Mary J. Ortner

Spectroscopic studies of rat mast cells, mouse mastocytoma cells, and compound 48/80—III: Evidence for a protein binding site for compound 48/80

Abstract The interaction of compound 48/80 with mast cells, mastocytoma cells, isolated membranes and liposomes has been studied using electron spin resonance (e.s.r.) and light-scattering techniques. Unlike spin-labeled stearic acid, spin-labeled 48/80 (SL-48/80) bound to mastocytoma cells in a manner that was unaffected by the non-ionic detergent, Triton X-100. In contrast, sodium dodecylsulfate (SDS), a protein denaturant, altered the SL-48/80 binding site(s), causing an apparent increase in the motion of the spin label. Treatment of SL-48/80-labeled mast cells with trypsin also increased the apparent motion of the bound label. Binding of 48/80 to mast cells, mastocytoma cells, erythrocyte ghosts, and isolated mastocytoma cell membranes increased their respective light-scattering properties. In contrast, the Rayleigh scatter of protein-free liposomes was unaffected by 48/80 treatment. Cationic, anionic, and non-ionic detergents also increased the light-scattering properties of mastocytoma cells, in contrast to the effect of several other cationic drugs, including histamine liberators. Increased light-scattering occurred after 48/80 treatment of inhibited mast cells (treated at 5°), indicating that the effect was not a consequence of degranulation. Untreated cells heated above 40° showed a similar increase in light scattering that became irreversible after reaching 45°. This indicates that cells treated with 48/80 resemble those in which the proteins have been denatured by heat. The light-scattering and the e.s.r. data, therefore, are consistent with the hypothesis that 48/80 binds to proteins in biological membranes.

THE EFFECT OF PROTOPORPHYRIN ON HISTAMINE SECRETION BY RAT PERITONEAL MAST CELLS: A DUAL PHOTOTOXIC REACTION

Abstract— Protoporphyrin‐induced phototoxicity in rat peritoneal mast cells was manifested either by inhibition of 48/80‐stimulated histamine secretion or by cell lysis. At a protoporphyrin concentration of 100ng/m/ (0.17 μM), histamine secretion was completely inhibited after 30min illumination. After initiation, the inhibited state progressed in the dark, and was irreversible, however, it did not develop into cell lysis. More severe phototoxic reactions in mast cells could not be produced by increasing the PP concentration or the incubation time; however, cell lysis was evoked by increasing the light intensity between 180–950W/m2, using a light source with emission maxima in the 350–470nm region. Dual phototoxic effects could also be demonstrated in erythrocytes by manipulating the illumination conditions. Increased resistance to osmotic lysis was seen under moderate conditions, and decreased resistance and cell lysis were seen under severe conditions. In the absence of protoporphyrin, the effect of light alone on mast cells was similar to protoporphyrin‐phototoxicity, although the light intensities required were higher both for inhibition (60–130W/m2) and lysis (280–950W/m2). The data therefore indicate that certain cell functions can be specifically disrupted by phototoxic reactions that are not cytotoxic; however, phototoxic reactions that lead to severe membrane protein denaturation and cell lysis also occur. The manifestation of these dual effects depends on the intensity of illumination in the 350–470nm region.

Spectroscopic studies of rat mast cells, mouse mastocytoma cells, and compound 48/80—I: A spin-label study of membrane fluidities and the effect of 48/80

Abstract Purified rat mast cells, mixed peritoneal leukocytes and mouse mastocytoma cells take up spin-labeled membrane probes (5-doxyl, 12-doxyl and 16-doxyl stearic acids and their methyl stearate counterparts) in a manner similar to that of other cell types. The electron spin resonance spectra of these probes suggest that the membrane fluidities in the regions probed are similar in both mast cells and other normal leukocytes of the rat peritoneal cavity. However, the membranes of the mastocytoma cells were significantly more fluid in all of the regions probed. Membrane fluidity differences reported by the methyl stearate probes were greater than those reported by the corresponding fatty acid probes. This suggests that the chemical nature of the probe may contribute significantly to the apparent reported fluidity. The histamine liberator, compound 48/80, did not affect the spectra of any of the probes at concentrations that were magnetically dilute; however, it reduced the degree of spin exchange at high probe concentrations in mastocytoma cells. This suggests that 48/80 may increase the volume of the mastocytoma cell membranes, resulting in reduced intermolecular interactions among the probe molecules.

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 α-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 α-helical conformation of spectrin is altered by high levels of microwave radiation.

Studies on acute in vivo exposure of rats to 2450-MHz microwave radiation. 1. Mast cells and basophils

Eight-hour continuous exposure to 2450-MHz microwave radiation in rats was carried out at incident power densities that cause no apparent temperature increase (2 and 10 mW/cm/sup 2/). The peritoneal mast cells were unchanged in their viability, percentage, toluidine blue metachromasia, histamine content, or size. Isolated mast cells from control and irradiated rats responded in a similar manner to drugs that stimulate histamine secretion via both the chemical (compound 48/80, a condensation product of p-methoxy-N-methyl phenethylamine and formaldehyde) and immunological (concanavalin A) pathways. Rats subjected to both levels of irradiation were similar to the controls in their initial blood pressures and their hyposensitive response to 48/80.

The oxidation of endogenous ascorbic acid during histamine secretion by rat peritoneal mast cells

Abstract Endogenous ascorbic acid is oxidized to the free radical species by rat mast cells during histamine secretion. This antioxidant may function as a radical scavenger of Superoxide and other membrane-damaging radicals known to be liberated by this process. The high levels of ascorbic acid in mast cells may, therefore, function to protect the cell membrane from oxidative damage and thereby promote cell survival after an extensive secretory response.

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.

Studies on acute in vivo exposure of rats to 2450-MHz microwave radiation. III. Biochemical and hematologic effects.

Male rats were exposed to 2450-MHz cw microwave radiation for 8 hr at incident power densities of 0 (sham), 2, or 10 mW/cm/sup 2/. Following exposure, rats were killed by decapitation, and blood samples were collected for determination of hematocrit, hemoglobin, red and white cell count, and differential white cell percentages. The total red and white cell counts were not affected by either exposure level. The blood hemoglobin level was also unaffected by the 8-hr microwave exposure, having a value of approximately 15.5 g% for all three groups. The percentages of lymphocytes and neutrophils for both exposed groups was similar to those of the sham group. The other cell types were also unchanged by the microwave exposure. None of the serum biochemistries examined were affected by either microwave exposure level. These data therefore demonstrate that acute (8 hr) exposure to 2450-MHz cw microwave radiation has no effect on the hematologic and biochemical parameters examined.

Studies on Acute in Vivo Exposure of Rats to 2450-MHz Microwave Radiation: II. Effects on Thyroid and Adrenal Axes Hormones

The effects of 8 hr continuous exposure of rats to 2450-MHz (cw) microwave radiation were studied at incident power densities of 0, 2 and 10 mW/cm/sup 2/. Thyroid axis function, as measured by serum thyroxine (T/sub 4/) and triiodothyronine (T/sub 3/) as well as T/sub 3/ uptake, free thyroxine index, and adjusted-T/sub 4/ values was not altered by the experimental conditions. Adrenal axis activity was also unaffected in rats exposed to 2 mW/cm/sup 2/ microwave radiation for 8 hr. In the 10 mW/cm/sup 2/ group, the serum corticosterone levels were less than in the 0 or 2 mW/cm/sup 2/ groups (7.8 versus 9.9 ..mu..g/dl). The experimental protocol increased serum corticosterone levels (7.0 versus 9.9 ..mu..g/dl for the nonhandled and sham-exposed groups, respectively); however, the corticosterone concentration in the 10 mW/cm/sup 2/ group was similar to that in the untreated (nonhandled) controls (7.8 versus 7.0 ..mu..g/dl). This modified adrenal axis function gives further support to the concept that microwave radiation affects endocrine function.

Spectroscopic studies of rat mast cells, mouse mastocytoma cells, and compound 48/80—II: The synthesis and some binding properties of spinlabeled 48/80☆

Abstract Compound 48/80 has been spin labeled (SL-48/80), purified and its interaction with mastocytoma cells studied. The labeled drug was similar to the unlabeled compound with regard to biological activity and binding to biological membranes. The electron spin resonance (E.S.R.) spectra of membrane bound SL-48/80 were indicative of a bound probe in an environment which is more restricted than that of bound stearic acid spin labels, since the low field extremum was broad and the high field extremum was not discernible. Although ferricyanide ions broadened the E.S.R. signal of free SL-48/80, this agent did not affect the spectrum of SL-48/80 bound to mastocytoma cells. This would suggest that SL-48/80 binds in a region of the cell that is not in contact with the extracellular aqueous environment. Competition studies showed that SL-48/80 could be displaced by high concentrations of unlabeled 48/80 and polymyxin B.