Macrophage activation by synthetic peptides. II. The effect of oxidative burst products on the fluorescence kinetics of macrophages stained with fluorescein diacetate

Our previous study has shown that the intensity of macrophages in a medium containing fluorescein diacetate (FDA) retains a certain constant level ready to fall upon the addition of stimulants. The decrease in fluorescence was attempted to explain by secretion of oxygen metabolites during the activation process. The decrease in fluorescence is much smaller in the medium containing catalase and superoxide dismutase. Hydrogen peroxide (50 nM and higher) causes a reversible decrease in the fluorescent intensity of resting macrophages. These experiments suggest that the oxidative burst products may serve as regulators of the feedback control during macrophage activation.     

Phloretin-induced changes of lipophilic ion transport across the plasma membrane of mammalian cells.

The adsorption of the hydrophobic anion [W(CO)(5)CN](-) to human lymphoid Jurkat cells gave rise to an additional anti-field peak in the rotational spectra of single cells, indicating that the cell membrane displayed a strong dielectric dispersion in the kilohertz to megahertz frequency range. The surface concentration of the adsorbed anion and its translocation rate constant between the two membrane boundaries could be evaluated from the rotation spectra of cells by applying the previously proposed mobile charge model. Similar single-cell electrorotation experiments were performed to examine the effect of phloretin, a dipolar molecule known to influence the dipole potential of membranes, on the transport of [W(CO)(5)CN](-) across the plasma membrane of mammalian cells. The adsorption of [W(CO)(5)CN](-) was significantly reduced by phloretin, which is in reasonable agreement with the known phloretin-induced effects on artificial and biological membranes. The IC(50) for the effect of phloretin on the transport parameters of the lipophilic ion was approximately 10 microM. The results of this study are consistent with the assumption that the binding of phloretin reduces the intrinsic dipole potential of the plasma membrane. The experimental approach developed here allows the quantification of intrinsic dipole potential changes within the plasma membrane of living cells.     

Electron and proton transport across the plasma membrane

Transplasma membrane electron transport in both plant and animal cells activates proton release. The nature and components of the electron transport system and the mechanism by which proton release is activated remains to be discovered. Reduced pyridine nucleotides are substrates for the plasma membrane dehydrogenases. Both plant and animal membranes have unusual cyanide-insensitive oxidases so oxygen can be the natural electron acceptor. Natural ferric chelates or ferric transferrin can also act as electron acceptors. Artificial, impermeable oxidants such as ferricyanide are used to probe the activity. Since plasma membranes containb cytochromes, flavin, iron, and quinones, components for electron transport are present but their participation, except for quinone, has not been demonstrated. Stimulation of electron transport with impermeable oxidants and hormones activates proton release from cells. In plants the electron transport and proton release is stimulated by red or blue light. Inhibitors of electron transport, such as certain antitumor drugs, inhibit proton release. With animal cells the high ratio of protons released to electrons transferred, stimulation of proton release by sodium ions, and inhibition by amilorides indicates that electron transport activates the Na⁺/H⁺ antiport. In plants part of the proton release can be achieved by activation of the H⁺ ATPase. A contribution to proton transfer by protonated electron carriers in the membrane has not been eliminated. In some cells transmembrane electron transport has been shown to cause cytoplasmic pH changes or to stimulate protein kinases which may be the basis for activation of proton channels in the membrane. The redox-induced proton release causes internal and external pH changes which can be related to stimulation of animal and plant cell growth by external, impermeable oxidants or by oxygen.     

Glucose transport across plasma membrane in human platelets

Studies have been carried out in the presence of 2-deoxyglucose, by utilizing a technique of platelet rapid filtration. Kinetic data suggest that glucose uptake across plasma membrane is the rate limiting step in its utilization. 2-deoxyglucose is transported by facilitated diffusion. L-glucose is transferred at only 1/1200 of the rate of glucose. Transport system shows high affinity for substrate. Transport is inhibited by cytochalasin B, phloretin and N-ethylmaleimide. Cytochalasin E does not affect 2-deoxyglucose uptake. Diamide can have activating or inhibitory effect. t-Butyl hydroperoxide is always activating. Insulin has no effect on rate transport. D-glucose, 3-O-methylglucose, non radioactive 2-deoxyglucose and D-mannose are strong competitors, whereas D-galactose and D-fructose compete weakly with 2-deoxyglucose transport.     

Peptide transport across the animal cell plasma membrane: recent developments.

Transfer of intact peptides across the plasma membrane of animal cells, especially in the small intestine and the kidney, is a well established phenomenon. This process plays an important role in the maintenance of protein nutrition. Evidence is accumulating which suggests that the process may also have a great potential for pharmacological and clinical applications. It is therefore important to understand various aspects of peptide transport such as its function, chemical nature of the transport protein and its gene, the operational mechanisms and their regulation, and the relevance of the transport system to health and disease. Recent years have witnessed considerable progress in the field. The driving force for the transport system has been identified as the proton motive force which makes the system unique and distinct from the majority of solute transport systems in animal cells which are driven by a sodium motive force. A great deal is now known on the chemical nature of the active site. The protein responsible for the transport process in the small intestine has been purified and characterized. The system has been successfully expressed in its functional form in Xenopus laevis oocytes by microinjection into the oocytes of poly(A)+ mRNA isolated from intestinal mucosal cells. There is no doubt that the coming years will bring even more exciting information on the transport system, especially in areas such as hormonal regulation, clinical applicability and cloning, and characterization of the gene encoding the transport system.     

Transport of heptafluorostearate across model membranes. Membrane transport of long-chain fatty acid anions I

Heptafluorostearic acid, an isogeometric derivative of stearic acid, has a pK(a) value of about 0.5. To evaluate the suitability of heptafluorostearate as model compound for anions of long-chain fatty acids in membrane transport, monolayer and liposome studies were performed with lipid mixtures containing phospholipids;-cholesterol-heptafluorostearate or stearate (100:40:20 molar ratios). Transfer of heptafluorostearate and stearate from liposomes to bovine serum albumin (BSA) was followed by measuring the intrinsic fluorescence of BSA. The percentage of heptafluorostearate, equivalent to the amount placed in their outer monolayer, transferred from liposomes (120;-130 nm diameter) to BSA was 55.7 +/- 3.7% within 10 min at 25 degrees C and 55 +/- 2% within 5 min at 37 degrees C. Slow transfer of 22.7 +/- 2.5% of heptafluorostearate at 25 degrees C followed with a half-life of 2.3 +/- 0.4 h and of 20 +/- 4% at 37 degrees C with a half-life of 0.9 +/- 0.1 h until the final equilibrium distributions between BSA and liposomes were reached, 79 +/- 6% to 21 +/- 5% at 25 degrees C and 75 +/- 5% to 25 +/- 4% at 37 degrees C. The pseudounimolecular rate constants for flip-flop of heptafluorostearate equal k(FF,25) = 0.24 +/- 0.05 h(-) and k(FF,37) = 0.6 +/- 0.1 h(-), respectively. By comparison, transfer of stearate required only 3 min to reach equilibrium distribution.The difference between heptafluorostearate and stearate may be explained by a rapid flip-flop movement of the un-ionized fatty acids which exist in different concentrations in accordance with their pK(a) values. Half-life of flip-flop of heptafluorostearate makes it suitable to study mediated membrane transport of long-chain fatty acid anions.     

Rapid transport of phospholipids across the plasma membrane of Leishmania infantum

The internalization of fluorescent phospholipid analogs of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM) in Leishmania infantum promastigotes was studied. We observed a rapid inward redistribution of NBD-PC, -PE, and -PS across the plasma membrane at 28 and 4 degrees C. This internalization was shown to be independent of the endocytic activity of parasites. Rapid inward movement was coupled to an energy-dependent transporter because it was almost inhibited by depletion of cellular ATP and was blocked after pretreatment with N-ethylmaleimide (NEM). In contrast, NBD-SM traverses the plasma membrane by passive flip-flop. By comparing this pattern of phospholipid transbilayer movement with those known from other eukaryotic cells, candidate lipid transporters are discussed.     

Control of phosphate transport across the plasma membrane of Chara corallina

This paper examines the control of phosphate uptake into Chara corallina. Influxes of inorganic phosphate (Pi) into isolated single internodal cells were measured with ³²Pi. Pretreatment of cells without Pi for up to 10 d increased Pi influx. However, during this starvation the concentrations of Pi in both the cytoplasm and the vacuole remained quite constant. When cells were pre-treated with 0.1 mM Pi, the subsequent influx of Pi was low. Under these conditions the Pi concentrations in the cytoplasm was almost the same as that of Pi-starved cells, but vacuolar Pi increased with time. Transfer of cells from medium containing 0.1 mM Pi to Pi-free medium induced an increase of Pi influx within 3 d irrespective of the concentration of Pi in the vacuole.During Pi starvation, neither the membrane potential nor the cytoplasmic pH changed. Manipulation of the cytoplasmic pH by weak acids or ammonium decreased the Pi influx slightly.Pi efflux was also measured, using cells loaded with ³²Pi. Addition of a low concentration of Pi in the rinsing medium rapidly and temporarily induced an increase in the efflux.The results show that Pi influx is controlled by factors other than simple feedback from cytoplasmic or vacuolar Pi concentrations or thermodynamic driving forces for H⁺-coupled Pi uptake. It is suggested that uptake of Pi is controlled via the concentration of Pi in the external medium through induction or repression of two types of plasma membrane Pi transporters.Key words: Chara corallina, membrane transport, phosphate influx, phosphate starvation      

Macrophage activation by mycobacterial water soluble compounds and synthetic muramyl dipeptide.

The adjuvant effects of mycobacteria can be replaced by more chemically defined isolates of the cell walls including a water soluble fraction (WSA) and by the synthetic analog N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP), which is the minimal structure required for adjuvanticity. These compounds can directly activate macrophages as determined by an increase in spreading and adherence and by an elevated synthesis of the enzyme collagenase. Moreover, this increase in collagenase production is modulated by enhanced production of prostaglandins that influences intracellular levels of cyclic AMP. In addition, both MDP and WSA induced macrophages to produce a biologically active mediator that triggers quiescent fibroblasts into active proliferation. It thus appears that a mechanism for mycobacterial adjuvant action as determined with MDP and WSA is via activation of macrophages, which may then precipitate a multiplicity of other reactions resulting in enhanced immune phenomena. Furthermore, the granulomatous and fibrotic reactions associated with mycobacterial infection may be a consequence of this direct activation of macrophages.     

Inhibition of mycolic acid transport across the Mycobacterium tuberculosis plasma membrane

New chemotherapeutics active against multidrug-resistant Mycobacterium tuberculosis are urgently needed. We report on the identification of an adamantyl urea compound that shows potent bactericidal activity against M. tuberculosis and a unique mode of action, namely the abolition of the translocation of mycolic acids from the cytoplasm, where they are synthesized to the periplasmic side of the plasma membrane and are in turn transferred onto cell wall arabinogalactan or used in the formation of virulence-associated, outer membrane, trehalose-containing glycolipids. Whole-genome sequencing of spontaneous-resistant mutants of M. tuberculosis selected in vitro followed by genetic validation experiments revealed that our prototype inhibitor targets the inner membrane transporter MmpL3. Conditional gene expression of mmpL3 in mycobacteria and analysis of inhibitor-treated cells validate MmpL3 as essential for mycobacterial growth and support the involvement of this transporter in the translocation of trehalose monomycolate across the plasma membrane.     

Macrophage activation by synthetic peptides. III. Changes in the membrane potential, Ca2+ content and the regulatory decrease of macrophage volume under the action of tuftsin and its antagonist

With the use of oxonol voltage-sensitive fluorescent dye it has been shown that the stimulation of macrophages (MP) with tuftsin results in a two-phase change in membrane potential: depolarization followed by hyperpolarization of plasma membrane. The pattern of changes in membrane potential depends on Na+ concentration in the medium and is disturbed with binding of cytoplasmic Ca2+. Fluorescent signal obtained from MP loaded with Ca(2+)-activated photoprotein obelin points to a significant increase in the concentration of cytoplasmic Ca2+ under the influence of tuftsin on cells: the source for Ca2+ being the medium. The rate of regulatory voltage decrease in MP increases under the influence of tuftsin: the effect of this peptide being similar to that of calcium ionophore. All these findings taken together enable us to suggest a phenomenological scheme of transmembrane ion signals arising during stimulation of MP with tuftsin: the receptor-mediated calcium channel provides a rise in cytoplasmic Ca2+ which opens non-selective cation channels for Na+ ions to activate eventually Ca(2+)-dependent K(+)-transport.     

NBD-labeled phosphatidylcholine and phosphatidylethanolamine are internalized by transbilayer transport across the yeast plasma membrane

The internalization and distribution of fluorescent analogs of phosphatidylcholine (M-C6-NBD-PC) and phosphatidylethanolamine (M-C6-NBD-PE) were studied in Saccharomyces cerevisiae. At normal growth temperatures, M-C6-NBD-PC was internalized predominantly to the vacuole and degraded. M-C6-NBD-PE was internalized to the nuclear envelope/ER and mitochondria, was not transported to the vacuole, and was not degraded. At 2 degrees C, both were internalized to the nuclear envelope/ER and mitochondria by an energy-dependent, N-ethylmaleimide-sensitive process, and transport of M-C6-NBD-PC to and degradation in the vacuole was blocked. Internalization of neither phospholipid was reduced in the endocytosis-defective mutant, end4-1. However, following pre-incubation at 37 degrees C, internalization of both phospholipids was inhibited at 2 degrees C and 37 degrees C in sec mutants defective in vesicular traffic. The sec18/NSF mutation was unique among the sec mutations in further blocking M-C6-NBD-PC translocation to the vacuole suggesting a dependence on membrane fusion. Based on these and previous observations, we propose that M-C6-NBD-PC and M-C6-NBD-PE are transported across the plasma membrane to the cytosolic leaflet by a protein-mediated, energy-dependent mechanism. From the cytosolic leaflet, both phospholipids are spontaneously distributed to the nuclear envelope/ER and mitochondria. Subsequently, M-C6-NBD-PC, but not M-C6-NBD-PE, is sorted by vesicular transport to the vacuole where it is degraded by lumenal hydrolases.     

Lack of response of murine peritoneal macrophages to in vitro activation by muramyl dipeptide (MDP). I. Macrophage activation by MDP is species dependent

Peritoneal exudate macrophages from mice, rats, and guinea pigs were assessed using six different parameters of macrophage activation to determine whether the cells were stimulated under similar experimental conditions. Peritoneal exudate macrophages from mice, irrespective of strain, were far less responsive to a variety of in vitro stimulatory effects of N-acetylmuramyl-L-alanyl-D-isoglutamine than those from rats or guinea pigs, while no significant differences were noted with the reactivity to stimulation by endotoxic lipopolysaccharide. We conclude that macrophage activation by MDP in vitro is species dependent.     

Changes in the membrane potential, the cytoplasmic Ca2+ concentration and in the transport of fluorescein anions across the macrophage plasma membrane under the influence of O2(-) and H2O2 in small concentrations]

It has been shown that under the influence of superoxide anion (300 nM) and hydrogen peroxide (100 nM) on murine peritoneal macrophages, the depolarization of membrane takes place, and a change in the rate of fluorescein anion efflux from the cells occurs. Hydrogen peroxide (but not superoxide anions) causes a transient increase in the cytoplasmic Ca2+ concentration. These changes are regarded as early signs of macrophage activation. It is assumed that macrophage activation with reactive oxygen intermediates at such a low concentration might be interpreted as follows: a small portion of stimulated phagocytes activates the whole population with the aid of the products of their own oxidative burst.