Cyanine dye fluorescence used to measure membrane potential changes due to the assembly of complement proteins C5b-9

Summary The fluorescent potentiometric indicator diS–C₃-(5) has been used to investigate changes in membrane potential due to assembly of the C5b-9 membrane attack complex of the complement system. EAC1-7 human red blood cells and resealed erythrocyte ghosts—bearing membrane-assembled C5b67 complexes—were generated by immune activation in C8-deficient human serum. Studies performed with these cellular intermediates revealed that the membrane potential of EAC1-7 red cells and ghosts is unchanged from control red cells (–7 mV) and ghosts (0 mV), respectively. Addition of complement proteins C8 and C9 to EAC1-7 red cells results in a dose-dependent depolarization of membrane potential which precedes hemolysis. This prelytic depolarization of membrane potential—and the consequent onset of hemolysis—is accelerated by raising external [K⁺], suggesting that the diffusional equilibration of transmembrane cation gradients is rate limiting to the cytolytic event. In the case of EAC1-7 resealed ghosts suspended at either high external [K⁺] or [Na⁺], no change in membrane potential (from 0 mV) could be detected after C8/C9 additions. When the membrane potential of the EAC1-7 ghost was displaced from 0 mV by selectively increasing the K⁺ conductance with valinomycin, a dose-dependent depolarization of the membrane was observed upon addition of C8 and C9. In these experiments, lytic breakdown of the ghost membranes was <5%. Conclusions derived from this study include: (i) measured prelytic depolarization of the red cell Donnan potential directly confirms the colloid-osmotic theory of immune cytolysis. (ii) The diffusional transmenbrane equilibration of Na⁺ and K⁺ through the C5b-9 pore results in a dose-dependent depolarization of the membrane potential (E _m ) which appears to be rate-limiting to cytolytic rupture of the target erythrocyte. (iii) Enhanced immune hemolysis observed in high K⁺ media cannot be attributed to cation-selective conductance across the C5b-9 pore, and is probably related to the nearequilibrium condition of potassium-containing red cells when suspended at high external K⁺. These experiments demonstrate that carbocyanine dye fluorescent indicators can be used to monitor electrochemical changes arising from immune damage to the plasma membrane under both cytolytic and noncytolytic conditions. Potential application of this method to the detection of sublytic pathophysiological changes in the plasma membrane of complement-damaged cells are discussed.     

Permeability studies on red cell membranes of dog, cat, and beef

Water permeability coefficients of dog, cat, and beef red cell membranes have been measured under an osmotic pressure gradient. The measurements employed a rapid reaction stop flow apparatus with which cell shrinking was measured under a relative osmotic pressure gradient of 1.25 to 1.64 times the isosmolar concentration. For the dog red cell the osmotic permeability coefficient is 0.36 cm⁴/(sec, osmol). The water permeability coefficient for the dog red cell under a diffusion gradient was also measured (rate constant = 0.10/msec). The ratio between the two permeabilities was used to calculate an equivalent pore radius of 5.9 A. This value agrees welt with an equivalent pore radius of 6.2 A obtained from reflection coefficients of nonelectrolyte water-soluble molecules, and is consistent with data on the permeability of the dog red cell membrane to glucose. These data provide evidence supporting the existence of equivalent pores in single biological membranes.     

Biophysical properties of human astrocytic brain tumor cells in cell culture

For malignant cells cultured from a human astrocytoma, electrophysiological characteristics of the plasma membrane included specific resistivity of 446.82 ± 279.5 ohm·cm², specific capacitance of 0.758 ± 0.52 microfarads/cm², time constant 0.318± 0.10 msec. The resting membrane potential averaged-14.07 ± 7.4 mV; the mean input resistance 8.1 ± 4.0 megohms. The average cell area was 1638 ± 585 ±² for contactual and 1919 ± 989 ±² for noncontactual cells. Changes in input resistance and resting membrane potential were observed with increasing time in culture, possibly reflecting cell cycling. There did not appear to be electrical coupling in this cell line.     

The entrance of water into beef and dog red cells

The rate constants for diffusion of THO across the red cell membrane of beef and dog, and the rate of entrance of water into the erythrocytes of these species under an osmotic pressure gradient have been measured. For water entrance into the erythrocyte by diffusion the rate constants are 0.10 ± 0.02 msec.^-1 (beef) and 0.14 ± 0.03 msec.^-1 (dog); the permeability coefficients for water entrance under a pressure gradient of 1 osmol./cm³ are 0.28 See PDF for Equation These values permit the calculation of an equivalent pore radius for the erythrocyte membrane of 4.1 A for beef and 7.4 A for dog. In the beef red cell the change in THO diffusion due to osmotically produced cell volume shifts has been studied. The resistance to THO diffusion increases as the cell volume increases. At the maximum volume, (1.06 times normal), THO diffusion is decreased to 0.84 times the normal rate. This change in diffusion is attributed to swelling of the cellular membrane.     

Cholesterol promotes hemifusion and pore widening in membrane fusion induced by influenza hemagglutinin

Cholesterol-specific interactions that affect membrane fusion were tested for using insect cells; cells that have naturally low cholesterol levels (< 4 mol %). Sf9 cells were engineered (HAS cells) to express the hemagglutinin (HA) of the influenza virus X-31 strain. Enrichment of HAS cells with cholesterol reduced the delay between triggering and lipid dye transfer between HAS cells and human red blood cells (RBC), indicating that cholesterol facilitates membrane lipid mixing prior to fusion pore opening. Increased cholesterol also increased aqueous content transfer between HAS cells and RBC over a broad range of HA expression levels, suggesting that cholesterol also favors fusion pore expansion. This interpretation was tested using both trans-cell dye diffusion and fusion pore conductivity measurements in cholesterol-enriched cells. The results of this study support the hypothesis that host cell cholesterol acts at two stages in membrane fusion: (1) early, prior to fusion pore opening, and (2) late, during fusion pore expansion.     

Preserved function of the plasma membrane calcium pump of red blood cells from diabetic subjects with high levels of glycated haemoglobin

The activity of the plasma membrane Ca²⁺-pump decreases steeply throughout the 120 days lifespan of normal human red blood cells. Experiments with isolated membrane preparations showed that glycation of a lysine residue near the catalytic site of the pump ATPase had a powerful inhibitory effect. This prompted the question of whether glycation is the mechanism of age-related decline in pump activity in vivo. It is important to investigate this mechanism because the Ca²⁺ pump is a major regulator of Ca²⁺ homeostasis in all cells. Its impaired activity in diabetic patients, continuously exposed to high glycation rates, may thus contribute to varied tissue pathology in this disease. We measured Ca²⁺-pump activity as a function of red cell age in red cells from diabetics continuously exposed to high glucose concentrations, as documented by their high mean levels of glycated haemoglobin. The distribution of Ca²⁺-pump activities was indistinguishable from that in non-diabetics, and the pattern of activity decline with cell age in the diabetics’ red cells was identical to that observed in red cells from non-diabetics. These results indicate that in intact cells the Ca²⁺ pump is protected from glycation-induced inactivation.     

Cell Communication in the Basal Cells of the Human Epidermis

Electrotonic spread can be measured in the basal cells of the human epidermis. The communication between neighboring cells is high, whereas no leak to the intercellular spaces could be detected. The specific resistance of the membranes between the cells is about 10 Ωcm². This finding suggests that for those particles that are able to pass the cell membrane the intracellular path through the epidermis is at least as suitable as the path through the intercellular spaces.     

The failure of hydrodynamic analysis to define pore size in cell membranes

The equivalent pore theory predicts that the size of water transporting pores can be calculated from the ratio of osmotic (Pf, cm . s-1) to diffusive (Pd, cm . s-1) water permeability. Determinations of Pf and Pd in human red cells within the last thirty years have increased the ratio of Pf to Pd. According to the equivalent pore theory the pore diameter has increased from 9 A to 25 A. A pore diameter of 25 A is not compatible with the permeability characteristics of the red cell membrane. We conclude that the equivalent pore theory fails to determine pore size in red blood cells. We suggest that water transporting pores in human red cells transport water molecules in a single file fashion.     

A single-cell technique for the measurement of membrane potential, membrane conductance, and the efflux of rapidly penetrating solutes in Amphiuma erythrocytes

We describe a single-cell technique for measuring membrane potential, membrane resistance, and the efflux of rapidly penetrating solutes such as Cl and H2O. Erythrocytes from Amphiuma means were aspirated into a Sylgard (Dow Corning Corp.)-coated capillary. The aspirated cell separated a solution within the capillary from a solution in the bath. Each of these two solutions was contiguous with approximately 5% of the total membrane surface. Microelectrodes placed concentrically within the capillary permit the measurement of intracellular voltage, specific membrane resistance, and the electrical seal between the two solutions. The intracellular voltage averaged -17.7 mV (pH 7.6) and changed as either intra- or extracellular chloride was varied. The average specific membrane resistance measured by passing current across the exposed membrane surface was 110 ohm-cm2. 36Cl and tritiated H2O fluxes (0.84 +/- 0.05 x 10(-6) M . cm-2 . min-1 and 6.4 +/- 1.5 x 10(-3) M . cm-2 . min-1, respectively) were determined by noting the rate at which isotope leaves the cell and crosses the membrane exposed to the bath. Our measured values for the flux of 36Cl and tritiated H2O approximate reported values for free-floating cells. 36Cl efflux, in addition, is inhibited by 4-acetamido-4'-isothiocyano-stilbene 2,2'-disulfonic acid (SITS) and furosemide, known inhibitors of the anion exchange mechanism responsible for the rapid anion fluxes of red blood cells. One can also demonstrate directly that > 89% of 36Cl efflux is "electrically silent" by analyzing the flux in the presence of an imposed transcellular voltage.     

Water diffusion permeability of human erythrocytes studied by a pulsed gradient NMR technique

Water diffusion permeability of human erythrocytes has been measured by NMR using a pulsed magnetic field gradient technique. The measurement of exchange rates was based on restricted diffusion of water molecules within red blood cells. This method avoids addition of paramagnetic ions, such as Mn²⁺ and is used in vivo.The mean lifetime of water inside human erythrocytes was found to be 17 ms at 24°C. A sulfhydryl reagent, known to inhibit water osmotic permeability, reduced significantly water diffusion across the red cell membrane.     

Reflection coefficients of permeant molecules in human red cell suspensions

The Staverman reflection coefficient, sigma for several permeant molecules was determined in human red cell suspensions with a Durrum stopped-flow spectrophotometer. This procedure was first used with dog, cat, and beef red cells and with human red cells. The stopped-flow technique used was similar to the rapid-flow method used by those who originally reported sigma measurements in human red cells for molecules which rapidly penetrate the red cell membrane. The sigma values we obtained agreed with those previously reported for most of the slow penetrants, except malonamide, but disagreed with all the sigma values previously reported for the rapid penetrants. We were unable to calculate an "equivalent pore radius" with our sigma data. The advantages of our equipment and our experimental procedure are discussed. Our sigma data suggest that sigma is indirectly proportional to the log of the nonelectrolyte permeability coefficient, omega. Since a similar trend has been previously shown for log omega and molar volume of the permeant molecules, a correlatioo was shown between sigma and molar volume suggesting the membrane acts as a sieve.     

Electrical properties of soybean plasma membrane measured in heterotrophic suspension callus

Previous work on heterotrophic suspension-cultured cells has failed to detect the electrogenic processes normally associated with the plasma membranes of non-animal cells. This study reports measurements on heterotrophic cells from soybean (Glycine max L.) suspension cultures, which are shown to be amenable to impalement with microelectrodes. The plasma membrane clearly exhibits fundamental characteristics which are common to many other plant cell types: (i) a resting membrane potential significantly more negative than-100mV (measured value:121±4mV); (ii) obvious electrogenic activity, as evidenced by the marked depolarization of the membrane (87±6mV) by cyanide, and by the fact the membrane potential was frequently more negative than the equilibrium potential for K⁺; (iii) a finite permeability to K⁺ ions; (iv) electrophoretic transport of glucose. The development of a recording medium consisting primarily of 1:5 diluted growth medium was critical for successful impalement of these cells. It is proposed that the novel identification of electrogenic processes in heterotrophic suspension-cultured cells results from the deployment of electrodes with relatively dilute filling solutions, thus avoiding substantial changes in intracellular ion concentrations.The overwhelming majority of cells in soybean suspension cultures exist in small clusters, and the possibility of intercellular coupling potentially precludes assessment of membrane specific resistance and current density. Furthermore, as with most higher-plant cells, the vacuole occupies a large fraction of the intracellular volume. However, a model in which the measuring electrode is cytosolically located and the cells are electrically well-coupled is the only one which satisfactorily generates values for membrane specific resistance in a manner which is not strongly dependent on the number of cells in the cluster: other models in which the electrode tip is located in the vacuole and-or the impaled cell is electrically isolated from the others do not seem to apply. The measured values of membrane specific resistance are in the range 5.4 to 8.4 ·m², which is in excellent agreement with comparable measurements on other plant and fungal cells. The results are discussed with respect to mechanisms of transmembrane signalling in soybean, as well as to general electrophysiological studies on higher-plant cells in suspension culture and in tissues.Abbreviations and symbols R_m membrane resistance - r_p plasma-membrane resistivity - SRB Soybean Recording Buffer - V_m membrane potential     

The Na:K pump in red cells is electrogenic.

The membrane potential, E, of the red cell measured with a fluorescent dye, 3,3'-dipropylthiadicarbocyanine iodide, hyperpolarizes when the Na:K pump is activated by adding external K and depolarizes upon the subsequent addition of ouabain. The electrogenic pump is optimally observed in cells where internal Na+ has been raised, SO2-(4) has replaced Cl-, and SO2-(4) permeability has been inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS)). The change in E associated with the electrogenic component is about 6 mV in human red cells, somewhat smaller in sheep, and larger in duck and Amphiuma red cells. The membrane resistance, Rm, can be estimated from the pump-dependent change in E and from the current flow assumed to be one-third the ouabain-sensitive Na efflux. In human red cells, Rm is about 1 X 10(6) ohm-cm2. Rm calculated from the residual DIDS-insensitive SO2-(4) flux is also about 1 X 10(6) ohm-cm2. The closeness of these two values of Rm is paralleled in the other three types of red cells (even though the absolute values of Rm vary among the four types by a factor of 10), indicating that the net current flow across the membrane can be accounted for by the net transport of Na by the pump.     

Role of membrane proteins and lipids in water diffusion across red cell membranes

When human red cells are treated with the mercurial sulfhydryl reagent, $\text{p-}\text{chloromercuribenzene sulfonate}$, osmotic water permeability is suppressed and only diffusional water permeability remains (Macey, R.I. and Farmer, R.E.L. (1970) Biochim. Biophys. Acta 211, 104–106). It has been suggested that the route for the remaining water permeation is by diffusion through the membrane lipids. However, after making allowance for the relative lipid area of the membrane, the water diffusion coefficient through lipid bilayers which contain cholesterol is too small by a factor of two or more. We have measured the permeability coefficient of normal human red cells by proton $\text{T}{}_1$ NMR and obtained a value of 4.0 · 10^?3 cm · s^?1, in good agreement with published values. In order to study permeation-through red cell lipids we have perturbed extracted red cell lipids with the lipophilic anesthetic, halothane, and found that halothane increases water permeability. The same concentration of halothane has no effect on the water permeability of human red cells, after maximal pCMBS inhibition. In order to compare halothane mobility in extracted red cell membrane lipids with that in red cell ghost membranes, we have studied halothane quenching of $\text{N-}\text{phenyl}\text{-1-}\text{naphthylamine}$ by equilibrium fluorescence and fluorescence lifetime methods. Since halothane mobility is similar in these two preparations, we have concluded that the primary route of water diffusion in pCMBS-treated red cells is not through membrane lipids, but rather through a membrane protein channel.     

Mechanism of human erythrocyte hemolysis induced by short-chain phosphatidylcholines and lysophosphatidylcholine

The incorporation and accumulation of a certain amount of short-chain phosphatidylcholine or lysophosphatidylcholine into lipid bilayers of erythrocyte membranes is the first step causing membrane perturbation in the process of hemolysis. Accumulation of dilauroylglycerophosphocholine into membranes makes human erythrocytes "permeable cells"; Ions such as Na+ or K+ can permeate through the membrane, though large molecules such as hemoglobin can not. The "pore" formation was partially reproduced in liposomes prepared from lipids extracted from human erythrocyte membranes; C12:0PC induced the release of glucose from liposomes but did not significantly induce the release of dextran. It was suggested that the phase boundary between dilauroylglycerophosphocholine and the host membrane bilayer or dilauroylglycerophosphocholine rich domain itself behaves as "pores." Erythrocytes could expand to 1.5 times the original cell volume without any appreciable hemolysis when incubated with C12:0PC at 37 degrees C. The capacity of the erythrocytes to expand was temperature dependent. The capacity may play an important role in the resistance of the cells against lysis. The "permeable cell" stage could be hardly observed when erythrocytes were treated with didecanoylglycerophosphocholine and lysophosphatidylcholine. Perturbation induced by accumulation of didecanoylglycerophosphocholine or lysophosphatidylcholine may cause non specific destruction of membranes rather than formation of a kind of "pore."     

Changes in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy.

Cells can be transiently permeabilized by exposing them briefly to an intense electric field (a process called "electroporation"), but it is not clear what structural changes the electric field induces in the cell membrane. To determine whether membrane pores are actually created in the electropermeabilized cells, rapid-freezing electron microscopy was used to examine human red blood cells which were exposed to a radio-frequency electric field. Volcano-shaped membrane openings appeared in the freeze-fracture faces of electropermeabilized cell membranes at intervals as short as 3 ms after the electrical pulse. We suggest that these openings represent the membrane pathways which allow entry of macromolecules (such as DNA) during electroporation. The pore structures rapidly expand to 20-120 nm in diameter during the first 20 ms of electroporation, and after several seconds begin to shrink and reseal. The distribution of pore sizes and pore dynamics suggests that interactions between the membrane and the submembrane cytoskeleton may have an important role in the formation and resealing of pores.     

In situ solid-state NMR study of antimicrobial peptide interactions with erythrocyte membranes

Antimicrobial peptides are promising therapeutic agents to mitigate the global rise of antibiotic resistance. They generally act by perturbing the bacterial cell membrane and are thus less likely to induce resistance. Because they are membrane-active molecules, it is critical to verify and understand their potential action toward eukaryotic cells to help design effective and safe drugs. In this work, we studied the interaction of two antimicrobial peptides, aurein 1.2 and caerin 1.1, with red blood cell (RBC) membranes using in situ ³¹P and ²H solid-state NMR (SS-NMR). We established a protocol to integrate up to 25% of deuterated fatty acids in the membranes of ghosts, which are obtained when hemoglobin is removed from RBCs. Fatty acid incorporation and the integrity of the lipid bilayer were confirmed by SS-NMR and fluorescence confocal microscopy. Leakage assays were performed to assess the lytic power of the antimicrobial peptides. The in situ perturbation of the ghost membranes by aurein 1.2 and caerin 1.1 revealed by ³¹P and ²H SS-NMR is consistent with membrane perturbation through a carpet mechanism for aurein 1.2, whereas caerin 1.1 acts on RBCs via pore formation. These results are compatible with fluorescence microscopy images of the ghosts. The peptides interact with eukaryotic membranes following similar mechanisms that take place in bacteria, highlighting the importance of hydrophobicity when determining such interactions. Our work bridges model membranes and in vitro studies and provides an analytical toolbox to assess drug toxicity toward eukaryotic cells.     

Further characterization of the effects of alpha-1-acid glycoprotein on the passage of human erythrocytes through micropores

Effects of human alpha-1-acid glycoprotein (AG) on the passage of human red blood cell(s) (RBC) through membrane filters with micropores were examined in vitro. RBCs, with a mean major diameter of 7.2 micron, that had been suspended at 1% in physiological phosphate-buffered saline (PBS), were filtered through membrane filters of various pore diameters under positive pressure. The percentages of cells that passed through the micropores and of cells hemolyzed during filtration were determined. RBCs suspended in PBS did not pass through micropores that had an average pore diameter of 3 micron; instead hemolysis took place. Neither temperature nor applied pressure affected cell passage; but when AG at 0.1 mg/ml or above was added to an RBC-suspension, it promoted cell passage through the 3 micron micropores and reduced the degree of hemolysis. The effects of AG were dose dependent up to a concentration of 0.5 mg/ml. The addition of AG to an RBC-suspension that contained 90% human serum had the same additive effects. Washing AG-treated RBCs with normal saline produced a marked decrease in cell passage through the 3 micron pores. Fluorescence antibody staining revealed that the exogenous AG was localized on the membrane surface of the RBCs. Our results suggest that the AG bound to the surface of the RBCs acts as a lubricant between the RBCs and the wall of the micropore; this would facilitate RBC-passage through the micropores.     

Crossflow filtration of yeast broth cultivated in molasses

A broth of yeast cells cultivated in molasses was crossfiltered with a thin-channel module. The permeation flux gradually decreased at a constant cell concentration. The flux was much lower than that obtained for yeast broth cultivated in yeast extract, polypeptone, and dextrose (YPD) medium during the filtration. The flux did not depend on the membrane pore size (0.45 to 5 mum). The steady-state flux was one-twentieth that calculated for a cake filtration mode from the amount of cake per unit filtration area and the specific resistance of the cake measured in a dead-end filtration apparatus. The lower flux was due to small particles (most of which were less than 1 mum in diameter) in the molasses. The mehanism of crossflow filtration of broths of yeast cells cultivated in molasses was clarified by analysis of the change in flux with time and observations with scanning electron microscopy. At the initial stage of crossflow filtration the yeast cells and particles from the molasses were deposited on the membrane to form the molasses were deposited on the membrane to form a cake in a similar way to dead-end filtration. After the deposition of cells onto the membrane ceased, the fine particles from molasses formed a thin layer, which had higher resistance than the cake formed next to the membrane. The backwashing method was effective to increase the flux. The flux increased low when the pore size was 0.45 to 0.08 mum, but using larger pores of 3 to 5 mum it returned almost to the bases line. (c) 1994 John Wiley & Sons, Inc.     

Red Cell Membrane Permeability Deduced from Bulk Diffusion Coefficients

The permeability coefficients of dog red cell membrane to tritiated water and to a series of[¹⁴C]amides have been deduced from bulk diffusion measurements through a "tissue" composed of packed red cells. Red cells were packed by centrifugation inside polyethylene tubing. The red cell column was pulsed at one end with radiolabeled solute and diffusion was allowed to proceed for several hours. The distribution of radioactivity along the red cell column was measured by sequential slicing and counting, and the diffusion coefficient was determined by a simple plotting technique, assuming a one-dimensional diffusional model. In order to derive the red cell membrane permeability coefficient from the bulk diffusion coefficient, the red cells were assumed to be packed in a regular manner approximating closely spaced parallelopipeds. The local steady-state diffusional flux was idealized as a one-dimensional intracellular pathway in parallel with a one-dimensional extracellular pathway with solute exchange occurring within the series pathway and between the pathways. The diffusion coefficients in the intracellular and extracellular pathways were estimated from bulk diffusion measurements through concentrated hemoglobin solutions and plasma, respectively; while the volume of the extracellular pathway was determined using radiolabeled sucrose. The membrane permeability coefficients were in satisfactory agreement with the data of Sha'afi, R. I., C. M. Gary-Bobo, and A. K. Solomon (1971. J. Gen. Physiol. 58:238) obtained by a rapid-reaction technique. The method is simple and particularly well suited for rapidly permeating solutes.