The relation of temperature and lipid composition to cell adhesion

We have examined as a function of temperature the effect of changes in the composition of the fatty acid chains of membrane phospholipids on the rate of cell to cell adhesion in the neuronal cell line B103. The rate of cell to cell adhesion in this cell line is highly temperature dependent but is not influenced by changes in the fatty acid composition of the plasma membrane generated by growing the cells either in the presence of oleic acid or elaidic acid. In contrast the temperature dependence of the rate of cell to cell adhesion, measured in a monolayer adhesion assay, is highly dependent on the shear force used during the assay. A two-step model of cell to cell adhesion involving multiple adhesion ligands is presented which can be used to explain these observations.     

Alanine transport by Chinese hamster ovary cells with altered phospholipid acyl chain composition

The Na+-dependent transport of alanine has been examined in Chinese hamster ovary (CHO) cells as a function of the fatty acid composition of their membrane lipids. Significant changes in the fatty acid composition of the CHO cell phospholipids were achieved by supplementation of the growth medium with specific saturated (palmitate) or monoenoic (oleate) free fatty acids. Arrhenius plots of the temperature-dependent uptake of alanine were constructed for cells of altered fatty acid composition. Alanine uptake was characterized by a single discontinuity in the Arrhenius plot. The temperature of this break was observed to be dependent upon the fatty acid composition of the cell phospholipids, ranging from 16 degrees C for cells enriched with oleate to 32 degrees C for cells enriched in palmitate. Calculation of the Km value for the uptake process showed no significant change with temperature or fatty acid supplementation. Correlations are made between the physical state of the membrane lipids and the temperature-dependence for alanine transport. The results are discussed in terms of membrane fatty acid composition, ordered in equilibrium fluid phase transitions and amino acid transport.     

Use of fluorescence polarization to monitor intracellular membrane changes during temperature acclimation. Correlation with lipid compositional and ultrastructural changes.

Fluorescence polarization of 1,6-diphenylhexatriene (DPH) was used to study the effects of temperature acclimation on Tetrahymena membranes. The physical properties of membrane lipids were found to be highly dependent on cellular growth temperature. DPH polarization in lipids from three different membrane fractions correlated well with earlier freeze-fracture and electron spin resonance observations showing that membrane fluidity progressively decreases in the order microsomes greater than pellicles greater than cilia throughout a wide range of growth temperatures. Changes in membrane lipid fluidity following a shift from high to low growth temperatures proceed rapidly in the microsomes, whereas there is a pronounced lag in the changes of peripheral cell membrane lipids. These data support previous observations that adaptive changes in membrane fluidity proceed via lipid modifications in the endoplasmic reticulum, followed by dissemination of lipid components to other cell membranes. The rapid changes in polarization observed in the microsomal lipids following a temperature shift correspond closely with the time-dependent alterations in both lipid fatty acid composition and freeze-fracture patterns of membrane particle distribution, suggesting that, in the endoplasmic reticulum, lipid phase separation is the primary cause of membrane particle rearrangements.     

Anatomic and molecular correlates of divergent selection for basal metabolic rate in laboratory mice

Proximal mechanisms describing the evolution of high levels of basal metabolic rate (BMR) in endotherms are one of the most intriguing problems of evolutionary physiology. Because BMR mostly reflects metabolic activity of internal organs, evolutionary increase in BMR could have been realized by an increase in relative organ size and/or mass-specific cellular metabolic rate. According to the "membrane pacemaker" theory of metabolism, the latter is mediated by an increase in the average number of double bonds (unsaturation index) in cell membrane fatty acids. To test this, we investigated the effect of divergent artificial selection for body-mass-corrected BMR on the mass of internal organs and the fatty acid composition of cell membranes in laboratory mice (Mus musculus). Mice from the high-BMR line had considerably larger liver, kidneys, heart, and intestines. In contrast, the unsaturation index of liver cell membranes was significantly higher in low-BMR mice, mainly because of the significantly higher content of highly polyunsaturated 22 : 6 docosahexanoic fatty acid. Thus, divergent selection for BMR did not affect fatty acyl composition of liver and kidney phospholipids in the direction predicted by the membrane pacemaker theory. We conclude that an intraspecific increase in BMR may rapidly evolve mainly as a result of the changes in size of internal organs, without simultaneous increase of the unsaturation index in cell membrane lipids.     

Effect of supplementation with exogenous fatty acid on the biological properties of a fatty acid requiring auxotroph ofSalmonella typhimurium

The effects of changes in fatty acid composition of the cell membrane on different biological functions ofSalmonella typhimurium have been studied with the help of a temperature sensitive fatty acid auxotroph which cannot synthesise unsaturated fatty acids at high temperature. On being shifted to nonpermissive temperature the cells continue growing for another one and half to two generations. The rates of protein and DNA syntheses run parallel to the growth rate but the rate of RNA synthesis is reduced. Further, there is a gradual reduction in the rate of transport of exogenous uridine and thymidine into the soluble pool. The transport process can be restored by supplementing the growth medium with cis-unsaturated fatty acids but not trans-unsaturated ones although the growth of the cells is resumed by supplementation with eithercis or trans-unsaturated fatty acids. However, supplementation withtrans, trans-unsaturated fatty acids leads to only partial recovery of the transport process. The rate of oxygen uptake is also affected in cells grown in the presence of thetrans-unsaturated fatty acids, elaidic acid and palmitelaidic acid. Analysis of cells grown under different fatty acid supplementation indicate that fatty acid composition of the cell membrane, especially the ratio of unsaturated to saturated fatty acids varies with temperature shift and supplementation of the growth media with fatty acids.     

Escherichia coli membrane fluidity as detected by excimerization of dipyrenylpropane: sensitivity to the bacterial fatty acid profile.

A coordinated study of membrane fluidity and fatty acid composition has been carried out in Escherichia coli W3110. The lipid acyl chain profile of the bacteria, altered by growing cells in steady state at 30, 37, 42, or 45 degrees C, was determined by gas chromatography of the fatty acid methyl esters. In parallel experiments, total membranes obtained from cells of the above-mentioned cultures were labeled with dipyrenylpropane and their relative fluidity was measured on the basis of the excimer to monomer fluorescence intensity ratio of the fluorophore. It has been found that, at constant assay temperature, fluidity determined with dipyrenylpropane decreases gradually with the growth temperature increment, from 30 to 45 degrees C. Interestingly, when fatty acid composition is taken into account, fluidity increases linearly in the range under study, with the proportion of unsaturated fatty acyl chains, both variables being highly correlated (0.924 相似文献   

Modification of the fatty acid composition of cultured human fibroblasts.

The fatty acid composition of human skin fibroblasts grown in 10% dialyzed fetal calf serum can be modified considerably by adding supplemental fatty acids to the culture medium. The degree of modification was dependent on the concentration of added fatty acid over the range tested, 2.5 X 10(-5) to 1 X 10(-4) M. At the higher concentration, the extent of the modifications was as those which can be produced in nonhuman or malignant cell lines. Although the greatest changes were produced in the neutral lipid fraction, the cellular phospholipids also exhibited appreciable modifications. The phospholipids isolated from a microsomal fraction prepared from the cell homogenate exhibited similar changes in fatty acyl composition. These findings indicate that the human fibroblast can tolerate considerable variability in fatty acid composition, even in membrane phospholipids. The triglyceride content of the cells increased when they were grown in the presence of added fatty acids, but the phospholipid and cholesterol content remained unchanged. Growth was not affected by either oleic or linoleic acids, but it was reduced up to 50% when palmitic linolenic, or arachidonic acid was added in concentrations of 5 X 10(-5) M or above. Extensive modifications in phospholipid fatty acid composition also were produced in confluent monolayers of these fibroblasts. This suggest that some membrane lipid turnover occurs even when the cultures are not rapidly growing. Fatty acid modifications also were produced in the commercially available IMR-90 strain of human lung fibroblasts, suggesting that the ability to tolerate considerable differences in fatty acid composition is not a special property of the skin fibroblast line that was isolated locally.     

Changes in fatty acid composition of Pseudomonas aeruginosa ATCC 15442 induced by growth conditions: consequences of resistance to quaternary ammonium compounds

The effects of changes in the fatty acid composition of Pseudomonas aeruginosa induced by growth conditions on its resistance to two quaternary ammonium compounds (QAC) were investigated. The temperature and growth phase were the most influential parameters affecting the fatty acid composition of this bacterium. Furthermore, the formation of saturated fatty acids and cyclopropane fatty acids was stimulated by increasing the temperature, whereas the proportion of unsaturated fatty acids fell. The degree of saturation and the proportion of cyclopropane fatty acids increased in the course of the exponential and stationary phases. These modifications mostly concerned the inner membrane of the bacterium. Resistance of P. aeruginosa to both QAC tested was not significantly influenced by temperature and growth phase variations. Thus, resistance to the two QAC did not seem to be dependent on modifications of the fatty acid composition of the inner membrane.     

Membrane fatty acid composition and endothelial cell functional properties

In order to study the influence of endothelial cell fatty acid composition on various membrane related parameters, several in vitro methods were developed for manipulating the fatty acid content of human endothelial cell membranes. Changes in membrane fatty acid profile were induced by using fatty acid modified lipoproteins or free fatty acids. The largest changes in endothelial fatty acid composition were obtained by culturing the cells in media supplemented with specific free fatty acids. An increase in arachidonic acid content of endothelial phospholipids was induced by supplementation with saturated fatty acids or with arachidonic acid itself. A decrease in arachidonic acid content was obtained by supplementation with other unsaturated fatty acids. Under the experimental conditions used endothelial cells showed a low desaturase activity and a high elongase activity. Considerable alterations in membrane fatty acid composition did not greatly influence certain membrane related parameters such as polymorphonuclear leukocyte adherence and endothelial cell procoagulant activity. In general, for fatty acid modified endothelial cells an association between endogenous arachidonic acid content and total production of eicosanoids was found. This study demonstrates that considerable changes in membrane fatty acid profile affect endothelial cell arachidonic acid metabolism, but it also illustrates homeostasis at the level of endothelial cell functional activity.     

Polyunsaturated fatty acid supplements modulate mast cell membrane microdomain composition

In the present study, the lipid raft composition of a canine mastocytoma cell line (C2) was analyzed. Lipid rafts were well separated from non-raft plasma membranes using a detergent-free isolation technique. To study the influence of n-3 and n-6 polyunsaturated fatty acids (PUFA) on raft fatty acid composition in comparison to non-raft cell membrane, C2 were supplemented with one of the following: α-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, linoleic acid or arachidonic acid. Enrichment of the culture medium with a specific PUFA resulted in an increase in the content of this fatty acid both in rafts and non-raft membranes. Contents of cholesterol and protein were found not to be affected by the changes in the fatty acid profiles. In conclusion, our data provide strong evidence that PUFA modulate lipid composition and physiological properties of membrane micro domains of mast cells which in turn may have effects on mast cell function.     

Modification of fatty acid composition of membrane phospholipid in hepatocyte monolayer with n-3, n-6 and n-9 fatty acids and its relationship to triacylglycerol production

The objective of these studies with rat hepatocytes in primary culture was to establish that: (a) membrane phospholipids would become enriched with the specific fatty acid supplemented to the media and (b) hepatocyte monolayer triacylglycerol synthetic rates were dependent on the type of fatty acid enrichment of the membrane phospholipids. Hepatocytes cultured in the absence of media lipid developed a phospholipid fatty acid composition which is indicative of an essential fatty acid deficiency. The extensive rise in 18:1(n - 9) content indicated that delta 9-desaturase was active. The fatty acid composition of phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol in the microsomal- and mitochondrial-enriched fractions was highly dependent upon the type of fatty acid supplemented to the medium. Incorporation of fatty acids into phospholipids was rapid, and a new steady-state in fatty acid composition was achieved within approx. 36 h. Changes in the fatty acid composition of these hepatocyte phospholipid subclasses resulting from media supplementation with 18:2/20:4(n-6) or 20:5(n-3) were similar, but not identical, to changes which occurred in vivo as a result of consuming diets rich in 18:2(n-6) or 20:5(n-3). Hepatocyte lipogenesis was highly dependent upon the type of fatty acid supplemented to the medium. Prior conditioning with 16:0 increased triacylglycerol synthesis and secretion. Secretion of triacylglycerol was reduced by polyenoic fatty acid enrichment with 20:5(n-3) greater than 20:4/18:2(n-6). The suppression of triacylglycerol synthesis by 20:5(n-3) was due to an increased (P less than 0.05) diacylglycerol specific activity, which indicates that 20:5(n-3) suppression of hepatic triacylglycerol production may be caused in part by the inhibition of diacylglycerol acyltransferase.     

Mutants of Bacillus species isolated on the basis of protonophore resistance are deficient in fatty acid desaturase activity.

The fatty acid desaturase activity in cell extracts of Bacillus subtilis was characterized and found to be O2 dependent, NADH dependent, and cyanide sensitive. In cell fractionation studies, only 10% of the desaturase activity was recovered in the membrane fraction; the addition of cytosolic factors, which by themselves were devoid of activity, restored membrane activity to the level found in the unfractionated cell extracts. NADH was preferred over NADPH as an electron donor, and palmitoyl-coenzyme A was used preferentially over stearoyl-coenzyme A as the straight-chain fatty acid substrate. An increase in desaturase activity was observed when either the growth or the assay temperature was lowered from 37 to 20 degrees C, although the assay temperature appeared to be the more important parameter. Three protonophore-resistant mutants of B. subtilis and a comparable mutant of Bacillus megaterium had been found to possess reduced levels of unsaturated fatty acids in their membrane phospholipids; their protonophore resistance was abolished when grown in the presence of an unsaturated fatty acid supplement. All of these strains were found to be either significantly deficient in or totally lacking desaturase activity in comparison with their wild-type parent strains. Full, protonophore-sensitive revertants of the mutants had levels of desaturase activity comparable to those of the wild-type. Temperature-sensitive revertants of two of the mutants, which grew at 32 degrees C but not at 26 degrees C in the presence of protonophore, exhibited desaturase activity comparable to that of the wild-type at 26 degrees C but lacked activity at 32 degrees C. These results indicate that the biochemical basis for protonophore resistance in these Bacillus mutants is a fatty acid desaturase deficiency.     

Cell surface lipids and adhesion. III. The effects on cell adhesion of changes in plasmalemmal lipids.

The two preceding papers of this series suggest that the state of the plasmalemmal lipids affects cell adhesion. Plasmalemmal composition was altered by the experimental incorporation of fatty acids into R1 and R2 positions in the phosphatidyl components of the cell surface. In this paper we report that: (1) If the incorporation is of long chain length fatty acids (saturated) cell adhesion rises. (2) If the incorporation is of unsaturated fatty acids cell adhesion falls as the unsaturation increases. (3) Incorporation has to be extensive to produce a large change in adhesion. (4) Changes in adhesion parallel the plasmalemmal incorporation but do not follow the total cell incorporation. Item (4) argues that it is plasmalemmal and not other membrane lipids that are involved in cell adhesion. Item (3) suggests that bulk membrane properties and not some very specific grouping are involved in the effects of lipids on adhesion. The similar extents of incorporation of the various different fatty acids and the negligible amounts of lysophospholipids in the membranes of cells that have incorporated fatty acids argue that the effects are not due to differential accumulations of these lysolipids when incubations are done with different fatty acids. The changes in adhesion cannot be accounted for by changes in surface charge density since the electrophoretic mobility of the cells is unchanged by these incubations. It is suggested that these effects on adhesion due to changes in plasmalemmal lipids can be explained either in terms of the action of intermembrane van der Waals--London (electrodynamic) forces in cell adhesion or of changes in surface fluidity. These alternatives are discussed.     

Kinetics of membrane immunoglobulin capping on murine B lymphocytes. Effects of phospholipid fatty acid replacement

Detailed analyses regarding the effects of temperature and phospholipid fatty acid replacement on the capping of membrane immunoglobulin (mIg) have been performed using a recently described flow cytometric procedure (Cuchens, M. A., and Buttke, T. M. (1984) Cytometry 5, 601-609). Purified murine B cells were incubated for 12-20 h in the presence of bovine serum albumin-complexed 80 microM stearic (18:0), oleic (cis-18:1), or linoleic (cis, cis-18:2) free fatty acids. Unmodified and free fatty acid-treated cells were stained with fluorescein-conjugated rabbit anti-mouse Ig and subjected to pulse-shape (width) analyses to follow the kinetics of mIg capping. In both unmodified and free fatty acid-treated cells, capping of mIg occurred at all temperatures between 17 and 37 degrees C, but the rate of cap formation was temperature dependent. Arrhenius plots of mIg capping were linear, with activation energies ranging from 14 to 23 kcal/mol depending on the saturated/unsaturated fatty acid ratio of B cell phospholipids. Ligand-induced redistribution of mIg thus appears to be sensitive to changes in membrane acyl chain composition.     

Control of fatty acid composition of Acholeplasma laidlawii membranes

The temperature-dependent pattern of incorporation of palmitate and oleate from the growth medium into Acholeplasma laidlawii membrane lipids correlates with the physical state of the membrane defined by calorimetry. Both the pattern and the state can be changed at will by changing the fatty acid composition of the membrane lipids. The ratio of palmitate to oleate incorporated is independent of temperature when the membrane bilayer is below its transition and fully ordered, but becomes temperature dependent upon the onset of the transition and continues to be temperature dependent when the membrane is above its transition and fully fluid. This behavior is mimicked by the physical binding of palmitate and oleate to bilayers of extracted membrane lipids and to bilayers of lecithin. Selective binding by membranes may provide a means for controlling lipid fatty acid composition without invoking an enzymatic mechanism.     

Biochemical studies on cell fusion. II. Control of fusion response by lipid alteration

The preceding communication (Roos, D.S. and P.W. Choppin, 1985, J. Cell Biol. 101:1578-1590) described the lipid composition of a series of mouse fibroblast cell lines which vary in susceptibility to the fusogenic effects of polyethylene glycol (PEG). Two alterations in lipid content were found to be directly correlated with resistance to PEG-induced cell fusion: increases in fatty acyl chain saturation, and the elevation of neutral glycerides, including an unusual ether-linked compound. In this study, we have probed the association between lipid composition and cell fusion through the use of fatty acid supplements to the cellular growth medium, and show that the fusibility of cells can be controlled by altering their acyl chain composition. The parental Clone 1D cells contain moderately unsaturated fatty acids with a ratio of saturates to polyunsaturates (S/P) approximately 1 and fuse virtually to completion following a standard PEG treatment. By contrast, the lipids of a highly fusion-resistant mutant cell line, F40, are highly saturated (S/P approximately 4). When the S/P ratio of Clone 1D cells was increased to approximate that normally found in F40 cells by growth in the presence of high concentrations of saturated fatty acids, they became highly resistant to PEG. Reduction of the S/P ratio of F40 cells by growth in cis-polyunsaturated fatty acids rendered them susceptible to fusion. Cell lines F8, F16, etc., which are normally intermediate between Clone 1D and F40 in both lipid composition and fusion response, can be altered in either direction (towards either increased or decreased susceptibility to fusion) by the addition of appropriate fatty acids to the growth medium. Although trans-unsaturated fatty acids have phase-transition temperatures roughly similar to saturated compounds, and might therefore be expected to affect membrane fluidity in a similar manner, trans-unsaturated fatty acids exerted the same effect as cis-unsaturates on the control of PEG-induced cell fusion. This observation suggests that the control of cell fusion by alteration of fatty acid content is not due to changes in membrane fluidity, and thus that the fatty acids are involved in some other way in the modulation of cell fusion.     

Modification of membrane phospholipid fatty acyl composition in a leukemic T cell line: effects on receptor mediated intracellular Ca2+ increase.

The effect of modifying fatty acyl composition of cellular membrane phospholipids on receptor-mediated intracellular free Ca2+ concentration ([Ca2+]i) increase was investigated in a leukemic T cell line (JURKAT). After growing for 72 h in medium supplemented with unsaturated fatty acids (UFAs) and alpha-tocopherol, the fatty acyl composition of membrane phospholipids in JURKAT cells was extensively modified. Each respective fatty acid supplemented in the culture medium was readily incorporated into phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine in the JURKAT cells. The total n-6 fatty acyl content was markedly reduced in phosphatidylinositol and phosphatidylcholine of cells grown in the presence of n-3 fatty acids (alpha-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid). Conversely, in the presence of n-6 fatty acids (linoleic acid and arachidonic acid), the total n-3 fatty acyl content was reduced in all the phospholipids examined. In n-3 and n-6 polyunsaturated fatty acid (PUFA) modified JURKAT cells, the total n-9 monounsaturated fatty acyl content in the phospholipids were markedly reduced. Changing the fatty acyl composition of membrane phospholipids in the JURKAT cells appears to have no affect on the presentation of the T cell receptor/CD3 complex or the binding of anti-CD3 antibodies (OKT3) to the CD3 complex. However, the peak increase in [Ca2+]i and the prolonged sustained phase elicited by OKT3 activation were suppressed in n-3 and n-6 PUFA but not in n-9 monounsaturated fatty acid modified cells. In Ca2+ free medium, OKT3-induced transient increase in [Ca2+]i representing Ca2+ release from the inositol 1,4,5-trisphosphate-sensitive Ca2+ stores, were similar in control and UFA modified cells. Using Mn2+ entry as an index of plasma membrane Ca2+ permeability, the rate of fura-2 fluorescence quenching as a result of Mn2+ influx stimulated by OKT3 in n-9 monounsaturated fatty acid modified cells was similar to control cells, but the rates in n-3 and n-6 PUFA modified cells were significantly lower. These results suggest that receptor-mediated Ca2+ influx in JURKAT cells is sensitive to changes in the fatty acyl composition of membrane phospholipids and monounsaturated fatty acids appears to be important for the maintenance of a functional Ca2+ influx mechanism.     

Thermal regulation of fatty acid synthetase from Brevibacterium ammoniagenes.

The fatty acid composition of Brevibacterium ammoniagenes was affected by the temperature of growth. As the growth temperature was lowered, the proportion of unsaturated fatty acids increased. The fatty acid synthetase obtained from B. ammoniagense produced oleic acid as well as saturated fatty acids. The ratio of unsaturated to saturated fatty acids synthesized by this enzyme in vitro was dependent on the temperature of the enzyme reaction but not on the growth temperature of B. ammoniagenes from which the enzyme was prepared. These results suggest that the changes of composition in cellular fatty acids reflect the temperature dependence of the fatty acid synthetase.     

The effects of exposure to exogenous fatty acids and membrane fatty acid modification on the electrical properties of NG108-15 cells

The role of membrane lipid composition in determining the electrical properties of neuronal cells was investigated by altering the available fatty acids in the growth medium of cultured neuroblastoma X glioma hybrid cells, clone NG108-15. Growth of the cells for several days in the presence of polyunsaturated fatty acids (linoleic, linolenic, and arachidonic) caused a pronounced decrease in the Na+ action-potential rate of rise (dV/dt) and smaller decreases in the amplitude, measured by intracellular recording. Oleic acid had no effect on the action potentials generated by the cells. In contrast, a saturated fatty acid (palmitate) and a trans monounsaturated fatty acid (elaidate) caused increases in both the rate of rise and the amplitude. No changes in the resting membrane potentials or Ca2+ action potentials of fatty acid-treated cells were observed. The membrane capacitance and time constant were not altered by exposure to arachidonate, oleate, or elaidate, whereas arachidonate caused a small increase in membrane resistance. Examination of the membrane phospholipid fatty acid composition of cells grown with various fatty acids revealed no consistent alterations which could explain these results. To examine the mechanism for arachidonate-induced decreases in dV/dt, the binding of 3H-saxitoxin (known to interact with voltage-sensitive Na+) channels was measured. Membranes from cells grown with arachidonate contained fewer saxitoxin binding sites, suggesting fewer Na+ channels in these cells. We conclude that conditions which lead to major changes in the membrane fatty acid composition have no effect on the resting membrane potential, membrane capacitance, time constant, or Ca2+ action potentials in NG108-15 cells. Membrane resistance also does not appear to be very sensitive to membrane fatty acid composition. However, changes in the availability of fatty acids and/or changes in the subsequent membrane fatty acid composition lead to altered Na+ action potentials. The primary mechanism for this alteration appears to be through changes in the number of Na+ channels in the cells.