Increased production of superoxide anion by macrophages exposed in vitro to muramyl dipeptide or lipopolysaccharide.

After in vitro exposure to lipopolysaccharide (LPS) or muramyl dipeptide (MDP), cultured resident mouse peritoneal macrophages were primed to display enhanced generation of superoxide anion (O2-) in response to stimulation by phorbol myristate acetate (PMA) or opsonized zymosan. Priming with LPS (1 microgram/ml) produced a sevenfold enhancement of PMA-stimulated O2- generation; priming was detected within 30 min and persisted for at least 4 d. Exposure to MDP (1 muM) primed the macrophages to double their O2- release; the response was first observed after 4 h and persisted for at least 3 d. The priming response was not observed with stereoisomers of MDP, which are inactive as adjuvants. LPS and MDP appeared to work directly on the macrophages rather than indirectly by interacting with adherent lymphocytes: (a) Addition of nonadherent cell populations that contained lymphocytes had no effect on the response. (b) The response was normal with cells from nude mice, which lack mature T lymphocytes. (c) Macrophages from C3H/HeJ mice, whose B lymphocytes fail to respond to LPS, were weak in their response to priming LPS; the addition of normal (C3Heb/FeJ) nonadherent cells had no effect on this weak response. (d) The macrophage-like cell line J774.1 also showed enhanced O2--generating capacity after a 4-h exposure to LPS or MDP. The O2--generating capacity of macrophages primed with LPS in vitro was equivalent to that previously observed with cells elicited in vivo by injection of LPS or activated by infection with Bacille Calmette-Guérin. The data suggest that previous exposure to bacterial products could prime macrophages to respond with increased production of toxic oxygen metabolites on contact with invading microorganisms or tumor cells.

Cholesterol hydroperoxide formation in red cell membranes and photohemolysis in erythropoietic protoporphyria.

3beta-Hydroxy-5alpha-hydroperoxy-Delta(6)-cholestene is produced in protoporphyrin-containing red blood cell ghosts irradiated with approximately 400-nanometer light in the presence of oxygen. Incorporation of this cholesterol photooxidation product into normal red blood cells leads to increased osmotic fragility and eventual hemolysis. These results may be relevant to photohemolysis associated with erythropoietic protoporphyria.

Formation of an intermediate in prostaglandin biosynthesis and its association with the platelet release reaction.

A compound that could be converted to prostaglandin F(2alpha) by mild chemical reduction was formed by human platelets in response to arachidonic acid, collagen, or L-epinephrine. It was present in maximal amounts at about 1 min after addition of arachidonic acid or collagen to platelet-rich plasma. Its initial formation appeared to precede platelet aggregation by these agents and was closely correlated with the release of adenine nucleotides and radioactive 5-hydroxytryptamine from platelets. Moreover, the compound was itself found outside the platelets. This compound is probably an endoperoxide intermediate in prostaglandin biosynthesis and may be a trigger for the platelet release reaction.

The effect of an NADH oxidase inhibitor (hydrocortisone) on polymorphonuclear leukocyte bactericidal activity.

Polymorphonuclear neutrophils (PMN) from patients with chronic granulomatous disease of childhood have impaired bactericidal activity and are deficient in diphosphopyridine nucleotide, reduced form of, (NADH) oxidase. Since hydrocortisone had been shown to inhibit NADH oxidation, experiments were undertaken to determine the effect of hydrocortisone on several parameters of human PMN function. The phagocytic and bactericidal capacity of PMN with or without hydrocortisone (2.1 mM) was determined by quantitation of cell-free, cell-associated, and total bacteria. Phagocytosis of Staphylococcus aureus and several gram-negative rods was unimpaired by the presence of hydrocortisone in the media. In contrast, killing of bacteria was markedly impaired by hydrocortisone. After 30 min of incubation, there were 20-400 times as many bacteria surviving in hydrocortisone-treated PMN as in simultaneously run controls without hydrocortisone. The defect of intracellular killing noted in the presence of hydrocortisone was not related to impaired degranulation. Quantitative kinetic studies of degranulation revealed no difference in the release of granule associated acid phosphatase in hydrocortisone-treated and control PMN after phagocytosis. Electron microscopy of PMN also indicated that the presence of hydrocortisone had no effect on the extent of degranulation after phagocytosis. These observations were confirmed by studies using histochemical techniques to detect lysosomal enzymes. After phagocytosis, hydrocortisone-treated PMN demonstrated less NADH oxidase activity, oxygen consumption, and hydrogen peroxide production than postphagocytic control PMN. In addition, Nitro blue tetrazolium dye reduction was diminished in hydrocortisone-treated PMN.Thus, impairment of NADH oxidase activity in normal human PMN by hydrocortisone results in reduced intracellular killing of bacteria, diminished postphagocytic oxygen consumption, decreased ability to reduce Nitro blue tetrazolium, and decreased hydrogen peroxide production. These abnormalities are similar to those seen in the PMN of patients with chronic granulomatous disease of childhood.

On the mechanism of enhanced monocyte and neutrophil cytotoxicity in severe psoriasis.

Monocyte and neutrophil function assessed as antibody-dependent cell-mediated cytotoxicity (ADCC) using IgG-sensitizing human erythrocytes as target cells was enhanced in patients with severe psoriasis when compared to healthy controls. We found significant correlation between increased monocyte ADCC and increased neutrophil ADCC, No differences in basal cAMP levels and cAMP responses during initiation of the ADCC reaction was observed between psoriatics and normals. Also degranulation determined as lysozyme release during ADCC was normal. In contrast, the increase in ADCC was significantly correlated to an enhanced hexose monophosphate shunt activation in the effector cells during the cytotoxic reaction. Activity of enzymes responsible for the respiratory burst was not altered in psoriasis since superoxide production after stimulation with phorbol myristate acetate was normal. Likewise, oxygen consumption and degranulation following phagocytosis of opsonized zymosan particles in neutrophils was found normal in psoriasis. Since monocytes showed increased binding of IgG-sensitized erythrocytes these data indicate that the enhanced monocyte and neutrophil ADCC is caused by an enhancement of the respiratory burst possibly induced by increased Fc receptor activity.

Selective inhibitory actions of sodium-p-benzyl-4-[1-oxo-2-(4-chlorobenzyl)-3-phenyl propyl] phenyl phosphonate (N-0161) and indomethacin on the biosynthesis of prostaglandins and thromboxanes from arachidonic acid.

1 Sodium p-benzyl-4-[1-oxo-2-(4-chlorobenzyl)-3-phenyl propyl]phenyl phosphonate (N-0164) selectively inhibited the formation of thromboxane-A(2) from prostaglandin endoperoxides by human platelet microsomes in a dose-dependent manner (IC(50) 2.2 x 10(-5) M or 11.6 mug/ml).2 N-0164 was approximately 15 to 20 times as potent as indomethacin as an inhibitor of thromboxane-A(2) formation. In contrast, indomethacin was 20 times as potent as N-0164 as an inhibitor of prostaglandin endoperoxide formation from arachidonic acid (IC(50) 2.6 x 10(-5) M or 9.4 mug/ml).3 Spiral strips of dog coronary arteries relaxed in the presence of prostaglandin endoperoxides and were contracted by prostaglandin E(2) and thromboxane-A(2) and were therefore used to distinguish between prostaglandins and their intermediate precursors, the endoperoxides.4 Neither indomethacin nor N-0164 (both 50 mug/ml) significantly inhibited the formation of prostaglandin-like activity from the endoperoxides following incubation with indomethacin-pretreated rabbit kidney medulla microsomes.5 It is not known whether this action of N-0164 is related to its ability to antagonize certain actions of prostaglandins (and related compounds) or whether N-0164 can penetrate the cell membrane to inhibit thromboxane formation in the intact cell.6 Selective inhibition of thromboxane formation by drugs such as N-0164 may be useful both clinically and as a pharmacological tool to elucidate the patho-physiological roles of the thromboxanes.

Morphology and function of Malpighian tubules and associated structures in the cockroach, Periplaneta americana.

This paper describes the different regions of the Malpighian tubules and the associated structures (ampulla, midgut, ileum) in the cockroach, Periplaneta americana. There are about 150 tubules in each insect. Each tubule consists of at least three parts. The short distal region is thinner than the other parts and is highly contractile. The middle region comprises most of the tubule length and is composed of primary and stellate cells. Primary cells contain numerous refractile mineral concretions, while stellate cells have smaller nuclei, fewer organelles, simpler brush border, and numerous multivesicular bodies. Symbiont protozoa are sometimes present within the lumen of the middle region near where it opens into the proximal region of the tubule. The latter is a short region that drains the tubular fluid into one of the six ampullae. These are contractile diverticula of the intestine located at the midgut-hindgut junction. The ampulla is highly contractile, and consists of a layer of epithelial cells surrounding a cavity that opens into the gut via a narrow slit lined by cells of unusual morphology. The proximal region of the tubule and the ampulla resemble the midgut in that they have similar micromal origin and reabsorptive function for the proximal region of the tubule and for the ampulla. A number of inclusions found within the tubule cells are described, including peroxisomes and modified mitochondria. Current theories of fluid transport are evaluated with regard to physiological and morphological characteristics of Malpighian tubules. The possible role of long narrow channels such as those between microvilli and within basal folds is considered, as is the mechanism by which these structures are formed and maintained. Also discussed is the role of peroxisomes and symbionts in the excretory process.

Effect of deuterium oxide on neutrophil oxidative metabolism, phagocytosis, and lysosomal enzyme release.

We have previously shown that deuterium oxide (D2O) enhances the oxidation of methionine, a myeloperoxidase (MPO) -mediated reaction, by human neutrophils during phagocytosis. However, D2O has no effect on the oxidation of methionine by the purified MPO-H2O2-Cl- system. To explain this observation, we studied the effect of D2O on the oxidative metabolism, phagocytosis, and lysosomal enzyme release by human neutrophils. D2O stimulated the hexose monophosphate shunt (HMS) activity of resting neutrophils in a dose-response fashion. In the presence of latex particles or phorbol myristate acetate (PMA), D2O brought about an exaggerated stimulation of the HMS activity. This enhancement of the HMS activity by D2O was markedly reduced when neutrophils form two patients with X-linked chronic granulomatous disease (CGD) were used, either in the presence or absence of latex particles or PMA. Superoxide and H2O2 production by neutrophils in the presence of latex particles or PMA were also stimulated by D2O. In contrast, D2O inhibited the ingestion of latex particles. D2O enhanced the extracellular release of MPO, but not lactate dehydrogenase, by neutrophils only in the simultaneous presence of cytochalasin B and latex particles. The enhancement of HMS activity and MPO release by D2O was partially inhibited by colchicine. Our results suggest that enhancement of neutrophil oxidative metabolism by D2O may in part explain the stimulation of methionine oxidation by phagocytosing neutrophils.

Preparation and purification of lipid hydroperoxides from arachidonic and gamma-linolenic acids.

Commercial soybean lipoxygenase may be used under carefully controlled reaction conditions to give high yields of lipid hydroperoxides. Lipid hydroperoxides so derived from gamma-linolenic or arachidonic acid may be purified by high pressure liquid chromatography. Thus, commercial lipoxygenase serves as a viable source for 100 mg quantities of lipid hydroperoxides.

Peroxide formation, vitamin E and myocardial damage in the rat.

Rats were fed diets containing 10% cod liver oil with or without dietary tocopherol for 16 or 32 weeks. Adipose tissue, skeletal muscle and myocardium were isolated. They were examined histologically and analyzed for autoxidation products and a number of energy metabolites. After 16 weeks small amounts of peroxides were present in adipose tissue as determined by the thiocyanate method. Ceroid pigment and slightly defective striation were observed in myocardium and to a lesser extent in skeletal muscle of the tocopherol deficient group. The ATP content was also significantly lower in this group. After 32 weeks, adipose tissue of the tocopherol deficient group contained large amounts of ceroid and had a high content of peroxides and other autoxidation products. More ceroid and a significantly higher peroxide estimate in lipid extracts were found in myocardium of the tocopherol deficient group as compared to the controls. Similar but weaker signs of peroxide occurrence and ceroid formation were obtained in skeletal muscle. The significance of the findings for myocardial function is discussed.

[Free radical lipid oxidation and several ways of regulating it with ascorbic acid]. / Svobodnoradikal'noe oskilenie lipidov i nekotorye puti ego reguliatsii askorbinovoi kislotoi.

The correlation between the content of primary molecular product of free radical fat acyls oxidation--hydrolipid peroxidation and ascorbic acid amount in guinea-pig tissue was presented by means of polarographic method. The data obtained led to the following conclusion: the importance of ascorbic acid as prooxidant in vivo is rather great and its deficiency is the reason for an increase of its content in the primary products of lipid free radical oxidation, which probably plays the main role in C-avitaminosis pathogenesis.

[Participation of phospholipases in the "repair" of photoreceptor membranes subjected to peroxidation]. / Ob uchastii fosfolipaz v "reparatsii" fotoretseptornykh membran, podvergshikhsia perekisnomu okisleniiu.

Results are presented showing that Ca++-activated endogenous phospholipases are able to catalyze hydrolysis of hydroperoxydiacyglycerophosphatides with subsequent formation of monoacyglycerophosphatides and free hydroperoxides of fatty acids in frog rod outer segment membranes. The role of endogenous phospholipases in the mechanisms of repair of photoreceptor membranes subjected to lipid peroxidation, as well as in "molecular substitution" of phospholipids under membrane renewal is discussed.

[Alteration of lipid peroxidation of rat liver endoplasmic reticulum in the presence of steroid hormones]. / Izmenenie perekisnogo okisleniia lipidov éndoplazmaticheskogo retikuluma pecheni krys v prisutstvii steroidnykh gormonov.

Effect of steroid hormones on peroxidation of lipids of rats' liver endoplasmic reticulum was studied. Accumulation of peroxidation products was measured by the change in chemoluminescence intensity and by the rate of malone dealdehydeformation. It was shown that typical antioxidants--estrogenes were the inhibitors of NADH-H and ascorbate-dependent systems of lipid peroxidation in rat liver microsomes, the enzymatic system being the most sensitive one.

[Role of superoxide anion-radicals and superoxide dismutase in the lipid photoperoxidation reactions of isolated chloroplasts]. / Rol' superoksidnykh anion-radikalov i superoksiddismutazy v reaktsiiakh fotopereokisleniia lipidov izolirovannykh khloroplastov.

Exogenous superoxide dismutase (SOD, erythrocuprein) inhibits the chlorophyll bleaching and photoperoxidation of polyunsaturated lipids in pea isolated chloroplasts. As SOD did not produce this effect in tris-HCl buffer, it is suggested that peroxidation is mediated by the OH radicals which may appear in illuminated chloroplasts with the participation of superoxide radicals and hydrogen peroxide. The physiological role of SOD is discussed in relation to the stability of cells photosynthesizing organisms towards light and oxygen effect.

[Role of lipid composition in the kinetics of free radical lipid oxidation in photoreceptor membranes]. / O roli lipidnogo sostava v kinetike svobodnoradikal'nogo okisleniia lipidov v membranakh fotoretseptorov.

The initial rate of lipid peroxidation (LPO) in photoreceptor membranes (PRM) of walleye pollock is 1.8--2.3 times higher than in frog PRM. Rhodopsin bleaching leads to an increase of LPO initial rate in PRM while having no effect on LPO in walleye pollack PRM. The content of unsaturated fatty acids in walleye pollock PRM is 1.4 times greater than in frog PRM. It is suggested that differences in LPO kinetics in PRM of walleye pollack and frog are caused by the differences in the lipid composition of the membranes (primarily by a high level of phospholipid molecular species with two unsaturated fatty acid residues in walleye pollock retina rod outer segments).

[Interrelationship between structural and functional modifications in the membranes of the sarcoplasmic reticulum following lipid peroxidation]. / Vzaimosviaz' strukturnykh i funktsional'nykh perestroek v membranakh sarkoplazmaticheskogo retikulums pri perekisnom okislenii lipidov.

Structural and functional modifications of sarcoplasmic reticulum membranes from skeletal muscles by molecular oxygen was studied. Lipid peroxidation (accumulation of 5-10 nmoles hydroperoxides/mg lipids) results in membrane permeability increase for Ca2+-ions whereas the activity of Ca2+-dependant ATPase preserved unchanged. In the temperature range 5-30 degrees C decrease of solubilization parameter alpha (for nitroxide radical 2,2,6,6-tetramethylpiperidin-1-oxyl) was registered while alpha reached the control values at temperatures higher than 30 degrees C. Further increase in lipoperoxide content (more than 20 nmoles/mg lipids) lead to inhibition of Ca2+dependant ATPase. In this case alpha was lower than in intack membranes in the whole temperature interval investigated. The loss of Ca2+-accumulating capacity is explained on the basis of peroxide clusters formation in lipid bilayer regions of sarcoplasmic membrane. One of the mechanisms of Ca2+-dependant ATPase inhibition after lipid peroxidation is the deficiency of polyunsaturated fatty acyls in microenvironment of the enzyme.

[Effect of alpha-tocopherol on phospholipid concentration and lipid peroxidation in rat brain tissue following a burn injury]. / Deistvie alpha-tokoferola na soderzhanie fosfolipidov i perekisnoe okislenie lipidov v tkani mozga krys posle ozhogovoi travmy.

Studies of the lipid peroxides and phospholipid content in the rat brain in different periods after burns showed that the total phospholipid content in all the periods is decreased. The daily intraperitoneal administration of alpha-tocopherol in a dose of 100 mg/kg against a background of the burn decreases sharply the phospholipid content. alpha-Tocopherol in a dose of 1 mg/kg, administered immediately after the trauma and then 3, 7, 12 and 17 days after burns restores the phospholipid content to normal. The described changes were connected mainly with the lecithin and phosphatidylethanolamine fractions.

[Acrylonitrile stimulation of lipid peroxidation in rat liver]. / Stimuliatsiia akrilonitrilom perekisnogo okisleniia lipidov v pecheni krys.

Acrylonitrile stimulated slightly the lipid peroxidation in isolated microsomes and exhibited distinct prooxidative effect in postmitochondrial supernatant, containing microsomes and cell juice. Acrylonitrile did not form a complex with cytochrome P-450, possessing distinct spectral properties of the I or II types, and did not stimulate the oxygen utilization by microsomes in presence of NADPH. The hepatotoxic effect of acrylonitrile appears to be due to its property to stimulate the formation of lipid peroxides in hepatocytes.