Nanosecond spectroscopy of retinol.

Nanosecond fluorescence spectroscopy was used to study the unique binding site of the retinol-binding protein (RBP) from human serum. At pH 7.4, the binding of retinol to RBP caused the following spectroscopic changes in the ligand: (a) an enhancement of the fluorescence decay time (gamma = 8 ns); and (b) an increase in the emission anisotropy (A = 0.29). Retinol in hexane has a fluorescent decay time of 4.2 ns and a low emission anisotropy (A = 0.02). The increase in the fluorescence decay time of bound retinol is not due to dielectric relaxation effects of polar groups, since nanosecond time-resolved emission spectra of either retinol in glycerol or retinol bound to RBP, failed to show any time-dependent shifts in emission maxima during the time period investigated 0 to 30 ns. The degree of rotational mobility of bound retinol was investigated by time emission anisotropy measurements. The observed rotational correlation time (theta = 7.2 ns) is consistent with a rigid compact macromolecule of 21,000 molecular weight.     

A decrease of lipid fluidity of the porcine intestinal brush-border membranes by treatment with malondialdehyde.

The effect of treatment of the porcine intestinal brush-border membranes with malondialdehyde (MDA) on their lipid fluidity was examined using a fluorescence probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). When the membranes were treated with MDA, the fluorescence anisotropy of DPH-labeled membranes increased and the amount of DPH molecules incorporated into the membranes decreased from 3.25 to 2.23 nmol/mg protein. In addition, the response of the fluorescence anisotropy of DPH-labeled membranes to benzyl alcohol, a well-known fluidizer, was markedly suppressed by treatment of the membranes with MDA. These results suggest that treatment of the membranes with MDA causes a decrease of the membrane lipid fluidity. This interpretation was further supported by the increase observed in the fluorescence anisotropy of DPH-labeled liposomes prepared from the extracted lipids of MDA-treated membranes. The results of SDS-polyacrylamide gel electrophoresis suggested that the formation of high-molecular-weight aggregates of the membrane proteins is not involved in the increase of the fluorescence anisotropy of DPH-labeled membranes by treatment with MDA. On the basis of these results, changes in the physical properties of the intestinal brush-border membranes by treatment with MDA are discussed.     

Fluorescence studies of the blood platelet membranes associated with fibrinogen

To follow microviscosity changes in membranes associated with fibrinogen binding to human platelets, specific fluorescent probes were used and their fluorescence anisotropy was analysed. The degree of fluorescence anisotropy of diphenylhexatriene, anilinonaphthalene sulfonate (ANS) and fluorescamine increased significantly when fibrinogen reacted with its membrane receptors. Fluorescence polarization analyses showed that fibrinogen binding to platelet membranes is accompanied by an increase in the membrane lipid rigidity. On the other hand, changes in the fluorescence anisotropy of membrane tryptophans and N-(3-pyrene)maleimide suggest augmented mobility of the membrane proteins. The binding of fibrinogen to the membrane receptors is not accompanied by any change in the fluorescence intensity of ANS attached to the membranes. This may suggest that covering of platelets with fibrinogen molecules does not influence the surface membrane charge.     

Nanosecond fluorescence anisotropy decays of 1,6-diphenyl-1,3,5-hexatriene in membranes.

Nanosecond decays of the fluorescence anisotropy, r, were studied for the emission of 1,6-diphenyl-1,3,5-hexatriene (DPH) embedded in a series of mixed multilamellar liposomes containing egg yolk phosphatidylcholine, phosphatidylethanolamine and cholesterol in varying molar ratios, as well as in membranes of intact cells and in virus envelopes. The relative contributions of the fast and the infinitely slow decaying component to the steady-state value r, of the fluorescence anisotropy were very similar for artifical and biological membranes. Angles, theta, of the cone, by which the motion of the fluorescent molecule is limited, were calculated from the intensity of the infinitely slow decaying anisotropy component and compared with steady-state fluorescence anisotropies and with 'microviscosities', (eta). An increase in (eta) from 1.5 to 5.2 P in our systems was accompanied by a decrease in theta from 49 degrees to 30 degrees while the decrease in the mean motional relaxation times, phi f, of the label molecule was not more than 1 ns and due mainly to changes in the potential, by which the diffusion of DPH in the membrane is restricted. From these observations we conclude that differences in the steady-state fluorescence anisotropy and in 'microviscosities' of cholesterol-containing membranes (r greater than 0.15) represent changes in the degree of static orientational constraint rather than changes in diffusion rates of the label.     

The effect of ATP on the cell membrane structure]

ATP influence on the structure of plasma membranes thymocytes of cattle was studied. Fluorescence anisotropy of tryptophan residues of membrane proteins, fluorescence anisotropy of 3-methoxybenzanetron and fluorescence intensity of 1-anilinonaphthalene-8-sulphonate were determined. Changes of tryptophan fluorescence anisotropy and of ANS fluorescence intensity were established. It is supposed that the observed changes are connected with the change of membrane proteins structure and plasma membrane charge.     

Influence of serosal Cl on transport properties and cation activities in frog skin

Summary Treatment of resident peritoneal macrophages of rats with small unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC SUV) potentiated their activation for tumor cell lysis by endotoxins. The fluorescence polarization of diphenylhexatriene (DPH) embedded in rough endoplasmic reticulum membranes isolated from DPPC SUV-treated macrophages was enhanced. The average fluorescence lifetime of DPH and the rotational correlation time deduced from anisotropy decay were unchanged, whereas the residual anisotropy and hence the order parameter were increased. The measurement of the fluorescence anisotropy of DPH as a function of the temperature showed a phase transition. No phase transition was observed in the rough endoplasmic reticulum membranes of macrophages either treated or not treated with cholesterol/DPPC SUV (1/1; mol/mol). The synergistic effect of DPPC SUV on the tumoricidal activity of macrophages induced by endotoxins appears to be correlated with the changes in the properties of the rough endoplasmic reticulum membranes. Both effects were transient; they had the same kinetics of induction and reversion, and they were both inhibited by cholesterol.     

Interphotoreceptor retinoid-binding protein is the physiologically relevant carrier that removes retinol from rod photoreceptor outer segments

Light detection by vertebrate rod photoreceptor outer segments results in the destruction of the visual pigment, rhodopsin, as its retinyl moiety is photoisomerized from 11-cis to all-trans. The regeneration of rhodopsin is necessary for vision and begins with the release of the all-trans retinal and its reduction to all-trans retinol. Retinol is then transported out of the rod outer segment for further processing. We used fluorescence imaging to monitor retinol fluorescence and quantify the kinetics of its formation and clearance after rhodopsin bleaching in the outer segments of living isolated frog (Rana pipiens) rod photoreceptors. We independently measured the release of all-trans retinal from bleached rhodopsin in frog rod outer segment membranes and the rate of all-trans retinol removal by the lipophilic carriers interphotoreceptor retinoid binding protein (IRBP) and serum albumin. We find that the kinetics of all-trans retinol formation in frog rod outer segments after rhodopsin bleaching are to a good first approximation determined by the kinetics of all-trans retinal release from the bleached pigment. For the physiological concentrations of carriers, the rate of retinol removal from the outer segment is determined by IRBP concentration, whereas the effect of serum albumin is negligible. The results indicate the presence of a specific interaction between IRBP and the rod outer segment, probably mediated by a receptor. The effect of different concentrations of IRBP on the rate of retinol removal shows no cooperativity and has an EC50 of 40 micromol/L.     

Decreased fluidity of isolated erythrocyte membranes in type 1 and type 2 diabetes. The effect of resorcylidene aminoguanidine.

Changes in the physico-chemical properties of erythrocyte membranes induced by nonenzymatic glycation as well as the possible prevention of their rise were studied. Using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH), fluorescence anisotropy values were determined in erythrocyte membranes isolated from type 1 and type 2 diabetic patients with and without complications. The mean anisotropy values for the groups of diabetic patients were significantly higher than those for the control group (p < 0.01). This indicated pathologically decreased fluidity in cell membranes in the diabetics regardless of the type of diabetes or the presence of complications. The fluorescence anisotropy positively correlated (p < 0.01) with clinical parameters, such as glycohaemoglobin and plasma cholesterol content, which are important for the monitoring of the compensation status of the diabetic patient. Our results support the suggestion that protein crosslinking and oxidative stress induced by nonenzymatic glycation contribute to changes in the physico-chemical properties of erythrocyte membranes. In vitro testing of a new potential drug resorcylidene aminoguanidine (RAG) showed its ability to increase significantly (p < 0.001), to various extent (p < 0.01), the fluidity of both diabetic and control erythrocyte membranes. Upon the administration of RAG, reduced fluorescence anisotropy values for the groups of diabetic patients approached the normal values obtained for the controls. This may play an important role in the improvement of impaired cell functions found in diabetes that are controlled by the cell membrane.     

A new analysis method for the membrane viscosity from steady-state fluorescence depolarization

The absolute value of the viscosity in membrane lipid bilayers, which is different from the microviscosity advocated by Shinitzky, could be calculated from steady-state fluorescence depolarization of a hydrocarbon fluorophore, 1,6-diphenyl-1,3,5-hexatriene (DPH). This method was based on the theory of time-resolved fluorescence anisotropy and empirical relationships between fluorescence life time and the anisotropy parameters such as half cone angle in wobbling motion and wobbling diffusion rate of the fluorescent probe. Obtained viscosity values of various membranes from this method were consistent with those from time resolved method within experimental error.     

Dynamics and heterogeneity of bovine hippocampal membranes: Role of cholesterol and proteins

The structural and dynamic consequence of alterations in membrane lipid composition (specifically cholesterol) in neuronal membranes is poorly understood. Previous work from our laboratory has established bovine hippocampal membranes as a convenient natural source for studying neuronal receptors. In this paper, we have explored the role of cholesterol and proteins in the dynamics and heterogeneity of bovine hippocampal membranes using fluorescence lifetime distribution analysis of the environment-sensitive fluorescent probe Nile Red incorporated into such membranes by the maximum entropy method (MEM), and time-resolved fluorescence anisotropy measurements. The peak position and the width of the lifetime distribution of Nile Red show a progressive reduction with increasing cholesterol depletion from native hippocampal membranes indicating that the extent of heterogeneity decreases with decrease in membrane cholesterol content. This is accompanied by a concomitant decrease of the fluorescence anisotropy and rotational correlation time. Our results point out that the microenvironment experienced by Nile Red is relatively insensitive to the presence of proteins in hippocampal membranes. Interestingly, Nile Red lifetime distribution in liposomes of lipid extracts is similar to that of native membranes indicating that proteins do not contribute significantly to the high level of heterogeneity observed in native membranes. These results could be relevant in understanding the neuronal diseases characterized by defective membrane lipid metabolism.     

Dynamics and heterogeneity of bovine hippocampal membranes: role of cholesterol and proteins

The structural and dynamic consequence of alterations in membrane lipid composition (specifically cholesterol) in neuronal membranes is poorly understood. Previous work from our laboratory has established bovine hippocampal membranes as a convenient natural source for studying neuronal receptors. In this paper, we have explored the role of cholesterol and proteins in the dynamics and heterogeneity of bovine hippocampal membranes using fluorescence lifetime distribution analysis of the environment-sensitive fluorescent probe Nile Red incorporated into such membranes by the maximum entropy method (MEM), and time-resolved fluorescence anisotropy measurements. The peak position and the width of the lifetime distribution of Nile Red show a progressive reduction with increasing cholesterol depletion from native hippocampal membranes indicating that the extent of heterogeneity decreases with decrease in membrane cholesterol content. This is accompanied by a concomitant decrease of the fluorescence anisotropy and rotational correlation time. Our results point out that the microenvironment experienced by Nile Red is relatively insensitive to the presence of proteins in hippocampal membranes. Interestingly, Nile Red lifetime distribution in liposomes of lipid extracts is similar to that of native membranes indicating that proteins do not contribute significantly to the high level of heterogeneity observed in native membranes. These results could be relevant in understanding the neuronal diseases characterized by defective membrane lipid metabolism.     

Determination of Fluorescence Polarization of Membrane Probes in Intact Erythrocytes: Possible Scattering Artifacts

The anisotropy of the fluorescence of diphenylhexatriene has been reported to be less in the membranes of intact erythrocytes than in erythrocyte ghost membranes or in membranes prepared from erythrocyte lipids. Evidence is presented that this may be an artifact due to the intense light scattering by the intact erythrocytes.     

A fluorescent sterol probe study of cholesterol/phospholipid membranes

The behavior of dehydroergosterol in -α-dimyristoylphosphatidylcholine (DMPC) unsonicated multilamellar liposomes was characterized by absorption spectroscopy and fluorescence measurements. Dehydroergosterol exhibited a lowered absorption coefficient in multilamellar liposomes whiel the steady-state fluorescence anisotropy of dehydroergosterol in these membranes decreased significantly with increasing dehydroergosterol concentration, suggesting membrane sterol-sterol interactions. The comparative steady-state anisotropy of 0.9 mole percent dehydroergosterol in multilamellar liposomes was lower than in small unilamellar vesicles suggesting different sterol environments for dehydroergosterol. Dehydroergosterol fluorescence lifetime was relatively independent of membrane sterol content and yielded similar values in sonicated and unsonicated model membranes. In multilamellar liposomes containing 5 mole percent cholesterol, the gel-to-liqui crystalline phase transition of DMPC detected by 0.9 mole percent dehydroergosterol was significantly broadened when compared to the phase transition detected by dehydroergosterol in the absence of membrane cholesterol (Smutzer, G. et al. (1986) Biochim. Biophys. Acta 862, 361–371). In multilamellar liposomes containing 10 mole percent cholesterol, the major fluorescence lifetime of dehydroergosterol did not detect the gel-to-liquid crystalline phase transition of DMPC. Time-correlated fluorescence anisotropy decays of dehydroergosterol in DMPC multilamellar liposomes in the absence and presence of 5 mole percent cholesterol exhibited a single rotational correlation time near one nanosecond that was relatively independent of temperature and low concentrations of membrane cholesterol. The limiting anisotropy of 0.9 mole percent dehydroergosterol decreased above the gel-to-liquid crystalline phase transition in membranes without cholesterol and was not significantly affected by the phase transition in membranes containing 5 mole percent cholesterol. These results suggested hindered rotational diffusion of dehydroergosterol in multilamellar liposomes. Lifetime and time-correlated fluorescence measurements of 0.9 mole percent dehydroergosterol in multilamellar liposomes further suggested this fluorophore was detecting physical properties of the bulk membrane phospholipids in membranes devoid of cholesterol and was detecting sterol-rich regions in membranes of low sterol concentration.     

Mechanism of vitamin A movement between rod outer segments, interphotoreceptor retinoid-binding protein, and liposomes

Vitamin A movement between rod outer segment (ROS) membranes, interphotoreceptor retinoid-binding protein (IRBP), and liposomes was examined by two different methods. Equilibrium exchange of all-trans-retinol was followed by assessing the transfer of [3H]retinol from liposomes to ROS membranes as compared to a nontransferable marker, [14C]triolein. In the absence of IRBP, a rapid, spontaneous transfer of [3H] retinol to the ROS membranes occurred. In the presence of 2 microM IRBP, retinol transfer decreased by approximately one-half, whereas a similar concentration of bovine serum albumin had no effect on this spontaneous transfer. Kinetics of retinol transfer between single unilamellar vesicles were determined by the method of fluorescence energy transfer. The first order rate constant for this transfer was 0.85 s-1 at 22 degrees C at either pH 7.4 or pH 2.8. This rate was not affected by varying the concentration of acceptor vesicles 50-fold or by varying their concentration 10-fold at a constant ratio of donor-to-acceptor vesicles. The presence of IRBP as an additional acceptor did not change the rate. The transfer was temperature-dependent with an activation energy of 7.8 kcal/mol. The transfer rate appeared to be an increasing exponential function of ionic strength since high concentrations of NaCl decreased the transfer rate significantly. The transfer rate of retinol from IRBP to single unilamellar vesicles also followed first order kinetics with a rate constant of 0.11 s-1 at 22 degrees C, which was approximately 8 times slower than that of transfer between vesicles. We conclude that the transfer of all-trans-retinol between liposomes and membranes can be accomplished rapidly via the aqueous phase, and that IRBP retards rather than facilitates this transfer process.     

Cholesterol dynamics in membranes.

Time-resolved fluorescence anisotropy of the sterol analogue, cholestatrienol, and 13C nuclear magnetic resonance (NMR) spin lattice relaxation time (T1c) measurements of [13C4] labeled cholesterol were exploited to determine the correlation times characterizing the major modes of motion of cholesterol in unsonicated phospholipid multilamellar liposomes. Two modes of motion were found to be important: (a) rotational diffusion and (b) time dependence of the orientation of the director for axial diffusion, or "wobble." From the time-resolved fluorescence anisotropy decays of cholestatrienol in egg phosphatidylcholine (PC) bilayers, a value for tau perpendicular, the correlation time for wobble, of 0.9 x 10(-9) s and a value for S perpendicular, the order parameter characterizing the same motion, of 0.45 s were calculated. Both tau perpendicular and S perpendicular were relatively insensitive to temperature and cholesterol content of the membranes. The T1c measurements of [13C4] labeled cholesterol did not provide a quantitative determination of tau parallel, the correlation time for axial diffusion. T1c from the lipid hydrocarbon chains suggested a value for tau perpendicular similar to that for cholesterol. Steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in a variety of pure and mixed lipid multilamellar liposomes. Both the lipid headgroups and the lipid hydrocarbons chains contributed to the determination of the sterol environment in the membrane, as revealed by these fluorescence measurements. In particular, effects of the phosphatidylethanolamine (PE) headgroup and of multiple unsaturation in the lipid hydrocarbon chains were observed. However, while the steady-state anisotropy was sensitive to these factors, the time-resolved fluorescence analysis indicated that tau perpendicular was not strongly affected by the lipid composition of the membrane. S perpendicular may be increased by the presence of PE. Both steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in three biological membranes: bovine rod outer segment (ROS) disk membranes, human erythrocyte plasma membranes, and light rabbit muscle sarcoplasmic reticulum membranes. In the ROS disk membranes the value for S perpendicular was marginally higher than in the PC membranes, perhaps reflecting the influence of PE. The dramatic difference noted was in the value for tau perpendicular. In both the ROS disk membranes and the erythrocyte membranes, tau perpendicular was one-third to one-fifth of tau perpendicular in the phospholipid bilayers. This result may reveal an influence of membrane proteins on sterol behavior.     

Microenvironment changes of human blood platelet membranes associated with fibrinogen binding

Alterations in the membrane organization caused by fibrinogen binding to human blood platelets and their isolated membranes were analyzed by fluorescence and electron spin resonance measurements. The degree of fluorescent anisotropy of DPH, ANS and fluorescamine increased significantly when fibrinogen reacted with its membrane receptors. Both fluorescence and ESR analyses showed that fibrinogen binding to platelet membranes is accompanied by an increase of the membrane lipid rigidity. This effect seems to be indirect in nature and is mediated by altered membrane protein interactions. As it has been shown that an increased membrane lipid rigidity leads to a greater exposure of membrane proteins, including fibrinogen receptors, this might facilitate a formation of molecular linkages between neighboring platelets. On the other hand, changes of fluorescence anisotropy of membrane tryptophans and N-(3-pyrene) maleimide suggest the augmented mobility of the membrane proteins. Evidence is presented which indicated that the binding of fibrinogen to the membrane receptors is not accompanied by any changes in the fluorescence intensity of ANS attached to the membranes. It may suggest that the covering of platelets with fibrinogen does not influence the surface membrane charge. In contrast to fibrinogen, calcium ions caused an increase of the fluorescence intensity resulting from the more efficient binding of ANS to the platelet membranes.     

Role of sterol structure in the thermotropic behavior of plasma membranes of Saccharomyces cerevisiae

Plasma membranes from Saccharomyces cerevisiae were prepared by a new procedure involving lyticase treatment of the yeast cells. The plasma membranes were right-side-out, closed vesicles of uniform appearance with a sterol to phospholipid molar ratio of 0.365. The thermotropic behavior of these plasma membranes from wild-type yeast and from sterol mutants was examined by differential scanning calorimetry, fluorescence anisotropy and Arrhenius kinetics of plasma membrane enzymes. While differential scanning calorimetry failed to demonstrate any lipid transition, fluorescence anisotropy data indicated that lipid transitions were occurring in the plasma membranes of the yeast sterol mutants but not the sterol wild-type. The temperature dependence of the plasma membrane enzymes, chitin synthase and Mg²⁺-ATPase, was also investigated. The Arrhenius kinetics of chitin synthase did not reveal any transitions in either the sterol mutant or wild-type plasma membranes, yet the Arrhenius kinetics of the Mg²⁺-ATPase suggested that lipid transitions were occurring in both cases.     

Effect of amyloid β-peptide on the fluidity of phosphatidylcholine membranes: Uses and limitations of diphenylhexatriene fluorescence anisotropy

There is accumulating evidence that peptide-induced perturbations in the order and dynamics of cellular membranes may play a role in the neurotoxicity of amyloid β-peptide (Aβ). Several studies have reported that Aβ decreases fluidity of membranes based on an Aβ-induced increase in the fluorescence anisotropy of diphenylhexatriene (DPH). However, the effect of Aβ on the membrane fluidity is still a subject of controversy, because other studies that employed pyrene as a fluorescent probe have shown that Aβ has the opposite effect. To reveal the reason for this discrepancy, we have examined the effect of Aβ on the fluidity of phosphatidylcholine membranes using spectroscopic methods. The fluorescence anisotropy of DPH is dramatically increased on addition of Aβ to DPH-containing phosphatidylcholine membranes. However, Aβ does not affect the Raman spectrum of the membrane, which is sensitive to the packing order of the hydrocarbon chains of lipids. We have also found that circular dichroism (CD) bands of DPH appear during incubation of DPH-containing membranes with Aβ, whereas DPH is an achiral molecule. The observed CD bands of DPH are induced by a chiral environment of Aβ but not by that of the lipids, because positive CD bands appear regardless of the d/l-chirality of phosphatidylcholine. The findings obtained from CD measurements provide evidence that DPH molecules translocate from the membrane to Aβ. The peptide-mediated extraction of DPH from the membrane may cause changes in the fluorescence anisotropy of DPH, even though Aβ does not affect the fluidity of membranes.     

Use of Steady-State Laurdan Fluorescence to Detect Changes in Liquid Ordered Phases in Human Erythrocyte Membranes

In artificial phospholipid bilayers, dual measurements of laurdan steady-state anisotropy and emission spectra can be used to identify the presence of liquid ordered phases. Human erythrocytes were used as a model to test whether similar measurements could be applied to biological samples. Specifically, laurdan anisotropy and emission spectra were obtained from native erythrocytes before and after treatment with calcium ionophore and from the microvesicles (known to be enriched in liquid ordered domains) shed from the cells during calcium entry. Spectral and anisotropy data were consistent with an increased order and reduced fluidity of erythrocyte membrane lipids upon ionophore treatment. Microvesicle membranes appeared more ordered than native erythrocytes and similar to ionophore-treated cells based on laurdan emission. In contrast, the anisotropy value was lower in microvesicles compared to ionophore-treated cells, suggesting greater probe mobility. Parallel measurements of diphenylhexatriene anisotropy corroborated the laurdan data. These results were consistent with the liquid ordered property of microvesicle membranes based on comparisons to behavior in artificial membranes. Two-photon microscopy was used to examine the distribution of laurdan fluorescence along the surface of erythrocyte membranes before and after ionophore treatment. A dual spatial analysis of laurdan anisotropy, as revealed by the distribution of laurdan emission spectra, and intensity excited by polarized light suggested that the plasma membranes of ionophore-treated erythrocytes may also exhibit elevated numbers of liquid ordered domains.     

Evidence for 21-aminosteroid association with the hydrophobic domains of brain microvessel endothelial cells.

Studies were conducted to demonstrate 21-aminosteroid distribution into the hydrophobic or lipid domains of biological membranes, a presumed site at which these compounds inhibit lipid peroxidation. Bovine brain microvessel endothelial cells (BMECs) were labeled with diphenylhexatriene fluorophores and interactions with cell membranes characterized with fluorescence anisotropy and lifetimes. Two 21-aminosteroids (U-74500A and U74006F) were shown to preferentially alter the fluorescence anisotropy and lifetime parameters of the diphenylhexatriene probe distributing into membranes throughout the BMECs. Little or no effect of the compounds was observed on the fluorescence parameters of the probe localized on the surface of BMEC plasma membranes. By contrast, cholesterol used as a positive control substantially altered the fluorescence parameters of BMECs labeled with either diphenylhexatriene probe. Results suggest 21-aminosteroid-induced changes in the molecular packing order and drug: fluorescent probe interactions in membrane hydrophobic (or lipid) domains throughout the BMEC. Concentrations of 21-aminosteroids altering the fluorescence parameters of diphenylhexatriene labeled BMECs correspond to those concentrations of 21-aminosteroids effective in vitro in inhibition of lipid peroxidation.