Modulation of P-glycoprotein-mediated drug transport by alterations in lipid fluidity of rat liver canalicular membrane vesicles.

P-glycoprotein (P-gp) is believed to function as an ATP-dependent efflux pump for natural product anti-cancer drugs in multidrug-resistant (MDR) tumor cells and in certain normal tissues. P-gp has been localized to the apical plasma membrane of the bile canaliculus where it has been shown to transport [3H]daunomycin. In this study, we investigated whether alterations in membrane lipid fluidity of canalicular membrane vesicles (CMV) could modulate the P-gp-mediated accumulation of [3H]daunomycin and [3H]vinblastine. Accumulation of both cytotoxic agents was stimulated by ATP, exhibited temperature dependence and osmotic sensitivity, and followed Michaelis-Menten kinetics. Alterations in CMV lipid fluidity were induced by the known fluidizers, 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate (A2C) and benzyl alcohol, and were assessed by fluorescence polarization techniques using the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). Both A2C (2.5-5.0 microM) and benzyl alcohol (10-20 mM) produced a dose-dependent increase in CMV lipid fluidity. Moreover, both fluidizers, at the above doses, significantly inhibited (p < 0.05) the ATP-dependent accumulation of [3H]daunomycin. [3H]Vinblastine accumulation was also inhibited by A2C (p < 0.05). Lower doses of A2C (0.6 microM) and benzyl alcohol (1 mM) failed to influence either lipid fluidity or P-gp-mediated drug accumulation. Kinetic analysis revealed that A2C (5.0 microM) noncompetitively inhibited [3H]daunomycin accumulation and uncompetitively inhibited [3H]vinblastine accumulation with apparent Ki values of approximately 1.5 and approximately 1.2 microM, respectively. Verapamil competitively inhibited P-gp-mediated accumulation of [3H]daunomycin but failed to alter the fluidity of CMV. Taken together, the present results demonstrate that while increases in membrane fluidity of CMV are not necessarily required to inhibit P-gp-mediated drug accumulation, they can inhibit these processes, at least in CMV. Alterations in the physical state of CMV, therefore, appear to be at least one important modulator of P-gp function.     

The effect of ACTH on rat brain synaptic plasma membrane lipid fluidity

The effect of ACTH on the lipid fluidity was examined in synaptic plasma membranes from rat forebrain. ACTH1-24 increased the fluidity of the synaptic plasma membranes in a dose-dependent way, the lowest effective dose being 10(-5) M. The shorter N-terminal fragment ACTH1-10 was not effective. The significance of this finding is discussed in relation to the known effects of ACTH on synaptic membrane phosphorylation.     

In vitro effect of free bile acids on the bile canalicular membrane phospholipids in the rat.

Liver cell plasma membranes of male rats were isolated and separated into two fractions, one rich in bile canalicular membranes (BCM) and the other comprising the rest of the plasma membrane (PM). Aliquots of BCM, PM, and microsomes were incubated with deoxycholic, chenodeoxycholic, or cholic acid at bile acid - membrane phospholipid mole ratios up to 100, and the phospholipid solubilization from the PM and from microsomes was linear and apparently nonselective, while that from BCM was biphasic and distinctly selective. Phosphatidyl choline and phosphatidyl ethanolamine made up 90% of the phospholipids solubilized from the BCM at a bile acid - membrane phospholipid mole ratio sufficient to solubilize about 50% of the total phospholipids of the BCM. Of particular interest was the observation that the molecular species and fatty acid composition of the phospholipids solubilized from the BCM under these experimental conditions were similar to those of bile obtained from the same animal, and were quite unlike those solubilized at higher bile acid - phospholipids mole ratios. The data are discussed in terms of the mechanism of the biliary secretion of phospholipids.     

The lipid fluidity of rat liver membrane subfractions

1. The lipid fluidity of three major rat liver plasma-membrane subfractions, as well as Golgi apparatus and endocytic fractions, was assessed with a fatty acid spin probe by using e.s.r. techniques. 2. The sinusoidal (blood-facing) plasma-membrane subfraction was the most fluid of the three plasma-membrane regions. Fractions originating from the bile-canalicular and contiguous (lateral) regions were most rigid. Endocytic fractions isolated (endosomes and diacytosomes) were of a similar fluidity to fractions originating from the sinusoidal plasma-membrane region. By far the most fluid fractions examined were derived from the Golgi-apparatus complex. 3. The three plasma-membrane subfractions each showed a different response to the bilayer-fluidizing effect of benzyl alcohol. 4. Arrhenius-type plots of the order parameter S and outer hyperfine splitting, 2T, identified lipid-phase separations in the plasma-membrane subfractions.     

A domain-specific marker for the hepatocyte plasma membrane. III. Isolation of bile canalicular membrane by immunoadsorption

Previous immunolabeling studies (Roman, L.M., and A.L. Hubbard, 1983, J. Cell Biol., 96:1548-1558; Roman, L.M., and A.L. Hubbard, 1984, J. Cell Biol., 98:1488-1496, companion paper) established leucine aminopeptidase (LAP) as a specific marker for the bile canalicular (BC) domain of the rat hepatocyte plasma membrane (PM). In this study, we have isolated membrane from a sonicated PM vesicle fraction using anti-LAP-coated Staphylococcus aureus cells as a solid-phase immunoadsorbent. The extent and specificity of the immunoadsorption were assessed by following the behavior of LAP (the BC marker) and 32P-labeled membrane phospholipids (a uniform membrane marker). The BC fraction obtained was significantly enriched in LAP (yield: greater than 70% of PM-LAP). Alkaline phosphatase, 5'-nucleotidase, and a 110,000-dalton glycoprotein, HA-4, were enriched in the BC fraction to the same extent as LAP (enzyme or antigen/LAP = 1.0). However, alkaline phosphodiesterase I was not enriched to the same degree (enzyme/LAP = 0.5). Contamination of this BC fraction by membrane derived from the sinusoidal domain and endoplasmic reticulum, as determined from the distribution of the asialoglycoprotein receptor and NADH cytochrome c reductase, respectively, was small (less than 13%).     

Isolation and partial characterization of a bile canalicular plasma membrane fraction from normal and regenerating rat liver.

A bile canalicular membrane fraction was isolated from 24-hour regenerating rat livers, and its properties were compared to those of homologous fractions prepared from the livers of sham-operated and unoperated controls. These canalicular membrane fractions were found to be closely related in terms of their morphology, their purity, their yield, and their qualitative protein banding profiles on sodium dodecyl sulfate-polyacrylamide gels. However, when a rigorous examination of plasma membrane enzyme marker activities was made, the regenerating liver membranes were shown to possess an increased specific activity of alkaline phosphatase and lower levels of Mg2+ ATPase and 5'-nucleotidase in comparison with control specific activity values.     

Effect of lipid composition on rat liver nuclear membrane fluidity

Nuclear membrane fluidity is measured in rat liver by use of the fluorescence anisotropy of two probes: diphenylhexatriene and its cationic derivative trimethylammonium-diphenylhexatriene. It has been shown that, in 2-month-old rat liver cells, the bilayer surface is less fluid than the hydrophobic core. The fluidity was higher in 6-day-old rat liver nuclei, in which both the amount of cholesterol and the cholesterol/phospholipid ratio decreased. The influence of the single phospholipids, and in particular of phosphatidylcholine, has been studied by increasing the phosphatidylcholine with a choline base exchange reaction in isolated nuclear membranes. After this reaction, the fluorescence anisotropy of the bilayer surface increased, whereas at the hydrophobic core it decreased. Analysis of fatty acid composition shows an increase of phosphatidylcholine unsaturated fatty acids. The results show that the fluidity of nuclear membranes changes in relation to the lipid content and to the fatty acid composition. The role of nuclear membrane fluidity in cell function is discussed. © 1997 John Wiley & Sons, Ltd.     

Mechanisms by which cAMP increases bile acid secretion in rat liver and canalicular membrane vesicles

Bile acid secretion induced by cAMP and taurocholate is associated with recruitment of several ATP binding cassette (ABC) transporters to the canalicular membrane. Taurocholate-mediated bile acid secretion and recruitment of ABC transporters are phosphatidylinositol 3-kinase (PI3K) dependent and require an intact microtubular apparatus. We examined mechanisms involved in cAMP-mediated bile acid secretion. Bile acid secretion induced by perfusion of rat liver with dibutyryl cAMP was blocked by colchicine and wortmannin, a PI3K inhibitor. Canalicular membrane vesicles isolated from cAMP-treated rats manifested increased ATP-dependent transport of taurocholate and PI3K activity that were reduced by prior in vivo administration of colchicine or wortmannin. Addition of a PI3K lipid product, phosphoinositide 3,4-bisphosphate, but not its isomer, phosphoinositide 4,5-bisphosphate, restored ATP-dependent taurocholate in these vesicles. Addition of a decapeptide that activates PI3K to canalicular membrane vesicles increased ATP-dependent transport above baseline activity. In contrast to effects induced by taurocholate, cAMP-stimulated intracellular trafficking of the canalicular ABC transporters was unaffected by wortmannin, and recruitment of multidrug resistance protein 2, but not bile salt excretory protein (bsep), was partially decreased by colchicine. These studies indicate that trafficking of bsep and other canalicular ABC transporters to the canalicular membrane in response to cAMP is independent of PI3K activity. In addition, PI3K lipid products are required for activation of bsep in the canalicular membrane. These observations prompt revision of current concepts regarding the role of cAMP and PI3K in intracellular trafficking, regulation of canalicular bsep, and bile acid secretion.     

Spin label studies on rat liver plasma membrane: calcium effects on membrane fluidity.

Spin-labeled stearic acid probes were incorporated into rat liver plasma membrane purified by a zonal centrifugation procedure. Temperature effects on the order parameter calculated from the spectra indicated the possible presence of a membrane phase transition at 20 degrees, in agreement with earlier studies in other systems. Addition of Ca2+ (1.96 mM) at 37 degrees increased the order parameter by 6.0 +/- 0.7%, suggesting a Ca2+-mediated decrease in membrane fluidity.     

Effect of docosahexaenoic acid on membrane fluidity and function in intact cultured Y-79 retinoblastoma cells.

Considerable metabolic energy is expended in ensuring that membranes possess a characteristic fatty acid composition. The nature of the specific requirement of the retina for high levels of docosahexaenoic acid (DHA) is as yet undefined. Previous work has speculated that DHA is required to maintain the fluid nature and permeability necessary for optimal retinal function. Cultured Y-79 retinoblastoma cells were grown in serum-containing media with and without supplemental DHA. Resultant changes in membrane fluidity were assessed using fluorescent probes. No differences were observed in rotational probe mobility as assessed by fluorescence polarization despite a fourfold increase in cellular DHA content. Lateral probe mobility as assessed by pyrene eximer formation was significantly enhanced in DHA-supplemented cells. Both the DHA content and total fatty acid unsaturation index in retinoblastoma cells were directly correlated with membrane fluidity as reported by eximer formation (Pearson's rho = 0.96 and 0.92, respectively). DHA supplementation also resulted in a significant increase in cellular choline uptake. We speculate that the effect of DHA content on retinal function may be mediated by changes in membrane fluidity and associated enzyme and transport activities.     

Postnatal maturation of rat intestinal membrane: lipid composition and fluidity.

1. We studied the lipid composition and the fluidity of small intestine brush border membrane (BBM) of rats of different age: 'very young' (5-7 weeks old), 'young' (9 weeks old), 'adult' (30 weeks old) and 'old' (85 weeks old). 2. Fluorescence anisotropy, as assessed by 1,6-diphenyl-1,3,5-hexatriene probe (DPH), was increased from very young to adult rats. 3. In agreement with these results the lipid composition in adult animals showed a lower lipid/protein ratio (derived mainly from a lower content of total polar lipids) and an increase of cholesterol esters and sphingomyelin (SM) saturation index. 4. A marked decrease of the order parameter was observed in the 'old' group, accompanied by a decreased cholesterol/phospholipid ratio. 5. The percentage distribution of membrane phospholipids significantly changed during development, but the modifications were not correlated with the anisotropy of DPH.     

A domain-specific marker for the hepatocyte plasma membrane. II. Ultrastructural localization of leucine aminopeptidase to the bile canalicular domain of isolated rat liver plasma membranes

Leucine aminopeptidase (LAP) is an integral membrane glycoprotein localized to the apical membrane domain of intestinal and kidney epithelial cells. By indirect immunofluorescence, we have shown that antibodies raised against rat intestinal LAP recognized a similar protein concentrated in the bile canalicular (BC) domain of the hepatocyte in situ (Roman, L.M., and A.L. Hubbard, 1983, J. Cell Biol., 96:1548-1558). We have extended this localization to the ultrastructural level. When a saponin-permeabilized, agarose-embedded plasma membrane (PM) fraction was incubated with affinity-purified anti-LAP, 85% of the protein A-gold particles associated with the three recognizable PM domains were present in the BC. The levels of labeling on the other two domains (sinusoidal and lateral) did not exceed that observed with nonimmune controls. The concentration of LAP in the BC domain in isolated PM sheets prompted us to use this antigen for the affinity isolation of BC membrane (Roman, L.M., and A.L. Hubbard, 1984, J. Cell Biol., 98:1497-1504, companion paper).     

A dietary regimen alters hepatocyte plasma membrane lipid fluidity and ameliorates ethinyl estradiol cholestasis in the rat

The beta-adrenergic receptor mediating the inhibition of sterol synthesis by catecholamines in freshly isolated human mononuclear leukocytes was defined pharmacologically by using selective beta 1- and beta 2-agonists and -antagonists. Incubation of cells for 6 h in a medium containing lipid-depleted serum resulted in a 3-fold increase in the incorporation of [14C]acetate or tritiated water into sterols. The beta-agonist (-)-isoproterenol was approximately equipotent with (-)-epinephrine and (-)-norepinephrine in suppressing sterol synthesis, yielding a sigmoidal log-dose-effect curve. Accordingly, the effects of the catecholamines were reversed by the beta-antagonist (+/-)-propranolol. The beta 2-agonists terbutaline and salbutamol inhibited sterol synthesis by 42 and 26%, respectively, at a concentration of 0.1 mmol/l. Contrary to that, the beta 1-agonists prenalterol and dobutamine had no effect. In accordance with the influence of the agonists, the beta 2-antagonist butoxamine, but not the beta 1-antagonists atenolol, metoprolol and practolol, reversed the catecholamine action on sterol synthesis. The results provide evidence that catecholamines may regulate sterol synthesis by stimulating beta 2-adrenergic receptors.     

Comparison of bile acid binding to sinusoidal and bile canalicular membranes isolated from rat liver

Simon et al. (J. Clin. Invest., 70 (1982) 401) studied cholate binding to crude liver plasma membrane vesicles and suggested that the binding may represent mainly the binding to the receptor (carrier) on the canalicular membrane. This hypothesis was supported by finding a good correlation between the number of cholate binding sites on liver plasma membrane and the maximal rate of biliary secretion (Tm) for taurocholate. We studied bile acid binding to sinusoidal and canalicular membrane vesicles isolated from rat liver by a rapid filtration technique. Scatchard analysis of the saturation kinetics showed both [3H]cholate and [3H]chenodeoxycholate bind to two classes of binding site on each membrane. However, little difference was observed between the binding to sinusoidal and canalicular membrane vesicles for each bile acid (cholate, Kd1 = 10.4 and 19.8 microM, n1 = 31.0 23.6 pmol/mg protein, Kd2 = 1.32 and 1.73 mM, n2 = 13.1 and 23.4 nmol/mg protein; and chenodeoxycholate, Kd1 = 0.207 and 0.328 microM, n1 = 36.7 and 27.4 pmol/mg protein, Kd2 = 1.16 and 2.26 mM, and n2 = 20.6 and 24.2 nmol/mg protein; numbers show the mean values sinusoidal and canalicular membrane vesicles, respectively). Chenodeoxycholate binding to sinusoidal membrane vesicles was markedly inhibited by cholate but not by Rose bengal, an organic anion dye. These studies indicate that both membranes (sinusoidal and canalicular membrane vesicles) have two kinds of binding site for bile acids, although no clear difference in the binding properties was observed between the two membranes. Consequently, the cholate binding Simon detected may represent the binding not only to canalicular membrane vesicles but also to sinusoidal membrane vesicles.     

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.     

Effects of trace elements on membrane fluidity.

According to the Fluid Mosaic Model, a biological membrane is a two-dimensional fluid of oriented proteins and lipids. The lipid bilayer is the basic structure of all cell and organelle membranes. Cell membranes are dynamic, fluid structures, and most of their molecules are able to move in the plane of the membrane. Fluidity is the quality of ease of movement and represents the reciprocal value of membrane viscosity. Fluid properties of biological membranes are essential for numerous cell functions. Even slight changes in membrane fluidity may cause aberrant function and pathological processes. Several evidences suggest that trace elements, e.g., iron, copper, zinc, selenium, chromium, cadmium, mercury and lead may influence membrane fluidity. The interaction of heavy metals with cellular membranes may contribute to explain, at least partially, the toxicity associated with these metals.     

Effects of cholesteryl esters on the accessibility of LH/hCG receptors and membrane lipid fluidity in rat testes

Incubation of rat testicular membranes with cholesteryl hemisuccinate resulted in an increase in both membrane lipid microviscosity and 125I-labelled hCG specific binding. The purpose of this investigation was to establish which functional groups of cholesteryl hemisuccinate are important for the stimulatory effects. The data obtained showed that only esters of cholesterol with dicarboxylic acids, not those of monocarboxylic acids, increase the accessibility of LH/hCG receptors and membrane rigidity. Experiments with cholesteryl sulfates showed that there are polar groups on C3 carbon of cholesterol having no stimulatory effect on receptors, although an increase in membrane rigidity occurred. The side-chain of cholesterol is important for the stimulatory action. Androstenolone hemisuccinate was ineffective in this respect. On the other hand, partially modified side-chains (hemisuccinates of beta-sitosterol and stigmasterol) did not result in a marked reduction of the stimulatory action. The carboxyl group of cholesteryl hemisuccinate must be 'free': its esterification abolishes the stimulatory effect of cholesteryl hemisuccinate on both the LH/hCG receptor and membrane microviscosity. These results suggest that an intact carboxyl group of ester and the side-chain of cholesterol are indispensable for the stimulatory effect of cholesteryl hemisuccinate on the accessibility of LH/hCG receptors.     

Effects of lindane on fluidity and lipid composition in rat renal cortex membranes.

The influence of lindane upon dynamic properties of plasma membranes from rat renal cortex has been investigated using a fluorescence polarization technique. Preincubation with lindane increased membrane fluidity in a manner that is dose-dependent. This increase was higher in brush border membranes than in basolateral membranes. However, a significant decrease of the membrane fluidity was found in brush border membranes when rats were injected with lindane for 12 days. A possible solution to this difference could involve a resistance to membrane disordering by lindane through a regulatory mechanism that would balance the amount of cholesterol and phospholipid classes in the renal cortex membranes of lindane-injected rats.     

Alternations in lipid membrane fluidity and the physical state of cell-surface sialic acid in zinc-deficient rat erythrocyte ghosts

Erythrocyte ghosts, prepared from the blood of rats fed zinc-deficient diets, were evaluated for membrane fluidity and surface sialic acid properties using spin-labeled probes and electron spin resonance (ESR) spectroscopy. These physical parameters of the erythrocyte ghosts from the zinc-deficient group were compared to those for erythrocyte ghosts obtained from ad libitum and pair fed controls consuming zinc-adequate diets. As the animals became progressively zinc deficient, the erythrocyte ghost membranes became more fluid than those from the control groups. In addition, the apparent rotational correlation time of Tempamine spin probes on surface sialic acid residues was smaller for the zinc deficient group, indicative of an increased rotational mobility of the spin label. These results suggest that zinc deficiency can have pronounced effects on the physical state of membrane bilayer lipids and cell surface carbohydrates and supports the view that many of the pathological signs of zinc deficiency are due to a general membrane defect.