Arginine supplementation of sickle transgenic mice reduces red cell density and Gardos channel activity

Blood ◽  
2002 ◽  
Vol 99 (4) ◽  
pp. 1103-1108 ◽  
Author(s):  
José R. Romero ◽  
Sandra M. Suzuka ◽  
Ronald L. Nagel ◽  
Mary E. Fabry
Keyword(s):  
Nitric Oxide ◽  
Cell Density ◽  
Sickle Cell ◽  
T Test ◽  
Red Cells ◽  
K Channel ◽  
Mean Corpuscular Hemoglobin ◽  
Red Cell ◽  
Plasma Arginine ◽  
Arginine Supplementation

Nitric oxide (NO), essential for maintaining vascular tone, is produced from arginine by nitric oxide synthase. Plasma arginine levels are low in sickle cell anemia, and it is reported here that low plasma arginine is also found in our sickle transgenic mouse model that expresses human α, human βS, and human βS-Antilles and is homozygous for the mouse βmajor deletion (S+S-Antilles). S+S-Antilles mice were supplemented with a 4-fold increase in arginine that was maintained for several months. Mean corpuscular hemoglobin concentration (MCHC) decreased and the percent high-density red cells was reduced. Deoxy K+ efflux is characteristic of red cells in sickle cell disease and contributes to the disease process by increasing the MCHC and rendering the cells more susceptible to polymer formation. This flux versus the room air flux was reduced in S+S-Antilles red cells from an average value of 1.6 ± 0.3 mmol per liter of red cells × minute (FU) in nonsupplemented mice to 0.9 ± 0.3 FU (n = 4, P < .02, paired t test) in supplemented mice. In room air, Vmax of the Ca++-activated K+ channel (Gardos) was reduced from 4.1 ± 0.6 FU (off diet) to 2.6 ± 0.4 FU (n = 7 and 8,P < .04, t test) in arginine-supplemented mice versus clotrimazole. In conclusion, the major mechanism by which arginine supplementation reduces red cell density (MCHC) in S+S-Antilles mice is by inhibiting the Ca++-activated K+ channel.

Arginine Supplementation of Sickle Transgenic Mice Leads to a Rapid Decrease in MCHC and Percent Dense Cells and a Slow Increase When Supplementation Is Discontinued.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3579-3579
Author(s):  
Mary E. Fabry ◽  
Zipora Etzion ◽  
Robert M. Bookchin ◽  
Sandra M. Suzuka ◽  
Ronald L. Nagel
Keyword(s):  
Nitric Oxide ◽  
Transgenic Mice ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
High Density ◽  
Red Cells ◽  
Red Cell ◽  
Red Cell Indices ◽  
Plasma Arginine ◽  
Arginine Supplementation

Abstract Nitric oxide (NO) is produced from arginine by nitric oxide synthase (NOS) and is essential to the maintenance of vascular tone. Plasma arginine levels are reduced in sickle cell anemia patients, and we have previously reported that S+S− Antilles mice have lower plasma arginine than control mice (C57BL). Long term arginine supplementation (5% arginine in chow) restored plasma arginine levels in S+S− Antilles mice to that found in C57BL mice, and, in S+S− Antilles mice, MCHC and the percent high density red cells were reduced. Our observation that Ca++-activated K+ channel [K(Ca) channel or Gardos channel] activity is reduced in supplemented vs non-supplemented S+S− Antilles mice can account for reduced red cell density and dense cell formation (Blood99: 1103, 2002). The time course of arginine induced changes in red cell indices in sickle transgenic mice has not yet been studied. Using the Advia 120 Hematology System, we found that, within 7 days after the onset of arginine supplementation, MCHC* (CHCM in the Advia system) fell from 33.4±0.5 g/dL to 31.0±0.2 (mean ±SD, P<0.000002, N=6) and the percent high density red cells (MCHC>37 g/dL) decreased from 10.5±4.3 % to 4.5±1.6 (P<0.01, N=6). MCV rose from 43.6±0.8 fL to 46.4±0.5 (P<0.00003, N=6). Reticulocyte counts and mean corpuscular hemoglobin did not vary significantly. Although plasma arginine levels increase within a few hours of oral administration and persist for only a few hours, no change in red cell indices was observed 7 days after discontinuing arginine supplementation, and the indices slowly returned to baseline levels after 40 days. In summary, MCHC* and the percent high density RBCs decrease significantly within 7 days of the onset of arginine supplementation. When arginine supplementation is withdrawn, the return to baseline values is much slower and required more than 40 days to complete. These observations have important implications for arginine supplementation in sickle cell anemia patients. In particular, the persistence of decreased MCHC* and reduced percent high density RBCs for an extended period of time after supplementation was withdrawn parallels our previously reported observation of persistent improvement of perfusion in sickle transgenic mice after supplementation was withdrawn and suggests that daily administration of arginine may not be necessary once an induction period has passed.

scholarly journals Calcium accumulated by sickle cell anemia red cells does not affect their potassium (86Rb+) flux components

Blood ◽  
1986 ◽  
Vol 67 (3) ◽  
pp. 710-715
Author(s):  
OE Ortiz ◽  
VL Lew ◽  
RM Bookchin
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Membrane Surface ◽  
Red Cells ◽  
Cell Fraction ◽  
K Channel ◽  
Ionophore A23187 ◽  
Na Pump ◽  
Before And After ◽  
86Rb Influx

We investigate here the hypothesis that the high Ca content of sickle cell anemia (SS) red cells may produce a sustained activation of the Ca2+-dependent K+ permeability (Gardos effect) and that the particularly high Ca levels in the dense SS cell fraction rich in irreversibly sickled cells (ISCs) might account for the Na pump inhibition observed in these cells. We measured active and passive 86Rb+ influx (as a marker for K+) in density-fractionated SS cells before and after extraction of their excess Ca by exposure to the Ca ionophore (A23187) and ethylene glycol tetra-acetic acid and with or without adenosine triphosphate depletion or addition of quinine. None of these maneuvers revealed any evidence of a Ca2+-dependent K leak in SS discocytes or dense cells. Na pump inhibition in the dense SS cells was associated with normal activation by external K+ and a low Vmax that persisted after Ca extraction from the cells. These results are consistent with our recent findings that the excess Ca in these cells is compartmentalized in intracellular inside-out vesicles and unavailable as free Ca2+ to the inner membrane surface. Although the steady-state free cytoplasmic Ca2+ in oxygenated SS cells must be below the levels needed to activate the K+ channel, possible brief activation of the channels of some SS cells resulting from transient elevations of cell Ca2+ during deoxygenation-induced sickling cannot be excluded. The dense, ISC-rich SS cell fraction showed a Ca2+-independent increase in the ouabain-resistant, nonsaturable component of 86Rb+ influx that, if uncompensated by Na+ gain, could contribute to the dehydration of these cells.

scholarly journals Reticulocyte Survival in Sickle Cell Anemia: Effect of Cyanate

Blood ◽  
1972 ◽  
Vol 40 (5) ◽  
pp. 733-739 ◽  
Author(s):  
Blanche P. Alter ◽  
Yuet Wai Kan ◽  
David G. Nathan
Keyword(s):  
Cell Survival ◽  
Sickle Cell ◽  
Fetal Hemoglobin ◽  
Red Cells ◽  
Red Cell ◽  
Hemoglobin Molecule ◽  
Red Cell Survival ◽  
And Control

Abstract Cyanate prevents sickling in vitro and apparently prolongs the survival of 51Cr-tagged sickle erythrocytes in vivo. Cautious interpretation is required because the effects of cyanate on 51Cr binding to sickle and fetal hemoglobin-containing red cells are unknown, and comparison of the effect of cyanate on sickle red cell survival to control red cell survival must be performed sequentially. We have studied the survival of sickle reticulocytes utilizing radioactive amino acids that are incorporated into hemoglobin. Two informed adult patients with sickle cell disease were studied. In each study, two 50-ml samples of blood were incubated separately with 14C- and 3H-leucine for 2 hr, after which 50 mM cyanate was added to one aliquot for 1 hr. The cells were then washed and reinfused. Frequent venous samples were obtained, and the specific activities of 14C and 3H in the hemoglobin were followed. The t ½ of the carbamylated cells was tripled, but remained below normal. This method provides a generally useful measurement of the influence of drugs bound to red cells on reticulocyte lifespan. The labels are incorporated into the hemoglobin molecule of the reticulocyte, and simultaneous comparison of the survivals of the same cohort of drug-treated and control cells is achieved.

scholarly journals Increased Ca++, Mg++, and Na+ + K+ ATPase activities in erythrocytes of sickle cell anemia

Blood ◽  
1982 ◽  
Vol 60 (6) ◽  
pp. 1332-1336 ◽  
Author(s):  
MG Luthra ◽  
DA Sears
Keyword(s):  
Enzyme Activity ◽  
Blood Cell ◽  
Atpase Activity ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Red Blood Cell ◽  
Calcium Binding ◽  
Red Cells ◽  
Low Density ◽  
Red Cell

Abstract To determine whether diminished activity of the Ca++ extrusion pump could account for the high levels of red blood cell (RBC) Ca++ in sickle cell anemia (SS), we measured calmodulin-sensitive Ca++ ATPase activity in normal and SS RBC. Hemolysates prepared with saponin were compared, since such preparations expressed maximum ATPase activities, exceeding isolated membranes or reconstituted systems of membranes plus cytosol, SS RBC hemolysates had greater Ca++ ATPase activity than normal hemolysates; they exhibited higher Mg++ and Na+ + K+ ATPase activities as well. Assays on density (age) fractions of SS and normal red cells demonstrated that all ATPase activities were highest in low density (young) cells, and activities in SS red cells exceeded those in normals in all fractions studied. Thus, when studied under conditions that maximize enzyme activity, Ca++ ATPase activity, like Mg++ and Na+ + K+ ATPase, is actually increased in SS RBC, probably due to the young red cell population present. The elevated Ca++ levels in these cells are more likely due to an increased Ca++ leak or abnormal calcium binding than to defective extrusion by the ATPase pump.

scholarly journals Red cells in sickle cell crisis: observations on the pathophysiology of crisis

Blood ◽  
1977 ◽  
Vol 49 (6) ◽  
pp. 967-979 ◽  
Author(s):  
EE Rieber ◽  
G Veliz ◽  
S Pollack
Keyword(s):  
Sickle Cell ◽  
Red Cells ◽  
Red Cell ◽  
Molecular Abnormality ◽  
Sickle Cell Crisis ◽  
Sickle Cell Crises

Abstract The pathophysiology of the occurrence and resolution of sickle cell crisis is unknown. The molecular abnormality is constant, while crisis is episodic. In the present study, red cell filterability and sickling with deoxygenation have been measured during sickle cell crises. Recovery from sickle crisis is associated with an increased filterability of the circulating red cell and a decreased susceptibility of the red cell to sickle with deoxygenation (p less than 0.05). The possibility that these changes are responsible for the resolution of crisis is suggested.

Determinants of Sickle Cell Density Distribution: A Study of Benin Twins.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1244-1244
Author(s):  
Mary E. Fabry ◽  
Anne C. Rybicki ◽  
Sandra M. Suzuka ◽  
M. Cherif Rahimy ◽  
Rajagopal Krishnamoorthy ◽  
...  
Keyword(s):  
Density Distribution ◽  
Cell Density ◽  
Sickle Cell ◽  
Dna Analysis ◽  
Complex Mixture ◽  
Density Gradients ◽  
Red Cell ◽  
Alpha Thalassemia ◽  
Gardos Channel ◽  
One Year

Abstract Red cell density distribution affects both hemolysis and vaso-occlusion; however, currently recognized factors cannot account for all of the variation seen. We hypothesized that a range of genetically controlled factors contributes to red cell density distribution and hemolysis, which has recently received a great deal of attention from Gladwin et al for its role in sickle cell anemia (SCA) and its impact on NO metabolism. Our previous studies have demonstrated that although RBC density distribution varies significantly from patient to patient, the pattern for individual patients is stable in the absence of disease. Some genetically determined factors that affect red cell density distribution have been defined, such as alpha-thalassemia and % HbF (Fabry et al, Blood, 1982); however, neither of these factors completely predicts density distribution. The study of identical twins offers the unique opportunity to minimize some of the genetic variability between individuals that may not be relevant to RBC density while allowing the remaining differences to be detected. Because SCA patients from the US have a complex mixture of Caucasian, other ethnicities, and genes from all parts of Africa including all of the sickle haplotypes, we have chosen to recruit our population from Benin that has a single beta-globin haplotype, thus further minimizing differences arising from admixture from inside and outside of Africa. We have collected samples from six sets of monozygous twins from Benin and validated their monozygosity by DNA analysis. Of the 6 twin sets analyzed to date, 4 have alpha-thalassemia. We compared density gradients on two separate occasions, approximately one year apart, for these twins and found that density gradients for both members of all twin sets without medical complications (malaria, painful crisis) were indistinguishable. This is not true for pairs of randomly chosen individuals even after their % HbF and alpha-thalassemia status has been determined and matched. After elimination of WBCs, we were able to isolate sufficient RNA to obtain microarray data without amplification. We compared subjects with a low % dense cells vs a high % dense cells. In the combinations that were analyzed, we found a consistent pattern of up- and down-regulated genes. Down-regulated genes included the Gardos channel (KCNN4), K-Cl cotransporter (KCC1), NOS3, CAIV, and PKC. Up-regulated genes included ferritin heavy chain (probably in mitochondria that are present in reticulocytes) and 2,3-bisphosphomutase that was elevated in twins with higher MCHC (density). The latter is consistent with our previous observations and those of Poillon et al that DPG can affect polymer formation and RBC density. We conclude that the study of twins demonstrates that there is a strong genetic component in the control of sickle cell density distribution and that a better understanding of factors controlling density distribution may lead to new forms of treatment.

2001 Lemieux Award Lecture Organic chemistry and hemoglobin: Benefits from controlled alteration

2002 ◽  
Vol 80 (3) ◽  
pp. 217-221 ◽  
Author(s):  
Ronald Kluger
Keyword(s):  
Organic Chemistry ◽  
Nitric Oxide ◽  
Blood Pressure ◽  
Oxygen Affinity ◽  
Red Cells ◽  
Red Cell ◽  
Native Protein ◽  
Cross Link ◽  
Cross Links ◽  
Internal Electron

Hemoglobin carries oxygen in circulation within red cells but does not function outside the cells because it fails not only to release oxygen but also dissociates into dimers that make up the tetrameric protein. Bifunctional anionic acylating agents that contain a structurally rigid bridge introduce cross-links that stabilize hemoglobin and alter its oxygen affinity so that it could be used to carry oxygen outside cells. Nitric oxide binds to hemoglobin and in circulation this causes undesirable increases in blood pressure. It had been reported that higher weight collections of hemoglobin do not cause vasoconstriction. Reagents with two pairs of reaction sites joined by a rigid link connect and cross-link two hemoglobins. The resulting bis-tetramers lack the cooperativity of the native protein and bind oxygen too tightly to be useful; occupation by oxygen blocks the sites from nitric oxide. Nitric oxide may be delivered from thionitrosyl groups, which occur in hemoglobin in the red cell. Cross-linked hemoglobin can be specifically nitrosylated. These species can then serve as circulating sources of nitric oxide resulting from an internal electron transfer.Key words: proteins, hemoglobin, cross-link, red cells, cooperativity, connecting.

scholarly journals The incidence of painful crisis in homozygous sickle cell disease: correlation with red cell deformability

Blood ◽  
1988 ◽  
Vol 72 (6) ◽  
pp. 2056-2059
Author(s):  
WM Lande ◽  
DL Andrews ◽  
MR Clark ◽  
NV Braham ◽  
DM Black ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Hemoglobin Concentration ◽  
Red Cells ◽  
Strong Positive Correlation ◽  
Red Cell ◽  
Cell Disease ◽  
Cell Deformability ◽  
Red Cell Deformability ◽  
Cellular Dehydration ◽  
Painful Crisis

To determine whether the vasoocclusive severity of homozygous sickle cell (SS) disease is influenced by cellular dehydration, we correlated the incidence of painful crisis with steady-state measurements of red cell hydration. Sixteen children with SS disease were followed for 3.3 to 8 years (mean, 6.8 years), and a single crisis rate was calculated for each patient. At the time of well visits, cellular hydration was assessed by measuring cell deformability, the percentage of red cells with a density greater than or equal to 1.1056 g/mL, and the percentage of irreversibly sickled cells (ISC). The incidence of painful crisis showed a strong positive correlation with Omax, a deformability measurement reflecting cellular hydration (r = .84, P less than .002), and with hemoglobin concentration (r = .59, P = .04). That is, higher crisis rates were observed in patients with less dehydrated, more deformable red cells and also in patients with higher hemoglobin concentrations. Furthermore, cell deformability and hemoglobin concentration were independent predictors of the incidence of painful crisis, which is consistent with separate effects of these two red cells parameters on vasoocclusive severity.

scholarly journals Increased Ca++, Mg++, and Na+ + K+ ATPase activities in erythrocytes of sickle cell anemia

Blood ◽  
1982 ◽  
Vol 60 (6) ◽  
pp. 1332-1336
Author(s):  
MG Luthra ◽  
DA Sears
Keyword(s):  
Enzyme Activity ◽  
Blood Cell ◽  
Atpase Activity ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Red Blood Cell ◽  
Calcium Binding ◽  
Red Cells ◽  
Low Density ◽  
Red Cell

To determine whether diminished activity of the Ca++ extrusion pump could account for the high levels of red blood cell (RBC) Ca++ in sickle cell anemia (SS), we measured calmodulin-sensitive Ca++ ATPase activity in normal and SS RBC. Hemolysates prepared with saponin were compared, since such preparations expressed maximum ATPase activities, exceeding isolated membranes or reconstituted systems of membranes plus cytosol, SS RBC hemolysates had greater Ca++ ATPase activity than normal hemolysates; they exhibited higher Mg++ and Na+ + K+ ATPase activities as well. Assays on density (age) fractions of SS and normal red cells demonstrated that all ATPase activities were highest in low density (young) cells, and activities in SS red cells exceeded those in normals in all fractions studied. Thus, when studied under conditions that maximize enzyme activity, Ca++ ATPase activity, like Mg++ and Na+ + K+ ATPase, is actually increased in SS RBC, probably due to the young red cell population present. The elevated Ca++ levels in these cells are more likely due to an increased Ca++ leak or abnormal calcium binding than to defective extrusion by the ATPase pump.

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