Academic literature on the topic 'Erythrocyte signaling pathways'

Activation of the G protein Gs results in increases in cAMP, a necessary step in the pathway for ATP release from rabbit and human erythrocytes. In all cells, the level of cAMP is the product of its synthesis by adenylyl cyclase and its hydrolysis by phosphodiesterases (PDEs). Both iloprost (Ilo), a PGI2 analog, and isoproterenol (Iso), a β-agonist, stimulate receptor-mediated increases in cAMP in rabbit and human erythrocytes. However, the specific PDEs associated with each of these signaling pathways in the erythrocyte have not been fully characterized. Previously, we reported that PDE3B is present in rabbit and human erythrocyte membranes and that PDE3 inhibitors potentiate Ilo-induced increases in cAMP. Here we report that inhibitors of either PDE2 or PDE4, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and rolipram, respectively, potentiate Iso-induced increases in cAMP in rabbit and human erythrocytes. Importantly, these inhibitors had no effect on cAMP increases associated with the incubation of erythrocytes with Ilo. In addition, we establish, for the first time, the presence of PDE2A protein in rabbit and human erythrocyte membranes. Finally, we determined that preincubation of human erythrocytes with EHNA and rolipram together potentiate Iso-induced ATP release, whereas preincubation with cilostazol enhances Ilo-induced release of ATP. These results are consistent with the hypothesis that, in rabbit and human erythrocytes, Ilo-induced increases in cAMP and ATP release are regulated by PDE3, whereas those associated with Iso are regulated by the activities of both PDE2 and PDE4. These studies demonstrate that PDE activity in these cells is localized to specific signaling pathways.

Increases in the second messenger cAMP are associated with receptor-mediated ATP release from erythrocytes. In other signaling pathways, cAMP-specific phosphodiesterases (PDEs) hydrolyze this second messenger and thereby limit its biological actions. Although rabbit and human erythrocytes possess adenylyl cyclase and synthesize cAMP, their PDE activity is poorly characterized. It was reported previously that the prostacyclin analog iloprost stimulated receptor-mediated increases in cAMP in rabbit and human erythrocytes. However, the PDEs that hydrolyze erythrocyte cAMP synthesized in response to iloprost were not identified. PDE3 inhibitors were reported to augment increases in cAMP stimulated by prostacyclin analogs in platelets and pulmonary artery smooth muscle cells. Additionally, PDE3 activity was identified in embryonic avian erythrocytes, but the presence of this PDE in mammalian erythrocytes has not been investigated. Here, using Western blot analysis, we determined that PDE3B is a component of rabbit and human erythrocyte membranes. In addition, we report that the preincubation of rabbit and human erythrocytes with the PDE3 inhibitors milrinone and cilostazol potentiates iloprost-induced increases in cAMP. In addition, cilostamide, the parent compound of cilostazol, potentiated iloprost-induced increases in cAMP in human erythrocytes. These findings demonstrate that PDE3B is present in rabbit and human erythrocytes and are consistent with the hypothesis that PDE3 activity regulates cAMP levels associated with a signaling pathway activated by iloprost in these cells.

The possible role of physiologic stress hormones in enhancing adhesion of sickle erythrocytes (SS RBCs) to endothelial cells (ECs) in sickle cell disease (SCD) has not been previously explored. We have now found that up-regulation of intracellular cyclic adenosine monophosphate (cAMP)–dependent protein kinase A (PKA) by epinephrine significantly increased sickle but not normal erythrocyte adhesion to both primary and immortalized ECs. Inhibition of serine/threonine phosphatases also enhanced sickle erythrocyte adhesion at least partially through a PKA-dependent mechanism. Adhesion was mediated through LW (intercellular adhesion molecule-4 [ICAM-4], CD242) blood group glycoprotein, and immunoprecipitation studies showed that LW on sickle but not on normal erythrocytes undergoes increased PKA-dependent serine phosphorylation as a result of activation. The major counter receptor for LW was identified as the αvβ3 integrin on ECs. These data suggest that adrenergic hormones such as epinephrine may initiate or exacerbate vaso-occlusion and thus contribute to the association of vaso-occlusive events with physiologic stress.

Abstract Sickle Cell Disease (SCD) is one of the most devastating hemolytic genetic disorders affecting millions worldwide. Erythrocytes possess high sphingosine kinase 1 (Sphk1) activity and are considered to be the major cell type for supplying plasma sphingosine-1-phosphate, a signaling lipid regulating multiple physiological and pathological functions. Recent studies revealed that erythrocyte SphK1 activity is up-regulated in sickle cell disease (SCD) and contributes to sickling and disease progression. However, how erythrocyte Sphk1 activity is regulated in SCD remains unknown. In an effort to identify specific factors and signaling pathways involved in regulation of erythrocyte SphK1 activities in SCD, we first chose to screen the effects of molecules known to induce SphK1 activities in other cell types and/or reported to be elevated in the circulation of SCD including tumor necrosis factor alpha (TNF-α), interleukin 1 (IL-1), endothelin 1 (ET-1), vascular endothelial growth factor (VEGF), S1P and adenosine, on the activities of SphK1 in cultured primary mouse normal erythrocytes. Among all of those molecules tested, we found that adenosine is a previously unidentified hypoxia inducible molecule directly inducing SphK1 activity in vitro in a time and dosage-dependent manner. Next, using four adenosine receptor deficient mice and pharmacological approaches, we determined that the A2B adenosine receptor (ADORA2B) is essential for adenosine-induced SphK1 activity in cultured primary mouse normal and sickle erythrocytes. Subsequently, we provided in vivo genetic evidence that adenosine deaminase (ADA)-deficiency leads to excess plasma adenosine and elevated erythrocyte SphK1 activity. Lowering adenosine by ADA enzyme therapy or genetic deletion of ADORA2B significantly reduced excess adenosine-induced erythrocyte SphK1 activity in ADA-deficient mice. Mechanistically, we revealed that PKA functions downstream of ADORA2B mediating ERK activation and subsequently underlying adenosine-induced SphK1 activities in cultured mouse erythrocytes. Finally, we conducted human translational studies and reported that adenosine signaling via ADORA2B directly increases SphK1 activity in cultured primary human normal and sickle erythrocytes in a PKA/ERK-dependent manner. Overall, our findings reveal a novel signaling network regulating erythrocyte SphK1 and highlight innovative mechanisms to control SphK1 activity in normal and sickle setting. Disclosures No relevant conflicts of interest to declare.

Ion channels in the plasma membrane serve multiple functions such as setting the membrane potential, adjusting the cell volume and the intracellular electrolyte concentrations or eliciting versatile cytosolic Ca2+ signals. Channel activities regulate many basic cellular processes. Among those are cell proliferation, migration, differentiation and apoptotic cell death. Although devoid of nuclei and mitochondria, mature mammalian erythrocytes maintain a full set of functional ion channels in their plasma membrane. This special issue of The Open Biology Journal focuses on ion channels in the membrane of mature human erythrocytes, their regulation and their putative functions. Mature human erythrocytes travel about 100 miles and circulate more than 100.000 times through the body during their normal life span of 120 ± 4 days. The most obvious task thereby is the transport of blood oxygen and carbon dioxide as well as buffering of the pH in the blood. These functions depend on carboanhydrase, hemoglobin and band 3 anion exchanger. The latter two are the most abundant proteins in the erythrocyte cytosol and membrane, respectively. Because of this high abundance of hemoglobin which accounts for 98% of the cytosol protein content and because of the substantial absence of intracellular organelles, mature human erythrocytes are commonly simplified to hemoglobincontaining sacks. In sharp contrast to this view, increasing numbers of proteins are identified in mature human erythrocytes. Among those are proteins that build up signaling cascades. Outside-in signaling via membrane receptors such as purinergic receptors, as well as inside-out signaling via release of, e.g., ATP has been reported (see the article of Duranton et al. in this special issue). Moreover, intracellular signaling molecules such as protein kinases, have been unequivocally demonstrated to be functional suggesting that mature human erythrocytes are endowed with complex signaling similar to nucleated cells. As an example, circulating erythrocytes are cellular sensors of the oxygen tension and mechanical stress. Decline in oxygen partial pressure results in release of ATP regulated, presumably, through Gs protein, adenylcyclase and protein kinase Adependent activation of the ABC transporter CFTR. The released ATP, in turn, triggers vessel dilatation via formation of nitric oxide by the endothelium. Nitric oxide also negatively feeds back to the erythrocyte where it down-regulates ATP release. Hence, by releasing ATP at decreased oxygen pressure erythrocytes adapt the microcirculation to the oxygen consumption. In addition to ATP release, signaling via adenylcyclase and protein kinase A activates a CFTR-associated anion channel in mature erythrocytes (reviewed by Bouyer et al. in this special issue). Moreover, mature human erythrocytes activate organic osmolyte and anion channels via ATP release and autocrine purinergic signaling which is reminiscent of the activation of osmolyte channels in nucleated cells upon cell swelling (reviewed by Duranton et al. in this special issue). This indicates that mature human erythrocytes functionally express ion channel-regulating pathways similar if not identical to those implemented in nucleated cells. Erythrocyte ion channels are also involved in sensing cellular stress. Shumilina and Huber report in this special issue that cellular stress such as oxidative stress activates ClC-2, a further type of anion channel in the erythrocyte membrane which participates in the cellular stress response. Being largely silent under resting conditions, erythrocyte channels may build up membrane conductances in the nS range upon various signals (such as oxidative stress). A strong activator of erythrocyte ion channel activity is the intraerythrocytic amplification of the malaria parasite Plasmodium falciparum. Accordingly, most of what we know about the electrophysiology of ClC-2, the CFTR-associated anion channel, and the organic osmolyte and anion channel in human erythrocytes came from whole-cell and single-channel recording in Plasmodium-infected cells. The data on ClC-2, the CFTR-associated channel and the osmolyte channel are discussed in this special issue by the articles of Shumilina and Huber, Bouyer et al., and Duranton et al., respectively. The fourth article in this special issue, the article of Lars Kästner, summarizes our current knowledge about the cation channels in erythrocytes. Mature human erythrocytes express KCa3.1 (Gardos) K+ channels, different types of nonselective cation channels including TRPC6 and NMDA receptors. In addition circumstantial evidence hints to the expression of the voltage-gated Ca2+ channel CaV2.1 in human erythrocytes. Besides the comprehensive catalogue of the erythrocyte channel types, the article of Lars Käster gives a rewarding compendium about the history of channel research in human erythrocytes. Taken together, this special issue provides an overview of the unexpected diversity of erythrocyte ion channels that endows these small enucleated cells with a toolkit for electrosignaling. This toolkit enables erythrocytes to quickly respond to internal or external stimuli with changes in cytosolic free Ca2+, de- or hyperpolarization of the membrane, cell swelling or shrinkage, or release of channel-permeable solutes such as ATP. Moreover, the ion channels are integral modules of complex programs such as oxygen-regulated ATP release. Malaria is one example how such programs are exploited by the parasite, to adapt the erythrocyte cytosol to its needs.

Abstract Sickle cell disease (SCD) is an autosomal-recessive hemolytic disorder with high morbidity and mortality. The pathophysiology of SCD is characterized by the polymerization of deoxygenated intracellular sickle hemoglobin, which causes the sickling of erythrocytes. The recent development of metabolomics, the newest member of the “omics” family, has provided a powerful new research strategy to accurately measure functional phenotypes that are the net result of genomic, transcriptomic, and proteomic changes. Metabolomics changes respond faster to external stimuli than any other “ome” and are especially appropriate for surveilling the metabolic profile of erythrocytes. In this review, we summarize recent pioneering research that exploited cutting-edge metabolomics and state-of-the-art isotopically labeled nutrient flux analysis to monitor and trace intracellular metabolism in SCD mice and humans. Genetic, structural, biochemical, and molecular studies in mice and humans demonstrate unrecognized intracellular signaling pathways, including purinergic and sphingolipid signaling networks that promote hypoxic metabolic reprogramming by channeling glucose metabolism to glycolysis via the pentose phosphate pathway. In turn, this hypoxic metabolic reprogramming induces 2,3-bisphosphoglycerate production, deoxygenation of sickle hemoglobin, polymerization, and sickling. Additionally, we review the detrimental role of an impaired Lands’ cycle, which contributes to sickling, inflammation, and disease progression. Thus, metabolomic profiling allows us to identify the pathological role of adenosine signaling and S1P-mediated erythrocyte hypoxic metabolic reprogramming and hypoxia-induced impaired Lands' cycle in SCD. These findings further reveal that the inhibition of adenosine and S1P signaling cascade and the restoration of an imbalanced Lands' cycle have potent preclinical efficacy in counteracting sickling, inflammation, and disease progression.

Invasion of the red blood cell (RBC) by the Plasmodium parasite defines the start of malaria disease pathogenesis. To date, experimental investigations into invasion have focused predominantly on the role of parasite adhesins or signaling pathways and the identity of binding receptors on the red cell surface. A potential role for signaling pathways within the erythrocyte, which might alter red cell biophysical properties to facilitate invasion, has largely been ignored. The parasite erythrocyte-binding antigen 175 (EBA175), a protein required for entry in most parasite strains, plays a key role by binding to glycophorin A (GPA) on the red cell surface, although the function of this binding interaction is unknown. Here, using real-time deformability cytometry and flicker spectroscopy to define biophysical properties of the erythrocyte, we show that EBA175 binding to GPA leads to an increase in the cytoskeletal tension of the red cell and a reduction in the bending modulus of the cell’s membrane. We isolate the changes in the cytoskeleton and membrane and show that reduction in the bending modulus is directly correlated with parasite invasion efficiency. These data strongly imply that the malaria parasite primes the erythrocyte surface through its binding antigens, altering the biophysical nature of the target cell and thus reducing a critical energy barrier to invasion. This finding would constitute a major change in our concept of malaria parasite invasion, suggesting it is, in fact, a balance between parasite and host cell physical forces working together to facilitate entry.

The pathogenicity of Plasmodium falciparum is due to the unique ability of infected erythrocytes (IRBCs) to adhere to vascular endothelium. We investigated whether adhesion of IRBCs to CD36, the major cytoadherence receptor on human dermal microvascular endothelial cells (HDMECs), induces intracellular signaling and regulates adhesion. A recombinant peptide corresponding to the minimal CD36-binding domain from P falciparum erythrocyte membrane protein 1 (PfEMP1), as well as an anti-CD36 monoclonal antibody (mAb) that inhibits IRBC binding, activated the mitogen-activated protein (MAP) kinase pathway that was dependent on Src-family kinase activity. Treatment of HDMECs with a Src-family kinase–selective inhibitor (PP1) inhibited adhesion of IRBCs in a flow-chamber assay by 72% (P < .001). More importantly, Src-family kinase activity was also required for cytoadherence to intact human microvessels in a human/severe combined immunodeficient (SCID) mouse model in vivo. The effect of PP1 could be mimicked by levamisole, a specific alkaline-phosphatase inhibitor. Firm adhesion to PP1-treated endothelium was restored by exogenous alkaline phosphatase. In contrast, inhibition of the extracellular signal–regulated kinase 1/2 (ERK 1/2) and p38 MAP kinase pathways had no immediate effect on IRBC adhesion. These results suggest a novel mechanism for the modulation of cytoadherence under flow conditions through a signaling pathway involving CD36, Src-family kinases, and an ectoalkaline phosphatase. Targeting endothelial ectoalkaline phosphatases and/or signaling molecules may constitute a novel therapeutic strategy against severe falciparum malaria.

Erythrocytes are the most abundant cell type in the human body, although considered as merely hemoglobin carriers for a long time. Extensive studies on its biochemical pathways, metabolism, and structure-activity relationship with a consistent number of publications demonstrated the presence of autocrine, paracrine, and endocrine hormone receptors. In this chapter, some of these hormones will be discussed, bringing attention to those that regulate erythrocyte survival, disease connection, and functionality.