RESUMO
Activation of hepatic stellate cells (HSCs) is a critical step in the development of liver fibrosis. During activation, HSCs lose their lipid droplets (LDs) containing triacylglycerols (TAGs), cholesteryl esters, and retinyl esters (REs). We previously provided evidence for the presence of two distinct LD pools, a preexisting and a dynamic LD pool. Here we investigate the mechanisms of neutral lipid metabolism in the preexisting LD pool. To investigate the involvement of lysosomal degradation of neutral lipids, we studied the effect of lalistat, a specific lysosomal acid lipase (LAL/Lipa) inhibitor on LD degradation in HSCs during activation in vitro The LAL inhibitor increased the levels of TAG, cholesteryl ester, and RE in both rat and mouse HSCs. Lalistat was less potent in inhibiting the degradation of newly synthesized TAG species as compared with a more general lipase inhibitor orlistat. Lalistat also induced the presence of RE-containing LDs in an acidic compartment. However, targeted deletion of the Lipa gene in mice decreased the liver levels of RE, most likely as the result of a gradual disappearance of HSCs in livers of Lipa-/- mice. Lalistat partially inhibited the induction of activation marker α-smooth muscle actin (α-SMA) in rat and mouse HSCs. Our data suggest that LAL/Lipa is involved in the degradation of a specific preexisting pool of LDs and that inhibition of this pathway attenuates HSC activation.
Assuntos
Células Estreladas do Fígado/metabolismo , Gotículas Lipídicas/metabolismo , Lisossomos/metabolismo , Esterol Esterase/metabolismo , Animais , Inibidores Enzimáticos/farmacologia , Feminino , Células Estreladas do Fígado/efeitos dos fármacos , Gotículas Lipídicas/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos , Ratos Wistar , Esterol Esterase/antagonistas & inibidores , Esterol Esterase/deficiência , Relação Estrutura-AtividadeRESUMO
Hepatic stellate cells (HSCs) play an important role in liver physiology and under healthy conditions they have a quiescent and lipid-storing phenotype. Upon liver injury, HSCs are activated and rapidly lose their retinyl ester-containing lipid droplets. To investigate the role of lecithin:retinol acyltransferase (LRAT) and acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) in retinyl ester synthesis and lipid droplet dynamics, we modified LC-MS/MS procedures by including multiple reaction monitoring allowing unambiguous identification and quantification of all major retinyl ester species. Quiescent primary HSCs contain predominantly retinyl palmitate. Exogenous fatty acids are a major determinant in the retinyl ester species synthesized by activated HSCs and LX-2 cells, indicating that HSCs shift their retinyl ester synthesizing capacity from LRAT to DGAT1 during activation. Quiescent LRAT-/- HSCs retain the capacity to synthesize retinyl esters and to store neutral lipids in lipid droplets ex vivo. The median lipid droplet size in LRAT-/- HSCs (1080nm) is significantly smaller than in wild type HSCs (1618nm). This is a consequence of an altered lipid droplet size distribution with 50.5±9.0% small (≤700nm) lipid droplets in LRAT-/- HSCs and 25.6±1.4% large (1400-2100nm) lipid droplets in wild type HSC cells. Upon prolonged (24h) incubation, the amounts of small (≤700nm) lipid droplets strongly increased both in wild type and in LRAT-/- HSCs, indicating a dynamic behavior in both cell types. The absence of retinyl esters and reduced number of lipid droplets in LRAT-deficient HSCs in vivo will be discussed.
Assuntos
Aciltransferases/metabolismo , Ésteres/metabolismo , Células Estreladas do Fígado/metabolismo , Gotículas Lipídicas/metabolismo , Lipídeos/fisiologia , Animais , Linhagem Celular , Diacilglicerol O-Aciltransferase/metabolismo , Humanos , Hepatopatias/metabolismo , Camundongos , Espectrometria de Massas em Tandem/métodosRESUMO
BACKGROUND: A link between lipid metabolism and disease has been recognized in cats. Since hepatic lipidosis is a frequent disorder in cats, the aim of the current study was to evaluate liver and plasma lipid dimorphism in healthy cats and the effects of gonadectomy on lipid profiling. From six female and six male cats plasma and liver lipid profiles before and after spaying/neutering were assessed and compared to five cats (three neutered male and two spayed female) diagnosed with hepatic lipidosis. RESULTS: Intact female cats had a significantly lower level of plasma triacylglycerides (TAG) and a higher liver level of the long chain polyunsaturated fatty acid arachidonic acid (AA) compared to their neutered state. Both male and female cats with lipidosis had a higher liver, but not plasma TAG level and an increased level of plasma and liver sphingomyelin compared to the healthy cats. CONCLUSION: Although lipid dimorphism in healthy cats resembles that of other species, intact female cats show differences in metabolic configuration that could predispose them to develop hepatic lipidosis. The increased sphingomyelin levels in cats with lipidosis could suggest a potential role in the pathogenesis of hepatic lipidosis in cats.
Assuntos
Doenças do Gato/metabolismo , Metabolismo dos Lipídeos , Lipidoses/veterinária , Fígado/metabolismo , Animais , Ácido Araquidônico/sangue , Doenças do Gato/sangue , Gatos , Feminino , Lipidoses/sangue , Lipidoses/metabolismo , Masculino , Orquiectomia/veterinária , Ovariectomia/veterinária , Fatores Sexuais , Esfingomielinas/sangue , Triglicerídeos/sangueRESUMO
Hepatic stellate cell (HSC) activation is a critical step in the development of chronic liver disease. During activation, HSCs lose their lipid droplets (LDs) containing triacylglycerol (TAG), cholesteryl esters (CEs), and retinyl esters (REs). Here we aimed to investigate which enzymes are involved in LD turnover in HSCs during activation in vitro. Targeted deletion of the Atgl gene in mice HSCs had little effect on the decrease of the overall TAG, CE, and RE levels during activation. However, ATGL-deficient HSCs specifically accumulated TAG species enriched in PUFAs and degraded new TAG species more slowly. TAG synthesis and levels of PUFA-TAGs were lowered by the diacylglycerol acyltransferase (DGAT)1 inhibitor, T863. The lipase inhibitor, Atglistatin, increased the levels of TAG in both WT and ATGL-deficient mouse HSCs. Both Atglistatin and T863 inhibited the induction of activation marker, α-smooth muscle actin, in rat HSCs, but not in mouse HSCs. Compared with mouse HSCs, rat HSCs have a higher turnover of new TAGs, and Atglistatin and the DGAT1 inhibitor, T863, were more effective. Our data suggest that ATGL preferentially degrades newly synthesized TAGs, synthesized by DGAT1, and is less involved in the breakdown of preexisting TAGs and REs in HSCs. Furthermore a large change in TAG levels has modest effect on rat HSC activation.
Assuntos
Diacilglicerol O-Aciltransferase/genética , Células Estreladas do Fígado/metabolismo , Lipase/genética , Triglicerídeos/biossíntese , Animais , Ésteres do Colesterol/genética , Ésteres do Colesterol/metabolismo , Inibidores Enzimáticos/administração & dosagem , Ácidos Graxos Insaturados/biossíntese , Células Estreladas do Fígado/patologia , Gotículas Lipídicas/metabolismo , Lipogênese/genética , Lipólise/genética , Camundongos , Camundongos Knockout , Compostos de Fenilureia/administração & dosagem , Ratos , Triglicerídeos/genéticaRESUMO
Hepatic stellate cell (HSC) activation is a critical step in the development of chronic liver disease. We previously observed that the levels of triacylglycerol (TAG) species containing long polyunsaturated fatty acids (PUFAs) are increased in in vitro activated HSCs. Here we investigated the cause and consequences of the rise in PUFA-TAGs by profiling enzymes involved in PUFA incorporation. We report that acyl CoA synthetase (ACSL) type 4, which has a preference for PUFAs, is the only upregulated ACSL family member in activated HSCs. Inhibition of the activity of ACSL4 by siRNA-mediated knockdown or addition of rosiglitazone specifically inhibited the incorporation of deuterated arachidonic acid (AA-d8) into TAG in HSCs. In agreement with this, ACSL4 was found to be partially localized around lipid droplets (LDs) in HSCs. Inhibition of ACSL4 also prevented the large increase in PUFA-TAGs in HSCs upon activation and to a lesser extent the increase of arachidonate-containing phosphatidylcholine species. Inhibition of ACSL4 by rosiglitazone was associated with an inhibition of HSC activation and prostaglandin secretion. Our combined data show that upregulation of ACSL4 is responsible for the increase in PUFA-TAG species during activation of HSCs, which may serve to protect cells against a shortage of PUFAs required for eicosanoid secretion.
Assuntos
Coenzima A Ligases/metabolismo , Ácidos Graxos Insaturados/metabolismo , Células Estreladas do Fígado/enzimologia , Triglicerídeos/metabolismo , Animais , Ácido Araquidônico/metabolismo , Linhagem Celular , Coenzima A Ligases/antagonistas & inibidores , Coenzima A Ligases/genética , Inibidores Enzimáticos/farmacologia , Células Estreladas do Fígado/efeitos dos fármacos , Humanos , Masculino , Fosfatidilcolinas/metabolismo , Interferência de RNA , Ratos Wistar , Rosiglitazona , Tiazolidinedionas/farmacologia , Fatores de Tempo , Transfecção , Regulação para CimaRESUMO
BACKGROUND: Feline hepatic lipidosis (FHL) is a common cholestatic disease affecting cats of any breed, age and sex. Both choline deficiency and low hepatic phosphatidylethanolamine N-methyltransferase (PEMT) activity are associated with hepatic lipidosis (HL) in humans, mice and rats. The PEMT expression is known to be upregulated by oestrogens, protecting the females in these species from the development of HL when exposed to choline deficient diets. The aim of the present study was to evaluate the influence of sex hormones on choline synthesis via the PEMT pathway in healthy male and female cats before and after spaying/neutering, when fed a diet with recommended dietary choline content. RESULTS: From six female and six male cats PEMT activity was assayed directly in liver biopsies taken before and after spaying/neutering, and assessed indirectly by analyses of PEMT-specific hepatic phosphatidylcholine (PC) species and plasma choline levels. Hepatic PEMT activity did not differ between intact female and male cats and no changes upon spaying/neutering were observed. Likewise, no significant differences in liver PC content and PEMT-specific polyunsaturated PC species were found between the sexes and before or after spaying/neutering. CONCLUSION: These results suggest that choline synthesis in cats differs from what is observed in humans, mice and rats. The lack of evident influence of sex hormones on the PEMT pathway makes it unlikely that spaying/neutering predisposes cats for HL by causing PC deficiency as suggested in other species.
Assuntos
Colina/metabolismo , Histerectomia/veterinária , Orquiectomia/veterinária , Ovariectomia/veterinária , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Animais , Gatos , Feminino , Regulação Enzimológica da Expressão Gênica/fisiologia , Fígado/enzimologia , Masculino , Fosfatidiletanolamina N-Metiltransferase/genética , Regulação para CimaRESUMO
Metabolic conditions characterized by elevated free fatty acid concentrations in blood and follicular fluid are often associated with impaired female fertility. Especially elevated saturated fatty acid levels can be lipotoxic for several somatic cell types. The aim of this study was to determine the impact of elevated free fatty acid concentrations in follicular fluid on neutral lipids (fatty acids stored in lipid droplets) inside cumulus cells and oocytes and their developmental competence. To this end, cows were exposed to a short-term fasting period during final oocyte maturation. This resulted in elevated, but distinct, free fatty acid concentrations in blood and follicular fluid and a rise in the concentrations of in particular fatty acids with a chain length of 14-18 carbon atoms. Interestingly, elevated free fatty acid concentrations in follicular fluid resulted in a massive increase in the level of neutral lipids in cumulus cells, whereas the level of neutral lipid in oocytes was hardly affected. Furthermore, competence of oocytes to develop to the blastocyst stage after fertilization and culture of cumulus-oocyte-complexes of the experimental and control group was not different. In conclusion these data suggest that short-term elevated free fatty acid concentrations in follicular fluid do not harm oocyte developmental competence. We propose that the involvement of high levels of mobilized oleic acid in follicular fluid in combination with the induced lipid storage in cumulus cells serves to prevent harmful saturated fatty acid exposure to the oocyte.
Assuntos
Células do Cúmulo/metabolismo , Ácidos Graxos/metabolismo , Metabolismo dos Lipídeos/fisiologia , Oócitos/metabolismo , Oogênese/fisiologia , Animais , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Bovinos , Células Cultivadas , Células do Cúmulo/citologia , Células do Cúmulo/efeitos dos fármacos , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/fisiologia , Ácidos Graxos/farmacologia , Feminino , Fertilização in vitro , Líquido Folicular/metabolismo , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oogênese/efeitos dos fármacosRESUMO
Mobilization of fatty acids from adipose tissue during metabolic stress will increase the amount of free fatty acids in blood and follicular fluid and, thus, may affect oocyte quality. In this in vitro study, the three predominant fatty acids in follicular fluid (saturated palmitic and stearic acid and unsaturated oleic acid) were presented to maturing oocytes to test whether fatty acids can affect lipid storage of the oocyte and developmental competence postfertilization. Palmitic and stearic acid had a dose-dependent inhibitory effect on the amount of fat stored in lipid droplets and a concomitant detrimental effect on oocyte developmental competence. Oleic acid, in contrast, had the opposite effect, causing an increase of lipid storage in lipid droplets and an improvement of oocyte developmental competence. Remarkably, the adverse effects of palmitic and stearic acid could be counteracted by oleic acid. These results suggest that the ratio and amount of saturated and unsaturated fatty acid is relevant for lipid storage in the maturing oocyte and that this relates to the developmental competence of maturing oocytes.
Assuntos
Ácido Oleico/metabolismo , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Ácido Palmítico/metabolismo , Ácidos Esteáricos/metabolismo , Animais , BovinosRESUMO
Vitamin A (retinol) is important for normal growth, vision and reproduction. It has a role in the immune response and the development of metabolic syndrome. Most of the retinol present in the body is stored as retinyl esters within lipid droplets in hepatic stellate cells (HSCs). In case of liver damage, HSCs release large amounts of stored retinol, which is partially converted to retinoic acid (RA). This surge of RA can mediate the immune response and enhance the regeneration of the liver. If the damage persists activated HSCs change into myofibroblast-like cells producing extracellular matrix, which increases the chance of tumorigenesis to occur. RA has been shown to decrease proliferation and metastasis of hepatocellular carcinoma. The levels of RA and RA signaling are influenced by the possibility to esterify retinol towards retinyl esters. This suggests a complex regulation between different retinoids, with an important regulatory role for HSCs.
Assuntos
Carcinoma Hepatocelular/patologia , Células Estreladas do Fígado/metabolismo , Neoplasias Hepáticas/patologia , Regeneração Hepática , Vitamina A/metabolismo , Carcinogênese/patologia , Ésteres/metabolismo , Matriz Extracelular/metabolismo , Células Estreladas do Fígado/citologia , Humanos , Gotículas Lipídicas/metabolismo , Fígado/citologia , Fígado/metabolismo , Fígado/patologia , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Hepatic lipidosis is increasing in incidence in the Western world, with cats being particularly sensitive. When cats stop eating and start utilizing their fat reserves, free fatty acids (FFAs) increase in blood, causing an accumulation of triacylglycerol (TAG) in the liver. OBJECTIVE: Identifying potential new drugs that can be used to treat hepatic lipidosis in cats using a feline hepatic organoid system. ANIMALS: Liver organoids obtained from 6 cats. METHODS: Eight different drugs were tested, and the 2 most promising were further studied using a quantitative TAG assay, lipid droplet staining, and qPCR. RESULTS: Both T863 (a diacylglycerol O-acyltransferase 1 [DGAT1] inhibitor) and 5-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR; an adenosine monophosphate kinase activator) decreased TAG accumulation by 55% (P < .0001) and 46% (P = .0003), respectively. Gene expression of perilipin 2 (PLIN2) increased upon the addition of FFAs to the medium and decreased upon treatment with AICAR but not significantly after treatment with T863. CONCLUSIONS AND CLINICAL IMPORTANCE: Two potential drugs useful in the treatment of hepatic lipidosis in cats were identified. The drug T863 inhibits DGAT1, indicating that DGAT1 is the primary enzyme responsible for TAG synthesis from external fatty acids in cat organoids. The drug AICAR may act as a lipid-lowering compound via decreasing PLIN2 mRNA. Liver organoids can be used as an in vitro tool for drug testing in a species-specific system and provide the basis for further clinical testing of drugs to treat steatosis.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Doenças do Gato/tratamento farmacológico , Diacilglicerol O-Aciltransferase/antagonistas & inibidores , Fígado Gorduroso/veterinária , Lipidoses/veterinária , Organoides/metabolismo , Ribonucleotídeos/farmacologia , Aminoimidazol Carboxamida/farmacologia , Animais , Doenças do Gato/metabolismo , Gatos , Ácidos Graxos não Esterificados/metabolismo , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/metabolismo , Lipidoses/tratamento farmacológico , Lipidoses/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologiaRESUMO
The mutant Chinese hamster ovary cell line MT58 contains a thermosensitive mutation in CTP:phosphocholine cytidylyltransferase, the regulatory enzyme in the CDP-choline pathway. As a result, MT58 cells have a 50% decrease in their phosphatidylcholine (PC) level within 24 h when cultured at the nonpermissive temperature (40 degrees C). This is due to a relative rapid breakdown of PC that is not compensated for by the inhibition of de novo PC synthesis. Despite this drastic decrease in cellular PC content, cells are viable and can proliferate by addition of lysophosphatidylcholine. By [(3)H]oleate labeling, we found that the FA moiety of the degraded PC is recovered in triacylglycerol. In accordance with this finding, an accumulation of lipid droplets is seen in MT58 cells. Analysis of PC-depleted MT58 cells by electron and fluorescence microscopy revealed a partial dilation of the rough endoplasmic reticulum, resulting in spherical structures on both sites of the nucleus, whereas the morphology of the plasma membrane, mitochondria, and Golgi complex was unaffected. In contrast to these morphological observations, protein transport from the ER remains intact. Surprisingly, protein transport at the level of the Golgi complex is impaired. Our data suggest that the transport processes at the Golgi complex are regulated by distal changes in lipid metabolism.
Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Fosfatidilcolinas/metabolismo , Animais , Células CHO , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colina-Fosfato Citidililtransferase/genética , Colina-Fosfato Citidililtransferase/metabolismo , Cricetinae , Cricetulus , Retículo Endoplasmático/ultraestrutura , Recuperação de Fluorescência Após Fotodegradação , Complexo de Golgi/ultraestrutura , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Lisofosfatidilcolinas/farmacologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Microscopia Confocal , Microscopia Imunoeletrônica , Mutação , Ácido Oleico/metabolismo , Transporte Proteico , Temperatura , Triglicerídeos/metabolismo , Trítio , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
During biomineralization the organism controls the nature, orientation, size and shape of the mineral phase. The aim of this study was to investigate whether proteins or vesicles that are constitutively released by growing ATDC5 cells have the ability to affect the formation of the calcium phosphate crystal. Therefore, subconfluent cultured ATDC5 cells were incubated for 1 h in medium without serum. Subsequently, medium was harvested and incubated for 24 h in the presence of additional Pi. This resulted in the formation of flat mineralizing structures (FMS), consisting of complex irregularly shaped flat crystals, which occasionally contained fiber-like structures ( approximately 40 microm in size). Without pre-incubation of medium with cells, only small punctate (dot like) calcium phosphate precipitates were observed. The formation of FMS was shown to be caused by soluble factors released by subconfluent ATDC5 cells. Proteomic analysis by mass spectrometry showed that FMS contained a specific set intracellular proteins, serum proteins, and extracellular matrix proteins. Bulk cytosolic proteins derived from homogenized cells or serum proteins did, however, not induce the formation of FMS. Conditioned medium from HeLa, CHO K1, RAW 264.7 and MDCK cells was also capable to form FMS under our experimental conditions. Therefore the formation of FMS seems to be caused by specific soluble factors constitutively released by ADTC5 and other cells. This in vitro model system can be used as a tool to identify factors that affect the shape of the biomineral phase.
Assuntos
Fosfatos de Cálcio/química , Condrócitos/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Cristalização , Eletroforese em Gel de Poliacrilamida , Células HeLa , Humanos , Camundongos , Microscopia Eletrônica , Proteínas/fisiologia , Espectrofotometria InfravermelhoRESUMO
Mineralization is an essential requirement for normal skeletal development, but under certain pathological conditions organs like articular cartilage and cardiovascular tissue are prone to unwanted mineralization. Recent findings suggest that the mechanisms regulating skeletal mineralization may be similar to those regulating pathological mineralization. In general, three forms of cell-mediated mineralization are recognized in an organism: intramembranous ossification, endochondral ossification and pathological mineralization. This review summarizes recent work that tried to elucidate how cell-mediated mineralization is initiated and regulated. To explain mineralization, several theories have been proposed. One theory proposes that mineralization is initiated within matrix vesicles (MVs). A second, not mutually exclusive, theory proposes that phosphate induces apoptosis, and that apoptotic bodies nucleate crystals composed of calcium and phosphate. A third theory suggests that mineralization is mediated by certain non-collagenous proteins, which associate with the extracellular matrix. Regardless of the way mineralization is initiated, the organism also actively inhibits mineralization by specific proteins and removal of an inhibitor may also induce mineralization. Although many studies greatly contributed to a better understanding of the mechanisms regulating cell-mediated mineralization, many questions remain about the mechanisms that trigger cell-mediated mineralization and how this process is regulated. Further investigation is necessary to develop in the future novel therapeutic strategies to prevent pathological mineralization.
Assuntos
Calcificação Fisiológica , Animais , Apoptose , Calcinose/etiologia , Vesículas Citoplasmáticas/metabolismo , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/fisiologia , Humanos , Camundongos , alfa-Fetoproteínas/fisiologiaRESUMO
Hepatic stellate cells (HSCs) are professional lipid-storing cells and are unique in their property to store most of the retinol (vitamin A) as retinyl esters in large-sized lipid droplets. Hepatic stellate cell activation is a critical step in the development of chronic liver disease, as activated HSCs cause fibrosis. During activation, HSCs lose their lipid droplets containing triacylglycerols, cholesteryl esters, and retinyl esters. Lipidomic analysis revealed that the dynamics of disappearance of these different classes of neutral lipids are, however, very different from each other. Although retinyl esters steadily decrease during HSC activation, triacylglycerols have multiple pools one of which becomes transiently enriched in polyunsaturated fatty acids before disappearing. These observations are consistent with the existence of preexisting "original" lipid droplets with relatively slow turnover and rapidly recycling lipid droplets that transiently appear during activation of HSCs. Elucidation of the molecular machinery involved in the regulation of these distinct lipid droplet pools may open new avenues for the treatment of liver fibrosis.
RESUMO
Sodium nitroprusside (SNP) is a nitric oxide (NO) donor drug, which is therapeutically used as a vasodilating drug in heart transplantations. In our previous study it was found that SNP at a concentration of 100 microM inhibited mineralization in a cell culture system, indicating that the beneficial effects of this drug may also include inhibition of vascular calcification. The aim of this study was to investigate which bioactive compounds generated from SNP inhibit mineralization. ATDC5 cells were grown for 14 days and mineralization was induced by addition of 5 mM phosphate for 24 h. Mineralization was determined by staining precipitated calcium with an alizarin red stain. It was found that the NO donors S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine were not able to inhibit mineralization and NO scavengers could not antagonize the inhibiting effect of SNP on mineralization. The iron chelator deferoxamine (200 microM) antagonized the inhibiting effect on mineralization mediated by SNP and ammonium iron sulfate inhibited mineralization in a dose-dependent manner (10-100 microM). Furthermore, iron ions (30 microM) were detected to be released from SNP in the cell culture. These data show that the iron moiety of sodium nitroprusside, rather than nitric oxide inhibits mineralization.
Assuntos
Ferro/farmacologia , Minerais/metabolismo , Doadores de Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Animais , Bepridil/farmacologia , Cálcio/análise , Cálcio/metabolismo , Catalase/farmacologia , Linhagem Celular , Óxidos N-Cíclicos/farmacologia , Relação Dose-Resposta a Droga , Compostos Férricos/farmacologia , Ferricianetos/farmacologia , Compostos Ferrosos/farmacologia , Sequestradores de Radicais Livres/farmacologia , Imidazóis/farmacologia , Ferro/química , Manitol/farmacologia , Estrutura Molecular , Óxido Nítrico/antagonistas & inibidores , Doadores de Óxido Nítrico/química , Nitroprussiato/química , Oxirredução , Penicilamina/análogos & derivados , Penicilamina/farmacologia , Compostos de Amônio Quaternário/farmacologia , Espécies Reativas de Oxigênio/antagonistas & inibidores , S-Nitrosoglutationa/farmacologia , Superóxido Dismutase/farmacologiaRESUMO
The anticancer drug hexadecylphosphocholine (HePC), an alkyl-lysophospholipid analog (ALP), has been shown to induce apoptosis and inhibit the synthesis of phosphatidylcholine (PC) in a number of cell lines. We investigated whether inhibition of PC synthesis plays a major causative role in the induction of apoptosis by HePC. We therefore directly compared the apoptosis caused by HePC in CHO cells to the apoptotic process in CHO-MT58 cells, which contain a genetic defect in PC synthesis. HePC-provoked apoptosis was found to differ substantially from the apoptosis observed in MT58 cells, since it was (i) not accompanied by a large decrease in the amount of PC and diacylglycerol (DAG), (ii) not preceded by induction of the pro-apoptotic protein GADD153/CHOP, and (iii) not dependent on the synthesis of new proteins. Furthermore, lysoPC as well as lysophosphatidylethanolamine (lysoPE) could antagonize the apoptosis induced by HePC, whereas only lysoPC was able to rescue MT58 cells. HePC also induced a rapid externalisation of phosphatidylserine (PS). These observations suggest that inhibition of PC synthesis is not the primary pathway in HePC-induced apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Fosfatidilcolinas/biossíntese , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Animais , Células CHO , Cricetinae , Fosfatidilcolinas/antagonistas & inibidoresRESUMO
Type II cGMP-dependent protein kinase (cGK II) is the protein product of one of two genes coding for cGKs in mammalian genomes. cGK II has a domain structure similar to cGK I (alpha or beta) consisting of an N-terminal regulatory domain, which contains a dimerization and an autoinhibitory region, two cGMP-binding domains and a C-terminal catalytic domain. However, the position of the high and low affinity cGMP-binding-domains in cGK II are reversed in comparison to cGK I. Moreover, the isoenzymes exhibit a different affinity towards various membrane permeable cGMP-analogs, allowing differentiation between the cGKs. Type II cGK is bound to the membrane by a myristoyl moiety. It has a distinct function and an expression pattern distinct from that of cGKI, being expressed predominantly in intestine, brain, and kidney. It is involved in regulating electrolyte and water secretion by epithelial tissues in response to the luminocrinic hormones guanylin and uroguanylin and in the secretory diarrhea provoked by heat-stable enterotoxins. Type II cGK also plays a role in the regulation of endochondral ossification by C-type natriuretic peptide, in renin secretion by the kidney, aldosterone secretion by the adrenal, and in the adjustment of the biological clock.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/fisiologia , GMP Cíclico/metabolismo , Processamento de Proteína Pós-Traducional , Trifosfato de Adenosina/fisiologia , Animais , Proteína Quinase Dependente de GMP Cíclico Tipo II , Proteínas Quinases Dependentes de GMP Cíclico/química , Proteínas Quinases Dependentes de GMP Cíclico/genética , Regulação Enzimológica da Expressão Gênica , HumanosRESUMO
For an insight regarding the control of PtdEtn (phosphatidylethanolamine) synthesis via the CDPethanolamine pathway, rat liver cDNA encoding ECT (CTP:phosphoethanolamine cytidylyltransferase) was transiently or stably transfected in Chinese-hamster ovary cells and a rat liver-derived cell line (McA-RH7777), resulting in a maximum of 26- and 4-fold increase in specific activity of ECT respectively. However, no effect of ECT overexpression on the rate of [3H]ethanolamine incorporation into PtdEtn was detected in both cell lines. This was explored further in cells overexpressing four times ECT activity (McA-ECT1). The rate of PtdEtn breakdown and PtdEtn mass were not changed in McA-ECT1 cells in comparison with control-transfected cells. Instead, an accumulation of CDPethanolamine (label and mass) was observed, suggesting that in McA-ECT1 cells the ethanolaminephosphotransferase-catalysed reaction became rate-limiting. However, overexpression of the human choline/ethanolaminephosphotransferase in McA-ECT1 and control-transfected cells had no effect on PtdEtn synthesis. To investigate whether the availability of DAG (diacylglycerol) limited PtdEtn synthesis in these cells, intracellular DAG levels were increased using PMA or phospholipase C. Exposure of cells to PMA or phospholipase C stimulated PtdEtn synthesis and this effect was much more pronounced in McA-ECT1 than in control-transfected cells. In line with this, the DAG produced after PMA exposure was consumed more rapidly in McA-ECT1 cells and the CDPethanolamine level decreased accordingly. In conclusion, our results suggest that the supply of CDPethanolamine, via the expression level of ECT, is an important factor governing the rate of PtdEtn biosynthesis in mammalian cells, under the condition that the amount of DAG is not limiting.
Assuntos
Cistina Difosfato/análogos & derivados , Cistina Difosfato/metabolismo , Diglicerídeos/metabolismo , Etanolaminas/metabolismo , Nucleotidiltransferases/metabolismo , Fosfatidiletanolaminas/biossíntese , Animais , Células CHO , Linhagem Celular Tumoral , Colina/metabolismo , Cricetinae , Citidina Trifosfato/metabolismo , Etanolamina/metabolismo , Humanos , Fígado/enzimologia , Nucleotidiltransferases/genética , Fosfatidilcolinas/biossíntese , Fosfatidilcolinas/metabolismo , RNA Nucleotidiltransferases , Ratos , Acetato de Tetradecanoilforbol/farmacologia , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Fosfolipases Tipo C/metabolismoRESUMO
Surfactant protein C (SP-C) is a small lipopeptide of which the main part consists of a typical valyl-rich transmembrane domain. The protein is expressed as a propeptide (proSP-C) which is processed and sorted via the regulated secretory pathway to the lamellar body, where mature SP-C is stored before secretion into the alveolar space. In this study we investigated the identity of the compartment to which proSP-C is sorted in cells that do not have a regulated secretory pathway, such as CHO cells. By electron microscopy we determined that proSP-C was localized in an uncommon membrane compartment with very regular morphology, which was not present in control cells. This membrane compartment is not influenced by the palmitoylation of proSP-C and is probably derived from the endoplasmic reticulum. However, proSP-C chimeras with artificial transmembrane domains induced a membrane compartment with a different morphology. Therefore we propose that the typical amino acid sequence of the transmembrane domain of proSP-C plays a role in membrane formation and morphology, which may be relevant under physiological conditions.
Assuntos
Células CHO/fisiologia , Membranas Intracelulares/metabolismo , Peptídeos/fisiologia , Proteína C Associada a Surfactante Pulmonar/fisiologia , Sequência de Aminoácidos , Animais , Células CHO/citologia , Células CHO/ultraestrutura , Cricetinae , Meios de Cultura/química , Meios de Cultura/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Membranas Intracelulares/ultraestrutura , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteína C Associada a Surfactante Pulmonar/genética , Proteína C Associada a Surfactante Pulmonar/metabolismo , Surfactantes Pulmonares/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Hexadecylphosphocholine (HePC, Miltefosine) is an antitumour phospholipid and known inducer of apoptosis in human breast cancer cells. The mechanism underlying the induction of cell death by HePC, however, is not clear yet. In this study, we have investigated the cytotoxic effects of HePC on canine mammary tumour cells (CMTs) in vitro. Upon addition of HePC, CMTs rapidly exhibited several features that resembled apoptotic cell death. Cells showed externalization of phosphatidylserine, a hallmark of apoptosis, within 5 min after addition of HePC at concentrations as low as 10 microM. Furthermore, rapid swelling of mitochondria was observed. Rounding and detachment of cells followed within 30 min. However, fragmentation of nuclear DNA could not be observed. Overall, HePC was shown to induce a type of cell death in CMTs that in some aspects resembles apoptosis, though the process proceeds much more rapidly than reported for other tumour cell lines.