17 results on '"Akompong T"'
Search Results
2. Glucocorticoids inhibit soluble phospholipase C activity and cytosolic guanine nucleotide regulatory protein-alpha i immunoreactivity in spleen.
- Author
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Akompong, T, primary, Spencer, R L, additional, and McEwen, B S, additional
- Published
- 1993
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3. Immunological analysis of beta-thalassemic mouse intestinal proteins reveals up-regulation of sucrase-isomaltase in response to iron overload.
- Author
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Akompong, Thomas, Ramm, Elizabeth, Akompong, T, Ramm, E, Chang, C, Yu, Z K, and Wessling-Resnick, M
- Subjects
THALASSEMIA ,IA antigens ,ANIMAL models in research ,IRON metabolism ,ANIMAL experimentation ,COMPARATIVE studies ,HOMEOSTASIS ,IMMUNOBLOTTING ,INTESTINAL absorption ,INTESTINES ,IRON in the body ,RESEARCH methodology ,MEDICAL cooperation ,MICE ,MONOCLONAL antibodies ,NUCLEOTIDE separation ,RESEARCH ,RESEARCH funding ,WESTERN immunoblotting ,EVALUATION research ,BETA-Thalassemia - Abstract
Maintenance of iron homeostasis must balance the demand for iron due to heme synthesis, which is driven by hematopoiesis, and the restricted intestinal uptake of iron, which otherwise limits absorption of this toxic element. The consequences of perturbed iron homeostasis are witnessed in inherited forms of beta-thalassemia in which erythroid hyperplasia results in enhanced intestinal iron absorption despite tissue iron overload. To gain a better understanding of intestinal factors that are induced when iron homeostasis is disrupted, a panel of monoclonal antibodies that recognize intestinal microvillous membrane proteins of the beta-thalassemic Hbbd(th3)/Hbbd(th3) mouse was established. The monoclonal antibodies were screened by differential Western blotting against normal and beta-thalassemic mouse intestine to identify antigens modulated in the disease state. Here we report the initial characterization of one immunoreactive species that is up-regulated in beta-thalassemic mouse intestine and the tentative identification of this antigen as sucrase-isomaltase. Studies in Caco-2 cells revealed the rather unexpected finding that expression of this intestinal hydrolase is increased in response to iron toxicity. [ABSTRACT FROM AUTHOR]
- Published
- 1999
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4. Stable expression of a new chimeric fluorescent reporter in the human malaria parasite Plasmodium falciparum.
- Author
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Kadekoppala, M, Kline, K, Akompong, T, and Haldar, K
- Abstract
Stable transfection of a new, chimeric reporter in the human malaria parasite Plasmodium falciparum confers green fluorescence and methotrexate resistance that can be quantitated by Western blotting and flow cytometry. This provides a sensitive, live reporter for exploitation of genomic and high-throughput assays for the identification of new pathogenic determinants.
- Published
- 2000
5. Artemisinin and its derivatives are transported by a vacuolar-network of Plasmodium falciparum and their anti-malarial activities are additive with toxic sphingolipid analogues that block the network
- Author
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Akompong, T., VanWye, J., Ghori, N., and Haldar, K.
- Published
- 1999
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6. Phorbol esters stimulate non-transferrin iron uptake by K562 cells.
- Author
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Akompong, T, Inman, R S, and Wessling-Resnick, M
- Abstract
Characterization of non-transferrin (non-Tf) iron transport by K562 cells has revealed unique properties relative to iron uptake mechanisms present in other cell types (Inman, R. S., and Wessling-Resnick, M. (1993) J. Biol. Chem. 268, 8521-8528). Since treatment of K562 cells with phorbol esters promotes stable megakaryocytic differentiation, we examined the uptake of non-Tf iron in response to protein kinase C activation. Treatment of K562 cells with phorbol esters increased the cellular uptake of 55Fe 4-6-fold compared with untreated cells. The phorbol ester-induced stimulation of 55Fe uptake was time- and dose-dependent, with significantly enhanced transport observed only after prolonged administration of 50 nM phorbol 12,13-dibutyrate (> 8 h). These effects can be attributed to an increased Vmax of transport (14.0 +/- 5 versus 0.6 +/- 0.2 pmol/min/10(6) cells) as well as a 6-fold increase in the apparent Km (1.2 +/- 0.4 versus 0.2 +/- 0.06 microM). It is thought that the reduction of Fe3+ to Fe2+ is required as a first step in the uptake mechanism, and the associated ferrireductase activity of K562 cells is also enhanced with phorbol ester treatment by 5-10-fold (337 +/- 53 versus 43 +/- 3 pmol/min/10(6) cells). Bryostatin-1, a protein kinase C activator that fails to induce differentiation of K562 cells, did not promote this effect, indicating that the enhanced transport activity is dependent on the differentiation response. The idea that synthesis of a new class of transporters is responsible for this effect is supported by the observation that actinomycin D blocks up-regulation of non-Tf iron transport. The increased transport and ferrireductase activity induced upon differentiation also correlate with the appearance of saturable iron-binding sites on the surface of K562 cells with Kd approximately 0.4 microM. These results indicate that non-Tf iron transport activity and the expression of cell-surface iron-binding proteins can be controlled by environmental factors that promote megakaryocytic differentiation of K562 cells.
- Published
- 1995
7. Mechanism of transferrin receptor down-regulation in K562 cells in response to protein kinase C activation.
- Author
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Schonhorn, J E, Akompong, T, and Wessling-Resnick, M
- Abstract
Treatment with phorbol esters increases endocytosis of the transferrin receptor in K562 cells (Klausner, R. D., Harford, J., and van Renswoude, J. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3005-3009). In this report, we demonstrate that this effect is reversible within early times of protein kinase C activation (< 2 h) but that prolonged exposure to phorbol esters results in a net loss of receptors. These effects are not due to the differentiation response of K562 cells to phorbol esters since bryostatin-1 also down-regulates the endocytosis of the transferrin receptor and shut downs receptor synthesis, but does not induce differentiation (Hocevar, B. A., Morrow, D. M., Tykocinski, M. L., and Fields, A. P. (1992) J. Cell Sci. 101, 671-679). We have characterized the early stages of receptor down-regulation which occur due to stimulation of receptor internalization from the cell surface. The fact that fluid-phase pinocytosis is also enhanced upon protein kinase C activation indicates that this effect is not specific for the transferrin receptor itself, but is a rather general cellular response to tumor-promoting phorbol esters. The fate of down-regulated transferrin receptors was followed in morphological and subcellular fractionation studies that demonstrate localization of this pool of receptors in early endocytic and recycling compartments. Our results exclude the possibility that transferrin receptor down-regulation results in trafficking of the receptor to lysosomal compartments for degradation. This idea is consistent with the observations that the time course of transferrin receptor degradation is not enhanced in stimulated K562 cells, while transferrin receptor synthesis is shut down. Our results rigorously demonstrate that activation of protein kinase C down-regulates the K562 cell transferrin receptor in two stages: acute regulation of early steps in endocytosis that results in an immediate reduction of approximately 40% in cell surface number of receptors and a more chronic reduction in transferrin receptor synthesis upon prolonged exposure to phorbol esters (> 15 h).
- Published
- 1995
8. Identification of a stomatin orthologue in vacuoles induced in human erythrocytes by malaria parasites. A role for microbial raft proteins in apicomplexan vacuole biogenesis.
- Author
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Hiller NL, Akompong T, Morrow JS, Holder AA, and Haldar K
- Subjects
- Amino Acid Sequence, Animals, Blood Proteins genetics, Blotting, Western, Caveolin 1, Caveolins metabolism, Cell Membrane metabolism, Fluorescent Antibody Technique, Indirect, Green Fluorescent Proteins, Humans, Luminescent Proteins metabolism, Membrane Proteins chemistry, Membrane Proteins metabolism, Models, Biological, Molecular Sequence Data, Phylogeny, Plasmodium falciparum metabolism, Plasmodium falciparum pathogenicity, Protein Structure, Tertiary, Recombinant Fusion Proteins metabolism, Recombinant Proteins metabolism, Sequence Homology, Amino Acid, Tetanus Toxin chemistry, Time Factors, Vacuoles metabolism, Blood Proteins chemistry, Erythrocytes metabolism, Erythrocytes microbiology, Membrane Microdomains metabolism
- Abstract
When the human malaria parasite Plasmodium falciparum infects erythrocytes, proteins associated with host-derived detergent-resistant membrane (DRM) rafts are selectively recruited into the newly formed vacuole, but parasite proteins that contribute to raft-based vacuole development are unknown. In mammalian cells, DRM-associated integral membrane proteins such as caveolin-1 and flotillin-1 that form oligomers have been linked to the formation of DRM-based invaginations called caveolae. Here we show that the P. falciparum genome does not encode caveolins or flotillins but does contain an orthologue of human band 7 stomatin, a protein known to oligomerize, associate with non-caveolar DRMs and is distantly related to flotillins. Stomatins are members of a large protein family conserved in evolution and P. falciparum (Pf) stomatin appears to be a prokaryotic-like molecule. Evidence is presented that it associates with DRMs and may oligomerize, suggesting that these features are conserved in the stomatin family. Further, Pfstomatin is an integral membrane protein concentrated at the apical end of extracellular parasites, where it co-localizes with invasion-associated rhoptry organelles. A resident rhoptry protein, RhopH2 also resides in DRMs. This provides the first evidence that rhoptries of an apicomplexan parasite contain DRM rafts. Further, when the parasite invades erythrocytes, rhoptry Pfstomatin and RhopH2 are inserted into the newly formed vacuole. Thus, like caveolin-1 and flotillin-1, a stomatin may also associate with non-clathrin coated, DRM-enriched vacuoles. We propose a new model of invasion and vacuole formation involving DRM-based interactions of both host and parasite molecules.
- Published
- 2003
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9. Erythrocyte G protein-coupled receptor signaling in malarial infection.
- Author
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Harrison T, Samuel BU, Akompong T, Hamm H, Mohandas N, Lomasney JW, and Haldar K
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- Adrenergic beta-2 Receptor Agonists, Adrenergic beta-2 Receptor Antagonists, Adrenergic beta-Agonists pharmacology, Adrenergic beta-Antagonists pharmacology, Alprenolol pharmacology, Animals, Catecholamines metabolism, Cyclic AMP metabolism, Erythrocyte Membrane metabolism, Erythrocytes metabolism, GTP-Binding Protein alpha Subunits, Gs chemistry, Humans, Malaria metabolism, Membrane Microdomains metabolism, Mice, Parasitemia, Peptide Fragments pharmacology, Plasmodium falciparum growth & development, Propranolol pharmacology, Purinergic P1 Receptor Agonists, Purinergic P1 Receptor Antagonists, Receptors, Purinergic P1 metabolism, Signal Transduction, Stereoisomerism, Vacuoles parasitology, Erythrocytes parasitology, GTP-Binding Protein alpha Subunits, Gs metabolism, Malaria parasitology, Plasmodium berghei physiology, Plasmodium falciparum physiology, Receptors, Adrenergic, beta-2 metabolism
- Abstract
Erythrocytic mechanisms involved in malarial infection are poorly understood. We have found that signaling via the erythrocyte beta2-adrenergic receptor and heterotrimeric guanine nucleotide-binding protein (Galphas) regulated the entry of the human malaria parasite Plasmodium falciparum. Agonists that stimulate cyclic adenosine 3',5'-monophosphate production led to an increase in malarial infection that could be blocked by specific receptor antagonists. Moreover, peptides designed to inhibit Galphas protein function reduced parasitemia in P. falciparum cultures in vitro, and beta-antagonists reduced parasitemia of P. berghei infections in an in vivo mouse model. Thus, signaling via the erythrocyte beta2-adrenergic receptor and Galphas may regulate malarial infection across parasite species.
- Published
- 2003
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10. A bifunctional molecule that displays context-dependent cellular activity.
- Author
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Braun PD, Barglow KT, Lin YM, Akompong T, Briesewitz R, Ray GT, Haldar K, and Wandless TJ
- Subjects
- Amino Acid Sequence, Animals, Antimalarials chemistry, Antimalarials metabolism, Cell Line, Female, Folic Acid Antagonists chemistry, Folic Acid Antagonists metabolism, Humans, Kinetics, Ligands, Methotrexate chemistry, Methotrexate metabolism, Molecular Sequence Data, Plasmodium falciparum drug effects, Plasmodium falciparum enzymology, Tacrolimus Binding Proteins chemistry, Tacrolimus Binding Proteins metabolism, Tetrahydrofolate Dehydrogenase metabolism, Uterus cytology, Uterus drug effects, Antimalarials pharmacology, Folic Acid Antagonists pharmacology, Methotrexate analogs & derivatives, Methotrexate pharmacology, Peptidylprolyl Isomerase
- Abstract
The cell-permeable dihydrofolate reductase inhibitor methotrexate was covalently linked to a ligand for the protein FKBP to create a bifunctional molecule called MTXSLF. The covalent tether between the two ligands was designed to be prohibitively short, so that unfavorable protein-protein interactions between DHFR and FKBP preclude formation of a trimeric complex. In vitro and in vivo experiments demonstrate that MTXSLF is an effective inhibitor of human DHFR, but that efficacy is decreased in the presence of human FKBP due to the high concentration of FKBP and its tight affinity for MTXSLF. MTXSLF also inhibits Plasmodium falciparum DHFR in vitro, but a low concentration of the weaker binding Plasmodium FKBP has no effect on the inhibitory potency of MTXSLF in vivo. These studies illustrate a potentially general strategy for modulating the biological activity of synthetic molecules that depends on the ligand-binding properties of a nontarget protein.
- Published
- 2003
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11. Trans expression of a Plasmodium falciparum histidine-rich protein II (HRPII) reveals sorting of soluble proteins in the periphery of the host erythrocyte and disrupts transport to the malarial food vacuole.
- Author
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Akompong T, Kadekoppala M, Harrison T, Oksman A, Goldberg DE, Fujioka H, Samuel BU, Sullivan D, and Haldar K
- Subjects
- Animals, Blotting, Western, Catalysis, Chloroquine metabolism, Cytoplasm metabolism, Electrophoresis, Polyacrylamide Gel, Epitopes, Flow Cytometry, Hemeproteins metabolism, Humans, Inhibitory Concentration 50, Microscopy, Electron, Scanning, Microscopy, Fluorescence, Models, Biological, Plasmids metabolism, Protein Structure, Tertiary, Protein Transport, Time Factors, Transfection, Transgenes, Erythrocytes parasitology, Plasmodium falciparum metabolism, Protein Biosynthesis, Proteins chemistry
- Abstract
The heme polymer hemozoin is produced in the food vacuole (fv) of the parasite after hemoglobin proteolysis and is the target of the drug chloroquine. A candidate heme polymerase, the histidine-rich protein II (HRPII), is proposed to be delivered to the fv by ingestion of the infected-red cell cytoplasm. Here we show that 97% of endogenous Plasmodium falciparum (Pf) HRPII (PfHRPII) is secreted as soluble protein in the periphery of the red cell and avoids endocytosis by the parasite, and 3% remains membrane-bound within the parasite. Transfected cells release 90% of a soluble transgene PfHRPIImyc into the red cell periphery and contain 10% membrane bound within the parasite. Yet these cells show a minor reduction in hemozoin production and IC(50) for chloroquine. They also show decreased transport of resident fv enzyme PfPlasmepsin I, the endoplasmic reticulum (ER) marker PfBiP, and parasite-associated HRPII to fvs. Instead, all three proteins accumulate in the ER, although there is no defect in protein export from the parasite. The data suggest that novel mechanisms of sorting (i) soluble antigens like HRPII in the red cell cytoplasm and (ii) fv-bound membrane complexes in the ER regulate parasite digestive processes.
- Published
- 2002
- Full Text
- View/download PDF
12. Protein and lipid trafficking induced in erythrocytes infected by malaria parasites.
- Author
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Haldar K, Mohandas N, Samuel BU, Harrison T, Hiller NL, Akompong T, and Cheresh P
- Subjects
- Animals, Biological Transport, Active, Blood Proteins metabolism, Host-Parasite Interactions, Humans, Malaria, Falciparum blood, Malaria, Falciparum parasitology, Membrane Microdomains metabolism, Models, Biological, Signal Transduction, Vacuoles metabolism, Vacuoles parasitology, Erythrocytes metabolism, Erythrocytes parasitology, Plasmodium falciparum pathogenicity, Protozoan Proteins blood
- Abstract
The human malaria parasite Plasmodium falciparum develops in a parasitophorous vacuolar membrane (PVM) within the mature red cell and extensively modifies structural and antigenic properties of this host cell. Recent studies shed significant new, mechanistic perspective on the underlying processes. There is finally, definitive evidence that despite the absence of endocytosis, transmembrane proteins in the host red cell membrane are imported in to the PVM. These are not major erythrocyte proteins but components that reside in detergent resistant membrane (DRM) rafts in red cell membrane and are detected in rafts in the PVM. Disruption of either erythrocyte or vacuolar rafts is detrimental to infection suggesting that raft proteins and lipids are essential for the parasitization of the red cell. On secretory export of parasite proteins: an ER secretory signal (SS) sequence is required for protein secretion to the PV. Proteins carrying an additional plastid targeting sequence (PTS) are also detected in the PV but subsequently delivered to the plastid organelle within the parasite, suggesting that the PTS may have a second function as an endocytic sorting signal. A distinct but yet undefined peptidic motif underlies protein transport across the PVM to the red cell (although all of the published data does not yet fit this model). Further multiple exported proteins transit through secretory 'cleft' structures, suggesting that clefts may be sorting compartments assembled by the parasite in the red cell.
- Published
- 2002
- Full Text
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13. Gametocytocidal activity and synergistic interactions of riboflavin with standard antimalarial drugs against growth of Plasmodium falciparum in vitro.
- Author
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Akompong T, Eksi S, Williamson K, and Haldar K
- Subjects
- Animals, Cell Division drug effects, Drug Synergism, Parasitic Sensitivity Tests, Photosensitizing Agents pharmacology, Plasmodium falciparum cytology, Antimalarials pharmacology, Plasmodium falciparum drug effects, Riboflavin pharmacology
- Abstract
Our previous studies have shown that riboflavin has activity against Plasmodium falciparum asexual-stage parasites in vitro. In the present study we examine the gametocytocidal activity of riboflavin and the interaction of riboflavin with some commonly used antimalarial drugs against the asexual forms of P. falciparum in vitro. The addition of riboflavin to P. falciparum cultures killed gametocytes at all stages, even those at late stages (III to V), which are not affected by many of the commonly used antimalarials. Combinations of riboflavin with mefloquine, pyrimethamine, and quinine showed a marked potentiation of the activities of these drugs against asexual-stage parasites in vitro. The combination of riboflavin with artemisinin was additive, while that with chloroquine was mildly antagonistic. High doses of riboflavin are used clinically to treat several inborn errors of metabolism with no adverse side effects. Its efficacy in combination with standard antimalarial drugs in treating and preventing the transmission of P. falciparum malaria can therefore be evaluated in humans.
- Published
- 2000
- Full Text
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14. In vitro activity of riboflavin against the human malaria parasite Plasmodium falciparum.
- Author
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Akompong T, Ghori N, and Haldar K
- Subjects
- Animals, Erythrocytes chemistry, Hemeproteins pharmacology, Humans, Methemoglobin metabolism, Plasmodium falciparum chemistry, Antimalarials pharmacology, Plasmodium falciparum drug effects, Riboflavin pharmacology
- Abstract
The human malaria parasite Plasmodium falciparum digests hemoglobin and polymerizes the released free heme into hemozoin. This activity occurs in an acidic organelle called the food vacuole and is essential for survival of the parasite in erythrocytes. Since acidic conditions are known to enhance the auto-oxidation of hemoglobin, we investigated whether hemoglobin ingested by the parasite was oxidized and whether the oxidation process could be a target for chemotherapy against malaria. We released parasites from their host cells and separately analyzed hemoglobin ingested by the parasites from that remaining in the erythrocytes. Isolated parasites contained elevated amounts (38.5% +/- 3.5%) of oxidized hemoglobin (methemoglobin) compared to levels (0.8% +/- 0.2%) found in normal, uninfected erythrocytes. Further, treatment of infected cells with the reducing agent riboflavin for 24 h decreased the parasite methemoglobin level by 55%. It also inhibited hemozoin production by 50% and decreased the average size of the food vacuole by 47%. Administration of riboflavin for 48 h resulted in a 65% decrease in food vacuole size and inhibited asexual parasite growth in cultures. High doses of riboflavin are used clinically to treat congenital methemoglobinemia without any adverse side effects. This activity, in conjunction with its impressive antimalarial activity, makes riboflavin attractive as a safe and inexpensive drug for treating malaria caused by P. falciparum.
- Published
- 2000
- Full Text
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15. Metabolic depletion inhibits the uptake of nontransferrin-bound iron by K562 cells.
- Author
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Gutierrez JA, Inman RS, Akompong T, Yu J, and Wessling-Resnick M
- Subjects
- 2,4-Dinitrophenol pharmacology, Adenosine Triphosphate deficiency, Adenosine Triphosphate physiology, Antimycin A analogs & derivatives, Antimycin A pharmacology, Biological Transport, Active, Carrier Proteins metabolism, Energy Metabolism drug effects, HeLa Cells drug effects, HeLa Cells metabolism, Humans, Hydrogen-Ion Concentration, K562 Cells drug effects, Membrane Potentials, NADH, NADPH Oxidoreductases metabolism, Rotenone pharmacology, FMN Reductase, Iron metabolism, Iron-Binding Proteins, K562 Cells metabolism, Ubiquitin-Conjugating Enzymes, Uncoupling Agents pharmacology
- Abstract
The effect of metabolic inhibitors on nontransferrin bound iron transport by K562 cells was investigated. Incubation with 1 microM rotenone, 10 microM antimycin, or 0.5 mM 2,4-dinitrophenol effectively reduced ATP levels by approximately 50%. Both the rate and extent of Fe+3 uptake were impaired in ATP-depleted cells, which display a reduced Vmax for uptake. K562 cell ferrireductase activity was also lowered by metabolic inhibitors, suggesting that the apparent energy requirements for transport reside in the reduction of Fe+3 to Fe+2. However, ATP depletion was found to inhibit the rate and extent of Fe+2 uptake as well. Thus, the transbilayer passage of Fe+2 and/or Fe+3 appears to be an energy-requiring process. These features possibly reflect properties of the transport mechanism associated with a recently identified K562 cell transport protein, called SFT for "Stimulator of Fe Transport," since exogenous expression of its activity is also affected by ATP depletion.
- Published
- 1998
- Full Text
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16. Cytosolic phospholipase C activity: II. Relationship to concanavalin A-induced phosphatidylinositol-turnover in splenocytes.
- Author
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Akompong T, Spencer RL, and McEwen BS
- Subjects
- Animals, Cell Division drug effects, Concanavalin A pharmacology, Cytosol metabolism, Dexamethasone pharmacology, Enzyme Induction drug effects, GTP-Binding Proteins metabolism, In Vitro Techniques, Kinetics, Male, Models, Biological, Phosphatidylinositol Diacylglycerol-Lyase, Phosphoric Diester Hydrolases metabolism, Rats, Sodium Fluoride pharmacology, Solubility, Spleen cytology, Spleen drug effects, Substrate Specificity, Tetradecanoylphorbol Acetate pharmacology, Trifluoperazine pharmacology, Type C Phospholipases biosynthesis, Vanadates pharmacology, Phosphatidylinositols metabolism, Spleen metabolism, Type C Phospholipases metabolism
- Abstract
We have described in the first paper the coupling between cytosolic Gi alpha and cytosolic PLC activity in a cell free preparation. In order to establish the functional significance of the cytosolic Gi alpha coupled soluble PLC, we examined the effects of DEX, NaF, and trifluoperizine (TFP) on concanavalin A (Con A)-induced PI-turnover in intact splenocytes and, in parallel, on soluble PLC activity in cytosol preparations. Cytosolic PLC activity was measured with [3H]PI and [3H]PIP2 as substrates. 1) The Con A-induced increase (2-4 fold) in PI-turnover in intact splenocytes was paralleled by an 1.2-5-fold increase in soluble PLC activity in vitro. Con A administration also increased cytosolic Gi alpha immunoreactivity 3-6-fold as expected if cytosolic Gi alpha was coupled to soluble PLC activation. 2) DEX (10(-7) M), administered 6 h prior to Con A administration, inhibited the Con A-induced increase in PI-turnover in intact splenocytes. This was paralleled by DEX inhibition of the Con A-induced increase in soluble PLC activity measured in vitro and cytosolic Gi alpha immunoreactivity. 3) We have demonstrated in the first paper that NaF and TFP inhibited soluble PLC activity. Here we show that NaF and TFP inhibited the Con A-induced increase in PI-turnover extending the similarities between soluble PLC activity and Con A-stimulated PLC activity in intact splenocytes. 4) In order to examine whether or not the Con A-induced PLC was similar to PLC gamma, we measured PI-turnover induced by Con A or NaVO3 in combination with DEX and PMA. Whereas the Con A-induced PI-turnover was significantly inhibited (40-60%) by DEX, the NaVO3-induced PI-turnover was not affected by DEX. The Con A-induced PI-turnover was not affected by PMA (50 nM), but the NaVO3-induced PI-turnover was increased over 2-fold by PMA (50 nM), suggesting that the Con A-induced PLC in intact splenocytes is different from NaVO3-induced PLC. Based on these results a model for the sequential activation of substrate-specific PLCs in splenocyte by mitogen is presented.
- Published
- 1994
- Full Text
- View/download PDF
17. Cytosolic phospholipase C activity: I. Evidence for coupling with cytosolic guanine nucleotide-binding protein, Gi alpha.
- Author
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Akompong T, Spencer RL, and McEwen BS
- Subjects
- Animals, Antibodies, Base Sequence, Cell Line, Cytosol metabolism, Enzyme Activation drug effects, GTP-Binding Proteins genetics, Guanine Nucleotides pharmacology, In Vitro Techniques, Male, Molecular Sequence Data, Oligonucleotides, Antisense genetics, Pertussis Toxin, Phosphatidylinositol Diacylglycerol-Lyase, Phosphoprotein Phosphatases antagonists & inhibitors, Phosphoric Diester Hydrolases metabolism, Rats, Rats, Sprague-Dawley, Sodium Fluoride pharmacology, Solubility, Spleen drug effects, Spleen metabolism, Substrate Specificity, Type C Phospholipases immunology, Virulence Factors, Bordetella pharmacology, GTP-Binding Proteins metabolism, Type C Phospholipases metabolism
- Abstract
In a previous report we showed that glucocorticoid inhibition of cytosolic PLC activity correlated with a reduction in cytosolic Gi alpha levels, suggesting that there may be a functional relationship between cytosolic PLC and cytosolic Gi alpha. In order to establish the nature of the coupling between cytosolic Gi alpha and cytosolic PLC we examined the effects of G-protein activators, and inhibitors on cytosolic PLC activity from rat splenocytes and the rat lymphoma cell line Nb 2, with [3H] PI and [3H]PIP2 as substrates. 1) Neither GTP nor its nonhydrolyzable analogue, GTP gamma S, at 100 microM had any effect on the calcium stimulated as well as the basal PLC activity. 2) However, affinity purified antibodies to Gi alpha 1 and Gi alpha 2 inhibited soluble PLC activity, by 85% and 55%, respectively, with PI as substrate; with PIP2 as substrate, soluble PLC activity was inhibited 50-70% by antibodies to Gi1, whereas antibodies to Gi2 had little effect. 3) Administration of Gi alpha 1 antisense oligonucleotides to splenocytes for 48 h produced 25-40% decrease in cytosolic Gi alpha 1 levels compared to control. The soluble PLC activity with both PI and PIP2 as substrates was also reduced by 25-50% compared to control conditions. This suggest that cytosolic Gi alpha is associated with the activation of splenocyte soluble PLC. 4) Pertussis toxin administered in vivo significantly reduced cytosolic Gi alpha immunoreactivity and soluble PLC activity when PI was used as substrate, providing additional evidence that cytosolic Gi alpha is associated with the activation of soluble PLC. 5) Another agent that has been used extensively to define G-protein coupled processes is NaF/AlCl3. NaF (5 mM; with or without AlCl3) inhibited soluble PLC activity with PIP2 as substrate, in contrast to the stimulatory effect that has been reported in the activation of membrane PLC. 6) Because NaF can act as a protein phosphatase inhibitor, we also tested the effects of trifluoperizine (50 microM, TFP), an inhibitor of protein phosphatase 2B; TFP (50 microM) significantly inhibited soluble PLC activity when PI was used as substrate. These results suggest a direct involvement of cytosolic Gi alpha in the activation of soluble PLC from splenocytes. Other questions pertaining to the functional significance, the nature, and possible substrate preference of the splenocyte Gi alpha coupled PLC is addressed in the second paper.
- Published
- 1994
- Full Text
- View/download PDF
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