Masanobu Miyahara

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.

Selective interaction of cytoskeletal proteins with liposomes

The initial step in cell activation by a ligand is believed to be its binding to specific receptors on the cell surface [I], and a ligand-induced redistribution or cross-linking of the receptor proteins is considered to be a step in the generation of a transmembrane signal [2]. It has been proposed that transmembrane surface proteins associate with components of the cytoskeletal network at the inner face of the cell membrane [3]. Therefore, a network of cytoskeletal structures on the inner face of membrane could interfere with the topology of receptor moieties on the outer surface of membrane [4]. In this case, it is considered that the hydrophobic interaction between membrane lipids and cytoplasmic proteins has an important role in the generation of the transmembrane signal. In attempting to understand this role, the interaction between phospholipid vesicles and cytoplasmic proteins was studied and it was found that many of the cytoskeletal proteins such as tubulin [5], actin, a-actinin and myosin associated strongly with liposomes made from dimyristoy1 or dipalmitoyl phosphatidylcholine vesicles [6]. In this report, we describe the association of actomyosin complex of polymorphonuclear leukocytes (PMN) and purified muscle actin with phospholipid vesicles and the formation of protein-liposome recombinants depending on the disorder of the phospholipid matrix. Liposomes were made with dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), cholesterol, cetylamine and dicetylphosphate as described in [6]. Unilamellar vesicles containing carboxyfluorescein (CF) were prepared by the method of Klausner et al. [lo] for the measurement of phase transition release (PTR). Phospholipids were dissolved in chloroform-methanol (2: 1) and the thin filmed lipids were hydrated by vortex-mixing and sonicated with a sonicator (Branson, type 185) in the presence of CF (0.1 M) at 25’C for DMPC and 45°C for DPPC. The CF-containing vesicles were centrifuged at 100 X g for 5 min and unilamellar vesicles were fractionated in a Sepharose 4B column (1.5 X 25 cm) with a 0.1 M NaCl-20 mM phosphate buffer (pH 6.8) eluant at 4°C.

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

Ca2+‐binding protein Protein‐lipid interaction Liposome Polymorphonuclear leukocyte Phosphatidylserine Cardiolipin

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.

Radiation-induced Damage to Mitochondrial d-β-hydroxybutyrate Dehydrogenase and Lipid Peroxidation

Radiation-induced damage to the reconstituted system of membrane-bound enzyme, D-beta-hydroxybutyrate dehydrogenase obtained from rat liver mitochondria, was investigated in relation to the lipid peroxidation of membranes. The activity of D-beta-hydroxybutyrate dehydrogenase in fresh mitochondria was very low in general and was not affected by irradiation because of little incorporation of substrates into mitochondria. However, the enzyme activity in one-day-aged mitochondria or submitochondrial particles was five times higher than that of fresh mitochondria and decreased with increasing radiation dose accompanying the increase in peroxidation of membrane lipids. The activity of D-beta-hydroxybutyrate dehydrogenase in the reconstituted system of the purified enzyme with irradiated liver microsomes or irradiated liposomes was decreased considerably in comparison with either unirradiated control or irradiated enzyme. Therefore, the radiation-induced decrease in the enzyme activity was thought to be caused mainly by peroxidation of membrane lipids and not to be due to direct damage by radiation to the enzyme molecule itself. Irradiation of microsomes, a component of the reconstituted system, caused decreases in phosphatidylcholine and phosphatidylethanolamine content and an increase in lysophosphatidylcholine content. In addition, arachidonic acid contents in phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine were also markedly decreased with increasing radiation dose. These results are discussed in terms of a mechanism involving radiation-induced damage to membrane function and structures.

Mitochondrial damage in galactosamine-induced liver intoxication in rats

Mitochondria isolated from livers of rats which received D-galactosamine (375 mg/kg body wt., four times) demonstrated a marked decrease in respiratory control ratios, the ADP/O ratios, and state 3 respiration rates and an increase in state 4 respiration rates. The aberration was profound with site I being altered prior to sites II and III. Quantitation of phospholipids revealed a reduction of total phospholipids per mg protein with decreases in phosphatidylcholine and phosphatidylethanolamine contents. Caldiolipin was the only phospholipid which remained unaltered. Fatty acid composition was altered in these phospholipids; caldiolipin was altered most severely, showing reductions in linoleic and arachidonic acids, and an elevation in saturated fatty acids and in some other small components of fatty acids. In phosphatidylethanolamine, palmitic acid decreased, whereas stearic and docosahexenoic acids increased. These changes were smaller in phosphatidylcholine fatty acids. These mitochondria were also characterized by an altered composition in high molecular weight polypeptide components. By experiments with normal mitochondria in vitro, galactosamine, but not other aminohexoses, was proved to be an uncoupling agent of the oxidative phosphorylation system. Electron microscopic observation demonstrated that both in vivo and in vitro treatments with galactosamine induced marked disorganization of mitochondrial structures. These results suggest that mitochondrial damage is also included in galactosamine-induced hepatic lesion.

Inhibition of mitochondrial energy transfer reactions by 5,5′-dithiobis(2-nitrobenzoic acid), Ellman's reagent

Abstract The role of -SH groups in mitochondrial energy transfer was studied by using 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) 1 , the specific reagent for the oxidation of -SH groups in biological materials. Observations on rat liver mitochondria have revealed that DTNB inhibits ADP-stimulated and arsenate-stimulated respiration without giving any effects on State-4 respiration, the ADP/O ratio, and the DNP-stimulated respiration. The activity of DNP-stimulated ATPase is arrested by DTNB with a slight enhance of the activity of Mg 2+ -stimulated ATPase, but the ATP-P i exchange reaction is suppressed only partially. Ca 2+ -stimulated respiration is also inhibited by DTNB. The results indicate that in intact mitochondria -SH groups to be attacked by DTNB will not be essential for the segment of the energy transfer pathway concerned with the ATP-P i exchange but essential for that concerned with the DNP-stimulated ATPase. Thus, it is suggested that the effecting site of DTNB is located between the sites to be stimulated by DNP and sensitive to oligomycin in the pathway of oxidative phosphorylation.

Neutrophil specific 33 kDa protein: its Ca2+- and phospholipid-dependent intracellular translocation.

A 33 kDa protein from neutrophils has been shown to associate reversibly with phosphatidylserine containing liposomes in a Ca2+‐dependent manner. The protein was purified from guinea pig neutrophils. Immunoblotting and cytochemical studies with polyclonal and monoclonal antibodies to the protein revealed that the protein is commonly distributed in neutrophil cytoplasm of different animal species. The protein was translocated to the plasma membrane by treatment with stimuli. Thus the 33 kDa protein is neutrophil specific and may be involved in transmembrane signaling.

Swelling-induced O−2 generation in guinea-pig neutrophils

Without the addition of any exogenous stimuli, neutrophils generated O2- and then ceased in a reversible manner that correlated with cellular swelling and contraction. The nature of the possible mechanism responsible for this O2- generation was studied and compared with that observed in the triggering of stimulant-dependent O2- generation (respiratory burst). The swelling-induced O2- generation was inhibited by diphenyliodonium, and was independent of the functional distortion of mitochondrial and/or microsomal electron transport and xanthine oxidase. This suggested that such generation was involved in respiratory-burst oxidase activation; however, this generation was not accompanied by any new phosphorylation of the 47-kDa protein or of tyrosine proteins. Dihydrocytochalasin B potentiated the O2- generation. The cellular swelling produced a priming effect on the triggering of respiratory burst with different stimuli. Cellular contraction, conversely, suppressed the respiratory burst. The structural specificity of the swelling-induced plasma membrane modulation for the O2- generation was suggested by the finding that modulation of plasma membrane structures by various non-ionic detergents per se inhibited O2- generation. Lipophilic and positively-charged agents inhibited the generation and this inhibition was abrogated by negatively-charged, but not by non-ionic agents. Negatively-charged agents potentiated the O2- generation. These results suggest that both the interaction of the plasma membrane with the cytoskeleton and an increase in net negative charges at the plasma membrane play important role in evoking O2- generation; this is discussed and compared with the signal transduction reported previously for respiratory burst.

Permeability changes of phospholipid liposomes caused by pancreatic phospholipase A2: analysis by means of phase transition release

Many biological messages are recognized by the binding of the ligands to specific receptors of the outer surface of the cell membrane [l-4]. These bindings then initiate certain chemical and physical changes in the membrane. One of the early changes in membrane state is the activation of phospholipase A2 (PLA2) [5]. This reaction has many important functions such as in biosynthesis of prostaglandins, in physicochemical changes of biomembranes and in regulation of the activities of other membrane bound enzymes. Therefore, the activation of membrane bound PLA2 has a key role in the mechanisms of cell activation. One of the important features of the regulation of PLA2 is the dependency of activation on membrane structure. Many investigations were carried out on the analogies with much better defined systems using soluble phospholipase, though this system is essentially different from those occurring in natural membranes. In those studies, it was proposed that a particular region of PLA2, so called interface recognition site, is involved in interaction with phospholipid structure [6]. Moreover, it was considered that phosphatidylcholine can be hydrolysed only near the transition temperature where lipid in liquid crystalline phase and in the gel phase coexist ]7,81. We have therefore applied the phase transition release technique to the study of the activation mechanism as related to the changes in physiological characteristics of the membrane and bring evidence that the maximal carboxyfluorescein release Phospholipase A2 (Phospholipid liposome) Dipalmitoylphosphatidylcholine