RESUMO
Pitx2, a paired-like homeodomain transcription factor, is expressed in post-mitotic neurons within highly restricted domains of the embryonic mouse brain. Previous reports identified critical roles for PITX2 in histogenesis of the hypothalamus and midbrain, but the cellular identities of PITX2-positive neurons in these regions were not fully explored. This study characterizes Pitx2 expression with respect to midbrain transcription factor and neurotransmitter phenotypes in mid-to-late mouse gestation. In the dorsal midbrain, we identified Pitx2-positive neurons in the stratum griseum intermedium (SGI) as GABAergic and observed a requirement for PITX2 in GABAergic differentiation. We also identified two Pitx2-positive neuronal populations in the ventral midbrain, the red nucleus, and a ventromedial population, both of which contain glutamatergic precursors. Our data suggest that PITX2 is present in regionally restricted subpopulations of midbrain neurons and may have unique functions that promote GABAergic and glutamatergic differentiation.
Assuntos
Ácido Glutâmico/metabolismo , Proteínas de Homeodomínio/metabolismo , Mesencéfalo/citologia , Mesencéfalo/embriologia , Neurônios/fisiologia , Fatores de Transcrição/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Diferenciação Celular/fisiologia , Proteínas de Homeodomínio/genética , Mesencéfalo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Transdução de Sinais , Fatores de Transcrição/genética , Transcrição Gênica , Proteína Homeobox PITX2RESUMO
A single intravenous injection of 0.1 mg of heat-killed Bacillus Calmette Guérin (BCG) in 0.1 ml of Bayol F produced an accumulation of activated alveolar macrophages (BCG induced). Cells were collected 3.5-4.0 wk after injection. Phospholipases A and three lysosomal marker enzymes (acid phosphatase, beta-glucuronidase, and lysozyme) were measured in homogenates, and the distribution of the phospholipases A and lysosomal, mitochondrial, and microsomal marker enzymes were examined after sucrose gradient centrifugation of a postnuclear (1,000 g) supernatant. Homogenates of normal and BCG-induced macrophages contained phospholipases A(1) and A(2) which had optimal activity at pH 4.0 in the presence of 2.0 mM ethylenediaminetetraacetate (EDTA). These activities were inhibited 50-70% by 2.0 mM CaCl(2). Homogenates of BCG-induced macrophages had specific activities of beta-glucuronidase, acid phosphatase, and lysozyme, which were increased 1.5- to 3.0-fold over the controls, whether expressed as activity per mg protein or activity per 10(7) cells. The specific activities of the phospholipases A, on the other hand, were consistently lower than those of the control. Distribution of the phospholipases A and the lysosomal marker enzymes after sucrose gradient centrifugation suggested that the phospholipases A active at pH 4.0 in the presence of EDTA are of lysosomal origin since: (a) BCG treatment caused a selective increase in the density of particles which contained both the phospholipases A and three lysosomal marker enzymes; and (b) since the density of mitochondria and microsomes were not affected by BCG treatment. The increase in the density of lysosomes seen here may be related to previously described morphologic changes of BCG-induced alveolar macrophages.
Assuntos
Lisossomos/enzimologia , Macrófagos/enzimologia , Fosfolipases/análise , Fosfatase Ácida/análise , Animais , Fracionamento Celular , Centrifugação com Gradiente de Concentração , Ácido Edético , Complexo IV da Cadeia de Transporte de Elétrons/análise , Glucuronidase/análise , Concentração de Íons de Hidrogênio , Injeções Intravenosas , Microssomos/análise , Mitocôndrias/análise , Muramidase/análise , Mycobacterium bovis , Alvéolos Pulmonares , CoelhosRESUMO
Functional tests such as Motor Function Measure-32 (MFM-32), supine to stand, ascend/descend stairs permit the assessment of task-specific motor function in neuromuscular disease (NMD). The 6-min walk test (6MWT), though functional, is primarily used to assess endurance and disease progression in children with neuromuscular disorders. Barriers to 6MWT administration, in this population, can include reduced attention span due to age and inability to tolerate test length due to weakness. We propose task-specific functional deficits are related to endurance. Additionally, the 2-min walk test (2MWT) could effectively replace the 6MWT in this population. Seventy-seven participants, ages 5-18, with a variety of neuromuscular disorders performed the 6MWT, timed functional tests (TFT), and the MFM-32. Correlation and paired t-test analyses were used to compare the distance walked in the first 2 min (2MWD) to the distance walked in the entire 6 min (6MWD) and to the functional outcome measures above. The 2MWD strongly correlated with 6MWD and the other outcome measures. Paired t-test analysis also showed that the 2MWD did not differ from the distance walked in the last 2 min of the 6MWT. Although equivalence testing could not reject the claim that this difference exceeded the upper practical limit of 9.5 m, it only showed a modest overestimation of the 4-6MWD compared with the 2MWD. Together, our results support the ability of the 2MWD to predict the 6MWD, specifically in the pediatric neuromuscular disease population.
Assuntos
Doenças Neuromusculares/diagnóstico , Teste de Caminhada/métodos , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Doenças Neuromusculares/complicações , Avaliação de Resultados em Cuidados de Saúde , Resistência Física/fisiologia , Fatores de TempoRESUMO
Two classes of aminoacyl fucosides termed FL3 and FL4 were studied as possible markers of tumorigenic and metastatic potential in herpes simplex virus type 2 transformed rat cells. In the present study, clonal cell lines of transformed highly tumorigenic and metastatic (t-REF-G-1.1), weakly tumorigenic and nonmetastatic (t-REF-G-2.1), nontumorigenic (t-REF-G-2.0), and secondary nontransformed rat embryo fibroblast cells were labeled with [3H]fucose, and cell extracts were analyzed for ratio of radioactivity incorporated into FL3 and FL4. Results indicated that, in extracts from t-REF-G-2.0 and nontransformed rat embryo fibroblast cells, the ratios of FL4/FL3 were 5.78 and 5.71, respectively. In contrast, t-REF-G-2.1 cells exhibited a FL4/FL3 ratio of 1.45, while t-REF-G-1.1 cells exhibited a FL4/FL3 ratio of 0.74. In subclonal cell lines isolated from TPA-treated and mock-treated t-REF-G-2.1 cells, the FL4/FL3 ratios correlated with the tumorigenic and metastatic potential of these subclones in newborn syngeneic White Buffalo rats. These data suggested that alterations in fucose-labeled components can be used to predict the tumorigenic and metastatic potential of herpes simplex virus type 2-transformed rat cells.
Assuntos
Aminoglicosídeos/análise , Transformação Celular Viral , Fucose/metabolismo , Neoplasias/metabolismo , Lesões Pré-Cancerosas/metabolismo , Animais , Linhagem Celular , Cromatografia em Camada Fina , Suscetibilidade a Doenças , Fucose/análise , Ratos , Simplexvirus , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Mitonchondria isolated from the Morris hepatoma 7777 demonstrated a markedly different phospholipid composition from those of control mitochondria, both with respect to the amounts of the various types present and the fatty acid composition. The level of polyunsaturated fatty acids in the mitochondrial phospholipids was lowered, wheras there was an increase in the level of monounsaturated fatty acids. Moreover, the usual distribution of saturated fatty acids at position 1 and polyunsaturated fatty acids at position 2 does not exist in hepatoma phospholipids; a high percentage of monounsaturated fatty acids was found at both positions. The cardiolipin content was lower in hepatoma mitochondria (3.7%) than in livers of animals with hepatomas (5.2%). There was, however, some compensation in the amount of acidic phospholipids in these mitochondria due to an increase in phosphatidylserine (4.9% versus 1.3%). The force-area curves of the hepatoma phospholipids spread on a monomolecular film demonstrated a smaller area per molecule than those from liver mitochondria. The zeta potential of liposomes of the hepatoma phospholipids (-45) was less than those of control mitochondria (-81), as determined by microelectrophoresis. The calcium-stimulated phospholipase A activity of the hepatoma mitochondria appeared to be more readily expressed than the same activity in liver organelles. The maximal activity was lower, however, than that noted in liver mitochondria. Furthermore, by following the incorporation of [3H]ethanolamine into mitochondria phospholipids, it was established that the conversion of glycerophosphorylethanolamine to glycerophosphorylcholine was increased in the hepatoma. These observations suggest dramatic changes in phospholipid metabolism in the hepatoma, at the level of both the endoplasmic reticulum and the mitochondrion. Accompanying the changes in phospholipid compositon and metabolism were alterations in mitochondrial energy-linked processes. The hepatoma mitochondria demonstrated lower respiratory control ratios even when isolated in an isotonic solution containing 1mM ethylenediaminetetraacetate and bovine serum albumin (0.5 mg/ml). This was due to increased state 4 respiration.
Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Mitocôndrias Hepáticas/metabolismo , Fosfolipídeos/metabolismo , Animais , Carcinoma Hepatocelular/ultraestrutura , Ácidos Graxos Insaturados/metabolismo , Glicerofosfatos/biossíntese , Fígado/metabolismo , Neoplasias Hepáticas/ultraestrutura , Masculino , Mitocôndrias Hepáticas/enzimologia , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/ultraestrutura , Consumo de Oxigênio , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Fosfolipases/metabolismo , RatosRESUMO
Knowledge of the PLD superfamily is rapidly expanding and new insights into the mechanism and regulation of the superfamily are rapidly emerging. The recent structural analysis and use of mutant proteins suggest a mechanism that involves two active sites acting in concert. While a number of residues are required for activity, it appears most likely that a histidine is the residue that becomes covalently linked to phosphatidate in catalysis. Evidence for these proposals is covered in this article.
Assuntos
Fosfolipase D/genética , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Modelos Moleculares , Dados de Sequência Molecular , Fosfolipase D/química , Fosfolipase D/metabolismo , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Recent work within our laboratory has focused on the enzymes we hypothesize are involved in the biosynthesis of bis(monoacylglycerol)phosphate from phosphatidylglycerol. Here we describe a transacylase, active at acidic pH values, isolated from a macrophage-like cell line, RAW 264.7. This enzyme acylates the head group glycerol of sn-3:sn-1' lysophosphatidylglycerol to form sn-3:sn-1' bis(monoacylglycerol)phosphate. Here we demonstrate that this enzyme uses two lysophosphatidylglycerol molecules, one as an acyl donor and another as an acyl acceptor, and that the acyl contributions from all other lipids tested are comparatively minor. This enzyme prefers saturated acyl chains to monounsaturates, 16 and 18 carbon fatty acids over 14 carbon fatty acids, and saturated acyl chains at the sn-1 position to monounsaturated acyl chains on the sn-2 carbon of lysophosphatidylglycerol. We present data which show the transacylase activity depends on the presence of a lipid-water interface and the lipid polymorphic state.
Assuntos
Aciltransferases/metabolismo , Lisofosfolipídeos/biossíntese , Aciltransferases/isolamento & purificação , Animais , Lisofosfolipídeos/metabolismo , Macrófagos/enzimologia , Camundongos , Monoglicerídeos , Fosfatidilgliceróis/metabolismo , Células Tumorais CultivadasRESUMO
The plasma membrane of rat liver contains an enzyme which is stimulated by heparin and hydrolyzes liposomes composed of phosphoglycerides as well as mono-and diacylglycerol (Waite, M. and Sisson, P (1973) J.Biol, Chem. 248, 7985-7902). Further, in liposomes this enzyme catalyzes a transacylation in which the acyl group is removed from position 1 of an acyl glyceride donor, and combined with the hydroxy group of a variety of acyl acceptors. To investigate the possible role of this enzyme in lipoprotein metabolism, we have incorporated specific labeled glycerides into lipoproteins. The high density, low and very low lipoproteins were then separated by molecular sieving and characterized by their physical and chemical properties. Using the labeled substrates as model lipoproteins, we found the following: 1)The enzyme is capable of hydrolyzing monoacylglycerol, diacylglycerophosphoethanolamine and diacylglycerophosphocholine; monoacylglycerol however is the preferred substrate in all three lipoprotein fractions. 2)Relative to the activity found on liposomes, the transacylation activity is low. 3)The specific radioactivity of the substrates in fraction B (low density lipoprotein) did not change during the reaction, which indicates that the labeled lipid is not a pool separate from the endogenous lipid of the lipoprotein.
Assuntos
Aciltransferases/metabolismo , Lipoproteínas/sangue , Animais , Membrana Celular/enzimologia , Colesterol/análise , Glicerídeos , Cinética , Fígado/enzimologia , Fosfolipídeos/análise , RatosRESUMO
The hydrolysis of phospholipid mixtures by purified rat hepatic lipase, also known as hepatic triglyceride lipase, was studied in a Triton X-100/lipid mixed micellar system. Column chromatography of the mixed micelles showed elution of Triton X-100 and binary lipid mixtures of phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine as a single peak. This indicated that the mixed micelles were homogenous and contained all components in the designated molar ratios. The molar ratio of Triton X-100 to lipid was kept constant at 4 to 1. Labeling one lipid with 3H and the other lipid with 14C enabled us to determine the hydrolysis of both components of these binary lipid mixed micelles. We found that the hydrolysis of phosphatidylcholine was activated by the inclusion of small amounts of phosphatidic acid (2.5-fold), phosphatidylethanolamine (1.5-fold) or phosphatidylserine (1.4-fold). The maximal activation of phosphatidylcholine hydrolysis was observed when 5 mol% of phosphatidylethanolamine, 7.5 mol% phosphatidic acid or 5 mol% phosphatidylserine was added to Triton X-100 mixed micelles. The hydrolysis of phosphatidic acid was activated 30%, and that of phosphatidylserine was inhibited 30% when the molar proportion of phosphatidylcholine was less than 50 mol%. The hydrolysis of phosphatidylethanolamine was slightly activated when the mol% of phosphatidylcholine was below 5. The hydrolysis of phosphatidylserine was inhibited by phosphatidylethanolamine when the mol% of the latter was 50 or less whereas phosphatidylethanolamine hydrolysis was not affected by phosphatidylserine. Under the conditions used sphingomyelin and cholesterol did not have a significant effect on the hydrolysis of the phospholipids studied. In agreement with our previous study (Kucera et al. (1988) J. Biol. Chem. 263, 1920-1928) these studies show that the phospholipid polar head group is an important factor which influences the action of hepatic lipase and that the interfacial properties of the substrate play a role in the expression of the activity of this enzyme. The molar ratios of phosphatidic acid, phosphatidylethanolamine and phosphatidylserine which activated phosphatidylcholine hydrolysis correspond closely to the molar ratios of these lipids found in the surface lipid film of lipoproteins e.g., high density lipoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Lipase/metabolismo , Fígado/enzimologia , Fosfolipídeos/metabolismo , Animais , Cromatografia em Gel , Ativação Enzimática/efeitos dos fármacos , Hidrólise , Micelas , Octoxinol , Ácidos Fosfatídicos/metabolismo , Ácidos Fosfatídicos/farmacologia , Fosfatidilcolinas/metabolismo , Fosfatidilcolinas/farmacologia , Fosfatidiletanolaminas/metabolismo , Fosfatidiletanolaminas/farmacologia , Fosfatidilserinas/metabolismo , Fosfatidilserinas/farmacologia , Polietilenoglicóis , Coelhos , RatosRESUMO
The effect of apolipoproteins A-I, A-II, C-II, C-III and E on the hydrolysis of phosphatidylcholine and triacylglycerol by hepatic lipase was studied. Hepatic lipase catalyzed phospholipid hydrolysis was 1.8-fold activated by apolipoprotein E while the other apolipoproteins did not affect the hydrolysis by this enzyme. Triacylglycerol hydrolysis by hepatic lipase was 1.5-fold activated by apolipoprotein E while the other apolipoproteins inhibited hepatic lipase. These results suggest that lipoproteins containing apolipoprotein E may be preferred substrates for hepatic lipase.
Assuntos
Apolipoproteínas E/fisiologia , Lipase/metabolismo , Fígado/enzimologia , Fosfatidilcolinas/metabolismo , Animais , Apolipoproteínas/fisiologia , Catálise , Ativação Enzimática , Hidrólise , Técnicas In Vitro , Lipase/antagonistas & inibidores , Ratos , Triglicerídeos/metabolismoRESUMO
Cells of the marine sponge, Microciona prolifera, the most ancient of the animal cells which clump on recognition, resemble neutrophils and platelets in undergoing stimulus-response coupling when exposed to Ca2+ ionophores and phorbol esters. We have studied lipid content and remodelling in sponge cells by thin-layer, gas-liquid, and high-performance liquid chromatography (HPLC) analyses supplemented by ultraviolet and mass spectroscopy. Phosphatidylcholine (PC) (35.6%), phosphatidylethanolamine (PE) (27.4%) and phosphatidylserine (PS) (21.4%) constituted the bulk of phospholipids detected. The major fatty acids were all polyenoic; 22:6 (22%), 26:2 (17%) and 26:3 (15%). Arachidonic acid (20:4), present as 2.7% of total phospholipid, and docosahexanoic acid (22:6) were found to elicit aggregation of sponge cells when added (10 microM) in synergy with ionomycin (1 microM), resembling in their effects those of phorbol esters (but not phorbol) and 1-oleyl-2-acetylglycerol (OAG). Moreover, 20:4 and 22:6, as well as phorbol ester and OAG, overcame the block to aggregation imposed by colchicine and vinblastine. Kinetic studies of lipid remodelling showed that aggregating cells diverted [14C]22:6 or [14C]20:4 from triacylglycerol into diacylglycerol and phospholipids; appearance of label in phosphatidic acid and phosphatidylinositol (PI) anteceded labeling of phosphatidylcholine. In unstimulated cells, [14C]22:6 was rapidly incorporated into phosphatidylcholine with little accumulation in phosphatidate. Although 22:6 and 20:4 resembled OAG and phorbol esters in overcoming the effects of colchicine and vinblastine (which had no effects on overall lipid metabolism), they did not reverse the block to aggregation of nordihydroguaiaretic acid (NDGA) (which inhibited lipid metabolism). Under none of these circumstances was 22:6 or 20:4 converted to cyclooxygenase or lipoxygenase products in the course of aggregation: all labeled acyl groups remained present as unmodified fatty acids on alkaline hydrolysis. These data not only extend the observations of Muller et al. (J. Biol. Chem. 262 (1987) 9850-9858) on the role of phosphoinositides and C kinase in marine sponge cell aggregation, but also demonstrate that sponges form diacylglycerols in the process. We suggest that exogenous 22:6 and 20:4 (like phorbol esters or OAG) can substitute for endogenous diacylglycerol in the activation of protein kinase C.
Assuntos
Ácidos Araquidônicos/farmacologia , Agregação Celular/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Metabolismo dos Lipídeos , Poríferos/fisiologia , Animais , Ácido Araquidônico , Cálcio/farmacologia , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Colchicina/farmacologia , Éteres/farmacologia , Ácidos Graxos/análise , Cromatografia Gasosa-Espectrometria de Massas , Ionomicina , Masoprocol/farmacologia , Ésteres de Forbol/farmacologia , Fosfolipídeos/análise , Poríferos/efeitos dos fármacos , Vimblastina/farmacologiaRESUMO
Bis(monoacylglycerol)phosphate (BMP) in macrophages is known to rapidly turn over its acyl moiety(s) located at primary positions of the glycerols, yet the glycerols and phosphate remain stable within the BMP molecule. Here we examine whether the phospholipase A1 isolated from rat-liver lysosomes is capable of deacylating BMP. By comparison with the precursor of BMP, phosphatidylglycerol, BMP is a very poor substrate for the phospholipase A1. We conclude, therefore, that a direct deacylation of the acyl groups at the primary alcohol level of the glycerol probably does not occur, but postulate that transacylations may occur to account for the removal of the acyl moiety.
Assuntos
Glicerofosfatos/metabolismo , Lisossomos/enzimologia , Fosfolipases A/metabolismo , Animais , Fígado/enzimologia , Fosfatidilgliceróis/metabolismo , Fosfolipases A1 , Ratos , Especificidade por SubstratoRESUMO
We examined the uptake of radiolabeled lysophospholipids and oleic acid by Bacillus Calmette-Guerin-induced rabbit alveolar macrophages either in the presence or absence of challenge particles. There was no difference in the uptake and metabolism of lysophospholipids by control or challenged cells for incubation periods up to 5 h. When incubated with [3H]oleic acid, challenged cells consistently exhibited a slightly greater uptake of radioactivity. Extraction of the whole cells revealed that the greater amount of radioactivity found in the challenged cells primarily was in triacylglycerol. There was no marked difference in the amount of radioactivity associated with the phospholipids in the whole cell extracts from control and challenged cells. When the macrophages were pre-labeled for 15 min with [3H]oleic acid and then reincubated in fresh medium in the presence or absence of autoclaved Escherichia coli B, more radioactivity was retained by the challenged cells, again in the form of triacylglycerol. Only in isolated plasma membrane fractions did we observe a difference in the amount of radioactivity associated with phospholipids from control and challenged cells. Plasma membranes isolated from Bacillus Calmette-Guerin-induced rabbit alveolar macrophages that had been incubated for 6 h with [3]oleic acid in the presence of E. coli B contained significantly higher level of radioactivity in all lipids than plasma membranes from control cells. Since the greatest and the most consistent difference between control and challenged cells is associated with the triacylglycerol molecule, it is postulated that this molecule may serve as a precursor in the synthesis of alveolar macrophage phospholipids, both by the reacylation pathway and the de novo pathway. It is possible that the high level of radiolabeled phospholipid found in the plasma membrane arose via the de novo pathway following the cleavage of an acyl group as we have found cytidine diphosphocholine phosphotransferase in the plasma membrane fraction (Wang, P., DeChatelet, L.R., and Waite, M. (1977) Biochim. Biophys. Acta 450, 311--321).
Assuntos
Vacina BCG , Macrófagos/metabolismo , Lipídeos de Membrana/metabolismo , Mycobacterium bovis , Animais , Transporte Biológico , Membrana Celular/metabolismo , Escherichia coli , Ácidos Graxos/metabolismo , Feminino , Glicerídeos/biossíntese , Cinética , Ácidos Oleicos/metabolismo , Fosfolipídeos/biossíntese , Alvéolos Pulmonares , Coelhos , Triglicerídeos/biossínteseRESUMO
The rabbit alveolar macrophage is capable of renewing its plasma membrane by at least two metabolic pathways. It contains (1) a monoacylphospholipid acyltransferase, which catalyzes the synthesis of diacylphospholipids by recycling monoacylphospholipids produced by the action of phospholipases and (2) a cytidine diphosphocholine phosphotransferase (CDPcholine phosphotransferase), which catalyzes the last step in the synthesis de novo of diacylglycerophosphocholine. These activities have been characterized in the cell homogenate with respect to time, protein, pH optimum (for CDPcholine phosphotransferase), substrate specificity (for monoacylphospholipid acyltransferase) and cation requirement ( for CDPcholine phosphotransferase). Monoacylphospholipid acyltransferase activity is localized solely in the endoplasmic reticulum. On the other hand, the CDPcholine phosphotransferase activity can be measured in the endoplasmic reticulum and in the plasma membrane, characterized by both differential and gradient sedimentation techniques. In addition to the normal route of phospholipid synthesis in the endoplasmic reticulum, the rabbit alveolar macrophage may thus possess the capacity for in situ synthesis of phospholipids of plasma membrane as a mechanism for membrane renewal following phagocytosis.
Assuntos
Aciltransferases/metabolismo , Vacina BCG , Macrófagos/enzimologia , Fosfolipídeos/metabolismo , Fosfotransferases/metabolismo , Alvéolos Pulmonares/enzimologia , Animais , Humanos , Cinética , Neutrófilos/enzimologia , CoelhosRESUMO
Dissociated embryonic chick limb cells will undergo terminal differentiation in culture. The addition of whole or delipidated mouse serum to the cultures will, however, inhibit the chondrogenic potential of the cells. The inhibitory serum readily stimulates increased incorporation of arachidonic acid and palmitic acid into triacylglycerol in the treated cells, while the incorporation of arachidonate into various phospholipids is significantly lowered. In contrast mouse serum has no effect on the incorporation of inorganic [32P]phosphate into phospholipid. We interpret the patterns of incorporation of these lipid substrates as indicating that mouse serum modulates the deacylation-reacylation cycle of phospholipids (Lands' cycle), which is primarily responsible for the incorporation of arachidonic acid. This finding suggests that there may be a decrease in membrane fluidity which might play a key role in cellular regulation and differentiation.
Assuntos
Metabolismo dos Lipídeos , Triglicerídeos/biossíntese , Animais , Sangue , Diferenciação Celular , Células Cultivadas , Embrião de Galinha , Meios de Cultura , Extremidades/embriologia , Fluidez de Membrana , Camundongos , Fosfolipídeos/metabolismoRESUMO
We have undertaken a study to resolve the conflicting reports on the substrate specificity of the lipolytic enzyme(s) released by heparin from liver and report the following: (1) Heparin perfusates from liver contain an enzyme(s) capable of degrading triacylglycerol, diacylglycerophosphorylethanolamine and monoacylglycerol, whereas a heparin-solubilized fraction from liver plasma membranes hydrolyzes diacylglycerophosphorylethanolamine and monoacylglycerol only; (2) The lipolytic activities for the two sources behave differently on gel filtration but have the same behavior on heparin-Sepharose affinity chromatography; (3) Treatment of the preparation from the plasma membrane with Triton X-100 followed by heparin-Sepharose affinity chromatography produces forms of the enzyme(s) that now have activity on triacylglycerol This study suggests that the enzyme(s) from the two sources may be the same and that some change occurs when the enzyme is released from the intact liver.
Assuntos
Fígado/enzimologia , Aciltransferases/análise , Animais , Hidrolases de Éster Carboxílico/análise , Membrana Celular/enzimologia , Heparina/farmacologia , Monoacilglicerol Lipases/análise , Perfusão , RatosRESUMO
1. During in vitro incubation of liposomes or unilamellar vesicles prepared from egg-yolk or rat-liver phosphatidylcholine with human, monkey or rat plasma the phospholipid becomes associated with a high molecular weight protein-containing component. 2. The phosphatidylcholine . protein complex thus formed co-chromatographs with high-density lipoprotein on Ultrogel AcA34 and has the same immunoelectrophoretic properties as this lipoprotein. 3. Release of the phosphatidylcholine from liposomes was also observed when liposomes were incubated with pure monkey high-density lipoproteins. Under those conditions some transfer of protein from the lipoprotein to the liposomes was observed as well. 4. The observed release of phospholipid from the liposomes is a one-way process, as the specific radioactivity of liposome-associated phosphatidylcholine remained constant during incubation with plasma. 5. It is concluded that either the lipoprotein particle takes up additional phospholipid or that a new complex is formed from protein constituents of the lipoprotein and the liposomal phosphatidylcholine. 6. Massive release of entrapped 125I-labeled albumin from the liposome during incubation with plasma suggests that the observed release of phosphatidylcholine from the liposomes has a highly destructive influence on the liposomal structure. 7. Our results are discussed with special reference to the use of liposomes as intravenous carriers of drugs and enzymes.
Assuntos
Lipoproteínas HDL/sangue , Lipossomos/sangue , Fosfatidilcolinas/sangue , Animais , Haplorrinos , Humanos , Técnicas In Vitro , Lipossomos/uso terapêutico , Veículos Farmacêuticos , RatosRESUMO
We investigated the metabolism by hepatocyte suspensions of the acylglycerols in lipoprotein remnants as well as those associated with albumin and low or high density lipoproteins. Remnants, albumin and plasma lipoproteins, rich in monoacylglycerol were prepared by short-term incubations of radio-labeled chylomicra or very low density lipoproteins with extrahepatic lipoprotein lipase in the presence of albumin and low and high density lipoproteins. We demonstrated that liver parenchymal cells contain an active monoacylglycerol acyltransferase that is located on the extracellular surface of the cell plasma membrane. Further, the enzyme is capable of degrading the monoacylglycerol in all the above forms. Triacylglycerol in intact chylomicra and very low density lipoproteins were not metabolized by the cells to any appreciable degree. The degradation of the remnant triacylglycerol appeared to depend solely on the activity of the lipoprotein lipase bound to the lipoprotein remnants. Little uptake of intact lipoprotein acylglycerols by the hepatocytes was observed; instead, hydrolysis of the substrates in the medium always preceded the uptake of the products. The products were then utilized for the synthesis of triacylglycerol and phospholipid within the cells.
Assuntos
Glicerídeos/metabolismo , Lipoproteínas/metabolismo , Fígado/metabolismo , Animais , Membrana Celular/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Glicerol/metabolismo , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Fígado/citologia , Modelos Biológicos , Ácidos Oleicos/metabolismo , Fosfolipídeos/metabolismo , Ratos , Soroalbumina Bovina/metabolismoRESUMO
The tumor-promoting phorbol diester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), stimulates Madin-Darby canine (MDCK) cells to deacylate cellular phospholipid and to produce prostaglandins. We have used this system to characterize the kinetics of deacylation of [3H]arachidonate and the further metabolism of arachidonate by the cyclooxygenase system. Stimulation of the appearance of [3H]arachidonic acid in extracellular fluids was found to be maximal 2 h after treatment with TPA and its subsequent removal. The production of prostaglandins then followed for up to 24 h. Phospholipase activity was not inhibited by indomethacin over the range of 0.01-100 micrograms/ml. In contrast, prostaglandin synthesis was inhibited at 1 microgram/ml indomethacin. Further, there was a significant stimulation of deacylation within 15 min in the presence of TPA that increased to nearly 30% of the total radioactivity within 1 h. Likewise, stimulation of prostaglandin production was detected within 15 min, but, unlike the deacylation process, did not increase significantly during TPA treatment. The source of arachidonic acid in the early stimulation period was found to be primarily phosphatidylethanolamine, but phosphatidylcholine and phosphatidylinositol were also deacylated. The results presented here argue that the phospholipase and cyclooxygenase are not tightly coupled in this system. Furthermore, we conclude that the earliest effect of TPA with regard to increased prostaglandin production in the MDCK cell is the direct stimulation of phospholipase activity.
Assuntos
Rim/metabolismo , Forbóis/farmacologia , Fosfolipídeos/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Animais , Ácido Araquidônico , Ácidos Araquidônicos/metabolismo , Linhagem Celular , Cães , Indometacina/farmacologia , Rim/efeitos dos fármacos , Cinética , Prostaglandinas/biossínteseRESUMO
We report here that both the hepatic lipase and lipoprotein lipase demonstrate specificity towards the acyl group present on monoacylglycerols. We found that unsaturated glycerides are more readily degraded than saturated glycerides. However, the basis for this specificity appears to be different for each enzyme. The activity of the hepatic lipase, but not the lipoprotein lipase, could be stimulated by Triton X-100 and phosphoglycerides. We interpret these results to show that while both the lipoprotein lipase and hepatic lipase are sensitive to the physical state of the substrate (as shown by fluorescence depolarization), the lipoprotein lipase also has a low affinity for monoacylglycerols that contain a saturated acyl group. In the course of this study we also obtained evidence that some type of phase separation occurs when mixtures of saturated and unsaturated monoacylglycerols are prepared.