12-O-tetradecanoyl phorbol 13-acetate stimulates the myristylation of an approximately 82 kDa protein in HL-60 cells.

We have studied protein acylation using [3H]myristate in the two leukemia cell lines HL-60 and HL-60 Blast II. The latter is a variant which does not differentiate after treatment with 12-O-tetradecanoyl phorbol 13-acetate (TPA). The acylation profiles of the two cell lines as examined by SDS-PAGE differed. TPA induced the myristylation of an approximately 82 kDa protein in the sensitive cells, but not in the resistant cells. Myristic acid was shown to be covalently linked to these proteins. Analysis of the cell lipids labelled with [3H]myristate showed that in contrast to observations with the proteins, the changes induced by TPA were observed in both TPA-sensitive and TPA-resistant cells. We conclude that the induction of myristylation may be an important step in the mechanism of differentiation.

[Comparative metabolism of phospholipids of osmoregulation effector organs in European eel (Anguilla anguilla)]. / Métabolisme comparé des phospholipides des organes effecteurs DE L'osmorégulation chez l'anguille européenne (Anguilla anguilla)

The phospholipid composition from various organs of the fresh water eel, such as gill, kidney, gut, liver and muscle, were determined by thin-layer chromatography. The major phosphatides found in these tissues were PC, PE and SPH and minor constituents PS, PI, DPG, AP and also LPC in the gut. A greater percentage of PS and SPH occurs in the osmoregulatory effector organs such as gill, kidney, and gut. From in vivo comparative kinetic studies of the 32P incorporation into the phospholipids, between 6 and 48 hours, certain remarkable features of phospholipid metabolism have been found in these tissues. A low uptake of inorganic 32P into the tissue lipid phosphorus was observed in the eel at 15 degrees C. The specific activity of the lipid phosphorus increased continuously in all tissues during 48 hours after 32P injection. During this experimental period, phosphatidic acid and phosphatidyl inositol fractions were labelled most rapidly in gill, kidney and gut, while the specific activity of the phosphatidyl choline fraction remained low in these organs. In liver, the rate of PC formation appears to be faster than the PI and PE biosynthesis. In gill and gut, the PE showed greater turnover than the PC as measured by 32P incorporation. In the eel, an euryhalin fish, the DPG of osmoregulatory effector organs has a high specific activity at all times. PS showed only a high specific activity in the gill. Labelling of SPH occured slowly in the various tissues only becoming evident after 24 hours. The results are compared with those published for other poikilotherm and homeotherm vertebrates. Relative differences between the turnover of various tissue phosphatides are discussed with of reference to the general scheme on phospholipid biosynthesis and to the physiological functions of the various organs.

Glycerol and fatty acid kinetics in rainbow trout: effects of endurance swimming

Continuous infusions of 2-[3H]glycerol and 1-[14C]palmitate were performed in vivo in rainbow trout to measure the effects of prolonged swimming on (1) the rate of appearance of glycerol (Ra glycerol or lipolytic rate), (2) the rate of appearance of non-esterified fatty acids (Ra NEFA) and (3) the rate of triacylglycerol:fatty acid cycling (TAG:FA cycling or re-esterification). Our goals were to test the hypothesis that sustained exercise for up to 4 days causes the progressive mobilization of triacylglycerol reserves to supply fuel to contracting muscles, and to assess whether TAG:FA cycling plays a role in the regulation of NEFA availability in teleosts. Contrary to expectation, the rates of lipolysis and fatty acid release in resting trout are not affected by endurance exercise. Unlike mammals, which increase the rate of lipolysis by two- to fourfold during submaximal exercise, these active teleosts do not mobilize triacylglycerol reserves beyond resting levels to supply more NEFAs to working muscles. Furthermore, they maintain Ra glycerol and Ra NEFA well in excess of oxidative fuel requirements even at rest. More than two-thirds of the NEFAs produced are re-esterified, but the results show that TAG:FA cycling is not involved in the regulation of NEFA availability during or after swimming. We propose that the observed high rates of re-esterification represent an important feature of ectothermic metabolism that allows the restructuring of membrane phospholipids to be synchronized with frequent changes in body temperature.

Changes in biophysical parameters and in phospholipid composition associated with resistance to doxorubicin.

Friend leukemia cells (FLC) resistant to different concentrations of doxorubicin were used to investigate the biochemical and biophysical changes associated with resistance. We have found that fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene analyzed on single cell level increased in resistant as compared to sensitive FLC. Furthermore, phospholipid analysis of sensitive and doxorubicin-resistant cells revealed changes in ratios of phosphatidyl-choline to phosphatidyl-ethanolamine and phosphatidyl-choline to sphingomyelin. These results correlate with decreased electrophoretic mobility in resistant cells. Our results indicate that changes in cell structure occur with the level of resistance to doxorubicin. These changes are probably the consequence rather than the cause of resistance.

Effects of L-thyroxine on incorporation of (32)P into phospholipids of freshwater eels.

The effects of L-thyroxine on phospholipid biosynthesis, via (32)P incorporation, were studied in gill, kidney, liver and muscle tissue of eels acclimatized at 11 degrees C. L-thyroxine treatment had no effect on tissue content of lipid, inorganic and organic acid-soluble phosphorus. Only an increase of the specific radioactivities of lipid, inorganic and organic acid-soluble phosphorus was observed in the muscle. Percentage distribution of (32)P among classes of phospholipid were significantly altered in liver and muscle, without change in phospholipid composition. A specific effect of L-thyroxine on (32)P incorporation into phosphatidic acid in muscle and liver has been shown. As expected by the higher specific radioactivity of muscle inorganic and organic acid-soluble phosphorus, the increased incorporation of (32)P into phosphatidic acid probably results from a higher specific radioactivity of muscle ATP phosphorus.

Differential labelling of sphingolipids by [3H]serine and ([3H]methyl)-methionine in fish leukocytes.

Long chain bases are constituents of all sphingolipids and their biosynthesis is presumed to occur via the initial condensation of serine with palmitoyl-CoA. The biosynthesis of phytosphingosine, a long chain base containing three hydroxyl groups, has been less studied than sphingosine but is assumed to occur by hydroxylation of sphinganine. We report in this paper that the label from ([3H]methyl)-methionine is preferentially incorporated into phytosphingosine bases of neutral glycosphingolipids, whereas the label from [3H]serine is mainly incorporated into the sphingoid base of sphingomyelin. These results show that in fish leukocytes the biosynthesis of individual sphingoid bases and their downstream sphingolipid products follow different pathways of metabolism. Our observations suggest that in fish leukocytes the synthesis of the constitutive long chain bases of sphingomyelin and complex glycosphingolipids is coordinately regulated and may be localized in separate compartments.

Sphingomyelin metabolism is linked to salt transport in the gills of euryhaline fish.

By in vivo and in vitro studies of L-(3-3H)serine and [9,10(n)-3H]palmitic acid incorporation into phospholipids, we show a change in the renewal of the ceramide moiety of sphingomyelin in the gills of euryhaline fish (sea bass and eels) when the animals were subjected to abrupt alterations in environmental salinity. In vivo, decrease of the salinity from sea water (salinity 3.7%) to diluted sea water (salinity 1%) induced an increase of label incorporation into gill sphingomyelin. The same was true when gills from sea water-adapted sea bass or sea water-adapted eels were incubated in diluted sea water. A decrease in free ceramides synthesis was also observed in the gills of sea water-adapted sea bass when the salinity of the incubation medium was reduced. Direct inhibition of Na+/K(+)-ATPase activity with ouabain decreased the sphingomyelin synthesis in the gills of sea bass during in vitro incubation in diluted sea water, whereas treatment with furosemide stimulated sphingomyelin synthesis in the same gills incubated in sea water. These findings indicate that changes in Na+ fluxes modify the sphingomyelin turnover and control the production of free ceramides and sphingosine in gill cells of euryhaline fish. In view of the well-known effects of these sphingomyelin degradation products on isolated tumor cell differentiation, we suggest that they play a very important role in modulating chloride cell distribution and metabolism of fish gills during abrupt changes in environmental salinity.

Interferon alpha/beta induces changes in the metabolism of polyenoic phospholipids and diacylglycerols in the livers of suckling mice.

Suckling mice were injected daily from birth for 10 days with potent preparations of mouse interferon alpha/beta. Interferon treatment resulted in a markedly lower concentration of polyunsaturated fatty acids (20:4 omega 6 and 22:6 omega 3) in the two principal liver phospholipids, phosphatidylcholine and phosphatidylethanolamine, than in livers of control-treated mice. This effect appeared to correlate with a low level of synthesis of polyunsaturated phospholipids in the livers of interferon-treated mice. Thus, in control mice, synthesis of species of polyunsaturated phospholipids increased markedly in the first 10 days of life, whereas in 10-day-old interferon-treated mice, the level of synthesis of species of polyunsaturated phospholipids was comparable to that in newborn mice. In parallel, a marked increase in the diacylglycerol content without change of its renewal was observed in the livers of interferon-treated mice. We suggest that interferon treatment results in an inhibition of one of the processes that leads to activation of the enzymatic systems responsible for the synthesis of species of polyunsaturated phosphatidylcholine and phosphatidylethanolamine in the liver of suckling mice. It seems likely that these results are related to the inhibition of liver cell maturation and the marked cell necrosis that are observed in interferon-treated suckling mice.

Effect of exercise on the plasma nonesterified fatty acid composition of dogs and goats: species with different aerobic capacities and diets.

The goals of this study were to determine: (i) whether mammals mobilize particular nonesterified fatty acids (NEFA) preferentially during locomotion, (ii) if differences in aerobic capacity or diet can affect the pattern of NEFA mobilization and (iii) which individual NEFA are most representative of total NEFA concentration changes, to use them as tracers for turnover studies. Individual NEFA were measured in trained dogs and goats (VO2max dog/VO2max goat = 2.2; where VO2max = maximal oxygen consumption) during treadmill exercise at 40 and 60% VO2max. Important interspecies differences in individual NEFA concentrations could be attributed to differences in aerobic capacity. The more aerobic species (dog) had much higher plasma NEFA concentrations for all but one NEFA (18:0), when compared with the low-aerobic species (goat). In addition, exercise caused a large increase in concentration of individual NEFA in the dogs, with the largest increases seen in 18:1 (150% above resting values) and 16:0 (60% increase), but it had no effect in goats. Therefore, the aerobic species has a much higher ability for mobilizing and transporting NEFA in plasma than its low-aerobic counterpart. Two NEFA accounted for more than half total plasma NEFA in both species, 18:1 (about 35% total NEFA) and 16:0 (20%). Calculation of variability in percent composition reveals that oleate and palmitate also closely reflect changes in total NEFA and are therefore the most appropriate tracers for in vivo kinetic studies in exercising mammals.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular pharmacology of anthracycline resistance and its circumvention.

Adriamycin-resistant cells express a multiple drug resistance phenotype characterized by cross-resistance to compounds of related and unrelated structure and action. The pharmacological determinants of this resistance, such as decreased drug uptake and/or decreased drug retention, are associated with biochemical alterations in the cells. To overcome multiple drug resistance, a calcium-channel blocking agent, verapamil, was used, which acted by increasing the amount of drug retained in resistant cells and consequently enhanced the cytotoxic effectiveness. The basis for this enhanced retention and cytotoxicity is not known. Whether these compounds sensitize the cells to the action of, or potentiate the effect of, anticancer drugs remains to be determined. The preliminary data tend to support the second possibility.

Phospholipid composition and metabolism of crustacean gills as related to changes in environmental salinities: relationship between Na+-K+-ATPase activity and phospholipids.

The phospholipid composition and metabolism are studied in crustacean gills. It is reported that branchiae are rich in PC, PE and DPG and abundant in long chain polyunsaturated fatty acids (20:4 omega 6 and 20:5 omega 3 acids). The pathways of phospholipids synthesis appear similar to those described for vertebrates. It is demonstrated that there exist significant differences in the level of phosphatides between the anterior and posterior gills of Eriocheir sinensis. No matter what the salinity, the three more posterior located gills of Chinese crabs are shown to contain much more unsaturated phospholipids (PE and DPG). This is particularly true when animals are acclimated to dilute media. Moreover, lipids of posterior gills appear more fluid than the anterior ones as reported by the values of the degree of fluorescence polarization and the index of unsaturation of fatty acids. It is suggested that these lipid changes may indicate the existence of a functional difference between the various branchiae of euryhaline Eriocheir sinensis with respect to their ability to transport salt. It is shown that the renewal of DPG and PS is increased in posterior gills isolated from freshwater Chinese crabs. It is postulated that the enhanced formation of DPG in posterior gills is an indicator of an increased synthesis of mitochondria having as principal function to produce the necessary energy for the Na+ uptake. An attempt is made to correlate the PS metabolism and the Na+-K+-ATPase activity which is particularly located in the mitochondrial fractions of the three pairs of posterior gills.

Relationship between membrane sterol composition and responsiveness to 12-O-tetradecanoylphorbol 13-acetate in HL-60 human promyelocytic leukaemia cell lines.

We have examined the sterol composition and metabolism of promyelocytic leukaemia cell lines (HL-60) after treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). A variant cell line (Blast II cells) which is resistant to TPA was used as control. Analysis of the sterols of TPA-sensitive cells radiolabelled with [3H]leucine, [14C]acetate or [14C]pyruvate showed a high incorporation into cholesterol and a low incorporation in lanosterol + dihydrolanosterol. The inverse relationship was observed in TPA-resistant cells. Experiments with other cellular variants representing TPA-sensitive and TPA-resistant classes gave similar results. Analysis of the cellular sterol composition by gas chromatography confirmed that TPA-resistant cells are particularly rich in lanosterol/dihydrolanosterol. TPA treatment enhanced the incorporation of [14C]pyruvate into the sterol fraction of both cell types. This was accompanied by an alteration of incorporation into several lipids, particularly phospholipids. Pulse-chase studies with [14C]acetate revealed that TPA induced the release of radioactive lipids into the medium from HL-60 and Blast II cells. However this treatment released phospholipids from the TPA-sensitive cells and sterols and fatty acids from the TPA-resistant cells. We conclude that the sterol composition can regulate specific biochemical processes in the membrane and can be considered as a factor that plays a role in the responsiveness of HL-60 cells to TPA.

[Influence of the acclimation temperature on the phospholipid metabolism of the fresh water ell (Anguilla anguilla) (author's transl)]. / Influence de la température d'acclimatation sur le métabolisme des phospholipides de l'anguille (Anguilla anguilla) en eau douce

Lowering of the acclimation temperature of the eel induced a reduction of 32P incorporation into the lipid phosphorus of various tissues. This effect was partly the result of a slight decrease of the inorganic phosphate exchange and metabolism of tissues. 2. The variation of the 32P incorporation into the various phosphatides was different according to the tissue ; gill and kidney phospholipids being the most effected. At high temperature (22 degrees C), the turnover of sphingomyelin and phosphatidyl-choline was four or seven fold higher in all tissues than at low temperature. At low temperature (12 degrees C), the incorporation of 32P into gill and kidney phosphatidylinositol and gill diphosphatidyl-glycerol was the same as at high temperature. The kidney diphosphatidyl-glycerol turnover was affected to the same extent as the other phosphatides by high acclimation temperature. The gill phosphatidyl-ethanolamines had a relatively higher turnover at low temperature. 3. The acclimation of the eel at low temperature provoked an increase of the turnover of phosphatidyl-ethanolamine and diphosphatyl-glycerol into the gill, parallel with an increase of some enzymatic activities. 4. The variation of the acclimation temperature may impose restrictions on the turnover of some phosphatides. There is a limited action of the temperature on the turnover of metabolically active phosphatides of the osmoregulatory organs, such as gill phosphatidyl-inositol and diphosphatidyl-glycerol or kidney phosphatidyl-inositol. 5. The significance of these findings is discussed in relation to the role of phospholipids in the cell and to the physiological functions of the various organs of the eel.