RESUMO
Glucocorticoids have a wide range of effects in mammalian tissues. In this study we investigated the hypothesis that some of the long-term effects of glucocorticoids on immune cell function may occur by regulating phospholipase C (PLC) signal transduction systems that are known to play a role in immune cell activation. PLC activity was measured in vitro with [3H]phosphatidylinositol and [3H]phosphatidylinositol 4,5-bisphosphate as substrates. Although the guanine nucleotide regulatory protein, Gi, is a membrane-associated protein, our unpublished observations show significant Gi levels in the cytosol of several tissues, including the spleen, where the highest levels were detected. We measured cytosolic Gi alpha immunoreactivity after hormone treatment to establish the relationship between the regulation of cytosolic PLC activity and cytosolic Gi alpha levels. GTP and its nonhydrolyzable analogs have been shown in some instances to regulate soluble PLC activity. In vivo administration of dexamethasone (DEX; 5 mg/ml, sc) to rats for 24 h reduced soluble PLC activity from spleen by 25-50%. In the same tissue, cytosolic Gi alpha immunoreactivity was decreased by 60-70%. The time dependency and receptor specificity of the glucocorticoid effects observed in vivo were investigated further in isolated splenocytes. Treatment of intact splenocytes with DEX (10(-8) and 10(-7) M) for 48 h inhibited calcium-stimulated cytosolic PLC activity by 80-90%; cytosolic Gi alpha immunoreactivity was also reduced by about 90%. In a time course experiment with DEX (10(-7) M) in splenocytes, significant effects were apparent by 12 h after steroid treatment and were maximal by 48 h. When splenocytes were coincubated with DEX (10(-8) M) and the glucocorticoid receptor antagonist RU 486 (10(-7) M), the effects of DEX on soluble PLC activity and cytosolic Gi alpha immunoreactivity were both blocked, suggesting that the effects were mediated by DEX activation of the classical intracellular glucocorticoid receptor. The effects of glucocorticoids reported here may represent one way by which these hormones act to modulate immune cell function.
Assuntos
Corticosterona/farmacologia , Dexametasona/farmacologia , Proteínas de Ligação ao GTP/antagonistas & inibidores , Baço/metabolismo , Fosfolipases Tipo C/antagonistas & inibidores , Adrenalectomia , Animais , Cálcio/farmacologia , Membrana Celular/metabolismo , Citosol/metabolismo , Masculino , Mifepristona/farmacologia , Fosfatidilinositol 4,5-Difosfato , Fosfatos de Fosfatidilinositol/metabolismo , Ratos , Ratos Sprague-Dawley , Baço/efeitos dos fármacos , Fosfolipases Tipo C/metabolismoRESUMO
Ca2+/calmodulin-dependent processes are altered by manipulations of the hypothalamic-pituitary-adrenal axis. In particular, adrenalectomy (ADX) attenuates hippocampal, but not cortical, calmodulin-dependent adenylate cyclase activity measured during the active (waking) phase of rats. The involvement of calmodulin- and guanine nucleotide (G)-binding proteins in the effects of ADX on the activity of calmodulin-dependent adenylate cyclase were investigated. In hippocampal membranes, inclusion of the GTP antagonist guanosine 5'-O-(2-thiodiphosphate) (250 microM) caused pronounced inhibition of calmodulin-stimulated adenylate cyclase activity. Guanosine 5(1)-O-(2-thiodiphosphate) had much smaller effects on calmodulin-independent (basal and forskolin-stimulated) enzyme activity. Substitution of Mn2+ for Mg2+ in the assay medium increased basal and forskolin-stimulated adenylate cyclase activity, but abolished calmodulin-dependent activation of this enzyme in both hippocampal and cortical membranes. These treatments blunted ADX-induced attenuation of hippocampal adenylate cyclase. ADX, with or without corticosterone administration (40 mg/kg, sc, once daily), failed to alter either Gi alpha or Gs alpha membrane protein content in either hippocampus or cortex. The levels of major membrane calmodulin-binding proteins in hippocampus and cortex also were not significantly altered by ADX. These results confirm that hormonal and biochemical regulation of calmodulin-dependent adenylate cyclase is distinct from that of other adenylate cyclase family members. Changes in Gs alpha and Gi alpha protein content alone cannot account for the effects of ADX on this enzyme. Overall, our studies suggest that the effects of ADX on calmodulin-dependent adenylate cyclase may occur through a reduction in the absolute amount of the catalytic subunit or an alteration(s) in the efficiency of coupling between adenylate cyclase and its modulators.
Assuntos
Adenilil Ciclases/metabolismo , Córtex Suprarrenal/enzimologia , Adrenalectomia , Calmodulina/metabolismo , Corticosterona/farmacologia , Hipocampo/enzimologia , Animais , Calmodulina/farmacologia , Proteínas de Ligação a Calmodulina/isolamento & purificação , Proteínas de Ligação a Calmodulina/metabolismo , Membrana Celular/enzimologia , Colforsina/farmacologia , Eletroforese em Gel de Poliacrilamida , Proteínas de Ligação ao GTP/metabolismo , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacologia , Hipocampo/efeitos dos fármacos , Magnésio/farmacologia , Masculino , Manganês/farmacologia , Especificidade de Órgãos , Ratos , Ratos Sprague-Dawley , Valores de Referência , Tionucleotídeos/farmacologiaRESUMO
There is great need to identify and characterize drug targets and chemotherapeutic strategies against malaria. Here we show that a vacuolar-network induced by the human malaria parasite Plasmodium falciparum, is a major import pathway for artemisinin, a leading, new anti-malarial that is known to be effective against drug resistant strains. We also show that artemisinin-treatment induces aberrant, budding of a vacuolar-network membrane protein and its antimalarial activity is additive with toxic sphingolipid analogues that block the network. The data suggest that artemisinin alters membrane protein export from the vacuolar-network and combinations with anti-network reagents have the potential to provide powerful new chemotherapy for drug resistant malaria.
Assuntos
Antimaláricos/metabolismo , Artemisininas , Plasmodium falciparum/metabolismo , Sesquiterpenos/metabolismo , Esfingolipídeos/farmacologia , Vacúolos/metabolismo , Animais , Antimaláricos/farmacologia , Transporte Biológico , Cloroquina/farmacologia , Resistência a Medicamentos , Sinergismo Farmacológico , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Malária Falciparum/parasitologia , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/metabolismo , Sesquiterpenos/farmacologia , Vacúolos/efeitos dos fármacosRESUMO
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.
Assuntos
Antimaláricos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Riboflavina/farmacologia , Animais , Eritrócitos/química , Hemeproteínas/farmacologia , Humanos , Metemoglobina/metabolismo , Plasmodium falciparum/químicaRESUMO
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.
Assuntos
Fosfatidilinositóis/metabolismo , Baço/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Concanavalina A/farmacologia , Citosol/metabolismo , Dexametasona/farmacologia , Indução Enzimática/efeitos dos fármacos , Proteínas de Ligação ao GTP/metabolismo , Técnicas In Vitro , Cinética , Masculino , Modelos Biológicos , Fosfatidilinositol Diacilglicerol-Liase , Diester Fosfórico Hidrolases/metabolismo , Ratos , Fluoreto de Sódio/farmacologia , Solubilidade , Baço/citologia , Baço/efeitos dos fármacos , Especificidade por Substrato , Acetato de Tetradecanoilforbol/farmacologia , Trifluoperazina/farmacologia , Fosfolipases Tipo C/biossíntese , Vanadatos/farmacologiaRESUMO
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.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Anticorpos , Sequência de Bases , Linhagem Celular , Citosol/metabolismo , Ativação Enzimática/efeitos dos fármacos , Proteínas de Ligação ao GTP/genética , Nucleotídeos de Guanina/farmacologia , Técnicas In Vitro , Masculino , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/genética , Toxina Pertussis , Fosfatidilinositol Diacilglicerol-Liase , Fosfoproteínas Fosfatases/antagonistas & inibidores , Diester Fosfórico Hidrolases/metabolismo , Ratos , Ratos Sprague-Dawley , Fluoreto de Sódio/farmacologia , Solubilidade , Baço/efeitos dos fármacos , Baço/metabolismo , Especificidade por Substrato , Fosfolipases Tipo C/imunologia , Fatores de Virulência de Bordetella/farmacologiaRESUMO
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.
Assuntos
Antimaláricos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Riboflavina/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Sinergismo Farmacológico , Testes de Sensibilidade Parasitária , Fármacos Fotossensibilizantes/farmacologia , Plasmodium falciparum/citologiaRESUMO
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.
Assuntos
Plasmodium falciparum/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Western Blotting , Resistência a Medicamentos , Citometria de Fluxo , Corantes Fluorescentes , Expressão Gênica , Genes Reporter , Humanos , Imunossupressores/farmacologia , Metotrexato/farmacologia , Modelos Genéticos , Plasmídeos , Plasmodium falciparum/genética , Proteínas Recombinantes de Fusão/genéticaRESUMO
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.
Assuntos
FMN Redutase , Ferro/metabolismo , Dibutirato de 12,13-Forbol/farmacologia , Transporte Biológico/efeitos dos fármacos , Membrana Celular/enzimologia , Dactinomicina/farmacologia , Ativação Enzimática , Humanos , Metais/farmacologia , NADH NADPH Oxirredutases/metabolismo , Proteína Quinase C/metabolismo , Transferrina/metabolismo , Células Tumorais CultivadasRESUMO
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).
Assuntos
Regulação para Baixo , Proteína Quinase C/metabolismo , Receptores da Transferrina/metabolismo , Briostatinas , Compartimento Celular , Endocitose , Ativação Enzimática , Humanos , Lactonas/farmacologia , Macrolídeos , Dibutirato de 12,13-Forbol/farmacologia , Receptores da Transferrina/biossíntese , Receptores da Transferrina/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais CultivadasRESUMO
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.
Assuntos
Intestinos/enzimologia , Sobrecarga de Ferro/enzimologia , Complexo Sacarase-Isomaltase/metabolismo , Talassemia beta/enzimologia , Animais , Anticorpos Monoclonais , Northern Blotting , Western Blotting , Células CACO-2 , Homeostase , Humanos , Absorção Intestinal , Ferro/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
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.