Endothelin-1 Receptor Antagonists Regulate Cell Surface-Associated Protein Disulfide Isomerase In Sickle Cell Disease

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 265-265 ◽  
Author(s):  
Gregory N Prado ◽  
Jessica Alves ◽  
Anna J Hernandez ◽  
Enrique R Maldonado ◽  
Rodeler Youte ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Cell Surface ◽  
Sickle Cell ◽  
Hydration Status ◽  
Channel Activity ◽  
Cell Disease ◽  
Receptor Antagonists ◽  
Sickle Erythrocyte ◽  
Gardos Channel ◽  
Sickle Erythrocytes

Abstract Abstract 265 Erythrocyte hydration status and endothelial cell activation have been proposed as important contributors to vaso-occlusion and impaired blood flow in the pathophysiology of sickle cell disease (SCD). However, the physiological mechanism(s) that mediate the interplay between erythrocytes hydration status and the endothelium in SCD are unclear. We have recently reported a role for dual endothelin-1 receptor antagonists in improving sickle erythrocyte hydration status and K+ transport in vivo via modulation of Gardos channel activity (Rivera A., 2008, Amer J Physiol). The Gardos channel is an important contributor to sickle erythrocyte dehydration that maybe modulated by protein disulfide isomerase (PDI). PDI in leukocytes has been reported to catalyze disulfide interchange reactions, mediate redox modifications and has been observed to be up-regulated under hypoxic conditions. We report the detection of PDI by western blot analyses in membranes from both human and mouse sickle erythrocytes. We observed greater levels of cell surface associated PDI in sickle vs Hb A-containing erythrocytes. We also quantified PDI activity and observed a significant correlation between Gardos channel activity and cell surface associated PDI activity in human sickle erythrocytes and Hb A-containing cells (n=40, r2=0.3046, p=0.0002). In fact, closer examination revealed that sickle erythrocyte membranes had higher PDI activity than Hb A-containing erythrocyte membranes (5.07±0.4 vs 1.30±0.1%, n=22 and 18, respectively p<0.0001). Similar results were observed in membrane preparations of erythrocytes isolated from the BERK sickle transgenic mouse model when compared with wild-type controls. Consistent with a functional role for PDI in Gardos channel activation, we also observed that sickle erythrocytes incubated in cycles of oxygenation/de-oxygenation for 3 hr in the presence of PDI antibodies were associated with reduced sickle dense cell formation. Similar results were observed with bacitracin, another PDI inhibitor. We then treated BERK mice with dual ET-1 receptor antagonists (BQ123/BQ788) for 14 days and measured erythrocyte surface associated PDI activity. We observed that as with Gardos channel activity, cell surface associated PDI activity was significantly reduced following treatment with BQ123/BQ788 (8.80±0.5 to 6.4±0.6%, n=3 P<0.02). These changes were associated with an increase in erythrocyte MCV (31.3±1.63 to 40.4±0.35 fL, n=3, p<0.002) and a decrease in MCHC (40.4±0.8 to 27.4±3 g/dL, n=3, p<0.003). We then studied the direct effects of ET-1 on the human endothelial cell line, EA.hy926 (EA), as well as in primary cultures of BERK mouse aortic endothelial cells (BMAEC). Using quantitative RT-PCR with Taqman chemistries and GAPDH and beta-actin as endogenous controls, we observed that stimulation of EA cells with 100nM ET-1 for 4 hr was associated with increased mRNA expression of PDI levels that was 1.89 fold greater than vehicle treated cells (n=6, P<0.04). Similar results were observed on PDI mRNA expression in BMAEC isolated and cultured for 10 days then incubated with 100 nM ET-1 for 4 hr. Thus, our results strongly implicate cell surface associated PDI in cellular hydration status and its regulation by ET-1. We posit that aberrant regulation of PDI activity and/or its expression and secretion from either erythrocytes or endothelial cells represent a novel target aimed at ameliorating the complications associated with the pathophysiology of Sickle Cell Disease. Supported by NIH R01HL090632 to AR. Disclosures: No relevant conflicts of interest to declare.

Regulation of Erythrocyte Volume by Cell Surface Associated Protein Disulfide Isomerase.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 149-149
Author(s):  
Jose R. Romero ◽  
Manuel D. Bicho ◽  
Lin-Chien Pong ◽  
Joel Greenbowe ◽  
Alicia Rivera
Keyword(s):  
Sickle Cell Disease ◽  
Cell Surface ◽  
Sickle Cell ◽  
Protein Disulfide Isomerase ◽  
Red Cells ◽  
Channel Activity ◽  
Cell Disease ◽  
Gardos Channel ◽  
Sickle Erythrocytes ◽  
Protein Disulfide

Abstract Acute and chronic clinical manifestations of sickle cell disease (SCD) are based on vaso-occlusion and impaired blood flow. Dense erythrocytes are believed to be important contributors to the vaso-occlusive manifestations of SCD. However, the physiological regulation of erythrocyte hydration status in SCD is not entirely clear. The Gardos channel and K/Cl cotransport are major contributors to sickle erythrocyte dehydration. Protein disulfide isomerase (PDI) on the cell surface catalyzes disulfide formation, causes redox modifications and has been observed to be up-regulated under hypoxic conditions. We now report the detection of PDI in red cells. Western blot analysis revealed a prominent band, migrating at 55 kDa, in ghost preparation from both sickle and normal erythrocytes. To evaluate the role of PDI in Gardos channel activation, we measured charybdotoxin-sensitive K+ influx in the presence of bacitracin, a well-known blocker of PDI activity. When sickle erythrocytes are exposed to bacitracin, Gardos channel activity is significantly reduced (1.4 ±0.2 to 0.8± 0.05 mmol/L cell x min, n=6, P&lt;0.01). We also observed that Gardos channel activity was attenuated by 0.25 mM bacitracin and was maximally inhibited by 3 mM in sickle and normal erythrocytes. Similar results were observed with phenyl arsine oxide and acetyl-thyroxin, two other well-known inhibitors of cell surface PDI activity. We then studied the effects of PDI inhibition on red cell density profiles of sickle and normal human red cells. Analysis of the baseline density profile indicates that erythrocytes have a median density of 1.10 g/mL. This value significantly increased to 1.125 g/mL (n=2) after 3 h of oxygenation/de-oxygenation cycles. However, in the presence of 3 mM bacitracin, the cellular density profile markedly shifted to the left (1.098 g/mL) following deoxygenation/oxygenation. We also investigated the reductive capacity of freshly isolated human erythrocytes by the ferrocyanide-production method. The reductive capacity of normal Hb A red cells was significantly lower than in Hb S containing cells (2.8 ± 1.1 vs 5.1 ± 1.3 mmol/L cell x h, n=18, p&lt;0.0001). Similar results were observed in Santillies and NYKO1, two transgenic mouse models of sickle cell disease, when compared to C57 mice. These results strongly implicate cell surface associated PDI in cellular dehydration and formation of dense sickle erythrocytes by activating K+ efflux via the Gardos channel and suggest that aberrant regulation of PDI activity and/or its expression may contribute to the pathophysiology of Sickle Cell Disease.

Aldosterone Promotes Inflammation and Endothelial Cell-Sickle Cell Adhesion: Potential for a New Therapeutic Target in Sickle Cell Disease

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 125-125 ◽  
Author(s):  
Wilbur Aaron Lam ◽  
Wendy R Hansen ◽  
James Huang ◽  
William Mentzer ◽  
Bertram Harold Lubin ◽  
...  
Keyword(s):  
Heart Failure ◽  
Cell Adhesion ◽  
Endothelial Cell ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Dependent Manner ◽  
Cell Disease ◽  
Vascular Effects ◽  
Sickle Erythrocyte ◽  
Sickle Erythrocytes

Abstract Sickle cell disease is fundamentally an inflammatory state, and endothelial activation and dysfunction have significant roles in the pathophysiology of this disease. In the last decade, research in the cardiovascular field has proven that the hormone aldosterone, canonically viewed as a regulator of renal electrolyte handling and blood pressure, also has direct, pro-inflammatory effects on the vascular endothelium that are independent of its classical effects. Excessive aldosterone is now known to cause microvascular damage, vascular inflammation, oxidative stress and endothelial dysfunction although the molecular mechanisms remain poorly understood (Brown, Hypertension 2008). In addition, aldosterone decreases endothelial cell production of nitric oxide and upregulates VCAM-1 and ICAM-1 production, leading to increased leukocyte-endothelial cell adhesion (Oberleithner, PNAS, 2007; Krug, Hypertension 2007). In animal models, aldosterone-mediated vascular injury in the brain, heart, and kidneys leads to stroke, myocardial injury, and renal damage (Marney, Clin Sci 2007). In addition, several large clinical trials have shown that aldosterone-antagonizing medications decrease mortality in patients with renal and heart failure, due in part to the blocking of the inflammatory vascular effects of this hormone (Pitt, N Engl J Med, 2003). Although the vascular effects of aldosterone are similar to those that occur in sickle cell disease, no published studies to date have investigated the possible interactions between aldosterone and sickle cell disease. Furthermore, the efficacy of aldosterone-antagonists as a potential therapy/prophylaxis for sickle cell complications has not been evaluated. We found that patients with Hemoglobin SS (n=21) have abnormally elevated aldosterone plasma levels, as measured with ELISA, that range from 1.5–40 times (median: 8.6 times) higher than normal levels, similar in range to those of patients with heart failure (Struthers, Eur J of Heart Failure 2004). In addition, aldosterone levels in sickle cell patients positively correlated with secretory phospholipase A2 levels (R=0.43, p&lt;0.05), a known biomarker for predicting acute chest syndrome. To determine how aldosterone affects endothelialsickle cell adhesion, we exposed human umbilical vein endothelial cells (HUVECs) and sickle erythrocytes and leukocytes isolated from patient samples to varying physiologic concentrations (1.0–100 nM) of aldosterone ex vivo for 2 hours and then utilized static and dynamic flow adhesion assays. We found that aldosterone increases sickle erythrocyte (but not normal erythrocytes), neutrophil and mononuclear cell (monocytes + lymphocytes) adhesion to endothelial cells in a dose-dependent manner (compared to controls, p&lt;0.05 for all concentrations between 1–10 nM, p&lt;0.001 for all concentrations &gt;10nM) in static conditions. Compared to controls, endothelial-sickle blood cell adhesion increased up to 100 times with aldosterone exposure. Similarly, under physiologic flow conditions (shear stress: 1 dyne/cm2), endothelial cell exposure to aldosterone increased capture of sickle erythrocytes and leukocytes in a dose dependent manner (compared to controls, p&lt;0.05 for all concentrations &gt;10 nM). Furthermore, measurements with atomic force microscopy (AFM), a highly sensitive tool used to measure and track cell adhesion and deformability at the single cell level, revealed that the adhesive force between single sickle cell erythrocytes and HUVECs increases over time with aldosterone exposure. With the addition of spironolactone, an aldosterone antagonist, all adhesive interactions decreased to near baseline levels/controls (p&gt;0.3 for all comparisons with baseline levels/controls) as measured with static and dynamic flow adhesion assays and AFM. To investigate the underlying mechanisms of these phenomena, fluorescence imaging revealed increased reactive oxygen species production and expression of VCAM-1 and ICAM-1 in HUVECs exposed to aldosterone for only 2 hr when compared to controls. Aldosterone exposure did not affect sickle erythrocyte or leukocyte deformability as measured with ektacytometry and AFM, respectively. Taken together, these results suggest that aldosterone may play an important role in sickle cell vasculopathy and the high levels of this hormone may provide an effective therapeutic target for this disease.

Pharmacological Inhibition of Calpain-1 Prevents Red Cell Dehydration and Reduces Gardos Channel Activity in a Mouse Model of Sickle Cell Disease. Identification of Druggable Protease Target

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 852-852
Author(s):  
Lucia De Franceschi ◽  
Alessandro Mattè ◽  
Carlo Brugnara ◽  
Angela Siciliano ◽  
Mariarita Bertoldi ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Cysteine Proteases ◽  
Calcium Pump ◽  
Membrane Association ◽  
Red Cell ◽  
Channel Activity ◽  
Cell Disease ◽  
Left Shift ◽  
Gardos Channel

Abstract Abstract 852 Sickle cell disease (SCD) is a hereditary red cell disorder characterized by the presence of pathological HbS, which polymerizes upon deoxygenation promoting red blood cell (RBC) dehydration and sickling. In SCD, the dense RBCs play a crucial role in the pathogenesis of sickle cell related organ damage and clinical manifestations. Although progress has been made in pathogenesis of SCD, the treatment options for SCD have limited pharmacological tools for clinical practice. Calpains are ubiquitous calcium-activated cysteine proteases, causing controlled proteolysis of protein substrates with regulatory functions. RBCs express only calpain-1, whose physiological function remains poorly understood. Gene inactivation of mouse calpain-1 revealed differential regulation of RBC calcium pump and enhanced RBC hydration. To investigate the relevance of these findings in SCD, we used BDA-410, a novel orally active inhibitor of calpain-1. Using the sickle (SAD) mouse, a model for human sickle cell disease, BDA-410 was administrated at the dosage of 30 mg/K/d by gavage to wild-type (WT) and sickle cell (SAD) mice. Animals were divided into 4 groups of 6 mice each: two groups from each strain were treated with BDA-410 for 14 days along with vehicle controls. Mice at baseline, day 7, and day 14 of BDA-410 treatment were evaluated for hematological parameters including the RBC density profile with phthalate density curves, RBC cation content, and Ca2+ activated K+ channel (Gardos channel) activity. BDA-410 induced a significant increase in Hct in both WT and SAD mice with no significant changes in Hb levels and an associated increased in MCV. The red cell K+ content increased significantly in SAD RBCs at day 7 and 14 of inhibitor treatment as compared to untreated SAD mice; whereas no major changes were observed in the WT RBCs. The mean corpuscular Hb concentration (CHCM) decreased in both WT and SAD mice treated with BDA-410. A left-shift in the RBC density curves was observed in SAD mice; whereas this left-shift was limited to a sub-fraction of denser red cells in the WT mice. The activity of the Gardos channel was significantly reduced in BDA-410 treated SAD mice compared to the untreated SAD group, while no significant differences were observed in the WT mice. Since the membrane-association of Peroxiredoxin-2 (Prx2) is increased in SCD RBCs, and has been correlated with Gardos channel activity, we evaluated Prx2-membrane association. BDA-410 treatment induced a significant reduction in the amount of Prx2 translocated to the membrane in both WT and SAD mice. Moreover, when we exposed WT and SAD mice to hypoxia (8% oxygen) for 48 hours, followed by 2 hours of re-oxygenation to mimic sickle cell related vaso-occlusive events, BDA-410 treatment prevented the hypoxia induced K+ loss and RBC dehydration in SAD mice. Identification of RBC membrane substrates in the calpain-1 null mice suggests that proteolytic modification of clapain targets as calcium pump, Prx2, and the Gardos channel protein may underpin some of the protective effects of BDA-410 in SAD mice. These results suggest that the inhibition of calpain-1 may offer a new therapeutic strategy to ameliorate hematological phenotype of SCD. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Pharmacological inhibition of calpain‐1 prevents red cell dehydration and reduces Gardos channel activity in a mouse model of sickle cell disease

2012 ◽  
Vol 27 (2) ◽  
pp. 750-759 ◽  
Author(s):  
Lucia De Franceschi ◽  
Robert S. Franco ◽  
Mariarita Bertoldi ◽  
Carlo Brugnara ◽  
Alessandro Matté ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Mouse Model ◽  
Sickle Cell ◽  
Red Cell ◽  
Channel Activity ◽  
Cell Disease ◽  
Pharmacological Inhibition ◽  
Gardos Channel ◽  
Cell Dehydration

Protein Disulfide Isomerase in Sickle Cell Disease

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2166-2166
Author(s):  
Daniel Gil de Lamadrid ◽  
Yaritza Inostroza-Nieves ◽  
Joshua Cazares ◽  
Isamar Alicea ◽  
Analaura Santiago ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Mouse Models ◽  
Sickle Cell ◽  
Protein Disulfide Isomerase ◽  
Channel Activity ◽  
Cell Disease ◽  
Disulfide Isomerase ◽  
Gardos Channel ◽  
Sickle Erythrocytes ◽  
Protein Disulfide

Abstract Protein disulfide isomerase (PDI), a multifunctional oxidoreductase that regulates thrombus formation, leukocyte adherence to the endothelium and nitric oxide delivery, is present at high levels and regulates KCNN4 (Gardos Channel) activity in erythrocytes from humans with Sickle Cell Disease (SCD) and sickle mouse models. We reported evidence of elevated erythrocyte-surface associated and circulating PDI activity in humans and mouse models of SCD when compared to either wild-type mice, transgenic mice expressing normal human globins or otherwise healthy individuals; results that suggest a novel role for PDI in the pathophysiology of SCD through unclear mechanisms. We studied the effects of deoxygenation (5% O2) on PDI activity in endothelial cells and sickle erythrocytes from humans with SCD and two sickle transgenic knockout mouse models expressing human sickle hemoglobin, BERK and βSAntilles. Our data shows that deoxygenation of human and mouse sickle erythrocytes increased surface-associated reductive capacity that was sensitive to monoclonal antibodies against PDI (mAb PDI [RL-90]). We then studied sickle human erythrocytes and showed that PDI inhibitors (quercentin-3-rutinoside [Q3R] and mAb PDI) significantly reduced deoxy-stimulated sickle cell dehydration and Gardos channel activity (n=5; P <0.03). We characterized erythrocyte density with a phthalate density-distribution assay, generated density distribution curves and calculated the D50. Both mAb PDI and Q3R significantly reduced D50 when compared to vehicle (1.113±0.002 to 1.102±0.001 [P <0.0001]; and 1.101±0.002 [P <0.0001]; respectively). In contrast, incubation with exogenous PDI (3 nM) increased cellular dehydration (from D50= 1.110 ± 0.001 to D50= 1.115 ±0.001, P <0.01). We also measured the effect of hypoxia and endothelin-1 (ET-1; 10nM) in the human vascular endothelial cell line, EA.hy926. We observed that hypoxia induced PDI secretion that was further enhanced by co-incubation with ET-1 (10nM; n=3; P <0.05). The selective inhibitor of ET-1 subtype B receptor antagonist, BQ788 blocked ET-1 stimulated PDI increases in these cells as was previously reported in red blood cells. Consistent with these results hypoxia was associated with increased mRNA expression of MCP-1, VEGF-a but not ICAM by qRT-PCR and Taqman probes (n=3; P <0.05). Of importance we observed that early cultures of mouse aortic endothelial cells from BERK mice showed similar results. We then evaluated the effects of PDI on erythrocyte hemolysis by exposing cells from patients with SCD to 20 mM 2-2'-azo-bis- (2-amidinopropane) dihydrochloride (AAPH) with or without Q3R or mAb PDI. AAPH-induced hemolysis was dose-dependently blocked by Q3R (IC50: 4.1nM; n=6, P <0.0001 compared to vehicle) or mAb PDI (IC50: 11nM; n=3). Irrelevant IgG did not affect hemolysis under these conditions. Experiments performed in blood from BERK or βSAntilles mice showed similar results. We then studied sickle mice that express varying levels of HbF; BERK (<1% HbF), BERKγM (25% HbF), and BERKγH (45% HbF). BERKγH had the lowest circulating and cell associated PDI activity among the three mouse types that was associated with lower circulating ET-1 levels (n=2; P <0.05). Consistent with these results we observed an inverse correlation between levels of HbF and PDI activity in cells from humans with SCD (n=4). Thus we posit that in SCD elevated erythrocyte PDI activity is important for cell survival and stability and that its inhibition may represent a novel therapeutic approach for improving both the hematological and vascular complications of SCD as it may not only increase sickle erythrocyte survival but may likewise interfere with cellular adhesion leading to reduced vaso-occlusive episodes. [Supported by NIH: HL090632 (AR) and HL096518 (JRR)] Disclosures No relevant conflicts of interest to declare.

Decreased erythrocyte nicotinamide adenine dinucleotide redox potential and abnormal pyridine nucleotide content in sickle cell disease

Blood ◽  
1988 ◽  
Vol 71 (2) ◽  
pp. 512-515 ◽  
Author(s):  
CR Zerez ◽  
NA Lachant ◽  
SJ Lee ◽  
KR Tanaka
Keyword(s):  
Sickle Cell Disease ◽  
Redox Potential ◽  
Sickle Cell ◽  
Nicotinamide Adenine Dinucleotide ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Pyridine Nucleotide ◽  
Nicotinamide Adenine ◽  
Pyridine Nucleotides ◽  
Cell Disease ◽  
Sickle Erythrocyte

Abstract RBCs from individuals with sickle cell disease are more susceptible to oxidant damage. Because key antioxidant defense reactions are linked to the pyridine nucleotides nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), we tested the hypothesis that the RBC redox potential as manifested by the NADH/[NAD+ + NADH] and NADPH/[NADP+ + NADPH] ratios is decreased in sickle erythrocytes. Our data demonstrate that sickle RBCs have a significant decrease in the NADH/[NAD+ + NADH] ratio compared with normal RBCs (P less than .00005). Interestingly, sickle RBCs also had a significant increase in total NAD content compared with normal RBCs (P less than .00005). In contrast, although sickle RBCs had a significant increase in the total NADP content compared with normal RBCs (P less than .00005), sickle RBCs had no significant alteration in the NADPH/[NADP+ + NADPH] ratio. High reticulocyte controls demonstrated that these changes were not related to cell age. Thus, sickle RBCs have a decrease in NAD redox potential that may be a reflection of their increased oxidant sensitivity. The changes in these pyridine nucleotides may have further metabolic consequences for the sickle erythrocyte.

Regulation of Human Sickle Erythrocyte Hydration Status by Endothelin-1 Is Mediated by Casein Kinase II.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3020-3020
Author(s):  
Jose R. Romero ◽  
Corey Hoehn ◽  
Jessica Alves ◽  
Alicia Rivera
Keyword(s):  
Sickle Cell ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Cytoskeletal Proteins ◽  
Hydration Status ◽  
Hemoglobin S ◽  
Endothelin 1 ◽  
Sickle Erythrocyte ◽  
Cellular Dehydration ◽  
Sickle Erythrocytes

Abstract Abstract 3020 Poster Board II-996 In Sickle Cell Disease, K+ efflux and osmotically induced water loss leads to erythrocyte dehydration, an important step in the formation of hemoglobin S polymers and irreversible sickle cells. We have previously reported that activation of sickle erythrocyte endothelin-1 (ET-1) receptors is partially responsible for dense irreversible sickle cell formation thru mechanisms that are still undefined. Casein Kinase II (CK2), a serine/threonine kinase that phosphorylates acidic proteins, has been shown to regulate calmodulin activity and cytoskeletal proteins. Thus, we posit that CK2 may regulate cellular hydration status. CK2 activity is modulated by apigenin, emodin, heparin, and ornithine decarboxylase. Our results indicate that CK2 activity in erythrocyte membranes increases in the presence of ET-1 (2.8 ± 0.1 to 4.9 ± 0.01 nmol/min/mL * 106 cell) an event that is blunted by pre-incubation with the ET-1 B receptor blocker, BQ788 (2.5 ± 0.1 nmol/min/mL * 106 cell). Our data also indicate that 20 μM apigenin blocks ET-1 induced CK2 activity in sickle erythrocytes (2.8 ± 0.1 to 2.7 ± 0.4 nmol/min/mL * 106 cell). We then examined the role of CK2 activation in cellular dehydration. We incubated sickle erythrocytes for 3 hours in deoxygenation-oxygenation cycles in the presence or absence of 20μM apigenin or 2μM 4,5,6,7-tetrabromobenzotriazole (TBB), another specific CK2 inhibitor, and measured the changes in erythrocyte density by the phthalate oil method. We observed that inhibition of CK2 led to the inhibition of deoxygenation-stimulated cellular dehydration in sickle erythrocytes by apigenin (D50= 1.106 to 1.100 g/mL) or TBB (D50 =1.097 g/mL). Apigenin also inhibited the volume-stimulated K+ efflux in a dose-dependent manner (IC50 = 18 ± 1 μM). These results strongly implicate CK2 as an intermediate of the ET-1 stimulated cellular volume regulatory systems in erythrocytes and suggest that regulation of CK2 activity may lead to reduction of hemoglobin S polymerization in vivo. Supported by NIH R01-HL09632 to AR. Disclosures No relevant conflicts of interest to declare.

Senicapoc, a Gardos Channel Inhibitor Developed to Treat Sickle Cell Disease, Exhibits Antimalarial Activity

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 743-743
Author(s):  
Venee N. Tubman ◽  
Pedro Mejia ◽  
Boris E. Shmukler ◽  
Seth L. Alper ◽  
James R. Mitchell ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Antimalarial Activity ◽  
Fixed Ratio ◽  
Parasite Growth ◽  
Cell Disease ◽  
Gardos Channel ◽  
Potent Inhibition ◽  
Ic50 Value

Abstract BACKGROUND: The inhibitor of the erythrocyte Gardos channel (KCNN-4, IK-1), senicapoc, was developed for treatment of sickle cell disease. Activation of the Gardos channel constitutes a major potassium leak pathway contributing to cellular dehydration. Administration of senicapoc to patients with sickle cell disease was well tolerated and reduced hemolysis through attenuation of sickle red cell dehydration, but failed to reduce the frequency of vaso-occlusive pain crises (Ataga KI et al. Br J Haematol 2011;153:92-104). Red cell volume regulation is critical both to the pathology of sickle cell disease and to the growth of Plasmodium, the parasites that cause malaria, since Plasmodium depends upon ion flow across the host membrane for growth (Glushakova S et al. Curr Biol 2010; 20:1117-1121). We tested the hypothesis that senicapoc-induced blockade of the Gardos channel inhibits intraerythrocytic growth and development of malarial parasites. METHODS: Blood type O+ erythrocytes were infected with P. falciparum strains 3D7, W2mef, and 7G8, and P. knowlesi strains H-1 and YH-1. Parasites were cultured in the presence of senicapoc and in parallel, with drugs with known antimalarial properties (e.g. mefloquine, chloroquine, clotrimazole, and dihydroartemisinin). Parasites were also cultured in the presence of each of eight senicapoc analogs with variable inhibitory potency for the Gardos channel. To define interactions between these drugs, parasites were cultured in fixed-ratio combinations of senicapoc and senicapoc analogs using the fixed-ratio isobologram method. Parasite growth and maturation were determined by microscopy and flow cytometry. The effect of senicapoc in vivo was determined using wild-type C57Bl/6 mice along with IK-1+/+ and IK-1-/- pups on a C57Bl/6 background. Mice were infected with the murine parasite, P. yoelii and treated with senicapoc or vehicle control (methylcellulose 0.5%). RESULTS: We observed that senicapoc blocked the intraerythrocyte growth of P. falciparum 3D7 with an IC50 value of 7 uM and P. knowlesi H1 with an IC50 value of 18 uM. Senicapoc inhibited growth leading to death of intracellular parasites at and beyond the late trophozoite stage, demonstrating minimal effect on ring stage parasites, and an intermediate effect on mature schizonts. Analogs with IC50 values for inhibition of parasite growth similar to senicapoc were potent inhibitors of the Gardos channel. These analogs also shared common elements of the molecular structure. Combined treatment with senicapoc and analogs with potent inhibition of the Gardos channel demonstrated an additive effect on inhibition of parasite growth. Both additive and synergistic interactions were demonstrated when parasites were cultured with senicapoc and analogs without potent inhibition of the Gardos channel. In the murine model, C57Bl/6 mice treated with senicapoc exhibited suppression in parasite growth, confirming the efficacy of senicapoc against Plasmodia in vivo. Parasite growth was suppressed in IK+/+ mice treated with senicapoc, though the effect was not sustained through the period of treatment. Surprisingly, parasite growth was also suppressed in IK-/-mice treated with senicapoc, though the response was delayed. The kinetics of parasite growth differed between mice with and without the Gardos channel. CONCLUSIONS: Senicapoc demonstrates antimalarial activity against P. falciparum and P. knowlesiin vitro and in vivo against P. yoelii in mice. Our in vivo studies suggest a mechanism for inhibition of parasite growth in mice that includes both Gardos-dependent and independent components. The excellent safety profile of senicapoc and its long half-life in humans demonstrated in clinical trials suggest its possible utility in antimalarial development either as a lead compound or in combination with other antimalarials. Disclosures No relevant conflicts of interest to declare.

Pharmacological modification of oxygen affinity improves deformability of deoxygenated sickle erythrocytes: a possible therapeutic approach to sickle cell disease

1989 ◽  
Vol 76 (4) ◽  
pp. 357-362 ◽  
Author(s):  
A. J. Keidan ◽  
M. C. Sowter ◽  
C. S. Johnson ◽  
S. S. Marwah ◽  
J. Stuart
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Oxygen Affinity ◽  
Saturation Curve ◽  
Cell Disease ◽  
Filtration Method ◽  
Sickle Cells ◽  
Sickle Erythrocytes ◽  
Haemoglobin S

1. The formation of polymerized haemoglobin S in sickle cells is critically dependent on the concentration of deoxygenated haemoglobin so that compounds which increase the oxygen affinity of haemoglobin S are potential anti-sickling agents. 2. BW12C [5-(2-formyl-3-hydroxyphenoxy)pentanoic acid] and BWA589C [4-(2-formyl-3-hydroxyphenoxymethyl)benzoic acid] are aromatic benzaldehydes that cause a dose-dependent left-shift of the oxygen saturation curve of haemoglobin S by stabilization of its oxy-(R)-conformation. 3. A 5 μm pore filtration method, which is highly sensitive to polymerization of haemoglobin S, was used to demonstrate a significant improvement in the deformability of deoxygenated sickle erythrocytes at concentrations (0.75–1.5 mmol/l) of BW12C and BWA589C that are achievable in vivo. Both compounds may therefore be of value for the treatment of sickle cell disease. 4. Filtration of sickle cells through pores of 5μm diameter is a sensitive technique for evaluating the rheological effects of potential anti-sickling compounds.

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