RESUMO
We previously reported that 2-arachidonoylglycerol (2-AG) synthesis by diacylglycerol lipase (DAGL) and lysophosphatidate phosphohydrolase (LPAP) and hydrolysis by monoacylglycerol lipase (MAGL) in rod outer segments (ROS) from bovine retina were differently modified by light applied to the retina. Based on these findings, the aim of the present research was to evaluate whether 2-AG metabolism could be modulated by proteins involved in the visual process. To this end, ROS kept in darkness (DROS) or obtained in darkness and then subjected to light (BROS) were treated with GTPγS and GDPßS, or with low and moderate ionic strength buffers for detaching soluble and peripheral proteins, or soluble proteins, respectively. Only DAGL activity was stimulated by the application of light to the ROS. GTPγS-stimulated DAGL activity in DROS reached similar values to that observed in BROS. The studies using different ionic strength show that (1) the highest decrease in DROS DAGL activity was observed when both phosphodiesterase (PDE) and transducin α (Tα) are totally membrane-associated; (2) the decrease in BROS DAGL activity does not depend on PDE association to membrane, and that (3) MAGL activity decreases, both in DROS and BROS, when PDE is not associated to the membrane. Our results indicate that the bioavailability of 2-AG under light conditions is favored by G protein-stimulated increase in DAGL activity and hindered principally by Tα/PDE association with the ROS membrane, which decreases DAGL activity.
Assuntos
Ácidos Araquidônicos , Endocanabinoides , Glicerídeos , Segmento Externo da Célula Bastonete , Animais , Endocanabinoides/metabolismo , Ácidos Araquidônicos/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Bovinos , Glicerídeos/metabolismo , Transdução de Sinal Luminoso , Transducina/metabolismo , Luz , Lipase Lipoproteica/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Visão Ocular/fisiologia , Visão Ocular/efeitos dos fármacosRESUMO
The aim of the present research was to evaluate if the endocannabinoid system (enzymes and receptors) could be modulated by light in rod outer segment (ROS) from bovine retina. First, we analyzed endocannabinoid 2-arachidonoylglycerol (2-AG) metabolism in purified ROS obtained from dark-adapted (DROS) or light-adapted (LROS) retinas. To this end, diacylglycerol lipase (DAGL), monoacylglycerol lipase (MAGL), and lysophosphatidate phosphohydrolase (LPAP) enzymatic activities were analyzed using radioactive substrates. The protein content of these enzymes and of the receptors to which cannabinoids bind was determined by immunoblotting under light stimulus. Our results indicate that whereas DAGL and MAGL activities were stimulated in retinas exposed to light, no changes were observed in LPAP activity. Interestingly, the protein content of the main enzymes involved in 2-AG metabolism, phospholipase C ß1 (PLCß1), and DAGLα (synthesis), and MAGL (hydrolysis), was also modified by light. PLCß1 content was increased, while that of lipases was decreased. On the other hand, light produced an increase in the cannabinoid receptors CB1 and CB2 and a decrease in GPR55 protein levels. Taken together, our results indicate that the endocannabinoid system (enzymes and receptors) depends on the illumination state of the retina, suggesting that proteins related to phototransduction phenomena could be involved in the effects observed.
Assuntos
Endocanabinoides/metabolismo , Luz , Segmento Externo da Célula Bastonete/metabolismo , Segmento Externo da Célula Bastonete/efeitos da radiação , Animais , Bovinos , Lipase Lipoproteica/metabolismo , Modelos Biológicos , Monoacilglicerol Lipases/metabolismo , Fosfolipase C beta/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Canais de Cátion TRPV/metabolismoRESUMO
Even in immortalized cell lines, circadian clocks regulate physiological processes in a time-dependent manner, driving transcriptional and metabolic rhythms, the latter being able to persist without transcription. Circadian rhythm disruptions in modern life (shiftwork, jetlag, etc.) may lead to higher cancer risk. Here, we investigated whether the human glioblastoma T98G cells maintained quiescent or under proliferation keep a functional clock and whether cells display differential time responses to bortezomib chemotherapy. In arrested cultures, mRNAs for clock (Per1, Rev-erbα) and glycerophospholipid (GPL)-synthesizing enzyme genes, 32P-GPL labeling, and enzyme activities exhibited circadian rhythmicity; oscillations were also found in the redox state/peroxiredoxin oxidation. In proliferating cells, rhythms of gene expression were lost or their periodicity shortened whereas the redox and GPL metabolisms continued to fluctuate with a similar periodicity as under arrest. Cell viability significantly changed over time after bortezomib treatment; however, this rhythmicity and the redox cycles were altered after Bmal1 knock-down, indicating cross-talk between the transcriptional and the metabolic oscillators. An intrinsic metabolic clock continues to function in proliferating cells, controlling diverse metabolisms and highlighting differential states of tumor suitability for more efficient, time-dependent chemotherapy when the redox state is high and GPL metabolism low.
Assuntos
Antineoplásicos/farmacologia , Bortezomib/farmacologia , Proliferação de Células/efeitos dos fármacos , Relógios Circadianos/efeitos dos fármacos , Glioblastoma/metabolismo , Neurônios/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Relógios Circadianos/fisiologia , Glioblastoma/genética , Humanos , Neurônios/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , FosforilaçãoRESUMO
Phosphatidic acid (PA) is an important bioactive lipid that mediates chilling responses in barley. Modifications in the lipid composition of cellular membranes during chilling are essential to maintain their integrity and fluidity. First, we investigated the molecular species of PA present in leaves and roots by ESI-MS/MS, to evaluate the modifications that occur in response to chilling. We demonstrated that PA pools in leaves differ from PA fatty acid composition in roots. Compared with plants grown at 25⯰C, the short-term and long-term chilling for 3â¯h and 36â¯hâ¯at 4⯰C not produced significant changes in PA molecular species. The endogenous DAG and PA phosphorylation by in vitro DAG and PA kinase activities showed higher activity in leaves compared with that in root, and they showed contrasting responses to chilling. Similarly, PA removal by phosphatidate phosphohydrolase was tested, showing that this activity was specifically increased in response to chilling in roots. The findings presented here may be helpful to understand how the PA signal is modulated between tissues, and may serve to highlight the importance of knowing the basal PA pools in different plant organs.
Assuntos
Temperatura Baixa , Hordeum/metabolismo , Ácidos Fosfatídicos/metabolismo , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Diglicerídeos/metabolismo , Análise Fatorial , Hordeum/enzimologia , Monoglicerídeos/metabolismo , Solubilidade , Espectrometria de Massas por Ionização por Electrospray , Água/químicaRESUMO
Alzheimer's disease (AD) is the most prevalent disorder of senile dementia mainly characterized by amyloid-beta peptide (Aß) deposits in the brain. Cannabinoids are relevant to AD as they exert several beneficial effects in many models of this disease. Still, whether the endocannabinoid system is either up- or down-regulated in AD has not yet been fully elucidated. Thus, the aim of the present paper was to analyze endocannabinoid 2-arachidonoylglycerol (2-AG) metabolism in cerebral cortex synaptosomes incubated with Aß oligomers or fibrils. These Aß conformations were obtained by "aging" the 1-40 fragment of the peptide under different agitation and time conditions. A diminished availability of 2-AG resulting from a significant decrease in diacylglycerol lipase (DAGL) activity was observed in the presence of large Aß1-40 oligomers along with synaptosomal membrane damage, as judged by transmission electron microscopy and LDH release. Conversely, a high availability of 2-AG resulting from an increase in DAGL and lysophosphatidic acid phosphohydrolase activities occurred in the presence of Aß1-40 fibrils although synaptosomal membrane disruption was also observed. Interestingly, neither synaptosomal mitochondrial viability assayed by MTT reduction nor membrane lipid peroxidation assayed by TBARS formation measurements were altered by Aß1-40 oligomers or fibrils. These results show a differential effect of Aß1-40 peptide on 2-AG metabolism depending on its conformation.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Ácidos Araquidônicos/metabolismo , Endocanabinoides/metabolismo , Glicerídeos/metabolismo , Fragmentos de Peptídeos/metabolismo , Sinaptossomos/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/ultraestrutura , Animais , Córtex Cerebral/metabolismo , Córtex Cerebral/ultraestrutura , Humanos , Peroxidação de Lipídeos , Lipase Lipoproteica/metabolismo , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/ultraestrutura , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/patologia , Ratos Wistar , Sinaptossomos/ultraestruturaRESUMO
The aim of this work was to study how age-related changes could modify several enzymatic activities that regulate lipid mediator levels in nuclei from rat cerebellum and how these changes are modulated by all-trans retinoic acid (RA), docosahexaenoic acid (DHA) and arachidonic acid (AA). The higher phosphatidate phosphohydrolase activity and lower diacylglycerol lipase (DAGL) activity observed in aged animals compared with adults could augment diacylglycerol (DAG) availability in the former. Additionally, monoacylglycerol (MAG) availability could be high due to an increase in lysophosphatidate phosphohydrolase (LPAPase) activity and a decrease in monocylglycerol lipase activity. Interestingly, RA, DHA and AA were observed to modulate these enzymatic activities and this modulation was found to change in aged rats. In adult nuclei, whereas RA led to high DAG and MAG production through inhibition of their hydrolytic enzymes, DHA and AA promoted high MAG production by LPAPase and DAGL stimulation. In contrast, in aged nuclei RA caused high MAG generation whereas DHA and AA diminished it through LPAPase activity modulation. These results demonstrate that aging promotes a different nuclear lipid metabolism as well as a different type of non-genomic regulation by RA, DHA and AA, which could be involved in nuclear signaling events.
Assuntos
Envelhecimento , Ácido Araquidônico/química , Núcleo Celular/metabolismo , Ácidos Docosa-Hexaenoicos/química , Metabolismo dos Lipídeos , Tretinoína/química , Animais , Diglicerídeos/química , Glicerofosfatos/química , Homeostase , Hidrólise , Lipase/metabolismo , Monoglicerídeos/química , Ratos , Ratos Wistar , Transdução de SinaisRESUMO
2-Arachidonoylglycerol (2-AG) is one of the principal endocannabinoids involved in the protection against neurodegenerative processes. Cannabinoids primarily interact with the seven-segment transmembrane cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2), both of which are expressed in the central nervous system (CNS). The level of 2-AG is controlled through key enzymes responsible for its synthesis or degradation. We have previously observed a deregulation of 2-AG metabolism in physiological aging. The aim of this study was to analyze how 2-AG metabolism is modulated by CB1/CB2 receptors during aging. To this end, both CB1 and CB2 receptor expression and the enzymatic activities (diacylglycerol lipase (DAGL), lysophosphatidate phosphohydrolase (LPAase) and monoacylglycerol lipase (MAGL)) involved in 2-AG metabolism were analyzed in the presence of cannabinoid receptor (CBR) agonists (WIN and JWH) and/or antagonists (SR1 and SR2) in synaptosomes from adult and aged rat cerebral cortex (CC). Our results demonstrate that: (a) aging decreases the expression of both CBRs; (b) LPAase inhibition, due to the individual action of SR1 or SR2, is reverted in the presence of both antagonists together; (c) LPAase activity is regulated mainly by the CB1 receptor in adult and in aged synaptosomes while the CB2 receptor acquires importance when CB1 is blocked; (d) modulation via CBRs of DAGL and MAGL by both antagonists occurs only in aged synaptosomes, stimulating DAGL and inhibiting MAGL activities; (e) only DAGL stimulation is reverted by WIN. Taken together, the results of the present study show that CB1 and/or CB2 receptor antagonists trigger a significant modulation of 2-AG metabolism, underlining their relevance as therapeutic strategy for controlling endocannabinoid levels in physiological aging.
Assuntos
Envelhecimento/metabolismo , Ácidos Araquidônicos/metabolismo , Endocanabinoides/metabolismo , Glicerídeos/metabolismo , Receptores de Canabinoides/fisiologia , Animais , Membrana Celular/metabolismo , Córtex Cerebral/metabolismo , Lipase Lipoproteica/metabolismo , Monoacilglicerol Lipases/metabolismo , Fosfatidato Fosfatase/metabolismo , Ratos , Ratos Wistar , Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/antagonistas & inibidores , Receptor CB2 de Canabinoide/metabolismo , Receptores de Canabinoides/metabolismo , Sinaptossomos/metabolismoRESUMO
Circadian clocks regulate the temporal organization of several biochemical processes, including lipid metabolism, and their disruption leads to severe metabolic disorders. Immortalized cell lines acting as circadian clocks display daily variations in [(32)P]phospholipid labeling; however, the regulation of glycerophospholipid (GPL) synthesis by internal clocks remains unknown. Here we found that arrested NIH 3T3 cells synchronized with a 2 h-serum shock exhibited temporal oscillations in a) the labeling of total [(3)H] GPLs, with lowest levels around 28 and 56 h, and b) the activity of GPL-synthesizing and GPL-remodeling enzymes, such as phosphatidate phosphohydrolase 1 (PAP-1) and lysophospholipid acyltransferases (LPLAT), respectively, with antiphase profiles. In addition, we investigated the temporal regulation of phosphatidylcholine (PC) biosynthesis. PC is mainly synthesized through the Kennedy pathway with choline kinase (ChoK) and CTP:phosphocholine cytidylyltranferase (CCT) as key regulatory enzymes. We observed that the PC labeling exhibited daily changes, with the lowest levels every ~28 h, that were accompanied by brief increases in CCT activity and the oscillation in ChoK mRNA expression and activity. Results demonstrate that the metabolisms of GPLs and particularly of PC in synchronized fibroblasts are subject to a complex temporal control involving concerted changes in the expression and/or activities of specific synthesizing enzymes.
Assuntos
1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Colina Quinase/metabolismo , Ritmo Circadiano , Fibroblastos/metabolismo , Glicerofosfolipídeos/biossíntese , Fosfatidato Fosfatase/metabolismo , Animais , Células Cultivadas , Relógios Circadianos , Fibroblastos/citologia , Fibroblastos/enzimologia , Camundongos , Células NIH 3T3 , Proteínas Associadas a PancreatiteRESUMO
The aim of the present research was to analyze the pathways for phosphatidic acid metabolism in purified nuclei from liver. Lipid phosphate phosphatase, diacylglycerol lipase, monoacylglycerol lipase and PA-phospholipase type A activities were detected. The presence of lysophosphatidic acid significantly reduced DAG production while sphingosine 1-phoshate and ceramide 1-phosphate reduced MAG formation from PA. Using different enzymatic modulators (detergents and ions) an increase in the PA metabolism by phospholipase type A was observed. Our findings evidence an active PA metabolism in purified liver nuclei which generates important lipid second messengers, and which could thus be involved in nuclear processes such as gene transcription.
Assuntos
Núcleo Celular/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Ácidos Fosfatídicos/metabolismo , Animais , Cálcio/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Ceramidas/metabolismo , Diglicerídeos/metabolismo , Immunoblotting , Lipase Lipoproteica/metabolismo , Lisofosfolipídeos/metabolismo , Magnésio/farmacologia , Masculino , Microscopia Eletrônica , Monoacilglicerol Lipases/metabolismo , Monoglicerídeos/metabolismo , Octoxinol/farmacologia , Fosfatidato Fosfatase/metabolismo , Fosfolipases A/metabolismo , Ratos , Ratos Wistar , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMO
Phosphatidic acid (PA) is the common lipid product in abscisic acid (ABA) and gibberellic acid (GA) response. In this work we investigated the lipid metabolism in response to both hormones. We could detect an in vivo phospholipase D activity (PLD, EC 3.1.4.4). This PLD produced [(32)P]PA (phosphatidic acid) rapidly (minutes) in the presence of ABA, confirming PA involvement in signal transduction, and transiently, indicating rapid PA removal after generation. The presence of PA removal by phosphatidate phosphatase 1 and 2 isoforms (E.C. 3.1.3.4) was verified in isolated aleurone membranes in vitro, the former but not the latter being specifically responsive to the presence of GA or ABA. The in vitro DGPP phosphatase activity was not modified by short time incubation with GA or ABA while the in vitro PA kinase - that allows the production of 18:2-DGPP from 18:2-PA - is stimulated by ABA. The long term effects (24 h) of ABA or GA on lipid and fatty acid composition of aleurone layer cells were then investigated. An increase in PC and, to a lesser extent, in PE levels is the consequence of both hormone treatments. ABA, in aleurone layer cells, specifically activates a PLD whose product, PA, could be the substrate of PAP1 and/or PAK activities. Neither PLD nor PAK activation can be monitored by GA treatment. The increase in PAP1 activity monitored after ABA or GA treatment might participate in the increase in PC level observed after 24 h hormone incubation.
Assuntos
Ácido Abscísico/farmacologia , Giberelinas/farmacologia , Hordeum/metabolismo , Ácidos Fosfatídicos/metabolismo , Difosfatos/metabolismo , Glicerol/análogos & derivados , Glicerol/metabolismo , Hordeum/efeitos dos fármacos , Proteínas Associadas a Pancreatite , Fosfatidato Fosfatase/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
One of the principal monoacylglycerol (MAG) species in animal tissues is 2-arachidonoylglycerol (2-AG), and the diacylglycerol lipase (DAGL) pathway is the most important 2-AG biosynthetic pathway proposed to date. Lysophosphatidate phosphatase (LPAase) activity is part of another 2-AG-forming pathway in which monoacylglycerol lipase (MAGL) is the major degrading enzyme. The purpose of this study was to analyze the manner in which DAGL, LPAase, and MAGL enzymes are modified in the central nervous system (CNS) during aging. To this end, diacylglycerols (DAGs) and MAGs of different composition were used as substrates of DAGL and MAGL, respectively. All enzymatic activities were evaluated in membrane and soluble fractions as well as in synaptic terminals from the cerebral cortex (CC) of adult and aged rats. Results related to 2-AG metabolism show that aging: (a) decreases DAGL-α expression in the membrane fraction whereas in synaptosomes it increases DAGL-ß and decreases MAGL expression; (b) decreases LPAase activity in both membrane and soluble fractions; (c) decreases DAGL and stimulates LPAase activities in CC synaptic terminals; (d) stimulates membrane-associated MAGL-coupled DAGL activity; and (e) stimulates MAGL activity in CC synaptosomes. Our results also reveal that during aging the net balance between the enzymatic activities involved in 2-AG synthesis and breakdown is low availability of 2-AG in CC membrane fractions and synaptic terminals. Taken together, our results lead us to conclude that these enzymes play crucial roles in the regulation of 2-AG tissue levels during aging.
Assuntos
Envelhecimento/fisiologia , Ácidos Araquidônicos/metabolismo , Endocanabinoides/metabolismo , Glicerídeos/metabolismo , Animais , Córtex Cerebral/enzimologia , Córtex Cerebral/metabolismo , Diglicerídeos/metabolismo , Ácidos Graxos/metabolismo , Immunoblotting , Lipase Lipoproteica/metabolismo , Masculino , Monoacilglicerol Lipases/metabolismo , Monoglicerídeos/metabolismo , Ratos , Ratos Wistar , Sinaptossomos/enzimologia , Sinaptossomos/metabolismoRESUMO
We analyzed lipid kinase and lipid phosphatase activities and determined endogenous phytohormone levels by liquid chromatography-tandem mass spectrometry in root and coleoptile tissues following germination of barley (Hordeum vulgare) seeds. The enzymes showing highest activity in aleurone cells were diacylglycerol kinase (DAG-k, EC 2.7.1.107) and phosphatidate kinase (PA-k). The ratio of gibberellins (GAs) to abscisic acid (ABA) was 2-fold higher in aleurone than in coleoptile or root tissues. In coleoptiles, phosphatidylinositol 4-kinase (PI4-k, EC 2.7.1.67) showed the highest enzyme activity, and jasmonic acid (JA) level was higher than in aleurone. In roots, activities of PI4-k, DAG-k, and PA-k were similar, and salicylic acid (SA) showed the highest concentration. In the assays to evaluate the hydrolysis of DGPP (diacylglycerol pyrophosphate) and PA (phosphatidic acid) we observed that PA hydrolysis by LPPs (lipid phosphate phosphatases) was not modified; however, the diacylglycerol pyrophosphate phosphatase (DGPPase) was strikingly higher in coleoptile and root tissues than to aleurone. Relevance of these findings in terms of signaling responses and seedling growth is discussed.
Assuntos
Cotilédone/metabolismo , Hordeum/enzimologia , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/metabolismo , Sementes/metabolismo , 1-Fosfatidilinositol 4-Quinase/metabolismo , Diacilglicerol Quinase/metabolismo , Difosfatos/metabolismo , Germinação/fisiologia , Glicerol/análogos & derivados , Glicerol/metabolismo , Glicerofosfatos/metabolismo , Hordeum/crescimento & desenvolvimento , Hordeum/metabolismo , Fosfatidato Fosfatase/metabolismo , Ácidos Fosfatídicos/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Proteínas de Plantas/metabolismo , Pirofosfatases/metabolismo , Transdução de SinaisRESUMO
Lipid kinases and phosphatases play essential roles in signal transduction processes involved in cytoskeletal rearrangement, membrane trafficking, and cellular differentiation. Phosphatidic acid (PtdOH) is an important mediator lipid in eukaryotic cells, but little is known regarding its regulation in the parasite Trypanosoma cruzi, an agent of Chagas disease. In order to clarify the relationship between PtdOH metabolism and developmental stages of T. cruzi, epimastigotes in culture were subjected to hyperosmotic stress (~1,000 mOsm/L), mimicking the environment in the rectum of vector triatomine bugs. These experimental conditions resulted in differentiation to an intermediate form between epimastigotes and trypomastigotes. Morphological changes of epimastigotes were correlated with an increase in PtdOH mass accomplished by increased enzyme activity of diacylglycerol kinase (DAGK, E.C. 2.7.1.107) and concomitant decreased activity of phosphatidate phosphatases type 1 and type 2 (PAP1, PAP2, E.C. 3.1.3.4). Our results indicate progressive increases of PtdOH levels during the differentiation process, and suggest that the regulation of PtdOH metabolism is an important mechanism in the transition from T. cruzi epimastigote to intermediate form.
Assuntos
Doença de Chagas/parasitologia , Ácidos Fosfatídicos/metabolismo , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/crescimento & desenvolvimento , Sequência de Aminoácidos , Diacilglicerol Quinase/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas Associadas a Pancreatite , Fosfatidato Fosfatase/metabolismo , Trypanosoma cruzi/metabolismoRESUMO
This paper explores the way ceramide, sphingosine, ceramide 1-phosphate, and sphingosine 1-phosphate modulate the generation of second lipid messengers from phosphatidic acid in two experimental models of the central nervous system: in vertebrate rod outer segments prepared from dark-adapted retinas as well as in rod outer segments prepared from light-adapted retinas and in rat cerebral cortex synaptosomes under physiological aging conditions. Particular attention is paid to lipid phosphate phosphatase, diacylglycerol lipase, and monoacylglycerol lipase. Based on the findings reported in this paper, it can be concluded that proteins related to phototransduction phenomena are involved in the effects derived from sphingosine 1-phosphate/sphingosine or ceramide 1-phosphate/ceramide and that age-related changes occur in the metabolism of phosphatidic acid from cerebral cortex synaptosomes in the presence of either sphingosine 1-phosphate/sphingosine or ceramide 1-phosphate/ceramide. The present paper demonstrates, in two different models of central nervous system, how sphingolipids influence phosphatidic acid metabolism under different physiological conditions such as light and aging.
RESUMO
The aim of the present research was to analyse the pathways for phosphatidic acid metabolism in purified nuclei from cerebellar cells. Lipid phosphate phosphatase and diacylglyceride lipase activities were detected in nuclei from cerebellar cells. It was observed that DAGL activity makes up 50% of LPP activity and that PtdOH can also be metabolised to lysophosphatidic acid. With a nuclear protein content of approximately 40 µg, the production of diacylglycerol and monoacylglycerol was linear for 30 min and 5 min, respectively, whereas it increased with PtdOH concentrations of up to 250 µM. LysoPtdOH, sphingosine 1-phosphate and ceramide 1-phosphate, which are alternative substrates for LPP, significantly reduced DAG production from PA. DAG and MAG production increased in the presence of Triton X-100 (1 mM) whereas no modifications were observed in the presence of ionic detergent sodium deoxycholate. Ca²+ and Mg²+ stimulated MAG production without affecting DAG formation whereas fluoride and vanadate inhibited the generation of both products. Specific PtdOH-phospholipase A1 and PtdOH-phospholipase A2 were also detected in nuclei. Our findings constitute the first reported evidence of active PtdOH metabolism involving LPP, DAGL and PtdOH-selective PLA activities in purified nuclei prepared from cerebellar cells.
Assuntos
Núcleo Celular/metabolismo , Cerebelo/citologia , Redes e Vias Metabólicas , Ácidos Fosfatídicos/metabolismo , Animais , Cálcio/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Ceramidas/metabolismo , Cerebelo/efeitos dos fármacos , Cerebelo/enzimologia , Cerebelo/metabolismo , Detergentes/farmacologia , Diglicerídeos/biossíntese , Diglicerídeos/metabolismo , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Lisofosfolipídeos/metabolismo , Magnésio/farmacologia , Masculino , Monoglicerídeos/biossíntese , Monoglicerídeos/metabolismo , Fosfolipases A1/metabolismo , Fosfolipases A2/metabolismo , Ratos , Ratos Wistar , Fluoreto de Sódio/farmacologia , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Fatores de Tempo , Vanadatos/farmacologiaRESUMO
Rod outer segments (ROSs) are specialized light-sensitive organelles in vertebrate photoreceptor cells. Lipids in ROS are of considerable importance, not only in providing an adequate environment for efficient phototransduction, but also in originating the second messengers involved in signal transduction. ROSs have the ability to adapt the sensitivity and speed of their responses to ever-changing conditions of ambient illumination. A major contributor to this adaptation is the light-driven translocation of key signaling proteins into and out of ROS. The present review shows how generation of the second lipid messengers from phosphatidylcholine, phosphatidic acid, and diacylglycerol is modulated by the different illumination states in the vertebrate retina. Findings suggest that the light-induced translocation of phototransduction proteins influences the enzymatic activities of phospholipase D, lipid phosphate phosphatase, diacylglyceride lipase, and diacylglyceride kinase, all of which are responsible for the generation of the second messenger molecules.
Assuntos
Metabolismo dos Lipídeos , Lipídeos/fisiologia , Segmento Externo da Célula Bastonete/enzimologia , Segmento Externo da Célula Bastonete/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Animais , Diglicerídeos/metabolismo , Humanos , Transdução de Sinal Luminoso , Ácidos Fosfatídicos/metabolismo , Fosfatidilcolinas/metabolismo , Transporte ProteicoRESUMO
ABA plays an important regulatory role in seed germination because it inhibits the response to GA in aleurone, a secretory tissue surrounding the endosperm. Phosphatidic acid (PA) is a well-known intermediary in ABA signaling, but the role of diacylglycerol pyrophosphate (DGPP) in germination processes is not clearly established. In this study, we show that PA produced by phospholipase D (E.C. 3.1.4.4) during the antagonist effect of ABA in GA signaling is rapidly phosphorylated by phosphatidate kinase (PAK) to DGPP. This is a crucial fact for aleurone function because exogenously added dioleoyl-DGPP inhibits secretion of alpha-amylase (E.C. 3.2.1.1). Aleurone treatment with ABA and 1-butanol results in normal secretory activity, and this effect is reversed by addition of dioleoyl-DGPP. We also found that ABA decreased the activity of an Mg2+-independent, N-ethylmaleimide-insensitive form of phosphatidate phosphohydrolase (PAP2) (E.C. 3.1.3.4), leading to reduction of PA dephosphorylation and increased PAK activity. Sequence analysis using Arabidopsis thaliana lipid phosphate phosphatase (LPP) sequences as queries identified two putative molecular homologues, termed HvLPP1 and HvLPP2, encoding putative Lpps with the presence of well-conserved structural Lpp domains. Our results are consistent with a role of DGPP as a regulator of ABA antagonist effect in GA signaling and provide evidence about regulation of PA level by a PAP2 during ABA response in aleurone.
Assuntos
Difosfatos/farmacologia , Giberelinas/farmacologia , Glicerol/análogos & derivados , Hordeum/efeitos dos fármacos , Hordeum/enzimologia , Sementes/efeitos dos fármacos , Sementes/enzimologia , alfa-Amilases/metabolismo , 1-Butanol/farmacologia , Ácido Abscísico/farmacologia , Sequência de Aminoácidos , Arabidopsis/enzimologia , Diacilglicerol Quinase/metabolismo , Inibidores Enzimáticos/farmacologia , Glicerol/farmacologia , Dados de Sequência Molecular , Fosfatidato Fosfatase/química , Fosfatidato Fosfatase/metabolismo , Ácidos Fosfatídicos/metabolismo , Ácidos Fosfatídicos/farmacologia , Fosfolipase D/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Filogenia , Proteínas Quinases/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The aim of the present research was to evaluate the generation of [2-3H]diacylglycerol ([2-3H]DAG) from [2-3H]-Phosphatidic acid ([2-3H]PA) by lipid phosphate phosphatases (LPPs) at different concentrations of lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P), and ceramide 1-phosphate (C1P) in purified ROS obtained from dark-adapted retinas (DROS) or light-adapted retinas (BLROS) as well as in ROS membrane preparations depleted of soluble and peripheral proteins. Western blot analysis revealed the presence of LPP3 exclusively in all membrane preparations. Immunoblots of entire ROS and depleted ROS did not show dark-light differences in LPP3 levels. LPPs activities were diminished by 53% in BLROS with respect to DROS. The major competitive effect on PA hydrolysis was exerted by LPA and S1P in DROS and by C1P in BLROS. LPPs activities in depleted ROS were similar to the activity observed in entire DROS and BLROS, respectively. LPA, S1P and C1P competed at different extent in depleted DROS and BLROS. Sphingosine and ceramide inhibited LPPs activities in entire and depleted DROS. Ceramide also inhibited LPPs activities in entire and in depleted BLROS. Our findings are indicative of a different degree of competition between PA and LPA, S1P and C1P by LPPs depending on the illumination state of the retina.
Assuntos
Ceramidas/fisiologia , Lisofosfolipídeos/fisiologia , Fosfatidato Fosfatase/metabolismo , Ácidos Fosfatídicos/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Esfingosina/análogos & derivados , Adaptação Ocular/fisiologia , Animais , Western Blotting , Soluções Tampão , Bovinos , Separação Celular , Ceramidas/metabolismo , Adaptação à Escuridão/fisiologia , Eletroforese em Gel de Poliacrilamida , Hidrólise , Espécies Reativas de Oxigênio/metabolismo , Segmento Externo da Célula Bastonete/enzimologia , Esfingosina/metabolismo , Esfingosina/fisiologiaRESUMO
Insulin receptor associated with the cerebral cortex (CC) has been shown to be involved in brain cognitive functions. Furthermore, deterioration of insulin signaling has been associated with age-related brain degeneration. We have reported previously that aging stimulates phospholipase D/phosphatidate phosphohydrolase 2 (PLD/PAP2) pathway in CC synaptosomes from aged rats, generating a differential availability of their reaction products: diacylglycerol (DAG) and phosphatidic acid (PA). The aim of this work was to determine the effect of aging on DAG kinase (DAGK), as an alternative pathway for PA generation, and to evaluate the effect of insulin on PLD/PAP2 pathway and DAGK. PLD, PAP2, and DAGK activities were measured using specific radiolabeled substrates in CC synaptosomes from adult (4 months old) and aged rats (28 months old). In adult animals, in the presence of the tyrosine phosphatase inhibitor (sodium o-vanadate), insulin stimulated PLD activity at 5 min incubation. DAGK activity was also increased at the same time of incubation and PAP2 was inhibited. In aged animals, PLD activity was not modified by the presence of insulin plus vanadate, PAP2 was inhibited, and DAGK was stimulated by the hormone. Insulin, vanadate, and the combination of both induced protein tyrosine phosphorylation in adult CC synaptosomes. Aged rats showed a lower level of protein phosphorylation with respect to adult rats. Our results show that insulin modulates PA and DAG availability through the regulation of PLD/PAP2 and DAGK pathways in adult rat CC synaptosomes. Additionally, we demonstrated that PA and DAG generation is regulated differentially by insulin during aging.
Assuntos
Envelhecimento/fisiologia , Diacilglicerol Quinase/metabolismo , Insulina/fisiologia , Fosfatidato Fosfatase/metabolismo , Fosfolipase D/metabolismo , Sinaptossomos/enzimologia , Animais , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , Diacilglicerol Quinase/efeitos dos fármacos , Diglicerídeos/metabolismo , Inibidores Enzimáticos/farmacologia , Fosfatidato Fosfatase/efeitos dos fármacos , Ácidos Fosfatídicos/metabolismo , Fosfolipase D/efeitos dos fármacos , Ratos , Ratos Wistar , Receptor de Insulina/efeitos dos fármacos , Receptor de Insulina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Sinaptossomos/efeitos dos fármacos , Vanadatos/farmacologiaRESUMO
The present study demonstrates that the biosynthesis of phospholipids in the inner nuclear layer cells of the chicken retina displays daily rhythms under constant illumination conditions. The vertebrate retina contains circadian oscillators and photoreceptors (PRCs) that temporally regulate its own physiology and synchronize the whole organism to the daily environmental changes. We have previously reported that chicken photoreceptors and retinal ganglion cells (RGCs) present significant daily variations in their phospholipid biosynthesis under constant illumination conditions. Herein, we demonstrate that cell preparations highly enriched in inner nuclear layer cells also exhibit a circadian-regulated phospholipid labeling after the in vivo administration of [(32)P]phosphate or [(3)H]glycerol both in animals maintained under constant darkness or light for at least 48h. In constant darkness, there was a significant incorporation of both precursors into phospholipids with the highest levels of labeling around midday and dusk. In constant light, the labeling of (32)P-phospholipids was also significantly higher during the day and early night whereas the incorporation of [(3)H]glycerol into phospholipids, that indicates de novo biosynthesis, was greater during the day but probably reflecting a higher precursor availability at those phases. We also measured the in vitro activity of phosphatidate phosphohydrolase and diacylglycerol lipase in preparations obtained from the dark condition. The two enzymes exhibited the highest activity levels late in the day. When we assessed the in vitro incorporation of [(14)C]oleate into different lysophospholipids from samples collected at different phases in constant darkness, reaction catalyzed by lysophospholipid acyltransferases II, labeling showed a complex pattern of daily activity. Taken together, these results demonstrate that the biosynthesis of phospholipids in cells of the chicken retinal inner nuclear layer exhibits a daily rhythmicity under constant illumination conditions, which is controlled by a circadian clock.