestation of the altered cerebral lipid composition commonly observed during ischemia.     

The relationship between plasma potassium concentration and muscle torque during recovery following intense exercise

The present study investigated the relationship between plasma potassium ion concentration ([K⁺]) and skeletal muscle torque during three different 15-min recovery periods after fatigue induced by four 30-s sprints. Four males and one female completed the multiple sprint exercise on three separate days; recovery was passive, i.e. no cycling exercise (PRec), active cycling at 30% peak oxygen consumption O_2peak (30% Rec) and active cycling at 60% O_2peak (60% Rec). Plasma [K⁺] was measured from blood sampled from an antecubital vein of subjects at rest and at 0, 3, 5, 10 and 15 min into each recovery. Isokinetic leg strength was measured at rest and at 1, 6, 11 and 16 min during each recovery. Following the exhaustive sprints, [K⁺] increased significantly from an average mean (SEM) resting value of 3.81 (0.07) mmol · l⁻¹ to 4.48 (0.19) mmol · l⁻¹ (P < 0.01). In all recovery conditions, plasma [K⁺] returned to resting levels within 3 min following the fourth sprint. However, in the two active recovery conditions plasma [K⁺] increased over the remainder of the recovery periods to 4.36 (0.12) mmol · l⁻¹ in the 30% Rec condition and 4.62 (0.12) mmol · l⁻¹ in the 60% Rec condition, the latter being significantly higher than the former (P < 0.01). The maximum torque measured following the sprints decreased significantly, on average, to 61.1 (8.36)% of peak levels (P < 0.01). After 15 min of recovery, maximum torque was highest in the 30% Rec condition at 92.13 (3.06)% of peak levels (P < 0.01), compared to 85.23 (3.64)% and 85.71 (0.82)% for the PRec and 60% Rec conditions, respectively. In contrast to the significant differences in plasma [K⁺] across all three recovery conditions, muscle torque recovery was significantly different in only the 30% Rec condition. In summary, recovery of peak levels of muscle torque following fatiguing exercise does not appear to follow changes in plasma [K⁺]. Accepted: 18 October 1996     

Influence of cerebral ischemia and post-ischemic reperfusion on mitochondrial oxidative phosphorylation

Unilateral ischemia in the right cerebral hemisphere of the rat was induced by ligation of the right common carotid artery coupled with controlled hemorrhage to produce hypotension (25±8 mm/Hg). Where indicated after 30 min of ischemia, the withdrawn blood was reinfused to restore arterial pressure to normal. Mitochondria isolated from the ipsilateral hemisphere after 30 min of ischemia showed significantly lower respiratory rates than the organelles isolated from the contralateral side. Oxidation of NAD⁺-linked substrates was more sensitive to inhibition in ischemia (30%) than was of ferrocytochromec (12%), succinate oxidation being intermediate. The activities of membrane-bound dehydrogenases (both NADH and succinate-linked) were also significantly lowered. Ischemia did not affect the cytochrome content of mitochondria. Respiratory activity (NAD⁺-linked) of mitochondria isolated from the ipsilateral hemisphere was twice as sensitive to inhibition by fatty acid as was of preparations from the contralateral side. Mitochondria isolated from cerebral cortex after 90 min of post-ischemic reperfusion showed no significant improvement in the rate of substrate oxidation. Adenine nucleotide translocase activity and energy-dependent Ca²⁺ uptake, both of which decreased significantly in mitochondria isolated from the ischemic brain, showed little recovery, on reperfusion. These observations suggested the strong possibility that the deleterious effects of ischemia on mitochondrial respiratory function might be mediated by free fatty acids that are known to accumulate in large amounts in ischemic tissues. The pattern of inhibition of ATPase activity was consistent with this view.     

Influence of Tl+ on mitochondrial permeability transition pore in Ca2+-loaded rat liver mitochondria

The Tl⁺-induced opening of the MPTP in Ca²⁺-loaded rat liver mitochondria energized by respiration on the substrates succinate or glutamate plus malate was recorded as increased swelling and dissipation of mitochondrial membrane potential as well as decreased state 4, or state 3, or 2,4-dinitrophenol-stimulated respiration. These effects of Tl⁺ increased in nitrate media containing monovalent cations in the order of Li⁺ < NH₄⁺ ≤ Na⁺ < K⁺. They were potentiated by inorganic phosphate and diminished by the MPTP inhibitors (ADP, CsA, Mg²⁺, Li⁺, rotenone, EGTA, and ruthenium red) both individually and more potently in their combinations. Maximal swelling of both non-energized and energized Ca²⁺-loaded mitochondria in rotenone-free media is an indication of Ca²⁺ uptake driven by respiration on mitochondrial endogenous substrates. It is suggested that Tl⁺ (distinct from Cd²⁺, Hg²⁺, and other heavy metals and regardless of the used respiratory substrates) can stimulate opening of the MPTP only in the presence of Ca²⁺. We discuss the possible participation of Ca²⁺-binding sites, located near the respiratory complex I and the adenine nucleotide translocase, in inducing opening of the MPTP.     

Interdependence of mitochondrial ATP production and extramitochondrial ATP utilization in intact spermatozoa

The dependence of both respiration and total activity of ATP-consuming reactions on the cellular adenine nucleotide pattern was investigated in intact bovine spermatozoa. ATP consumption was manipulated by inhibition with vanadate and activation with caffeine, leading to a decrease or increase in the rate of respiration up to 70% or 20%, respectively. Oligomycin blocked the respiration to the same extent as did vanadate, suggesting that the total extramitochondrial ATP-consuming activity is vanadate-sensitive. The major part of ATP utilization must be linked to dynein ATPase, since inhibition of (Na⁺, K⁺) ATPase by ouabain showed only a small effect on respiration (−17%). Being a potent inhibitor of dynein ATPase, vanadate drastically reduced the amount of motile cells, whereas caffeine tended to increase the intensity of motion. The effects of vanadate or caffeine on respiration were paralleled by changes in cellular ATP, reflecting the response of mitochondrial respiration on the cellular ATP/ADP ratio. Respiration was found to depend on changes in the ATP/ADP ratio in the range from about 3 (+ caffeine) to 9 (+ vanadate). The range of response of ATP consumption to the ATP/ADP ratio was determined by varying the mitochondrial ATP production via the concentration of lactate which was used as substrate. The measured effects on both respiratory rate and ATP/ADP ratio suggested that ATP consumption was markedly dependent on ATP/ADP ratios below 5. It is concluded that lactate concentrations above 1 mM sufficiently supply bovine spermatozoa with substrate and the energy turnover is mainly limited by the activity of dynein ATPase rather than by the capacity of mitochondrial oxidative phosphorylation.     

Preconditioning Prevents the Inhibition of Na+,K+-ATPase Activity after Brain Ischemia

Application of single transient forebrain ischemia (ISC) in adult Wistar rats, lasting 2 or 10 min, caused inhibition of Na⁺,K⁺-ATPase activity in cytoplasmic membrane fractions of hippocampus and cerebral cortex immediately after the event. In the 2-min ISC group followed by 60 min of reperfusion, the enzyme inhibition was maintained in the cortex, while there was an increase in hippocampal enzyme activity; both effects were over 1 day after the event. However, in the 10-min ISC group enzyme inhibition had been maintained for 7 days in both cerebral structures. Interestingly, ischemic preconditioning (2-min plus 10-min ISC, with a 24-hour interval in between) prevented the inhibitory effect of ischemia/reperfusion on Na⁺,K⁺-ATPase activity observed either after a single insult of 2 min or 10 min ischemia. We suggest that the maintenance of Na⁺,K⁺-ATPase activity afforded by preconditioning be related to cellular neuroprotection.     

Regulation of pyruvate oxidation in blowfly flight muscle mitochondria: Requirement for ADP

Blowfly (Phormia regina) flight muscle mitochondria oxidized pyruvate (+ proline) in the presence of either ADP (coupled respiration) or carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP-uncoupled respiration). There was an absolute requirement for ADP (K_m = 8.0 μm) when pyruvate oxidation was stimulated by FCCP in the presence of oligomycin. This requirement for ADP was limited to the oxidation of pyruvate; uncoupled α-glycerolphosphate oxidation proceeded maximally even in the absence of added ADP. Atractylate inhibited uncoupled pyruvate oxidation whether added before (>99%) or after (95%) initiation of respiration with FCCP. In the presence of FCCP, oligomycin, and limiting concentrations of ADP (less than 110 μm), there was a shutoff in the uptake of oxygen. This inhibition of respiration was completely reversed by the addition of more ADP. Plots of net oxygen uptake as a function of the limiting ADP concentration were linear; the observed ADP/O ratio was 0.22 ± 0.025. An ADP/O ratio of 0.2 was predicted if phosphorylation occurred only at the succinyl-CoA synthetase step of the tricarboxylate cycle. Experiments performed in the presence of limiting concentrations of ADP, and designed to monitor changes in the mitochondrial content of ADP and ATP, demonstrated that the shutoff in oxygen uptake was not due to the presence of a high intramitochondrial concentration of ATP. Indeed, ATP, added to the medium prior to the addition of FCCP, inhibited uncoupled pyruvate oxidation; the apparent K_I was 0.8 mm. These results are consistent with the hypothesis that it is the intramitochondrial ATP/ADP ratio that is one of the controlling factors in determining the rate of flux through the tricarboxylate cycle. Changes in the mitochondrial content of citrate, isocitrate, α-ketoglutarate, and malate during uncoupled pyruvate oxidation in the presence of a limiting concentration of ADP were consistent with the hypothesis that the mitochondrial NAD⁺-linked isocitric dehydrogenase is a major site for such control through the tricarboxylate cycle.     

Sildenafil citrate concentrations not affecting oxidative phosphorylation depress H2O2 generation by rat heart mitochondria

Sildenafil citrate (Viagra) is a potent and specific inhibitor of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5), which exhibits cardioprotective action against ischemia/reperfusion injury in intact and isolated heart. The mechanism of its cardioprotective action is not completely understood, but some results suggested that sildenafil exerts cardioprotection through the opening of mitochondrial ATP-sensitive K⁺ channels (mitoK_ATP). However, the impact of sildenafil citrate per se on isolated heart mitochondrial function is unknown. The goal of this study was to investigate the influence of the compound on mitochondrial function (bioenergetics, Ca²⁺-induced mitochondrial permeability transition, and hydrogen peroxide (H₂O₂) generation) in an attempt to correlate its known actions with effects on heart mitochondria. It was observed that sildenafil citrate concentrations of up to 50 μM did not significantly affect glutamate/malate-supported respiration in states 2, 3, 4, oligomycin-inhibited state 3, and uncoupled respiration. The respiratory control ratio (RCR), the ADP to oxygen ratio (ADP/O), the transmembrane potential (ΔΨ), the phosphorylation rate, and the membrane permeability to H⁺, K⁺ and Ca²⁺ were not affected either. However, sildenafil citrate decreased H₂O₂ generation by mitochondria respiring glutamate/malate, and also decreased the formation of superoxide radical (O₂^•−) generated in a hypoxantine/xantine oxidase system. It was concluded that sildenafil citrate concentrations of up to 50 μM do not affect either rat heart mitochondrial bioenergetics or Ca²⁺-induced mitochondrial permeability transition, but it depresses H₂O₂ generation by acting as a superoxide dismutase (SOD)-mimetic. By preventing reactive oxygen species (ROS) generation, sildenafil citrate may preserve heart mitochondrial function.