The role of hydroxyl radicals and calcium ions in the priming of a respiratory burst in neutrophils and the increase in luminol-dependent blood chemiluminescence on exposure to combined magnetic fields with a very weak low-frequency alternating component

The intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA AM) used at low concentrations (1.0 and 2.5 μM) was shown to block the priming effect of weak combined static (42 μT) and low-frequency collinear alternating (1.0, 4.4, and 16.5 Hz; 0.86 μT) magnetic fields. This blockage was inferred from a greater increase in chemiluminescence observed for a mouse neutrophil suspension exposed to combined magnetic fields in response to the bacterial peptide N-formyl–Met–Leu–Phe added in the presence of luminol. Similar results were obtained for the effect of BAPTA AM on luminol-dependent chemiluminescence of whole blood. The priming effect of weak combined magnetic fields on the respiratory burst in neutrophils did not depend on the presence of extracellular Ca²⁺ and was not affected by the hydroxyl radical scavenger dimethyl sulfoxide used at 0.025–1.0 mM.     

The Role of Oxygen in the Priming of Neutrophils on Exposure to a Weak Magnetic Field

A preliminary mild partial degassing of a neutrophil suspension at an atmospheric gas pressure of 640 mm Hg was accompanied by a decrease in oxygen to 412 ng-atom O/mL and was shown to cause a significant (fourfold) decrease in neutrophil priming index on exposure to combined weak magnetic fields (a static magnetic field of 42 μT and a low-frequency collinear alternating magnetic field of 860 nT; 1, 4.4, and 16.5 Hz) but did not affect the cell potential to generate a respiratory burst in response to an activator (the peptide N-formyl–Met–Leu–Phe) in the control. A partial replacement of the air mixture with carbogen, xenon, or sulfur hexafluoride reduced the intensity of luminol-dependent chemiluminescence of the samples.     

Priming of the respiratory burst in neutrophils exposed to a combination of weak constant and alternating low-frequency magnetic fields in vitro

An hour-long exposure of peritoneal neutrophils of mice to a combination of a weak constant magnetic field (42 μT) and low-frequency alternating magnetic fields collinear to the weak constant magnetic field (frequencies 1, 4.4, and 16.5 Hz, total amplitude 0.86 μT) at physiological temperatures promoted a significant increase in chemiluminescence of cells in response to subsequent exposure to low concentrations of respiratory burst activators (formylated peptide N-formyl-Met–Leu–Phe or phorbol ester phorbol-12-myristate-13-acetate) in the presence of luminol. The response of human neutrophils isolated from peripheral blood to the pretreatment with combined magnetic fields followed by exposure to the activator N-formyl-Met–Leu–Phe was similar to the response of mouse neutrophils.     

Effect of chronic ethanol treatment on the t-butyl hydroperoxide-dependent lipid peroxidation in rat liver

1. The effect of chronic ethanol consumption on the level of the t-butyl hydroperoxide (Bu'OOH)-induced lipid peroxidation in rat liver homogenate and subcellular fractions was measured using chemiluminescence technique and malondialdehyde formation. 2. It was shown that under the action of ethanol the rate of lipid peroxidation was decreased in the whole and "postnuclear" liver homogenates. 3. Ethanol significantly decreased the intensity of lipid peroxidation in microsomes, but did not affect the Bu'OOH-dependent process in mitochondria. 4. The level of lipid peroxidation was reduced after incubation of the total particulate fraction (mitochondria plus microsomes) with the undialysed cytosol from ethanol-treated rat liver. Dialysis of the cytosol prevented depressive effect of ethanol treatment on lipid peroxidation. 5. Reduced glutathione (0.1-1.0 mM) was shown to decrease the rate of lipid peroxidation in rat liver microsomes, but did not affect its level in mitochondria. 6. Pyrazole injections to rats reduced and phenobarbital treatment increased the level of the Bu'OOH-dependent lipid peroxidation in liver microsomes. 7. The data obtained indicate that the Bu'OOH-dependent lipid peroxidation is not an appropriate marker of the ethanol-induced oxidative stress in rat liver cells.     

NADPH-oxidase activity and lipid peroxidation in neutrophils from rats fed fat-rich diets

In order to investigate the effect of fat-rich diets on neutrophil functions, 21 day-aged rats were fed for 6 weeks with a control diet consisting of a regular laboratory rodent chow (4 per cent final fat content), a control diet supplied with soybean oil (15 per cent final fat content), or a control diet supplied with coconut oil (15 per cent final fat content). Glycogen-elicited peritoneal neutrophils from rats fed soybean and coconut oil-enriched diets presented a reduction in spontaneous and PMA-stimulated H2O2 generation relative to neutrophils from rats fed the control diet. The activity of superoxide dismutase, glutathione peroxidase and catalase did not change in animals fed fat-rich diets. In addition, the capacity to generate O2-, spontaneously or in response to PMA, did not change in neutrophils from animals fed fat-rich diets. Values attained matched those observed in animals fed the control diet, regardless of the method used to measure O2-, the superoxide dismutase-inhibitable reduction of cytochrome c or the lucigenin-dependent chemiluminescence. However, the initial rate of O2- generation both in resting neutrophils and in PMA-stimulated cells was significantly reduced when animals were fed with coconut or soybean oil-enriched diets due, at least in part, to a reduction in the activity of glucose-6-phosphate dehydrogenase. The concentration of thiobarbituric acid reactive substances, an index of lipid peroxidation, was increased in animals fed both fat-rich diets. This was accompanied by an increase in arachidonic acid content in these cells. Results presented suggest that lipid peroxidation in neutrophils from animals fed fat-rich diets may be associated with a consumption of H2O2 yielding more reactive oxygen-derived species such as the hydroxyl radical.     

Antioxidant capacity of allopurinol in biological systems

The antioxidant capacity of allopurinol was investigated in three biological systems by measurements of visible chemiluminescence, oxygen uptake and production of thiobarbituric acid reactive substances (TBARS). The addition of allopurinol to rat brain homogenates undergoing autoxidation and erythrocyte ghost membranes supplemented with 2,2'-azo-bis-(2-amidinopropane), in concentrations up to 2 mM, has a negligible effect on lipid peroxidation development. In erythrocyte ghost membranes exposed to gamma irradiation (9.5 Gy/min), allopurinol inhibits the radiation-induced lipid peroxidation with a Q(1/2) of 2.0 mM. It is suggested that allopurinol may have an alternative antioxidant pathway of action in biological systems, probably through a scavenging action upon hydroxyl radicals.     

Relative efficacies of indole antioxidants in reducing autoxidation and iron-induced lipid peroxidation in hamster testes

Increased iron stores are associated with free radical generation and carcinogenesis. Lipid peroxidation is involved in DNA damage, thus indirectly participating in the early steps of tumor initiation. Melatonin and structurally related indoles are effective in protecting against oxidative stress. The aim of the study was to compare the relative efficacies of melatonin, N-acetylserotonin (NAS), indole-3-propionic acid (IPA), and 5-hydroxy-indole-3-acetic acid (5HIAA) in altering basal and iron-induced lipid peroxidation in homogenates of hamster testes. To determine the effect of the indoles on the autoxidation of lipids, homogenates were incubated in the presence of each agent in concentrations of 0.0, 0.01, 0.05, 0.1, 0.25, 0.5, 0.75, 1.0, 2.0, 2.5, or 5.0 mM. To study their effects on induced lipid peroxidation, homogenates were incubated with FeSO(4) (30 microM + H(2)O(2) (0.1 mM) + each of the indoles in the same concentrations as above. The degree of lipid peroxidation was expressed as concentrations of malondialdehyde + 4-hydroxyalkenals (MDA + 4-HDA) per mg protein. The indoles decreased both basal and iron-related lipid peroxidation in a concentration-dependent manner. Melatonin reduced basal MDA + 4-HDA levels when used at the concentrations of 0.25 mM or higher, and prevented iron-induced lipid peroxidation at concentrations of 1.0, 2.0, 2.5, or 5.0 mM. The lowest effective concentrations of NAS required to lower basal and iron-related lipid peroxidation were 0.05 mM and 0.25 mM, respectively. IPA, only when used in the highest concentrations of 2.5 mM or 5 mM inhibited basal lipid peroxidation levels and it was ineffective on the levels of MDA + 4-HDA due to iron damage. 5HIAA reduced basal lipid peroxidation when used at concentrations of 0.25 mM or higher, and it prevented iron-induced lipid peroxidation only at the highest applied concentration (5 mM). In conclusion, melatonin and related indoles at pharmacological concentrations protect against both the autoxidation of lipids as well as induced peroxidation of lipids in testes. In doing so, these agents would be expected to reduce testicular cancer that is initiated by products of lipid peroxidation.     

Pholasin chemiluminescence detects mostly superoxide anion released from activated human neutrophils

The bioluminescent oxygen metabolite indicator protein pholasin was characterized with respect to the type and location of reactive oxygen metabolites detected in suspensions of stimulated human neutrophils. Whereas pholasin detected reactive oxygen metabolites from neutrophil suspensions stimulated with soluble agents, particulate stimulants were apparently not effective triggering agents for pholasin-dependent neutrophil chemiluminescence. Neutrophils stimulated with fMet-Leu-Phe (1 to 100 nmol/l) showed maximum pholasin-dependent chemiluminescence 45 to 60s after stimulation. The time of maximum chemiluminescence was virtually independent of fMet-Leu-Phe concentration. In contrast, the time to reach maximum light emission increased from 60s with 100 nmol/l phorbol ester to 295s with 1 nmol/l phorbol ester. Significant inhibition of stimulated chemiluminescence was caused by both superoxide dismutase (20 μg/ml, 80% inhibition) and reduction of the oxygen concentration in the incubation medium to less than 0.5 μmol/l (95% inhibition). In contrast, the myeloperoxidase inhibitor sodium azide (0.1 nmol/l) afforded only 50% inhibition of the pholasin-dependent neutrophil chemiluminescence. Our results show that pholasin detects superoxide radicals released from cells stimulated by soluble stimulants but not intracellular oxidative activity elicited by particulate stimulants.     

The priming of neutrophils by lipopolysaccharide for production of intracellular platelet-activating factor. Potential role in mediation of enhanced superoxide secretion

LPS priming of the neutrophil results in enhanced release of superoxide upon subsequent stimulation, but the mechanism of this effect remains obscure. The recent recognition that neutrophils synthesize and retain platelet-activating factor within the cell led us to hypothesize that enhanced synthesis of platelet-activating factor in the LPS-primed cell might account for the observed effects of lipopolysaccharide. Using human neutrophils isolated on plasma-Percoll gradients, we found that incubation with 100 ng/ml LPS for 60 min resulted in a small but significant increase in intracellular platelet-activating factor assessed after lipid extraction, TLC, and bioassay. The further stimulation of primed neutrophils with FMLP resulted in a marked increase in neutrophil platelet-activating factor compared with non-LPS-treated controls. The priming effect of LPS was time dependent (30 to 60 min), dose dependent, and inhibited at 0 degree C and did not require protein synthesis. Platelet-activating factor so generated was not released but rather retained within the neutrophil, and the molecular species of platelet-activating factor produced was predominantly 1-O-hexadecyl-2-acetyl-sn-3-phosphorylcholine. Platelet-activating factor production in LPS-treated neutrophils was also enhanced by PMA, suggesting that receptor-mediated events could not account exclusively for the enhancement. Considering the ability of nanomolar concentrations of exogenously added platelet-activating factor to prime the neutrophil for enhanced release of superoxide, the rapid intracellular accumulation of platelet-activating factor that accompanies stimulation of an LPS-primed cell by FMLP may modulate the secretory events that accompany such stimulation.     

Variations of the effect of insulin on neutrophil respiratory burst. The role of tyrosine kinases and phosphatases

The priming effect of insulin on the fMLP-induced respiratory burst of mouse neutrophils as well as the involvement of tyrosine protein kinases and phosphatases in this process have been studied. Peritoneal evoked neutrophils of NMRI strain mice were incubated with 0.01-100 nM insulin for 1-60 min at 22, 30, or 37°C and activated by 0.1-50 M N-formyl-methionyl-leucyl-phenylalanine (fMLP). The production of reactive oxygen species (ROS) by neutrophils was monitored by luminol-dependent chemiluminescence. We found that ¹²⁵I-labeled insulin binding by mouse neutrophils occurred with saturation and high affinity. Insulin itself did not change the basal level of the ROS production but could modulate fMLP-induced respiratory burst. The effect of insulin depended on temperature and duration of pretreatment of the neutrophils with insulin and the concentration combination of the insulin and fMLP. The tyrosine kinase inhibitor tyrphostin 51 decreased the fMLP-induced respiratory burst significantly. Insulin did not change the fMLP response of neutrophils pretreated with tyrphostin. However, the effect of tyrphostin on the response to 50 M fMLP was considerably decreased in neutrophils treated with insulin. There was no such effect during activation by 5 M fMLP, for which the priming effect of insulin was not observed. Insulin did not increase the fMLP-induced respiratory burst in neutrophils treated with the protein phosphatase inhibitors orthovanadate and pyrophosphate. If the inhibitors were added after insulin, the combined effect was nearly additive. It is possible that priming by insulin of the fMLP-induced respiratory burst is triggered by tyrosine phosphorylation, realized with its participation, and involves the signaling pathways initiated by tyrosine phosphorylation but subsequently is not dependent on the latter. The role of protein phosphatases in priming by insulin is of little importance. The data indirectly confirm the idea that priming of the neutrophil respiratory burst is a result of crosstalk of signaling pathways of the insulin and fMLP receptors with the participation of tyrosine phosphorylation.     

Comparison of potential protective effects of melatonin, indole-3-propionic acid, and propylthiouracil against lipid peroxidation caused by potassium bromate in the thyroid gland

Potassium bromate (KBrO3) is a prooxidant and carcinogen, inducing thyroid tumors. Melatonin and indole-3-propionic acid (IPA) are effective antioxidants. Some antioxidative effects of propylthiouracil (PTU)--a thyrostatic drug--have been found. The aim of the study was to compare protective effects of melatonin, IPA, and PTU against lipid peroxidation in the thyroids, collected from rats treated with KBrO3, and in homogenates of porcine thyroids, incubated in the presence of KBrO3. Wistar rats were administered KBrO3 (110 mg/kg b.w., i.p., on the 10th day of the experiment) and/or melatonin, or IPA (0.0645 mmol/kg b.w., i.p., twice daily, for 10 days), or PTU (0.025% solution in drinking water, for 10 days). Homogenates of porcine thyroids were incubated for 30 min in the presence of KBrO3 (5 mM) plus one of the antioxidants: melatonin (0.01, 0.1, 0.5, 1.0, 5.0, 7.5 mM), or IPA (0.01, 0.1, 0.5, 1.0, 5.0, 7.5, 10.0 mM), or PTU (0.01, 0.1, 0.5, 1.0, 5.0, 7.5, 10.0 mM). The level of lipid peroxidation products (MDA + 4-HDA) was measured spectrophotometrically in thyroid homogenates. In vivo pretreatment with either melatonin or with IPA or with PTU decreased lipid peroxidation caused by KBrO3--injections in rat thyroid gland. Under in vitro conditions, PTU (5.0, 7.5, and 10.0 mM), but neither melatonin nor IPA, reduced KBrO3-related lipid peroxidation in the homogenates of porcine thyroids. In conclusion, melatonin and IPA may be of great value as protective agents under conditions of exposure to KBrO3.     

Human granulocyte-macrophage colony-stimulating factor and other cytokines prime human neutrophils for enhanced arachidonic acid release and leukotriene B4 synthesis

Human recombinant granulocyte-macrophage CSF (GM-CSF) "primes" neutrophils for enhanced biologic responses to a number of secondary stimuli. Here, we examined the properties of neutrophil priming by GM-CSF and other growth factors such as human rTNF and granulocyte CSF. Although GM-CSF has a negligible direct effect on [3H]arachidonic acid release, it enhances or "primes" neutrophils for three- to fivefold increased release of [3H]arachidonic acid, induced by 1.0 microM A23187 and the chemotactants FMLP, platelet-activating factor, and leukotriene B4 (LTB4) (all 0.1 microM). The priming effects of GM-CSF were concentration- and time-dependent (maximum 100 pM, 1 h at 23 degrees C), and consistent with the determined dissociation constant of the human GM-CSF receptor. Indomethacin (10(-8) M), cycloheximide (100 micrograms/ml), and pertussis toxin (200 ng/ml, 2 h at 37 degrees C) had no effect on GM-CSF-, A23187, or platelet-activating factor-induced [3H]arachidonic acid release. The lipoxygenase inhibitor, nordihydroguaiaretic acid, however, totally abolished A23187-induced [3H]arachidonic acid release from both diluent- and GM-CSF-treated neutrophils. Consistent with this observation, we found that GM-CSF-pretreated neutrophils synthesize increased levels of LTB4 after stimulation with A23187 and chemotactic factors. GM-CSF enhances neutrophil arachidonic acid release and LTB4 synthesis, and thereby may amplify the inflammatory response to chemotactic factors and other physiologically relevant stimuli.     

4-Hydroxy-2,3-trans-nonenal stimulates microsomal lipid peroxidation by reducing the glutathione-dependent protection

Glutathione (GSH) protects liver microsomes against lipid peroxidation. This is probably due to the reduction of vitamin E radicals by GSH, a reaction catalyzed by a membrane-bound protein. Pretreatment of liver microsomes with 0.1 or 1mM 4-hydroxy-2,3-trans-nonenal (HNE), a major product of lipid peroxidation, reduces the GSH-dependent protection. GSH and vitamin E concentrations are not affected by this pretreatment. Pretreatment with 0.1 mM N-ethyl maleimide (NEM), a synthetic sulfhydryl reagent, resulted in a reduction similar to that with HNE of the GSH-dependent protection against lipid peroxidation. The reduction of the GSH-dependent protection by HNE and NEM is probably the result of inactivation of the membrane-bound protein by covalent binding to an essential SH group on the protein. If the GSH-dependent protection would proceed via the microsomal GSH transferase, pretreatment with NEM, which activates the microsomal GSH transferase, should enhance the GSH-dependent protection. Actually a decrease in the GSH-dependent protection is found. Apparently the GSH-dependent protection does not proceed via the microsomal GSH transferase. Also the microsomal phospholipase A2 is not involved, since addition of 0.1 mM mepacrine, an inhibitor of phospholipase A2, did not preclude the GSH-dependent protection. Once the process of lipid peroxidation, either in vivo or in vitro, has started, the protection of liver microsomes by GSH is less effective. This might be the result of formed HNE. In this way an endproduct of lipid peroxidation stimulates the process that generates this product.     

Pentoxifylline Pretreatment Enhances the Oxidative Burst Activity of Human Peripheral Blood Mononuclear Cells

The effect of pentoxifylline pretreatment on the lucigenin-augmented chemiluminescence and dismutase-inhibitable superoxide production of human neutrophils and mononuclear cells (MNCs) was studied. Pentoxifylline at 20–2,000 μg/ml enhanced the lucigenin-augmented chemiluminescence (118–165% of the control, P < 0.01) of phorbol myristate acetate (PMA)-stimulated MNC. Pentoxifylline at 20–2,000 μg/ml increased the MNC superoxide production, i.e., 142–171% of the control (P < 0.05) using PMA stimulation and 145–159% of the control (P < 0.01) using opsonized zymosan stimulation. In contrast, pentoxifylline (up to 2,000 μg/ml) did not influence the lucigenin-augmented chemiluminescence and superoxide production of human neutrophils, stimulated by either PMA or opsonized zymosan. These results suggest that pentoxifylline is an immunomodulator and may have potential usefulness in the enhancement of immune defenses in compromised hosts.     

Priming action of inositol hexakisphosphate (InsP6) on the stimulated respiratory burst in human neutrophils.

After priming by a number of different host, bacterial and chemical agents, human neutrophils may be stimulated to produce a greater respiratory burst than would be elicited by the stimulus alone. Other neutrophil functions may be similarly enhanced by pre-exposure to a priming agent. We describe here a new extracellular role for inositol hexakisphosphate (InsP6) as a priming agent for a variety of human neutrophil functional responses. Preincubation of the cells with InsP6 alone (up to 250 microM) has no stimulatory effect upon the basal production of reactive oxygen intermediates but the response to a subsequent stimulus (FMLP, PMA or phagocytic particles) is substantially enhanced. Levels 100-200% higher than 'stimulus only' controls have been recorded. Peak enhancement of the FMLP-induced oxidative response occurs after 1-2 min preincubation with InsP6 and the effect is dose-dependent (maximum at approx. 100 microM InsP6). As others have shown FMLP stimulation of superoxide anion production has no external Ca2+ dependence but the presence of low levels of Ca2+ and Mg2+ (0.1 mM) during priming appears to be an essential requirement for full expression. Reports of intracellular concentrations of InsP6 in mammalian cells in the 30-100 microM range suggest that the local release of this inositol polyphosphate from damaged or effect cells could have a physiologically important modulatory role on neutrophil functions.     

The effect of weak magnetic fields on the production of reactive oxygen species in neutrophils

It was shown that a 1-h-long exposure of mouse peritoneal neutrophils to a combination of a weak constant magnetic field (42 μT) and low-frequency alternating magnetic fields collinear to the weak constant magnetic field (the sum of the frequencies 1.0, 4.4, and 16.5 Hz; amplitude, 0.86 μT) at physiological temperatures caused an increase in the intracellular production of reactive oxygen species, as measured by the changes in fluorescence of the products of 2,7-dichlorodihydrofluorescein and dihydrorhodamine 123 oxidation. The effect of weak magnetic fields was significantly more pronounced in the presence of low concentrations of respiratory burst activators (N-formyl-Met–Leu–Phe or phorbol 12-meristate-13-acetate).

     

Effects of D-Glucose on Chemokinesis and Resting Production of Reactive Oxygen Species in Neutrophil Granulocytes of Lean or Obese-Hyperglycemic Mouse

The response to D-glucose (0–21 mM) was studied in neutrophil granulocytes from obese, hyperglycemic and hyperinsulinemic Umeå ob/ob mice and their lean, littermate controls in order to further elucidate the effects of in vivo and in vitro hyperglycemia on neutrophil function. Neutrophil random locomotion on glass and neutrophil resting luminol-enhanced chemiluminescence in cell suspension were studied. Random locomotion was stimulated by D-glucose in neutrophils from both Umeå ob/ob and control mice but the locomotive activity in Umeå ob/ob mouse neutrophils was significantly higher than that found in the controls at 4–21 mM glucose. In both types of mice, the stimulatory effect of D-glucose on random locomotion was diminished at 21 mM glucose (not significantly different from that at 0 mM glucose). Resting chemiluminescence from mouse neutrophils was also stimulated by glucose but here the magnitude of response was similar in neutrophils from both types of mice. These results indicate that chronic hyperglycemia and hyperinsulinemia in the Umeå ob/ob mouse may be associated with an increased neutrophil random locomotive activity but a similar resting production of reactive oxygen species, as compared with neutrophils from control mice at physiological and hyperglycemic glucose concentrations in vitro.     

Relationships between formaldehyde metabolism and toxicity and glutathione concentrations in isolated rat hepatocytes

The metabolism and toxicity of formaldehyde (CH2O) in isolated rat hepatocytes was found to be dependent upon the intracellular concentration of glutathione (GSH). Using hepatocytes depleted of GSH by treatment with diethyl maleate (DEM), the rate of CH2O (5.0 mM) disappearance was significantly decreased. Formaldehyde decreased the concentration of GSH in hepatocytes, probably by the extrusion of the CH2O-GSH adduct, S-hydroxymethylglutathione. Formaldehyde toxicity was potentiated in cells pretreated with 1.0 mM DEM as measured by the loss of membrane integrity (NADH stimulation of lactate dehydrogenase (LDH) activity) and an increase in lipid peroxidation (formation of thiobarbituric acid-reactive compounds). This potentiation of toxicity was both CH2O concentration-dependent and time-dependent. There was an excellent correlation between the increase in lipid peroxidation and the decrease in cell viability. L-Methionine (1.0 mM) both protected the cells from toxicity caused by the combination of 8.0 mM CH2O and 1.0 mM DEM and increased the cellular GSH concentration. The antioxidants, ascorbate, butylated hydroxytoluene (BHT) and alpha-tocopherol (10, 25 and 125 microM), all exhibited dose-dependent protection against toxicity produced by 8.0 mM CH2O and 1.0 mM DEM. At toxic concentrations of CH2O (10.0-13.0 mM), administered by itself, lipid peroxidation did not increase concomitantly with the decrease in cell viability and the addition of antioxidants (125 microM) did not influence CH2O toxicity. These results suggest that CH2O toxicity in GSH-depleted hepatocytes may be mediated by free radicals as a result of the effect of CH2O on a critical cellular pool of GSH. However, cells with normal concentrations of GSH are damaged by CH2O by a different mechanism.     

The effect of diuretics and vasopressin on the osmotic permeability of the frog urinary bladder.

1. Large concentrations (in mM) of ethacrynic acid (0.1), furosemide (1.0), theophylline (5.0) and osmotic diuretics (100.0) sharply increased the flux of water along an osmotic gradient through the frog urinary bladder wall. Spironolactone (0.1), and hydrochlorothiazide (5.0) showed only a weak action on osmotic permeability. MercusalR, clopamide and triamterene did not affect water transport. 2. The presence of 0.2--1.0 mU/ml vasopressin (ADH) after pretreatment with a diuretic did not result in summation of the effects of both drugs used. 0.01--0.1 mM ethacrynic acid and 0.01 mM MercusalR significantly decreased the reaction to ADH. 1.0 mM furosemide, 0.1 mM spironolactone, 0.01 mM clopamide and 0.8 mM acetazolamide did not change the reaction to ADH. A reduction in the cellular response to ADH and a decrease in the osmotic permeability of the tubular wall may be responsible in part for the diuretic action of ethacrynic acid and MercusalR.     

Melatonin preserves arachidonic and docosapentaenoic acids during ascorbate-Fe2+ peroxidation of rat testis microsomes and mitochondria

The pineal hormone melatonin (N-acetyl, 5-methoxytryptamine) was recently accepted to act as an antioxidant under both in vivo and in vitro conditions. In this study, we examined the possible preventive effect of melatonin on ascorbate-Fe(2+) lipid peroxidation of rat testis microsomes and mitochondria. Special attention was paid to the changes produced on the highly polyunsaturated fatty acids C20:4 n6 and C22:5 n6. The lipid peroxidation of testis microsomes or mitochondria produced a significant decrease of C20:4 n6 and C22:5 n6. The light emission (chemiluminescence) used as a marker of lipid peroxidation was similar in both kinds of organelles when the control and peroxidized groups were compared. Both long chain polyunsaturated fatty acids were protected when melatonin was incorporated either in microsomes or mitochondria. The melatonin concentration required to inhibit by 100% the lipid peroxidation process was 5.0 and 1.0mM in rat testis microsomes and mitochondria, respectively. IC 50 values calculated from the inhibition curve of melatonin on the chemiluminescence rates were higher in microsomes (4.98 mM) than in mitochondria (0.67 mM). The protective effect observed by melatonin in rat testis mitochondria was higher than that observed in microsomes which could be explained if we consider that the sum of C20:4 n6+C22:5 n6 in testis microsomes is two-fold greater than present in mitochondria.