RESUMO
Eric Newsholme's laboratory was the first to show glutamine utilization by lymphocytes and macrophages. Recently, we have found that neutrophils also utilize glutamine. This amino acid has been shown to play a role in lymphocyte proliferation, cytokine production by lymphocytes and macrophages and phagocytosis and superoxide production by macrophages and neutrophils. Knowledge of the metabolic fate of glutamine in these cells is important for the understanding of the role and function of this amino acid in the maintenance of the proliferative, phagocytic and secretory capacities of these cells. Glutamine and glucose are poorly oxidized by these cells and might produce important precursors for DNA, RNA, protein and lipid synthesis. The high rate of glutamine utilization and its importance in such cells have raised the question as to the source of this glutamine, which, according to current evidence, appears to be muscle.
Assuntos
Glutamina/metabolismo , Linfócitos/metabolismo , Macrófagos/metabolismo , Neutrófilos/metabolismo , Divisão Celular/fisiologia , Humanos , Sistema Imunitário/citologia , Sistema Imunitário/metabolismo , Sistema Imunitário/fisiologia , Fagocitose/fisiologiaRESUMO
Eric Newsholme's laboratory was the first to show glutamine utilization by lymphocytes and macrophages. Recently, we have found that neutrophils also utilize glutamine. This amino acid has been shown to play a role in lymphocyte proliferation, cytokine production by lymphocytes and macrophages and phagocytosis and superoxide production by macrophages and neutrophils. Knowledge of the metabolic fate of glutamine in these cells is important for the understanding of the role and function of this amino acid in the maintenance of the proliferative, phagocytic and secretory capacities of these cells. Glutamine and glucose are poorly oxidized by these cells and might produce important precursors for DNA, RNA, protein and lipid synthesis. The high rate of glutamine utilization and its importance in such cells have raised the question as to the source of this glutamine, which, according to current evidence, appears to be muscle
Assuntos
Humanos , Glutamina/metabolismo , Sistema Imunitário/citologia , Linfócitos/metabolismo , Macrófagos/metabolismo , Neutrófilos/metabolismo , Divisão Celular/fisiologia , Sistema Imunitário/metabolismo , Sistema Imunitário/fisiologia , Fagocitose/fisiologiaRESUMO
Mg(2+)-dependent vanadate-sensitive glutathione S-conjugate ATPase (GS-X pump) activity is a common feature of some ATP-binding cassette (ABC) transporters, such as the multidrug resistance-associated protein (MRP1) gene product, that exports biologically active electrophiles after their conjugation with intracellular glutathione (GSH) from normal and cancer cells. Antitumor electrophiles (e.g. naturally occurring cyclopentenone prostaglandins and anticancer chemicals) can be intracellularly conjugated with GSH via a glutathione S-transferase catalyzed reaction and be eliminated through GS-X pumps thus threatening cancer chemotherapeutics. Since different sensitivities to antitumor electrophiles are shown by different cell types, the ability of several human cancer cell lines to produce and export S-(2,4-dinitrophenyl)-glutathione (DNP-SG) conjugate through the GS-X pump, using whole cells and inside-out membrane vesicle preparations, is investigated. Different cancer cell lines exhibited characteristically different GS-X pump activity. In particular, HEp-2 larynx carcinoma cells possess an elevated DNP-SG export rate through the GS-X pump compared with HeLa, K562, U937 or HL-60 cells, which exhibit the lowest activity. The differences in DNP-SG export rates are not due to decreased glutathione S-transferase activity or impaired de novo synthesis of GSH. The findings suggest that the GS-X pump may be involved in the modulation of the biological activity of both naturally occurring electrophiles and anticancer drugs. The differential expression of GS-X pumps may lead to an improved understanding of multidrug resistance and may be exploited in the development of new therapeutic strategies for the treatment of cancer patients.
Assuntos
Adenosina Trifosfatases/biossíntese , Glutationa/metabolismo , Neoplasias/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Transporte Biológico , Humanos , Neoplasias/tratamento farmacológico , Células Tumorais CultivadasRESUMO
A severe complication in late-stage cancer patients is host immunosuppression. It is suggested that overproduction of the highly cytostatic and cytotoxic antiproliferative cyclopentenone prostaglandins (CP-PGs) within the plasma of cancer-bearing subjects may contribute to immunosuppression. Lymphoid tissues of Walker 256 tumor-bearing rats accumulate large amounts of CP-PGs while the tumor tissue itself does not. Moreover, tumor cells may present differential sensitivity to CP-PGs due to the expression of the multidrug resistance-associated protein (MRP1) gene product which shows a Mg(2+)-dependent vanadate-sensitive glutathione S-conjugate export ATPase (GS-X pump) activity that extrudes CP-PGs from cells as glutathione S-conjugates. In this study, the possibility that deficient GS-X pump activity in immune cells that may be involved in the accumulation of CP-PGs is investigated. Rat lymph node lymphocytes do not exhibit any notable activity even when mitogen-stimulated. Conversely, although rat peritoneal resident (quiescent) or thioglycollate-stimulated (inflammatory) macrophages exhibit low GS-X pump activity, Bacillus Calmette-Guérin (BCG)-activated macrophages show a notable rise in the activity of the ATPase, suggesting that the cellular activation state may modulate GS-X pump activity/expression and that, under appropriate stimuli (e.g., during immune response) macrophages may provide a self-defense against electrophilic CP-PGs by forming GS-conjugates that can be extruded from cells through the GS-X pump. ras oncogene expression may be linked with MRP1/GS-X pump expression/activity, since C2C12 promyoblasts transformed by v-H-ras transfection doubled GS-X pump activity. These results support the proposition that the accumulation of CP-PGs and the immunosuppression of tumor-bearing subjects may be attributed to a lack of GS-X pump activity/expression in lymphocytes.
Assuntos
Adenosina Trifosfatases/metabolismo , Glutationa/metabolismo , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Animais , Comunicação Celular , Humanos , Linfócitos/metabolismo , Ativação de Macrófagos , Macrófagos/metabolismo , Ratos , Células Tumorais CultivadasRESUMO
A major feature of macrophage metabolism is its capacity to produce and export cholesterol. Several reports have shown that the manipulation of lymphocyte cholesterol content elicits important changes in lymphocyte proliferation. These findings lead to an inquiry as to whether macrophage-derived cholesterol released into the lymphocyte surroundings may be transferred to the latter thus affecting lymphocyte function. In this study, cholesterol transfer from macrophages to lymphocytes was examined in vitro using rat cells in culture. The findings indicate that there may be a significant transfer of cholesterol from [4-14C]cholesterol labeled resident peritoneal macrophages to mesenteric lymph node resting lymphocytes (up to 173.9 +/- 2.7 pmol/10(7) lymphocytes/10(7) macrophages when co-cultivated for 48 h), in a lipoprotein-dependent manner. This represents the mass transfer of ca. 17 nmoles of cholesterol molecules per 10(7) lymphocytes from 10(7) macrophages (calculated on the basis of specific radioactivity incorporated into macrophages after the pre-labelling period), which suggests that macrophages are capable of replacing the whole lymphocyte cholesterol pool every 21 h. Moreover, an 111%-increase in the total cholesterol content of lymphocytes was found after co-cultivation with macrophages for 48 h. When compared to peritoneal cells, monocytes/macrophages obtained from circulating blood leukocytes presented a much higher cholesterol transfer capacity to lymphocytes (3.06 +/- 0.10 nmol/10(7) lymphocytes/10(7) macrophages co-cultivated for 24 h). Interestingly, inflammatory macrophages dramatically reduced their cholesterol transfer ability (by up to 91%, as compared to resident macrophages). Cholesterol transfer may involve a humoral influence, since it is not only observed when cells are co-cultivated in a single-well chamber system (cells in direct contact), but also in a two-compartment system (where cells can communicate but not by direct contact). Co-cultivation with macrophages decreased the basal incorporation of [2-14C]thymidine into lymphocyte DNA and the addition of cholesterol to lymphocyte culture media also impaired the lymphocyte proliferative response to the mitogens concanavalin A (Con A) and bacterial lipopolysaccharide (LPS). The above results suggest that macrophages may transfer cholesterol to lymphocytes (from both lymph nodes and blood), thus regulating lymphocyte function by raising the intracellular cholesterol content and suppressing lymphocyte proliferative activity. If this is so, a modulatory role for the transfer of cholesterol in both physiological (e.g. immune response) and pathological conditions (e.g. atherosclerosis) may be postulated. This hypothesis is currently under investigation in our laboratory.
Assuntos
Colesterol/metabolismo , Linfócitos/metabolismo , Macrófagos Peritoneais/metabolismo , Animais , Contagem de Células , Células Cultivadas , Ésteres do Colesterol/metabolismo , Técnicas de Cocultura , DNA/biossíntese , Lipoproteínas/metabolismo , Linfonodos/imunologia , Ativação Linfocitária , Linfócitos/citologia , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos Peritoneais/citologia , Masculino , Ratos , Ratos Wistar , Tioglicolatos/farmacologiaRESUMO
Hydrogen peroxide (H2O2) perfused into the aorta of the isolated rat heart induces a positive inotropic effect, with cardiac arrhythmia such as extrasystolic potentiation or cardiac contractures, depending on the dose. The last effect is similar to the "stone heart" observed in reperfusion injury and may be ascribed to lipoperoxidation (LPO) of the membrane lipids, to protein damage, to reduction of the ATP level, to enzymatic alterations and to cardioactive compounds liberated by LPO. These effects may result in calcium overload of the cardiac fibers and contracture ("stone heart"). Hearts from male Wistar rats (300-350g) were perfused at 31°C with Tyrode, 0.2 mM trolox C, 256 mM H2O2 or trolox C + H2O2. Cardiac contractures (baseline elevation of the myograms obtained) were observed when hearts were perfused with H2O2 (Tyrode: 5.9 + 3.2; H2O2: 60.5 + 13.9 percent of the initial value); perfusion with H2O2 increased the LPO of rat heart homogenates measured by chemiluminescence (Tyrode: 3,199 + 259; H2O2: 5,304 + 133 cps mg protein(-1) 60 min(-1), oxygen uptake (Tyrode: 0.44 + 0.1; H2O2: 3.2 + 0.8 nmol min(-1) mg protein(-1) and malonaldehyde (TBARS) foramtion (Tyrode: 0.12 + 0; H2O2: 0.37 + 0.1 nmol/ml). Previous perfusion with 0.2 mM trolox C reduced the LPO (Chemiluminescence: 4,098 + 531), oxygen uptake (0.51 + 0) and TBARS (0.13 + 0) bud did not prevent the H2O2-induced contractures (33.3 + 16 percent). ATP (Tyrode: 2.84 + 0; H2O2: 0.57 + 0) and glycogen levels (Tyrode: 0.46 + 0; H2O22: 0.26 + 0) were reduced by H2O2. Trolox did not prevent these effects (ATP: 0.84 + 0 and glycogen: 0.27 + 0). Trolox C is known to be more effective than alpha-tocopherol or gamma-tocopherol in reducing LPO though it lacks the phytol portion of vitamin E to be fixed to the cell membranes. Trolox C, unlike vitamin A, did not prevent the glycogen reduction induced by H2O2. Trolox C induced a positive chronotropic effect that resulted in higher energy consumption. The reduction of energy level seemed to be more important than LPO in the mechanism of H2O2-induced contracture.
Assuntos
Ratos , Animais , Masculino , Antioxidantes/farmacologia , Peróxido de Hidrogênio/farmacologia , Contração Miocárdica/efeitos dos fármacos , Vitamina E/farmacologia , Ratos WistarRESUMO
[14C]-labelled palmitic acid (PA), oleic acid (OA), linoleic (LA) and arachidonic (AA) acids were transferred from macrophages (M phi) to lymphocytes (LY) when equal numbers of the two cell types were co-cultured. The relative degree and amounts of the fatty acids transferred from M phi to LY are as follow: AA (368.57 +/- 21.62) = OA (274.52 +/- 15.41) > LA (42.11 +/- 8.31) = PA (36.53 +/- 2.45). The transfer units are nmol/10(10) M phi/10(10) LY and the values are mean +/- SEM for 7 experiments. The [14C]-radioactivity transferred was mainly directed to the phospholipid fraction of the lymphocytes (85% by PA, 86% by LA, 83% by OA and 79% by AA). In the same order as above, phosphatidylcholine was the phospholipid moiety most heavily labelled (82% by PA, 71% by LA, 66% by OA and 47% by AA). The amount of [14C]-radioactivity transferred to stimulated lymphocytes of thioglycollate treated animals remained unchanged for LA, PA and AA but reduced for OA (71%). The significance of these observations for the immune functions of the cells and resolution of the question of whether some of the [14C]-isotope transfer involves a component of exchange or is unequivocally net fatty acid mass transfer are still being investigated.
Assuntos
Ácidos Graxos/metabolismo , Linfócitos/metabolismo , Macrófagos/metabolismo , Animais , Transporte Biológico , Radioisótopos de Carbono/metabolismo , Separação Celular , Células Cultivadas , Centrifugação Isopícnica , Técnicas de Cocultura , Masculino , Fosfolipídeos/isolamento & purificação , Ratos , Ratos Wistar , Tioglicolatos/farmacologiaRESUMO
Lipogenesis is essential for the rapid proliferation of cells and it is established that the biosynthesis of selected lipids precedes S-phase, DNA synthesis and the initiation of cell division. Pyruvate was previously shown to be an important lipid precursor for lymphocytes, macrophages and tumour cells. This study now reports the role of prostaglandins (PG) on the regulation of lipogenesis from [3-14C] pyruvate in 24-h cultured lymphocytes. It is shown that indomethacin (10 microM) and ibuprofen (10 microM), both inhibitors of PG biosynthesis, increased [3-14C] pyruvate incorporations into phospholipid and cholesterol fractions in resting lymphocytes but reduced its incorporation into these fractions in concanavalin A (Con A)-stimulated lymphocytes and tumour cells. These two agents also affected [2-14C] thymidine incorporation into the DNA of these cells in the same manner. Lipids produced from [3-14C] pyruvate and exported into the cell culture medium were also measured. The PG biosynthesis inhibitions reduced the transfer to culture medium of arachidonic acid, phospholipids, cholesterol and fatty acids higher than C20 by lymphocytes and tumour cells. Although macrophages are not proliferative cells, the cytoplasmic export measurement is important because these cells have a high capacity for lipid secretion. The results show that the PG biosynthesis inhibitors do not affect the export of phospholipids and cholesterol in macrophages. They do however markedly change the export of arachidonic acid and fatty acids higher than C20 produced from [3-14C] pyruvate in macrophages. It is also reported that a cell-stimulating response diminished the above fatty acid outcome in resting cells and augmented it in thioglycollate-stimulated macrophages. The findings suggest that PG modulation of lipogenesis may depend on cell cycle phase as well as the intrinsic lipid metabolic diversity and capacity.
Assuntos
Lipídeos/biossíntese , Linfócitos/metabolismo , Macrófagos Peritoneais/metabolismo , Neoplasias/metabolismo , Prostaglandinas/fisiologia , Acetatos/metabolismo , Animais , Ácido Araquidônico/metabolismo , Radioisótopos de Carbono , Células Cultivadas , Concanavalina A/farmacologia , DNA/metabolismo , DNA de Neoplasias/metabolismo , Células HeLa , Humanos , Ibuprofeno/farmacologia , Indometacina/farmacologia , Células KB , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Neoplasias Experimentais/metabolismo , Prostaglandinas/biossíntese , Piruvatos/metabolismo , Ácido Pirúvico , Ratos , Timidina/metabolismo , Células Tumorais CultivadasRESUMO
1. The effect of age and Walker 256 tumor on maximal phosphate-dependent glutaminase activity of rat immune tissue was determined. Glutaminase is a key enzyme in the metabolism of glutamine, an important fuel for normal and neoplastic cells. 2. Maximal activity of phosphate-dependent glutaminase was measured in immune tissues and tumors of Walker 256 tumor-bearing young (28 days old), mature (3 months old) and aged (15 months old) Wistar rats. The following tissues were examined: thymus, spleen, mesenteric lymph nodes and tumor. 3. Tumor implantation for 14 days reduced glutaminase activity in the thymus and mesenteric lymph nodes. Tumor glutaminase activity was lowest in aged rats and highest in the mature group. 4. Comparison of glutaminase activity in immune and tumor tissues suggested the flux of glutamine between these tissues in the 3 groups. Glutaminase activity was 2.8-fold higher in immune tissues in aged rats (2.58 +/- 0.35 vs 0.93 +/- 0.16 mumol min-1 g tissue wet weight-1, mean +/- SEM, 5 rats), and 1.9- (4.14 +/- 0.47 vs 8.36 +/- 1.29 mumol min-1 g tissue wet weight-1, mean +/- SEM, 5 rats) and 2.5-fold increased (2.41 +/- 0.20 vs 5.92 +/- 0.22 mumol min-1 g tissue wet weight-1, mean +/- SEM, 5 rats) in tumor tissue in the mature and young groups, respectively. These results suggest the deviation of glutamine flux from defense cells to the neoplastic tissue in tumor-bearing young and mature rats and may partially explain the slow cancer growth in elderly patients.
Assuntos
Envelhecimento/metabolismo , Carcinoma 256 de Walker/enzimologia , Glutaminase/metabolismo , Linfonodos/enzimologia , Baço/enzimologia , Timo/enzimologia , Animais , Imuno-Histoquímica , Masculino , Mesentério , Transplante de Neoplasias , Fosfatos/metabolismo , Ratos , Ratos WistarRESUMO
1. The effect of age and Walker 256 tumor on maximal phosphate-dependent glutaminase activity of rat immune tissue was determined. Glutaminase is a key enzyme in the metabolism of glutamine, an important fuel for normal and neoplastic cells. 2. Maximal activity of phosphate-dependent glutaminase was measured in immune tissues and tumors of Walker 256 tumor-bearing young (28 days old), mature (3 months old) and aged (15 months old) Wistar rats. The following tissues were examined: thymus, spleen, mesenteric lymph nodes and tumor. 3. Tumor implantation for 14 days reduced glutaminase activity in the thymus and mesenteric lymph nodes. Tumor glutaminase activity was lowest in aged rats and highest in the mature group. 4. Comparison of glutaminase activity in immune and tumor tissues suggested the flux of glutamine between these tissues in the 3 groups. Glutaminase activity was 2.8-fold higher in immune tissues in aged rats (2.58 +/- 0.35 vs 0.93 +/- 0.16 mumol min-1 g tissue wet weight-1, mean +/- SEM, 5 rats), and 1.9- (4.14 +/- 0.47 vs 8.36 +/- 1.29 mumol min-1 g tissue wet weight-1, mean +/- SEM, 5 rats) and 2.5-fold increased (2.41 +/- 0.20 vs 5.92 +/- 0.22 mumol min-1 g tissue wet weight-1, mean +/- SEM, 5 rats) in tumor tissue in the mature and young groups, respectively. These results suggest the deviation of glutamine flux from defense cells to the neoplastic tissue in tumor-bearing young and mature rats and may partially explain the slow cancer growth in elderly patients
Assuntos
Animais , Masculino , Envelhecimento/metabolismo , /enzimologia , Glutaminase/metabolismo , Linfonodos/enzimologia , Baço/enzimologia , Timo/enzimologia , Imuno-Histoquímica , Mesentério , Transplante de Neoplasias , Fosfatos/metabolismo , Ratos , Ratos WistarRESUMO
Although several studies have shown the effect of cytokines on islet B cell function, the role of circulating cells in the control of insulin secretion has not been investigated. The effect of lymphocyte administration on plasma glucose and insulin levels was examined in male Wistar albino rats weighing 180-200 g. The animals were anesthetized and the jugular vein was cannulated for saline or lymphocyte injection (10(6) cells in 1 ml) and blood collection after 5, 10, 15, 20, 30, 45 and 60 min. A marked increase in plasma insulin levels (180 microU/ml as compared to 56 microU/ml in the control group, at 20 min) and an unexpected increase (P < 0.05) in blood glucose levels (at 60 min only) were observed after lymphocyte administration. Similar experiments undertaken with simultaneous administration of inhibitors of arachidonic acid metabolism, indomethacin and nordihydroguairetic acid at 1 mg/kg body weight, suggest that this effect of lymphocytes is not mediated by prostaglandins or leukotrienes.
Assuntos
Glicemia/metabolismo , Insulina/sangue , Linfócitos/fisiologia , Animais , Peso Corporal , Injeções Intravenosas , Veias Jugulares , Masculino , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Althugh several studies have shown the effect of cytokines on islet B cell function, the role of circulating cells in the control of insulin secretion has not been investigated. The effect of lymphocyte administration on plasma glucose and insulin levels was examined in male Wistar albino rats weighing 180-200g. The animals were anesthetized and the jugular vein was cannulated for saline or lymphocyte injection (10**6 cells in 1 ml) and blood collection after 5, 10, 15, 20, 30, 45, and 60 min. A marked increase in plasma insulin levels (180*U/ml as compared to 56*U/ml in the control group, at 20 min) and an unexpected increase (P<0.05) in blood glucose levels (at 60 min only) were observed after lymphocyte administration. Similar experiments undertaken with simultaneous administration of inhibitors of arachidonic acid metabolism, indomethacin and nordihydroguairetic acid at 1 mg/Kg body weight, suggest that this effect of lymphocytes is not mediated by prostaglandins or leukotrienes
Assuntos
Ratos , Animais , Masculino , Glucose/sangue , Insulina/sangue , Linfócitos/fisiologia , Peso Corporal , Injeções Intravenosas , Veias Jugulares , Ratos Wistar , Fatores de TempoRESUMO
Several studies have shown the relationship between prostaglandins (PGs) and cell proliferation. Some PGs may trigger cell division or are involved in this process. This study analyzes the effect of PG biosynthesis inhibitors on tumor growth in vivo and cachexia in Walker 256 tumor-bearing rats. Indomethacin markedly inhibited tumor growth (95.5%) while ibuprofen and aspirin reduced tumor growth by 73.9% and 59.4%, respectively. In addition, all drug-treated rats partially recovered body weight and food intake as compared to the saline-treated group. These findings suggest that PG synthesis inhibitors improve cancer cachexia.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Carcinoma 256 de Walker/patologia , Antagonistas de Prostaglandina/farmacologia , Animais , Aspirina/farmacologia , Peso Corporal , Caquexia/tratamento farmacológico , Divisão Celular/efeitos dos fármacos , Ibuprofeno/farmacologia , Indometacina/farmacologia , Masculino , Transplante de Neoplasias , Prostaglandinas E/sangue , RatosRESUMO
Several studies have shown the relationship between prostaglandins (PGs) and cell proliferation. Some PGs may trigger cell dibision or are involved in this process. This a=study analyzes the effect of PG biosyntheseis inhibitors on tumor growth in vivo and cachexia in Walker 256 tumor-bearing rats. Indomethacin markedly inhibited tumor growth (95.5% while ibuprofen and aspirin reduced tumor growth by 73.9% and 59.4%, respectively. In addition, all drug-treated rats partially recovered body weight and food intake as compared to the saline-treated group. These findings suggest that PG synthesis inhibitors improve cancer cachexia