Regulation of pyruvate dehydrogenase in rat heart. Mechanism of regulation of proportions of dephosphorylated and phosphorylated enzyme by oxidation of fatty acids and ketone bodies and of effects of diabetes: role of coenzyme A, acetyl-coenzyme A and reduced and oxidized nicotinamide-adenine dinucleotide.

The proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart was decreased by alloxan-diabetes or by perfusion with media containing acetate, n-octanoate or palmitate. The total activity of the dehydrogenase was unchanged. 2. Pyruvate (5 or 25mM) or dichloroacetate (1mM) increased the proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart, presumably by inhibiting the pyruvate dehydrogenase kinase reaction. Alloxan-diabetes markedly decreased the proportion of active dehydrogenase in hearts perfused with pyruvate or dichloroacetate. 3. The total activity of pyruvate dehydrogenase in mitochondria prepared from rat heart was unchanged by diabetes. Incubation of mitochondria with 2-oxo-glutarate plus malate increased ATP and NADH concentrations and decreased the proportion of active pyruvate dehydrogenase. The decrease in active dehydrogenase was somewhat greater in mitochondria prepared from hearts of diabetic rats than in those from hearts of non-diabetic rats. Pyruvate (0.1-10 mM) or dichloroacetate (4-50 muM) increased the proportion of active dehydrogenase in isolated mitochondria presumably by inhibition of the pyruvate dehydrogenase kinase reaction. They were much less effective in mitochondria from the hearts of diabetic rats than in those of non-diabetic rats. 4. The matrix water space was increased in preparations of mitochondria from hearts of diabetic rats. Dichloroacetate was concentrated in the matrix water of mitochondria of non-diabetic rats (approx. 16-fold at 10 muM); mitochondria from hearts of diabetic rats concentrated dichloroacetate less effectively. 5. The pyruvate dehydrogenase phosphate phosphatase activity of rat hearts and of rat heart mitochondria (approx. 1-2 munit/unit of pyruvate dehydrogenase) was not affected by diabetes. 6. The rate of oxidation of [1-14C]pyruvate by rat heart mitochondria (6.85 nmol/min per mg of protein with 50 muM-pyruvate) was approx. 46% of the Vmax. value of extracted pyruvate dehydrogenase (active form). Palmitoyl-L-carnitine, which increased the ratio of [acetyl-CoA]/[CoA] 16-fold, inhibited oxidation of pyruvate by about 90% without changing the proportion of active pyruvate dehydrogenase.     

Endotoxemia reduces skeletal muscle protein synthesis in neonates

Protein synthesis in skeletal muscle is reduced by as much as 50% as early as 4 h after a septic challenge in adults. However, the effect of sepsis on muscle protein synthesis has not been determined in neonates, a highly anabolic population whose muscle protein synthesis rates are elevated and uniquely sensitive to insulin and amino acid stimulation. Neonatal piglets (n = 10/group) were infused for 8 h with endotoxin [lipopolysaccharide (LPS), 0 and 10 microg. kg(-1). h(-1)]. Plasma amino acid and glucose concentrations were kept at the fed level by infusion of dextrose and a balanced amino acid mixture. Fractional protein synthesis rates were determined by use of a flooding dose of [(3)H]phenylalanine. LPS infusion produced a septic-like state, as indicated by an early and sustained elevation in body temperature, heart rate, and plasma tumor necrosis factor-alpha, interleukin-1, cortisol, and lactate concentrations. Plasma levels of insulin increased, whereas glucose and amino acids decreased, suggesting the absence of insulin resistance. LPS significantly reduced protein synthesis in longissimus dorsi muscle by only 11% and in gastrocnemius by only 15%, but it had no significant effect in masseter and cardiac muscles. LPS increased protein synthesis in the liver (22%), spleen (28%), kidney (53%), jejunum (19%), diaphragm (21%), lung (50%), and skin (13%), but not in the stomach, pancreas, or brain. These findings suggest that, when substrate supply is maintained, skeletal muscle protein synthesis in neonates compared with adults is relatively resistant to the catabolic effects of sepsis.     

Conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active complex by the phosphate reaction in heart mitochondria is inhibited by alloxan-diabetes or starvation in the rat.

1. The conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active (dephosphorylated) complex by pyruvate dehydrogenase phosphate phosphatase is inhibited in heart mitochondria prepared from alloxan-diabetic or 48h-starved rats, in mitochondria prepared from acetate-perfused rat hearts and in mitochondria prepared from normal rat hearts incubated with respiratory substrates for 6 min (as compared with 1 min). 2. This conclusion is based on experiments with isolated intact mitochondria in which the pyruvate dehydrogenase kinase reaction was inhibited by pyruvate or ATP depletion (by using oligomycin and carbonyl cyanide m-chlorophenylhydrazone), and in experiments in which the rate of conversion of inactive complex into active complex by the phosphatase was measured in extracts of mitochondria. The inhibition of the phosphatase reaction was seen with constant concentrations of Ca2+ and Mg2+ (activators of the phosphatase). The phosphatase reaction in these mitochondrial extracts was not inhibited when an excess of exogenous pig heart pyruvate dehydrogenase phosphate was used as substrate. It is concluded that this inhibition is due to some factor(s) associated with the substrate (pyruvate dehydrogenase phosphate complex) and not to inhibition of the phosphatase as such. 3. This conclusion was verified by isolating pyruvate dehydrogenase phosphate complex, free of phosphatase, from hearts of control and diabetic rats an from heart mitochondria incubed for 1min (control) or 6min with respiratory substrates. The rates of re-activation of the inactive complexes were then measured with preparations of ox heart or rat heart phosphatase. The rates were lower (relative to controls) with inactive complex from hearts of diabetic rats or from heart mitochondria incubated for 6min with respiratory substrates. 4. The incorporation of 32Pi into inactive complex took 6min to complete in rat heart mitocondria. The extent of incorporation was consistent with three or four sites of phosphorylation in rat heart pyruvate dehydrogenase complex. 5. It is suggested that phosphorylation of sites additional to an inactivating site may inhibit the conversion of inactive complex into active complex by the phosphatase in heart mitochondria from alloxan-diabetic or 48h-starved rats or in mitochondria incubated for 6min with respiratory substrates.     

Expression of endothelin-1 system in a pig model of endotoxic shock

Endothelin (ET)-1 is a potent vasoconstrictive peptide and it is involved in the pathogenesis of septic shock. Blockade of ET-1 receptors abolishes the LPS-induced pulmonary hypertension and worsens the LPS-dependent systemic hypotension, but the role of ET-1 in sepsis remains uncertain. To determine the role of ET-1 in cardiovascular and respiratory derangement in a porcine model of endotoxemic shock we evaluated ET-1 plasma levels and ET-1 mRNA and protein levels in lung, liver, and heart as well as Endothelin Converting Enzyme-1, ET(A) and ET(B) receptors mRNA in the same tissues. Twelve piglets were randomised to sham operated or to LPS-treated (40 microg/kg/h for 4 h) groups. During the experiment, respiratory and circulatory parameters have been recorded and blood samples collected. At the end of the experiment the animals were sacrificed and tissue samples collected for real-time quantitative PCR and ELISA test. LPS infusion evokes a large increase in ET-1 plasma concentration, and in tissues mRNA levels, associated with an increase in pulmonary arterial pressure, as well as in pulmonary and systemic vascular resistances, and a decrease in stroke volume. LPS infusion caused also a derangement of respiratory mechanics, evidenced by an increase in resistance and a decrease in compliance of the respiratory system. ET(A) and ET(B) receptor mRNA levels were markedly decreased in liver and lung and slightly increased in heart, evidencing that ET receptor subtypes were differentially regulated in the major organs of endotoxin treated pigs. In conclusion our data show the presence of a continuative and differentially regulated stimulating mechanism of ET-1 expression during pig endotoxaemia as well as a fundamental role of ET-1 system in the cardiovascular and respiratory derangement.     

Effect of endotoxin on protein degradation and lipid peroxidation of erythrocytes.

Sepsis has often been associated with infection due to endotoxin (LPS) produced from gram-negative bacteria. Microcirculatory failure is one of the ultimate causes of septic shock. We studied the effect of endotoxin on the protein breakdown and lipid peroxidation of erythrocyte. In vivo (20 ug LPS/100 g) studies in rats showed increased tyrosine production from erythrocyte, as an index of protein degradation in erythrocyte. In vitro studies using 25 microg to 250 microg LPS per ml also showed similar type of increased effect of endotoxin in protein degradation. Washed erythrocyte devoid of plasma and leucocytes did not show any increased effect after endotoxin treatment. Lipid peroxidation was also increased after endotoxin treatment. However, protein degradation was more prominent than lipid peroxidation. We concluded therefore that the protein degradation and lipid peroxidation of erythrocytes caused by endotoxin are probably related to the production of septic shock.     

Endotoxemia causes central downregulation of sympathetic vasomotor tone in healthy humans

Experimental endotoxemia as a model of the initial septic response affects the autonomic nervous system with profound cardiovascular sequelae. Whether the postsynaptic sympathoneural activity to the muscle vascular bed is altered in the early septic phase remains to be determined. The present study aimed to elucidate the early effects of LPS on muscle sympathetic nerve activity (MSNA) and cardiovascular regulation in healthy humans. Young, healthy volunteers randomly received either an LPS bolus (4 ng/kg body wt, n = 11) or placebo (saline; n = 7). Experimental baroreflex assessment (baseline measurements followed by infusion of vasoactive drugs nitroprusside/phenylephrine) was done prior to and 90 min following LPS or placebo challenge. MSNA, heart rate, blood pressure, and blood levels of catecholamines, TNF-alpha and IL-6 were measured sequentially. Endotoxin but not placebo-induced flu-like symptoms and elevated cytokine levels. In contrast to placebo, LPS significantly suppressed MSNA burst frequency 90 min after injection [mean +/- SE: 12.1 +/- 2.9 vs. 27.5 +/- 3.3 burst/min (post- vs. pre-LPS); P < 0.005] but increased heart rate [78.4 +/- 3.1 vs. 60.6 +/- 2.0 beats/min (post- vs. pre-LPS); P < 0.001]. Baseline blood pressure was not altered, but baroreflex testing demonstrated a blunted MSNA response and uncoupling of heart rate modulation to blood pressure changes in the endotoxin group. We conclude that endotoxin challenge in healthy humans has rapid suppressive effects on postsynaptic sympathetic nerve activity to the muscle vascular bed and alters baroreflex function which may contribute to the untoward cardiovascular effects of sepsis.     

Control of reversible intracellular transfer of reducing potential.

Isolated rat liver mitochondria were incubated in the presence of a reconstituted malate-aspartate shuttle under carboxylating conditions in the presence of glutamate, octanoyl-carnitine and pyruvate, or a preset lactate/pyruvate ratio. The respiration and attendant energy state were varied with soluble F1-ATPase. Under these conditions reducing equivalents are exported due to pyruvate carboxylation. This was shown by lactate production from pyruvate and by a substantial increase in the lactate/pyruvate ratio. This led to a competition between malate export and energy-driven malate cycling via the malate-aspartate shuttle, resulting in a lowered redox segregation of the NAD systems between the mitochondrial and extramitochondrial spaces. If pyruvate carboxylation was blocked, this egress of reducing equivalents was also blocked, leading to an elevated value of redox segregation, delta G(redox) (in kJ) = -5.7 log(NAD+/NADHout)/(NAD+/NADHin) being then equal to approximately one-half of the membrane potential, in accordance with electrogenic glutamate/aspartate exchange. Reconstitution of malate-pyruvate cycling led to a further kinetic decrease in the original malate-aspartate shuttle-driven value of delta G(redox). Therefore, the value of segregation of reducing potential between mitochondria and cytosol caused by glutamate/aspartate exchange can be diminished kinetically by processes exporting reducing equivalents from mitochondria, such as pyruvate carboxylation and pyruvate cycling.     

Post-mortem changes in structure and function of ox muscle mitochondria. 1. Electron microscopic and polarographic investigations

Electron microscopy shows that intact mitochondria can be isolated from neck-muscle stored at 144h post-mortem at 4°. Isolated mitochondria, all in the condensed configuration, have clearly defined outer and inner membranes, outer compartments and intracristal spaces; a larger proportion of swollen ones was isolated from the 144h than from the 120 h post-mortem tissue.Mitochondria from 96 h tissue still retained the following % of the initial values for the ADP/O ratio, respiratory control index (RCI) and state 3 respiratory rate observed for 0–5h tissue: malate+pyruvate, 100, 72 and 53; succinate, 80, 30 and 74; ascorbate+ tetramethyl-p-phenylencdiamine (TMPD), 92, 88 and 72.Both the succinate and ascorbate-TMPD oxidase systems appear to have a critical storage time of about 70 h, whereas the malate+pyruvate system has one of about 96 h. Asharp decline of the ADP/O ratio, RCI and the state 3 respiratory rate occurred after this time, but these three parameters were better preserved in the ascorbate-TMPD oxidase system.The oxidation of the citric acid cycle intermediates in the neck-muscle mitochondria thus shows a higher sensitivity to post-mortem ageing with respect to cytochrome oxidase activity. This is probably due to post-mortem muscle acidification.     

Effects of chronic caloric restriction on mitochondrial respiration in the ischemic reperfused rat heart

Dietary restriction increases life span and delays the development of age-related diseases in rodents. We have recently demonstrated that chronic dietary restriction is beneficial on recovery of heart function following ischemia. We studied whether the metabolic basis of this benefit is associated with alterations in mitochondrial respiration. Male Wistar rats were assigned to an ad libitum-fed (AL) group and a food restricted (FR) group, in which food intake was reduced to 55% of the amount consumed by the AL group. Following an 8-month period of restricted caloric intake, isolated working hearts perfused with glucose and high levels of fatty acids were subjected to global ischemia followed by reperfusion. At the end of reperfusion, total heart mitochondria was respiration was assessed in the presence of pyruvate, tricarboxylic acid intermediates, and palmitoylcarnitine. Recovery of heart function following ischemia was greater in FR hearts compared to AL hearts. Paralleling these changes in heart function was in increase in state 3 respiration with pyruvate. The respiratory control ratios in the presence of pyruvate and tricarboxylic acid intermediates were higher in FR hearts compared to AL hearts, indicating well-coupled mitochondria. Overall energy production, expressed as the ADP:O ratio and the oxidative phosphorylation rate, was also improved in FR hearts. Our results indicate that the beneficial effect of FR on recovery of heart function following ischemia is associated with changes in mitochondrial respiration.     

Modulation of muscle protein synthesis by insulin is maintained during neonatal endotoxemia

Sepsis promotes insulin resistance and reduces protein synthesis in skeletal muscle of adults. The effect of sepsis on insulin-stimulated muscle protein synthesis has not been determined in neonates, a highly anabolic population that is uniquely sensitive to insulin. Overnight fasted neonatal pigs were infused for 8 h with endotoxin [lipopolysaccharide (LPS), 0 and 10 mug.kg(-1).h(-1)]. Glucose and amino acids were maintained at fasting levels, insulin was clamped at either fasting or fed (2 or 10 muU/ml) levels, and fractional protein synthesis rates were determined at the end of the infusion. LPS infusion induced a septic-like state, as indicated by a sustained elevation in body temperature, heart rate, and cortisol. At fasting insulin levels, LPS reduced fractional protein synthesis rates in gastrocnemius muscle (-26%) but had no effect on the masseter and heart. By contrast, LPS stimulated liver protein synthesis (+28%). Increasing insulin to fed levels accelerated protein synthesis rates in gastrocnemius (controls by +38%, LPS by +60%), masseter (controls by +50%, LPS by +43%), heart (controls by +34%, LPS by +40%), and diaphragm (controls by +54%, LPS by +29%), and the response to insulin was similar in LPS and controls. Insulin did not alter protein synthesis in liver, kidney, or jejunum in either group. These findings suggest that acute endotoxemia lowers basal fasting muscle protein synthesis in neonates but does not alter the response of protein synthesis to insulin.     

Amino acids augment muscle protein synthesis in neonatal pigs during acute endotoxemia by stimulating mTOR-dependent translation initiation

In skeletal muscle of adults, sepsis reduces protein synthesis by depressing translation initiation and induces resistance to branched-chain amino acid stimulation. Normal neonates maintain a high basal muscle protein synthesis rate that is sensitive to amino acid stimulation. In the present study, we determined the effect of amino acids on protein synthesis in skeletal muscle and other tissues in septic neonates. Overnight-fasted neonatal pigs were infused with endotoxin (LPS, 0 and 10 microg.kg(-1).h(-1)), whereas glucose and insulin were maintained at fasting levels; amino acids were clamped at fasting or fed levels. In the presence of fasting insulin and amino acids, LPS reduced protein synthesis in longissimus dorsi (LD) and gastrocnemius muscles and increased protein synthesis in the diaphragm, but had no effect in masseter and heart muscles. Increasing amino acids to fed levels accelerated muscle protein synthesis in LD, gastrocnemius, masseter, and diaphragm. LPS stimulated protein synthesis in liver, lung, spleen, pancreas, and kidney in fasted animals. Raising amino acids to fed levels increased protein synthesis in liver of controls, but not LPS-treated animals. The increase in muscle protein synthesis in response to amino acids was associated with increased mTOR, 4E-BP1, and S6K1 phosphorylation and eIF4G-eIF4E association in control and LPS-infused animals. These findings suggest that amino acids stimulate skeletal muscle protein synthesis during acute endotoxemia via mTOR-dependent ribosomal assembly despite reduced basal protein synthesis rates in neonatal pigs. However, provision of amino acids does not further enhance the LPS-induced increase in liver protein synthesis.     

Effects of iNOS-related NO on hearts exposed to liposoluble iron

Inducible nitric oxide synthase (iNOS) protects heart against ischemia/reperfusion injury. However, it is unknown whether the beneficial effects of iNOS are mediated by the interaction of NO with radical oxygen species (ROS). To address this issue, we examined the effects of liposoluble iron-induced ROS generation in isolated perfused hearts from rats treated with lipopolysaccharide (LPS). LPS administration (10 mg/kg, i.p., 6 h before heart removal) induced iNOS expression and increased NO production as indicated by a 3-fold elevation of nitrite level in coronary effluents relative to control hearts. An enhanced expression of hemeoxygenase 1 protein was also observed in septic hearts compared to control. Iron-induced perfusion and contractile deficits were ameliorated by LPS with more important coronary than myocardial benefits. In iron-loaded hearts, oxidative stress as measured by the 2,3 dihydroxybenzoic acid/salicylic acid concentration ratio in cardiac tissue was 23% lower in septic than in control heart although the difference did not reach significance. In addition, the presence of the NO synthase inhibitor N-nitro-L-arginine in the perfusion medium totally blocked NO production but did not reverse the protective effects of LPS. The results indicate that LPS protects from iron-induced cardiac dysfunction by mechanisms independent on ex vivo NO production and suggest that NO acts as a trigger rather than a direct mediator of the cardioprotective effects of LPS in heart exposed to iron.     

Studies on the effects of lipopolysaccharide on lipid peroxidation of erythrocyte and its reversal by mannitol and glycerol.

Gram-negative sepsis often produces endotoxin (LPS) which causes infection. Reduction in tissue perfusion due to microcirculatory failure may lead to septic shock. We studied the effect of LPS on lipid peroxidation of erythrocyte. In vitro studies using 50 microg to 250 microg LPS/ml blood showed increased lipid peroxidation of erythrocyte in a dose-dependent manner. The increased effect of lipid peroxidation does not occur with LPS when erythrocytes were washed to remove plasma and leukocytes. Mannitol and glycerol, known scavengers of hydroxyl radical, arrest the elevation in lipid peroxidation of erythrocytes after LPS treatment. Hemolysis of erythrocytes was reduced with low doses of LPS. Plasma lipid peroxidation was elevated after treatment of blood with LPS. From the results we suggest that the peroxidation of erythrocyte lipid caused by LPS may probably play a role in the production of septic shock.     

Diaphragm and cardiac mitochondrial creatine kinases are impaired in sepsis.

Previous studies indicate that ATP formation by the electron transport chain is impaired in sepsis. However, it is not known whether sepsis affects the mitochondrial ATP transport system. We hypothesized that sepsis inactivates the mitochondrial creatine kinase (MtCK)-high energy phosphate transport system. To examine this issue, we assessed the effects of endotoxin administration on mitochondrial membrane-bound creatine kinase, an important trans-mitochondrial ATP transport system. Diaphragms and hearts were isolated from control (n = 12) and endotoxin-treated (8 mg.kg(-1).day(-1); n = 13) rats after pentobarbital anesthesia. We isolated mitochondria using techniques that allow evaluation of the functional coupling of mitochondrial creatine kinase MtCK activity to oxidative phosphorylation. MtCK functional activity was established by 1) determining ATP/creatine-stimulated oxygen consumption and 2) assessing total creatine kinase activity in mitochondria using an enzyme-linked assay. We examined MtCK protein content using Western blots. Endotoxin markedly reduced diaphragm and cardiac MtCK activity, as determined both by ATP/creatine-stimulated oxygen consumption and by the enzyme-linked assay (e.g., ATP/creatine-stimulated mitochondrial respiration was 173.8 +/- 7.3, 60.5 +/- 9.3, 210.7 +/- 18.9, was 67.9 +/- 7.3 natoms O.min(-1).mg(-1) in diaphragm control, diaphragm septic, cardiac control, and cardiac septic samples, respectively; P < 0.001 for each tissue comparison). Endotoxin also reduced diaphragm and cardiac MtCK protein levels (e.g., protein levels declined by 39.5% in diaphragm mitochondria and by 44.2% in cardiac mitochondria; P < 0.001 and P = 0.009, respectively, comparing sepsis to control conditions). Our data indicate that endotoxin markedly impairs the MtCK-ATP transporter system; this phenomenon may have significant effects on diaphragm and cardiac function.     

Rapid preparation of subsarcolemmal and interfibrillar mitochondrial subpopulations from cardiac muscle

• 1.1. Subsarcolemmal and interfibrillar mitochondria were prepared with complete recovery from rabbit and porcine heart muscle by upward-flotation during 60 sec of Percollö density gradient centrifugation.
• 2.2. Mitochondrial subpopulations were identified and characterized according to buoyant density, electron-microscopy, marker enzyme activities and respiratory performance.
• 3.3. ADP-induced state 3-respiration related to latent citrate synthase activity as a marker for structurally intact mitochondria was not significantly different in both mitochondrial subtypes.

     

Effect of endogenous nitric oxide on energy metabolism of rat heart mitochondria during ischemia and reperfusion

The effects of ischemia and postischemic reperfusion on the functions of the heart and its mitochondria were studied with special attention to the effect of nitric oxide (NO) by treatment of rat hearts with the nitric oxide synthase (NOS) inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) or its noninhibitory isomer N^G-nitro-D-arginine methyl ester (D-NAME). NO generated during reperfusion caused increase in coronary flow (CF), but had no effect on the left ventricular pressure (LVP) or heart rate (HR). The ATP level of the heart decreased during ischemia and was not completely restored by introduction of oxygen during reperfusion due to damage of complexes I and II of the respiratory chain of mitochondria by NO. Inhibition of the respiratory chain resulted in generation of hydrogen peroxide, and NO and NO-derived species generated after production of NO caused further damage of various proteins in mitochondria, such as complexes I and II of the respiratory chain and pyruvate dehydrogenase (PDH). These results suggested that NO generated on reperfusion was the primary cause of mitochondrial dysfunction by damage of complexes I and II of the respiratory chain, with consequent increase of CF in the heart.     

Thermoregulatory responses of rats to conventional preparations of lipopolysaccharide are caused by lipopolysaccharide per se-- not by lipoprotein contaminants

LPS preparations cause a variety of body temperature (T(b)) responses: monophasic fever, different phases of polyphasic fever, and hypothermia. Conventional (c) LPS preparations contain highly active lipoprotein contaminants (endotoxin proteins). Whereas LPS signals predominantly via the Toll-like receptor (TLR) 4, endotoxin proteins signal via TLR2. Several TLR2-dependent responses of immunocytes to cLPS in vitro are triggered by endotoxin proteins and not by LPS itself. We tested whether any T(b) response to cLPS from Escherichia coli 055:B5 is triggered by non-TLR4-signaling contaminants. A decontaminated (d) LPS preparation (free of endotoxin proteins) was produced by subjecting cLPS to phenol-water reextraction. The presence of non-TLR4-signaling contaminants in cLPS (and their absence in dLPS) was confirmed by showing that cLPS (but not dLPS) induced IL-1beta expression in the spleen and increased serum levels of TNF-alpha and IL-1beta of C3H/HeJ mice; these mice bear a nonfunctional TLR4. Yet, both cLPS and dLPS caused cytokine responses in C3H/HeOuJ mice; these mice bear a fully functional TLR4. We then studied the T(b) responses to cLPS and dLPS in Wistar rats preimplanted with jugular catheters. At a neutral ambient temperature (30 degrees C), a low (0.1 microg/kg iv) dose of cLPS caused a monophasic fever, whereas a moderate (10 microg/kg iv) dose produced a polyphasic fever. In the cold (20 degrees C), a high (500 microg/kg iv) dose of cLPS caused hypothermia. All T(b) responses to dLPS were identical to those of cLPS. We conclude that all known T(b) responses to LPS preparations are triggered by LPS per se and not by non-TLR4-signaling contaminants of such preparations.     

Refined anaesthesia for implantation of engineered experimental aortic valves in the pulmonary artery using a right heart bypass in sheep

The feasibility of an anaesthetic protocol developed for surgery during right heart bypass in sheep is reported. Seven female Suffolk sheep, weighing 25-35 kg, were selected for the study. Premedication consisted of midazolam and methadone (both 0.1 mg kg(-1) intravenously). Anaesthesia was induced with propofol (2-4 mg kg(-1)) and maintained with isoflurane in oxygen and continuous rate infusions of propofol (5-7 mg kg(-1 )h(-1)) and fentanyl (5 microg kg(-1) bolus, 5 microg kg(-1) h(-1)). Cisatracurium (0.2 mg kg(-1)) provided muscle relaxation. A standard roller pump was used for the extracorporeal circulation. Drugs administered to maintain blood pressure and heart rate within acceptable levels included phenylephrine (3-4 microg kg(-1)), ephedrine (0.1-0.2 mg kg(-1)), nitroglycerine (50-150 microg kg(-1) h(-1)) and metoprolol succinate (30-80 microg kg(-1)). Electrolytes were infused as needed. Postoperative analgesia was provided by an intercostal block (15 mL 0.5% bupivacaine + epinephrine), carprofen (4 mg kg(-1)) and an opioid (methadone 0.1 mg kg(-1) or buprenorphine 0.01 mg kg(-1)). One sheep became hypoxic during the bypass (PaO(2) 47.7 mmHg). Irregularities of the electrocardiogram were observed during manipulation of the heart in all animals. During the initial phase of the bypass, blood pressure decreased in all sheep, accompanied by dilatation of the heart and large intrathoracic veins in five sheep. With appropriate treatment, blood pressure was restored and easily maintained until the end of the bypass. Weaning from the bypass, using an infusion of nitrates, was smooth. One sheep required a blood transfusion because of severe blood loss and another sheep died postoperatively from respiratory complications. Minor irregularities of the electrocardiogram observed during manipulation of the heart were not life threatening and required no treatment. Decreases in blood pressure at the beginning of the bypass can be expected and require treatment. Nitrates are useful in avoiding volume overload during weaning. The anaesthetic protocol is acceptable for surgery under right heart bypass in sheep.     

Stimulation of the respiratory and phosphorylating activities in rat brain mitochondria by idebenone (CV-2619), a new agent improving cerebral metabolism

The effects of 6-(10-hydroxydecyl)-2,3-dimethoxy-5-methyl-1,4-benzoquinone (CV-2619) on the respiration and non-respiratory oxygen consumption induced by ascorbate and Fe2+ in rat brain mitochondria were studied. When CV-2619 (100 and 300 mg/kg) was orally administered to rats for 3 days, it increased the state 3 respiration stimulated by ADP, slightly decreased the state 4 respiration after the consumption of ADP and resulted in a significant increase of the respiratory control index (RCI) by 14-19% for glutamate oxidation (p less than 0.01) and 10-17% for succinate oxidation (p less than 0.05), respectively. The RCI increasing effect of CV-2619 was dose dependent, but the compound had no effect on the ADP/O ratio. CV-2619 significantly suppressed by about 10% the non-respiratory oxygen consumption (p less than 0.02), which closely associated with non-enzymatic reactions such as lipid peroxidation, membrane lysis and swelling of mitochondria. Thus, CV-2619 may contribute to stimulate the net ATP formation by the well-coupling of electron and energy transfer, and by the reduction of non-respiratory oxygen consumption in cerebral metabolism.     

Gut mucosal damage during endotoxic shock is due to mechanisms other than gut ischemia.

Whether the gut alterations seen during sepsis are caused by microcirculatory hypoxia or disturbances in cellular metabolic pathways associated with mitochondrial respiration remains controversial. We hypothesized that hypoperfusion or hypoxia and local production of nitric oxide might play an important role in the development of gut mucosal injury during endotoxic shock and investigated their roles by using differing levels of fluid resuscitation and occlusion of the superior mesenteric artery (SMA). Anesthetized New Zealand rabbits were allocated to group I (sham, n = 8); group II [low-dose endotoxin (LPS, Escherichia coli-055:B5, 150 microg/kg)/fluid resuscitation (12 ml x kg(-1) x h(-1)); n = 8]; group III [high-dose LPS (1 mg/kg)/fluid resuscitation (12 ml x kg(-1) x h(-1)); n = 8]; group IV [high-dose LPS (1 mg/kg)/hypovolemia (4 ml x kg-1 x h(-1) fluids); n = 8]; and group V [SMA ligation/fluid resuscitation (12 ml x kg(-1) x h(-1)); n = 4]. Luminal gut lactate concentrations and PCO2 gap increased in groups IV and V (P < 0.05), reflecting alterations in gut perfusion. Interestingly, significant histological alterations were observed in all LPS groups but not in group V. Blood and luminal gut nitrate/nitrite concentrations increased only in group IV. The mechanism of gut injury in endotoxic shock seems unrelated to hypoxia and release of nitric oxide. Gut dysfunction may occur as a result of so-called "cytopathic hypoxia."