Reduced Lactate Dehydrogenase Release Research Articles

Glycine minimizes reperfusion injury in a low-flow, reflow liver perfusion model in the rat

This study investigated the effects of glycine on reperfusion injury in a low-flow, reflow liver perfusion model. With this protocol, livers were perfused at low flow rates of approximately 1 ml.g-1. min-1 for 75 min, which caused cells in pericentral regions of the liver lobule to become anoxic because of insufficient delivery of oxygen. When normal flow rates (approximately 4 ml.g-1.min-1) were restored for 40 min, an oxygen-dependent reperfusion injury occurred. Upon reflow, lactate dehydrogenase (LDH), a cytosolic enzyme, and malondialdehyde (MDA), an end product of lipid peroxidation, were released into the effluent perfusate. LDH increased from basal levels of approximately 1-35 IU.g-1.h-1 in livers from control rats. Glycine (0.06-2.00 mM) minimized enzyme release in a dose-dependent manner (half-maximal decrease = 133 microM), with maximal values only reaching 5 IU.g-1.h-1 when glycine was increased to 2 mM. Reflow for 40 min after 75 min of low-flow hypoxia caused death in approximately 30% of previously anoxic parenchymal cells in pericentral regions; however, infusion of glycine (2 mM) decreased cell death to less than 10%. Strychnine (1 mM), which was found to mimic the cytoprotective effect of glycine in proximal renal tubules, also reduced LDH release to 11 IU.g-1.h-1 in this study. Bile was released at rates of approximately 42 microliters.g-1.h-1 in livers from control rats, but values were not altered significantly by glycine. Maximal MDA production during reperfusion decreased by 35% with 0.6 mM of glycine. Trypan blue distribution time, an indicator of hepatic microcirculation, was reduced significantly by glycine at 5 and 40 min after reflow, but changes were about twofold greater at later time points compared with earlier ones (half-maximal decrease = 225 microM). Time for oxygen to reach steady state upon reflow was reduced by glycine in a dose-dependent manner, and the rates of entry and exit of a dye confined to vascular space (fluorescein dextran) were increased two- to threefold by glycine, respectively. Taken together, these data indicate that a reperfusion injury that occurs in previously hypoxic pericentral regions of the liver upon reintroduction of oxygen is minimized by glycine, possibly by action on a glycine-sensitive anion channel to improve microcirculation during the reperfusion period.

A hydroxylated analog of the β-adrenoceptor antagonist, carvedilol, affords exceptional antioxidant protection to postischemic rat hearts

The antioxidant and cardioprotective effects of the β-adrenoceptor antagonist, carvedilol, and its hydroxylated analog. BM-910228, were compared using the postischemic rat heart model. Hearts were infused with either agent (0.01, 0.10, or 10 nM final, or drug-free infusate) for 10 min prior to 30 min global ischemia, and also during the initial 15 min of reperfusion. Recovery of postischemic hemodynamic parameters (left ventricular systolic and developed pressures, mean diastolic pressure, cardiac output, coronary flow rate, and cardiac pressure-volume work), and the extent of postischemic tissue lactate dehydrogenase (LDH) loss, lipid hydroperoxide (LOOH) formation, and lipid peroxidation (LPO)-derived free radical production were assessed and compared among the treatment groups. The depressive pharmacological properties (β and α-blockade) of both agents masked the extent of postischemic hemodynamic recovery, except at the lowest dose (10 pM) of the analog, which provided significant improvements in systolic and developed pressures, and cardiac work. Treatment with both agents provided significant dose-dependent reductions in postischemic LOOH formation and lipid alkoxyl radical production, as determined by electron spin resonance spectroscopy and α-phenyl- tert-butylnitrone (PBN) spin trapping (PBN/alkoxyl adduct hyperfine splitting α N = 13.63 G and α H = 1.93 G). Although both agents reduced oxidative injury, the hydroxylated analog was clearly the superior antioxidant (equipotent at doses two to three orders of magnitude lower) compared to the parent compound. This was also reflected with respect to drug-mediated improvement in myocardial preservation (reduced LDH release), which paralleled the antioxidant protective effects. Because neither agent displayed significant primary radical scavenging ability at doses (≤ 10 nM), which did provide substantial inhibition of postischemic LOOH and alkoxyl formation, our data suggest that the antioxidant properties of carvedilol and its analog are mediated primarily through a LPO chain-breaking mechanism. Moreover, the significant antioxidant protection afforded by the analog BM-910228 at subnanomolar levels places this agent into an exclusive category reserved for exceptionally potent antioxidants.

Escherichia coli Endotoxin-Mediated Endothelial Injury Is Modulated by Glutathione Ethyl Ester

The mechanisms involved in endotoxin-induced endothelial injury are not fully understood. Oxidant stress is thought to play a role in cell damage after endotoxin exposure. Glutathione may ameliorate these affects. Glutathione ethyl ester (GSE) was used in bovine pulmonary artery endothelial cell (BPAEC) cultures to determine the potential for attenuation of endotoxin-induced injury. GSE (0.05-25 mM) was preincubated with BPAEC for 4 h before endotoxin exposure. Fresh media containing GSE and Escherichia coli endotoxin (0.05 microgram/mL) were then placed on the BPAEC and incubated for 18 h. GSE, at doses of 5 and 25 mM, attenuated endotoxin-induced injury, as reflected by a significant reduction in lactate dehydrogenase release. This was paralleled by a significant increase in endotoxin-stimulated prostaglandin E2 and prostacyclin release. Thus, GSE attenuates endotoxin-induced injury of BPAEC in culture and alters BPAEC prostaglandin metabolism.

Substrate Accessibility to Cytosolic Aspartate Aminotransferase Improves Posthypoxic Recovery of Isolated Rat Heart

The effects of aspartate (Asp) and 2-oxoglutarate (2-OG) on metabolism and function of isolated rat heart during hypoxia and reoxygenation were studied. Hearts were subjected to oxygenated perfusion with Krebs-Henseleit buffer supplied with 11 mM glucose (30 min) and anoxic perfusion with the buffer saturated with N-2 (20 min), followed by reoxygenation (30 min). The substrate concentrations in the perfusate were 3.5 mM each. The additives had no effect on the energy metabolism and function of the oxygenated heart despite a two-fold rise in myocardial Asp and 2-OG. Substrate supplementation during anoxic perfusion resulted in reduced lactate dehydrogenase release and less depression of cardiac function. Prevention of Asp, glutamate, and 2-OG degradation in hypoxic myocardium was accompanied by relief of glycolytic flux and better preservation of ATP, phosphocreatine (PCr), and total creatine (Cr). Reoxygenation without the additives after supplemented anoxic perfusion failed to improve recovery of high-energy phosphates and cardiac function compared to control. However, during reoxygenation with the additives the treated hearts showed less cell membrane damage and enhanced recovery of contractile and pump function. These effects were associated with higher myocardial contents of ATP, PCr, and adenine nucleotides and a smaller Cr loss during reoxygenation. A more effective restoration of oxidative metabolism was related to promoted glucose oxidation due to replenishment of the malate-aspartate shuttle reactants. The results substantiate the use of substrates of cytosolic aspartate aminotransferase for myocardial protection against hypoxia/reoxygenation stress.

Hypoxic preconditioning preserves antioxidant reserve in the working rat heart

The aim was to examine whether intracellular antioxidants play a role in myocardial preservation following hypoxic preconditioning. Isolated working rat hearts were subjected to 30 min ischaemia and 30 min reperfusion. Control hearts were compared to hearts preconditioned with 10 min hypoxia. Left ventricular function and lactate dehydrogenase (LDH) release were measured in each group. Ascorbate dependent (ADAR) and thiol dependent (TDAR) components of the endogenous myocardial antioxidant reserve were assessed using electron spin resonance spectroscopy. a Hypoxic preconditioning had no effect on left ventricular function after 10 min reoxygenation. During reperfusion, the hypoxically preconditioned hearts had a significantly increased survival rate, aortic flow, developed pressure, and dP/dtmax, and a reduced lactate dehydrogenase release, compared to non-preconditioned controls (P < 0.05). Preconditioned hearts also had significantly higher preservation of baseline ADAR (79%) and TDAR (96%) compared with control hearts, (70%) and (77%), respectively (P < 0.05). Hypoxic preconditioning enhances functional recovery and reduces cell necrosis following global ischaemia in the working rat heart. This phenomenon may, in part, be mediated through enhanced ascorbate and thiol components of the antioxidant reserve.

Acute pneumocyte injury, poly(ADP-ribose) polymerase activity, and pyridine nucleotide levels after in vitro exposure of murine lung slices to cyclophosphamide

Cyclophosphamide (CYC) is a metabolically activated, DNA-alkylating, antitumor agent that causes pulmonary fibrosis. BALB/cN (B) mice are sensitive and C57B1/6N (C) mice are resistant to CYC-induced fibrosis. Pulmonary bioactivation may contribute to strain sensitivity. Therefore, we tested the intrinsic susceptibility of murine lung slices to cell injury by direct exposure to CYC for 2–8 hr. Injury was measured by release of lactate dehydrogenase (LDH). DNA damage activates the nuclear enzyme poly(ADP-ribose) polymerase (PAP, EC 2.4.2.30), causing depletion of its substrate, NAD. NAD can also be decreased by phosphorylation to NADP, as seen with oxidative stress. Depletion of NAD can lead to loss of ATP. Thus, we measured LDH release, PAP activation, NAD, NADP and ATP in slices incubated with or without the PAP-inhibitor, 3-aminobenzamide (3-AB). CYC (0.1 to 1.0 mg/mL for 4–8 hr) caused LDH release in slices from both murine strains, but LDH release was significantly greater in B lung slices than in C slices. After an 8-hr incubation 63.9±3.7% (mean±SEM) of total LDH was released from B lung slices with 1.0 mg CYC/mL, whereas only 45.8±2.6% was released from C lung slices (P<0.05). 3-AB reduced LDH release to 44.7±2.4% in B slices and 28.1±2.0% in C slices (P<0.05 vs CYC only). PAP activity in nuclei isolated from CYC-treated B lung slices was increased 2- to 4-fold after 2 hr of incubation with 0.5 and 1.0 mg CYC/mL. PAP activation was delayed and reduced with incubation in 3-AB. PAP was activated 2-fold in nuclei from C slices treated with 0.5 mg CYC/mL for 2 hr. NAD was decreased at 2 and 24 hr in B slices treated with 0.5 and 1.0 mg CYC/mL, and at 4 hr with 0.1 mg CYC/mL. NAD depletion occurred only at 4 hr in the resistant C slices treated with 1.0 mg CYC/mL. CYC increased NADP by a similar extent in B and C lung slices. In B slices, NAD losses were approximately 4 times the increases in NADP. CYC did not decrease ATP in B slices and ATP dropped 25% only after 4 hr in the resistant C slices. We conclude that CYC is directly toxic to lung tissue and observe that strain sensitivity in vitro mirrors the sensitivity to fibrosis in vivo. PAP activation and oxidate stress may contribute to this toxicity.

Pyruvate increases threshold for preconditioning in globally ischemic rat hearts.

Isolated rat hearts can be protected by preconditioning, although this has not been found when they are perfused with pyruvate. We addressed the question of whether pyruvate could increase the threshold for preconditioning in isolated rat hearts and whether this could be overcome with increased durations of ischemia. A protocol of four periods of 5 min of ischemic preconditioning (4 x 5 min) protected hearts (improved recovery of function, reduced lactate dehydrogenase release) not perfused with pyruvate from a subsequent 30-min period of global ischemia, but did not protect pyruvate-perfused hearts. Pilot studies indicated that hearts perfused in the presence of pyruvate must be ischemic for approximately 40% longer to produce equivalent ischemic damage in nonpyruvate-treated hearts. Thus the preconditioning period of 5 min was increased by approximately 40% to 7 min to produce equivalent degrees of preconditioning. Hearts preconditioned with the 4 x 7 min protocol with pyruvate were significantly protected against a subsequent severe global ischemia (enhanced recovery of function, reduced lactate dehydrogenase release). High-energy phosphates were measured at the end of the preconditioning protocol (before final global ischemia) to determine whether there was a correlation between cardioprotection and high-energy phosphate levels. There was no correlation between ATP, ADP, or AMP levels and the efficacy of preconditioning. However, an increase in creatine phosphate was associated with cardioprotection, although the importance of this in mediating preconditioning is doubtful. Thus the ability to precondition rat hearts is somewhat dependent on their energy source, but this appears to be due to changes in the severity of the ischemic preconditioning event.

Effects of GM1 ganglioside on cardiac function following experimental hypoxia-reoxygenation

Rat hearts made hypoxic for 20 min by perfusion with 95% N 2/5% CO 2 and reoxygenated for 20 min in a Langerdorff apparatus showed a dose-dependent reduction of lactate dehydrogenase release when incubated with ganglioside GM1 (0.1–10 mu;M). The decline of contractile force during hypoxia was also reduced dose dependently in the presence of GM1. Similar effects were observed in hearts obtained from animals treated i.p. with 40 mg/kg GM1 for 14 days. The levels of Na +, K +-ATPase in ventricular tissue were also reduced after hypoxia-reoxygenation and the reduction was prevented in hearts from GM1-treated animals. GM1 (1–30mu;M) reduced the functional response to field stimulation of adrenergic nerve terminals in isolated atria. Rat atria made hypoxic in glucose-free media maintained normal stores of tissue noradrenaline in the presence of 1 mu;M GM1. In the rabbit, GM1 (40 mg/kg i.p. for 4 days) reduced the alterations of the ST segment of the ECG during acute occlusion of the left descending and circumflex coronaries artery. In conclusion, ganglioside GM1 reduces some effects of hypoxia-reoxygenation in the heart, through still unknown mechanisms.

Protective effect of serotonin (5-HT2) receptor antagonists in ischemic rat hearts.

Serotonin (5-HT) may play a role in exacerbating thrombosis and coronary spasm during myocardial ischemia, but its role in mediating myocardial damage directly is not clear. We determined the effect of the 5-HT2 receptor antagonists cinanserin (0.1-10 microM), ketanserin (0.3-10 microM), and LY 53857 (1-10 microM) on time to contracture, recovery of contractile function, and lactate dehydrogenase (LDH) release after 25-min global ischemia and 30-min reperfusion in isolated rat heart. All 5-HT2 antagonists significantly increased time to contracture in a concentration-dependent manner (EC25 = 1.6, 5.5, and 6.1 microM for cinanserin, ketanserin, and LY 53857, respectively). These compounds also significantly reduced LDH release and improved recovery of contractile function during reperfusion. 5-HT > or = 30 microM significantly reduced time to contracture, indicating a proischemic effect. The proischemic effect of 5-HT was abolished by ketanserin and cinanserin. Inhibition of 5-HT synthesis by parachlorophenylalanine resulted in significant cardioprotection, further indicating the involvement of 5-HT in the pathogenesis of ischemia in this model. Although cinanserin and ketanserin had alpha 1-adrenoceptor blocking effects, LY 53857 was devoid of this activity at concentrations exhibiting cardioprotection. Therefore, 5-HT may exacerbate ischemic injury in rat heart, and this exacerbation appears to be mediated specifically by 5-HT2 receptors.

Chronic Ethanol Exposure Potentiates NMDA Excitotoxicity in Cerebral Cortical Neurons

The effect of acute and chronic ethanol exposure on excitotoxicity in cultured rat cerebral cortical neurons was examined. Neuronal death was quantitated by measuring the accumulation of lactate dehydrogenase (LDH) in the culture media 20 h after exposure to NMDA. Addition of NMDA (25-100 microM) to the culture dishes for 25 min in Mg(2+)-free buffer resulted in a dose-dependent increase in LDH accumulation. Phase-contrast microscopy revealed obvious signs of cellular injury as evidenced by granulation and disintegration of cell bodies and neuritic processes. Chronic exposure of neuronal cultures to ethanol (100 mM) for 96 h followed by its removal before NMDA exposure, significantly increased NMDA-stimulated LDH release by 36 and 22% in response to 25 microM and 50 microM NMDA, respectively. Neither basal LDH release nor that in response to maximal NMDA (100 microM) stimulation was altered by chronic alcohol exposure. In contrast to the effects of chronic ethanol on NMDA neurotoxicity, inclusion of ethanol (100 mM) only during the NMDA exposure period significantly reduced LDH release by approximately 50% in both control and chronically treated dishes. This reduction by acute ethanol was also observed under phase-contrast microscopy as a lack of development of granulation and a sparing of disintegration of neuritic processes. These results indicate that chronic exposure of ethanol to cerebral cortical neurons in culture can sensitize neurons to excitotoxic NMDA receptor activation.

Effect of calmodulin and protein kinase C inhibitors on globally ischemic rat hearts.

Several calmodulin inhibitors have been reported to be cardioprotective, but the ability of these compounds to inhibit protein kinase C (PKC) suggests that calmodulin inhibition may not be the sole mechanism responsible. To distinguish between the effects, we determined the cardioprotective activity of several calmodulin inhibitors with differing PKC inhibitory potencies in isolated globally ischemic rat hearts. Twenty-five minutes of global ischemia caused significant myocardial dysfunction, contracture formation, and lactate dehydrogenase (LDH) release on reperfusion in vehicle-treated hearts. The calmodulin inhibitors trifluoperazine, W-7, calmidazolium, W-13, and CGS 9343B improved postischemic contractile function and/or reduced LDH release. They also reduced preischemic cardiac function, although cardioprotection did not appear to be correlated with cardiodepression. Calmodulin inhibitors increased preischemic coronary flow (CF) and decreased heart rate (HR), but controlling these parameters did not affect the cardioprotection. Pretreatment of ischemic hearts with trifluoperazine was associated with preservation of myocardial ATP. Pretreatment of ischemic rat hearts with the PKC inhibitors staurosporine, calphostin C, polymyxin B, and H-7 did not result in cardioprotection. Thus, calmodulin inhibition causes cardioprotection that appears to be independent of PKC inhibition.

Prostaglandin but not cimetidine reduces spontaneous degeneration of isolated gastric gland cells.

We studied the effect of either placebo, 16,16-dimethyl-prostaglandin E2 (16,16-dimethyl-PGE2), or cimetidine on spontaneous degeneration of isolated rat gastric glands maintained in vitro in a basic oxygenated medium for 24 h. We assessed the viability of gland cells with fast green exclusion, measured release of lactate dehydrogenase (LDH) into the medium, and assessed the cell ultrastructure using a scanning electron microscope. Gastric glands incubated in medium for 6, 12, and 24 h underwent spontaneous degeneration reflected by a decrease in cell viability, increase in LDH release into the medium, and ultrastructural cell damage. 16,16-Dimethyl-PGE2 either at 0.1, 1, or 10 micrograms/ml significantly reduced the decrease in cell viability, increasing cell survival; reduced LDH release into the medium; and ultrastructural damage. Incubation with cimetidine at 1 or 10 micrograms/ml did not affect cell viability at 6, 12, or 24 h, whereas 100 micrograms/ml reduced cell viability (vs. placebo) at 12 and 24 h. LDH release and ultrastructural damage were not affected (not reduced) by cimetidine. Our study indicates that 16,16-dimethyl-PGE2, but not cimetidine, directly protects isolated gastric gland cells against degeneration in vitro, under conditions independent of systemic, neural, and hormonal factors.

Reduction of ischemic damage in isolated rat hearts by the potassium channel opener, RP 52891

Potassium channel activators have been shown to protect ischemic myocardium. We studied the ability of the novel potassium channel activator, RP 52891, to also reduce ischemic damage in isolated globally ischemic rat hearts. RP 52891 (1–100 μM) was given before the hearts were subjected to 25 nun of ischemia and 30 min of reperfusion. Before ischemia, RP 52891 reduced contractile function only at the highest concentration (100 μM). Significant reductions in ischemie damage were observed at 3 μM and higher concentrations. RP 52891 improved reperfusion contractile function and reduced lactate dehydrogenase release. Contracture was significantly reduced by RP 52891 during reperfusion. The protective effects of RP 52891 were completely reversed by glyburide and sodium 5-hydroxyde-canoate, both Mockers of ATP-sensitive potassium channels. Thus, RP 52891 has direct cardioprotective efficacy, which may be related to activation of ATP-sensitive potassium channels.

Ruthenium Red Improves Postischemic Contractile Function in Isolated Rat Hearts

The effect of ruthenium red (inhibitor of mitochondrial calcium entry) on reperfusion contractile function and enzyme release was determined in isolated perfused rat hearts and compared with that of diltiazem. The hearts were made ischemic for 25 min and reperfused for 30 min. They were pretreated with 1-10 microM ruthenium red, 1 microM diltiazem, or vehicle. All concentrations of ruthenium red significantly improved reperfusion contractile function without affecting lactate dehydrogenase (LDH) release or contracture. Diltiazem significantly improved reperfusion function and reduced LDH release and contracture formation. Ruthenium red still improved function even when given only during reperfusion. Diltiazem increased reperfusion oxygen consumption and efficiency of oxygen utilization whereas ruthenium red improved efficiency without an increase in oxygen consumption. Diltiazem significantly increased reperfusion functional reserve in these hearts, although ruthenium red did not. Ruthenium red reduced coronary flow and contractile function in nonischemic myocardial tissue and the reduced function appeared to be secondary to the reduced coronary flow as well as to a direct negative inotropic effect. Thus, ruthenium red improved reperfusion contractile function and oxygen efficiency; this may be related to its ability to block mitochondrial calcium uptake.

The effect of halothane, isoflurane, and verapamil on ischemic-isolated rabbit renal tubules.

The effects of the volatile anesthetics halothane and isoflurane, and the calcium entry blocker verapamil, were studied in isolated rabbit renal tubules under nonischemic and simulated ischemic conditions. Isolated rabbit renal tubules were subjected to zero (control), 30 (I-30), or 60 (I-60) minutes of simulated ischemia following the method of Weinberg. Following the ischemic period, tubules were reoxygenated in a Gilson respirometer (simulated reperfusion) and treated with either halothane (1%) or isoflurane (1%) in the controls and at I-30, or halothane (1%, 2%, 4%) or verapamil (5 microM, 15 microM, 30 microM) at I-60. Tubules were analyzed for lactate dehydrogenase (LDH) release (measuring cell membrane integrity), intracellular potassium and adenosine triphosphate (ATP), and oxygen consumption (cellular respiratory rate). In nonischemic tubules, exposure to 1% isoflurane caused significantly reduced LDH release compared with that released by controls, indicating cell membrane protection, whereas 1% halothane had no effect on these cells. With 30 min of ischemia, 1% isoflurane was associated with significantly higher cellular LDH release and lower ATP concentration, suggesting increased cellular damage. Halothane (1%) was associated with only an increased ATP concentration in tubules exposed to 30 min of ischemia. Following 60 min of ischemia, halothane (4%) decreased LDH release by 45% (29.2 +/- 2.3% vs. 47.0 +/- 9.6% without halothane). Tubules exposed to halothane also had higher intracellular potassium and ATP concentrations, and increased respiratory rates. Halothane (2%) was less protective and only increased the ATP concentration. The release of LDH was not statistically different with or without 2% halothane.(ABSTRACT TRUNCATED AT 250 WORDS)

Dissociation of Cardiodepression from Cardioprotection with Calcium Antagonists

This study was performed to determine if cardiodepression can be dissociated from cardioprotection with calcium antagonists and which one (diltiazem, nifedipine, or verapamil) can maximally protect ischemic myocardium at a given level of cardiodepression. Isolated rat hearts were subjected to 0.1, 0.5, or 1.0 microM diltiazem, verapamil, or nifedipine 10 min before global ischemia. Ischemia was maintained for 25 min, at which time reperfusion was instituted for 30 min. Pre- and postischemia function, flow, and lactate dehydrogenase (LDH) release were measured. All three drugs reduced preischemic function and improved postischemic function and reflow in a dose-dependent fashion. LDH release and contracture were also mitigated with all drugs. When the efficacy of these drugs was expressed as the ratio of LDH release versus preischemic, postdrug function (ability of drug to reduce LDH release at a given level of cardiodepression), diltiazem had a significantly lower ratio as compared with verapamil or nifedipine. When similar experiments were performed with various concentrations of calcium in the perfusion buffer (2.50, 1.25, 0.75, 0.50, 0.41 mM CaCl2) administered 10 min before ischemia and reperfusion with normal (1.25 mM) buffer, preischemic function was reduced in a concentration-dependent fashion. Despite severe reductions in function at the concentration of 0.50 mM CaCl2, LDH release was not reduced. The concentration of 0.41 mM CaCl2, which depressed function to the same degree as 0.50 mM CaCl2, reduced LDH release. This reduction in LDH release, however, was not as great as that which occurred with the high dose of the calcium antagonists. Reperfusion with 0.41 mM calcium buffer, however, nearly abolished LDH release. Thus, although all three calcium antagonists reduced the severity of ischemia, diltiazem reduces it with the lowest cost in cardiac function. Reduction in extracellular calcium reduces cardiac function, but reductions in severity of ischemia, as measured by LDH release, do not parallel these changes.

Adenosine, deoxyadenosine, and deoxyguanosine induce DNA cleavage in mouse thymocytes.

When thymocytes were cultured with adenosine, deoxyadenosine, or deoxyguanosine at 1 mM for 24 h, DNA cleavage at internucleosomal sites with multiples of approximately 180 bp was induced, followed by lactate dehydrogenase release into the medium. In the presence of coformycin, an adenosine deaminase inhibitor, or formycin B, a purine nucleoside phosphorylase inhibitor, DNA cleavage was induced by these nucleosides at concentrations of less than 50 microM. Other purine and pyrimidine ribo- and deoxyribonucleosides did not induce DNA cleavage or LDH release. Because thymocyte nuclei contain a Ca2+,Mg2+-dependent endonuclease, which preferentially cuts DNA in its linker regions, DNA fragmentation induced by the three purine nucleosides was suggested to occur through increased activity of the endonuclease. The DNA cleavage induced by the nucleosides required protein phosphorylation and synthesis, inasmuch as it was inhibited by an inhibitor of protein kinases, H-7, and by an inhibitor of protein synthesis, cycloheximide. The inhibition of DNA cleavage was accompanied by a reduction in lactate dehydrogenase release, suggesting a causal relationship between DNA cleavage and cell death. The DNA cleavage and subsequent cell lysis might be related to the selective thymocyte deletion observed in patients with adenosine deaminase or purine nucleoside phosphorylase deficiency.

Prostaglandin protection of human isolated gastric glands against indomethacin and ethanol injury. Evidence for direct cellular action of prostaglandin.

Isolated human gastric glands from surgical specimens were preincubated in an oxygenated medium with placebo or 16,16 dimethyl prostaglandin E2 (dmPGE2) and incubated at 37 degrees C in either medium alone, medium containing 4.43 mM indomethacin or medium containing 8% ethanol. We assessed the viability of gland cells with fast green exclusion, release of lactate dehydrogenase (LDH) into the medium, and ultrastructural damage by scanning and transmission electron microscopy. Both indomethacin and ethanol significantly reduced the viability of placebo-pretreated glands, increased LDH release into the medium, and produced prominent ultrastructural damage. DmPGE2 significantly reduced both indomethacin and ethanol-induced injury, increased the number of viable cells, reduced LDH release, and diminished the extent of ultrastructural damage. These studies indicate that PG protection of gastric mucosal cells has a direct cellular action that is not limited to replacement of depleted endogenous PGs. PG protection in our experiments did not depend on PG's previously described systemic actions, such as protection of the microvessels, preservation of the mucosal blood flow, or stimulation of bicarbonate and mucus secretion.

Protective effects of amiodarone pretreatment on mitochondrial function and high energy phosphates in ischaemic rat heart

The effects of the antianginal and antiarrhythmic drug amiodarone on mitochondrial function and high-energy phosphate content were assessed during normothermic ischaemic cardiac arrest and reperfusion in Langendorff-perfused rat heart. Total ischaemia for 30 min at 37 degrees C produced highly significant changes in mitochondrial oxidative phosphorylation and high-energy phosphate content. Pretreatment of the rats with one single dose of amiodarone (20 mg/kg i.v., 30 min before killing) markedly attenuated the deleterious effect of ischaemia on mitochondrial function and slightly reduced ATP depletion. In normally perfused hearts, amiodarone pretreatment did not modify any parameter of mitochondrial respiratory function nor did it influence high-energy phosphate or glycogen content. After reperfusion for 15 min, amiodarone-treated hearts showed improved recovery of mitochondrial oxidative phosphorylation and tissue high-energy phosphate content as compared to control hearts. Pretreatment of hearts with amiodarone did not reduce ischaemia-induced leakage of total adenylic nucleotides but highly significantly reduced lactate dehydrogenase release during reperfusion. These results indicate that amiodarone could exert substantial protection on the infarcting myocardium.

Sodium Arachidonate Induced In Vitro Polymorphonuclear Leukocyte Aggregation

Human polymorphonuclear leukocytes (PMNs) aggregated to sodium arachidonate (AA) in a dose dependent manner. Other agents such as epinephrine, or ADP were unable to initiate aggregation. The cells released lactate dehydrogenase (LDH) during aggregation, and electron microscopy demonstrated distinct morphologic changes in PMNs aggregated with AA. Indomethacin enhanced AA induced aggregation, whereas 1-methylimidazole inhibited aggregation and LDH release. Preincubation with cytochalasin B (CTB) did not enhance AA induced aggregation, nor did it reduce LDH release. Also, the effects of indomethacin or 1-methylimidazole on AA induced aggregation were unaltered by the presence of CTB. The results indicate that 1) LDH release occurs as a function of the aggregation process rather than a non-specific effect, 2) competition between cyclooxygenase and lipoxygenase occurs and 3) shifting this competition in favor of lipoxygenase enhances the PMN aggregation process.