Energy Charge Potential Research Articles

Analysis of changes in energy and redox states in HepG2 hepatoma and C6 glioma cells upon exposure to cadmium

The energy and redox states of the HepG2 hepatoma and the C6 glioma cells were studied by quantifying the levels of ATP, ADP, AMP, GSH, and GSSG. These values were used to calculate the energy charge potential (ECP = [ATP + 0.5ADP]/TAN), total adenosine nucleotides (TAN = ATP + ADP + AMP), total glutathione (TG = [GSH + GSSG]/TAN), and the redox state (GSH/GSSG ratio). For comparison between cell types, the level of each energy metabolite (ATP, ADP, and AMP) was normalized against TAN of the respective cell. The results showed that ATP:ADP:AMP ratio was 0.76:0.11:0.13 for the HepG2 cells and 0.80:0.11:0.09 for the C6 glioma cells. ECP was 0.81 +/- 0.01 and 0.85 +/- 0.01 for the HepG2 and the C6 glioma cells, respectively. GSH/GSSG ratio was 2.66 +/- 0.16 and 3.63 +/- 0.48 for HepG2 and C6 glioma cells, respectively. TG was 3.2 +/- 0.54 for the HepG2 cells and 2.43 +/- 0.18 for the C6 glioma cells, indicating that the level of total glutathione is more than two to three times higher than the total energy metabolites in these cell lines. Following a 3-h incubation in medium containing different concentrations of Cd, there was a dose-dependent decrease in cell viability. The 3-h LC50 for the HepG2 cells was 0.5 mM and that for the C6 glioma cells was 0.4 mM. Cellular TAN decreased with a decrease in cell viability. Upon careful analysis of the energy state, there was a significant increase in relative amount of ATP and decrease in ADP and AMP in both cells as Cd concentration increased from 0 to 0.1, 0.2, and 0.6 mM. However, ECP in both cell lines increased, which indicated that the level of high energy phosphate was adequate. There was also a significant increase in TG and a significant decrease in GSH/GSSG in the C6 glioma cells when cells were exposed to as low as 0.1 mM Cd, which suggested that the cellular redox state was compromised. The HepG2 cells, on the other hand, showed no significant change in both TG and GSH/GSSG level until Cd concentration reached 0.6 mM. Results of the present study also showed that there were differences between the two cells in response to the same level of Cd exposure. The C6 glioma cells were more sensitive to Cd-induced injuries. Although there was a decrease in total amount of high energy phosphate as cell viability decreased, the surviving cells were not devoid of high energy phosphates. The relative abundance of ATP amongst the adenosine nucleotide pool and the increase in ECP could be interpreted as a way the cells signal the conservation of energy utilization in response to the damaged mitochondrial function. This move for energy conservation might be the cause of eventual cell death through the process of apoptosis.

Cardiac Mechanical Dysfunction Induced by Ischemia‐reperfusion in Perfused Heart Isolated from Stroke‐prone Spontaneously Hypertensive Rats

We investigated the difference in mechanical function after ischemia and reperfusion between Wistar‐Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) or stroke‐prone SHR (SHRSP) using the isolated working heart model, in order to examine postischemic mechanical dysfunction in the severely hypertrophied heart. Systolic blood pressure of SHRSP was higher than that of SHR and WKY, and the left ventricular wall in SHRSP was thicker than in WKY. Mechanical dysfunction of the heart during reperfusion following ischemia (11 min) in SHRSP was severer than that in SHR and WKY, and recovery of the cardiac energy charge potential (ECP) level in SHRSP was lower than that in SHR and WKY. Twenty‐five, 12 and 11 min‐ischemia in WKY, SHR and SHRSP, respectively, caused a similar level of cardiac mechanical damage. Also, the ECP levels were almost equivalent among them at the end of 20 min reperfusion following each time of ischemia. Under each ischemic condition, a Ca2 +‐channel blocker, diltiazem, and an adenosine potentiator, dilazep, produced a beneficial effect on the post‐ischemic dysfunction in SHR and WKY. However, neither cardioprotective drug led to recovery of the mechanical dysfunction of the heart during reperfusion following ischemia in SHRSP. Thus, the severely hypertrophied heart such as that in SHRSP was more susceptible to cardiac reperfusion dysfunction, than the moderately hypertrophied heart such as that in SHR. These results suggest that the cardioprotective effects of drugs may be deteriorated in severe hypertrophied hearts.

Determination of adenosine nucleotides in cultured cells by ion-pairing liquid chromatography–electrospray ionization mass spectrometry

A method using ion-pairing liquid chromatography–mass spectrometry (MS) was developed for analyzing adenosine 5 ′-monophosphate (AMP), adenosine 5 ′-diphosphate (ADP), and adenosine 5 ′-triphosphate (ATP) in cellular extracts. Dimethylhexylamine (DMHA) was used as ion-pairing agent to retain and separate the analytes on a reversed-phase microbore column with a gradient program. Positive-ion electrospray ionization–MS was applied for the detection because of the use of the ion-pairing agent. Adduct ions of DMHA with AMP, ADP, and ATP were found to be the most intensive peaks and thus selected as quantitative ions. An external calibration method with linear ranges from 0.1 to 20 μM for AMP, 2 to 20 μM for ADP, and 2.5 to 20 μM for ATP was used for the quantitation. The method was applied to determine concentrations of AMP, ADP, and ATP in extracts of cultured rat C6 glioma cells that were pretreated with various concentrations of Zn. The detected levels of the adenosine nucleotides have been used to calculate total adenosine nucleotide and energy charge potential. Changes in cellular energy status upon exposure to increasing concentration of Zn in the culture medium were analyzed. The results indicated that the addition of Zn in a range of 40 to 120 μg/ml cause a gradual increased in energy charge potential of the cells.

Determination of Energy Charge Potential in the C6 Glioma and the HepG-2 Cell Culture

The energy charge potential (ECP) is the ratio among adenosine 5'-triphosphate (ATP), -diphosphate (ADP), and -monophosphate (AMP); it reflects the high-energy-bond content in biological systems. Under normal physiological conditions, the value of ECP is between 0.7 and 1.0. In this study, two different methods were used to extract adenosine nucleotides (ATP, ADP, and AMP) for determination of the ECP in the C6 glioma and the HepG-2 human hepatoma cell lines. In the first method, cells were removed from culture plates by trypsinization before the nucleotides were extracted using perchloric acid. Some of the trypsinized cells were used for a cell count, thereby allowing the calculation of an average nucleotide level per cell. In the second method, perchloric acid was added directly to the culture plate immediately after the medium was removed. This method resulted in an instant termination of any enzymatic process that might degrade the nucleotides and alter their levels in cells. Adenosine nucleotides were determined by high-performance liquid chromatography. The direct extraction method yielded a total adenosine nucleotide (TAN) level more than twofold higher than that obtained following trypsinization. For the C6 glioma cells, the ratio of ATP to ADP to AMP in the TAN pool was 0.70:0.15:0.15, as compared to 0.36:0.27:0.37 when cells were extracted following trypsinization. Similarly, for the HepG-2 cells, the ratio was 0.62:0.15:0.23 following direct extraction, as compared to 0.15:0.28:0.57 following trypsinization. Using the results from the direct extraction method, the value of the cellular ECP was 0.78 ± 0.06 for the C6 glioma cells and 0.70 ± 0.06 for the HepG-2 cells. Compared to the ECP values obtained using the trypsinization method (0.50 ± 0.05 and 0.29 ± 0.12, respectively), the ECP values derived using the direct extraction method were significantly greater in both cell lines and were suggestive of the energy status of normal, healthy cells.

Modulation of Energy Metabolism in C6 Glioma Cells as Possible Mechanism Contributing to Zinc Neurotoxicity

Elevated tissue levels of zinc (Zn) have been associated with neurodegenerative diseases such as global ischemia, seizure, and Alzheimer's. The mechanism of action of Zn in causing neuronal injury is not clear. One of the possible mechanisms is the ability of Zn to alter cellular energy metabolism. Using the C6 glioma cell as a model, the present study aimed to determined the effects of increasing concentrations of Zn on cellular energy states, as defined by the levels of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine 5'-monophosphate (AMP), the total adenosine nucleotides (TAN) (TAN = ATP + ADP + AMP), and the energy charge potential (ECP = [ATP + 0.5 ADP]/TAN). Uptake of Zn was visualized by the appearance of N-(6-methoxy-8-quinolyl)-p-toluene sulfonamide (TSQ)-stained fluorescent granules after a 3-h exposure to Zn in the medium. At [Zn] = 1 mM, cells appeared apoptotic. Levels of ATP and TAN decreased as the level of Zn increased. The change mirrors the increase in cell death as determined by the trypan blue exclusion test. However, when the ratio of ATP:ADP:AMP within the TAN was calculated, the percentage of ATP in the TAN increased significantly, while that of AMP decreased. The change in the relative AMP level mirrored the change in cell viability as measured by the MTT assay, which indicated a decreased in mitochondrial activity. Cellular ECP increased significantly from 0.85 ± 0.007 to 0.92 ± 0.04. The elevated ECP and relative ATP level, together with a significant decrease in the relative AMP level, are all indicators of inhibition of cellular metabolism. These results support the notion that acute exposure of C6 glioma cells to a high concentration of Zn might initially result in a decrease in relative AMP and an inhibition of mitochondrial activity. However, the ultimate toxic action of Zn on the C6 glioma cells appears to be due to a gradual inhibition of energy utilization, leading to cell shrinkage and apoptosis.

Enteral glutamine administration prevents the decrease in cell energy charge potential produced in ileum after a skin burn in the rat

Enteral glutamine administration prevents the decrease in cell energy charge potential produced in ileum after a skin burn in the rat

Enteral Glutamine Administration Prevents the Decrease in Cell Energy Charge Potential Produced in Ileum After a Skin Burn in the Rat

A skin burn produces a decrease in cell energy charge potential (ECP), a marker of cell function, in the liver and other organs. The process appears to be oxidant induced because reduced glutathione (GSH) appears to be protective. The gut mucosal barrier is also known to be altered after burns and appears to be improved by glutamine, which is a source of energy and GSH production. Neither the relationship between the gut barrier change and the ECP of the gut barrier nor the mechanism of glutamine protection has been defined. The effect of a 20% total body surface area, full-thickness burn in a rat on ECP and GSH content in the ileum and the liver and the effect of oral glutamine (1 g/kg/d) on any changes were studied. Three groups of rats (14 rats per group) were studied: a control group, a group with burns alone, and a group with burns that were given glutamine. Half of the rats were killed 1 day after the burn, and the other half were killed 6 days after the burn. ECP did not decrease 1 day after the burn, but 6 days after the burn, ECP decreased from a control of 0.53+/-0.06 to 0.41+/-0.01 in the liver and from 0.64+/-0.04 to 0.29+/-0.04 in the ileum. In the ileum, glutamine prevented the decrease of the ECP (which increased to 0.58+/-.03), but in the liver, glutamine did not alter the ECP (which remained at 0.42+/-0.05). The GSH content was unchanged after the burn injury in both organs compared with the value before the burn injury. It can be concluded that a delayed decrease in ECP in the ileum occurs after a burn injury. Orally administered glutamine prevents the decrease in ECP, probably by providing cell energy rather than by increasing gut GSH (antioxidant) activity.

Substrates for endogenous metabolism by mature boar spermatozoa

Washed boar spermatozoa incubated in the absence of exogenous substrates maintained a high energy charge potential (ECP) for at least 10 h. Addition of bromopyruvate, an inhibitor of stage 2 of the glycolytic pathway, at any time during the incubation caused an immediate decrease in the ECP, indicating that the mobilization of endogenous compounds requires this section of the pathway for the production of lactate, the major mitochondrial substrate for ATP production. Some of the sources of the metabolic substrates have been identified, by NMR and metabolic studies, as di- or triglycerides, to produce glycerol, and membrane phospholipids for the production of glycerol 3-phosphate. Acetylcarnitine contributes acetyl groups early in the incubation; glycerylphosphorylcholine is degraded to glycerol 3-phosphate and choline after about 5 h, and acetate also accumulates after about 5 h. The presence of phosphorylcholine and phosphorylethanolamine later in the incubation indicates that phospholipids are also degraded to glycerol.

Substrates for endogenous metabolism by mature boar spermatozoa

Washed boar spermatozoa incubated in the absence of exogenous substrates maintained a high energy charge potential (ECP) for at least 10 h. Addition of bromopyruvate, an inhibitor of stage 2 of the glycolytic pathway, at any time during the incubation caused an immediate decrease in the ECP, indicating that the mobilization of endogenous compounds requires this section of the pathway for the production of lactate, the major mitochondrial substrate for ATP production. Some of the sources of the metabolic substrates have been identified, by NMR and metabolic studies, as di- or triglycerides, to produce glycerol, and membrane phospholipids for the production of glycerol 3-phosphate. Acetylcarnitine contributes acetyl groups early in the incubation; glycerylphosphorylcholine is degraded to glycerol 3-phosphate and choline after about 5 h, and acetate also accumulates after about 5 h. The presence of phosphorylcholine and phosphorylethanolamine later in the incubation indicates that phospholipids are also degraded to glycerol.

Differential effects of acute morphine administrations on polymorphonuclear cell metabolism in various mouse strains

This paper shows that an acute morphine treatment dose-depently alters the energetic and oxidative metabolism of polymorphonuclear leukocytes obtained from BALB/c and DBA/2 mice, while phagocytic cells from C57BL/6 were not affected. In sensitive mouse strains, i.e. BALB/c and DBA/2, morphine decreased both ATP concentration and energy charge potential. At the same time, ATP catabolic products, i.e. nucleosides (inosine+adenosine) and oxypurines (hypoxanthine+xanthine+uric acid), significantly increased, indicating an imbalance between energy production and consumption. Morphine treatment also induced malondialdehyde and Superoxide anions production in leukocyte cells from sensitive mice. The opiate antagonist naloxone blocked morphine-induced modifications by the lower morphine dose. The same parameters in cells from C57BL/6 mice were not affected. These findings confirm that: i) the phagocytic cells are an important target for the in vivo effects of morphine, and ii) the genotype-dependent variation influences the immunological responsiveness to opiates.

Catecholamines Potentiate Amyloid β-Peptide Neurotoxicity: Involvement of Oxidative Stress, Mitochondrial Dysfunction, and Perturbed Calcium Homeostasis

Oxidative stress and mitochondrial dysfunction are implicated in the neuronal cell death that occurs in physiological settings and in neurodegenerative disorders. In Alzheimer's disease (AD) degenerating neurons are associated with deposits of amyloid β-peptide (Aβ), and there is evidence for increased membrane lipid peroxidation and protein oxidation in the degenerating neurons. Cell culture studies have shown that Aβ can disrupt calcium homeostasis and induce apoptosis in neurons by a mechanism involving oxidative stress. We now report that catecholamines (norepinephrine, epinephrine, and dopamine) increase the vulnerability of cultured hippocampal neurons to Aβ toxicity. The catecholamines were effective in potentiating Aβ toxicity at concentrations of 10–200 μM, with the higher concentrations (100–200 μM) themselves inducing cell death. Serotonin and acetylcholine were not neurotoxic and did not modify Aβ toxicity. Levels of membrane lipid peroxidation, and cytoplasmic and mitochondrial reactive oxygen species, were increased following exposure to neurons to Aβ, and catecholamines exacerbated the oxidative stress. Subtoxic concentrations of catecholamines exacerbated decreases in mitochondrial energy charge and transmembrane potential caused by Aβ, and higher concentrations of catecholamines alone induced mitochondrial dysfunction. Antioxidants (vitamin E, glutathione, and propyl gallate) protected neurons against the damaging effects of Aβ and catecholamines, whereas the β-adrenergic receptor antagonist propanolol and the dopamine (D1) receptor antagonist SCH23390 were ineffective. Measurements of intracellular free Ca2+([Ca2+]i) showed that Aβ induced a slow elevation of [Ca2+]iwhich was greatly enhanced in cultures cotreated with catecholamines. Collectively, these data indicate a role for catecholamines in exacerbating Aβ-mediated neuronal degeneration in AD and, when taken together with previous findings, suggest roles for oxidative stress induced by catecholamines in several different neurodegenerative conditions.

Endischemic oxygen persufflation to improve viability of marginally preserved donor livers.

The quality of cold-stored donor livers slowly declines beyond approximately 12 h, although these organs may still be used for clinical transplantation. The aim of the present study was to improve the energetic status and viability of long-term-preserved livers by short-term gaseous oxygen insufflation prior to implantation of the organ using a technique that has already been shown to promote aerobic energy metabolism during hypothermia. Livers from ten male Wistar rats were isolated, rinsed blood-free. Five livers (group 1) were stored for 48 h at 4 degrees C in UW preservation solution, and five livers (group 2) were isolated and stored in the same manner for 47 h, and were then, during the last 60 min of the preservation period, connected to a persufflation device and gaseous oxygen was introduced into the organ via the inferior caval vein, with the liver still immersed in cold UW solution. This technique of endischemic gaseous oxygenation resulted in a significant normalization of vascular resistance upon isolated reperfusion in vitro and a reduction in hepatic efflux of alanine aminotransferase as well as glutamate dehydrogenase, which led to improved recovery of the reperfused grafts of group 2 as evidenced by an elevated energy charge potential at the end of the reperfusion period. In conclusion, the technique described seemed effective in enhancing the preoperative viability of marginal donor grafts.

Metabolite concentrations in skeletal muscle of different aged rats submitted to hypoxia and pharmacological treatment with nicergoline.

The energy metabolism of the gastrocnemius and soleus muscles in young-adult, mature, and senescent rats was evaluated after 72 h of continuous exposure to normobaric hypoxia or normoxia. The effects of treatment with the alpha-adrenergic antagonist nicergoline were also investigated. In the gastrocnemius muscle we evaluated the concentrations of some significative metabolites involved in anaerobic glycolysis and the Krebs' cycle, free amino acids related to the Krebs' cycle, ammonia, some energy mediators, and the energy store creatine phosphate. In the soleus muscle a selection of these was evaluated. In both muscles aging was similarly characterized by a decrease in muscular creatine phosphate concentration, while the energy mediators and the energy charge potential remained unchanged. Singly, some gastrocnemius muscle metabolites showed linear changes in their concentrations with aging, while for the soleus muscle the only linear change regarded glucose-6-phosphate. Continuous normobaric hypoxia induced greater changes at the age of 4 and 24 months than at 12 months. Chronic treatment with nicergoline modified the influence of hypoxic conditions on muscle metabolites concentrations only in some cases, regardless of the age of the animals. Further investigations are necessary before any firm conclusions can be drawn about the pharmacological activity of nicergoline on hypoxia in aged rats.

Endischemic oxygen persufflation to improve viability of marginally preserved donor livers

Abstract The quality of colD-stored donor livers slowly declines beyond approximately 12 h, although these organs may still be used for clinical transplantation. The aim of the present study was to improve the energetic status and viability of long-term-preserved livers by short-term gaseous oxygen insufflation prior to implantation of the organ using a technique that has already been shown to promote aerobic energy metabolism during hypothermia. Livers from ten male Wistar rats were isolated, rinsed blooD-free. Five livers (group 1) were stored for 48 h at 4°C in UW preservation solution, and five livers (group 2) were isolated and stored in the same manner for 47 h, and were then, during the last 60 min of the preservation period, connected to a persufflation device and gaseous oxygen was introduced into the organ via the inferior caval vein, with the liver still immersed in cold UW solution. This technique of endischemic gaseous oxygenation resulted in a significant normalization of vascular resistance upon isolated reperfusion in vitro and a reduction in hepatic efflux of alanine aminotransferase as well as glutamate dehydrogenase, which led to improved recovery of the reperfused grafts of group 2 as evidenced by an elevated energy charge potential at the end of the reperfusion period. In conclusion, the technique described seemed effective in enhancing the preoperative viability of marginal donor grafts.

Energy charge potential and glutathione levels as predictors of outcome following burn injury complicated by endotoxemia.

We studied the effect of water-soluble antioxidants geared at restoring glutathione levels on oxygen consumption, cell energetics as measured by energy charge potential (ECP), glutathione levels, and mortality, in response to a 20% total body surface area (TBSA) third degree burn injury combined with endotoxemia, five days after burn in a rat model. The 20% TBSA third degree burn injury was not fatal for the six day study period. Oxygen consumption as well as red blood cell ECP remained unchanged from control values. Liver ECP was significantly reduced; however, liver glutathione was significantly increased. The 20% TBSA burn injury combined with endotoxemia produced a 60% mortality rate. Twenty-four hour survivors (40%) demonstrated a significant decrease in oxygen consumption, red blood cell ECP, and liver ECP. Liver glutathione was significantly decreased compared with burn but was not significantly decreased compared with control. Nonsurvivors of the burn injury combined with endotoxin (60%) demonstrated a significant reduction in liver glutathione levels compared with survivors. Oxygen consumption and ECP could not be measured in the nonsurvivors due to the rapid loss of ATP in the moribund state that occurred by 4 h postinjury. Antioxidants produced 100% survival, attenuated in the fall in liver ECP, and restored red blood cell ECP and liver glutathione levels to normal values. We conclude that a modest burn injury combined with endotoxemia produces a liver glutathione debt, oxygen debt, an energy deficit, and 60% mortality. The mechanism of injury is oxidant related as antioxidants prevented mortality restored liver glutathione levels, and prevented or attenuated the decrease in ECP. A decrease in ECP and glutathione levels appear to be more sensitive indicators of outcome than the presence of an oxygen debt. The survivors, in both burn plus endotoxin groups treated with or without antioxidants was comparable, indicating a critical value for oxygen consumption exists before death occurs.

Studies of repeated administration of FK-506 on myocardial metabolism in rats.

The effect of a 14-day administration of FK-506 at 1 or 3 mg/kg on plasma lipids and myocardial energy and glucose metabolism in rats was investigated. FK-506 increased the level of blood glucose in rats when given daily at an oral dosage of 3 mg/kg for 14 days. The plasma level of total cholesterol was significantly increased by FK-506 at either 1 or 3 mg/kg, whereas that of triglyceride was not changed. There was no significant difference in energy charge potential and the lactate/pyruvate ratio of the myocardial tissue between FK-506- and solvent-treated groups. Because FK-506 administration for 14 consecutive days did not disturb myocardial energy and glucose metabolism, its cardiotoxic side effects were not recognized in the present experimental toxicological evaluation of rats.

Effects of increasing times of incomplete cerebral ischemia upon the energy state and lipid peroxidation in the rat.

Different times of incomplete cerebral ischemia (2, 4, 6, 8, 10 and 30 min) were induced by bilateral common carotid artery occlusion in anesthetized rats to evaluate the time course of changes in lipid peroxidation and energy metabolism. Analysis of malondialdehyde (used to assess the levels of lipid peroxidation), ascorbic acid, oxypurines, nucleosides, nicotinic coenzymes and high-energy phosphates, was carried out by high-performance liquid chromatography on neutralized perchloric acid extract of brain tissue. Under the present experimental conditions, malondialdehyde, nicotinic coenzymes and ATP catabolites (oxypurines and nucleosides) were affected by increasing times of ischemia, with respect to control sham-operated rats. In particular, the concentration of malondialdehyde, undetectable in control brains, increased from 1.26 nmol/g wet weight after 2 min of carotid clamping to 13.42 nmol/g wet weight at the end of 30 min of incomplete cerebral ischemia. The presence of oxidative stress was further supported by ascorbic acid depletion, which was particularly significant after 10 and 30 min of incomplete ischemia. Carotid clamping provoked an imbalance between energy production and consumption that was evidenced by a reduction in ATP and GTP concentrations and an increase in ATP degradation products such as AMP, oxypurines and nucleosides. A decrement in the sum of adenine nucleotides and the energy charge potential indicated a progressive malfunctioning of energy-producing metabolic cycles. A possible contribution to such a severe change in energy state might be related to depletion of NAD and NADP, particularly noticeable after the longest incomplete brain ischemia times, that should have provoked a consequent lessening of oxido-reductive reactions. Bilateral carotid clamping causes a significant reduction in brain oxygen and substrate supply that results in inhibition of energy metabolism and triggering of oxygen-radical-induced lipid peroxidation.

Endogenous energy production by mature boar spermatozoa.

When incubated in the absence of exogenous substrates, washed boar spermatozoa maintained a high energy charge potential (ECP) for at least 5 h. Addition of 3-chloro-1-hydroxypropanone, an inhibitor of triosephosphate isomerase and glyceraldehyde 3-phosphate dehydrogenase, at any time caused the ECP to decline and fructose-1,6-bisphosphate, dihydroxyacetone phosphate and glycerol to accumulate. There appear to be two endogenous substrates that are degraded ultimately to produce the triosephosphates which allow the cells to produce lactate for the mitochondrial synthesis of ATP. One substrate generates minor amounts of glycerol 3-phosphate whereas the other substrate degrades to glycerol and may be di-glycerides, or tri-glycerides, or both.

Effector mechanism of adenosine in acute ischemic preconditioning of skeletal muscle against infarction.

We used adenosine A1 receptor agonist N6-1(phenyl-2R-isopropyl)-adenosine (PIA), A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and ATP-sensitive K+ (KATP) channel blockers sodium 5-hydroxydecanoate (5-HD) and glibenclamide (Glib), as probes to investigate the role and mechanism of adenosine in ischemic preconditioning (IPC) of noncontractile skeletal muscle against infarction, using the pig latissimus dorsi muscle flap model. Except for Glib, all drugs were delivered to each muscle flap by 10-min local intra-arterial infusion to avoid systemic effects. Muscle flaps that were subjected to 4 h of global ischemia and 48 h of reperfusion sustained 40 +/- 2% infarction. IPC with three cycles of 10 min ischemia and reperfusion, preischemic adenosine, or PIA treatment reduced (P < 0.05) muscle infarction to 24 +/- 2, 18 +/- 2, and 24 +/- 2%, respectively. The anti-infarction effect of IPC and adenosine was blocked by DPCPX, 5-HD, and Glib (P < 0.05). Preischemic adenosine treatment also maintained higher muscle contents of phosphocreatine, ATP, and energy charge potential and lower muscle contents of dephosphorylated metabolites and lactate during ischemia and a lower muscle myeloperoxidase (MPO) activity during reperfusion compared with the control (P < 0.05). Preischemic adenosine treatment did not increase muscle content of adenosine during ischemia or reperfusion. Furthermore, adenosine given at the onset of reperfusion was not effective in attenuating muscle MPO activity or infarction. Taken together, these observations indicate that adenosine, through A1 receptors, initiates the mechanism of IPC with postreceptor involvement of KATP channels in skeletal muscle. However, adenosine is unlikely to play a key role in the effector mechanism. Presently, the cause and role of energy sparing and neutrophil inhibitory effects associated with the anti-infarction effect of preischemic adenosine treatment are unknown.

Role of ATP-sensitive K+ channels in ischemic preconditioning of skeletal muscle against infarction.

We studied the role and mechanism of ATP-sensitive K+ (KATP) channels in ischemic preconditioning (IPC) of skeletal muscle against infarction in vivo. Surgically denervated, noncontractile latissimus dorsi muscle flaps in pentobarbitone-anesthetized pigs were assigned to nine groups: control; IPC (3 cycles of 10-min ischemia/reperfusion); preischemic lemakalim (LMK, 0.18 mg/muscle); postischemic LMK; sodium 5-hydroxydecanoate (5-HD, 27 mg/muscle) before IPC; glibenclamide (Glib 0.3 mg/kg iv) before IPC; 5-HD before preischemic LMK; 5-HD before ischemia; and Glib before ischemia. Except for Glib, all drugs were delivered to each muscle by 10-min local intraarterial infusion to avoid systemic effects. All muscle flaps underwent 4 h of global ischemia. Infarction was assessed at 48 h of reperfusion. In a separate study, muscle biopsies were taken before, during, and after ischemia for assay of high-energy phosphate and lactate contents and myeloperoxidase (MPO) activity. It was observed that muscle infarction in the IPC (24 +/- 2%) and preischemic LMK (21 +/- 2%) groups were smaller (P < 0.05) than that in the control (42 +/- 2%). The anti-infarction effect of IPC and LMK was blocked by 5-HD or Glib. IPC and preischemic LMK caused a higher (P < 0.05) muscle content of ATP and energy charge potential, a lower (P < 0.05) muscle content of lactate during ischemia, and a lower (P < 0.05) muscle MPO activity throughout 16 h of reperfusion compared with the control. These observations indicated for the first time that KATP channels are also involved in the anti-infarction effect of IPC in noncontractile skeletal muscle in vivo. Presently, the cause and importance of energy-sparing and neutrophil-inhibitory effects of IPC and LMK are not known.