scholarly journals Inhibition of the Aquaporin-1 Cation Conductance by Selected Furan Compounds Reduces Red Blood Cell Sickling

2022 ◽  
Vol 12 ◽  
Author(s):  
Pak Hin Chow ◽  
Charles D. Cox ◽  
Jinxin V. Pei ◽  
Nancy Anabaraonye ◽  
Saeed Nourmohammadi ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Water Channel ◽  
Ion Conductance ◽  
Aquaporin 1 ◽  
Hypoxic Conditions ◽  
Combined Application ◽  
Cation Conductance ◽  
Osmotic Water ◽  
Combined Features

In sickle cell disease (SCD), the pathological shift of red blood cells (RBCs) into distorted morphologies under hypoxic conditions follows activation of a cationic leak current (Psickle) and cell dehydration. Prior work showed sickling was reduced by 5-hydroxylmethyl-2-furfural (5-HMF), which stabilized mutant hemoglobin and also blocked the Psickle current in RBCs, though the molecular basis of this 5-HMF-sensitive cation current remained a mystery. Work here is the first to test the hypothesis that Aquaporin-1 (AQP1) cation channels contribute to the monovalent component of Psickle. Human AQP1 channels expressed in Xenopus oocytes were evaluated for sensitivity to 5-HMF and four derivatives known to have differential efficacies in preventing RBC sickling. Ion conductances were measured by two-electrode voltage clamp, and osmotic water permeability by optical swelling assays. Compounds tested were: 5-HMF; 5-PMFC (5-(phenoxymethyl)furan-2-carbaldehyde); 5-CMFC (5-(4-chlorophenoxymethyl)furan-2-carbaldehyde); 5-NMFC (5-(2-nitrophenoxymethyl)-furan-2-carbaldehyde); and VZHE006 (tert-butyl (5-formylfuran-2-yl)methyl carbonate). The most effective anti-sickling agent, 5-PMFC, was the most potent inhibitor of the AQP1 ion conductance (98% block at 100 µM). The order of sensitivity of the AQP1 conductance to inhibition was 5-PMFC > VZHE006 > 5-CMFC ≥ 5-NMFC, which corresponded with effectiveness in protecting RBCs from sickling. None of the compounds altered AQP1 water channel activity. Combined application of a selective AQP1 ion channel blocker AqB011 (80 µM) with a selective hemoglobin modifying agent 5-NMFC (2.5 mM) increased anti-sickling effectiveness in red blood cells from human SCD patients. Another non-selective cation channel known to be expressed in RBCs, Piezo1, was unaffected by 2 mM 5-HMF. Results suggest that inhibition of AQP1 ion channels and capacity to modify hemoglobin are combined features of the most effective anti-sickling agents. Future therapeutics aimed at both targets could hold promise for improved treatments for SCD.

scholarly journals Role of Calcium in Phosphatidylserine Externalisation in Red Blood Cells from Sickle Cell Patients

Anemia ◽  
2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Erwin Weiss ◽  
David Charles Rees ◽  
John Stanley Gibson
Keyword(s):  
Red Blood Cells ◽  
Sickle Cell ◽  
Blood Cells ◽  
Phosphatidylserine Exposure ◽  
Channel Inhibition ◽  
Cation Conductance ◽  
Gardos Channel ◽  
Cation Permeability

Phosphatidylserine exposure occurs in red blood cells (RBCs) from sickle cell disease (SCD) patients and is increased by deoxygenation. The mechanisms responsible remain unclear. RBCs from SCD patients also have elevated cation permeability, and, in particular, a deoxygenation-induced cation conductance which mediates entry, providing an obvious link with phosphatidylserine exposure. The role of was investigated using FITC-labelled annexin. Results confirmed high phosphatidylserine exposure in RBCs from SCD patients increasing upon deoxygenation. When deoxygenated, phosphatidylserine exposure was further elevated as extracellular [] was increased. This effect was inhibited by dipyridamole, intracellular chelation, and Gardos channel inhibition. Phosphatidylserine exposure was reduced in high saline. levels required to elicit phosphatidylserine exposure were in the low micromolar range. Findings are consistent with entry through the deoxygenation-induced pathway (), activating the Gardos channel. [] required for phosphatidylserine scrambling are in the range achievablein vivo.

scholarly journals Transepithelial water and urea permeabilities of isolated perfused Munich-Wistar rat inner medullary thin limbs of Henle's loop

2014 ◽  
Vol 306 (1) ◽  
pp. F123-F129 ◽  
Author(s):  
C. Michele Nawata ◽  
Kristen K. Evans ◽  
William H. Dantzler ◽  
Thomas L. Pannabecker
Keyword(s):  
Water Channel ◽  
Wistar Rat ◽  
Structural Features ◽  
Urea Transporter ◽  
Outer Medulla ◽  
Aquaporin 1 ◽  
Passive Mechanism ◽  
Short Loop ◽  
Osmotic Water ◽  
Gene Expression Levels

To better understand the role that water and urea fluxes play in the urine concentrating mechanism, we determined transepithelial osmotic water permeability ( Pf) and urea permeability ( Purea) in isolated perfused Munich-Wistar rat long-loop descending thin limbs (DTLs) and ascending thin limbs (ATLs). Thin limbs were isolated either from 0.5 to 2.5 mm below the outer medulla (upper inner medulla) or from the terminal 2.5 mm of the inner medulla. Segment types were characterized on the basis of structural features and gene expression levels of the water channel aquaporin 1, which was high in the upper DTL (DTLupper), absent in the lower DTL (DTLlower), and absent in ATLs, and the Cl-1 channel ClCK1, which was absent in DTLs and high in ATLs. DTLupper Pf was high (3,204.5 ± 450.3 μm/s), whereas DTLlower showed very little or no osmotic Pf (207.8 ± 241.3 μm/s). Munich-Wistar rat ATLs have previously been shown to exhibit no Pf. DTLupper Purea was 40.0 ± 7.3 × 10−5 cm/s and much higher in DTLlower (203.8 ± 30.3 × 10−5 cm/s), upper ATL (203.8 ± 35.7 × 10−5 cm/s), and lower ATL (265.1 ± 49.8 × 10−5 cm/s). Phloretin (0.25 mM) did not reduce DTLupper Purea, suggesting that Purea is not due to urea transporter UT-A2, which is expressed in short-loop DTLs and short portions of some inner medullary DTLs close to the outer medulla. In summary, Purea is similar in all segments having no osmotic Pf but is significantly lower in DTLupper, a segment having high osmotic Pf. These data are inconsistent with the passive mechanism as originally proposed.

Expression of Aquaporin-1 in the Peritoneal Tissues: Localization and Regulation by Hyperosmolality

2002 ◽  
Vol 22 (3) ◽  
pp. 307-315 ◽  
Author(s):  
Tomoko Ota ◽  
Michio Kuwahara ◽  
Shuling Fan ◽  
Yoshio Terada ◽  
Takashi Akiba ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Water Permeability ◽  
Water Channel ◽  
Mesothelial Cells ◽  
Scattering Method ◽  
Osmotic Water Permeability ◽  
Peritoneal Mesothelial Cells ◽  
Aquaporin 1 ◽  
Osmotic Water ◽  
Aqp1 Expression

Objective The purpose of this study was to determine the localization of the aquaporin-1 (AQP1) water channel in peritoneal tissues and the effect of hyperosmolality on the peritoneal expression and function of AQP1. Methods Immunohistochemical localization of AQP1 was identified in rat peritoneal tissues. Cultured rat peritoneal mesothelial cells (RPMCs) were exposed to hyperosmolality by adding 4% glucose to the culture medium. After 1 hour, 4 hours, 24 hours, and 48 hours, AQP1 was identified by semiquantitative immunoblot and immunocytochemistry. Osmotic water permeability was measured using a light-scattering method. Results Immunohistochemistry of rat peritoneal tissues showed the presence of AQP1 in mesothelial cells, venular endothelial cells, and capillary endothelial cells, but not in arteriole and interstitial cells. Semiquantitative immunoblot revealed that exposure to hyperosmolality significantly increased AQP1 expression after 24 hours in whole RPMC lysates (3.3-fold at 24 hours and 3.9-fold at 48 hours). Consistent with the immunoblot, osmotic water permeability of RPMC was augmented 1.7-fold and 2.7-fold after 1 hour and 24 hours, respectively, in a hyperosmotic environment. In RPMC membrane fractions, AQP1 expression was significantly increased after 1 hour of exposure to hyperosmolality (3.9-fold at 1 hour, 7.1-fold at 4 hours, and 8.7-fold at 24 hours). Immunocytochemistry of RPMCs showed that AQP1 was gradually redistributed from the perinuclear area to the peripheral cytoplasm, and then to the plasma membrane after a 1-hour hyperosmotic challenge, suggesting hyperosmolality-induced translocation of AQP1. Upregulation of AQP1 was also observed in the omentum of rats loaded intraperitoneally with hyperosmotic dialysate every day for 10 weeks. Conclusion AQP1 is widely distributed in the peritoneal cavity and may provide the major aqueous pathway across the peritoneal barrier. In addition, our findings suggested that hyperosmolality increases AQP1-dependent water permeability in peritoneal tissues by regulating the translocation and synthesis of AQP1 protein.

scholarly journals Oxidation induces a Cl − ‐dependent cation conductance in human red blood cells

2002 ◽  
Vol 539 (3) ◽  
pp. 847-855 ◽  
Author(s):  
Christophe Duranton ◽  
Stephan M. Huber ◽  
Florian Lang
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Human Red Blood Cells ◽  
Cation Conductance

scholarly journals Aquaporin-1 Facilitates Transmesothelial Water Permeability: In Vitro and Ex Vivo Evidence and Possible Implications in Peritoneal Dialysis

2021 ◽  
Vol 22 (22) ◽  
pp. 12535
Author(s):  
Francesca Piccapane ◽  
Andrea Gerbino ◽  
Monica Carmosino ◽  
Serena Milano ◽  
Arduino Arduini ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Peritoneal Dialysis ◽  
Tight Junctions ◽  
Water Transport ◽  
Water Flux ◽  
Water Channel ◽  
Bathing Solution ◽  
Ussing Chambers ◽  
Aquaporin 1 ◽  
Osmotic Water

We previously showed that mesothelial cells in human peritoneum express the water channel aquaporin 1 (AQP1) at the plasma membrane, suggesting that, although in a non-physiological context, it may facilitate osmotic water exchange during peritoneal dialysis (PD). According to the three-pore model that predicts the transport of water during PD, the endothelium of peritoneal capillaries is the major limiting barrier to water transport across peritoneum, assuming the functional role of the mesothelium, as a semipermeable barrier, to be negligible. We hypothesized that an intact mesothelial layer is poorly permeable to water unless AQP1 is expressed at the plasma membrane. To demonstrate that, we characterized an immortalized cell line of human mesothelium (HMC) and measured the osmotically-driven transmesothelial water flux in the absence or in the presence of AQP1. The presence of tight junctions between HMC was investigated by immunofluorescence. Bioelectrical parameters of HMC monolayers were studied by Ussing Chambers and transepithelial water transport was investigated by an electrophysiological approach based on measurements of TEA+ dilution in the apical bathing solution, through TEA+-sensitive microelectrodes. HMCs express Zo-1 and occludin at the tight junctions and a transepithelial vectorial Na+ transport. Real-time transmesothelial water flux, in response to an increase of osmolarity in the apical solution, indicated that, in the presence of AQP1, the rate of TEA+ dilution was up to four-fold higher than in its absence. Of note, we confirmed our data in isolated mouse mesentery patches, where we measured an AQP1-dependent transmesothelial osmotic water transport. These results suggest that the mesothelium may represent an additional selective barrier regulating water transport in PD through functional expression of the water channel AQP1.

scholarly journals The Capacity of Red Blood Cells to Reduce Nitrite Determines Nitric Oxide Generation under Hypoxic Conditions

PLoS ONE ◽  
2014 ◽  
Vol 9 (7) ◽  
pp. e101626 ◽  
Author(s):  
Marcel H. Fens ◽  
Sandra K. Larkin ◽  
Bryan Oronsky ◽  
Jan Scicinski ◽  
Claudia R. Morris ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Hypoxic Conditions

scholarly journals Combined use of micro-focused ultrasound and autologous blood cells in correction of involutive skin changes

2019 ◽  
Vol 2 (26) ◽  
pp. 105-109
Author(s):  
Zh. Yu. Yusova ◽  
T. V. Stepanova ◽  
P. A. Belkov
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Focused Ultrasound ◽  
Clinical Effect ◽  
Autologous Blood ◽  
Study Data ◽  
Skin Changes ◽  
Combined Application ◽  
Combined Use ◽  
Qualitative Characteristics

A comparative study of micro-focused ultrasound in monotherapy and combined use with autologous blood cells was conducted. In assessing the effectiveness of the procedures studied the qualitative characteristics of the skin and ultrasound scanning data. All patients (35) with involutional changes of the skin at the procedures were divided into tho groups: combined application of ultrasound macrofocusrange with autologous red blood cells and macrofocusrange ultrasound as monotherapy. The results of the study, data were obtained that showed a more pronounced clinical effect in patients treated with the combined use of ultrasound macrofocusrange with autologous red blood cells.

Imaging Flow Cytometry Documents Incomplete Resistance Of F-Cells To Hypoxia-Induced Sickling In Blood Samples From Patients With Sickle Cell Anemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 183-183
Author(s):  
Kleber Yotsumoto Fertrin ◽  
Eduard J. van Beers ◽  
Leigh Samsel ◽  
Laurel G. Mendelsohn ◽  
Rehan Saiyed ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Red Blood Cells ◽  
Cell Count ◽  
Sickle Cell Anemia ◽  
Blood Cells ◽  
Blood Samples ◽  
Imaging Flow Cytometry ◽  
Hypoxic Conditions ◽  
Round Shape ◽  
F Cells

Abstract Fetal hemoglobin (HbF) production induced by hydroxyurea is the mainstay of treatment for sickle cell anemia (SCA). Increased HbF production correlates with a higher number of HbF-containing red blood cells (RBCs) called F-cells. Successful treatment with hydroxyurea is associated with an increased number of F-cells, less hemolysis, improvement of anemia, and decreased frequency of vaso-occlusive crises in SCA patients. Comparison of in vitro sickling among blood specimens from sickle mouse models and from patients with different HbF levels has provided compelling evidence that increasing the percentage of circulating F-cells is associated with improvement of hemolytic biomarkers. While it has been demonstrated that higher HbF content prolongs sickle RBC survival, there is only indirect evidence of the response to hypoxia of F-cells compared to non-F-cells. We investigated the influence of HbF content on sickling through our recently developed Sickle Imaging Flow Cytometry Assay (SIFCA). SIFCA allows simultaneous analysis of both expression of intracellular proteins and morphological features of each cell in a robust, reproducible, operator-independent sickling assay. Peripheral venous blood samples were collected upon written consent from adult SCA patients with a wide range of HbF percentages (HbF range 2.0-26.9%) (n=15, nine on hydroxyurea treatment). RBC pellets were used to prepare 1% suspensions that were subjected to deoxygenation for 2 hours at 2% oxygen. RBCs were then labeled for HbF using a standard protocol for F-cell quantitation and a minimum of 20,000 cells were analyzed by imaging flow cytometry (ImageStreamX Mk II, Amnis Corporation), allowing the correlation between shape change and intensity of HbF expression for each RBC. We confirmed previous observations using conventional flow cytometry that F-cell count percentages significantly correlate with mean HbF determined by HPLC (r2P=0.9700, 95% CI 0.9098-0.9902, P<0.0001). F-cell count by SIFCA correlated highly with conventional F-cell flow cytometry by an independent CLIA-certified facility (r2P =0.9976, 95% CI 0.9861-0.9996, P<0.0001). SIFCA morphological analysis showed that the percentage of non-F-cells sickling upon deoxygenation was significantly higher than among F-cells (17.75% [95% CI 12.5-23.00] vs. 12.41% [95% CI 8.67-16.15], P=0.0015), a 1.498-fold difference (95% CI 1.228-1.768). Image analysis also allowed us to identify the presence of F-cells that still sickle despite their high HbF content, as well as non-F-cells that are resistant to sickling (Figure 1). Transmission electron microscopy of F-cells enriched by fluorescence activated cell sorting confirmed that sickled F-cells contained hemoglobin S polymers. In summary, we have documented for the first time at the individual RBC level that human F-cells are less prone to sickle under hypoxia ex vivo than non-F-cells. This study also illustrates the power of imaging flow cytometry to characterize predisposition to sickling in populations of red blood cells from the same patient, and would be suitable for use as a supportive biomarker assay in clinical trials investigating the efficacy of novel HbF inducers and their anti-sickling effect in a single assay. While the finding that F-cells sickle less than non-F-cells is not unexpected, it seems surprising to us that the difference in hypoxia-induced sickling between F-cells and non-F-cells is so small. This finding emphasizes the need to characterize additional RBC features that render individual cells more susceptible or resistant to sickling. Identification of factors besides HbF that modulate sickle hemoglobin polymerization may help design novel therapies for hydroxyurea-resistant SCA patients.Figure 1Sample images showing non-F-cells (left column) and F-cells (right column) as they appear on imaging flow cytometry. Under hypoxic conditions, non-F-cells are expected to sickle (panel A), while F-cells are expected to maintain a round shape (panel B). Nevertheless, round erythrocytes can be found among non-F-cells (panel C), as well as typically sickled F-cells (panel D).Figure 1. Sample images showing non-F-cells (left column) and F-cells (right column) as they appear on imaging flow cytometry. Under hypoxic conditions, non-F-cells are expected to sickle (panel A), while F-cells are expected to maintain a round shape (panel B). Nevertheless, round erythrocytes can be found among non-F-cells (panel C), as well as typically sickled F-cells (panel D). Disclosures: No relevant conflicts of interest to declare.

scholarly journals Effect of Storage on Levels of Nitric Oxide Derivatives in Blood Components.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2884-2884
Author(s):  
Fabiola G. Rizzatti ◽  
David Stroncek ◽  
Melissa Qazi ◽  
Nathawut Sibmooh ◽  
Barbora Piknova ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Red Blood Cells ◽  
Whole Blood ◽  
Blood Cells ◽  
Vascular System ◽  
Oxides Of Nitrogen ◽  
Blood Components ◽  
Transfusion Therapy ◽  
Hypoxic Conditions ◽  
Nitric Oxide Metabolites

Abstract The important role of erythrocytes in nitric oxide (NO) physiology changed the traditional view of the red blood cells (RBC) as only a carrier of oxygen and carbon monoxide. Nitrite is a primary oxidative NO metabolite and is considered a major intravascular storage pool for NO. In the vascular system, erythrocytes are the major storage sites of nitrite, which can be activated to NO by deoxyhemoglobin, but also are responsible for its rapid destruction after reaction with oxyhemoglobin.The purpose of this study was to quantify the nitric oxide metabolites, nitrite and nitrate, in red blood cells (RBCs) stored as packed cells or whole blood and to evaluate their levels with the time of storage. Whole blood, leukoreduced, and non-leukoreduced packed RBCs were obtained from healthy volunteer donors and were stored in polyvinyl chloride (PVC) bags to up to 42 days at 4°C. Sequential aliquots were taken from the bags using a liquid transfer set to maintain sterile conditions. Nitrite and nitrate were measured in the whole blood and in RBC components using reductive gas phase chemiluminescence. Nitrite concentrations decreased during the storage in the three blood components analyzed. The nitrite concentration in RBCs before storage was 202±45 nM, but fell rapidly upon storage. In the leukoreduced RBCs, nitrite levels were 81±36 nM on day 1 and 51±8 nM on day 42. The concentration of nitrate remained stable during blood storage, 30±14 uM on day 1 and 33±5 uM on day 42 of storage. The pH decreased slightly in all three blood components during storage, from pH 6.7±0.05 on day 1 to 6.5±0 on day 42. The blood pO2 before storage was 40.5±1.5 and increased to 251±4 mmHg on day 42, presumably due to the diffusion of oxygen from the room air. In control experiments, PVC bags were filled with normal saline used for medical purposes and stored up to 42 days at 4°C in room air; nitrite concentrations gradually increased while nitrate values remained stable. Similar results were observed for nitrite and nitrate concentrations in the non-leukoreduced RBCs and whole blood. Both cells and saline controls maintained in an argon chamber at 4°C for 42 days showed decreased levels of nitrite when compared to the bags stored in room air under the same temperature. Our results show that nitrite levels fall in hemocomponents during blood bank storage, nitrate remains stable, while pH decreases and pO2 increases. The decrease in nitrite levels could be explained either by its reaction with oxyhemoglobin, resulting in nitrate and methemoglobin, or with deoxyhemoglobin. The diffusion of oxides of nitrogen gases through the PVC bags could in part explain why nitrite levels do not completely disappear in the RBCs stored for transfusion, under standard transfusion medicine conditions. As erythrocytes may contribute to the control of blood flow and oxygen delivery through reduction of nitrite to NO under hypoxic conditions, our findings may provide insight into the vasodynamic effects of blood transfusion. These measurements of NO derivatives may have implication for transfusion therapy, explaining some adverse effects of RBC transfusion and/or optimizing the preservation of stored hemocomponents.

scholarly journals GBT440 reverses sickling of sickled red blood cells under hypoxic conditions in vitro

2018 ◽  
Vol 10 (2) ◽  
Author(s):  
Kobina Dufu ◽  
Donna Oksenberg
Keyword(s):  
Sickle Cell Disease ◽  
Red Blood Cells ◽  
Sickle Cell ◽  
Blood Cells ◽  
Oxygen Affinity ◽  
Microvascular Blood Flow ◽  
Cell Disease ◽  
Hypoxic Conditions

Sickle cell disease is characterized by hemolytic anemia, vasoocclusion and early mortality. Polymerization of hemoglobin S followed by red blood cell sickling and subsequent vascular injury are key events in the pathogenesis of sickle cell disease. Sickled red blood cells are major contributors to the abnormal blood rheology, poor microvascular blood flow and endothelial injury in sickle cell disease. Therefore, an agent that can prevent and or reverse sickling of red blood cells, may provide therapeutic benefit for the treatment of sickle cell disease. We report here that GBT440, an anti-polymerization agent being developed for the chronic treatment of sickle cell disease, increases hemoglobin oxygen affinity and reverses in vitro sickling of previously sickled red blood cells under hypoxic conditions. Our results suggest that besides preventing sickling of red blood cells, GBT440 may mitigate vasoocclusion and microvascular dysfunction by reversing sickling of circulating sickled red blood cells in vivo.

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