Krebs-Henseleit Bicarbonate Research Articles

Effect of Trolox C on hypoxia/reoxygenation-induced injury in isolated perfused rat liver

Livers isolated from 18 hours fasted rats were subjected to N(2) hypoxia (for 45 min) followed by reoxygenation (for 45 min). The perfusion medium used was Krebs-Henseleit bicarbonate buffer (KHBB, pH 7.4). Lactate and alanine were added as gluconeogenic and ureagenic substrates and Trolox C was also added to perfusate. Oxygen consumption, lactate dehydrogenase (LDH), alanine transaminase (ALT), total glutathione, oxidized glutathione, bile flow, glucose and urea were measured. After hypoxia oxygen consumption significantly dropped but Trolox C had no influence on this decrease. ALT and LDH were significantly increased by hypoxia/reoxygenation. This increase was markedly attenuated in the presence of Trolox C. The total glutathione and oxidized glutathione efflux increased following hypoxia, which were prevented by the treatment of Trolox C. Bile flow rate decreased following hypoxia/reoxygenation but did not continue to decrease in the reoxygenation phase by Trolox C. Following hypoxia/reoxygenation glucose and urea releases decreased. Trolox C had no influence on inhibition of glucose and urea production. These results suggest that Trolox C protected the liver cells against hypoxia/reoxygenation injury, yielding further evidence for a causative role of oxidative stress in this model.

Influences of dietary omega-3 polyunsaturated fatty acids on the recovery of cardiac and renal functions after preservation in hyperlipidemic rats.

The effects of a soybean oil diet and a high-cholesterol oil (HC) diet, and an HC diet with eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) supplementation, on basal and postpreservative cardiac function of the hearts and on postpreservative renal function of the kidneys from older rats were examined. Groups 1 through 4 of 100-week-old rats were fed either soybean oil, HC, HC with EPA, or HC with DHA, respectively, for 12 weeks. Blood was collected for analysis of plasma fatty acids, and the heart and left kidney were removed from the rat. In experiment 1, the heart was perfused on a Langendorff apparatus. After evaluation of the cardiac function of each rat, the heart was stored in histidine-tryptophan-ketoglutarate solution for 8 hr at 4 degrees C. The heart was reperfused and the recovery of cardiac function was evaluated. The coronary perfusate during reperfusion was collected to measure 6-keto prostaglandin F1alpha and thromboxane B2. Coronary flow (CF) perfused with Krebs-Henseleit bicarbonate (KHB) solution containing 5-hydroxytryptamine (5-HT) and nitroglycerin were evaluated in the Langendorff mode with atrial pacing (330 beats/min). In experiment 2, the excised left kidney was immediately flushed and preserved with University of Wisconsin solution for 8 hr at 4 degrees C. The kidney was then reperfused with KHB solution and renal function was evaluated. The plasma and cardiac EPA levels in group 3 were significantly higher than the levels found in the other groups. The plasma and cardiac ratios of EPA to arachidonic acid were significantly higher in groups 3 and 4 than in groups 1 and 2. There were no significant differences in basal cardiac function among any of the diet-fed rats. The percentage values of the recovery of aortic flow, cardiac output (CO), and left ventricular max dp/dt in group 3 and CO in group 4 were significantly higher than in group 2. In addition, the recovery of CF in group 3 tended to be higher than in group 2 (P=0.07). The percentage values of the recovery of aortic flow, CF, CO, and left ventricular max dp/dt in group 1 were significantly lower than in the other dietary groups. CF reperfused with KHB solution containing 5-HT was significantly higher in group 3 than in groups 1 and 2. CF reperfused with KHB solution containing 5-HT was significantly higher in group 4 than in group 1. CF reperfused with KHB solution containing nitroglycerin in group 3 tended to be higher than in groups 1 and 2 (P=0.07). The thromboxame B2 concentrations in the coronary perfusate during reperfusion in groups 3 and 4 were significantly lower than in groups 1 and 2. Fractional sodium reabsorption in group 3 was significantly higher than in group 2. Inulin clearance in groups 3 and 4 was significantly higher than in group 1. The postpreservative urinary flow in group 3 was significantly higher than in groups 1 and 2. The urinary flow was significantly higher in group 4 than in group 1. These results suggest that EPA administration may attenuate preservation and reperfusion injury and improve the recovery of cardiac and renal functions in hyperlipidemic and older rats. DHA administration may also show beneficial effects on kidney preservation in hyperlipidemic rats.

Effect of prolonged hypothermic ischemia and reperfusion on oxygen consumption and total mechanical energy in rat myocardium: participation of mitochondrial oxidative phosphorylation.

To reduce ischemia-reperfusion injury of hearts in open heart surgery and transplantation, it is important to know the critical period of ischemia in which donor hearts can sustain their function satisfactorily. Cardiac function has been deduced from oxygen consumption (VO2) and mechanical parameters such as pressure-volume area (PVA). Inhibited mitochondrial oxidative phosphorylation during ischemia indicates that ATP production is uncoupled from VO2. Therefore, both mitochondrial oxidative phosphorylation and total mechanical energy should be examined to evaluate cardiac function after ischemia and reperfusion. Isolated rat hearts were stored in Euro-Collins solution at 4 degrees C for 8, 12, and 24 hr and reperfused in a working mode with a modified Krebs-Henseleit bicarbonate solution. PVA and VO2 were examined in isovolumic contraction, and ventricular contractility and total mechanical energy were assessed, respectively, by the end-systolic elastance (Ees) and PVA. Mitochondrial oxidative phosphorylation in the presence of succinate and mitochondrial lipid peroxide levels were estimated in similarly treated rat hearts. Ees was decreased by ischemia without significant difference. The VO2 to PVA ratio remained linear, although VO2 at null PVA and the VO2 to PVA ratio significantly increased after 12 hr of ischemia. Mitochondrial oxidative phosphorylation was decreased significantly by reperfusion after 12 hr of ischemia. Mitochondrial lipid peroxide levels were increased significantly after 12 hr of ischemia. In isolated rat hearts, decreased efficiency for energy conversion from consumed oxygen to cardiac performance occurs between 8 and 12 hr of hypothermic ischemia, which was coincident with disturbed mitochondrial oxidative phosphorylation, to which lipid peroxidation may contribute.

Tumor necrosis factor-induced, superoxide-mediated neutrophil accumulation in cold ischemic/reperfused rat liver

The mechanisms of hepatic ischemia/reperfusion injury are complicated and multifactorial. This study was designed to examine superoxide generation and neutrophil accumulation in cold ischemic-reperfused rat livers after elimination of Kupffer cells and to determine the role of superoxide/tumor necrosis factor (TNF) interactions. Rat Kupffer cells were eliminated by liposome-encapsulated dichloromethylene diphosphonate injected intravenously. Livers from control and treated rats were isolated and preserved in University of Wisconsin solution (4°C) for 0, 12, and 24 hours and then perfused for 60 minutes with oxygenated Krebs-Henseleit bicarbonate buffer (37°C) by adding neutrophils into the perfusate. Superoxide generation was measured by using real-time chemiluminescence (CL) during perfusion, and neutrophil accumulation was assessed by measuring myeloperoxidase activity in the liver tissue. In the control livers, CL intensity markedly increased on reoxygenation, and after neutrophil infusion it increased again with a lag period of 10 minutes. Total CL intensity and myeloperoxidase activity increased with the duration of cold preservation. TNF release into the effluent perfusate was detectable only after 24 hours of preservation, and lactate dehydrogenase release was high. Elimination of Kupffer cells attenuated CL intensity and TNF and lactate dehydrogenase release and resulted in reduced myeloperoxidase activity. Electron microscopy revealed amelioration of hepatocyte swelling and endothelial cell disruption when Kupffer cells were eliminated. After 24 hours of preservation, superoxide generation was inhibited in the control livers by anti-TNF antiserum, whereas TNF release was not inhibited by superoxide dismutase. These results suggest that TNF induces superoxide generation by Kupffer cells, which mediates neutrophil accumulation and causes cellular injury in the initial phase of reperfusion.

Tumor necrosis factor-induced, superoxide-mediated neutrophil accumulation in cold ischemic/reperfused rat liver

The mechanisms of hepatic ischemia/reperfusion injury are complicated and multifactorial. This study was designed to examine superoxide generation and neutrophil accumulation in cold ischemic-reperfused rat livers after elimination of Kupffer cells and to determine the role of superoxide/tumor necrosis factor (TNF) interactions. Rat Kupffer cells were eliminated by liposome-encapsulated dichloromethylene diphosphonate injected intravenously. Livers from control and treated rats were isolated and preserved in University of Wisconsin solution (4 degrees C) for 0, 12, and 24 hours and then perfused for 60 minutes with oxygenated Krebs-Henseleit bicarbonate buffer (37 degrees C) by adding neutrophils into the perfusate. Superoxide generation was measured by using real-time chemiluminescence (CL) during perfusion, and neutrophil accumulation was assessed by measuring myeloperoxidase activity in the liver tissue. In the control livers, CL intensity markedly increased on reoxygenation, and after neutrophil infusion it increased again with a lag period of 10 minutes. Total CL intensity and myeloperoxidase activity increased with the duration of cold preservation. TNF release into the effluent perfusate was detectable only after 24 hours of preservation, and lactate dehydrogenase release was high. Elimination of Kupffer cells attenuated CL intensity and TNF and lactate dehydrogenase release and resulted in reduced myeloperoxidase activity. Electron microscopy revealed amelioration of hepatocyte swelling and endothelial cell disruption when Kupffer cells were eliminated. After 24 hours of preservation, superoxide generation was inhibited in the control livers by anti-TNF antiserum, whereas TNF release was not inhibited by superoxide dismutase. These results suggest that TNF induces superoxide generation by Kupffer cells, which mediates neutrophil accumulation and causes cellular injury in the initial phase of reperfusion. (Hepatology 1997 Jul;26(1):113-20)

The Effect of Hypertonic Sodium and Dantrolene on Propranolol Cardiotoxicity

To evaluate whether measures that lower cytosolic calcium (Ca) can reverse propranolol (PROP) toxicity in the isolated, perfused rat heart. Isolated rat hearts were perfused on a Langendorff apparatus with Krebs-Henseleit-bicarbonate (KHB) buffer solution. Toxicity was produced by perfusing the hearts with PROP (5 micrograms/mL) for 30 minutes. Subsequently, the hearts were treated for 30 minutes with buffer containing PROP plus experimental treatment. Three treatments were chosen: hypertonic sodium (Na) (160 mmol), to stimulate Na-Ca exchange, dantrolene (DAN) (10 mumol), to inhibit Ca release from sarcoplasmic reticulum, and combined hypertonic Na and DAN. The hearts were paced after 20 minutes of treatment. Heart rate (HR), left ventricular peak systolic pressure (LVP), the first derivative of LVP (dP/dt), and coronary flow were measured. PROP decreased HR and rendered the hearts refractory to pacing. PROP did not alter dP/dt. PROP increased LVP consistent with increased cytosolic Ca. Combined hypertonic Na and DAN treatment restored the ability to pace PROP-toxic hearts to the basal HR. Individually, hypertonic Na or DAN treatment partially restored the ability to pace toxic hearts. As experimental treatments increased HR, dP/dt and LVP decreased, consistent with decreased cytosolic Ca availability. These data are consistent with the hypothesis that bradycardia during beta-blocker cardiotoxicity is mediated by altered Ca homeostasis.

Generation of free radicals during anoxia and reoxygenation in perfused osteoblastlike cells.

Sensitivity to ischemia and reperfusion injury is a main problem afflicting tissues exposed to a prolonged period of oxygen deprivation. The generation of oxygen free radicals, in particular, is considered a major cause of postischemic reperfusion injury. However, studies on the mechanisms of production of free radicals are limited by the difficulty to measure in real time their formation and to discriminate between the different oxyradical species. The aim of this study was to determine whether the formation of oxygen free radicals occurs in murine osteoblastlike cells (MC3T3-E1) exposed to anoxia and reoxygenation and to explore its relation to the reoxygenation injury. Cells were cast in agarose and perfused with oxygenated Krebs-Henseleit bicarbonate buffer. Anoxia was obtained by shifting the gas phase of the media to 95% N2-5% CO2. Oxygen free radicals were detected by enhanced chemiluminescence: anion superoxide or hydrogen peroxide was measured by adding lucigenin or luminol plus horseradish peroxidase to the media, respectively. Cell injury was assessed by the rate of lactate dehydrogenase release. During the control period, lucigenin and luminol plus horseradish chemiluminescences were 15 +/- 1 nA per chamber and 20 +/- 2 nA per chamber, respectively. and lactate dehydrogenase release was 10 +/- 1 mU per minute. During anoxia, both chemiluminescences dropped to background levels, although lactate dehydrogenase release increased progressively to 38 +/- 7 mU per minute. During reoxygenation, O2 formation increased sharply to 45 +/- 6 nA and decreased to control levels; H2O2 production increased slowly, reaching 42 +/- 7 nA at the end of the reoxygenation period; lactate dehydrogenase declined progressively to control values. These results show that osteoblastlike cells produce measurable amounts of superoxide and hydrogen peroxide radicals during reoxygenation. Because lactate dehydrogenase release did not appear to relate to chemiluminescence, oxyradical flux may serve as a signal for other events that eventually lead to cell injury.

Pharmacokinetics and hepatotoxicity of diclofenac using an isolated perfused rat liver

Pharmacokinetics and hepatotoxicity of diclofenac was studied in a recirculating model of isolated perfused rat liver. Ten male Sprague-Dawley rat (weighing 230-330 g) livers were perfused for 2 h with 250 mL Krebs-Henseleit bicarbonate buffer that contained 10.75 mg (group A, n = 5) and 1.075 mg (group B, n = 5) of diclofenac (approximately 100 and 10 times the therapeutic dose in man, respectively). Samples were collected from the efflux at regular time intervals for the determination of diclofenac concentrations by a high performance liquid chromatography (HPLC) method. Pharmacokinetic analyses were carried out using a computer program. To establish viability of the liver and toxicity of the drug, enzyme activity measurements of lactate dehydrogenase (LDH), aspartate aminotransferase (SGOT) and piruvate aminotransferase (SGPT) were performed by a spectrophotometric method. Oxygen consumption was also recorded during the entire perfusion period. Both groups presented bicompartmental kinetics. Concentration profiles showed that group B had a better metabolizing capacity, reflected in a 85.54 +/- 37.05 min half-life, a 0.52 +/- 0.19 mL min-1 g-1 liver clearance and a 0.517 +/- 0.188 extraction ratio, compared to group A, which presented a 123.95 +/- 88.13 min half-life, a 0.1164 +/- 0.067 mL min-1 g-1 liver clearance (P < 0.002) and a 0.116 +/- 0.680 extraction ratio (P < 0.002). LDH activity showed a significant increase in group A at 90 min in comparison with the control group, while in group B this increase was significantly higher at 10 min (P < 0.004). The aminotransferase levels did not show a significant increase. According to these results, diclofenac would not have a direct hepatotoxic effect, even at doses 100 times higher than therapeutic ones.

Rat hepatocytes isolated from alcohol-induced fatty liver have an increased sensitivity to anoxic injury.

The aim of this study was to determine whether rat hepatocytes isolated from steatotic or nonsteatotic livers have different thresholds for injury due to anoxia-reoxygenation. Rats were fed ethanol or control diets for 8 weeks. Histology showed that more than 75% of the hepatocytes in alcohol-fed and less than 3% in control animals contained fatty vacuoles. The glycogen content was significantly reduced in steatotic livers. Isolated hepatocytes were cast in agarose gel threads and perfused with Krebs-Henseleit bicarbonate buffer. Cell viability was determined by Trypan Blue (TB) exclusion; cell injury was determined by lactate dehydrogenase (LDH) release; and superoxide anion (O2.-) was determined by lucigenin-enhanced chemiluminescence (LCL). During the pre-anoxic basal perfusion the following occurred: viability was 86% +/- 1% and 85% +/- 1%; LDH release was 16 +/- 3 and 15 +/- 3 mU/min; and LCL was 4 +/- 1 and 5 +/- 1 nA in steatotic and nonsteatotic hepatocytes, respectively. Cell viability decreased slightly under 4 hours of aerobic perfusion without differences between the two groups. In contrast, fatty hepatocytes died much faster than did control hepatocytes during anoxia; after 3 hours viability was 17% +/- 8% vs. 60% +/- 2% (P < .001), respectively. With reoxygenation following 2 hours of anoxia, the changes in viability, in LDH release, and in LCL were similar in both groups. These results indicate that in hepatocytes isolated from alcohol-fed rats when compared with control hepatocytes: 1) cell viability under aerobic conditions is not influenced; 2) anoxic injury is significantly increased; 3) a reduction in the hepatic glycogen stores, which may contribute to the enhanced sensitivity to anoxia, can be demonstrated; and 4) O2.- generation and cell injury occurring immediately after reoxygenation do not appear to be affected.

Kupffer Cells Generate Superoxide Anions and Modulate Reperfusion Injury in Rat Livers After Cold Preservation

ABSTRACT This study was designed to determine the source of reactive oxygen species (ROS) and whether Kupffer cells modulate the injury of perfused rat liver after cold preservation. The livers of male Lewis rats pretreated with schizophyllan glucan (SPG) (10 mg/kg, SPG group) or gadolinium chloride (5 mg/kg; Gd group) and untreated rats (control group) were preserved in University of Wisconsin solution for 0, 12, and 24 hours at 4 degrees C and then perfused for 60 minutes with oxygenated Krebs–Henseleit bicarbonate buffer. Real–time chemiluminescence (CL) of the liver during perfusion was measured using a sensitive photomultiplier, and reperfusion injury was assessed by measuring lipid peroxidation and lactate dehydrogenase release. CL intensity reached a peak within 5 minutes of reperfusion, and superoxide dismutase completely inhibited this CL in all groups. In the control group, the total CL intensity was high after 24 hours of preservation. It significantly ( P &lt; .05 vs. control group) increased in the SPG group, while it decreased in the Gd group after 12 and 24 hours of preservation. The total CL intensity decreased by 70% (when diphenyliodonium chloride (100 micromol/L; an NADPH oxidase inhibitor) was added to the perfusate before preservation of untreated rats. Lipid peroxidation and lactate dehydrogenase release significantly ( P &lt; .05) deteriorated in the SPG group, while they ameliorated in the Gd group after 24 hours of preservation. These results demonstrate that Kupffer cells primarily generate superoxide anions and modulate the organ injury in the initial phase of reperfusion after cold preservation.

Kupffer cells generate superoxide anions and modulate reperfusion injury in rat livers after cold preservation

This study was designed to determine the source of reactive oxygen species (ROS) and whether Kupffer cells modulate the injury of perfused rat liver after cold preservation. The livers of male Lewis rats pretreated with schizophyllan glucan (SPG) (10 mg/kg, SPG group) or gadolinium chloride (5 mg/kg; Gd group) and untreated rats (control group) were preserved in University of Wisconsin solution for 0, 12, and 24 hours at 4 degrees C and then perfused for 60 minutes with oxygenated Krebs-Henseleit bicarbonate buffer. Real-time chemiluminescence (CL) of the liver during perfusion was measured using a sensitive photomultiplier, and reperfusion injury was assessed by measuring lipid peroxidation and lactate dehydrogenase release. CL intensity reached a peak within 5 minutes of reperfusion, and superoxide dismutase completely inhibited this CL in all groups. In the control group, the total CL intensity was high after 24 hours of preservation. It significantly (P < .05 vs. control group) increased in the SPG group, while it decreased in the Gd group after 12 and 24 hours of preservation. The total CL intensity decreased by 70% (when diphenyliodonium chloride (100 micromol/L; an NADPH oxidase inhibitor) was added to the perfusate before preservation of untreated rats. Lipid peroxidation and lactate dehydrogenase release significantly (P < .05) deteriorated in the SPG group, while they ameliorated in the Gd group after 24 hours of preservation. These results demonstrate that Kupffer cells primarily generate superoxide anions and modulate the organ injury in the initial phase of reperfusion after cold preservation.(Hepatology 1997 Feb;25(2):356-60)

Differential effects of steroids on vascular tone and smooth muscle cell Ca2+-homeostasis

The protective effects of 17p oestradiol against the onset of atherosclerosis in women can only partly be explained by altered lipid metabolism. The vessel wall itself is also a target organ for sex steroids. 17p oestradiol-induced increases in the expression of endothelial cell nitric oxide synthase [l] and proliferation of endothelial cells [2], as well as the decrease in the proliferation of smooth muscle cells [3] require de now protein synthesis. These effects of 17p oestradiol appear to be mediated by the classical ICI 182,780-sensitive nuclear oestrogen receptors. In contrast, 17p oestradiol induces a rapid and reversible endotheliumindependent relaxation of preconstricted vascular rings [4]. This latter response to 17p oestradiol is independent of changes in protein synthesis and has been attributed to an alteration of smooth muscle cell Ca2'-homeostasis, presumably involving a reduction in Ca2' influx through voltage operated L-type channels [5]. Recent studies have shown that progesterone also acts as a vasodilator [6], suggesting that the novel steroid receptor in the plasmalemma of cells is less selective than classical, nuclear ones. In the present study we have investigated the effects of 17poestradiol and progesterone on coronary vascular tone in isolated perfised Langendofl rat hearts [7] and on intracellular Ca2'-homeostasis in a rat fetal aortic smooth muscle cell line A7r5. Isolated perfused hearts: Male Sprague-Dawley rats (>350g) were killed by cervical dislocation, the heart excised and the aorta cannulated. The coronary vasculature was perfised retrogradely through the aorta at a constant flow (10 ml.min-') with a heated (37°C) Krebs-Henseleit bicarbonate buffer [7]. Coronary perfision pressure (CPP) was recorded as an index of vascular resistance. As CPP is generally low in isolated hearts perfised with a blood-free solution, CPP was raised with the thromboxane A2 mimetic U46619 (5 nM). Bolus injections (10 pl) of bradykinin and sodium nitroprusside (a donor of nitric oxide in aqueous solution) were then administered into the coronary perfusate. Close-arterial injections of bradykinin (BK, 0.1 nmol) and sodium nitroprusside (SNP, 10 nmol) caused a rapid and transient fall in CPP: 4 1 s mmHg (meankSE, n=15) for BK and 41k3 mmHg (n=15) for S N P . These vasodilator responses to BK and S N P confirmed the integrity of endothelial and smooth muscle cell layers [7]. 17p oestradiol and progesterone caused a rapid, dose-dependent (Fig. 1) and transient reduction in CPP. At the maximal dose used (1 00 nmol), 17p oestradiol and progesterone reduced CPP by 34+3 mmHg (n=15) and 19M mmHg (n=8), respectively. Disruption of the endothelium with 1.5 ml air reversed the normal vasodilator response to BK (0.1 nmol) to a vasoconstriction (754 mmHg, n=4), but did not alter the vasodilator responses to SNP (10 nmol, 50+7 mmHg), 17p oestradiol (100 nmol, 3 7 H mmHg, n=4) or progesterone (100 nmol, 20*6 mmHg, n=4). These findings suggest that 17p oestradiol and progesterone are acting directly on coronary vascular smooth muscle cells 40 1

Uncoupling effects of diclofenac and aspirin in the perfused liver and isolated hepatic mitochondria of rat

Gluconeogenesis, glycolysis and glycogenolysis were studied in rat perfused liver following the infusion of various concentrations of diclofenac and aspirin, two non-steroidal anti-inflammatory drugs (NSAIDs). Glucose synthesis was measured in livers isolated from 48-h fasted rats perfused with Krebs-Henseleit bicarbonate buffer containing l-lactate (2 mM) and pyruvate (0.1 mM) as precursors. Both diclofenac (0.01–0.1 mM) and aspirin (1–10 mM) had an inhibitory effect on gluconeogenesis (GNG). The inhibition was dose-dependent and reversible. For the estimation of glycogenolysis and glycolysis, the rates of glucose release and of lactate and pyruvate production were measured in livers of well-fed rats perfused with substrate-free buffer. Infusion of diclofenac (0.1 mM) or aspirin (5 mM) strongly stimulated glycogenolysis and glycolysis (GGL/GL). In general, an increased oxygen consumption by the liver tissue was also noted in both types of experiments, as deduced from the continuous monitoring of oxygen concentration changes in the effluent. Such a pattern of response can be attributed to the uncoupling effects of the two drugs on oxidative phosphorylation. Measurements of respiration rates and membrane potential in isolated liver mitochondria submitted to various concentrations of diclofenac and aspirin confirms this assumption. Thus, 0.01 to 0.2 mM diclofenac stimulates state-4 respiration and slightly inhibits state 3, decreasing the respiratory control ratio, while the membrane potential is decreased or collapsed (depending on the drug concentration). Similar effects are recorded for aspirin at higher concentrations (0.2–5 mM), even though state 3 is not affected in this case. Arguments are presented that the concentrations of the drugs used largely correspond to the pharmacological doses employed in antipyretic and anti-inflammatory treatments. Therefore, a greater consideration should be given to the uncoupling effect, at least from the toxicological viewpoint.

An Important Role for Pentose Cycle in the Synthesis of Citrulline and Proline from Glutamine in Porcine Enterocytes

This study was designed to determine a role of pentose cycle in the provision of NADPH for the synthesis of citrulline and proline from glutamine in porcine enterocytes. Enterocytes from 4-day-old pigs were incubated at 37°C for 0 to 30 min in Krebs–Henseleit bicarbonate buffer (pH 7.4) containing 2 mMglutamine and 5 mMglucose in the presence of 0, 0.1, or 0.25 mMdehydroepiandrosterone (DHEA), a potent inhibitor of glucose-6-phosphate dehydrogenase which is the key regulatory enzyme of pentose cycle. The activity of this cycle was estimated with the use of [1-14C]glucose and [6-14C]glucose. In some experiments, the medium included 2 mMornithine and 2 mMNH4Cl (no glutamine). About 14% of glucose taken up by enterocytes was metabolized via pentose cycle. The flux from glucose into this cycle was decreased by 70 and 86%, respectively, in the presence of 0.1 and 0.25 mMDHEA compared with its absence. DHEA inhibited the synthesis of ornithine, citrulline, arginine, and proline from glutamine in a concentration-dependent manner, but had no effect on the formation of citrulline and arginine from ornithine. However, DHEA decreased the synthesis of proline from ornithine by 79%. DHEA had no effect on cellular ATP concentrations. These results provide the first line of evidence suggesting that glucose metabolism via pentose cycle plays an important role in providing NADPH for the conversion of glutamine into pyrroline-5-carboxylate in porcine enterocytes, which may explain the glucose-dependent synthesis of citrulline and proline from glutamine in these cells.

The role of a hydroxyl radical scavenger (nicaraven) in recovery of cardiac function following preservation and reperfusion.

We investigated the efficacy in reducing myocardial preservation and reperfusion (P/R) injury of direct hydroxyl radical scavenging by nicaraven as compared with scavenging of both superoxide radicals and hydrogen peroxides by superoxide dismutase (SOD) and catalase (CAT), respectively. Isolated rat hearts were mounted on a Langendorff (L) apparatus to estimate the baseline aortic flow (AF), coronary flow (CF), cardiac output (CO), systolic pressure (SP), aortic mean pressure (MP), rate pressure product, and LV dp/dt. They were divided into 3 groups: group 1, 12 hr storage in HTK solution; group 2, 12 hr storage in HTK solution containing 2.5x10(5) U/L SOD and 2x10(5) U/L mg/L CAT; and Group 3, 12 hr storage in HTK solution containing 10(-3) M nicaraven. SOD, CAT, and nicaraven were administered intraperitoneally before harvesting. Hearts were stored in each preservation solution at 4, and then reperfused. Postpreservative function and concentrations of leaked enzymes were measured. The hearts were switched back to the L-mode and paced at 330 beats/min. CF following perfusion with Krebs-Henseleit bicarbonate buffer (KHB) solution containing 10(-6) M 5-hydroxytryptamine (5-HT) or 10(-5) M nitroglycerin (NTG) then evaluated. The myocardial water content also was measured. The recovery of CF, CO, SP, MP, and LV dp/dt was significantly greater in group 3 than in group 1. The recovery of CF was superior to that in group 2 (P<0.05). There were no significant differences in the recovery of cardiac function between groups 1 and 2. 5-HT caused a decrease in CF in each group, however, CF in group 3 was higher than that in group 1 (P<0.05). NTG caused no significant differences among the groups. There were no significant differences in leaked enzymes and myocardial water content among the three groups. These results suggest that nicaraven protects against myocardial P/R injury through its hydroxyl radical scavenging activity, and that therapy with oxygen-free radical scavengers should be directed toward inactivation of hydroxyl radicals rather than superoxide radicals and/or hydrogen peroxides.

Effect of the potassium-channel opener nicorandil as an adjunct to cardioplegia on myocardial preservation in isolated rabbit hearts.

We studied the effect of nicorandil on the hemodynamic, biochemical, and ultrastructural changes in rabbit hearts (n = 50) rendered cardioplegic with a single injection of Bretschneider's HTK solution over 30 min or 60 min at 37 degrees C or 15 degrees C, followed by reperfusion at 37 degrees C for 60 min. Particular attention was focused on the aspects of dose-response relationship, temperature sensitivity, and ischemic tolerance. Isolated hearts were prepared for modified Langendorff circulation using modified Krebs-Henseleit bicarbonate solution bubbled with a 95% O2(-5)% CO2 gas mixture, to which nicorandil (0, 0.1, 1, and 5 mM) was added. The optimal concentration of nicorandil was 1mM, which increased the recovery of left ventricular (LV) function, affecting coronary flow and the myocardial cyclic adenosine monophosphate, but not the myocardial concentrations of adenine nucleotide compounds or total calcium. These effects were abolished by the addition of glibenclamide to the HTK, but they were not diminished by a high potassium (K+) concentration of 20mM. The addition of nicorandil 1mM to the HTK at 15 degrees C did not improve the recovery of LV function. Our result suggested that nicorandil used adjunctly prevents LV functional depression after 30min, and possibly 60min of cardioplegia at 37 degrees C, and that this effect is not disturbed by a high K+ concentration up to 20 mM. However, nicorandil has temperature sensitivity whereby it loses its efficacy at 15 degrees C.

Comparison of Isolated Hepatocytes and Tissue Slices for Study of Liver Hypothermic Preservation/Reperfusion Injury

Simple models are needed that effectively test the variables that may be important in liver preservation. Two such models are isolated hepatocytes and tissue slices. In this study the effects of hypothermic preservation on the viability of hepatocytes (HC) and tissue slices (TS) from rat livers were measured by LDH leakage after cold storage and rewarming. We compared how glycine, calcium, and fasting, shown previously to affect preservation injury in hepatocytes, affected both HC and TS viability. Hepatocytes were cold-stored in University of Wisconsin organ preservation solution for up to 48 h and rewarmed in Krebs–Henseleit Bicarbonate (KHB) for 120 min. Tissue slices were studied in two ways. Either livers were cold-stored intact and then tissue slices (TS-A) prepared and rewarmed in KHB, or tissue slices were prepared from the fresh liver, cold-stored, and then rewarmed (TS-B). The latter method may be similar to cold storage of HC. Freshly prepared samples (HC, TS-A, or TS-B) showed <15% LDH leakage during the rewarming phase. Cold storage for 24 h resulted in <30% LDH leakage in all preparations. After 48 h cold storage there was a significant increase in LDH leakage (HC, 65.1 ± 5.1%; TS-A, 52.9 ± 0.8%, TS-B, 53.6 ± 2.6%). Glycine (3 mM) or calcium (1.5 mM) included in the KHB significantly reduced LDH leakage from 48 h cold-stored HC to 20.7 ± 1.8 and 26.3 ± 2.4%, respectively. These agents caused a smaller decrease in LDH release from tissue slices (around 40%). Hepatocytes appear more susceptible to preservation/reperfusion damage than the more structurally intact tissue slices as suggested by the greater release of LDH. Another difference was that the agents which improved preservation quality of HC were not as effective in TS. Hepatocytes may be more vulnerable to preservation/reperfusion damage because of the harsh methods used in their preparation. The damage induced during preparation appears amenable to suppression by glycine or calcium. Tissue slices, which are intact pieces of liver tissue, may be more suitable for studies related to development of better methods for liver preservation. The intact cells in TS have not been exposed to harsh conditions and maintain a more natural cell–cell relationship.

Effect of Cardioplegic Arrest and Reperfusion on Coronary Reserve and Autoregulation

The effects of cardioplegic arrest and reperfusion on the coronary vasculature remain to be characterized. This study was designed to investigate changes in coronary reserve and autoregulation after hypothermic cardioplegic arrest and reperfusion. Isolated rabbit hearts were perfused in a retrograde manner with Krebs-Henseleit bicarbonate buffer solution at a pressure of 80 cm H2O. Baseline measurements were performed for (1) coronary flow; (2) vasodilatory response to 5-hydroxytryptamine (10(-7) mol/L) and nitroglycerin (10(-4) mol/L); (3) autoregulatory capacity, quantified as closed-loop gains; and (4) isovolemic left ventricular function. Hearts were then subjected to cardioplegic arrest for 90 minutes. Twenty minutes after reperfusion, measurements were repeated. Coronary flow decreased significantly after reperfusion (6.2 +/- 1.1 versus 5.3 +/- 1.1 mL.min-1.g-1; p < 0.01). The response to 5-hydroxytryptamine as percentage increase of flow decreased significantly after reperfusion (134.0% +/- 12.0% versus 109.1% +/- 6.8%; p < 0.01). However, there was no significant change in the response to nitroglycerin after reperfusion (121.3% +/- 17.6% versus 136.6% +/- 13.3%). The closed-loop gain demonstrated negative values before arrest but became positive after reperfusion, indicating loss of autoregulation after reperfusion. There was no significant change in left ventricular function. The coronary flow reserve in response to 5-hydroxytryptamine and autoregulation were impaired after cardioplegic arrest and reperfusion, whereas nitroglycerin-induced vasodilatory response and left ventricular function were preserved.

Effects of concanavalin A and phorbol myristate acetate on glutamine metabolism and proliferation of porcine intestinal intraepithelial lymphocytes.

This study was designed to determine the effects of concanavalin A (ConA) (a T-cell mitogen) and phorbol myristate acetate (PMA) (an activator of protein kinase C) plus ionomycin (Iono) on glutamine metabolism and proliferation of porcine intestinal intraepithelial lymphocytes (IEL). IEL were prepared from jejunum of 29-day-old pigs weaned at 21 days of age. Cells were cultured at 37 degrees C for 48 hr in RPMI-1640 medium containing 10 mM D-glucose, 0 to 4 mM L-glutamine, 0 to 5 micrograms/ml ConA, or 20 ng/ml PMA + 375 ng/ml Iono. The medium was also supplemented with 0 or 0.1 mM adenosine, guanosine, inosine, uridine or cytosine to study the effect of nucleosides or bases on IEL proliferation. IEL proliferation was assessed by pulsing with 3H-thymidine for 18 hr. Glutamine metabolism was studied in incubated IEL in the presence of Krebs-Henseleit bicarbonate buffer containing 5 mM D-glucose and 1 mM L-[U-14C]glutamine. PMA+Iono markedly stimulated 3H-thymidine incorporation and glutamine metabolism to ammonia, glutamate, aspartate and CO2. When stimulated by PMA+Iono, rates of 3H-thymidine incorporation and glutamine metabolism were much lower in IEL than in mesenteric lymph node lymphocytes. Glutamine was required for IEL proliferation, and it could not be replaced by adenosine, guanosine, inosine, uridine or cytosine, suggesting that porcine IEL cannot interconvert purine and pyrimidine nucleotides. Porcine IEL poorly or not at all responded to ConA stimulation, in contrast to lymph node lymphocytes, in terms of both [3H]thymidine uptake and glutamine metabolism.

Effect of Phosphodiesterase III-Inhibitor (E-1020) Adjunct to Bretschneider's HTK Cardioplegic Solution on Myocardial Preservation in Rabbit Heart

We examined the hypothesis that an increase in the myocardial cyclic adenosine monophosphate (cAMP) by a phosphodiesterase III inhibitor, E-1020, may ameliorate the hemodynamic and biochemical changes in rabbit hearts after cardioplegic arrest, and that this enzyme-mediated process is temperature-sensitive. Sixty-one male Japanese white rabbits weighing 2.8 to 3.5 kg were used. Isolated hearts were prepared for modified Langendorff circulation using modified Krebs-Henseleit bicarbonate solution bubbled with a 95% O2-5% CO2 gas mixture. Thirty or sixty minutes of cardioplegia at 37 degrees C, or thirty minutes of cardioplegia at 15 degrees C was followed by normothermic reperfusion for 60 minutes. E-1020, 0.1 mmol/L was added to the cardioplegic solution (Bretschneider's HTK solution). The left-ventricular function was measured with a latex balloon placed in the left-ventricular cavity. The myocardial cAMP was higher, the total myocardial calcium was lower in hearts with E-1020 than in hearts with HTK alone (p < 0.05). E-1020 at 0.1 mmol/L did increase the myocardial concentration of cAMP and the functional recovery, and prevented the increase in the myocardial total calcium. Temperature affected the myocardium to preserve myocardial concentrations of adenine nucleotide compounds and cAMP. Our results suggest that a 0.1 mmol/L E-1020 adjunct to HTK solution at 37 degrees C completely prevents left-ventricular functional depression during 30 min of cardioplegia induced with non-oxygenated HTK, but decreases its potential efficacy at 15 degrees C. The protective effects disappear if the ischemic period lasts 60 min at 37 degrees C.