Stimulation Of Hexose Monophosphate Shunt Activity Research Articles

Favism: effect of divicine on rat erythrocyte sulfhydryl status, hexose monophosphate shunt activity, morphology, and membrane skeletal proteins.

Favism is an acute anemic crisis that can occur in susceptible individuals who ingest fava beans. The fava bean pyrimidine aglycone divicine has been identified as a hemotoxic constituent; however, its mechanism of toxicity remains unknown. We have shown recently that divicine can induce a favic-like response in rats and that divicine is directly toxic to rat red cells. In the present study, we have examined the effect of hemotoxic concentrations of divicine on rat erythrocyte sulfhydryl status, hexose monophosphate (HMP) shunt activity, morphology, and membrane skeletal proteins. In vitro exposure of rat red cells to divicine markedly stimulated HMP shunt activity and resulted in depletion of reduced glutathione with concomitant formation of glutathione-protein mixed-disulfides. Examination of divicine-treated red cells by scanning electron microscopy revealed transformation of the cells to an extreme echinocytic morphology. SDS-PAGE and immunoblotting analysis of the membrane skeletal proteins indicated that hemotoxicity was associated with the apparent loss of skeletal protein bands 2.1, 3, and 4.2, and the appearance of membrane-bound hemoglobin. Treatment of divicine-damaged red cells with dithiothreitol reversed the protein changes, which indicated that the observed alterations were due primarily to the formation of disulfide-linked hemoglobin-skeletal protein adducts. The data suggest that oxidative modification of hemoglobin and membrane skeletal proteins by divicine may be key events in the mechanism underlying favism.

Nitric oxide attenuates cellular hexose monophosphate shunt response to oxidants in articular chondrocytes and acts to promote oxidant injury.

Nitric oxide (NO) has been implicated in both cartilage degradation and cell survival. Importantly, NO has been shown, in a cell-type-dependent manner, to directly cause cell death or indirectly promote cell death by compromising the ability of cells to detoxify intra- or extracellular oxidants. In this study we examined the role of NO in the survival of bovine chondrocytes exposed to catabolic cytokines (interleukin-1 (IL-1); tumor necrosis factor [TNF]) with or without the addition of an exogenous oxidant stress (e.g., H2O2, HOOCl, etc.). The exposure of chondrocytes to a mixture of IL-1 and TNF (IL-1/TNF) results in the release of NO but did not alter cell viability. However, there was evidence of NO-dependent oxidative responses in the IL-1/TNF group, as we observed an increased level of intracellular oxidants as well as the appearance of a 55 kD nitrated protein which reflects the formation of peroxynitrite. We next analyzed viability with H2O2. The LD50 for IL-1/TNF-treated cells was 0.1 mM (vs. 1 mM for control). The enhanced sensitivity was completely reversed when cells were incubated with the NO synthase inhibitor 1-n5-1-iminoethylornithine (NIO). To test whether cell death was caused by compromising the ability of cells to detoxify extracellular oxidants, we examined the hexose monophosphate shunt (HMPS) response in cells given H2O2. Treatment of control cells with H2O2 resulted in a fourfold increase in HMPS activity. In contrast, IL-1/TNF cells exhibited no increase in HMPS activity. The attenuation of stimulated HMPS activity was reversed by the coaddition of NIO. Thus, these data indicate that 1) endogenous NO mediates cytokine-dependent susceptibility to oxidant injury and 2) this effect is in part due to impaired activation of the HMPS. In inflamed joints replete with cytokines and oxidants, NO may contribute to chondrocyte death and progressive joint destruction.

Effects of colchicine and vincristine on polymorphonuclear leucocyte function in vitro

The effect of the drugs colchicine and vincristine on normal polymorphonuclear leucocytes (PMNs) was investigated in vitro. These drugs are known to interfere with the organization of microtubules both in the mitotic spindle of dividing cells, causing metaphase arrest, and in the cytoplasm of other non-dividing cells affecting a number of normal cellular functions. PMNs were obtained from healthy volunteers by centrifugation of heparinized blood over Ficoll Isopaque gradients followed by dextran sedimentation and hypotonic lysis of residual erythrocytes. The preparations were greater than 95% pure PMNs, and cell viability was 99% as assessed by Trypan blue exclusion. Four aspects of PMN function were studied in vitro—chemotaxis, phagocytic activity, hexosemonophosphate (HMP) shunt activity and superoxide production. Chemotaxis was measured in a Boyden chamber system using 3.0 Millipore filters and zymosan activated serum as the chemotactic stimulus. Phagocytosis was measured by the ability of PMNs to ingest heat killed candida albicans and the phagocytic index was expressed as the number of yeast cells per PMN. HMP shunt activity in both resting and stimulated cells was determined by measuring the oxidation of glucose – 1 – 14C with release of 14CO2. Superoxide production by PMNs was measured on the basis of its ability to reduce cytochrome C. Ten separate experiments were performed and the results obtained for PMNs exposed to either colchicine (2.5 × 10-4M) or vincristine (1 × 10-6’M) were compared to controls. Chemotaxis, phagocytic activity and superoxide production were all significantly reduced in the presence of either colchicine or vincristine. Stimulated HMP shunt activity was also reduced, but resting PMNs were not affected. The data indicate that these drugs have multiple effects on PMN function which may be important in the in vivo PMN response. The effect of the drugs colchicine and vincristine on normal polymorphonuclear leucocytes (PMNs) was investigated in vitro. These drugs are known to interfere with the organization of microtubules both in the mitotic spindle of dividing cells, causing metaphase arrest, and in the cytoplasm of other non-dividing cells affecting a number of normal cellular functions. PMNs were obtained from healthy volunteers by centrifugation of heparinized blood over Ficoll Isopaque gradients followed by dextran sedimentation and hypotonic lysis of residual erythrocytes. The preparations were greater than 95% pure PMNs, and cell viability was 99% as assessed by Trypan blue exclusion. Four aspects of PMN function were studied in vitro—chemotaxis, phagocytic activity, hexosemonophosphate (HMP) shunt activity and superoxide production. Chemotaxis was measured in a Boyden chamber system using 3.0 Millipore filters and zymosan activated serum as the chemotactic stimulus. Phagocytosis was measured by the ability of PMNs to ingest heat killed candida albicans and the phagocytic index was expressed as the number of yeast cells per PMN. HMP shunt activity in both resting and stimulated cells was determined by measuring the oxidation of glucose – 1 – 14C with release of 14CO2. Superoxide production by PMNs was measured on the basis of its ability to reduce cytochrome C. Ten separate experiments were performed and the results obtained for PMNs exposed to either colchicine (2.5 × 10-4M) or vincristine (1 × 10-6’M) were compared to controls. Chemotaxis, phagocytic activity and superoxide production were all significantly reduced in the presence of either colchicine or vincristine. Stimulated HMP shunt activity was also reduced, but resting PMNs were not affected. The data indicate that these drugs have multiple effects on PMN function which may be important in the in vivo PMN response.

Effect of mega doses of vitamin C on bactericidal ativity of leukocytes

Effect of ingesting mega doses of ascorbic acid was studied on the leukocyte function in five normal human subjects. During the first 15 days the subjects received daily supplements of 200 mg of ascorbic acid, and during the next 2 weeks they were given 2 g of vitamin C per day. Supplementation of 200 mg as well as 2 g of ascorbic acid stimulated hexose monophosphate shunt activity of resting leukocytes indicating an increase in resting metabolism. Intakes of 200 mg of ascorbic acid per day did not affect bacterial killing by leukocytes. On the other hand, daily intakes of 2 g of ascorbic acid for 2 weeks significantly impaired bactericidal activity. Four weeks after withdrawal of the viatmin supplementation, bactericidal activity returned to normal.

Abnormal erythrocyte metabolism in hepatic disease

Erythrocyte (RBC) metabolic studies were done on 114 patients with severe hepatic disease. Heinz body formation after incubation of RBCs with acetyl phenylhydrazine was found to be significantly higher in patients than in controls. RBC-reduced glutathione levels were lower than those of controls both before and after incubation with acetyl phenylhydrazine, and patients with the highest Heinz body counts had the lowest reduced glutathione levels. RBC methylene blue-stimulated hexose monophosphate (HMP) shunt metabolism and glucose recycling through the shunt were significantly lower in patients with active hepatic disease than in controls. There was no difference in resting HMP shunt activity or in resting recycling of glucose. Despite impairment of shunt metabolism, total glucose consumption was greater in patients than in controls. The patients with the lowest stimulated HMP shunt metabolism and glucose recycling had the highest Heinz body counts, lowest reduced glutathione, and highest total glucose consumption. A continuum of abnormal shunt metabolism was seen, from a mild reduction of stimulated HMP shunt activity to a severe combined decrease in both the HMP shunt and glucose recycling. When measured, glutathione reductase, glutathione peroxidase, glucose-6-phosphate dehydrogenase, and transketolase were normal or increased. Sequential studies were done on 11 patients who had abnormal metabolic studies. Coincident with improvement of HMP shunt metabolism, the Heinz body counts became lower, reduced glutathione higher, hematocrit higher, and liver function improved. Impaired HMP shunt metabolism appears to be a common, acquired RBC abnormality in patients with severe, active liver disease.

Abnormal Erythrocyte Metabolism in Hepatic Disease

Erythrocyte (RBC) metabolic studies were done on 114 patients with severe hepatic disease. Heinz body formation after incubation of RBCs with acetyl phenylhydrazine was found to be significantly higher in patients than in controls. RBC-reduced glutathione levels were lower than those of controls both before and after incubation with acetyl phenylhydrazine, and patients with the highest Heinz body counts had the lowest reduced glutathione levels. RBC methylene blue-stimulated hexose monophosphate (HMP) shunt metabolism and glucose recycling through the shunt were significantly lower in patients with active hepatic disease than in controls. There was no difference in resting HMP shunt activity or in resting recycling of glucose. Despite impairment of shunt metabolism, total glucose consumption was greater in patients than in controls. The patients with the lowest stimulated HMP shunt metabolism and glucose recycling had the highest Heinz body counts, lowest reduced glutathione, and highest total glucose consumption. A continuum of abnormal shunt metabolism was seen, from a mild reduction of stimulated HMP shunt activity to a severe combined decrease in both the HMP shunt and glucose recycling. When measured, glutathione reductase, glutathione peroxidase, glucose-6-phosphate dehydrogenase, and transketolase were normal or increased. Sequential studies were done on 11 patients who had abnormal metabolic studies. Coincident with improvement of HMP shunt metabolism, the Heinz body counts became lower, reduced glutathione higher, hematocrit higher, and liver function improved. Impaired HMP shunt metabolism appears to be a common, acquired RBC abnormality in patients with severe, active liver disease.

Stimulation by propylthiouracil of the hexose monophosphate shunt in human polymorphonuclear leucocytes during phagocytosis.

The effect of propylthiouracil on glucose metabolism in human polymorphonuclear leucocytes was studied. At a therapeutically achievable concentration (0.1 mM), propylthiouracil stimulated hexose monophosphate shunt activity in normal leucocytes during phagocytosis but not in resting cells. However, in the presence of hydrogen peroxide it stimulated hexose monophosphate shunt activity in resting cells, and in the soluble fraction when reduced glutathione and reduced nictotinamide adenine dinucleotide phosphate (NADPH) were also present. Propylthiouracil had nor effect on glucose-1-C oxidation in either phagocytosing or resting leucocytes obtained from two male patients with chronic granulomatous disease. Stimulation of the hexose monophosphate shunt activity in normal leucocytes during phagocytosis also was demonstrated with methimazole, thiouracil and thiourea, but not with adenine, uracil or urea. There was an apparent minimal common structure requirement in thriourea. Propylthiouracil had no effect on phagocytosis, formate oxidation, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase or catalase activities. Thus, the stimulation of the hexose monophosphate shunt activity by propylthiouracil is dependent on hydrogen peroxide and is best explained by its stimulation or participation in the glutathione cycle.

Enhancement of random migration and chemotactic response of human leukocytes by ascorbic acid.

Incubation of human leukocytes with ascorbic acid at neutral pH and at concentrations 10-50 times that of normal blood levels augmented both the in vitro random migration and chemotaxis of the cells by 100-300% without influencing their phagocytic capacity. Enhancement of mobility by ascorbate was evident for isolated neutrophils, eosinophils, and mono-nuclear leukocytes and was independent of the specific chemotactic stimulus. Stimulation by ascorbate of the hexose monophosphate shunt of adherent neutrophils and augmentation by ascorbate of neutrophil mobility had comparable dose-response relationships, could be reversed by washing the cells, and were both suppressed by preincubation of the neutrophils with 6-aminonicotinamide, but not with the neutrophil-immobilizing factor. Glutathione, the proposed intermediate for ascorbate action, similarly stimulated hexose monophosphate shunt activity and enhanced migration. The enhancement in vitro of leukocyte mobility by ascorbate at concentrations found in some normal tissues, therefore, appears to be dependent upon stimulation of the leukocyte hexose monophosphate shunt.