Investigation of phospholipids of the pulmonary extracellular lining by electron paramagnetic resonance. The effects of phosphatidylglycerol and unsaturated phosphatidylcholines on the fluidity of dipalmitoyl phosphatidylcholine.

The membranous structures of the pulmonary extracellular lining were removed from the lungs of rabbits by pulmonary lavage and isolated by differential centrifugation. This membranous fraction contained 93% of the total extracellular phospholipids present in lavage effluents and consisted of membranous vesicles, membrane fragments, tubular myelin and secreted lamellar bodies. The fraction was rich in phosphatidylcholine (79.4%) containing 85.2% palmitic acid in the 1-position and 57.4% palmitic acid in the 2-position. Phosphatidylglycerol was the next most abundant phospholipid, accounting for 9.4% of the total. E.p.r. spectra, obtained by using 5-doxylmethylstearate as a probe, showed that the extracellular phospholipids of the pulmonary lining were organized into structures which were much more fluid than erythrocyte-ghost membranes. The fluidity of phosphatidylcholine isolated from the membranous fraction was similar to that of the fraction itself, indicating that the minor phospholipids had very little influence on the fluidity of the major phospholipid. At physiological temperature, the fluidity of dipalmitoyl phosphatidylcholine was relatively low, but could be markedly increased by the presence of 1-palmitoyl-2-oleoyl phosphatidylcholine or phosphatidylglycerol (10%). Protein present in the extracellular phospholipid fraction did not affect the fluidity of the fraction. These studies indicate that the unsaturated phosphatidylcholines could play a major role in determining the fluidity of the important surface-tension-lowering phospholipids such as dipalmitoyl phosphatidylcholine.

Isolation and characterization of rabbit lung lamellar bodies.

A method has been devised for the isolation of a highly purified preparation of lamellar bodies from rabbit lung. The purity of the preparation was confirmed by electron microscopy, marker enzymes, phospholipid composition, and isopycnic centrifugation on continuous density sucrose gradients. Contamination of the lamellar bodies by such subcellular components as mitochondria, nuclei, lysosomes and plasma membranes could be excluded; however, reduced nicotinamide adenine dinucleotide phosphate (NADPH) cytochrome c reductase, an enzyme specific for the endoplasmic reticulum components was a persistent contaminant in the preparation of the isolated lamellar bodies. When the lamellar bodies were subject to isopycnic centrifugation, all of the NADPH cytochrome c reductase activity was associated with the lamellar bodies in the low density peak; no reductase activity could be detected in the region of the density gradient demonstrated to localize microsomes. Use of 3H-radiolabeled microsomes confirmed that all of the NADPH cytochrome c reductase activity present in the lamellar body preparations could be accounted for by microsomal contamination. When lamellar bodies or liposomal membranes synthesized from the total phospholipid fraction of lamellar bodies were analyzed by the electron paramagnetic resonance probe, 5-dioxyl-methylstearate, they exhibited a high degree of fluidity at physiological temperature. This was in contrast to the low fluidity of liposomal membranes composed of pure dipalmitoylphosphatidylcholine, the major component (50%) of rabbit lamellar body phospholipids. Furthermore, the major temperature-dependent phase transition in lamellar body membranes occurred at a different temperature (30.5 degrees C) from that of dipalmitoyl-phosphatidylcholine (41.0 degrees C). It is clear, therefore, that the membrane fluidity of lamellar bodies must be highly influenced by the minor lipid component.

The effect of 2450-MHz microwave radiation during microtubular polymerization in vitro.

Exposure to 2450-MHz (cw) microwave radiation causes inhibition of cell division in intact cells and varied in vivo biological effects in both avian and mammalian species. Because these reported effects may result from alterations in the dynamics of microtubule formation, we studied the effects of simultaneous microwave exposure (2450 MHz, cw) during each of the three critical stages of the intracellar polymerization cycle. In addition, using circular dichroism spectroscopy, we studied the effect of microwave irradiation on the secondary structure of purified tubulin polypeptides. These studies were accomplished using specially constructed exposure systems that permit the continuous recording of turbidometric or circular dichroism measurements during simultaneous exposure to microwaves. The baseline turbidity of microtubular protein did not change under the influence of microwave radiation (20 or 200 mW/g SAR) and irradiation had no effect on the light-scattering properties of the depolymerized protein. EGTA-induced polymerization and cold-induced depolymerization patterns were also similar for both control and microwave-irradiated samples. The circular dichroism spectrum of purified tubulin also did not appear to be influenced by microwave irradiation, indicating a lack of effect on the protein secondary structure. The data suggest that the cellular effects of microwaves are not due to changes in microtubular proteins or their rate of polymerization.

A circular dichroism study of human erythrocyte ghost proteins during exposure to 2450 MHz microwave radiation.

The effect of 2450 MHz microwave radiation on the proteins of human erythrocyte ghosts has been investigated using circular dichroism spectroscopy. A specially constructed waveguide inserted into the spectropolarimeter allowed the continuous recording of optical activity before, during and after microwave irradiation. The data indicate that high levels of microwave radiation (600 mW/g, specific absorption rate) induce decreases in alpha-helical conformation that may result from both thermal vibrations and increased strain on the intramolecular hydrogen bonds that maintain secondary structure. The latter effect may result from differential intramolecular interactions with the oscillating electric field. Spectrin (bands 1 and 2) isolated from the ghosts was more sensitive to microwave irradiation than intact ghosts, and spectrin-depleted vesicles were the least sensitive. The data, therefore, indicate that the alpha-helical conformation of spectrin is altered by high levels of microwave radiation.

A novel method for the study of fluorescent probes in biological material during exposure to microwave radiation.

Instrumentation has been developed which allows the monitoring of fluorescence in erythrocyte ghost membranes before, during, and after exposure to microwave radiation. Using non-fluorescent, UV-transmitting fiber optic cables, excitation light of specific wavelengths was delivered to a stirred sample undergoing irradiation (2450 MHz, CW) within a fluid-filled, temperature-controlled waveguide. Fluorescence was collected using an identical cable and transferred through appropriate filters to standard detecting, amplification and recording devices. We have used the fluorescent probe, 1-anilino-8-naphthalene sulfonate (ANS) to monitor the effect of microwave radiation on the binding of calcium to erythrocyte ghosts. Microwave radiation at specific absorption rates of 10 and 200 mW/g had no effect on the binding of ANS to the membranes. Dose-response curves also showed no influence of microwaves on calcium binding between 2.0 and 10.0 x 10(-4) M. In addition, experiments studying fluorescence energy transfer between intrinsic tryptophan residues and membrane bound ANS showed that intermolecular distances between donor and acceptor are also unaffected by microwave radiation. We have thus shown that 2450 MHz microwave radiation at the specific absorption rates used does not interfere with the binding of calcium to erythrocyte ghosts or alter intermolecular distances between intrinsic molecules and bound ANS.

The effect of concentration on the binding of compound 48/80 to rat mast cells: a fluorescence microscopy study.

A fluorescent analog of the chemical histamine liberator, compound 48/80, has been synthesized by the covalent attachment of rhodamine to the 48/80 polymer (R-48/80). The histamine liberating characteristics of this analog were similar to those of the parent compound. The binding characteristics of R-48/80 to rat peritoneal mast cells were then studied using fluorescence microscopy. At concentrations that caused minimal secretory stimulation (less than 1.0 microgram/ml), R-48/80 bound to the mast cell surface in a diffuse manner, with no indication of patching or capping. When the cells were incubated at higher concentrations, where non-cytotoxic histamine secretion was stimulated, the drug bound heavily to the exposed granules, but not to unexposed granules or other cell organelles. At cytotoxic concentrations, R-48/80 caused extensive cell clumping, with the drug bound to masses of cell debris and released granules. Therefore, although R-48/80 binds initially to the cell membrane, its primary binding site at concentrations that induce secretion becomes the mast cell granule. The properties of these granules should thus be considered when studying the binding of compound 48/80 or other cationic drugs to rat peritoneal mast cells.

The effect of 2450 MHz microwave radiation on histamine secretion by rat peritoneal mast cells.

Isolated rat peritoneal mast cells actively secrete histamine in response to reaginic or chemical stimulation. Mast cells were irradiated in a waveguide microwave exposure chamber at 2450 MHz with power absorptions of 8.2 and 41.0 mW/g for periods up to 3 h. These levels of microwave absorption caused no change in the morphological characteristics or viability of the cells. Irradiated mast cells were stimulated with compound 48/80, a potent, noncytotoxic histamine releasing agent. The dose response curves showed that neither prior nor simultaneous irradiation of mast cells at 37 degrees C affected 48/80-induced secretion. However, microwave power absorptions of 41.0 mW/g inhibited secretion at 44.0 degrees C. Precise measurements of the effect of heat on secretion indicated that this level of inhibition could have been produced by a radiation induced increase in cell temperature between 0.4 and 0.9 degrees C above ambient levels. Alternatively, the heat stress produced at 44 degrees C may have sensitized the cells to the electromagnetic effects of the microwave radiation. Rat peritoneal mast cells can therefore be useful as a model for the study of functioning secretory cells during microwave irradiation and can also be used to monitor the synergistic effects of cell heating during in vitro exposure.