P.R. Miles

Cytosolic factors which affect microsomal lipid peroxidation in lung and liver

Abstract Studies were carried out to determine the effects of lung and liver cytosol on pulmonary and hepatic mierosomal lipid peroxidation, to determine the cytosolic concentrations of various substances which affect lipid peroxidation, and to determine which of these substances is responsible for the effects of the cytosol on lipid peroxidation. Lung cytosol inhibits both enzymatic (NADPH-induced) and nonenzymatic (Fe2+-induced) lung microsomal lipid peroxidation. In contrast, liver cytosol stimulates lipid peroxidation in hepatic microsomes during incubation alone, enhances Fe2+-stimulated lipid peroxidation, and has no effect on the NADPH-induced response. Substances which are known to be involved in inhibition of lipid peroxidation, including glutathione, glutathione reductase, glutathione peroxidase, and superoxide dismutase, are found in greater concentrations in liver cytosol than in lung cytosol. However, ascorbate is found in approximately equal concentrations in pulmonary and hepatic cytosol. Most of the effects of the cytosol on lipid peroxidation seem to be due to ascorbate and glutathione. For example, ascorbate, in concentrations found in lung cytosol, inhibits lung microsomal lipid peroxidation to about the same extent as the cytosol. The effects of liver cytosol on hepatic microsomal lipid peroxidation can be duplicated by concentrations of ascorbate and glutathione normally found in the cytosol; i.e., ascorbate stimulates and glutathione inhibits lipid peroxidation with the net effect being similar to that of liver cytosol. The results indicate that ascorbate has opposite effects on pulmonary and hepatic microsomal lipid peroxidation and suggest that ascorbate plays a major role in protecting pulmonary tissue against the harmful effects of lipid peroxidation.

The relationship between chemiluminescence and lipid peroxidation in rat hepatic microsomes

Abstract Studies were carried out to determine the relationship between NADPH- and ascorbate-initiated chemiluminescence (CL) and lipid peroxidation (LP) in rat hepatic microsomes. NADPH-initiated CL and LP become maximal 15 min after addition of NADPH to the microsomes and ascorbate-initiated CL and LP become maximal 90 to 120 min following addition of ascorbate. There are four lines of evidence to indicate that both NADPH- and ascorbate-initiated chemiluminescence are related to lipid peroxidation. (i) The time courses for the increases in CL and in LP are identical. (ii) There is a linear relationship between total (integral) or maximal CL and LP. (iii) Drug substrates which inhibit LP also inhibit CL in a quantitatively similar manner. (iv) Inhibitors of lipid peroxidation, such as Co2+, Mn2+, Hg2+, para-chloromercuribenzenesulfonic acid, and EDTA, also inhibit chemiluminescence. The results of these experiments indicate that chemiluminescence initiated in hepatic microsomes by either NADPH or ascorbate is directly proportional to lipid peroxidation.

The alveolar type II epithelial cell: a multifunctional pneumocyte.

The epithelial surface of the alveoli is composed of alveolar type I and type II cells. Alveolar type I cells comprise 96% of the alveolar surface area. These cells are extremely thin, thus, minimizing diffusion distance between the alveolar air space and pulmonary capillary blood. Type II cells are spherical pneumocytes which comprise only 4% of the alveolar surface area, yet they constitute 60% of alveolar epithelial cells and 10-15% of all lung cells. Four major functions have been attributed to alveolar type II cells: (1) synthesis and secretion of surfactant; (2) xenobiotic metabolism; (3) transepithelial movement of water; and (4) regeneration of the alveolar epithelium following lung injury. Therefore, alveolar type II cells play important roles in normal pulmonary function and in the response of the lung to toxic compounds which may cause lung damage. Techniques have now been developed to isolate and purify alveolar type II epithelial cells from lung tissue. Such cellular preparations afford bioassay systems to monitor the effects of occupational or environmental pollutants on alveolar pneumocytes and should yield important information concerning the etiology of pulmonary disease in the alveolar region of the lung.

Luminol-dependent chemiluminescence analysis of cellular and humoral defects of phagocytosis using a chem-glo photometer

Abstract In summary, this report describes the conditions under which luminol has been utilized to measure phagocytosis-associated metabolic events in activated human PMNs and rabbit and dog alveolar macrophages. We feel that this system may have wide applicability to both clinical and experimental situations. Some possible applications are shown in the following table. Potential Applications of Luminol-Amplified Chemiluminescence Cell type Applications Human polymorphonuclear leukocytes (1) Detection of bactericidal defects, particularly chronic granulomatous disease (2) Detection of host opsonic defects (both immunoglobulin and complement −C3b opsonic defects). (3) Analysis of drug effects on host cellular and opsonic defenses ( 9,11 ). (4) Characterization of bacteria or other particulate matter in terms of ability to generate opsonic activity and/or be ingested by phagocytic cells ( 3,7 ). Alveolar macrophages (1) Detection of environmental pollutant effects on respiratory defense mechanisms (against both particulate and soluble matter). (2) Analysis of drug effects on respiratory defense mechanisms, particularly drugs administered in the treatment of respiratory diseases.

Effects of heavy metal ions on selected oxidative metabolic processes in rat alveolar macrophages

The effects of four heavy metal cations, Cd2+, Hg2+, Ni2+, and Pb2+, on oxygen consumption, glucose metabolism, and the release of active oxygen species (as measured by chemiluminescence) were studied in rat alveolar macrophages at rest (no phagocytosis) and during phagocytosis. All four heavy metals depress the oxygen consumption and glucose metabolism which occurs in alveolar macrophages at rest by about 60–95%. During phagocytosis there is release of reactive forms of oxygen from the cells, a two- to threefold increase in oxygen consumption, but no change in glucose metabolism from that which occurs in resting cells. The metals inhibit the release of active oxygen from the cells and the oxygen consumption which occurs during phagocytosis by 75–85%. The ED50 values, i.e., the concentrations of metals which produce one-half of the maximal effects, indicate that the mechanism for release of active oxygen is affected by much lower concentrations of metals than is oxygen consumption. Also, experiments with trypan blue provide evidence that the metals can affect oxidative metabolism without causing gross membrane damage. The results of these experiments indicate that heavy metals inhibit oxidative metabolic processes in alveolar macrophages and, thus, may diminish the antibacterial activity of these cells.

Inhibition of hepatic microsomal lipid peroxidation by drug substrates without drug metabolism

Experiments were performed to study the mechanism of action of drug substrates on lipid peroxidation in rat hepatic microsomes. Addition of the drug substrates, aniline, β-diethylaminoethyl diphenylpropylacetate (SKF-525A), aminopyrine, benzo[a]pyrene or ethylmorphine, to hepatic microsomes causes almost complete inhibition of NADPH-induced (enzymatic) lipid peroxidation. These substrates also produce similar inhibition of ascorbate-induced (non-enzymatic) lipid peroxidation in microsomes in which drug-metabolizing enzymes were inactivated by heat treatment. The substrate concentrations producing half-maximal inhibition (K12 are also similar for NADPH- and ascorbate-induced lipid peroxidation. Addition of metyrapone, an inhibitor of drug metabolism, has no effect on either the K12 values or on the maximal substrate inhibition of NADPH-induced lipid peroxidation. All five drug substrates also inhibit Fe2+-stimulated oxidation of linoleic acid. These results demonstrate that inhibition of lipid peroxidation in hepatic microsomes by drug substrates is independent of drug metabolism and is probably due to the antioxidant properties of the substrates.

Chemiluminescence associated with phagocytosis of foreign particles in rabbit alveolar macrophages.

Abstract Rabbit alveolar macrophages exhibit a chemiluminescent response which is associated with phagocytosis of zymosan and polystyrene-butadiene particles. The chemiluminescence reaches a peak in 15 to 25 minutes and then gradually diminishes over the next 1 to 3 hours. During the time of maximal light emission there appears to be no actual uptake of particles, but the response is dependent upon the particle concentration. The metabolic inhibitor, DNP (2,4-dinitrophenol), causes a rapid inhibition of the chemiluminescent response. The addition of ATP to the medium prior to exposure of the cells to particles causes the chemiluminescent response to be greatly diminished, i.e., 0.3mM ATP virtually abolishes the response. These experiments suggest that some metabolic response of the cell to phagocytosis is responsible for the chemiluminescence.

Factors Which Affect Superoxide Anion Release from Rat Alveolar Macrophages

In order to investigate some of the characteristics of superoxide anion release from alveolar macrophages, the effects of substances known to influence superoxide release from polymorphonuclear leukocytes (PMN) were studied in rat alveolar macrophages. There is a relatively small, but constant, amount of superoxide released from alveolar macrophages at rest. The amount released increases 5- to 6-fold and becomes maximal in about 20-30 min following exposure to unopsonized zymosan particles. The rate of superoxide release is maximal only 2 min after exposure of the cells to particles, i.e., long before particle uptake is complete. In addition to particles, release of superoxide anion can be stimulated by phorbol-12-myristate-13-acetate (PMA). Lectins and chemotactic factors, which stimulate release in PMN, have little or no effect in alveolar macrophages. Superoxide release during exposure to zymosan appears to be dependent upon extracellular Ca++. Also, the release mechanism can be affected by the addition of cyclic AMP or various protein modifiers to the medium. Since many of these findings differ from those reported by others for PMN, the control of superoxide anion release from alveolar macrophages and PMN is probably different.

Incorporation of [3H]palmitate into disaturated phosphatidylcholines in alveolar type II cells isolated by centrifugal elutriation

In order to study synthesis of pulmonary surfactant materials, we measured incorporation of [3H]palmitate into disaturated phosphatidylcholines (PC) in alveolar type II cells isolated by centrifugal elutriation. The time course for this process is not linear and, at high external palmitate levels (1 mM), incorporation is maximal in 4-5 h. Incorporation is dependent on extracellular palmitate with a Vmax (at 1 mM) of 1.66 nmol palmitate incorporated into disaturated PC/4.2 X 10(5) cells per 2 h and a K1/2 of 0.1 mM palmitate. Addition of an optimal amount of extracellular choline (0.05 mM) increases Vmax and decreases K1/2 for palmitate. Incorporation of palmitate is dependent upon cell number, inhibited by extracellular Ca2+ and stimulated by external Mg2+. Cholinergic and beta-adrenergic agonists do not increase incorporation. Pulmonary lavage fluid inhibits incorporation of palmitate into disaturated PC, suggesting there is negative feedback involved. Disaturated PC which has been recently synthesized (i.e., over a 2 h period) is broken down intracellularly by type II cells when they are suspended in palmitate-free medium. These results indicate that (1) several factors, such as substrate levels, cell number, Ca2+, Mg2+ and amount of surfactant present, are involved in the regulation of palmitate incorporation into disaturated PC; (2) disaturated PC which has been recently synthesized may be broken down by type II cells; and (3) surfactant synthesis in freshly isolated cells differs slightly from that reported by other investigators in type II cells maintained in primary cell culture.

Alterations in rat alveolar surfactant phospholipids and proteins induced by administration of chlorphentermine

Chlorphentermine is a cationic amphiphilic drug which produces a phospholipid storage disorder in rat lungs. Experiments were carried out to characterize changes in the composition of acellular alveolar lavage materials and to study possible mechanisms by which pulmonary surfactant phospholipidosis is produced by administration of the drug. Following ten daily injections of chlorphentermine (25 mg/kg body weight), there are 12.2- and 13.6-fold increases of pulmonary lavage total phospholipids and disaturated phosphatidylcholines (disaturated PC), respectively. In addition, there is a 2.8-fold increase in total protein and a 12.7-fold increase in the surfactant apoprotein group with molecular weights from 28,000 to 32,000. We measured incorporation of labeled palmitate, choline and glycerol into disaturated PC in type II cells and alveolar macrophages isolated from control and chlorphentermine-treated animals. The drug does not affect the incorporation of labeled substrates into disaturated PC in either cell type. However, in alveolar macrophages there is a decrease in the rate of intracellular degradation of recently synthesized disaturated PC in chlorphentermine-treated animals. The drug also inhibits the phospholipase-induced catabolism of rat surfactant disaturated PC which occurs during incubation of alveolar lavage fluid in vitro at 37 degrees C. When the lavage fluid is divided into subfractions by differential centrifugation, a larger percentage of the phospholipid is distributed in the less sedimentable subfractions in chlorphentermine-treated animals relative to controls, suggesting the accumulation of older surfactant materials. These results suggest that chlorphentermine-induced phospholipidosis of pulmonary surfactant materials is due to decreased rates of phospholipid degradation.