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.     

Intravital research on hydrophobic interactions in protein fibrils using a fluorescence polarization method

Fluorescent probe of acridine orange (AO) is known to be bound exclusively by materials of thick protofibril muscle fibres. The dependence of fluorescence anisotropy of AO in frog muscle fibres on the temperature has been studied. It has been found that the fluorophore orientation decreases with temperature, i.e. here the adsorbent cold denaturation takes place. This phenomenon was demonstrated for living, skinned and glycerinated muscle fibres. It shows that hydrophobic interactions contribute much to stabilization of the myosin structure. After contraction of glycerinated muscle fibres at the action of ATP, the AO fluorescence anisotropy grows independently of the temperature which is indicative of structural rearrangements in materials of thick protofibrils upon contraction.     

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.     

Effect of temperature on the formation and inactivation of syringomycin E pores in human red blood cells and bimolecular lipid membranes

The effects of temperature on the formation and inactivation of syringomycin E (SRE) pores were investigated with human red blood cells (RBCs) and lipid bilayer membranes (BLMs). SRE enhanced the RBC membrane permeability of 86Rb and monomeric hemoglobin in a temperature dependent manner. The kinetics of 86Rb and hemoglobin effluxes were measured at different temperatures and pore formation was found to be only slightly affected, while inactivation was strongly influenced by temperature. At 37 degrees C, SRE pore inactivation began 15 min after and at 20 degrees C, 40 min after SRE addition. At 6 degrees C, below the phase transition temperature of the major lipid components of the RBC membrane, no inactivation occurred for as long as 90 min. With BLMs, SRE induced a large current that remained stable at 14 degrees C, but at 23 degrees C it decreased over time while the single channel conductance and dwell time did not change. The results show that the temperature dependent inactivation of SRE pores is due to a decrease in the number of open pores.     

Kinetics of opening and closure of syringomycin E channels formed in lipid bilayers

A cyclic lipodepsipeptide, syringomycin E (SME), incorporated into planar lipid membranes forms two types of channels ("small" and "large") different in their conductance by approximately a factor of six (Biophys. J. 74:2918-2925 (1998)). We analysed the dynamics of the SME-induced transmembrane current under voltage-clamp conditions to clarify the mechanisms of formation of these channels. The voltage-dependent opening/closure of SME channels in lipid bilayers are interpreted in terms of transitions between three types of clusters including 6-7 SME molecules and some lipid molecules. The initial cluster, the precursor of the other two, was in equilibrium with SME monomer molecules at the membrane surface. The other two types of clusters (State 1 and State 2) were formed from the precursor and also during their interconversions (the consecutive-parallel mechanism of transitions). State 1 was a non-conducting state in equilibrium with small channels, which partially determined the ionic conductance of lipid bilayers modified by SME. State 2 corresponded to large SME channels, major contributors to the conductance of a bilayer. The results of the theoretical analysis based on the chemical kinetics concepts were consistent with experimental observations. Such properties of the SME-induced channels as cluster organisation, voltage dependence and the existence of a non-conducting state are all features shared by many ion channels in biological membranes. This makes it possible to use SME channels as a model to study naturally occurring ion channels.     

The measurement of the macrophage membrane potential by using an oxonol fluorescent probe]

Technical questions of macrophage (MP) membrane potential measuring with a probe bis(1,3-dibutyl barbiturate) trimethineoxonol (diBA-C4 (3)) have been elaborated. Measurements were made of single adherent cells. It was shown that at a high concentration of probe in the medium (900 nM) the fluorescent signal well traces the depolarization of membrane, whereas at a low concentration of probe (110 nM) the hyperpolarization is detected more effectively. To find out the reasons for this difference, measurements were made of dye distribution between the cell and the medium measured as well as of the kinetics of probe efflux from MP in the dye-free medium. The gradient of dye concentration on the cell-medium interface appeared to depend on the concentration of diBA-C4 (3) in the medium. Using gramicidin D and Na- and Cl-free solutions, the calibration of fluorescent signal was done; the value of K+ equilibrium potential of MP was -66 - -71 mV. The effect of quinidine and the binding of intracellular calcium result in a significant depolarization of MP membrane; a conclusion is made of the significant contribution of Ca(+)-dependent K(+)-channels to the maintenance of the MP resting potential.