Inhibition of oxidative injury of biological membranes by astaxanthin.

The value of astaxanthin, a carotenoid pigment, in the treatment of oxidative injury is assessed. Astaxanthin protects the mitochondria of vitamin E-deficient rats from damage by Fe2(+)-catalyzed lipid peroxidation both in vivo and in vitro. The inhibitory effect of astaxanthin on mitochondrial lipid peroxidation is stronger than that of alpha-tocopherol. Thin layer chromatographic analysis shows that the change in phospholipid components of erythrocytes from vitamin E-deficient rats induced by Fe2+ and Fe3(+)-xanthine/xanthine oxidase system was significantly suppressed by astaxanthin. Carrageenan-induced inflammation of the paw is also significantly inhibited by administration of astaxanthin. These data indicate that astaxanthin functions as a potent antioxidant both in vivo and in vitro.

Halothane, an inhalation anesthetic, activates protein kinase C and superoxide generation by neutrophils.

The rate of superoxide generation of guinea pig intraperitoneal neutrophils by a chemotactic peptide or 12-O-tetradecanoylphorbol-13-acetate (TPA) was increased by 2-bromo-2-chloro-1,1,1,-trifluoroethane (halothane), an inhalation anesthetic. This increase was inhibited by 1-(5-isoquinolinesulfonyl)methylpiperazine dihydrochloride (H-7), a specific inhibitor of Ca2+- and phospholipid-dependent protein kinase C (PKC). Halothane was found to significantly activate partially purified PKC. The activation required phosphatidylserine (PS) and Ca2+. Dioleoylglycerol- or TPA-activated PKC activity was further increased by halothane. The cytoplasmic proteins of guinea pig neutrophils phosphorylated by halothane-activated PKC were similar to those phosphorylated by PMA-activated PKC. The phosphorylation of a 48 kDa protein, a phosphorylated protein required for NADPH oxidase activation, was also increased by halothane. These data suggest that the increase of superoxide production by halothane is correlated with its activation of PKC.

Association of a myristoylated protein with a biological membrane and its increased phosphorylation by protein kinase C.

A hydrophilic enzyme, lysozyme, was myristoylated in vitro by the N-hydroxysuccinimide ester of myristic acid, and the monomyristoylated lysozyme was isolated by CM-cellulose cation-exchange column chromatography. The monomyristoylated lysozyme associated with phospholipid vesicles, whereas the association of native lysozyme was negligible. The membrane-associated monomyristoylated lysozyme was phosphorylated with partially purified rat brain Ca2+- and phospholipid-dependent protein kinase (protein kinase C) in the presence of Ca2+, phosphatidylserine and phorbolmyristate acetate. Thus, the myristoylated lysozyme became a substrate of protein kinase C through its hydrophobic association with the membrane. The present results suggest that the myristoylation of cytoplasmic proteins may have an important role in signal transduction.

Charge-dependent regulation of NADPH oxidase activities in intact and subcellular systems of polymorphonuclear leukocytes.

It has been reported that respiratory bursts with N-formylmethionylleucylphenylalanine, A23187, phorbol ester and fatty acids are switched off and on by modulating the net charges of plasma membranes in guinea-pig neutrophils (Miyahara, M. et al. (1987), Biochim. Biophys. Acta, 929, 253-262). In the present study, this was further extended in cells treated with protein kinase C inhibitors which completely suppressed the phorbol ester-dependent respiratory burst. This suggested that the initiation of the respiratory burst, which is generally accepted as linked to protein kinase C activation, might also be implicated in the net charge changes of plasma membranes. The above results were also supported by data obtained with a cell-free system reconstituted with plasma membranes and cytosolic fractions from unstimulated neutrophils, guanosine 5'-[gamma-thio]triphosphate and NADPH. Arachidonate stimulated NADPH oxidase activity accompanied by a marked phosphorylation of membrane proteins. The phosphorylation was sensitive to H-7, but it did not appear to be essential for the respiratory burst, because the oxidase activation was insensitive to H-7. Pretreating the plasma membranes with positively charged cetylamine inhibited the oxidase activation by arachidonate. These results suggest that a charge-dependent process, which does not use protein kinase C, may play an important role in the reaction leading to NADPH oxidase activation, and this may be related to the interaction of plasma membranes with the cytosolic activation factor.

Stimulative effect of chlordane on the various functions of the guinea pig leukocytes.

The effects of the chlordane-related compounds, cis-chlordane, trans-chlordane, heptachlor, and heptachlor epoxide, on stimulation responses of guinea pig polymorphonuclear leukocytes (PMNs) were examined. Treatment of PMN with these compounds stimulated superoxide (O2-) generation, altered membrane potential, and increased intracellular Ca2+ concentration ([Ca2+]i). However, there was no definite tendency among the stimulating effects of chlordane-related compounds, therefore the relationship between the effect and molecular structure of these substances remains unknown. Of these response reactions of PMN stimulated by chlordane-related compounds, stimulation of O2- generation lagged behind others. Increase in [Ca2+]i was due both to acceleration of extracellular Ca2+ penetration and to Ca2+ release from the intracellular pool. These results indicate that chlordane-related compounds stimulate PMN and suggest a causal relationship between the stimulation of O2- generation by these substances and their toxicity.

Lipocortin-like 33 kDa protein of guinea pig neutrophil. Its distribution and stimulation-dependent translocation detected by monoclonal anti-33 kDa protein antibody.

33 kDa protein of neutrophil is a lipocortin-like protein, as proposed from its biochemical properties, amino acid composition, and the homology of its amino acid sequence to human lipocortin I. The localization and translocation of 33 kDa protein (p33) in blood cells of guinea pig were studied by immunoblotting (Western blotting) and immunocytochemical fluorescence methods using polyclonal and monoclonal mouse anti-p33 antibodies. The protein was determined to be present only in the cytoplasm of neutrophils, but not in cells such as the monocyte, lymphocyte, platelet, and other bone marrow cells. The translocation of the protein from cytoplasm to cell membrane was coupled with the increase in intracellular calcium ion and in superoxide generation induced by a chemotactic factor. These findings suggest that p33 may have an important role not only in the regulatory mechanism of phospholipase A2 (PLA2) activity but also in other transmembrane signaling.

Activation of protein kinase C with cardiolipin-containing liposomes in relation to membrane-protein interaction.

Many cytoplasmic proteins, including Ca2+- and phospholipid-dependent protein kinase (protein kinase C) of polymorphonuclear leukocytes (PMNs) associate in Ca2+-dependent manner with phospholipid liposomes containing cardiolipin (CL), as in the case of phosphatidylserine (PS)-containing liposomes. A crude protein kinase C fraction was purified by association of the enzyme with CL-containing liposomes (flotation method). The partially purified protein kinase C from rat brain or guinea pig PMN was activated by the CL-containing liposomes in the presence of dioleoylglycerol (DG) and Ca2+. This activation was analogous to that of PS. The half maximum activity was obtained with 20 microM CL in the presence of 1 microM Ca2+ and 5 microM DG. Many of the cytoplasmic proteins which associate with CL-containing liposomes were preferentially phosphorylated by membrane-associated protein kinase C in the presence of DG and Ca2+. These results suggest that the association of cytoplasmic protein kinase C with the membrane has an important role in regulation of protein kinase C activity in relation to the association of other cytoplasmic proteins to the membrane.

Mechanism of polymorphonuclear leukocyte activation by myristate. Involvement of calcium ion and protein kinase C.

The stimulative effects of myristate on the superoxide generation and depolarization of membrane potential of polymorphonuclear leukocytes (PMN) are particularly strong, yet myristate does not affect the intracellular free Ca2+ level ([Ca2+]i) in the presence of 1 microM free calcium in calcium-EGTA buffer. The half maximum concentration of myristate was 10 microM. Myristate inhibited the transitory changes in [Ca2+]i induced by formylmethionyl-leucyl-phenylalanine (FMLP), but stimulated further the FMLP-induced superoxide generation; these effects are similar to those of phorbol myristate acetate (PMA). The myristate-induced superoxide generation was partially inhibited by H-7, a specific inhibitor of protein kinase C. Myristate stimulated the activity of Ca2+- and phospholipid-dependent protein kinase (protein kinase C) in a concentration-dependent manner in the presence of 10(-6) M Ca2+. The Ka was 100 microM. These results suggested that there is no relation between the superoxide generation and the [Ca2+]i change in PMNs and that the effects of myristate are similar to those of PMA against PMN.

Charge-dependent regulation of NADPH oxidase activity in guinea-pig polymorphonuclear leukocytes.

The mechanism of respiratory burst was studied by modulating membrane surfaces with lipophilic ions in guinea-pig polymorphonuclear leukocytes and their subcellular membranes. Positively charged alkylamines in concentration ranges of 0.5 to 15 microM (ED50 values) inhibited the O2- generation with phorbol 12-myristate 13-acetate, N-formylmethionylleucylphenylalanine, A23187, myristate and arachidonate in intact cells, and the inhibition was relieved by negatively charged agents. A similar molecular size of alkylalcohols had no effects. A similar charge-dependent O2- generation was also observed with fatty acids in subcellular membrane fractions prepared from unstimulated control cells, and this was insensitive to H-7 and W-7. These results suggest that triggering of NADPH oxidase activation involves a reaction(s) that is regulated by membrane charges.

Inhibitory effect of cholesterol on interaction between cytoplasmic actin and liposomes, and restorative effect of high osmotic pressure.

Although cholesterol is one of the major components of plasma membranes in eukaryotic cells, very little is known about its role in biological membranes. We reported previously (Okimasu et al., Cell Struct. Funct. 11, 273-283, 1986) that introduction of cholesterol into the liposomal membrane caused a decrease in membrane permeability, especially by the binding of cytoplasmic proteins to the liposomal membrane. The present study was carried out to further clarify the biochemical function of cholesterol in the membrane-protein interactions, especially under high osmotic pressure. The association of membranes with cytoplasmic proteins and their permeability were decreased by the introduction of cholesterol, but its effects were diminished in a hypertonic medium. The protein species associated with cholesterol-containing liposomes vary depending on the sort of hypertonic condition. It was suggested that since the degree of lipid packing by the cholesterol was reduced by the locally increased curvature in the lipid bilayer under high osmotic pressure, some cytoplasmic proteins can penetrate into the liposomal membrane.

Fluorescence and electron microscopic study of intracellular F-actin in concanavalin A-treated and cytochalasin B-treated Ehrlich ascites tumor cells.

To investigate the involvement of actin filaments in concanavalin A (Con A)-induced cap formation and cytochalasin B (CB)-induced zeiotic knob migration, the distribution of F-actin was studied in Con A-treated and CB-treated Ehrlich ascites tumor cells (EATC) by fluorescence microscopy using heavy meromyosin conjugated with a fluorescent dye, N-(7-dimethylamino-4-methylcoumarinyl) maleimide, (DACM-HMM). In non-treated cells, the diffuse fluorescence of DACM-HMM was observed in the cytoplasm, particularly intensely under the plasma membrane and around the nucleus. In Con A- and CB-treated cells, the fluorescence was seen at Con A-induced-capped and CB-induced-knob-accumulated regions. This fluorescence was more intense in CB-treated cells. To study the actin filaments in these fluorescent regions more clearly, the soluble components of the cells were eliminated by treatment with Triton X-100 or saponin solution containing a low concentration of glutaraldehyde, and the detergent-treated and saponin-treated cells were observed under a transmission electron microscope. Concentrated actin filaments were observed directly beneath the Con A-induced capping area and CB-induced zeiotic knob-accumulation area. The area of concentrated actin filaments appeared to correspond to the electron dense area observed in the identical region in the cells fixed without detergent treatment. More actin filaments were observed in CB-treated cells than in Con A-treated ones.

Cytoplasmic proteins, association with phospholipid vesicles and its dependency on cholesterol.

Since the plasticity of the plasma membrane is correlated with changes in its dynamic behavior and biological functions, the content of cholesterol in this membrane is thought to have an important role in the regulation of cell metabolism. The research reported here was done to clarify the role of cholesterol in the interaction of the innersurface of plasma membrane with cytoplasmic proteins in relation to the regulatory mechanism of signal transduction. Many cytoplasmic proteins of EATC or rat liver cells were found to associate with DPPC liposomes and they induced a transient increase in membrane permeability at the phase transition temperature of the liposomal lipid. The association and the permeability increase were inhibited by the introduction of cholesterol into DPPC liposomes, and the sensitivity of individual proteins to the action of cholesterol differed. F-actin, but not G-actin, associated selectively with the liposomes. Also, the main endothermic peak of the unilamellar DPPC liposomes was shifted from 37 degrees C to 43 degrees C by this protein association, evidence of transformation from a unito multilamellar structure. The introduction of cholesterol into the liposomal membrane caused a reduction in the energy content of the phase transition and in the inhibition of protein-membrane interaction. We concluded that the cholesterol in the plasma membrane contributes to the regulation of cell surface signal transduction.

Calcium-dependent association of 33 kDa protein in polymorphonuclear leukocytes with phospholipid liposomes containing phosphatidylserine or cardiolipin.

The effects of lipids and Ca2+ on the association of cytoplasmic proteins of polymorphonuclear leukocytes (PMN) with dipalmitoylphosphatidylcholine liposomes containing phosphatidylserine (PS) or cardiolipin (CL) were examined. In the presence of Ca2+, a cytoplasmic protein (33 kDa) of PMN was found to be a major component that bound to these liposome membranes. The maximum association with the liposomes was obtained at the molecular concentration of 25% CL and 50% PS; the required concentrations of Ca2+ were in the nanomolar and micromolar ranges for CL and PS, respectively. The liposome-associated 33 kDa protein was released from liposomes by an addition of EGTA. These results suggest that the 33 kDa protein reversibly associates to the cytoplasmic surface of PMN plasma membranes where PS localizes depending on a small change in intracellular concentration of free Ca2+.

Improvement of the anoxia-induced mitochondrial dysfunction by membrane modulation.

The mitochondrial dysfunction induced by anoxia in vitro was improved with chlorpromazine, cepharanthine, bromophenacyl bromide, and mepacrine without affecting phospholipid or adenine nucleotide metabolisms. The drugs inhibited lipid peroxidation by Fe2+, mitochondrial disruption by Ca2+, and membrane perturbation by lysolecithin, and retained the activity to control H+ permeability across mitochondrial membranes. The drugs appeared to preserve the functions by acting to suppress the development of membrane deterioration which may have resided in the deenergization of mitochondria in the absence of oxygen.

Stimulation of phospholipase A2 activity by high osmotic pressure on cholesterol-containing phospholipid vesicles.

The hydrolytic action of porcine pancreatic phospholipase A2 (PLA2) on cholesterol-containing dipalmitoylphosphatidylcholine liposomes was studied under various osmotic conditions by means of the phase transition release (PTR) technique. A reduced rate in the release of carboxyfluorescence (CF) from the cholesterol-containing liposomes was observed with treatment by PLA2 concomitant with the decrease in its enzymatic activity. However, the reduced PLA2 activity was elevated about 5-fold in medium of high osmotic pressure. This evidence suggests that high osmotic pressure might be responsible for the molecular packing at the bilayer surface or the high curvature of liposomal membrane structure.

Developmental changes in mitochondrial components in liver of newborn rats.

Chronological studies on the respiratory function and membrane components of newborn rat liver mitochondria revealed that perinatal mitochondria retained considerably well-coupled oxidative phosphorylation that functioned in sites II and III prior to site I. The ratio of protein to phospholipid contents (mg/mumol) was low in these mitochondria, in which phospholipids were characterized by their high phosphatidylethanolamine contents (43% of the total phospholipids) and low cardiolipin contents (6% of the total), as compared to those of the adult. The fatty acid composition in major phospholipid species was also different from those of the adult. These characteristics altered with development, and the carbamylphosphate synthetase contents markedly increased from 12 to 20% of the total polypeptides in 10 days postnatal time. From these results structural and functional completion of mitochondria accompanying development is discussed from a mitochondrial biogenesis viewpoint.

Inhibition of phospholipase A2 and platelet aggregation by glycyrrhizin, an antiinflammation drug.

We studied the effect of glycyrrhizin, a compound known as an anti-inflammatory and antiallergic drug, on the membrane permeability change induced by phospholipase A2 (PLA2) and on platelet aggregation. Glycyrrhizin was found to inhibit the PLA2-induced carboxyfluorescein (CF) release from D,L-dipalmitoyl phosphatidylcholine (DPPC) liposomes. Part of this inhibitory effect of glycyrrhizin on PLA2 is accounted for by the physical state of the substrate, the DPPC liposome membrane. Glycyrrhizin also inhibited collagen-induced platelet aggregation in a concentration dependent manner, which may in part account for its inhibitory effect on PLA2.

Measurement of membrane potential in polymorphonuclear leukocytes and its changes during surface stimulation.

The membrane potential of guinea pig polymorphonuclear leukocytes has been assessed with two indirect probes, tetraphenylphosphonium (TPP+) and 3,3'-dipropylthiadicarbocyanine (disS-C3-(5)). The change in TPP+ concentration in the medium was measured with a TPP+-selective electrode. By monitoring differences in accumulation of TPP+ in media containing low and high potassium concentrations, a resting potential of -58.3 mV was calculated. This potential is composed of a diffusion potential due to the gradient of potassium, established by the Na+, K+ pump, and an electrogenic potential. The chemotactic peptide fMet-Leu-Phe elicits a rapid efflux of accumulated TPP+ (indicative of depolarization) followed by its reaccumulation (indicative of repolarization). In contrast, stimulation with concanavalin A results in a rapid and sustained depolarization without a subsequent repolarization. The results obtained with TPP+ and diS-C3-(5) were comparable. Such changes in membrane potential were observed in the absence of extracellular sodium, indicating that an inward movement of sodium is not responsible for the depolarization. Increasing potassium levels, which lead to membrane depolarization, had no effect on the oxidative metabolism in nonstimulated or in fMet-Leu-Phe-stimulated cells. Therefore, it seems unlikely that membrane depolarization per se is the immediate stimulus for the respiratory burst.