scholarly journals Endothelin‐1 receptor antagonists regulate cell surface‐associated protein disulfide isomerase in sickle cell disease

2013 ◽  
Vol 27 (11) ◽  
pp. 4619-4629 ◽  
Author(s):  
Gregory N. Prado ◽  
Jose R. Romero ◽  
Alicia Rivera
Keyword(s):  
Sickle Cell Disease ◽  
Cell Surface ◽  
Sickle Cell ◽  
Protein Disulfide Isomerase ◽  
Cell Disease ◽  
Receptor Antagonists ◽  
Disulfide Isomerase ◽  
Endothelin 1 ◽  
Protein Disulfide

Aldosterone Stimulates Neutrophils Leading To Increased β-Glucuronidase, Protein Disulfide Isomerase and Myeloperoxidase Secretion

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2271-2271
Author(s):  
Arelys Ramos-Rivera ◽  
Alicia Rivera ◽  
Enrique D. Machado-Fiallo ◽  
Josue A. Benabe-Carlo ◽  
Gregory N. Prado ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Respiratory Burst ◽  
Protein Disulfide Isomerase ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Cell Disease ◽  
Rt Pcr ◽  
Disulfide Isomerase ◽  
Protein Disulfide

Abstract Aldosterone (ALDO) has been shown to play an important role in inflammatory responses in addition to its well described effects on sodium homeostasis via activation of the mineralocorticoid receptor (MR). However, its effects on polymorphonuclear leukocytes (PMNC) are not well described. We isolated untouched circulating human PMNC by immunomagnetic isolation following density gradient sedimentation with PolymorphPrep from otherwise healthy subjects. Flow cytometric analyses showed greater than 97% of PMNC were positive for the myeloid-neutrophil markers, CD45, CD16 and CD66b. We show that PMNC express MR by western blot and RT-PCR analyses. We incubated PMNC with ALDO (10–9–10–7M) for 30 min and observed a dose-dependent rise in β–glucuronidase release with an EC50 of 6.11 nM (P<0.001, n=3), an event that was blocked by pre-incubation of cells with 1μM canrenoic acid (CA), an MR antagonist (P<0.04, n=3). In addition, our results show that incubation of human PMNC with 10-8M ALDO likewise led to increases in myeloperoxidase ([MPO], P<0.05, n=3) and protein disulfide isomerase ([PDI], P<0.01, n=4), a multifunctional enzyme of the thioredoxin superfamily that mediates redox modifications, regulates KCNN4 channel and erythrocyte volume and is up-regulated under hypoxic conditions (Prado, 2013 FASEB J). We then studied the effects of ALDO on HL-60, a human promyelocytic cell line, induced to differentiate into neutrophil-like cells by incubation for 5 days with 1.3% DMSO. Our results likewise show an increase in MPO responses upon 10–8M ALDO stimulation as compared to vehicle (AUC: 1090±147 to 505±48, P<0.02, n=3). We have recently reported that aldosterone stimulates increases of striatin, a scaffolding protein that interacts with caveolin-1, and co-precipitates with striatin and as such may facilitate cross talk of signaling complexes. As there are no pharmacological inhibitors of striatin we used a molecular approach to reduce striatin levels. In differentiated HL-60 cells, siRNA against striatin led to reduced MPO responses (AUC: 590±14 to 528±13, P<0.05, n=3) that were associated with significantly reduced striatin mRNA levels but not when cells were transfected with scrambled siRNA as determined by quantitative RT-PCR with ABI TaqMan detection probes and β-microglobulin used as an endogenous control (P<0.01, n=3). These results suggest that striatin plays an important role in ALDO-stimulated degranulation responses. Of importance we also observed that incubation with ALDO (10–9–10–7M) in differentiated HL60 cells led to increases in the oxidative-respiratory burst [superoxide production] in a dose- and time-dependent manner (P<0.01, n=4). Consistent with these results, we observed that ALDO likewise led to significant increases in the oxidative-respiratory burst in human PMNC (P<0.01, n=3). As there is evidence that activated neutrophils, MPO and PDI are elevated in Sickle Cell Disease, we studied the in vivo effects of MR blockade in BERK sickle transgenic mice, a model of increased oxidative stress. Sickle mice were randomized to receive either normal rodent chow or chow containing eplerenone (156 mg/kg per day), an MR receptor antagonist, and tap water ad libitum for 14 days at which time the mice were sacrificed and blood collected. We observed that mice on eplerenone had significantly lower plasma PDI activity than mice on regular chow (63.7 ± 8.7 control diet to 47.9 ± 2.4 eplerenone, Relative Fluorescence Units [RFU]; P<0.005, n=6 and 9) and lower MPO levels (AUC: 214±11 to 73±20, P<0.03, n=3); events that were associated with increases in both erythrocyte MCV (41.3±2.5 vs 47.4±1.1 fL, P<0.03, n=7) and reticulocyte MCV (53.6.3±2.8 vs 60.1±0.6 fL, P<0.02, n=7). Thus, our results suggest that MR activation by ALDO is a novel mechanism for neutrophil stimulation and as such represents a novel therapeutic target aimed at ameliorating the vascular complications of Sickle Cell Disease. Supported by NIH R01HL090632 (AR) and R01HL096518 (JRR). 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.

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 Endothelin-1-Mediated Reactive Oxygen Species Production by Protein Disulfide Isomerase in Endothelial Cells: Role in Sickle Cell Disease

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1073-1073
Author(s):  
Patricia Neuman ◽  
Gregory N. Prado ◽  
Philip Asamoah ◽  
Daphne Diaz ◽  
Jose R Romero ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Ros Production ◽  
Cell Disease ◽  
Rt Pcr ◽  
Endothelin 1 ◽  
Endogenous Controls ◽  
Protein Disulfide

Abstract Abstract 1073 Endothelial cell activation and increased levels of endothelin-1 (ET-1) have been proposed as important contributors to vaso-occlusion in the pathophysiology of Sickle Cell Disease (SCD). We have reported a role for ET-1 receptor antagonists in improving hematological complications of two transgenic mouse models of SCD in vivo (Rivera A., 2008, Amer J Physiol). Activation of endothelial cells leads to, among other factors, increased levels of reactive oxygen species (ROS) and protein disulfide isomerase (PDI). PDI mediates redox modifications, catalyzes disulfide interchange reactions in the plasma membrane and is up-regulated under hypoxic conditions as commonly observed in SCD. We tested the hypothesis that ET-1 would regulate PDI activity in endothelial cells. We now report on the detection of PDI and ET-1 receptor B by western blot analyses in membranes from the human endothelial cell line, EA.hy926 (EA). We studied the effects of ET-1 on cellular ROS production by fluorimetry of DCFDA-AM loaded EA cells and observed that 10−7 M ET-1 for 60 mins led to significant increases in cellular ROS production (1720±78 vs 1447±86 Relative Fluorescent Units (RFU), P<0.001, n=6) that were significantly blocked by ET-1 receptor B antagonist, 10−6 M BQ-788 (1720±78 vs 1426±55 RFU, P<0.0007, n=3). We also tested the effects of the specific ET-1 receptor B agonist, 10−7 M BQ-3020 on cellular ROS production and observed similar results (1748±194 vs 1447±86 RFU, P<0.02, n=3). We then studied the effect of a well-described PDI inhibitor, monoclonal antibodies to PDI (RL90), on ET-1 stimulated ROS production and showed that RL90 likewise blocked ET-1 stimulated ROS production in EA cells (P<0.001, n=3), thus suggesting a role for PDI in ET-1-stimulated ROS production. To further explore the role of PDI on ROS production, we employed a molecular approach and show that siRNA against PDI likewise led to reduced ET-1-stimulated ROS production (P<0.0001, n=2) that was associated with significantly reduced PDI mRNA levels in siRNA transfected cells, but not when cells were transfected with scrambled siRNA as determined by quantitative RT-PCR with ABI TaqMan detection probes and GAPDH and β2 microglobulin as endogenous controls. We then optimized conditions to measure PDI activity using fluorescently labeled GSSG conversion to GSH in supernatants of ET-1-stimulated cells. We observed that incubation of EA cells for 60 mins with 10−7 M ET-1 was associated with increased extracellular PDI activity (232±183 to 664±94 (RFU), n=3, P<0.02) that was blunted by PDI siRNA knockdown (1731±147 to 757±141 RFU, n=2) when compared to scrambled siRNA transfected cells. Consistent with these data we observed increases in PDI protein levels in supernatants of EA cells stimulated with ET-1 by ELISA. Moreover, incubation of EA cells for 12 hr with 10−8 M ET-1 led to increases in cell-associated PDI levels by western blot analyses. We then tested the in vivo effects of ET receptor antagonist on plasma PDI activity in the BERK mouse model of SCD. We blocked ET-1 receptors in vivo by treatment with BQ-788 and BQ-123 (360mg/Kg/Day) for 14 days and observed a reduction of plasma PDI activity when compared to vehicle treatment (67.7±3 to 34.3±6, RFU, P<0.03, n=3). We also characterized the effects of ET-1 on PDI expression in EA cells, using quantitative RT-PCR with ABI TaqMan probes and GAPDH and β-actin as endogenous controls and observed that stimulation of EA cells with 10−8 M ET-1 for 4 hr was associated with increased PDI mRNA expression levels that were 1.89 fold greater than vehicle treated cells (n=6, P<0.04). Thus, our results provide evidence to suggest that endothelin-1 receptor blockade leads to improved measures of ROS via regulation of PDI and thus implicate PDI as a novel regulator of Sickle Cell Disease pathophysiology. Disclosures: No relevant conflicts of interest to declare.

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.

scholarly journals Sodium phenyl butyrate downregulates endothelin-1 expression in cultured human endothelial cells: Relevance to sickle-cell disease

2007 ◽  
Vol 82 (5) ◽  
pp. 357-362 ◽  
Author(s):  
Marie-Hélène Odièvre ◽  
Manuel Brun ◽  
Rajagopal Krishnamoorthy ◽  
Claudine Lapouméroulie ◽  
Jacques Elion
Keyword(s):  
Endothelial Cells ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Cell Disease ◽  
Human Endothelial Cells ◽  
Endothelin 1

Protein Disulfide Isomerase Regulates Sickle Erythrocyte Volume Via ET-1 Dependent Casein Kinase II Mechanism

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2114-2114
Author(s):  
Shirley D Valentin-Berrios ◽  
Jose R Romero ◽  
Alicia Rivera
Keyword(s):  
Plasma Membrane ◽  
Sickle Cell ◽  
Protein Disulfide Isomerase ◽  
Disulfide Isomerase ◽  
Sickle Erythrocyte ◽  
Cellular Dehydration ◽  
Sickle Erythrocytes ◽  
Protein Disulfide

Abstract Abstract 2114 Disordered K+ efflux and osmotically induced water loss leads to red blood cell (RBC) dehydration and plays a role in the pathophysiology of Sickle Cell Disease. We previously reported that activation of endothelin-1 (ET-1) receptors in sickle erythrocyte was partially responsible for dense sickle cell formation. However, the mechanism by which ET-1 regulates RBC volume remains unclear. Serine/threonine kinases have been shown to regulate K+ transport in RBC. Casein Kinase II (CK2), a serine/threonine kinase, phosphorylates acidic proteins, regulates calmodulin activity and cytoskeletal proteins and is present in RBC. CK2 activity is blocked by apigenin, emodin, heparin, and ornithine decarboxylase. Previous reports have shown a role for flavonoids such as apigenin as substrates for erythrocyte plasma membrane oxidoreductases. We recently observed a role for Protein Disulfide Isomerase (PDI) in regulating cellular hydration and K+ efflux in human RBC. PDI catalyzes disulfide interchange reactions in the plasma membrane, mediates redox modifications and is up-regulated under hypoxic conditions. However the relationship between CK2 and PDI in the setting of cellular hydration status is un-explored. Our results indicate that erythrocyte membrane CK2 activity increases when sickle cells are incubated with 500 nM ET-1 for 30 min (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, n=3, p<0.04) and 20 μM apigenin (2.7 ± 0.4 nmol/min/mL * 106 cell, n=3, p<0.04). We examined the role of CK2 activation on 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), a specific CK2 inhibitor, and measured the changes in erythrocyte density by phthalate oil density analysis. We observed that inhibition of CK2 led to reduced deoxygenation-stimulated cellular dehydration in sickle erythrocytes by apigenin (D50= 1.106 to 1.100 g/mL) or TBB (D50 =1.097 g/mL). We then studied the role of CK2 inhibitors on PDI activity by Insulin Turbidity Assay and observed that apigenin and TBB led to significant reductions in PDI activity in vitro (64% and 42% respectively). We also studied the effects of the flavonoids: naringenin, naringin, apigenin and rutin on PDI activity and observed reductions in PDI activity that were greater with apigenin>rutin>TBB>naringin>naringenin (n=2, P<0.05). Furthermore, we observed that K+ flux via Gardos channel activation is correlated with PDI activity in vitro in sickle erythrocytes. Taken together our results implicate CK2 and PDI as intermediate regulators of ET-1 stimulated cellular volume systems in red blood cells. Supported by NIH R01-HL09632 to AR. Disclosures: No relevant conflicts of interest to declare.

Blockade of the Mineralocorticoid Receptor in Sickle Transgenic Mice Reduces Circulating Protein Disulfide Isomerase and Gardos Channel Activity

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 999-999
Author(s):  
Christopher Vega ◽  
Gregory N. Prado ◽  
Philip Asamoah ◽  
Patricia Neuman ◽  
Jose R Romero ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transgenic Mice ◽  
Protein Disulfide Isomerase ◽  
Mineralocorticoid Receptor ◽  
Hematological Parameters ◽  
Channel Activity ◽  
Cell Disease ◽  
Disulfide Isomerase ◽  
Gardos Channel ◽  
Protein Disulfide

Abstract Abstract 999 Endothelin-1 (ET-1), erythrocyte sickling and endothelial cell activation have been proposed as important contributors to the pathophysiology of sickle cell disease (SCD). We have provided evidence for the use of ET-1 receptor antagonists in improving hematological parameters in two transgenic mouse models of SCD (Rivera A., 2008, Amer J Physiol). However, the mechanisms that mediate the interplay between red blood cells (RBC) and the endothelium in SCD remain unresolved. Activation of endothelial cells leads to, among other factors, increased levels of protein disulfide isomerase (PDI). PDI catalyzes disulfide interchange reactions, mediates redox modifications and has been observed to be up-regulated under hypoxic conditions. We now report that circulating PDI activity is increased in BERK sickle transgenic mice when compared to wild-type controls. The mineralocorticoid receptor (MR) is a member of the steroid family of nuclear receptors that function as a transcription factor that upon binding to the hormone responsive element of genes such as edn1, the gene for ET-1, leads to increased ET-1 expression. In vivo, blockade of MR has been shown to reduce circulating ET-1 levels and kidney ET-1 mRNA expression. We hypothesized that MR blockade of BERK sickle transgenic mice would lead to reduced PDI activity and improved hematological parameters. Sickle mice were randomized to receive either normal rodent chow or chow containing eplerenone (156 mg/kg per day), an MR receptor antagonist, and tap water ad libitum for 14 days at which time the mice were sacrificed and tissues and blood collected. Plasma PDI activity was calculated by optimization of fluorescently labeled GSSG conversion to GSH. We observed that mice on eplerenone had significantly lower plasma PDI activity than mice on regular chow (63.7 ± 8.7 control diet to 47.9 ± 2.4 eplerenone, Relative Fluorescence Units; P<0.005, n=6 and 9, respectively). We also studied RBC Gardos channel activity in these mice and observed a significant reduction in clotrimazole-sensitive K+ efflux following MR blockade (2.49±0.5 control and 1.37±0.3 mmol/1013 cells × hr; P<0.04 n= 5 and 7 respectively). MR blockade was associated with increases in both erythrocyte MCV (41.3±2.5 vs 47.4±1.1 fL, P<0.03, n=7) and reticulocyte MCV (53.6.3±2.8 vs 60.1±0.6 fL, P<0.02, n=7) as determined by an ADVIA 120 hematology analyzer. In contrast no significant effects on MCHC levels were observed under these conditions. We then studied ET-1 gene expression using quantitative RT-PCR with ABI Taqman chemistries and GAPDH and β-actin as endogenous controls. We observed that MR blockade was associated with reduced expression of ET-1 mRNA in heart tissue (0.654 ± 0.233, ΔΔCT, relative to mice on regular chow, P<0.04, n=5 and n=7) but not lung tissue. Western blot analyses in membranes from human and mouse sickle erythrocytes and endothelial cells revealed the presence of both MR and PDI proteins. We then studied the effects of ET-1 in early cultures of BERK mouse aortic endothelial cells (MAEC). We observed that stimulation of MAEC cells with 100nM ET-1 for 4 hr was associated with increased mRNA expression of PDI levels that was 1.71 fold greater than vehicle treated cells (n=4, P<0.05). Thus, our results suggest that MR blockade reduces ET-1 levels leading to reduced Gardos and PDI activity in Sickle mice. These effects on PDI activity and Gardos channel regulation may represent a novel mechanism for protective effects of MR blockade aimed at ameliorating vascular complications of Sickle Cell Disease. Supported by NIH R01HL090632 to AR. Disclosures: No relevant conflicts of interest to declare.

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