RESUMO
Endothelin1 (ET-1) is a 21-amino acid peptide produced by the vascular endothelium under hypoxia, that acts locally as regulator of vascular tone and inflammation. The role of ET-1 in Plasmodium falciparum malaria is unknown, although tissue hypoxia is frequent as a result of the cytoadherence of parasitized red blood cell (pRBC) to the microvasculature. Here, we show that both synthetic and endothelial-derived ET-1 are removed by parasitized RBC (D10 and W2 strains, chloroquine sensitive, and resistant, resp.) and native haemozoin (HZ, malaria pigment), but not by normal RBC, delipidized HZ, or synthetic beta-haematin (BH). The effect is dose dependent, selective for ET-1, but not for its precursor, big ET-1, and not due to the proteolysis of ET-1. The results indicate that ET-1 binds to the lipids moiety of HZ and membranes of infected RBCs. These findings may help understanding the consequences of parasite sequestration in severe malaria.
Assuntos
Endotelina-1/metabolismo , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Hemeproteínas/metabolismo , Malária Falciparum/sangue , Plasmodium falciparum/metabolismo , Linhagem Celular , Células Endoteliais , Endotelina-1/química , Endotelina-1/genética , Hemeproteínas/química , Humanos , Lipídeos , Malária Falciparum/parasitologia , Plasmodium falciparum/química , Reação em Cadeia da PolimeraseRESUMO
Two new quinolizidinyl-alkyl derivatives of 7-chloro-4-aminoquinoline, named AM-1 and AP4b, which are highly effective in vitro against both the D10 (chloroquine [CQ] susceptible) and W2 (CQ resistant) strains of Plasmodium falciparum and in vivo in the rodent malaria model, have been studied for their ability to bind to and be internalized by normal or parasitized human red blood cells (RBC) and for their effects on RBC membrane stability. In addition, an analysis of the heme binding properties of these compounds and of their ability to inhibit beta-hematin formation in vitro has been performed. Binding of AM1 or AP4b to RBC is rapid, dose dependent, and linearly related to RBC density. Their accumulation in parasitized RBC (pRBC) is increased twofold compared to levels in normal RBC. Binding of AM1 or AP4b to both normal and pRBC is higher than that of CQ, in agreement with the lower pKa and higher lipophilicity of the compounds. AM1 or AP4b is not hemolytic per se and is less hemolytic than CQ when hemolysis is accelerated (induced) by hematin. Moreover, AM-1 and AP4b bind heme with a stoichiometry of interaction similar to that of CQ (about 1:1.7) but with a lower affinity. They both inhibit dose dependently the formation of beta-hematin in vitro with a 50% inhibitory concentration comparable to that of CQ. Taken together, these results suggest that the antimalarial activity of AM1 or AP4b is likely due to inhibition of hemozoin formation and that the efficacy of these compounds against the CQ-resistant strains can be ascribed to their hydrophobicity and capacity to accumulate in the vacuolar lipid (elevated lipid accumulation ratios).
Assuntos
Aminoquinolinas/metabolismo , Aminoquinolinas/farmacologia , Antimaláricos/metabolismo , Antimaláricos/farmacologia , Eritrócitos/efeitos dos fármacos , Heme/metabolismo , Plasmodium falciparum/fisiologia , Aminoquinolinas/efeitos adversos , Aminoquinolinas/química , Antimaláricos/efeitos adversos , Antimaláricos/química , Células Cultivadas , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Humanos , Malária Falciparum/tratamento farmacológico , Estrutura MolecularRESUMO
We provide two simple low-cost and low-tech procedures to measure with good precision and accuracy the binding and internalization into human erythrocytes of chloroquine and other aminoquinolines. The methods are based on the high fluorescence of the quinoline ring and are complementary. Method A evaluates residual drugs in the supernatants of treated erythrocytes, whereas method B quantifies the total uptake by whole cells and the fraction bound to the membranes. Drug uptake is dose dependent and related to the number of erythrocytes. These assays could be useful when studying the cell interaction of quinoline-type compounds not available in the radioactive form.
Assuntos
Aminoquinolinas/farmacocinética , Eritrócitos/metabolismo , Fluorometria/métodos , Aminoquinolinas/química , Transporte Biológico , Cloroquina/química , Cloroquina/farmacocinética , Humanos , Projetos de PesquisaRESUMO
Erythrocyte glyceraldehyde-3-phosphate dehydrogenase (G3PD), is a glycolytic enzyme normally inhibited upon binding to the anion transporter Band 3 and activated when free in the cytosol. We have previously reported that ferric protoporphyrin IX (FP) enhances G3PD activity in human erythrocytes (RBC). This could be due to two mechanisms considered in this work: Band 3 tyrosine phosphorylation or oxidative damage of specific G3PD binding sites in the membrane. In both cases binding of G3PD to the membrane would be prevented, leading to the enhancement of G3PD activity. Here, we show that FP induces a dose- and time-dependent phosphorylation of tyrosine 8 and 21 of Band 3, as confirmed by the recruitment of SHP2 phosphatase to the membrane. It appears that Band 3 phosphorylation is due to the oxidation of critical sulfydryl groups of a membrane phosphatase (PTP). Data on membrane localization, Mg2+ dependence, sensitivity to thiol oxidizing agents and protection by N-acetylcysteine (NAC) and DTT strongly suggest the involvement of PTP1B, the major PTP of human RBC associated to and acting on Band 3. However, FP activates G3PD even when Band 3 phosphorylation is inhibited, therefore phosphorylation is not the mechanism underlying G3PD activation by FP. The capacity of NAC of counteracting the stimulatory activity of FP, supports the hypothesis that FP might induce the oxidative damage of specific G3PD binding sites in the membrane, causing the displacement of the enzyme into the cytosol and/or the release from its binding site and therefore its activation.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Membrana Eritrocítica/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Hemina/farmacologia , Tirosina/metabolismo , Western Blotting , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Membrana Eritrocítica/enzimologia , Membrana Eritrocítica/metabolismo , Eritrócitos/citologia , Eritrócitos/enzimologia , Eritrócitos/metabolismo , Humanos , FosforilaçãoRESUMO
In falciparum malaria, the deformability of the entire erythrocyte population is reduced in proportion to disease severity, and this compromises microcirculatory blood flow through vessels partially obstructed by cytoadherent parasitized erythrocytes. The cause of rigidity of uninfected erythrocytes in not known but could be mediated by malaria heme products. In this study, we show that red blood cell deformability (RBC-D), measured by laser-assisted optical rotational cell analyzer, decreased in a dose-dependent manner after incubation with hemin and hydrogen peroxide but not with hemoglobin or beta-hematin. Hemin also reduced mean red cell volume. Albumin decreased and N-acetylcysteine (NAC) both prevented and reversed rigidity induced by hemin. Hemin-induced oxidative damage of the membrane seems to be a more important contributor to pathology than cell shrinkage because the antioxidant NAC restored RBC-D but not red blood cell volume. The findings suggest novel approaches to the treatment of potentially lethal malaria.
Assuntos
Albuminas/farmacologia , Deformação Eritrocítica/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Hemeproteínas/farmacologia , Malária Falciparum/sangue , Acetilcisteína/farmacologia , Albuminas/metabolismo , Anemia/sangue , Anemia/parasitologia , Antioxidantes/farmacologia , Eritrócitos/patologia , Glutationa/metabolismo , Glutationa/farmacologia , Hemeproteínas/metabolismo , Hemina/metabolismo , Hemina/farmacologia , Hemoglobinas/metabolismo , Hemoglobinas/farmacologia , Humanos , Peróxido de Hidrogênio/farmacologiaRESUMO
Erythrocyte glyceraldehyde-3-phosphate dehydrogenase (G3PD) is a glycolytic enzyme containing critical thiol groups and whose activity is reversibly inhibited by binding to the cell membrane. Here, we demonstrate that the insertion of ferriprotoporphyrin IX (FP) into the red cell membranes exerts two opposite effects on membrane bound G3PD. First, the enzyme is partially inactivated through oxidation of critical thiols. Dithiothreitol restores part of the activity, but some critical thiols are irreversibly oxidized or crosslinked to products of FP-induced lipid peroxidation. Second, G3PD binding to the membrane is modified and the enzyme is activated through displacement into the cytosol and/or release from its binding site.
Assuntos
Eritrócitos/enzimologia , Gliceraldeído 3-Fosfato Desidrogenase (NADP+)/metabolismo , Hemina/metabolismo , Membrana Celular/metabolismo , Membrana Eritrocítica/enzimologia , Eritrócitos/citologia , Humanos , Oxirredução , Ligação Proteica , Compostos de Sulfidrila/químicaRESUMO
Malaria-associated acute lung injury (MA-ALI) and its more severe form malaria-associated acute respiratory distress syndrome (MA-ARDS) are common, often fatal complications of severe malaria infections. However, little is known about their pathogenesis. In this study, biochemical alterations of the lipid composition of the lungs were investigated as possible contributing factors to the severity of murine MA-ALI/ARDS. C57BL/6J mice were infected with Plasmodium berghei NK65 to induce lethal MA-ARDS, or with Plasmodium chabaudi AS, a parasite strain that does not induce lung pathology. The lipid profile of the lung tissue from mice infected with Plasmodium berghei NK65 developing MA-ALI/ARDS, but not that from mice without lung pathology or controls, was characterized by high levels of phospholipids -mainly phosphatidylcholine- and esterified cholesterol. The high levels of polyunsaturated fatty acids and the linoleic/oleic fatty acid ratio of the latter reflect the fatty acid composition of plasma cholesterol esters. In spite of the increased total polyunsaturated fatty acid pool, which augments the relative oxidability of the lung membranes, and the presence of hemozoin, a known pro-oxidant, no excess oxidative stress was detected in the lungs of Plasmodium berghei NK65 infected mice. The bronchoalveolar lavage (BAL) fluid of Plasmodium berghei NK65 infected mice was characterized by high levels of plasma proteins. The phospholipid profile of BAL large and small aggregate fractions was also different from uninfected controls, with a significant increase in the amounts of sphingomyelin and lysophosphatidylcholine and the decrease in phosphatidylglycerol. Both the increase of proteins and lysophosphatidylcholine are known to decrease the intrinsic surface activity of surfactant. Together, these data indicate that an altered lipid composition of lung tissue and BAL fluid, partially ascribed to oedema and lipoprotein infiltration, is a characteristic feature of murine MA-ALI/ARDS and possibly contribute to lung dysfunction.
Assuntos
Lipídeos/química , Pulmão/patologia , Malária/complicações , Surfactantes Pulmonares/metabolismo , Síndrome do Desconforto Respiratório/complicações , Síndrome do Desconforto Respiratório/patologia , Animais , Membrana Celular/metabolismo , Modelos Animais de Doenças , Feminino , Fibrose , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Plasmodium berghei/fisiologia , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/patologia , Síndrome do Desconforto Respiratório/metabolismoRESUMO
BACKGROUND: Endothelial dysfunction in widely diffuse disorders, such as atherosclerosis, hypertension, diabetes and senescence, is associated with nitric oxide (NO) deficiency. Here, the behavioural and molecular consequences deriving from NO deficiency in human umbilical vein endothelial cells (HUVECs) were investigated. RESULTS: Endothelial nitric oxide synthase (eNOS) was chronically inhibited either by N(G)-Nitro-L-arginine methyl ester (L-NAME) treatment or its expression was down-regulated by RNA interference. After long-term L-NAME treatment, HUVECs displayed a higher migratory capability accompanied by an increased Vascular Endothelial Growth Factor (VEGF) and VEGF receptor-2 (kinase insert domain receptor, KDR) expression. Moreover, both pharmacological and genetic inhibition of eNOS induced a state of pseudohypoxia, revealed by the stabilization of hypoxia-inducible factor-1α (HIF-1α). Furthermore, NO loss induced a significant decrease in mitochondrial mass and energy production accompanied by a lower O(2) consumption. Notably, very low doses of chronically administered DETA/NO reverted the HIF-1α accumulation, the increased VEGF expression and the stimulated migratory behaviour detected in NO deficient cells. CONCLUSION: Based on our results, we propose that basal release of NO may act as a negative controller of HIF-1α levels with important consequences for endothelial cell physiology. Moreover, we suggest that our experimental model where eNOS activity was impaired by pharmacological and genetic inhibition may represent a good in vitro system to study endothelial dysfunction.
Assuntos
Movimento Celular , Endotélio Vascular/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Óxido Nítrico/metabolismo , Apoptose , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Inativação Gênica , Humanos , NG-Nitroarginina Metil Éster/farmacologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/genética , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
In falciparum malaria, both infected and uninfected red cells have structural and functional alterations. To investigate the mechanisms of these modifications, we studied the effects of two Plasmodium falciparum haem products (haematin and malaria pigment in the synthetic form beta-haematin) on isolated human red blood cells (RBCs) and purified RBC ghosts. A dose- and time-dependent incorporation of haematin into RBC ghosts and intact cells was observed, which was in proportion to the extent of haematin- induced haemolysis. RBCs pre-incubated with haematin were more sensitive to haemolysis induced by hypotonic shock, low pH, H2O2 or haematin itself. Haemolysis was not related to membrane lipid peroxidation and only partially to oxidation of protein sulphydryl groups and it could not be prevented by scavengers of lipid peroxidation or hydroperoxide groups. N-acetylcysteine partly protected the oxidation of SH groups and significantly reduced haemolysis. In contrast, beta-haematin was neither haemolytic nor oxidative towards protein sulphydryl groups. Beta-haematin did destabilise the RBC membrane, but to a lesser extent than haematin, inducing increased susceptibility to lysis caused by hypotonic medium, H2O2 or haematin. This study suggests that the destabilising effect of haematin and, to a much less extent, beta-haematin on the RBC membrane does not result from oxidative damage of membrane lipids but from direct binding or incorporation which may affect the reciprocal interactions between the membrane and cytoskeleton proteins. These changes could contribute to the reduced red cell deformability associated with severe malaria.
Assuntos
Eritrócitos/parasitologia , Plasmodium falciparum/patogenicidade , Animais , Antioxidantes/farmacologia , Membrana Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/parasitologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Glutationa/metabolismo , Hemeproteínas/toxicidade , Hemina/toxicidade , Hemólise/efeitos dos fármacos , Hemorreologia , Humanos , Técnicas In Vitro , Peroxidação de Lipídeos/efeitos dos fármacos , Proteínas de Membrana/metabolismo , OxirreduçãoRESUMO
Heme (ferric protoporphyrin IX, FP) dissolves very rapidly into the lipid phase of membranes, and a large number of studies have focused attention on its possible toxic effect in whole cells or isolated membranes. However, because of its molecular structure and reactivity, different problems can be encountered during the course of studying biological samples containing FP. In this article, we discuss important interferences by FP and artifacts that can affect the experimental values. First, FP interferes with the Lowry's protein determination; therefore, membranes containing FP are overestimated in their protein content determined by this procedure. Second, freezing membranes at -20 degrees C artifactually increases the local concentration of FP, thereby enhancing FP-induced lipid peroxidation. Third, in the presence of thiol compounds such as N-acetyl cysteine, FP is degraded to products that interfere with the thiobarbituric acid assay, one of the most widely used methods to measure the extent of lipoperoxidation.
Assuntos
Artefatos , Heme/análise , Acetilcisteína/química , Ácido Araquidônico/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Eritrocítica/efeitos dos fármacos , Congelamento , Heme/química , Heme/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Lipídeos de Membrana/química , Micelas , Proteínas/análise , SolubilidadeRESUMO
Red blood cells infected withPlasmodium falciparum(IRBCs) undergo changes primarily in their membrane composition that contribute to malaria pathogenesis. However, all manifestations (eg, anemia) cannot be accounted for by IRBCs alone. Uninfected erythrocytes (URBCs) may play a role, but they have been under-researched. We wanted to document changes in the erythrocyte membrane that could contribute to URBC reduced life span and malaria-associated anemia. Human erythrocytes were cultured withP falciparumand washed at the trophozoite stage. IRBCs and URBCs were separated on Percoll density gradient, thus obtaining erythrocyte fractions of different densities/ages. IRBC- and URBC-purified membranes were analyzed and compared with control normal erythrocytes (NRBCs) of the same age, from the same donor, kept in the same conditions.P falciparumaccelerated aging of both IRBCs and URBCs, causing a significant shift in the cell population toward the denser (old) fraction. Protein, phospholipid, and cholesterol content were reduced in IRBCs and young URBCs. Young and medium uninfected fractions had higher levels of lipid peroxidation and phospholipid saturation (because of the loss of polyunsaturated fatty acids, PUFAs) and lower phosphatidylserine. In IRBCs, thiobarbituric reactive substances (TBARSs) were higher, and PUFAs and phosphatidylserine lower than in NRBCs and URBCs. In comparison, trophozoite membranes had lower phospholipid (particularly sphingomyelin and phosphatidylserine) and cholesterol content and a higher degree of saturation. Parasite-induced peroxidative damage might account for these modifications. In summary, we demonstrated that membrane damage leading to accelerated senescence of both infected and uninfected erythrocytes will likely contribute to malaria anemia.