Role of membrane lipids in the interaction of daunomycin with plasma membranes from tumor cells: implications in drug-resistance phenomena

Equilibrium binding studies on the interaction between the anthracycline daunomycin and plasma membrane fractions from daunomycin-sensitive and -resistant murine leukemia P-388 cells are presented. Drug binding constants (KS) are 15,000 and 9800 M-1 for plasma membranes from drug-sensitive and drug-resistant cells, respectively. Drug binding to the membranes is not affected by either (i) thermal denaturation of membrane proteins or (ii) proteolytic treatment with trypsin, thus suggesting that the protein components of the membranes do not have a major role in determining the observed drug binding. Also, fluorescence resonance energy transfer between tryptophan and daunomycin in the membranes indicates that interaction of protein components with the drug should not be responsible for the observed differences in drug binding exhibited by plasma membranes from drug-sensitive and -resistant cells. Plasma membranes from drug-sensitive cells contain more phosphatidylserine and slightly less cholesterol than membranes from drug-resistant cells. Differences in the content of the acidic phospholipid between the two plasma membranes seem to produce a different ionic environment at membrane surface domains, as indicated by titration of a membrane-incorporated, pH-sensitive fluorescence probe. The possible role of membrane lipids in modulating drug binding to the membranes was tested in equilibrium binding studies using model lipid vesicles made from phosphatidylcholine, phosphatidylserine, and cholesterol in different proportions. The presence of phosphatidylserine greatly increases both the affinity and the stoichiometry of daunomycin binding to model lipid vesicles. The similarity between the effects of phosphatidylserine and other negatively charged compounds such as dicetyl phosphate, cardiolipin, or phosphatidic acid suggests that electrostatic interactions are important in the observed binding of the drug.(ABSTRACT TRUNCATED AT 250 WORDS)     

Association of daunomycin to membrane domains studied by fluorescence resonance energy transfer

1,6-Diphenyl-1,3,5-hexatriene and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene are fluorophores used to explore different hydrophobic domains of membrane bilayers (Andrich, M.P. and Vanderkooi, J.M. (1976) Biochemistry 15, 1257-1265; Prendergast, F.G., Haugland, R.P. and Callahan, P.J. (1981) Biochemistry 20, 7333-7338). Fluorescence resonance energy transfer between these fluorophores, acting as energy donors, and the anthracycline, daunomycin, as the acceptor, was used to analyze the interaction of the drug with natural membranes, and its relative location within the membrane bilayer. The transfer process was demonstrated by: (1) emission fluorescence of the acceptor when the samples were excited at the excitation maximum of the donor (360 nm); and (2) progressive quenching of the energy donor (at 428 nm) when in the presence of increasing acceptor concentration. Also, the disruption of the energy transfer by solubilization of the membrane with Triton X-100 evidences a role for the membrane in providing the appropriate site(s) for energy transfer to occur. At moderately low daunomycin/membrane lipid ratios, the different efficiencies of resonance energy transfer between the two donors and daunomycin predicts a preferential, but not exclusive, location of the drug at membrane 'surface' domains, i.e., those regions of the bilayer explored by the 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene probe. In support of this observation, a large fraction (approx. 75%) of membrane-associated daunomycin was rapidly sequestered away from the membrane upon addition of excess DNA, which forms high-affinity complexes with daunomycin (Chaires, J.B., Dattagupta, n. and Crothers, D.M. (1982) Biochemistry 21, 3927-3932), thus acting as a drug 'sink'. Also, a large fraction of drug was accessible to fluorescence quenching by iodide, a collisional water-soluble quencher. On the other hand, a smaller population of the membrane-associated daunomycin was characterized by slow sequestering by the added DNA and inaccessibility to quenching by iodide. We conclude that the daunomycin, which is only slowly sequestered, is located deep within the hydrophobic domains of the bilayer, likely to be those probed by 1,6-diphenyl-1,3,5-hexatriene.     

Verapamil reverses the ultrastructural alterations in the plasma membrane induced by drug resistance.

Two P388 cell sublines with different levels of resistance to daunomycin (DNM), P388/20 and P388/100 cells (approximately 20- and 100-fold resistance, respectively), undergo a significant (approximately 2-fold) increase in the number of intramembrane particles (IMPs) present at their plasma membrane, as compared to that exhibited by the parental, drug-sensitive P388 (P388/S) cell line. Regardless of the level of resistance, incubation of drug-resistant cells with verapamil, a well known reverting agent of anthracycline resistance, restores the morphology of the plasma membrane in these cells, yielding a pattern in which the number and size distribution of IMPs at both leaflets of the bilayer, become undistinguishable from those displayed by drug-sensitive cells. Furthermore, verapamil did not affect the ultrastructural organization of the plasma membrane of drug-sensitive cells. It is possible that the alterations in the structural organization of the plasma membrane of the antineoplastic-resistant tumor cells, might represent a reliable 'marker' for early diagnosis of drug resistance.     

Equilibrium, kinetic and photoaffinity labeling studies of daunomycin binding to P-glycoprotein-containing membranes of multidrug-resistant Chinese hamster ovary cells

The binding of daunomycin and its Bolton-Hunter derivative iodomycin to plasma membranes isolated from multidrug-resistant Chinese hamster ovary cells (CHO B30) and their drug-sensitive parents (B1) was investigated. The thermodynamics and kinetics of equilibrium binding monitored by fluorescence titrations and temperature-jump relaxation spectrometry were compared with the specificity of covalent photolabeling with [3H]daunomycin and [125I]iodomycin. The facts that the uptake of anthracycline from aqueous solution into the CHO membranes was not accompanied by any substantial increase of fluorescence anisotropy nor by any spectral shift of the fluorescence emission spectrum and that the partition ratio into the membrane was 20-30-fold higher when compared to a lecithin bilayer, provided evidence that the non-covalent drug binding sites are constituted by polar protein domains without any substantial contribution from the surrounding lipids. Photoaffinity labeling with nanomolar concentrations of anthracycline and equilibrium binding curves independently showed that a 150-170-kDa plasma membrane glycoprotein (P-glycoprotein), whose overexpression is the major difference between B1 and B30 membranes, provides the binding sites of highest affinity for daunomycin and iodomycin (K approximately equal to 4 x 10(7) M-1). Comparison of photolabeling and equilibrium data suggested that the same binding sites on P-glycoprotein were most probably being monitored. The photolabeling of P-glycoprotein by iodomycin was inhibited in a dose-dependent manner by other compounds to which multi-drug-resistant cells are either resistant or collaterally sensitive with the following orders of effectiveness: vinblastine greater than verapamil greater than nitrendipine greater than daunomycin much greater than colchicine. Temperature-jump experiments covering the time range of 1 microseconds to 1 s revealed a single concentration-dependent relaxation time of 10-30 microseconds. The association of daunomycin with its binding sites in the membranes was found to be a diffusion-controlled process with kon rates of 2-4 X 10(9) M-1 s-1. Therefore, the selectivity of drug binding was entirely reflected in the dissociation rates.     

Flow cytometry is a new method for the characterization of intestinal plasma membrane.

The highly differentiated plasma membrane of rabbit enterocytes constitutes an interesting model for membrane studies. Flow cytometry allows combined measurements of the size of membrane vesicles by light-scatter and fluorescence polarization at a single-particle level. The degree of fluorescence polarization of 1,6-diphenylhexa-1,3,5-triene was determined at 4, 18 and 37 degrees C in the brush-border and basolateral plasma membranes. The fluorescence polarization was considerably higher in brush-border than in basolateral membranes. After incubation with dimethyl sulphoxide, the membrane fluidity decreased in both types of membranes. Moreover, a time-effect study of dimethyl sulphoxide showed changes in fluorescence polarization. Only in brush-border membrane a temporary fluid phase was observed. The different properties of the two membrane domains in relation to the lipid-protein dynamics of enterocytes are discussed.     

Kinetic parameters for the uptake of anthracycline by drug-resistant and drug-sensitive K562 cells.

Fluorescence-emission spectra from anthracycline-treated cells suspended in buffer have been used to measure the uptake of three anthracycline derivatives: adriamycin, 4'-O-tetrahydropyranyladriamycin and aclacinomycin in drug-sensitive and drug-resistant K562 cells. The initial rate of uptake and the kinetics of active efflux under the effect of an integral membrane glycoprotein, P-glycoprotein, have been measured as a function of temperature. The activation energies for the passage of the drugs through the plasma membrane have been calculated. In the case of 4'-O-tetrahydropyranyladriamycin, the activation energies for the passive diffusion of the drug equal 45 kJ.mol-1 and 37 kJ.mol-1 for sensitive and resistant cells, respectively. The activation energy for the active efflux of 4'-O-tetrahydropyranyladriamycin equal 25 kJ.mol-1.     

Effects of pH on the kinetics of the interaction between anthracyclines and lipid bilayers

In the present study we have analyzed the kinetics of the initial steps (first 10 seconds) of the interaction between the anthracycline daunomycin (DNM) and artificial lipid vesicles bearing opposite surface charge. The process can be monitored by measuring the fluorescence increase of the drug accompanying its association with the lipid bilayers. The results indicated that DNM consistently interacts to a larger extent with the liposomes having negative surface charge than with those having positive surface charge, suggesting the involvement of electrostatic components in the interaction. In contrast, DNM associates with the vesicles bearing positive surface charge 2 – 3 times faster (in terms of the apparent rate constants describing the process of interaction) than with those having negative surface charge, an observation probably related to the more fluid physical state of the former. Regarding the rate of access of DNM to the vesicles, rather than depending on the surface charge of the vesicles, this is critically affected by the ionization state of the drug, i. e. by the pH. Thus, the rate at which the interaction proceeds is increased nearly 15-fold when the pH of the medium increases from 7.0 to 8.3, regardless of the surface charge of the liposomes. On this basis, and taking into account the fact that the anthracyclines enter the cells by passive diffusion, possible effects of pH on the transport of these drugs through the membranes of tumor cells are discussed. Received: 3 December 1996 / Accepted: 4 July 1997     

Interaction of amphotericin B and its N-fructosyl derivative with murine thymocytes: a comparative study using fluorescent membrane probes

The polyene antibiotics amphotericin B (AmB) and N-(1-deoxy-D-fructos-1-yl)amphotericin (N-Fru-AmB) have different activity towards murine thymocytes (N-Fru-AmB is less toxic but is a potent immunomodulator). The interactions of the drugs with these cells have been studied by fluorescence methods. Fluorescence energy transfer from 1-[4-(trimethylammonio) phenyl]-6-phenylhexa-1,3,5-triene, p-toluenesulfonate (TMA-DPH) to polyenes was used to follow the binding of the two drugs to the plasma membrane. The results, confirmed by circular dichroism measurements, indicate that at saturation the ratio AmB bound/plasma membrane lipid is low (less than 1 molecule of polyene for 170 lipids). The slightly higher binding of AmB as compared to N-Fru-AmB demonstrates that affinity of the antibiotic for plasma membrane does not account for the activity of the polyenes towards lymphocytes. The effect of the two polyenes on membrane fluidity was studied by steady-state fluorescence anisotropy. The results suggest that AmB strongly perturbs the structure of the membrane whereas only a slight decrease of the anisotropy is observed with N-Fru-AmB in the range of concentration where the biological activity has been demonstrated. Polyene location was further investigated by comparing the energy transfer efficiency obtained with TMA-DPH and with the parental compound 1,6-diphenylhexa-1,3,5-triene, p-toluene sulfonate (DPH). While AmB binds to plasma membrane, as well as to intracellular structures, N-Fru-AmB seems to accumulate into the cell and bind to intracellular membrane structures.     

Effect of Escherichia coli lipopolysaccharide on the microviscosity of liver plasma membranes and hepatocyte suspensions and monolayers

The fluorescence probe 1,6-diphenylhexa-1,3,5-triene (DPH) was used for monitoring structural perturbations induced by lipopolysaccharide (LPS) of Escherichia coli (0111:B4) in plasma membranes of rat liver. Changes in microviscosity were observed in plasma membrane preparations from control rats after treatment with LPS and in plasma membrane preparations from liver perfused with LPS. In both systems fluorescence polarization was measured from which microviscosity was calculated. This parameter increases with LPS treatment. From temperature dependence studies was inferred that LPS interaction with plasma membrane preparations induces an increase of both the polarization term (r0/r-1)-1 and flow activation energy (delta E). Addition of LPS to hepatocyte suspensions also induces an increase on microviscosity and a delay in the fall of microviscosity induced by a temperature rise in hepatocyte monolayers grown on microcover slides. These data suggest that LPS interaction can be attributed to its binding to membrane hydrophobic regions in a non-specific manner.     

Immunocytochemical localization of P170 at the plasma membrane of multidrug-resistant human cells

P170 (P-glycoprotein) is a membrane protein found in high levels in multidrug-resistant cultured cell lines. We have localized this protein using monoclonal antibody MRK16 by immunofluorescence and electron microscopy in the multidrug-resistant human carcinoma cell line KB-C4. The P170 determinant recognized by antibody MRK16 was found on drug-resistant KB-C4 cells, but not on parental drug-sensitive KB-3-1 cells. The determinant was present on the external surface of the plasma membrane and on the luminal side of Golgi stack membranes. P170 was excluded from coated pits at the plasma membrane and absent from endocytic vesicles and lysosomes. This determinant was detected only in small amounts in the endoplasmic reticulum. The high protein concentration of P170 in the plasma membrane is consistent with a role of this protein as a drug efflux pump at the cell surface.     

Phospholipid and cholesterol levels in the tumor cell plasma membranes with different sensitivity to doxorubicin

The investigation is aimed to study qualitative and quantitative composition of phospholipids, cholesterol content and lipids unsaturation index in plasma membranes of Guerin's carcinoma cells sensitive or resistant to doxorubicin. The comparison of infrared spectra and phospholipids unsaturation index showed that the unsaturation level of fatty acids in plasma membrane from resistant cells was lower than that from sensitive carcinoma cells. 31P-NMR spectroscopy of plasma membranes phospholipids shows the increase of phosphatidylserine and sphingomyeline content in plasma membrane isolated from resistant tumor as compared with sensitive tumor. The levels of phosphatidylcholine and phosphatidylethanolamine were equal in drug-resistant and drug-sensitive carcinoma strains. Changes in plasma membrane from resistant cells result in elevation of plasma membrane microviscosity and phosphatidylserine level increase can suggest the activation of P-glycoprotein-mediated efflux of doxorubicin.     

A fluorescent hydrophobic probe used for monitoring the kinetics of exocytosis phenomena

A fluorescence method is presented for quantitatively analyzing exocytosis phenomena and monitoring their kinetics. The method is based on the particular properties of a hydrophobic fluorescent probe, 1-[4-(trimethylammonio)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) [Prendergast, F.G., Haugland, R.P., & Callahan, P.J. (1981) Biochemistry 20, 7333-7338; Kuhry, J.G., Fonteneau, P., Duportail, G., Maechling, C., & Laustriat, G. (1983) Cell Biophys. 5, 129-140; Kuhry, J.G., Duportail, G., Bronner, C., & Laustriat, G. (1985) Biochim. Biophys. Acta 845, 60-67]. When this probe is interacted with intact resting cells in aqueous suspensions, it labels solely the membranes that are in contact with the external medium and is incorporated into them according to a partition equilibrium; i.e., the amount of the probe incorporated is proportional to the available membrane surface. TMA-DPH is highly fluorescent in membranes and not at all in water. Thus, a measurement of the TMA-DPH fluorescence intensity provides a signal proportional to the membrane surface. In secretory cells, the membrane surface available for the probe is increased upon fusion of the membrane of the secretory granules with the cell plasma membranes, directly or via intergranule fusion. Thus, when these cells are stimulated, more TMA-DPH is incorporated than in resting cells since the probe is allowed to also interact with the granule membranes now connected with the external medium by pores. This process results in a proportional increase in the TMA-DPH fluorescence intensity. The response was found to be very rapid and able to follow accurately the exocytosis kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resolution of plasma membrane lipid fluidity in intact cells labelled with diphenylhexatriene

The partitioning of fluorescence probes into intracellular organelles poses a major problem when fluorescence methods are applied to evaluate the fluidity properties of cell plasma membranes with intact cells. This work describes a method for resolution of fluidity parameters of the plasma membrane in intact cells labelled with the fluorescence polarization probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The method is based on selective quenching, by nonradiative energy transfer, of the fluorescence emitted from the plasma membrane after tagging the cell with a suitable membrane impermeable electron acceptor. Such selective quenching is obtained by chemical binding of 2,4,6-trinitrobenzene sulfonate (TNBS), or by incorporation of $\text{N-}\text{bixinoyl}$ glucosamine (BGA) to DPH-labelled cells. The procedures for determination of lipid fluidity in plasma membranes of intact cells by this method are simple and straightforward.     

Plasma membrane fluidity measurements in intact endothelial cells: effect of hyperoxia on fluorescence anisotropies of 1-[4-(trimethylamino)phenyl]-6-phenyl hexa-1,3,5-triene

Fluorescence anisotropy measurements are widely used as sensitive indicators of cell membrane fluidity. 1-[4-(trimethylamino)phenyl]-6-phenyl hexa-1,3,5-triene (TMA-DPH) is a cationic fluorescent aromatic hydrocarbon that anchors at the lipid-water interface of membrane lipid bilayers. Its uptake into porcine pulmonary artery and aortic endothelial cells was monitored and the probe remained specifically localized on the cell surface for at least 4 h. It can therefore be recommended for use for specific plasma membrane lipid fluidity measurements in these cells. The effect of hyperoxia on plasma membrane fluidity was measured by using TMA-DPH. In both cell types, hyperoxic damage resulted in decreases in plasma membrane fluidity. Recovery was achieved 48 h after a 42-h hyperoxic exposure. These results indicate that TMA-DPH is a sensitive probe of plasma membrane lipid domains of pulmonary artery and aortic endothelial cells and that hyperoxia causes reversible changes in the physical state of superficial lipid domains of the plasma membrane of these cells.     

Comparison of the membrane transport of anthracycline derivatives in drug-resistant and drug-sensitive K562 cells.

One of the phenotypes of multidrug resistance is characterized by a decrease in the intracellular concentration of drug in resistant cells as compared to sensitive cells. This is correlated with the presence in the membrane of resistant cells of a 150-180-kDa glycoprotein, P-glycoprotein, responsible for an active efflux of the drug. The fluorescence emission spectra from anthracycline-treated cells suspended in buffer have been used to compare the membrane transport of five anthracycline derivatives: adriamycin, daunorubucin, 4'-o-tetrahydropyranyladriamycin, carminomycin and aclacinomycin in drug-sensitive and drug-resistant K562 cells. The initial rate of uptake of these five drugs has been measured as a function of the extracellular pH, pHe. The data show that the uptake occurs through free permeation of the neutral form of the drug. For each drug an influx coefficient kpHe, characteristic of the drug and of the cell type has been defined and calculated: k+(7.2) = V+/[D]e.n where V+ and [D]e are the initial rate of uptake and the concentration of drug in the medium at pHe = 7.2 respectively and n is the number of cells. This coefficient is characteristic of a passive diffusion of the neutral form of the drug through the lipid bilayer. Efflux coefficients k-(7.2)- at pHi = 7.2 (the intracellular pH value) have also been calculated. In the case of sensitive cells, k+(7.2) and k-(7.2)- are equal. For resistant cells, the efflux coefficient is composed of two terms: (a) (k-)p corresponding to the passive diffusion of the neutral form of the drug and (k-)p = k+; (b) (k-)a corresponding to an active efflux mediated by the P-glycoprotein. Our data suggest that the anthracycline derivatives efflux actively in the neutral form.     

Fluorescence probes in metastatic B16 melanoma membranes

Fluorescence probe molecules, trans-parinaric acid and 1,6-diphenylhexatriene, were utilized to characterize the structure of plasma membranes, microsomes and mitochondria from B16 melanoma cells. High metastatic B16-F10 and low metastatic B16-F1 melanoma cell lines had markedly different membrane structures. The fluorescence polarization, fluorescence lifetime and limiting anisotropy of trans-parinaric acid were significantly lower ($\text{P < 0.05}$) in all three membrane fractions of the B16-F1 cell line than in the corresponding membranes of the B16-F10 cell line. These data indicated less restriction to rotational motion in the solid lipid domains of B16-F1 cell membranes preferentially sensed by trans-parinaric acid. The limiting anisotropy of both trans-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene was significantly lower in the outer monolayer than the inner monolayer of the plasma membrane of B16-F1 cells but not in B16-F10 cells. A breakpoint in Arrhenius plots of fluorescence near 30–34°C indicated the presence of a phase separation that was assigned to the inner monolayer of the plasma membrane. However, no differences in this breakpoint temperature were noted between the B16-F1 and B16-F10 melanoma membranes. Thus, more fluid solid membrane domains and a distinct transbilayer fluidity difference were characteristic of plasma membranes from low metastatic B16-F1 melanoma cells in contrast to high metastatic B16-F10 melanoma cells.     

Transbilayer effects of ethanol on fluidity of brain membrane leaflets

Previous work on membrane effects of ethanol focused on fluidization of the bulk membrane lipid bilayer. That work was extended in the present study to an examination of ethanol's effect on lipid domains. Two independent methods were developed to examine the effects of ethanol on the inner and outer leaflets of synaptic plasma membranes (SPM). First, differential polarized phase and modulation fluorometry and selective quenching of diphenyl-1,3,5-hexatriene (DPH) were used to examine individual leaflets. Both limiting anisotropy and rotational relaxation time of DPH in SPM indicated that the outer leaflet was more fluid than the inner leaflet. Second, plasma membrane sidedness selective fluorescent DPH derivatives, cationic 1-[4-(trimethylammonio)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) and anionic 3-[p-6-phenyl)-1,3,5-hexatrienyl]phenylpropionic acid (PRO-DPH), confirmed this transmembrane fluidity difference. TMA-DPH and PRO-DPH preferentially localized in the inner and outer leaflets of SPM, respectively. Ethanol in vitro had a greater fluidizing effect in the outer leaflet as compared to the inner leaflet. Thus, ethanol exhibits a specific rather than nonspecific fluidizing action within transbilayer SPM domains. This preferential fluidization of the SPM outer leaflet may have a role in ethanol affecting transmembrane signaling in the nervous system.     

Vinblastine photoaffinity labeling of a high molecular weight surface membrane glycoprotein specific for multidrug-resistant cells

Photoactive radioactive analogues of vinblastine were used to photoaffinity label membranes of Chinese hamster lung drug-sensitive (DC-3F), multidrug-resistant sublines selected for resistance to vincristine (DC-3F/VCRd-5L) or actinomycin D (DC-3F/ADX), and revertant (DC-3F/ADX-U) cells. A radiolabeled doublet (150-180 kDa) consisting of a major and minor band which was barely detectable in parental drug-sensitive cells was increased up to 150-fold in the drug-resistant variants but only 15-fold in the revertant cells. Photoaffinity labeling in the presence of 200-fold excess vinblastine reduced radiolabeling of the 150-180-kDa species up to 96%, confirming its Vinca alkaloid binding specificity. The radiolabeled doublet comigrated with a Coomassie Blue stained polypeptide doublet in the drug-resistant cells and was immunoprecipitated with polyclonal antibody which is specific for the 150-180-kDa surface membrane glycoprotein in multidrug-resistant cell lines. The identification of this Vinca alkaloid acceptor in multidrug-resistant plasma cell membranes suggests the possibility of a direct functional role for the 150-180-kDa surface membrane protein in the development of multidrug resistance.     

Temperature optimum of insulin-stimulated 2-deoxy-D-glucose uptake in rat adipocytes. Correlation of cellular transport with membrane spin-label and fluorescence-label data.

The effects of temperature alterations between 22 degrees C and 48 degrees C on basal and insulin-stimulated 2-deoxy-D-[1-14C]glucose uptake were examined in isolated rat adipocytes. A distinct optimum was found near physiological temperature for uptake in the presence of maximally effective insulin concentrations where insulin stimulation and hexose uptake were both conducted at each given assay temperature. Basal uptake was only subtly affected. Control and maximally insulin-stimulated cells incubated at 35 degrees C subsequently exhibited minimal temperature-sensitivity of uptake measured between 30 and 43 degrees C. The data are mostly consistent with the concept that insulin-sensitive glucose transporters are, after stimulation by insulin, functionally similar to basal transporters. Adipocyte plasma membranes were labelled with various spin- and fluorescence-label probes in lipid structural studies. The temperature-dependence of the order parameter S calculated from membranes labelled with 5-nitroxide stearate indicated the presence of a lipid phase change at approx. 33 degrees C. Membranes labelled with the fluorescence label 1,6-diphenylhexa-1,3,5-triene, or the cholesterol-like spin label nitroxide cholestane, reveal sharp transitions at lower temperatures. We suggest that a thermotropic lipid phase separation occurs in the adipocyte membrane that may be correlated with the temperature-dependence of hexose transport and insulin action in the intact cells.     

Membrane Anomalies in Huntington''s Disease Fibroblasts

Plasma membranes, microsomes, and mitochondria were isolated from paired, passage number matched, cultured human fibroblasts. The cells were obtained from skin biopsies of Huntington's disease (HD) subjects and from sex and age matched controls. All fibroblasts were cultured in identical media for three to seven passages. Enrichment of surface marker enzymes such as Na+,K+-ATPase indicated a 10-fold purification of the isolated plasma membrane. The specific activity of Na+,K+-ATPase was 62 and 82% greater in the crude homogenate and isolated plasma membrane, respectively, of HD fibroblasts than in control fibroblasts. The specific activity of plasma membrane Na+,K+-ATPase was correlated with lipid composition and with membrane structure as determined by measurement of the rotational relaxation time and limiting anisotropy of fluorescence probe molecules. Major alterations in the structure of the plasma membranes in HD fibroblasts were not noted. The rotational relaxation time and limiting anisotropy of 1,6-diphenyl-1,3,5-hexatriene and of trans-parinaric acid were not significantly different between the plasma membrane, microsomes, or mitochondria of HD versus those of control fibroblasts. trans-Parinaric acid demonstrated the coexistence of fluid and solid domains in all three subcellular membrane fractions of the normal and HD skin fibroblasts. Lastly, both trans-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene displayed characteristic breakpoints in Arrhenius plots of absorbance corrected fluorescence in plasma membranes, microsomes, and mitochondria. In all cases, similar breakpoint temperatures, indicative of phase alterations, were noted near 20 degrees and 30 degrees C. These breakpoints were unaltered in HD. In summary, the data do not support the concept of major membrane structural defects in HD.