The penetration of local anesthetics into the red blood cell membrane as studied by fluorescence quenching.

The local anesthetics procaine and tetracaine were found to quench the fluorescence of the probes N-octadecyl naphthyl-2-amine 6-sulfonic acid and 12-(9-anthroyl)stearic acid in the presence of erythrocyte membranes. This quenching was shown to be due to the aromatic amine of the procaine and tetracaine molecules. Lidocaine, an active anesthetic that does not contain an aromatic amine in the same position as does procaine and tetracaine did not quench either of the fluorophores. The preferential quenching of the fluorescent probes by procaine and tetracaine indicated a greater accessibility of tetracaine than of procaine to the hydrocarbon region of the membrane and a greater accessibility of procaine than of tetracaine at the membrane's surface. The addition of calcium was found to reverse the quenching of 12-(9-anthroyl)stearic acid by tetracaine in the presence of red cell membranes.     

Platelet activation studied by fluorescence polarization

Platelet activation was elevated by changes in the fluorescence anisotropy of the sulfhydryl-reactive fluorescent probe, (5-[2-(iodoacetyl) aminacetyl]aminonaphthalene-1-sulfonic acid. The membrane-permeable fluorophore was shown to bind to a multitude of cytoplasmic and membrane proteins. Platelets were stimulated by addition of thrombin, arachidonic acid or ADP under conditions that did not induce aggregation. A sudden increase in the fluorescence anisotropy, r of moderate degree (25-33%) occurred during the first 60 s after exposure of platelets to the aggregating agents and was sustained during the entire period of observation (15-18 min). Phenylmethylsulfonyl thrombin was unable to produce these changes in fluorescence anisotropy. Preincubation of platelets with colchicine reduced r within 30-60 s after platelets were exposed to thrombin. These findings are interpreted as an indication of a general decrease in the 'motional freedom' of the fluorophores and indirectly their ligand molecules.     

Carbocyanine dye usage in demarcating boundaries of the aged human red nucleus

Background

Though the adult human magnocellular Red nucleus (mNr) is essentially vestigial and its boundaries with neighbouring structures have never been well demarcated, human studies in utero have shown a well developed semilunar mNr wrapping around the caudal parvicellular Red nucleus (pNr), similar to what is seen in quadrupeds. In the present study, we have sought to better delineate the morphological determinants of the adult human Red nucleus (ahRn).

Methods and Findings

Serial sections of ahRn show fine myelinated fibers arising from pNr and turning toward the central tegmental tract. DiI was deposited within a well restricted region of ahRn at the fasciculus retroflexus level and the extent of label determined. Nissl-stained serial sections allowed production of a 3-D mNr model, showing rudimentary, vestigial morphology compared with its well developed infant homologue. DiI within this vestigial mNr region at the level of the oculomotor nerve showed labeled giant/large mNr neurons, coarse fiber bundles at the ventral tegmental decussation and lateral lemniscal label.

Conclusions

Large amounts of DiI and a long incubation time have proven useful in aged human brain as a marker of long axons and large cell bodies of projecting neurons such as the rubrospinal projection and for clarifying nuclear boundaries of closed nuclei (e.g., the large human pNr). Our 3D model of adult human mNr appeared shrunken in shape and axially rotated compared with the infant mNr, the rotation being a common feature among mammalian mNr.     

Tumor promoters induce membrane changes detected by fluorescence polarization.

The probe, 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to determine if tumor promoting agents alter cell membranes. The active tumor promoters TPA (12-0-tetra-decanoyl-phorbol-13-acetate), PDD (phorbol-12,13-didecanoate) and PDB (phorbol-12,13-dibenzoate) were found to decrease fluorescence polarization of DPH in rat embryo cells, whereas the inactive tumor promoting compounds phorbol and 4α-PDD failed to induce this change.     

The orientation of insulin receptors in the red cell membrane

High-affinity binding of insulin to receptors in human erythrocyte membranes occurred at the external surface, but not at the cytoplasmic surface of the plasma membrane, as assessed by insulin binding to right-side-out and inside-out membrane vesicles. Even after prolonged (3 h) incubation at 22°C, binding at the cytoplasmic membrane aspect remained negligible. The data indicate that the insulin receptor displays its hormone-binding site exclusively toward the extracellular space and that transmembrane mobility (“flip-flop”) of the receptor from one to the other membrane leaflet is severely restricted.     

Changes in membrane properties during energy depletion-induced cell injury studied with fluorescence microscopy.

The changes in membrane structural properties occurring during the process of ATP depletion-induced cell injury in adherent human astrocytoma cells (UC-11 MG) were studied with two epifluorescence techniques: 1) steady-state fluorescence anisotropy (r) to examine microstructural changes in the membrane phospholipids and 2) fluorescence redistribution after photobleaching (FRAP) to examine membrane fluidity changes. A new method for r measurement was established that provides the unique advantage of simultaneously monitoring both vertical and horizontal polarized fluorescence emissions needed for the calculation of r. In this study, r in the astrocytoma cells labeled with 1-(4-trimethylammonium phenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate was shown to remain stable for up to 90 min. However, when the cells were treated with 75 microM iodoacetic acid (IAA), a metabolic inhibitor that induces rapid depletion of cellular ATP, r continually decreased, indicating a decrease in membrane lipid order and perturbation of the bilayer structure. This decrease in r could be prevented by the pretreatment of cells with lipophilic antioxidants such as tirilazad or gossypol. Tirilazad itself caused a significant increase in r, suggesting that tirilazad intercalates into the membrane bilayer and profoundly increases the lipid order in uninjured cells. Gossypol, however, did not exhibit this property. Further investigations into these phenomena with FRAP confirmed the r results and indicated that membrane fluidity increased while its structure became less rigid during the process of ATP-induced cell injury. In addition, lipophilic antioxidants prevented the membrane structural aberrations induced by IAA. Experimental results suggest that different mechanisms of cytoprotective action may exist for tirilazad and the antioxidant gossypol. Gossypol appears to prevent or delay the observed cell injury entirely because of its antioxidant action, whereas tirilazad's protection is mediated not only via its antioxidant activity, but also by its ability to increase cell membrane lipid order.     

Cell membrane orientation visualized by polarized total internal reflection fluorescence.

In living cells, variations in membrane orientation occur both in easily imaged large-scale morphological features, and also in less visualizable submicroscopic regions of activity such as endocytosis, exocytosis, and cell surface ruffling. A fluorescence microscopic method is introduced here to visualize such regions. The method is based on fluorescence of an oriented membrane probe excited by a polarized evanescent field created by total internal reflection (TIR) illumination. The fluorescent carbocyanine dye diI-C(18)-(3) (diI) has previously been shown to embed in the lipid bilayer of cell membranes with its transition dipoles oriented nearly in the plane of the membrane. The membrane-embedded diI near the cell-substrate interface can be fluorescently excited by evanescent field light polarized either perpendicular or parallel to the plane of the substrate coverslip. The excitation efficiency from each polarization depends on the membrane orientation, and thus the ratio of the observed fluorescence excited by these two polarizations vividly shows regions of microscopic and submicroscopic curvature of the membrane, and also gives information regarding the fraction of unoriented diI in the membrane. Both a theoretical background and experimental verification of the technique is presented for samples of 1) oriented diI in model lipid bilayer membranes, erythrocytes, and macrophages; and 2) randomly oriented fluorophores in rhodamine-labeled serum albumin adsorbed to glass, in rhodamine dextran solution, and in rhodamine dextran-loaded macrophages. Sequential digital images of the polarized TIR fluorescence ratios show spatially-resolved time-course maps of membrane orientations on diI-labeled macrophages from which low visibility membrane structures can be identified and quantified. To sharpen and contrast-enhance the TIR images, we deconvoluted them with an experimentally measured point spread function. Image deconvolution is especially effective and fast in our application because fluorescence in TIR emanates from a single focal plane.     

Segmental flexibility in Escherichia coli ribosomal protein S1 as studied by fluorescence polarization.

Ribosomal protein S1 covalently reacts with approximately one equivalent of iodoacetylethylenediamine (1,5-napthol sulfonate (IAEDANS) or iodoacetylaminofluorescein (IAAF). The product AEDANS-S1 can bind to 30S ribosomal subunits lacking S1 as shown by polyacrylamide-agarose gel electrophoresis AEDANS-S1 and AAF-S1 when added back to S1-depleted 30S subunits modulate poly(U)-dependent polyphenylalanine synthesis in the presence of IF3 in a very similar way to unmodified S1. AEDANS-S1 also stimulates RI7-dependent fMet-tRNA binding to 1.0M NH4C1 washed ribosomes whereas AAF-S1 does not. Both static and nanosecond fluorescence polarization techniques were used to study the rotational motions of AEDANS-S1. Several previous studies had indicated that S1 is a highly extended protein which can be modeled by a prolate ellipsoid with an axial ratio of 10 to 1. However, the rotational correlation time we find is about half that expected for such a particle. This suggests that S1 is a flexible protein with at least two domains that can rotate independently.     

Rotational dynamics of curved DNA fragments studied by fluorescence polarization anisotropy

The rotational dynamics of short DNA fragments with or without intrinsic curvature were studied using time-resolved phase fluorimetry of intercalated ethidium with detection of the anisotropy. Parameters determined were the spinning diffusion coefficient of the DNA fragments about the long axis and the zero-time ethidium fluorescence anisotropy. We find a significant decrease in the spinning diffusion coefficient for all curved fragments compared to the straight controls. This decrease is likewise evident in rotational diffusion coefficients computed from DNA structures obtained by a curvature prediction program for these sequences. Using a hinged-cylinder model, we can identify the change in rotational diffusion coefficient with a permanent bend of 13-16 degrees per helix turn for the sequences studied. Moreover, for some of the curved fragments an increased flexibility has to be assumed in addition to the permanent bend in order to explain the data.     

Interaction of retinol and intestinal microvillus membranes studied by fluorescence polarization

The interaction of retinol and microvillus membranes prepared from the duodenum, jejunum and ileum of the rat can be studied readily by fluorescence and fluorescence polarization. A characteristic pattern of increased retinol anisotropy in membranes from more distal segments is demonstrated. Studies with trypsin indicate that the membrane proteins influence retinol fluorescence intensity and anisotropy.     

Physical state of bulk and protein-associated lipid in nicotinic acetylcholine receptor-rich membrane studied by laurdan generalized polarization and fluorescence energy transfer.

The spectral properties of the fluorescent probe laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) were exploited to learn about the physical state of the lipids in the nicotinic acetylcholine receptor (AChR)-rich membrane and compare them with those in reconstituted liposomes prepared from lipids extracted from the native membrane and those formed with synthetic phosphatidylcholines. In all cases redshifts of 50 to 60 nm were observed as a function of temperature in the spectral emission maximum of laurdan embedded in these membranes. The so-called generalized polarization of laurdan exhibited high values (0.6 at 5 degrees C) in AChR-rich membranes, diminishing by approximately 85% as temperature increased, but no phase transitions with a clear Tm were observed. A still unexploited property of laurdan, namely its ability to act as a fluorescence energy transfer acceptor from tryptophan emission, has been used to measure properties of the protein-vicinal lipid. Energy transfer from the protein in the AChR-rich membrane to laurdan molecules could be observed upon excitation at 290 nm. The efficiency of this process was approximately 55% for 1 microM laurdan. A minimum donor-acceptor distance r of 14 +/- 1 A could be calculated considering a distance 0 < H < 10 A for the separation of the planes containing donor and acceptor molecules, respectively. This value of r corresponds closely to the diameter of the first-shell protein-associated lipid. A value of approximately 1 was calculated for Kr, the apparent dissociation constant of laurdan, indicating no preferential affinity for the protein-associated probe, i.e., random distribution in the membrane. From the spectral characteristics of laurdan in the native AChR-rich membrane, differences in the structural and dynamic properties of water penetration in the protein-vicinal and bulk bilayer lipid regions can be deduced. We conclude that 1) the physical state of the bulk lipid in the native AChR-rich membrane is similar to that of the total lipids reconstituted in liposomes, exhibiting a decreasing polarity and an increased solvent dipolar relaxation at the hydrophilic/hydrophobic interface upon increasing the temperature; 2) the wavelength dependence of laurdan generalized polarization spectra indicates the presence of a single, ordered (from the point of view of molecular axis rotation)-liquid (from the point of view of lateral diffusion) lipid phase in the native AChR membrane; 3) laurdan molecules within energy transfer distance of the protein sense protein-associated lipid, which differs structurally and dynamically from the bulk bilayer lipid in terms of polarity and molecular motion and is associated with a lower degree of water penetration.     

Radiation effects on erythrocyte membrane structure studied by the intrinsic fluorescence.

The changes in the intrinsic fluorescence, primarily from tryptophan residues, of sheep erythrocyte membranes following X-irradiation (0--4000 R) were investigated. The experiments showed that there was (1) a decrease in the intensity of fluorescence with increasing dose of X-rays, (2) a small shift of fluorescence emission to longer wavelengths, (3) a decrease in the fluorescence polarization, and that (4) treatment of membranes with a perturbing solvent, 2-chloroethanol, can eliminate the effects of X-rays. The amount of tryptophan in the membranes was not altered after X-irradiation. It was also shown that sulphydryl reagents, N-ethylmaleimide and 2,2'-dithiodipyridine, induced similar fluorescence changes. From these results it was concluded that the fluorescence changes could result from a change in the environment surrounding tryptophan residues, from being relatively non-polar to being more polar, implying that conformational changes of membrane proteins are brought about by low doses of X-rays.     

Alterations in membrane fluidity during keratinocyte differentiation measured by fluorescence polarization

Summary The epidermis shows a distinctive pattern of differentiation wherein keratinocytes proliferate in the basal cell layer and mature into spinous and granular cells. Using a discontinuous density-gradient centrifugation method, guinea-pig keratinocytes were separated into high (HDF), intermediate (IDF), and low (LDF) density fractions. Morphological and flow cytometrical observations demonstrated that HDF, IDF, and LDF were basal, spinous, and granular cell-rich fractions, respectively. Membrane fluidity of the fractionated keratinocytes was measured by diphenylhexatriene fluorescence polarization. Polarization (p)-value of keratinocytes was negatively correlated with temperature. At each temperature, HDF cells showed a lower p-value than IDF or HDF cells except at 40° C. Since a low p-value indicates a high degree of Brownian motion, membrane fluidity is higher in basal cells and lower in spinous and granular cells. Our results indicate that membrane fluidity of guinea-pig keratinocytes decreases during their maturation.     

Measurement of single macromolecule orientation by total internal reflection fluorescence polarization microscopy

A new approach is presented for measuring the three-dimensional orientation of individual macromolecules using single molecule fluorescence polarization (SMFP) microscopy. The technique uses the unique polarizations of evanescent waves generated by total internal reflection to excite the dipole moment of individual fluorophores. To evaluate the new SMFP technique, single molecule orientation measurements from sparsely labeled F-actin are compared to ensemble-averaged orientation data from similarly prepared densely labeled F-actin. Standard deviations of the SMFP measurements taken at 40 ms time intervals indicate that the uncertainty for individual measurements of axial and azimuthal angles is approximately 10 degrees at 40 ms time resolution. Comparison with ensemble data shows there are no substantial systematic errors associated with the single molecule measurements. In addition to evaluating the technique, the data also provide a new measurement of the torsional rigidity of F-actin. These measurements support the smaller of two values of the torsional rigidity of F-actin previously reported.     

The pH dependence of red cell membrane transport of titratable anions studied by NMR spectroscopy

The effects of varying extracellular pH on the rates of uptake of titratable anions by human erythrocytes under conditions of constant intracellular pH have been determined for a series of highly related anions, the phosphate "analogs." These compounds are simply substituted phosphorus oxyacids, differing in the number and acidity of titratable protons: phosphate (HPO4(2-), pKa 6.8); phosphite (HPO3(2-), pKa 6.4); hypophosphite (H2PO2-); methylphosphonate ((CH3)PO3(2-), pKa 7.4); dimethylphosphinate ((CH3)2PO2-); fluorophosphate [PO3F2-, pKa 4.7); and thiophosphate (HSPO3(2-), pKa 5.5). Suspensions of intact, Cl(-)-loaded erythrocytes (intracellular pH, 7.2) were incubated at 37 degrees C in isotonic buffers (pH 4-8) containing 60 mM phosphate analog for specified time intervals, whereupon influx was halted by the addition of 1 mM 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), an inhibitor of anion exchange. The intracellular anion concentrations were determined from 31P or 19F nuclear magnetic resonance spectra from the erythrocyte suspensions. The influx rates for the titratable phosphate analogs exhibited bimodal pH dependence, reaching maximal levels at pH values that increased with increasing anion pK. This pH-dependent behavior is consistent with a transport channel that contains a titratable regulatory site which interacts with the translocated anion. Based upon the Henderson-Hasselbalch equation, the probability that a titratable anion will have an electric charge of equal magnitude to that of the titratable carrier is highest at a pH value exactly midway between the pK of the regulatory site and that of the anion. The pH maxima observed for the phosphate analogs indicate a pK for this site of 5.5 at 37 degrees C. Intracellular pH changes associated with influx indicated that transport of the "fast" anion phosphite is largely in monoionized form. Intracellular pH changes associated with transport of slow anions were predominantly determined by partial ionic equilibrium effects and did not indicate the ionization state of the transported anion.     

Deoxygenation affects fluorescence photobleaching recovery measurements of red cell membrane protein lateral mobility.

We have used the fluorescence photobleaching recovery technique to study the dependence on oxygen tension of the lateral mobility of fluorescently labeled band 3, the phospholipid analogue fluorescein phosphatidylethanolamine, and glycophorins in normal red blood cell membranes. Band 3 protein and sialic acid moieties on glycophorins were labeled specifically with eosin maleimide and fluorescein thiosemicarbazide, respectively. The band 3 diffusion rate increased from 1.7 x 10(-11) cm2 s-1 to 6.0 x 10(-11) cm2 s-1 as oxygen tension was decreased from 156 to 2 torr, and a further increase to 17 x 10(-11) cm2 s-1 occurred as oxygen tension was decreased from 2 to 0 torr. The fractional mobility of band 3 decreased from 58 to 32% as oxygen tension was decreased from 156 to 0 torr. The phospholipid diffusion coefficient remained constant as oxygen tension was decreased from 156 to 20 torr, but increased from 2.3 x 10(-9) cm2 s-1 to 7.1 x 10(-9) cm2 s-1 as oxygen tension was decreased from 20 to 0 torr. Neither the diffusion coefficient nor the fractional mobility of glycophorins changed significantly at low oxygen tension. Under non-bleaching excitation conditions, intensities of fluorescence emission were identical for oxygenated and deoxygenated eosin-labeled RBCs. Deoxygenated eosin-labeled RBCs required 160-fold greater laser intensities than did oxygenated RBCs to achieve comparable extents of photobleaching, however. Oxygen seems to act as a facilitator of fluorophore photobleaching and may thereby protect the fluorescently labeled red cell membrane from photodamage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Homogeneity and variability in the structure of azurin molecules studied by fluorescence decay and circular polarization.

The fluorescence decay of apoazurin derived from Pseudomonas aeruginosa is monoexponential. By this criterion the population of molecules of apoazurin is homogeneous. The emission anisotropy factor and the absorption anisotropy factor at the red edge of the absorption band assume similar values, showing that the tryptophan residue in apoazurin has the same asymmetric environment both in the ground and excited states. This finding suggests tight packing of the protein at the tryptophan environment. Native azurin does not decay monoexponentially. Moreover, comparison between the quantum yield calculated from the decay kinetics and the one measured directly shows that the majority of the azurin molecules are not fluorescent. There is thus variability in the structure of azurin molecules with an equilibration time that is longer than the fluorescence lifetime. Different asymmetric environment was found for the tryptophan residue in oxidized and reduced holoprotein and in apoazurin, as studied by the circular polarization of the fluorescence. D(2)O increases the fluorescence lifetime of apoazurin by 6 percent, compared to the lifetime in H(2)O solution; therefore water molecules may have access to the tryptophan residue, though the latter is situated in a hydrophobic environment.     

Glucose induces membrane changes detected by fluorescence polarization in endocrine pancreatic cells

Pancreatic endocrine cells prepared from rat islets were labelled with 1,6-diphenyl-1,3,5-hexatriene and examined in a microviscosimeter. Glucose caused a dose-related decrease in fluorescence polarization. This decrease was observed within 1 min after increasing the concentration of glucose. These findings suggest that glucose affects membrane viscosity in pancreatic endocrine cells. At a glucose concentration of 16.7 mM, the estimated viscosity was 15 ± 3 per cent lower than basal value (2.01 ± 0.12 P).     

Myoepithelial cells in human thymus: staining, polarization and fluorescence microscopic studies.

Myoid cells in human thymus were studied around the turn of the century, and alterations in patients with cardiovascular disease were reported. It was therefore deemed of interest to reinvestigate these long forgotten cells. The configurational staining, polarization and fluorescence microscopic properties of smooth myofibrils in thymic epithelial cells were identical with those of classical myoepithelial cells, smooth muscle, and A bands of striated muscle. Cross-striated myoid cells could not be found in thymus of children. Myoepithelial cells formed a layer at the surface of thymic lobules; others were scattered throughout the cortex and medulla. In addition, the medulla contained seemingly hypertrophic myoepithelial cells. Hassall's corpuscles consisted of layers of myoepithelial cells. Hammar (1905) regarded epithelial cells with smooth myofibrils in human thymus as equivalents of the cross-striated myoid cells in lower vertebrates. The myoepithelial cells observed in this study are apparently identical with the smooth myoid cells of early anatomists; the hypertrophic myoepithelial cells correspond to the unicellular Hassall's corpuscles. The functions of these cells are not yet clear; the wide variations from case to case in the same age group indicate that the myoepithelial cells are affected by a variety of diseases.