ABSTRACT
Mono- and diglycerides play a crucial role in the food industry as multifunctional food additives and emulsifiers. Their importance stems from their unique properties, which allow them to improve the quality, texture, and stability of various food products. Here, results of the kinetic modeling of the mono- and diglycerides synthesis mediated by the lipase Lipozyme® TL 100 L immobilized on the clayey support Spectrogel® type C are reported. The support was characterized by TEM, SEM, and FTIR. Firstly, the influence of pH and lipase load on the immobilization process was analyzed, resulting in an enzymatic activity of 93.2 ± 0.7 U g-1 under optimized conditions (170.9 U g-1 of lipase and pH of 7.1). Afterward, the effects of reaction temperature and concentration of immobilized biocatalyst in the feedstock conversion were evaluated. At optimized parameters, a triglycerides conversion of 97% was obtained at 36.5 °C, 7.9 vol.% of enzyme, a glycerol to feedstock molar ratio of 2:1, and 2 h. The optimized conditions were used to determine the kinetic constants of the elementary reactions involved in the glycerolysis, where a fit superior to 0.99 was achieved between experimental values and predicted data.
Subject(s)
Enzymes, Immobilized , Lipase , Lipase/chemistry , Lipase/metabolism , Enzymes, Immobilized/chemistry , Kinetics , Diglycerides/chemistry , Diglycerides/biosynthesis , Clay/chemistry , Hydrogen-Ion Concentration , Temperature , Models, ChemicalABSTRACT
The glycerophospholipids phosphatidylethanolamine, phosphatidylglycerol (PG), and cardiolipin (CL) are major structural components of bacterial membranes. In some bacteria, phosphatidylcholine or phosphatidylinositol and its derivatives form part of the membrane. PG or CL can be modified with the amino acid residues lysine, alanine, or arginine. Diacylglycerol is the lipid anchor from which syntheses of phosphorus-free glycerolipids, such as glycolipids, sulfolipids, or homoserine-derived lipids initiate. Many membrane lipids are subject to turnover and some of them are recycled. Other lipids associated with the membrane include isoprenoids and their derivatives such as hopanoids. Ornithine-containing lipids are widespread in Bacteria but absent in Archaea and Eukarya. Some lipids are probably associated exclusively with the outer membrane of many bacteria, i.e. lipopolysaccharides, sphingolipids, or sulfonolipids. For certain specialized membrane functions, specific lipid structures might be required. Upon cyst formation in Azotobacter vinelandii, phenolic lipids are accumulated in the membrane. Anammox bacteria contain ladderane lipids in the membrane surrounding the anammoxosome organelle, presumably to impede the passage of highly toxic compounds generated during the anammox reaction. Considering that present knowledge on bacterial lipids was obtained from only a few bacterial species, we are probably only starting to unravel the full scale of lipid diversity in bacteria. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.
Subject(s)
Bacteria/metabolism , Diglycerides/biosynthesis , Glycerophospholipids/biosynthesis , Lipogenesis , Membrane Lipids/biosynthesis , Diglycerides/chemistry , Diglycerides/classification , Glycerophospholipids/chemistry , Glycerophospholipids/classification , Membrane Lipids/chemistry , Membrane Lipids/classification , Molecular Structure , Structure-Activity RelationshipABSTRACT
The diacylglycerols (DAG) are emulsifiers with high added value used as functional additives in food, medicine, and cosmetic industries. In glycerolysis of oils for the production of DAG, the immiscibility between the substrates (glycerol and oil phases) has to be overcome, for example, by the addition of a food grade surfactant like Tween 65. The main objective of this work was to optimize the process conditions for obtaining diacylglycerols rich in the omega-3 eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, through the enzymatic glycerolysis of fish oil, in systems with Tween 65 and without this surfactant, using Lipozyme TL(®) IM as biocatalyst. The experiments were performed according to predetermined conditions varying the temperature, enzyme load, the oil to glycerol molar ratio and, when added, the surfactant load. After 6 h of reaction, it was possible to produce up to 20.76 and 13.76% of diacylglycerols from fish oil in the reactions with and without Tween 65, respectively.
Subject(s)
Diglycerides/biosynthesis , Fish Oils/metabolism , Glycerol/metabolism , Lipase/metabolism , Surface-Active Agents/chemistry , Catalysis , HydrolysisABSTRACT
Diacylglycerol (DAG) rich oils have an organoleptic property like that of regular edible oils, but these oils do not tend to be accumulated as fat. Palm oil ranks first in the world in terms of edible oil production owing to its low cost. The aim of this study was to propose a new methodology to produce diacylglycerol by hydrolysis of palm oil using Lipozyme RM IM commercial lipase as a catalyst under ultrasound irradiation. The reactions were carried out at 55 °C with two different methods. First, the reaction system was exposed to ultrasonic waves for the whole reaction time, which led to enzymatic inactivation and water evaporation. Ultrasound was then used to promote emulsification of the water/oil system before the hydrolysis reaction, avoiding contact between the probe and the enzymes. An experimental design was used to optimize the ultrasound-related parameters and maximize the hydrolysis rate, and in these conditions, with a change in equilibrium, DAG production was evaluated. Better reaction conditions were achieved for the second method: 11.20 wt.% (water+oil mass) water content, 1.36 wt.% (water+oil mass) enzyme load, 12 h of reaction time, 1.2 min and 200 W of exposure to ultrasound. In these conditions diacylglycerol yield was 34.17 wt.%.
Subject(s)
Diglycerides/biosynthesis , Lipase/metabolism , Plant Oils/metabolism , Sonication , Biocatalysis , Diglycerides/chemistry , Hydrolysis , Palm Oil , Plant Oils/chemistryABSTRACT
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.
Subject(s)
Cell Nucleus/metabolism , Cerebellum/cytology , Metabolic Networks and Pathways , Phosphatidic Acids/metabolism , Animals , Calcium/pharmacology , Cell Nucleus/drug effects , Cell Nucleus/enzymology , Ceramides/metabolism , Cerebellum/drug effects , Cerebellum/enzymology , Cerebellum/metabolism , Detergents/pharmacology , Diglycerides/biosynthesis , Diglycerides/metabolism , Hydrogen-Ion Concentration , In Vitro Techniques , Lysophospholipids/metabolism , Magnesium/pharmacology , Male , Monoglycerides/biosynthesis , Monoglycerides/metabolism , Phospholipases A1/metabolism , Phospholipases A2/metabolism , Rats , Rats, Wistar , Sodium Fluoride/pharmacology , Sphingosine/analogs & derivatives , Sphingosine/metabolism , Time Factors , Vanadates/pharmacologyABSTRACT
Distilled glycerides are obtained through distillation of the system mono-diglycerides which is produced from the esterification reaction between a triglyceride with glycerol. In this work, monoglycerides (MG) and diglycerides (DG) are produced through lipase-catalyzed glycerolysis of soybean oil using Candida antarctica B in a solvent-free system. To separate the products of the reaction in order to obtain essentially MG and an oil of DG, it is necessary to use a suitable process in order to preserve the stability of the components and to keep the products free of inappropriate solvents. So, after 24 h of enzymatic reaction, the mixture of acylglycerols and fatty acids was distilled into a centrifugal molecular distiller, since it provides a free solvent and lower temperature environment to increase the desired product concentration. Starting from a material with 25.06% of triglycerides (TG), 46.63% of DG, 21.72% of MG, 5.38% of free fatty acids (FFA), and 1.21% of glycerol, the MG purity in the distillate stream was 80% at evaporator temperature (T (E)) equal to 250 degrees C and feed flow rate (Q) equal to 10.0 mL/min. At these conditions, the MG recovery was 35%. The material collected in the residue stream presented DG-enriched oil with TG unhydrolyzed, residual MG, and low acidity (29.83% of TG, 53.20% of DG, 15.64% of MG, and 1.33% of FFA), which is suitable to replace TG oil in the human diet.
Subject(s)
Chemical Fractionation/methods , Diglycerides/biosynthesis , Diglycerides/isolation & purification , Glycerol/metabolism , Lipase/metabolism , Monoglycerides/biosynthesis , Monoglycerides/isolation & purification , Biocatalysis , Candida/enzymology , Centrifugation/methods , Chromatography, High Pressure Liquid , Diglycerides/chemistry , Enzymes, Immobilized/metabolism , Fungal Proteins , Monoglycerides/chemistry , Particle Size , Soybean Oil/chemistry , Soybean Oil/metabolism , TemperatureABSTRACT
In the bacterial model organism Escherichia coli only the three major membrane lipids phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin occur, all of which belong to the glycerophospholipids. The amino acid-containing phosphatidylserine is a major lipid in eukaryotic membranes but in most bacteria it occurs only as a minor biosynthetic intermediate. In some bacteria, the anionic glycerophospholipids phosphatidylglycerol and cardiolipin can be decorated with aminoacyl residues. For example, phosphatidylglycerol can be decorated with lysine, alanine, or arginine whereas in the case of cardiolipin, lysine or d-alanine modifications are known. In few bacteria, diacylglycerol-derived lipids can be substituted with lysine or homoserine. Acyl-oxyacyl lipids in which the lipidic part is amide-linked to the alpha-amino group of an amino acid are widely distributed among bacteria and ornithine-containing lipids are the most common version of this lipid type. Only few bacterial groups form glycine-containing lipids, serineglycine-containing lipids, sphingolipids, or sulfonolipids. Although many of these amino acid-containing bacterial membrane lipids are produced in response to certain stress conditions, little is known about the specific molecular functions of these lipids.
Subject(s)
Amino Acids/metabolism , Bacteria/metabolism , Membrane Lipids/metabolism , Bacteria/enzymology , Cardiolipins/biosynthesis , Cardiolipins/metabolism , Diglycerides/biosynthesis , Diglycerides/metabolism , Glycerophospholipids/biosynthesis , Glycerophospholipids/metabolism , Membrane Lipids/biosynthesis , Membrane Lipids/chemistry , Phosphatidylglycerols/biosynthesis , Phosphatidylglycerols/metabolism , Serine C-Palmitoyltransferase/classification , Serine C-Palmitoyltransferase/metabolism , Sphingolipids/biosynthesis , Sphingolipids/metabolismABSTRACT
Age-related changes in insulin action on diacylglycerol (DAG) degradation was studied in rat cerebral cortex synaptosomes. The generation of monoacylglycerol (MAG) and water soluble products (WSP, glycerol plus glycerol-3-phosphate) from DAG was studied in cerebral cortex (CC) synaptosomes from adult (4-month-old) and aged (28-month-old) rats. Additionally, the effect of porcine insulin and tyrosine phosphorylation was evaluated in the same group of animals. In this study we demonstrate that the age-related increase in WSP generation was accompanied by unmodified MAG levels. In the presence of diacylglycerol lipase (DAG lipase) inhibitor, RHC-80267, a lower inhibitory effect on MAG production was observed in CC synaptosomes from aged rats with respect to that in adult membranes. Under these experimental conditions, WSP formation was only diminished in aged membranes. Insulin stimulated MAG and WSP formation at long incubation times (30 min) in adult animals, while it had an inhibitory effect in aged animals. Insulin plus vanadate (as tyrosine-phosphatase inhibitor) inhibited MAG production at short incubation times whereas the same effect was observed in aged animals at long times of incubation. WSP formation was stimulated by insulin plus vanadate both in adult and aged animals at 30 min of incubation. Our results show that insulin differentially modulates MAG and WSP production from exogenous PA in CC synaptosomes from aged rats compared with adult rats.
Subject(s)
Aging , Diglycerides/biosynthesis , Hydrolysis , Insulin/metabolism , Phosphatidic Acids/metabolism , Animals , Cerebral Cortex/enzymology , Diglycerides/metabolism , Lipoprotein Lipase/antagonists & inhibitors , Male , Monoglycerides/biosynthesis , Rats , Rats, Wistar , Synaptosomes/enzymologyABSTRACT
Esterification of glycerol with conjugated linoleic acid (CLA) was carried out in hexane. Lipase from Rhizomucor miehei provided a high degree of esterification (80%) in 8 h at 50 degrees C when used at 15% (w/w) in a system containing a 1:2 molar ratio of glycerol to free fatty acids. Esterification levels >80% were obtained in 8 h at 40 degrees C with 15% (w/w) lipase from Candida antarctica at the same molar ratio of reactants. The extent of esterification of CLA was >90% after 4 h of reaction at 50 degrees C with a 5% (w/w) loading of either R. miehei or C. antarctica lipase, together with a 1:1 molar ratio of substrates. Both enzymes incorporated the original CLA as acylglycerol residues in primarily 1,3-diacylglycerol and 1-monoacylglycerol. The CLA-rich acylglycerols can be employed as emulsifiers or as substitutes for natural fats and oils.
Subject(s)
Diglycerides/biosynthesis , Glycerides/biosynthesis , Glycerol/metabolism , Linoleic Acids, Conjugated/metabolism , Lipase/metabolism , Chromatography, High Pressure Liquid , Enzymes, Immobilized , Food Technology/methods , Glycerides/chemistry , Hexanes , Rhizomucor/enzymology , TemperatureABSTRACT
Intestinal extracts of Triatoma infestans induce cell differentiation of Trypanosoma cruzi epimastigotes into the infective metacyclic form. Part of this effect can be explained by the presence of haemoglobin fragments, which stimulate trypanosomal adenylate cyclase. In this work we examined the metacyclogenic activity of lipids present in this intestinal extract. We found that lipid extracts of the intestinal extract have significant stimulatory effects that reside with the free-fatty-acid fraction, especially oleic acid. These compounds stimulate de novo diacylglycerol formation and protein kinase C activity in the parasite. Moreover, metacyclogenesis is stimulated by phorbol esters and cell-permeant diacylglycerol, while protein kinase C down-regulation or incubation with inhibitors of this kinase abrogates this effect. These results indicate that free fatty acids are a novel signal, inducing metacyclogenesis, acting through a pathway involving diacylglycerol biosynthesis and protein kinase C activation.
Subject(s)
Cell Differentiation/drug effects , Fatty Acids, Nonesterified/pharmacology , Sulfonamides , Trypanosoma cruzi/drug effects , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Animals , Bucladesine/pharmacology , Cell Division/drug effects , Cyclic AMP-Dependent Protein Kinases/metabolism , Diglycerides/biosynthesis , Diglycerides/pharmacology , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Fatty Acids, Nonesterified/pharmacokinetics , Intestines/chemistry , Isoquinolines/pharmacology , Oleic Acid/pharmacokinetics , Oleic Acid/pharmacology , Phosphorylation/drug effects , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Triatoma/chemistry , Trypanosoma cruzi/cytology , Trypanosoma cruzi/metabolismABSTRACT
Apoptosis is a means by which organisms dispose of unwanted cells without inducing an inflammatory response. Alterations in apoptosis is a common process by which cells become cancerous. Paradoxically, many cancer chemotherapeutics preferentially kill cancer cells by inducing apoptosis. Diacylglycerol is a lipid second messenger that regulates cell growth and apoptosis and is produced during signal transduction by hydrolysis of membrane phospholipids. Protein kinase Cs are a family of diacyglycerol responsive enzymes that are recruited to cellular membranes as a consequence of diacylglycerol production where they phosphorylate specific target proteins responsible for regulating cell growth. In this review, we will first summarize our current understanding of the role of specific proteins kinase C isoforms in the induction of cell growth/apoptosis. Subsequently, we will discuss how insights gained in lipid-mediated regulation of protein kinase Cs promotes our understanding of the role specific family members play in regulating cell growth. Finally, other diacylglycerol binding proteins involved in regulating apoptosis will be discussed.
Subject(s)
Antibodies, Antiphospholipid/biosynthesis , Apoptosis/physiology , Diglycerides/physiology , Phospholipids/biosynthesis , Protein Kinase C/physiology , Animals , Carrier Proteins/physiology , Cell Division , Diglycerides/biosynthesis , Enzyme Activation , Farnesol/metabolism , Farnesol/pharmacology , Humans , Phosphorylation , Protein Isoforms/metabolism , Protein Isoforms/physiology , Protein Kinase C/metabolism , Second Messenger Systems , Signal TransductionABSTRACT
Similar to those of other species, the Harderian glands of armadillo produce an abundant lipid secretion, most of which is composed of 1-alkyl-2,3-diacylglycerol. Biosynthesis of this component is apparently performed with the participation of one cytosolic pool of acyl-CoA and another of free fatty acids. The acyl-CoA-binding protein (ACBP) is present at a concentration at least 7-fold that of the heart-type fatty acid-binding protein (H-FABP), though lower than that in other armadillo organs such as liver and brain. The ACBP complete amino acid sequence was determined by Edman degradation of peptides generated by cleavage of the protein with cyanogen bromide, endopeptidase Glu-C, and trypsin. ACBP consists of 86 residues and has a calculated molecular mass of 9783 Da, taking into account that an acetyl group is blocking the N-terminus. Identity percentages between armadillo Harderian gland ACBP and other known ACBPs show that the protein belongs to the liver-specific ACBP isoform (L-ACBP). The fact that the ACBP concentration is higher than that of FABP suggests that the Harderian gland is able to store acyl-CoA amounts in ACBP larger than those of fatty acids in H-FABP for 1-alkyl-2,3-diacylglycerol synthesis.
Subject(s)
Armadillos/metabolism , Carrier Proteins/chemistry , Harderian Gland/chemistry , Lipid Metabolism , Neoplasm Proteins , Tumor Suppressor Proteins , Amino Acid Sequence , Animals , Carrier Proteins/isolation & purification , Carrier Proteins/physiology , Chromatography, Gel , Chromatography, Thin Layer , Cytosol/chemistry , Diazepam Binding Inhibitor , Diglycerides/biosynthesis , Fatty Acid-Binding Protein 7 , Fatty Acid-Binding Proteins , Female , Harderian Gland/metabolism , Humans , Mammals/metabolism , Molecular Sequence Data , Organ Specificity , Sequence Alignment , Species SpecificityABSTRACT
[(14)C]Oleic acid injected into the hemocoel of Rhodnius prolixus females was shown to rapidly associate with lipophorin particles. Half of the lipophorin-associated [(14)C]oleic acid was transferred in about 5 min to different organs, but the midgut was the main organ to take it up on day 10 after a blood meal. The rate of [(14)C]oleic acid incorporation by the midgut was high up to 15 min after injection and then declined. The [(14)C]oleic acid incorporated by the midgut was found in phospholipids (58.6%) and neutral lipids (37.4%). The midgut capacity to incorporate [(14)C]oleic acid varied on different days after a meal: it increased up to day 10 and then decreased. The fate of the [(14)C]lipids synthesized by the midgut was followed and it was observed that 10 days after feeding diacylglycerol was the main lipid released to hemolymph and that most of phospholipids and triacylglycerols remained associated with the midgut. The metabolism of free fatty acids in Rhodnius prolixus females is discussed in the context of major biological events that follow a blood meal such as digestion and oogenesis.
Subject(s)
Carrier Proteins/metabolism , Hemolymph/chemistry , Lipoproteins/metabolism , Oleic Acid/metabolism , Rhodnius/metabolism , Animals , Carrier Proteins/blood , Chromatography, Thin Layer/veterinary , Diglycerides/biosynthesis , Diglycerides/blood , Electrophoresis, Polyacrylamide Gel/veterinary , Female , Lipoproteins/blood , Oleic Acid/blood , Phosphatidylcholines/biosynthesis , Phosphatidylcholines/blood , Phosphatidylethanolamines/biosynthesis , Phosphatidylethanolamines/blood , Phosphatidylserines/biosynthesis , Phosphatidylserines/blood , Scintillation Counting/veterinary , Time Factors , Triglycerides/biosynthesis , Triglycerides/bloodABSTRACT
The activities of the enzymatic systems involved in the activation and degradation of fatty acids, and in the synthesis of triacylglycerols and phospholipids were studied in vitro using total cellular homogenate and subcellular fractions of eggs of the shrimp Macrobrachium borellii at different developing stages. Egg development was divided into seven stages based on morphological features of the embryo. Palmitoyl-CoA ligase activity increased as the embryo developed and showed its maximum at stage V. An increase in the synthesis of triacylglycerols and diacylglycerols was also observed at this stage. Diacylglycerylethers were synthesized more actively during the first stages of development. The higher specific activity observed in total homogenate than in microsomal fraction suggested that their synthesis was not exclusively microsomal. Phospholipid synthesis was very active all along development, reflecting active membrane biosynthesis. The highest activity of the cytosolic triacylglycerol lipase was observed at stage V. Fatty acid degradation, measured as mitochondrial beta-oxidation activity, did not vary significantly during development. We conclude that both the anabolic and catabolic processes concerning lipid metabolism are very active, with values similar to those described for adult hepatopancreas, revealing the major role of lipids during shrimp embryogenesis energetics, and that the highest activities of lipid synthesis-hydrolysis take place at stage V when embryos are under active organogenesis. J. Exp. Zool. 286:231-237, 2000.
Subject(s)
Coenzyme A Ligases/metabolism , Embryo, Nonmammalian/enzymology , Embryonic and Fetal Development/physiology , Lipase/metabolism , Lipid Metabolism , Palaemonidae , Repressor Proteins , Saccharomyces cerevisiae Proteins , Animals , Cell Fractionation , Diglycerides/biosynthesis , Female , Palaemonidae/embryology , Palaemonidae/metabolism , Triglycerides/biosynthesisABSTRACT
The involvement of the phospholipase C (PLC) pathway in the non-genomic regulation of duodenal cell Ca2+ concentration by 17beta-oestradiol was investigated. The PLC inhibitors neomycin (0.5 mM) and U-73122 (2 microM) suppressed the stimulatory effect of 0.1 nM 17beta-oestradiol on the 45Ca2+ influx into enterocytes isolated from rat duodenum. The hormone (1 pM to 10 nM) increased the formation of 1,2-diacylglycerol in a biphasic pattern, characterized by an early peak at 45 s (+82%) and a later peak at 5 min (+46%). Both PLC inhibitors suppressed the first peak but were unable to block the 17beta-oestradiol effect at 5 min. 17beta-Oestradiol also increased the generation of inositol 1,4,5-trisphosphate within 15 s, with maximal stimulation at 30 s. 17beta-Oestradiol induced a rapid (30 s) and sustained (up to 5 min) increase in the intracellular Ca2+ concentration ([Ca2+]i) of fura 2-loaded enterocytes. The fast rise in [Ca2+]i was specific because other sex steroid hormones were without effect and could be blocked to a great extent by U-73122 (by 86% at 1 min). The effects of 17beta-oestradiol on enterocyte [Ca2+]i were decreased significantly (by 75%) in a Ca2+-free extracellular medium but a pronounced increase in [Ca2+]i was obtained after readmission of Ca2+ to the medium. The latter change was suppressed by 10 microM La3+, whereas nitrendipine (1 microM) and verapamil (10 microM) separately were without effect. The permeability of the 17beta-oestradiol-induced Ca2+ influx pathway to Mn2+ was increased 2.8-fold by treatment with oestrogen. These results suggest the operation of a PLC-dependent store-operated Ca2+ channel mechanism in 17beta-oestradiol regulation of enterocyte extracellular Ca2+ influx.
Subject(s)
Calcium/metabolism , Duodenum/drug effects , Estradiol/pharmacology , Animals , Cells, Cultured , Diglycerides/biosynthesis , Duodenum/enzymology , Duodenum/metabolism , Enzyme Activation , Enzyme Inhibitors/pharmacology , Estrenes/pharmacology , Female , Inositol 1,4,5-Trisphosphate/biosynthesis , Protein Kinase C/metabolism , Pyrrolidinones/pharmacology , Rats , Rats, Wistar , Type C Phospholipases/antagonists & inhibitors , Type C Phospholipases/metabolismABSTRACT
Displacement curves of 125I-Endothelim-1 (ET-1) binding to rat adrenal cells with unlabeled ET-1, and the ET-1 receptor-related peptides sarafotoxin and BQ-123, show that rat adrenal cortex possess, as its bovine counterpart, two different receptors to ET-1 named ET-A and ET-B. Binding of ET-1 to its rat adrenal receptors stimulates i) aldosterone production, in vivo and in vitro ii) calcium influx, which is mediated through voltage dependent- and receptor operated- calcium channels, iii) cholesterol uptake, iv) stimulation of Na+/K+-ATPase and iv) diacylglycerol production. While the last effect is mediated through ET-A receptors the others involve binding of ET-1 to ET-B receptors. Finally, ouabain potentiates the ET-1-mediated stimulation of aldosterone production, suggesting that the effect of the peptidic hormone on Na+/K+-ATPase could act as a negative feedback mechanism.
Subject(s)
Endothelin-1/pharmacology , Zona Glomerulosa/metabolism , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Aldosterone/biosynthesis , Animals , Calcium/metabolism , Calcium Channels/metabolism , Cattle , Cells, Cultured , Cholesterol/metabolism , Diglycerides/biosynthesis , Endothelin-1/metabolism , Male , Ouabain/pharmacology , Peptides, Cyclic/pharmacology , Rats , Receptor, Endothelin A , Receptor, Endothelin B , Receptors, Endothelin/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Verapamil/pharmacology , Viper Venoms/metabolism , Viper Venoms/pharmacology , Zona Glomerulosa/cytology , Zona Glomerulosa/drug effectsABSTRACT
Treatment with 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3) (1-12 h, 10(-10) M) stimulates DNA synthesis in proliferating myoblasts, with an early response at 2-4 h of treatment followed by a maximal effect at 10 h. To investigate the mechanism involved in the mitogenic action of the hormone we studied the possible activation of intracellular messengers by 1,25(OH)2D3. The initial phase of stimulation of [3H]thymidine incorporation into DNA by the sterol was mimicked by the protein kinase C activator tetradecanoylphorbol acetate (TPA) in a manner which was dose dependent and specific as the inactive analog 4 alpha-phorbol was without effect. Maximal responses to TPA (100 nM) were obtained at 4 h. Staurosporine, a protein kinase C inhibitor, blocked the effect of 1,25(OH)2D3 on myoblast proliferation at 4 h. In addition, a fast (1-5 min) elevation of diacylglycerol levels and membrane-associated protein kinase C activity was observed in response to 1,25(OH)2D3. The adenylate cyclase activator forskolin (20 microM) and dibutyryl-cAMP (50 microM) increased DNA synthesis reproducing the second 1,25(OH)2D3-dependent stimulatory phase at 10 h. Inhibitors of protein kinase A blocked the increase in muscle cell DNA synthesis induced by 1,25(OH)2D3 at 10 h. Significant increases in cyclic AMP levels were detected in myoblasts treated with the sterol for 1-10 h. The calcium channel antagonist nifedipine (5-10 microM) abolished both the effects of 4-h treatment with 1,25(OH)2D3 or TPA and 10-h treatment with 1,25(OH)2D3 or dibutyryl-cAMP. Similar to the calcium channel agonist Bay K8644, 1,25(OH)2D3 stimulated myoblast 45Ca uptake and its effects were blocked by nifedipine.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Calcitriol/pharmacology , Muscles/cytology , Protein Kinase C/physiology , Protein Kinases/physiology , 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester/pharmacology , Alkaloids/pharmacology , Animals , Bucladesine/pharmacology , Calcium/physiology , Calcium Channels/physiology , Cell Division/drug effects , Cells, Cultured , Chick Embryo , Colforsin/pharmacology , DNA/biosynthesis , Diglycerides/biosynthesis , Dose-Response Relationship, Drug , Muscles/drug effects , Muscles/enzymology , Muscles/metabolism , Nifedipine/pharmacology , Protein Kinase C/antagonists & inhibitors , Staurosporine , Tetradecanoylphorbol Acetate/pharmacology , Time FactorsABSTRACT
The phospholipid composition as well as the in vivo [14C]glycerol uptake in lipids was found to be similar in the toad brain and retina. The choroid lipid labeling was markedly different. An in vitro time-course study of [14C]glycerol incorporation in toad retina lipids disclosed that under the conditions of these experiments: (1) retina is able to rapidly synthesize phosphatidic acid from the radioactive precursor; (2) the sequence phosphatidic acid-diacylglycerol-triacylglycerol operates; (3) a high rate of phosphatidylinositol de novo biosynthesis takes place; (4) phosphoglycerides of choline and of ethanolamine are also heavily labeled after a lag period; (5) in vivo labeling profiles resembled those obtained in vitro mainly regarding phosphatidylinositol biosynthesis; and (6) the presence of glycerol kinase in the CNS is suggested.