The effect of propranolol on gene expression during the blood alcohol cycle of rats fed ethanol intragastrically

Propranolol, a beta adrenergic blocker prevents the blood alcohol (BAL) cycle in rats fed ethanol intragastrically at a constant rate by preventing the cyclic changes in the metabolic rate caused by fluctuating levels of norepinephrine released into the blood. The change in the rate of metabolism changes the rate of alcohol elimination in the blood which causes the BAL to cycle. Microarray analysis of the livers from the rats fed ethanol and propranolol showed similar changes in clusters of functionally related gene expressions. The controls and the trough of the cycle differed dramatically from the cluster pattern seen in the rats at the peaks of the blood alcohol cycle. The changes in gene expression induced by ethanol were similar when propranolol was fed without ethanol especially with the changes in the kinases and phosphatases, Toll-like receptor signaling and cytokine-cytokine receptor interaction were also changed.The changes in gene expression caused by ethanol and propranolol feeding are alike probably because both drugs induce β adrenergic receptor desensitization.     

Oxidative phosphorylation in the skeletal muscles of rabbits during dinitrophenol hyperthermia

After intramuscular injection of 2,4-dinitrophenol (DNP) into rabbits in doses of 10, 15, 25, and 30 mg/kg the degree of elevation of the body temperature, the increase in the oxygen consumption, and the degree of uncoupling of oxidative phosphorylation in skeletal muscle homogenates were found to increase with the dose. In the course of dinitrophenol hyperthermia (following injection of 25 mg/kg DNP) the changes in body temperature and oxygen consumption of the animals followed a parallel course with the changes in the level of oxidative phosphorylation in the skeletal muscles.     

The effect of Viagra (sildenafil citrate) on liver injury caused by chronic ethanol intragastric feeding in rats

Rats fed with ethanol and a nutritious diet intragastrically develop liver pathologic changes associated with cyclic elevation of blood and urinary ethanol levels (BAL and UAL cycle). At the peaks of the UAL cycle, the livers are hypoxic. When the liver portal hepatic blood flow is temporarily clamped for 2 min and then released, the livers at the peak UAL fail to recover completely compared to the control livers and the livers at the UAL cycle troughs. Viagra was fed to the ethanol-fed rats to enhance the effects of nitric oxide. Since nitric oxide is known to increase hepatic blood flow, it was anticipated that Viagra would prevent the liver hypoxia at the UAL cycle peaks and also improve the post-clamp recovery from the post-clamp ischemia challenge. Viagra tended to improve the post-clamp recovery of the liver surface pO2 levels of the ethanol-fed rats probably by slowing O2 consumption as result of NO inhibition of mitochondrial cytochrome c oxidase activity. However, Viagra increased the pathology score when fed with ethanol. For this reason, Viagra is a two-edged sword. On the one hand, it tended to be protective in the post-ischemic injury in the ethanol-fed rats and on the other hand, it enhanced the liver injury caused by ethanol. Viagra did not affect the UAL cycle.     

DNP-induced dissipation of ATP in anoxic ventricular muscle

1. During aerobic incubation in 5 mM glucose medium, 10(-5) M-DNP reduced the action potential duration and amplitude and the developed tension of guinea-pig ventricular muscle more rapidly and to a greater extent than anoxia.2. The DNP effect on electrical and mechanical activity was even more pronounced following prolonged anoxic incubation. Since the action potential duration and developed tension of anoxic ventricular muscle have previously been shown to be dependent on glycolytic ATP, and since the effects of DNP could not be duplicated with NaCN, it was concluded that DNP was exerting an effect in addition to its uncoupling of oxidative phosphorylation.3. Anoxic muscle was incubated with 10(-4) M-IAA or with 10(-4) M-IAA + 10(-4) M-DNP. The ATP content of IAA-treated muscle was significantly lower than control but in the presence of both IAA and DNP there was a further reduction in ATP and an increased lactate production.4. Sodium azide (10(-2) M), a potent inhibitor of mitochondrial ATPase, did not prevent the reduction of ATP in DNP-treated anoxic muscle.5. Ouabain (10(-7) M) partially prevented the rapid decline of action potential duration and developed tension of DNP-treated anoxic muscle. In addition, the glycoside partially blocked the DNP-induced break-down of ATP and stimulation of lactate production.6. Oligomycin (10 mug/ml.) partially prevented the reduction in action potential duration and developed tension of DNP-treated anoxic muscle.7. It was concluded that DNP induces an ;energy leak' by actively promoting the hydrolysis of an high energy glycolytic intermediate at least one step beyond the sites of ATPase inhibition by ouabain and oligomycin.     

The effect of rotenone on the urinary ethanol cycle in rats fed ethanol intragastrically

Chronic ethanol ingestion alters mitochondrial function in the liver including inhibition of complex I of the electron transport chain. This leads to a shift in the NAD/NADH ratio to the reduced state when blood ethanol levels are high. Rotenone also inhibits complex I and induces a reduced state. The combination of ethanol feeding and rotenone toxicity should amplify the reduced state and block the cyclic increase and decrease in the rate of metabolism in the liver. The change in the redox state occurs during the urinary ethanol cycle in the intragastric tube feeding rat model of alcoholic liver disease. To test this hypothesis, rats were fed ethanol with rotenone and the 24-h urinary ethanol levels were measured daily. When ethanol was fed alone, the urinary ethanol cycle occurred. However, when ethanol was fed with rotenone the cycle was prevented and the urinary ethanol levels remained at the 200-mg% range. The rats fed ethanol or fed ethanol plus rotenone had the same increase in the pathology score and ALT elevations in the blood. Rotenone fed alone had the same normal values as the dextrose pair fed control rats. The results indicate that the UAL cycle is driven by fluctuation in the NAD/NADH ratio. When this fluctuation is blocked by rotenone, the cycle does not occur. It is concluded that the urinary ethanol cycle is dependent on cyclic fluctuation of the NAD/NADH ratio, which regulates the rate of ethanol elimination.     

The modeling of oxidative phosphorylation in skeletal muscle

A computer model of oxidative phosphorylation was developed in isolated muscle mitochondria [Korzeniewski and Mazat: Biochem J 319: 143-148, 1996] and in intact skeletal muscle [Korzeniewski and Zoladz: Biophys Chem 92: 17-34, 2001]. Within this model the dependence on different metabolite concentrations of the rate of each enzymatic reaction, process and flux is described by an appropriate kinetic equation. The changes of metabolite concentrations over time are described by a set of ordinary differential equations. The model has been very extensively tested by a comparison of computer simulations with a broad set of experimental results concerning various kinetic properties of the oxidative phosphorylation system. Next the model was used for theoretical studies on the regulation of oxidative phosphorylation in intact muscle cells. The model decidedly supports the so-called parallel-activation mechanism or each-step-activation mechanism of adjusting the rate of ATP supply to the current energy demand [Korzeniewski: Biochem J 330: 1189-1195, 1998; Korzeniewski: Biochem J 375: 799-804, 2003]. Because of this mechanism, not only ATP usage, but also the substrate dehydrogenation system and all oxidative phosphorylation complexes (complex I, complex III, complex IV, ATP synthase, ATP/ADP carrier, phosphate carrier) are directly (and not by changes in metabolite concentrations) activated by some intracellular factor(s) related to muscle contraction, probably by calcium ions, during the transition from rest to work. This mechanism is able to account for several kinetic properties of oxidative phosphorylation that cannot be explained by other mechanisms postulated in the literature. Thus the discussed kinetic model of oxidative phosphorylation has appeared to be a very useful research tool.     

Dynamics of lymphocyte receptor affinity for hapten during the primary immune response

Changes in affinity of antigen-binding receptors of lymphocytes for dinitrophenol (DNP) were studied during the primary immune response in CBA mice. The process was evaluated by the method of inhibition of rosette formation with DNP-ovalbumin-sheep's red cells complex by hapten (DNP-ε-lysine). An increase in affinity of immunoglobulin receptors on the lymphocyte surface for DNA was shown, starting from the 10th day after immunization. The question of the role of affinity and density of receptors on the surface of the immunocyte is discussed.     

Production of reactive oxygen species in brain mitochondria: contribution by electron transport chain and non-electron transport chain sources

Overwhelming evidence has accumulated indicating that oxidative stress is a crucial factor in the pathogenesis of neurodegenerative diseases. The major site of production of superoxide, the primary reactive oxygen species (ROS), is considered to be the respiratory chain in the mitochondria, but the exact mechanism and the precise location of the physiologically relevant ROS generation within the respiratory chain have not been disclosed as yet. Studies performed with isolated mitochondria have located ROS generation on complex I and complex III, respectively, depending on the substrates or inhibitors used to fuel or inhibit respiration. A more "physiological" approach is to address ROS generation of in situ mitochondria, which are present in their normal cytosolic environment. Hydrogen peroxide formation in mitochondria in situ in isolated nerve terminals is enhanced when complex I, complex III, or complex IV is inhibited. However, to induce a significant increase in ROS production, complex III and complex IV have to be inhibited by >70%, which raises doubts as to the physiological importance of ROS generation by these complexes. In contrast, complex I inhibition to a small degree is sufficient to enhance ROS generation, indicating that inhibition of complex I by approximately 25-30% observed in postmortem samples of substantia nigra from patients suffering from Parkinson's disease could be important in inducing oxidative stress. Recently, it has been described that a key Krebs cycle enzyme, alpha-ketoglutarate dehydrogenase (alpha-KGDH), is also able to produce ROS. ROS formation by alpha-KGDH is regulated by the NADH/NAD+ ratio, suggesting that this enzyme could substantially contribute to generation of oxidative stress due to inhibition of complex I. As alpha-KGDH is not only a generator but also a target of ROS, it is proposed that alpha-KGDH is a key factor in a vicious cycle by which oxidative stress is induced and promoted in nerve terminals.     

Generation of adenosine tri-phosphate in Leishmania donovani amastigote forms

Leishmania, the causative agent of various forms of leishmaniasis, is the significant cause of morbidity and mortality. Regarding energy metabolism, which is an essential factor for the survival, parasites adapt to the environment under low oxygen tension in the host using metabolic systems which are very different from that of the host mammals. We carried out the study of susceptibilities to different inhibitors of mitochondrial electron transport chain and studies on substrate level phosphorylation in wild-type L. donovani. The amastigote forms of L. donovani are independent on oxidative phosphorylation for ATP production. Indeed, its cell growth was not inhibited by excess oligomycin and dicyclohexylcarbodiimide, which are the most specific inhibitors of the mitochondrial Fo/F1-ATP synthase. In contrast, mitochondrial complex I inhibitor rotenone and complex III inhibitor antimycin A inhibited amastigote cell growth, suggesting the role of complex I and complex III in cell survival. Complex II appeared to have no role in cell survival. To further investigate the site of ATP production, we studied the substrate level phosphorylation, which was involved in the synthesis of ATP. Succinate-pyruvate couple showed the highest substrate level phosphorylation in amastigotes whereas NADH-fumarate and NADH-pyruvate couples failed to produce ATP. In contrast, NADPH-fumarate showed the highest rate of ATP formation in promastigotes. Therefore, we can conclude that substrate level phosphorylation is essential for the survival of amastigote forms of Leishmania donovani.     

Increased thyroxine secretion following administration of dinitrophenol to rats

1. Feeding male rats 0.2% dinitrophenol (DNP) in the diet caused weight loss, a significant fall in the plasma protein bound iodine concentration, and a rise in the thyroxine secretion rate. Thyroxine concentration was raised in the liver and intestinal tract with contents, reduced in the carcass, and unchanged in the pelt.2. Radio-iodine uptake by the thyroid gland was unchanged but there was a significant fall in the thyroid/serum activity ratio.3. Five hours after tracer radiothyroxine, DNP treated rats had a reduced fraction of dose in the plasma, with raised concentrations in the liver and intestines.4. Fractional turnover rates of radiothyroxine in plasma, liver and carcass were raised in DNP treated animals.5. DNP caused a significant increase in the biliary clearance of radiothyroxine, when measured immediately after administration of tracer and 4 hr later.6. It is postulated that DNP displaces thyroxine from its plasma binding proteins and that the displaced hormone is excreted in the bile. This wastage together with increased demand resulting from DNP induced hypermetabolism results in increased output of thyroxine by the thyroid gland.     

24-Hour rhythm in gene expression of nitric oxide synthase and heme-peroxidase in anterior pituitary of ethanol-fed rats

Chronic exposure of rats to ethanol results in significant changes in pituitary hormone secretion. However, identification of the site(s) and mechanism of action of ethanol to induce these effects remains elusive. Free radical damage at the adenohypophyseal level may play a role in the decline in serum gonadotropin levels in ethanol-fed rats. Since 24-h changes in redox state occurred, we analyzed the 24-h changes in pituitary gene expression of the prooxidant enzymes nitric oxide synthase (NOS) 1 and 2, and of heme oxygenase-1 (HO-1) enzyme, and in plasma NO(2)(-) and NO(3)(-) (NO(x)) levels, in ethanol and control rats. Male rats, 35-day-old, received a liquid diet for 4 weeks. The ethanol-fed group received a similar diet to controls except for that maltose was isocalorically replaced by ethanol. Animals were killed at six time intervals during a 24-h cycle. Anterior pituitary mRNA levels encoding NOS1, NOS2 and HO-1 were measured by real-time PCR analysis. Plasma NO(x) concentration was determined by the Griess reaction. Ethanol feeding of prepubertal rats changed significantly the 24-h pattern of expression of NOS1, NOS2 and HO-1 in the adenohypophysis and augmented NOS2 and HO-1 mRNA levels. Peak values for the three enzymes in ethanol-fed rats occurred at the beginning of the scotophase (i.e., at 21:00 h). Ethanol feeding augmented mean values plasma NO(x) levels with a maximum at 13:00 h while in controls a biphasic pattern was observed, with peaks at 09:00 h and 17:00-21:00 h. One of the mechanisms by which ethanol augments oxidative damage in the adenohypophysis may include overproduction of nitric oxide and carbon monoxide.     

The mechanism of cortisone's effect of hyperthermia in rabbits induced by 2,4-dinitrophenol

Summary The paper deals with the study of the mechanism of antipyrogenic action exerted by cortisone in dinitrophenol hyperthermia. The author proceeded from the fact that dinitrophenol hyperthermia is connected with a considerable intensification of the oxidative processes in the tissues, while the activity of the thermoregulating centers is directed towards discharge of heat from the body. If cortisone depresses the condition of these centers, hyperthermia would be more pronounced against the background of its action. However, cortison administration in a dose of 25 mg leads to the reduction of the hyperthermia intensity in rabbits in comparison with controls. Since the action of dinitrophenol is intensified by thyrotoxin, whereas cortisone reduces the production of the latter, experiments were staged on thyroidectomized rabbits. Hyperthermia was intensified in these animals by cortisone administration. Hence the antipyrogenic effect of cortisone tends to remove its depressing effect on the thermoregulating centers.(Presented by Active Member AMN SSSR A. I. Nesterov) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 51, No. 3, pp. 49–52, March, 1961     

A renewed focus on the interplay between viruses and mitochondrial metabolism

Mitochondria fulfil several key functions within cellular metabolic and antiviral signalling pathways, including their central role in ATP generation. Viruses, as intracellular parasites, require from their cellular host the building blocks for generation of their viral progeny and the energy that drives viral replication and assembly. While some viruses have adopted ways to manipulate the infected cell such that cellular metabolism supports optimal virus production, other viruses simply exhaust cellular resources. The association of viruses with mitochondria is influenced by several important factors such as speed of the viral replication cycle and viral dependence on cellular enzymes and metabolites. This review will highlight the complex interconnectivity of viral life cycles with the three main mitochondrial metabolic pathways, namely β-oxidation, the tricarboxylic (TCA) cycle, and oxidative phosphorylation. This interconnectivity has the potential to reveal interesting points for antiviral therapy with either prometabolites or antimetabolites and highlights the importance of the viral association with mitochondrial metabolism.     

Reduced intestinal epithelial mitochondrial function enhances in vitro interleukin-8 production in response to commensal <Emphasis Type="Italic">Escherichia coli</Emphasis>

Uncoupling of oxidative phosphorylation in epithelial mitochondria results in decreased epithelial barrier function as characterized by increased internalization of non-invasive Escherichia coli and their translocation across the epithelium. We hypothesized that the increased burden of intracellular commensal bacteria would activate the enterocyte, with the potential to promote inflammation. Treatment of human colon-derived epithelial cell lines in vitro with dinitrophenol (DNP) and commensal E. coli (strains F18, HB101) provoked increased production of interleukin (IL-8), which was not observed with conditioned medium from the bacteria, lipopolysaccharide or inert beads. The IL-8 response was inhibited by co-treatment with cytochalasin-D (blocks F-actin rearrangement), chloroquine (blocks phagosome acidification) and a MyD88 inhibitor (blocks TLR signaling), consistent with TLR-signaling mediating IL-8 synthesis subsequent to bacterial internalization. Use of the mitochondria-targeted antioxidant, mitoTEMPO, or U0126 to block ERK1/2 MAPK signalling inhibited DNP+E. coli-evoked IL-8 production. Mutations in the NOD2 (the intracellular sensor of bacteria) or ATG16L1 (autophagy protein) genes are susceptibility traits for Crohn’s, and epithelia lacking either protein displayed enhanced IL-8 production in comparison to wild-type cells when exposed to DNP?+?E coli. Thus, metabolic stress perturbs the normal epithelial–bacterial interaction resulting in increased IL-8 production due to uptake of bacteria into the enterocyte: this potentially pro-inflammatory event is enhanced in cells lacking NOD2 or ATG16L1 that favor increased survival of bacteria within the enterocyte. We speculate that by increasing epithelial permeability and IL-8 production, reduced mitochondria function in the enteric epithelium would contribute to the initiation, pathophysiology, and reactivation of inflammatory disease in the gut.     

Glutathione recycling is attenuated by acute ethanol feeding in rat liver.

The mechanism for ethanol-induced oxidative stress has been disputed because of the controversies on modulation of radical generating and scavenging activities by ethanol. In the present work, we attempted to clarify the acute effect of ethanol on the radical generating system as well as the radical scavenging system. For that purpose, chow-fed rats were given ethanol (5 g/kg) or isocaloric glucose solution by intragastric intubation and placed at 32 degrees C for 6 hr. Acute ethanol administration enhanced the expression of cytochrome P450 II E1(CYP II E1) in the liver and attenuated the activities of hepatic glutathione peroxidase (GPx) and reductase (GR). It also caused a significant increase in the level of hepatic thiobarbituric acid reactive substances (TBARS), an indicator of lipid peroxidation. On the other hand, acute ethanol feeding had no effect on the activities of catalase, xanthine oxidase (XO), glutathione transferase (GST) and glucose-6-phosphate dehydrogenase (G6PDH). From this result, it is suggested that acute ethanol administration causes the oxidative tissue damage by CYP II E1-associated radical generation and the decreased radical scavenging function due to the reduced activities of hepatic glutathione recycling system such as GPx and GR.     

Mechanoenergetic estimation of multiple cross-bridge steps per ATP in a beating heart

The efficiency from the ventricular O(2) consumption (VO(2)) to the total mechanical energy (TME) generated by ventricular contraction has proved relatively constant at approximately 35%, independent of the loading and contractile conditions in a canine heart. TME is the sum of the external mechanical work for ejecting a stroke volume against the afterload and of the mechanical potential energy for developing ventricular pressure in each beat. The approximately 35% VO(2)-to-TME efficiency indicates an also constant approximately 60% ATP-to-TME efficiency in a beating heart, based on the nominal approximately 60% VO(2)-to-ATP efficiency in the myocardial oxidative phosphorylation. I newly attempted to explain the load-independent approximately 60% ATP-to-TME efficiency by the recently reported approximately 7-10 nm unitary step size and approximately 0.8-1.5 pN unitary force of a cross-bridge (CB) at the molecular level in in vitro motility assays. This single CB behavior suggests that its unitary cycle could generate a mechanical energy of approximately 0.6-1.5x10(-20) J at most. From the nominal free energy of approximately 10x10(-20) J per ATP, the efficiency from one ATP to the CB unitary cycle would then be approximately 6-15%. This low efficiency is only approximately 1/10-1/4 of the approximately 60% ATP-to-TME efficiency at the heart level. This discrepancy suggests that each CB would repeat the unitary cycle at least approximately 4-10 times per ATP to achieve the high constant ATP-to-TME efficiency in a beating heart. This seems to represent a considerable mechanoenergetic advantage of the heart at the integrative heart level as compared to the molecular CB level.     

Actions of dinitrophenol and some other metabolic inhibitors on cortical neurones

1. In cats under methoxyflurane, DNP and other metabolic inhibitors were tested on cortical neurones by iontophoresis from micropipettes.2. DNP, dinitro-o-cresol, iodoacetate, pentachlorophenol and oligomycin (uncouplers or inhibitors of oxidative phosphorylation), as well as moderate anoxia, blocked selectively and reversibly spontaneous firing and discharges evoked by ACh; responses evoked by glutamate were facilitated by moderate doses of DNP and blocked only by large amounts.3. Azide, cyanide, ouabain and strophanthidine had a mainly excitatory effect; the cardiac glycosides tended to depress more strongly responses to glutamate.4. Intracellular observations showed that DNP causes a sharp fall in electrical excitability, associated with a hyperpolarization and fall in membrane resistance.5. The hyperpolarizing action of DNP had a mean reversal level (E(DNP)) nearly 30 mV more negative than the resting potential; E(DNP) was identical with the mean reversal level for the depolarizing action of ACh, measured on the same cells.6. DNP had its usual hyperpolarizing effect on neurones whose IPSPs had been made positive by raising the internal [Cl]; the mean E(IPSP) was over 30 mV more positive than E(DNP).7. It is concluded that DNP lowers excitability by raising the membrane conductance to K(+) (g(K)) and that it blocks ACh responses selectively because ACh has a precisely opposite action on these neurones.8. In the Discussion, it is suggested that the rise in g(K) is mediated by an increase in internal free Ca(2+), caused by a slowing of mitochondrial activity, and that a similar mechanism may play a significant role in general anaesthesia.     

Glucose transporter protein 1 expression in mucoepidermoid carcinoma of salivary gland: correlation with grade of malignancy

Mucoepidermoid carcinoma (MEC), the most common primary salivary malignancy, shows great variability in clinical behaviour, thus demanding investigation to identify of prognostic markers. Since Warburg’s studies, unrestricted cell growth during tumorigenesis has been linked to altered metabolism, implying hypoxic stimulation of glycolysis and diminished contribution of mitochondrial oxidative phosphorylation to cellular ATP supply. Hypothesizing that the study of MEC metabolic status could lead to the discovery of prognostic markers, we investigated by immunohistochemistry the expression of glucose transporter 1 (Glut‐1), mitochondrial antigen and peroxiredoxin I (Prx I) in samples of MEC from different histological grades. Our results showed that mitochondrial antigen and Prx I were expressed in the majority of the MEC cases independent of the histological grade. In contrast Glut‐1 expression increased significantly as the tumours became more aggressive. These results suggested that oxidative phosphorylation may contribute to ATP supply in all stages of MEC progression, and that the relative contribution of glycolysis over mitochondria for cellular ATP supply increases during MEC progression, favouring growth under low oxygen concentration. In addition, the observed high Prx I protein levels could provide protection to tumour cells against reactive oxygen species generated as a consequence of mitochondrial function and hypoxia‐reoxygenation cycling. Altogether our findings suggest that upregulation of Glut‐1 and Prx I constitute successful adaptive strategies of MEC cells conferring a growth advantage over normal salivary gland cells in the unstable oxygenation tumour environment.     

Influence of hyposensitization of ATP level and CO2 production of mast cells in anaphylaxis.

Anaphylaxis in a glucose-free medium containing pyruvate caused a release of histamine and a significant decrease in the ATP level of rat mast cells. The fall was maximal after 10 min and it was found to reverse after 22 min. Glucose completely counteracted the ATP fall without changing the anaphylactic histamine release. Furthermore, the oxidative metabolism of exogenous pyruvate to CO2 was stimulated in the mast cell. A high level of protection of mast cells to antigen challenge was obtained following hyposensitization and only a small amount of the intracellular histamine was released in contrast to non-hyposensitized cells. Hyposensitization counteracted the ATP fall by antigen challenge but the increase in oxidative metabolism remained unchanged. The results indicate that hyposensitization exerts effects in the mast cell consistent with a reduced ATP utilization or with a reduced uncoupling of oxidative phosphorylation. The mechanism of the hyposensitization must be due to inhibition of one or more of the cellular steps leading to histamine release and subsequent morphological changes of the cell or to uncoupling of oxidative phosphorylation.