RESUMO
Acute, inflammatory conditions associated with dysregulated complement activation are characterized by significant increases in blood concentration of reactive oxygen species (ROS) and ATP. The mechanisms by which these molecules arise are not fully understood. In this study, using luminometric- and fluorescence-based methods, we show that ligation of glycophorin A (GPA) on human red blood cells (RBCs) results in a 2.1-fold, NADPH-oxidase-dependent increase in intracellular ROS that, in turn, trigger multiple downstream cascades leading to caspase-3 activation, ATP release, and increased band 3 phosphorylation. Functionally, using 2D microchannels to assess membrane deformability, GPS-ligated RBCs travel 33% slower than control RBCs, and lipid mobility was hindered by 10% using fluorescence recovery after photobleaching (FRAP). These outcomes were preventable by pretreating RBCs with cell-permeable ROS scavenger glutathione monoethyl ester (GSH-ME). Our results obtained in vitro using anti-GPA antibodies were validated using complement-altered RBCs isolated from control and septic patients. Our results suggest that during inflammatory conditions, circulating RBCs significantly contribute to capillary flow dysfunctions, and constitute an important but overlooked source of intravascular ROS and ATP, both critical mediators responsible for endothelial cell activation, microcirculation impairment, platelet activation, as well as long-term dysregulated adaptive and innate immune responses.
Assuntos
Eritrócitos/metabolismo , Glicoforinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Trifosfato de Adenosina/metabolismo , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Caspase 3/metabolismo , Membrana Eritrocítica/metabolismo , Eritrócitos/efeitos dos fármacos , Humanos , Metabolismo dos Lipídeos , Oxirredução , Fenótipo , Fosforilação , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Human antidepressants have been previously shown to induce foot detachment from the substrate in aquatic snails. Prior to foot detachment, antidepressants also affect snail crawling speed. We tested two commonly prescribed antidepressants, venlafaxine ("Effexor") and fluoxetine ("Prozac") on crawling speed and time to reach the air-water interface in two species of marine snail, the oyster drill Urosalpinx cinerea and the American starsnail Lithopoma americanum. Exposure to venlafaxine increased crawling speed in both species, while fluoxetine slowed them down. Our lowest LOEC (lowest observed effect concentration) was 31.3 µg/L venlafaxine in Urosalpinx. Similarly, snails (L. americanum) exposed to venlafaxine tended to move faster and more often to the air-water interface, but exposure to fluoxetine slowed them down. Our lowest LOEC was 345 µg/L fluoxetine in Lithopoma. These results indicate that venlafaxine boosts locomotion, while fluoxetine reduces it, and both behaviors are preludes to foot detachment. The different effects of these two antidepressants on snail locomotion suggest differing physiological mechanisms of action in marine snails as well as possible ecological consequences.
Assuntos
Antidepressivos/toxicidade , Cicloexanóis/toxicidade , Fluoxetina/toxicidade , Caramujos/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Locomoção/efeitos dos fármacos , Especificidade da Espécie , Cloridrato de VenlafaxinaRESUMO
Humans and other higher primates are unique among mammals in using complement receptor 1 (CR1, CD35) on red blood cells (RBC) to ligate complement-tagged inflammatory particles (immune complexes, apoptotic/necrotic debris, and microbes) in the circulation for quiet transport to the sinusoids of spleen and liver where resident macrophages remove the particles, but allow the RBC to return unharmed to the circulation. This process is called immune-adherence clearance. In this study we found using luminometric- and fluorescence-based methods that ligation of CR1 on human RBC promotes ATP release. Our data show that CR1-mediated ATP release does not depend on Ca(2+) or enzymes previously shown to mediate an increase in membrane deformability promoted by CR1 ligation. Furthermore, ATP release following CR1 ligation increases the mobility of the lipid fraction of RBC membranes, which in turn facilitates CR1 clustering, and thereby enhances the binding avidity of complement-opsonized particles to the RBC CR1. Finally, we have found that RBC-derived ATP has a stimulatory effect on phagocytosis of immune-adherent immune complexes.