Wendy Jessup

Membrane proteins are critical targets in free radical mediated cytolysis.

The hypothesis that proteins are critical targets in free radical mediated cytolysis was tested using U937 mononuclear phagocytes as targets and iron together with hydrogen peroxide to generate radicals. Those conditions which, after a lag of approx. 30 min, led to drastic lysis were also associated with very rapid membrane depolarisation. Conversely, when the early membrane depolarisation was prevented (by the addition of chelator and catalase), so was lysis. A similar correlation between early membrane depolarisation and subsequent lysis was also observed when the cells were exposed to a toxin from Actinobacillus actinomycetemcomitans. Those conditions of radical attack which led to lysis normally caused substantial lipid peroxidation. However, depolarisation and subsequent lysis were not prevented even when lipid peroxidation was completely suppressed by exogenous antioxidant. ATP levels were not grossly affected within the critical first 30 min period. These data exclude lipids and ATP as the target for lytic damage. We argue therefore that proteins are probably amongst the primary targets in cytolysis by radicals.

The modification of low density lipoprotein by the flavonoids myricetin and gossypetin

Myricetin and gossypetin, two hexahydroxylated flavonoids, are capable of modifying low density lipoprotein (LDL) to increase greatly its uptake by macrophages. When human 125I-labelled LDL was incubated with 100-1000 microM myricetin or gossypetin, it was subsequently endocytosed much faster by mouse peritoneal macrophages. This modification did not occur at a concentration of 10 microM. Nine other flavonoids containing up to five hydroxyl substituents did not modify LDL to any great extent at 100 microM. The modification of LDL by 100 microM myricetin was time-dependent and complete by 6 hr. Flavonoids can sometimes act as pro-oxidants but myricetin did not act by oxidizing the LDL, as the LDL lipid hydroperoxide content was not increased by myricetin, nor did it promote the depletion of the endogenous antioxidant alpha-tocopherol in the LDL. High concentrations of myricetin caused the aggregation of LDL particles, as judged by light microscopy, agarose gel electrophoresis, retention by a membrane filter and sedimentability by centrifugation. SDS-PAGE indicated that the apolipoprotein B-100 molecules of LDL particles were covalently crosslinked. The uptake and degradation by macrophages of myricetin-modified 125I-labelled LDL reached saturation at about 10 micrograms protein/mL, suggesting the existence of a high affinity uptake process for the modified LDL. The uptake of myricetin-modified 125I-labelled LDL was not competed for by a large excess of non-labelled native LDL or acetylated LDL. We conclude that myricetin and gossypetin at high concentrations are capable of modifying LDL by a novel non-oxidative mechanism to a form taken up by macrophages by a high affinity process.

A continuous-flow automated assay for iodometric estimation of hydroperoxides

An iodometric method for the analysis of hydroperoxides has been automated to allow analysis of aqueous biological samples (containing less than 20 mg/ml protein) and lipid hydroperoxide extracts. The evolution of triiodide ions is measured spectrophotometrically at 360 nm. Dependent on the type of sample, 30-60 samples can be analyzed per hour and the system allows detection of less than 100 pmol of peroxide. The assay is linear over a range of 100 pmol to 25 nmol. The sample volume used routinely was 80 microliters.

Secretion by mononuclear phagocytes of lysosomal hydrolases bearing ligands for the mannose-6-phosphate receptor system of fibroblasts: Evidence for a second mechanism of spontaneous secretion?

Abstract β-Hexosaminidase secreted by peritoneal macrophages in response to stimulation by zymosan or NH 4 Cl, or spontaneously by a macrophage-like cell line (P388D 1 ), is susceptible to receptor-mediated endocytosis by human fibroblasts. This endocytosis is almost completely blocked by exogenous mannose-6-phosphate and therefore seems to depend on a mannose-6-phosphate ligand on the enzyme. It is suggested that macrophage lysosomal enzyme packaging may involve mannose-6-phosphate recognition markers, and that a continuous hypersecretion mechanism may exist which does not depend on a defect in this ligand.

The effect of weak bases on lysosomal enzyme secretion by mononuclear phagocytes

NH4Cl induces a dose-dependent secretion of lysosomal enzymes by mouse peritoneal macrophages and human peripheral blood monocytes. The mechanism of NH4Cl-stimulated hexosaminidase release is distinct from that initiated by the inflammatory stimulus, zymosan. The spontaneous lysosomal secretion of the continuous murine macrophage-like cell line, P388D1, is inhibited by up to 50% in the presence of NH4Cl and other weak bases.

Inhibition of some spontaneous secretory processes in macrophages and fibroblasts by ammonium chloride

Ammonium chloride inhibits the spontaneous secretion of lysozyme by mouse macrophages and the murine macrophage-like cell line P388D1 and the spontaneous secretion of lysosomal enzymes by P388D1 cells, normal human fibroblasts and the hypersecretory mucolipidosis II (I-cell) fibroblasts. NH4Cl can also inhibit protein synthesis, but this is shown to occur mainly at higher concentrations, or after longer periods of exposure, than are needed for the inhibition of the spontaneous secretory processes. It is confirmed that this amine can also interfere with the continuous endocytosis of fluid in the murine cell types. The nature of the inhibitions is discussed.

Lysosomal enzyme secretion by cystic fibrosis fibroblasts is normal

Lysosomal enzyme secretion by skin fibroblasts derived from cystic fibrosis homozygotes and normal donors of the same range of age, sex and culture passage number were compared. No differences were detected between these groups in their rates of spontaneous lysosomal enzyme release. Incubation of either normal or cystic fibrosis fibroblasts with cystic fibrosis serum did not alter these secretion rates. The lysosomal enzyme secretion of both normal and cystic fibrosis fibroblasts responded similarly to incubation with NH4Cl, monensin, A23187, trifluoperazine or Cytochalasin B. It was concluded that lysosomal enzyme hypersecretion, which has previously been suggested to occur in cystic fibrosis, is unlikely to provide a reliable diagnostic test for the disease.

Modulation of exocytosis of previously pinocytosed fluid by human fibroblasts

A slow phase of linear exocytosis by fibroblasts of previously endocytosed fluid, succeeding the initial burst of release, is described. This is not due to cell lysis, is virtually abolished at 4 degrees C, and is retarded when the lysosomal system is expanded. It may reflect membrane recycling.

Lysosomal hydrolases in macrophages exposed to swainsonine

Swainsonine reversibly inhibits macrophage lysosomal acid alpha-mannosidase in vitro. When supplied to cultured cells for periods of up to 24 h, swainsonine penetrates the cells and produces a dose- and time-dependent inhibition of cellular alpha-mannosidase. Exposure of macrophages to swainsonine for 24 h, followed by continued incubation in the absence of this agent, produces elevated cellular activity of alpha-mannosidase, relative to unexposed controls; prolonged incubation of macrophage cultures with swainsonine for 1-2 weeks results also in significant increases in cell protein, lactate dehydrogenase activity and in that of another lysosomal enzyme, beta-hexosaminidase.

Endocytosis and intracellular protein degradation in cystic fibrosis fibroblasts.

Normal rates of pinocytosis of [3H]sucrose were measured in cystic fibrosis fibroblasts, and were not affected by the addition of cystic fibrosis serum. Bulk protein degradation (a significant proportion of which occurs intralysosomally following autophagy) and its regulation by growth state was apparently identical in normal and cystic fibrosis cultures.