RESUMEN
Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol to yield phosphatidic acid. To date, very little is known about the regulation of DGK activity. We have previously identified the DGKtheta isotype, which is predominantly expressed in brain (Houssa, B., Schaap, D., van der Wal, J., Goto, K., Kondo, H., Yamakawa, A., Shibata, M., Takenawa, T., and Van Blitterswijk, W. J. (1997) J. Biol. Chem. 272, 10422-10428). We now report that DGKtheta binds specifically to activated RhoA in transfected COS cells as well as in nontransfected neuronal N1E-115 cells. Binding is abolished by a point mutation (Y34N) in the effector loop of RhoA. DGKtheta does not bind to inactive RhoA, nor to the other Rho-family GTPases, Rac or Cdc42. Like active RhoA, DGKtheta localizes to the plasma membrane. Strikingly, the binding of activated RhoA to DGKtheta completely inhibits DGK catalytic activity. Our results suggest that DGKtheta is a downstream effector of RhoA and that its activity is negatively regulated by RhoA. Through accumulation of newly produced diacylglycerol, RhoA-mediated inhibition of DGKtheta may lead to enhanced PKC activity in response to external stimuli.
Asunto(s)
Diacilglicerol Quinasa/metabolismo , Proteínas de Unión al GTP/metabolismo , Animales , Catálisis , Línea Celular , Unión Proteica , Fracciones Subcelulares/enzimologíaRESUMEN
The role of diacylglycerol (DG) formation from phosphatidylcholine in mitogenic signal transduction is poorly understood. We have generated this lipid at the plasma membrane by treating Rat-1 fibroblasts with bacterial phosphatidylcholine-specific phospholipase C (PC-PLC). This treatment leads to activation of mitogen-activated protein kinase (MAPK). However, unlike platelet-derived growth factor (PDGF) or epidermal growth factor (EGF), PC-PLC fails to activate Ras and to induce DNA synthesis, and activates MAPK only transiently (<45 min). Down-regulation of protein kinase C (PKC) -alpha, -delta and -epsilon isotypes has little or no effect on MAPK activation by either PC-PLC or growth factors. However, Ro 31-8220, a highly selective inhibitor of all PKC isotypes, including atypical PKC-zeta but not Raf-1, blocks MAPK activation by PDGF and PC-PLC, but not that by EGF, suggesting that atypical PKC mediates the PDGF and PC-PLC signal. In line with this, PKC-zeta is activated by PC-PLC and PDGF, but not by EGF, as shown by a kinase assay in vitro, using biotinylated epsilon-peptide as a substrate. Furthermore, dominant-negative PKC-zeta inhibits, while (wild-type) PKC-zeta overexpression enhances MAPK activation by PDGF and PC-PLC. The results suggest that DG generated by PC-PLC can activate the MAPK pathway independent of Ras and phorbol-ester-sensitive PKC but, instead, via PKC-zeta.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Diglicéridos/metabolismo , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Procesamiento Proteico-Postraduccional , Fosfolipasas de Tipo C/metabolismo , Animales , Células COS , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Fibroblastos/metabolismo , Sustancias de Crecimiento/farmacología , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Indoles/farmacología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Proteína Quinasa 1 Activada por Mitógenos , Mitosis/efectos de los fármacos , Ésteres del Forbol/farmacología , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/genética , Proteínas Tirosina Quinasas/metabolismo , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Transfección , Proteínas ras/fisiologíaRESUMEN
The role of phosphatidylcholine (PC) hydrolysis in activation of the mitogen-activated protein kinase (MAPK) pathway by platelet-derived growth factor (PDGF) was studied in Rat-1 fibroblasts. PDGF induced the transient formation of phosphatidic acid, choline, diacylglycerol (DG), and phosphocholine, the respective products of phospholipase D (PLD) and phospholipase C (PC-PLC) activity, with peak levels at 5-10 min. PLD-catalyzed transphosphatidylation (with n-butyl alcohol) diminished DG formation at 5 min but not at later stages of PDGF stimulation. Phorbol ester-induced down-regulation of protein kinase C (PKC) completely blocked PLD activation but not the formation of DG and phosphocholine at 10 min of PDGF stimulation. Collectively, these data indicate that PDGF activates both PLD and PC-PLC. In contrast, epidermal growth factor did not activate PC-PLC in these cells, and it activated PLD only weakly. DG formation by itself, through Bacillus cereus PC-PLC treatment of cells, was sufficient to mimic PDGF in activation of MAPK independent of phorbol ester-sensitive PKC. Since PKC down-regulation blocked PDGF-induced PLD but not MAPK activation, we conclude that PLD is not involved in MAPK signaling. In contrast, MAPK activation by exogenous (bacterial) PLD was not affected by PKC down-regulation, indicating that signals evoked by exogenous PLD differ from endogenous PLD. D609 (2-10 microg/ml), an inhibitor of PC-PLC, blocked PDGF- but not epidermal growth factor-induced MAPK activation. However, D609 should be used with caution since it also affects PLD activity. The results suggest that PC-PLC rather than PLD plays a critical role in the PDGF-activated MAPK pathway.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Fosfatidilcolinas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Transducción de Señal , Fosfolipasas de Tipo C/metabolismo , Animales , Hidrocarburos Aromáticos con Puentes/farmacología , Células Cultivadas , Diglicéridos/metabolismo , Regulación hacia Abajo , Activación Enzimática , Factor de Crecimiento Epidérmico/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Hidrólisis , Norbornanos , Inhibidores de Fosfodiesterasa/farmacología , Fosfolipasa D/metabolismo , Ratas , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Tiocarbamatos , Tionas/farmacología , Fosfolipasas de Tipo C/antagonistas & inhibidoresRESUMEN
The regulation of diacylglycerol (DG) kinase activity was studied in fibroblasts and Jurkat T cells. We questioned whether enzyme activity only depends on substrate availability or whether it requires receptor stimulation. To this end, we raised DG levels up to 15-fold by treatment of cells with bacterial phosphatidylinositol-specific phospholipase C (PLC). In detergent cell lysates, DG kinase was readily capable of converting this surplus of DG to phosphatidic acid (PA), but in intact cells the enzyme remained inactive. Stimulation of fibroblasts with bradykinin or endothelin and Jurkat cells with anti-CD3 resulted in DG kinase-mediated formation of PA, but its level was unaffected by PLC pretreatment. Likewise, in streptolysin O-permeabilized fibroblasts, where bradykinin stimulation in the presence of [gamma-32P]ATP and guanosine 5'-O-(thiotriphosphate) generates [32P]PA exclusively via DG kinase, PLC pretreatment did not affect the amount of [32P]PA formed. We conclude that DG kinase acts on DG generated by receptor stimulation, but not on DG generated by exogenous PLC. We propose a model in which DG kinase physically associates with endogenous PLC. Within this complex, receptor-induced DG would then be transmitted ("channeled") from endogenous PLC to the active site of DG kinase, whereas excess DG generated randomly in the plasma membrane by bacterial PLC is inaccessible to this catalytic site.
Asunto(s)
Diglicéridos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Bradiquinina/farmacología , Células Cultivadas , Diacilglicerol Quinasa , Humanos , Técnicas In Vitro , Lípidos de la Membrana/metabolismo , Ácidos Fosfatidicos/metabolismo , Fosfolipasa D/fisiología , Proteína Quinasa C/fisiología , Ratas , Transducción de Señal , Fosfolipasas de Tipo C/farmacologíaRESUMEN
Synthetic peptides corresponding to the pseudosubstrate domains of protein kinase C (PKC) have been used as specific inhibitors of PKC in in vitro assays and permeabilized cell systems. However, their use in vivo was hampered by the impermeability of the plasma membrane for such peptides. Here, we show that N-myristoylation of the PKC pseudosubstrate nonapeptide Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln permits its use as an inhibitor of PKC in intact cells. The myristoylated peptide, myr-psi PKC, inhibits phosphorylation of the myristoylated alanine-rich C kinase substrate protein, as induced by 12-O-tetradecanoyl-phorbol-13-acetate, and the activation of phospholipase D by bradykinin, which strictly depends on PKC. Half-maximal inhibition is obtained at concentrations of 8 and 20 microM, respectively. An N-myristoylated peptide derived from an inhibitor protein of the cAMP-dependent protein kinases, Myr-Gly-Arg-Arg-Asn-Ala-Ile-His-Asp-Ile, was ineffective. These results show that myr-psi PKC is a selective and cell-permeable inhibitor of PKC.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Ácidos Mirísticos , Oligopéptidos/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Secuencia de Aminoácidos , Bradiquinina/farmacología , Activación Enzimática/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Ácido Mirístico , Ácidos Mirísticos/química , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Oligopéptidos/química , Fosfolipasa D/antagonistas & inhibidores , Fosfolipasa D/metabolismo , Fosforilación , Proteínas/metabolismo , Especificidad por Sustrato , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
Lysophosphatidic acid (LPA) is a simple phospholipid that possesses hormone- and growth-factor-like properties. LPA initiates its action by inducing GTP-dependent phosphoinositide hydrolysis and inhibiting adenylate cyclase [van Corven, Groenink, Jalink, Eichholtz & Moolenaar (1989) Cell 59, 45-54]. Here we show that LPA stimulates rapid breakdown of phosphatidylcholine (PC) in Rat-1 fibroblasts. LPA-induced PC breakdown occurs through activation of phospholipase D (PLD), as measured by the formation of free choline and phosphatidic acid and by transphosphatidylation in the presence of butan-1-ol. LPA also stimulates generation of diacylglycerol, but there is no detectable formation of phosphocholine, suggesting that a PC-specific phospholipase C (PLC) is not involved. The response to LPA was compared with that to endothelin, a potent inducer of phospholipid hydrolysis but a poor mitogen for Rat-1 cells. Our results indicate that: (1) LPA is less efficient than endothelin in inducing phosphoinositide and PC breakdown; (2) LPA-induced PLD activation is short-lived, levelling off after 2 min, whereas the endothelin-stimulated increase in PLD activity persists for at least 1 h; (3) the effect of LPA on PLD, like that of endothelin, is blocked by long-term pretreatment of the cells with phorbol ester, suggesting that PLD activation occurs through a protein kinase C-dependent mechanism. Furthermore, our results support the notion that there is no simple causal relationship between the degree of agonist-induced phospholipid hydrolysis and the magnitude of the mitogenic response.
Asunto(s)
Endotelinas/farmacología , Lisofosfolípidos/farmacología , Fosfatidilcolinas/metabolismo , Fosfolipasa D/metabolismo , Toxina de Adenilato Ciclasa , Animales , Células Cultivadas , ADN/biosíntesis , Regulación hacia Abajo , Activación Enzimática , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fosfatos de Inositol/metabolismo , Proteína Quinasa C/metabolismo , Ratas , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Lysophosphatidic acid (3-sn-lysophosphatidic acid; LPA) can activate cells similar to hormones and growth factors. We have considered the question whether metabolic conversion of LPA taken up by the cell could be of any importance in this activation. Addition of [14C-glycerol]LPA to quiescent Rat-1 fibroblasts resulted in rapid formation of [14C]monoacylglycerol (MG), closely followed by accumulation of [14C]triacylglycerol. Only very little [14C]diacylglycerol and [14C]phosphatidic acid was formed (approx. 100-fold less than MG). MG, when added exogenously to cells, lacks detectable biological activity. The results suggest that LPA itself, rather than one of its metabolites is the biologically active principle.
Asunto(s)
Fibroblastos/metabolismo , Lisofosfolípidos/metabolismo , Animales , Células Cultivadas , Humanos , Cinética , RatasRESUMEN
Bradykinin (BK) and phorbol 12-myristate 13-acetate (PMA) both stimulate the hydrolysis of phosphatidylcholine (PC) in human fibroblasts, resulting in the formation of phosphatidic acid (PA) and diacylglycerol (DG) (Van Blitterswijk, W.J., Hilkmann, H., de Widt, J., and Van der Bend, R.L. (1990) J. Biol. Chem. 266, 10337-10343). Stimulation with BK resulted in the rapid and synchronous formation of [3H]choline and [3H]myristoyl-PA from the correspondingly prelabeled PC, indicative of phospholipase D (PLD) activity. In the presence of ethanol or n-butanol, transphosphatidylation by PLD resulted in the formation of [3H]phosphatidylethanol or - butanol, respectively, at the cost of PA and DG formation. This suggests that PC-derived DG is generated via a PLD/PA phosphohydrolase pathway. A more pronounced but delayed formation of these products was observed by PMA stimulation. The Ca2+ ionophore ionomycin also activated PLD and accelerated (synergized) the response to PMA. Both [3H] choline and [3H]phosphocholine were released into the extracellular medium in a time- and stimulus-dependent fashion, without apparent changes in the high intracellular levels of [3H]phosphocholine. The protein kinase C (PKC) inhibitors staurosporin and 1-O-hexadecyl-2-O-methylglycerol inhibited BK- and PMA-induced activation of PLD. Down-regulation of PKC by long-term pretreatment of cells with phorbol ester caused a dramatic drop in background [3H]choline levels, while subsequent stimulation with BK, ionomycin, or PMA failed to increase these levels and failed to induce transphosphatidylation. From these results we conclude that PLD activation is entirely mediated by (downstream of) PKC. Unexpectedly, however, BK stimulation of these PKC-depleted cells caused a marked generation of DG from PC within 15 s, which was not seen in BK-stimulated control cells, suggesting PC breakdown by a phospholipase C (PLCc). We conclude that cells stimulated with BK generate DG via both the PLCc and the PLD/PA hydrolase pathway, whereas PMA stimulates mainly the latter pathway. BK stimulation of normal cells leads to activation of PKC and, by consequence, to attenuation of the level of PLCc-generated DG and to stimulation of the PLD pathway, whereas the reverse occurs in PKC-down-regulated cells.
Asunto(s)
Bradiquinina/farmacología , Fosfatidilcolinas/metabolismo , Fosfolipasa D/metabolismo , Proteína Quinasa C/metabolismo , Fosfolipasas de Tipo C/metabolismo , Colina/metabolismo , Diglicéridos/biosíntesis , Regulación hacia Abajo , Etanol/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Éteres de Glicerilo/farmacología , Humanos , Hidrólisis , Ionomicina/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Stimulation of human fibroblasts with bradykinin (BK) results in the generation of diacylglycerol (DG) and phosphatidic acid (PA). Prelabeling of the cells with [3H]arachidonic acid and [14C]palmitic acid allowed us to quantitate these lipid second messengers and to determine their origin, i.e. DGi and PAi from 3H-enriched inositol phospholipids, and DGc and PAc from 14C-enriched phosphatidylcholine, respectively. BK elicited a biphasic DG response: a first peak at 10-15 s, containing DGi, followed by a second peak at 10-30 min, which is mainly DGc. The latter did not result from de novo lipid biosynthesis. BK also generated free [3H]arachidonate and, to a lesser extent, mono[3H]arachidonoylglycerol. BK stimulation rapidly increased PAi, much more so than PAc, suggesting that DGi, rather than DGc, is the preferred substrate for the enzyme DG kinase. Short pretreatment of the cells with phorbol 12-myristate 13-acetate (PMA) abolished the BK-induced breakdown of phosphoinositides, but did not affect the second-phase DGc level. PMA alone also elicited DGc formation, but more slowly, suggesting a different mechanism. Down-regulation of protein kinase C (PKC) by long term treatment with phorbol ester, prior to BK stimulation, resulted in (i) enhanced DGi and decreased PAi formation, suggesting that DG kinase activity is positively controlled by PKC; (ii) the unexpected manifestation of rapidly formed DGc; (iii) no change in the DGc levels obtained after 30-min BK stimulation, but complete suppression of PMA-induced DGc formation. In contrast, two inhibitors of PKC, staurosporin and 1-O-hexadecyl-2-O-methylglycerol, inhibited both BK- and PMA-induced DGc formation at 30 min, leaving the rapid response towards BK unaffected. The results suggest that the BK-induced rapid and later-phase DG formation and the PMA-induced DG formation are differentially controlled by PKC via mechanisms that differ in the susceptibility to down-regulation or inhibition of PKC.
Asunto(s)
Bradiquinina/farmacología , Diglicéridos/biosíntesis , Fosfatidilcolinas/metabolismo , Fosfatidilinositoles/metabolismo , Proteína Quinasa C/metabolismo , Alcaloides/farmacología , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Regulación hacia Abajo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Éteres de Glicerilo/farmacología , Humanos , Proteína Quinasa C/antagonistas & inhibidores , Estaurosporina , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Diacylglycerol (DG) kinase attenuates the level of the second messenger DG in signal transduction, and therefore possibly modulates protein kinase C (PKC). DG kinase was purified to homogeneity from human white blood cells, showing an Mr of 86 kDa as determined by SDS-PAGE and gel filtration. Two amino acid sequences of tryptic peptides from DG kinase were determined and degenerate oligonucleotides were prepared and used in the polymerase chain reaction. An amplified DNA fragment was subsequently used to clone the full-length human DG kinase cDNA. This sequence is the human homolog of a porcine DG kinase cDNA sequence reported recently. The sequence contains a double EF-hand structure typical for Ca2+ binding proteins. DG kinase further contains a double cysteine repeat that is present in all PKC isoforms, where it constitutes the phorbol ester (and most likely diacylglycerol) binding site. Therefore we speculate that the double cysteine repeat in DG kinase is involved in DG binding. DG kinase is transcribed as a single mRNA of 3.2 kb, that is highly expressed in T-lymphocytes. The human DG kinase cDNA when transfected in mammalian cells (COS-7) results in a 6-7-fold increase of DG kinase activity.
Asunto(s)
Fosfotransferasas/aislamiento & purificación , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Proteínas de Unión al Calcio/genética , Clonación Molecular , ADN/genética , Diacilglicerol Quinasa , Expresión Génica , Humanos , Leucocitos/enzimología , Datos de Secuencia Molecular , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , ARN Mensajero/genética , Secuencias Reguladoras de Ácidos Nucleicos , Especificidad por Sustrato , PorcinosRESUMEN
Mice of the GR/A strain were fed four different isocaloric semipurified diets, enriched in either (1) saturated fatty acids (palm oil), or (2) polyunsaturated fatty acids (corn oil), or (3) palm oil plus cholesterol, or (4) a fat-poor diet containing only a minimal amount of essential fatty acids. We have studied the effects of these dietary lipids on the density profile and composition of the plasma lipoproteins and on the lipid composition and fluidity of (purified) lymphoid cell membranes in healthy mice and in mice bearing a transplanted lymphoid leukemia (GRSL). Tumor development in these mice occurred in the spleen and in ascites. While the fatty acid composition of the VLDL-triacylglycerols still strongly resembled the dietary lipids, the effects of the diets decreased in the order VLDL-triacylglycerols greater than HDL-phospholipids greater than plasma membrane phospholipids. Diet-induced differences in the latter fraction were virtually confined to the content of oleic acid and linoleic acid, and they were too small to affect the membrane fluidity, as measured by fluorescence polarization using the probe 1,6-diphenyl-1,3,5-hexatriene. Healthy mice were almost irresponsive to dietary cholesterol, but in the tumor bearers, where lipoprotein metabolism has been shown to be disturbed, the cholesterol diet caused a substantial increase in the low- and very-low density regions of both blood and ascites plasma lipoproteins. The cholesterol-rich diet also increased the cholesterol/phospholipid molar ratio and lipid structural order (decreased fluidity) in GRSL ascites cell membranes, but not in the splenic GRSL cell membranes. We conclude that the composition of plasma lipoproteins and cell membrane lipids in GR/A mice is subject to exquisite homeostatic control. However, in these low-responders to dietary lipids the development of an ascites tumor may lead to increased responsiveness to dietary cholesterol. The elevated level of membrane cholesterol thus obtained in GRSL ascites cells did not affect the expression of various cell surface antigens or tumor cell growth.
Asunto(s)
Grasas de la Dieta/farmacología , Leucemia Experimental/metabolismo , Lipoproteínas/sangre , Fluidez de la Membrana/efectos de los fármacos , Animales , Líquido Ascítico/metabolismo , Membrana Celular/metabolismo , Colesterol/metabolismo , Difenilhexatrieno , Ácidos Grasos/metabolismo , Lipoproteínas HDL/metabolismo , Lipoproteínas VLDL/metabolismo , Masculino , Lípidos de la Membrana/metabolismo , Ratones , Trasplante de Neoplasias , Espectrometría de Fluorescencia , Bazo/metabolismoRESUMEN
In our search for the mechanisms by which the drug 1-O-alkyl-2-O-methylglycero-3-phosphocholine (AMG-PC) inhibits tumor growth and metastasis, we have detected a metabolite, 1-O-alkyl-2-O-methylglycerol (AMG), in membranes of MO4 mouse fibrosarcoma cells grown in the presence of the drug. Synthetic AMG inhibited the activation of highly purified human protein kinase C by diacylglycerol in the presence of phosphatidylserine. Furthermore, AMG also inhibited the receptor-specific binding of 3H-phorbol-12,13-dibutyrate to human HL-60 promyeloid leukemia cells in a dose-dependent fashion. AMG-PC was not effective or much less so in these assays. We suggest that interaction of the metabolite AMG with protein kinase C may inhibit stimulus-response coupling in tumor cells and may thus potentially contribute to the mechanism by which AMG-PC exerts its anticancer activities.
Asunto(s)
Antineoplásicos/farmacología , Éteres de Glicerilo/farmacología , Éteres Fosfolípidos/farmacología , Proteína Quinasa C/metabolismo , Células Tumorales Cultivadas/efectos de los fármacos , Animales , Línea Celular , Fibrosarcoma , Ratones , Ratones Endogámicos C3H , Invasividad Neoplásica , Ésteres del Forbol/metabolismo , Células Tumorales Cultivadas/enzimologíaRESUMEN
The membrane fluidity of murine lymphoid GRSL tumor cells has been shown to depend on their site of growth in the host. Tumor cells located in ascites, in contrast to those in the enlarged spleen, show a very high plasma membrane fluidity, mainly due to a decreased level of cellular (membrane) cholesterol. Yet, the rate of cholesterol biosynthesis in the tumor cells as estimated by the activity of HMG-CoA reductase and the incorporation of [14C]acetate into cholesterol was extremely high when compared to various lymphoid cells in normal control mice. Also the rate of biosynthesis and the cholesterol content in liver and spleen of tumor-bearing mice were substantially higher than in the organs of control mice. Blood plasma cholesterol, however, was decreased in tumor-bearing mice as a result of altered lipoprotein patterns. Outgrowth of the tumor was accompanied by a strong reduction in plasma high-density lipoproteins. Low-density lipoproteins became transiently increased, but eventually all lipoproteins, and consequently the plasma cholesterol content decreased to very low levels, especially so in the ascites plasma. The low transfer of [14C]cholesteryl ester-labeled lipoproteins between blood and ascites plasma after either intravenous or intraperitoneal injection suggested a hampered flow between the two compartments. Also apparent differences in cholesteryl ester fatty acid composition between lipoproteins of the blood and ascites plasma indicated the lack of a rapid equilibration between the two compartments. The results suggest that the limited availability of lipoproteins as an additional source of cholesterol to the rapidly growing ascites cells promotes their high membrane fluidity.
Asunto(s)
Colesterol/metabolismo , Leucemia Experimental/metabolismo , Lipoproteínas/sangre , Fluidez de la Membrana , Acetatos/metabolismo , Animales , Ascitis/metabolismo , Fenómenos Químicos , Química , Colesterol/biosíntesis , Colesterol/sangre , Ácidos Grasos/análisis , Homeostasis , Hidroximetilglutaril-CoA Reductasas/metabolismo , Leucemia Experimental/sangre , Hígado/metabolismo , Lípidos de la Membrana/fisiología , Ratones , Proteínas de Neoplasias/metabolismo , Trasplante de Neoplasias , Bazo/metabolismo , Timo/metabolismoRESUMEN
Outgrowth of the transplanted GRSL lymphoma in GR mice yielded several-fold increased blood plasma levels of low- and very-low-density lipoproteins, while high-density lipoproteins were strongly reduced. Changes in cholesteryl ester fatty acid profiles indicated an accumulation of HDL-like particles rather than LDL in the low-density fractions. By intravenous injection of [14C]cholesteryl ester-labeled HDL into tumor-bearing mice, conversion of HDL into lipoproteins of low density was demonstrated.