Effect of ethylene oxide and propylene oxide block copolymers on the permeability of bilayer lipid membranes to small solutes including doxorubicin

The effects of ethylene oxide and propylene oxide block copolymers (pluronics) on the permeability of several weak acids and bases through bilayer lipid membranes have been studied by the methods of monitoring (1) pH shifts near planar bilayers, (2) doxorubicin fluorescence quenching inside liposomes, and (3) current transients in the presence of hydrophobic anions. It has been shown that pluronics facilitate the permeation of comparatively large molecules (such as 2-n-undecylmalonic acid and doxorubicin) across lipid bilayers, while the permeation of small solutes (such as ammonium and acetic acid) remains unaffected. Pluronics also accelerate the translocation of large hydrophobic anions (tetraphenylborate). The effect of pluronics correlates with the content of propylene oxide units: it is enhanced when the portion of polypropylene oxide block in the copolymer is increased. The action of the pluronic on lipid membrane permeability differs from the effect of the conventional detergent Triton X-100, which does not affect doxorubicin transport if added at concentrations similar to those used for pluronics. It has been proposed that pluronics accelerate the processes of solute diffusion within lipid bilayers (in a structure-dependent manner) rather than influencing the rate of solute adsorption/desorption on the membrane surface. We suppose that the effect of pluronics on doxorubicin permeation across lipid bilayers along with the known effect on the multidrug resistance protein determines its influence on the therapeutic activity of anthracycline drugs.     

Contact-mediated changes in the fluidity of membrane lipids in normal and malignant transformed mammalian fibroblasts

The degree of microviscosity, gh, (fluidity/rigidity behavior) of membrane lipids of normal and transformed mammalian fibroblasts obtained from mice, hamsters and rats was quantitatively monitored by fluorescence polarization, P, analysis of the fluorescent probe 1,6-diphenyl 1,3,5-hexatriene (DPH) when embedded in lipid regions of cellular membranes of intact viable cells. Analysis of membrane microviscosity of six different cell populations and of individual cells in each cell population have indicated that the membrane microviscosity of all cell types, both normal and transformed fibroblasts, changes as a function of the cell density in the growing cultures. The membrane microviscosity was found to be low (high lipid fluidity) in sparse conditions but high (high lipid rigidity) in dense conditions. The induced changes in membrane microviscosity are practically reversible for all cell types and a complete reversion can be obtained within a few hours after changing the cell density conditions from sparse to dense and vice versa.Comparative studies with normal and transformed fibroblasts have shown that transformed fibroblasts have a more rigid lipid layer in their cellular membranes than normal or untransformed fibroblasts. The difference in membrane microviscosity between transformed and normal fibroblasts is higher in confluent conditions as compared with subconfluent cultures. These differences in the degree of fluidity of membrane lipids that are controlled by possible differences in the cellto-cell contact in normal and transformed fibroblasts may play a major role in determining the growth behavior of normal and malignant cells that are growing as a solid tissue and may have a direct effect on the control mechanisms that determine the presence or absence of the “density dependent inhibition” of growth.     

Effect of nitric oxide on viscosity of nerve cell membranes

The influence of nitric oxide on the microviscosity of nerve cell membranes was investigated by resonance Raman (RR) spectroscopy. Changes in membrane viscosity were estimated from the resonance Raman-spectra of carotenoids localized in the axon plasmatic membrane and membranes of subcellular vesicles (cytosomes). For the nerve fibre, the extracellular addition of nitric oxide donor, sodium nitroprusside (0.5 mM), caused an increase in the 1526 cm(-1) band relative half-width and the modification of 1160 cm-1 band structure. Moreover, sodium nitroprusside led to an increase in the I1526/I1160 ratio by 13% in 25 min and a decrease in this ratio by 10% in 50 min. In the case of cytosomes, sodium nitroprusside (0.5 mM) resulted in the reduction of the I1526/I1160 ratio by 8% in 25 and 50 min. It was shown that the neuron rhythmic activity correlated with the I1526/I1160 ratio and cytosome membrane microviscosity. We suppose that nitric oxide causes a conformational transition of carotenoids in the axon plasmatic membrane and the membranes of cytosomes. This process can be due to nitric oxide-induced changes of the membrane microviscosity or potential.     

Lipid composition determines interaction of liposome membranes with Pluronic L61

Triblock copolymers of ethylene oxide (EO) and propylene oxide (PO) of EO(n/2)PO(m)EO(n/2) type (Pluronics) demonstrate a variety of biological effects that are mainly due to their interaction with cell membranes. Previously, we have shown that Pluronics can bind to artificial lipid membranes and enhance accumulation of the anti-tumor drug doxorubicin (DOX) inside the pH-gradient liposomes and transmembrane migration (flip-flop) of NBD-labeled phosphatidylethanolamine in the liposomes composed from one component-lecithin. Here, we describe the effects caused by insertion of other natural lipids in lecithin liposomes and the significance of the lipid composition for interaction of Pluronic L61 with the membrane. We used binary liposomes consisting of lecithin and one of the following lipids: cholesterol, phosphatidylethanolamine, ganglioside GM1, sphingomyelin, cardiolipin or phosphatidic acid. The influence of the additives on (1) membrane microviscosity; (2) binding of Pluronic L61; (3) the copolymer effect on lipid flip-flop and membrane permeability towards DOX was studied. The results showed that insertion of sphingomyelin and cardiolipin did not influence membrane microviscosity and effects of Pluronic on the membrane permeability. Addition of phosphatidic acid led to a decrease in microviscosity of the bilayer and provoked its destabilization by the copolymer. On the contrary, cholesterol increased microviscosity of the membrane and decreased binding of Pluronic and its capacity to enhance flip-flop and DOX accumulation. Analogous tendencies were revealed upon incorporation of egg phosphatidylethanolamine or bovine brain ganglioside GM1. Thus, a reverse dependence between the microviscosity of membranes and their sensitivity to Pluronic effects was demonstrated. The described data may be relevant to mechanisms of Pluronic L61 interaction with normal and tumor cells.     

Importance of cholesterol-phospholipid interaction in determining dynamics of normal and abetalipoproteinemia red blood cell membrane

Acanthocytic red blood cells in patients with abetalipoproteinemia have a decrease membrane fluidity that is associated with increased sphingomyelin/phosphatidylcholine (SM/PC) ratios. Here we describe studies designed to gain better insight into (i) the interrelationship between the composition of lipoprotein and red blood cell membrane in abetalipoproteinemia patients and normal controls; and (ii) how the differences in lipid composition of the red blood cell membrane affect its fluidity. The increased SM/PC ratio found in abetalipoproteinemia plasma high density lipoproteins (HDL) (3 times greater than controls) was paralleled by an increase in this ratio in acanthocytic red cells, but to a lesser degree (almost twice greater than control red cells). Cholesterol/phospholipid mole ratios (C/P) were increased 3-fold in abetalipoproteinemia HDL, but only slightly increased in red cells compared to controls values. As in the controls, 80-85% of abetalipoproteinemia red cell sphingomyelin was found to be in the outer half of the erythrocyte membrane. Membrane fluidity was defined in terms of microviscosity (eta) between 5 and 42 degrees C by the fluorescent polarization of 1,6-diphenylhexatriene (DPH) present in erythrocyte ghost membranes. At all temperatures, membrane microviscosity was higher in abetalipoproteinemia ghosts than controls, but these differences decreased at higher temperatures (12.34 vs 9.79 poise, respectively at 10 degrees C; 4.63 vs 4.04 poise at 37 degrees C). These differences were eliminated after oxidation of all membrane cholesterol to cholest-4-en-3-one by incubation with cholesterol oxidase. Following cholesterol oxidation, the membrane microviscosity decreased in patient ghosts more than in normal red blood cells so that at all temperatures no significant differences were present relative to control ghosts, in which the apparent microviscosity was also diminished but to a lesser degree. Therefore, although increased SM/PC ratios in abetalipoproteinemia may be responsible for decreased erythrocyte membrane fluidity, these effects are dependent upon normal interactions of cholesterol with red cell phospholipid.     

Importance of cholesterol-phospholipid interaction in determining dynamics of normal and abetalipoproteinemia red blood cell membrane

Acanthocytic red blood cells in patients with abetalipoproteinemia have a decreased membrane fluidity that is associated with increased sphingomyelin/phosphatidylcholine (SM/PC)§ ratios. Here we describe studies designed to gain better insight into (i) the interrelationship between the composition of lipoprotein and red blood cell membrane in abetalipo-proteinemia patients and normal controls; and (ii) how the differences in lipid composition of the red blood cell membrane affect its fluidity. The increased SM/PC ratio found in abetalipoproteinemia plasma high density lipoproteins (HDL) (3 times greater than controls) was paralleled by an increase in this ratio in acanthocytic red cells, but to a lesser degree (almost twice greater than control red cells). Cholesterol/phospholipid mole ratios (C/P) were increased 3-fold in abetalipoproteinemia HDL, but only slightly increased in red cells compared to controls values. As in the controls, 80–85% of abetalipo-proteinemia red cell sphingomyelin was found to be in the outer half of the erythrocyte membrane. Membrane fluidity was defined in terms of microviscosity ({ie116-1}) between 5 and 42°C by the fluorescent polarization of 1,6-diphenylhexatriene (DPH) present in erythrocyte ghost membranes. At all temperatures, membrane microviscosity was higher in abetalipoproteinemia ghosts than controls, but these differences decreased at higher temperatures (12.34 vs 9.79 poise, respectively, at 10°C; 4.63 vs 4.04 poise at 37°C). These differences were eliminated after oxidation of all membrane cholesterol to cholest-4-en-3-one by incubation with cholesterol oxidase. Following cholesterol oxidation, the membrane microviscosity decreased in patient ghosts more than in normal red blood cells so that at all temperatures no significant differences were present relative to control ghosts, in which the apparent microviscosity was also diminished but to a lesser degree. Therefore, although increased SM/PC ratios in abetalipoproteinemia may be responsible for decreased erythrocyte membrane fluidity, these effects are dependent upon normal interactions of cholesterol with red cell phospholipid.     

Polarization of plasma membrane microviscosity during endothelial cell migration

Cell movement is characterized by anterior-posterior polarization of multiple cell structures. We show here that the plasma membrane is polarized in moving endothelial cells (EC); in particular, plasma membrane microviscosity (PMM) is increased at the cell leading edge. Our studies indicate that cholesterol has an important role in generation of this microviscosity gradient. In vitro studies using synthetic lipid vesicles show that membrane microviscosity has a substantial and biphasic influence on actin dynamics; a small amount of cholesterol increases actin-mediated vesicle deformation, whereas a large amount completely inhibits deformation. Experiments in migrating ECs confirm the important role of PMM on actin dynamics. Angiogenic growth factor-stimulated cells exhibit substantially increased membrane microviscosity at the cell front but, unexpectedly, show decreased rates of actin polymerization. Our results suggest that increased PMM in lamellipodia may permit more productive actin filament and meshwork formation, resulting in enhanced rates of cell movement.     

Modulation of membrane composition of swine vascular smooth muscle cells by homologous lipoproteins in culture

Swine vascular smooth muscle cells were exposed to homologous low-density or high-density lipoprotein fractions for 24 h. Total cell membranes were isolated from the post-nuclear supernatant of the cell homogenates, fractionated by sucrose density gradient centrifugation and characterized by enzyme assays. The membrane fraction with the lowest density was enriched in plasma membrane marker enzymes. Cholesterol analysis showed that cells exposed to low-density lipoprotein had higher cholesterol-to-protein ratios in total cells, total cell membranes and individual membrane fractions than had the cells exposed to high-density lipoproteins. Cholesterol-to-phospholipid ratios of the plasma membrane-enriched fraction from cells exposed to low-density lipoprotein were higher than the same membrane fraction of cells exposed to high-density lipoprotein. Studies with iodinated lipoproteins showed that these compositional changes could not be due to lipoprotein contamination. Membrane microviscosity was determined by fluorescence depolarization with diphenylhexatriene and the microviscosity of the plasma membrane-enriched fraction was different in the cells exposed to the two different lipoprotein fractions. This difference in membrane microviscosity was significant only when the medium cholesterol content was 40 μg per ml or greater; cells exposed to low-density lipoprotein gave membranes with higher microviscosity.These results demonstrate that the properties of vascular smooth muscle cell membranes are influenced by exposure of the cells to homologous lipoprotein fractions.     

Comparative Membrane Microviscosity of Fish and Mammalian Rhabdoviruses Studied by Fluorescence Depolarization

The microviscosity of the hydrophobic region of the membrane of infectious hematopoietic necrosis virus was determined using fluorescence depolarization analysis of the probe 1,6-diphenyl-1,3,5-hexatriene and was found to be much lower at 37 C than that of another rhabdovirus, vesicular stomatitis virus. However, the microviscosity of this fish virus at 18 C, the temperature at which it was grown, corresponded to the microviscosity of vesicular stomatitis virus at 37 C. Data obtained with the fish virus host cell (chinook salmon embryo cells) grown at 18 C suggest that its membranes have a lower microviscosity than either L-929 or BHK-21 cells (the vesicular stomatitis virus host cells) grown at 37 C.     

Membrane microviscosity regulates endothelial cell motility

Endothelial cell (EC) movement is an initiating and rate-limiting event in the neogenesis and repair of blood vessels. Here, we explore the hypothesis that microviscosity of the plasma membrane (PM) is a key physiological regulator of cell movement. Aortic ECs treated with membrane-active agents, such as alpha-tocopherol, cholesterol and lysophospholipids, exhibited a biphasic dependency on membrane microviscosity, in which moderate increases enhanced EC migration, but increases beyond a threshold markedly inhibited migration. Surprisingly, angiogenic growth factors, that is, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), also increased membrane microviscosity, as measured in live cells by fluorescence recovery after photobleaching (FRAP). The localization of Rac to the PM was modified in cells treated with membrane-active agents or growth factors, suggesting a molecular mechanism for how membrane microviscosity influences cell movement. Our data show that angiogenic growth factors, as well as certain lipophilic molecules, regulate cell motility through alterations in membrane properties and the consequent relocalization of critical signalling molecules to membranes.     

Enhancement in ‘apparent’ membrane microviscosity during differentiation of embryonal carcinoma cells induced by retinoids

Upon differentiation of embryonal carcinoma cells induced by retinoids (10⁻⁷ M) the ‘apparent’ membrane microviscosity increases dramatically. Only biologically active retinoids induce differentiation and cause an enhancement in microviscosity. Several embryonal carcinoma cell lines have a relatively lower ‘apparent’ microviscosity than their differentiated derivatives, suggesting that this may be a general property of these cells. At higher concentrations retinoids cause a reduction in ‘apparent’ membrane microviscosity of various cells. This change occurs whether the analogue is biologically active or not, indicating the non-specific nature of this action.     

Nitric oxide scavenging by red blood cells as a function of hematocrit and oxygenation

The reaction rate between nitric oxide and intraerythrocytic hemoglobin plays a major role in nitric oxide bioavailability and modulates homeostatic vascular function. It has previously been demonstrated that the encapsulation of hemoglobin in red blood cells restricts its ability to scavenge nitric oxide. This effect has been attributed to either factors intrinsic to the red blood cell such as a physical membrane barrier or factors external to the red blood cell such as the formation of an unstirred layer around the cell. We have performed measurements of the uptake rate of nitric oxide by red blood cells under oxygenated and deoxygenated conditions at different hematocrit percentages. Our studies include stopped-flow measurements where both the unstirred layer and physical barrier potentially participate, as well as competition experiments where the potential contribution of the unstirred layer is limited. We find that deoxygenated erythrocytes scavenge nitric oxide faster than oxygenated cells and that the rate of nitric oxide scavenging for oxygenated red blood cells increases as the hematocrit is raised from 15% to 50%. Our results 1) confirm the critical biological phenomenon that hemoglobin compartmentalization within the erythrocyte reduces reaction rates with nitric oxide, 2) show that extra-erythocytic diffusional barriers mediate most of this effect, and 3) provide novel evidence that an oxygen-dependent intrinsic property of the red blood cell contributes to this barrier activity, albeit to a lesser extent. These observations may have important physiological implications within the microvasculature and for pathophysiological disruption of nitric oxide homeostasis in diseases.     

Enveloped viruses as model membrane systems: microviscosity of vesicular stomatitis virus and host cell membranes.

The fluorescence probe 1,6-diphenyl-1,3,5-hexatriene was used to study and compare the dynamic properties of the hydrophobic region of vesicular stomatitis virus grown on L-929 cells, plasma membrane of L-929 cells prepared by two different methods, liposomes prepared from virus lipids and plasma membrane lipids, and intact L-929 cells. The rate of penetration of the probe into the hydrophobic region of the lipid bilayer was found to be much faster in the lipid vesicle bilayer as compared with the intact membrane, but in all cases the fluorescence anisotropy was constant with time. The L-cell plasma membranes, the vesicles prepared from the lipids derived from the plasma membranes, and intact cells are found to have much lower microviscosity values than the virus or virus lipid vesicles throughout a wide range of temperatures. The microviscosity of plasma membrane and plasma membrane lipid vesicles was found to depend on the procedure for plasma membrane preparation as the membranes prepared by different methods had different microviscosities. The intact virus and liposomes prepared from the virus lipids were found to have very similar microviscosity values. Plasma membrane and liposomes prepared from plasma membrane lipids also had similar microviscosity values. Factors affecting microviscosity in natural membranes and artificially mixed lipid membranes are discussed.     

Changes in membrane fluidity of erythrocytes during cell maturation

The measurements of the fluorescence polarization of perylene embedded in erythrocyte membranes were carried out with normal and reticulocyte-rich blood, and the microviscosity of erythrocyte membranes was calculated from the polarization degree. In intact cells, reticulocyte membranes had a significantly lower microviscosity than normal erythrocyte membranes, while in ghosts no significant difference in membrane microviscosity was observed between reticulocytes and mature erythrocytes.     

Lateral mobility of a lipid analog in the membrane of irreversible sickle erythrocytes

The major feature of sickle cell anemia is the tendency of erythrocytes to sickle when exposed to decreased oxygen tension and to unsickle when reoxygenated. Irreversible sickle cells (ISCs) are sickle erythrocytes which retain bipolar elongated shapes despite reoxygenation. ISCs are believed to owe their biophysical abnormalities to acquired membrane alterations which decrease membrane deformability. While increased membrane surface viscosity has been measured in ISCs, the lateral dynamics of membrane lipids in these cells have not heretofore been examined. We have measured the lateral diffusion of the lipid analog 3,3'-dioctadecylindocyanine iodide (DiI) in the plasma membrane of intact normal erythrocytes, reversible sickle cells (RSCs), and irreversible sickle cells by fluorescence photobleaching recovery (FPR). The diffusion coefficients +/- standard errors of the mean of DiI in intact normal red blood cells (RBCs), RSCs, and ISCs at 37 degrees C are (8.06 +/- 0.29) X 10(-9) cm2 X s-1, (7.74 +/- 0.22) X 10(-9) cm2 X s-1, and (7.29 +/- 0.24) X 10(-9) cm2 X s-1, respectively. A similar decrease in the diffusion coefficient of DiI in the plasma membranes of the three cell types was observed at 4, 10, 17, 23, and 30 degrees C. ANOVA analysis of the changes in DiI diffusion showed significant differences between the RBC and ISC membranes at all temperatures examined. The characteristic breaks in Arrhenius plots of the diffusion coefficients for the RBCs, RSCs, and ISCs occurred at 20, 19, and 18.6 degrees C, respectively. Photobleaching recovery data were used to estimate (Boullier, J.A., Melnykovich, G. and Barisas, B.G. (1982) Biochim. Biophys. Acta 692, 278-286) the microviscosities of the plasma membranes of the three cell types at 25 degrees C. We find significant differences between our microviscosity values and those obtained in previous fluorescence depolarization studies. However, both methods indicate qualitatively similar differences in membrane microviscosity among the various cell types.     

Correlation between cell density, membrane fluidity, and the availability of transferrin receptors in Friend erythroleukemic cells

Undifferentiated Friend erythroleukemic cells (FL-cells) acquire membrane microviscosity (eta), in accord with the culture cell density. At low cell density eta (21 degrees) approximately 2.8 poise, whereas at confluency it increases to eta (21 degrees) approximately 5.3 poise. Concomitantly, the total number of available transferring receptors per cell decreases by about 80% upon increase in cell density. Modulation of membrane microviscosity, by artificial alteration of the membrane cholesterol level, mediates similar modulations of the availability of the transferrin receptors. The correlation between the availability of the transferring receptors and the membrane lipid fluidity may take part in the overt decrease in iron uptake by erythroid cells along the erythropoiesis pathway.     

Comparing beta-carotene,vitamin E and nitric oxide as membrane antioxidants

Singlet oxygen initiates lipid peroxidation via a nonfree radical mechanism by reacting directly with unsaturated lipids to form lipid hydroperoxides (LOOHs). These LOOHs can initiate free radical chain reactions leading to membrane leakage and cell death. Here we compare the ability and mechanism by which three small-molecule membrane antioxidants (beta-carotene, alpha-tocopherol and nitric oxide) inhibit lipid peroxidation in membranes. We demonstrate that beta-carotene provides protection against singlet oxygen-mediated lipid peroxidation, but does not slow free radical-mediated lipid peroxidation. Alpha-Tocopherol does not protect cells from singlet oxygen, but does inhibit free radical formation in cell membranes. Nitric oxide provides no direct protection against singlet oxygen exposure, but is an exceptional chain-breaking antioxidant as evident from its ability to blunt oxygen consumption during free radical-mediated lipid peroxidation. These three small-molecule antioxidants appear to have complementary mechanisms for the protection of cell membranes from detrimental oxidations.     

The study of rous sarcoma virus-transformed baby hamster kidney cells using fluorescent probes.

The fluorescent probes pyrene, pyrene butyric acid and N-phenyl 1-naphthylamine have been used to investigate the changes that accompany in vitro transformation of a baby hamster kidney cell line using Rous sarcoma virus. The fluorescent probes which reside in the membrane were used to compare the changes in microviscosity and polarity of the membranes of normal cells with two transformed cell lines. The spectrofluorimetric data indicate that following transformation the probe N-phenyl 1-naphthylamine resides in a more polar environment. However, using the probe pyrene, the yield of excimer indicates decreased mobility of this probe in the membrane of transformed cells. The data also indicate differences between the two transformed cell lines. Laser photolysis was used to study the lifetime of the pyrene probes and the quenching of the pyrene fluorescence in the membrane by several different quenching molecules. The data indicate differences between the three cell lines and suggest that transformation decreases movement within the membrane.     

Nitric oxide and pH modulation in gynaecological cancer

Nitric oxide plays several roles in cellular physiology, including control of the vascular tone and defence against pathogen infection. Neuronal, inducible and endothelial nitric oxide synthase (NOS) isoforms synthesize nitric oxide. Cells generate acid and base equivalents, whose physiological intracellular concentrations are kept due to membrane transport systems, including Na⁺/H⁺ exchangers and Na⁺/HCO₃^? transporters, thus maintaining a physiological pH at the intracellular (~7.0) and extracellular (~7.4) medium. In several pathologies, including cancer, cells are exposed to an extracellular acidic microenvironment, and the role for these membrane transport mechanisms in this phenomenon is likely. As altered NOS expression and activity is seen in cancer cells and because this gas promotes a glycolytic phenotype leading to extracellular acidosis in gynaecological cancer cells, a pro‐inflammatory microenvironment increasing inducible NOS expression in this cell type is feasible. However, whether abnormal control of intracellular and extracellular pH by cancer cells regards with their ability to synthesize or respond to nitric oxide is unknown. We, here, discuss a potential link between pH alterations, pH controlling membrane transport systems and NOS function. We propose a potential association between inducible NOS induction and Na⁺/H⁺ exchanger expression and activity in human ovary cancer. A potentiation between nitric oxide generation and the maintenance of a low extracellular pH (i.e. acidic) is proposed to establish a sequence of events in ovarian cancer cells, thus preserving a pro‐proliferative acidic tumour extracellular microenvironment. We suggest that pharmacological therapeutic targeting of Na⁺/H⁺ exchangers and inducible NOS may have benefits in human epithelial ovarian cancer.     

Comparison between internal microviscosity of low-density erythrocytes and the microviscosity of hemoglobin solutions: an electron paramagnetic resonance study.

The hypothesis that the internal viscosity of erythrocytes is governed by the intracellular hemoglobin (Hb) concentration is examined. Here viscosity is determined by labeling of the cytoplasmic reduced glutathione with the spin label maleimido-Tempo. Erythrocyte populations with different Hb concentrations in isosmotic conditions were obtained through incomplete lysis, followed by cell resealing, and discontinuous density gradient separation. This procedure maintains normal cell shape and volume. Microviscosity of membrane-free Hb solutions was measured by addition of spin labeled glutathione. It was found that microviscosity values are similar for the erythrocyte cytoplasm and for Hb solutions of equivalent concentrations, showing that the erythrocyte membrane does not have any influence on internal microviscosity. The dependence of the microviscosity on the concentration of Hb solutions was compared with results of macroscopic viscosity obtained by other authors. It is concluded that microviscosity is sensitive to individual properties of the Hb molecule (intrinsic viscosity), but that it is not sensitive to intermolecular interactions. As the microviscosity behavior as a function of Hb concentration is the same in Hb solutions as in the erythrocyte cytoplasm, the inferences regarding macroscopic viscosity in Hb solutions could be translated to the rheological properties of the erythrocyte cytoplasm. Thus, these properties could be predicted from the values of the mean corpuscular Hb concentration.