Minor effects of bulk viscosity on lipid translational diffusion measured by the excimer formation technique

We have investigated the effect of bulk viscosity on lipid translational diffusion using the excimer formation technique. In contrast to a study by Vaz et al. (1987), performed with the fluorescence recovery after photobleaching technique, we observed only a minor decrease of less than a factor of two for pyrene labelled phosphatidylcholine in glycerinated phosphatidylcholine bilayer membranes compared to an aqueous dispersion. Even the diffusion of pyrene labelled gangliosides with an oligosaccharide head-group that protrudes from the membrane surface is not strongly restricted by the increased bulk viscosity. We conclude that the viscosity of the fluid bounding the lipid bilayers is of minor importance for the diffusion of membrane lipids.Abbreviations DPPC 1-2 dipalmitoyl-sn-glycero-3-phosphocholine - DSPC 1-2 distearoyl-sn-glycero-3-phosphocholine - PyPC 1-acyl-2-[10(-1-pyrene)decanoyl]-sn-glycero-3-phosphocholine - PyG_M1 N-12-(1-pyrene)dodecanoyl-lyso G_M1 - PyG_M2 N-12-(1-pyrene)dodecanoyl-lyso G_M2 - PyG_M3 N-12-(1-pyrene) dodecanoyl-lyso G_M3 - I_M fluorescence intensity of the monomeric pyrene probe - I_D fluorescence intensity of the excimer     

Role of polyunsaturated fatty acids and lipid peroxidation in LM fibroblast plasma membrane transbilayer structure

The effects of polyunsaturated fatty acids and lipid peroxidation on LM fibroblast plasma membrane individual leaflet sterol distribution and structural order were examined. The cytofacial (inner) leaflet was more rigid and contained more sterol than the exofacial (outer) leaflet. The static (limiting anisotropy) and dynamic (rotational relaxation time) structural components of diphenylhexatriene (DPH) motion in each leaflet were determined by phase and modulation fluorometry measurements combined with leaflet-specific quenching by trinitrophenyl groups. Polyunsaturated fatty acids, incorporated into the membrane phospholipids by culture medium supplementation, decreased the limiting anisotrophy of DPH in the cytofacial but not the exofacial leaflet thereby abolishing the transbilayer difference in fluidity. Peroxidation by Fe(II) + H2O2 resulted in a rigidification (increase in limiting anisotropy and rotational relaxation time) of the plasma membrane exofacial leaflet, regardless of whether the membranes contained saturated and monounsaturated fatty acids or were enriched in either linoleate or linolenate. The structure of the cytofacial leaflet reported by DPH was unaffected. Plasma membrane transbilayer sterol distribution, measured by leaflet-specific quenching of dehydroergosterol fluorescence, indicated that 20-28% of the sterol was localized in the exofacial leaflet. Polyunsaturated fatty acid supplementation of LM fibroblasts resulted in a complete reversal of plasma membrane transbilayer sterol distribution (72-76% exofacial leaflet). Sterol transbilayer distribution between the membrane leaflets was completely resistant to alteration by exposure to crosslinking agents and peroxidation in control plasma membranes and by peroxidation in linoleate- or linolenate-supplemented membranes.     

Influence of chain length of pyrene fatty acids on their uptake and metabolism by Epstein-Barr-virus-transformed lymphoid cell lines from a patient with multisystemic lipid storage myopathy and from control subjects.

The uptake and intracellular metabolism of 4-(1-pyrene)butanoic acid (P4), 10-(1-pyrene)decanoic acid (P10) and 12-(1-pyrene)dodecanoic acid (P12) were investigated in cultured lymphoid cell lines from normal individuals and from a patient with multisystemic lipid storage myopathy (MLSM). The cellular uptake was shown to be dependent on the fatty-acid chain length, but no significant difference in the uptake of pyrene fatty acids was observed between MLSM and control lymphoid cells. After incubation for 1 h the distribution of fluorescent fatty acids taken up by the lymphoid cell lines also differed with the chain length, most of the fluorescence being associated with phospholipid and triacylglycerols. In contrast with P10 and P12, P4 was not incorporated into neutral lipids. When the cells were incubated for 24 h with the pyrene fatty acids, the amount of fluorescent lipids synthesized by the cells was proportional to the fatty acid concentration in the culture medium. After a 24 h incubation in the presence of P10 or P12, at any concentration, the fluorescent triacylglycerol content of MLSM cells was 2-5-fold higher than that of control cells. Concentrations of pyrene fatty acids higher than 40 microM seemed to be more toxic for mutant cells than for control cells. This cytotoxicity was dependent on the fluorescent-fatty-acid chain length (P12 greater than P10 greater than P4). Pulse-chase experiments permitted one to demonstrate the defect in the degradation of endogenously biosynthesized triacylglycerols in MLSM cells (residual activity was around 10-25% of controls on the basis of half-lives and initial rates of P10- or P12-labelled-triacylglycerol catabolism); MLSM lymphoid cells exhibited a mild phenotypic expression of the lipid storage (less severe than that observed in fibroblasts). P4 was not utilized in the synthesis of triacylglycerols, and thus did not accumulate in MLSM cells: this suggests that natural short-chain fatty acids might induce a lesser lipid storage in this disease.     

Lipid peroxidation causes an increase of lipid order and a decrease of 5'-nucleotidase activity in the liver plasma membrane.

The effect of peroxidation on 5'-nucleotidase activity as well as on membrane microviscosity has been investigated in liver plasma membranes from Wistar rats. The peroxidation was performed with 100 microM H2O2 and 200 microM FeSO4 and/or with 5 mM t-butylhydroperoxide. Treatment of the membranes with these oxidizing agents resulted in an elevation of the transition temperatures of the polarization of the lipid fluorescent probes 1,6 diphenyl-1,3,5 hexatriene (DPH), 3-p-(6-phenyl) 1,3,5 hexatriene phenylpropionic acid (PA-DPH) as well as of the fluorescent thiol reagent N-(1-pyrene) maleimide (1-PM). The peroxidation resulted in a decrease of the activity of 5'nucleotidase. Our data support that the increase of membrane microviscosity of the lipid domain regulates the activity of 5'-nucleotidase.     

Fluorescent reagents for in vitro studies of lipid-linked steps of bacterial peptidoglycan biosynthesis: derivatives of UDPMurNAc-pentapeptide containing d-cysteine at position 4 or 5

UDPMurNAc-L-Ala-gamma-D-Glu-X-D-Ala-DAla (X = L-Lys or m-DAP) is the cytoplasmic precursor for the lipid-linked cycle of bacterial peptidoglycan biosynthesis, consisting of at least four enzymatic reactions, which are targets for antibacterial agents. Fluorescent derivatives of the UDPMurNAc-pentapeptide labelled at the 3rd, 4th, and 5th position of the peptide chain were prepared chemoenzymatically, in order to study the reactions catalysed by enzymes in this cycle. Derivatives labelled on the epsilon-amino group of the 3rd amino acid (N-dansyl, N-fluorescamine and N-phthalaldehyde) were prepared by chemical modification. Two methods were developed for preparation of analogues of UDPMurNAc-pentapeptide containing D-cysteine at position 4 or 5: either by MurF-catalysed ligation of the UDPMurNAc-tripeptide to synthetic D-Ala-D-Cys or D-Cys-D-Ala dipeptides; or by enzymatic synthesis of D-Ala-D-Cys by ligase VanD. D-Cys-containing UDPMurNAc-pentapeptides were labelled with pyrene maleimide, to give 4-pyrene and 5-pyrene labelled derivatives. The fluorescent UDPMurNAc-pentapeptides were processed as substrates by Escherichia coli MraY or E. coli membranes, giving 1.5-150-fold changes in fluorescence upon transformation to lipid intermediate I. Subsequent processing to lipid intermediate II gave rise only to small changes in fluorescence. Pyrene-labelled lipid intermediates I and II can be generated using Micrococcus flavus membranes, enabling the study of the later lipid-linked steps.     

Changes in the fluorescence parameters of bound N-(1-pyrene) maleimide by lipid peroxidation of intestinal brush-border membranes

Using a fluorogenic thiol reagent, N-(1-pyrene)maleimide (NPM), we have examined of lipid peroxidation on the microenvironment around SH groups of the membrane proteins in porcine intestinal brush-border membrane vesicles. The lipid peroxidation of the membranes was performed with various concentrations of t-butylhydroperoxide (t-BuOOH) in the presence of 100 microM ascorbic acid and 10 microM Fe2+. Treatment of NPM-labeled membranes with these oxidizing agents resulted in a decrease of the fluorescence lifetime, suggesting modification of the environmental properties around the bound dye. Measurement of the steady-state fluorescence anisotropy of the labeled membranes indicated restriction of the motion of the bound dye by the lipid peroxidation membranes. This interpretation was further supported by an elevation of the transition temperature of the anisotropy, a decrease in the quenching rate constant of the fluorescence with acrylamide and a decrease in the SH reactivity of the membrane proteins for NPM by lipid peroxidation. Based on these results, the possibility of conformation changes in the vicinity of SH groups in the membrane proteins associated with lipid peroxidation has been discussed.     

Monitoring the location profile of fluorophores in phosphatidylcholine bilayers by the use of paramagnetic quenching

Spin probes differing in the position of their paramagnetic centre are used to quench the fluorescence of pyrene derivatives and chlorophylls incorporated into dimyristoyl phosphatidylcholine membranes. Pyrene butyric acid and pyrene decanoic acid with known orientation relative to the membrane surface are investigated. The quenching efficiency of fatty acid spin probes is dependent on the position of the nitroxide radical group in the fatty acid chain. Using this short fange interaction we developed a spectroscopic method to characterize the molecular arrangement within the lipid membrane. Applied to chlorophyll-containing vesicles, we were able to characterize the orientation of the porphyrin ring within the membrane. Moreover, the chlorophyll fluorescence is also quenched by a water-soluble spin label. Therefore the porphyrin ring appears to be orientated in the polar head group region of the lipid layer, but not to be protruding out into the water phase.This conclusion is confirmed by the use of pyrene derivatives. Fluorescence quenching by a water-soluble spin label within the lipid matrix is observed even in the rigid state of the membrane. Fluorescence lifetime measurements suggest the existence of two different quenching mechanisms: (1) a static quenching occurring below the lipid phase transition temperature, and (2) an additional dynamic quenching taking place in the fluid state of the lipid bilayer.     

Monitoring the location profile of fluorophores in phosphatidylcholine bilayers by the use or paramagnetic quenching.

Spin probes differing in the position of their paramagnetic centre are used to quench the fluorescence of pyrene derivatives and chlorophylls incorporated into dimyristoyl phosphatidylcholine membranes. Pyrene butyric acid and pyrene decanoic acid with known orientation relative to the membrane surface are investigated. The quenching efficiency of fatty acid spin probes is dependent on the position of the nitroxide radical group in the fatty acid chain. Using this short range interaction we developed a spectroscopic method to chlorophyll-containing vesicles, we were able to characterize the orientation of the porphyrin ring within the membrane. Moreover, the chlorophyll fluorescence is also quenched by a water-soluble spin label. Therefore the porphyrin ring appears to be orientated in the polar head group region of the lipid layer, but not to be protruding out into the water phase. This conclusion is confirmed by the use of pyrene derivatives. Fluorescence quenching by a water-soluble spin label within the lipid matrix is observed even in the rigid state of the membrane. Fluorescence lifetime measurements suggest the existence of two different quenching mechanisms: (1) a static quenching occurring below the lipid phase transition temperature, and (2) an additional dynamic quenching taking place in the fluid state of the lipid bilayer.     

Uptake and degradation of virus-associated fluorescent sulfatide by skin fibroblasts

A fluorescent derivative of cerebroside sulfate, pyrene-dodecanoyl sphingosylgalactosylsulfate (P12-CS) was incorporated into the envelope of vesicular stomatitis virus (VSV). When the P12-CS-containing virus was incubated for 24 h with skin fibroblasts, up to 40% of the sulfatide was located in the cells--a value at least 10 fold greater than that observed using sulfatide suspensions without virus. In a similar experiment, in which the 24 h 'pulse' was followed by a 48 h 'chase', about 15-20% of the virus-associated fluorescence was recovered in the skin fibroblasts. Of the latter, about one-third was present as desulfated degradation products of P12-CS. The high uptake and degradation of the virus-associated sulfatide by intact skin fibroblasts suggested that enveloped viruses could be used for introducing other lipids into cells. This could be utilized for studying lipid catabolism and diagnosing lipid storage disorders in intact living cells.     

Protein-dependent Reduction of the Pyrene Excimer Formation in Membranes

The presence of proteins in lipid bilayers always decreases the excimer formation rate of pyrene and pyrene lipid analogues in a way that is related to the protein-to-lipid ratio. Energy transfer measurements from intrinsic tryptophans to pyrene have shown (Engelke et al., 1994), that in microsomal membranes, the excimer formation rate of pyrene and pyrene fatty acids is heterogeneous within the membrane plane, because a lipid layer of reduced fluidity surrounds the microsomal proteins. This study investigates whether of not liposomes prepared from egg yolk phosphatidylcholine with incorporated gramicidin A give results comparable to those from microsomal membranes. The results indicate that the influence of proteins on the lipid bilayer cannot be described by one unique mechanism: Small proteins such as gramicidin A obviously reduce the excimer formation rate by occupying neighboring positions of the fluorescent probe and thus decrease the pyrene collision frequency homogeneously in the whole membrane plane, while larger proteins are surrounded by a lipid boundary layer of lower fluidity than the bulk lipid. The analysis of the time-resolved tryptophan fluorescence of gramicidin A incorporated liposomes reveals, that the tryptophan quenching by pyrene is stronger for tryptophans located closely below the phospholipid headgroup region because of the pyrene enrichment in this area of the lipid bilayer. Received: 29 December 1996/Revised: 15 May 1996     

Lipid peroxidation increases the molecular order of microsomal membranes

The effect of enzymatic lipid peroxidation on the molecular order of microsomal membranes was evaluated by ESR spectroscopy using the spin probes 5-, 12-, and 16-doxyl-stearic acid. Rat liver microsomal membranes were peroxidized by the NADPH-dependent reaction in the presence of the chelate ADP-Fe3+. Peroxidation resulted in a preferential depletion of polyenoic fatty acids and an increase in the percentage composition of shorter fatty acyl chains. There was no change in the cholesterol/phospholipid ratio of the peroxidized microsomes. The molecular order of both control and peroxidized membranes decreased toward the central region of the bilayer, and the order parameter (S) of each probe was temperature dependent. Peroxidation of the microsomal membrane lipids resulted in an increase in the order parameter determined with the three stearic acid spin probes. Of the three probes, 12-doxylstearic acid was the most sensitive to the changes in membrane organization caused by peroxidation. These data indicate that ESR spectroscopy is a sensitive method of detecting changes in membrane order accompanying peroxidation of membrane lipids.     

2 pathways of polycyclic hydrocarbon hydroxylation in endoplasmic reticulum membranes

The effect of pro-oxidant (ions of iron) and antioxidants (alpha-tocopherol, propylgallate) on hydroxylation of polycyclic hydrocarbon benz(a)-pyrene and the effect of hydroxylation process on lipid peroxidation have been studied. The role of allyl radicals formed in the fatty acid chains is discussed. The binding of oxygen radicals (formation of peroxy radicals) is regarded only as on of the possible reactions of the radical utilization. It is assumed that other reactions involving lipid (allyl) radicals, in particular, hydroxylation of benz(alpha)pyrene may occur in microsomal membranes.     

Transbilayer asymmetry of pyrene mobility in human spherocytic red cell membranes.

The diffusion-dependent formation of pyrene excimers (excited dimers) was studied in normal and spherocytic red cell membranes. Pyrene emission was alternatively quenched in either bilayer half by non radiative energy transfer to haemoglobin. Pyrene excimer to monomer fluorescence intensity ratio, I'/I, was 0.35 +/- 0.03 (S.E.) in washed red blood cells obtained from normal donors (n = 8) and 0.45 + 0.03 (n = 13) in the corresponding isolated, haemoglobin-free resealed membranes (P less than 0.02). In the spherocytic condition the respective values were 0.28 +/- 0.01 (n = 9) and 0.53 +/- 0.03 (n = 9), P less than 0.001. In contrast to the decrease of I'/I in red cells as compared to isolated membranes, being 22% in normal cells and 47% in spherocytic ones, haemoglobin added to the exofacial side of isolated membranes, respectively, reduced I'/I by 18% and 5%. In normal red cell membranes, pyrene mobility appears to be higher in the inner monolayer than in the outer one. In spherocytic membranes our results indicate an enhanced transmembrane asymmetry in lipid monolayer fluidity, probably due to a defect of the membrane protein skeleton organization.     

Differential modulation of human small intestinal brush-border membrane hemileaflet fluidity affects leucine aminopeptidase activity and transport of D-glucose and L-glutamate.

The fluidity of the exofacial (outer) and cytofacial (inner) leaflets of human proximal small intestinal brush-border membrane vesicles was studied by selective quenching by trinitrophenyl groups, steady-state fluorescence polarization, and differential polarized phase fluorometry techniques, utilizing the lipid soluble fluorophore 1,6-diphenyl-1,3,5-hexatriene. Differences in the hemileaflet's phospholipid composition were also analyzed by trinitrophenylation of aminophospholipids and phospholipase A2 treatment of these preparations. The results of these studies demonstrated that the inner leaflet of these membranes was less fluid than its outer counterpart. Phosphatidylserine was located mainly in the inner hemileaflet, whereas phosphatidylethanolamine and phosphatidylcholine were more symmetrically distributed between the hemileaflets of this membrane. Moreover, in vitro addition of 2-[(2-methoxyethoxy)ethyl]-cis-8-(2-octylcyclopropyl)octanoate (final concentration, 7.5 microM) preferentially fluidized the cytofacial leaflet and concomitantly increased Na(+)-gradient-dependent D-glucose uptake, but decreased Na+, K+-dependent L-glutamic acid uptake in these membrane vesicles. In vitro addition of benzyl alcohol (final concentration, 25 mM) preferentially fluidized the exofacial leaflet and decreased leucine aminopeptidase activity in these preparations. These results, therefore, demonstrate that the hemileaflets of human small intestinal brush-border membranes have different phospholipid compositions and fluidities. Alterations of either the exofacial or cytofacial leaflet fluidity, moreover, modulate protein-mediated activities in a distinct manner.     

Differential effect of lipid peroxidation on membrane fluidity as determined by electron spin resonance probes

The effect of lipid peroxidation on membrane fluidity was examined in sonicated soybean phospholipid vesicles. Following iron/ascorbate dependent peroxidation, the vesicles were labeled with a series of doxyl stearate spin probes which differed in the site of attachment of the nitroxide free radical to the fatty acid. Comparison of motional and partitioning parameters derived from electron spin resonance spectra of the probes indicated that the membranes were less fluid following peroxidation. However, the magnitude of the fluidity decrease was markedly dependent on the intramembrane location, as well as on the extent of lipid peroxidation. The effect of lipid peroxidation on fluidity was maximal in the membrane microenvironment sampled by 12-doxyl stearate, whereas other regions of the bilayer were less affected. These findings indicate that lipid peroxidation leads to an alteration of the transbilayer fluidity gradient.     

Effect of diamide on protein oxidation and physico-chemical properties of lipids in erythrocyte membranes

The effect of diamide on the physicochemical state of proteins and lipids of human erythrocyte membrane was studied. It was found that diamide at a concentration of 1 mM decreases the content of the SH-groups of membrane proteins by approximately 50%, resulting in enhanced vesiculation of erythrocytes upon metabolic exhaustion of cells. It was shown using fluorescein isothiocyanate-labeled concanavalin A and 4,4'-diisothiocyano-2,2'-stilbene disulfonate that diamide changes the structural state of the main integral protein of erythrocyte membranes, the band 3 protein. Changes in the microviscosity of the membrane lipid bilayer depending on diamide concentration were determined from the changes in the fluorescence parameters of the lipophilic probes (pyrene and 1,6-diphenyl-3,5-hexatriene). The level of lipid peroxidation products in membranes remained unchanged. It follows from these data that the SH-oxidizing agent diamide does not directly interact with the lipid bilayer of membrane and produces changes in the physicochemical state of lipids presumably by disrupting protein-lipid interactions that take place upon oxidation of the SH-groups and cross-linking of membrane proteins.     

In vitro effect of AlCl3 on human erythrocytes: Changes in membrane morphology and functionality

Aluminum belongs to a group of potential toxic elements capable of penetrating the human body. In this paper, the effect of aluminum concentrations on red blood cell membranes using different fluorescent probes able to localize in various parts of the phospholipid bilayer (TMA-DPH, laurdan and pyrene) were studied. Our results confirm that human erythrocytes exposed to aluminum undergo physico-chemical modifications at the membrane level. A decrease in fluorescence anisotropy of TMA-DPH and in the polarity of the lipid bilayer with a concomitant shift toward a gel phase was observed, and the pyrene excimerization coefficient (k_ex) increased.Furthermore, the presence of aluminum induced lipid peroxidation and reduced the activity of erythrocyte antioxidant enzymes (SOD, CAT and GSHPx). Al-induced morphological changes on the erythrocyte membrane surface were monitored using atomic force microscopy. These results provide further information on the target of action of different aluminum amounts.     

Partitioning and membrane disordering effects of dopamine antagonists: influence of lipid peroxidation, temperature, and drug concentration.

The effect of four dopamine antagonists (spiperone, haloperidol, pimozide, and domperidone) on the lipid order of caudate nucleus microsomal membranes and on liposomes from membrane lipid extracts was evaluated and related to the partition coefficients (Kp) of the drugs. Lipid membrane order was determined by fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe of the membrane core and 1-[4-(trimethylammonium)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) as a probe of the membrane surface. Dopamine antagonists decrease the fluorescence polarization of both probes, indicating that they disorder the membrane lipids at different depths. Pimozide and domperidone, the drugs with higher Kp values, are more effective at decreasing the polarization of DPH, a probe of the membrane core, than that of TMA-DPH. In contrast, spiperone and haloperidol, which have lower values for Kp, induce more significant decreases in TMA-DPH depolarization, a probe of the membrane surface. These findings indicate that higher partition coefficients of the drugs are directly correlated with an increase of fluidity in the hydrophobic core of brain membranes. Ascorbate/Fe(2+)-induced membrane lipid peroxidation increases membrane order. Membrane lipid peroxidation decreases the partition coefficients of the dopamine antagonists tested. Increasing temperature (4-37 degrees C) decreases membrane order, but temperature effect is less evident after lipid peroxidation. The disordering effect of dopamine antagonists increases with increasing drug concentrations (1-15 microM), a maximum being observed at 10 microM. However, this effect is also less evident after membrane lipid peroxidation. We can conclude that dopamine antagonists and membrane lipid peroxidation affect membrane lipid order and that the action of these drugs is dependent on initial bilayer fluidity. Membrane lipid peroxidation increases membrane order while dopamine antagonists show a disordering effect of membrane phospholipids. This disordering effect can indirectly influence the activity of membrane proteins and it is one of the mechanisms through which membrane function can be altered by these drugs.     

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.     

Charged anesthetics selectively alter plasma membrane order

Although indirect evidence supporting differential lipid fluidity in the two monolayers of plasma membranes has accumulated, unambiguous demonstration of this difference has been difficult to obtain. In the present study, the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH), selective quenching of fluorescence by trinitrophenyl groups, and differential polarized phase fluorescence techniques were used to directly examine the static (order) and dynamic (rotational rate) components of lipid motion in the exofacial and cytofacial leaflets of LM fibroblast plasma membranes. The limiting anisotropy (0.137), the order parameter (0.590), and the rotational relaxation time (1.20 ns) of DPH in the plasma membranes (inner plus outer leaflet) indicated rapid but restricted probe motion in the lipid environment. However, the statics and dynamics of DPH motion in the individual monolayers were significantly (p less than 0.025) different. The limiting anisotropy, order parameter, and rotational relaxation time of DPH in the cytofacial monolayer were 0.036, 0.08, and 0.16 ns, respectively, greater than calculated for the exofacial monolayer of the LM plasma membrane. At appropriate concentrations, phenobarbital and, to a lesser degree, pentobarbital preferentially reduced the limiting anisotropy of DPH calculated for the exofacial leaflet while prilocaine reduced the limiting anisotropy of DPH in the cytofacial leaflet of LM fibroblast plasma membranes. In contrast, the putative cytofacial anesthetic procaine failed to show any preference for either leaflet. Arrhenius plots of DPH fluorescence in LM plasma membranes showed a prominent characteristic break point near 30-32 degrees C. Phenobarbital, pentobarbital, and procaine did not affect this break point while prilocaine selectively abolished it. The break point was therefore assigned to the inner monolayer of the LM plasma membrane.